A fully integrated microfluidic chip

Abstract

The present invention provides a fully integrated microfluidic chip, belonging to the field of microfluidic technology, including: a lysis chamber; a nucleic acid purification module, the nucleic acid purification module is arranged on the side close to the rotation center compared to the lysis chamber, the nucleic acid purification module has at least one purification chamber, the purification chamber includes a working chamber and a sealing chamber, the distal side of the sealing chamber of the purification chamber is connected to the proximal side of the working chamber, and the proximal end of the lysis chamber is connected to the sealing chamber of the primary purification chamber through a first valve; a sealing liquid storage chamber, the distal end of the sealing liquid storage chamber is connected to the sealing chamber of the final purification chamber through a second valve; a downstream module, the downstream module is connected to the distal end of the final purification chamber through a third valve. The present invention uses centrifugal force as the driving force of the liquid reagent, by filling the liquid reagent into each functional chamber and the nucleic acid extract moving from the distal chamber to the proximal chamber, and using the binding force of the centrifugal force on the reagent in each chamber to avoid cross contamination of adjacent chambers.

Description

Technical Field

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

[0002] With the continuous development and innovation of microfluidic technology, the application of microfluidic chips is gradually becoming a hot topic in various research fields. Due to the advantages of microfluidic chips such as miniaturization, integration and high automation, they have been widely used in the field of biomedical testing (including biochemistry, immunology and molecular diagnosis, etc.). Microfluidic chip kits used to load and run various detection reagents are gradually becoming the most effective carrier form for achieving "point-of-care testing" (POCT) at the patient's bedside.

[0003] To meet the detection needs, microfluidic chip test kits need to perform various processing, mixing, separation and transfer operations on liquid components such as samples and reagents. Among them, the diversion, quantification and sequential release of liquid reagents are important links for various microfluidic chip test kits to achieve internal fluid manipulation and operation, and are often also the key to ensuring the sensitive and efficient operation of microfluidic chips. For example, when there are multiple parallel reaction chambers (such as multiple PCR amplification chambers) downstream, accurate and consistent quantification and diversion are often required upstream. For example, when sequential reactions of reagents are required downstream (such as nucleic acid extraction and purification), sequential release of reagents is often required upstream.

[0004] At present, although some microfluidic solutions have made certain design optimizations and performance improvements in subdivided technical strategies such as quantitative diversion and sequential release, the microfluidic field still has a lot of room for technical improvement and demand in terms of the accuracy of quantitative diversion and the feasibility and simplicity of sequential release operations. There is a risk of cross-contamination between adjacent chambers of existing microfluidic chips, and in order to achieve accurate detection results, the chambers are set larger, which makes it more difficult to miniaturize the chip. Summary of the invention

[0005] The purpose of the present invention is to provide a fully integrated microfluidic chip. Centrifugal force is used as the driving force for liquid reagents, and the liquid reagents are filled into each functional cavity; in the nucleic acid cleavage and purification chamber, the nucleic acid extract moves from the distal chamber to the proximal chamber, and the binding force of the centrifugal force on the reagents in each chamber is used to avoid cross contamination of adjacent chambers, and the reagents in the nucleic acid are further discharged based on the capillary channel, so that the chamber space can be set smaller, and the chip can be miniaturized.

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

[0007] A fully integrated microfluidic chip comprises: a lysis chamber; a nucleic acid purification module, wherein the nucleic acid purification module is arranged on a side closer to a rotation center than the lysis chamber, the nucleic acid purification module has at least one purification chamber, the purification chamber comprises a working chamber and a sealing chamber, the distal side of the sealing chamber of the purification chamber is communicated with the proximal side of the working chamber, the proximal end of the lysis chamber is communicated with the sealing chamber of a primary purification chamber through a first valve; a sealing liquid storage chamber, wherein the distal end of the sealing liquid storage chamber is communicated with the sealing chamber of a final purification chamber through a second valve; and a downstream module, wherein the downstream module is communicated with the distal end of the final purification chamber through a third valve.

[0008] Furthermore, when there are two or more purification chambers, the purification chambers are arranged close to the rotation center in sequence along the purification direction; adjacent purification chambers are connected through a fourth valve; the first valve and the fourth valve connecting the lysis chamber and each purification chamber are respectively connected to two sides of the same sealed chamber.

[0009] Furthermore, it also includes a reagent chamber, which is connected to the proximal end of the corresponding purification chamber through a fifth valve.

[0010] Furthermore, the first valve and the fourth valve are composed of a capillary channel, the capillary channel includes a first channel extending radially along the microfluidic chip and a second channel along the axis of the microfluidic chip, the width of the second channel is smaller than the first channel; the first channel is connected to the proximal end of the lysis chamber or the purification chamber, and the second channel is connected to the sealed chamber of the purification chamber.

[0011] Furthermore, the width of the second channel is smaller than the width of the sealed cavity, and the connection position between the second channel and the sealed cavity is located at an upper position.

[0012] Furthermore, it also includes a sample cavity; the microfluidic chip is arranged in two layers; the lysis cavity, nucleic acid purification module, downstream module, sealing liquid storage cavity and final reagent storage cavity are arranged in one layer; the sample cavity and other reagent storage cavities are arranged in another layer.

[0013] Furthermore, the sample chamber is located at a corresponding position of the primary purification chamber; except for the final reagent storage chamber, other reagent storage chambers are located at corresponding positions of the downstream purification chamber; preferably, other reagent storage chambers are located at corresponding positions of adjacent downstream purification chambers.

[0014] Furthermore, the sealing liquid storage chamber and the final reagent storage chamber are arranged in a fan-shaped manner on the same radius; the downstream module is arranged farther away from the rotation center than the lysis chamber; the outer contours of the sealing liquid storage chamber, the final reagent storage chamber, the nucleic acid purification module and the downstream module are arranged in a fan-shaped manner on the microfluidic chip.

[0015] Furthermore, the lysis chamber and the purification chamber are fan-shaped; the working chambers of the lysis chamber and the purification chamber are along the direction of the nucleic acid lysis and purification process, and the volume of the downstream working chamber is not greater than the volume of the upstream working chamber.

[0016] Furthermore, the downstream module 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 a sixth valve; at least one nucleic acid distribution chamber is connected through a distribution channel, the upstream of the distribution channel is connected to the distal end of the final purification chamber through a third valve, and the proximal end of the distribution channel is provided with a return air passage, and the return air passage is connected to the proximal end of the sealing liquid storage chamber.

[0017] Compared with the prior art, the beneficial technical effects of the present invention include:

[0018] 1. The present invention provides a sealing liquid storage chamber in which sealing liquid is stored. When the lysis chamber and the purification chamber are filled with corresponding reagents, the sealing liquid seals each chamber to prevent reagents in different chambers and nucleic acid adsorption devices from running around and causing contamination during circulation in different chambers.

[0019] 2. Along the direction of the nucleic acid extraction process, each chamber is arranged in sequence toward the rotation center. The above arrangement has the following effects: the farther away from the rotation center, the greater the centrifugal force, and the more significant the separation effect of the sealing liquid and the reagent or nucleic acid extract, including a good separation effect at the interface between the nucleic acid extract and the sealing liquid, thereby avoiding the mixing of part of the nucleic acid extract into the sealing liquid during the nucleic acid extraction process, resulting in cross-contamination of adjacent chambers. On the other hand, after the nucleic acid in the lysis chamber is lysed, it is transferred from the upstream chamber to the downstream chamber, for example, from the lysis chamber to the primary purification chamber, and moves toward the rotation center. The sealing liquid itself has the ability to repel reagents. The superposition of centrifugal force during the transfer process further reduces the problem of cross-contamination between adjacent chambers due to nucleic acid transfer.

[0020] 3. Since the nucleic acid in the chamber far away from the rotation center contains more impurities, the repulsive effect of the sealing liquid plus the large centrifugal force removes a large amount of impurities in the nucleic acid, reducing the burden of subsequent cleaning. Therefore, the downstream chamber space can be set smaller, while ensuring the nucleic acid purification effect, the size of the cavity is reduced to achieve miniaturization; at the same time, the above effect leads to a small size of the final purification chamber, which is equivalent to concentrating the nucleic acid and improving the accuracy of subsequent detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 A top view of a fully integrated microfluidic chip provided by an embodiment of the present invention;

[0023] Figure 2 A schematic diagram of another first valve structure provided by an embodiment of the present invention;

[0024] Figure 3 for Figure 1 A partial enlarged view of the

[0025] Figure 4 A bottom view of a fully integrated microfluidic chip provided by an embodiment of the present invention;

[0026] Figure 5 A schematic diagram of two stacked layers of a fully integrated microfluidic chip provided by an embodiment of the present invention;

[0027] Figure 6 Schematic diagram of different stages of the microfluidic chip provided in an embodiment of the present invention, a is the addition of the sample to be tested, reagents and isolation liquid, b is the sample release and liquid reagent dissolution preparation, c is the sealed mineral oil release filling, dg are nucleic acid extraction and purification, h is the distribution of the purified nucleic acid sample, and i is the nucleic acid sample entering the amplification reaction chamber to start amplification and signal detection.

[0028] Figure markings: 01, first valve; 010, first channel; 011, second channel; 02, second valve; 03, third valve; 04, fourth valve; 05, fifth valve; 06, sixth valve; 10, lysis chamber; 100, sample chamber; 1000, sample addition port; 110, purification chamber; 1100, working chamber; 1101, sealing chamber; 12, sealing liquid storage chamber; 13, downstream module; 130, nucleic acid distribution chamber; 131, amplification reaction chamber; 132, distribution channel; 14, reagent storage chamber; 15, return air passage. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 the internal communication of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0034] In the embodiment of the present invention, the "distal end" and the "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.

[0035] The embodiment of the present invention provides a fully integrated microfluidic chip, such as Figure 1As shown, it includes: a lysis chamber 10; a nucleic acid purification module, which is arranged on the side close to the rotation center O compared to the lysis chamber 10, and the nucleic acid purification module has at least one purification chamber 110, and the purification chamber 110 includes a working chamber 1100 and a sealing chamber 1101, the distal side of the sealing chamber 1101 of the purification chamber 110 is connected with the proximal side of the working chamber 1100, and the proximal end of the lysis chamber 10 is connected with the sealing chamber 1101 of the primary purification chamber 110 through a first valve 01; a sealing liquid storage chamber 12, the distal end of the sealing liquid storage chamber 12 is connected with the sealing chamber 1101 of the final purification chamber 110 through a second valve 02; a downstream module 13, and the downstream module 13 is connected with the distal end of the final purification chamber 110 through a third valve 03.

[0036] Figure 1 Three purification chambers 110 are shown. Based on the needs of the nucleic acid purification process, the purification chamber 110 can be set to one or more. The purification chamber 110 includes a working chamber 1100 and a sealing chamber 1101. During operation, the working chamber is filled with reagents for cleaning and eluting nucleic acid cleavage products. The sealing chamber is filled with a sealing liquid. The sealing liquid is used to seal the reagents in the working chamber to prevent cross-contamination between different reagents in adjacent purification chambers and between the nucleic acid purification module and the cleavage chamber. It can be understood that the reagents do not have to completely fill the working chamber, and the remaining cavities can be filled with sealing liquid. Secondly, the corresponding reagents or nucleic acids can be injected into the cleavage chamber 10 and the purification chamber 110 in advance. Under the action of centrifugal force or opening the second valve 02, the sealing liquid in the sealing liquid storage chamber 12 passes through the second valve 02, the fourth valve 04 and the first valve 01 in turn to seal the working liquid in each chamber. Figure 1 The lysis chamber 10 is not provided with a sealed chamber, but a sealed chamber can be provided based on actual conditions. Since the lysis chamber 10 belongs to the first process and the direction of movement of the nucleic acid proposed in this application is toward the rotation center, the lysis chamber does not need to be provided with a sealed chamber to prevent cross contamination between adjacent chambers, thereby reducing the difficulty of microfluidic chip processing and improving production efficiency.

[0037] It should be pointed out that for the addition of nucleic acid samples, the nucleic acid samples or reagents can be added to the corresponding lysis chamber or purification chamber in advance, or an independent reagent storage chamber or sample chamber can be set up. In the embodiment of the present invention, an independent sample chamber and a lysis chamber connected thereto are set up to lyse the nucleic acid sample, and then the nucleic acid is subjected to processes such as impurity removal, cleaning and elution in the purification 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 the nucleic acid can be adsorbed and the nucleic acid can be transferred between different chambers under external drive. The "sealing liquid" is a hydrophobic liquid with a density less than the density 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 components of the specific sealing liquid are not limited here.

[0038] The "primary purification chamber" and "final purification chamber" in the present invention refer to the purification chambers at the first and last positions in the purification process. When there is only one purification chamber, the primary purification chamber and the final purification chamber are the same purification chamber. The sealing chamber and the working chamber are substantially one cavity. The sealing chamber is used to store the sealing liquid, and the working chamber is used to receive the reagent. Preferably, before the sealing liquid is filled, the lysis chamber and the purification chamber are first filled with nucleic acid samples and reagents, and the sealing liquid is filled after the filling is completed. In order to avoid the sealing liquid from being violently stirred with the reagent or nucleic acid sample during the filling process to cause emulsification, the depth of the sealing chamber (i.e., perpendicular to the paper surface) is less than the depth of the working chamber. Preferably, the width of the sealing chamber 1101 is at least twice its depth, and the depth of the working chamber is at least three times the depth of the sealing chamber. The depth of the working chamber is less than 10 mm, and the depth of the sealing chamber 1101 is 0.1-3 mm.

[0039] The present invention provides a sealing liquid storage chamber 12, in which sealing liquid is stored. When the lysis chamber and the purification chamber are filled with corresponding reagents, the sealing liquid seals each chamber to prevent reagents in different chambers and samples from running around and causing contamination during the circulation of samples in different chambers. Secondly, along the direction of the nucleic acid extraction process, each chamber is arranged toward the rotation center in sequence. The above arrangement has the following effects: the farther away from the rotation center, the greater the centrifugal force, and the more significant the separation effect of the sealing liquid and the reagent or nucleic acid extract, including a good separation effect at the interface between the nucleic acid extract and the sealing liquid, which prevents part of the nucleic acid extract from mixing with the sealing liquid during the nucleic acid extraction process and causing cross-contamination of adjacent chambers. On the other hand, after the nucleic acid in the lysis chamber is lysed, it is transferred from the upstream chamber to the downstream chamber, for example, from the lysis chamber to the primary purification chamber, and moves toward the rotation center. The sealing liquid itself has the ability to repel reagents. The superposition of centrifugal force during the transportation process further reduces the problem of cross-contamination between adjacent chambers due to nucleic acid transfer. Thirdly, since the nucleic acid in the chamber far away from the rotation center contains more impurities, the repulsive effect of the sealing liquid plus the large centrifugal force causes a large amount of impurities in the nucleic acid to be removed, reducing the burden of subsequent cleaning. Therefore, the downstream chamber space can be set smaller, while ensuring the nucleic acid purification effect, the size of the cavity is reduced to achieve miniaturization; at the same time, the above effect leads to a small size of the final purification chamber, which is equivalent to concentrating the nucleic acid and improving the accuracy of subsequent detection.

[0040] When there are two or more purification chambers 110, the purification chambers 110 are sequentially arranged close to the rotation center O along the purification direction; adjacent purification chambers 110 are connected through the fourth valve 04; the first valve 01 and the fourth valve 04 connecting the lysis chamber 10 and each purification chamber 110 are respectively connected to the two sides of the same sealed chamber 1101. By connecting the first valve 01 and the fourth valve 04 to the two sides of the same sealed chamber 1101, the cleaning intensity for nucleic acid can be improved. After the nucleic acid is cleaned in one of the chambers, it needs to be transferred from one side to the other side to achieve the transfer of nucleic acid, which increases the running path of nucleic acid and improves the cleaning intensity. In particular, in the working chamber 1100 of the purification chamber 110, some reagents may not participate in the cleaning or elution of nucleic acid due to the "dead zone" of the fluid. By extending the running track of nucleic acid in the working chamber 1100, the formation of dead zone is avoided and the mixing degree of nucleic acid and reagent is improved.

[0041] Figure 1 A connection method of the first valve 01 and the fourth valve 04 is given: the proximal end of the upstream cavity is connected to the sealed cavity adjacent to the same side through the fourth valve or the first valve. This method reduces the difficulty of chip processing, but due to the short path of the nucleic acid, the cleaning effect of impurities contained in the nucleic acid is limited. Another preferred embodiment is: the proximal end of the upstream cavity is connected to the sealed cavity adjacent to different sides through the fourth valve or the first valve. Taking the first valve 01 as an example, Figure 2As shown, the lysis chamber 10 and the purification chamber 110 are connected between adjacent chambers through the first valve 01, and the proximal end of the lysis chamber 10 is connected to the adjacent sealed chambers on different sides through the first valve 01. This embodiment can increase the flow path of the nucleic acid and improve the centrifugal force and the sealing liquid's removal effect on reagents and impurities.

[0042] Specifically, Figure 1 As shown, it also includes a reagent storage chamber 14, which is connected to the proximal end of the corresponding purification chamber 110 through a fifth valve 05. Other reagent storage chambers are not shown in the figure. By setting up an independent reagent storage chamber 14, not only the system integration is improved, but also the miniaturization of the product is facilitated, and the problem of difficulty in packaging when storing reagents in the purification chamber and increasing the labor intensity of workers by manually adding reagents is solved.

[0043] Specifically, Figure 3 As shown, the first valve 01 and the fourth valve 04 are composed of capillary channels. Taking the first valve 01 as an example, the capillary channel includes a first channel 010 extending radially along the microfluidic chip and a second channel 011 along the axis of the microfluidic chip. The width L2 of the second channel 011 is smaller than the width L1 of the first channel 010. The first channel 010 is connected to the proximal end of the lysis chamber 10 or the purification chamber 110, and the second channel 011 is connected to the sealed chamber 1101 of the purification chamber 110. The first valve 01 and the fourth valve 04 in the present application are channels with a rectangular cross-sectional shape. The capillary channel allows magnetic beads with nucleic acids to pass through. The effect achieved by the capillary channel of this shape is that there is enough sealing liquid in the first channel 010 to discharge the reagents mixed in the nucleic acids. On the other hand, the width of the first channel 010 is larger than that of the second channel 011, which prevents extremely small reagent droplets from escaping due to the small centrifugal force, thereby increasing the probability of flowing into the downstream chamber. Setting the width of the second channel 011 smaller further reduces the possibility of very small droplets of reagent entering the downstream chamber. Preferably, the width L1 of the first channel 010 is 1.5-2.5 times the width L2 of the second channel.

[0044] Specifically, Figure 3, the width L2 of the second channel 011 is smaller than the width L3 of the sealing cavity 1101, and the connection position between the second channel 011 and the sealing cavity 1101 is located at the upper part. The direction of travel of the nucleic acid is toward the side of the rotation center, and the sealing liquid fills the purification cavity and / or the lysis cavity under the action of centrifugal force, and the liquid flows from the proximal end to the distal end. As the radius increases, the centrifugal force on the sealing liquid increases, and a suction effect is generated, so the second channel 011 is connected to the upper part of the sealing cavity 1101 to prevent the downstream reagent from entering the upstream cavity under the influence of the sealing liquid; secondly, the first valve or fourth valve structure proposed in the present application can play a buffering role for the sealing liquid. First, the second channel 011 with a smaller width has a flow-blocking effect on the sealing liquid, and after the sealing liquid in the second channel enters the first channel, the impulse of the liquid is reduced due to the increase in size, which avoids the degree of stirring between the sealing liquid and the reagent or lysis liquid, and avoids emulsification. Preferably, the width L2 of the second channel 011 is 0.2-0.8 times the width L3 of the sealed cavity 1101 .

[0045] Specifically, Figure 1 and Figure 4 As shown, the microfluidic chip is arranged in two layers; the lysis chamber 10, nucleic acid purification module, downstream module 13, sealing liquid storage chamber 12 and final reagent storage chamber 14 are arranged in one layer; the sample chamber 100 and other reagent storage chambers 14 are arranged in another layer. The above arrangement can reduce the chip size and realize chip miniaturization. Figure 1 As shown, a sample adding port 1000 is also provided on the sample chamber.

[0046] like Figure 5 As shown, the dotted line indicates the arrangement of the cavity in another layer. It can be seen that the sample cavity 100 is located at the corresponding position of the primary purification cavity 110; except for the final reagent storage cavity 110, the other reagent storage cavities 14 are respectively located at the corresponding positions of the adjacent downstream purification cavity 110. Through the above-mentioned structural arrangement, when preparing the microfluidic chip, it is easy to form and not easy to deform, avoiding the problems of torsion deformation of the prepared product due to thermal stress; in addition, compared with the corresponding purification cavity 110, the other reagent storage cavity 14 is closer to the center of the circle, which is easy to release all the reagents in the other reagent storage cavity 14, thereby improving the stability of the system.

[0047] Specifically, Figure 1 or Figure 5 As shown, the sealing liquid storage chamber 12 and the final reagent storage chamber 14 are arranged in a fan-shaped manner on the same radius; the downstream module 13 is arranged farther away from the rotation center O than the lysis chamber 10; the outer contours of the sealing liquid storage chamber 12, the final reagent storage chamber 14, the nucleic acid purification module and the downstream module 13 are arranged in a fan-shaped manner on the microfluidic chip.

[0048] Specifically, Figure 5 As shown, the lysis chamber 10 and the purification chamber 110 are fan-shaped; the working chambers of the lysis chamber 10 and the purification chamber 110 are along the direction of the nucleic acid lysis and purification process, and the volume of the downstream working chamber is not greater than the volume of the upstream working chamber. In another embodiment, the volumes of the working chambers of the lysis chamber 10 and the purification chamber 110 decrease successively along the direction of the nucleic acid lysis and purification process. In the technical solution provided by the present invention, the volume ratio of the lysis chamber 10 to the final purification chamber can even be as low as 20:1, that is, the nucleic acid collected in 20 volumes of the nucleic acid lysis chamber can be concentrated into 1 volume of solution. Preferably, the lysis chamber 10 and the purification chamber 110 have the same depth, and the closer to the rotation center O, the shorter the arc length of the purification chamber 110, and the fan ring widths of the lysis chamber 10 and the purification chamber 110 are the same. In the present application, as the lysis and purification process flow, the corresponding chambers are arranged in sequence toward the center. At this time, there is a problem. The closer to the rotation center, the smaller the chamber space that can be set, the smaller the amount of reagent filled, and the weakened ability to purify nucleic acids. Methods for solving the above problems include: setting the fan angle α to a larger angle to increase the volume of the purification chamber and improve the cleaning ability of nucleic acids, or increasing the fan ring width or depth of the chamber closer to the rotation center. In the present application, the above-mentioned purification process is used, and the direction of the nucleic acid cleavage and purification process is toward the rotation center. In the early stage, the impurities are strongly removed by combining the larger centrifugal force with the sealing liquid of the present application. As the nucleic acid cleavage and purification process proceeds, although the impurity removal ability gradually weakens, it still meets the requirements for cleaning and eluting the nucleic acid in a smaller purification chamber. Under the existing cavity depth, the angle α of the technical solution proposed in the present application is less than 45°, thereby realizing the miniaturization of the chip; and the elution of nucleic acids in the final purification chamber is equivalent to concentrating the nucleic acid sample, thereby improving the accuracy of subsequent detection.

[0049] Specifically, Figure 5 As shown, the downstream module 13 includes: at least one nucleic acid distribution chamber 130 and a matching amplification reaction chamber 131, the distal end of the nucleic acid distribution chamber 130 is connected to the proximal end of the amplification reaction chamber 131 through the sixth valve 06; at least one nucleic acid distribution chamber 130 is connected through a distribution channel 132, the upstream of the distribution channel 132 is connected to the distal end of the final purification chamber 110 through the third valve 03, and the proximal end of the distribution channel 132 is provided with a return air passage 15, and the return air passage 15 is connected to the proximal end of the sealing liquid storage chamber 12. The return air passage facilitates the smooth filling of the downstream module.

[0050] It should be pointed out that in the embodiment of the present invention, the sealing liquid storage chamber 12 is set to be relatively large and the return air passage 15 is connected to the proximal end of the sealing liquid storage chamber 12. On the one hand, the sealing liquid storage chamber is filled with more sealing liquid, which is convenient for filling the lysis chamber 10 and the purification chamber 110. After the filling is completed, some sealing liquid is allowed to remain in the sealing liquid storage chamber 12, which reduces the quantitative accuracy requirements of the sealing liquid during the preparation of the microfluidic chip. After the lysis and purification of the nucleic acid is completed, the excess sealing liquid flows into the distribution channel through the third valve 03 to seal the distributed nucleic acid. Avoid cross-contamination of nucleic acids in different reaction pools during the reaction process.

[0051] The first valve 01, the second valve 02, the third valve 03, the fourth valve 04, the fifth valve 05 and the sixth valve 06 in the present application all use capillary channels; the cross-sectional size of the capillary channels ranges from hundreds of micrometers to millimeters, and the sizes of the capillary channels at different layers are limited to ensure smooth preparation. Preferably, the third valve uses a phase change valve or a mechanical valve. The present invention belongs to a fully 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.

[0052] In order to better understand the design ideas and operating principles of this technical solution, the specific implementation methods and operating procedures for realizing a fully integrated microfluidic chip are described as follows:

[0053] (1) Add the sample to be tested, reagents and sealing liquid. Figure 6 As shown in a, the sample to be tested, buffer solution (dark gray filled part) and sealed mineral oil (light gray filled part in the figure) are added to the sample chamber, sealing liquid storage chamber and reagent storage 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). Since some reagent storage chambers and sample chambers are located on the back of the microfluidic chip, they are not displayed.

[0054] (2) Sample release and liquid reagent dissolution preparation. Figure 6As shown in b, a centrifugal speed V1 is applied to the fully integrated microfluidic chip, and the sample is released from the buffer chamber into the lysis chamber to dissolve and disperse the dry magnetic beads pre-placed therein for nucleic acid adsorption. At the same time, the buffer solution is released into the nucleic acid purification chamber to complete the dissolution and preparation of the dry reagents pre-placed in the latter for nucleic acid cleaning and elution.

[0055] (4) Seal mineral oil release filling. Figure 6 As shown in c, a centrifugal speed V2 is applied to the fully integrated microfluidic chip, and the sealed mineral oil is released from the sealing liquid storage chamber to completely fill the sealing cavity in the annular purification cavity, thereby achieving isolation and sealing of the nucleic acid sample lysis cavity and the nucleic acid purification cavity.

[0056] (5) Nucleic acid extraction and purification. Figure 6 As shown in d-6g, the fully integrated microfluidic chip is controlled to cooperate with the external magnet, so that the magnetic beads adsorbed with nucleic acids in the sample lysis chamber are transferred into the nucleic acid purification 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 purification chamber for storage.

[0057] (6) Distribution of purified nucleic acid samples. Figure 6 As shown in h, a centrifugal speed V3 is applied to the fully integrated microfluidic chip, so that the purified nucleic acid sample in the final nucleic acid purification chamber fills each nucleic acid distribution chamber in turn along the distribution channel, and the sealing mineral oil then completes the sealing of the proximal inlet of each nucleic acid distribution chamber.

[0058] (7) The nucleic acid sample enters the amplification reaction chamber to start amplification and signal detection. Figure 6 As shown in Fig. 1, a centrifugal speed V4 is applied to the fully integrated microfluidic chip, so that the sample nucleic acid in each nucleic acid distribution cavity enters the amplification reaction cavity corresponding to the respective distal end, and at the same time, the sealing mineral oil in the upstream first sealing cavity and the second sealing cavity is also filled down, and the sealing 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.

[0059] The above description is only an embodiment of the present application and is not intended to limit the protection scope 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 protection scope of the present application.

Claims

1. A fully integrated microfluidic chip, characterized in that: include: Lysis chamber; A nucleic acid purification module, wherein the nucleic acid purification module is arranged on a side closer to the rotation center than the lysis chamber, and the nucleic acid purification module has at least one purification chamber, wherein the purification chamber comprises a working chamber and a sealing chamber, wherein the distal side of the sealing chamber of the purification chamber is communicated with the proximal side of the working chamber, and the proximal end of the lysis chamber is communicated with the sealing chamber of the primary purification chamber through a first valve, and during the nucleic acid extraction and purification process, the nucleic acid moves from the lysis chamber to the nucleic acid purification module; A sealing liquid storage chamber, wherein the distal end of the sealing liquid storage chamber is connected to the sealing chamber of the final purification chamber through a second valve, and under the action of centrifugal force, the sealing liquid in the sealing liquid storage chamber fills the sealing chamber, wherein the sealing liquid is a hydrophobic liquid, has a density less than that of the liquid in the first working chamber, is inert, and will not react with substances in the system; A downstream module, wherein the downstream module is connected to the distal end of the final purification chamber through a third valve; The first valve is composed of a capillary channel, the capillary channel includes a first channel extending radially along the microfluidic chip and a second channel along the axis of the microfluidic chip, the width of the second channel is smaller than the first channel; The first channel is connected to the proximal end of the lysis chamber, and the second channel is communicated with the sealing cavity of the purification chamber.

2. The fully integrated microfluidic chip according to claim 1, characterized in that: When there are two or more purification chambers, the purification chambers are sequentially arranged close to the rotation center along the purification direction; adjacent purification chambers are connected through a fourth valve; The first valve and the fourth valve connecting the lysis chamber and each purification chamber are respectively connected to two sides of the same sealed chamber.

3. The fully integrated microfluidic chip according to claim 1, characterized in that: The device also comprises a reagent chamber, which is communicated with the proximal end of the corresponding purification chamber through a fifth valve.

4. The fully integrated microfluidic chip according to claim 2, characterized in that: The fourth valve is composed of a capillary channel, which includes a first channel extending radially along the microfluidic chip and a second channel along the axis of the microfluidic chip, wherein the width of the second channel is smaller than that of the first channel; the first channel is connected to the sealed cavity of the upstream purification cavity, and the second channel is connected to the sealed cavity of the downstream purification cavity.

5. The fully integrated microfluidic chip according to claim 4, characterized in that: The width of the second channel is smaller than the width of the sealed cavity, and the connection position between the second channel and the sealed cavity is located at an upper position.

6. The fully integrated microfluidic chip according to claim 1, characterized in that: Also included is a sample chamber; The microfluidic chip is configured as two layers; The lysis chamber, nucleic acid purification module, downstream module, sealing liquid storage chamber and final reagent storage chamber are arranged in one layer; The sample chamber and other reagent storage chambers are arranged on another layer.

7. The fully integrated microfluidic chip according to claim 6, characterized in that: The sample chamber is located at a corresponding position of the primary purification chamber; Except for the final reagent storage chamber, other reagent storage chambers are located at corresponding positions of the downstream purification chamber.

8. The fully integrated microfluidic chip according to claim 1, characterized in that: The sealing liquid storage chamber and the final reagent storage chamber are arranged in a fan-shaped ring shape on the same radius; The downstream module is arranged farther from the rotation center than the lysis chamber; The outer contours of the sealing liquid storage chamber, the final reagent storage chamber, the nucleic acid purification module and the downstream module are arranged in a fan shape on the microfluidic chip.

9. The fully integrated microfluidic chip according to claim 1, characterized in that: The lysis chamber and purification chamber are fan-shaped; The working chambers of the lysis chamber and the purification chamber are along the direction of the nucleic acid lysis and purification process, and the volume of the downstream working chamber is not greater than the volume of the upstream working chamber.

10. The fully integrated microfluidic chip according to claim 1, characterized in that: The downstream module comprises: at least one nucleic acid distribution chamber and an amplification reaction chamber matched therewith, wherein the distal end of the nucleic acid distribution chamber is connected to the proximal end of the amplification reaction chamber via a sixth valve; At least one nucleic acid distribution chamber is connected through a distribution channel, the upstream of the distribution channel is connected to the distal end of the final purification chamber through a third valve, and the proximal end of the distribution channel is provided with a return air passage, which is connected to the proximal end of the sealing liquid storage chamber.

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