Highly integrated microfluidic chip

Abstract

The present invention provides a highly integrated microfluidic chip and belongs to the technical field of microfluidics. The highly integrated microfluidic chip comprises: a central layer, comprising an isolation liquid cavity and a reagent cavity; and a first ring layer, arranged on the side of the central layer away from a rotation center and comprising a plurality of quantitative distribution cavities relatively close to the rotation center and a corresponding number of nucleic acid extraction cavities relatively far from the rotation center, wherein the nucleic acid extraction cavities comprise a first working cavity and a first sealed cavity, and the first sealed cavity is arranged on the side of the first working cavity close to the rotation center. By means of a reusable isolation liquid, sealing and isolation for nucleic acid extraction and purification using a magnetic bead method and sample target nucleic acid amplification are successively realized. The present invention not only ensures the reliability of nucleic acid extraction and purification processes, but also eliminates the problem of inter-well crosstalk, which may occur during nucleic acid amplification.

Description

A highly integrated microfluidic chip Technical Field

[0001] This application relates to the field of microfluidics, and in particular to a highly integrated microfluidic chip. Background Technology

[0002] With the development of microfluidic technology and diagnostic testing technology, the application of microfluidic chips based on microfluidic technology in in vitro diagnostics (IVD) and other fields has become increasingly in-depth and widespread. Microfluidic chips are characterized by miniaturization, integration, and high automation, which makes the diagnostic testing process based on them more convenient, efficient, and cost-effective compared to traditional diagnostic testing processes. Nucleic acid amplification detection (such as PCR, LAMP, and RPA) is a highly sensitive biotechnology that performs qualitative or quantitative analysis of trace target gene sequences under specific enzyme catalysis systems. In recent years, its deep integration and application with microfluidic chips (such as point-of-care testing, POCT) has yielded a series of achievements and advancements.

[0003] Currently, microfluidic chips used for nucleic acid amplification detection can be broadly classified into three types based on their main functions and degree of internal integration. The first type involves adding a pre-treated target gene template for direct amplification and detection, and can be called a pure amplification chip. These chips only have amplification and detection capabilities and cannot directly detect raw samples; therefore, they often require additional nucleic acid extraction instruments. The second type involves adding a simple raw sample, releasing nucleic acid, and then directly amplifying and detecting; this can be called an extraction-free amplification chip or a one-step extraction amplification chip. These chips are generally only suitable for raw samples with simple components, weak inhibition and interference, or samples with high target content. They also have high requirements for the performance of the nucleic acid release agent. Therefore, these chips may experience missed detections or false detections when processing raw samples with complex components or containing inhibitory or interfering components. The third type involves adding the raw sample, performing a complete nucleic acid extraction and purification process, and then amplifying and detecting; this can be called a fully extracted or fully integrated amplification and detection chip. These chips integrate the entire process of nucleic acid extraction and purification, and nucleic acid amplification and detection, offering higher compatibility for complex sample types and higher detection sensitivity and reliability. In summary, fully extracted or fully integrated nucleic acid amplification detection chips are gradually becoming the mainstream development trend for integrated and portable nucleic acid amplification detection. However, these chips often have higher design requirements and more complex manufacturing processes.

[0004] Currently available fully integrated nucleic acid amplification detection products, such as cartridge-based systems like Cepheid's GeneXpert and Biocartis's Idylla systems, and BOE's NAT-3000 system, all use plunger pumps or external air pumps to provide positive and negative pressure to drive the fluid. They also use rotary switching valves to selectively connect internal flow channels. These cartridge-based products are often complex in structure and have a large number of components, resulting in higher costs. Chip-based products, such as Bio-Rad's DxLab-2A system and Chenghui Medical's CQ-0403 system, use centrifugal silicon membrane / column extraction for nucleic acid. Silicon membranes / columns are prone to clogging, affecting the entire extraction process. They also have interconnected and complex siphon channels to assist in liquid release, resulting in poor fluid stability or requiring complex pre-treatment for hydrophilicity. It is particularly noteworthy that, currently, regardless of the type of fully integrated nucleic acid amplification detection product, the liquid reagents required for nucleic acid extraction and purification must be pre-sealed and stored or additionally removed and added before use. Pre-sealing and storing liquid reagents inside the cartridge / chip places high demands on the relevant processes and is prone to leakage. At the same time, an appropriate and efficient liquid reagent release method must be considered to ensure the normal functioning of the reagents. Adding additional liquid reagents before use will greatly increase the complexity of pipetting operations and place more stringent requirements on the overall integrated sealing of the product and the design of the supporting operating system. Summary of the Invention

[0005] The purpose of this invention is to provide a highly integrated microfluidic chip. By utilizing a reusable insulating liquid, it achieves sequential sealed isolation between the magnetic bead-based nucleic acid extraction and purification process and the amplification of the target nucleic acid in the sample. This ensures the reliability of the nucleic acid extraction and purification process while eliminating potential crosstalk between wells during nucleic acid amplification. Furthermore, multiple reagents can be prepared online with a single sample addition operation, reducing manual steps and workload, while also avoiding the risk of cross-contamination between reagents. In addition, the design of multiple reagent chambers is eliminated, enabling chip miniaturization.

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

[0007] A highly integrated microfluidic chip includes: a central layer comprising an isolation liquid chamber and a reagent chamber; a first concentric layer disposed on a side of the central layer away from the rotation center, comprising multiple nucleic acid extraction chambers located relatively far from the rotation center, each nucleic acid extraction chamber comprising a first working chamber and a first sealing chamber, the first sealing chamber being disposed on a side of the first working chamber closer to the rotation center, the reagent chamber being connected to the nucleic acid extraction chamber; the isolation liquid chamber being controlled by a third valve to fill the first sealing chamber with an isolation liquid; and a second concentric layer disposed on a side of the first concentric layer away from the rotation center, comprising at least one downstream chamber, the proximal end of the downstream chamber being connected to the distal end of the last nucleic acid extraction chamber corresponding to the nucleic acid extraction step via a fourth valve; the reagent chamber and the nucleic acid extraction chamber are connected by opening the third valve before, after, or simultaneously with the transfer of reagents, and subsequently opening the fourth valve.

[0008] Furthermore, the first concentric layer includes a plurality of quantitative dispensing chambers relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers relatively far from the rotation center; the distal ends of the plurality of quantitative dispensing chambers are respectively connected to the proximal ends of the first sealing chambers of the corresponding nucleic acid extraction chambers through a second valve, and the reagent chamber is connected to the quantitative dispensing chamber through a first valve.

[0009] Furthermore, a fifth valve is provided between adjacent nucleic acid extraction chambers, and the fifth valve is located between adjacent first sealing chambers.

[0010] Furthermore, the central layer also includes a sample chamber; the first ring layer also includes a sample lysis chamber, the sample lysis chamber including a second sealing chamber near the rotation center and a second working chamber away from the rotation center, the proximal end of the second sealing chamber being connected to the distal end of the sample chamber via a sixth valve; one end of the third valve is connected to the distal end of the isolation liquid chamber, and the other end of the third valve is connected to the proximal end of the second sealing chamber or the first sealing chamber.

[0011] Furthermore, a seventh valve is provided between the second sealing cavity and the first sealing cavity; the nucleic acid adsorption device can bypass the fifth and seventh valves.

[0012] Furthermore, the downstream chamber includes: at least one nucleic acid dispensing chamber and a matching amplification reaction chamber; the distal end of the nucleic acid dispensing chamber is connected to the proximal end of the amplification reaction chamber via an eighth valve; at least one of the nucleic acid dispensing chambers is connected via a dispensing channel, the dispensing channel being a gradually spiral or arc-shaped structure, and the upstream end of the dispensing channel is connected to the distal end of the nucleic acid extraction chamber via a fourth valve.

[0013] Furthermore, it also includes a return air chamber, which is arranged around the rotation center along with the downstream chamber. One end of the return air chamber is connected to the proximal end of the distribution channel, and the other end of the return air chamber is connected to the proximal end of the quantitative distribution chamber.

[0014] Furthermore, it also includes a waste liquid chamber, which is located at the distal end and between the return gas chamber and the downstream chamber; the end of the quantitative distribution chamber is provided with an overflow chamber, the distal end of which is connected to the proximal end of the waste liquid chamber.

[0015] Furthermore, the isolation liquid chamber, reagent chamber, and sample chamber are arranged around the rotation center; buffer chambers are also provided on the pipelines of the third valve and the sixth valve, and the buffer chamber and quantitative dispensing chamber are arranged around the rotation center; the sample lysis chamber and multiple nucleic acid extraction chambers are arranged around the rotation center.

[0016] Furthermore, each nucleic acid extraction chamber has a second valve with different opening conditions based on the steps of the nucleic acid extraction process; the second valve corresponding to the nucleic acid extraction chamber located upstream of the step opens later than the second valve corresponding to the nucleic acid extraction chamber located downstream of the step.

[0017] Furthermore, the nucleic acid extraction chamber is provided with three valves, the first valve, the second valve, the third valve, the fifth valve, the sixth valve and the seventh valve are all capillary channels; the fourth valve is a phase change valve or a mechanical valve.

[0018] Another aspect of the present invention provides a highly integrated microfluidic chip, comprising: a central layer including a reagent chamber and a sample chamber arranged around a rotation center; a first concentric layer disposed on a side of the central layer away from the rotation center, including a plurality of nucleic acid extraction chambers located relatively far from the rotation center, the reagent chambers being connected to the nucleic acid extraction chambers; and a sample lysis chamber connected to the sample chambers via a sixth valve; the sample lysis chamber and the plurality of nucleic acid extraction chambers being arranged around the rotation center; and a second concentric layer disposed on a side of the first concentric layer away from the rotation center, including at least one downstream chamber, the upstream end of which is connected to the distal end of the last nucleic acid extraction chamber corresponding to the nucleic acid extraction step via a fourth valve, the downstream chamber being arranged around the rotation center.

[0019] Furthermore, the system includes: a first concentric layer comprising multiple quantitative dispensing chambers relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers relatively far from the rotation center; the distal ends of the multiple quantitative dispensing chambers are respectively connected to the corresponding nucleic acid extraction chambers via second valves; the quantitative dispensing chambers are connected to the reagent chambers via first valves; a buffer chamber is provided on the pipeline of the sixth valve, and the buffer chambers and quantitative dispensing chambers are arranged around the rotation center; the second concentric layer also includes a return gas chamber, which is arranged around the rotation center along with the downstream chamber; one end of the return gas chamber is connected to the proximal end of the downstream chamber, and the other end of the return gas chamber is connected to the proximal end of the quantitative dispensing chamber.

[0020] Furthermore, the microfluidic chip is stepped, with each step being cylindrical and symmetrical about the axis of rotation; the central layer is located at the top of the stepped structure, the first concentric layer is located below the central layer, and the second concentric layer is located below the first concentric layer.

[0021] Furthermore, it also includes an isolation liquid chamber, which is disposed on the central layer and arranged around the rotation center along with the reagent chamber and the sample chamber; the isolation liquid chamber is connected to the proximal end of the sample lysis chamber or the nucleic acid extraction chamber through a third valve, and a buffer chamber is provided on the pipeline of the third valve, and the corresponding buffer chambers on the sixth valve and the third valve are arranged around the rotation center along with the quantitative dispensing chamber.

[0022] Furthermore, the nucleic acid extraction chamber includes a first working chamber and a first sealing chamber, the first sealing chamber being disposed on the side of the first working chamber near the rotation center; the sample lysis chamber includes a second sealing chamber on the side near the rotation center and a second working chamber on the side away from the rotation center; a fifth valve is disposed between adjacent nucleic acid extraction chambers, the fifth valve being disposed between adjacent first sealing chambers, and a seventh valve is disposed between the second sealing chamber and the first sealing chamber.

[0023] Furthermore, it also includes a nucleic acid adsorption device that can bypass the fifth and seventh valves.

[0024] Furthermore, it also includes a waste liquid chamber, which is located at the distal end and between the return gas chamber and the downstream chamber; the end of the quantitative distribution chamber is provided with an overflow chamber, the distal end of which is connected to the proximal end of the waste liquid chamber.

[0025] Furthermore, the downstream chamber includes: at least one nucleic acid dispensing chamber and a matching amplification reaction chamber; the distal end of the nucleic acid dispensing chamber is connected to the proximal end of the amplification reaction chamber via an eighth valve; at least one of the nucleic acid dispensing chambers is connected via a dispensing channel, the dispensing channel being a gradually spiral or arc-shaped structure, and the upstream end of the dispensing channel is connected to the distal end of the nucleic acid extraction chamber via a fourth valve.

[0026] Furthermore, it also includes a second return gas passage, one end of which is connected to the proximal end of the quantitative dispensing chamber, and the other end of which is connected to the proximal end of the reagent chamber.

[0027] Furthermore, each nucleic acid extraction chamber has a second valve with different opening conditions based on the steps of the nucleic acid extraction process; the second valve corresponding to the nucleic acid extraction chamber located upstream of the step opens later than the second valve corresponding to the nucleic acid extraction chamber located downstream of the step.

[0028] Furthermore, the nucleic acid extraction chamber is provided with three valves, the first valve, the second valve, the third valve, the fifth valve, the sixth valve and the seventh valve are all capillary channels; the fourth valve is a phase change valve or a mechanical valve.

[0029] This invention provides a highly integrated microfluidic chip, which has the following advantages compared to existing technologies:

[0030] This invention utilizes an isolation liquid chamber to store an isolation liquid. After the first working chamber of the nucleic acid extraction chamber is filled with reagents, this liquid fills the first sealing chamber, achieving a seal between different nucleic acid extraction chambers and preventing reagent contamination from mixing. Secondly, after the nucleic acid extraction process is completed, a fourth valve is opened. Under centrifugal force, the liquid containing nucleic acid from the first working chamber of the terminal nucleic acid extraction chamber flows first through the fourth valve into the downstream chamber. After the liquid is filled, the isolation liquid then enters the downstream chamber, sealing it and preventing cross-contamination of aerosols generated during the amplification reaction. This also ensures the absolute amount of reaction reagents in the downstream chamber, improving detection accuracy. It can be seen that this application achieves isolation between multiple steps and different chambers using the same isolation liquid, improving the utilization efficiency of the isolation liquid and the overall accuracy of the microfluidic chip detection. Finally, the highly integrated microfluidic chip provided by this invention integrates the online preparation process of multiple reagents, avoiding repetitive pipetting operations and reducing the operator's workload and the probability of contamination. Attached Figure Description

[0031] 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.

[0032] Figure 1 is a structural diagram of a highly integrated microfluidic chip provided in an embodiment of the present invention;

[0033] Figure 2 is a partial structural diagram of the highly integrated microfluidic chip provided in an embodiment of the present invention;

[0034] Figure 3 is a diagram of the downstream cavity structure of the highly integrated microfluidic chip provided in an embodiment of the present invention;

[0035] Figure 4 is a side view of a highly integrated microfluidic chip provided in an embodiment of the present invention;

[0036] Figure 5 is a schematic diagram of different stages of nucleic acid detection provided in the embodiment of the present invention. a is the addition of the sample to be tested, reagents and isolation solution, b is the quantitative buffer solution, c is the sample release and liquid reagent dissolution and preparation, d is the sealing mineral oil release and filling, eh is the nucleic acid extraction and purification, i is the distribution of purified nucleic acid sample, and j is the nucleic acid sample entering the amplification reaction chamber to start amplification and signal detection.

[0037] Reference numerals: 01, First valve; 001, First mass section; 02, Second valve; 002, Second mass section; 03, Third valve; 003, Third mass section; 04, Fourth valve; 05, Fifth valve; 06, Sixth valve; 07, Seventh valve; 08, Eighth valve; 1, Central layer; 10, Isolation liquid chamber; 11, Reagent chamber; 12, Sample chamber; 13, Second return gas passage; 2, First concentric ring; 20, Quantitative distribution chamber; 200, Overflow dam; 21, Nucleic acid extraction chamber; 210, First working chamber; 211, First sealing chamber; 212, First protrusion; 213, Second protrusion; 22, Sample lysis chamber; 220, Second working chamber; 221, Second sealing chamber; 23, Overflow chamber; 24, Buffer chamber; 3, Second concentric ring; 30, Downstream chamber; 300, Nucleic acid distribution chamber; 31. Amplification reaction chamber; 32. Dispensing channel; 33. Collection chamber; 34. Gas return chamber; 35. Waste liquid chamber; 4. Nucleic acid adsorption device. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. Example

[0043] This invention discloses a highly integrated microfluidic chip, as shown in Figure 1, comprising: a central layer 1, which includes an isolation liquid chamber 10 and a reagent chamber 11; a first concentric layer 2, disposed on the side of the central layer 1 away from the rotation center O, which includes a plurality of nucleic acid extraction chambers 21 relatively far from the rotation center O, each nucleic acid extraction chamber 21 including a first working chamber 210 and a first sealing chamber 211, the first sealing chamber 211 being disposed on the side of the first working chamber 210 closer to the rotation center O; and the reagent chamber 11 and... The nucleic acid extraction chamber 21 is connected; the isolation liquid chamber 10 controls the filling of the first sealing chamber 211 with isolation liquid through the third valve 03; the second ring 3 is arranged on the side away from the rotation center O relative to the first ring 2, including at least one downstream chamber 30, the proximal end of the downstream chamber 30 is connected to the distal end of the last nucleic acid extraction chamber 21 corresponding to the nucleic acid extraction process through the fourth valve 04; the reagent chamber 11 and the nucleic acid extraction chamber 21 complete the transfer of reagents, the third valve 03 is opened before, after or simultaneously, and the fourth valve 04 is opened subsequently.

[0044] This invention establishes an isolation liquid chamber 10, which stores an isolation liquid. After the first working chamber 210 of the nucleic acid extraction chamber is filled with reagents, it is used to fill the first sealing chamber 211, thereby sealing different nucleic acid extraction chambers and preventing reagents from mixing and causing contamination in different nucleic acid extraction chambers 21. Secondly, after the nucleic acid extraction process is completed, the fourth valve is opened. Under the action of centrifugal force, the liquid containing nucleic acid in the first working chamber 210 of the terminal nucleic acid extraction chamber 21 flows into the downstream chamber 30 through the fourth valve 04. After the liquid is filled, the isolation liquid then enters the downstream chamber 30 to seal the downstream chamber, preventing cross-contamination of aerosols generated during the amplification reaction and ensuring the absolute amount of reaction reagents in the downstream chamber, thus improving the accuracy of detection. It can be seen that this application achieves isolation of multiple steps and different chambers through the same isolation liquid, improving the utilization efficiency of the isolation liquid and the accuracy of the entire microfluidic chip detection.

[0045] It should be noted that the opening of the third valve in this invention is not affected by the transfer of reagents between the reagent chamber and the nucleic acid extraction chamber. Even if the third valve is opened before the reagent transfer, the density of the insulating liquid is less than that of the reagent, and under the action of centrifugal force, the insulating liquid can seal the reagent in the nucleic acid extraction chamber. Preferably, the third valve 03 is opened after the transfer of reagents between the reagent chamber and the nucleic acid extraction chamber is completed. In this way, there will be no mixing between the insulating liquid and the reagent, which will lead to the problem of emulsification at the interface between the insulating liquid and the reagent.

[0046] Specifically, the first concentric layer includes multiple quantitative dispensing chambers 20 relatively close to the rotation center O and a corresponding number of nucleic acid extraction chambers 21 relatively far from the rotation center O; the distal ends of the multiple quantitative dispensing chambers 20 are respectively connected to the proximal ends of the first sealing chambers 211 of the corresponding nucleic acid extraction chambers 21 via second valves 02, and the reagent chamber 11 is connected to the quantitative dispensing chambers 20 via first valves 01. Preferably, the valves are opened in the following order: first valve 01, second valve 02, third valve 03, and fourth valve 04.

[0047] The present invention connects the reagent chamber 11 to multiple quantitative distribution chambers 20 through a first valve 01. Under the action of centrifugal force, the quantitative distribution chambers 20 quantify the reagents. Then, each quantitative distribution chamber 20 delivers the quantified reagents to the nucleic acid extraction chamber 21 through a corresponding pipeline via a second valve 02, thereby improving the reagent distribution accuracy.

[0048] This invention incorporates multiple valves, such as a first valve, a second valve, a third valve, and a fourth valve. The form and combination of these valves are not limited, as long as they open and close at the required steps. They can be active or passive valves. Specifically, they can employ manually operated mechanical valves or phase change valves, or capillary channels that open or close under centrifugal force. To reduce production costs while maintaining detection accuracy, capillary channels are preferred for control. Specifically, capillary tubes, ranging in size from hundreds of micrometers to millimeters, are used. Different diameters and lengths allow for opening or closing under varying centrifugal forces. Phase change valves refer to valves that can open and close in response to changes in external conditions.

[0049] The "isolation liquid" is a hydrophobic liquid with a density lower than that of the liquid in the first working chamber. It is also inert and will not react with substances in the system, such as mineral oil. The specific composition of the isolation liquid is not specified here.

[0050] It should be noted that, regarding the addition of nucleic acid samples, the nucleic acid samples can be added to the nucleic acid extraction chamber corresponding to the first step of the extraction process in advance, or a separate sample chamber can be set up for addition. In this embodiment of the invention, an independent sample chamber and a corresponding sample lysis chamber are set up to lyse the nucleic acid samples. Subsequently, the nucleic acid is subjected to impurity removal, washing, and elution processes in the nucleic acid extraction chamber. The movement of nucleic acid substances in each chamber is transferred using a nucleic acid adsorption device, such as magnetic beads or polymer materials, as long as they can adsorb nucleic acids and achieve the transfer of nucleic acids between different chambers under external drive.

[0051] The terms "distal end" and "proximal end" are derived by comparing their 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.

[0052] Specifically, as shown in Figure 1, a fifth valve 05 is provided between adjacent nucleic acid extraction chambers, and the fifth valve 05 is located between adjacent first sealing chambers 211. By placing the fifth valve in the first sealing chamber 211, the purification of nucleic acid during the transfer process between different nucleic acid extraction chambers 21 can be further enhanced. Since the insulating liquid in the first sealing chamber 211 is incompatible with the liquid in the first working chamber 210, when nucleic acid is transferred from the upstream nucleic acid extraction chamber to the downstream nucleic acid extraction chamber, the nucleic acid material will be transferred through the fifth valve 05 between the first sealing chambers 211. At this time, the insulating liquid will repel the reagents remaining in the nucleic acid, so that the reagents are removed during the process of entering the insulating liquid, thereby preventing the reagents in the upstream nucleic acid extraction chamber from being carried into the downstream nucleic acid extraction chamber during the nucleic acid transfer process.

[0053] Specifically, as shown in Figure 1, the central layer 1 of the present invention further includes a sample chamber 12; the first ring layer 2 further includes a sample lysis chamber 22, the sample lysis chamber 22 including a second sealing chamber 221 near the rotation center O and a second working chamber 220 away from the rotation center O, the proximal end of the second sealing chamber 221 being connected to the distal end of the sample chamber 12 via a sixth valve 06; one end of the third valve 03 is connected to the distal end of the isolation liquid chamber 10, and the other end of the third valve 03 is connected to the proximal end of the second sealing chamber 221. It can be understood that the other end of the third valve 03 can be connected to the proximal end of the first sealing chamber 211. By placing the sample chamber, reagent chamber, and isolation liquid chamber on the same chip, the chip integration is improved, the chip pre-detection processing steps are reduced, and the complexity of operation and the probability of misoperation caused by multiple pipetting are avoided. Preferably, the sixth valve 06 is opened before the third valve 03.

[0054] In this embodiment of the invention, the central layer 1, the first concentric layer 2, and the second concentric layer 3 are divided based on the area marked by the circular dashed line, and each area has a corresponding chamber to meet the requirements of the detection process.

[0055] Specifically, as shown in Figures 1 and 2, the isolation liquid chamber 10, reagent chamber 11, and sample chamber 12 in this invention are arranged in a fan-shaped circle around the rotation center O. The side furthest from the rotation center O is spiral-shaped, and the side furthest from the rotation center O is arc-shaped, which can also perform the corresponding functions, but its effect is less than that of the spiral-shaped arrangement. The farthest ends of the chambers from the rotation center are used to connect the first valve 01, the third valve 03, and the sixth valve 06, respectively. The quantitative dispensing chamber 20 is arranged around the rotation center O. The quantitative dispensing chamber 20 is based on a large fan-shaped cavity, with a quantitative overflow on the side furthest from the rotation center. A dam 200, the upper and lower sides (perpendicular to the paper plane) of the quantitative overflow dam 200 are connected to the upper and lower surfaces of the fan-shaped cavity to divide the fan-shaped cavity into several quantitative dispensing chambers 20, as shown in Figure 2. The upper surface of the quantitative overflow dam 200 is sloping and flows in the direction A of the reagent flow. The angle α between the upper surface of the overflow dam 200 and the radial direction Xr centered at the rotation center O is greater than 90°. The upper surface of the quantitative overflow dam 200 can be a sloping surface as shown in Figure 2, or it can be an arc surface, but the angle between the tangent at any point of the arc surface and the radial direction Xr is greater than 90°. Furthermore, in order to accurately quantify each quantitative dispensing chamber 20, the maximum distance L1 between the upper surface of the upstream overflow dam 200 and the rotation center O is less than the minimum distance L2 between the upper surface of the downstream overflow dam 200 and the rotation center O. Therefore, when the reagent fills the quantitative distribution chamber 20 along the flow direction A, the upstream quantitative distribution chamber is filled first, and the filling amount is the volume formed by the cylindrical surface swept by the line connecting the highest point of the corresponding overflow dam and the rotation center O as the radius and the inner surface of the upstream quantitative distribution chamber. After the upstream quantitative distribution chamber is filled, the next quantitative distribution chamber is filled. At this time, the filling amount is the volume formed by the cylindrical surface swept by the line connecting the highest point of the corresponding overflow dam and the rotation center O as the radius and the inner surface of the next quantitative distribution chamber, and so on. It can be seen that the shape and position of the overflow dam can limit the amount of liquid distributed in each quantitative distribution chamber, reducing the difficulty of chip design and manufacturing.

[0056] Specifically, a seventh valve 07 is provided between the second sealing cavity 221 and the first sealing cavity 211; a nucleic acid adsorption device 4 is provided, which can pass through the fifth valve 05 and the seventh valve 07. Preferably, the nucleic acid adsorption device 4 in this invention is a magnetic bead, which can pass through the fifth valve 05 and the seventh valve 07 under the control of external magnetic force to achieve flow in different chambers. The fifth valve 05 and the seventh valve 07 are capillary channels, which can be capillary tubes or capillary channels with other cross-sectional shapes, as long as their maximum cross-sectional distance is between several hundred micrometers and millimeters.

[0057] The first working chamber 210 is connected to the first sealing chamber 211, and the second working chamber 220 is connected to the second sealing chamber 221. It should be noted that the volume of the first working chamber 210 is large enough to fully receive the reagents dispensed by the corresponding quantitative dispensing chamber 20. The arrangement of the first working chamber 210 and the first sealing chamber 211, as well as the second working chamber 220 and the second sealing chamber 221, can be structurally configured based on actual needs. In this embodiment, as shown in Figure 2, the nucleic acid extraction chamber 21 is formed by isolating multiple first protrusions 212 on the side wall of a single fan-shaped chamber away from the rotation center O. In this embodiment, two first protrusions 212 are provided to form three nucleic acid extraction chambers 21. This embodiment also includes a second protrusion 213 for isolating the sample lysis chamber 22 from the nucleic acid extraction chamber 21. A fifth valve 05 is provided on the first protrusion 212, and a seventh valve 07 is provided on the upper part of the second protrusion 213. It should be noted that the seventh valve in this invention is a capillary channel with a flow channel size of hundreds of micrometers to several millimeters. The first working chamber 210, the first sealing chamber 211, the second working chamber 220, and the second sealing chamber 221 are chambers with different thicknesses. The first sealing chamber 211 and the second sealing chamber 221 are thinner (the thickness direction is perpendicular to the paper surface), which facilitates the complete filling of the second sealing chamber 221 of the sample lysis chamber and the first sealing chamber 211 of the nucleic acid extraction chamber with the isolation liquid. The second working chamber 220 and the first working chamber 210 are thicker so that they can accommodate more reagents. The above-mentioned size design not only facilitates processing, but also avoids the chip from vibrating due to an imbalance of the center of gravity during rotation caused by too much isolation liquid. It should be noted that in this embodiment of the invention, the fifth valve 05 and the seventh valve 07 are capillary channels with a size of several hundred micrometers to several millimeters. The first sealing cavity and the second sealing cavity can also have a thickness of several hundred micrometers to several millimeters. Here, the fifth valve and the seventh valve should be understood as structures with a small size that prevent the flow of the insulating liquid during nucleic acid extraction. The side of the fifth valve 05 and the seventh valve 07 away from the rotation center O is inclined or arc-shaped, and along the direction of the nucleic acid extraction process, the distance L3 between the side of the fifth valve 05 and the rotation center O gradually decreases. The above arrangement is mainly to facilitate the removal of the reagents carried by the insulating liquid during the transfer of nucleic acid.

[0058] In this embodiment of the invention, the nucleic acid extraction process includes lysing the nucleic acid sample in the sample lysis chamber 22, and adsorbing the lysed nucleic acid with magnetic beads. Optionally, the sample lysis chamber 22 may be pre-set with reagents required for lysis. As shown in Figure 2, along the counterclockwise direction, the magnetic beads adsorbed with nucleic acid enter the nucleic acid extraction chamber 21. The nucleic acid extraction chamber 21 is provided with three chambers for the first washing, the second washing, and nucleic acid elution, respectively. Each nucleic acid extraction chamber is pre-set with a drying reagent to achieve the corresponding effect. The reagent chamber 11 is filled with buffer solution. The buffer solution is released into the nucleic acid extraction chamber 21 after being quantified by the quantitative distribution chamber 20 for precise preparation with the reagents in each extraction chamber.

[0059] As shown in Figure 3, the downstream chamber 30 includes: at least one nucleic acid distribution chamber 300 and a matching amplification reaction chamber 31; the distal end of the nucleic acid distribution chamber 300 is connected to the proximal end of the amplification reaction chamber 31 via an eighth valve 08; at least one of the nucleic acid distribution chambers 300 is connected via a distribution channel 32, which is a gradually spiral or arc-shaped channel, and the upstream of the distribution channel 32 is connected to the distal end of the nucleic acid extraction chamber 21 via a fourth valve 04. After the nucleic acid is eluted in the third nucleic acid extraction chamber 21, the fourth valve 04 is opened. The elution solution and nucleic acid gradually fill the nucleic acid distribution chamber 300 through the fourth valve until the excess nucleic acid is filled into the end collection chamber 33. The collection chamber 33 is set to be large enough to ensure that the elution solution containing nucleic acid in the end nucleic acid extraction chamber 21 does not completely fill the collection chamber 33. Then, the isolation solution in the first sealing chamber 211 and the second sealing chamber 221 fills the distribution channel through the fourth valve. Under the action of centrifugal force, the elution solution containing nucleic acid in the nucleic acid distribution chamber 300 enters the amplification reaction chamber 31. The isolation solution fills the nucleic acid distribution chamber to isolate each amplification reaction chamber 31.

[0060] Furthermore, to improve control smoothness and address the issue of strictly controlling the amount of insulating liquid added, this embodiment of the invention also includes a return gas chamber 34. The return gas chamber 34 and the downstream chamber 30 are arranged around the rotation center O. One end of the return gas chamber 34 is connected to the proximal end of the distribution channel 32, and the other end is connected to the proximal end of the quantitative distribution chamber 20. With this arrangement, excess insulating liquid can be transported to the return gas chamber through the flow channel, while gas in the return gas chamber is transferred to the quantitative distribution chamber 20 through the channel. The quantitative distribution chamber is connected to the fluid (insulating liquid and elution buffer containing nucleic acids), thereby forming a circular pathway with the fluid, improving the smoothness and controllability of the fluid flow.

[0061] Specifically, it also includes a waste liquid chamber 35, which is located at the distal end and between the return gas chamber 34 and the downstream chamber 30; an overflow chamber 23 is provided at the end of the metering chamber 20, and the distal end of the overflow chamber 23 is connected to the proximal end of the waste liquid chamber 35. The distance between the side of the overflow chamber 23 away from the rotation center and the rotation center is greater than the minimum distance between the top surface of the overflow dam 200 that makes up the overflow chamber 23 and the rotation center. This arrangement improves the rationality of the layout within a limited space.

[0062] As shown in Figures 1 and 2, the isolation liquid chamber 10, reagent chamber 11, and sample chamber 12 are arranged in a circle around the rotation center. A buffer chamber 24 is also provided on the pipelines of the third valve 03 and the sixth valve 06. The buffer chamber 24 and the quantitative dispensing chamber 20 are arranged in a circle around the rotation center O. The sample lysis chamber 22 and multiple nucleic acid extraction chambers 21 are arranged in a circle around the rotation center O. The "circle arrangement" refers to the arrangement of corresponding concentric layers to improve the stability of the nucleic acid detection chip during rotation. The deviation of the chip's center of mass from the rotation axis affects the stability during rotation, including the static center of mass and the change in the center of mass during liquid flow. This invention improves the stability of the microfluidic chip during use by adopting a layered distribution of different chambers, thereby increasing the coincidence of the center of mass with the rotation axis of the nucleic acid detection chip in both static and dynamic states.

[0063] Specifically, each nucleic acid extraction chamber 21 has a second valve 02 with different opening conditions based on the steps of the nucleic acid extraction process; the second valve 02 corresponding to the nucleic acid extraction chamber 21 located upstream of the step opens later than the second valve 02 corresponding to the nucleic acid extraction chamber 21 located downstream of the step. This is designed to prevent the buffer solution in the quantitative dispensing chamber 20 from breaking down before quantification is completed, thus preventing inaccurate quantification volume. The second valve 02 uses a capillary tube. Firstly, the capillary tube can provide greater resistance to meet the precise control requirements of each step in this application; secondly, different resistances can be achieved by controlling the cross-sectional size or length of the capillary tube. In this embodiment, capillary tubes with the same cross-sectional size are used, and as shown in Figure 2, capillary tubes with different lengths connected to the second valve 02 of the nucleic acid extraction chamber 21 in the counterclockwise direction are used, reducing the difficulty of preparation.

[0064] Specifically, the nucleic acid extraction chamber 21 is provided with three valves: the first valve 01, the second valve 02, the third valve 03, the fifth valve 05, the sixth valve 06, and the seventh valve 07 all adopt capillary channels; the fourth valve adopts a phase change valve or a mechanical valve. The cross-sectional dimensions of the above-mentioned capillaries range from hundreds of micrometers to millimeters. By limiting the size of different layers of capillaries, the preparation process is ensured to proceed smoothly. This invention pertains to a highly integrated microfluidic chip. The capillary tubes can be connected or closed at different rotational speeds. Therefore, to ensure smooth detection, the difference in the cross-sectional dimensions of the capillary tubes is set relatively large to control the opening and closing of the valve body at multiple rotational speeds. Since the number of capillary tubes used as valve bodies cannot be excessive, capillary tubes occupy less space than existing mechanical methods or phase change valves and only require different rotational speeds to provide centrifugal forces to achieve liquid transfer. This allows for chip miniaturization and low cost. However, under siphon action, they can easily lead to inaccurate quantification. Mechanical valves or phase change valves can achieve complete opening or closing of the valve body under external control conditions, but they occupy a large amount of space and require external driving devices or manual operation. This invention provides a matching method for the aforementioned valve bodies to achieve high integration, high detection accuracy, precise process control, and low cost.

[0065] Example 1

[0066] This invention also provides a highly integrated microfluidic chip, as shown in Figures 1-3, comprising: a central layer 1, which includes a reagent chamber 11 and a sample chamber 12, arranged around a rotation center; a first ring layer 2, located on the side of the central layer 1 away from the rotation center O, which includes multiple nucleic acid extraction chambers 21 located relatively far from the rotation center O, the reagent chambers 11 being connected to the nucleic acid extraction chambers 21; and a sample lysis chamber 22, which is connected to the sample chamber 12 via a sixth valve 06; the sample lysis chamber 22 and the multiple nucleic acid extraction chambers 21 are arranged around the rotation center O; and a second ring layer 3, located on the side of the first ring layer 2 away from the rotation center O, which includes at least one downstream chamber 30, the proximal end of which is connected to the distal end of the last nucleic acid extraction chamber 21 corresponding to the nucleic acid extraction step via a fourth valve 04, the downstream chamber 30 being arranged around the rotation center O.

[0067] The technical solution provided by the embodiments of the present invention integrates the reagent preparation process, avoids pipetting operations, reduces the operational intensity of operators and the probability of contamination; secondly, by setting the cavity around the rotation center, the centroid of the nucleic acid detection chip can be stabilized in both static and dynamic states, avoiding vibration caused by the centroid shifting off the rotation axis during rotation.

[0068] Specifically, it includes: a first concentric layer comprising multiple quantitative dispensing chambers 20 relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers 21 relatively far from the rotation center, the distal ends of the multiple quantitative dispensing chambers 20 being connected to the corresponding nucleic acid extraction chambers 21 via second valves 02; the quantitative dispensing chambers 20 being connected to the reagent chambers 11 via first valves 01; a buffer chamber 24 being provided on the pipeline of the sixth valve 06, the buffer chamber 24 and the quantitative dispensing chambers 20 being arranged around the rotation center O; the second concentric layer further includes a return gas chamber 34, the return gas chamber 34 and the downstream chamber 30 being arranged around the rotation center O, one end of the return gas chamber 34 being connected to the proximal end of the downstream chamber 30, and the other end of the return gas chamber 34 being connected to the proximal end of the quantitative dispensing chambers 20.

[0069] In this embodiment of the invention, the rotation axis is the axis of rotation when the microfluidic chip rotates, and the rotation center O is located on the rotation axis.

[0070] Specifically, as shown in Figure 4, the microfluidic chip is stepped, with each step being cylindrical and symmetrical about the rotation axis X; the central layer 1 is located at the top of the stepped structure, the first ring layer 2 is located below the central layer 1, and the second ring layer 3 is located below the first ring layer 2. The above structure simultaneously achieves: 1. Improved stability of the microfluidic chip under both static and dynamic conditions; specifically, it can be seen that the central layer 1, the first ring layer 2, and the second ring layer 3 constitute the first mass part 001 in the vertical direction, the first ring layer 2 and the second ring layer 3 constitute the second mass part 002 in the vertical direction, and the second ring layer constitutes the third mass part 003. The mass of the first mass part 001 is greater than that of the second mass part 002, and the mass of the second mass part 002 is greater than that of the third mass part 003. Through the above arrangement, the first mass part 001 has a larger proportion, while the mass of the second mass part 002 or the third mass part 003 has a smaller proportion. Even if there is a slight centroid shift in the second mass part 002 or the third mass part 003, it will be corrected by the first mass part; 2. Improved mechanical strength of the microfluidic chip itself with less material cost; Compared with the prior art, the embodiment of the present invention integrates the quantitative dispensing cavity 20 and nucleic acid extraction. The cavity 21 reduces the pipetting work in the prior art, but increases the size of the microfluidic chip. If its strength is increased and deformation is prevented, the product needs to be thicker or more reinforcing ribs need to be added, which greatly increases the weight and cost. However, the present application can reduce the cost of materials while maintaining strength through the above structural design. 3. The vertical height is increased, which lengthens the length required for capillary design and reduces the difficulty of design and subsequent processing. If a planar microfluidic chip is used, the capillary is set on a plane. The length change can only be achieved by increasing the degree of bending of the capillary. However, for the highly integrated nucleic acid detection chip of the present invention, if it is only achieved in the above way, the size will be too large. Moreover, a large number of capillaries require high control and processing accuracy to achieve specific process steps. The present application adopts a stepped microfluidic chip, which increases the design space of the capillary and reduces the difficulty of design and subsequent processing.

[0071] Optionally, as shown in Figures 1-3, an isolation liquid chamber 10 is also included. The isolation liquid chamber 10 is disposed on the central layer 1 and is arranged around the rotation center along with the reagent chamber 11 and the sample chamber 12. The isolation liquid chamber 10 is connected to the proximal end of the sample lysis chamber 22 via a third valve 03. A buffer chamber 24 is provided on the pipeline of the third valve 03. The corresponding buffer chambers 24 on the sixth valve 06 and the third valve 03 are arranged around the rotation center O along with the quantitative dispensing chamber 20. It should be noted that the isolation liquid chamber can also be connected to the proximal end of the nucleic acid extraction chamber via the third valve.

[0072] The nucleic acid extraction chamber 21 includes a first working chamber 210 and a first sealing chamber 211, with the first sealing chamber 211 located on the side of the first working chamber 210 near the rotation center O. The sample lysis chamber 22 includes a second sealing chamber 221 located on the side near the rotation center O and a second working chamber 220 located on the side away from the rotation center O. A fifth valve 05 is provided between adjacent nucleic acid extraction chambers 21, with the fifth valve 05 located between adjacent first sealing chambers 211. A seventh valve 07 is provided between the second sealing chamber 221 and the first sealing chamber 211.

[0073] Similar to Example 1, it also includes a nucleic acid adsorption device 4, which can bypass the fifth valve 05 and the seventh valve 07.

[0074] It also includes a waste liquid chamber 35, which is located at the distal end and between the return gas chamber 34 and the downstream chamber 30; the end of the quantitative distribution chamber 20 is provided with an overflow chamber 23, the distal end of which is connected to the proximal end of the waste liquid chamber 35.

[0075] The downstream chamber 30 includes: at least one nucleic acid distribution chamber 300 and a matching amplification reaction chamber 31; the distal end of the nucleic acid distribution chamber 300 is connected to the proximal end of the amplification reaction chamber 31 via an eighth valve 08; at least one of the nucleic acid distribution chambers 300 is connected via a distribution channel 32, the distribution channel 32 being a gradually spiral or arc-shaped, and the upstream end of the distribution channel 32 is connected to the distal end of the nucleic acid extraction chamber 21 via a fourth valve 04.

[0076] As shown in Figure 3, a second return gas passage 13 is also included. One end of the second return gas passage 13 is connected to the proximal end of the quantitative dispensing chamber, and the other end of the second return gas passage 13 is connected to the proximal end of the reagent chamber 11. By simply setting up the return gas chamber and the second return gas passage, the unidirectional movement of the microfluidic chip can be achieved. Fewer structural features are required, thus reducing the area occupied by the microfluidic chip. This is particularly crucial for the highly integrated microfluidic chip of this application, where structural omissions are essential while maintaining detection accuracy.

[0077] Specifically, each nucleic acid extraction chamber 21 has a second valve 02 with different opening conditions based on the steps of the nucleic acid extraction process; the second valve 02 corresponding to the nucleic acid extraction chamber 21 located upstream of the step opens later than the second valve 02 corresponding to the nucleic acid extraction chamber 21 located downstream of the step.

[0078] Specifically, the nucleic acid extraction chamber 21 is provided with three valves: the first valve 01, the second valve 02, the third valve 03, the fifth valve 05, the sixth valve 06, and the seventh valve 07 all adopt capillary channels; the fourth valve adopts a phase change valve or a mechanical valve.

[0079] Example 2

[0080] This invention provides a method for using the highly integrated microfluidic chip described above, including:

[0081] (1) Add the test sample, reagents and isolation solution. As shown in Figure 5a, add the test sample (black filled part in the figure), buffer solution (dark gray filled part) and sealing mineral oil (light gray filled part in the figure) to the sample chamber, isolation solution chamber and reagent chamber respectively. At this time, the microfluidic chip has been pre-filled with fully dry reagents for nucleic acid extraction, purification and nucleic acid amplification detection, including magnetic beads (black clumps of particles in the figure).

[0082] (2) Buffer quantification. As shown in Figure 5b, a centrifugal speed V1 is applied to the highly integrated microfluidic chip, the buffer is released from the reagent chamber, the quantitative dispensing chamber completes the accurate quantification of the buffer (dark gray filled part), the excess buffer enters the waste liquid chamber, and at the same time the sample in the sample chamber fills the buffer chamber.

[0083] (3) Sample release and liquid reagent dissolution preparation. As shown in Figure 5c, a centrifugal speed V2 is applied to the highly integrated microfluidic chip, and the sample is released from the buffer chamber into the sample lysis chamber, where the pre-placed dry magnetic beads for nucleic acid adsorption are dissolved and dispersed. At the same time, the pre-quantified buffer solution in the quantitative dispensing chamber is released into the nucleic acid extraction chamber, completing the dissolution and preparation of the pre-placed dry reagent for nucleic acid washing and elution in the latter. During this process, the fourth valve remains closed.

[0084] (4) Sealing mineral oil release and filling. As shown in Figure 5d, a centrifugal speed V3 is applied to the highly integrated microfluidic chip, and the sealing mineral oil is released from the isolation liquid chamber, completely filling the annular first and second sealing chambers, thus achieving isolation and sealing of the nucleic acid sample lysis chamber and the nucleic acid extraction chamber. During this process, the seventh and fifth valves remain open, and the fourth valve remains closed.

[0085] (5) Nucleic acid extraction and purification. As shown in Figures 5e-5h, the highly integrated microfluidic chip and the external magnet work together to transfer the magnetic beads with nucleic acid adsorbed in the sample lysis chamber into the nucleic acid extraction chamber to complete the first cleaning, the second cleaning and nucleic acid elution. Finally, the magnetic beads after nucleic acid elution are transferred back to the upstream nucleic acid extraction chamber for storage. During this process, the fifth or seventh valve is opened or closed as needed, while the fourth valve remains closed.

[0086] (6) Dispensing of purified nucleic acid samples. As shown in Figure 5i, a centrifugal speed V4 is applied to the highly integrated microfluidic chip, so that the purified nucleic acid sample in the terminal nucleic acid extraction chamber fills each nucleic acid dispensing chamber in sequence along the dispensing channel. The sealing mineral oil then seals the proximal entrance of each nucleic acid dispensing chamber. During this process, the seventh valve, the fifth valve and the fourth valve remain open.

[0087] (7) Nucleic acid samples enter the amplification reaction chamber to begin amplification and signal detection. As shown in Figure 5j, a centrifugal speed V5 is applied to the highly integrated microfluidic chip, so that the sample nucleic acids in each nucleic acid distribution chamber enter the amplification reaction chamber corresponding to their respective distal ends. At the same time, the sealing mineral oil in the upstream first and second sealing chambers is also filled in. The mineral oil seals the amplification reaction chamber. After the sample nucleic acid dissolves the primers and enzyme system inside the latter, a suitable temperature is applied to start the amplification reaction and the detection of the amplification signal.

[0088] 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 highly integrated microfluidic chip, characterized in that, include: The central layer includes an isolation liquid chamber and a reagent chamber; The first concentric layer is located on the side of the central layer away from the rotation center. It includes multiple nucleic acid extraction chambers that are relatively far from the rotation center. Each nucleic acid extraction chamber includes a first working chamber and a first sealing chamber. The first sealing chamber is located on the side of the first working chamber closer to the rotation center. The reagent chamber is connected to the nucleic acid extraction chamber. The isolation liquid chamber is controlled by a third valve to fill the first sealing chamber with isolation liquid. The second concentric layer is arranged on the side away from the rotation center relative to the first concentric layer, and includes at least one downstream chamber. The proximal end of the downstream chamber is connected to the distal end of the last nucleic acid extraction chamber corresponding to the nucleic acid extraction process through a fourth valve. The reagent chamber and the nucleic acid extraction chamber open the third valve before, after, or simultaneously with the transfer of reagents, and subsequently open the fourth valve. A fifth valve is provided between adjacent nucleic acid extraction chambers, and the fifth valve is located between adjacent first sealing chambers.

2. The highly integrated microfluidic chip according to claim 1, characterized in that, include: The first concentric layer includes multiple quantitative dispensing cavities that are relatively close to the rotation center and a corresponding number of nucleic acid extraction cavities that are relatively far from the rotation center; The distal ends of the multiple quantitative dispensing chambers are respectively connected to the proximal ends of the first sealed chambers of the corresponding nucleic acid extraction chambers via second valves, and the reagent chamber is connected to the quantitative dispensing chambers via first valves.

3. The highly integrated microfluidic chip according to claim 2, characterized in that, The central layer also includes a sample chamber; The first concentric layer further includes a sample lysis chamber, which includes a second sealed chamber on the side closer to the rotation center and a second working chamber on the side farther from the rotation center. The proximal end of the second sealed chamber is connected to the distal end of the sample chamber through a sixth valve. One end of the third valve is connected to the distal end of the isolation liquid chamber, and the other end of the third valve is connected to the proximal end of the second sealing chamber or the first sealing chamber.

4. The highly integrated microfluidic chip according to claim 3, characterized in that, A seventh valve is provided between the second sealing cavity and the first sealing cavity; A nucleic acid adsorption device, which can bypass the fifth and seventh valves.

5. The highly integrated microfluidic chip according to claim 3, characterized in that, The downstream chamber includes: at least one nucleic acid dispensing chamber and a matching amplification reaction chamber; The distal end of the nucleic acid distribution chamber is connected to the proximal end of the amplification reaction chamber via an eighth valve; At least one of the nucleic acid dispensing chambers is connected by a dispensing channel, which is an asymptotic spiral or an arc shape, and the upstream end of the dispensing channel is connected to the distal end of the nucleic acid extraction chamber via a fourth valve.

6. The highly integrated microfluidic chip according to claim 5, characterized in that, It also includes a return air chamber, which is arranged around the rotation center along with the downstream chamber. One end of the return air chamber is connected to the proximal end of the distribution channel, and the other end of the return air chamber is connected to the proximal end of the quantitative distribution chamber.

7. The highly integrated microfluidic chip according to claim 6, characterized in that, It also includes a waste liquid chamber, which is located at the distal end and between the return gas chamber and the downstream chamber; An overflow chamber is provided at the end of the quantitative dispensing chamber, and the distal end of the overflow chamber is connected to the proximal end of the waste liquid chamber.

8. The highly integrated microfluidic chip according to claim 7, characterized in that, The isolation liquid chamber, reagent chamber, and sample chamber are arranged in a circle around the rotation center; The pipelines of the third valve and the sixth valve are also equipped with buffer chambers, and the buffer chambers and the metering distribution chambers are arranged around the rotation center. The sample lysis chamber and multiple nucleic acid extraction chambers are arranged in a circle around the rotation center.

9. The highly integrated microfluidic chip according to claim 3, characterized in that, Each nucleic acid extraction chamber has a second valve with different opening conditions based on the steps of the nucleic acid extraction process; the second valve corresponding to the nucleic acid extraction chamber located upstream of the step opens later than the second valve corresponding to the nucleic acid extraction chamber located downstream of the step.

10. The highly integrated microfluidic chip according to claim 9, characterized in that, The nucleic acid extraction chamber is provided with three valves, and the first valve, second valve, third valve, fifth valve, sixth valve and seventh valve all adopt capillary channels; The fourth valve is either a phase change valve or a mechanical valve.

11. A highly integrated microfluidic chip, characterized in that, include: The central layer includes a reagent chamber and a sample chamber, which are arranged around the center of rotation. The first concentric layer, located on the side of the central layer away from the rotation center, includes multiple nucleic acid extraction chambers relatively far from the rotation center, with the reagent chamber connected to the nucleic acid extraction chambers; it also includes a sample lysis chamber, which is connected to the sample chamber via a sixth valve; the sample lysis chamber and the multiple nucleic acid extraction chambers are arranged in a circle around the rotation center; The second concentric layer is arranged on the side away from the rotation center relative to the first concentric layer, and includes at least one downstream chamber. The upstream of the downstream chamber is connected to the distal end of the last nucleic acid extraction chamber corresponding to the nucleic acid extraction process through a fourth valve. The downstream chamber is arranged around the rotation center. The nucleic acid extraction chamber includes a first working chamber and a first sealing chamber, the first sealing chamber being located on the side of the first working chamber near the rotation center; a fifth valve is provided between adjacent nucleic acid extraction chambers, the fifth valve being located between adjacent first sealing chambers; The first sealed cavity is used to fill the insulating liquid.

12. The highly integrated microfluidic chip according to claim 11, characterized in that, include: The first concentric layer includes multiple quantitative dispensing chambers relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers relatively far from the rotation center. The distal ends of the multiple quantitative dispensing chambers are respectively connected to the corresponding nucleic acid extraction chambers through a second valve. The quantitative dispensing chambers are connected to the reagent chambers through a first valve. A buffer chamber is provided on the pipeline of the sixth valve. The buffer chambers and quantitative dispensing chambers are arranged around the rotation center. The second concentric ring also includes a return air chamber, which is arranged around the rotation center along with the downstream chamber. One end of the return air chamber is connected to the proximal end of the downstream chamber, and the other end of the return air chamber is connected to the proximal end of the quantitative dispensing chamber.

13. The highly integrated microfluidic chip according to claim 12, characterized in that, The microfluidic chip is stepped, with each step being cylindrical and symmetrical about the axis of rotation. The central layer is located at the top of the stepped structure, the first concentric layer is located below the central layer, and the second concentric layer is located below the first concentric layer.

14. The highly integrated microfluidic chip according to claim 13, characterized in that, It also includes an isolation liquid chamber, which is disposed on the central layer and arranged around the rotation center along with the reagent chamber and the sample chamber; The isolation chamber is connected to the proximal end of the sample lysis chamber or nucleic acid extraction chamber via a third valve. A buffer chamber is provided on the pipeline of the third valve. The corresponding buffer chambers on the sixth valve and the third valve are arranged around the rotation center along with the quantitative dispensing chamber.

15. The highly integrated microfluidic chip according to claim 14, characterized in that, The sample lysis chamber includes a second sealed chamber on the side close to the rotation center and a second working chamber on the side away from the rotation center; A seventh valve is provided between the second sealing cavity and the first sealing cavity.

16. The highly integrated microfluidic chip according to claim 15, characterized in that, It also includes a nucleic acid adsorption device that can bypass the fifth and seventh valves.

17. The highly integrated microfluidic chip according to claim 16, characterized in that, It also includes a waste liquid chamber, which is located at the distal end and between the return gas chamber and the downstream chamber; An overflow chamber is provided at the end of the quantitative dispensing chamber, and the distal end of the overflow chamber is connected to the proximal end of the waste liquid chamber.

18. The highly integrated microfluidic chip according to claim 17, characterized in that, The downstream chamber includes: at least one nucleic acid dispensing chamber and a matching amplification reaction chamber; The distal end of the nucleic acid distribution chamber is connected to the proximal end of the amplification reaction chamber via an eighth valve; At least one of the nucleic acid dispensing chambers is connected by a dispensing channel, which is an asymptotic spiral or an arc shape, and the upstream end of the dispensing channel is connected to the distal end of the nucleic acid extraction chamber via a fourth valve.

19. The highly integrated microfluidic chip according to claim 12, characterized in that, It also includes a second return gas passage, one end of which is connected to the proximal end of the quantitative dispensing chamber, and the other end of which is connected to the proximal end of the reagent chamber.

20. The highly integrated microfluidic chip according to claim 12, characterized in that, Each nucleic acid extraction chamber has a second valve with different opening conditions based on the steps of the nucleic acid extraction process; the second valve corresponding to the nucleic acid extraction chamber located upstream of the step opens later than the second valve corresponding to the nucleic acid extraction chamber located downstream of the step.

21. The highly integrated microfluidic chip according to claim 20, characterized in that, The nucleic acid extraction chamber is provided with three valves, and the first valve, second valve, third valve, fifth valve, sixth valve and seventh valve all adopt capillary channels; The fourth valve is either a phase change valve or a mechanical valve.

Images