A microfluidic device for extracting DNA

Abstract

The present invention provides a microfluidic device for DNA extraction. The microfluidic device comprises: an inlet layer, a buffer layer, and a pneumatic layer, which are stacked and sealed in sequence; an electromagnetic plate is provided between the buffer layer and the pneumatic layer; the inlet layer is provided with multiple main channels and three blister ports; the inlet of each main channel is connected to a syringe pump; the buffer layer is provided with segmented buffer channels; each buffer channel segment has multiple channels, and each buffer channel segment corresponds to a pop-up blister; the buffer channels are connected to the main channels; the inlet of the buffer channels is connected to the pop-up blister; the pop-up blister is provided on the top of the inlet layer and connected to the inlet of the buffer channel through the blister port; each pop-up blister is provided with a different type of buffer; each buffer channel segment has multiple converging nodes; each converging node is provided with a pop-up valve; the pop-up valve is pneumatically controlled by the pneumatic layer. The present invention can reduce extraction costs while ensuring low extraction time and high throughput.

Description

Technical Field

[0001] The present invention relates to the field of DNA extraction, in particular to a microfluidic device for extracting DNA. Background Art

[0002] Friedrich Miescher first isolated DNA from human white blood cells in 1869, and today many diagnostic and research laboratories extract DNA routinely found in a variety of cell types, including bacteria, fungi, malarial parasites, plants, animals, and humans.

[0003] DNA extraction is often the first step in molecular studies related to the following: 1. Disease diagnosis; 2. Mutation detection and cancer gene analysis; 3. Genetic biomarker identification and forensics; 4. Genetic engineering and cloning; 5. Parentage testing / lineage tracing; 6. Sex determination in many ornamental birds, such as lovebirds; 7. DNA sequencing (Sanger and next-generation sequencing).

[0004] When DNA extraction is used in bacteria, fungi, plants, animals, or humans, multiple samples are often run simultaneously. Therefore, the DNA extraction process must be cost-effective, rapid, and able to provide large quantities of high-quality DNA. Current methods for DNA extraction can be divided into three main categories based on the separation agent used (chemical, centrifugal, or magnetic): classical (manual) chemical methods; modern (semi-automated / centrifugal) spin column methods; and modern (fully automated) magnetic methods.

[0005] Based on user-friendliness and the number of DNA samples that can be run in parallel at any given time, methods can be categorized as either low-throughput or high-throughput. With the increasing burden on diagnostic laboratories to process population samples and the need to analyze DNA for a variety of purposes, the adoption of time-saving, high-throughput, and low-cost methods is almost inevitable. Current methods offer either low-cost or high-throughput, but not both.

[0006] There are many commercially available kits that allow users to efficiently extract DNA from sample cells using the principles of lysis, binding, washing, and elution (Carpi FM, Di Pietro F, Vincenzetti S, Mignini F, Napolioni V. Human DNA extraction methods: patents and applications. Recent Pat DNA Gene Seq. 2011; 5(1): 1–7); Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009; 2009: 574398).

[0007] Most DNA extraction methods use a set of lysis, binding, wash, and elution buffers that many researchers have used (Shi R, Lewis RS, Panthee DR (2018) Filter paper-based spin column method for cost-efficient DNA or RNA purification. PLOS ONE 13(12):e0203011. https: / / doi.org / 10.1371 / journal.pone.0203011), or use magnetic beads alongside these buffers.

[0008] In the fastest DNA extraction method currently available, magnetic beads are released into a whole cell lysate to initiate the DNA capture and binding process. The use of magnetic beads for DNA extraction first emerged in the 1990s, as evidenced by the US patent granted for “DNA purification and separation using magnetic beads.” The method remains largely unchanged and relies on the use of commercially available (https: / / www.cytivalifesciences.com / en / us / news-center / magnetic-beads-a-simple-guide-10001) coated magnetic beads (20 μm carboxylate-modified magnetite beads) that reversibly bind nucleic acids simply by adjusting buffer conditions. After binding the DNA, an external magnetic field draws the beads to the outer edge of the channel, immobilizing them. While the beads are immobilized, the bead-bound DNA is retained during the wash steps. Adding elution buffer and removing the magnetic field releases the DNA as a purified sample, ready for quantification and analysis.

[0009] Comparing the main DNA extraction methods currently used by researchers or diagnostic laboratories around the world, Table 1 shows the parameters of different DNA extraction methods provided by the present invention. Figure 1 Schematic diagram of different DNA extraction methods. Figure 1 (a) Schematic diagram of DNA extraction based on the phenol-chloroform (organic) method, which usually takes 1-2 days. Figure 1 (b) is a schematic diagram of the silica gel-based (semi-automatic) method for DNA extraction, which usually takes 1-2 hours. Figure 1 (c) is a schematic diagram of the magnetic bead-based (automatic) DNA extraction method, which usually takes less than 20 minutes, as shown in Table 1 and Figure 1 shown.

[0010] Table 1

[0011]

[0012]

[0013]

[0014] As can be seen from the above, the magnetic bead-based method takes the shortest time to extract DNA. However, most high-throughput methods utilize the flexibility of magnetic bead assays and require the installation of expensive robots to use mechanized DNA extraction platforms, which is extremely costly.

[0015] One approach to lowering the cost per test is to provide the robot free of charge under a long-term contract with the end user, allowing the end user to continue using the same closed-platform robot and purchasing DNA extraction tests from the same manufacturer. This allows for a price of $5 to $10 per test. Some open-platform platforms have improved mechanisms to allow any manufacturer's test to be run on the robot, but even here, cost remains a concern. Furthermore, these robotic platforms are not feasible for small- to medium-sized users, such as academic researchers, who sometimes use tens to hundreds of samples per day but cannot afford to install commercial-scale robots. Summary of the Invention

[0016] The purpose of the present invention is to provide a microfluidic device for extracting DNA to solve the problem of not being able to take into account the cost while ensuring low time consumption and high throughput.

[0017] To achieve the above object, the present invention provides the following solutions:

[0018] A microfluidic device for extracting DNA, comprising: an inlet layer, a buffer layer, and a pneumatic layer, which are stacked and sealed in sequence; an electromagnetic plate is provided between the buffer layer and the pneumatic layer;

[0019] The inlet layer is provided with a plurality of main channels and three bubble cap ports; the inlet of each main channel is connected to a syringe pump; the syringe pump is used to inject the lysate sample into the main channel;

[0020] The buffer layer is provided with a segmented buffer channel; each segment of the buffer channel is provided with multiple channels, and each segment of the buffer channel corresponds to one pop-up blister; the buffer channel is connected to the main channel; the inlet of the buffer channel is connected to the pop-up blister; the pop-up blister is provided on the top of the inlet layer and is connected to the inlet of the buffer channel through the blister opening; each pop-up blister is provided with a different type of buffer; the buffers include a magnetic binding buffer, a washing buffer, and an elution buffer;

[0021] Each section of the buffer channel is provided with a plurality of convergence nodes; a pop-up valve is provided at the convergence node; and the pop-up valve is pneumatically controlled by the pneumatic layer.

[0022] Optionally, the length of the confluence layer, the buffer layer, and the pneumatic layer are all 10 cm, and the thickness of the layer are all 1 mm.

[0023] Optionally, a collection device is provided at the outlet of each main channel; the collection device is used to collect DNA samples.

[0024] Optionally, a pop-up bubble cap filled with a magnetic binding buffer, a pop-up bubble cap filled with a washing buffer, and a pop-up bubble cap filled with an elution buffer are sequentially arranged along the direction from the entrance to the exit of the main channel;

[0025] Along the direction from the entrance of the main channel to the exit of the main channel, the pop-up bubble cap containing the magnetic binding buffer, the pop-up bubble cap containing the washing buffer, and the pop-up bubble cap containing the elution buffer are opened in sequence to release the magnetic binding buffer, the washing buffer, and the elution buffer in sequence.

[0026] Optionally, the main channels include 8 channels; and each section of the buffer channels includes 8 channels.

[0027] Optionally, each section of the buffer channel is provided with an 8-pin channel separator with 3 pop-up valves.

[0028] Optionally, the pop-up valve is a one-way valve, and the pop-up valve is used to limit the flow into one buffer channel or into 8 buffer channels at the same time.

[0029] Optionally, the pop-up valve is a T-shaped freely movable polydimethylsiloxane structure.

[0030] Optionally, after use, clean with 1%-2% bleach, rinse with double-distilled water, and air-dry.

[0031] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects: the present invention provides a microfluidic device for extracting DNA, which seals and stacks the input layer, the buffer layer and the pneumatic layer in sequence, and uses a magnetic bead method to simultaneously collect high-quality and high-quantity DNA using multiple channels. Moreover, due to the provision of an electromagnetic plate and a magnetic binding buffer, the magnetic bead method further reduces the DNA extraction time, and can extract high-throughput pure DNA in a short time without the need for expensive robot assistance, thereby reducing the extraction cost while ensuring low time consumption and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Schematic diagram of different DNA extraction methods; Figure 1 (a) Schematic diagram of DNA extraction based on the phenol-chloroform (organic) method; Figure 1 (b) Schematic diagram of DNA extraction based on silica gel (semi-automatic) method; Figure 1 (c) Schematic diagram of DNA extraction based on magnetic beads (automatic) method;

[0034] Figure 2 This is a schematic diagram of the exploded structure of the microfluidic device for DNA extraction provided by the present invention;

[0035] Figure 3 This is a flow chart of the DNA extraction method based on the microfluidic device for DNA extraction provided by the present invention. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] The purpose of the present invention is to provide a microfluidic device for extracting DNA, which can reduce the extraction cost while ensuring low time consumption and high throughput.

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] like Figure 2As shown, the present invention provides a microfluidic device for extracting DNA (hereinafter referred to as RapidμFDNA), comprising: an inlet layer 1, a buffer layer 2, and a pneumatic layer 3, which are sealed and stacked in sequence; an electromagnetic plate is provided between the buffer layer and the pneumatic layer; a plurality of main channels 5 and three bubble cap ports 4 are provided on the inlet layer; the inlet of each main channel 5 is connected to a syringe pump 6; the syringe pump 6 is used to inject a lysate sample into the main channel 5; a segmented buffer channel 7 is provided on the buffer layer; each segment of the buffer channel 7 has multiple channels, and a segment of the buffer channel 7 is connected to the bubble cap 4. One of the pop-up bubbles corresponds to the above-mentioned one; the buffer channel 7 is connected to the main channel 5; the entrance of the buffer channel 7 is connected to the pop-up bubble; the pop-up bubble is arranged on the top of the confluence layer, and is connected to the entrance of the buffer channel 7 through the bubble mouth 4; each of the pop-up bubbles is provided with different types of buffer solutions; the buffer solutions include magnetic binding buffer solution, washing buffer solution and elution buffer solution; each section of the buffer channel 7 is provided with a plurality of convergence nodes; a pop-up valve 8 is provided at the convergence node; the pop-up valve 8 is pneumatically controlled by the pneumatic layer.

[0040] In practical applications, the length of the inlet layer, the buffer layer, and the pneumatic layer are all 10 cm, and the thickness is 1 mm.

[0041] In practical applications, a collection device 9 is provided at the outlet of each main channel 5 ; the collection device is used to collect DNA samples.

[0042] In actual application, along the direction from the entrance of the main channel 5 to the exit of the main channel 5, a pop-up blister P2 filled with magnetic binding buffer, a pop-up blister P3 filled with washing buffer, and a pop-up blister P4 filled with elution buffer are arranged in sequence; along the direction from the entrance of the main channel 5 to the exit of the main channel 5, the pop-up blister P2 filled with magnetic binding buffer, the pop-up blister P3 filled with washing buffer, and the pop-up blister P4 filled with elution buffer are opened in sequence to release the magnetic binding buffer, the washing buffer, and the elution buffer in sequence.

[0043] In practical applications, the main channels 5 include 8 channels; and each section of the buffer channels 7 includes 8 channels.

[0044] In practical applications, each section of the buffer channel 7 is provided with an 8-pin channel separator with three pop-up valves 8 .

[0045] In practical applications, the pop-up valve 8 is a one-way valve, and the pop-up valve 8 is used to limit the flow into one buffer channel 7 or into eight buffer channels 7 at the same time.

[0046] In practical applications, the pop-up valve 8 is a T-shaped freely movable polydimethylsiloxane structure.

[0047] In practical applications, after use, clean it with 1%-2% bleach, rinse it with double distilled water and then air dry it.

[0048] In practical applications, the fabrication of RapidμFDNA primarily involves microfluidic chips made of polydimethylsiloxane (PDMS), which can be prepared using standard industrial techniques used by other companies: photolithography, photomasks, and photoresists.

[0049] Each layer is made of a 1-millimeter-thick cover layer of polydimethylsiloxane (PDMS). The channels of buffer layer 2 must connect to the main channels 5 that flow into layer 1, while the pneumatic controllers of pneumatic layer 3 connect to the valves of buffer layer 2. Therefore, the overlapping parts of the design must be perfectly aligned for these channels to work together. Each layer is 10 centimeters long and 1 millimeter thick, giving a total thickness of 3 millimeters and a length of 10 centimeters for all three layers.

[0050] These three layers are interconnected and work together as a single unit. They are sealed together through a process called plasma sealing. Inlet Layer 1 is the primary "feeder" layer, delivering cell lysate into the device, while Buffer Layer 2 is the "buffer" layer, adding the required buffer to Inlet Layer 1 at the correct time. Finally, Pneumatic Layer 3 is the "pneumatic" layer, containing the pneumatic controls for Buffer Layer 2's valves and the control mechanism for blister ejection and buffer release. These layers are sandwiched together and sealed, ultimately forming a single unit.

[0051] Branch channels in buffer layer 2 are responsible for directing buffer from the pop-up window to channels 1, 2, 3, 4, 5, 6, 7, or all 8. Once the valve determines how many channels are needed, the buffer is transferred to those channels, and then that specific buffer is gradually added to the main channel 5 in the sink layer 1.

[0052] Finally, Pneumatic Layer 3 is the "pneumatic" layer, containing the pneumatic controls for the valves in Buffer Layer 2, as well as the blister. These three layers are sandwiched together and sealed to form a single unit. Pneumatic Layer 3 controls the pneumatic valves found in Buffer Layer 2.

[0053] The valves of the buffer layer 2 are controlled by the pneumatic controller of the pneumatic layer 3.

[0054] “Stacking” refers to the “sandwich” design of the microfluidic device, connecting three layers together.

[0055] The channels in the confluence layer 1 are continuous, with feeder inlets and outlets. Buffer layer 2 adds specific buffers at specific points into the channels of confluence layer 1. The blisters contain the buffers that need to be released into the channels of buffer layer 2, which ultimately bring the buffers into the channels of confluence layer 1 for mixing.

[0056] The blisters (P2, P3, P4) are basically containers / reservoirs containing buffers (chemicals) required for the DNA extraction process. The buffers from P2, P3, P4 are released under the pneumatic controller located in the pneumatic layer 3 of the device.

[0057] These three liquids are called buffers: P2 contains magnetic binding buffer, P3 contains wash buffer, and P4 contains elution buffer.

[0058] The buffer is initially released into the channels of buffer layer 2 but gradually enters the channels of sink layer 1 where mixing occurs.

[0059] There's only one electromagnet beneath pneumatic layer 3, but its magnetic force propagates to both buffer layer 2 and inlet layer 1. This magnetic force attracts magnetic particles from the magnetic binding buffer released by P2, once they interact with the lysate in inlet layer 1. These magnetic particles bind to DNA, so the magnet attracts both the magnetic particles and the DNA. In doing so, the magnetic particles separate the DNA from the lysate fed into the channels of inlet layer 1.

[0060] Inlet layer 1: This layer is the "main layer" and has a buffer inlet and is divided into 8 channels. Syringe pump 6 (containing lysate samples 1-8) delivers the lysate into the microfluidic device.

[0061] Once the lysate flows into the channel, three bubbles pop out of Buffer 2, pushing the binding, wash, and elution buffers into the channel in sequence. At the end of the process, pure DNA can be collected from the collection port. The electromagnet in Buffer 2 provides the function of separating the DNA from the original lysate.

[0062] Buffer layer 2: The buffer inlet is a pop-up blister containing 50 μL of magnetic binding buffer containing fine magnetic beads. Once the lysed samples (1 to 8) are loaded onto the chip and pushed into the chip channel by the positive pressure controller, the blister release automatically releases the magnetic beads. This mechanism introduced here allows users to extract from 8 independent samples simultaneously using one or 8 channels.

[0063] To this end, the present invention provides an 8-pin channel separator with three valves, allowing users to select the number of channels required for DNA extraction. These valves are T-shaped, freely movable PDMS structures that can be closed or opened at will.

[0064] Once added to the pre-lysate sample, the magnetic beads specifically bind to the DNA in the lysate. The electromagnetic plate beneath the chip is switched to "ON," forcing the DNA-bound magnetic beads into the channel. The electromagnet in buffer layer 2 separates the DNA from the original lysate. As the positive pressure increases and the magnetism gradually releases, the DNA attracted to the magnetic beads moves forward into the channel. At this point, the magnetic binding buffer in bubble P2 of the buffer layer is released.

[0065] Buffer layer 2 also contains a pop-up valve 8 with an 8-pin channel separator. The pop-up bubbler contains 100 μL of wash buffer, and 12.5 μL (100 / 8) of wash buffer is added to each channel, depending on the number of channels used. The channels in buffer layer 2 taper upward and connect to the main channels in the inlet layer 1. When magnetic beads bind to DNA in the inlet layer 1 channel, buffer layer 2 introduces a channel containing wash buffer, washing the magnetic beads and DNA to remove any debris, waste products, or other nucleic acids. This step allows users to obtain high-purity DNA free of contamination.

[0066] The buffer layer 2 also contains a pop-up valve 8 with an 8-pin channel separator, which contains three valves for controlling the flow of buffer 4 into the channels. The pop-up bubbler contains 100uL of elution buffer, which separates the magnetic beads from the DNA captured by the beads, allowing users to obtain pure DNA from the lysed sample at the end of the chip. One to eight collection tubes connected to the outlet allow users to collect 1-8 pure DNA samples from the RapiduF DNA chip.

[0067] Pneumatic Layer 3 is the pneumatic control layer that regulates the one-way valves in Buffer Layer 2. These pneumatic valves allow the user to restrict flow into one channel or into all eight channels simultaneously. Valve control allows the user to run a single or eight samples for DNA extraction.

[0068] The inlet layer 1 is responsible for introducing the original cell lysate into the straight channel. The buffer layer 2 sequentially adds magnetic beads, washing and elution buffers to the lysed sample and uses electromagnetics to capture DNA. The pneumatic layer 3 provides pneumatic control for the valves in the buffer layer 2. These three layers are manufactured separately but need to be connected and work together, because the buffer layer 2 and the pneumatic layer 3 ultimately flush their substances into the main channel of the inlet layer 1. The overall workflow is as follows Figure 3 shown.

[0069] Materials and equipment:

[0070] 1. One 30 ml bottle of Lysis Buffer P1 (this lysis buffer is used to isolate DNA from non-blood cells, such as fungi, bacteria, or plant cells) and one 25 ml bottle of Buffer P0 (this buffer is a mixed solution that lyses blood cells to recover DNA).

[0071] 2. Three blisters containing buffers P2-P4 and pop-up.

[0072] 3. Pop-up blister P2 contains 50uL of magnetic beads, pop-up blister P3 contains 100uL of magnetic binding buffer, and pop-up blister P4 contains 100uL of elution buffer.

[0073] 4. A microfluidic device with 8 sample input ports, a built-in electromagnet and circuitry to manage the release of the blister pop-up contents at the desired time.

[0074] 5. A pressure pump provides positive pressure to all eight sample input ports, pushing the buffer forward in the microfluidic device.

[0075] 6. Silicone tubing for connecting the pressure pump to the device and the 8 collection tubes.

[0076] For all samples, dissolve 1.5 mg of Proteinase K powder in 1.5 ml of deionized water to make aliquots of a 1 mg / ml Proteinase K solution, which should be sufficient for 75 reactions.

[0077] a. Starting sample 1 (blood):

[0078] Add 200 μl of unclotted blood into a 1.5 ml Eppendorf tube.

[0079] Add 200 μl of buffer and vortex, incubate at room temperature for 5 min with occasional inversion.

[0080] Centrifuge at 5000 rpm for 10 minutes. Discard 250 μl of the supernatant using a pipette tip, while avoiding the loss of the clearly visible white thread-like cells.

[0081] Add another 200 μl of buffer to the tube and incubate for 5 minutes, occasionally inverting, and centrifuge at 5000 rpm for 10 minutes. The white line should fall to the bottom of the tube.

[0082] The supernatant was discarded and 20 μl of proteinase K was added to the tube containing the cell pellet.

[0083] Unclotted blood includes fresh blood and blood stored in tubes containing EDTA or citrate.

[0084] b. Starting sample 2 (cultured cells):

[0085] Centrifuge no more than 5 x 106 cells at 8000 rpm for at least 10 minutes. Discard the supernatant and resuspend the cell pellet in 20 μl of proteinase K.

[0086] c. Starting sample 3 (bacterial cells):

[0087] Centrifuge no more than 2 x 109 cells at 8000 rpm for 10 minutes.

[0088] Discard the supernatant and resuspend the cell pellet in 20 μl of proteinase K.

[0089] d. Starting Sample 4 (Mammalian and Rodent Tail Tissue):

[0090] 10 mg of spleen and 20 mg of other mammalian tissues were ground.

[0091] Recommended specimen lengths are 0.6 cm for rat tails and 0.5 cm for mouse tails.

[0092] Both rodent tissues can then be frozen in liquid nitrogen and ground using a pestle and mortar; other options for processing rodent tissue include disrupting the tissue using a homogenizer. All samples should be suspended in 20 μl of proteinase K.

[0093] After pretreatment, incubate the samples at 30°C for 5 minutes. All samples should be incubated in the refrigerator until DNA extraction.

[0094] 1. The preprocessing step is followed by the main extraction procedure, which consists of the following steps:

[0095] 1) Add 10 μg / ml RNAse A to buffer P1 and vortex to mix (RNAse A is not provided in the Rapid μF DNA kit).

[0096] 2) Add 200 μl of buffer P1 (containing RNAse A) to the pretreated DNA sample mixture and pulse vortex for 15 seconds to make a homogenous solution.

[0097] 3) Incubate the solution in a 56°C water bath for 5 minutes.

[0098] 4) Add 400 μl of 50% ethanol and mix using a pipette or vortex. (Note: For blood samples, add 200 μl of 96% ethanol.)

[0099] 5) Transfer the sample to a 1.5 mL Eppendorf tube and centrifuge at 10,000 RPM for 3 minutes. After centrifugation, transfer approximately 500 μL of the supernatant to RapidμF DNA as follows:

[0100] 2. Magnetic binding: (Steps 2 to 5 are fully automated and performed in the RapidμF DNA device).

[0101] 1) 50uL of magnetic beads were added to the buffer to form magnetic binding buffer, and 50uL of magnetic beads in magnetic binding buffer were added to the lysate which was pumped into RapidμF DNA via the electronically controlled blister P2 release pump.

[0102] 2) Magnetic beads preferentially bind to DNA particles in the lysate.

[0103] 3) When the lysate is pumped in and the magnetic beads are released, the external electromagnet turns on and attracts the bound DNA particles and fixes them to the bottom of the RapidμF DNA.

[0104] 3. Washing: (Steps 2 to 5 are fully automated and performed in the RapidμF DNA device).

[0105] 1) As the magnet gradually closes and releases the bound DNA particles, the next bubble electronically releases wash buffer, which removes any contaminants and cell debris from the DNA.

[0106] 2) The magnet remains on during the entire washing process.

[0107] 3) With the magnet fully closed, the liquid is pushed into the next elution stage.

[0108] 4. Elution: (Steps 2 to 5 are fully automated and performed in the RapidμF DNA device).

[0109] 1) The liquid containing the magnetic bead-bound high-purity DNA is separated from the magnetic beads by releasing the elution buffer from the next blister.

[0110] 2) During the elution process, the electromagnet automatically turns off, separating the magnetic beads from the DNA particles.

[0111] 5. Collection: (Steps 2 to 5 are fully automated and performed in the RapidμF DNA device).

[0112] 1) At the end of all the first 4 steps required for the DNA extraction process, collect the DNA from the sample in a collection tube.

[0113] 2) The collected DNA can be stored at -20℃ for up to 3 months.

[0114] 6. Embedded electronic control system.

[0115] 1) Each blister contains enough reagent for all 8 channels. The mainboard divides the liquid in each blister into 1, 2, 4, or 8 channels. This number is the same for each blister type, so 3+4 valves are required.

[0116] 2) Because a c.1 T magnet is used, no ferrous (magnetic) material can be placed close to the main microfluidic device, which means that the actual extraction of the fluid must be done beforehand and then transferred to the main PDMS chip.

[0117] 3) Pneumatic switches can be used to control fluid flow on the master PDMS. The master PDMS contains pressurized reservoirs corresponding to each blister pack, and it is known that the flow of actual blister packs is uneven. These areas are flushed between uses to keep them clean.

[0118] 4) The timing of fluid injection from the blister array is controlled by control software. This software is designed to meet the precise timing requirements. However, if a mechanical failure occurs, the process will be stopped. The timing of the release of the buffer from each pop-up blister is 30 seconds.

[0119] 5) The microfluidic device is controlled using a small, low-power embedded system controller, such as any low-power ARM embedded system, including the Raspberry Pi 4B or Nvidia Jetson. The microfluidic chip moves the buffer (liquid) to the reservoir and measures the liquid to ensure the correct amount is transferred.

[0120] 6) A controller board capable of activating the blister pack is connected to the microcontroller and used in conjunction with a motor controller (shield) board to inject liquid (buffer) from the blister pack. Because there are four timing points, three control circuits need to be switched. This can be achieved using an L298N H-bridge motor driver or a MOSFET driver (such as the TB6612FNG).

[0121] 7) In addition, there are two main valves that need to be controlled, meaning there are six valves to switch. Furthermore, three valve switches are required to select test channels 1, 2, 4, or 8 on the device. This can be done using an IC2 relay board, such as SKU: EP-0099.

[0122] 8) The microcontroller software also measures and uses the buffer to provide feedback on the success of the added functionality.

[0123] 9) A matching power supply is also required.

[0124] The present invention can simultaneously extract DNA from eight samples with high throughput, without the need for expensive robotic assistance; it can rapidly separate and extract DNA, with DNA separation taking less than 10 minutes; the equipment can be cleaned and repeated multiple times; the present invention can obtain high-quality and high-quantity DNA; the current test cost of each DNA extraction is approximately US$5-10, but the present invention can reduce the cost to US$1-2, making the present invention extremely low-cost.

[0125] The present invention is a 3-layer segmented PDMS microfluidic chip, all three layers are sandwiched between each other and work as a whole, and the three layers will be sealed together.

[0126] The release of liquid from the blister poppers and valves will be controlled by pneumatic controllers placed in layer C. These pneumatic controllers will send electrical pulses to the valves and blister poppers.

[0127] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0128] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only intended to help understand the method and core concept of the present invention. At the same time, those skilled in the art will find that the specific implementation methods and application scopes may vary based on the concept of the present invention. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims

1. A microfluidic device for extracting DNA, characterized in that include: The stacked inlet layer, buffer layer and pneumatic layer are sealed in sequence; an electromagnetic plate is provided between the buffer layer and the pneumatic layer; The inlet layer is provided with a plurality of main channels and three bubble cap ports; the inlet of each main channel is connected to a syringe pump; the syringe pump is used to inject the lysate sample into the main channel; The buffer layer is provided with a segmented buffer channel; each segment of the buffer channel is provided with multiple channels, and one segment of the buffer channel corresponds to a pop-up blister; the buffer channel is connected to the main channel; the entrance of the buffer channel is connected to the pop-up blister; the pop-up blister is provided on the top of the inlet layer and is connected to the entrance of the buffer channel through the blister opening; each pop-up blister is provided with a different type of buffer; the buffers include magnetic binding buffer, washing buffer and elution buffer; there is only one electromagnet under the pneumatic layer, and the magnetic force of the electromagnet is transmitted to the buffer layer and the inlet layer; the magnetic force is used to attract magnetic particles in the magnetic binding buffer released by the pop-up blister containing the magnetic binding buffer, and once the magnetic particles bind to the lysate in the inlet layer, the magnetic particles bind to the DNA, so the electromagnet attracts the magnetic particles and the DNA, and the magnetic particles separate the DNA from the lysate fed into the inlet layer channel; Each section of the buffer channel is provided with multiple convergence nodes; a pop-up valve is provided at the convergence node; the pop-up valve is pneumatically controlled by the pneumatic layer; the main channels include 8; each section of the buffer channel includes 8; each section of the buffer channel is provided with an 8-pin channel separator with 3 pop-up valves.

2. The microfluidic device for extracting DNA according to claim 1, characterized in that The length of the inflow layer, the buffer layer and the pneumatic layer are all 10 cm, and the thickness is 1 mm.

3. The microfluidic device for extracting DNA according to claim 1, characterized in that A collection device is provided at the outlet of each main channel; the collection device is used to collect DNA samples.

4. The microfluidic device for extracting DNA according to claim 1, characterized in that A pop-up blister containing a magnetic binding buffer, a pop-up blister containing a washing buffer, and a pop-up blister containing an elution buffer are sequentially arranged along a direction from the entrance of the main channel to the exit of the main channel; Along the direction from the entrance of the main channel to the exit of the main channel, the pop-up bubble cap containing the magnetic binding buffer, the pop-up bubble cap containing the washing buffer, and the pop-up bubble cap containing the elution buffer are opened in sequence to release the magnetic binding buffer, the washing buffer, and the elution buffer in sequence.

5. The microfluidic device for extracting DNA according to claim 1, characterized in that The pop-up valve is a one-way valve, and is used to limit flow into one buffer channel or into eight buffer channels at the same time.

6. The microfluidic device for extracting DNA according to claim 5, characterized in that The pop-up valve is a T-shaped freely movable polydimethylsiloxane structure.

7. The microfluidic device for extracting DNA according to claim 1, characterized in that After use, clean with 1%-2% bleach, rinse with double-distilled water, and air dry.

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