Microfluidic device for capturing short cell-free DNA and method of making the same
By coating the capture layer of a microfluidic device with carboxyl-modified nanowires, the problem of cumbersome and unsatisfactory short free DNA capture steps in existing technologies is solved, achieving efficient and rapid DNA capture, reducing costs and supporting automation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CITY UNIVERSITY OF HONG KONG SHENZHEN FUTIAN RESEARCH INSTITUTE
- Filing Date
- 2022-12-30
- Publication Date
- 2026-07-21
AI Technical Summary
Existing kits have cumbersome capture procedures for short cell-free DNA and their capture ability is not ideal, resulting in the loss of a large amount of short cell-free DNA.
A microfluidic device is designed to enhance the capture capacity of short free DNA by coating a capture layer with carboxyl-modified nanowires, utilizing the high specific surface area of the nanowires and the increased number of capture groups.
This method enables simple and efficient capture of short cell-free DNA, reduces operational steps, improves capture capacity, and lowers costs, with the potential for automated capture.
Smart Images
Figure CN115845945B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microfluidics technology, specifically relating to a microfluidic device for capturing short free DNA and its preparation method. Background Technology
[0002] Cell-free DNA (CFU) is DNA that exists outside of cells, primarily as DNA-protein complexes in various body fluids. Short CFU has been shown to be closely related to disease progression. Because it carries an individual's genetic information, it has been used for prenatal screening and tumor detection. Detection methods generally fall into two categories: one is based on polymerase chain reaction (PCR) to indirectly or directly quantify a specific fragment; the other involves library construction and sequencing of the fragment to detect mutation sites. Both methods require capturing CFU in body fluids; however, existing kits for capturing short CFU are cumbersome and inconvenient, and their capture capacity is not ideal, resulting in significant loss of short CFU.
[0003] To address this, the present invention proposes a microfluidic device for capturing short free DNA and its preparation method. Summary of the Invention
[0004] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide a microfluidic device for capturing short free DNA and a method for preparing the same.
[0005] In one aspect, the present invention provides a microfluidic device for capturing short free DNA, the microfluidic device comprising: a lower cover plate, a capture layer, a channel layer, and an upper cover plate stacked together; wherein, The flow channel layer has multiple liquid flow channels on the side facing the capture layer; The capture layer is coated with carboxyl-modified nanowires for capturing short free DNA on the side facing the flow channel layer.
[0006] Optionally, the trapping layer is coated with carboxyl-modified nanowires on the side facing the flow channel layer using the following method: Nanowires were fabricated on the trapping layer; Carboxyl groups are modified on the surface of the nanowires.
[0007] Optionally, the lower cover plate, the trapping layer, and the upper cover plate are made of polymethyl methacrylate; The nanowire raw material is a dichloromethane solution of polymethyl methacrylate; The flow channel layer is made of polydimethylsiloxane.
[0008] Optionally, the microfluidic device includes at least one liquid inlet and multiple liquid outlets; The at least one liquid inlet and the plurality of liquid outlets are respectively connected to the liquid flow channel; and / or, The plurality of liquid channels are spaced apart, and the width of each liquid channel ranges from 400μm to 600μm, and the interval between the plurality of liquid channels ranges from 400μm to 600μm.
[0009] In another aspect, the present invention provides a method for preparing a microfluidic device for capturing short free DNA, the method being used to prepare the microfluidic device described above, the method comprising: It provides a lower cover plate, a capture layer, a flow channel layer, and a top cover plate; Carboxyl-modified nanowires for capturing short free DNA were fabricated on the capture layer. The trapping layer has one side with carboxyl-modified nanowires attached to the channel layer with the liquid flow channel on the other side. The lower cover plate and the upper cover plate are respectively stacked on the side opposite to the capture layer and the flow channel layer.
[0010] Optionally, the preparation of carboxyl-modified nanowires on the trapping layer includes: Nanowires are fabricated on the trapping layer; Carboxyl groups are modified on the surface of the nanowires.
[0011] Optionally, the fabrication of nanowires on the trapping layer includes: A pre-prepared solution with the same composition as the capture layer is spin-coated onto a pre-prepared template; Soften the capture layer; The preset template is thermally bonded to the softened trapping layer to prepare nanowires on the trapping layer.
[0012] Optionally, the modification of the nanowire surface with carboxyl groups includes: A layer of gold nanoparticles is formed on the surface of the nanowires, and an allyl mercaptan solution is added to allow the gold nanoparticles to undergo a coordination reaction with the allyl mercaptan solution. Then, acrylic acid, ammonium persulfate, and a solvent are added to carry out a polymerization reaction to modify carboxyl groups on the surface of the nanowires.
[0013] Optionally, the concentration of the allyl mercaptan solution ranges from 0.4M to 0.6M, the amount of acrylic acid added ranges from 0.4g to 0.6g, the amount of ammonium persulfate added ranges from 15mg to 25mg, and the amount of solvent added ranges from 30g to 60g.
[0014] Optionally, the coordination reaction time ranges from 0.5 h to 1.5 h; the polymerization reaction time ranges from 2 h to 5 h.
[0015] This invention proposes a microfluidic device for capturing short free DNA and its preparation method. By coating a capture layer with carboxyl-modified nanowires, the high specific surface area of the nanowires can increase the contact area between the short free DNA and the capture surface of the capture layer, thereby improving the capture ability of short free DNA. Attached Figure Description
[0016] Figure 1 This is a schematic diagram of the overall structure of a microfluidic device, a microfluidic liquid flow channel pattern diagram, and related channel parameters according to an embodiment of the present invention; Figure 2 For the present invention Figure 1 A schematic diagram of relevant channel parameters on the flow channel layer; Figure 3 This is a flowchart illustrating a method for fabricating a microfluidic device according to another embodiment of the present invention. Figure 4 Here is a physical image and related parameters of polymethyl methacrylate nanowires according to another embodiment of the present invention; Figure 5 Infrared characterization images of polymethyl methacrylate nanowires and carboxyl-modified nanowires according to another embodiment of the present invention; Figure 6 Contact angle measurement of polymethyl methacrylate nanowires and carboxyl-modified nanowires according to another embodiment of the present invention; Figure 7 This is the result of the capture rate varying with different ratios of the working buffer in Example 2 of the present invention; Figure 8 This is the result of the capture rate varying with different nanowire heights in Example 3 of the present invention; Figure 9 The results show the capture rate variation with different nanowire diameters in Example 3 of this invention. Detailed Implementation
[0017] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of the present invention.
[0018] Unless otherwise specifically stated, the technical or scientific terms used in this invention should be understood in their ordinary sense by one of ordinary skill in the art to which this invention pertains. The terms "comprising" or "including," as used in this invention, do not limit the shapes, numbers, steps, actions, operations, components, elements, and / or groups thereof mentioned, nor do they exclude the appearance or inclusion of one or more other different shapes, numbers, steps, actions, operations, components, elements, and / or groups thereof. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number and order of the indicated technical features.
[0019] In some descriptions of the invention, unless otherwise explicitly specified and limited, terms such as "installation," "connection," "linking," or "fixing" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect through an intermediate medium, which can be the internal connection of two elements or the interaction between two elements. Furthermore, terms such as "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are used only to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.
[0020] like Figure 1 As shown, one aspect of the present invention provides a microfluidic device for capturing short free DNA. The microfluidic device 100 includes: a lower cover plate 110, a capture layer 120, a channel layer 130, and an upper cover plate 140 stacked together; wherein, the capture layer 120 has a plurality of liquid channels on the side facing the channel layer 130; and the channel layer 130 is coated with carboxyl-modified nanowires for capturing short free DNA on the side facing the capture layer 120.
[0021] In this embodiment, by coating the capture layer with carboxyl-modified nanowires, the high specific surface area of the nanowires can increase the contact area between short free DNA and the capture surface of the capture layer, thereby improving the capture ability of short free DNA.
[0022] Furthermore, the carboxyl-modified nanowires of this embodiment are modified by polymerizing carboxyl groups on the nanowires, which can further increase the number of capturing groups for short free DNA, thereby further improving the ability to capture short free DNA.
[0023] Specifically, the present invention can use the following method to coat the side of the trapping layer facing the flow channel layer with carboxyl-modified nanowires, including: preparing nanowires on the trapping layer; and modifying the surface of the nanowires with carboxyl groups.
[0024] The preparation of nanowires on the trapping layer includes the following steps: spin-coating a preset solution onto a preset template (e.g., a nano-alumina single-hole plate); softening the trapping layer; and thermally bonding the preset template onto the softened trapping layer to prepare nanowires on the trapping layer.
[0025] It should be noted that the preset solution in this embodiment is made of the same material as the capture layer. For example, the capture layer is made of polymethyl methacrylate, and the preset solution is a dichloromethane solution of polymethyl methacrylate. That is, a nano-alumina single-channel plate is used as a mold, and the dichloromethane solution of polymethyl methacrylate is spin-coated on the mold. The spin-coated nano-alumina single-channel plate is then attached to a polymethyl methacrylate microfluidic plate to prepare nanowires. After preparation, the mold is removed.
[0026] In addition, modifying the surface of nanowires with carboxyl groups includes the following steps: spraying a layer of gold nanoparticles onto the surface of the trapping layer coated with nanowires, and adding an allyl mercaptan solution, so that the gold nanoparticles and the allyl mercaptan solution undergo a coordination reaction to form Au-S bonds; then, adding acrylic acid, ammonium persulfate and solvent to carry out a polymerization reaction at 70°C, that is, acrylic acid undergoes a linear polymerization reaction between its different molecules under the excitation of ammonium persulfate, and acrylic acid polymerizes with double bonds. This reaction occurs on the gold nanoparticles of the nanowires to modify the surface of the nanowires with carboxyl groups.
[0027] It should be noted that, during the reaction of nano-gold and allyl mercaptan, the optimal concentration of the allyl mercaptan ethanol solution is 0.5M, and the preferred amounts of acrylic acid, ammonium persulfate solid, and water solvent added during the polymerization reaction are 0.5g, 20mg, and 50g, respectively. The preferred coordination reaction time between nano-gold and allyl mercaptan is 1 hour, and the preferred polymerization reaction time is 3 hours.
[0028] In this embodiment, a capture layer coated with carboxyl-modified nanowires is bonded together with a flow channel layer to form a closed flow channel. When short free DNA passes through, it is captured by the nanowires on the capture layer.
[0029] Furthermore, the microfluidic device of this embodiment includes a liquid inlet and multiple liquid outlets; at least one liquid inlet and multiple liquid outlets are respectively connected to a liquid flow channel to introduce liquid into the liquid flow channel through the liquid inlet and to draw liquid out of the liquid flow channel to the outside through the multiple liquid outlets for liquid analysis.
[0030] Furthermore, in this embodiment, the flow channel layer is provided with multiple liquid flow channels, which are spaced apart. The width of each liquid flow channel ranges from 400μm to 600μm, for example, 500μm. The spacing between the multiple liquid flow channels also ranges from 400μm to 600μm, for example, 500μm. Figure 2As shown.
[0031] It should be noted that in this embodiment, the lower cover plate, the trapping layer, and the upper cover plate are made of polymethyl methacrylate (PMMA), i.e., a PMMA microfluidic plate, and the channel layer is made of polydimethylsiloxane (PDMS), i.e., a PDMS microfluidic plate. In other words, in this embodiment, the nanowire-containing side of the PMMA microfluidic plate is bonded to the channel-containing side of the PDMS microfluidic plate to form a closed liquid channel. A larger PMMA microfluidic plate without nanowires (the upper and lower cover plates) is then screwed together on both the top and bottom sides to integrate them into a single microfluidic device.
[0032] In this embodiment, the microfluidic device captures short free DNA at a flow rate of approximately 200 μL / mL.
[0033] This invention uses polymethyl methacrylate to prepare nanowires and modifies the nanowires with carboxyl groups. By integrating the modified nanowires onto a microfluidic device, a simple and efficient capture of short free DNA can be achieved. This microfluidic device can overcome the shortcomings of current kits, such as long time consumption and cumbersome process in capturing short free DNA. It achieves ideal short free DNA capture capability, is fast and simple, and has low cost. Based on the characteristics of microfluidics, it is expected to realize the automation of short free DNA capture.
[0034] like Figure 3 As shown, in another aspect, the present invention provides a method S200 for preparing a microfluidic device for capturing short free DNA. This method is used to prepare the microfluidic device described above. The preparation method S200 includes steps S210 to S240: S210 provides a lower cover plate, a capture layer, a flow channel layer, and an upper cover plate.
[0035] In this embodiment, the lower cover plate is a polymethyl methacrylate microfluidic plate, the flow channel layer is a polydimethylsiloxane microfluidic plate containing liquid flow channels, the capture layer is a polymethyl methacrylate microfluidic plate, and the upper cover plate is a polymethyl methacrylate microfluidic plate.
[0036] S220. Prepare carboxyl-modified nanowires on the capture layer for capturing short free DNA.
[0037] Specifically, nanowires are fabricated on the trapping layer; carboxyl groups are modified on the surface of the nanowires.
[0038] The preparation of nanowires on the trapping layer includes: spin-coating a preset solution (a dichloromethane solution of polymethyl methacrylate as the raw material for nanowires) onto a preset template (a nano-alumina single-hole plate); softening the trapping layer; and thermally bonding the preset template onto the softened trapping layer to prepare nanowires on the trapping layer.
[0039] In addition, modifying the surface of nanowires with carboxyl groups includes: spraying a layer of gold nanoparticles onto the surface of the nanowires, then coordinating the gold nanoparticles with an allyl mercaptan solution, and then adding acrylic acid, ammonium persulfate and a solvent (e.g., water) to carry out a polymerization reaction at 70°C, which is equivalent to the polymerization of acrylic acid with double bonds to modify the surface of nanowires with carboxyl groups.
[0040] It should be noted that the concentration of the allyl mercaptan solution ranges from 0.4M to 0.6M (e.g., 0.5M), the amount of acrylic acid added ranges from 0.4g to 0.6g (e.g., 0.5g), the amount of ammonium persulfate added ranges from 15mg to 25mg (e.g., 20mg), the solvent is water, and the amount of the solvent added ranges from 30g to 60g (e.g., 50g); the coordination reaction time ranges from 0.5h to 1.5h (e.g., 1h), and the polymerization reaction time ranges from 2h to 5h (e.g., 3h).
[0041] S230. The side of the trapping layer with the carboxyl-modified nanowires is attached to the side of the flow channel layer with the liquid flow channel to seal the liquid flow channel.
[0042] S240. The lower cover plate and the upper cover plate are stacked on the side opposite to the flow channel layer and the capture layer, respectively. The four microfluidic plates are fixed by screws and other fasteners to form a microfluidic device.
[0043] The present invention provides a simple process for forming a microfluidic device, which can effectively improve the ability to capture fragmented free DNA, and the capture process is simple and fast.
[0044] The following will further illustrate the preparation method and specific applications of the microfluidic device for capturing short free DNA with reference to several specific embodiments: Example 1 This example illustrates a method for fabricating a microfluidic device, including the following steps: S1. Fabricating nanowires on the trapping layer, including: Approximately 1 mL of a 20% polymethyl methacrylate (PMMA) solution in dichloromethane was spin-coated onto a nano-alumina single-hole plate at 1000 rpm and dried overnight. A PMMA plate was softened by heating it to 280°C on a plate heater, and the PMMA-containing nano-alumina single-hole plate was then thermally bonded onto the PMMA plate, allowing it to cool naturally at room temperature. The bonded PMMA plate was treated with 1M sodium hydroxide until numerous bubbles appeared on the surface. The surface alkali was washed away with water, and the plate was then placed in a supersaturated copper sulfate aqueous solution containing a small amount of hydrochloric acid for 10 minutes. The copper was washed away with water, and the plate was placed in 8% hydrochloric acid and incubated overnight in a 70°C water bath. The acid was washed away with water, and the plate was dried. The surface morphology was observed under an electron microscope. Figure 4 As shown.
[0045] S2. Modifying the surface of nanowires with carboxyl groups, including: A layer of gold nanoparticles was sprayed onto the surface of the nanowires from step S1 and baked at 95°C for 5 min. A 0.5 M allyl mercaptan ethanol solution was added, and a coordination reaction was carried out at room temperature for 1 h. Then, a polymerization reaction was performed with acrylic acid and double bonds at 70°C for 3 h, thereby modifying the nanowire surface with carboxyl groups. Infrared characterization and contact angle measurements were performed on the nanowires before and after carboxyl group modification (e.g.,...). Figure 5 and Figure 6 As can be seen, the surface of the carboxyl-modified nanowires is hydrophilic, indicating that the carboxyl modification on the nanowire surface plays a role in capturing short free DNA. The preferred amounts of acrylic acid, ammonium persulfate solid, and water added during the polymerization reaction are 0.5 g, 20 mg, and 50 g, respectively.
[0046] S3. Stack the upper cover plate, the flow channel layer, the capture layer, and the lower cover plate to form a microfluidic device, including: The capture layer with modified carboxyl groups from step S2 is attached to the channel-containing side of a polydimethylsiloxane microfluidic plate (channel plate) to form a liquid channel. A larger polymethyl methacrylate plate (upper and lower cover plates) without nanowires is then screwed onto the top and lower sides to integrate the microfluidic device. In this embodiment, the microfluidic device has one liquid inlet and multiple liquid outlets. The width of the liquid channel is 500 μm, and the spacing between the liquid channels is 500 μm (see...). Figure 2 ).
[0047] It should be noted that the preparation of the polydimethylsiloxane microfluidic plate in this embodiment was carried out in accordance with the instructions of the SYLGARD 184 kit.
[0048] Example 2 This example demonstrates the process for optimizing the ethanol to polyethylene glycol ratio in the working buffer of a microfluidic device, including the following steps: The microfluidic devices containing nanowires with different parameters from Example 1 were used to capture short, cell-free DNA artificially incorporated into plasma. Specifically, an 81 bp DNA fragment was incorporated into plasma in a total volume of 700 μL. After pretreatment of the DNA-incorporated plasma, appropriate amounts of sodium chloride, magnesium chloride, ethanol, and polyethylene glycol were added to form mixtures containing different proportions of ethanol and polyethylene glycol, with a final volume of 2 mL. The mixture was then passed through a nanowire-containing microfluidic device to capture the 81 bp DNA at a flow rate of approximately 200 μL / min. The capture rate was calculated by comparing the 81 bp DNA content before and after passing through the nanowire-containing microfluidic device using quantitative real-time PCR. The optimal ethanol to polyethylene glycol ratio in the working buffer was 0.75:1 (see [link to example]). Figure 7 ).
[0049] Example 3 This example illustrates the optimization process for nanowire parameters in a microfluidic device, including the following steps: Nanowire microfluidic devices with different parameters were prepared according to Example 1 to capture short free DNA artificially incorporated into plasma. Specifically, an 81 bp DNA fragment was incorporated into plasma in a total volume of 700 μL. After pretreatment of the DNA-coated plasma, appropriate amounts of sodium chloride, magnesium chloride, ethanol, and polyethylene glycol were added, resulting in a final volume of 2 mL. The mixture was then passed through a nanowire-containing microfluidic device to capture the 81 bp DNA at a flow rate of approximately 200 μL / min. The capture rate was calculated by comparing the 81 bp DNA content before and after passing through the nanowire-containing microfluidic device using quantitative real-time PCR. The optimal parameters for the nanowires were a height of 50 μm and a diameter of 70 nm (see Example 1). Figure 8 and Figure 9 ).
[0050] Example 4 This example illustrates the application process of a microfluidic device, including the following steps: The microfluidic device containing carboxyl-modified nanowires from Example 1 was used to capture short, artificially incorporated DNA fragments from plasma. Specifically, an 81 bp DNA fragment was incorporated into plasma in a total volume of 700 μL. After pretreatment of the DNA-incorporated plasma, appropriate amounts of sodium chloride, magnesium chloride, ethanol, and polyethylene glycol were added to form a mixture with a final volume of 2 mL. The mixture was then passed through the nanowire-containing microfluidic device to capture the 81 bp DNA at a flow rate of approximately 200 μL / min. The capture rate was calculated to be 71.5% by comparing the 81 bp DNA content before and after passing through the nanowire-containing microfluidic device using quantitative real-time PCR.
[0051] Example 5 This example illustrates the application process of a microfluidic device, including the following steps: The microfluidic device containing carboxyl-modified nanowires from Example 1 was used to capture short, artificially incorporated DNA from plasma. An 81 bp DNA fragment was incorporated into the plasma, with a total volume of 700 μL. After pretreatment of the DNA-incorporated plasma, appropriate amounts of sodium chloride, magnesium chloride, ethanol, and polyethylene glycol were added to form a mixture with a final volume of 2 ml. The mixture was then passed through the nanowire-containing microfluidic device to capture the 81 bp DNA at a flow rate of 200 μL / min. The capture rate was calculated to be 75.4% by comparing the 81 bp DNA content before and after passing through the nanowire-containing microfluidic device using quantitative real-time PCR.
[0052] Example 6 This example illustrates the application process of a microfluidic device, including the following steps: The microfluidic device containing carboxyl-modified nanowires from Example 1 was used to capture short, artificially incorporated DNA from plasma. An 81 bp DNA fragment was incorporated into the plasma, with a total volume of 700 μL. After pretreatment of the DNA-incorporated plasma, appropriate amounts of sodium chloride, magnesium chloride, ethanol, and polyethylene glycol were added to form a mixture with a final volume of 2 ml. The mixture was then passed through the nanowire-containing microfluidic device to capture the 81 bp DNA at a flow rate of 200 μL / min. The capture rate was calculated to be 68.2% by comparing the 81 bp DNA content before and after passing through the nanowire-containing microfluidic device using quantitative real-time PCR.
[0053] This invention proposes a microfluidic device for capturing short free DNA and its preparation method, which has the following beneficial effects: First, this invention utilizes the advantage of nanowires having a high specific surface area, coating them onto the capture layer to increase the contact area between short free DNA and the capture surface, thereby improving the capture ability of short free DNA.
[0054] Secondly, by modifying carboxyl groups on nanowires, this invention can increase the number of capturing groups for short free DNA, thereby further improving the capturing ability of short free DNA.
[0055] Third, this invention integrates nanowires into a microfluidic device, which can overcome the shortcomings of current kits that have long steps and cumbersome operation processes for capturing short free DNA. The microfluidic device of this invention has a strong ability to capture short free DNA, the capture process is fast and simple, and the cost is low. Based on the characteristics of microfluidics, it is expected to realize the automation of capturing short free DNA.
[0056] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.
Claims
1. A microfluidic device for capturing short cell-free DNA, characterized in that, The microfluidic device comprises: a lower cover plate, a capture layer, a flow channel layer, and an upper cover plate stacked together; wherein... The flow channel layer has multiple liquid flow channels on the side facing the capture layer; The capture layer is coated with carboxyl-modified nanowires for capturing short free DNA on the side facing the channel layer; wherein both the capture layer and the nanowires are made of polymethyl methacrylate, and the carboxyl groups on the surface of the nanowires are formed by the following method: A layer of gold nanoparticles is formed on the surface of the nanowires, and an allyl mercaptan solution is added to allow the gold nanoparticles to undergo a coordination reaction with the allyl mercaptan solution. Then, acrylic acid, ammonium persulfate, and a solvent are added to carry out a polymerization reaction to modify carboxyl groups on the surface of the nanowires.
2. The microfluidic device according to claim 1, characterized in that, The lower cover plate and the upper cover plate are made of polymethyl methacrylate; The flow channel layer is made of polydimethylsiloxane.
3. The microfluidic device according to claim 1 or 2, characterized in that, The microfluidic device includes at least one liquid inlet and multiple liquid outlets; The at least one liquid inlet and the plurality of liquid outlets are respectively connected to the liquid flow channel; and / or, The plurality of liquid channels are spaced apart, and the width of each liquid channel ranges from 400μm to 600μm, and the interval between the plurality of liquid channels ranges from 400μm to 600μm.
4. A method for preparing a microfluidic device for capturing short cell-free DNA, characterized in that, The preparation method is used to prepare the microfluidic device according to any one of claims 1 to 3, and the preparation method includes: It provides a lower cover plate, a capture layer, a flow channel layer, and a top cover plate; Preparing carboxyl-modified nanowires for capturing short free DNA on the capture layer includes: preparing nanowires on the capture layer; A layer of gold nanoparticles is formed on the surface of the nanowires, and an allyl mercaptan solution is added to allow the gold nanoparticles to undergo a coordination reaction with the allyl mercaptan solution. Then, acrylic acid, ammonium persulfate and solvent are added to carry out a polymerization reaction to modify carboxyl groups on the surface of the nanowires. The trapping layer has one side with carboxyl-modified nanowires attached to the channel layer with the liquid flow channel on the other side. The lower cover plate and the upper cover plate are respectively stacked on the side opposite to the capture layer and the flow channel layer.
5. The preparation method according to claim 4, characterized in that, The fabrication of nanowires on the trapping layer includes: A pre-prepared solution with the same composition as the capture layer is coated onto a pre-prepared template; Soften the capture layer; The preset template is thermally bonded to the softened trapping layer to prepare nanowires on the trapping layer.
6. The preparation method according to claim 4, characterized in that, The concentration of the allyl mercaptan solution ranges from 0.4M to 0.6M, the amount of acrylic acid added ranges from 0.4g to 0.6g, the amount of ammonium persulfate added ranges from 15mg to 25mg, and the amount of solvent added ranges from 30g to 60g.
7. The preparation method according to claim 4, characterized in that, The coordination reaction time ranges from 0.5 h to 1.5 h; the polymerization reaction time ranges from 2 h to 5 h.