Centrifugal microfluidic chip and microfluidic system

Abstract

The application discloses a centrifugal microfluidic chip and a microfluidic system, and relates to the technical field of microfluidic chips.The chip comprises a substrate and a sample processing unit, a liquid storage unit, a transfer unit, a reaction unit and a waste liquid pool integrated on the substrate.The sample processing unit realizes accurate sample quantification through a sample quantitative pool, an auxiliary quantitative pipeline and an overflow through hole;the liquid storage unit comprises a sample adding pool, a cleaning pool and an independent substrate storage cavity connected in series, and realizes reagent quantification in cooperation with an overflow structure;the transfer unit realizes time sequence control release of multiple liquids through a liquid buffer cavity and a switch valve, and distributes the liquids to each reaction cavity through a distribution pipeline;the waste liquid pool adopts a two-stage structure of a buffer cavity and a collection cavity to prevent backflow pollution of waste liquid.The application utilizes centrifugal force to drive liquid flow, realizes accurate quantification of samples and reagents and multi-index parallel detection, effectively avoids cross contamination between reaction cavities, is simple to operate, low in cost and suitable for instant test scenes.

Description

Technical Field

[0001] This invention relates to the technical field of microfluidic chips, and particularly to a centrifugal microfluidic chip and a microfluidic system. Background Technology

[0002] Centrifugal microfluidic chips are miniature analytical devices that use centrifugal force to drive the flow of liquid in microchannels. Due to their advantages such as not requiring external pumps and valves, simple structure, and easy integration, they have received widespread attention in the field of in vitro diagnostics (IVD), especially in point-of-care testing (POCT) scenarios.

[0003] In immunoassay, the combined detection of multiple disease biomarkers can provide richer diagnostic information. In traditional single-chamber multi-marker immunoassay protocols, antigens, antibodies, and reagents corresponding to different markers are mixed in the same reaction space, making antigen-antibody cross-reactions between different markers difficult to avoid.

[0004] Therefore, there is an urgent need to develop a centrifugal microfluidic chip for multi-step reactions to achieve cross-reaction-free multi-index immune detection. Summary of the Invention

[0005] In view of this, the present invention provides a centrifugal microfluidic chip to achieve cross-reactive multi-index immunoassay, thereby improving the universality and reliability of POCT detection. Furthermore, a microfluidic system utilizing the aforementioned centrifugal microfluidic chip is also provided.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A centrifugal microfluidic chip includes a substrate and a sample processing unit, a liquid storage unit, a transfer unit, a reaction unit, and a waste liquid pool integrated on the substrate.

[0008] The sample processing unit includes a sample quantification cell, an auxiliary quantification pipeline, and a sample dilution chamber; the proximal end of the sample quantification cell is connected to an inlet and a first overflow orifice, which is connected to the outside atmosphere; the distal end of the sample quantification cell is connected to the auxiliary quantification pipeline, which is connected to the proximal end of the sample dilution chamber, and the cross-section of the auxiliary quantification pipeline is smaller than the cross-sectional area of ​​the sample quantification cell.

[0009] The liquid storage unit includes a sample addition pool, a substrate storage chamber, at least one washing pool, and multiple liquid storage pools arranged circumferentially; the sample addition pool and the washing pool are connected in series; a first liquid addition port is provided at the proximal end of the sample addition pool, the distal end of the sample addition pool is connected to the sample dilution chamber, a second liquid addition port is connected at the proximal end of the substrate storage chamber, and the liquid storage pools are respectively connected at the distal ends of the substrate storage chamber and the washing pool.

[0010] The transfer unit includes a liquid buffer chamber, and the distal end of the sample dilution chamber and the distal end of the liquid storage tank are configurably connected to the corresponding liquid buffer chamber.

[0011] The liquid buffer chambers are all connected to the reaction chambers of the reaction unit through distribution pipes. There are at least two reaction chambers, which are arranged at circumferential intervals along the distribution pipes and are all connected to the distribution pipes.

[0012] The waste liquid tank includes a waste liquid buffer chamber and a waste liquid collection chamber that are connected. The waste liquid buffer chamber is located at the proximal end of the waste liquid collection chamber. The proximal end of the waste liquid buffer chamber is connected to the reaction chamber, and the distal end of the waste liquid buffer chamber is connected to the waste liquid collection chamber.

[0013] Optionally, in the above-mentioned centrifugal microfluidic chip, the number of the cleaning tanks is two, including a first cleaning tank and a second cleaning tank connected in series radially, wherein the first cleaning tank is connected in series with the sample addition tank.

[0014] The first and second cleaning tanks are each connected to one of the aforementioned liquid storage tanks;

[0015] Furthermore, a first through hole is provided at the proximal end of the cleaning pool at the end.

[0016] Optionally, in the centrifugal microfluidic chip described above, the distal end of the substrate storage chamber is connected to the corresponding liquid storage tank via a first switching valve.

[0017] Optionally, in the above-mentioned centrifugal microfluidic chip, the liquid buffer chamber is divided into a first liquid buffer chamber, a second liquid buffer chamber, and a third liquid buffer chamber; the first liquid buffer chamber, the second liquid buffer chamber, and the third liquid buffer chamber are arranged circumferentially.

[0018] The distal end of the sample dilution chamber is connected to the proximal end of the first liquid buffer chamber via a second switching valve; the distal end of the storage tank connected to the washing tank is connected to the proximal end of the second liquid buffer chamber via corresponding switching valves; and / or, the distal end of the storage tank connected to the substrate storage chamber is connected to the proximal end of the third liquid buffer chamber via a fifth switching valve.

[0019] Optionally, in the above-mentioned centrifugal microfluidic chip, the distribution channel is a reciprocating arc-shaped channel along the circumferential direction, and the distribution channel includes a connected proximal segment and a distal segment, and the connection between the proximal segment and the distal segment forms a bending region, and both the proximal segment and the distal segment are arc-shaped structures.

[0020] The proximal segment gradually moves away from the rotation center of the substrate from the initial region of the proximal segment toward the bending region, and the distal segment gradually moves away from the rotation center of the substrate from the bending region toward the end region of the distal segment.

[0021] The distal end of all the liquid buffer chambers is connected to the proximal section of the distribution pipe, and the proximal end of all the reaction chambers is connected to the distal section of the distribution pipe.

[0022] Optionally, in the above-mentioned centrifugal microfluidic chip, the waste liquid pool further includes a first waste liquid chamber and a second waste liquid chamber, and the proximal end of the first waste liquid chamber is connected to a third through hole;

[0023] The distal end of the distal section is connected to the proximal end of the first waste liquid chamber, the bend of the distribution pipe is connected to the proximal end of the second waste liquid chamber, and the flow area at the connection between the second waste liquid chamber and the bend is smaller than the flow area at the connection between the distribution pipe and the reaction chamber.

[0024] The reaction chamber is connected to the region between the bent region and the end region.

[0025] Optionally, in the above-mentioned centrifugal microfluidic chip, the second waste liquid chamber has a double-layer structure, including a first chamber and a second chamber distributed radially along the substrate. The proximal end of the first chamber is connected to the distribution channel, the distal end of the first chamber is connected to the proximal end of the second chamber, and the flow area at the connection between the first chamber and the bend region is smaller than the flow area at the connection between the reaction chamber and the distribution channel.

[0026] Optionally, in the centrifugal microfluidic chip described above, the distal end region of the distal segment is connected to the proximal end of the first waste liquid chamber via a curved second transfer channel, which extends radially along the substrate and reciprocates in the circumferential direction of the substrate.

[0027] Optionally, in the centrifugal microfluidic chip described above, the proximal end of the reaction chamber is connected to the distal end of the distribution pipe via a connecting pipe;

[0028] The depth of the reaction chamber in the substrate is greater than the depth of the connecting pipe in the substrate.

[0029] Optionally, in the centrifugal microfluidic chip described above, the flow area of ​​the connecting pipe gradually decreases and then gradually increases from the distribution pipe toward the reaction chamber.

[0030] Optionally, in the centrifugal microfluidic chip described above, the waste liquid buffer chamber is connected to the reaction chamber through a third transfer pipe, and the third transfer pipe extends radially along the substrate, and the third transfer pipe is a pipe that reciprocates in the circumferential direction of the substrate.

[0031] Optionally, in the above-mentioned centrifugal microfluidic chip, the volume of the waste liquid buffer chamber is smaller than the volume of the waste liquid collection chamber.

[0032] Optionally, in the above-mentioned centrifugal microfluidic chip, the sample inlet, the first liquid inlet, and the second liquid inlet are all located on the back side of the substrate.

[0033] Optionally, the centrifugal microfluidic chip described above further includes a blood cell storage structure, wherein the blood cell storage structure is disposed at the distal end of the sample quantification cell, and the proximal end of the blood cell storage structure is connected to the distal end of the sample quantification cell.

[0034] A microfluidic system includes a centrifugal microfluidic chip, wherein the centrifugal microfluidic chip is any one of the centrifugal microfluidic chips described above.

[0035] Optionally, the microfluidic system also includes a tray, which is arranged opposite to the centrifugal microfluidic chip along the thickness direction of the centrifugal microfluidic chip;

[0036] The tray is equipped with a magnetic suction device, which is configured to correspond one-to-one with the reaction chamber of the centrifugal microfluidic chip.

[0037] This invention discloses a centrifugal microfluidic chip, wherein the number of reaction chambers is at least two, both located between a first waste liquid chamber and a second waste liquid chamber. Each reaction chamber is connected to a dispensing pipe for parallel detection of multiple indicators. After liquid is dispensed from the reaction chambers, the liquid path connection between each reaction chamber can be severed using the dispensing pipe. Each reaction chamber can correspond to a different detection target. This configuration allows multiple reaction chambers to be physically independent, connected only upstream of each chamber via dispensing pipes. Furthermore, the dispensing pipes isolate the reaction chambers after each liquid addition, ensuring complete isolation during the incubation phase and fundamentally avoiding cross-reaction problems between antibodies and antigens targeting different targets in multi-indicator immunoassay.

[0038] In addition, lyophilized reagents can be pre-stored in the reaction chamber so that all reaction chambers can use the same set of sample processing units and liquid storage units, which helps to improve the space utilization of centrifugal microfluidic chips and can also expand the number of indicators to achieve multi-indicator detection.

[0039] In addition, the sample processing unit is equipped with a sample quantification chamber, an auxiliary quantification channel, and a first overflow orifice. The cross-section of the auxiliary quantification channel is smaller than that of the sample quantification chamber. It utilizes capillary force to confine the liquid within the sample quantification chamber, and excess sample is discharged through the first overflow orifice during sample addition, thus achieving accurate sample quantification without relying on precision pipettes or specialized operators. Simultaneously, the sample addition chamber, washing chamber, and substrate storage cavity in the liquid storage unit, in conjunction with the overflow orifice, achieve liquid quantification, enabling precise measurement of samples and various reagents on the chip. This significantly lowers the operational threshold for on-site testing and enhances the universality and reliability of POCT testing. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is a front view of the centrifugal microfluidic chip disclosed in an embodiment of the present invention;

[0042] Figure 2a This is a liquid flow diagram of the first working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0043] Figure 2b This is a liquid flow diagram of the second working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0044] Figure 2c This is a liquid flow diagram of the third working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0045] Figure 2d This is a liquid flow diagram of the fourth working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0046] Figure 2e This is a liquid flow diagram of the fifth working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0047] Figure 2fThis is a liquid flow diagram of the sixth working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0048] Figure 2g This is a liquid flow diagram of the seventh working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0049] Figure 2h This is a liquid flow diagram of the eighth working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0050] Figure 2i This is a liquid flow diagram of the ninth working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0051] Figure 2j This is a liquid flow diagram of the tenth working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0052] Figure 2k This is a liquid flow diagram of the eleventh working stage of the centrifugal microfluidic chip in Embodiment 1 of the present invention;

[0053] Figure 3a This is a partial structural diagram of the microfluidic chip system in Embodiment 2 of the present invention;

[0054] Figure 3b This is a front view of a partial structure of the microfluidic chip system in Embodiment 2 of the present invention;

[0055] Figure 4a This is a liquid flow diagram of the first working stage of the centrifugal microfluidic chip in Embodiment 3 of the present invention;

[0056] Figure 4b This is a liquid flow diagram of the second working stage of the centrifugal microfluidic chip in Embodiment 3 of the present invention;

[0057] Figure 4c This is a liquid flow diagram of the third working stage of the centrifugal microfluidic chip in Embodiment 3 of the present invention;

[0058] Figure 4d This is a liquid flow diagram of the fourth working stage of the centrifugal microfluidic chip in Embodiment 3 of the present invention. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] It should be noted that, for ease of description, only the parts relevant to the application are shown in the accompanying drawings. Unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0061] As indicated in this application and claims, unless the context clearly indicates otherwise, the words "a," "an," "a," and / or "the" are not specifically singular and may include the plural. Generally, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements. An element defined by the phrase "comprising an..." does not exclude the presence of other identical elements in the process, method, product, or apparatus that includes the element.

[0062] Hereinafter, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

[0063] like Figure 1 As shown in the figure, this application discloses a centrifugal microfluidic chip 1000, which includes a substrate and a sample processing unit, a liquid storage unit, a transfer unit, a reaction unit and a waste liquid pool integrated on the substrate.

[0064] The sample processing unit includes an inlet 101, a sample quantification chamber 103, a first overflow orifice 102, an auxiliary quantification pipeline 104, a blood cell storage structure 105, and a sample dilution chamber 106.

[0065] In this embodiment, the sample inlet 101 is located on the back of the substrate. The sample inlet 101 has a tapered countersunk hole structure. Optionally, the taper of the tapered countersunk hole is 30° to 60°. The larger end of the tapered countersunk hole faces the outside of the substrate, and the smaller end connects to the proximal end of the sample quantitative cell 103. That is, the sample inlet 101 is chamfered. With this configuration, the sample inlet 101 can be adapted to the outlet of pipette tips and Pasteur pipettes of different specifications. The chamfering further guides the liquid to flow smoothly, avoiding liquid residue or splashing at the sample inlet during sample addition, thereby reducing the difficulty of operation and allowing non-professionals to complete the sample addition operation using crude tools such as Pasteur pipettes.

[0066] The sample quantitative cell 103 is a groove structure with a quantitative chamber opened on the substrate, and the proximal end of the sample quantitative cell 103 is connected to the above-mentioned inlet 101 and the first overflow hole 102.

[0067] The first overflow orifice 102 penetrates the substrate and is connected to the outside atmosphere. Optionally, the diameter of the first overflow orifice 102 is larger than the diameter of the sample inlet 101. The first overflow orifice 102 is used to discharge excess sample exceeding the volume of the sample quantitative cell 103. With this configuration, excess sample can be smoothly discharged from the first overflow orifice 102 during sample addition, avoiding inaccurate quantification due to poor discharge. In addition, the first overflow orifice 102 is connected to the outside atmosphere, allowing gas in the sample quantitative cell 103 to be discharged in a timely manner during sample addition and centrifugation, ensuring smooth liquid flow.

[0068] In this embodiment, the distal end of the sample quantitative cell 103 is connected to the auxiliary quantitative pipe 104. The cross-sectional area of ​​the auxiliary quantitative pipe 104 is smaller than that of the sample quantitative cell 103. Optionally, the cross-sectional area of ​​the auxiliary quantitative pipe 104 is 1 / 50 to 1 / 10 of the cross-sectional area of ​​the sample quantitative cell 103, but it is not limited to this.

[0069] Because the cross-sectional area of ​​the auxiliary quantitative pipe 104 is very small, the surface tension of its inner wall can bind the liquid sample in the sample quantitative pool 103 within the sample quantitative pool 103, thereby achieving sample quantification.

[0070] The auxiliary quantitative conduit 104 is connected to the proximal end of the sample dilution chamber 106. This configuration allows the auxiliary quantitative conduit 104 to utilize capillary force to confine the liquid within the sample dilution chamber 103 during sample addition. This prevents the liquid from prematurely flowing into the downstream chamber due to gravity, even if the added sample volume far exceeds the volume of the sample dilution chamber 103. Only after applying sufficient centrifugal force can the liquid overcome the capillary force and enter the auxiliary quantitative conduit 104, thus achieving stepwise control of quantification followed by transfer and ensuring quantitative accuracy.

[0071] Optionally, the first overflow orifice 102 is positioned on the substrate higher than the connection point between the auxiliary quantitative channel 104 and the sample quantitative cell 103, meaning the first overflow orifice 102 is closer to the rotation center of the substrate. With this configuration, excess sample is preferentially discharged from the higher-positioned first overflow orifice 102 under the influence of gravity during sample addition, rather than entering the lower-positioned auxiliary quantitative channel 104, further ensuring quantitative accuracy.

[0072] The blood cell storage structure 105 is located at the distal end of the sample quantification pool 103, and the proximal end of the blood cell storage structure 105 is connected to the distal end of the sample quantification pool 103.

[0073] Optionally, the blood cell storage structure 105 is a narrow groove extending along the centrifugal force direction. The width of the groove can be from 0.5 mm to 2 mm, and the volume of the groove is set to accommodate all the blood cells in a single test sample. With this configuration, when the sample is a whole blood sample, the blood cells, due to their higher density, are thrown into the blood cell storage structure 105 and accumulate under high centrifugal speed, while the plasma, due to its lower density, enters the sample dilution chamber 106 through the auxiliary quantitative channel 104, thus achieving plasma separation from the whole blood sample. This eliminates the need for pre-centrifugation of the whole blood sample, simplifying the operation process.

[0074] The sample dilution chamber 106 is used to mix the sample, and its volume is larger than that of the sample quantification chamber 103. This configuration provides ample space for mixing the sample and the diluent, which is beneficial for thorough mixing of the sample and the diluent during high-speed and low-speed oscillation.

[0075] The liquid storage unit includes a sample addition tank 201, a substrate storage chamber 204, a first cleaning tank 202, and a second cleaning tank 203 arranged circumferentially. In addition, the liquid storage unit also includes a first liquid storage tank 211, a second liquid storage tank 212, and a third liquid storage tank 213 arranged circumferentially.

[0076] Please continue reading. Figure 1 The sample loading tank 201, the first cleaning tank 202, and the second cleaning tank 203 are all U-shaped tanks connected in series. Each U-shaped tank has a U-shaped bend structure, and the bottom of the bend structure of each U-shaped tank is located on the same arc trajectory, with the center of rotation of the substrate as the center. This configuration allows the U-shaped bend structure to effectively trap liquid in the centrifugal force field, preventing premature outflow before reaching the predetermined centrifugal speed. The fact that the bottom of each U-shaped tank is on the same arc trajectory ensures that the liquid in each U-shaped tank experiences the same centrifugal acceleration under centrifugal force, guaranteeing that the liquid transfer in each U-shaped tank is completed simultaneously and synchronously, improving operational consistency and controllability.

[0077] The liquid storage unit can also be equipped with an additional number of cleaning tanks.

[0078] A first liquid inlet 301 is provided at the proximal end of the sample loading chamber 201. Optionally, the first liquid inlet 301 is located on the back side of the substrate. In this example, the first liquid inlet 301 is a conical countersunk hole structure with a taper of 30° to 60°. The larger end of the conical countersunk hole faces outward from the substrate, and the smaller end connects to the proximal end of the sample loading chamber 201. In this embodiment, the depth of the first liquid inlet 301 can be 1 mm to 3 mm, and the edge of the first liquid inlet 301 is chamfered. With this configuration, the structure of the first liquid inlet 301 is the same as that of the sample inlet 101, and it is also compatible with pipettes and Pasteur pipettes for liquid addition, further reducing the difficulty of operation.

[0079] The end of the sample addition tank 201 is connected to the beginning of the first cleaning tank 202 via a pipe 302, and the end of the first cleaning tank 202 is connected to the beginning of the second cleaning tank 203 via a pipe 302, thus realizing the sequential connection of the three U-shaped tanks. The end of the second cleaning tank 203 is provided with a first through hole 601, which is connected to the outside atmosphere.

[0080] During the process of adding liquid to the first liquid inlet 301, the liquid sequentially fills the sample loading tank 201, the first cleaning tank 202, and the second cleaning tank 203 through the pipe 302. Excess liquid overflows from the first through hole 601, thereby achieving quantitative liquid distribution. In this embodiment, the sample loading tank 201, the first cleaning tank 202, and the second cleaning tank 203 share a single liquid inlet and an overflow hole, which simplifies the structure. Furthermore, the sample loading tank 201, the first cleaning tank 202, and the second cleaning tank 203 are arranged in series, occupying less radial space on the centrifugal microfluidic chip, which is beneficial for the miniaturization and integration of the centrifugal microfluidic chip.

[0081] In this embodiment, the distal end of the sample loading chamber 201 is connected to the sample dilution chamber 106, the distal end of the first washing chamber 202 is connected to the first storage tank 211, and the distal end of the second washing chamber 203 is connected to the second storage tank 212 via first transfer pipes 303. That is, the distal end of the sample loading chamber 201 is connected to the sample dilution chamber 106 via one first transfer pipe 303, the distal end of the first washing chamber 202 is connected to the first storage tank 211 via another first transfer pipe 303, and the distal end of the second washing chamber 203 is connected to the second storage tank 212 via yet another first transfer pipe 303.

[0082] With this configuration, the liquid in each U-shaped pool can be transferred to its corresponding downstream chamber or storage pool during high-speed centrifugation, realizing the synchronous transfer of different liquids in the same centrifugation step and simplifying the operation process.

[0083] A second liquid inlet 312 is connected to the proximal end of the substrate storage cavity 204. Optionally, the second liquid inlet 312 is located on the back side of the substrate. In this example, the second liquid inlet 312 is a conical countersunk hole structure with a taper of 30° to 60°. The edges of the second liquid inlet 312 are chamfered. With this configuration, the structure of the second liquid inlet 312 is the same as that of the sample inlet 101, and it is also compatible with pipettes and Pasteur pipettes for liquid addition, further reducing the difficulty of operation.

[0084] The substrate storage chamber 204 is an elongated chamber extending in the direction of centrifugal force. A fourth through hole 604 is provided at the distal end of the substrate storage chamber 204. Optionally, the fourth through hole 604 penetrates the substrate to discharge excess substrate exceeding the volume of the substrate storage chamber 204. During the process of adding liquid to the second liquid inlet 312, excess liquid is discharged from the fourth through hole 604, thereby achieving quantitative distribution of the substrate.

[0085] In this embodiment, the substrate storage chamber 204 is a U-shaped structure independent of the series structure of the washing chamber and the sample addition chamber 201. It has an independent liquid addition port and an overflow hole, which allows the addition and quantitative operation of the substrate to be carried out independently without being affected by the general solution addition operation. This facilitates the preparation of centrifugal microfluidic chips and reagent preloading before the experiment.

[0086] It should be noted that, in order to ensure the smooth flow of liquid, the first liquid storage tank 211, the second liquid storage tank 212 and the third liquid storage tank 213 in this embodiment are all provided with a second through hole 602 at their proximal ends to discharge the gas in the first liquid storage tank 211, the second liquid storage tank 212 and the third liquid storage tank 213, so as to ensure the smooth flow of liquid.

[0087] The distal end of the substrate storage chamber 204 is connected to the third liquid storage tank 213 via a first switching valve 705. Optionally, the first switching valve 705 is a column valve. In this embodiment, the first switching valve 705 is a normally closed valve, which can be opened when the column valve moves in a direction perpendicular to the substrate.

[0088] After the first switching valve 705 is opened, the substrate in the substrate storage chamber 204 is transferred to the third liquid reservoir 213 under centrifugal force. With this configuration, the substrate can be pre-loaded into the substrate storage chamber 204 during the centrifugal microfluidic chip fabrication stage. During use, the substrate can be released simply by opening the first switching valve 705, eliminating the need for on-site substrate addition, further simplifying the operation and reducing operational errors.

[0089] The transfer unit includes a first liquid buffer chamber 304, a second liquid buffer chamber 307, a third liquid buffer chamber 308, a fourth transfer pipe 305, a distribution pipe 306, and a second transfer pipe 311.

[0090] The first liquid buffer chamber 304, the second liquid buffer chamber 307, and the third liquid buffer chamber 308 are disposed circumferentially on the substrate. The shape and size of the first liquid buffer chamber 304, the second liquid buffer chamber 307, and the third liquid buffer chamber 308 can be configured according to different needs and are not limited here.

[0091] The distal end of the sample dilution chamber 106 is connected to the proximal end of the first liquid buffer chamber 304 via a second switching valve 701. When the second switching valve 701 is opened, liquid enters the first liquid buffer chamber 304 from the sample dilution chamber 106 under centrifugal force. This configuration allows the second switching valve 701 to control the release of the sample diluent, ensuring that the sample diluent is fully mixed in the dilution chamber before release, thus guaranteeing sufficient reaction time between the sample and the diluent. Simultaneously, the first liquid buffer chamber 304 serves as an intermediate storage chamber for liquid transfer from the sample dilution chamber 106 to the distribution pipe 306, acting as a buffer and flow stabilizer to prevent uneven distribution due to unstable flow rate when the liquid directly enters the distribution pipe 306.

[0092] The distal end of the first liquid storage tank 211 is connected to the proximal end of the second liquid buffer chamber 307 via a third switching valve 702. When the third switching valve 702 is opened, the liquid is transferred from the first liquid storage tank 211 to the second liquid buffer chamber 307 under centrifugal force. In this configuration, the first liquid storage tank 211 serves as a temporary storage chamber for the first cleaning solution, allowing the first cleaning solution to be stored in the first liquid storage tank 211 after high-speed centrifugation. When cleaning is required, it is released by opening the third switching valve 702, achieving time-sharing release control of the cleaning solution.

[0093] The distal end of the second liquid storage tank 212 is connected to the proximal end of the second liquid buffer chamber 307 via a fourth switching valve 703. When the fourth switching valve 703 is opened, the liquid is transferred from the second liquid storage tank 212 to the second liquid buffer chamber 307 under centrifugal force. With this configuration, the second liquid storage tank 212 serves as a temporary storage chamber for the second cleaning fluid, enabling the time-separated release of the second and first cleaning fluids, allowing the two cleaning operations to be performed independently at different times.

[0094] The distal end of the third storage tank 213 is connected to the proximal end of the third liquid buffer chamber 308 via a fifth switching valve 704. When the fifth switching valve 704 is opened, the liquid is transferred from the third storage tank 213 to the third liquid buffer chamber 308 under centrifugal force. In this configuration, the third storage tank 213 serves as a temporary storage chamber for the substrate solution, allowing the substrate solution to be released into the reaction chamber only after the reaction is complete. This avoids premature contact between the substrate and the reaction system in the reaction chamber, ensuring the timing control of the substrate catalytic reaction.

[0095] Optionally, the second switching valve 701, the third switching valve 702, the fourth switching valve 703, and the fifth switching valve 704 are all column valves. The second switching valve 701, the third switching valve 702, the fourth switching valve 703, and the fifth switching valve 704 are opened and closed by a pin device on the instrument, which is simple and reliable to operate.

[0096] The distal ends of the first liquid buffer chamber 304, the second liquid buffer chamber 307, and the third liquid buffer chamber 308 are respectively connected to the distribution pipe 306 via the fourth transfer pipe 305. This configuration allows the three liquid buffer chambers to share the same distribution pipe 306, reducing the number of pipes on the centrifugal microfluidic chip, simplifying its structure, and facilitating miniaturization and low-cost manufacturing of the centrifugal microfluidic chip.

[0097] In this embodiment, the distribution pipe 306 is a reciprocating arc-shaped pipe along the circumference. In the example, the distribution pipe 306 includes a connected proximal section and a distal section.

[0098] In this section, the proximal segment, being closer to the rotation center, forms a bend at the junction with the distal segment, and both the proximal and distal segments are arc-shaped. The proximal segment gradually moves away from the rotation center of the substrate from its initial region towards the bend, while the distal segment gradually moves away from the rotation center of the substrate from its bend towards its final region.

[0099] The distal ends of all liquid buffer chambers are connected to the proximal section of distribution pipe 306, that is, the distal ends of the first liquid buffer chamber 304, the second liquid buffer chamber 307, and the third liquid buffer chamber 308 are all connected to the proximal section of distribution pipe 306. The proximal ends of all reaction chambers 402 are connected to the distal section of distribution pipe 306.

[0100] In this embodiment, both the proximal and distal sections of the distribution pipe 306 are arc-shaped and extend circumferentially, allowing the liquid to be distributed sequentially to each reaction chamber when flowing along the distribution pipe 306 under centrifugal force. The bend at the connection between the proximal and distal sections changes the direction of liquid flow, altering the flow velocity and direction as the liquid enters the distal section, which is beneficial for the uniform distribution of liquid among the reaction chambers. Simultaneously, the distal ends of each liquid buffer chamber are connected to the proximal section of the distribution pipe 306, and the proximal ends of each reaction chamber are connected to the distal section of the distribution pipe 306, enabling liquids from different sources (sample diluent, washing solution, substrate solution) to enter each reaction chamber through the same distribution pipe 306, achieving resource sharing and simplifying the centrifugal microfluidic chip structure.

[0101] The end of the distribution pipe 306 is the second transfer pipe 311, which extends radially along the substrate and bends back and forth in the circumferential direction of the substrate. The second transfer pipe 311 is connected to the first waste liquid chamber 503 of the waste liquid pool.

[0102] In this embodiment, the second transfer channel 311 bends back and forth in the circumferential direction of the substrate, increasing the total length and flow resistance of the second transfer channel 311. This results in the liquid flowing at a significantly slower speed in the second transfer channel 311 than in the distribution channel 306. When excess liquid enters the second transfer channel 311, due to the high flow resistance, the liquid continues to flow slowly in the second transfer channel 311, generating a continuous traction force that gradually pulls the residual liquid in the distribution channel 306 into the first waste liquid chamber 503, ultimately causing the distribution channel 306 to be completely emptied. This structure can achieve physical isolation between the reaction chambers 402, completely eliminating liquid crosstalk between different reaction chambers 402 caused by sharing the second transfer channel 311.

[0103] It should be noted that a third through hole 603 is provided at the proximal end of the first waste liquid chamber 503 to discharge the gas in the first waste liquid chamber 503 and ensure that the liquid can smoothly enter the first waste liquid chamber 503.

[0104] The bend in the distribution pipe 306 connects to the proximal end of the second waste liquid chamber 504 in the waste liquid pool. Optionally, the proximal end of the second waste liquid chamber 504 is connected via a connecting pipe, and the flow area of ​​the connecting pipe is smaller than the flow area at the connection between the distribution pipe 306 and the reaction chamber 402. This arrangement ensures that the resistance at the connecting pipe is greater than the resistance at the connection between the distribution pipe 306 and the reaction chamber 402. During the filling process of the reaction chamber 402 by the liquid in the distribution pipe 306, the liquid will not enter the connecting pipe. This ensures that the liquid preferentially flows along the distribution pipe 306 into each reaction chamber 402 of the reaction unit, rather than into the second waste liquid chamber 504, guaranteeing normal liquid distribution. Furthermore, residual liquid in the distribution pipe 306 and the fourth transfer pipe 305, etc., enters the second waste liquid chamber 504 through the bend under greater centrifugal force, preventing residual liquid from being ejected and causing contamination during subsequent operations.

[0105] In some embodiments, the second waste liquid chamber 504 has a double-layer structure, including a first chamber and a second chamber distributed radially along the substrate.

[0106] In this design, the proximal end of the first chamber connects to the bend in the distribution pipe 306, while the distal end of the first chamber connects to the second chamber via a pipe with a flow area smaller than that of the distribution pipe 306. This arrangement ensures that during the transfer of liquid from the reaction chamber 402 to the waste liquid collection chamber 502, residual liquid in the first liquid buffer chamber 304 and the fourth transfer pipe 305 is transferred into the second waste liquid chamber 504 under centrifugal force. The double-layer structure of the second waste liquid chamber 504 allows the waste liquid to first enter the first chamber and then slowly flow into the second chamber for storage through a narrower pipe. Because the second chamber is located at the distal end and the flow area of ​​the connecting pipe is very small, once the waste liquid enters the second chamber, it is less likely to be ejected during the oscillation or low-speed centrifugation of the centrifugal microfluidic chip, thus avoiding contamination of other areas of the centrifugal microfluidic chip. This structural design fully considers the operational characteristics of the centrifugal microfluidic chip, which requires multiple oscillations and variable-speed centrifugation during the detection process, ensuring the cleanliness of the centrifugal microfluidic chip throughout the entire operation.

[0107] The reaction unit in this embodiment includes a connecting pipe 401 and at least two reaction chambers 402.

[0108] The proximal ends of each reaction chamber 402 are connected to the distal ends of the distribution pipe 306 via connecting pipe 401.

[0109] In this embodiment, the flow area of ​​the connecting pipe 401 gradually decreases and then gradually increases from the distribution pipe 306 toward the reaction chamber 402. The variable cross-section structure of the connecting pipe 401, which gradually decreases and then gradually increases, serves both a throttling and buffering function, preventing liquid from rushing into the reaction chamber 402 at excessively high flow rates and causing reagent splashing or loss, while also avoiding excessive resistance to liquid flow.

[0110] The reaction chamber 402 is deeper in the substrate than the connecting pipe 401, which gives the reaction chamber 402 a larger volume and a larger inner wall area. This is beneficial for the fixation of captured antibodies and the containment of reaction reagents, and helps to prevent crosstalk between adjacent reaction chambers 402.

[0111] In this embodiment, there are at least two reaction chambers 402, both located between the first waste liquid chamber 503 and the second waste liquid chamber 504. Each reaction chamber 402 is connected to a dispensing pipe 306 for parallel detection of multiple indicators. The reaction chambers 402 are arranged circumferentially at intervals, and each chamber corresponds to a different detection target. During the centrifugal microfluidic chip fabrication stage, capture antibodies are immobilized and sealed on the inner wall of each reaction chamber 402. Then, lyophilized enzyme-labeled detection antibodies are placed in each reaction chamber 402, with each chamber corresponding to a different target. This arrangement ensures that the multiple reaction chambers 402 are physically independent, connected only to the upstream via the dispensing pipe 306. The dispensing pipe 306 can be completely emptied after each liquid addition, ensuring complete isolation of each reaction chamber 402 during the incubation stage. This fundamentally avoids the cross-reaction problem between antibodies and antigens of different targets in multi-indicator immunoassay.

[0112] The waste liquid tank includes a waste liquid buffer chamber 501, a waste liquid collection chamber 502, a first waste liquid chamber 503, and a second waste liquid chamber 504.

[0113] The waste liquid buffer chamber 501 and the waste liquid collection chamber 502 are interconnected. Specifically, the waste liquid buffer chamber 501 is located at the proximal end of the waste liquid collection chamber 502. The proximal end of the waste liquid buffer chamber 501 is connected to the reaction chamber 402 through the third transfer pipe 309, and the distal end of the waste liquid buffer chamber 501 is connected to the waste liquid collection chamber 502 through the fifth transfer pipe 310.

[0114] In this embodiment, the volume of the waste liquid buffer chamber 501 is smaller than the volume of the waste liquid collection chamber 502. That is, the volume of the waste liquid collection chamber 502 is sufficient to hold all the waste liquid, so no waste liquid is stored in the waste liquid buffer chamber 501.

[0115] It should be noted that a high centrifugal speed is required to transfer the liquid from reaction chamber 402 to waste liquid collection chamber 502, but waste liquid collection chamber 502 lacks a ventilated structure connecting it to the outside. If waste liquid collection chamber 502 were connected to the outside, some liquid would prematurely enter waste liquid collection chamber 502 due to gravity or centrifugal force during the liquid distribution process into reaction chamber 402, resulting in insufficient liquid volume in reaction chamber 402, incomplete dissolution of lyophilized reagents, and uncontrollable reaction volume. Therefore, the waste liquid collection chamber 502 in this application is a sealed structure. During centrifugal waste discharge, the gas in waste liquid collection chamber 502 is compressed. If a waste liquid buffer chamber 501 is not provided, during centrifugal stopping or deceleration, the compressed gas in waste liquid collection chamber 502 will force a small amount of waste liquid back into reaction chamber 402 along the fifth transfer pipe 310, causing contamination of reaction chamber 402. In this embodiment, the volume of the waste liquid collection chamber 502 is sufficient to hold all the waste liquid. The waste liquid buffer chamber 501 does not store waste liquid. Therefore, the compressed gas only pushes the gas in the waste liquid buffer chamber 501 back into the reaction chamber 402, and does not push the waste liquid back, thereby effectively preventing the waste liquid from flowing back and contaminating the reaction chamber 402.

[0116] Optionally, the third transfer channel 309 extends radially along the substrate, and the third transfer channel 309 is a channel that bends back and forth in the circumferential direction of the substrate. That is, the third transfer channel 309 is a channel that bends back and forth in the circumferential direction. In this way, the flow resistance of the channel is increased, and it is avoided that during the process of distributing liquid into the reaction chamber 402, some liquid will enter the waste liquid collection chamber 502 in advance due to gravity or centrifugal force, which would result in insufficient liquid volume in the reaction chamber 402, incomplete dissolution of the lyophilized reagent, and uncontrollable reaction volume.

[0117] The connection relationship of the first waste liquid chamber 503 in this embodiment can be referred to the relevant description in the transfer unit of the above embodiment, and will not be repeated here.

[0118] The connection relationship and structure of the second waste liquid chamber 504 in this embodiment can be referred to the relevant description in the transfer unit of the above embodiment, and will not be repeated here.

[0119] The above content describes the specific structure of the centrifugal microfluidic chip. The following describes the application of the centrifugal microfluidic chip through different embodiments.

[0120] Example 1

[0121] This embodiment uses the aforementioned centrifugal microfluidic chip to achieve multi-index immunoassay without antigen-antibody cross-reactivity, and has strong index expansion and versatility. The following is a detailed description in conjunction with the accompanying drawings.

[0122] like Figure 2aAs shown, in the centrifugal microfluidic chip fabrication stage, capture antibodies are immobilized and sealed on the inner walls of each reaction chamber 402. Then, lyophilized enzyme-labeled detection antibodies are placed in each reaction chamber 402, with each chamber corresponding to a different target. A single-component chemiluminescent substrate is added to the substrate storage chamber 204 through the second liquid inlet 312, and excess liquid is discharged through the fourth through-hole 604, thereby achieving quantification of the substrate.

[0123] like Figure 2b As shown, before the experiment begins, a certain volume of sample solution is first added to the sample inlet 101 using a Parbich tube or pipette and then to the sample quantification cell 103. Excess liquid overflows from the first overflow hole 102. The liquid is constrained by the auxiliary quantitative pipe 104 and only fills the U-shaped area of ​​the sample quantification cell 103, thereby achieving sample quantification.

[0124] A certain volume of universal solution is added to the first inlet 301 using a Pasteur tube or pipette. This universal solution will serve as the sample diluent and washing solution during the immunoassay process. The universal solution sequentially fills the sample loading chamber 201, the first washing chamber 202, and the second washing chamber 203 through the pipe 302. Excess liquid overflows from the first through-hole 601, thus achieving quantitative determination of the universal solution.

[0125] like Figure 2c As shown, the centrifugal microfluidic chip is placed on the instrument, and the device on the instrument opens the first switch valve 705. Under the action of centrifugal force, the substrate in the substrate storage chamber 204 is transferred to the third liquid storage tank 213.

[0126] like Figure 2d As shown, the centrifugal microfluidic chip is centrifuged at high speed. Liquid in sample loading chamber 201 enters sample dilution chamber 106 through first transfer pipe 303. Liquid in first washing chamber 202 enters first storage chamber 211 through first transfer pipe 303. Liquid in second washing chamber 203 enters second storage chamber 212 through first transfer pipe 303. If the sample is a whole blood sample, blood cells accumulate in blood cell storage structure 105 under high centrifugation speed, while plasma enters sample dilution chamber 106 through auxiliary quantitative pipe 104.

[0127] High-speed and low-speed switching oscillations ensure that the sample and diluent are thoroughly mixed in the sample dilution chamber 106.

[0128] like Figure 2eAs shown, when the second switch valve 701 is opened, the liquid in the sample dilution chamber 106 is transferred to the first liquid buffer chamber 304 through the second switch valve 701 at a certain centrifugal speed, and then sequentially enters each reaction chamber 402 through the fourth transfer pipe 305 and the distribution pipe 306. Excess liquid enters the first waste liquid chamber 503 through the second transfer pipe 311. The liquid flow in the second transfer pipe 311 is slower, which can draw the liquid in the distribution pipe 306 to the first waste liquid chamber 503, thereby completely emptying the distribution pipe 306, and the reaction chambers 402 are not connected to each other.

[0129] After the sample enters each reaction chamber 402, the lyophilized reagent of the enzyme-labeled detection antibody is dissolved, forming a sandwich structure of "capture antibody-antigen-detection antibody" on the inner wall of the reaction chamber 402. The reactions in each reaction chamber 402 do not interfere with each other, thus avoiding the problem of antigen-antibody cross-reaction in multi-index immunoassay.

[0130] like Figure 2f As shown, after a period of reaction, the centrifugal speed is increased, and the reacted liquid flows from the reaction chamber 402 through the third transfer pipe 309 into the waste liquid buffer chamber 501, and then from the waste liquid buffer chamber 501 through the fifth transfer pipe 310 into the waste liquid collection chamber 502. During this process, the liquid remaining in the first liquid buffer chamber 304 and the fourth transfer pipe 305 is transferred into the second waste liquid chamber 504. The double-layer structure of the second waste liquid chamber 504 allows the waste liquid to be stored in the second chamber of the second waste liquid chamber 504, while the first chamber of the second waste liquid chamber 504 does not store waste liquid, thus preventing the waste liquid from being thrown out during the centrifugal microfluidic chip oscillation process.

[0131] like Figure 2g As shown, when the third switch valve 702 is opened, the liquid in the first storage tank 211 is transferred to the second liquid buffer chamber 307 through the third switch valve 702 at a certain centrifugal speed, and then enters each reaction chamber 402 in sequence through the fourth transfer pipe 305 and the distribution pipe 306. The excess liquid enters the first waste liquid chamber 503 through the second transfer pipe 311. At this time, the distribution pipe 306 is completely emptied.

[0132] like Figure 2h As shown, after a period of time, the centrifugal speed is increased, and the liquid after the reaction enters the waste liquid buffer chamber 501 through the third transfer pipe 309 from each reaction chamber 402, and then enters the waste liquid collection chamber 502 through the fifth transfer pipe 310 from the waste liquid buffer chamber 501, completing the first cleaning.

[0133] like Figure 2iAs shown, when the fourth switch valve 703 is opened, the liquid in the second storage tank 212 is transferred to the second liquid buffer chamber 307 through the fourth switch valve 703 at a certain centrifugal speed, and then enters each reaction chamber 402 in sequence through the fourth transfer pipe 305 and the distribution pipe 306. The excess liquid enters the first waste liquid chamber 503 through the second transfer pipe 311. At this time, the distribution pipe 306 is completely emptied.

[0134] like Figure 2j As shown, after a period of time, the centrifugal speed is increased, and the reacted liquid enters the waste liquid buffer chamber 501 through the third transfer pipe 309 from each reaction chamber 402, and then enters the waste liquid collection chamber 502 through the fifth transfer pipe 310 from the waste liquid buffer chamber 501, completing the second cleaning.

[0135] like Figure 2k As shown, when the fifth switch valve 704 is opened, the substrate solution in the third storage tank 213 is transferred to the third liquid buffer chamber 308 through the fifth switch valve 704 at a certain centrifugal speed, and then enters each reaction chamber 402 in sequence through the fourth transfer pipe 305 and the distribution pipe 306. Excess liquid enters the first waste liquid chamber 503 through the second transfer pipe 311. At this time, the distribution pipe 306 is completely emptied, and there is no crosstalk between each reaction chamber 402.

[0136] After the substrate has been catalyzed for a period of time, the signal intensity in each reaction chamber 402 is detected to obtain the detection results.

[0137] Example 2

[0138] The centrifugal microfluidic chip 1000 of this embodiment has the same structure as the centrifugal microfluidic chip 1000 of Embodiment 1. The difference is that the reagents pre-placed in the reaction chamber 402 of the centrifugal microfluidic chip 1000 are different, and a tray 2000 is used in conjunction with it. That is, the microfluidic system includes the centrifugal microfluidic chip 1000 and the tray 2000 disclosed above.

[0139] Please see Figure 3a and Figure 3b In this embodiment, during the preparation stage of the centrifugal microfluidic chip 1000, magnetic beads coated with capture antibodies and lyophilized reagents containing enzyme-labeled detection antibodies are placed in each reaction chamber 402, with each reaction chamber 402 corresponding to a different target. To fix the magnetic beads in the reaction chamber 402 during the reaction, a tray 2000 with a built-in magnetic suction element 2001 is placed below the centrifugal microfluidic chip 1000. For example, the magnetic suction element 2001 is a magnet. Optionally, the magnet is positioned one-to-one with the reaction chamber 402 and is arranged radially outside the reaction chamber 402.

[0140] The remaining operational steps are the same as in Example 1, including sample quantification, universal solution quantification, sample dilution, sample distribution to each reaction chamber, immunoassay, two washes, substrate addition, and signal detection. After the sample enters each reaction chamber 402, the magnetic beads are fixed inside the reaction chamber 402 by magnets, forming a sandwich structure of "capture antibody-antigen-detection antibody". During waste liquid transfer, the magnetic beads are fixed inside the reaction chamber 402 by magnets and will not be carried away.

[0141] With this configuration, the magnetic beads, as solid-phase carriers, have a larger specific surface area, enabling them to capture more antigen molecules and improve detection sensitivity. The tray 2000, used in conjunction with the centrifugal microfluidic chip 1000, fixes the magnetic beads within the reaction chamber 402 during the reaction, cleaning, and waste liquid transfer processes. This ensures reaction efficiency while preventing the loss of detection signal caused by the magnetic beads being carried away during the cleaning process.

[0142] Example 3

[0143] This embodiment uses the aforementioned centrifugal microfluidic chip to achieve highly sensitive and specific multi-index nucleic acid detection.

[0144] Please see Figures 4a to 4d Before the reaction began, RPA reaction buffer lyophilized pellets and RPA reaction enzyme lyophilized pellets were placed in sample dilution chamber 106. Primers targeting different targets were dried and fixed in each reaction chamber 402. CRISPR / Cas12a system lyophilized pellets targeting different targets were placed in waste collection chamber 502.

[0145] like Figure 4a As shown, when using it, a certain volume of sample solution is added to the sample addition cell 201. The sample addition process is the same as in Example 1 and will not be repeated here.

[0146] like Figure 4b As shown, the centrifugal microfluidic chip is centrifuged at high speed, and the sample solution enters the sample dilution chamber 106 through the first transfer channel 303. The RPA reaction buffer lyophilized bulb and the RPA reaction enzyme lyophilized bulb are dissolved and the RPA reaction occurs at a certain temperature, thereby achieving pre-amplification of multiple target molecules. With this setup, the RPA reaction, as an isothermal amplification technology, does not require thermal cycling equipment and can achieve exponential amplification of nucleic acids under isothermal conditions, making it suitable for use in POCT scenarios.

[0147] like Figure 4cAs shown, when the second switch valve 701 is opened, the liquid in the sample dilution chamber 106 is transferred to the first liquid buffer chamber 304 through the second switch valve 701 at a certain centrifugal speed, and then sequentially enters each reaction chamber 402 through the fourth transfer pipe 305 and the distribution pipe 306. Excess liquid enters the first waste liquid chamber 503 through the second transfer pipe 311. At this time, the distribution pipe 306 is completely emptied, and the reaction chambers 402 are not connected to each other.

[0148] After the pre-amplified sample solution enters each reaction chamber 402, the primers fixed in the chamber dissolve. Nested RPA amplification is achieved through primer design, improving specificity. This setup allows nested RPA amplification to perform a second round of amplification using internal primers on top of the pre-amplification, significantly improving amplification specificity and effectively avoiding false positives caused by non-specific amplification.

[0149] like Figure 4d As shown, after the reaction is completed, the centrifugal speed is increased, and the liquid after the reaction enters the waste liquid buffer chamber 501 through the third transfer pipe 309 from each reaction chamber 402, and then enters the waste liquid collection chamber 502 through the fifth transfer pipe 310 from the waste liquid buffer chamber 501.

[0150] The solution dissolved the CRISPR / Cas12a lyophilized pellets in the waste collection chamber 502. If the target molecule is present in the solution, the trans-cleavage activity of the Cas12a enzyme will be activated, causing the Cas12a enzyme to cleave the reporter probe and generate a fluorescent signal. The signal intensity in the waste collection chamber 502 is then detected after a period of time. This setup gives the CRISPR / Cas12a system extremely high specificity, enabling accurate identification of the target nucleic acid sequence. Combined with RPA pre-amplification, it achieves highly sensitive and specific nucleic acid detection.

[0151] In this embodiment, by combining nested RPA amplification with the CRISPR / Cas12a system on a centrifugal microfluidic chip, high-sensitivity and high-specificity multi-index nucleic acid detection is achieved.

[0152] In summary, the centrifugal microfluidic chip of this embodiment has a fixed flow channel structure. By simply changing the type of reagent pre-set in each reaction chamber 402, the switching between immunoassay and nucleic acid assay can be achieved. It has strong versatility and scalability and can be applied to a variety of in vitro diagnostic assays.

[0153] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0154] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A centrifugal microfluidic chip, characterized in that, It includes a substrate and a sample processing unit, a liquid storage unit, a transfer unit, a reaction unit, and a waste liquid tank integrated on the substrate; The sample processing unit includes a sample quantification chamber (103), an auxiliary quantification pipeline (104), and a sample dilution chamber (106). The proximal end of the sample quantification chamber (103) is connected to an inlet (101) and a first overflow orifice (102), which is connected to the outside atmosphere. The distal end of the sample quantification chamber (103) is connected to the auxiliary quantification pipeline (104), which is connected to the proximal end of the sample dilution chamber (106). The cross-section of the auxiliary quantification pipeline (104) is smaller than the cross-sectional area of ​​the sample quantification chamber (103). The liquid storage unit includes a sample loading tank (201), a substrate storage chamber (204), and at least one washing tank arranged circumferentially, as well as multiple liquid storage tanks; the sample loading tank (201) and the washing tank are connected in series; the proximal end of the sample loading tank (201) is provided with a first liquid inlet (301), the distal end of the sample loading tank (201) is connected to the sample dilution chamber (106), the proximal end of the substrate storage chamber (204) is connected to a second liquid inlet (312), and the distal end of the substrate storage chamber (204) and the distal end of the washing tank are respectively connected to the liquid storage tanks; The transfer unit includes a liquid buffer chamber, and the distal end of the sample dilution chamber (106) and the distal end of the liquid storage tank are configurably connected to the corresponding liquid buffer chamber. The liquid buffer chambers are all connected to the reaction chambers (402) of the reaction unit through the distribution pipes (306). There are at least two reaction chambers (402), which are arranged at intervals along the circumference of the distribution pipes (306) and are all connected to the distribution pipes (306). The waste liquid pool includes a waste liquid buffer chamber (501) and a waste liquid collection chamber (502) that are connected. The waste liquid buffer chamber (501) is located at the proximal end of the waste liquid collection chamber (502). The proximal end of the waste liquid buffer chamber (501) is connected to the reaction chamber (402). The distal end of the waste liquid buffer chamber (501) is connected to the waste liquid collection chamber (502).

2. The centrifugal microfluidic chip according to claim 1, characterized in that, The number of the cleaning tanks is two, including a first cleaning tank (202) and a second cleaning tank (203) connected in series radially, with the first cleaning tank (202) connected in series with the sample addition tank (201); The first cleaning tank (202) and the second cleaning tank (203) are respectively connected to one of the liquid storage tanks; Furthermore, a first through hole (601) is provided at the proximal end of the cleaning pool at the end.

3. The centrifugal microfluidic chip according to claim 1, characterized in that, The distal end of the substrate storage chamber (204) is connected to the corresponding liquid storage tank via a first switching valve (705).

4. The centrifugal microfluidic chip according to claim 1, characterized in that, The liquid buffer chamber is divided into a first liquid buffer chamber (304), a second liquid buffer chamber (307), and a third liquid buffer chamber (308); the first liquid buffer chamber (304), the second liquid buffer chamber (307), and the third liquid buffer chamber (308) are arranged circumferentially; The distal end of the sample dilution chamber (106) is connected to the proximal end of the first liquid buffer chamber (304) via a second switching valve (701); the distal end of the storage tank connected to the washing tank is connected to the proximal end of the second liquid buffer chamber (307) via corresponding switching valves; and / or, the distal end of the storage tank connected to the substrate storage chamber (204) is connected to the proximal end of the third liquid buffer chamber (308) via a fifth switching valve (704).

5. The centrifugal microfluidic chip according to any one of claims 1 to 4, characterized in that, The distribution pipe (306) is a reciprocating arc pipe along the circumferential direction, and the distribution pipe (306) includes a connected proximal section and a distal section, and the connection between the proximal section and the distal section forms a bending area, and both the proximal section and the distal section are arc-shaped structures. The proximal segment gradually moves away from the rotation center of the substrate from the initial region of the proximal segment toward the bending region, and the distal segment gradually moves away from the rotation center of the substrate from the bending region toward the end region of the distal segment. The distal end of all the liquid buffer chambers is connected to the proximal section of the distribution pipe (306), and the proximal end of all the reaction chambers (402) is connected to the distal section of the distribution pipe (306).

6. The centrifugal microfluidic chip according to claim 5, characterized in that, The waste liquid pool also includes a first waste liquid chamber (503) and a second waste liquid chamber (504), and the proximal end of the first waste liquid chamber (503) is connected to a third through hole (603). The distal end of the distal section is connected to the proximal end of the first waste liquid chamber (503), the bend of the distribution pipe (306) is connected to the proximal end of the second waste liquid chamber (504), and the flow area at the connection between the second waste liquid chamber (504) and the bend is smaller than the flow area at the connection between the distribution pipe (306) and the reaction chamber (402). The reaction chamber (402) is connected to the region between the bent region and the end region.

7. The centrifugal microfluidic chip according to claim 6, characterized in that, The second waste liquid chamber (504) has a double-layer structure, including a first chamber and a second chamber distributed radially along the substrate. The proximal end of the first chamber is connected to the distribution pipe (306), and the distal end of the first chamber is connected to the proximal end of the second chamber. The flow area at the connection between the first chamber and the bend area is smaller than the flow area at the connection between the reaction chamber (402) and the distribution pipe (306).

8. The centrifugal microfluidic chip according to claim 6, characterized in that, The distal end region of the distal segment is connected to the proximal end of the first waste liquid chamber (503) via a curved second transfer conduit (311) that extends radially along the substrate and bends circumferentially on the substrate.

9. The centrifugal microfluidic chip according to claim 5, characterized in that, The proximal end of the reaction chamber (402) is connected to the distal end of the distribution pipe (306) via a connecting pipe (401); The depth of the reaction chamber (402) in the substrate is greater than the depth of the connecting pipe (401) in the substrate.

10. The centrifugal microfluidic chip according to claim 9, characterized in that, The flow area of ​​the connecting pipe (401) gradually decreases and then gradually increases from the distribution pipe (306) toward the reaction chamber (402).

11. The centrifugal microfluidic chip according to any one of claims 1 to 5, characterized in that, The waste liquid buffer chamber (501) is connected to the reaction chamber (402) through a third transfer pipe (309), and the third transfer pipe (309) extends radially along the substrate and is a pipe that bends back and forth in the circumferential direction of the substrate.

12. The centrifugal microfluidic chip according to claim 11, characterized in that, The volume of the waste liquid buffer chamber (501) is smaller than the volume of the waste liquid collection chamber (502).

13. The centrifugal microfluidic chip according to any one of claims 1 to 5, characterized in that, The sample inlet (101), the first liquid inlet (301), and the second liquid inlet (312) are all located on the back side of the substrate.

14. The centrifugal microfluidic chip according to any one of claims 1 to 5, characterized in that, Also includes: A blood cell storage structure (105) is disposed at the distal end of the sample quantification pool (103), and the proximal end of the blood cell storage structure (105) is connected to the distal end of the sample quantification pool (103).

15. A microfluidic system, characterized in that, Includes a centrifugal microfluidic chip (1000), wherein the centrifugal microfluidic chip (1000) is the centrifugal microfluidic chip (1000) as described in any one of claims 1 to 14.

16. The microfluidic system according to claim 15, characterized in that, It also includes a tray (2000) for being arranged opposite to the centrifugal microfluidic chip (1000) along the thickness direction of the centrifugal microfluidic chip (1000); The tray (2000) is provided with a magnetic suction element (2001), and the magnetic suction element (2001) is provided in a one-to-one correspondence with the reaction chamber (402) of the centrifugal microfluidic chip (1000).

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