A microfluidic DNA extraction device

Abstract

The application discloses a micro-fluidic DNA extraction device, and belongs to the technical field of micro-fluidics. The device comprises a multi-stage extraction system and a collection unit. The output end of the front-stage extraction system is connected to the input end of the rear-stage extraction system through a siphon channel, and the output end of the last-stage extraction system is connected to the collection unit. Each extraction system and the collection unit are arranged on a disc-shaped centrifugal plate. The distance between each extraction system and the rotation center of the disc-shaped centrifugal plate decreases in turn, and the distance between the collection unit and the rotation center of the disc-shaped centrifugal plate is the smallest. The lowest centrifugal rotation speed required by each extraction system to realize the function increases in turn. The micro-fluidic DNA extraction device provided by the application has high integration, can automatically realize DNA extraction, and has simple manufacturing process and effectively controlled manufacturing cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microfluidic technology, and more particularly relates to a centrifugal microfluidic DNA extraction device. BACKGROUND

[0002] Traditional DNA extraction methods require multiple shaking, centrifugation, transfer, etc., and are complex, time-consuming, costly, and have low integration. It is of great practical significance to realize high-throughput, integrated and automated DNA extraction process. Microfluidic devices have the advantages of small size, low sample and reagent consumption, low analysis cost, fast analysis speed, and easy integration and automation of operation process, which opens up a new way for efficient and rapid DNA extraction and analysis. The extraction technology of DNA based on microfluidic devices has developed to a certain extent, but the current microfluidic chips still have problems such as difficulty in processing complex samples (such as tissues), low automation and integration. SUMMARY

[0003] In view of the shortcomings and deficiencies of the existing microfluidic DNA extraction device, the present application provides a microfluidic DNA extraction device with integration and automation, and the manufacturing process is simple, and the manufacturing cost can be effectively controlled.

[0004] The technical scheme adopted by the present application is as follows:

[0005] A microfluidic DNA extraction device, comprising a multi-stage extraction system and a collection unit, the output end of the front-stage extraction system is connected to the input end of the rear-stage extraction system through a siphon channel, and the output end of the last-stage extraction system is connected to the collection unit;

[0006] Each extraction system is composed of a pretreatment unit, a reaction unit and a separation and purification unit connected in sequence; the pretreatment unit is used for pre-mixing the reagent stored therein or the reagent stored therein and the product from the previous stage to obtain a mixed solution; the reaction unit is used for fully mixing and reacting the mixed solution from the pretreatment unit to obtain a reaction solution; the separation and purification unit is used for separating and purifying the reaction solution from the reaction unit to obtain a layered product; and the collection unit is used for collecting and processing the target substance in the layered product obtained by the last-stage extraction system;

[0007] Each extraction system and the collection unit are arranged on a disc-shaped centrifugal plate, the distance between each extraction system and the rotation center of the disc-shaped centrifugal plate decreases in sequence, and the distance between the collection unit and the rotation center of the disc-shaped centrifugal plate is the smallest, so that the lowest centrifugal rotation speed required for each extraction system to realize its function increases in sequence.

[0008] As the preferred embodiment of the present application, the pre-treatment unit comprises a storage chamber, a buffer chamber, a first capillary valve, a siphon channel and a variable cross-section channel; the storage chamber is connected to the inlet of the buffer chamber, and under the action of centrifugal force, the reagent in the storage chamber can enter the buffer chamber; the outlet of the buffer chamber is connected to one end of the first capillary valve, and the other end of the first capillary valve is connected to the small cross-section port of the variable cross-section channel through the siphon channel, which is used to transfer the liquid passing through the first capillary valve to the variable cross-section channel, and the variable cross-section channel has the function of preventing liquid backflow; the large cross-section port of the variable cross-section channel serves as the outlet of the pre-treatment unit; the first capillary valve opens when the centrifugal speed is higher than or equal to a threshold value, and closes when the centrifugal speed is lower than the threshold value.

[0009] As the preferred embodiment of the present application, the reaction unit is a zigzag-shaped mixed channel arranged along the radial direction of the disc-shaped centrifugal plate.

[0010] As the preferred embodiment of the present application, the mixed channel has an inwardly concave quadrilateral structure and a semicircular structure on its outer wall, and the quadrilateral baffles and semicircular blocks arranged alternately are formed in the mixed channel in sequence; the quadrilateral baffles are inclined towards the direction of liquid flow.

[0011] As the preferred embodiment of the present application, the separation and purification unit comprises two connected first and second separation chambers, and a second capillary valve connected to the bottom of the first separation chamber; the inlet of the first separation chamber is connected to the outlet of the reaction unit through a siphon channel, the top of the second separation chamber is connected to the top of the first separation chamber, and under the action of centrifugal force, the liquid from the reaction liquid in the reaction unit flows into the second separation chamber from the first separation chamber, and the layered product after separation and purification enters the second capillary valve from the bottom of the first separation chamber; the second capillary valve is connected to the inlet of the pre-treatment unit of the next-stage extraction system or to the inlet of the collection unit through a siphon channel, and the bottom of the second separation chamber is closed; the second capillary valve opens when the centrifugal speed is higher than or equal to a threshold value, and closes when the centrifugal speed is lower than the threshold value.

[0012] As the preferred embodiment of the present application, the first separation chamber and the second capillary valve are arranged along the radial direction of the disc-shaped centrifugal plate, and the second capillary valve is located away from the center of the disc-shaped centrifugal plate.

[0013] As the preferred embodiment of the present application, the collection unit comprises a storage chamber, a buffer chamber, a mixed channel and a collection chamber connected in sequence and arranged along the radial direction of the disc-shaped centrifugal plate; the storage chamber is used to store DNA washing liquid, and the buffer chamber is provided with an interface for connecting the last-stage extraction system; the layered product obtained from the last-stage extraction system is preliminarily mixed with the DNA washing liquid in the buffer chamber, and is fully mixed in the mixed channel, and the target substance is collected by the collection chamber.

[0014] As a preferred embodiment of the present invention, the extraction system has 3-5 stages, with the pretreatment unit in each stage being closest to the center of the disc centrifuge plate and the separation and purification unit being furthest from the center of the disc centrifuge plate.

[0015] As a preferred embodiment of the present invention, the working sequence of the multi-cascade extraction system and the collection unit is designed as follows: the multi-cascade extraction system starts working sequentially according to the connection order;

[0016] When the rotation speed is 0, the device does not work; as the rotation speed is increased to the minimum speed required for the first-stage extraction system to work, the first-stage extraction system starts to work. After the fluid is separated and purified in the first-stage extraction system, the fluid enters the second-stage extraction system. The rotation speed is then increased to the minimum speed required for the second-stage extraction system to work, and the second-stage extraction system starts to work. Similarly, until the last-stage extraction system has finished working, the rotation speed is increased until the collection unit starts working, and the extract is collected. The device is then complete.

[0017] As a preferred embodiment of the present invention, the working timing design of the extraction system at each level is as follows:

[0018] 1) Increase the rotation speed to the minimum speed required to open the storage chamber of the pretreatment unit, and the fluid enters the buffer chamber through the storage chamber;

[0019] 2) Increase the rotation speed to the minimum speed required to open the first capillary valve of the pretreatment unit. After the first capillary valve opens, the rotation speed decreases to 0 after the fluid passes through the first capillary valve, and the fluid enters the variable cross-section channel through the siphon channel.

[0020] 3) Increase the rotation speed. Under the action of centrifugal force, the fluid enters the reaction unit from the variable cross-section channel for a thorough mixing reaction. Then, reduce the rotation speed to 0, and the fluid enters the separation and purification unit through the siphon channel.

[0021] 4) Increase the rotation speed again and maintain it for a certain period of time. Different solutions will separate into layers due to the different centrifugal forces they are subjected to, thereby achieving separation and purification.

[0022] 5) Increase the rotation speed to the minimum speed required to open the second capillary valve of the separation and purification unit. The separated and purified stratified product enters the siphon channel through the second capillary valve. Then reduce the rotation speed to 0. The stratified product enters the next stage extraction system or the collection unit through the siphon channel.

[0023] The present invention provides a centrifugal microfluidic DNA extraction device in which the mixing and separation of internal fluids are achieved by centrifugation. Therefore, the device is controlled by a centrifuge, and the flow of fluid in the collection unit and the pretreatment unit, reaction unit and separation and purification unit of each extraction system is driven by centrifugal force.

[0024] The capillary valve used in this invention allows liquid to pass through when the centrifugal extraction device reaches a certain rotational speed. When the centrifugal speed is lower than this value, the liquid cannot pass through the capillary valve. Therefore, the opening and closing of the capillary valve is driven by centrifugal force. Furthermore, when the capillary valve size and solution parameters are determined, the centrifugal speed at which the capillary valve opens is also determined. In addition, the capillary valve also has a buffering effect, preventing reagents from the storage chamber and reaction solutions from the previous stage from failing to mix and react due to their temporal sequence.

[0025] This invention incorporates periodic baffles and inclined baffles within the mixing channel of the reaction unit. This induces a larger radial velocity component in the fluid within the microchannel, effectively stretching the fluid contact surface, generating lateral flow and vortex zones, and inducing chaotic convection, thereby significantly improving the mixing performance of the mixing channel. Furthermore, the mixing channel is radially distributed within the device, and the fluid flow within the mixing channel is driven by centrifugal force.

[0026] In summary, compared with the prior art, the technical solution conceived by the present invention has the following beneficial effects:

[0027] (1) The microfluidic DNA extraction device of the present invention adopts a multi-stage cascade structure, which integrates functions such as mixing, transfer and separation purification, and can meet the general biological process. In addition, the structure ensures the forward progress of the process and has the characteristics of high integration. Furthermore, the microfluidic DNA extraction device of the present invention is a centrifugal microfluidic device, which relies on rotational centrifugation for operation. It is simple and easy to operate and the whole process is automated.

[0028] (2) The microfluidic DNA extraction device of the present invention sets periodic baffles and blocks in the mixing channel, causing the originally parallel fluid layers to stretch and recombine. Even under laminar flow conditions, the fluid can generate a diffusion characteristic close to turbulent flow state in the microchannel, thereby causing the fluid to generate a large radial velocity in the mixing channel, which effectively stretches the contact surface of the fluid, generates lateral flow and vortex zone, induces chaotic convection, and thus significantly improves the mixing performance of the mixing channel.

[0029] (3) The microfluidic DNA extraction device of the present invention can be equipped with multiple extraction systems that are symmetrical about the device center on a disc-shaped centrifuge plate (e.g., attached). Figure 1 (To provide two sets of schematic diagrams), and with identical functions, resource utilization and work efficiency are improved; moreover, as a microfluidic chip, the device is designed with an anti-backflow structure to ensure the reliability of the process.

[0030] (4) The microfluidic DNA extraction device of the present invention has a simple structure and can be mass-produced. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the microfluidic DNA extraction device provided in an embodiment of the present invention;

[0032] Figure 2 This is a schematic diagram of the preprocessing unit provided by the present invention;

[0033] Figure 3 This is a schematic diagram of the reaction unit provided by the present invention;

[0034] Figure 4 This is a schematic diagram of the separation and purification unit provided by the present invention;

[0035] Figure 5 This is a schematic diagram of the collection unit provided by the present invention;

[0036] Figure 6 This is the simulation result of the mixing effect of the mixing channel - the radius curve of the semi-circular block;

[0037] Figure 7 This is the simulation result of the blending effect of the mixing channel - the width curve of the quadrilateral baffle;

[0038] Figure 8 This is the simulation result of the critical pressure when the capillary valve opens versus the capillary valve width curve.

[0039] Figure 9 This is a rotation timing diagram of the microfluidic DNA extraction device provided in the embodiments of the present invention during operation;

[0040] Figure 10 This is a graph showing the relationship between the maximum slope of the hypotenuse of the trapezoidal channel and the contact angle.

[0041] Explanation of reference numerals in the attached figures:

[0042] 1 is the storage chamber, 2 is the buffer chamber, 3 is the first capillary valve, 4 is the variable cross-section channel, 5 is the purification and separation unit, 501 is the first separation chamber, 502 is the second separation chamber, 503 is the second capillary valve, 6 is the siphon channel, 7 is the mixing channel, 8 is the collection chamber, 9 is the semi-circular block, and 10 is the quadrilateral baffle. Detailed Implementation

[0043] To more clearly illustrate the objectives, technical solutions, and advantages of this invention, further explanation is provided below in conjunction with the accompanying drawings. It should be understood that the principles described herein are for explaining the invention, but are not intended to limit it.

[0044] The microfluidic DNA extraction device of the present invention includes a pretreatment unit, a reaction unit, a purification and separation unit, and a collection unit. The pretreatment unit, reaction unit, and purification and separation unit constitute a series of systems. This device is a five-stage cascaded system. Specifically, the last stage system consists of the collection unit. Each stage from the first to the fourth stage is composed of a pretreatment unit, a reaction unit, and a purification and separation unit connected sequentially. The reaction solution output from the previous stage system is mixed into the pretreatment unit of the next stage system for further processing.

[0045] The pretreatment unit mixes and pretreats the reagents from storage chamber 1 and the reaction liquid or storage chamber from the previous stage in buffer chamber 2, and then sequentially transports the resulting mixture to the reaction unit through the first capillary valve 3, siphon channel 6, and variable cross-section channel 4. The reaction unit fully mixes and reacts the mixture through the mixing channel, and then transports the resulting reaction liquid to the separation and purification unit through the siphon channel. The separation and purification unit separates and purifies the reaction liquid obtained from the reaction unit. Different solutions are separated into layers due to different centrifugal forces, thus achieving separation and purification. The collection unit collects and processes the target substance obtained through the separation and purification unit.

[0046] Specifically, such as Figure 1 and 2 As shown, in this embodiment of the invention, the preprocessing unit includes:

[0047] Storage chamber 1 is used to pre-store the reagents or solutions required for the reaction; buffer chamber 2 is used to pre-mix the reagents from storage chamber 1 and the reaction liquid from the previous stage; first capillary valve 3 is a fluid valve device that controls the flow of the mixed solution into the next stage system; siphon channel 6 is used to transfer liquid; variable cross-section channel 4 is used to prevent liquid backflow.

[0048] like Figure 1 and 3 As shown, the reaction unit includes:

[0049] Mixing channel 7 is equipped with periodic baffles and blocks, which cause the originally parallel fluid layers to stretch and reorganize. Even under laminar flow conditions, the fluid can generate diffusion characteristics in the microchannel that are close to turbulent flow, thereby causing the fluid to generate a large radial velocity in the mixing channel. This effectively stretches the fluid contact surface, generates lateral flow and vortex zones, and induces chaotic convection, thus significantly improving the mixing performance of the mixing channel.

[0050] like Figure 1 and 4 As shown, the purification and separation unit 5 includes:

[0051] The microfluidic DNA extraction device provided by this invention consists of two interconnected first separation chambers 501 and second separation chambers 502, and a second capillary valve 503 connected to the bottom of the first separation chamber. The device operates by centrifugal rotation. Under centrifugal force, the liquid from the reaction solution of the reaction unit flows from the first separation chamber 501 into the second separation chamber 502. Solutions or substances of different components will separate due to stratification. The separated and purified stratified products enter the second capillary valve 503 from the bottom of the first separation chamber 501. The second capillary valve 503 is opened when the centrifugal speed is higher than or equal to the centrifugal speed threshold and closed when the centrifugal speed is lower than the centrifugal speed threshold, thereby selecting the desired substance or solution to achieve separation and purification.

[0052] like Figure 1 and 5 As shown, the collection unit includes:

[0053] Collection chamber 8 is used to collect the target substance.

[0054] The variable cross-section channel proposed in this invention allows liquid to flow smoothly into the next large chamber without centrifugal force, thus eliminating the valve effect and preventing liquid backflow. Fluid simulations were performed in the finite element software Multiphysics COMSOL, yielding a curve showing the relationship between the maximum slope of the trapezoidal channel's hypotenuse and the contact angle under the condition of eliminating the valve effect, as shown below. Figure 10 As shown.

[0055] In one specific embodiment of the invention, the mixing channel is used for thorough mixing of the liquid. Figure 3 This is a schematic diagram of the reaction unit provided by the present invention, namely a schematic diagram of the mixing channel, including semi-circular blocking blocks 9 and quadrilateral baffles 10 alternately arranged on the mixing channel.

[0056] To verify the mixing effect of the mixing channel, fluid simulation of a typical device design was performed using the finite element software Multiphysics COMSOL. The mixing effect of the mixing channel under different semi-circular block radii is shown in the following figures, with a quadrilateral baffle width of 75 μm. Figure 6 As shown; the mixing effect of the mixing channel under different quadrilateral baffle thicknesses when the radius of the semi-circular block is 0.1mm. Figure 7 As shown. The relative concentration variance is used to measure the mixing effect, with a value of 1 indicating no mixing at all and a value of 0 indicating complete mixing.

[0057] To verify the valve effect of the capillary valve, a typical device design was simulated using the finite element software Multiphysics COMSOL. The results showed the relationship between the theoretical critical pressure and the simulated value versus the capillary valve width when the capillary valve height was 100 μm. Figure 8As shown.

[0058] The calculation of the theoretical critical pressure value is shown in Formula 1:

[0059]

[0060] Where P s γ is the critical pressure, d is the surface tension coefficient, h is the cross-sectional width, and θ is the cross-sectional height.

[0061] In one specific embodiment of the present invention, the structural design process of the above-mentioned microfluidic DNA extraction device includes:

[0062] (1) Assembly of the devices: The storage chamber, buffer chamber, first capillary valve, siphon channel and variable cross-section channel are connected in sequence to form a pretreatment unit; the mixing channel forms a reaction unit; the first separation chamber, second separation chamber and second capillary valve form a separation and purification unit; the mixing channel and collection chamber are connected to form a collection unit. The pretreatment unit, reaction unit and separation and purification unit are connected in sequence to form a primary system to realize a typical biological reaction or biological process in one step. Moreover, each structure (such as flow channel, capillary valve and mixing channel) is distributed radially, so as to maximize the use of the driving effect of centrifugal force.

[0063] DNA extraction typically involves multiple biological reactions, thus requiring a multi-stage system. A siphon channel connects each stage of the system, transferring fluid from the radially distal stage to the radially proximal stage, while maximizing the use of space within the device. The microfluidic DNA extraction device provided in this embodiment features a five-stage cascade system (considering the collection unit as the fifth stage), with each system connected via a siphon channel, and the distance from the device center decreasing sequentially.

[0064] (2) Functional Implementation of the Device: General biological reactions or processes involve operations such as mixing, purification, and separation, and require the reaction to be irreversible in the forward direction. The structural design method of the microfluidic DNA extraction device provided by this invention corresponds to the process requirements in terms of structural design: the pretreatment unit corresponds to the pretreatment function, the reaction unit corresponds to the mixing function, the separation and purification unit corresponds to the separation and purification function, and the collection unit corresponds to the collection function. Moreover, in the structural design of the device: on the one hand, siphon channels are used to connect and communicate with each device and each level of the system, so that the biological process proceeds in the forward direction, which meets the requirements of general biological processes; on the other hand, variable cross-section channels are used to connect the unit structures and each level of the system to ensure that the fluid does not flow back or backflow, and that the functions of each unit structure and each system do not interfere with each other.

[0065] (3) Timing design method of the device: The microfluidic DNA extraction device of the present invention belongs to the centrifugal microfluidic device. The core components of the device, such as capillary valves, mixing channels, separation structures, etc., rely on centrifugal rotation for operation.

[0066] This invention provides a structural design method for a microfluidic DNA extraction device. In terms of spatial distribution, the distance between each system level and the device center decreases sequentially. Combined with the device dimensions, the minimum centrifugal rotation speed required for each system to function increases sequentially. This ensures that during centrifugal rotation, each system level triggers its function sequentially with increasing rotation speed, and the next lower-level system cannot begin operation while each level is in operation.

[0067] The timing sequence of the microfluidic DNA extraction device provided by this invention is as follows: When the rotation speed is 0, all stages of the system are inactive; as the rotation speed increases to the minimum required for the first-stage system to operate, the first-stage system begins to operate, while the second, third, fourth, and fifth-stage systems remain inactive; after the fluid completes separation and purification in the first-stage system, all the fluid enters the second-stage system, and the rotation speed is increased again to the minimum required for the second-stage system to operate, at which point the second-stage system begins to operate, while the third, fourth, and fifth-stage systems remain inactive; after the fluid completes separation and purification in the second-stage system, all the fluid enters the third-stage system, and the rotation speed is increased again to the minimum required for the third-stage system to operate, at which point the third-stage system begins to operate, while the fourth and fifth-stage systems remain inactive; after the fluid completes separation and purification in the third-stage system, all the fluid enters the fourth-stage system, and the rotation speed is increased again to the minimum required for the fourth-stage system to operate, at which point the fourth-stage system begins to operate, while the fifth-stage system remains inactive; after the fluid completes separation and purification in the fourth-stage system, all the fluid enters the fourth-stage system, and the rotation speed is increased again to the minimum required for the fifth-stage system to operate, at which point the fifth-stage system begins to operate; finally, the fluid is collected in the fifth-stage system, and the device operation is complete.

[0068] The timing sequence of each stage of the system is as follows: The rotational speed is increased to the minimum required to open storage chamber 1, allowing fluid to enter buffer chamber 2. Then, the rotational speed is increased again to the minimum required to open the first capillary valve 3, allowing fluid to pass through it. The rotational speed is then reduced to 0, allowing fluid to pass through siphon channel 6 and variable cross-section channel 4. The rotational speed is then increased again, and under centrifugal force, the fluid enters mixing channel 7 for thorough mixing. The rotational speed is then reduced to 0 again, and the fluid enters purification and separation unit 5 through the siphon channel. The rotational speed is then increased again to maintain a certain value for separation and extraction. After a certain time, separation and purification are completed. The rotational speed is then increased to the minimum required to open the second capillary valve 503, allowing fluid to enter the siphon channel through the second capillary valve 503. Finally, the rotational speed is reduced to 0, and the fluid enters the next stage of the system through the siphon channel.

[0069] like Figure 9 The diagram shows the operating parameters of a five-stage system for a microfluidic DNA extraction device. In a specific embodiment of the present invention, the method for DNA extraction using the last four stages of the microfluidic DNA extraction device includes the following steps:

[0070] (1) Single cell preparation: Set the device speed to zero, add an appropriate amount of trypsin solution and cell tissue fluid to the storage chamber 1 of the second-stage system, and correspondingly add an appropriate amount of cell lysis solution, DNA precipitation solution and DNA washing solution to the storage chamber 1 of the subsequent third, fourth and fifth-stage systems, respectively.

[0071] The rotation speed is increased to 1200 rpm, and the trypsin solution and cell tissue fluid enter the buffer chamber 2 through the storage chamber. Then, as the rotation speed is increased to 1280 rpm, the first capillary valve 3 opens, and the mixture of trypsin solution and cell tissue fluid passes through the first capillary valve 3. The rotation speed is then reduced to 0, and the mixture passes through the siphon channel 6 and the variable cross-section channel 4. The rotation speed is then increased to 800 rpm, and under centrifugal force, the mixture enters the mixing channel 7 for thorough mixing. The rotation speed is then reduced to 0 again, and the mixture enters the purification and separation unit 5 through the siphon channel. The rotation speed is then increased again to 800 rpm for separation and extraction. After a certain period, the separation and purification are completed, and the rotation speed is increased to 1000 rpm. The second capillary valve 503 opens, and the precipitate, i.e., the single-cell fluid, enters the siphon channel. The rotation speed is then reduced to 0, and the single-cell fluid enters the next stage system through the siphon channel.

[0072] (2) Cell lysis: The single-cell fluid obtained in step (1) enters the next stage system. The rotation speed is then increased to 1400 rpm, and the cell lysis solution enters the buffer chamber 2 together with the cell lysis solution through the storage chamber 1. Then, the rotation speed is increased to 1550 rpm, the first capillary valve 3 opens, and the mixture of single-cell fluid and cell lysis solution passes through the first capillary valve 3. Then, the rotation speed is reduced to 0, and the mixture passes through the siphon channel 6 and the variable cross-section channel 4. Then, the rotation speed is increased to 800 rpm, and under the action of centrifugal force, the mixture enters the mixing channel 7 for thorough mixing reaction. Then, the rotation speed is reduced to 0 again, and the mixture enters the purification and separation unit 5 through the siphon channel. Then, the rotation speed is increased to 800 rpm again for separation and extraction. After a certain period of time, the separation and purification are completed, the rotation speed is increased to 1100 rpm, the second capillary valve 503 opens, and the precipitate, i.e., the cell lysis solution, enters the siphon channel. Then, the rotation speed is reduced to 0, and the cell lysis solution enters the next stage system through the siphon channel.

[0073] (3) DNA Extraction: The cell lysate obtained in step (2) enters the next stage system. The rotation speed is then increased to 1690 rpm, and the DNA precipitate enters the buffer chamber 2 together with the cell lysate through storage chamber 1. The rotation speed is then increased to 1800 rpm, the first capillary valve 3 opens, and the mixture of DNA precipitate and cell lysate passes through the first capillary valve 3. The rotation speed is then reduced to 0, and the mixture passes through the siphon channel 6 and the variable cross-section channel 4. The rotation speed is then increased to 800 rpm, and under centrifugal force, the mixture enters the mixing channel 7 for thorough mixing. The rotation speed is then reduced to 0 again, and the mixture enters the purification and separation unit 5 through the siphon channel. The rotation speed is then increased to 800 rpm again for separation and extraction. After a certain period, the separation and purification are completed, the rotation speed is increased to 1100 rpm, the second capillary valve 503 opens, and the precipitate, i.e., the DNA precipitate, enters the siphon channel. The rotation speed is then reduced to 0, and the DNA precipitate enters the next stage system through the siphon channel.

[0074] (4) DNA collection: The DNA precipitate obtained in step (3) enters the next stage, and then the rotation speed is increased to 2500 rpm. The DNA washing solution in the storage chamber 1 of this stage enters the buffer chamber 2 to wash the DNA precipitate. Finally, the DNA enters the collection chamber 8, and the rotation speed is reduced to 0 to complete the entire DNA extraction process.

[0075] In one specific embodiment of the present invention, a preprocessing step can be added before the second-level system, which can be designed according to actual needs. Figure 9 The document also provides the control parameters and procedures for executing the first-level system:

[0076] The rotation speed is increased to 950 rpm, storage chamber 1 opens, and fluid enters buffer chamber 2. Then, as the rotation speed is increased to 1050 rpm, the first capillary valve 3 opens, and fluid passes through it. The rotation speed is then reduced to 0, and fluid passes through siphon channel 6 and variable cross-section channel 4. The rotation speed is then increased to 800 rpm, and under centrifugal force, the fluid enters mixing channel 7 for thorough mixing. The rotation speed is then reduced to 0 again, and the mixture enters purification and separation unit 5 through siphon channel. The rotation speed is then increased to 800 rpm again for separation and extraction. After a certain period, separation and purification are completed. The rotation speed is increased to 1000 rpm, the second capillary valve 503 opens, and the precipitated stratified product enters the siphon channel. The rotation speed is then reduced to 0, and the stratified product enters the next stage system through the siphon channel.

[0077] Those skilled in the art should understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic DNA extraction device, characterized in that, It includes a multi-cascaded extraction system and a collection unit. The output of the previous extraction system is connected to the input of the next extraction system through a siphon channel, and the output of the last extraction system is connected to the collection unit. Each extraction system consists of a pretreatment unit, a reaction unit, and a separation and purification unit connected in sequence. The pretreatment unit is used to premix the reagents stored in it or the reagents stored in it and the product from the previous stage to obtain a mixture. The reaction unit is used to fully mix and react the mixture from the pretreatment unit to obtain a reaction solution. The separation and purification unit is used to separate and purify the reaction solution from the reaction unit to obtain a stratified product. The collection unit is used to collect and process the target substances in the stratified products obtained from the final extraction system; Each extraction system and collection unit is arranged on a disc-shaped centrifuge plate. The distance between each extraction system and the center of rotation of the disc-shaped centrifuge plate decreases sequentially, and the distance between the collection unit and the center of rotation of the disc-shaped centrifuge plate is the smallest. This makes the minimum centrifugal rotation speed required for each extraction system to function sequentially increase. In addition, the pretreatment unit in each extraction system is closest to the center of the disc-shaped centrifuge plate, while the separation and purification unit is farthest from the center of the disc-shaped centrifuge plate. The pretreatment unit includes a storage chamber (1), a buffer chamber (2), a first capillary valve (3), a siphon channel (6), and a variable cross-section channel (4). The storage chamber (1) is connected to the inlet of the buffer chamber (2). Under the action of centrifugal force, the reagent in the storage chamber (1) can enter the buffer chamber (2). The outlet of the buffer chamber (2) is connected to one end of the first capillary valve (3). The other end of the first capillary valve (3) is connected to the small cross-section port of the variable cross-section channel (4) through the siphon channel (6). The large cross-section port of the variable cross-section channel (4) serves as the outlet of the pretreatment unit. The first capillary valve (3) is opened when the centrifugal speed is higher than or equal to the centrifugal speed threshold and closed when the centrifugal speed is lower than the centrifugal speed threshold. The reaction unit is a serrated mixing channel (7) arranged radially along the disc-shaped centrifuge plate; the outer wall of the mixing channel (7) is provided with an inwardly concave quadrilateral structure and a semi-circular structure, and alternating quadrilateral baffles (10) and semi-circular blocks (9) are formed in the mixing channel (7); the quadrilateral baffles (10) are inclined in the direction of liquid flow. The separation and purification unit includes two interconnected first separation chambers (501) and second separation chambers (502), and a second capillary valve (503) connected to the bottom of the first separation chamber. The first separation chamber and the second capillary valve are arranged radially along the disc-shaped centrifuge plate, and the second capillary valve is located away from the center of the disc-shaped centrifuge plate. The inlet of the first separation chamber is connected to the outlet of the reaction unit through a siphon channel, and the top of the second separation chamber is connected to the top of the first separation chamber. Under the action of centrifugal force, the liquid from the reaction liquid of the reaction unit flows from the first separation chamber into the second separation chamber, and the separated and purified stratified product enters the second capillary valve from the bottom of the first separation chamber. The second capillary valve is connected to the inlet of the pretreatment unit of the next stage extraction system or the inlet of the collection unit through a siphon channel, and the bottom of the second separation chamber is closed. The second capillary valve is opened when the centrifugal speed is higher than or equal to the centrifugal speed threshold and closed when the centrifugal speed is lower than the centrifugal speed threshold.

2. The microfluidic DNA extraction device according to claim 1, characterized in that, The extraction system described is of level 3-5.

3. The microfluidic DNA extraction device according to claim 1, characterized in that, The working sequence design of the multi-cascade extraction system and collection unit is as follows: the multi-cascade extraction system starts working sequentially according to the connection order; When the rotation speed is 0, the device does not work; as the rotation speed is increased to the minimum speed required for the first-stage extraction system to work, the first-stage extraction system starts to work. After the fluid is separated and purified in the first-stage extraction system, the fluid enters the second-stage extraction system. The rotation speed is then increased to the minimum speed required for the second-stage extraction system to work, and the second-stage extraction system starts to work. Similarly, until the last-stage extraction system has finished working, the rotation speed is increased until the collection unit starts working, and the extract is collected. The device is then complete.

4. The microfluidic DNA extraction device according to claim 3, characterized in that, The timing design of the extraction system at each level is as follows: 1) Increase the rotation speed to the minimum speed required to open the storage chamber (1) of the pretreatment unit, and the fluid enters the buffer chamber (2) through the storage chamber (1); 2) Increase the rotation speed to the minimum speed required to open the first capillary valve (3) of the pretreatment unit. After the first capillary valve (3) opens, the rotation speed decreases to 0 after the fluid passes through the first capillary valve (3). The fluid enters the variable cross-section channel (4) through the siphon channel (6). 3) Increase the rotation speed. Under the action of centrifugal force, the fluid enters the reaction unit from the variable cross-section channel (4) to carry out a full mixing reaction. Then the rotation speed is reduced to 0, and the fluid enters the separation and purification unit through the siphon channel. 4) Increase the rotation speed again and maintain it for a certain period of time. Different solutions will separate into layers due to the different centrifugal forces they are subjected to, thereby achieving separation and purification. 5) Increase the rotation speed to the minimum required to open the second capillary valve (503) of the separation and purification unit. The separated and purified stratified product enters the siphon channel through the second capillary valve (503). Then reduce the rotation speed to 0. The stratified product enters the next stage extraction system or the collection unit through the siphon channel.

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