Centrifugal micro-fluidic chip

Abstract

The invention provides a centrifugal microfluidic chip, which belongs to the technical field of microfluidics and comprises a chip body. An upstream cavity, wherein the upstream cavity is located on the chip body; the downstream cavity is located on the chip body, and one side of the distal end of the upstream cavity is communicated with the downstream cavity through a flow channel; and the repeated switching valve is arranged on the flow channel, a phase change material is arranged in the repeated switching valve, and the upstream cavity and the downstream cavity can be repeatedly blocked or communicated by controlling heating and the rotating speed. The device is composed of the three channels, the phase-change material and the energy storage device connected with the phase-change material, controllable batch release and precise quantitative release of upstream liquid can be conveniently achieved, the structure composition is simple, the production and manufacturing cost can be conveniently reduced, and the complexity of fluid controlled release is simplified.

Description

Technical Field

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

[0002] In recent years, with the in-depth cross-disciplinary development of physics, material chemistry, mechanical engineering, biotechnology and other disciplines, human beings' manipulation technology of tiny fluids (referred to as "microfluidic technology") has continued to develop and innovate, keeping pace with the times. Due to the characteristics of miniaturization, integration and high automation, microfluidic chips have been widely used in fields such as biomedical testing and synthetic biology. Microfluidic chips used to load and run various types of micro-volume fluid reagents are gradually developing into the most effective carrier form for efficient in vitro diagnosis and high-precision biochemical synthesis. Among them, an important research field of high-precision synthetic biology is synthetic genomics, such as the synthesis of oligonucleotides of specific sequences, which has a very wide range of applications in molecular biology fields such as gene sequencing and gene chips.

[0003] The in situ synthesis of specific sequence oligonucleotides is a high-throughput synthesis of oligonucleotides on a solid-state chip substrate, which has the advantages of high throughput and high precision. Microfluidic chips can flexibly release, transfer, mix and separate liquid components such as reagents and samples. Therefore, the in situ synthesis of oligonucleotides based on microfluidic chips is also more flexible and efficient. The efficient and accurate processing of liquid behavior by microfluidic chips relies on various microvalves integrated inside them. These microvalves and microvalve-related components are the key to guiding liquid flow or determining liquid movement patterns. Microfluidic chips used for in situ synthesis of oligonucleotides of specific sequences (taking the most commonly used phosphoramidite chemistry as an example) need to functionally realize the repeated cyclic release of liquid reagents such as deprotecting agents, active nucleotide monomers, oxidants and blocking agents. Therefore, they have higher requirements for the selection of microvalve types and the implementation of valve control methods. The valve control methods used in current synthetic process methods (such as mechanical valves, pneumatic valves, etc.) generally face problems such as complex component composition, bulky external equipment, and high cost. Paraffin valve, as the most typical and most commonly used phase change valve, is characterized by simple structure and easy opening and closing. Except for simple heating modules, it generally does not require additional complex component modules, and has almost no effect on the activity, detection and synthesis reactions of various biochemical reagents. It is a relatively ideal microfluidic valve.

[0004] However, there are still some problems in the use of paraffin valves in the field of microfluidic chips. For example, the inability to open and close repeatedly limits their application, the cavities created by the cooling and shrinkage of the paraffin affect the airtightness and reliability of the valve body, and the overly complex paraffin valve assembly structure. These problems often lead to functional limitations in the chip design process, further increasing the cost and complexity of chip manufacturing or use, and even affecting the normal fluid operation of the microfluidic chip. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a centrifugal microfluidic chip. Through the three-channel, phase change material and energy storage device connected thereto, the controllable batch release and precise quantitative release of upstream liquid can be conveniently realized, and its structure is simple, which is convenient for reducing production costs and simplifying the complexity of fluid controlled release.

[0006] The present invention adopts the following technical solution: A centrifugal microfluidic chip comprises: a chip body; an upstream cavity, the upstream cavity is located on the chip body; a downstream cavity, the downstream cavity is located on the chip body, and one side of the distal end of the upstream cavity is connected to the downstream cavity through a flow channel; a repeated switching valve, the repeated switching valve is arranged on the flow channel, a phase change material is arranged inside, and the upstream cavity and the downstream cavity can be repeatedly blocked or connected by controlling heating and rotation speed.

[0007] Furthermore, the repeated switching valve includes: three channels, the first channel of the three channels is connected to the upstream cavity through the flow channel, the second channel of the three channels is connected to the downstream cavity through the flow channel, and the distal end of the third channel of the three channels is connected to the first channel and the second channel; an energy storage device, the energy storage device is arranged closer to the rotation center than the three channels, and the proximal end of the third channel is connected to the side of the energy storage device away from the rotation center.

[0008] Furthermore, the connecting intersection of the first channel, the second channel and the third channel is located at a portion of the three channels far away from the rotation center.

[0009] Furthermore, the inner diameters of the first channel and the second channel gradually increase as they move away from the connecting intersection.

[0010] Furthermore, the first channel and the second channel are bent toward the rotation center with the connected intersection as a base point to form a bending area; when the repeated switching valve is closed, the position of the phase change material in the first channel and the second channel does not exceed the bending area; the inner diameter of the third channel gradually decreases as it moves away from the connected intersection.

[0011] Furthermore, the energy storage device is a cavity structure.

[0012] Furthermore, the connection points of the first channel, the second channel and the third channel are connected in an arc shape; the minimum cross-sectional position of the first channel and the minimum cross-sectional position of the second channel form a minimum radius with the rotation center; the inner surface of the three-way channel in the area outside the two minimum radius lines is subjected to non-infiltrative treatment.

[0013] Furthermore, a buffer channel is provided between the proximal end of the third channel and the energy storage device.

[0014] Further, the relationship between the minimum value of the minimum cross-sectional area of ​​the buffer channel and the third channel, the minimum cross-sectional area of ​​the first channel and the minimum cross-sectional area of ​​the second channel satisfies the following formula: ; Wherein, S1 is the minimum cross-sectional area of ​​the buffer channel, S2 is the minimum cross-sectional area of ​​the third channel, S3 is the minimum cross-sectional area of ​​the first channel, and S4 is the minimum cross-sectional area of ​​the second channel.

[0015] Furthermore, when it is necessary to block the flow channel, the phase change material is heated, and the centrifugal microfluidic chip is controlled to rotate. At a first preset speed, the liquid phase change material moves from the proximal end to the distal end to seal the flow channel between the upstream cavity and the downstream cavity; then the heating is stopped to wait for the liquid phase change material to solidify; when it is necessary to connect the upstream cavity and the downstream cavity, the solid phase change material is heated, and under the action of negative pressure, the phase change material transformed from solid to liquid flows toward the energy storage device through the third channel, and when the upstream cavity and the downstream cavity are connected, the heating of the phase change material is stopped to wait for the liquid phase change material to solidify; or, while heating the solid phase change material, the rotation speed of the centrifugal microfluidic chip is controlled to ensure that the phase change material transformed from solid to liquid flows through the third channel toward the energy storage device at a certain flow rate, and when the upstream cavity and the downstream cavity are connected, the heating of the phase change material is stopped to wait for the liquid phase change material to solidify.

[0016] The present invention provides a centrifugal microfluidic chip, which has the following beneficial technical effects compared with the prior art: (1) First, by controlling the centrifugal force and heating conditions, the repeated connection and closing of the switching valve are achieved. The reaction liquid is added drop by drop to ensure the normal progress of the reaction process. By repeatedly closing the switching valve, it is ensured that the reaction in the downstream chamber will not be affected by the liquid in the upstream chamber. The controllable batch release and precise quantitative release of the upstream liquid make the detection results more accurate.

[0017] (2) Secondly, when the phase change material melts, the valve is switched on and off using the stable centrifugal force at a certain speed and the constant gas pressure generated by the volume change, making the valve control method more efficient, stable and reliable. Based on the balance between the centrifugal force and the gas pressure, the repeated switching of the valve can be realized, thereby controlling the batch release and precise quantitative release of the liquid reagent, making the microfluidic chip design more functional and meeting various microfluidic control requirements. The repeated switching valve has a simple structure and an efficient and concise operation process, with extremely low requirements on operators and hardware systems, avoiding the complex components and operation processes of other active valve control methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 A structural diagram of a centrifugal microfluidic chip provided in Example 1 of the present invention; Figure 2 A schematic diagram of the structure of the open state of the repeated switching valve provided in Example 1 of the present invention; Figure 3 A schematic structural diagram of another repeated switching valve in an open state provided in Example 1 of the present invention; Figure 4 A schematic diagram of the structure of the closed state of the repeated switching valve provided in Example 1 of the present invention; Figure 5 A schematic structural diagram of a closed state of a repeated switching valve when the third channels provided in Example 1 of the present invention have equal diameters; Figure 6 A schematic structural diagram of another repeated switching valve closed state provided in Example 1 of the present invention; Figure 7 A schematic diagram of a three-channel structure provided in Example 1 of the present invention; Figure 8 A schematic diagram of a three-channel non-invasive treatment area provided in Example 1 of the present invention; Fig. 9 A schematic structural diagram of a closed state of a repeated switching valve with a buffer channel provided in Example 1 of the present invention; Fig.10 This is a structural diagram of a centrifugal microfluidic chip for in situ synthesis of oligonucleotides provided in Example 2 of the present invention.

[0020] Figure numerals: 1. Chip body; 01. Deprotection agent storage chamber; 02. Nucleotide monomer storage chamber; 020. Adenine A storage chamber; 021. Thymine T storage chamber; 022. Cytosine C storage chamber; 023. Guanine G storage chamber; 03. Oxidant storage chamber; 04. Sealing agent storage chamber; 05. Fluid channel; 06. Oligonucleotide synthesis chamber; 07. Waste liquid tank; 2. Upstream cavity; 3. Downstream cavity; 4. Flow channel; 5. Repeated switching valve; 50. Phase change material; 51. Three-way; 510. First channel; 511. Second channel; 512. Third channel; 52. Energy storage device; 53. Buffer channel. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Example 1

[0027] The embodiment of the present invention discloses a centrifugal microfluidic chip, such as Figure 1 and 2 As shown, Figure 2 The middle black represents solid phase change material, including: a chip body 1; an upstream cavity 2, the upstream cavity 2 is located on the chip body 1; a downstream cavity 3, the downstream cavity 3 is located on the chip body 1, and the distal end of the upstream cavity 2 is connected to the downstream cavity 3 through a flow channel 4; a repeated switching valve 5, the repeated switching valve 5 is arranged on the flow channel 4, and a phase change material 50 is arranged inside. By controlling the heating and rotation speed, the upstream cavity 2 and the downstream cavity 3 can be repeatedly blocked or connected.

[0028] The chip body 1 is provided with a mounting hole for connecting with the device. Under the drive of the device, the chip body 1 is driven to rotate. The upstream cavity 2 and the downstream cavity 3 are in an upstream and downstream relationship. The liquid in the upstream cavity can be repeatedly dripped into the downstream cavity 3 by repeatedly switching the valve 5 to complete the relevant reaction. The upstream cavity 2 and the downstream cavity 3 can be any step required in the biochemical reaction, used for the transfer or reaction of the liquid. The upstream cavity 2 and the downstream cavity 3 can be set with corresponding reagents or magnetic beads to accelerate the reaction based on the specific reaction conditions, which will not be repeated here.

[0029] The upstream chamber 2 and the downstream chamber 3 are in an upstream and downstream relationship in the process steps. The upstream chamber 2 and the downstream chamber can be located on the same radius or on different radii with the rotation center O as the center. Figure 1 In this embodiment, the distance between the downstream cavity 3 and the rotation center O is greater than the distance between the upstream cavity 2 and the rotation center O.

[0030] In the present invention, the "distal end" and the "proximal end" are determined relative to the rotation center O. The phase change material 50 in the present invention can be a wax, for example, the wax can be paraffin, microcrystalline wax, synthetic wax or natural wax. Alternatively, the phase change material can also be a gel or a thermoplastic resin. The gel can be polyacrylamide, polyacrylate, polymethacrylate, or polyvinylamine. Thermoplastic resins can be cyclic olefin copolymers (COC), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polyoxymethylene (POM), perfluoroalkoxy (PFA), polyvinyl chloride (PVC), polypropylene (PP), polyethylene terephthalate (PET), polyetheretherketone (PEEK), polyacrylate (PA), polysulfone (PSU) or polyethylene diene fluoride (PVDF), etc.

[0031] The centrifugal microfluidic chip provided by the present invention, firstly, realizes the repeated connection and closing of the repeated switching valve 5 by controlling the two conditions of centrifugal force and heating, and the reaction liquid is dripped one by one to ensure the normal progress of the reaction process, and by closing the repeated switching valve 5, it is ensured that the reaction in the downstream cavity 3 will not be affected by the liquid in the upstream cavity 2, and the upstream liquid can be controlled to release in batches and release in a precise and quantitative manner, so that the detection result is more accurate. Secondly, when the phase change material melts, the constant gas pressure generated by the stable centrifugal force and volume change at a certain speed is used to control the switch of the valve, so that the valve control method is more efficient, stable and reliable; based on the balance relationship between centrifugal force and gas pressure, the repeated switching valve 5 is repeatedly switched, and the batch release and precise and quantitative release of the liquid reagent can be controlled, so that the microfluidic chip design is more functional and meets various microfluid control requirements; the repeated switching valve has a simple structure, an efficient and concise operation process, and extremely low requirements for operators and hardware systems, avoiding the complex components and operation processes of other active valve control methods.

[0032] It is understandable that the present application does not limit the heating method, as long as the heat source cooperates with the phase change material to achieve the phase change material being converted into liquid under heating conditions.

[0033] Specifically, Figure 1 and 2As shown, the repeated switching valve 5 includes: a three-way passage 51, wherein the first channel 510 of the three-way passage 51 is connected to the upstream cavity 2 through the flow channel 4, the second channel 511 of the three-way passage 51 is connected to the downstream cavity 3 through the flow channel 4, and the distal end of the third channel 512 of the three-way passage 51 is connected to the first channel 510 and the second channel 511; an energy storage device 52, wherein the energy storage device 52 is arranged closer to the rotation center O than the three-way passage 51, and the proximal end of the third channel 512 is connected to a side of the energy storage device 52 away from the rotation center. Compared with the third channel 512, the energy storage device 52 is arranged near the proximal end, which can meet the requirements of blocking or opening the connection between the upstream cavity 2 and the downstream cavity 3 while reducing the probability of bubbles appearing in the phase change material. Specifically, the energy storage device stores energy in the form of negative pressure, and the negative pressure acts on the surface of the liquid phase change material. The gas inside the liquid phase change material escapes from the phase change material under the negative pressure, preventing the gas in the phase change material from merging, so that the phase change material is divided into multiple sections by bubbles, which seriously affects the sealing effect.

[0034] As another embodiment of the present invention, Figure 3 As shown, the first channel 510 and the second channel 511 in the three channels 51 extend in a direction away from the rotation center O, and can also achieve blocking and connecting the upstream cavity 2 and the downstream cavity 3. It should be pointed out that in the embodiment of the present invention, there is no specific limitation on the extending direction of the first channel 510 and the second channel 511, as long as the repeated switching valve 5 can achieve blocking and connecting the flow channel 4; secondly, the first channel 510 and the second channel 511 can be symmetrically arranged or asymmetrically arranged, preferably symmetrically arranged.

[0035] As a preferred embodiment, Figure 2 As shown, the connecting intersection S of the first channel 510, the second channel 511 and the third channel 512 is located at the portion of the three channels far from the rotation center O. This structure can further improve the sealing effect of the phase change material on the channel. Figure 4As shown, light gray represents liquid phase change material. When the phase change material is heated to melt, under the condition that the chip body 1 rotates, the centrifugal force drives the liquid phase change material 50 to move in a direction away from the rotation center O. At this time, the liquid phase change material is deposited near the connecting intersection S. Since the liquid phase change material flows in a direction away from the rotation center O, a negative pressure is generated in the energy storage device 52. The distance between the liquid surface in the third channel 512 and the rotation center is smaller than the distance between the liquid surface and the rotation center of the first channel 510 and the second channel 511. Under the action of centrifugal force, the liquid phase change material deposited at the connecting intersection S drives the gas inside it to move toward the liquid surface, that is, it moves in the channel toward the direction close to the rotation center, thereby preventing the gas inside the phase change material from gathering. Secondly, the centrifugal force has a compacting effect on the phase change material. The volume of the liquid phase change material solidified and shrunk is offset by the volume increased by the phase change material after the centrifugal force is removed, thereby preventing the liquid phase change material from solidifying and shrinking, thereby preventing the first channel 510 and the second channel 511 from being effectively sealed.

[0036] Specifically, Figure 4 As shown, the inner diameters of the first channel 510 and the second channel 511 gradually increase as they move away from the connecting intersection S. As the liquid phase change material solidifies, the relatively long axial lengths of the first channel 510 and the second channel 511 will produce a larger contraction length, while the radial contraction is smaller. By setting the inner diameters of the first channel 510 and the second channel 511 away from the connecting intersection S, the inner diameters gradually increase, so that the phase change materials with larger inner diameters at both ends after solidification and contraction can form tensile stress and closely contact the inner walls of the first channel 510 and the second channel 511 to form a good seal.

[0037] To facilitate processing, in the embodiment of the present invention, the cross-sections of the flow channel 4, the first channel 510, the second channel 511 and the third channel 512 are preferably circular, but may also be other shapes.

[0038] The first channel 510 and the second channel 511 are bent toward the rotation center O with the connection intersection S as the base point to form a bending area; when the repeated switching valve 5 is closed, the position of the phase change material 50 in the first channel 510 and the second channel 511 does not exceed the bending area; the inner diameter of the third channel 512 gradually decreases as it moves away from the connection intersection S. For the reasons mentioned above, when the first channel 510 and the second channel 511 are bent toward the rotation center O, Figure 4As shown, during the solidification process, the gradually solidified phase change material in the first channel 510 and the second channel 511 is compressed toward the connected intersection S by the combined force F under the action of centrifugal force, and the gradually solidified phase change material with variable diameter compacts the tube wall of the first channel and the second channel under the action of pressure, thereby improving the sealing effect; secondly, when the third channel 512 is an equal-diameter channel, there is a possibility that under the conditions of centrifugal force and solidification and shrinkage of the liquid phase change material, there is still a possibility that the partially solidified phase change material on both sides of the first channel 510 and the second channel 511 cannot compensate for the pores generated by the solidification of the core under the constraint of the inner wall of the variable diameter channel, which easily causes negative pressure cracks in the core. The cracks are very likely to absorb local small bubbles, thereby merging and becoming larger, so that the phase change material is separated by bubbles, resulting in a poor sealing effect and a small number of repeated uses, such as Figure 5 As shown, in order to solve the above problems, the present invention proposes that the inner diameter of the third channel 512 gradually decreases as it moves away from the connecting intersection S. On the one hand, the phase change material located inside the third channel 512 close to the rotation center O is easy to solidify, and the phase change material located inside the third channel 512 away from the rotation center O takes a longer time to solidify. The liquid phase change material at the bottom of the third channel 512 compensates for the cracks generated during the solidification and shrinkage of the liquid phase change material in the first channel 510 and the second channel 511, thereby improving the sealing effect of the liquid phase change material on the three channels 51.

[0039] It is understandable that the first channel 510 and the second channel 511 can be bent upward in an arc shape. Figure 1 , 2 As shown in FIG. 4 , it can also be bent upward in a straight line, such as Figure 6 shown.

[0040] In order to improve the shrinkage effect of the liquid phase change material in the third channel 512 on the first channel 510 and the second channel 511, as shown in FIG. Figure 7 As shown, the relationship between the cross-sectional area of ​​the connection between the third channel 512 and the first channel 510 and the second channel 511 and the minimum cross-sectional area of ​​the first channel 510 and the second channel 511 satisfies the following formula: ; Among them, S3 is the minimum cross-sectional area of ​​the first channel, S4 is the minimum cross-sectional area of ​​the second channel, S5 is the cross-sectional area of ​​the third channel at the connection between the first channel and the second channel, or S5 is the maximum cross-sectional area of ​​the third channel. The above-mentioned size limitation ensures the shrinkage compensation effect.

[0041] The energy storage device 52 of the present application is negative pressure energy storage. Specifically, under the action of centrifugal force, when the liquid phase change material flows from the proximal end to the distal end, the negative pressure in the energy storage device 52 increases. The energy storage device in the present invention is a cavity structure sealed by a phase change material. In order to improve the energy storage effect, the cavity structure has a certain volume. It should be pointed out that the volume of the cavity structure will affect the selection of the speed range and the speed control accuracy; when the volume of the cavity structure is small, a larger speed is required to increase the centrifugal force and drive the liquid phase change material to flow from the proximal end to the distal end. The speed selection range is large, but the speed control accuracy is high. A slight change in the speed will also affect the position and state of the liquid phase change material in the channel, and the energy consumption is large; the larger the volume of the cavity structure, the smaller the energy consumption, but the speed selection range is small, the control accuracy is relatively low, and the position of the phase change material in the channel changes greatly; and setting the cavity structure to a certain volume, under the condition of moderate energy consumption, the liquid phase change material can be controlled in the channel at a lower speed. On the other hand, when the chip body is in a stationary state, the space reserved in the energy storage device 52 is small. Even if the repeated switching valve 5 is repeated for many times, and part of the gas leaks into the cavity in the energy storage device 52, it can still be used for a long time. Through measurement, when the liquid phase change material fills one-third of the volume of the energy storage device, it can be repeated more than 1,000 times and the blocking and connection effects are good.

[0042] The above technical solution can meet the repeated blocking and connection between the upstream cavity 2 and the downstream cavity 3, but there is also a problem. For certain special reactions, the interior of the reaction cavity is required to be composed of reaction substances and no impurities can be mixed in. For example, inert phase change materials should not be mixed into the reaction system. In order to ensure that the liquid phase change material is not mixed into the downstream cavity during the use of the repeated switching valve 5, the present invention proposes a corresponding technical solution.

[0043] like Figure 8 As shown, the connection between the first channel 510, the second channel 511 and the third channel 512 is connected in an arc shape; the minimum cross-section S3 position of the first channel 510 and the minimum cross-section S4 position of the second channel 511 form minimum radii R1 and R2 with the rotation center O; the inner surface of the three-channel channel outside the line connecting the two minimum radii R1 and R2 is treated with non-infiltration, such as the area indicated by the oblique lines in the figure. During the actual use of the product, the liquid phase change material is easy to adhere at the position of the connecting intersection S, and the phase change material adhered to the bottom of the three-channel 51 enters the downstream cavity 3 under the flushing of the liquid discharged from the upstream cavity 2, thereby causing pollution to the reaction system of the downstream cavity 3. By performing non-infiltration treatment on the lower surface, the internal bonding force of the liquid phase change material with a certain viscosity is greater than its bonding force with the inner wall of the channel, which is conducive to absorbing the liquid phase change material into the energy storage device 52 and the third channel 512 through negative pressure.

[0044] Non-wetting treatment can be achieved by coating the inner wall of the channel with a coating that is non-wetting with the phase change material, or by setting micro-grooves on the inner wall of the channel. Since the liquid phase change material has a certain surface tension, the existence of surface tension makes it impossible for the liquid phase change material to infiltrate the inside of the micro-grooves, thereby reducing the bonding area with the inner wall of the channel, making the bonding force between the liquid phase change material and the inner wall of the channel smaller.

[0045] It should be noted that the three-way channel in the area within the line connecting the two minimum radii R1 and R2 is not subjected to non-wetting treatment. The main reason is that if tiny grooves are provided on the inner wall of the channel, the sealing effect of the phase change material on the three-way channel will be destroyed.

[0046] like Fig. 9 As shown, a buffer channel 53 is provided between the proximal end of the third channel 512 and the energy storage device 52. The above arrangement can further reduce the probability of liquid phase change material remaining at the connecting intersection S. Specifically, after the channel surface is subjected to non-wetting treatment, the internal bonding force of the liquid phase change material with a certain viscosity is greater than its bonding force with the inner wall of the channel. However, if the negative pressure of the energy storage device 52 is large, the flow rate of the liquid phase change material is too fast, resulting in the internal bonding force of the local liquid phase change material being less than its bonding force with the inner wall of the channel, so that part of the liquid phase change material remains on the channel surface. By providing a buffer channel, the flow rate of the liquid phase change material can be further reduced, and the probability of the phase change material adhering to the inner wall of the channel can be reduced.

[0047] In order to achieve the buffering effect, the buffer channel may be set longer to increase the friction between the liquid phase change material and the channel, or the tube diameter may be limited to increase the movement resistance of the buffer channel.

[0048] Specifically, the relationship between the minimum value of the minimum cross-sectional area of ​​the buffer channel 53 and the third channel 512 and the minimum cross-sectional area S3 of the first channel 510 and the minimum cross-sectional area S4 of the second channel 511 satisfies the following formula: ; In the formula, S1 is the minimum cross-sectional area of ​​the buffer channel, S2 is the minimum cross-sectional area of ​​the third channel, S3 is the minimum cross-sectional area of ​​the first channel, and S4 is the minimum cross-sectional area of ​​the second channel. Through the above-mentioned size limitation, when the chip body 1 is in a static state, the suction of the liquid phase change material by the negative pressure will not cause the flow rate of the liquid phase change material to be too fast, so that the phase change material remains at the connection intersection S.

[0049] The operating principle of the centrifugal microfluidic chip proposed in the present invention is as follows: (1) When the flow channel needs to be blocked, the phase change material is heated, and the centrifugal microfluidic chip is controlled to rotate. At a preset speed, the liquid phase change material moves from the proximal end to the distal end to seal the flow channel between the upstream cavity and the downstream cavity; then the heating is stopped and the liquid phase change material is allowed to solidify.

[0050] It should be pointed out that heating the phase change material and controlling the rotation of the centrifugal microfluidic chip include two control states: a. heating the phase change material, and after the phase change material changes from solid to liquid, controlling the rotation of the centrifugal microfluidic chip; b. controlling the rotation of the centrifugal microfluidic chip while heating the phase change material, and after the phase change material changes from solid to liquid, the liquid phase change material moves toward the distal end; the technical solutions protected by the present invention include the above two situations.

[0051] Secondly, in the technical solution protected by the present application, "then stop heating and wait for the liquid phase change material to solidify" also includes two scenarios: then stop heating, wait for the liquid phase change material to solidify, and control the centrifugal microfluidic chip to stop rotating or keep rotating.

[0052] (2) When it is necessary to connect the upstream cavity 2 and the downstream cavity 3, the solid phase change material is heated. Under the action of negative pressure, the phase change material changes from solid to liquid and flows toward the energy storage device through the third channel. At this time, the chip body 1 does not need to rotate. Under the action of negative pressure, the liquid phase change material is sucked to connect the upstream cavity and the downstream cavity. When the upstream cavity and the downstream cavity are connected, the heating of the phase change material is stopped. After the liquid phase change material solidifies, the centrifugal microfluidic chip can be controlled to stop rotating or keep rotating; or, while the solid phase change material is heated, the rotation speed of the centrifugal microfluidic chip is controlled to ensure that the phase change material changes from solid to liquid flows toward the energy storage device at a certain flow rate through the third channel. When the upstream cavity and the downstream cavity are connected, the heating of the phase change material is stopped. After the liquid phase change material solidifies, the centrifugal microfluidic chip can be controlled to stop rotating or keep rotating.

[0053] Example 2

[0054] like Fig.10As shown, the embodiment of the present invention adopts the repeated switching valve 5 of Example 1. The centrifugal microfluidic chip described in this scheme is used for in situ synthesis of oligonucleotides, and is mainly composed of a raw material / reagent storage chamber located on the chip body 1, related fluid channels, a repeated switching valve 5 and a waste liquid pool. The remaining supporting functional components and the specific synthesis mechanism are not shown and described in detail here, which is in line with the conventional cognition of professionals in this field. The deprotecting agent storage chamber 01, the nucleotide monomer storage chamber 02, the oxidant storage chamber 03 and the sealing agent storage chamber 04 are all located at the position closest to the rotation center O, and the oligonucleotide synthesis chamber 06 is connected to the deprotecting agent storage chamber 01, the nucleotide monomer storage chamber 02, the oxidant storage chamber 03 and the sealing agent storage chamber 04 through several fluid channels 05. The nucleotide monomer storage chamber 02 is actually composed of 4 sub-storage chambers, namely, adenine A storage chamber 020, thymine T storage chamber 021, cytosine C storage chamber 022 and guanine G storage chamber 023, each of which is connected to the oligonucleotide synthesis chamber 06 through its own separate fluid channel 05. The oligonucleotide synthesis chamber 06 is located farther away from the rotation center O than the other storage chambers, and the distal end of the oligonucleotide synthesis chamber 06 is connected to the waste liquid pool 07 at the further end through the fluid channel 05. All fluid channels 05 are provided with a repeated switching valve 5.

[0055] In the specific implementation mode involved in this proposal, the in situ synthesis of oligonucleotides of specific sequence is mainly achieved by phosphoramidite chemistry. The synthesis process mainly includes four basic steps, namely deprotection reaction, active nucleotide monomer coupling, oxidation stabilization reaction and capping reaction. The in situ synthesis of oligonucleotides of specific sequence on the microfluidic chip can be achieved by repeating the four basic steps multiple times. The specific chemical reaction process and mechanism are not described in detail here. Only the fluid release process of the microfluidic chip and the corresponding fluid regulation mechanism are described in detail.

[0056] The following are the specific steps for regulating the repeated switching of the repeating switching valve 5 structure to achieve in situ synthesis of oligonucleotides with specific sequences: (1) A series of first protected nucleotide molecules are fixed in advance in the oligonucleotide synthesis chamber 06, and corresponding liquid reagents are added to all liquid reagent storage chambers. At this time, all the repeat switching valves 5 are in a completely closed state, that is, the upstream chamber and the downstream chamber are completely sealed and isolated by the closed repeat switching valves 5; (2) Only the area of ​​the repeated switching valve 5 between the deprotection agent storage chamber 01 and the oligonucleotide synthesis chamber 06 is continuously and locally heated, while the centrifugal microfluidic chip remains stationary or rotates at a low speed, and the heating is stopped after the melted phase change material enters the energy storage device 52. After the phase change material cools and solidifies, the repeated switching valve 5 is opened; (3) The centrifugal microfluidic chip is rotated at a certain speed to apply centrifugal force, so that the deprotecting agent in the deprotecting agent storage chamber 01 is partially released into the oligonucleotide synthesis chamber 06; (4) After the deprotecting agent partially enters the oligonucleotide synthesis chamber 06, only the area of ​​the repeated switching valve 5 between the deprotecting agent storage chamber 01 and the oligonucleotide synthesis chamber 06 is continuously locally heated again. At the same time, the centrifugal microfluidic chip enters the three-way passage under the action of centrifugal force after the phase change material in the energy storage device melts at a preset speed. At this time, the heating is gradually stopped while maintaining the rotation speed. After the phase change material cools and solidifies, the repeated switching valve 5 is closed again; (5) a nucleotide molecule deprotection reaction occurs in the oligonucleotide synthesis chamber 06; (6) Only the area of ​​the repetitive switching valve 5 between the oligonucleotide synthesis chamber 06 and the waste liquid pool 07 is continuously and locally heated, while the centrifugal microfluidic chip remains stationary or rotates at a low speed. After the melted phase change material enters the energy storage device 52 of the repetitive switching valve 5, the heating is stopped. After the phase change material cools and solidifies, the repetitive switching valve 5 is opened; (7) The centrifugal microfluidic chip rotates at a certain speed to apply centrifugal force, so that the waste liquid after the nucleotide molecule deprotection reaction in the oligonucleotide synthesis chamber 06 is completely released into the waste liquid pool 07; (8) After the deprotection reaction waste liquid completely enters the waste liquid pool 07, only the area of ​​the repeated switching valve 5 between the oligonucleotide synthesis chamber 06 and the waste liquid pool 07 is continuously locally heated again, and the centrifugal microfluidic chip keeps rotating at a high speed. After the phase change material in the energy storage device melts, it enters the three-way passage under the action of centrifugal force. At this time, the heating is gradually stopped while maintaining the rotation speed. After the phase change material cools and solidifies, the repeated switching valve 5 is closed again; (9) At this time, the first step of the four basic steps in the in situ synthesis of oligonucleotides, the deprotection reaction step, has been completed. At this time, all the repeated switching valves 5 are in a completely closed state, and the first series of nucleotide molecules pre-fixed in the oligonucleotide synthesis chamber 06 have been deprotected; (10) Repeat the above steps (2) to (8) to selectively open and close the nucleotide monomer storage chamber 02 in a timely manner to complete the second link of the four basic links in the in situ synthesis of oligonucleotides - the active nucleotide monomer coupling link. At this time, all the repeated switching valves 5 are in a completely closed state, and the first required nucleotide molecule has been coupled in the oligonucleotide synthesis chamber 06; (11) Repeat the above steps (2) to (8), selectively open and close the oxidant storage chamber 03 at the appropriate time, and complete the third link of the four basic links in the in situ synthesis of oligonucleotides - the oxidation stabilization reaction link. At this time, all the repeated switching valves 5 are in a completely closed state, and the stabilization treatment after the coupling of the first desired nucleotide molecule has been completed in the oligonucleotide synthesis chamber 06; (12) Repeat the above steps (2) to (8), selectively open and close the blocking agent storage chamber 04 at the appropriate time, and complete the fourth step of the four basic steps in the in situ synthesis of oligonucleotides - the capping reaction step. At this time, all the repeated switching valves 5 are in a completely closed state, and the first nucleotide that has not been coupled to the desired nucleotide molecule has been blocked in the oligonucleotide synthesis chamber 06 to prevent the sequence with the deletion mutation from continuing to extend; (13) The above four basic steps of in situ synthesis of oligonucleotides are repeated, and different types of active nucleotide monomers in the nucleotide monomer storage chamber 02 are selectively released to finally complete the in situ synthesis of oligonucleotides of specific sequence on the centrifugal microfluidic chip.

[0057] 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 centrifugal microfluidic chip, characterized in that: include: Chip body; An upstream cavity, the upstream cavity is located on the chip body; A downstream cavity, wherein the downstream cavity is located on the chip body, and a distal end of the upstream cavity is connected to the downstream cavity through a flow channel; A repeated switching valve is arranged on the flow channel, and a phase change material is arranged inside the valve. By controlling heating and rotation speed, the upstream cavity and the downstream cavity can be repeatedly blocked or connected.

2. The centrifugal microfluidic chip according to claim 1, characterized in that: The repeated switching valve comprises: Three channels, wherein a first channel of the three channels is connected to an upstream cavity through a flow channel, a second channel of the three channels is connected to a downstream cavity through a flow channel, and a distal end of a third channel of the three channels is connected to the first channel and the second channel; An energy storage device is arranged closer to the rotation center than the three passages, and a proximal end of the third passage is connected to a side of the energy storage device away from the rotation center.

3. The centrifugal microfluidic chip according to claim 2, characterized in that: The connecting intersection of the first channel, the second channel and the third channel is located at a portion of the three channels away from the rotation center.

4. The centrifugal microfluidic chip according to claim 3, characterized in that: The inner diameters of the first channel and the second channel gradually increase as they move away from the connecting intersection.

5. The centrifugal microfluidic chip according to claim 4, characterized in that: The first channel and the second channel are bent toward the rotation center with the connected intersection as a base point to form a bending area; When the repeated switching valve is closed, the position of the phase change material in the first channel and the second channel does not exceed the bending area; As the third channel moves away from the connected intersection, Its inner diameter gradually decreases.

6. The centrifugal microfluidic chip according to claim 2, characterized in that: The energy storage device is a cavity structure.

7. The centrifugal microfluidic chip according to claim 5, characterized in that: The connection points of the first channel, the second channel and the third channel are connected in an arc shape; The minimum cross-sectional position of the first channel and the minimum cross-sectional position of the second channel form a minimum radius with the rotation center; The inner surface of the three-way channel outside the area connecting the two minimum radius lines is subjected to non-wetting treatment.

8. The centrifugal microfluidic chip according to claim 5, characterized in that: A buffer channel is arranged between the proximal end of the third channel and the energy storage device.

9. The centrifugal microfluidic chip according to claim 8, characterized in that: The relationship between the minimum value of the minimum cross-sectional area of ​​the buffer channel and the third channel, the minimum cross-sectional area of ​​the first channel and the minimum cross-sectional area of ​​the second channel satisfies the following formula: ； Wherein, S1 is the minimum cross-sectional area of ​​the buffer channel, S2 is the minimum cross-sectional area of ​​the third channel, S3 is the minimum cross-sectional area of ​​the first channel, and S4 is the minimum cross-sectional area of ​​the second channel.

10. The centrifugal microfluidic chip according to any one of claims 2 to 9, characterized in that: When the flow channel needs to be blocked, the phase change material is heated, and the centrifugal microfluidic chip is controlled to rotate. At a first preset speed, the liquid phase change material moves from the proximal end to the distal end to seal the flow channel between the upstream cavity and the downstream cavity; then the heating is stopped and the liquid phase change material is allowed to solidify; When it is necessary to connect the upstream cavity and the downstream cavity, the solid phase change material is heated, and under the action of negative pressure, the phase change material transformed from solid to liquid flows toward the energy storage device through the third channel. When the upstream cavity and the downstream cavity are connected, the heating of the phase change material is stopped, and the liquid phase change material is allowed to solidify; or, while the solid phase change material is heated, the rotation speed of the centrifugal microfluidic chip is controlled to ensure that the phase change material transformed from solid to liquid flows toward the energy storage device through the third channel at a certain flow rate. When the upstream cavity and the downstream cavity are connected, the heating of the phase change material is stopped, and the liquid phase change material is allowed to solidify.

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