Microfluidic chip for nucleic acid extraction and purification

Abstract

The present invention provides a microfluidic chip for nucleic acid extraction and purification and belongs to the technical field of microfluidics. The microfluidic chip comprises: a storage portion, comprising a sample cavity, a reagent cavity, and an isolation liquid cavity; and a nucleic acid extraction and purification portion, arranged on the side of the storage portion away from a rotation center and comprising at least two working cavities and a sealed cavity that is arranged on the side of the working cavities close to the rotation center. Different working cavities are connected by means of a first valve body arranged in the sealed cavity. The distal ends of the sample cavity, the reagent cavity, and the isolation liquid cavity are connected to the proximal end of the sealed cavity by means of a second valve body. By means of a centrifugal force and an Euler force, a liquid reagent is enabled to fill a space and be released completely and to be well mixed with magnetic beads. Mineral oil is used in conjunction with a unique cavity structure and a segmented pressure-blocking valve structure design to ensure that the liquid components in the cavities are isolated from one another, thereby ensuring a good nucleic acid extraction effect on the premise that the space is well utilized.

Description

A microfluidic chip for nucleic acid extraction and purification Technical Field

[0001] This application relates to the field of microfluidics, and more particularly to a microfluidic chip for nucleic acid extraction and purification. Background Technology

[0002] Molecular genetics is a branch of genetics that studies the mechanisms of biological inheritance and variation at the molecular level. Its main research content covers the nature, function, and changes of genes. Nucleic acids (including DNA and RNA) are the most important research objects in the field of molecular genetics, and the methods for obtaining them have always been a focus of research. Exploring a simple and efficient method for nucleic acid extraction and purification is of great significance for molecular genetics research and biomedical detection (especially molecular diagnostics).

[0003] Currently, the most common methods for nucleic acid extraction and purification can be divided into three categories: organic chemical reagent extraction, centrifugal column adsorption, and magnetic bead adsorption. Organic chemical reagent extraction generally requires the use of large amounts of hazardous chemicals (some containing chlorine), and manual operation is time-consuming, labor-intensive, and inefficient. Centrifugal column adsorption also faces the problem of low sample throughput, and the adsorption column is prone to clogging, affecting the entire extraction process. Compared with the other two methods, magnetic bead adsorption has higher efficiency and reproducibility, higher sample compatibility, and is more conducive to the large-scale and automated implementation of nucleic acid extraction and purification processes. Most of the fully automated nucleic acid extraction instruments or platforms currently on the market operate using magnetic bead adsorption.

[0004] However, current automation strategies in nucleic acid extraction and purification still face significant challenges. For example, they often require complex auxiliary devices or components, reliance on bulky and inefficient robotic arms, or sophisticated and expensive pipetting equipment. Microfluidic chips, on the other hand, manipulate minute volumes (from microliters to milliliters) of fluid on solid substrates, ultimately achieving specific functions or objectives through fluid movement. Due to their advantages of miniaturization, integration, and high automation, they hold great promise for application in nucleic acid extraction and purification. Currently, there are several publicly disclosed technical solutions using traditional pneumatically driven microfluidic chips as carriers for automated nucleic acid extraction and purification. For example, Chinese invention patents CN 113817577 A, CN 112322453 A, and CN 108300640 A each disclose a microfluidic chip structure based on magnetic bead adsorption for nucleic acid extraction. The working principle of these solutions is to use pressure changes as the driving force to achieve mixing and transfer of magnetic beads in various liquid reagents. However, a significant limitation of these solutions is the need for an externally supplied, relatively stable gas pressure. Excessive or insufficient pressure, or excessively rapid or slow pressure application, can easily lead to uncontrolled microfluidics. Therefore, they often require an additional, high-precision syringe pump or pneumatic pump, which may make the entire system overly complex. Automated nucleic acid extraction and purification solutions based on next-generation digital microfluidics (DMF) technology have also been reported, such as patent CN 115820399. A discloses an integrated nucleic acid analysis system based on digital microfluidics (DMF), which includes a nucleic acid extraction and purification method driven by dielectric wetting effect. This method can efficiently transfer and mix samples with smaller volumes, avoiding the dead volume problem that traditional microfluidics may face. It reduces sample and reagent costs and meets shorter time requirements. However, its drawbacks include the need for a relatively precise voltage control system and logic circuits, resulting in higher manufacturing costs. It is also limited by the sample processing volume and the overall size of the digital microfluidic device. Centrifugal disc-type microfluidic chips have unique advantages in space utilization and driving methods, and they also have applications in nucleic acid extraction and purification, such as patents CN 217600742 U, CN 115926928 A, and CN 109609345. Each of A and B disclosed a nucleic acid extraction chip design. These chips cleverly utilize the relative motion between magnetic beads and various liquid reagents or samples to adsorb, clean, and elute nucleic acid samples. Since they do not involve active liquid transfer, the design and implementation of such schemes are relatively simple and efficient. However, these schemes also have some risks and shortcomings. For example, in order to prevent crosstalk between the liquids in each chamber, complex barrier valves need to be used between the chambers. The lack of disturbance in the liquids in each chamber leads to poor mixing of the magnetic beads in the liquids of each chamber. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic chip for nucleic acid extraction and purification. It primarily relies on centrifugal force and Euler force to achieve complete filling and release of liquid reagents and thorough mixing with magnetic beads. Mineral oil, combined with a unique chamber structure and segmented pressure valve design, ensures the isolation of liquid components between chambers. This design simplifies the process, reduces costs, and simplifies operation, while significantly enhancing the stability and reliability of the nucleic acid extraction process, guaranteeing excellent nucleic acid extraction results with extremely high space utilization.

[0006] The present invention adopts the following technical solution:

[0007] A microfluidic chip for nucleic acid extraction and purification includes: a storage section comprising a sample chamber, a reagent chamber, and an isolation liquid chamber; and a nucleic acid extraction and purification section disposed on a side of the storage section away from the rotation center, the nucleic acid extraction and purification section comprising at least two working chambers and a sealed chamber disposed on the side of the working chambers closer to the rotation center, the different working chambers being connected by a first valve body disposed in the sealed chamber, and the distal ends of the sample chamber, reagent chamber, and isolation liquid chamber being connected to the proximal end of the sealed chamber by a second valve body.

[0008] Furthermore, the width of the sealing cavity is not less than the height of the sealing cavity, and the height of the working cavity is not less than the height of the sealing cavity.

[0009] Furthermore, the sealing cavity is in the shape of a spiral ring or a circular ring, and the working cavity is located on the outer edge of the sealing cavity away from the rotation center. Based on the extraction and purification process, the working cavities of the corresponding processes are sequentially arranged along the sealing cavity. The isolation liquid cavity is connected to the proximal end of the sealing cavity through a second valve body.

[0010] Furthermore, the first valve body is a first slit of a certain size, and the cross-section of the first slit gradually decreases along the flow direction of the extraction and purification process; the inner wall of the first slit on the side away from the rotation center is a plane or an arc surface, and the distance between the plane or the arc surface and the rotation center gradually decreases along the flow direction of the extraction and purification process.

[0011] Furthermore, the difference between the two ends of the inner wall of the first slit on the side away from the rotation center and the rotation center is not less than 1 / 2 of the width of the sealing cavity.

[0012] Furthermore, at least one flow-blocking dam is provided inside the sealing cavity, and the flow-blocking dam itself or the flow-blocking dam and the inner wall of the sealing cavity form a second slit; for a single working cavity, the projection of the flow-blocking dam rotating about the center of rotation completely covers the first slit of the first valve body connected to the working cavity.

[0013] Furthermore, the first slit has a first width, the second slit has a second width, the first width is less than the second width, and the first width of the first slit is not greater than the height of the sealing cavity; the flow-blocking dam is connected to the inner wall of the sealing cavity on the side near the rotation center, and the flow-blocking dam is located at or beyond the connection between the working cavity and the sealing cavity on the side away from the rotation center.

[0014] Furthermore, the flow-blocking dams are uniformly arranged along the direction of the spiral or circular ring, and the sum of the lengths of at least one side of the outer edge of the flow-blocking dam in the sealing cavity corresponding to a single working cavity is at least 2 / 5 of the length of the outer edge of the sealing cavity corresponding to the single working cavity.

[0015] Furthermore, the working chamber is connected to a bubble generating chamber via a third valve; the side of the bubble generating chamber away from the rotation center is connected to the inner wall of the working chamber via the third valve; the relationship between the volume V1 of the bubble generating chamber and the volume V2 of the working chamber satisfies the following equation:

[0016]

[0017] Where m1 is the mass of the magnetic beads added, and m0 is the standard mass. This is a correction factor.

[0018] Furthermore, the isolation liquid chamber is arranged in a ring shape at the proximal end of the microfluidic chip; the sample chamber and at least one reagent chamber are fan-shaped and arranged around the outer side of the isolation liquid chamber; at least two working chambers are fan-shaped and arranged on the outer edge of the sealing chamber, around the rotation center; the reagent chamber and the sample chamber are respectively connected to the proximal end of the sealing chamber corresponding to the working chamber through a second valve.

[0019] Alternatively, the isolation liquid chamber, sample chamber, and at least one reagent chamber may be fan-shaped, arranged in a circle around the rotation center; at least two of the working chambers may be fan-shaped, located on the outer edge of the sealed chamber, arranged in a circle around the rotation center, and the reagent chamber and sample chamber may be connected to the proximal end of the corresponding sealed chamber of the working chamber via a second valve body. Compared with the prior art, the beneficial technical effects of the present invention include:

[0020] 1. The various chambers are arranged in a circular pattern around the center of the disc base. Magnetic bead transfer is achieved through the relative circular motion between the rotating disc and a fixed magnet, making the magnetic bead transfer operation more stable, automated, and efficient. Furthermore, fluid flow and magnetic bead transfer are more reliable under the drive of stable centrifugal force. This invention proposes setting all chambers in the nucleic acid extraction process in the same circumferential direction. Magnetic beads can be transferred between chambers simply by rotating the chip, thereby realizing the nucleic acid purification process. The structure is simple and easily automated.

[0021] 2. The adsorption, cleaning and elution of nucleic acids are achieved by sequentially transferring magnetic beads in a specific liquid cavity. The waste liquid reagents after nucleic acid extraction and purification are stored in situ, eliminating the need for an additional waste liquid pool design. The overall space utilization of the chip is high, and the design of a waste liquid pool is avoided, thus realizing chip miniaturization.

[0022] 3. Using mineral oil as the sealing medium between the sample processing chambers has no impact on the nucleic acid extraction and purification process. Simultaneously, the transfer of magnetic beads in the mineral oil repels and removes residual liquid from the previous chamber, resulting in better washing and nucleic acid elution. This invention creatively proposes a sealed chamber design. By placing the sealed chamber near the proximal end of the working chamber, circumferential area is saved, achieving miniaturization. Furthermore, the height difference between the sealed chamber and the working chamber, combined with the surface tension of the medium, further removes residual liquid, improving the purity of nucleic acid extraction.

[0023] 4. The sealed cavity is a flat, spiral ring-shaped structure proximal to each sample processing chamber, and it is designed with multiple segmented flow-blocking valves inside. This prevents cross-contamination caused by the mixing of liquid reagents in the sample processing chambers with the mineral oil in the sealed mineral oil chamber during magnetic bead mixing, thus enhancing the reliability of nucleic acid extraction and purification using the magnetic bead method. The present invention proposes an arrangement where each working chamber is independently located at the distal end, and the sealed cavity is located at the proximal end. The connection of the sealed cavities enables communication between multiple working chambers. This arrangement prevents liquid cross-contamination caused by the Euler force generated during chip rotation and allows for the transfer of the nucleic acid extraction device between the working chambers. Furthermore, the segmented flow-blocking valves in the sealed cavity further reduce the possibility of cross-contamination and improve system stability.

[0024] 5. The bubbles generated by the bubble generation chamber provide cavity space for the liquid reagents in each sample processing chamber. Furthermore, the Euler force generated by the forward and reverse rotation of the chip ensures thorough mixing of the liquid reagents and magnetic beads in each sample processing chamber, guaranteeing good nucleic acid extraction and purification results without the need for additional mixing components. This invention creatively proposes a method for achieving reagent mixing within the chamber without the need for a stir bar. To prevent liquid cross-contamination between multiple working chambers, this invention fills the sealed chambers with mineral oil. However, this introduces another problem: when the chamber is completely filled with liquid, reagent mixing becomes difficult. This invention creatively proposes a bubble generator. Under centrifugal force, the volume of the bubble generator is compressed and energy is stored. When the centrifugal force stops, the gas in the bubble generator is rapidly discharged, forming bubbles in the reaction chamber. When mixing is required, by changing the chip speed or rotation direction, the bubbles will stir within the chamber under Euler force, achieving liquid mixing. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 is a microfluidic chip structure diagram for nucleic acid extraction and purification provided in an embodiment of the present invention;

[0027] Figure 2 is the AA view in Figure 1;

[0028] Figure 3 is the BB view in Figure 1;

[0029] Figure 4 is a rotational projection view based on the rotation center provided in an embodiment of the present invention;

[0030] Figure 5 is a schematic diagram of another flow-blocking dam structure provided in an embodiment of the present invention;

[0031] Figure 6 is a schematic diagram of a single nucleic acid extraction and purification unit provided in an embodiment of the present invention;

[0032] Figure 7 is a microfluidic chip structure diagram for nucleic acid extraction and purification provided in an embodiment of the present invention;

[0033] Figure 8 is a schematic diagram of different stages of nucleic acid detection provided in the embodiments of the present invention. a) Reagent pre-positioning and sample injection; b) Reagent centrifugation and mineral oil sealing; c) Bubble cavity generation; d) Thorough mixing of liquid reagent and magnetic beads in the working chamber for sample lysis and nucleic acid adsorption; e) Collection and transfer of magnetic beads in the working chamber for sample lysis and nucleic acid adsorption; f) Magnetic beads entering the mineral oil-sealed cavity near the center of the working chamber for magnetic bead washing; g) Thorough mixing of liquid reagent and magnetic beads in the working chamber for magnetic bead washing; h) Thorough mixing of liquid reagent and magnetic beads in the working chamber for secondary magnetic bead washing; i) Thorough mixing of liquid reagent and magnetic beads in the working chamber for nucleic acid elution; j) Completion of nucleic acid extraction and purification.

[0034] Reference numerals: 01, First valve body; 010, First slit; 011, Inner wall of the slit; 012, Non-obstruction zone; 02, Second valve body; 03, Third valve body; 1, Storage section; 10, Sample chamber; 11, Reagent chamber; 12, Isolation liquid chamber; 2, Nucleic acid extraction and purification section; 20, Sealing chamber; 200, Flow dam; 201, Second slit; 21, Working chamber; 22, Bubble generation chamber. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0036] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0037] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0038] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of this invention is in use. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. In addition, the terms "first," "second," and "third," etc., are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0039] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0040] In this embodiment of the invention, "distal end" and "proximal end" are determined by comparing the distances relative to the rotation center O. For example, the side of a chamber closer to the rotation center is the proximal end, and the side farther from the rotation center is the distal end.

[0041] This invention discloses a microfluidic chip for nucleic acid extraction and purification, as shown in Figure 1, comprising: a storage unit 1, which includes a sample chamber 10, a reagent chamber 11, and an isolation liquid chamber 12; and a nucleic acid extraction and purification unit 2, which is disposed on the side of the storage unit away from the rotation center O. The nucleic acid extraction and purification unit 2 includes at least two working chambers 21 and a sealing chamber 20 disposed on the side of the working chambers 21 near the rotation center O. The different working chambers 21 are connected by a first valve body 01 disposed in the sealing chamber 20. The distal ends of the sample chamber 10, reagent chamber 11, and isolation liquid chamber 12 are connected to the proximal end of the sealing chamber 20 by a second valve body 02.

[0042] As shown in Figure 1, this embodiment of the invention includes four working chambers 21 and a sealing chamber 20 located on the inner edge of each working chamber. The liquid in the sample chamber 10 enters the rightmost working chamber through the second valve body 02. This working chamber is used for sample lysis and nucleic acid adsorption. Subsequently, the working chambers in the counterclockwise direction are used for the first washing, the second washing, and nucleic acid elution. This embodiment of the invention also includes three reagent chambers for storing reagents with different functions. These chambers are connected to the proximal ends of the sealing chambers 20 corresponding to the respective working chambers 21 through three second valve bodies 02. However, it should be noted that one reagent chamber 11 can be provided to achieve any of the three functions of the above-mentioned reagent chambers 11. Alternatively, one reagent chamber 11 can be provided, and the same number of quantitative chambers as the working chambers 21 can be provided. The quantified reagent is then filled into the corresponding working chamber 21. The working chamber 21 contains a pre-prepared lyophilized reagent, which dissolves with the reagent to form a reagent solution with the corresponding function.

[0043] This invention incorporates multiple valves, such as a first valve body, a second valve body, and a third valve body (described later). The form and combination of the valves are not limited, as long as they open and close at the required steps. These can be active or passive valves. Specifically, they can employ manually operated mechanical valves or phase-change valves, or capillary tubes that open or close under centrifugal force. To reduce production costs while maintaining detection accuracy, capillary tubes are preferred for control. The capillary tubes range in size from hundreds of micrometers to micrometers, with different diameters and lengths to achieve opening or closing effects under varying centrifugal forces.

[0044] The isolation liquid chamber is used to store the isolation liquid. The "isolation liquid" is a hydrophobic liquid with a density lower than that of the liquid in the working chamber and is inert, so it will not react with substances in the system, such as mineral oil, paraffin, etc. The specific composition of the isolation liquid is not limited here.

[0045] The movement of nucleic acid substances in each chamber is achieved by transferring them using a nucleic acid adsorption device, such as magnetic beads or polymer materials, as long as they can adsorb nucleic acids and transfer them between different chambers under external drive.

[0046] The technical solution provided in this invention relies on centrifugal force and Euler force to achieve complete filling and release of liquid reagents and thorough mixing with magnetic beads. The use of mineral oil, combined with a unique chamber structure and segmented pressure valve structure design, ensures that the liquid components between chambers are isolated from each other. While simplifying the design, reducing costs, and facilitating operation, it greatly enhances the stability and reliability of the nucleic acid extraction process, and ensures good nucleic acid extraction results under the premise of extremely high space utilization.

[0047] As shown in Figure 2, the width L of the sealing cavity 20 is not less than the height H of the sealing cavity 20, and the height H1 of the working cavity 21 is not less than the height H of the sealing cavity. Since the insulating liquid in the insulating liquid cavity 12 isolates and seals multiple working cavities 21, the cross-section of the sealing cavity is set to be small to reduce the amount of insulating liquid added. Secondly, during the addition of the insulating liquid, the insulating liquid flows in the sealing cavities 20 corresponding to the multiple working cavities 21, which presents a chance of mixing during the filling process. Thirdly, if the cross-sectional dimensions of the sealing cavity 20 and the working cavity 21 are similar, the two liquids are prone to mixing, increasing the risk of cross-contamination between multiple working cavities 21. To solve the above problems, this application sets the cross-section of the sealing cavity 20 to be elongated, and the cross-sectional dimensions are significantly different from those of the working cavity 21, further reducing the disturbance of the reagents in the working cavity 21 by the insulating liquid during the filling process and avoiding cross-contamination problems between reagents in different working cavities 21. Preferably, the width L of the sealing cavity 20 is at least twice its height H, and the height H1 of the working cavity 21 is at least three times the height H of the sealing cavity; the height of the working cavity is less than 10 mm, and the height H of the sealing cavity 20 is 0.1-3 mm. The insulating liquid and reagent are prone to mixing at higher acceleration / deceleration and lower rotational speeds. This is because at higher acceleration / deceleration, speed fluctuations have a greater impact on the interface between the reagent and the insulating liquid; while at lower speeds, the interface stability between the insulating liquid and the reagent is relatively poor. Therefore, both conditions place higher demands on the control of the chip rotational speed. This application, through the above structural design, can achieve effective separation of the insulating liquid and reagent under existing experimental conditions, that is, effective separation of the insulating liquid and reagent can still be achieved at higher or lower rotational speeds, and the interface stability of the insulating liquid and reagent is increased.

[0048] Specifically, as shown in Figure 1, the sealing cavity 20 is in the shape of a spiral ring or a circular ring. The working cavity 21 is located on the outer edge of the sealing cavity 20 away from the rotation center O. Based on the extraction and purification process, the working cavities 21 of the corresponding processes are sequentially arranged along the sealing cavity 20. The isolation liquid cavity 12 is connected to the proximal end of the sealing cavity 20 through the second valve body 02. The spiral ring design ensures that the isolation liquid fills the sealing cavity 20 from the distal end to the proximal end, thereby ensuring that no air bubbles or cavities are generated during the entire filling process. Secondly, if the sealing cavity 20 of the upstream working chamber 21 is filled first, the interface stability between the isolation liquid and the reagent in the upstream working chamber is poor, and it is easy to carry the reagent in the upstream working chamber 21 into the downstream working chamber 21 during the filling process, forming crosstalk.

[0049] To further improve the isolation effect between different working chambers 21, and without using an externally triggered valve body, as shown in Figures 1 and 3, the first valve body 01 is a first slit 010 of a certain size. Along the flow direction of the extraction and purification process, the cross-section of the first slit 010 gradually decreases; the inner wall 011 of the slit away from the rotation center of the first slit 010 is a plane or an arc surface, and the distance L1 between the plane or the arc surface and the rotation center gradually decreases along the flow direction of the extraction and purification process. First, the above settings increase the resistance of the isolation liquid during flow, reduce its flow rate, and decrease the probability of reagents in the upstream working chamber being carried into the downstream working chamber. Second, the isolation liquid is an oil phase, which has poor compatibility with the reagents. The inner wall 011 of the first slit 010 away from the rotation center is a plane or an arc surface, and the distance L1 between the plane or arc surface and the rotation center gradually decreases along the flow direction of the extraction and purification process, making it more difficult for the reagents to flow into the downstream working chamber through the first valve body 01. In addition, if the microfluidic chip is rotating, the high-density reagents will adhere tightly to the working chamber 21 under the action of centrifugal force, further reducing the crosstalk problem between reagents in different working chambers. The above settings can ensure that no crosstalk problem will occur at a rotation speed of 100 r / min or higher.

[0050] As shown in Figure 1, the difference between the distances L2 and L3 between the two ends of the inner wall of the first slit away from the rotation center and the rotation center is not less than 1 / 2 of the width L4 of the sealing cavity. Experimental results show that when the above dimensional conditions are met, the chip will not experience crosstalk even when stationary, and the sealing performance of the insulating liquid is stable.

[0051] As shown in Figures 1-4, at least one flow-blocking dam 200 is provided inside the sealing cavity 20. The flow-blocking dam 200 itself or the flow-blocking dam 200 and the inner wall of the sealing cavity 20 form a second slit 201. For a single working cavity 21, the projection of the flow-blocking dam 200 rotating about the rotation center O completely covers the first slit 010 of the first valve body 01 connected to the working cavity. The above arrangement avoids the appearance of non-flow-blocking areas in a single working cavity, which can lead to excessive flow velocity of the isolation liquid and easy entrapment of reagents. As shown in Figures 2 and 3, the projection of the flow-blocking dam 200 rotating about the rotation center O cannot completely cover the first slit 010 of the first valve body 01 connected to the working cavity, as shown in Figure 4, where a non-flow-blocking area 012 exists.

[0052] Specifically, as shown in Figures 2 and 3, the first slit 010 has a first width W1, and the second slit 201 has a second width W2. The first width W1 is smaller than the second width W2, and the first width W1 of the first slit 010 is not greater than the height of the sealing cavity 20. The flow-blocking dam 200 is connected to the inner wall of the sealing cavity 20 on the side near the rotation center O, and the flow-blocking dam 200 is located at or beyond the connection between the working cavity and the sealing cavity on the side away from the rotation center O. As shown in Figure 1 or 2, it can be seen that the flow-blocking dam 200 is located at the connection between the working cavity and the sealing cavity on the side away from the rotation center O. As shown in Figure 5, the flow-blocking dam 200 extends beyond the connection between the working cavity and the sealing cavity on the side away from the rotation center O. Secondly, the first width W1 of the first slit 010 is limited, and its minimum value is limited. If the first width is too small, the resistance to the isolation liquid will be too large. Under the condition of high rotation speed, the isolation liquid will be transmitted through the working chamber, which will increase the disturbance between the isolation liquid and the reagent in the working chamber. There is a problem that the isolation liquid will wrap the reagent and carry the reagent into the downstream working chamber. Preferably, the first width W1 of the first slit 010 is not greater than 0.8 times the height H of the sealing cavity 20. The width design of the first slit needs to consider the flow of the isolation liquid in two scenarios: (1) During the filling process of the isolation liquid, the width of the first slit affects the resistance of the isolation liquid to flow through the first valve body. The greater the resistance, the lower the flow rate of the isolation liquid. The low flow rate of the isolation liquid has little impact on the interface between the isolation liquid and the reagent, thus avoiding the reagent in the upstream working cavity from flowing into the downstream working cavity during the filling process; (2) After the filling of the isolation liquid is completed, the isolation liquid seals the working cavity to ensure that different working cavities do not cross-flow. This application limits the width of the slit on the one hand and the structure of the first valve on the other hand, especially the wall surface of the first slit 010 away from the rotation center. The combination of the two improves the effectiveness of the isolation of different working cavities during subsequent use.

[0053] As shown in Figures 1 and 6, taking one of the working chambers 21 as an example, the flow-blocking dams 200 are uniformly arranged along the spiral or circular direction. The sum of the lengths of one side of the outer edge of at least one flow-blocking dam 200 in the sealing cavity 20 corresponding to a single working chamber 21 is at least 2 / 5 of the length of the outer edge of the sealing cavity corresponding to that single working chamber. As shown in Figure 6, the sum of the lengths of one side of the outer edge of two flow-blocking dams 200, L6 + L7, is at least 2 / 5 of the length L5 of the outer edge of the sealing cavity 20 corresponding to a single working chamber 21. Preferably, the flow-blocking dams 200 have the same structure, so L6 = L7. The flow-blocking dams play a stabilizing role for the reagent in the working chamber, restricting the free surface of the reagent and reducing the excessive free surface of the reagent near the rotation center, which can easily cause large fluctuations during the acceleration and deceleration of the microfluidic chip.

[0054] Specifically, as shown in Figure 6, the working chamber 21 is connected to the bubble generating chamber 22 via a third valve; the side of the bubble generating chamber 22 away from the rotation center O is connected to the inner wall of the working chamber 21 via a third valve body 03; the relationship between the volume V1 of the bubble generating chamber 22 and the volume V2 of the working chamber 21 satisfies the following equation:

[0055]

[0056] Where m1 is the mass of the magnetic beads added, and m0 is the standard mass. This is a correction factor. In this embodiment of the invention, m0 takes a value of 1-3µg. The value is between 0.01 and 0.05. If the working chamber and the sealed chamber are completely filled, there will be poor mixing between the reagent and the magnetic beads adsorbed with nucleic acid, resulting in poor cleaning or elution effects. To improve the cleaning or elution effect, it is necessary to increase the number of times the microfluidic chip rotates back and forth, which reduces the processing efficiency. Based on the above considerations, this embodiment of the invention connects a bubble generating chamber to the working chamber and limits the volume of the bubble generating chamber so that the generated bubble size meets the following requirements: the bubble size is small enough to prevent the gas from moving rapidly to the reagent surface or entering the isolation liquid under the action of centrifugal force; the bubble size is large enough to ensure that the bubble, magnetic beads, and reagent can be well mixed and stirred.

[0057] Specifically, the isolating liquid chamber 12 is arranged in a ring shape near the proximal end of the microfluidic chip; the sample chamber 10 and at least one reagent chamber 11 are fan-shaped and arranged around the outer side of the isolating liquid chamber 12; at least two working chambers 21 are fan-shaped and arranged on the outer edge of the sealing chamber 20, around the rotation center O. The reagent chamber 11 and sample chamber 10 are respectively connected to the proximal end of the sealing chamber corresponding to the working chamber through the second valve body 02. Through the above structural design, the center of mass of the microfluidic chip provided in this embodiment of the invention is kept as close as possible to the rotation axis, improving the stability of the microfluidic chip during use; secondly, the reasonable layout improves the compactness of the prepared microfluidic chip and reduces the manufacturing cost.

[0058] In another preferred embodiment, as shown in FIG7, the isolation liquid chamber 12, the sample chamber 10 and at least one reagent chamber 11 are fan-shaped and arranged around the rotation center O; at least two of the working chambers 21 are fan-shaped and arranged on the outer edge of the sealing chamber 20, around the rotation center O; the reagent chamber 11 and the sample chamber 10 are respectively connected to the proximal end of the sealing chamber 20 corresponding to the working chamber 21 through the second valve body 02.

[0059] The method of using the microfluidic chip for nucleic acid extraction and purification provided in this embodiment of the invention includes the following steps:

[0060] 1. Reagent preparation and sample injection. As shown in Figure 8a, the working chamber for nucleic acid adsorption in the microfluidic chip is pre-filled with magnetic beads (small white granular objects in the figure) for nucleic acid extraction, and sufficient mineral oil for sealing is added to the isolation liquid chamber (light gray shaded area in the figure). According to the extraction and purification process, appropriate amounts of sample, first washing solution, second washing solution and elution solution (dark gray shaded area in the figure) are added to the sample chamber and the three reagent chambers in sequence.

[0061] 2. Reagent centrifugation and mineral oil sealing. As shown in Figure 8b, a centrifugal speed V1 is applied to the microfluidic chip. The sample, first washing solution, second washing solution, and elution solution in the sample chamber are filled into their respective working chambers at the distal end under the action of centrifugal force. At the same time, the mineral oil in the isolation chamber (light gray shaded area in the figure) enters the sealing chamber (dark gray shaded area in the figure) to form an isolation seal for the liquids in the multiple working chambers.

[0062] 3. Bubble cavity generation. As shown in Figure 8c, the rotation speed of the microfluidic chip is further increased to the centrifugal speed V2. Under the action of centrifugal force, the volume in the bubble generation cavity is compressed and stored. When the centrifugal force stops instantly, the gas in the bubble generator will be quickly discharged (larger white circle in the figure) and enter the interior of each working chamber to form a bubble cavity.

[0063] 4. Magnetic bead mixing and nucleic acid adsorption. As shown in Figure 8d, by rapidly switching the centrifugation direction of the microfluidic chip, the air bubble cavity in the nucleic acid adsorption working chamber, under the action of Euler force, thoroughly mixes the sample and magnetic beads in the nucleic acid adsorption working chamber, thereby enabling the magnetic beads to adsorb nucleic acids.

[0064] 5. Magnetic Bead Collection and Transfer. As shown in Figure 8e, the nucleic acid adsorption working chamber is rotated to the fixed position of the magnet (not shown in the figure; at this time, the magnet is located at the starting end of the nucleic acid adsorption working chamber and directly below the sealed chamber). By rotating the chip, a relative displacement occurs between the nucleic acid adsorption working chamber and the fixed magnet. During this process, the magnetic beads in the working chamber will accumulate towards the mineral oil in the sealed chamber. During the accumulation process, the water molecules on the surface of the magnetic beads are dehydrated under the action of the oil-water interfacial tension, resulting in less liquid residue on the surface of the magnetic beads. At the same time, the accumulated magnetic beads move downstream towards the working chamber under the action of magnetic force.

[0065] 6. The magnetic beads pass through the first valve body and enter the downstream working chamber. As shown in Figure 8f, as the chip rotates, the fixed magnet drags the gathered magnetic beads through the first valve body. Based on the combined action of the first slit on the first valve body and the surface tension of the mineral oil, the residual liquid on the surface of the magnetic beads is dehydrated again, and the liquid on the surface of the magnetic beads is trapped in the upstream working chamber. After the residual liquid is filtered out, the magnetic beads enter the downstream working chamber.

[0066] 7. Transfer the magnetic beads after the first cleaning. As shown in Figure 8g, repeat steps 4-6 to ensure that the first washing solution is fully mixed with the magnetic beads. After the first cleaning of the magnetic beads is completed, transfer the magnetic beads to the downstream working chamber.

[0067] 8. After the first cleaning, the magnetic beads are transferred. Repeat step 7 to perform a second cleaning and transfer the magnetic beads to the downstream working chamber.

[0068] 9. Nucleic acid elution. As shown in Figure 8i, repeat step 4 in sequence. By rapidly switching the centrifugation direction of the microfluidic chip, the air bubble cavity in the nucleic acid elution working chamber is subjected to Euler force to fully mix the eluent and magnetic beads in the nucleic acid elution working chamber, thereby achieving nucleic acid elution.

[0069] 10. Magnetic bead removal. As shown in Figure 8j, after nucleic acid elution is complete, rotate the nucleic acid elution working chamber to the fixed position of the magnet (not shown in the figure; at this time, the magnet is located at the very end of the nucleic acid elution working chamber and directly below the sealed chamber). Rotate the chip in the opposite direction, and the magnetic beads in the elution working chamber will be enriched and move towards the upstream working chamber. Subsequently, the magnet enters the upstream working chamber in the opposite direction through the first valve body. At this time, nucleic acid extraction and purification are completed.

[0070] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A microfluidic chip for nucleic acid extraction and purification, characterized in that, include: The storage unit includes a sample chamber, a reagent chamber, and an isolation liquid chamber; The nucleic acid extraction and purification unit is located on the side furthest from the rotation center from the storage unit. The nucleic acid extraction and purification unit includes at least two working chambers and a sealed chamber located on the side of the working chambers closer to the rotation center. The different working chambers are connected by a first valve body located in the sealed chamber. The distal ends of the sample chamber, reagent chamber, and isolation liquid chamber are connected to the proximal end of the sealed chamber by a second valve body. The width of the sealing cavity is not less than the height of the sealing cavity, and the height of the working cavity is at least three times the height of the sealing cavity; The sealing cavity is in the shape of a spiral ring or a circular ring. The working cavity is located on the outer edge of the sealing cavity away from the rotation center. Based on the extraction and purification process, the working cavities of the corresponding processes are sequentially arranged along the sealing cavity. The isolation liquid cavity is connected to the proximal end of the sealing cavity through a second valve body. The isolation liquid is a hydrophobic liquid with a density lower than that of the liquid in the working cavity and is inert, so it will not react with the substances in the system. The first valve body is a first slit of a certain size, and the cross-section of the first slit gradually decreases along the flow direction of the extraction and purification process; The inner wall of the first slit on the side away from the rotation center is a plane or an arc surface, and the distance between the plane or the arc surface and the rotation center gradually decreases along the flow direction of the extraction and purification process; The difference in distance between the two ends of the inner wall of the first slit on the side away from the rotation center and the rotation center is not less than 1 / 2 of the width of the sealing cavity.

2. The microfluidic chip according to claim 1, characterized in that, At least one flow-blocking dam is provided inside the sealed cavity, and the flow-blocking dam itself or the flow-blocking dam and the inner wall of the sealed cavity form a second slit; For a single working chamber, the projection of the flow-blocking dam rotating about the center of rotation completely covers the first slit of the first valve body connected to the working chamber.

3. The microfluidic chip according to claim 2, characterized in that, The first slit has a first width, the second slit has a second width, the first width is less than the second width, and the first width of the first slit is not greater than the height of the sealing cavity; The flow-blocking dam is connected to the inner wall of the sealing cavity on the side closer to the rotation center, and the flow-blocking dam is located at or beyond the connection between the working cavity and the sealing cavity on the side farther from the rotation center.

4. The microfluidic chip according to claim 3, characterized in that, The flow-blocking dams are uniformly arranged along the spiral or circular direction, and the sum of the lengths of at least one side of the outer edge of the flow-blocking dam in the sealing cavity corresponding to a single working cavity is at least 2 / 5 of the length of the outer edge of the sealing cavity corresponding to the single working cavity.

5. The microfluidic chip according to claim 1, characterized in that, The working chamber is connected to a bubble generating chamber via a third valve body; The side of the bubble generating chamber away from the center of rotation is connected to the inner wall of the working chamber via a third valve body; The relationship between the volume V1 of the bubble generating chamber and the volume V2 of the working chamber satisfies the following equation: ； Where m1 is the mass of the magnetic beads added, and m0 is the standard mass, with a value ranging from 1 to 3 µg. This is a correction factor, with a value ranging from 0.01 to 0.

05.

6. [Correction 21.10.2025 according to Rule 91] The microfluidic chip according to claim 1 is characterized in that, The isolation fluid cavity is arranged in a ring shape at the proximal end of the microfluidic chip; The sample chamber and at least one reagent chamber are fan-shaped and arranged around the outside of the isolation liquid chamber; At least two of the working chambers are configured in a fan shape and are located on the outer edge of the sealed chamber, arranged around the rotation center. The reagent chamber and the sample chamber are respectively connected to the proximal end of the sealed chamber corresponding to the working chamber through a second valve body. Alternatively, the isolation liquid chamber, sample chamber, and at least one reagent chamber may be fan-shaped and arranged around the center of rotation; At least two of the working chambers are configured in a fan shape and are located on the outer edge of the sealed chamber, arranged around the rotation center. The reagent chamber and the sample chamber are respectively connected to the proximal end of the sealed chamber corresponding to the working chamber through a second valve body.

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