Preparation method of MOFs coating microneedle and application of MOFs coating microneedle in rapid DNA extraction

Abstract

The invention relates to a preparation method of an MOFs (Metal-Organic Frameworks) coating microneedle. The preparation method comprises the following steps: preparing a PDMS (Polydimethylsiloxane) female mold with a microneedle model area; preparing a polystyrene polymer solution by using N, N-dimethylformamide, pouring the polystyrene polymer solution to a microneedle model area, and drying and demolding to obtain a polystyrene microneedle; preparing an MOFs precursor solution; a polystyrene microneedle is inversely arranged on the liquid level of the MOFs precursor solution, the solution is kept still for 3-7 days under the room temperature condition, and a single layer of compact and uniform MOFs coating grows on the surface of the polystyrene microneedle in situ; and taking out, washing and airing to obtain the MOFs coating microneedle with the in-situ growth MOFs coating. The method disclosed by the invention has specific enrichment and extraction capability on DNA and can be repeatedly used, and a rapid DNA extraction kit is developed to realize repeatable extraction of DNA in DNA liquid or plant samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of microneedles, and in particular relates to a method for preparing a MOFs-coated microneedle and an application thereof in rapid DNA extraction. Background Art

[0002] DNA extraction has great application prospects in molecular biology detection methods, and has important application value in many fields such as disease diagnosis, environmental monitoring, and food safety. However, in disease diagnosis, the enrichment and extraction technology of DNA in tissue fluid has a significant impact on its detection efficiency. Traditional DNA extraction methods are complicated and use the toxic reagent chloroform, while commercially available DNA extraction kits are time-consuming, usually taking 3 to 5 hours to complete an effective extraction process. Therefore, the development of a simple DNA enrichment and extraction technology is of great significance for achieving rapid DNA extraction.

[0003] Microneedles are fine, needle-like micrometer-scale arrays ranging in height from 25 to 1500 μm. Since their development, microneedles have been widely used in drug delivery, tissue fluid sampling, immunotherapy, and vaccine delivery due to their excellent mechanical properties, minimal invasiveness, and painlessness. In tissue fluid sampling, hydrogel microneedles, due to their excellent swelling capacity, can rapidly aspirate skin tissue fluid, allowing subsequent in vitro detection of target analytes. Patent CN 118222561A discloses a method for rapidly extracting genomic DNA from mushrooms using polymer microneedles. Polyvinyl alcohol polymer microneedles are directly inserted into fresh mushroom tissue to extract intracellular DNA and other molecules. The extracted DNA is then collected using a buffered wash and analyzed by real-time fluorescence PCR using primer sequences. Because this method utilizes the swelling properties of the polymer to extract tissue fluid, the extracted fluid contains not only DNA but also other macromolecular proteins, small molecules, and other substances. This increases the operational complexity of specific DNA extraction and detection. In addition, invention patent CN 118530986 A discloses a rapid DNA extraction kit based on an acidic composite microneedle patch and its applications. In this invention, chitosan is used to protonate its amino groups under acidic conditions, resulting in positive charges. This allows for high-affinity binding to naturally negatively charged DNA, enabling DNA capture. However, the acidic environment (pH = 4.0-5.5) is not suitable for DNA extraction from tissue fluid, and the efficiency of DNA extraction through electrostatic interactions is low. Invention patent CN 112321870 B discloses a polyvinyl alcohol microneedle based on metal oxides, which utilizes the interaction between the metal oxides and the DNA phosphate backbone to enhance DNA extraction. This invention achieves DNA enrichment to a certain extent, but the preparation process of metal oxide-doped polymer microneedles is relatively complex, requiring polymer dissolution and microneedle preparation at 90°C-100°C. Furthermore, the uniformity of the metal oxide doping is not controllable, and due to the swelling properties of the polymer, reproducible DNA extraction from the same microneedle array is not possible. In addition, invention patent CN 114652672 A discloses a method for preparing a metal organic framework (MOF) microneedle patch with a core-shell structure. In this invention, the MOFs material is first synthesized, and then the MOFs material is added to the polymer solution required for preparing the microneedles in a certain proportion to prepare a core-shell microneedle with MOFs loading, and it is applied to the extraction of interstitial fluid and drug delivery. This invention loads MOFs inside the microneedle, solving the problem of high brittleness and poor mechanical strength of MOF. However, the MOFs loading process of this method is cumbersome, and the MOFs material must be synthesized first and then combined with the polymer solution to prepare the core-shell microneedle, which adds a certain degree of difficulty to the operation steps.In addition, since MOFs are added to polymer solutions to prepare core-shell microneedles, during the drying process of the microneedles, most of the MOFs particles will gather at the tip of the microneedles due to gravity, which will make it impossible to control the uniformity of the loading rate of MOFs between the microneedle bodies.

[0004] In summary, existing microneedle DNA extraction methods have the following problems: the swelling method results in poor DNA-specific extraction, the electrostatic interaction method's acidic environment limits its application in tissue fluids, the metal oxide method's microneedle preparation process is complex, and the microneedles prepared by these methods are not reusable and can only be used once. Furthermore, adding MOFs to a microneedle polymer solution to prepare MOF-loaded core-shell microneedles has problems such as the inability to determine the MOF loading rate and the inability to achieve uniform MOF loading. Summary of the Invention

[0005] Purpose of the Invention: This invention proposes a method for preparing microneedles with dense and uniform MOF coatings and their application in rapid DNA extraction. Under mild conditions, MOFs are grown directly on the surface of polystyrene microneedles via in situ growth, resulting in reusable, single-layer MOF-coated microneedles with specific DNA enrichment and extraction capabilities. Furthermore, a rapid DNA extraction kit is developed to enable reproducible DNA extraction from liquid or plant samples.

[0006] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] The first aspect of the present invention provides a method for preparing MOFs-coated microneedles, comprising the following steps:

[0008] S1. Prepare a mold according to the designed microneedle size, prepare a PDMS master mold based on the mold, and then remove the mold to form a PDMS master mold with a microneedle model area;

[0009] S2. Using N,N-dimethylformamide as a solvent to prepare a polystyrene polymer solution of a certain concentration, pouring it into the microneedle model area of ​​the PDMS master mold, and drying and demolding to obtain polystyrene microneedles. The polystyrene microneedles have a structure in which multiple needles are arrayed on a substrate;

[0010] S3, preparing a MOFs precursor solution with a fixed ratio;

[0011] S4. Inverting the polystyrene microneedles obtained in step S2, placing the needle bodies in the MOFs precursor solution with the bottom surface of the substrate above the liquid surface of the MOFs precursor solution, and keeping the solution undisturbed at room temperature for 3 to 7 days to allow a dense and uniform MOFs coating to grow in situ on the surface of the polystyrene microneedles;

[0012] S5. Take out the polystyrene microneedles, rinse them with ethanol, then rinse them with deionized water, and dry them to obtain MOFs-coated microneedles with in situ grown MOFs coatings.

[0013] Furthermore, the average molecular weight of the polystyrene is 260,000; and the certain concentration is a mass ratio of the polystyrene polymer to N,N-dimethylformamide of 15% to 25%.

[0014] Furthermore, MOFs is any one of UiO-66-COOH, UiO-66-NH2, ZIF-8, ZIF-67, MIL-53(Al), and Eu-MOF.

[0015] Preferably,

[0016] The concentration of UiO-66-COOH is anhydrous zirconium chloride: 1,2,4-benzenetricarboxylic acid: formic acid: deionized water in a mass volume ratio of 350-700 mg: 350-700 mg: 7 mL: 16 mL.

[0017] The concentration of UiO-66-NH2 is anhydrous zirconium chloride: 2-aminoterephthalic acid: formic acid: deionized water: ethanol mass volume ratio = 235~470mg:181~362mg:7mL:16mL:20mL;

[0018] The ZIF-8 concentration is Zn(NO3)2·6H2O:2-methylimidazole:methanol mass volume ratio = 73.5~147mg:164~328mg:14mL;

[0019] The ZIF-67 concentration is Co(NO3)2·6H2O:2-methylimidazole:methanol mass volume ratio = 72.8~145.6mg:164~328mg:14mL;

[0020] The concentration of MIL-53(Al) is Al(NO3)3·9H2O: sodium terephthalate: deionized water (mass volume ratio = 250-500 mg: 140-280 mg: 7 mL);

[0021] The Eu-MOF concentration is Eu(NO3)3·6H2O: disodium terephthalate: deionized water mass volume ratio = 89.2~178.4 mg: 210~420 mg: 16 mL.

[0022] The second aspect of the present invention provides an application of the preparation method of the MOFs-coated microneedles in rapid DNA extraction.

[0023] The third aspect of the present invention is a rapid DNA extraction kit, comprising MOFs-coated microneedles obtained by the preparation method according to claim 1, a cleaning solution and an eluent.

[0024] Furthermore, the steps for using the DNA rapid extraction kit are as follows:

[0025] (1) Place the MOFs-coated microneedle into the liquid sample or press it onto the sample surface, maintain this operation for 15 to 20 minutes, and extract the DNA from the sample to be tested;

[0026] (2) peeling the MOFs-coated microneedles from the sample surface, washing the impurities adsorbed on the surface of the MOFs-coated microneedles with deionized water, and then rinsing the microneedles with 10 mM phosphate buffer to collect the extracted target DNA;

[0027] (3) performing PCR on the extracted target DNA using the corresponding primer sequences to obtain amplified DNA;

[0028] (4) After the target DNA is extracted, the MOFs-coated microneedles are thoroughly cleaned with eluent and deionized water. After completion, the next extraction can be repeated.

[0029] Furthermore, the kit is used to quickly extract DNA from liquid samples or plant leaves.

[0030] Furthermore, the kit can be reused 5 to 7 times.

[0031] Beneficial Effects: The present invention can produce microneedles with a uniform and dense monolayer MOF coating at room temperature. The method involves placing a polystyrene microneedle upside down in a MOF precursor solution, with the needle body positioned within the solution and the bottom surface of the substrate above the MOF precursor solution. The solution is then kept undisturbed at room temperature for 3 to 7 days. This means that the in situ growth period is longer than the time it takes for the MOF precursor solution to generate MOF particles, thereby ensuring the in situ growth of a dense and uniform monolayer MOF coating on the surface of the polystyrene microneedle. π-π interactions are generated between the polystyrene and the organic ligands in the MOF precursor solution, making it easier for the ligands to bind to the polystyrene surface. Metal-organic frameworks can then be grown in situ on the polystyrene surface without any modification of the polystyrene. Subsequently, coordination occurs between the metal ions and the ligands, leading to coordination-driven self-assembly on the polystyrene surface to form small nuclei. As the reaction time increases, the small nuclei slowly grow to form MOF particles, ultimately forming a uniform and dense monolayer modified layer on the polystyrene microneedle surface. In addition, solid surface potential results show that the polystyrene surface is slightly negatively charged, which promotes the migration of positively charged metal ions to the polystyrene surface and further promotes the coordination between the metal ions and the surface organic ligands. This invention solves the current problem of uncontrollable uniformity of MOFs loading on microneedles.

[0032] Secondly, due to the many excellent properties of MOFs, such as rich functional groups, high porosity, large specific surface area, metal ion clusters, etc., the obtained MOFs coating microneedles have excellent binding ability to DNA chains, but have extremely weak binding ability to large molecular proteins (such as bovine serum albumin) and small molecules (such as rhodamine). This makes the MOFs coating microneedles prepared by the present invention expected to be used for rapid extraction of DNA from multiple types of test samples. It has the advantages of simple operation, minimally invasive, and no need for in vitro purification, which is convenient for subsequent detection and solves the problem of impure interstitial fluid targets extracted by hydrogels using swelling and water absorption properties. Finally, due to the excellent stability of the MOFs coating, the prepared MOFs microneedles can achieve repeated DNA extraction after being washed with phosphate buffer, which solves the problem that current hydrogel microneedles or metal oxide-loaded hydrogel microneedles can only be used for one-time extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the preparation and application process of metal-organic framework-coated microneedles;

[0034] Figure 2 Effect of precursor concentration on the density of UiO-66-COOH coating; A is the in-situ growth modification with a 4-fold dilution of the precursor concentration; B is the in-situ growth modification with a 2-fold dilution of the precursor concentration; C is the in-situ growth modification with the original precursor concentration;

[0035] Figure 3 A is a UiO-66-COOH coated microneedle grown in situ close to the surface of the precursor solution; B is a UiO-66-COOH coated microneedle modified by sinking to the bottom of the precursor solution;

[0036] Figure 4 The stability of the UiO-66-COOH coated microneedles before and after piercing simulated skin and pig skin; A is the coating distribution before piercing; B is the coating distribution after piercing agarose; C is the coating distribution after piercing pig skin;

[0037] Figure 5 Three-dimensional isosurface plots at different angles between a polystyrene molecule (n=1) and the organic ligand 1,2,4-benzenetricarboxylic acid molecule; the green portion is the IGMH isosurface;

[0038] Figure 6 Zeta potential diagram of the in situ growth system; DI is deionized water; HCOOH is formic acid; DI+HCOOH is deionized water and formic acid; PS MNs is the solid potential of the polystyrene microneedle surface;

[0039] Figure 7Optical photos of DNA extraction media from metal-organic framework-coated microneedles; a: polystyrene microneedle; b: UiO-66-COOH-coated microneedle; c: UiO-66-NH2-coated microneedle;

[0040] Figure 8 is the scanning electron microscopy image of PS MNs;

[0041] Figure 9 Scanning electron micrograph of UiO-66-COOH coated microneedles;

[0042] Figure 10 This is a scanning electron micrograph of the UiO-66-NH2 coated microneedles;

[0043] Figure 11 This is a scanning electron micrograph of ZIF-8 coated microneedles;

[0044] Figure 12 This is a scanning electron micrograph of the ZIF-67 coating;

[0045] Figure 13 is a scanning electron micrograph of MIL-53(Al) coated microneedles;

[0046] Figure 14 This is a scanning electron micrograph of Eu-MOF coated microneedles;

[0047] Figure 15 This is a time-dependent dynamic diagram of the formation process of UiO-66-COOH coated microneedles;

[0048] Figure 16 The surface coating of UiO-66-COOH coated microneedles, A is in stirring state; B is in static state; Figure 17 The results of DNA extraction from pepper leaves using UiO-66-COOH coated microneedles are shown in Figure 1. 1 is a blank group; 2 is extraction using polystyrene microneedles; 3 is extraction using UiO-66-COOH coated microneedles.

[0049] Figure 18 Figure 1 shows the results of DNA extraction from tomato leaves using UiO-66-COOH coated microneedles; Figure 1 shows the blank group; Figure 2 shows extraction using polystyrene microneedles; Figure 3 shows extraction using UiO-66-COOH coated microneedles;

[0050] Figure 19 Figure 1 shows the results of DNA extraction from pepper leaves using UiO-66-COOH-coated microneedles repeated seven times.

[0051] Figure 20This is the result of DNA extraction from milk using UiO-66-COOH coated microneedles. In the figure, 1 is the blank group; 2 is the polystyrene microneedle extraction; and 3 is the UiO-66-COOH coated microneedle extraction. DETAILED DESCRIPTION

[0052] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0053] In the embodiments of the present invention, the polystyrene used is provided by Thermo Fisher Scientific; N,N-dimethylformamide, acetonitrile, acetic acid, anhydrous methanol, and anhydrous ethanol are all provided by Tianjin Fuyu Fine Chemical Co., Ltd.; formic acid (purity 99%) is provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; anhydrous zirconium chloride, 2-aminoterephthalic acid, polyacrylic acid, aluminum nitrate nonahydrate (Al(NO3)3·9H2O), disodium terephthalate, and uranium nitrate hexahydrate (Eu(NO3)3·6H2O) are provided by Beijing Inokai Technology Co., Ltd.; 2-methylimidazole and 1,2,4-benzenetricarboxylic acid are provided by Shanghai MacLean Biochemical Technology Co., Ltd.; terephthalic acid, zinc nitrate hexahydrate (Zn(NO3)2·6H2O), and cobalt nitrate hexahydrate (Co(NO3)2·6H2O) are provided by Sinopharm Chemical Reagent Co., Ltd.

[0054] The present invention provides a method for preparing microneedles with a uniform and dense single-layer MOFs coating by in-situ growing a metal organic framework material on the surface of polystyrene at room temperature without any modification of the polystyrene. Figure 1 As shown, the following steps are included:

[0055] S1. Prepare a mold according to the designed microneedle size, prepare a PDMS master mold based on the mold, and then remove the mold to form a PDMS master mold with a microneedle model area;

[0056] 1.1 Designing the Dimensions of the Microneedle Model: In this example, SolidWorks software was used to design the dimensions of the microneedle model. The microneedle dimensions can be flexibly designed. The microneedle height can be designed to be 800 to 1500 μm, and the microneedles can be conical or quadrangular pyramidal. The base diameter can be 400 to 800 μm, and the interneedle spacing can be 600 to 2000 μm. The polystyrene microneedle dimensions can be flexibly designed as needed.

[0057] 1.2 Prepare a PDMS master mold: 3D print the microneedle model according to its dimensions, and after curing, obtain a mold for the microneedle model. Prepare a PDMS solution by mixing polydimethylsiloxane (PDMS) and a curing agent in a mass ratio of 10:1. Place the 3D-printed microneedles, needle-side up, in a 3cm Petri dish. Pour the PDMS solution into the dish, vacuum-freeze until it is clear, and cure at 80°C for 30 minutes. After cooling to room temperature, demold the PDMS master mold (remove the 3D-printed mold) to obtain the PDMS master mold with the microneedle model area.

[0058] S2. Use N,N-dimethylformamide as a solvent to prepare a polystyrene polymer solution of a certain concentration, pour it into the microneedle model area of ​​the PDMS master mold, and after drying and demolding, obtain a polystyrene microneedle structure with multiple needle bodies arranged in an array above the substrate; this embodiment does not limit the specific array arrangement structure, and it can be a directional array, a circular array, etc.

[0059] S3, preparing a MOFs precursor solution with a fixed ratio;

[0060] MOFs is any one of UiO-66-COOH, UiO-66-NH2, ZIF-8, ZIF-67, MIL-53 (Al), and Eu-MOF. Due to the many excellent properties of MOFs, such as rich functional groups, high porosity, large specific surface area, metal ion clusters, etc., the obtained MOFs-coated microneedles have specific binding ability to DNA chains, but have extremely weak binding ability to large molecular proteins (such as bovine serum albumin) and small molecules (such as rhodamine). The extracted DNA is more specific to DNA than the swelling method and has less impurity content. As an in situ array DNA extraction medium, MOFs-coated microneedles extract DNA through multiple interaction forces between MOF and DNA. The interaction force is mainly based on the coordination effect between the MOFs skeleton and the phosphate group of the DNA chain. This makes the MOFs-coated microneedles prepared by the present invention expected to be used for rapid extraction of DNA from multiple types of test samples, with the advantages of simple operation, minimal invasiveness, and no need for in vitro purification, which is convenient for subsequent detection.

[0061] Different MOFs precursor solutions were prepared according to different MOFs synthesis conditions. For example, UiO-66-COOH was anhydrous zirconium chloride (ZrCl4) and 1,2,4-benzenetricarboxylic acid (BDC-COOH) with a molar ratio of 1:1, and a solvent formic acid: deionized water volume ratio of 3.5:8; UiO-66-NH2 was anhydrous zirconium chloride: 2-aminoterephthalic acid with a molar ratio of 1:1, and a solvent formic acid: deionized water: ethanol volume ratio of 3.5:8:10; ZIF-8 was Zn( NO3)2·6H2O:2-methylimidazole molar ratio = 1:8, the solvent is anhydrous methanol; ZIF-67 is Co(NO3)2·6H2O:2-methylimidazole molar ratio = 1:8, the solvent is anhydrous methanol; MIL-53(Al) is sodium terephthalate:Al(NO3)3·9H2O molar ratio = 1:2, the solvent is deionized water; Eu-MOF is disodium terephthalate:Eu(NO3)3·6H2O molar ratio = 5:1, the solvent is deionized water.

[0062] like Figure 2 As shown, the density of MOF coatings can be controlled by adjusting the concentration of the MOF precursor. As the precursor concentration increases, the coating density increases. When the UiO-66-COOH precursor concentration is diluted 4-fold for in situ growth, the resulting UiO-66-COOH coating on the polystyrene microneedle surface has an extremely low density and fails to form a dense layer structure. However, when the UiO-66-COOH precursor concentration is diluted 2-fold for in situ growth, the resulting UiO-66-COOH coating on the polystyrene microneedle surface is uniform and dense, growing across the entire polystyrene microneedle surface. When the UiO-66-COOH precursor concentration is not diluted and in situ growth is performed directly, the resulting UiO-66-COOH coating on the polystyrene microneedle surface is even denser. To obtain a dense and uniform MOF coating, the precursor solution can be diluted from the original precursor concentration to 2-fold before in situ growth modification.

[0063] S4. Placing the polystyrene microneedle obtained in step S2 into a MOFs precursor solution with the bottom surface of the substrate located above the liquid surface of the MOFs precursor solution. Keeping the solution still at room temperature for 3 to 5 days allows a dense and uniform MOFs coating to be grown in situ on the surface of the polystyrene microneedle.

[0064] The average molecular weight of the polystyrene is 260,000.

[0065] During this step, it is important to ensure that the polystyrene microneedles are placed in the MOF precursor solution, with the bottom surface of the substrate above the MOF precursor solution. This means that the polystyrene microneedles are inverted onto the MOF precursor solution surface. This is the only way to achieve a dense and uniform in-situ MOF coating on the polystyrene microneedles. This does not mean that the polystyrene microneedles are suspended in the MOF precursor solution or sink to the bottom. Specifically, as the MOF precursor solution remains stationary for an extended period of time, some organic ligands bind to the polystyrene microneedles, while others bind to metal complexes to form MOF particles. These particles then settle to the bottom of the solution due to gravity.

[0066] When the polystyrene microneedle is inverted on the surface of the MOFs precursor solution, the surface of the polystyrene microneedle will only have a uniform MOFs coating obtained by in situ growth; while the polystyrene microneedle located at the bottom or middle of the solution not only has an in situ grown MOFs coating on its surface, but also the MOFs particles formed in the solution due to gravity will be deposited on the surface of the polystyrene microneedle to form a multi-layer MOFs coated microneedle. It is impossible to determine whether the MOFs on the surface of the polystyrene microneedle are in situ grown MOFs coatings or deposited MOFs particles. The in situ grown MOFs coating is an uneven coating.

[0067] The scanning electron microscope image of the modified coating on the surface of the MOFs-coated microneedles is shown in Figure 1. Figure 3 As shown. It is obvious that the former can obtain a single-layer modified UiO-66-COOH coated microneedle, while the latter can obtain a multi-layer modified UiO-66-COOH coated microneedle. The single-layer UiO-66-COOH coated microneedle was inserted into 2% agarose simulated skin and pig skin to investigate the coating stability. The results are shown in Figure 2. Figure 4 As shown in the figure. After piercing simulated skin, the single-layer coating did not fall off due to friction between the needle and the skin. However, after piercing pig skin, due to the high friction between the raised portions of the microneedle surface and the skin, only a very small portion of the raised coating fell off, while the coating remained evenly distributed over a large area, confirming the excellent stability of the in situ grown single-layer MOF coating. However, after piercing agarose and pig skin, the modified MOFs deposited at the bottom of the precursor solution fell off severely. This is because the MOF particles take precedence over the in situ grown MOF coating, resulting in the majority of the microneedle surface being deposited MOF particles, which is prone to falling off.

[0068] This example uses UiO-66-COOH as an example to illustrate the mechanism of the formation of a single-layer MOFs on the surface of polystyrene. Due to the π-π interaction between polystyrene and the ligand 1,2,4-benzenetricarboxylic acid (the simulation results are shown in Figure 2), the formation of a single-layer MOFs on the surface of polystyrene is shown in Figure 2. Figure 5 As shown in the figure, the isosurface of the complex system of polystyrene molecules and the organic ligand 1,2,4-benzenetricarboxylic acid is very smooth. In the IGMH diagram, the dimer has a large green flat area, which is basically parallel to the benzene rings of the two, confirming the π-π stacking characteristics between the two), making it easier for the organic ligand to bind to the polystyrene surface. Subsequently, a coordination effect occurs between the metal ions and the organic ligands, forming small crystal nuclei on the polystyrene surface. In addition, the solid surface potential results show that the polystyrene surface is slightly negatively charged, which further promotes the positively charged metal ions to tend to bind to polystyrene and coordinate with its surface ligands (Zeta potential data bar graph as shown in the figure). Figure 6 As the reaction time increases, the small crystal nuclei gradually grow into large crystal particles, forming a dense and uniform single-layer MOF coating on the polystyrene surface, solving the current problem of uncontrollable uniformity of MOFs loading on microneedles.

[0069] S5. Take out the polystyrene microneedles, first use a dropper to absorb anhydrous ethanol to rinse the surface of the polystyrene coated microneedles three times, then rinse the surface of the polystyrene coated microneedles with deionized water three times, and after drying, obtain the coated microneedles with in situ grown MOFs coating.

[0070] In summary, the present invention is a method for directly growing MOFs in situ on the surface of microneedles under mild conditions to form a uniform and dense MOFs coating. There is no need to first synthesize MOFs and then perform secondary loading. By utilizing the specific enrichment and extraction ability of MOFs for DNA, a DNA rapid extraction kit based on MOFs-coated microneedles can be developed. Due to its large number of metal coordination sites, metal-organic framework materials can form coordination effects with a large number of phosphate groups in DNA chains. Metal-organic framework material-coated microneedles can achieve rapid and specific DNA extraction. Deionized water in the kit is used as a cleaning agent to clean impurities on the surface of the microneedles. Phosphate buffer is used as a DNA eluent to collect the target DNA extracted by the MOF microneedles for elution for downstream detection. Due to the insolubility of polystyrene solid microneedles and the excellent stability of the MOFs coating, the extraction medium based on MOF-coated microneedles can achieve repeated DNA extraction multiple times, reducing usage costs and preparation costs.

[0071] Therefore, the present invention also proposes the application of the preparation method of MOFs-coated microneedles in rapid DNA extraction, which can be used to prepare DNA rapid extraction kits or DNA rapid extraction microneedle pens and other products.

[0072] A rapid DNA extraction kit includes MOF-coated microneedles, deionized water (cleaning solution), and phosphate buffer (elution solution). The MOF-coated microneedles serve as the extraction medium for the in situ array DNA extraction kit, deionized water serves as the cleaning solution, and phosphate buffer serves as the DNA elution solution.

[0073] The MOF-coated microneedles in the kit can be a four-partition MOF-coated microneedle (in situ array DNA extraction medium), a bottle of deionized water, and a bottle of phosphate buffer. The four-partition MOF-coated microneedles are used for DNA extraction, with each partition being used for multiple, rapid extractions of DNA from the sample being tested. The deionized water is used to clean impurities from the microneedle surface after DNA extraction, and the phosphate buffer is used to elute the target DNA.

[0074] The steps for using the DNA rapid extraction kit are as follows:

[0075] (1) Place the MOFs-coated microneedle into a liquid sample or press it onto the surface of a sample (e.g., a plant leaf) for 15–20 min to extract DNA from the sample.

[0076] (2) The MOFs-coated microneedles were peeled off the surface of the target sample, impurities adsorbed on the surface of the MOFs-coated microneedles were washed with deionized water, and then the microneedles were rinsed with 15 μL of 10 mM phosphate buffer to collect the extracted target DNA;

[0077] (3) Perform PCR on the extracted target DNA using the corresponding primer sequence to obtain amplified DNA.

[0078] (4) After the target DNA is extracted, the surface of the coated microneedle is washed three times with a phosphate eluent, and then the surface of the coated microneedle is washed three times with deionized water. After the extraction medium (MOF-coated microneedle) is thoroughly cleaned, the second extraction can be repeated, and so on. A 3×3 MOFs-coated microneedle can achieve multiple target DNA extractions. Preferably, 5 to 7 times. Example 1 Preparation of UiO-66-COOH-coated microneedles

[0079] 1. Preparation of PS MNs: Before preparing MOFs-coated microneedles, solid polystyrene microneedles, referred to as PS MNs, were prepared in a PDMS master mold using a 15% polystyrene polymer solution (dissolved in N,N-dimethylformamide).

[0080] 2. Preparation of UiO-66-COOH precursor solution: Weigh 700 mg of anhydrous zirconium chloride and add it to 7 mL of formic acid. After sonication to dissolve it, add 16 mL of deionized water and mix well to obtain solution 1. Weigh 700 mg of 1,2,4-benzenetricarboxylic acid (BDC-COOH) and add it to the above solution 1. Sonication to dissolve the BDC-COOH until it is completely dissolved to obtain a white UiO-66-COOH precursor solution.

[0081] 3. Preparation of UiO-66-COOH-coated microneedles: Pour the prepared mixed solution into a 10 cm disposable culture dish, and place the previously prepared PS MNs on the surface of the solution (with the microneedle tips facing downward and ensuring there are no bubbles between the needles. The needles should not sink to the bottom of the container or float in the middle of the container). After this operation is completed, seal the culture dish with parafilm to prevent the evaporation of the formic acid solvent.

[0082] 4. Formation of Coated Microneedles: The culture dish prepared in the previous step was left undisturbed at room temperature for 5 days to allow the in situ growth of UiO-66-COOH on the microneedle surface, forming a UiO-66-COOH coating. During this growth process, π-π interactions between the organic ligands (1,2,4-benzenetricarboxylic acid) and polystyrene, which form the UiO-66-COOH, initially caused it to adhere to the polystyrene microneedle surface. Coordination between the metal ions and the organic ligands, as well as weak electrostatic interactions between the metal ions and polystyrene, caused the metal ions to coordinate with the organic ligands on the polystyrene microneedle surface, driving self-assembly to form small UiO-66-COOH crystal nuclei. These small nuclei gradually grew into UiO-66-COOH particles, subsequently forming a uniform UiO-66-COOH coating.

[0083] 5. Washing of coated microneedles: Remove the modified microneedles from the growth environment and wash the microneedle surface three times with deionized water to remove unreacted monomers and unbound UiO-66-COOH particles on the surface. After the water on the microneedle surface evaporates, store the obtained UiO-66-COOH coated microneedles at room temperature for later use.

[0084] Optical photographs of PS MNs are shown in Figure 2. Figure 7 As shown in a, it is transparent; after modification with the UiO-66-COOH coating, a white coating can be clearly observed on the surface of the microneedle ( Figure 7 b); Figure 8 Scanning electron microscope images show that the surface of the polystyrene microneedles without MOFs coating is smooth. Figure 9 Scanning electron microscope images show that a dense and uniform single-layer MOFs coating can be observed on the polystyrene surface after modification of the UiO-66-COOH coating, proving the successful preparation of the in situ grown UiO-66-COOH coated microneedles of the present invention.

[0085] Example 2 Preparation of UiO-66-NH2 Coated Microneedles

[0086] 1. Preparation of PS MNs: Before preparing MOFs-coated microneedles, solid polystyrene microneedles, referred to as PS MNs, were prepared in a PDMS master mold using a 15% polystyrene polymer solution (dissolved in N,N-dimethylformamide).

[0087] 2. Preparation of UiO-66-NH2 precursor solution: Weigh 470 mg of anhydrous zirconium chloride and add it to 7 mL of formic acid. After sonication to dissolve it, add 16 mL of deionized water and 20 mL of anhydrous ethanol and mix well to obtain solution 1. Weigh 362 mg of 2-aminoterephthalic acid (BDC-NH2) and add it to the above solution 1 three times while sonicating to dissolve it. After the BDC-NH2 is completely dissolved, a brown UiO-66-NH2 precursor solution is obtained.

[0088] 3. Preparation of UiO-66-NH2-coated microneedles: The mixed solution prepared above was quickly poured into a 10 cm disposable culture dish, and the previously prepared PS MNs were placed above the solution surface (the microneedle tips were facing downward, and there were no bubbles between the needle bodies. They should not sink to the bottom of the container or float in the middle of the container). After this operation was completed, the culture dish was sealed with parafilm to prevent the volatilization of formic acid and anhydrous ethanol solvents.

[0089] 4. Formation of Coated Microneedles: The culture dish prepared in the previous step was left undisturbed at room temperature for 5 days to allow the in situ growth of UiO-66-NH2 on the microneedle surface, forming a UiO-66-NH2 coating. During this growth process, π-π interactions between the organic ligand 2-aminoterephthalic acid (2-ATA) and polystyrene initially caused the UiO-66-NH2 to adhere to the polystyrene microneedle surface. Coordination between the metal ion and the organic ligand, as well as weak electrostatic interactions between the metal ion and polystyrene, caused the metal ion to coordinate with the organic ligand on the polystyrene microneedle surface, driving self-assembly to form small UiO-66-NH2 nuclei. These nuclei gradually grew into UiO-66-NH2 particles, subsequently forming a uniform UiO-66-NH2 coating.

[0090] 5. Washing of coated microneedles: The modified microneedles were taken out of the growth environment, and the surface of the microneedles was washed three times with anhydrous ethanol to remove the unreacted MOF precursor. The surface of the microneedles was then washed three times with deionized water to remove the UiO-66-NH2 particles that were not firmly bound to the surface. After the water on the surface of the microneedles evaporated, the obtained UiO-66-NH2 coated microneedles were stored at room temperature for use.

[0091] After modification with UiO-66-NH2 coating, a light yellow coating can be clearly observed on the surface of the microneedle ( Figure 7 c), Scanning electron microscopy of UiO-66-NH2 coated microneedles Figure 10 Compared with the unmodified MOFs-coated polystyrene microneedle surface ( Figure 8 ), uniform and dense UiO-66-NH2 particles distributed in a single layer can be clearly observed on the surface of the UiO-66NH2 coated microneedles, proving the successful preparation of the UiO-66-NH2 coated microneedles.

[0092] Example 3 Preparation of ZIF-8 coated microneedles

[0093] 1. Preparation of PS MNs: Before preparing MOFs-coated microneedles, solid polystyrene microneedles, referred to as PS MNs, were prepared in a PDMS master mold using a 15% polystyrene polymer solution (dissolved in N,N-dimethylformamide).

[0094] 2. Preparation of ZIF-8 precursor solution: Weigh 147 mg of Zn(NO₃)₂·6H₂O into 7 mL of methanol and dissolve under sonication to obtain Solution 1. Weigh 328 mg of 2-methylimidazole into 7 mL of methanol and dissolve under sonication to obtain Solution 2. Mix the two solutions to obtain the precursor solution for synthesizing ZIF-8.

[0095] 3. Preparation of ZIF-8-coated microneedles: Pour the mixed solution prepared above into a 10 cm disposable culture dish, and place the previously prepared PS MNs on the surface of the ZIF-8 precursor solution (the microneedle tip faces downward, and ensure that there are no bubbles between the needles. The needles should not sink to the bottom of the container or float in the middle of the container). After this operation is completed, seal the culture dish with parafilm to prevent the evaporation of the methanol solvent.

[0096] 4. Formation of ZIF-8-Coated Microneedles: The culture dish prepared in the previous step was left undisturbed at room temperature for three days to allow the ZIF-8 to grow in situ on the microneedle surface, forming a ZIF-8 coating. During this growth process, weak electrostatic interactions between the metal ions forming the ZIF-8 and the polystyrene initially caused it to adhere to the polystyrene microneedle surface. The coordination between the organic ligand 2-methylimidazole and the metal ions then drove self-assembly to form small ZIF-8 nuclei on the polystyrene microneedle surface. These nuclei gradually grew into ZIF-8 particles, subsequently forming a uniform ZIF-8 coating.

[0097] 5. Washing of ZIF-8 coated microneedles: The modified microneedles were removed from the growth environment and the surface of the microneedles was washed three times with methanol to remove the unreacted monomers on the surface. The surface of the microneedles was then washed three times with deionized water to remove the ZIF-8 particles that were not firmly bound. After the moisture on the surface of the microneedles evaporated, the obtained ZIF-8 coated microneedles were stored at room temperature for use.

[0098] Scanning electron microscopy of the ZIF-8 coated microneedle surface Figure 11 Compared with the unmodified MOFs-coated polystyrene microneedle surface ( Figure 8 ), uniform and dense ZIF-8 particles distributed in a single layer can be clearly observed on the surface of the ZIF-8 coated microneedles, proving the successful preparation of the ZIF-8 coated microneedles.

[0099] Example 4 Preparation of ZIF-67 Coated Microneedles

[0100] 1. Preparation of PS MNs: Before preparing MOFs-coated microneedles, solid polystyrene microneedles, referred to as PS MNs, were prepared in a PDMS master mold using a 15% polystyrene polymer solution (dissolved in N,N-dimethylformamide).

[0101] 2. Preparation of ZIF-67 precursor solution: Weigh 145.6 mg of Co(NO₃)₂·6H₂O into 7 mL of methanol and dissolve it by sonication to obtain Solution 1. Weigh 328 mg of 2-methylimidazole into 7 mL of methanol and dissolve it by sonication to obtain Solution 2. The two solutions were mixed to obtain the precursor solution for synthesizing ZIF-67.

[0102] 3. Preparation of ZIF-67-coated microneedles: Pour the mixed solution prepared above into a 10 cm disposable culture dish, and place the previously prepared PS MNs on the surface of the ZIF-67 precursor solution (the microneedle tip faces downward, and ensure that there are no bubbles between the needles. It should not sink to the bottom of the container or float in the middle of the container). After this operation is completed, seal the culture dish with parafilm to prevent the evaporation of the methanol solvent.

[0103] 4. Formation of ZIF-67-Coated Microneedles: The culture dish prepared in the previous step was left undisturbed at room temperature for three days to allow ZIF-67 to grow in situ on the microneedle surface, forming a ZIF-67 coating. During this growth process, weak electrostatic interactions between the metal ions forming ZIF-67 and the polystyrene initially caused it to adhere to the polystyrene microneedle surface. The coordination between the organic ligand 2-methylimidazole and the metal ions then drove self-assembly to form small ZIF-8 crystal nuclei on the polystyrene microneedle surface. These nuclei gradually grew into ZIF-8 particles, subsequently forming a uniform ZIF-8 coating.

[0104] 5. Washing of ZIF-67 coated microneedles: Remove the modified microneedles from the growth environment and wash the surface of the microneedles three times with methanol to remove unreacted monomers on the surface. Then, wash the surface of the microneedles three times with deionized water to remove ZIF-67 particles that are not firmly bound. After the moisture on the surface of the microneedles evaporates, store the obtained ZIF-67 coated microneedles at room temperature for use.

[0105] Scanning electron microscopy of the ZIF-67 coated microneedle surface Figure 12 Compared with the unmodified MOFs-coated polystyrene microneedle surface ( Figure 8 ), uniform and dense ZIF-67 particles distributed in a single layer can be clearly observed on the surface of the ZIF-67 coated microneedles, proving the successful preparation of the ZIF-67 coated microneedles.

[0106] Example 5 Preparation of MIL-53 (Al) coated microneedles

[0107] 1. Preparation of PS MNs: Before preparing MOFs-coated microneedles, solid polystyrene microneedles, referred to as PS MNs, were prepared in a PDMS master mold using a 15% polystyrene polymer solution (dissolved in N,N-dimethylformamide).

[0108] 2. Preparation of MIL-53(Al) Precursor Solution: Weigh 840 mg of sodium terephthalate into 15 mL of deionized water and dissolve under sonication to obtain Solution 1. Weigh 1.5 g of Al(NO₃)₃·9H₂O into 6 mL of deionized water and dissolve under sonication to obtain Solution 2. With stirring at room temperature, Solution 1 was added dropwise to Solution 2 to obtain the precursor solution for synthesizing MIL-53(Al).

[0109] 3. Preparation of MIL-53(Al)-coated microneedles: Pour the mixed solution prepared above into a 10 cm disposable culture dish, and place the previously prepared PS MNs on the surface of the MIL-53(Al) precursor solution (the microneedle tip should face downward, and ensure that there are no bubbles between the needles. The needles should not sink to the bottom of the container or float in the middle of the container). After this operation is completed, seal the culture dish with a sealing film to prevent evaporation of water in the reaction solution.

[0110] 4. Formation of MIL-53(Al)-Coated Microneedles: The culture dish prepared in the previous step was left undisturbed at room temperature for 5 days to allow MIL-53(Al) to grow in situ on the needle surface, forming a MIL-53(Al) coating. During the growth process, π-π interactions between terephthalic acid, the organic ligand that forms MIL-53(Al), and polystyrene initially cause it to adhere to the polystyrene microneedle surface. Coordination between the metal ions and the organic ligands, as well as weak electrostatic interactions between the metal ions and polystyrene, cause it to coordinate with the organic ligands on the polystyrene microneedle surface, driving self-assembly to form small MIL-53(Al) crystal nuclei. These small nuclei gradually grow into MIL-53(Al) particles, subsequently forming a uniform MIL-53(Al) coating.

[0111] 5. Washing of MIL-53 (Al) coated microneedles: The modified coated microneedles were removed from the growth environment and the surface of the microneedles was washed three times with deionized water to remove unreacted monomers and unbound MIL-53 (Al) particles on the surface. After the moisture on the surface of the microneedles evaporated, the obtained MIL-53 (Al) coated microneedles were stored at room temperature for later use.

[0112] Scanning electron microscopy of MIL-53(Al) coated microneedles Figure 13 Compared with the unmodified MOFs-coated polystyrene microneedle surface ( Figure 8), a uniform and dense MIL-53(Al) coating distributed in a single layer can be clearly observed on the surface of the MIL-53(Al) coated microneedle, proving the successful preparation of the MIL-53(Al) coated microneedle.

[0113] Example 6 Preparation of Eu-MOF Coated Microneedles

[0114] 1. Preparation of PS MNs: Before preparing MOFs-coated microneedles, solid polystyrene microneedles, referred to as PS MNs, were prepared in a PDMS master mold using a 15% polystyrene polymer solution (dissolved in N,N-dimethylformamide).

[0115] 2. Preparation of Eu-MOF precursor solution: Weigh 420 mg of disodium terephthalate into 2 mL of deionized water and sonicate to dissolve it to obtain Solution 1. Weigh 178.4 mg of Eu(NO₃)₃·6H₂O into 14 mL of deionized water to obtain Solution 2. Under constant stirring, Solution 1 was added dropwise to Solution 2 to obtain a white Eu-MOF precursor solution.

[0116] 3. Preparation of Eu-MOF-coated microneedles: The prepared precursor solution was poured into a 10 cm disposable culture dish, and the pre-prepared PS MNs were placed on top of the Eu-MOF precursor solution (the microneedle tip should face downward, and there should be no bubbles between the needles. The needles should not sink to the bottom of the container or float in the middle of the container). After this operation is completed, the culture dish is sealed with sealing film to prevent evaporation of water in the reaction solution.

[0117] 4. Formation of Eu-MOF-Coated Microneedles: The culture dish prepared in the previous step was left undisturbed at room temperature for 5 days to allow Eu-MOF to grow in situ on the microneedle surface, forming a Eu-MOF coating. During this growth process, π-π interactions between the organic ligands (terephthalic acid) and polystyrene, which form the Eu-MOF, initially attach to the polystyrene microneedle surface. Coordination between the metal ions and the organic ligands, as well as weak electrostatic interactions between the metal ions and polystyrene, then coordinate with the organic ligands on the polystyrene microneedle surface, driving self-assembly to form small Eu-MOF crystal nuclei. These crystal nuclei gradually grow into Eu-MOF particles, subsequently forming a uniform Eu-MOF coating.

[0118] 5. Washing of Eu-MOF coated microneedles: Remove the modified microneedles from the growth environment and wash the surface of the microneedles three times with deionized water to remove unreacted monomers and loosely bound Eu-MOF particles. After the moisture on the surface of the microneedles evaporates, store the resulting Eu-MOF coated microneedles at room temperature for later use.

[0119] Scanning electron microscopy of Eu-MOF coated microneedles Figure 14Compared with the unmodified MOFs-coated polystyrene microneedle surface ( Figure 8 ), a uniform and dense Eu-MOF coating distributed in a single layer can be clearly observed on the surface of the Eu-MOF coated microneedles, proving the successful preparation of the Eu-MOF coated microneedles.

[0120] Comparative Example 1

[0121] The difference between this comparative example 1 and example 1 is that in step 4, the coating is left undisturbed for 1, 2, 3, 4, 5, 7, 8 and 14 days. The effect of the in-situ growth time on the coating growth is investigated. Figure 15 As shown in the figure, no UiO-66-COOH particles grew on the surface of the polystyrene microneedles during the first two days of the reaction. Starting from the third to fourth day of the reaction, UiO-66-COOH particles gradually formed on the surface of the polystyrene microneedles. From the fifth to seventh day of the reaction, the density of the UiO-66-COOH particles increased, and UiO-66-COOH particles continued to form on the polystyrene surface, subsequently growing into a UiO-66-COOH coating, resulting in a uniform and dense UiO-66-COOH-coated microneedle. When the reaction time was extended to 8-14 days, the density of the UiO-66-COOH coating formed on the microneedle surface did not change, indicating that a uniform and dense UiO-66-COOH coating can be formed within 7 days.

[0122] This indicates that insufficient in situ growth time will lead to reduced uniformity of the generated coating, and ultimately the density of the UiO-66-COOH coating will be low. There will be fewer UiO-66-COOH particles on the surface of the polystyrene microneedles, and there will be less extraction medium for binding to DNA, which will reduce the binding efficiency to DNA and ultimately reduce the in situ DNA sampling efficiency of the UiO-66-COOH coated microneedles.

[0123] Comparative Example 2

[0124] The difference between this comparative example and Example 1 is that in step 4, the solution is in a stirring state at room temperature rather than a static and undisturbed state. Figure 16 It can be concluded that disturbance of the precursor solution is not conducive to the growth of MOFs coatings on the polystyrene microneedle surface. Under stirring, the density of the UiO-66-COOH coating is low because the disturbance of the solution affects the growth of UiO-66-COOH nuclei on the polystyrene microneedle surface. However, when the precursor solution is static, the resulting UiO-66-COOH coating is dense and uniform, which facilitates the slow growth of nuclei on the microneedle surface. Therefore, it can be concluded that stirring the solution affects the coating density, which in turn reduces the formation of UiO-66-COOH particles on the polystyrene microneedle surface, ultimately leading to reduced DNA extraction efficiency from the coated microneedles.

[0125] Example 7: Application in extracting DNA from plant leaves

[0126] The kit consists of a four-partition MOF-coated microneedle (in situ array DNA extraction medium), a bottle of deionized water, and a bottle of phosphate buffered saline. The four-partition MOF-coated microneedle is used for DNA extraction, with each partition capable of multiple, rapid extractions of DNA from the sample being tested. The deionized water is used to clean impurities from the microneedle surface after DNA extraction, and the phosphate buffered saline is used to elute the target DNA.

[0127] Kit Usage:

[0128] a. Press the MOFs-coated microneedle onto the sample surface (such as a pepper leaf or tomato leaf) and maintain this operation for 15 minutes to extract DNA from the sample;

[0129] b. Peel off the MOFs-coated microneedles, clean the surface of the microneedles with deionized water to remove adsorbed impurities, and then rinse the microneedles with 15 μL of 10 mM phosphate buffer to collect the extracted target DNA. Store at 2-8°C until ready for use.

[0130] c. Perform PCR on the extracted target DNA using the corresponding primer sequences to verify the amplification effect, indicating the effectiveness of the extraction kit.

[0131] The primer sequences for amplifying the rbcL gene in plants (fresh pepper leaves, tomato leaves) are as follows:

[0132] Rbcla-F (5′-ATGTCACCACAAACAGAGACTAAAGC-3′)

[0133] rbcLajf634R (5′-GAAACGGTCTCTCCAACGCAT-3′)

[0134] The PCR reaction system is as follows:

[0135] Reagent Type Concentration (μmol / L) Sample volume (μL) 2X Taq Master Mix - 10 Upstream primer 10 1 Downstream primer 10 1 DNA template - 2 <![CDATA[ddH2O]]> - 6

[0136] The rbcL gene PCR reaction procedure is as follows:

[0137]

[0138] d. After target DNA extraction is complete, the extraction medium (MOF-coated microneedles) is thoroughly rinsed with phosphate eluent and deionized water. Once the surface moisture is dried, the extraction can be repeated a second time, and so on. A single 3×3 MOFs-coated microneedle can achieve multiple target DNA extractions.

[0139] The DNA extraction kit based on UiO-66-COOH coated microneedles is used to extract target DNA from plant samples (fresh pepper leaves, tomato leaves): the UiO-66-COOH coated microneedles are inserted into the pepper leaves for extraction for 15 minutes, the coated microneedles are peeled off with tweezers, and the surface adsorbed impurities are cleaned with deionized water. Then, 15 μL of eluent is used to elute the target DNA on the surface of the coated microneedles multiple times, the eluted DNA is collected, and amplified under the above-mentioned PCR conditions. 6% SDS-PAGE gel electrophoresis is used to verify the effectiveness of the MOF coated microneedle DNA extraction kit prepared by the present invention in extracting target DNA from mushrooms. Target DNA can be obtained in fresh pepper and tomato leaves of plants, Figure 17 This is the result of DNA extraction from pepper leaves using UiO-66-COOH coated microneedles; Figure 18 Figure 3 shows the results of DNA extraction from tomato leaves using UiO-66-COOH-coated microneedles. Figure 1 shows a blank control; 2 shows extraction using polystyrene microneedles; and 3 shows extraction using UiO-66-COOH-coated microneedles. Gel electrophoresis results show that in both plant leaves, target DNA bands were obtained after PCR amplification using both uncoated polystyrene and UiO-66-COOH-coated microneedles. However, the bands obtained with the UiO-66-COOH-coated microneedles were darker, indicating that under the same conditions, the amount of DNA extracted by the coated microneedles was much higher than that extracted by the uncoated polystyrene microneedles, demonstrating the superior DNA extraction capabilities of the coated microneedles.

[0140] Based on the reusability of the DNA extraction kit for UiO-66-COOH coated microneedles: DNA extraction was repeated 7 times in fresh pepper leaves, and the DNA templates were collected 7 times for repeated extraction. Amplification was performed under the above PCR conditions, and 6% SDS-PAGE gel electrophoresis was used to verify the reusability of the MOF coated microneedles prepared by the present invention. The repeated extraction results are shown in Figure 2. Figure 19 As shown, it can be seen that the UiO-66-COOH coated microneedles of the same array still have obvious target bands after repeatedly extracting the target DNA from fresh pepper leaves for 7 times, demonstrating its excellent extraction ability.

[0141] The same method was used to examine the number of times the microneedle DNA extraction kits in Examples 1 to 6 could be reused. The results were as follows:

[0142] Original concentration The concentration is 1 / 2 of the original concentration UiO-66-COOH coated microneedles (Example 1) 7 6 <![CDATA[UiO-66-NH2 Coated Microneedles (Example 2)]]> 7 6 ZIF-8 coated microneedles (Example 3) 6 5 ZIF-67 coated microneedles (Example 4) 6 5 MIL-53 (Al) coated microneedles (Example 5) 6 5 Eu-MOF coated microneedles (Example 6) 6 5

[0143] Example 8 is applied to extract DNA from liquid samples

[0144] The kit consists of a four-partition MOF-coated microneedle (in situ array DNA extraction medium), a bottle of deionized water, and a bottle of phosphate buffered saline. The four-partition MOF-coated microneedle is used for DNA extraction, with each partition capable of multiple, rapid extractions of DNA from the sample being tested. The deionized water is used to clean impurities from the microneedle surface after DNA extraction, and the phosphate buffered saline is used to elute the target DNA.

[0145] Kit Usage:

[0146] a. Place the MOFs-coated microneedles into milk and maintain this operation for 15 minutes to extract DNA from the sample;

[0147] b. Peel off the MOFs-coated microneedles, clean the surface of the microneedles with deionized water to remove adsorbed impurities, and then rinse the microneedles with 15 μL of 10 mM phosphate buffer to collect the extracted target DNA. Store at 2-8°C until ready for use.

[0148] c. Perform PCR on the extracted target DNA using the corresponding primer sequences to verify the amplification effect, indicating the effectiveness of the extraction kit.

[0149] The primer sequences for amplifying (milk) β-lactoglobulin in liquid samples are as follows:

[0150] Upstream primer (5′-GCATTCACTGGGTGTCTGGA-3′)

[0151] Downstream primer (5′-GCATCTCGAGTGCCCCTTATT-3′)

[0152] The PCR reaction system is as follows:

[0153] Reagent Type Concentration (μmol / L) Sample volume (μL) 2X Taq Master Mix - 10 Upstream primer 10 1 Downstream primer 10 1 DNA template - 2 <![CDATA[ddH2O]]> - 6

[0154] The β-lactoglobulin PCR reaction procedure is as follows:

[0155]

[0156] d. After target DNA extraction is complete, the extraction medium (MOF-coated microneedles) is thoroughly rinsed with phosphate eluent and deionized water. Once the surface moisture is dried, the extraction can be repeated a second time, and so on. A single 3×3 MOFs-coated microneedle can achieve multiple target DNA extractions.

[0157] The DNA extraction kit based on UiO-66-COOH coated microneedles is used to extract target DNA from liquid samples (milk): the UiO-66-COOH coated microneedles are placed in 100 μL of milk for extraction for 5 minutes, the coated microneedles are taken out with tweezers, and the grease and protein on the surface are cleaned with deionized water. Then, 15 μL of eluent is used to elute the target DNA on the surface of the coated microneedles multiple times, the eluted DNA is collected, and amplified under the above-mentioned PCR conditions. 6% SDS-PAGE gel electrophoresis was used to verify the effectiveness of the MOF coated microneedle DNA extraction kit prepared by the present invention in extracting target DNA from milk. Figure 20 Figure 3 shows the results of target DNA extraction from milk using UiO-66-COOH-coated microneedles. Figure 1 shows a blank group; 2 shows extraction using polystyrene microneedles; and 3 shows extraction using UiO-66-COOH-coated microneedles. Gel electrophoresis results show that the target DNA bands obtained through PCR amplification after extraction using unmodified polystyrene microneedles are very faint, while those obtained through PCR amplification using UiO-66-COOH-coated microneedles are darker. This indicates that under the same conditions, the amount of DNA extracted by the coated microneedles is much higher than that extracted by the unmodified polystyrene microneedles, demonstrating the excellent DNA extraction capabilities of the coated microneedles.

[0158] The MOFs-coated microneedles prepared by this invention are expected to be used for rapid DNA extraction from a variety of test samples. Their simplicity, minimal invasiveness, and the absence of in vitro purification facilitate subsequent testing and address the current issue of impure interstitial fluid extraction using hydrogels' swelling and water absorption properties. Finally, the MOFs microneedles can be used for repeated DNA extraction after washing with phosphate buffer, resolving the issue of single-use extractions limited to conventional hydrogel or metal oxide-loaded hydrogel microneedles.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing MOFs-coated microneedles, characterized in that: The steps include: S1. Prepare a mold according to the designed microneedle size, prepare a PDMS master mold based on the mold, and then remove the mold to form a PDMS master mold with a microneedle model area; S2. Using N,N-dimethylformamide as a solvent to prepare a polystyrene polymer solution of a certain concentration, pouring it into the microneedle model area of ​​the PDMS master mold, and drying and demolding to obtain polystyrene microneedles. The polystyrene microneedles have a structure in which multiple needles are arrayed on a substrate; S3, preparing a MOFs precursor solution with a fixed ratio; S4. Inverting the polystyrene microneedles obtained in step S2, placing the needle bodies in the MOFs precursor solution with the bottom surface of the substrate above the liquid surface of the MOFs precursor solution, and keeping the solution undisturbed at room temperature for 3 to 7 days to allow a dense and uniform MOFs coating to grow in situ on the surface of the polystyrene microneedles; S5. Take out the polystyrene microneedles, rinse them with ethanol, then rinse them with deionized water, and dry them to obtain MOFs-coated microneedles with in situ grown MOFs coatings.

2. The method for preparing a MOFs-coated microneedle according to claim 1, wherein: The average molecular weight of the polystyrene is 260,000; the certain concentration is a mass ratio of the polystyrene polymer to N,N-dimethylformamide of 15% to 25%.

3. The method for preparing a MOFs-coated microneedle according to claim 1, wherein: MOFs is any one of UiO-66-COOH, UiO-66-NH2, ZIF-8, ZIF-67, MIL-53(Al), and Eu-MOF.

4. The method for preparing a MOFs-coated microneedle according to claim 3, wherein: The concentration of UiO-66-COOH is anhydrous zirconium chloride: 1,2,4-benzenetricarboxylic acid: formic acid: deionized water in a mass volume ratio of 350-700 mg: 350-700 mg: 7 mL: 16 mL. The concentration of UiO-66-NH2 is anhydrous zirconium chloride: 2-aminoterephthalic acid: formic acid: deionized water: ethanol mass volume ratio = 235~470mg:181~362mg:7mL:16mL:20mL; The ZIF-8 concentration is Zn(NO3)2·6H2O:2-methylimidazole:methanol mass volume ratio = 73.5~147mg:164~328mg:14mL; The ZIF-67 concentration is Co(NO3)2·6H2O:2-methylimidazole:methanol mass volume ratio = 72.8~145.6mg:164~328mg:14mL; The concentration of MIL-53(Al) is Al(NO3)3·9H2O: sodium terephthalate: deionized water (mass volume ratio = 250-500 mg: 140-280 mg: 7 mL); The Eu-MOF concentration is Eu(NO3)3·6H2O: disodium terephthalate: deionized water mass volume ratio = 89.2~178.4 mg: 210~420 mg: 16 mL.

5. Use of the preparation method of the MOFs-coated microneedle according to claim 1 in rapid DNA extraction.

6. A rapid DNA extraction kit, characterized in that: The invention comprises a MOFs-coated microneedle obtained by the preparation method according to claim 1, and a cleaning solution and an eluent.

7. The rapid DNA extraction kit according to claim 6, characterized in that The steps for use are: (1) Place the MOFs-coated microneedle into the liquid sample or press it onto the sample surface, maintain this operation for 15 to 20 minutes, and extract the DNA from the sample to be tested; (2) peeling the MOFs-coated microneedles from the sample surface, washing the impurities adsorbed on the surface of the MOFs-coated microneedles with deionized water, and then rinsing the microneedles with 10 mM phosphate buffer to collect the extracted target DNA; (3) performing PCR on the extracted target DNA using the corresponding primer sequences to obtain amplified DNA; (4) After the target DNA is extracted, the MOFs-coated microneedles are thoroughly cleaned with eluent and deionized water. After completion, the next extraction can be repeated.

8. The rapid DNA extraction kit according to claim 6, characterized in that The kit is used for rapidly extracting DNA from liquid samples or plant leaves.

9. The rapid DNA extraction kit according to claim 6, characterized in that The kit can be reused 5 to 7 times.

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