Centrifugal micro-fluidic chip

Abstract

The utility model relates to the technical field of nucleic acid detection, and provides a centrifugal micro-fluidic chip which comprises a body provided with a rotating shaft key groove used for being connected with a power shaft; a detection module is arranged in the body and comprises a functional chamber and a reagent storage chamber; the functional chamber comprises a sample adding chamber, a cracking chamber, a purification chamber, an amplification chamber, a fluorescence detection chamber, a dilution chamber and a test strip pre-storage tank which are sequentially arranged in the direction gradually away from the center of the body; the reagent storage chamber comprises a lysis solution pre-storage chamber, a washing solution pre-storage chamber, an eluent pre-storage chamber, an amplification reagent pre-storage chamber and a diluent storage chamber. The centrifugal micro-fluidic chip provided by the utility model is provided with the functional chamber with more complete functions and the reagent storage chamber matched with the functional chamber, and different detection methods can be selected according to different detection samples, so that the diversity of user selection is increased, the universality and inclusiveness of the chip are improved, and the detection efficiency is favorably improved.

Description

Technical Field

[0001] The utility model relates to the technical field of nucleic acid detection, in particular to a centrifugal microfluidic chip. Background Art

[0002] As an important component of molecular diagnosis, nucleic acid detection can provide accurate diagnostic basis for the detection of pathogens. The centrifugal microfluidic chip in the prior art includes a sample loading chamber, a reagent chamber, a detection chamber and a waste liquid chamber. During detection, the sample is added to the sample loading chamber, and then the centrifugal force is used to drive the sample to flow through different reagent chambers in sequence for release, amplification and mixing, and then the sample is detected in the detection chamber, and the waste liquid after detection is finally discharged into the waste liquid chamber. However, in actual detection, different samples have different detection requirements, and the detection methods used are also different. For example, some samples may need to be lysed, washed, eluted, purified, diluted and other steps before detection. However, the existing centrifugal microfluidic cores are usually designed for specific detection objects, resulting in poor versatility. Utility Model Content

[0003] Therefore, the technical problem to be solved by the present invention is that the existing centrifugal microfluidic core is usually designed for a specific detection object, resulting in poor versatility, and thus a centrifugal microfluidic chip is provided.

[0004] In order to solve the above technical problems, the technical solution of the utility model is as follows:

[0005] The utility model provides a centrifugal microfluidic chip, comprising: a body, on which a rotating shaft keyway for connecting to a power shaft is arranged; a detection module is arranged in the body, and the detection module comprises a functional chamber and a reagent storage chamber; the functional chamber comprises a sample loading chamber, a lysis chamber, a purification chamber, an amplification chamber, a fluorescence detection chamber, a dilution chamber and a test strip pre-storage groove which are arranged in sequence in a direction gradually away from the center of the body; the reagent storage chamber comprises a lysis solution pre-storage chamber, a washing solution pre-storage chamber, an eluent pre-storage chamber, an amplification reagent pre-storage chamber and a diluent storage chamber; wherein the lysis solution pre-storage chamber is connected to the lysis chamber; the washing solution pre-storage chamber is connected to the purification chamber; the eluent pre-storage chamber is connected to the purification chamber; the amplification reagent pre-storage chamber is connected to the amplification chamber; and the diluent storage chamber is connected to the dilution chamber.

[0006] Furthermore, the detection module also includes a washing waste liquid transfer chamber, an elution product transfer chamber, an amplification product transfer channel and an amplification product transfer chamber; the washing waste liquid transfer chamber is connected to the purification chamber; the inlet of the elution product transfer chamber is connected to the purification chamber, and the outlet of the elution product transfer chamber is connected to the amplification chamber; the inlet of the amplification product transfer channel is connected to the amplification chamber, and the outlet of the amplification product transfer channel is divided into a first branch and a second branch, the first branch is connected to the fluorescence detection chamber, the second branch is connected to the inlet of the amplification product transfer chamber, and the outlet of the amplification product transfer chamber is connected to the dilution chamber.

[0007] Furthermore, the detection module further comprises a metering chamber, an inlet of the metering chamber is connected to the first branch, and an outlet of the metering chamber is connected to the fluorescence detection chamber.

[0008] Furthermore, the main body includes an upper cover plate and a channel bottom plate which are stacked; the shaft keyway passes through the upper cover plate and the channel bottom plate in sequence; and the functional chamber and the reagent storage chamber are arranged on the channel bottom plate.

[0009] Furthermore, the upper cover plate is provided with a sample adding hole and a reagent adding hole; the sample adding hole is adapted to the sample adding chamber; and the reagent adding hole is adapted to the reagent storage chamber.

[0010] Furthermore, the centrifugal microfluidic chip also includes a perspective mirror, which is arranged in the test strip pre-storage groove and located above the test strip in the test strip pre-storage groove; and the upper cover plate is provided with an observation port compatible with the perspective mirror.

[0011] Furthermore, the centrifugal microfluidic chip also includes a double-sided pressure-sensitive adhesive pad, and the upper cover plate and the channel bottom plate are connected via the double-sided pressure-sensitive adhesive pad.

[0012] Furthermore, an exhaust microchannel adapted to the functional chamber and the reagent storage chamber is provided on the channel bottom plate, and an exhaust hole is provided on the upper cover plate; one end of the exhaust hole is connected to the exhaust microchannel, and the other end is connected to the external environment.

[0013] Furthermore, different functional chambers, as well as the reagent storage chamber and the functional chamber are all connected by capillary channels; the capillary channels are provided with siphon channel valves and / or capillary channel valves.

[0014] Furthermore, a plurality of the detection modules are arrayed in the main body along the circumferential direction of the main body.

[0015] The technical solution of the utility model has the following advantages:

[0016] The centrifugal microfluidic chip provided by the utility model has a functional chamber with more complete functions and a reagent storage chamber adapted thereto, and different detection methods can be selected for different detection samples, thereby increasing the diversity of user selection, improving the versatility and inclusiveness of the chip, and being conducive to improving detection efficiency; moreover, the setting of the fluorescence detection chamber and the test strip pre-storage slot can realize quantitative and / or qualitative analysis of the detection results, and the results are intuitive, thereby increasing the scope of application of detection occasions, which is of great significance for medical testing and disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the utility model or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the utility model. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 It is a schematic diagram of a centrifugal microfluidic chip in an embodiment of the utility model from one viewing angle;

[0019] Figure 2 This is a schematic diagram of the centrifugal microfluidic chip in an embodiment of the utility model from another perspective;

[0020] Figure 3 It is an exploded schematic diagram of a centrifugal microfluidic chip in an embodiment of the utility model;

[0021] Figure 4 It is a schematic diagram of the bottom surface of the upper cover plate in the centrifugal microfluidic chip in the embodiment of the utility model;

[0022] Figure 5 It is a schematic diagram of the top surface of the channel bottom plate in the centrifugal microfluidic chip in the embodiment of the utility model;

[0023] Figure 6 It is a schematic diagram of a detection module in a centrifugal microfluidic chip in an embodiment of the present utility model.

[0024] Description of reference numerals:

[0025] 1. Upper cover; 2. Channel bottom plate; 3. Rotating shaft keyway; 4. Observation port; 5. Positioning hole; 6. Double-sided pressure-sensitive adhesive pad; 7. Test strip; 8. Periscope; 9. Sample addition hole; 10. Exhaust hole; 11. Detection module; 12. Sample addition chamber; 13. Lysis chamber; 14. Purification chamber; 15. Amplification chamber; 16. Fluorescence detection chamber; 17. Dilution chamber; 18. Test strip pre-storage tank; 19. Lysis chamber liquid pre-storage chamber; 20, washing liquid pre-storage chamber; 21, elution liquid pre-storage chamber; 22, amplification reagent pre-storage chamber; 23, diluent storage chamber; 24, washing waste liquid transfer chamber; 25, elution product transfer chamber; 26, amplification product transfer channel; 27, amplification product transfer chamber; 28, metering chamber; 29, exhaust microchannel; 30, capillary channel; 31, siphon channel valve; 32, capillary channel valve. DETAILED DESCRIPTION

[0026] The technical solution of the utility model will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the utility model, not all of the embodiments. Based on the embodiments of the utility model, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0027] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0028] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installation", "connection" and "connection" 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 components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0029] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] like Figures 1 to 6As shown, this embodiment provides a centrifugal microfluidic chip, including: a body, on which a rotating shaft keyway 3 for connecting to a power shaft is arranged; a detection module 11 is arranged in the body, and the detection module 11 includes a functional chamber and a reagent storage chamber. For example, the body includes an upper cover plate 1 and a channel bottom plate 2 arranged in layers; the rotating shaft keyway 3 can be the center of the body, and pass through the upper cover plate 1 and the channel bottom plate 2 in sequence; the functional chamber and the reagent storage chamber are both arranged on the channel bottom plate 2. Among them, the functional chamber includes a sample loading chamber 12, a lysis chamber 13, a purification chamber 14, an amplification chamber 15, a fluorescence detection chamber 16, a dilution chamber 17 and a test strip pre-storage tank 18 which are arranged in sequence along a direction gradually away from the center of the body; the reagent storage chamber includes a lysis solution pre-storage chamber 19, a washing solution pre-storage chamber 20, an eluent pre-storage chamber 21, an amplification reagent pre-storage chamber 22 and a diluent storage chamber 23; wherein the lysis solution pre-storage chamber 19 is connected to the lysis chamber 13; the washing solution pre-storage chamber 20 is connected to the purification chamber 14; the eluent pre-storage chamber 21 is connected to the purification chamber 14; the amplification reagent pre-storage chamber 22 is connected to the amplification chamber 15; and the diluent storage chamber 23 is connected to the dilution chamber 17.

[0031] The centrifugal microfluidic chip provided in this embodiment has a functional chamber with more complete functions and a reagent storage chamber adapted thereto, and different detection methods can be selected for different detection samples, which increases the diversity of user choices, improves the versatility and inclusiveness of the chip, and is conducive to improving detection efficiency; moreover, the setting of the fluorescence detection chamber 16 and the test strip pre-storage groove 18 can realize quantitative and / or qualitative analysis of the test results, the results are intuitive, and the scope of application of the detection occasions is increased, which is of great significance for medical testing and disease prevention and control.

[0032] Among them, the detection module 11 also includes a washing waste liquid transfer chamber 24, an elution product transfer chamber 25, an amplification product transfer channel 26 and an amplification product transfer chamber 27; the washing waste liquid transfer chamber 24 is connected to the purification chamber 14; the inlet of the elution product transfer chamber 25 is connected to the purification chamber 14, and the outlet of the elution product transfer chamber 25 is connected to the amplification chamber 15; the inlet of the amplification product transfer channel 26 is connected to the amplification chamber 15, and the outlet of the amplification product transfer channel 26 is divided into a first branch and a second branch, the first branch is connected to the fluorescence detection chamber 16, the second branch is connected to the inlet of the amplification product transfer chamber 27, and the outlet of the amplification product transfer chamber 27 is connected to the dilution chamber 17. For example, the above-mentioned different functional chambers, as well as each reagent storage chamber and its adapted functional chamber can be connected by a capillary channel 30; the capillary channel 30 is provided with a siphon channel valve 31 and / or a capillary channel valve 32, for example, the siphon channel valve 31 is formed by a capillary channel 30 bent in an arc shape, and the capillary channel valve 32 is a rectangular groove provided on the capillary channel 30. With such a configuration, the sequential release, mixing, and transportation transfer of different liquids can be achieved by utilizing centrifugal force.

[0033] Among them, the capillary channel 30 between the sample addition chamber 12 and the lysis chamber 13 may not be provided with the siphon channel valve 31 and the capillary channel valve 32; the capillary channel valve 31 may be provided on the capillary channel 30 between the lysis chamber 13 and the lysis solution pre-storage chamber 19; the capillary channel valve 31 may be provided on the capillary channel 30 between the lysis chamber 13 and the purification chamber 14; the capillary channel valve 32 and the siphon channel valve 31 may be provided on the capillary channel 30 between the lysis chamber 13 and the washing solution pre-storage chamber 20; the capillary channel valve 32 and the siphon channel valve 31 may be provided on the capillary channel 30 between the purification chamber 14 and the washing waste liquid transfer chamber 24; the capillary channel valve 32 and the siphon channel valve 31 may be provided on the capillary channel 30 between the dilution chamber 17 and the test strip pre-storage tank 18; the capillary channel valve 32 and the siphon channel valve 31 may be provided on the capillary channel 30 between the amplification product transfer chamber 27 and the amplification product transfer channel 26 1; the capillary channel 30 between the metering chamber 28 and the fluorescence detection chamber 16 may not be provided with the siphon channel valve 31 and the capillary channel valve 32; the capillary channel valve 32 and the siphon channel valve 31 may be provided on the capillary channel 30 between the amplification chamber 15 and the amplification product transfer channel 26; the capillary channel valve 31 and the capillary channel valve 32 may not be provided on the capillary channel 30 between the amplification chamber 15 and the amplification reagent pre-storage chamber 22; the capillary channel valve 31 and the capillary channel valve 32 may not be provided on the capillary channel 30 between the amplification chamber 15 and the elution product transfer channel 26; A siphon channel valve 31 and a capillary channel valve 32 can be set on the capillary channel 30 between the transfer chambers 25; a siphon channel valve 31 and a capillary channel valve 32 can be set on the capillary channel 30 between the purification chamber 14 and the elution product transfer chamber 25; a siphon channel valve 31 and a capillary channel valve 32 can be set on the capillary channel 30 between the purification chamber 14 and the eluent pre-storage chamber 21, and two groups of siphon channel valves 31 and capillary channel valves 32 are alternately set in series.

[0034] The detection module 11 further includes a metering chamber 28, the inlet of the metering chamber 28 is connected to the first branch, and the outlet of the metering chamber 28 is connected to the fluorescence detection chamber 16. A plurality of fluorescence detection chambers 16 may be provided in the same detection module 11, and correspondingly, a plurality of metering chambers 28 are also provided, so that the metering chambers 28 correspond to the fluorescence detection chambers 16 one by one.

[0035] Among them, the upper cover plate 1 is provided with a sample addition hole 9 and a reagent addition hole; the sample addition hole 9 is adapted to the sample addition chamber 12 for adding samples into the sample addition chamber 12; the reagent addition hole is adapted to the reagent storage chamber for adding required reagents into each reagent storage chamber.

[0036] The centrifugal microfluidic chip further includes a perspective mirror 8, which is arranged in the test strip pre-storage slot 18 and is located above the test strip 7 in the test strip pre-storage slot 18; the upper cover plate 1 is provided with an observation port 4 adapted to the perspective mirror 8. When in use, the test strip 7 is installed in the test strip pre-storage slot 18 in advance, and then the perspective mirror 8 is installed in the test strip pre-storage slot 18 and the test strip 7 is pressed. The test strip 7 is generally composed of a sample pad, a test line, a quality control line, an adsorption pad and other parts, and is suitable for quickly and intuitively displaying nucleic acid test results.

[0037] The centrifugal microfluidic chip further comprises a double-sided pressure-sensitive adhesive pad 6, and the upper cover plate 1 and the channel bottom plate 2 are connected via the double-sided pressure-sensitive adhesive pad 6 to improve the integration and sealing performance of the chip.

[0038] Among them, the channel bottom plate 2 is provided with an exhaust microchannel 29 adapted to the functional chamber and the reagent storage chamber, and the upper cover plate 1 is provided with an exhaust hole 10; one end of the exhaust hole 10 is connected to the exhaust microchannel 29, and the other end is connected to the external environment.

[0039] Among them, along the circumferential direction of the main body, a plurality of detection modules 11 are arranged in an array inside the main body, so that the simultaneous detection of multiple sample fluxes can be realized.

[0040] The upper cover plate 1 and the channel bottom plate 2 may also be provided with positioning holes 5 and mounting holes for easy assembly.

[0041] When assembling the centrifugal microfluidic chip: first place the corresponding test strip 7 on the bottom surface of the test strip pre-storage slot 18 of the channel bottom plate 2, install the perspective mirror 8 on the test strip pre-storage slot 18 and the upper surface of the test strip 7, use the double-sided pressure-sensitive adhesive pad 6 to bond the lower surface of the upper cover plate 1 and the upper surface of the channel bottom plate 2 together, and align the positions of the positioning holes 5, the exhaust holes 10, the reagent addition holes, the shaft keyway 3, the functional chamber, the reagent storage chamber, the capillary channel 30, the exhaust microchannel 29, etc.

[0042] Among them, the bonding method of the centrifugal microfluidic chip can be packaged by hot pressing bonding, ultrasonic bonding, etc. in addition to adhesive packaging.

[0043] Among them, since the hydrophilicity of polymer microfluidic chip materials is poor, the liquid infiltration, spreading and fluidity in the microchannel are poor, which reduces the detection accuracy and sensitivity. Therefore, the wall surface of each component can be pre-treated with hydrophilicity, such as using a hydrophilic reagent (2% Tween-20 ethanol solution) for hydrophilic pre-treatment before detection.

[0044] Among them, the centrifugal microfluidic chip can use oxygen plasma and other technologies to treat the chip surface to produce negatively charged hydroxyl groups on its surface, and then modify the chip surface through electrostatic action to inhibit the nonspecific adsorption of biomolecular proteins on the chip surface.

[0045] Among them, chambers, micropores or microchannels of different sizes and shapes on the centrifugal microfluidic chip can be manufactured by processes such as laser etching, CNC micromilling, 3D printing or photolithography.

[0046] Among them, the centrifugal microfluidic chip can be equipped with a corresponding controller, and the controller is electrically connected to the motor control module, the magnet control module, the photoelectric control module and the temperature control module respectively.

[0047] Among them, the motor control module can be located at the bottom of the chip, and cooperate with the chip through the shaft key to control the rotation speed and rotation acceleration of the chip. The magnet control module can be connected to an external magnet or a magnet-like body (such as a magnetic induction coil, etc.), and the magnet or magnet-like body can be located on the outer wall of the purification chamber 14. The photoelectric control module can be connected to the excitation light source and the photoelectric sensor, and the fluorescence detection chamber 16 can be located between the excitation light source and the photoelectric sensor. The temperature control module is connected to the temperature sensor, the heating plate or the heating film, and the temperature sensor, the heating plate or the heating film should be located on the outer wall of the amplification chamber 15 on the chip. The heating method can be local heating or constant temperature heating in the incubation chamber, and on this basis, additional air ducts, fans, or other ventilation and heat dissipation devices can be connected to achieve rapid temperature reduction and reduce liquid evaporation.

[0048] Among them, the centrifugal microfluidic chip can be used in conjunction with a computer or a smart phone, such as by programming and designing supporting application software or APP, connecting the machine through Bluetooth or WIFI to control the machine to perform different operations, and also by using grayscale images, binarization processing, Hough circle transform, and deep learning algorithms to program and design related image recognition software, so that users can more accurately identify the "CT" line detection results on the test strip 7.

[0049] Among them, the working principle of the centrifugal microfluidic chip is: through the cooperation of microfluidic channels of different sizes and shapes, siphon channel valves 31 and capillary channel valves 32, sequential connection between different chambers is realized, the bonded chip is assembled on the motor shaft through the cooperation of keys and key slots, the chip is controlled to work at the specified speed and acceleration through the program burned in the single-chip microcomputer, and the centrifugal force is used to realize the sequential release, mixing and transportation transfer of different liquids. At the same time, corresponding constant temperature heating devices, external magnet control devices and optical path detection devices are equipped according to functional requirements.

[0050] Among them, the lysis method can be physical lysis (such as high temperature, magnetic stirring, ultrasound, etc.) or chemical lysis (such as lysis solution, etc.). The purification method can be a magnetic bead method or a column membrane method, etc. The amplification method can be an isothermal amplification method (such as loop-mediated isothermal amplification method, etc.), and the centrifugal microfluidic chip can perform different operations according to user needs to achieve different functions, such as direct amplification of nucleic acids, amplification after lysis and purification, single-flux or multi-flux combined detection of single or multiple samples, qualitative or quantitative detection based on lateral flow immunoassay strips 7 and immunofluorescence, etc.

[0051] Through force analysis, it can be known that the flow of liquid on the centrifugal microfluidic chip is mainly controlled by centrifugal force, Coriolis force, Euler force, etc. The chip combines the siphon channel valve 31 with the capillary channel valve 32 to control the sequential flow of the liquid. The critical breakthrough angular velocity ω of this type of valve can be obtained by the following formula:

[0052]

[0053] Where ρ is the density of the microfluid, r1 and r2 are the radial inner diameter and radial outer diameter respectively, Δr = |r1-r2 is the fluid filling length, that is, the maximum deviation of the liquid surface of the microfluid in the radial direction, is the theoretical radius of the capillary, i.e., the average value of the radial inner diameter and the radial outer diameter, indicating the distance between the center of the microfluidic and the center of the chip. al is the surface tension of the fluid, θ is the contact angle of the fluid, and D h is the hydraulic diameter of the flow channel at the expansion part.

[0054] The mixing of liquids on the centrifugal microfluidic chip adopts a low-speed mixing method. The operation cycle T0 and the maximum speed V under different displacement ΔX and acceleration A are MAX It can be calculated by the following formula:

[0055]

[0056] Next, we will give an example of how to use the centrifugal microfluidic chip:

[0057] Embodiment 1:

[0058] This embodiment uses a centrifugal microfluidic detection chip for detection using a magnetic bead method, which utilizes the superparamagnetic properties of the magnetic beads and the specific nucleic acid binding properties of their surface modified coatings to perform the relevant purification process. The specific working process is as follows:

[0059] Step 1 (chip manufacturing and packaging): The various components of the centrifugal microfluidic chip are manufactured by laser etching, CNC micro-milling, 3D printing or photolithography. After the chip is prepared, it is first pre-treated with a hydrophilic reagent (2% Tween-20 in ethanol). At the same time, the chip surface can be treated with oxygen plasma and other technologies to produce negatively charged hydroxyl groups on the surface of the chip, and then the chip surface is modified by electrostatic action to inhibit the non-specific adsorption of biomolecule proteins on the chip surface. Then, the test strip 7 and the perspective mirror 8 are pre-placed at the corresponding positions between the channel bottom plate 2 and the upper cover plate 1. Finally, the various components of the chip are bonded by adhesive packaging, hot pressing bonding or ultrasonic bonding. In the chip bonding and packaging process, the lower surface of the upper cover plate 1, the pressure-sensitive adhesive pad and the positioning holes 5, the exhaust holes 10, the reagent addition holes, the shaft key slot 3, the chamber and the microchannel on the upper surface of the channel bottom plate 2 should be connected one by one.

[0060] Step 2 (preparing various required buffers and reagents): for example, sample buffer, nucleic acid lysis extraction solution (containing micro-nano superparamagnetic beads with specific adsorption properties), washing buffer, elution buffer, LAMP reaction mixture (or mix freeze-dried beads) and dilution buffer, etc. In particular, if mix freeze-dried reagent beads are used, the freeze-dried beads should be pre-buried in the amplification chamber 15 on the channel bottom plate 2 before chip bonding.

[0061] Step 3 (machine assembly and startup): After packaging the chip, add the prepared buffer solution and reagents into their respective chambers through the corresponding reagent addition holes, and then install them statically at the designated position of the machine shaft. Select the detection mode as "magnetic bead method" and start the machine. At this time, the machine triggers the corresponding program pre-burned in the microcontroller to work.

[0062] Step 4 (adding sample): the motor controls the chip to centrifuge at 1050 rpm / min in clockwise direction for 20 seconds, and the sample buffer is enriched and transferred from the sample adding chamber 12 to the lysis chamber 13; the motor is controlled to stop rotating and stand for 10 seconds.

[0063] Step 5 (adding lysis solution): the motor control chip centrifuges counterclockwise at a speed of 1050 rpm / min for 20 seconds, controls the motor to stop rotating, and stands for 10 seconds. At this time, the nucleic acid lysis extract is transferred from the lysis solution pre-storage chamber 19 to the lysis chamber 13 through the siphon effect of the siphon channel valve 31.

[0064] Step 6 (lysis and nucleic acid adsorption): The motor controls the chip at ΔX = 5°, A = 10000 rpm / s 2 、T0=0.3s、V MAX=750rpm / min parameter control chip clockwise / counterclockwise cycle rotation for 15s, the sample and nucleic acid lysis extract solution are fully mixed in the lysis chamber 13, the sample is fully lysed, the target nucleic acid molecules are released and specifically bind to the superparamagnetic beads in the lysis solution; the motor is controlled to stop rotating and stand for 10s.

[0065] Step 7 (lysate sample transfer): the motor controls the chip to centrifuge at a speed of 1050 rpm / min in a clockwise direction for 20 seconds, and the lysed sample solution is transferred from the lysate chamber 13 to the purification chamber 14; the motor is controlled to stop rotating and stand for 10 seconds.

[0066] Step eight (washing): the external magnet control module controls the movement of the magnet or magnet-like substance and gradually approaches the outer wall of the purification chamber 14. Under the action of the external magnetic field, the superparamagnetic beads are adsorbed to the wall edge of the purification chamber 14 and remain stationary; the motor control chip is centrifuged at a speed of 1600 rpm / min for 10 seconds in a counterclockwise direction. The washing buffer in the washing liquid pre-storage chamber 20 breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and stand for 5 seconds. The siphon channel valve 31 produces a siphon effect, and the washing buffer gradually fills the entire capillary channel 30. The motor control chip is centrifuged again at a speed of 1600 rpm / min for 20 seconds in a counterclockwise direction, and the washing buffer gradually transfers to the purification chamber 14; the motor is controlled to stop rotating and stand for 10 seconds; the motor control chip is ΔX=5°, A=10000rpm / s 2 、T0=0.3s、V MAX =750rpm / min parameter control chip clockwise / counterclockwise cycle rotation for 15s, the washing buffer and the lysed sample solution are fully mixed, the relevant impurities and pollutants are washed away, and the superparamagnetic beads bound to the nucleic acid molecules remain fixed during the washing process; the motor is controlled to stop rotating and stand for 10s; the motor controls the chip to centrifuge at a speed of 1700rpm / min in a clockwise direction for 10s, and the waste liquid after washing breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and stand for 5s, and the siphon channel valve 31 produces a siphon effect, and the washing waste liquid gradually fills the entire capillary channel 30. The motor controls the chip to centrifuge at a speed of 1700rpm / min again in a clockwise direction for 20s, and the washing waste liquid gradually transfers to the washing waste liquid transfer chamber 24; the motor is controlled to stop rotating and stand for 10s.

[0067] Step nine (elution purification): the motor controls the chip to centrifuge at a speed of 2000rpm / min in a clockwise direction for 10s. The elution buffer in the eluent pre-storage chamber 21 breaks through the first capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and stand for 5s. The first siphon channel valve 31 produces a siphon effect, and the elution buffer gradually fills the upstream capillary channel 30. The motor controls the chip to centrifuge at a speed of 1350rpm / min in a clockwise direction for 15s. The elution buffer breaks through the second capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and stand for 5s. The second siphon channel valve 31 produces a siphon effect, and the elution buffer gradually fills the downstream capillary channel 30. The motor controls the chip to centrifuge at a speed of 1350rpm / min again in a clockwise direction for 10s, and the elution buffer gradually transfers to the purification chamber 14; the motor is controlled to stop rotating and stand for 10s; the motor controls the chip at ΔX=5°, A=10000rpm / s 2 、T0=0.3s、V MAX =750rpm / min, the chip is controlled to rotate clockwise / counterclockwise for 15s, the nucleic acid molecules adsorbed on the superparamagnetic beads are fully eluted, and the purified sample solution after elution is obtained; the motor is controlled to stop rotating and stand still for 10s.

[0068] Step 10 (purified sample transfer): the motor controls the chip to centrifuge at a speed of 1500 rpm / min in a counterclockwise direction for 10 seconds. The purified sample solution in the purification chamber 14 breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating. After standing for 5 seconds, the siphon channel valve 31 produces a siphon effect, and the purified sample solution gradually fills the entire capillary channel 30. The motor controls the chip to centrifuge again at a speed of 1500 rpm / min in a counterclockwise direction for 20 seconds. The purified sample solution gradually transfers to the elution product transfer chamber 25; the motor is controlled to stop rotating. Let it stand for 10 seconds; the motor controls the chip to centrifuge at a speed of 1400 rpm / min in a counterclockwise direction for 10 seconds. The purified sample solution in the elution product transfer chamber 25 breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and let it stand for 5 seconds. The siphon channel valve 31 produces a siphon effect, and the purified sample solution gradually fills the entire capillary channel 30. The motor controls the chip to centrifuge again at a speed of 1400 rpm / min in a counterclockwise direction for 20 seconds. The purified sample solution gradually transfers to the amplification chamber 15; the motor is controlled to stop rotating and let it stand for 10 seconds.

[0069] Step 11 (amplification): If the amplification chamber 15 has been pre-buried with freeze-dried beads for the LAMP reaction, skip this step. If the LAMP reaction mixture is pre-installed, the motor control chip should be centrifuged at a speed of 1050 rpm / min for 20 seconds in a clockwise direction to transfer the LAMP reaction mixture from the amplification reagent pre-storage chamber 22 to the amplification chamber 15; the motor is controlled to stop rotating and stand for 10 seconds; the motor control chip is set at ΔX = 5°, A = 10000 rpm / s 2 、T0=0.3s、V MAX =750rpm / min parameter control chip clockwise / counterclockwise cycle for 15s, LAMP reaction mixture (or mix freeze-dried pellets) and purified sample solution are fully mixed; the external temperature control module controls the heating plate or heating film to heat the amplification chamber 15, and uses the temperature sensor to detect the chamber temperature in real time, and feeds the temperature data back to the single-chip computer program in real time to adjust the PID temperature control parameters, so that the temperature of the amplification chamber 15 is always maintained at around 65°C; the motor is controlled to stop rotating and stand for 10 minutes, so that the target nucleic acid molecules have sufficient time for amplification reaction; the external temperature control module controls the heating plate or heating film to cool down quickly to prevent excessive evaporation of the solution.

[0070] Step 12 (transfer of amplified products): the motor controls the chip to centrifuge at a speed of 1300 rpm / min in a counterclockwise direction for 10 seconds. The reaction solution in the amplification chamber 15 breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and stand for 5 seconds. The siphon channel valve 31 produces a siphon effect, and the reaction solution gradually fills the entire capillary channel 30. The motor controls the chip to centrifuge at a speed of 1300 rpm / min for 20 seconds again in a counterclockwise direction. The reaction solution gradually transfers to the amplified product transfer channel 26; the motor is controlled to stop rotating and stand for 20 seconds to allow the reaction solution to be fully transferred to each metering chamber 28; the motor controls the chip to centrifuge at a speed of 500 rpm / min in a counterclockwise direction for 15 seconds. The reaction solution in each metering chamber 28 breaks through the capillary channel valve 32 under the action of centrifugal force and is transferred to each fluorescence detection chamber 16; the motor is controlled to stop rotating and stand for 10 seconds; the motor controls the chip to centrifuge at a speed of 1350rpm / min in a clockwise direction for 10 seconds, and the remaining reaction solution in the amplification product transfer channel 26 breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating and stand for 5 seconds, and the siphon channel valve 31 produces a siphon effect, and the reaction solution gradually fills the entire capillary channel 30. The motor controls the chip to centrifuge again at a speed of 1350rpm / min in a clockwise direction for 20 seconds, and the reaction solution gradually transfers to the amplification product transfer chamber 27; the motor is controlled to stop rotating and stand for 10 seconds.

[0071] Step 13 (dilution): The motor controls the chip to centrifuge at 1050 rpm / min in a clockwise direction for 40 seconds, and the reaction solution is transferred from the amplification product transfer chamber 27 to the dilution chamber 17, and the dilution buffer is transferred from the dilution liquid storage chamber 23 to the dilution chamber 17; the motor is controlled to stop rotating and stand for 10 seconds; the motor controls the chip at ΔX = 5°, A = 10000 rpm / s 2 、T0=0.3s、V MAX =750rpm / min parameter control chip clockwise / counterclockwise cycle rotation for 15s, the reaction solution and the dilution buffer solution are fully mixed; control the motor to stop rotating, and stand for 10s.

[0072] Step 14 (diluted product transfer): the motor controls the chip to centrifuge at a speed of 1050 rpm / min in a counterclockwise direction for 10 seconds. The reaction diluent in the dilution chamber 17 breaks through the capillary channel valve 32 under the action of centrifugal force and begins to transfer. The motor is controlled to stop rotating. After standing for 5 seconds, the siphon channel valve 31 produces a siphon effect, and the reaction diluent gradually fills the entire capillary channel 30. The motor controls the chip to centrifuge at a speed of 1050 rpm / min again in a counterclockwise direction for 20 seconds. The reaction diluent gradually transfers to the test strip pre-storage tank 18; the motor is controlled to stop rotating, and the detection is completed.

[0073] Step 15 (result display and analysis): The test results can be obtained by directly observing the "CT" line displayed on the test strip 7. If the results are difficult to distinguish with the naked eye, they can also be observed and distinguished with the aid of supporting image recognition software; if quantitative analysis of the test results is required, the photoelectric control module in the operating machine should be used to perform quantitative analysis of the fluorescent products of the reaction solution in the fluorescence detection chamber 16 on the chip.

[0074] Embodiment 2:

[0075] This embodiment is suitable for nucleic acid detection by direct amplification of samples, where the samples do not need to be lysed and purified or the relevant lysed and purified steps have been completed outside the chip. The specific working process is basically the same as that of the first embodiment, with the following differences:

[0076] The sample buffer added in step 2 is a sample solution that does not require lysis and purification; and in step 2, there is no need to add nucleic acid lysis extraction solution, washing buffer and elution buffer.

[0077] In step 3, select the detection mode as "direct amplification method".

[0078] Steps 5, 6, 8 and 9 can be omitted.

[0079] Embodiment three:

[0080] This embodiment uses solid phase extraction to detect nucleic acids, which uses the adsorption and desorption of analytes on a solid matrix to achieve purification and enrichment of liquid samples. The specific working process is basically the same as that of the first embodiment, with the following differences:

[0081] Step 1 Before chip bonding and packaging, the solid-state extraction matrix phase should be coated, filled or micro-processed on the inner wall or close to the inner wall of the purification chamber 14 to form a solid phase extraction adsorbent, and the gel-sol (sol-gel) technology can be used to fix the stationary phase particles and form a continuous filling column. The stationary phase can be C18 silane particles, polystyrene microbeads, silica particles or octadecylsiloxane (ODS) particles, and the recommended method can be a silica gel column method made of silica gel or glass fiber.

[0082] The nucleic acid lysis extraction solution in step 2 may not contain micro-nano superparamagnetic beads.

[0083] In step 3, select the detection mode as "solid phase extraction".

[0084] The nucleic acid adsorption process in step six should be performed after the sample solution is transferred to the purification chamber 14 in step seven.

[0085] The external magnet control module in steps eight and nine does not work.

[0086] Embodiment 4:

[0087] This embodiment provides a centrifugal microfluidic chip developed by combining multiple detection methods. The detection methods include the magnetic bead method, direct amplification method, solid phase extraction method, fluorescence detection method, lateral flow immunoassay strip 7 detection method and LAMP isothermal amplification methods, as well as methods combined with enzyme lysis, thermal lysis, ultrasonic lysis, filter membrane separation, and extraction-free detection using special sample release agents. The specific working processes, types of external devices and control modes under different detection methods are different, and targeted innovative designs need to be carried out in combination with the characteristics of different detection methods, but all can be executed on the centrifugal microfluidic chip in this application.

[0088] In summary, the centrifugal microfluidic chip in this application uses simple centrifugal force to realize a complete set of detection experimental processes on a tiny chip, and integrates multiple detection methods into a chip. Compared with the prior art: comprehensive considerations increase the diversity and inclusiveness of user selection, and by selecting different detection methods for the type of test samples, it is possible to achieve rapid detection of nucleic acids, improve detection efficiency, and achieve qualitative or quantitative analysis of test results. The results are intuitive, increasing the scope of application of the detection occasion (it can be used for on-site POCT qualitative rapid detection, and can also be used for qualitative or quantitative professional detection in laboratories, hospitals and other places), which is of great significance for medical inspection and disease prevention and control. At the same time, the centrifugal microfluidic chip, in conjunction with other external nucleic acid analysis and detection devices, can realize simultaneous detection of multiple samples and multiple indicators, and realize single-flux or multi-flux joint detection of single or multiple samples, which provides a method for effective screening and molecular diagnosis of pathogens, has good applicability and practicality, and is easy to promote.

[0089] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention of the utility model.

Claims

1. A centrifugal microfluidic chip, characterized in that: include: A main body, wherein the main body is provided with a rotating shaft keyway (3) for connecting to a power shaft; A detection module (11) is arranged in the body, and the detection module (11) comprises a functional chamber and a reagent storage chamber; The functional chamber comprises a sample loading chamber (12), a lysis chamber (13), a purification chamber (14), an amplification chamber (15), a fluorescence detection chamber (16), a dilution chamber (17) and a test strip pre-storage tank (18) which are arranged in sequence in a direction gradually away from the center of the body; The reagent storage chamber comprises a lysate pre-storage chamber (19), a washing solution pre-storage chamber (20), an eluent pre-storage chamber (21), an amplification reagent pre-storage chamber (22) and a diluent storage chamber (23); Wherein, the lysis solution pre-storage chamber (19) is connected to the lysis chamber (13); the washing solution pre-storage chamber (20) is connected to the purification chamber (14); the eluent pre-storage chamber (21) is connected to the purification chamber (14); the amplification reagent pre-storage chamber (22) is connected to the amplification chamber (15); and the diluent storage chamber (23) is connected to the dilution chamber (17).

2. The centrifugal microfluidic chip according to claim 1, characterized in that: The detection module (11) further comprises a washing waste liquid transfer chamber (24), an elution product transfer chamber (25), an amplification product transfer channel (26) and an amplification product transfer chamber (27); The washing waste liquid transfer chamber (24) is in communication with the purification chamber (14); The inlet of the elution product transfer chamber (25) is connected to the purification chamber (14), and the outlet of the elution product transfer chamber (25) is connected to the amplification chamber (15); The inlet of the amplification product transfer channel (26) is connected to the amplification chamber (15), and the outlet of the amplification product transfer channel (26) is divided into a first branch and a second branch, the first branch is connected to the fluorescence detection chamber (16), the second branch is connected to the inlet of the amplification product transfer chamber (27), and the outlet of the amplification product transfer chamber (27) is connected to the dilution chamber (17).

3. The centrifugal microfluidic chip according to claim 2, characterized in that: The detection module (11) further comprises a metering chamber (28), the inlet of the metering chamber (28) being connected to the first branch, and the outlet of the metering chamber (28) being connected to the fluorescence detection chamber (16).

4. The centrifugal microfluidic chip according to claim 1, characterized in that: The body comprises an upper cover plate (1) and a channel bottom plate (2) which are stacked; The rotating shaft keyway (3) sequentially passes through the upper cover plate (1) and the channel bottom plate (2); The functional chamber and the reagent storage chamber are arranged on the channel bottom plate (2).

5. The centrifugal microfluidic chip according to claim 4, characterized in that: The upper cover plate (1) is provided with a sample adding hole (9) and a reagent adding hole; The sample adding hole (9) is adapted to be arranged in the sample adding chamber (12); The reagent adding hole is adapted to be arranged in the reagent storage chamber.

6. The centrifugal microfluidic chip according to claim 5, characterized in that: It also includes a perspective mirror (8), which is arranged in the test strip pre-storage groove (18) and is located above the test strip (7) in the test strip pre-storage groove (18); The upper cover plate (1) is provided with an observation port (4) adapted to the perspective mirror (8).

7. The centrifugal microfluidic chip according to claim 4, characterized in that: It also comprises a double-sided pressure-sensitive adhesive pad (6), and the upper cover plate (1) and the channel bottom plate (2) are connected via the double-sided pressure-sensitive adhesive pad (6).

8. The centrifugal microfluidic chip according to claim 4, characterized in that: The channel bottom plate (2) is provided with an exhaust microchannel (29) adapted to the functional chamber and the reagent storage chamber, and the upper cover plate (1) is provided with an exhaust hole (10); One end of the exhaust hole (10) is connected to the exhaust microchannel (29), and the other end is connected to the external environment.

9. The centrifugal microfluidic chip according to claim 1, characterized in that: Different functional chambers, as well as the reagent storage chamber and the functional chamber are all connected by capillary channels (30); The capillary channel (30) is provided with a siphon channel valve (31) and / or a capillary channel valve (32).

10. The centrifugal microfluidic chip according to claim 1, characterized in that: Along the circumferential direction of the main body, a plurality of detection modules (11) are arranged in an array within the main body.

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