A microfluidic chip for nucleic acid extraction and purification

Abstract

The present invention provides a microfluidic chip for nucleic acid extraction and purification, belonging to the field of microfluidic technology, including: a storage part, the storage part includes a sample chamber, a reagent chamber and an isolation liquid chamber; a nucleic acid extraction and purification part, the nucleic acid extraction and purification part is arranged on the side away from the rotation center compared to the storage part, the nucleic acid extraction and purification part includes at least two working chambers and a sealing chamber arranged on the side of the working chamber close to the rotation center, the different working chambers are connected by a first valve body arranged in the sealing chamber, and the distal end of the sample chamber, the reagent chamber and the isolation liquid chamber are connected to the proximal end of the sealing chamber by a second valve body. Centrifugal force and Euler force are relied on to achieve complete filling and release of liquid reagents and mixing with magnetic beads, and mineral oil is combined with a unique chamber structure and a segmented pressure-blocking valve structure design to ensure that the liquid components between chambers are isolated from each other, ensuring a good nucleic acid extraction effect under the premise of high space utilization.

Description

Technical Field

[0001] The present application relates to the field of microfluidics technology, and in particular to a microfluidics chip for nucleic acid extraction and purification. Background Art

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

[0003] At present, the most commonly used methods for nucleic acid extraction and purification can be divided into three categories: organic chemical reagent extraction, centrifugal column adsorption and magnetic bead adsorption. Organic chemical reagent extraction generally requires the use of a large amount of hazardous chemicals (some containing chlorine), and manual operation is time-consuming and labor-intensive, with low efficiency. Centrifugal column adsorption also faces the problem of low sample processing throughput, and the adsorption column is easily clogged, affecting the entire extraction process. Compared with the other two methods, magnetic bead adsorption has higher work efficiency and repeatability, is more compatible with samples, and is easier to achieve scale and automation of nucleic acid extraction and purification processes. Most of the fully automatic nucleic acid extraction instruments or platforms currently on the market use magnetic bead adsorption.

[0004] However, the current automation strategies in the field of nucleic acid extraction and purification still face prominent problems, such as the need to use complex auxiliary devices or components, and to rely on bulky and inefficient robotic arms or sophisticated and expensive pipetting equipment. Microfluidic chips manipulate tiny volumes of fluid (from a few microliters to a few milliliters) on the surface of solid substrates, and ultimately rely on the operation of fluids to achieve certain functions or purposes. Due to its advantages of miniaturization, integration, and high automation, it also has great application prospects in the field of nucleic acid extraction and purification. At present, there are some public technical solutions using traditional air pressure-driven microfluidic chips as carriers for automated nucleic acid extraction and purification. For example, Chinese invention patents CN113817577 A, CN 112322453 A and CN 108300640 A each disclose a microfluidic chip structure for extracting nucleic acids based on magnetic bead adsorption. The working principle is to use air pressure changes as a driving force to achieve mixing and transfer of magnetic beads in various liquid reagents. However, a major limitation of this type of solution is that it requires external relatively stable gas pressure. Too high or too low pressure, too fast or too slow pressure application speed can easily lead to uncontrolled microfluidics. Therefore, it often also requires additional syringe pumps or air pressure pumps with a certain degree of precision, which may make the entire system too complicated. Automated nucleic acid extraction and purification solutions based on the new generation of digital microfluidics (DMF) technology have also been reported, such as patent CN 115820399. A discloses an integrated nucleic acid analysis system based on digital microfluidics (DMF), which includes a nucleic acid extraction and purification method using dielectric wetting effect as a driving force, and can manipulate smaller samples for efficient transfer and mixing. This solution does not have the dead volume problem of fluid that traditional microfluidics may face, which can reduce the cost of samples and reagents and meet shorter time requirements. However, its disadvantage is that it requires a more precise voltage control system and logic circuit, and the corresponding manufacturing cost is relatively high. It is also limited by the volume of sample processing and the overall volume of digital microfluidic equipment; centrifugal disc microfluidic chips have unique advantages in space utilization and driving mode, and they also have relevant applications in the field of nucleic acid extraction and purification, such as patent CN 217600742 U, patent CN 115926928A and patent CN 109609345 A each disclosed a nucleic acid extraction chip design, which cleverly uses the relative movement between magnetic beads and various liquid reagents or samples to adsorb, wash and elute nucleic acid samples. Since it does not involve the active transfer of liquid, the design and implementation of such schemes are relatively simple and efficient. However, these schemes currently also have some risks and shortcomings. For example, in order to prevent the liquids between chambers from cross-talking with each other, complex barrier valves need to be used between the chambers, and the magnetic beads are not mixed well in the liquids in each chamber because the liquids in each chamber are not disturbed. Summary of the invention

[0005] The purpose of the present invention is to provide a microfluidic chip for nucleic acid extraction and purification. The microfluidic chip mainly relies on centrifugal force and Euler force to achieve complete filling and release of liquid reagents and sufficient mixing with magnetic beads, and mineral oil is combined with a unique chamber structure and a segmented pressure-blocking valve structure design to ensure that the liquid components between chambers are isolated from each other. While simplifying the design, reducing costs, and facilitating operation, the stability and reliability of the nucleic acid extraction process are greatly enhanced, and a good nucleic acid extraction effect is guaranteed under the premise of extremely high space utilization.

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

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

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

[0009] Furthermore, the sealing chamber is in the shape of a spiral ring belt or a circular ring belt, the working chamber is arranged on the side of the outer edge of the sealing chamber away from the rotation center, and based on the flow of the extraction and purification process, the working chambers of the corresponding processes are arranged in sequence along the sealing chamber, and the isolation liquid chamber is connected to the proximal end of the sealing chamber through a second valve body.

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

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

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

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

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

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

[0016] ;

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

[0018] Further, the isolation liquid chamber is annularly arranged at the proximal end of the microfluidic chip; the sample chamber and at least one reagent chamber are fan-shaped and arranged around the outside of the isolation liquid chamber; at least two working chambers are fan-shaped and arranged on the outer edge of the sealed chamber and around the rotation center, and the reagent chamber and the sample chamber are respectively connected to the proximal end of the sealed chamber corresponding to the working chamber through a second valve;

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

[0020] 1. Various cavities are arranged in a circular ring with the center of the disc base as the center, and the magnetic beads are transferred by the relative circular motion between the disc and the fixed magnet when the disc rotates, so that the magnetic bead transfer operation is more stable, automated and efficient, and the fluid operation and magnetic bead transfer are more reliable under the drive of stable centrifugal force. The present invention proposes to set all chambers of the nucleic acid extraction process in the same circumferential direction, and the magnetic beads can be transferred in each chamber only by rotating the chip, thereby realizing the nucleic acid purification process, with a simple structure and easy automation.

[0021] 2. The magnetic beads are transferred sequentially in a specific liquid cavity to achieve the adsorption, cleaning and elution of nucleic acids. The waste liquid reagents after nucleic acid extraction and purification are stored in situ, without the need for an additional waste liquid pool design. The overall space utilization rate of the chip is high. Secondly, the design of a waste liquid pool is avoided, and the chip is miniaturized.

[0022] 3. The use of mineral oil as the isolation and sealing medium between each sample processing chamber has no effect on the process of nucleic acid extraction and purification. At the same time, the transfer of magnetic beads in mineral oil can repel and remove the residual liquid in the previous chamber carried by the magnetic beads, making the effect of magnetic bead washing and nucleic acid elution better. The present invention creatively proposes a structural design of the sealing chamber. By designing the sealing chamber at the proximal end of the working chamber, the area in the circumferential direction is saved and miniaturization is achieved. At the same time, through the height difference design between the sealing chamber and the working chamber, and in combination with the surface tension of the medium, the residual liquid can be further removed, thereby improving the purity of nucleic acid extraction.

[0023] 4. The sealed chamber is in the shape of a flat spiral ring at the proximal end relative to each sample processing chamber, and a plurality of segmented choke valves are designed inside it, which prevents the liquid reagents in each sample processing chamber from mixing with the mineral oil in the mineral oil sealed chamber during the magnetic bead mixing operation, thereby preventing cross-liquid contamination, and enhancing the reliability of magnetic bead nucleic acid extraction and purification. The working chambers proposed in the present invention are independently arranged at the distal end, the sealed chamber is arranged at the proximal end, and the arrangement structure that connects multiple working chambers is realized through the connection of the sealed chambers, which can prevent the Euler force generated by the chip during the rotation process from causing mutual cross-liquid between liquids, and can also realize the transfer of the nucleic acid extraction device between each working chamber; in addition, the present invention further reduces the possibility of cross-liquid cross-liquid by designing segmented choke valves in the sealed chamber, thereby improving the stability of the system.

[0024] 5. The bubbles generated by the bubble generating chamber provide cavity space for the liquid reagents in each sample processing chamber. The Euler force generated by the forward and reverse rotation of the chip can be further used to fully mix the liquid reagents and magnetic beads in each sample processing chamber, and good nucleic acid extraction and purification effects can be guaranteed without the need for additional mixing and stirring components. The present invention creatively proposes a method for mixing reagents in a cavity without a stirrer. In order to prevent the liquids from interpenetrating between multiple working chambers, the present invention fills the sealed chamber by filling with mineral oil, but at the same time brings another problem. When the liquid completely fills the cavity, the mixing of the reagents becomes difficult. The present invention creatively proposes a bubble generator. Under the action of centrifugation, the volume in the bubble generator is compressed and stored. When the centrifugal force stops, the gas in the bubble generator will be quickly discharged to form bubbles in the reaction chamber. When mixing is required, by changing the chip speed or rotation direction, the bubbles will be stirred in the cavity under the Euler force to achieve mixing of the liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

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

[0027] Figure 2 for Figure 1 AA view in;

[0028] Figure 3 for Figure 1 BB view in;

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

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

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

[0032] Figure 7 A structural diagram of another microfluidic chip for nucleic acid extraction and purification provided by an embodiment of the present invention;

[0033] Figure 8Schematic diagram of different stages of nucleic acid detection provided in an embodiment of the present invention, schematic diagram of different stages of nucleic acid detection provided in an embodiment of the present invention, a is related reagent presetting and sample injection, b is reagent centrifugal distribution and mineral oil sealing, c is bubble cavity generation, d is liquid reagent and magnetic beads fully mixed in the working chamber for sample lysis and nucleic acid adsorption, e is magnetic bead collection and transfer in the working chamber for sample lysis and nucleic acid adsorption, f is magnetic beads entering the mineral oil sealing chamber at the proximal end of the working chamber for magnetic bead washing, g is liquid reagent and magnetic beads fully mixed in the working chamber for magnetic bead washing, h is liquid reagent and magnetic beads fully mixed in the working chamber for secondary magnetic bead washing, i is liquid reagent and magnetic beads fully mixed in the working chamber for nucleic acid elution, and j is nucleic acid extraction and purification completed.

[0034] Figure numerals: 01, first valve body; 010, first slit; 011, inner wall of slit; 012, non-blocking area; 02, second valve body; 03, third valve body; 1, storage unit; 10, sample chamber; 11, reagent chamber; 12, isolation liquid chamber; 2, nucleic acid extraction and purification unit; 20, sealing chamber; 200, flow blocking dam; 201, second slit; 21, working chamber; 22, bubble generating chamber. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

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

[0042] from Figure 1It can be seen from the figure that four working chambers 21 and a sealed chamber 20 are provided in the embodiment of the present invention. The liquid in the sample chamber 10 enters the rightmost working chamber through the second valve body 02. The working chamber is used for sample lysis and nucleic acid adsorption. Then the counterclockwise working chambers are used for the first washing, the second washing and nucleic acid elution. Three reagent chambers are provided in the embodiment of the present invention for storing reagents with different functions, and are respectively connected to the proximal end of the sealed chamber 20 corresponding to the corresponding working chamber 21 through three second valve bodies 02. However, it should be pointed out that one reagent chamber 11 can be provided to realize any function of the above three reagent chambers 11, or one reagent chamber 11 can be provided. By providing the same number of quantitative chambers as the working chamber 21, the quantitative reagent is filled into the corresponding working chamber 21. The corresponding freeze-dried reagent is pre-set in the working chamber 21 in advance. The freeze-dried reagent and the reagent are dissolved to form a reagent solution with the corresponding function.

[0043] The present invention is provided with multiple valves, such as a first valve body, a second valve body, and a third valve body (which will be mentioned later). The form and combination of the valves are not limited, as long as they can be opened and closed in the required steps. They can be active valves or passive valves. Specifically, manually operated mechanical valves and phase change valves in the prior art are used, and capillaries that are opened or closed under centrifugal action can also be used. In order to reduce production costs while maintaining detection accuracy, it is preferred to use capillaries for control. The size of the capillaries is from hundreds of microns to microns, and different diameters and lengths can achieve the effect of opening or closing under different centrifugal forces.

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

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

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

[0047] like Figure 2As shown, the width L of the sealed cavity 20 is not less than the height H of the sealed cavity 20, and the height H1 of the working cavity 21 is not less than the height H of the sealed cavity. Since the isolation liquid in the isolation liquid cavity 12 isolates and seals the multiple working cavities 21, the cross-section of the sealed cavity is set to be smaller to reduce the amount of isolation liquid added; secondly, in the process of adding the isolation liquid, the isolation liquid flows in the sealed cavity 20 corresponding to the multiple working cavities 21, and there is a probability of mixing during the filling process; thirdly, if the cross-sectional dimensions of the sealed cavity 20 and the working cavity 21 are close, the two liquids are prone to blending, increasing the risk of liquid crosstalk between the multiple working cavities 21; in order to solve the above problems, the present application sets the cross-sectional dimension of the sealed cavity 20 to be a long strip, and the cross-sectional dimension is greatly different from that of the working cavity 21, further reducing the disturbance of the isolation liquid to the reagent in the working cavity 21 during the filling process, and avoiding the crosstalk problem of reagents in different working cavities 21. Preferably, the width L of the sealed cavity 20 is at least twice its height H, and the height H1 of the working cavity 21 is at least three times the height H of the sealed cavity; the height of the working cavity is less than 10 mm, and the height H of the sealed cavity 20 is 0.1-3 mm. The isolation liquid and the reagent are prone to mixing at higher accelerations and lower rotation speeds. The reason is that at higher accelerations and decelerations, the speed fluctuation has a great impact on the interface between the reagent and the isolation liquid; while at low speeds, the interface stability between the isolation liquid and the reagent is relatively poor. Therefore, both conditions put forward higher requirements for the control of the chip rotation speed. The present application can achieve the purpose of effectively separating the isolation liquid and the reagent under existing experimental conditions through the above-mentioned structural design, that is, the isolation liquid and the reagent can still be effectively separated at higher rotation speeds or lower rotation speeds, and the interface stability of the isolation liquid and the reagent is increased.

[0048] Specifically, Figure 1 As shown, the sealed cavity 20 is in the shape of a spiral ring belt or a circular ring belt, the working cavity 21 is arranged on the side of the outer edge of the sealed cavity 20 away from the rotation center O, and based on the process of extraction and purification, the working cavity 21 of the corresponding process is arranged in sequence along the sealed cavity 20, and the isolation liquid cavity 12 is connected to the proximal end of the sealed cavity 20 through the second valve body 02. The spiral ring belt design can ensure that the isolation liquid always fills the sealed cavity 20 gradually from the distal end to the proximal end, thereby ensuring that no bubbles or cavities are generated during the entire filling process; secondly, if the sealed cavity 20 of the upstream working chamber 21 is filled first, the interface stability between the isolation liquid and the reagent in the upstream working chamber is poor at this time, and it is easy to bring the reagent in the upstream working chamber 21 into the downstream working chamber 21 during the filling process to form crosstalk.

[0049] In order to further improve the isolation effect between different working chambers 21, a valve body triggered by the outside is not used. Figure 1 and 3As shown, the first valve body 01 is a first slit 010 with a certain size, and the cross-section of the first slit 010 gradually decreases along the flow direction of the extraction and purification process; the slit inner wall 011 of the first slit 010 away from the rotation center is a plane or an arc surface, and the distance L1 between the plane or the arc surface and the rotation center gradually decreases along the flow direction of the extraction and purification process. First, through the above-mentioned arrangement, the resistance of the isolation liquid in the flow process is increased, the flow rate of the isolation liquid is reduced, and the probability of the reagent in the upstream working chamber being brought into the downstream working chamber is reduced; secondly, the isolation liquid is an oil phase and has poor compatibility with the reagent. The inner wall 011 of the first slit 010 away from the rotation center is a plane or an arc surface, and the distance L1 between the plane or the arc surface and the rotation center gradually decreases along the flow direction of the extraction and purification process, and the difficulty of the reagent passing through the first valve body 01 into the downstream working chamber increases. In addition, if the microfluidic chip is in a rotating state, the reagent with a higher density fits tightly with the working chamber 21 under the action of centrifugal force, further reducing the crosstalk problem of the reagents in different working chambers. The above-mentioned arrangement can ensure that no crosstalk problem will occur when the rotation speed is above 100r / min.

[0050] like Figure 1 As shown, the difference between the distances L2 and L3 between the two ends of the inner wall of the first slit away from the rotation center and the rotation center is not less than 1 / 2 of the width L4 of the sealed cavity. Through experimental measurement, when the above conditional dimensions are met, the chip will not have crosstalk problems even in a stationary state, and the isolation of the isolation liquid is stable.

[0051] like Figure 1-4 As shown, at least one blocking dam 200 is provided in the sealed cavity 20, and the blocking dam 200 itself or the blocking dam 200 and the inner wall of the sealed cavity 20 form a second slit 201; for a single working cavity 21, the projection of the blocking dam 200 rotating about the rotation center O completely covers the first slit 010 of the first valve body 01 connected to the working cavity. The above arrangement avoids the occurrence of a non-blocking area in a single working cavity, and the isolation liquid flow rate is too high, which is easy to be involved in the reagent, such as Figure 2 and 3 As shown in the dam 200 and the first valve body 01, the projection of the dam 200 rotating around the rotation center O cannot completely cover the first slit 010 of the first valve body 01 connected to the working chamber, as shown in FIG. Figure 4 As shown, there is a non-blocking area 012.

[0052] Specifically, Figure 2 and 3As shown, the first slit 010 has a first width W1, the second slit 201 has a second width W2, the first width W1 is smaller than the second width W2, and the first width W1 of the first slit 010 is not greater than the height of the sealed cavity 20; the dam 200 is connected to the inner wall of the sealed cavity 20 on the side close to the rotation center O, and the dam 200 is located at or beyond the connection between the working cavity and the sealed cavity on the side away from the rotation center O. Figure 1 As shown in FIG. 2 , it can be seen that the baffle dam 200 is located at the connection between the working chamber and the sealing chamber, away from the rotation center O. Figure 5 As shown, the baffle 200 passes over the connection between the working chamber and the sealing chamber on the side away from the rotation center O. Secondly, the first width W1 of the first slit 010 is limited, and its minimum value is limited. If the first width is too small, the resistance to the isolation liquid is too large. Under the condition of a high rotation speed, the isolation liquid is transmitted through the working chamber, which increases the disturbance of the isolation liquid and the reagent in the working chamber, and there is a problem that the isolation liquid wraps the reagent and brings the reagent into the downstream working chamber. Preferably, the first width W1 of the first slit 010 is not greater than 0.8 times the height H of the sealing cavity 20. The width design of the first slit needs to consider the flow of the isolation liquid in two situations: (1) During the isolation liquid filling process, the width of the first slit affects the resistance of the isolation liquid to flow through the first valve body. The greater the resistance, the lower the isolation liquid flow rate. The low isolation liquid flow rate has little effect on the isolation liquid and reagent interface, thereby preventing the reagent in the upstream working chamber from flowing into the downstream working chamber during the filling process; (2) After the isolation liquid filling is completed, the isolation liquid seals the working chamber to ensure that different working chambers do not flow into each other. The present application limits the width of the slit on the one hand, and limits the structure of the first valve on the other hand, especially the wall of the first slit 010 away from the rotation center. The two are combined to improve the effectiveness of isolating different working chambers during subsequent use.

[0053] like Figure 1 and 6 As shown, taking one of the working chambers 21 as an example, the baffle dams 200 are evenly arranged along the spiral ring or the circular ring, and the sum of the lengths of one side of the outer edge of at least one baffle dam 200 in the sealed chamber 20 corresponding to the single working chamber 21 is at least 2 / 5 of the length of the outer edge of the sealed chamber corresponding to the single working chamber. Figure 6 As shown, the sum of the lengths L6+L7 of one side of the outer edge of the two baffles 200 is at least 2 / 5 of the length L5 of the outer edge of the sealed cavity 20 corresponding to a single working cavity 21. Preferably, the baffles 200 have the same structure, so L6=L7. The baffles stabilize the reagents in the working cavity, constrain the free surface of the reagents, and reduce the free surface of the reagents close to the rotation center. It is too large, which can easily cause large fluctuations during the acceleration and deceleration of the microfluidic chip.

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

[0055] ;

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

[0057] Specifically, the isolation liquid chamber 12 is annularly arranged at the proximal end of the microfluidic chip; the sample chamber 10 and at least one reagent chamber 11 are fan-shaped, arranged around the outside of the isolation liquid chamber 12; at least two working chambers 21 are fan-shaped, arranged on the outer edge of the sealing chamber 20, and arranged around the rotation center O, and the reagent chamber 11 and the sample chamber 10 are respectively connected to the proximal end of the sealing chamber corresponding to the working chamber through the second valve body 02. Through the above-mentioned structural setting, the center of mass of the microfluidic chip provided by the embodiment of the present invention is kept on the rotation axis as much as possible, which improves the stability of the microfluidic chip during use; secondly, through reasonable layout, the compactness of the prepared microfluidic chip is improved and the preparation cost is reduced.

[0058] In another preferred embodiment, Figure 7 As shown, the isolation liquid chamber 12, the sample chamber 10 and at least one reagent chamber 11 are fan-shaped and arranged around the rotation center O; at least two working chambers 21 are fan-shaped and arranged on the outer edge side of the sealing chamber 20, and arranged around the rotation center O. The reagent chamber 11 and the sample chamber 10 are respectively connected to the proximal end of the sealing chamber 20 corresponding to the working chamber 21 through the second valve body 02.

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

[0060] 1. Pre-set relevant reagents and inject samples. Figure 8 As shown in a, the working chamber for nucleic acid adsorption in the microfluidic chip is pre-filled with magnetic beads for nucleic acid extraction (smaller white granular objects in the figure), and a sufficient amount of mineral oil for sealing is added to the isolation liquid chamber (the light grey shaded part in the figure), and appropriate amounts of sample, first washing solution, second washing solution and eluent (dark grey shaded part in the figure) are added to the sample chamber and three reagent chambers in turn according to the extraction and purification process.

[0061] 2. Centrifugal distribution of reagents and mineral oil sealing. Figure 8 As shown in FIG. 2 , a centrifugal speed V1 is applied to the microfluidic chip, and the sample, the first washing liquid, the second washing liquid and the elution liquid in the sample chamber are respectively filled into the corresponding working chambers at their distal ends under the action of centrifugal force. At the same time, the mineral oil in the isolation liquid chamber (the light gray shaded part in the figure) enters the sealing chamber (the dark gray shaded part in the figure), forming an isolation seal for the liquids in the multiple working chambers.

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

[0063] 4. Mix the magnetic beads and adsorb the nucleic acid. Figure 8 As shown in Figure d, by quickly switching the centrifugal direction of the microfluidic chip, the bubble cavity in the nucleic acid adsorption working chamber is fully mixed with the sample and magnetic beads in the nucleic acid adsorption working chamber under the action of the Euler force, thereby achieving adsorption of nucleic acids by the magnetic beads.

[0064] 5. Magnetic beads collection and transfer. Figure 8 As shown in FIG. 5 , the nucleic acid adsorption working chamber is rotated to the fixed position of the magnet (not marked in the figure, at this time the magnet is located at the starting end of the nucleic acid adsorption working chamber and directly below the sealed chamber). By rotating the chip, a relative displacement occurs between the nucleic acid adsorption working chamber and the fixed magnet. During this process, the magnetic beads in the working chamber will gather toward the mineral oil in the sealed chamber. During the aggregation process, the water molecules on the surface of the magnetic beads are dehydrated under the action of the oil-water interfacial tension, so that less liquid remains on the surface of the magnetic beads. At the same time, the gathered magnetic beads move toward the downstream working chamber under the action of the magnetic force.

[0065] 6. The magnetic bead passes through the first valve body and enters the downstream working chamber. Figure 8As shown in FIG. 5 , as the chip rotates, the fixed magnet drags the gathered magnetic beads through the first valve body. Based on the combined effect of the first slit on the first valve body and the surface tension of the mineral oil, the residual liquid on the surface of the magnetic beads is dehydrated again, and the liquid on the surface of the magnetic beads is retained in the upstream working chamber. After the residual liquid is filtered out, the magnetic beads enter the downstream working chamber.

[0066] 7. After the first wash, transfer the magnetic beads. Figure 8 As shown in g, steps 4-6 are repeated to achieve sufficient mixing of the first washing solution and the magnetic beads. After the first washing of the magnetic beads is completed, the magnetic beads are transferred to the downstream working chamber.

[0067] 8. After the first washing, transfer the magnetic beads. Repeat step 7 to transfer the magnetic beads to the downstream working chamber after the second washing.

[0068] 9. Nucleic acid elution. Figure 8 As shown in FIG. 1 , the operation of step 4 is repeated in sequence. By quickly switching the centrifugal direction of the microfluidic chip, the bubble cavity in the nucleic acid elution working chamber is fully mixed with the eluent and the magnetic beads in the nucleic acid elution working chamber under the action of the Euler force, thereby achieving the elution of the nucleic acid.

[0069] 10. Remove the magnetic beads. Figure 8 As shown in FIG. 1 , after the nucleic acid elution is completed, the nucleic acid elution working chamber is rotated to the fixed position of the magnet (not marked in the figure, at this time the magnet is located at the very end of the nucleic acid elution working chamber and directly below the sealing chamber), and the chip is rotated in the reverse direction. The magnetic beads in the elution working chamber are enriched and move toward the upstream working chamber; then, the magnet passes through the first valve body in the reverse direction and enters the upstream working chamber. At this time, the nucleic acid extraction and purification are completed.

[0070] 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 microfluidic chip for nucleic acid extraction and purification, characterized in that: include: A storage unit, the storage unit comprising a sample chamber, a reagent chamber, and an isolation liquid chamber; A nucleic acid extraction and purification unit, which is arranged on a side farther from the rotation center than the storage unit, and comprises at least two working chambers and a sealing chamber arranged on a side of the working chamber close to the rotation center, wherein different working chambers are connected via a first valve body arranged in the sealing chamber, and the distal ends of the sample chamber, the reagent chamber and the isolation liquid chamber are connected to the proximal end of the sealing chamber via a second valve body; The width of the sealed cavity is not less than the height of the sealed cavity, and the height of the working cavity is at least three times the height of the sealed cavity; The sealing cavity is in the shape of a spiral ring belt or a circular ring belt, the working cavity is arranged on the side of the outer edge of the sealing cavity away from the rotation center, and based on the process of extraction and purification, the working chambers of the corresponding processes are arranged in sequence along the sealing cavity, the isolation liquid cavity is connected to the proximal end of the sealing cavity through the second valve body, and the isolation liquid is a hydrophobic liquid with a density less than that of the liquid in the working cavity, and is inert and will not react with substances in the system; The first valve body is a first slit of a certain size, and the cross section of the first slit gradually decreases along the flow direction of the extraction and purification process; The inner wall of the first slit on the side away from the rotation center is a plane or a curved surface, and the distance between the plane or the curved surface and the rotation center gradually decreases along the flow direction of the extraction and purification process; The difference in distance between two ends of the inner wall of the first slit away from the rotation center and the rotation center is not less than 1 / 2 of the width of the sealing cavity.

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

3. The microfluidic chip according to claim 2, characterized in that: The first slit has a first width, the second slit has a second width, the first width is smaller than the second width, and the first width of the first slit is not greater than the height of the sealed cavity; The baffle dam is connected to the inner wall of the sealing cavity at one side close to the rotation center, and the baffle dam is located at or beyond the connection between the working cavity and the sealing cavity at one side away from the rotation center.

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

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

05.

6. The microfluidic chip according to claim 1, characterized in that: The isolation liquid chamber is annularly arranged at the proximal end of the microfluidic chip; The sample chamber and at least one reagent chamber are fan-shaped and arranged around the outside of the isolation liquid chamber; At least two of the working chambers are arranged in a fan shape, arranged on the outer edge of the sealing chamber, and arranged around the rotation center. The reagent chamber and the sample chamber are respectively connected to the proximal end of the sealing chamber corresponding to the working chamber through the second valve body; or The isolation liquid chamber, the sample chamber and the at least one reagent chamber are fan-shaped and arranged in a circle around the rotation center; At least two of the working chambers are arranged in a fan shape, arranged on the outer edge of the sealing chamber, and arranged around the rotation center. The reagent chamber and the sample chamber are respectively connected to the proximal end of the sealing chamber corresponding to the working chamber through the second valve body.

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