Centrifugal micro-fluidic chip

Abstract

According to the centrifugal micro-fluidic chip provided by the utility model, the exhaust channel is led out from the fluid channel between the reaction cavity and the detection cavity, the exhaust direction of the exhaust channel is opposite to the rotating centrifugal direction, the exhaust port is connected with the outside, the waterproof breathable film covers the exhaust port, and the exhaust port is higher than the fluid channel; by combining the atmospheric pressure internal and external balance principle and the centrifugal driving performance, the flow of gas and liquid in the reaction cavity and the detection cavity can be effectively controlled, the sample amount entering each cavity can be effectively controlled, multiple detection of nucleic acid can be completed only through one-time centrifugation, a to-be-detected sample can stably remain and react after entering each cavity, the sample backflow is effectively prevented, and the detection efficiency is improved. The problem of mutual interference or pollution is avoided; meanwhile, one or more concentric sub-chips can be arranged outside the chip body, so that high-throughput detection of more samples and more detection items can be realized, and the detection efficiency is improved.

Description

Technical Field

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

[0002] Existing microfluidic chips have integrated multiple steps such as sample processing, nucleic acid amplification, and nucleic acid detection on a small chip, quickly completing the "sample in - result out" detection process. Since microfluidic chips are free from the dependence on precision pipetting devices and professionals, they have great potential in the field of nucleic acid sample detection.

[0003] Isothermal amplification has gained more and more attention in the field of instant testing due to its fast reaction speed and low requirements for instruments and equipment. However, due to the complexity of the samples to be tested, such as blood, sputum, urine, tissue, etc., it is usually necessary to react with the detection reagent to amplify the signal after isothermal amplification to achieve accurate detection, which is called nucleic acid multiplex detection. In order to achieve nucleic acid multiplex detection, a reaction chamber and a detection chamber must be provided in the microfluidic chip at the same time. The reaction chamber is used for amplification reaction, and the detection chamber is used for signal amplification. The sample to be tested needs to be transferred in a controllable manner between the reaction chamber and the detection chamber, and the cross-effect caused by sample reflux can be prevented.

[0004] Centrifugal microfluidic chips drive the flow of liquid by rotating the centrifugal microfluidic chip, which can use centrifugal force to manipulate liquid on a submillimeter scale. Compared with traditional microfluidic chips, the entire device is more compact and simple to handle, so it is gradually being used in the diagnosis field. In the centrifugal microfluidic chip, the microchannels are distributed along the radial direction of the disk. When the chip rotates, the liquid to be tested moves along multiple microchannels to each reaction pool under the action of centrifugal force, realizing the distribution and addition of the liquid to be tested.

[0005] At present, diagnostic technology usually requires multiple indicators to jointly determine whether a patient has a certain disease. For example, during clinical examinations, the same patient sample needs to be screened for multiple respiratory pathogens, and the above indicators vary according to individual conditions. However, high-throughput centrifugal microfluidic chips also have problems such as short and inaccurate control processes and complex structures. When detecting multiple indicators at the same time, the chambers in the centrifugal microfluidic chip are prone to uneven injection volumes, and may enter other chambers during the centrifugation process, or reflux after the centrifugation is completed, resulting in mutual interference and contamination. As a result, the existing centrifugal microfluidic chips can only perform simultaneous amplification of a single sample or a few detection indicators. For example, the nucleic acid multiple detection microfluidic chip provided by the patent with publication number CN220166182U has a low detection throughput and is difficult to complete the sample and multiple-indicator pathogen screening work in a short time. When faced with the amplification requirements of multiple samples and multiple indicators, it not only cannot provide efficient and rapid amplification work, but also faces high chip replacement costs.

[0006] CN115684014A provides a microfluidic chip for centrifugation. Although it can also achieve high-throughput detection of multiple indicators, the structure and operation process are relatively complicated. Ventilation holes are set on the bottom plate and the cover plate for ventilation of the entire chip. The sample enters the corresponding chamber through centrifugation. Since it is difficult to control the amount of sample entering the chamber each time through centrifugation, a transition pool needs to be set, and the sample volume is measured by the volume of the transition pool. Therefore, it needs to be centrifuged three times to make the liquid reach the target position for detection (the first stage centrifugation reaches the transition pool, the second stage centrifugation reaches the reaction chamber, and the third stage centrifugation reaches the detection chamber). Since the centrifugal speed of the latter stage needs to be significantly higher than that of the previous stage, the liquid can enter the next chamber. Therefore, the three-stage centrifugation will inevitably require higher centrifugal force centrifugation, which is not only more complicated to operate, but also the pre-embedded freeze-dried or vitrified reagents in the chamber are easily precipitated by high-speed centrifugation when the solution is not sufficient, resulting in reaction failure and serious impact on the detection results. At the same time, there are many holes in the chip, and the flow channel and the hole are very small, which makes it difficult to control the work error, resulting in the liquid being easily misplaced and out of control when adding samples.

[0007] Therefore, there is an urgent need to find a method with a simple structure that can accurately allow samples to enter the corresponding chambers with only one centrifugation, and can effectively control the amount of samples entering each chamber without the problem of sample backflow or leakage to other chambers, completely solving the problems of mutual interference and contamination, and can perform high-throughput detection of multiple detection items for single or multiple samples, and ensure the accuracy of the test results. Utility Model Content

[0008] In order to solve the above problems, the utility model provides a centrifugal microfluidic chip, which leads out an exhaust channel on the fluid channel between the reaction chamber and the detection chamber, and makes the exhaust direction of the exhaust channel opposite to the rotating centrifugal direction, the exhaust port is connected to the outside, the exhaust port is covered with a waterproof and breathable membrane, and the exhaust port is located higher than the fluid channel. Combined with the principle of internal and external balance of atmospheric pressure and centrifugal driving performance, the flow of gas and liquid in the reaction chamber and the detection chamber can be effectively controlled, and the amount of samples entering each chamber can be effectively controlled. Multiple nucleic acid detection can be completed with only one centrifugation. After entering each chamber, the sample to be tested can be stably retained and reacted, effectively preventing sample reflux, and eliminating mutual interference or contamination. At the same time, by arranging one or more concentric circle sub-chips outside the chip body, high-throughput detection of more samples and more detection items can be achieved, thereby improving detection efficiency.

[0009] On the one hand, the utility model provides a centrifugal microfluidic chip, which includes a chip body, on which a reaction chamber for sample nucleic acid amplification and a detection chamber for sample detection are provided; the reaction chamber and the detection chamber are connected by a fluid channel, and an exhaust channel is also provided on the fluid channel, and the exhaust channel is used to control the flow of gas in the reaction chamber or the detection chamber.

[0010] The centrifugal microfluidic chip provided by the utility model has multiple groups of detection units on the chip body, each group of detection units is provided with a reaction chamber and a detection chamber, and can be applied to the simultaneous detection of multiple different target analytes, such as the detection of multiple different nucleic acid targets, and each group of detection units can realize multiple nucleic acid detection. Of course, it can be understood that the centrifugal microfluidic chip of the present application is not only applicable to multiple nucleic acid detection, but also applicable to other types of molecular detection such as protein and antibody, the reaction chamber can be used for sample nucleic acid amplification, and the detection chamber is used for sample detection.

[0011] During use, the centrifugal microfluidic chip needs to be kept horizontal with the sample port facing upward. In some ways, the centrifugal microfluidic chip can be inserted into a matching detection device, so that the centrifugal microfluidic chip is kept horizontally placed with the sample port facing upward, and the rotation center is buckled and fixed with the rotating shaft of the detection device for centrifugation, so that the rotating shaft can drive the centrifugal microfluidic chip to rotate centrifugally; in some ways, the detection device can provide a heat source for the reaction chamber of the centrifugal microfluidic chip to heat it; in some ways, the detection device can also detect the fluorescent substance produced after the sample reaction in the centrifugal microfluidic chip and read the detection result.

[0012] Since centrifugal microfluidic chips need to control the flow of liquid through centrifugation, exhaust channels cannot be set for each reaction chamber and detection chamber. This is because when each chamber is equipped with an exhaust channel, it is difficult to control the flow of samples during the centrifugation process. The sample may directly pass through the reaction chamber to the detection chamber under the action of centrifugation, affecting the amplification and detection results. Therefore, existing centrifugal microfluidic chips usually only have air holes on the chip floor or cover. Such a setting is not suitable for nucleic acid multiple detection chips because multiple chambers need to be set up. It is difficult to exhaust the rear chamber during the centrifugation process. A larger centrifugal force is required to push the sample in. High-speed centrifugation can easily cause the freeze-dried reagents pre-placed in each chamber to be insufficiently dissolved and precipitate, which seriously affects the accuracy of the test results.

[0013] The centrifugal microfluidic chip provided by the utility model is provided with an exhaust channel on the fluid channel connected between the reaction chamber and the detection chamber in each detection unit, so that the reaction chamber and the detection chamber share one exhaust channel, and the structure of the exhaust channel is improved, and combined with the performance of centrifugation, the reaction chamber and the detection chamber can be smoothly exhausted when they need to be exhausted, and can be smoothly closed to prevent interference when they need to be closed for reaction, and the amount of samples entering the reaction chamber and the detection chamber can be effectively controlled. Multiple nucleic acid tests can be completed by performing one centrifugation, and the problem of leakage or sample reflux can be perfectly avoided. After the sample to be tested enters the corresponding chamber, it can be stably retained in the chamber without reflux and flowing into other chambers, thereby ensuring that each step of the reaction can be completed smoothly, providing guarantee for the accurate detection of the centrifugal microfluidic chip.

[0014] The fluid channel connecting the reaction chamber and the detection chamber refers to a channel connecting the outlet of the reaction chamber and the inlet of the detection chamber.

[0015] Furthermore, a rotation center is also provided on the chip body; the rotation center is used to connect with a centrifugal driving device, thereby driving the centrifugal microfluidic chip to rotate centrifugally.

[0016] Furthermore, the exhaust channel includes an inlet and an outlet, the inlet is the connection between the fluid channel and the exhaust channel, and the outlet is the other end of the exhaust channel; the outlet is closer to the rotation center than the inlet, so that the exhaust direction of the exhaust channel is opposite to or at least partially opposite to the centrifugal direction of rotation.

[0017] When the centrifugal microfluidic chip is centrifuged, the flow direction of the fluid sample under the centrifugal action is emitted from the center of the circle to the outside. In the centrifugal microfluidic chip provided by the utility model, the exhaust channel is led out from the fluid channel between the reaction chamber and the detection chamber, and then approaches the rotation center, so that the exhaust direction is opposite to the centrifugal direction. The fluid sample will not enter the exhaust channel during centrifugation, but will accurately enter the corresponding chamber along the centrifugal direction.

[0018] In some embodiments, the exhaust channel is led out from the middle of the fluid channel or toward a position close to the detection chamber, and the initial section of the leading-out section is an arc-shaped channel, and the angle between the arc-shaped channel and the fluid channel close to the reaction chamber is less than 90 degrees.

[0019] In some embodiments, the exhaust channel can be led out slightly closer to the detection chamber. Because the exhaust channel is closer to the detection chamber, the pressure that the sample needs to overcome to enter the detection chamber during centrifugation can be reduced, making it easier for the sample to enter the detection chamber during centrifugation.

[0020] When the exhaust channel is led out from the fluid channel, the smaller the angle between the initial section and the fluid channel close to the reaction chamber, the less likely the fluid is to enter the exhaust channel. However, this angle cannot be too small, otherwise it may cause exhaust difficulties. It is preferred to use an arc-shaped channel at one end for transition, and the angle between the arc-shaped channel and the fluid channel close to the reaction chamber is less than 90 degrees.

[0021] In some embodiments, the exhaust passage has an inner diameter smaller than that of the fluid passage.

[0022] Furthermore, the outlet of the exhaust passage is connected to the outside, and a waterproof breathable membrane is provided at the outlet connected to the outside.

[0023] The outlet of the exhaust channel is covered with a waterproof breathable membrane, which is a membrane that is permeable to air but not water, and can discharge gas from the reaction chamber or detection chamber, but liquid cannot pass through. The waterproof breathable membrane can help the sample in the reaction chamber or detection chamber to establish internal and external pressure balance, and can regulate the direction of liquid flow, so that the sample in the reaction chamber or detection chamber will not leak or reflux. In some methods, the waterproof breathable membrane is made of polymer material.

[0024] The flow process of the sample to be tested in the centrifugal microfluidic chip mainly consists of two steps: the first step is that the sample to be tested enters the reaction chamber through the main channel under the action of injection pressure for reaction; the second step is that after the reaction is completed, the sample to be tested flows out of the reaction chamber through low-speed centrifugation and enters the detection chamber for reaction and detection.

[0025] The sample is injected from the injection port into the main channel through a pipette gun or a syringe, and under the action of the injection pressure, it flows from the main channel into each reaction chamber, and then the injection port is sealed. In the first step, when the sample enters the reaction chamber, the gas in the reaction chamber needs to be discharged outward through the exhaust channel. Since the detection chamber is not provided with an exhaust channel, it is difficult for the sample to flow in. At the same time, since the exhaust direction of the exhaust channel is opposite to the centrifugal direction and is higher than the fluid channel, the inner diameter of the exhaust channel is smaller than the inner diameter of the fluid channel. Therefore, the sample can only reach the intersection of the fluid channel and the exhaust channel in the first step, and it cannot continue to move forward, thereby controlling the volume of the sample entering the reaction chamber. At this time, since the injection port is sealed, that is, only the exhaust channel of the entire microfluidic chip is connected to the atmosphere, and the rest of the parts are in a sealed state, and the pressure inside and outside are balanced. The sample in the reaction chamber can no longer flow from the reaction chamber to other areas, because it is necessary to have a large pressure to break the established internal and external pressure balance to flow out of the reaction chamber. Therefore, all samples will remain in the reaction chamber for reaction, and leakage or backflow is impossible.

[0026] In the second step, after the sample completes the reaction in the reaction chamber, it is centrifuged at a low speed (such as 100 to 1000 rpm), and the initial pressure balance is broken. The sample flows out of the reaction chamber under the action of centrifugal force. At this time, since the exhaust direction of the exhaust channel is opposite to the centrifugal direction, the sample will not enter the exhaust channel, and will all flow to the detection chamber, and the gas in the detection chamber will be discharged from the exhaust channel. At this time, the pressure in the detection chamber is also balanced, so that the sample in the detection chamber can no longer flow out of the detection chamber, and all remain in the detection chamber for reaction and detection.

[0027] Therefore, under the joint action of the exhaust channel and the fluid flow channel, the internal and external pressures of the samples in the reaction chamber or the detection chamber can be balanced, and the direction of liquid flow can be regulated to prevent leakage or backflow of the samples in the reaction chamber or the detection chamber. The amount of samples entering the reaction chamber or the detection chamber can also be effectively controlled, and multiple nucleic acid tests can be completed with only one low-speed centrifugation.

[0028] Furthermore, when the centrifugal microfluidic chip is placed horizontally, the outlet of the exhaust channel is located higher than the inlet.

[0029] The outlet of the exhaust channel is located higher than the inlet, which can further increase the difficulty of the fluid sample entering the exhaust channel, thereby helping to control the flow of liquid in the chip and facilitating the exhaust of gas from the reaction chamber and the detection chamber.

[0030] Furthermore, a gas permeable cavity is provided at the outlet of the exhaust channel, and the volume of the gas permeable cavity is not less than that of the reaction chamber.

[0031] The provision of the air permeable cavity can be used to help exhaust the reaction cavity and the detection cavity, making it easier for the sample to enter the reaction cavity, and also facilitates the transfer of gas in the detection cavity from the detection cavity to the air permeable cavity during the centrifugation process.

[0032] Furthermore, the chip body is circular; a main flow channel is also provided on the chip body; and the chip body, the circular ring and the rotation center are coaxially arranged.

[0033] Furthermore, the main flow channel is arranged in a wave-like manner and is bent into a ring, and the main flow channel is also provided with a sample addition port.

[0034] In some embodiments, the main flow channel adopts a wavy flow channel with a large curvature and evenly spaced, which can help maintain the air pressure balance in the chip, smoothly control the flow of liquid, and help prevent the various detection units from directly interfering with each other.

[0035] In some embodiments, the main channel is a connected circular closed loop, and only one opening needs to be provided in the main channel for sample injection.

[0036] In some methods, the surface of the sample loading port of the centrifugal microfluidic chip is covered with a transparent film to seal the sample. When loading the sample, the transparent film is pierced by a syringe to load the sample. After the sample fills the main channel, the surface of the sample loading hole needs to be covered with a transparent film to seal it.

[0037] Furthermore, it also includes one or more sub-chips, which are concentric ring chips located outside the chip body; the sub-chip is also provided with a main channel, a reaction chamber and a detection chamber; when the sub-chip body rotates centrifugally, the sub-chip can rotate centrifugally along with the chip body.

[0038] The centrifugal microfluidic chip provided by the utility model decomposes a traditional single centrifugal microfluidic chip into a chip body and a sub-chip. The chip body and the sub-chip operate independently and are compact and easy to operate. They can perform multi-index detection of multiple samples at the same time, greatly improving the detection efficiency and meeting the needs of fast, convenient, low-cost high-throughput diagnosis. It has important application value in the field of clinical diagnosis or rapid on-site screening.

[0039] The chip body and the sub-chip can be directly molded and manufactured in one chip and directly connected into one body, or they can be manufactured separately and fixed for use by means of snap-fit ​​connection or the like.

[0040] In some embodiments, in order to make the centrifugation process more stable and controllable, the chip body and the sub-chip are formed in an integral structure and cannot be disassembled.

[0041] In some methods, the sub-chip, the chip body and the entire microfluidic chip are all in the same plane. The sub-chip is located outside the chip body and has a larger diameter, so more groups of detection units can be set up. At the same time, the sub-chip can also be provided with a sample loading port and a waste liquid pool to independently perform multiple nucleic acid detections. It can detect samples different from the chip body, realize simultaneous detection of multiple samples, and can detect more indicators at the same time (each detection unit can detect a nucleic acid target), significantly increasing the detection throughput and improving the detection efficiency.

[0042] In some methods, the number of sub-chips and the number of detection units in each sub-chip or the chip itself can be flexibly set according to the number of samples to be tested and the index detection requirements, and the reaction chamber of each detection unit and the solid reagent built into the detection chamber can be flexibly set according to actual needs. For example, amplification primers for different respiratory targets can be built into different reaction chambers, and corresponding detection reagents can be built into corresponding detection chambers, so that multiple detection of respiratory pathogens can be flexibly achieved.

[0043] In some embodiments, the rotation center is a slot, and the slot enables the centrifugal microfluidic chip to be stuck on the rotating shaft of the centrifugal drive device (detection device) to perform centrifugal rotation.

[0044] In some embodiments, the slot is a circular slot with a protrusion, and the protrusion is in the shape of a triangle, trapezoid, etc. The rotating shaft on the detection device also has a fixed groove that matches the size of the slot, so that the centrifugal microfluidic chip is fixed on the rotating shaft through the slot and performs centrifugal rotation with the rotating shaft.

[0045] In some embodiments, the cross-section of the reaction chamber is circular.

[0046] The cross-sectional shape of the reaction chamber is set to be circular, so that the sample to be tested reacts and is discharged in the reaction chamber without leaving dead corners, and it is not easy to leave residues during the inflow and outflow processes.

[0047] In some embodiments, the reaction chamber is used for amplifying the target nucleic acid, and the reaction chamber contains a nucleic acid amplification reagent; the detection chamber is used for detecting the target nucleic acid, and the detection chamber contains a nucleic acid detection reagent.

[0048] In some embodiments, the reaction chamber can serve as an ERA amplification chamber, and the detection chamber can serve as an ERA detection chamber.

[0049] In some embodiments, the reaction chamber can be used as an ERA reaction chamber, and the detection chamber can be used as a CRISPR detection chamber.

[0050] In some embodiments, the reaction chamber can be used as an ERA reaction chamber, and the detection chamber can be used as an Ago detection chamber.

[0051] In some embodiments, the reaction chamber can be used as an ERA reaction chamber, and the detection chamber can be used as an RNase H detection chamber.

[0052] In some embodiments, the amplification reaction includes but is not limited to EPA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, RCR, etc., and the solid amplification reagent includes but is not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0053] In some embodiments, the cross-section of the detection chamber is approximately semicircular and larger than the reaction chamber, which can ensure that all samples in the reaction chamber enter the detection chamber.

[0054] In some embodiments, the detection reagents include but are not limited to various detection systems established by ERA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, PCR, hybridization probe technology, CRISPR, Ago, RNase H, etc., and the detection reagents are solid detection reagents, including but not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0055] On the other hand, the utility model provides a nucleic acid detection method, which uses the centrifugal microfluidic chip as described above for detection. When the outlet of the exhaust channel is connected to the outside, the detection method includes the following steps:

[0056] (1) Place the centrifugal microfluidic chip horizontally and add the sample to be tested from the sample injection port;

[0057] (2) The sample to be tested enters the reaction chamber of the centrifugal microfluidic chip from the sample loading area for nucleic acid amplification;

[0058] (3) clamping the card slot of the centrifugal microfluidic chip on the rotating shaft of the detection device;

[0059] (4) centrifuging to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction;

[0060] (5) Read the test results.

[0061] In step (2), the nucleic acid amplification is isothermal amplification, and a heating device is required to provide the reaction chamber with a suitable temperature required for isothermal amplification (such as 30-65° C.).

[0062] The centrifugal microfluidic chip for multiple nucleic acid detection provided by the utility model has the following beneficial effects:

[0063] 1. By leading out an exhaust channel on the fluid channel between the reaction chamber and the detection chamber, and making the exhaust direction of the exhaust channel opposite to the rotating centrifugal direction, and the position is higher than the fluid channel, the flow of gas and liquid in the reaction chamber and the detection chamber can be effectively controlled, and the amount of sample entering the reaction chamber can be effectively controlled. Only one low-speed centrifugation is required to complete multiple nucleic acid detection, which is more convenient to operate and the test results are more accurate;

[0064] 2. After entering each chamber, the sample to be tested can be stably retained and reacted, effectively preventing sample backflow, eliminating mutual interference or contamination, and providing guarantee for accurate detection;

[0065] 4. One or more concentric circle sub-chips are set outside the chip body to achieve high-throughput detection of more samples and more detection indicators, thereby improving detection efficiency;

[0066] 5. It has simple structure, compact size and convenient operation. It can meet the needs of rapid detection anytime, anywhere, and adapt to more simple detection environments. It truly achieves low-cost integration and is suitable for large-scale production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is a front view of the centrifugal microfluidic chip in Example 2;

[0068] Figure 2 It is a back view of the centrifugal microfluidic chip in Example 2;

[0069] Figure 3 is a stereoscopic diagram of the centrifugal microfluidic chip in Example 2;

[0070] Figure 4 Schematic diagram of the structure of the detection unit in the centrifugal microfluidic chip in Example 2;

[0071] Figure 5 It is a front view of the centrifugal microfluidic chip in Example 1 (including the sub-chip);

[0072] Figure 6 The back view of the centrifugal microfluidic chip in Example 1 (including the sub-chip);

[0073] Figure 7 A three-dimensional diagram of the centrifugal microfluidic chip in Example 1 (including a sub-chip);

[0074] Figure 8 Schematic diagram of the detection unit structure in the centrifugal microfluidic chip in Example 1 (including sub-chips). DETAILED DESCRIPTION

[0075] The preferred embodiments of the present invention are described in further detail below in conjunction with the accompanying drawings. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention. The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0076] In the description of the present utility model, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present utility model 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 a limitation on the present utility model. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present utility model, unless otherwise specified, "multiple" means two or more.

[0077] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" 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 by specific circumstances.

[0078] Example 1: Centrifugal microfluidic chip provided by the utility model

[0079] The structure of the nucleic acid multiplex detection centrifugal microfluidic chip provided in this embodiment is as follows Figures 1 to 4 As shown, Figure 1 It is a front view of the centrifugal microfluidic chip; Figure 2 It is the back view of the centrifugal microfluidic chip; Figure 3 is a three-dimensional image of a centrifugal microfluidic chip; Figure 4 Schematic diagram of the detection unit structure in the centrifugal microfluidic chip.

[0080] like Figures 1 to 3As shown, the centrifugal microfluidic chip 1 for multiple nucleic acid detection provided in this embodiment includes a chip body 3 with a rotation center 2. Multiple groups of detection units 8 can be set on the chip body 3 according to project needs or according to the size of the chip, so as to realize the simultaneous detection of multiple targets. Each group of detection units 8 is provided with a reaction chamber 4 and a detection chamber 5. The reaction chamber 4 is used for sample nucleic acid amplification, and the detection chamber 5 is used for sample detection. Therefore, each group of detection units can realize multiple nucleic acid detection. A main channel 16 is provided on the chip body 3, and a sample injection port 17 is provided on the main channel 16. The main channel 16 is a connected circular closed loop, and the sample injection port 17 is an opening for injection set in the circular closed loop of the main channel 16. The main channel 16 is arranged in a wave-like manner and bent into a circular ring. The chip body 3, the circular ring and the rotation center 2 are coaxially arranged. The reaction chamber 4 and the detection chamber 5 are connected by a fluid channel 6. The fluid channel 6 is also provided with an exhaust channel 7, which is led out from approximately the middle of the fluid channel 6. The exhaust channel 7 is used to control the flow of gas in the reaction chamber 4 or the detection chamber 5; the rotation center 2 is used to connect with the centrifugal drive device to drive the centrifugal microfluidic chip 1 to centrifuge.

[0081] During use, the centrifugal microfluidic chip 1 needs to be kept in a horizontal state and placed in a matching detection device (centrifugal drive device), with the sample port 9 placed horizontally facing upward, and the rotation center 2 is fastened and fixed to the rotating shaft used for centrifugation of the detection device, so that the rotating shaft can drive the centrifugal microfluidic chip 1 to rotate centrifugally; at the same time, the detection device can also provide a heat source for the reaction chamber 4 of the centrifugal microfluidic chip 1 for heating; the detection device can also detect the fluorescent substance produced after the sample reaction in the centrifugal microfluidic chip 1, and read the detection result.

[0082] The chip body 3 is circular in shape, and the number of detection units 8 in the chip body 3 can be flexibly set according to the number of indicators that need to be detected, and the solid reagents built into the reaction chamber 4 and the detection chamber 5 of each detection unit 8 can be flexibly set according to actual needs. For example, amplification primers for different respiratory targets are built into different reaction chambers 4, and corresponding detection reagents are built into corresponding detection chambers 5, so that multiple detections of respiratory pathogens can be flexibly achieved.

[0083] like Figures 1 to 4, an exhaust channel 7 is provided on the fluid channel 6 connected between the reaction chamber 4 and the detection chamber 5 in each detection unit 8 of the centrifugal microfluidic chip 1, so that the reaction chamber 4 and the detection chamber 5 share an exhaust channel 7. The fluid channel 6 is a channel connecting the outlet 9 of the reaction chamber 4 with the inlet 10 of the detection chamber 5. The exhaust channel 7 includes an inlet 11 and an outlet 12, the inlet 11 is the connection between the fluid channel 6 and the exhaust channel 7, and the outlet 12 is the other end of the exhaust channel 7; the outlet 12 is closer to the rotation center 2 than the inlet 11, so that the exhaust direction of the exhaust channel 7 is opposite to the rotation centrifugal direction or at least partially opposite. When the centrifugal microfluidic chip 1 is centrifuged, the flow direction of the fluid sample under the centrifugal action is emitted from the center of the circle outward. At this time, since the exhaust channel 7 is drawn out from the fluid channel 6 between the reaction chamber 4 and the detection chamber 5 and approaches the rotation center 2, the exhaust direction is opposite to the centrifugal direction. It is difficult for the fluid sample to enter the exhaust channel 7 during centrifugation, but it accurately enters the corresponding chamber along the centrifugal direction.

[0084] Preferably, Figure 4 As shown, when the centrifugal microfluidic chip 1 is placed horizontally, the outlet 12 of the exhaust channel 7 is higher than the inlet 11. Such a design can further increase the difficulty for the fluid sample to enter the exhaust channel 7, thereby helping to control the flow of liquid in the chip so that the sample cannot enter the exhaust channel 7. At the same time, it can also facilitate the discharge of gas from the reaction chamber 4 and the detection chamber 5, so that the centrifugal microfluidic chip 1 can perform multiple nucleic acid detection more accurately.

[0085] like Figure 4 As shown, the exhaust channel 7 is led out from the middle of the fluid channel or the position close to the detection chamber 5, and the initial section led out is an arc-shaped channel 13, and the angle 14 between the arc-shaped channel 13 and the fluid channel close to the reaction chamber 4 is less than 90 degrees. The exhaust channel 7 can be led out slightly close to the detection chamber 5, because the exhaust channel 7 is close to the detection chamber, which can reduce the pressure that the sample needs to overcome to enter the detection chamber 5 during centrifugation, and the sample is easier to enter the detection chamber 5 during centrifugation. When the exhaust channel 7 is led out from the fluid channel 6, the smaller the angle 14 between the initial section and the fluid channel close to the reaction chamber 4, the less likely the fluid is to enter the exhaust channel 6, but this angle 14 cannot be too small, otherwise it may cause exhaust difficulties. It is preferred to use an arc-shaped channel 13 at one end for transition, and the angle between the arc-shaped channel 13 and the fluid channel close to the reaction chamber 4 is less than 90 degrees. Preferably, the inner diameter of the exhaust channel 7 is smaller than the inner diameter of the fluid channel 6, which can further prevent the sample from entering the exhaust channel 7 during centrifugation.

[0086] like Figure 4, the outlet 12 of the exhaust channel 7 is connected to the outside world, and the outlet 12 connected to the outside world is covered with a waterproof breathable membrane 15. The waterproof breathable membrane 15 is a membrane that is breathable but not water-permeable, which can achieve the discharge of gas from the reaction chamber 4 or the detection chamber 5, but liquid cannot pass through. The waterproof breathable membrane 15 can help the sample in the reaction chamber 4 or the detection chamber 5 to establish internal and external pressure balance, and can regulate the flow direction of the liquid so that the sample in the reaction chamber 4 or the detection chamber 5 will not leak or reflux. The waterproof breathable membrane 15 can be made of polymer materials, such as high-grade synthetic glue, with good sealing, no back glue and residue, and does not react with reaction reagents or detection reagents. In this embodiment, the waterproof breathable membrane is preferably made of material (manufacturer: Membrane; Article No.: VET022H60). When this setting is adopted, the centrifugal microfluidic chip 1 only needs one centrifugation to complete multiple nucleic acid detection throughout the whole process. Preferably, a gas permeable cavity 30 is further provided at the outlet 12 of the exhaust channel 7 , and the volume of the gas permeable cavity 30 is not less than that of the reaction chamber 4 , and is used to help exhaust the reaction chamber 4 so that the sample can enter the reaction chamber 4 more easily.

[0087] The flow process of the sample to be tested in the centrifugal microfluidic chip 1 mainly includes two steps: in the first step, the sample to be tested enters the reaction chamber 4 through the main channel 16 for reaction under the action of injection pressure; in the second step, after the reaction is completed, the sample to be tested flows out of the reaction chamber 4 through low-speed centrifugation and enters the detection chamber 5 for reaction and detection.

[0088] The sample is injected from the sample injection port 17 into the wavy main channel 16 through a pipette gun or a syringe, and under the action of the injection pressure, flows from the main channel 16 into each reaction chamber 4, and then the sample injection port 17 is sealed. In the first step, when the sample enters the reaction chamber 4, the gas in the reaction chamber 4 needs to be discharged outward through the exhaust channel 7. Since the detection chamber 5 is not provided with an exhaust channel 7, it is difficult for the sample to flow in. At the same time, since the exhaust direction of the exhaust channel 7 is opposite to the centrifugal direction and is higher than the fluid channel 6, and the inner diameter of the exhaust channel 7 is smaller than the inner diameter of the fluid channel 6, the sample can only reach the intersection of the fluid channel 6 and the exhaust channel 7 (the inlet 11 of the exhaust channel 7) at most in the first step, and cannot continue to move forward, thereby controlling the volume of the sample entering the reaction chamber 4. At this time, since the sample adding port 17 is sealed, that is, only the exhaust channel 7 of the entire centrifugal microfluidic chip 1 is connected to the atmosphere, and the rest of the parts are sealed, and the pressure balance is achieved inside and outside. The sample in the reaction chamber 4 can no longer flow from the reaction chamber 4 to other areas, because it is bound to require a large pressure to break the established internal and external pressure balance to flow out of the reaction chamber 4. Therefore, all samples will remain in the reaction chamber 4 for reaction, and leakage or backflow is impossible. In the second step, after the sample completes the reaction in the reaction chamber 4, it is centrifuged at a low speed (such as 100 to 1000 rpm, preferably 200-800 rpm in this embodiment, more preferably 500-600 rpm), and the initial pressure balance is broken. The sample flows out of the reaction chamber 4 under the action of centrifugal force. At this time, since the exhaust direction of the exhaust channel 7 is opposite to the centrifugal direction, the sample will not enter the exhaust channel 7, and will all flow to the detection chamber 5, and the gas in the detection chamber 5 is discharged from the exhaust channel 7. At this time, the pressure in the detection chamber 5 is also balanced, so that the sample in the detection chamber 5 can no longer flow out of the detection chamber 5 and all remain in the detection chamber 5 for reaction and detection.

[0089] Therefore, when this setting is adopted, under the joint action of the exhaust channel 7 and the fluid flow channel 6, the internal and external pressure balance of the sample in the reaction chamber 4 or the detection chamber 5 can be established, and the direction of liquid flow can be regulated to prevent leakage or backflow of the sample in the reaction chamber 4 or the detection chamber 5. The amount of sample entering the reaction chamber 4 or the detection chamber 5 can also be effectively controlled, and multiple nucleic acid detection can be completed with only one low-speed centrifugation.

[0090] Preferably, the main flow channel 16 is a wavy flow channel with a large curvature and evenly spaced, which can help maintain the air pressure balance in the chip 1, smoothly control the flow of liquid, and help prevent the detection units 8 from directly interfering with each other.

[0091] The surface of the sample port 17 of the centrifugal microfluidic chip 1 is covered with a transparent film for sealing. When adding samples, the transparent film is pierced by a syringe to add samples. After the sample fills the main channel 16, the surface of the sample port 17 needs to be covered with a transparent film for sealing.

[0092] like Figures 1 to 4 , the rotation center 2 is a slot 28, and the slot 28 can be clamped on the rotating shaft of the detection device for centrifugal rotation, thereby driving the entire chip to rotate centrifugally. Preferably, the slot 28 is a circular slot with a protrusion 29, and the shape of the protrusion 29 is a triangle, a trapezoid, etc. The rotating shaft on the detection device also has a fixing groove that can match the size of the slot 28, so that the centrifugal microfluidic chip 1 is fixed on the rotating shaft through the slot 28, and centrifugally rotates with the rotating shaft.

[0093] Preferably, the cross-sectional shape of the reaction chamber 4 is circular, so that the sample to be tested reacts and is discharged in the reaction chamber 4 without leaving dead corners, and it is not easy to leave residues during the inflow and outflow processes. The cross-sectional shape of the detection chamber 5 is approximately semicircular and larger than the reaction chamber 4, which can ensure that all samples in the reaction chamber 4 enter the detection chamber 5.

[0094] In some embodiments, the reaction chamber 4 is used for amplifying the target nucleic acid, and the reaction chamber 4 has a built-in nucleic acid amplification reagent, and the detection chamber 5 is used for detecting the target nucleic acid, and the detection chamber 5 has a built-in nucleic acid detection reagent; in some embodiments, the reaction chamber 4 can be used as an ERA amplification chamber, and the detection chamber 5 can be used as an ERA detection chamber; in some embodiments, the reaction chamber 4 can be used as an ERA reaction chamber, and the detection chamber 5 can be used as a CRISPR detection chamber; in some embodiments, the reaction chamber 4 can be used as an ERA reaction chamber, and the detection chamber 5 can be used as an Ago detection chamber; in some embodiments, the reaction chamber 4 can be used as an ERA reaction chamber, and the detection chamber 5 can be used as an RNase H detection chamber; in some embodiments, the amplification reaction includes but is not limited to EPA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, RCR, etc., and the solid amplification reagent includes but is not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc. In some methods, the detection reagents include but are not limited to various detection systems established by ERA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, PCR, hybridization probe technology, CRISPR, Ago, RNase H, etc., and the detection reagents are solid detection reagents, including but not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0095] When the sample to be tested is added to the sample addition port 17 through a pipetting device (not shown in the figure), the sample to be tested can be evenly diverted to the branch channel 19 along the main channel 16, reach the reaction chamber 4, mix with the solid amplification reagent therein, and at a certain temperature, an amplification reaction occurs; while the amplification reaction is completed, the rotating shaft in the detection device rotates clockwise to drive the centrifugal microfluidic chip 1 to perform centrifugal motion, and the liquid in the reaction chamber 4 is transferred to the detection chamber 5, and a fluorescent detection reaction occurs. After the experiment is completed, the results can be observed by the naked eye through the perspective window of the detection device, or an RGB sensor is set in the detection device for automatic identification and automatic judgment of the results. Preferably, an LED lamp or a laser lamp is set in the detection device to irradiate the detection chamber 5 by releasing blue light, thereby improving the color development effect of the detection chamber 5 (when observed by the naked eye as green, it is judged that positive amplification has occurred, and when observed by the naked eye as dark orange, it is judged as negative). The above are all prior art and will not be repeated.

[0096] Preferably, the main channel 16, the branch channel 19 (including the fluid channel 6) and the permeable channel 7 all adopt a gradually narrowing microchannel structure to prevent the sample droplets from being unevenly distributed due to too fast sample droplets; the width of the main channel 16 ranges from 500μm to 1000μm, and the depth ranges from 500μm to 1000μm; the width of the branch channel 19 ranges from 400μm to 800μm, and the depth ranges from 150μm to 300μm; the width of the permeable channel 7 ranges from 50μm to 500μm, and the depth ranges from 50 to 350μm. In this embodiment, the widths of the main channel 16, the branch channel 19 and the permeable channel 7 are 800um, 500um and 300μm, respectively, and the depths are 500um, 200um and 100μm, respectively.

[0097] The centrifugal microfluidic chip 1 provided in this embodiment adopts materials with low cost, easy processing performance and good biocompatibility, such as polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), etc. The waterproof and breathable membrane is attached to the chip body to form a sealed space as a whole, effectively preventing liquid leakage. According to the detection needs, the reaction chamber 4 is equipped with a fixed amplification reagent for nucleic acid amplification, and the detection chamber 5 is equipped with a solid detection substance for detection, such as a freeze-dried ball, freeze-dried powder or a quality control internal standard reagent.

[0098] In this embodiment, the chip body 3 is provided with 34 reaction chambers 4 and 34 detection chambers 5 .

[0099] The centrifugal microfluidic chip 1 provided in this embodiment is combined by a centrifugal drive mode. While accurately controlling the re-dissolution and mixing process of samples and reagents, it has the characteristics of ultra-high throughput and can be used for single sample multi-index detection (such as detecting multiple different respiratory pathogen screening of a single patient sample).

[0100] Example 2: Centrifugal microfluidic chip containing a sub-chip

[0101] The structure of the nucleic acid multiplex detection centrifugal microfluidic chip containing the sub-chip provided in this embodiment is as follows Figures 5 to 8 As shown, Figure 5 It is a front view of the centrifugal microfluidic chip; Figure 6 It is the back view of the centrifugal microfluidic chip; Figure 7 is a three-dimensional image of a centrifugal microfluidic chip; Figure 8 Schematic diagram of the detection unit structure in the centrifugal microfluidic chip.

[0102] The centrifugal microfluidic chip 1 for multiple nucleic acid detection provided in this embodiment is different from the chip in Example 1 in that a sub-chip 27 is provided on the periphery of the chip body 3 .

[0103] like Figures 5 to 8 As shown, the centrifugal microfluidic chip 1 containing a sub-chip provided in this embodiment includes a chip body 3 with a rotation center 2, and a sub-chip 27 arranged outside the chip body 3. When the chip body 3 is centrifugally rotated, the sub-chip 27 can be centrifugally rotated along with the chip body 3. The chip body 3 and the sub-chip 27 are both provided with a main flow channel 16, and the main flow channel 16 is also provided with a sample injection port 17 (including a first sample injection port 171 in the chip body 3 and a second sample injection port 172 in the sub-chip 27).

[0104] The chip body 3 is circular, and the sub-chip 27 is located on the concentric circle outside the chip body 3. The chip body 3 and the sub-chip 27 can be directly integrally formed and cannot be disassembled. Of course, if necessary, sub-chips can continue to be set outside the sub-chip 27. The sub-chip 27, the chip body 3 and the entire microfluidic chip 1 are all in the same plane. The sub-chip 27 is located outside the chip body 3 and has a larger diameter. More groups of detection units 8 can be set. The sub-chip 27 and independently perform multiple nucleic acid detection, which can detect samples different from the chip body 3, realize the simultaneous detection of multiple samples, and can detect more indicators at the same time (each detection unit can detect a nucleic acid target), significantly increase the detection flux, and improve the detection efficiency. The number of sub-chips 27 and the number of detection units 8 in each sub-chip 27 or the chip body 3 can be flexibly set according to the number of samples to be tested and the index detection requirements, and the solid reagents built into the reaction chamber 4 and the detection chamber 5 of each detection unit 8 can be flexibly set according to actual needs. For example, amplification primers for different respiratory targets can be built into different reaction chambers 4, and corresponding detection reagents can be built into corresponding detection chambers 5, so that multiple detection of respiratory pathogens can be flexibly achieved.

[0105] like Figures 1 to 4 On the chip body 3 and the sub-chip 27, an exhaust channel 7 is provided on the fluid channel 6 connecting the reaction chamber 4 and the detection chamber 5 in each detection unit 8, so that the reaction chamber 4 and the detection chamber 5 share one exhaust channel 7. The fluid channel 6 is a channel connecting the outlet 9 of the reaction chamber 4 with the inlet 10 of the detection chamber 5. The exhaust channel 7 includes an inlet 11 and an outlet 12, the inlet 11 is the connection between the fluid channel 6 and the exhaust channel 7, and the outlet 12 is the other end of the exhaust channel 7; the outlet 12 is closer to the rotation center 2 than the inlet 11, so that the exhaust direction of the exhaust channel 7 is opposite to or at least partially opposite to the centrifugal direction of rotation. When the centrifugal microfluidic chip 1 is placed horizontally, the outlet 12 of the exhaust channel 7 on the chip body 3 and the sub-chip 27 is higher than the inlet 11.

[0106] The number of sub-chips 27, the number of reaction chambers 4 and detection chambers 5 provided on each sub-chip 27, and the built-in solid reagents can be flexibly set as needed. Preferably, the number of sub-chips 27 can be set to 2 to 5, and the number of amplification chambers 4 and detection chambers 5 can be set to 4 to 80. In this embodiment, the number of sub-chips 27 is 1, the chip body 3 is provided with 34 reaction chambers 4 and detection chambers 5, and the sub-chip is provided with 66 reaction chambers 4 and detection chambers 5.

[0107] The centrifugal microfluidic chip 1 provided in this embodiment is designed with multiple chips (chip body 3 and sub-chip 27), each chip operates independently and is combined with an active drive mode. While accurately controlling the re-dissolution and mixing process of samples and reagents, it has the characteristics of ultra-high throughput and can be used for multi-sample and multi-index detection (such as detecting multiple different respiratory pathogen screening fields of multiple patient samples).

[0108] Example 3: Method for using a centrifugal microfluidic chip for multiplex nucleic acid detection

[0109] The centrifugal microfluidic chip provided in Examples 1 and 2 can be applied to multiple nucleic acid detection, comprising the following steps:

[0110] Step 1: Add sample and re-dissolve

[0111] The pipette is used to add samples, and different test samples are drawn and injected into the chip body 3 and the sub-chip 27 through the first sample injection hole 171 and the second sample injection hole 172 respectively. Under the action of the exhaust channel 7, the liquid is evenly distributed to the branch channel 19 along the main channel 16 in the chip body 3 and the sub-chip 27, and enters the reaction chamber 4, so as to redissolve the solid amplification reagent in the reaction chamber 4 and stay in the reaction chamber 4 under the action of air pressure balance;

[0112] Step 2: Amplification reaction

[0113] Place the mixed centrifugal microfluidic chip 1 in parallel into the detection device, and fix the card slot 28 to the rotating shaft in the detection device. At this time, the chip is heated up, and the nucleic acid in the sample to be tested will undergo an amplification reaction at a certain temperature, and the amplification reaction includes but is not limited to a constant temperature amplification reaction and a PCR reaction;

[0114] Step 3: Detection reaction

[0115] After step 2 is completed, the centrifugal device is started. At this time, the centrifugal microfluidic chip 1 rotates in a clockwise direction (at a speed of 800 rpm), and the amplification product in the reaction chamber 4 flows into the corresponding detection chamber 5 under the action of centrifugal force, and a detection reaction occurs after mixing with the detection reagent. The detection reaction includes but is not limited to a variety of detection systems established by hybridization probe technology, CRISPR, Ago, RNaseH, etc.; during the transfer of the sample to be tested from the reaction chamber 4 and the detection chamber 5, the gas in the detection chamber 5 will enter the outside through the exhaust channel 7;

[0116] Step 4: Data reading

[0117] After step 3 is completed, the fluorescence intensity on the microfluidic chip detection channel is read by the optical device of the detection device to obtain the detection result.

[0118] Both the amplification reaction and the detection reaction need to be performed at a certain temperature. Therefore, in step 2, a heating device is required to provide a suitable temperature for the centrifugal microfluidic chip 1. For example, an isothermal amplification reaction needs to provide a reaction temperature of 30 to 65°C.

[0119] Example 5: Verification of the detection effect of the centrifugal microfluidic chip for multiple nucleic acid detection

[0120] This embodiment adopts embodiment 1 ( Figures 1 to 4 ) Provides centrifugal microfluidic cores for genotyping testing

[0121] ERA-CRISPR detection technology (enzymatic recombinant amplification combined with CRISPR detection technology) was used to detect respiratory pathogen nucleic acid (influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human rhinovirus, Mycoplasma pneumoniae, human metapneumovirus, parainfluenza virus) in blood specimens suspected of respiratory infection, and qPCR detection method (commercial reagent) was used for comparative verification. The oral swab was lysed by nucleic acid using a sample release agent, and the lysate obtained was used as a template. The same template loading amount was used to amplify and detect the sample using a microfluidic chip device and a qPCR detection method, respectively. In the ERA-CRISPR detection technology, the amplification chamber of the chip device was preloaded with ERA freeze-dried microspheres containing specific amplification primers, and the detection chamber was preloaded with freeze-dried microspheres containing a specific CRISPR detection system. Whether the sample contained the corresponding pathogenic microorganism nucleic acid was determined, and the results are shown in Table 1.

[0122] Table 1. Centrifugal microfluidic chip detection performance verification list

[0123]

[0124] As can be seen from Table 1, when other conditions are the same, the detection results of the centrifugal microfluidic chip device of Example 1 are consistent with the results of the qPCR detection method, but it is more convenient and quick to use, has a lower comprehensive use cost, and is more suitable for on-site detection in the wild and special environments.

[0125] 2. Using the centrifugal microfluidic chip (including sub-chip) provided in Example 2 for genotyping detection

[0126] This embodiment adopts embodiment 2 ( Figures 5 to 8) centrifugal microfluidic chip was used to perform typing tests on two samples at the same time, and the ERA-CRISPR detection technology (enzymatic recombinant amplification combined with CRISPR detection technology) was used to detect respiratory pathogen nucleic acids (influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human rhinovirus, Mycoplasma pneumoniae, human metapneumovirus, parainfluenza virus) in blood specimens suspected of respiratory tract infection, and the qPCR detection method (commercial reagent) was used for comparative verification. The oral swab was lysed by a sample release agent, and the lysate obtained was used as a template. The same template loading amount was used to amplify and detect the samples using a microfluidic chip device and a qPCR detection method, respectively. In the ERA-CRISPR detection technology, the amplification chamber in the chip device was pre-loaded with ERA freeze-dried microspheres containing specific amplification primers, and the detection chamber was pre-loaded with freeze-dried microspheres containing a specific CRISPR detection system. Whether the sample contained the corresponding pathogenic microorganism nucleic acid was determined, and the results are shown in Table 2.

[0127] Table 2. Centrifugal microfluidic chip detection performance verification list

[0128]

[0129] It can be seen from Table 2 that, under the same other conditions, the detection results of the centrifugal microfluidic chip device containing the sub-chip in Example 2 are consistent with the results of the qPCR detection method, and the throughput is higher, and two samples can be detected at the same time, which is more suitable for on-site detection in the wild and special environments.

[0130] Although the utility model is disclosed as above, the utility model is not limited thereto. For example, it can be expanded according to its scope of application in the field of microfluidics. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the utility model, so the scope of protection of the utility model should be based on the scope defined by the claims. The contents of the articles, patents, patent applications and all other documents and electronically available information described or recorded herein are included in their entirety to some extent for reference, just as each individual publication is specifically and individually pointed out for reference. The applicant reserves the right to incorporate any and all materials and information from any such articles, patents, patent applications or other documents into this application.

Claims

1. A centrifugal microfluidic chip, characterized in that: The chip body comprises a reaction chamber for sample nucleic acid amplification and a detection chamber for sample detection. The reaction chamber and the detection chamber are connected by a fluid channel. The fluid channel is also provided with an exhaust channel for controlling the flow of gas in the reaction chamber or the detection chamber.

2. The centrifugal microfluidic chip according to claim 1, characterized in that: A rotation center is also provided on the chip body; the rotation center is used to connect with the centrifugal driving device, so as to drive the centrifugal microfluidic chip to rotate centrifugally.

3. The centrifugal microfluidic chip according to claim 2, characterized in that: The exhaust channel includes an inlet and an outlet, the inlet is the connection between the fluid channel and the exhaust channel, and the outlet is the other end of the exhaust channel; the outlet is closer to the rotation center than the inlet, so that the exhaust direction of the exhaust channel is opposite to or at least partially opposite to the centrifugal direction of rotation.

4. The centrifugal microfluidic chip according to claim 3, characterized in that: The outlet of the exhaust channel is connected to the outside world, and a waterproof and breathable membrane is provided at the outlet connected to the outside world.

5. The centrifugal microfluidic chip according to claim 4, characterized in that: When the centrifugal microfluidic chip is placed horizontally, the outlet of the exhaust channel is located higher than the inlet.

6. The centrifugal microfluidic chip according to claim 5, characterized in that: A vent cavity is also provided at the outlet of the exhaust channel, and the volume of the vent cavity is not less than that of the reaction chamber.

7. The centrifugal microfluidic chip according to claim 6, characterized in that: The chip body is circular; a main flow channel is also arranged on the chip body; the chip body, the circular ring and the rotation center are arranged coaxially.

8. The centrifugal microfluidic chip according to claim 7, characterized in that: The main flow channel is arranged in a wave-like manner and is bent into a circular ring. The main flow channel is also provided with a sample adding port.

9. The centrifugal microfluidic chip according to claim 8, characterized in that: It also includes one or more sub-chips, which are concentric ring chips located outside the chip body; the sub-chip is also provided with a main channel, a reaction chamber and a detection chamber; when the chip body is centrifugally rotated, the sub-chip can be centrifugally rotated along with the chip body.

10. The centrifugal microfluidic chip according to claim 9, characterized in that: The rotation center is a slot, and the slot enables the centrifugal microfluidic chip to be stuck on the rotation axis of the centrifugal drive device, thereby performing centrifugal rotation.

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