A microfluidic chip and detection method for infectious disease detection

Abstract

The present invention relates to the field of microfluidic detection technology, and specifically to a microfluidic chip and detection method for infectious disease detection, comprising: a substrate layer, the first surface of the substrate layer comprising a first centrifugal zone, a second centrifugal zone and a third centrifugal zone arranged in sequence; wherein the first centrifugal zone comprises: a sample loading chamber, a first quantitative chamber and a second quantitative chamber arranged in sequence along a set centrifugal direction; the second centrifugal zone comprises: a first pipeline, and a first accommodating chamber connected to the side of the first pipeline through a first channel, and a second accommodating chamber connected to the first accommodating chamber through a second channel; the liquid inlet end of the first pipeline is connected to the first quantitative chamber; the third centrifugal zone comprises: a reaction chamber, and the reaction chamber is connected to the second accommodating chamber through a third channel. The microfluidic chip and detection method provided by the present invention have the advantages of synchronous sample division and delayed mixing; and can realize multiple parallel detections of the same sample to be tested under one sample loading.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic detection technology, and in particular to a microfluidic chip and a detection method for infectious disease detection. Background Art

[0002] Most highly pathogenic microorganisms have the characteristics of strong infectivity, rapid transmission, short incubation period and rapid onset. The etiology of the diseases they cause is complex, posing a great threat to human health and social stability. Conventional methods for detecting pathogenic microorganisms mainly include culture methods, immunological detection methods and molecular biology methods, but they all have many shortcomings due to their own technical limitations. For example, traditional bacterial culture methods have low specificity, are time-consuming and labor-intensive, have high requirements for the culture environment and operators, and cannot quickly detect results. Immunological detection methods, such as enzyme-linked immunosorbent assays, bioluminescence methods, and immunocolloidal gold techniques, generally require a large number of purified bacteria, or require samples to be concentrated and enriched, have high operating requirements, and are also difficult to obtain test results in a short time. PCR-related technologies require special instruments, primers, reagents, etc., which are not suitable for large-scale detection needs.

[0003] Microfluidics is a technology that uses microchannels to control and process very small amounts of fluids, and has great scientific and commercial potential. Microfluidic devices are small in size, low in energy consumption, and highly portable. They can complete biological or chemical experiments that can only be achieved with large instruments in conventional laboratories on a few square centimeters of chips. Through the realization of the concept of "Lab on a chip (LOC)" or "micro total analysis system (TAS)", microfluidic chip technology currently shows great application prospects in the fields of detection and diagnosis of pathogenic microorganisms, but there are also some shortcomings. The patent with publication number CN207586245U and name "Centrifugal Microfluidic Chip" discloses a centrifugal microfluidic chip that can detect multiple pathogenic microorganisms at the same time. In one embodiment, it is disclosed that reagents or drugs are pre-stored in the reaction pool, such as biological reagents for detecting highly pathogenic pathogenic microorganisms. If the sample to be tested contains the corresponding pathogenic microorganism, it reacts with the biological reagent in the reaction pool to generate a fluorescent signal. The content of the pathogenic microorganism in the sample to be tested is then calculated based on the fluorescent signal. It can be seen that the microfluidic chip disclosed in the patent is a chip for quantitative detection of pathogenic microorganisms. When it comes to result interpretation, other instruments are usually required, and it is not suitable for rapid and large-scale detection of pathogenic microorganisms. Another invention is that the microfluidic chip is difficult to repeat the reaction during the detection process, and the detection accuracy is limited.

[0004] For another example, CN115254220A also discloses a microfluidic chip and a detection method, which is divided into three layers: a main body layer, a cover layer, and a liquid capsule component layer; the main body layer is provided with a sample injection chamber, a whole blood separation structure, a mixing chamber, a liquid separation channel, a plurality of parallel reaction units and a substrate reagent chamber; the cover layer is used to seal the structural layer; and the liquid capsule component layer provides wet reagents. The whole blood separation structure is connected to the sample injection chamber and the mixing chamber; the liquid separation channel is connected to the mixing chamber; a plurality of immune response units are connected to the liquid separation channel and are distributed in parallel at equal angles along the axis of the rotation center, and are pre-installed with different types of solid reagents for immune response. The parallel reaction units can avoid cross contamination of reagents; a plurality of liquid capsule components are connected to the mixing chamber through drainage channels. However, this type of microfluidic detection chip is still relatively limited in detection accuracy, for example, there is a problem of insufficient response.

[0005] In summary, the applicant has noticed that existing detection chips still have defects in rapid, large-scale and accurate detection of pathogenic microorganisms, and it is necessary to propose new improvement strategies. Summary of the invention

[0006] The purpose of the present invention is to provide a microfluidic chip and detection method for infectious disease detection, which partially solves or alleviates the above-mentioned deficiencies in the prior art, and can quickly and in large quantities perform semi-quantitative detection of pathogenic microorganisms in unknown samples, while improving the reliability and accuracy of the detection process.

[0007] In order to solve the above-mentioned technical problems, the present invention specifically adopts the following technical solutions:

[0008] A first aspect of the present invention is to provide a microfluidic chip, comprising:

[0009] A substrate layer, wherein the first surface of the substrate layer comprises a first centrifugal zone, a second centrifugal zone and a third centrifugal zone arranged in sequence; wherein the first centrifugal zone comprises: a sample adding chamber, a first quantitative chamber and a second quantitative chamber arranged in sequence along a set centrifugal direction, wherein the sample adding chamber is used to add a first sample;

[0010] The second centrifugal zone comprises: a first pipe with a first depth H1 formed by being recessed inwardly along the first surface, a first channel with a second depth H2 formed by being recessed inwardly along the first surface, and a first accommodating cavity with a third depth H3 formed by being recessed inwardly along the first surface; wherein the second depth H2 is smaller than the first depth H1 and the third depth H3; the liquid inlet end of the first pipe is connected to the first quantitative cavity; the liquid inlet end and the liquid outlet end of the first channel are connected to the first pipe and the first accommodating cavity respectively;

[0011] A second accommodating chamber, wherein the second accommodating chamber is connected to the first accommodating chamber through a second channel, and an inner diameter of a liquid inlet end of the second channel is smaller than an inner diameter of a liquid outlet end of the second channel;

[0012] The third centrifugal zone includes: a reaction chamber, which is connected to the second containing chamber through a third channel;

[0013] The encapsulation layer can cover at least one of the cavities, and at least one air hole is arranged on the encapsulation layer.

[0014] In some preferred embodiments, the method comprises: a first pore with a first aperture arranged corresponding to the first containing cavity, a second pore with a second aperture arranged corresponding to the reaction cavity, and the first aperture is larger than the second aperture.

[0015] In some preferred embodiments, the invention further comprises: a third air hole having a third aperture arranged corresponding to the second accommodating cavity, and the first aperture is larger than the third aperture.

[0016] In some preferred embodiments, the first centrifugal zone further comprises: at least one intermediate cavity; wherein the intermediate cavity is connected to the sample addition cavity via at least one first capillary channel, and the intermediate cavity is connected to the first quantitative cavity via at least one second capillary channel.

[0017] In some preferred embodiments, the inner diameter of the pore gradually increases in a direction away from the corresponding cavity or the corresponding channel.

[0018] In some preferred embodiments, the invention further comprises: at least one sealing film, wherein the sealing film is used to seal the pores.

[0019] Furthermore, the first containing cavity is provided with magnetic beads coupled with proteins. The surfaces of the magnetic beads have carboxyl functional groups, which can form amide bonds with amino-containing proteins or polypeptides through a chemical reaction mediated by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), thereby achieving coupling with the proteins or polypeptides.

[0020] In some preferred embodiments, the particle size of the magnetic beads is about 1.5 μm. Before use, the beads are blocked with casein at 37° C. for 4 hours and then washed.

[0021] In some preferred embodiments, the microspheres are colored microspheres.

[0022] It is understandable that the type of protein or polypeptide coupled to the protein-coupled magnetic beads can be designed and replaced according to the antigen or antibody of the pathogenic microorganism to be detected.

[0023] Furthermore, a reaction buffer is disposed in the second containing chamber, and the reaction buffer comprises physiological saline or PBS buffer.

[0024] Furthermore, a density gradient solution is provided in the reaction chamber, wherein the density gradient solution comprises 0.01-0.05 g / L methanol, 5-15 g / L fetal bovine serum and 700-1500 mesh dextran gel.

[0025] In some preferred embodiments, the methanol may be set to 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L or 0.05 g / L.

[0026] In some preferred embodiments, the fetal bovine serum can be set to 5 g / L, 10 g / L or 15 g / L.

[0027] In some preferred embodiments, the dextran gel can be set to 700-1500 mesh, for example, 700-750 mesh, 750-800 mesh, 800-850 mesh, 850-900 mesh, 950-1000 mesh, 1000-1100 mesh, 1100-1200 mesh, 1200-1300 mesh, 1300-1400 mesh or 1400-1500 mesh.

[0028] Another invention of the present invention also provides a detection method.

[0029] S100, providing a chip; the chip comprises: a substrate layer, the first surface of the substrate layer comprises a first centrifugal zone, a second centrifugal zone and a third centrifugal zone arranged in sequence; wherein the first centrifugal zone comprises: a sample loading chamber, a first quantitative chamber and a second quantitative chamber arranged in sequence along a set centrifugal direction; the second centrifugal zone comprises: a first pipe connected to the first quantitative chamber through a second pipe, and a first accommodating chamber connected to the side of the first pipe through a first channel, and a second accommodating chamber connected to the first accommodating chamber through a second channel; the liquid inlet end of the first pipe is connected to the first quantitative chamber; the third centrifugal zone comprises: a reaction chamber, the reaction chamber is connected to the second accommodating chamber through a third channel; an encapsulation layer, the encapsulation layer can cover at least one of the chambers, and the encapsulation layer is provided with at least one first pore, a second pore and a third pore corresponding to the first accommodating chamber, the second accommodating chamber and the reaction chamber respectively;

[0030] Magnetic beads coupled with proteins are placed in advance in the first receiving chamber; reaction buffer is placed in advance in the second receiving chamber; and density gradient liquid is placed in advance in the reaction chamber;

[0031] S101, after the first sample is added to the sample adding chamber, the chip is centrifuged for a first time at a first centrifugal force in a first state; wherein, when the chip is in the first state, the first pore, the second pore and the third pore are kept closed; the first sample is separated into a second sample under the action of the primary centrifugal force, and the second sample can at least partially enter the second channel;

[0032] Wherein, the first centrifugal force is 500-550g, and the first time length is 5-10s;

[0033] S102, centrifuging the chip for a second time with a second centrifugal force in a second state; wherein, when the chip is in the second state, keeping the second pore and / or the third pore closed;

[0034] Wherein, the second centrifugal force is 65-75g, and the second time length is 1.5-2.5min;

[0035] S103, centrifuging the chip for a third time period with a third centrifugal force in a third state, wherein when the chip is in the third state, the third pore is kept open;

[0036] Wherein, the third centrifugal force is 200-250g, and the third time length is 2-3min.

[0037] Furthermore, S101 includes:

[0038] (1) Centrifugation is performed with a first-order centrifugal force F1. At this time, the first sample is separated under the action of the first-order centrifugal force F1 to obtain a second sample. At this time, under the action of the first-order centrifugal force F1, the maximum driving force exerted on the plasma at the first resistance point A during the centrifugation process is a first driving force f2, and the first driving force f2 satisfies the following first force action model:

[0039] f1-f2≥λ1;

[0040] Wherein, f1 is the first resistance that the plasma needs to overcome to pass through the first resistance point, λ1 is the set first difference threshold; the first resistance point is the position where the second pipeline is closest to the centrifugal center;

[0041] (2) Centrifuging with a second first-order centrifugal force F2 so that the second sample can overcome the first resistance and enter the first pipe; at this time, under the action of the second first-order centrifugal force F2, the maximum driving force received by the second sample at the second resistance point during the centrifugation process is a second thrust f4, and the second thrust f4 satisfies the following second force action model:

[0042] f3-f4≥λ2;

[0043] Among them, f3 is the second resistance that plasma needs to overcome to enter the first containing chamber, the second resistance point is the connection between the first channel and the first containing chamber, and λ2 is the set second difference threshold.

[0044] In some embodiments, S102 includes:

[0045] (1) Centrifugation is performed with the first and second centrifugal forces F3. At this time, the resistance that the second sample needs to overcome to pass through the third resistance point is the third resistance f5, and the third resistance satisfies the following third force action model:

[0046] 0<(f6-f5) / m≤λ3;

[0047] Wherein, f6 is the third thrust exerted on the second sample at the third resistance point; m is f6 or f5, and correspondingly, λ3 is the set first ratio threshold; at this time, the second sample enters the first containing chamber and is preliminarily mixed with the first reagent to form a solid-liquid mixture, and part of the solid-liquid mixture can enter the second containing chamber;

[0048] (2) Centrifuging with a second secondary centrifugal force F4 so that the solid-liquid mixture is mixed again in the second containing chamber, and the second secondary centrifugal force is greater than the first secondary centrifugal force.

[0049] In some embodiments, S103 includes:

[0050] (1) centrifuging the chip for a first period of time at a first third-level centrifugal force, so that a portion of the solid-liquid mixture passes through the third channel at a first speed and then enters the reaction chamber;

[0051] (2) centrifuging the chip for a second period of time at a second third-level centrifugal force, so that the remaining solid-liquid mixture passes through the third channel at a second speed and then enters the reaction chamber; wherein the first speed is less than the second speed;

[0052] (3) Centrifuging the chip for a third period of time at a third level of centrifugal force to allow the solid-liquid mixture to fully react with the second reagent, wherein the third level of centrifugal force is greater than or equal to the second level of centrifugal force.

[0053] Wherein, the second-stage centrifugal force F2 is greater than the first-stage centrifugal force F1.

[0054] In some preferred embodiments, the first centrifugal force is 550 g, and the first time length is 10 s.

[0055] In some preferred embodiments, the second centrifugal force is 75 g, and the second time length is 1.5 min.

[0056] In some preferred embodiments, the third centrifugal force is 250 and the second time length is 2 minutes.

[0057] It is worth noting that the sample detection method for infectious disease detection in the present invention can be divided into the following stages: sample separation (such as separating whole blood into plasma and red blood cells) - sample initial mixing (such as initial mixing of plasma and magnetic beads to form a solid-liquid mixture) - sample reaction (such as the reaction of the solid-liquid mixture with the detection reagent). Correspondingly, the present invention proposes a segmented reaction method that can guide the sample to perform relatively independent reactions at different stages in a fully connected space, so as to improve the efficiency of the detection reaction and the accuracy of the structure.

[0058] Beneficial technical effects:

[0059] The present invention proposes a microfluidic centrifugal chip with sample separation synchronization and mixing delay effect, especially the dual flow limiting design realized by the second channel flow limiting and the depth change design of the two ports of the first channel, which can avoid the sample from flowing into the first accommodating cavity too early on the basis of meeting the high centrifugal force sample separation. The dual flow limiting design is also called a strong flow limiting solution.

[0060] In other words, for the detection scenario of semi-quantitative detection of pathogenic microorganisms in unknown samples in large quantities, the present invention proposes a simplified flow-limiting sampling design, which can make the sample contents of different reaction groups relatively close, while ensuring that the reaction stages between the reaction groups have a high degree of synchronization, thereby improving the accuracy and reliability of semi-quantitative detection. In addition, this simplified flow-limiting sampling design can also reduce the difficulty of processing the microstructure of the chip.

[0061] Furthermore, the present invention also proposes a segmented mixing design for the solid-liquid mixing stage involving the sample and the first reagent (such as magnetic beads). That is, unlike the strong flow limiting scheme, the present invention provides a relative flow limiting scheme for the second centrifugal stage. On the one hand, through the second channel with gradually increasing inner diameter, the third channel with capillary design, and the multi-segment connection structure design of the second accommodating chamber arranged between the second and third channels, the solid-liquid mixture can produce differential motion under the guidance of different sections, and then alleviate the congestion risk that may be caused by the solid-liquid mixture with the help of differential motion; at the same time, through the two-stage centrifugal force setting in the second centrifugal stage, it can cooperate with the above-mentioned multi-segment connection structure to further guide the solid-liquid mixture to perform differential motion in different time periods and different sections. Therefore, under the coordinated design of the centrifugal structure and the centrifugal stage, on the basis of ensuring that the solid-liquid mixture can be fully mixed for a long time in the second centrifugal stage, it can not only avoid its premature entry into the reaction chamber due to long-term centrifugation, but also avoid the risk of clogging in the microstructure of the second centrifugal zone.

[0062] In the present invention, by combining the strong current limiting scheme with the relative current limiting scheme, the accuracy and reliability of the reaction can be improved on the basis of simplifying the microstructure design and processing of the chip.

[0063] Furthermore, for the third centrifugation stage, the present invention also proposes a differential speed control mechanism, preferably a three-stage centrifugal control, that is, the speed at which the solid-liquid mixture enters the reaction chamber can gradually change from small to large, which can alleviate the risk of blockage of the solid-liquid mixture on the one hand, and at the same time, through the segmented mixing and reaction of the solid-liquid mixture and the second reagent (such as density gradient liquid), it can also improve the accuracy and reliability of the reaction results.

[0064] Finally, the multiple groups of reaction chambers for column agglutination reactions set in the microfluidic centrifugal chip of the present invention can realize multiple parallel detections of the same sample to be tested under one sample addition, and can intuitively judge whether the sample to be tested contains pathogenic microorganisms by the agglutination or non-agglutination of colored magnetic beads. The design of performing multiple parallel detections on the same sample to be tested at the same time greatly saves the time cost of human detection (biological samples often require multiple parallel detections). Finally, combined with the differential control mechanism set in the third centrifugal stage of the method of the present invention, the sample to be tested is mixed with the coupled magnetic beads and then falls rhythmically into the final density gradient gel. The sample can be fully mixed and fully enter the next chamber for reaction, thereby improving the accuracy of the test results as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0066] Figure 1 is a top view of a substrate layer in an exemplary embodiment of the present invention;

[0067] Figure 2 is a schematic structural diagram of a first channel in an exemplary embodiment of the present invention;

[0068] Figure 3 is a perspective structural diagram of a reaction group in an exemplary embodiment of the present invention;

[0069] Figure 4 is a schematic structural diagram of a reaction group in an exemplary embodiment of the present invention;

[0070] Figure 5 is a schematic structural diagram of a substrate layer in an exemplary embodiment of the present invention;

[0071] Figure 6 It is a schematic structural diagram of a chip package with a sealing film in an exemplary embodiment of the present invention;

[0072] Figure 7 It is a schematic diagram of the structure after the sealing film is torn open in an exemplary embodiment of the present invention;

[0073] Figure 8 Schematic diagram of the connection relationship between the intermediate cavity and the first quantitative cavity in an exemplary embodiment of the present invention.

[0074] Summary of reference numerals:

[0075] The sample adding chamber 11, the first quantitative chamber 12, the second quantitative chamber 13, the first capillary channel 14, the intermediate chamber 15, the second capillary channel 16, the second pipeline 17, the first pipeline 21, the first accommodating chamber 22, the second accommodating chamber 23, the first channel 25, the second channel 24, the reaction chamber 31, the third channel 32, the first pore 41, the second pore 42, the third pore 43, the sealing membrane 5, the first waste liquid chamber 6, the second waste liquid chamber 7, the first resistance point A, the second resistance point B, the third resistance point C, and the fourth resistance point D. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0077] Herein, suffixes such as "module", "component" or "unit" used to represent elements are only used to facilitate the description of the present invention, and have no specific meanings by themselves. Therefore, "module", "component" or "unit" can be used mixedly.

[0078] In this document, the terms "upper", "lower", "inner", "outer", "front", "back", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0079] In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0080] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0081] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0082] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0083] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0084] In this document, unless otherwise clearly specified and limited, along the flow direction of liquid (such as sample, buffer, etc.) in the channel, the two ends (first end and second end) of the channel are referred to as the liquid inlet end and the liquid outlet end.

[0085] Herein, a cavity refers to a spatial region that can accommodate a liquid. A channel and a pipe refer to a structure having a certain length through which a liquid can flow to connect two cavities. In some embodiments herein, channels or pipes may be used in a mixed manner. It should be understood that the description of a channel or pipe is only for the convenience of explaining the scheme and cannot be understood as indicating or implying the difference in spatial regions.

[0086] See also Figure 1-Figure 8 As shown, the present invention provides a microfluidic detection chip with synchronous sample splitting and synchronous and delayed mixing. The multi-stage centrifugal zone design in the chip can realize relatively independent reaction processes in the fully connected channels and chambers to improve the reliability and accuracy of the reaction.

[0087] Embodiment 1

[0088] The present invention provides a microfluidic chip, comprising:

[0089] A substrate layer, wherein the first surface of the substrate layer comprises a first centrifugal zone, a second centrifugal zone and a third centrifugal zone arranged in sequence; wherein the first centrifugal zone comprises: a sample adding chamber 11, a first quantitative chamber 12 and a second quantitative chamber 13 arranged in sequence along a set centrifugal direction, wherein the sample adding chamber is used to add a first sample;

[0090] See also Figure 2 As shown, the second centrifugal zone includes: a first pipe 21 with a first depth H1 formed by being recessed inwardly along the first surface, a first channel 25 with a second depth H2 formed by being recessed inwardly along the first surface, and a first accommodating cavity 22 with a third depth H3 formed by being recessed inwardly along the first surface; wherein the second depth H2 is smaller than the first depth H1 and the third depth H3 (see Figure 2As shown, the opening sizes of the liquid inlet end and the liquid outlet end of the first channel 25 are respectively smaller than the size of the first pipe 21 connected thereto, and the size of the first accommodating chamber connected thereto); the liquid inlet end of the first pipe 21 is connected to the first quantitative chamber 12; the liquid inlet end and the liquid outlet end of the first channel 25 are respectively connected to the first pipe 21 and the first accommodating chamber 22; the second accommodating chamber 23 is connected to the first accommodating chamber through the second channel 24, and the inner diameter of the liquid inlet end of the second channel 24 is smaller than the inner diameter of the liquid outlet end of the second channel 24. For example, in some embodiments, the inner diameter of the second channel gradually increases from its liquid inlet end to its liquid outlet end.

[0091] Preferably, the width of the second accommodating cavity is greater than the width of the liquid outlet end of the second channel and the liquid inlet end of the third channel.

[0092] Preferably, the first pipe 21 is connected to the first quantitative chamber 12 through the second pipe 17. Preferably, the second pipe is a capillary pipe, in particular, a U-shaped capillary channel, and the U-shaped opening of the U-shaped capillary channel is arranged in a direction away from the centrifugal center, and the two ends of the U are connected to the upper and lower chambers.

[0093] The third centrifugal zone includes: a reaction chamber 31, and the reaction chamber 31 is connected to the second containing chamber through a third channel 32;

[0094] The encapsulation layer can cover at least one of the cavities, at least one channel and at least one pipeline, and the encapsulation layer is provided with at least one pore. For example, it can cover all cavities, pipelines and channels except the sample loading cavity.

[0095] Among them, a first accommodating chamber is connected with a second accommodating chamber and a reaction chamber in sequence to form a reaction group. Figure 1 As shown, a plurality of reaction groups are sequentially arranged along the axial direction of the first pipe 21 .

[0096] It is worth noting that, in this embodiment, a limited multi-segment mixing mechanism is formed by the design of a flow-limiting dual channel (i.e., the second channel and the third channel) and the cooperation of the second accommodating chamber of the series-connected flow-limiting dual channels. This limited multi-segment mixing mechanism can help the solid-liquid mixture to produce differential motion in each segment, so as to reduce the risk of blockage of the solid-liquid mixture during a long mixing process while promoting full mixing of the solid-liquid mixture.

[0097] From another perspective, the present invention can alleviate the clogging problem of solid-liquid mixture in a simple and low-cost manner and improve the adequacy of mixing by limited multi-segment setting without increasing the processing difficulty of the chip's microstructure through the control of limited segment design and coordinated differential centrifugation (see the detection method section for details).

[0098] In an exemplary embodiment, the first receiving chamber can be used to store magnetic beads coated with antibodies (equivalent to the first reagent), the second receiving chamber can be set as a cavity (or other reagents can be set in combination with reaction requirements), and a density gradient liquid (equivalent to the second reagent) can be set in the reaction chamber. Figure 1 The exemplary working process of the chip in the present invention is described with this exemplary embodiment:

[0099] 1. During the first centrifugal stage, the blood (equivalent to the first sample) is separated under the action of the primary centrifugal force. At this time, the red blood cells in the plasma are separated from the plasma, and the red blood cells enter the second quantitative chamber 13. At this time, part of the plasma may enter the first pipe 21 under the action of capillary force, or, in the later stage of the first stage of centrifugation, it may enter the first pipe 21 under the combined action of capillary force and centrifugal force. Among them, the depth of the first channel 25 is less than the first pipe 21 and the first accommodating chamber 22. At this time, the first channel 25 plays a certain degree of flow limiting role, so that the plasma can first fill the first pipe (or can also partially enter the first pipe) to avoid entering the first accommodating chamber too early (in other words, it can ensure the synchronous sampling of each chamber during the centrifugation process to a certain extent).

[0100] 2. During the second centrifugal stage, the plasma enters the first reaction chamber synchronously with the help of multiple first channels 25 under the action of secondary centrifugal force, and mixes and reacts with the magnetic beads coated with antibodies pre-stored therein to form a first mixture. The first mixture is further rushed into the second containing chamber 23 under the action of the secondary centrifugal force to perform secondary mixing therein to form a corresponding second mixture; the second mixture may also partially enter the reaction chamber 31 under the action of the secondary centrifugal force, wherein the third channel 32 can play a certain degree of flow limiting role thereon; wherein the inner diameter of the second channel 24 can play a certain protective role on the magnetic bead particles, preventing the liquid inside it from entering the second containing chamber too early.

[0101] 3. During the third centrifugation stage, the second mixture is mixed and reacted with the density gradient liquid pre-existing in the reaction chamber to complete the detection.

[0102] That is to say, the present invention provides a flow-limiting centrifugal structure capable of synchronous sample separation and delayed mixing, wherein the dual flow-limiting design of the second pipe 17 and the first channel 25 is used in combination to ensure the synchronous operation of the liquid in the first and second centrifugal stages, avoiding the reaction group near the sample loading chamber from entering the mixing stage first, thereby improving the reliability of the detection results.

[0103] In some embodiments, in order to ensure the synchronous operation of each reaction group, the first centrifugation stage can also be set as a multi-stage centrifugation in conjunction with the design of the current limiting structure. The following is an example of the preferred solution - two-stage centrifugation:

[0104] First, centrifugation is performed with the first-order centrifugal force F1. At this time, the blood sample is subjected to sample separation under the action of the first-order centrifugal force F1, such as separating red blood cells from plasma, and the plasma may partially enter the second pipe 17. At this time, under the action of the first-order centrifugal force F1, the maximum driving force received by the plasma at the first resistance point A during the centrifugation process is the first thrust f2, and the first thrust f2 satisfies the following first force action model:

[0105] f1-f2≥λ1;

[0106] The first resistance f1 is the resistance that the plasma needs to overcome to pass through the first resistance point (equivalent to the flow limiting effect of the second channel 17 on the plasma), and λ1 is the set first difference threshold; the first resistance point is the position of the second channel 17 closest to the centrifugal center;

[0107] Subsequently, centrifugation is performed with the second primary centrifugal force F2, so that the plasma can overcome the first resistance and enter the first pipe 21. At this time, under the action of the second primary centrifugal force F2, the maximum driving force received by the plasma at the second resistance point B during the centrifugation process is the second thrust f4, and the second thrust f4 satisfies the following second force action model:

[0108] f3-f4≥λ2;

[0109] Among them, f3 is the second resistance that plasma needs to overcome to enter the first containing chamber 22, the second resistance point is the connection between the first channel and the first containing chamber, and λ2 is the set second difference threshold.

[0110] Preferably, the second primary centrifugal force F2 is greater than the first primary centrifugal force F1. That is, the second force action model is preferably:

[0111] f3-f4≥λ2;

[0112] F2≥F1.

[0113] Furthermore, in some embodiments, the second centrifugation stage may also be set as multi-stage centrifugation, and the following is described using the preferred solution of two-stage centrifugation as an example:

[0114] First, centrifugation is performed with the first and second centrifugal forces F3. At this time, the resistance that the plasma needs to overcome to pass through the third resistance point C is the third resistance f5, and the third resistance satisfies the following third force action model:

[0115] (f6-f5) / m≤λ3;

[0116] Wherein, f6 is the maximum thrust force (also referred to as the third thrust force) exerted on the plasma at the third resistance point C; m is f6 or f5, and correspondingly, λ3 is the set first proportional threshold value;

[0117] Subsequently, centrifugation is performed with a second secondary centrifugal force F4. At this time, a part of the solid-liquid mixture formed by the plasma and the magnetic beads will be further mixed in the second containing chamber, or a part of the solid-liquid mixture may be able to smoothly pass through the fourth resistance point under the action of the second secondary centrifugal force and enter the reaction chamber.

[0118] In this embodiment, the segmented centrifugal mechanism designed with double containing chambers can prolong the mixing time of the solid-liquid mixture and can also perform a certain degree of diversion to reduce the risk of clogging the microstructure (especially the channel) during the long mixing process.

[0119] Preferably, the second secondary centrifugal force is greater than or equal to the first secondary centrifugal force. That is, in this embodiment, the plasma and the magnetic beads are preferably controlled to be relatively evenly mixed in the first accommodating chamber 22 to prevent the plasma from entering the second accommodating chamber 23 too early.

[0120] In this embodiment, the first-stage centrifugal force, the third-stage centrifugal force and the second-stage centrifugal force decrease in sequence.

[0121] The first duration, the second duration, and the third duration increase sequentially.

[0122] Furthermore, in order to optimize the synchronous sampling and mixing delay operations, the present invention also provides a graded pore design.

[0123] For example, in some embodiments, the method includes: a first air hole 41 with a first aperture arranged corresponding to the first accommodating chamber (for example, the first air hole can be arranged above the first accommodating chamber, or the first air hole can be arranged on the first channel 25, and is preferably arranged at a position adjacent to the first accommodating chamber 22), and a second air hole 42 with a second aperture arranged corresponding to the reaction chamber, and the first aperture is larger than the second aperture.

[0124] In some embodiments, it also includes: a third air hole with a third aperture arranged corresponding to the second accommodating cavity, and the first aperture is larger than the third aperture.

[0125] For example, in some embodiments, see Figure 5 As shown, the encapsulation layer is provided with a first pore 41 corresponding to the first accommodating cavity 22, a third pore 43 corresponding to the second accommodating cavity 23, and a second pore 42 corresponding to the reaction cavity 31. Preferably, the aperture of the first pore 41 is larger than the apertures of the second pore and the third pore.

[0126] In some embodiments, the first, second, and third air holes are respectively provided with different sealing layers (such as tearable films, piston rods, etc.).

[0127] In this embodiment, the graded design of the pore diameters can further assist in achieving graded centrifugal operation.

[0128] Specifically, the pores have different aperture settings, and an easy-tear film (or sealing film) can be set on them. Therefore, the user can more flexibly set the first resistance and the second resistance value by controlling the opening and closing of the pores, so as to complete the deployment of multi-level centrifugal force under the centrifugal force requirements of mixing and reaction requirements.

[0129] For example, when the first centrifugal stage with relatively large centrifugal force is being carried out, by sealing the subsequent multiple air holes, effective separation of whole blood can be ensured, and at the same time, whole blood and its separated samples can be prevented from entering the next stage too early.

[0130] In some embodiments, the inner diameter of the pore gradually increases in a direction away from the cavity or channel to prevent liquid from overflowing from the pore.

[0131] In some embodiments, the pores may include: pore channels extending from the cavity (or channel), and pore openings disposed in the packaging layer. Therefore, the pore channel design can further prevent leakage of the internal liquid.

[0132] It is worth noting that the present invention provides a multi-stage centrifugation scheme based on flexible regulation of microfluidic internal air pressure, and this centrifugation scheme has better characteristics such as sample separation synchronization and mixing delay.

[0133] For example, in some embodiments, the sealing film 5 on the pore is as follows: Figure 6 As shown, the membrane can be manually torn off by the inspector during the centrifugation process.

[0134] For another example, in some embodiments, when batch testing is involved, a robotic arm may be used to assist in the tearing operation. For example, the sealing film may also have a tearing portion (such as a handle structure, or it may have a certain adsorption property). When the gripper of the robotic arm grabs the handle or adsorbs / adheres to the sealing film, the tearing operation of one or more chips may be performed in sequence.

[0135] Alternatively, in some embodiments, a piston column can be used to block the pores; one end of the piston column is used to block the pores, and the other end is connected to the mechanical arm structure. When the chip is centrifuged horizontally, the piston can adjust the blocking state of the pores by adjusting the position in the vertical direction, that is, keep it open or closed. (Specifically, the mechanical arm structure can remove the piston column to open the pores).

[0136] See also Figure 8 As shown, in some embodiments, the first centrifugal zone further includes: at least one intermediate cavity 15; wherein the intermediate cavity 15 is connected to the sample addition cavity 11 through at least one first capillary channel 14, and the intermediate cavity 15 is connected to the first quantitative cavity 12 through at least one second capillary channel 16. In this embodiment, the buffer flow limiting design achieved by the cooperation of the intermediate cavity and the upper and lower capillary channels can enable the first sample to enter the first containing cavity only under the condition of sufficiently large centrifugal force. Especially when performing batch detection, this synchronization can ensure the accuracy and reliability of the detection results.

[0137] In some embodiments, the chip further includes: a first waste liquid chamber 6, wherein the first waste liquid chamber is connected to the output end of the first pipe 21, and the first waste liquid chamber may also be provided with at least one air hole.

[0138] In some embodiments, the chip further includes: a second waste liquid chamber 7, wherein the second waste liquid chamber is connected to the first quantitative chamber 12, and the second waste liquid chamber may further be provided with at least one air hole.

[0139] In some embodiments, one or more channels are implemented as capillary channels.

[0140] The present invention also provides a sample detection method, comprising the steps of:

[0141] S100, providing a chip; the chip comprises: a substrate layer, the first surface of the substrate layer comprises a first centrifugal zone, a second centrifugal zone and a third centrifugal zone arranged in sequence; wherein the first centrifugal zone comprises: a sample loading chamber 11, a first quantitative chamber 12 and a second quantitative chamber 13 arranged in sequence along a set centrifugal direction; the second centrifugal zone comprises: a first pipeline 21, and a first accommodating chamber 22 connected to the side of the first pipeline 21 through a first channel 25, and a second accommodating chamber 23 connected to the first accommodating chamber 22 through a second channel 24; the liquid inlet end of the first pipeline 21 is connected to the first quantitative chamber; the third centrifugal zone comprises: a reaction chamber 31, and the reaction chamber 31 is connected to the second accommodating chamber through a third channel 32; an encapsulation layer, the encapsulation layer can cover at least one of the chambers, at least one channel and at least one pipeline, and the encapsulation layer is provided with at least one first pore, a second pore and a third pore corresponding to the first accommodating chamber, the second accommodating chamber and the reaction chamber respectively;

[0142] S101, after the first sample is added to the sample adding chamber, the chip is centrifuged for a first time at a first-level centrifugal force in a first state; wherein when the chip is in the first state, the first pore, the second pore, and the third pore are kept closed;

[0143] In this process, the first sample (such as whole blood) can be separated under the action of centrifugal force, such as separating the whole blood into plasma (equivalent to the second sample) and red blood cells.

[0144] S102, in the second state, centrifuging the chip for a second time period with a secondary centrifugal force; wherein, when the chip is in the second state, the second pore and / or the third pore are kept closed; preferably, in some embodiments, during the sample separation process, the first pore can be kept open, and the second pore and the third pore are kept closed.

[0145] S103, in a third state, centrifuging the chip for a third time period with a third level of centrifugal force, wherein when the chip is in the third state, the third pore is kept open.

[0146] Further, in some embodiments, S101 uses a two-stage centrifugation method to ensure synchronous sample division. For example, S101 includes the steps of:

[0147] The chip is centrifuged with a first-level centrifugal force F1. At this time, the first sample is separated under the action of the first-level centrifugal force F1 to obtain a second sample. At this time, under the action of the first-level centrifugal force F1, the maximum driving force received by the second sample at the first resistance point A during the centrifugation process is a first thrust f2, and the first thrust f2 satisfies the following first force action model:

[0148] f1-f2≥λ1;

[0149] Wherein, f1 is the first resistance that the second sample needs to overcome to pass through the first resistance point, λ1 is the set first difference threshold; the first resistance point is the position of the second pipe closest to the centrifugal center;

[0150] (2) centrifuging the chip with a second first-level centrifugal force F2 so that the second sample can overcome the first resistance and enter the first channel (21); at this time, under the action of the second first-level centrifugal force F2, the maximum thrust exerted on the second sample at the second resistance point (B) during the centrifugation process is a second thrust f4, and the second thrust f4 satisfies the following second force action model:

[0151] f3-f4≥λ2;

[0152] Wherein, f3 is the second resistance that the second sample needs to overcome to enter the first containing chamber, the second resistance point is the connection between the first channel and the first containing chamber (or, at the port of the first channel 25 adjacent to the first containing chamber 22), and λ2 is the set second difference threshold;

[0153] Among them, the second-level centrifugal force F2 is greater than the first-level centrifugal force F1.

[0154] In this embodiment, a strong flow-limiting centrifugation scheme is adopted for the first centrifugation stage, that is, the centrifugal structure and centrifugal force are designed to prevent the second sample from entering the chamber of the next centrifugation stage as much as possible.

[0155] Further, in some embodiments, S102 includes:

[0156] (1) Centrifugation is performed with the first and second centrifugal forces F3. At this time, the mixture formed by mixing the plasma and the pre-stored reagent (also called: solid-liquid mixture) needs to overcome the resistance f5 to pass through the third resistance point C, and the third resistance satisfies the following third force action model:

[0157] (f6-f5) / m≤λ3;

[0158] Wherein, f6 is the maximum thrust force (also referred to as the third thrust force) exerted on the plasma at the third resistance point C; m is f6 or f5, and correspondingly, λ3 is the set first proportional threshold value;

[0159] The third resistance point C is the connection between the second channel and the first accommodating chamber (or, the liquid inlet end of the second channel 24).

[0160] (2) Subsequently, centrifugation is performed with a second secondary centrifugal force F4.

[0161] In some embodiments, under the action of the second secondary centrifugal force, a portion of the plasma can smoothly pass through the fourth resistance point under the action of the second secondary centrifugal force.

[0162] The fourth resistance point D is the connection point between the third channel 32 and the reaction chamber 31 (or, the liquid outlet end of the third channel 32 ).

[0163] It is worth noting that, unlike the strong flow-limiting centrifugal scheme in the first centrifugal stage, a relatively flow-limiting centrifugal scheme is adopted in this embodiment, that is, the centrifugal force in the second centrifugal stage is limited to a certain extent, only preventing the solid-liquid mixture from entering the next chamber too much at this stage. In other words, with the help of a multi-segment flow-limiting setting, a certain degree of diversion is set for the solid-liquid mixture, that is, it can accommodate part of the mixture to enter the next chamber to a certain extent, but at the same time, it can ensure that the mixture can be fully mixed through a multi-segment setting to ensure the reliability of subsequent test results. In this way, the risk of blockage of the solid-liquid mixture is alleviated on the basis of taking into account reliability.

[0164] From another perspective, the present invention also produces a synergistic effect by using a relatively limited flow centrifugal scheme in conjunction with a multi-segment connection structure, that is, the scheme is more conducive to simplifying the setting of the microstructure, such as only needing to set a limited centrifugal structure, that is, the shorter second channel and the third channel can meet the centrifugal requirements of the second centrifugal area, which is more helpful to alleviate the risk of blockage. In addition, when using the relatively limited flow centrifugal scheme in this embodiment, it is preferred to use a gel with a larger mesh size to ensure the adequacy of the reaction to a certain extent.

[0165] Of course, it is understandable that the actual centrifugal stage and the magnitude of the centrifugal force can be flexibly set by the test personnel in combination with the actual test requirements or test scenarios.

[0166] Preferably, the second secondary centrifugal force is greater than or equal to the first secondary centrifugal force. That is, in this embodiment, the plasma and the magnetic beads are preferably controlled to be relatively evenly mixed in the first accommodating chamber 22 to prevent the plasma from entering the second accommodating chamber 23 too early.

[0167] Further, in some embodiments, S103 includes:

[0168] (1) centrifuging the chip for a first period of time at a first third-level centrifugal force, so that a portion of the solid-liquid mixture passes through the third channel 32 at a first speed and then enters the reaction chamber 31;

[0169] (2) centrifuging the chip for a second period of time at a second third-level centrifugal force, so that the remaining solid-liquid mixture passes through the third channel 32 at a second speed and then enters the reaction chamber 31; wherein the first speed is less than the second speed;

[0170] (3) Centrifuging the chip for a third period of time at the third level of centrifugal force so that the solid-liquid mixture and the second reagent can fully react, and the third level of centrifugal force is greater than or equal to the second level of centrifugal force.

[0171] Preferably, in this embodiment, the three-level centrifugal force in the first, second and third time periods gradually increases.

[0172] It can be understood that, in the absence of conflict, the chip in this embodiment can adopt the solution described in any of the above embodiments, which will not be described in detail here.

[0173] Embodiment 2

[0174] This embodiment provides a specific example of detecting a sample to be tested containing pathogenic microorganisms.

[0175] S100, providing a chip, wherein each chamber in the chip is pre-filled with reagents;

[0176] S101, adding a whole blood sample to be tested, centrifuging the chip for a first time at a first-level centrifugal force in a first state, and separating the whole blood sample into plasma and blood cells after centrifugation, wherein the plasma enters a subsequent channel of the chip, and the blood cell sediment enters a second quantitative chamber.

[0177] S102, in the second state, the chip is centrifuged for a second time with a secondary centrifugal force, and the plasma to be tested enters the first receiving chamber after centrifugation; the first receiving chamber is provided with antigen-microspheres that can bind to the antibodies of pathogenic microorganisms in the sample to be tested. The microspheres are colored microspheres. The antibodies are captured by the antigen-microspheres to generate antibody-antigen-microsphere complexes. Optionally, a desiccant may also be provided in the first receiving chamber. The generated antibody-antigen-microsphere complex enters the second receiving chamber, and the buffer provided in the second receiving chamber can separate impurities that are not bound to the antigen-microspheres.

[0178] S103, in the third state, the chip is centrifuged for a third time with a third centrifugal force, and the antibody-antigen-microsphere complex enters the reaction chamber after centrifugation. The third centrifugal force is a differential control centrifugation, and the speed at which the solid-liquid mixture enters the reaction chamber is first set to be relatively slow, so that the coupled microspheres can gradually pass through the reaction channel without blocking the channel; the centrifugation is further accelerated to allow the fetal bovine serum (secondary antibody) contained in the density gradient liquid set in the reaction chamber to further bind to the antibody-antigen, and at this time, the microspheres undergo agglutination reaction and are fixed on the surface of the dextran gel. At this time, the colored microspheres on the surface of the dextran gel can be observed by the naked eye, and the reaction result is positive (+).

[0179] It is understood that if the whole blood sample to be tested does not have the target microorganism antibody, the microspheres will not undergo agglutination reaction and fall to the bottom of the dextran gel. The naked eye can clearly observe the clear accumulation band formed by the microspheres at the bottom of the agglutination, and the reaction is negative (-).

[0180] In order to facilitate understanding of the technical solution adopted in this application and the technical effects achieved, the capillary action process in the microfluidic chip will be described here. Since the capillary channel has a certain degree of capillary force, when the capillary channel is connected to the cavity storing the liquid, part of the liquid can enter the capillary channel autonomously under the action of the capillary force. By setting a size mutation at the capillary channel, for example, the inner diameter of the second channel gradually increases, the capillary action mutation can limit the flow of the liquid.

[0181] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0182] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A sample detection method for infectious disease detection, characterized in that: Includes steps: S100, providing a chip; the chip comprising: a substrate layer, wherein a first surface of the substrate layer comprises a first centrifugal zone, a second centrifugal zone and a third centrifugal zone arranged in sequence; wherein the first centrifugal zone comprises: a sample loading chamber (11), a first quantitative chamber (12) and a second quantitative chamber (13) arranged in sequence along a set centrifugal direction; the second centrifugal zone comprises: a first pipe (21) connected to the first quantitative chamber (12) via a second pipe (17), and a first accommodating chamber connected to a side of the first pipe via a first channel, and a second accommodating chamber connected to the first accommodating chamber via a second channel ; the liquid inlet end of the first pipe is connected to the first quantitative chamber; the third centrifugal zone comprises: a reaction chamber (31), the reaction chamber (31) is connected to the second accommodating chamber through a third channel (32); an encapsulation layer, the encapsulation layer can cover at least one corresponding chamber, at least one corresponding channel and at least one corresponding pipe, and the encapsulation layer is provided with at least one first pore, a second pore and a third pore corresponding to the first accommodating chamber, the second accommodating chamber and the reaction chamber respectively; a first reagent is provided in the first accommodating chamber (22); and a second reagent is provided in the reaction chamber (31); S101, after the first sample is added to the sample adding chamber, the chip is centrifuged for a first time at a first-level centrifugal force in a first state; wherein when the chip is in the first state, the first pore, the second pore and the third pore are kept closed; the first sample is separated from the second sample under the action of the first-level centrifugal force, and the second sample enters the first quantitative chamber (12), and the second sample can also at least partially enter the second channel; S102, centrifuging the chip for a second time with a secondary centrifugal force in a second state; wherein, when the chip is in the second state, the first pore is kept open, and the second pore and / or the third pore is closed; at this time, the second sample enters the first containing cavity and mixes with the first reagent to form a solid-liquid mixture; S103, in a third state, centrifuging the chip for a third time period with a third level of centrifugal force, wherein when the chip is in the third state, the third pore is kept open; and the solid-liquid mixture enters the reaction chamber (31) to react with the second reagent; Wherein, the first-level centrifugal force is greater than the third-level centrifugal force, and the third-level centrifugal force is greater than the second-level centrifugal force; Wherein, S101 includes: (1) The chip is centrifuged with a first-level centrifugal force F1. At this time, the first sample is separated under the action of the first-level centrifugal force F1 to obtain a second sample. At this time, under the action of the first-level centrifugal force F1, the maximum driving force received by the second sample at the first resistance point (A) during the centrifugation process is a first driving force f2, and the first driving force f2 satisfies the following first force action model: f1-f2≥λ1; Wherein, f1 is the first resistance that the second sample needs to overcome to pass through the first resistance point, λ1 is the set first difference threshold; the first resistance point is the position of the second pipe closest to the centrifugal center; (2) centrifuging the chip with a second first-level centrifugal force F2 so that the second sample can overcome the first resistance and enter the first channel (21); at this time, under the action of the second first-level centrifugal force F2, the maximum thrust exerted on the second sample at the second resistance point (B) during the centrifugation process is a second thrust f4, and the second thrust f4 satisfies the following second force action model: f3-f4≥λ2; Wherein, f3 is the second resistance that the second sample needs to overcome to enter the first containing chamber, the second resistance point is the connection between the first channel and the first containing chamber, and λ2 is the set second difference threshold; Wherein, the second-stage centrifugal force F2 is greater than the first-stage centrifugal force F1; Wherein, S102 includes: (1) Centrifugation is performed with the first and second centrifugal forces F3. At this time, the resistance that the second sample needs to overcome to pass through the third resistance point (C) is the third resistance f5, and the third resistance satisfies the following third force action model: 0<(f6-f5) / m≤λ3; Wherein, f6 is the third thrust exerted on the second sample at the third resistance point (C); m is f6 or f5, and correspondingly, λ3 is the set first ratio threshold; at this time, the second sample enters the first containing chamber and is preliminarily mixed with the first reagent to form a solid-liquid mixture, and part of the solid-liquid mixture can enter the second containing chamber; (2) Centrifuging with a second secondary centrifugal force F4 so that the solid-liquid mixture is mixed again in the second containing chamber, and the second secondary centrifugal force is greater than the first secondary centrifugal force.

2. A sample detection method according to claim 1, characterized in that: S103 includes: (1) centrifuging the chip for a first period of time at a first third-level centrifugal force, so that a portion of the solid-liquid mixture passes through the third channel (32) at a first speed and then enters the reaction chamber (31); (2) centrifuging the chip for a second period of time at a second third-level centrifugal force, so that the remaining solid-liquid mixture passes through the third channel (32) at a second speed and then enters the reaction chamber (31); wherein the first speed is less than the second speed; (3) Centrifuging the chip for a third period of time at a third level of centrifugal force to allow the solid-liquid mixture to fully react with the second reagent, wherein the third level of centrifugal force is greater than or equal to the second level of centrifugal force.

3. A sample detection method according to claim 1, characterized in that: The first pipe (21) is recessed inwardly along the first surface to a first depth H1, the first channel (25) is recessed inwardly along the first surface to a second depth H2, and the first accommodating cavity (22) is recessed inwardly along the first surface to a third depth H3; wherein the second depth H2 is smaller than the first depth H1 and the third depth H3, and the inner diameter of the liquid inlet end of the second channel (24) is smaller than the inner diameter of the liquid outlet end of the second channel (24).

4. A sample detection method according to claim 3, characterized in that: A first aperture of the first pore is larger than a second aperture of the second pore.

5. A sample detection method according to claim 3, characterized in that: The first aperture of the first air hole is larger than the third aperture of the third air hole.

6. A sample detection method according to claim 3, characterized in that: The first centrifugal zone further comprises: at least one intermediate chamber (15); wherein the intermediate chamber (15) is connected to the sample addition chamber (11) via at least one first capillary channel (14), and the intermediate chamber (15) is connected to the first quantitative chamber (12) via at least one second capillary channel; And / or, the inner diameter of the air hole gradually increases along the direction away from the corresponding cavity or the corresponding channel.

7. A sample detection method according to claim 3, characterized in that: The first containing cavity (22) is provided with magnetic beads coupled to proteins; and / or, The reaction chamber (31) is provided with a density gradient liquid, wherein the density gradient liquid comprises 0.01-0.05 g / L methanol, 5-15 g / L fetal bovine serum and 700-1500 mesh dextran gel.

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