A highly integrated microfluidic chip

Abstract

The present invention provides a highly integrated microfluidic chip, belonging to the field of microfluidic technology, comprising: a central layer, the central layer comprising an isolation liquid chamber and a reagent chamber; a first ring layer, the first ring layer being arranged at a side of the central layer away from the rotation center, comprising a plurality of quantitative distribution chambers relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers relatively far from the rotation center, the nucleic acid extraction chamber comprising a first working chamber and a first sealing chamber, the first sealing chamber being arranged at a side of the first working chamber close to the rotation center. With the aid of reusable isolation liquid, the magnetic bead method nucleic acid extraction and purification and sample target nucleic acid amplification are successively sealed and isolated, which not only ensures the reliability of the nucleic acid extraction and purification process, but also eliminates the problem of crosstalk between holes that may occur during the nucleic acid amplification process.

Description

Technical Field

[0001] The present application relates to the field of microfluidics technology, and in particular to a highly integrated microfluidics chip. Background Art

[0002] With the development of microfluidics and diagnostic testing technology, the application of microfluidics chips based on microfluidics in the fields of in vitro diagnosis (IVD) has become more and more in-depth and extensive. Microfluidics chips have the characteristics of miniaturization, integration and high automation, which makes the diagnostic testing process based on them more convenient, efficient and low-cost compared with traditional diagnostic testing processes. Nucleic acid amplification detection (such as PCR, LAMP and RPA, etc.) is a type of highly sensitive biotechnology that conducts qualitative or quantitative analysis of trace target gene sequences under a specific enzyme catalysis system. In recent years, its deep integration and application with microfluidics chips (such as point-of-care testing, POCT) have achieved a series of results and progress.

[0003] At present, according to the main functions and the degree of internal integration, microfluidic chips used for nucleic acid amplification detection can be roughly divided into three types. The first type is to add the processed target gene template directly for amplification detection, which can be called pure amplification chip. This type of chip only has the function of amplification detection and cannot directly detect the original sample. Therefore, it often needs to be equipped with additional nucleic acid extraction instruments for use; the second type is to add a simple original sample for nucleic acid release and then directly perform amplification detection, which can be called extraction-free amplification chip or one-step extraction amplification chip. This type of chip is generally only suitable for original samples with simple components, weak inhibition and interference or samples with high target content to be tested, and has high requirements for the performance of nucleic acid releasers. Therefore, this type of chip may have risks such as missed detection and false detection when processing some original samples with complex components and containing inhibitory or interfering components; the third type is to add the original sample and then perform amplification detection after full-process nucleic acid extraction and purification, which can be called full extraction or fully integrated amplification detection chip. This type of chip integrates the entire process of nucleic acid extraction and purification and nucleic acid amplification detection in terms of function, has higher compatibility for complex types of samples, and has higher detection sensitivity and reliability. In summary, full-extraction or fully-integrated nucleic acid amplification detection chips are gradually becoming the mainstream development trend of integrated and portable nucleic acid amplification detection, but such chips often also have higher design requirements and process technology difficulties.

[0004] Currently, the fully integrated nucleic acid amplification detection products on the market include cartridge-type products such as Cepheid's GeneXpert system, Biocartis's Idylla system, and BOE Zhiwei's NAT-3000 system. They all use plunger pumps or external air pumps to provide positive and negative pressures to drive the fluid, and use rotary switching valves to achieve selective connection of internal flow channels. These cartridge-type products often have complex structures and a large number of components, so their costs are often high. Chip-type products such as Bio-Gene's DxLab-2A system and Chenghui Medical's CQ-0403 system both use centrifugal silicon membrane / column method for nucleic acid extraction. The silicon membrane / column is easily clogged and affects the entire extraction process. At the same time, they are also designed with interconnected and complex siphon pipes to assist in liquid release, which makes the stability of fluid operation poor or requires complex hydrophilic treatment in advance. It is particularly noteworthy that, at present, no matter what type of fully integrated nucleic acid amplification detection product is used, the liquid reagents required for nucleic acid extraction and purification need to be sealed and pre-stored in advance or additionally removed and added before use. The sealed pre-storage of liquid reagents inside the cartridge / chip has high requirements for related processes and is prone to leakage of liquid reagents. At the same time, appropriate and efficient liquid reagent release methods must be considered to ensure the normal function of the reagents. The additional removal and addition of the required liquid reagents before use will greatly increase the complexity of the pipetting operation, and put forward 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 object of the present invention is to provide a highly integrated microfluidic chip. With the help of reusable isolation liquid, the magnetic bead method nucleic acid extraction and purification and sample target nucleic acid amplification are sealed and isolated successively, which not only ensures the reliability of the nucleic acid extraction and purification process, but also eliminates the problem of crosstalk between holes that may occur during the nucleic acid amplification process. In addition, only one sample addition operation is required to realize the online preparation of multiple reagents, which reduces the manual operation steps and reduces the work intensity, while avoiding the risk of mutual contamination of multiple reagents. In addition, the design of multiple reagent chambers is avoided, and the chip is miniaturized.

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

[0007] A highly integrated microfluidic chip comprises: a central layer, the central layer comprising an isolation liquid chamber and a reagent chamber; a first ring layer, the first ring layer is arranged on the side of the central layer away from the rotation center, and comprises a plurality of nucleic acid extraction chambers relatively far from the rotation center, the nucleic acid extraction chamber comprises a first working chamber and a first sealed chamber, the first sealed chamber is arranged on the side of the first working chamber close to the rotation center, and the reagent chamber is connected to the nucleic acid extraction chamber; the isolation liquid chamber controls the isolation liquid to fill the first sealed chamber through a third valve; a second ring layer, the second ring layer is arranged on the side of the first ring layer away from the rotation center, and comprises 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 third valve is opened before, after or simultaneously with the transfer of reagents between the reagent chamber and the nucleic acid extraction chamber, and the fourth valve is opened subsequently.

[0008] Further, it includes: the first circle layer includes a plurality of quantitative distribution chambers closer to the rotation center and a corresponding number of nucleic acid extraction chambers farther from the rotation center; the distal ends of the plurality of quantitative distribution chambers are respectively connected to the proximal ends of the first sealed chambers of the corresponding nucleic acid extraction chambers through the second valve, and the reagent chamber is connected to the quantitative distribution chamber through the first valve.

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

[0010] Furthermore, the central layer also includes a sample cavity; the first ring layer also includes a sample lysis cavity, the sample lysis cavity includes a second sealed cavity close to the rotation center and a second working cavity away from the rotation center, the proximal end of the second sealed cavity is connected to the distal end of the sample cavity through a sixth valve; one end of the third valve is connected to the distal end of the isolation liquid cavity, and the other end of the third valve is connected to the proximal end of the second sealed cavity or the first sealed cavity.

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

[0012] Furthermore, the downstream chamber includes: at least one nucleic acid distribution 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 through an eighth valve; at least one of the nucleic acid distribution chambers is connected through a distribution channel, the distribution channel is a gradually spiral line or arc shape, and the upstream of the distribution channel is connected to the distal end of the nucleic acid extraction chamber through a fourth valve.

[0013] Furthermore, it also includes a return air chamber, which is arranged in a circle around the rotation center with the downstream chamber, one end of the return air chamber is connected to the proximal end of the 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 arranged at the distal end and located between the return air chamber and the downstream chamber; an overflow chamber is arranged at the end of the quantitative distribution chamber, and the distal end of the overflow chamber is connected to the proximal end of the waste liquid chamber.

[0015] Furthermore, the isolation liquid chamber, reagent chamber and sample chamber are arranged in a circle around the rotation center; a buffer chamber is also arranged on the pipeline of the third valve and the sixth valve, and the buffer chamber and the quantitative distribution chamber are arranged in a circle around the rotation center; the sample lysis chamber and the multiple nucleic acid extraction chambers are arranged in a circle around the rotation center.

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

[0017] Furthermore, the nucleic acid extraction chamber is provided with three valves, and the first valve, the second valve, the third valve, the fifth valve, the sixth valve and the seventh valve all adopt capillary channels; the fourth valve adopts 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, the central layer comprising a reagent chamber and a sample chamber, the reagent chamber and the sample chamber are arranged in a circle around a rotation center; a first circle layer, arranged on the side of the rotation center away from the central layer, comprising a plurality of nucleic acid extraction chambers relatively far from the rotation center, the reagent chamber being connected to the nucleic acid extraction chamber; also comprising a sample lysis chamber, the sample lysis chamber being connected to the sample chamber through a sixth valve; the sample lysis chamber and the plurality of nucleic acid extraction chambers are arranged in a circle around the rotation center; a second circle layer, the second circle layer is arranged on the side of the rotation center away from the first circle layer, comprising at least one downstream chamber, the upstream of the downstream chamber being connected to the distal end of the last nucleic acid extraction chamber corresponding to the nucleic acid extraction process through a fourth valve, and the downstream chamber is arranged in a circle around the rotation center.

[0019] Further, it includes: the first circle layer includes a plurality of quantitative distribution chambers closer to the rotation center and a corresponding number of nucleic acid extraction chambers farther from the rotation center, and the distal ends of the plurality of quantitative distribution chambers are respectively connected to the corresponding nucleic acid extraction chambers through the second valve; the quantitative distribution chamber is connected to the reagent chamber through the first valve; a buffer chamber is arranged on the pipeline of the sixth valve, and the buffer chamber and the quantitative distribution chamber are arranged around the rotation center; the second circle layer also includes a return air chamber, and the return air chamber and the downstream chamber are arranged around the rotation center, one end of the return air chamber is connected to the proximal end of the end of the downstream chamber, and the other end of the return air chamber is connected to the proximal end of the quantitative distribution chamber.

[0020] Furthermore, the microfluidic chip is in a step-like shape, each step is cylindrical and symmetrical with respect to the rotation axis; the central layer is located at the top of the step, the first circle layer is located below the central layer, and the second circle layer is located below the first circle layer.

[0021] Furthermore, it also includes an isolation liquid chamber, which is arranged on the central layer and is arranged in a circle around the rotation center 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 arranged on the pipeline of the third valve, and the sixth valve and the corresponding buffer chamber on the third valve are arranged in a circle around the rotation center with the quantitative distribution chamber.

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

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

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

[0025] Furthermore, the downstream chamber includes: at least one nucleic acid distribution 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 through an eighth valve; at least one of the nucleic acid distribution chambers is connected through a distribution channel, the distribution channel is a gradually spiral line or arc shape, and the upstream of the distribution channel is connected to the distal end of the nucleic acid extraction chamber through a fourth valve.

[0026] Furthermore, it also includes a second air return passage, one end of which is connected to the proximal end of the quantitative distribution 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 process of the nucleic acid extraction process; the opening time of the second valve corresponding to the nucleic acid extraction chamber located upstream of the process is later than the opening time of the second valve corresponding to the nucleic acid extraction chamber downstream of the process.

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

[0029] The present invention provides a highly integrated microfluidic chip, which has the following beneficial technical effects compared with the prior art:

[0030] The present invention provides an isolation liquid chamber in which isolation liquid is stored. When the first working chamber in the nucleic acid extraction chamber is filled with reagents, the isolation liquid is used to fill the first sealing chamber to achieve sealing of different nucleic acid extraction chambers, thereby avoiding contamination caused by reagents in different nucleic acid extraction chambers. Secondly, when the nucleic acid extraction process is completed, the fourth valve is opened. Under the action of centrifugal force, the liquid with nucleic acid in the first working chamber of the terminal nucleic acid extraction chamber first flows into the downstream chamber through the fourth valve. When the liquid is filled, the isolation liquid then enters the downstream chamber to seal the downstream chamber, thereby avoiding cross-contamination of aerosols generated during the amplification reaction, while ensuring the absolute amount of reaction reagents in the downstream chamber, and improving the accuracy of detection. It can be seen that the present application realizes isolation of multiple steps and different chambers through the same isolation liquid, thereby improving the utilization efficiency of the isolation liquid and the accuracy of the entire microfluidic chip detection. Finally, the highly integrated microfluidic chip provided by the present invention integrates the online preparation process of multiple reagents, avoids repeated pipetting operations, and reduces the operating intensity of operators and the probability of contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

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

[0034] Figure 3 A structural diagram of the downstream cavity of a highly integrated microfluidic chip provided by an embodiment of the present invention;

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

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

[0037] Figure numerals: 01, first valve; 001, first mass portion; 02, second valve; 002, second mass portion; 03, third valve; 003, third mass portion; 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 air passage; 2, first circle layer; 20, quantitative distribution chamber; 200, overflow dam; 21, nucleic acid extraction chamber; 210, first working chamber; 211, first sealed chamber; 212, first protrusion; 213, second protrusion; 22, sample lysis chamber; 220, second working chamber; 221, second sealed chamber; 23, overflow chamber; 24, buffer chamber; 3, second circle layer; 30, downstream chamber; 300, nucleic acid distribution chamber; 31. Amplification reaction chamber; 32. Distribution channel; 33. Collection chamber; 34. Return air chamber; 35. Waste liquid chamber; 4. Nucleic acid adsorption device. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0039] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0041] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, or the positions or positional relationships in which the product of the invention is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific position, be constructed and operated in a specific position, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0042] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "install", "connect", and "connect" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be a connection between two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0043] Example 1

[0044] The embodiment of the present invention discloses a highly integrated microfluidic chip, such as Figure 1 As shown, it includes: a central layer 1, which includes an isolation liquid chamber 10 and a reagent chamber 11; a first ring layer 2, which is arranged on the side of the central layer 1 away from the rotation center O, and includes a plurality of nucleic acid extraction chambers 21 relatively far from the rotation center O, and the nucleic acid extraction chamber 21 includes a first working chamber 210 and a first sealed chamber 211, and the first sealed chamber 211 is arranged on the side of the first working chamber 210 close to the rotation center O, and the reagent chamber 11 is connected to the nucleic acid extraction chamber 21; the isolation liquid chamber 10 controls the isolation liquid to fill the first sealed chamber 211 through the third valve 03; the second ring layer 3, which is arranged on the side of the first ring layer 2 away from the rotation center O, and includes at least one downstream chamber 30, and 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 the reagent before, after or at the same time to open the third valve 03 and then open the fourth valve 04.

[0045] The present invention provides an isolation liquid chamber 10 in which isolation liquid is stored. When the first working chamber 210 in the nucleic acid extraction chamber is filled with reagents, the isolation liquid is used to fill the first sealed chamber 211 to achieve sealing of different nucleic acid extraction chambers, thereby avoiding contamination caused by the reagents in different nucleic acid extraction chambers 21 running around. Secondly, when the nucleic acid extraction process is completed, the fourth valve is opened. Under the action of centrifugal force, the liquid with nucleic acid in the first working chamber 210 of the terminal nucleic acid extraction chamber 21 first flows into the downstream chamber 30 through the fourth valve 04. When the liquid is filled, the isolation liquid then enters the downstream chamber 30 to seal the downstream chamber, thereby avoiding cross-contamination of aerosols generated during the amplification reaction. At the same time, the absolute amount of reaction reagents in the downstream chamber is guaranteed, thereby improving the accuracy of detection. It can be seen that the present application achieves multi-step isolation of different chambers through the same isolation liquid, thereby improving the utilization efficiency of the isolation liquid and the accuracy of the entire microfluidic chip detection.

[0046] It should be pointed out that the opening of the third valve in the present 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, since the density of the isolation liquid is less than that of the reagent, the isolation liquid can seal the reagents in the nucleic acid extraction chamber under the action of centrifugal force. Preferably, the third valve 03 is opened after the reagent chamber and the nucleic acid extraction chamber complete the transfer of reagents. In this way, no mixing flow will occur between the isolation liquid and the reagent, resulting in the problem of emulsification of the interface between the isolation liquid and the reagent.

[0047] Specifically, the first circle layer includes a plurality of quantitative distribution chambers 20 that are relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers 21 that are relatively far from the rotation center O; the distal ends of the plurality of quantitative distribution chambers 20 are respectively connected to the proximal ends of the first sealed chambers 211 of the corresponding nucleic acid extraction chambers 21 through the second valve 02, and the reagent chamber 11 is connected to the quantitative distribution chamber 20 through the first valve 01. Preferably, the valves are opened in the following order: the first valve 01, the second valve 02, the third valve 03 and the fourth valve 04.

[0048] The present invention connects the reagent chamber 11 with a plurality of quantitative distribution chambers 20 through a first valve 01, and quantifies the reagent in the quantitative distribution chamber 20 under the action of centrifugal force. Subsequently, each quantitative distribution chamber 20 transports the quantified reagent to the nucleic acid extraction chamber 21 through corresponding pipelines through the second valve 02, thereby improving the distribution accuracy of the reagent.

[0049] The present invention is provided with a plurality of valves, such as a first valve, a second valve, a third valve and a fourth valve, etc. The form and combination of the valves are not limited, as long as they can be opened and closed in the required steps, and can be active valves or passive valves. Specifically, a manually operated mechanical valve or a phase change valve in the prior art is used, or a capillary channel opened or closed under centrifugal action can be used. In order to reduce production costs while maintaining detection accuracy, it is preferred to use a capillary channel for control, specifically, such as a capillary tube, the size of the capillary tube is from hundreds of microns to millimeters, and different diameters and lengths can be used to achieve the effect of opening or closing under different centrifugal forces. The phase change valve refers to a valve body that can produce a closing and opening effect when external conditions change.

[0050] The "isolating liquid" is a hydrophobic liquid, whose density is less than that of the liquid in the first working chamber, and is inert and will not react with substances in the system, such as mineral oil, etc. The specific composition of the isolating liquid is not limited here.

[0051] It should be pointed out that for 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 corresponding sample chamber can be set up separately for addition. In the embodiment of the present invention, an independent sample chamber and a corresponding sample lysis chamber are set up to lyse the nucleic acid sample, and then the nucleic acid is subjected to impurity removal, cleaning and elution in the nucleic acid extraction chamber. The movement of nucleic acid substances in each chamber is transferred by a nucleic acid adsorption device, specifically, magnetic beads or polymer materials, as long as they can adsorb nucleic acids and realize the transfer of nucleic acids between different chambers under external drive.

[0052] The “distal end” and “proximal end” are obtained by comparing the distances relative to the rotation center O. For example, the side of a chamber close to the rotation center is the proximal end, and the side away from the rotation center is the distal end.

[0053] Specifically, Figure 1 As shown, a fifth valve 05 is provided between adjacent nucleic acid extraction chambers, and the fifth valve 05 is provided between adjacent first sealed chambers 211. By providing the fifth valve in the first sealed chamber 211, the purification of nucleic acid during the transfer process between different nucleic acid extraction chambers 21 can be further enhanced. Since the isolation liquid in the first sealed chamber 211 is incompatible with the liquid in the first working chamber 210, when the 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 sealed chambers 211. At this time, the isolation liquid will repel the reagents remaining in the nucleic acid, so that the reagents are separated in the process of entering the isolation liquid, thereby preventing the reagents in the upstream nucleic acid extraction chamber from being brought into the downstream nucleic acid extraction chamber during the nucleic acid transfer process.

[0054] Specifically, Figure 1As shown, the central layer 1 of the present invention also includes a sample cavity 12; the first ring layer 2 also includes a sample lysis cavity 22, the sample lysis cavity 22 includes a second sealed cavity 221 close to the rotation center O and a second working cavity 220 away from the rotation center O, the proximal end of the second sealed cavity 221 is connected to the distal end of the sample cavity 12 through the sixth valve 06; one end of the third valve 03 is connected to the distal end of the isolation liquid cavity 10, and the other end of the third valve 03 is connected to the proximal end of the second sealed cavity 221. It can be understood that the other end of the third valve 03 can be connected to the proximal end of the first sealed cavity 211. By arranging the sample cavity, the reagent cavity and the isolation liquid cavity on the same chip, the integration of the chip is improved, the pre-processing process of the chip detection is reduced, and the complexity of the operation and the probability of misoperation caused by multiple pipetting are avoided. Preferably, the opening order of the sixth valve 06 is before the third valve 03.

[0055] In the embodiment of the present invention, the central layer 1, the first circle layer 2 and the second circle layer 3 are divided based on the areas marked by circular dotted lines, and each area has a corresponding chamber to meet the requirements of the detection process.

[0056] Specifically, Figure 1 and 2 As shown, the isolation liquid chamber 10, the reagent chamber 11 and the sample chamber 12 in the present invention are chambers arranged in a fan-shaped circle around the rotation center, and the side away from the rotation center O is in a spiral shape. The side away from the rotation center O is in an arc shape and can also complete the corresponding function, but its effect is different from the spiral deviation. The chamber is used to connect the first valve 01, the third valve 03 and the sixth valve 06 at the farthest end from the rotation center. The quantitative distribution chamber 20 is arranged around the rotation center O. The quantitative distribution chamber 20 is provided with a quantitative overflow dam 200 on the side away from the rotation center on the basis of a larger fan-shaped cavity. The upper and lower sides (perpendicular to the paper direction) 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 a plurality of quantitative distribution chambers 20, as shown in FIG. Figure 2 As shown, the upper surface of the quantitative overflow dam 200 is sloped, and along the reagent flow direction A, the angle α between the upper surface of the overflow dam 200 and the radial direction Xr with the rotation center O as the center is greater than 90°. The upper surface of the quantitative overflow dam 200 can be as follows Figure 2The inclined surface shown in can also be an arc surface, but the angle between the tangent line at any point of the arc surface and the radial direction Xr is greater than 90°. Secondly, in order to accurately quantify each quantitative distribution 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 quantitative distribution chamber located upstream is filled first, and the filling amount is the volume formed by the cylindrical surface with a radius swept by the line connecting the highest point of the corresponding overflow dam and the rotation center O and the inner surface of the upstream quantitative distribution chamber; when 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 with a radius swept by the line connecting the highest point of the corresponding overflow dam and the rotation center O and the inner surface of the next quantitative distribution chamber. By analogy, it can be seen that the shape and position of the overflow dam can limit the distribution amount of liquid in each quantitative distribution chamber, reducing the difficulty of chip design and manufacturing.

[0057] Specifically, a seventh valve 07 is provided between the second sealed cavity 221 and the first sealed cavity 211; a nucleic acid adsorption device 4, which can pass through the fifth valve 05 and the seventh valve 07. Preferably, the nucleic acid adsorption device 4 in the present invention is a magnetic bead, which can pass through the fifth valve 05 and the seventh valve 07 under the control of an external magnetic force to achieve circulation in different chambers. The fifth valve 05 and the seventh valve 07 are capillary channels, which can be capillaries or capillary channels of other cross-sectional shapes, as long as the maximum cross-sectional distance is between hundreds of micrometers and millimeters.

[0058] The first working chamber 210 is connected to the first sealed chamber 211, and the second working chamber 220 is connected to the second sealed chamber 221. It should be noted that the volume of the first working chamber 210 is large enough to completely receive the quantitative reagent of the corresponding quantitative distribution chamber 20. The arrangement of the first working chamber 210 and the first sealed chamber 211 and the second working chamber 220 and the second sealed chamber 221 can be structurally arranged based on actual needs. In the embodiment of the present invention, Figure 2As shown, the nucleic acid extraction chamber 21 is formed by isolating a plurality of first protrusions 212 on the side wall of a whole fan-shaped chamber away from the rotation center O. In the embodiment of the present invention, two first protrusions 212 are provided to form a total of three nucleic acid extraction chambers 21. In the embodiment of the present invention, a second protrusion 213 is further provided to isolate 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 pointed out that the seventh valve in the present invention is a capillary channel with a flow channel size of hundreds of microns to several millimeters. The first working chamber 21 0 and the first sealed chamber 211 and the second working chamber 220 and the second sealed chamber 221 are chambers with different thicknesses. The first sealed chamber 211 and the second sealed chamber 221 are relatively thin (the thickness direction is perpendicular to the paper surface), which is convenient for the isolation liquid to completely fill the second sealed chamber 221 of the sample lysis chamber and the first sealed chamber 211 of the nucleic acid extraction chamber. The thickness of the second working chamber 220 and the first working chamber 210 is relatively thick so as to accommodate more reagents. The above-mentioned size design is not only convenient for processing, but also avoids the vibration caused by the imbalance of the center of gravity of the chip during the rotation due to the excessive isolation liquid. It should be pointed out that the fifth valve 05 and the seventh valve 07 in the embodiment of the present invention are capillary channels with a size of hundreds of micrometers to several millimeters, and the first sealed chamber and the second sealed chamber can also be hundreds of micrometers to several millimeters in thickness. Here, the fifth valve and the seventh valve should be understood as structures with a smaller size to prevent the isolation liquid from flowing during the nucleic acid extraction process. 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 seventh valve 07 away from the rotation center O and the rotation center O gradually decreases. The above-mentioned setting is mainly to facilitate the isolation liquid to separate the reagents carried by the nucleic acid during the transfer process.

[0059] In the embodiment of the present invention, the nucleic acid extraction process includes lysing the nucleic acid sample in the sample lysis chamber 22, and the magnetic beads adsorbing the lysed nucleic acid. Optionally, the sample lysis chamber 22 may be pre-set with reagents required for lysis; Figure 2 As shown, along the counterclockwise direction, the magnetic beads adsorbed with nucleic acid enter the nucleic acid extraction chamber 21, and the nucleic acid extraction chamber 21 is provided with three chambers for the first cleaning, the second cleaning and nucleic acid elution, respectively. Each nucleic acid extraction chamber is pre-set with a dry reagent to achieve the corresponding effect, and the reagent chamber 11 is filled with a buffer solution. The buffer solution is quantitatively distributed in the quantitative distribution chamber 20 and then released into the nucleic acid extraction chamber 21 for precise preparation with the reagents in each extraction chamber.

[0060] like Figure 3As shown, 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 through the eighth valve 08; at least one of the nucleic acid distribution chambers 300 is connected through a distribution channel 32, and the distribution channel 32 is in the shape of a gradually extending spiral or arc, and the upstream of the distribution channel 32 is connected to the distal end of the nucleic acid extraction chamber 21 through the fourth valve 04. After the nucleic acid is eluted by the eluent in the third nucleic acid extraction chamber 21, the fourth valve 04 is opened, and the eluent and the nucleic acid gradually fill the nucleic acid distribution chamber 300 through the fourth valve until the excess nucleic acid is filled into the collection chamber 33 at the end. The collection chamber 33 is larger to ensure that the eluent with nucleic acid in the nucleic acid extraction chamber 21 at the end cannot fill the collection chamber 33 at most. Subsequently, the isolation liquid in the first sealed chamber 211 and the second sealed chamber 221 fills the distribution channel through the fourth valve. Under the action of centrifugal force, the eluent with nucleic acid in the nucleic acid distribution chamber 300 enters the amplification reaction chamber 31, and the isolation liquid fills the nucleic acid distribution chamber to isolate each amplification reaction chamber 31.

[0061] Furthermore, in order to increase the smoothness of control and strictly control the amount of isolation liquid added, the embodiment of the present invention further includes a return air chamber 34, which is arranged in a circle around the rotation center O with the downstream chamber 30, and one end of the return air chamber 34 is connected to the proximal end of the end of the distribution channel 32, and the other end of the return air chamber is connected to the proximal end of the quantitative distribution chamber 20. Through the above arrangement, the excess isolation liquid can be transported to the return air chamber through the flow channel, and the gas in the return air chamber is transferred to the quantitative distribution chamber 20 through the channel. The quantitative distribution chamber is connected to the fluid (isolation liquid and elution liquid with nucleic acid), thereby forming an annular passage with the fluid, thereby improving the smoothness and controllability of the fluid flow.

[0062] Specifically, it also includes a waste liquid chamber 35, which is arranged at the distal end and located between the return air chamber 34 and the downstream chamber 30; the end of the quantitative distribution chamber 20 is provided with an overflow chamber 23, 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 constituting the overflow chamber 23 and the rotation center. The above arrangement improves the rationality of the layout in a limited space.

[0063] like Figure 1 and 2As shown, the isolation liquid chamber 10, the reagent chamber 11 and the sample chamber 12 are arranged in a circle around the rotation center; the pipelines of the third valve 03 and the sixth valve 06 are also provided with a buffer chamber 24, and the buffer chamber 24 and the quantitative distribution chamber 20 are arranged in a circle around the rotation center O; the sample lysis chamber 22 and the plurality of nucleic acid extraction chambers 21 are arranged in a circle around the rotation center O. The "one circle arrangement" is arranged into corresponding circles to improve the stability of the nucleic acid detection chip during the rotation process. The centroid deviation of the chip from the rotation axis will affect the stability during the rotation process, including the static centroid and the change of the centroid during the liquid flow process. The present invention improves the overlap between the centroid and the rotation axis of the nucleic acid detection chip in static and dynamic conditions by distributing different chambers in layers to improve the stability of the microfluidic chip during use.

[0064] Specifically, each nucleic acid extraction chamber 21 has a second valve 02 with different opening conditions based on the process of the nucleic acid extraction process; the opening time of the second valve 02 corresponding to the nucleic acid extraction chamber 21 located upstream of the process is later than the opening time of the second valve 02 corresponding to the nucleic acid extraction chamber 21 downstream of the process. The above-mentioned setting is to prevent the buffer solution in the quantitative distribution chamber 20 from being punctured before the quantitative determination is completed, thereby causing inaccurate quantitative volume. The second valve 02 adopts a capillary. First, the capillary can provide a large resistance to meet the precise control of each process of this application; secondly, different resistances can be achieved by controlling the cross-sectional size or length of the capillary. In the embodiment of the present invention, the same cross-sectional size is adopted. Figure 2 It can be seen that, in the counterclockwise direction, the capillaries of the second valve 02 connected to the nucleic acid extraction chamber 21 have different lengths, which reduces the difficulty of preparation.

[0065] 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 use capillary channels; the fourth valve uses a phase change valve or a mechanical valve. The cross-sectional size of the capillary tubes ranges from hundreds of micrometers to millimeters. By limiting the sizes of the capillaries at different layers, the preparation process is ensured to proceed smoothly. The present invention belongs to a highly integrated microfluidic chip. The capillary can be connected or closed at different rotational speeds. Therefore, in order to ensure the smooth progress of the detection process, the difference in the cross-sectional dimensions of the capillary is set to be relatively large to satisfy the control of the opening and closing of the valve body at multiple rotational speeds. Too many capillaries cannot be set as valve bodies. Compared with the existing mechanical method or phase change valve, the capillary occupies less space and only needs to provide different centrifugal forces at different rotational speeds to achieve liquid transfer. Therefore, the chip can be miniaturized and low-cost, but it is easy to cause quantitative inaccuracy under the action of siphon. Mechanical valves or phase change valves can achieve complete opening or closing of the valve body under external control conditions, but mechanical valves and phase change valves occupy a large space and require external drive devices or manual operation. The present invention provides a matching method for the above-mentioned valve body to achieve the purposes of high integration, high detection accuracy, precise process control, and low cost.

[0066] Example 2

[0067] The present invention also provides a highly integrated microfluidic chip. Figure 1-3 As shown, it includes: a central layer 1, the central layer 1 includes a reagent chamber 11 and a sample chamber 12, and the reagent chamber 11 and the sample chamber 12 are arranged in a circle around the rotation center; a first circle layer 2, which is arranged on the side of the central layer 1 away from the rotation center O, and includes a plurality of nucleic acid extraction chambers 21 relatively far from the rotation center, and the reagent chamber 11 is connected to the nucleic acid extraction chamber 21; it also includes a sample lysis chamber 22, and the sample lysis chamber 22 is connected to the sample chamber 12 through a sixth valve 06; the sample lysis chamber 22 and the plurality of nucleic acid extraction chambers 21 are arranged in a circle around the rotation center O; a second circle layer 3, the second circle layer 3 is arranged on the side of the first circle layer 2 away from the rotation center O, and includes 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 a fourth valve 04, and the downstream chamber 30 is arranged in a circle around the rotation center O.

[0068] The above-mentioned technical solution provided by the embodiment of the present invention integrates the reagent preparation process, avoids pipetting operations, reduces the operating intensity of operators and the probability of contamination; secondly, the cavity is arranged in a circle around the rotation center, which can stabilize the center of mass of the nucleic acid detection chip in static and dynamic states, and avoid vibration during rotation caused by the center of mass offset from the rotation axis.

[0069] Specifically, it includes: the first circle layer includes a plurality of quantitative distribution chambers 20 that are relatively close to the rotation center and a corresponding number of nucleic acid extraction chambers 21 that are relatively far from the rotation center, and the distal ends of the plurality of quantitative distribution chambers 20 are respectively connected to the corresponding nucleic acid extraction chambers 21 through the second valve 02; the quantitative distribution chamber 20 is connected to the reagent chamber 11 through the first valve 01; a buffer chamber 24 is arranged on the pipeline of the sixth valve 06, and the buffer chamber 24 and the quantitative distribution chamber 20 are arranged around the rotation center O; the second circle layer also includes a return air chamber 34, and the return air chamber 34 and the downstream chamber 30 are arranged around the rotation center O, one end of the return air chamber 34 is connected to the proximal end of the end of the downstream chamber 30, and the other end of the return air chamber 34 is connected to the proximal end of the quantitative distribution chamber 20.

[0070] In the embodiment of the present invention, the rotation axis is the rotation axis of the microfluidic chip when it rotates, and the rotation center O is located on the rotation axis.

[0071] Specifically, Figure 4As shown, the microfluidic chip is in a step shape, each step is cylindrical and symmetrical with respect to the rotation axis X; the central layer 1 is located at the top of the step, the first circle layer 2 is located below the central layer 1, and the second circle layer 3 is located below the first circle layer 2. The above structure can simultaneously achieve the following: 1. The stability of the microfluidic chip is improved in both static and dynamic conditions; specifically, it can be seen that the central layer 1, the first circle layer 2 and the second circle layer 3 constitute the first mass portion 001 in the vertical direction, the first circle layer 2 and the second circle layer 3 constitute the second mass portion 002 in the vertical direction, and the second circle layer constitutes the third mass portion 003. The mass of the first mass portion 001 is greater than that of the second mass portion 002, and the mass of the second mass portion 002 is greater than that of the third mass portion 003. Through the above arrangement, the first mass portion 001 accounts for a larger proportion, and the mass of the second mass portion 002 or the third mass portion 003 accounts for a smaller proportion. Even if there is a slight center of mass offset in the second mass portion 002 or the third mass portion 003, it will be corrected by the first mass portion; 2. Under the premise of lower material cost, the mechanical strength of the microfluidic chip itself is improved; compared with the prior art, the embodiment of the present invention integrates a quantitative distribution chamber 20 and a nucleic acid extraction chamber 21. Cavity 21 reduces the pipetting work in the prior art, but increases the size of the microfluidic chip. In order to increase its strength and prevent its deformation, the product needs to be set thicker or more reinforcing ribs are set, which greatly increases the quality and increases the cost. The present application can meet the strength while reducing the material use cost through the above-mentioned structural design; 3. The vertical height is increased, the required length of the capillary design is lengthened, and the difficulty of design and subsequent processing is reduced; if a planar microfluidic chip is used, the capillary is set on a plane, and the length change can only be achieved by increasing the bending degree of the capillary. However, for the highly integrated nucleic acid detection chip of the present invention, if it is only realized by the above-mentioned method, the problem of large size will appear, and more capillaries have high requirements for control accuracy and processing accuracy to realize 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.

[0072] Optional, such as Figure 1-3 The figure also includes an isolation liquid chamber 10, which is arranged on the central layer 1 and is arranged in a circle with the reagent chamber 11 and the sample chamber 12 around the rotation center; the isolation liquid chamber 10 is connected to the proximal end of the sample lysis chamber 22 through the third valve 03, and a buffer chamber 24 is arranged on the pipeline of the third valve 03. The sixth valve 06 and the corresponding buffer chamber 24 on the third valve 03 are arranged in a circle with the quantitative distribution chamber 20 around the rotation center O. It should be pointed out that the isolation liquid chamber can also be connected to the proximal end of the nucleic acid extraction chamber through the third valve.

[0073] The nucleic acid extraction chamber 21 includes a first working chamber 210 and a first sealed chamber 211, and the first sealed chamber 211 is arranged on the side of the first working chamber 210 close to the rotation center O; the sample lysis chamber 22 includes a second sealed chamber 221 close to the rotation center O and a second working chamber 220 away from the rotation center; a fifth valve 05 is arranged between adjacent nucleic acid extraction chambers 21, and the fifth valve 05 is arranged between adjacent first sealed chambers 211, and a seventh valve 07 is arranged between the second sealed chamber 221 and the first sealed chamber 211.

[0074] Similar to the first embodiment, the invention further comprises a nucleic acid adsorption device 4 , and the nucleic acid adsorption device 4 can pass through the fifth valve 05 and the seventh valve 07 .

[0075] It also includes a waste liquid chamber 35, which is arranged at the distal end and located between the return air chamber 34 and the downstream chamber 30; an overflow chamber 23 is arranged at the end of the quantitative distribution chamber 20, and the distal end of the overflow chamber 23 is connected to the proximal end of the waste liquid chamber 35.

[0076] 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 through the eighth valve 08; at least one of the nucleic acid distribution chambers 300 is connected through a distribution channel 32, and the distribution channel 32 is in the shape of a gradually extending spiral line or arc, and the upstream of the distribution channel 32 is connected to the distal end of the nucleic acid extraction chamber 21 through the fourth valve 04.

[0077] like Figure 3 As shown, it also includes a second air return passage 13, 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 11. The microfluidic chip can be moved forward only by setting the air return chamber and the second air return passage, and fewer structural features are designed, which correspondingly reduces the occupied area of ​​the microfluidic chip. Especially for the highly integrated microfluidic chip of the present application, it is particularly critical to omit the structure while meeting the detection accuracy.

[0078] Specifically, each nucleic acid extraction chamber 21 has a second valve 02 with different opening conditions based on the process of the nucleic acid extraction process; the opening time of the second valve 02 corresponding to the nucleic acid extraction chamber 21 located upstream of the process is later than the opening time of the second valve 02 corresponding to the nucleic acid extraction chamber 21 downstream of the process.

[0079] Specifically, the nucleic acid extraction chamber 21 is provided with three valves, and 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.

[0080] Example 3

[0081] The embodiment of the present invention provides a method for using the highly integrated microfluidic chip, including:

[0082] (1) Add the sample to be tested, reagents and isolation fluid. Figure 5 As shown in a, the sample to be tested (the black filled part in the figure), buffer (the dark gray filled part in the figure) and sealed mineral oil (the light gray filled part in the figure) are added to the sample chamber, isolation liquid chamber and reagent chamber respectively. At this time, the microfluidic chip has been pre-installed with fully dry reagents for nucleic acid extraction and purification and nucleic acid amplification detection, including magnetic beads (black lumpy particles in the figure).

[0083] (2) Buffer quantification. Figure 5 As shown in b, a centrifugal speed V1 is applied to the highly integrated microfluidic chip, the buffer is released from the reagent chamber, the quantitative distribution chamber completes the precise quantification of the buffer (the dark gray filled part), the excess buffer enters the waste liquid chamber, and the sample in the sample chamber is filled into the buffer chamber.

[0084] (3) Sample release and liquid reagent dissolution preparation. Figure 5 As shown in c, 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 to dissolve and disperse the dry magnetic beads pre-placed therein for nucleic acid adsorption. At the same time, the quantitative buffer solution in the quantitative distribution chamber is released into the nucleic acid extraction chamber to complete the dissolution and preparation of the dry reagents pre-placed in the latter for nucleic acid cleaning and elution. During this process, the fourth valve is always kept closed.

[0085] (4) Seal mineral oil release filling. Figure 5 As shown in FIG. d, a centrifugal speed V3 is applied to the highly integrated microfluidic chip, and the sealed mineral oil is released from the isolation liquid chamber, completely filling the annular first sealing chamber and the second sealing chamber, thereby achieving isolation and sealing of the nucleic acid sample lysis chamber and the nucleic acid extraction chamber. During this process, the seventh valve and the fifth valve are always kept open, and the fourth valve is always kept closed.

[0086] (5) Nucleic acid extraction and purification. Figure 5 As shown in e-5h, the highly integrated microfluidic chip is controlled to cooperate with the external magnet so that the magnetic beads adsorbed with nucleic acid in the sample lysis chamber are transferred into the nucleic acid extraction chamber in sequence to complete the first cleaning, the second cleaning and the nucleic acid elution; finally, the magnetic beads after the nucleic acid elution are transferred back to the upstream nucleic acid extraction chamber for storage. During this process, the fifth valve or the seventh valve is opened or closed according to demand, and the fourth valve is always kept closed.

[0087] (6) Distribution of purified nucleic acid samples. Figure 5As shown in FIG. 1 , 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 cavity fills each nucleic acid distribution cavity in turn along the distribution channel, and the sealing mineral oil then completes the sealing of the proximal inlet of each nucleic acid distribution cavity. During this process, the seventh valve, the fifth valve and the fourth valve are always kept open.

[0088] (7) The nucleic acid sample enters the amplification reaction chamber to start amplification and signal detection. Figure 5 As shown in FIG. 1 , a centrifugal speed V5 is applied to the highly integrated microfluidic chip, so that the sample nucleic acid in each nucleic acid distribution cavity enters into the amplification reaction cavity corresponding to the respective distal end, and at the same time, the sealed mineral oil in the upstream first sealed cavity and the second sealed cavity is also filled down, and the mineral oil seals the amplification reaction cavity. After the sample nucleic acid dissolves the primer and enzyme system inside the latter, a suitable temperature is applied to start the amplification reaction and the detection of the amplification signal.

[0089] The above description is only an embodiment of the present application and is not intended to limit the scope of protection of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of protection of the present application.

Claims

1. A highly integrated microfluidic chip, characterized in that: include: A central layer, the central layer comprising an isolation liquid cavity and a reagent cavity; A first ring layer, which is arranged on a side of the central layer far from the rotation center, and includes a plurality of nucleic acid extraction chambers that are relatively far from the rotation center, wherein the nucleic acid extraction chamber includes a first working chamber and a first sealing chamber, wherein the first sealing chamber is arranged on a side of the first working chamber close to the rotation center, and the reagent chamber is connected to the nucleic acid extraction chamber; the isolation liquid chamber controls the isolation liquid to fill the first sealing chamber through a third valve; A second ring layer, which is arranged at a side of the first ring layer away from the rotation center, and includes at least one downstream chamber, wherein 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 third valve is opened before, after or simultaneously with the transfer of the reagent between the reagent chamber and the nucleic acid extraction chamber, and the fourth valve is subsequently opened; A fifth valve is arranged between adjacent nucleic acid extraction chambers, and the fifth valve is arranged between adjacent first sealing chambers.

2. The highly integrated microfluidic chip according to claim 1, characterized in that: include: The first circle layer includes a plurality of quantitative distribution chambers closer to the rotation center and a corresponding number of nucleic acid extraction chambers farther from the rotation center; The distal ends of the plurality of quantitative distribution chambers are respectively connected to the proximal ends of the first sealed chambers of the corresponding nucleic acid extraction chambers through the second valve, and the reagent chamber is connected to the quantitative distribution chamber through the first valve.

3. The highly integrated microfluidic chip according to claim 2, characterized in that: The central layer also includes a sample cavity; The first ring layer further includes a sample lysis chamber, which includes a second sealed chamber on one side close to the rotation center and a second working chamber on one side far from the rotation center, and 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 cavity, and the other end of the third valve is connected to the proximal end of the second sealed cavity or the first sealed cavity.

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

5. The highly integrated microfluidic chip according to claim 3, characterized in that: The downstream chamber comprises: at least one nucleic acid distribution chamber and an amplification reaction chamber matched therewith; 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 distribution chambers is connected via a distribution channel, the distribution channel is in the shape of a gradually extending spiral line or arc, and the upstream of the distribution 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 in a circle with the downstream chamber around the rotation center. One end of the return air chamber is connected to the proximal end of the 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 arranged at the distal end and between the return air chamber and the downstream chamber; An overflow chamber is arranged at the end of the quantitative distribution 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, the reagent chamber and the sample chamber are arranged in a circle around the rotation center; The pipelines of the third valve and the sixth valve are also provided with a buffer chamber, and the buffer chamber and the quantitative distribution chamber are arranged in a circle around the rotation center; The sample lysis chamber and the plurality of 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 process of the nucleic acid extraction process; the opening time of the second valve corresponding to the nucleic acid extraction chamber located upstream of the process is later than the opening time of the second valve corresponding to the nucleic acid extraction chamber downstream of the process.

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, the second valve, the third valve, the fifth valve, the sixth valve and the seventh valve all adopt capillary channels; The fourth valve is a phase change valve or a mechanical valve.

11. A highly integrated microfluidic chip, characterized in that: include: A central layer, the central layer comprising a reagent chamber and a sample chamber, wherein the reagent chamber and the sample chamber are arranged in a circle around a rotation center; The first circle layer is arranged on the side of the central layer far from the rotation center, and includes a plurality of nucleic acid extraction chambers far from the rotation center, the reagent chamber is connected to the nucleic acid extraction chamber; and also includes a sample lysis chamber, which is connected to the sample chamber through a sixth valve; the sample lysis chamber and the plurality of nucleic acid extraction chambers are arranged in a circle around the rotation center; A second circle layer, which is arranged at a side away from the rotation center relative to the first circle layer, and includes at least one downstream chamber, wherein 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, and the downstream chamber is arranged in a circle around the rotation center; The nucleic acid extraction chamber comprises a first working chamber and a first sealing chamber, wherein the first sealing chamber is arranged on a side of the first working chamber close to the rotation center; a fifth valve is arranged between adjacent nucleic acid extraction chambers, and the fifth valve is arranged between adjacent first sealing chambers; The first sealed cavity is used to be filled with an isolation liquid.

12. The highly integrated microfluidic chip according to claim 11, characterized in that: include: The first circle layer includes a plurality of quantitative distribution chambers closer to the rotation center and a corresponding number of nucleic acid extraction chambers farther from the rotation center, and the distal ends of the plurality of quantitative distribution chambers are respectively connected to the corresponding nucleic acid extraction chambers through the second valve; the quantitative distribution chamber is connected to the reagent chamber through the first valve; a buffer chamber is arranged on the pipeline of the sixth valve, and the buffer chamber and the quantitative distribution chamber are arranged in a circle around the rotation center; The second circle layer also includes a return air cavity, which is arranged with the downstream chamber in a circle around the rotation center. One end of the return air cavity is connected to the proximal end of the downstream chamber end, and the other end of the return air cavity is connected to the proximal end of the quantitative distribution cavity.

13. The highly integrated microfluidic chip according to claim 12, characterized in that: The microfluidic chip is in a step-like shape, and each step is cylindrical and symmetrical with respect to the rotation axis; The central layer is located at the top of the step, the first circle layer is located below the central layer, and the second circle layer is located below the first circle layer.

14. The highly integrated microfluidic chip according to claim 13, characterized in that: It also includes an isolation liquid chamber, which is arranged on the central layer and is arranged in a circle around the rotation center 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 the third valve, a buffer chamber is arranged on the pipeline of the third valve, and the corresponding buffer chambers on the sixth valve and the third valve and the quantitative distribution chamber are arranged in a circle around the rotation center.

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

16. The highly integrated microfluidic chip according to claim 15, characterized in that: Also included is a nucleic acid adsorption device, which can bypass the fifth valve and the seventh valve.

17. The highly integrated microfluidic chip according to claim 16, characterized in that: It also includes a waste liquid chamber, which is arranged at the distal end and between the return air chamber and the downstream chamber; An overflow chamber is arranged at the end of the quantitative distribution 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 comprises: at least one nucleic acid distribution chamber and an amplification reaction chamber matched therewith; 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 distribution chambers is connected via a distribution channel, the distribution channel is in the shape of a gradually extending spiral line or arc, and the upstream of the distribution 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 air return passage, one end of which is connected to the proximal end of the quantitative distribution 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 process of the nucleic acid extraction process; the opening time of the second valve corresponding to the nucleic acid extraction chamber located upstream of the process is later than the opening time of the second valve corresponding to the nucleic acid extraction chamber downstream of the process.

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, the second valve, the third valve, the fifth valve, the sixth valve and the seventh valve all adopt capillary channels; The fourth valve is a phase change valve or a mechanical valve.

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