A multiplex nucleic acid detection chip

Abstract

This invention relates to the field of microfluidics, specifically to a multiplex nucleic acid detection chip. It includes: an upstream chamber; and multiple downstream chambers connected to the upstream chambers via dispensing channels. Each downstream chamber has a first quantitative reservoir. The dispensing channel has a spiral or arc-shaped structure and features steps away from the center of rotation based on its curvature and radius. Under centrifugal force, liquid in the upstream chamber sequentially fills the corresponding first quantitative reservoir through the dispensing channel. This enables simultaneous detection of multiple targets with a single sample addition, avoiding the need to add liquid reagents to multiple detection tubes, reducing manual operation and the risk of mismatch.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, specifically to a multiplex nucleic acid detection chip. Background Technology

[0002] With the development of microfluidic technology and diagnostic testing technology, the application of microfluidic chips based on microfluidic technology in in vitro diagnostics (IVD) and other fields has become increasingly in-depth and widespread. Microfluidic chips are characterized by miniaturization, integration, and high automation, which makes the diagnostic testing process based on them more convenient, efficient, and cost-effective compared to traditional diagnostic testing processes. Nucleic acid amplification detection (such as PCR, LAMP, and RPA) is a highly sensitive biotechnology that performs qualitative or quantitative analysis of trace target gene sequences under specific enzyme catalysis systems. In recent years, its deep integration and application with microfluidic chips (such as point-of-care testing, POCT) has yielded a series of achievements and advancements.

[0003] For multiplex nucleic acid detection, existing technologies require adding liquid reagents to multiple test tubes, which is labor-intensive and prone to mismatches. Microfluidic chips can be used to address these issues, but they also present challenges in multiplex nucleic acid detection. For example, multiplex nucleic acid detection requires multiple corresponding quantification chambers and matching reaction chambers. As the quantification chambers are filled sequentially, ensuring complete filling and maintaining identical nucleic acid content or concentration in each chamber is a critical problem that needs to be solved. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a multiplex nucleic acid detection chip. It enables simultaneous detection of multiple targets with a single sample addition, avoiding the need to add liquid reagents to multiple detection tubes, reducing manual operation and the potential risk of mismatch.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A multiplex nucleic acid detection chip, comprising: an upstream chamber; a downstream chamber, wherein the downstream chamber comprises a plurality of downstream chambers, and the plurality of downstream chambers are connected to the upstream chamber through a distribution channel; the downstream chamber has a first quantitative pool, the distribution channel has a spiral or arc-shaped structure, and a step away from the center of rotation is provided based on the arc and radius of the distribution channel; under the action of centrifugal force, the liquid in the upstream chamber sequentially fills the corresponding first quantitative pool through the distribution channel.

[0006] Furthermore, the cross-section of the step along the direction of liquid flow is arc-shaped.

[0007] Furthermore, the step cross-section is arc-shaped, and the radius r of the arc is related to the radial distance L of the step.

[0008] Furthermore, a flow control valve is provided between the upstream chamber and the distribution channel.

[0009] Furthermore, the radius r of the arc satisfies: ; in, L is the correction factor, L is the radial distance of the step, and L0 is the standard distance.

[0010] Furthermore, the area of ​​the opening of the first metering pool located downstream of the step is not greater than the area of ​​the opening of the first metering pool located upstream of the step.

[0011] Furthermore, the first metering pool is divided into several groups based on the steps. The first metering pools in the same group have the same depth, and the depth of the first metering pool in the upstream group is less than that in the downstream group.

[0012] Furthermore, there is a certain gap between the opening of the first metering cell and the depth of the first metering cell; The gap is related to the radial distance of the step.

[0013] Furthermore, the radial distance L of the step satisfies: 1mm≤L≤5mm.

[0014] Furthermore, the upstream chamber includes a sample loading cell, a lysis cell, and a mixing cell connected in sequence; the mixing cell is connected to the downstream chamber via a distribution channel.

[0015] Furthermore, the sample loading chamber includes: a sample loading port; an exhaust port; a first-level groove, which is disposed close to the surface of the chip; a second-level groove, which is disposed away from the surface of the chip and has a sealing film at its bottom; the bottom surface of the first-level groove is connected to the second-level groove; the cross-sectional area of ​​the first-level groove is larger than the cross-sectional area of ​​the second-level groove; the sample loading port and the exhaust port are connected to the bottom surface of the second-level groove; and the sealing film seals the sample loading port and the exhaust port.

[0016] Furthermore, the gap G satisfies the following equation: ; in L is the correction factor, and L is the radial distance of the step.

[0017] Furthermore, the location of the steps satisfies the following: ; Where β is the threshold, α is the arc value between the steps, and R is the distance between the first quantitative cell and the rotation center.

[0018] The beneficial effects of this invention are as follows: This invention achieves simultaneous detection of ultra-multiple targets with a single sample addition through the above-described scheme, avoiding the need to add liquid reagents to multiple detection tubes, reducing manual operation and the potential risk of mismatch. Furthermore, the amplification system, except for primers, is thoroughly mixed before distribution, resulting in better detection consistency. Secondly, each detection target reacts independently, avoiding the competitive inhibition problem inherent in single-tube multiplex amplification. However, there are still certain technical challenges in achieving ultra-multiple nucleic acid detection based on microfluidic chips. For nucleic acid detection, the amount of liquid and nucleic acid in the quantification chamber must be almost identical, with a deviation not exceeding [a certain value]. 3%, and as the liquid filling progresses, the frictional force exerted on the distribution channel wall on the liquid increases, causing a significant decrease in liquid velocity. The decrease in liquid velocity causes nucleic acid material to be adsorbed onto the distribution channel wall under the action of surface tension, resulting in a reduction in the amount of nucleic acid in the subsequent first quantitative pool. To solve the above problem, this application proposes to set a step away from the rotation center based on the curvature and radius of the distribution channel. Since the centrifugal force increases as the liquid moves away from the rotation center, the increased centrifugal force is used to overcome the frictional force on the distribution channel wall, increase the liquid flow velocity, and avoid the decrease in velocity causing nucleic acid material to be deposited on the channel wall. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the multiplex nucleic acid detection chip structure provided in an embodiment of the present invention; Figure 2 This is a partial enlarged view of the multiplex nucleic acid detection chip provided in an embodiment of the present invention; Figure 3 This is a partial cross-sectional view of the side of the multiplex nucleic acid detection chip provided in an embodiment of the present invention; Figure 4 This is a schematic diagram of the sample addition tank structure provided in an embodiment of the present invention.

[0020] Reference numerals: 01, flow control valve; 10, upstream chamber; 100, sample loading cell; 1000, sample loading port; 1001, vent hole; 1002, second-stage groove; 1003, first-stage groove; 101, lysis cell; 102, mixing cell; 11, downstream chamber; 110, first quantitative cell; 111, reaction cell; 112, second quantitative cell; 12, distribution channel; 13, step. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances. Furthermore, the terms "first," "second," and "third," etc., are used only for distinguishing descriptions and should not be construed as indicating or implying relative importance.

[0024] In this invention, unless otherwise expressly specified and limited, "above or below" a first feature may include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on" the first feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the first feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0025] This invention provides a multiplex nucleic acid detection chip, such as... Figure 1 As shown, it includes: an upstream chamber 10; and a downstream chamber 11, wherein the downstream chambers 11 include multiple downstream chambers 11, which are connected to the upstream chamber 10 through a distribution channel 12. Each downstream chamber 11 has a first metering pool 110. The distribution channel 12 has a spiral or arc-shaped structure, and a step 13 away from the center of rotation is provided based on the arc and radius of the distribution channel 12. Under the action of centrifugal force, the liquid in the upstream chamber 10 sequentially fills the corresponding first metering pool 110 through the distribution channel 12.

[0026] In this application, "multiple" refers to at least 10 layers. When the distribution channel is spiral-shaped, the connection points between the downstream chambers and the distribution channel are progressively farther away as the liquid fills. When the distribution channel is arc-shaped, the distance between the connection points between the downstream chambers and the distribution channel and the rotation center remains constant. The downstream chamber 11 is a collection of multiple chambers, such as including a first metering tank 110 and a subsequent reaction tank 111. It should be noted that, based on the curvature and radius of the distribution channel 12, steps are provided away from the rotation center. The curvature of the distribution channel refers to the curvature of the distribution channel relative to the rotation center, such as... Figure 1 As shown, the arc corresponding to the first step is α, and the radius R corresponds to the radius of the relative rotation center of the corresponding step. When the distribution channel is arc-shaped, the radius R is a fixed value. When the distribution channel is spiral-shaped, the radius R is the average of the minimum and maximum radii of the spiral, the minimum radius, or the maximum radius. Preferably, the average of the minimum and maximum radii is used. The radii are all calculated based on the side of the distribution channel away from the rotation center.

[0027] This invention achieves simultaneous detection of multiple targets with a single sample addition through the above-described scheme, avoiding the need to add liquid reagents to multiple detection tubes, reducing manual operation and the risk of mismatch. Furthermore, the amplification system, except for primers, is completely mixed before distribution, resulting in better detection consistency. Secondly, each detection target reacts independently, avoiding the competitive inhibition problem present in single-tube multiplex amplification. However, there are still some technical challenges in achieving multiplex nucleic acid detection based on microfluidic chips. For nucleic acid detection, the amount of liquid and nucleic acid in the quantification chamber must be almost identical, with a deviation not exceeding 3%. As liquid filling progresses, the frictional force on the distribution channel wall increases, leading to a significant decrease in liquid velocity. This decrease in flow velocity causes nucleic acid to be adsorbed onto the distribution channel wall under surface tension, reducing the amount of nucleic acid in the subsequent first quantification chamber. To address this issue, this application proposes a step based on the curvature and radius of the distribution channel, located away from the center of rotation. Since the centrifugal force increases as the liquid moves further away from the center of rotation, this increased centrifugal force overcomes the frictional force on the distribution channel wall, increasing the liquid flow velocity and preventing the decrease in velocity from causing nucleic acid deposition on the channel wall.

[0028] Appendix Figure 1 As shown, a flow control valve 01 is provided between the upstream chamber 10 and the distribution channel 12. The flow control valve can be a siphon valve, a burst valve, a mechanical rotary valve, or a phase change valve. Preferably, the flow control valve is a paraffin valve among phase change valves.

[0029] Specifically, such as Figure 2As shown, the cross-section of step 13 along the direction of liquid flow is arc-shaped. Due to the inertia of the liquid during flow, if step 13 were right-angled, the liquid might skip a hole due to inertia, meaning the downstream first metering pool adjacent to the step would not be filled.

[0030] Specifically, such as Figure 2 As shown, the cross-section of the step 13 is arc-shaped, and the radius of the arc is related to the radial distance L of the step.

[0031] The radius r of the arc satisfies: ; in, The correction factor is 2.2mm-2.3mm, L is the radial distance of the step (mm), and L0 is the standard distance (2mm), where L ≥ L0. The arc is tangent to the side of the distribution channel upstream of the step that is furthest from the rotation center.

[0032] Because of the viscosity between the arc of the step and the liquid, the step exerts a pulling force that alters the liquid's flow direction. If the arc radius is large, the liquid is constantly pulled by the step, resulting in a significant impact on the first metering tank adjacent to the step. After filling, the downstream first metering tank, adjacent to the step, may be underfilled due to excessive liquid surface fluctuations during filling. If the arc radius is too small, the inertial force exceeds the pulling force of the step, causing the liquid to detach from the step and skip over the adjacent first metering tank, thus filling the downstream first metering tank. This results in a skip-filling phenomenon. Therefore, it is necessary to control the arc radius.

[0033] Specifically, the area of ​​the opening of the first metering pool 110 located downstream of the step 13 is no greater than the area of ​​the opening of the first metering pool 110 upstream of the step 13. Since a larger centrifugal radius results in greater surface fluctuations in the first metering pool, and the liquid exhibits surface tension, the volume of overflowing liquid cannot be accurately quantified. Therefore, reducing the area of ​​the opening of the first metering pool can avoid the problem of inaccurate quantification caused by large surface fluctuations.

[0034] The first quantitative cell 110 is divided into several groups based on the step 13. The first quantitative cells 110 in the same group have the same depth, and the depth of the first quantitative cells 110 in the upstream group is less than the depth of the first quantitative cells 110 in the downstream group. In this application, "depth" refers to the direction perpendicular to the detection chip, i.e. Figure 1 The center is perpendicular to the paper surface, such as Figure 1 As shown, it includes three steps 13 for dividing the first metering pool 110 into four groups. Since the first metering pool needs to release liquid to the downstream chamber at the same rotation speed, the centroid of the first metering pool needs to be kept consistent. The above function is achieved by changing the depth of the first metering pool to avoid compressing the radial dimension of the steps.

[0035] like Figure 3 As shown, there is a certain gap G between the opening of the first metering cell 110 and the depth of the first metering cell 110; the gap G is related to the radial distance L of the step 13. Firstly, this gap is used to limit the area of ​​the opening of the first metering cell, forming an effect similar to a beer bottle, reducing the impact of liquid level fluctuations in the first metering cell on the metering accuracy. Secondly, this gap is beneficial for stabilizing the liquid level, as the liquid itself has surface tension, and the gap helps to smooth out the fluctuations in the liquid level.

[0036] Specifically, the radial distance L of the step satisfies: 1mm ≤ L ≤ 5mm. The step should not be too small, as this limits the increase in centrifugal force, nor should it be too large, as this further compresses the radial dimension after quantitative analysis, leading to increased depth and excessive thickness of the detection chip.

[0037] To better leverage the stabilizing effect of the gap on the liquid surface, the gap G satisfies the following equation: ; in The correction factor is 0.9mm-1.0mm, L is the radial distance of the step in mm, and L1 is the unit length in 1mm.

[0038] The location of the steps is further specified, and the location of the steps satisfies the following: ; Where β is the threshold, α is the radian value between the steps, and R is the distance between the first quantitative cell and the rotation center, in mm. Specifically, β takes the value of mm 1 / 2 .

[0039] It should be noted that the multiplex nucleic acid detection chip provided in the embodiments of the present invention has a size structure that can meet the rotation speed requirements of commonly used microfluidic chips, preferably with a rotation speed of 1500-6000 r / min.

[0040] This embodiment uses the aforementioned multiplex nucleic acid detection chip, specifically, as follows: Figure 1 As shown, it also includes: a sample addition cell 100, a lysis cell 101, a mixing cell 102, and a second quantitative cell 112. Valves are provided between the sample addition cell 100 and the lysis cell 101, and between the lysis cell 101 and the mixing cell 102. These valves can be valves commonly used in the field of microfluidics; preferably, they are... Figure 1 The chip has a capillary channel, and each reaction unit is connected in a certain order to form an independent reactor; the chip is sealed by means of bonding, ultrasonic welding or laser welding.

[0041] Specifically, such as Figure 4 As shown, the sample loading chamber 100 includes: a sample loading port 1000; a vent 1001; a first-stage groove 1003, which is disposed close to the surface of the chip; and a second-stage groove 1002, which is disposed away from the surface of the chip and has a sealing film at its bottom. Figure 4 (Removing the sealing film), the bottom surface of the first-level groove 1003 is connected to the second-level groove 1002, the cross-sectional area of ​​the first-level groove 1003 is larger than the cross-sectional area of ​​the second-level groove 1002, the sample inlet 1000 and the vent 1001 are connected to the bottom surface of the second-level groove 1002, and the sealing film seals the sample inlet 1000 and the vent 1001.

[0042] The sample loading port and vent are both located within the sample loading chamber, which is a two-stage recessed groove design. The sample loading port and vent are located on the bottom surface of the second-stage groove. First, the chip is positioned at the loading station, and the sample loading port and vent are sealed using an easily tearable aluminum foil film (sealing film). Second, when sample loading is required, a robotic arm grasps the chip and places it at the loading station. Simultaneously, a needle punctures the sample loading port and vent, and the sample is added to the chip through the loading port. Then, UV adhesive is injected into the sample loading chamber and sealed using UV curing. The specific principle is as follows: the UV adhesive first enters the sample loading port and vent in the second-stage groove, achieving a first-stage seal and effectively preventing sample backflow. Furthermore, UV adhesive continues to be filled until the first-stage groove is completely filled, sealing the entire sample loading chamber and achieving a second-stage seal. Because the second-stage sealing area is larger, it can withstand the pressure generated by the thermal expansion of the internal cavity of the chip during the later heating process. In addition, the second-stage seal completely fills the cavity of the sample cell with UV adhesive, which not only makes the appearance flat and beautiful, but more importantly, the chip has good deformation consistency, so that the chip can withstand higher temperatures without deformation.

[0043] The vent is a through-hole, which is generally cylindrical but can be of any shape. The vent is used to expel excess gas from the sample cell during sample addition, maintaining pressure balance within the chip. In this application, the vent is located in the mixing cell, independent of the sample addition port, making sample addition and gas expulsion more direct, efficient, and controllable, achieving more precise fluid manipulation.

[0044] The sample addition cell 100 is located in the direction closest to the center of the circle, and its downstream end is connected to the lysis cell.

[0045] In the distribution channel 12, the radius R of the step's arc is calculated using L as 3mm and a correction factor of 2.2mm. The calculated R is 3.27, and the value is set to 3.3. (Based on the formula...) The radius is 38mm, and α is calculated to be... Since the angle is 90°, three steps are sufficient. The value of G can be calculated using the formula.

[0046] The mixing chamber 102 is connected upstream to the distal end of the lysis chamber 101 via a valve, and downstream to the proximal end of the distribution channel 12 via a flow control valve 01. When the sample enters the lysis chamber 101 through the sample inlet of the sample loading chamber 100, it is completely mixed with the release agent in the lysis chamber 101 under centrifugation speed V1, and then comes into contact with the reaction system pre-preserved in the mixing chamber under centrifugation speed V2. This application proposes storing all amplification systems except primers in a dry form in the mixing chamber 102; preferably, the amplification system is in the form of lyophilized pellets, which is not only convenient for processing but also easy to dissolve.

[0047] The second quantitative cell 112 is connected to the side wall of the first quantitative cell 110 away from the center. It is a liquid weighing chamber that is evenly spaced along the side wall of the distribution cell. Its upstream is connected to the first quantitative cell 110, and its downstream is connected to the reaction cell 111 through a valve. The second quantitative cell 112 is designed to be narrower at the top and wider at the bottom, and the depth of the second quantitative cell 112 is not less than the depth of the first quantitative cell 110 (perpendicular to the plane of the paper). The volume of each second quantitative cell 112 can be the same or different as needed; however, the distal ends of all first quantitative cells 110 are at the same centrifugal radius. This design facilitates valve design and allows the quantified liquid to enter the second quantitative cell 112 simultaneously, improving the consistency of the detection. Furthermore, the volume of the liquid to be tested entering each quantitative cell is equal; in addition, the second quantitative cell corresponds one-to-one with the downstream reaction cell 111, and the reaction cell 111 corresponds one-to-one with the second quantitative cell. The reaction cell is pre-embedded with primers and reagents related to the amplification reaction. The reagents can be in the form of, but are not limited to, ointments, dry powders, granules, or films; moreover, the pre-embedded primers and reagents are different for each cell, allowing for the simultaneous detection of multiple pathogenic microorganisms. The reaction cell can be circular, elliptical, rectangular, or any other arbitrary shape.

[0048] The method of using the multiplex nucleic acid detection chip is as follows: Step 1: Use a pipette to add the sample to be tested to the sample loading port 1000, seal the sample loading port 1000 and the vent 1001, apply centrifugation speed V1, the sample to be tested is mixed with the release agent in the lysis cell 101, and the pathogen nucleic acid is released.

[0049] Step 2: Apply centrifugation speed V2, and the lysed sample breaks through the valve and enters the mixing tank 102, where it is mixed with the lyophilized microspheres that are pre-existing.

[0050] Step 3: Apply centrifugation speed V3, and the sample after being mixed with the reaction system breaks through the valve and enters the distribution channel 12. The liquid fills the first quantitative cell 110 sequentially along the distal end of the distribution channel 12 to achieve quantitative liquid measurement. Then apply centrifugation speed V4, and the liquid in the first quantitative cell 110 enters the second quantitative cell 112 to achieve precise liquid measurement.

[0051] Step 4: Apply centrifugation speed V5. After accurate quantification, the liquid enters reaction cell 111 through the valve. The liquid mixes with the reagents pre-set in the reaction cell and completes the corresponding amplification and detection.

[0052] The design of V1-V5 mentioned above is based on the structure of the detection chip itself, which is common knowledge in the field and will not be elaborated here.

[0053] Experiments revealed that the amount of nucleic acid in each reaction tank fluctuated very little, meeting the requirements and providing accurate measurement results.

[0054] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A multiplex nucleic acid detection chip, characterized in that, include: Upstream chamber; The downstream chamber includes multiple downstream chambers, which are connected to the upstream chamber through a distribution channel. Each downstream chamber has a first metering pool. The distribution channel has a spiral or arc-shaped structure and is provided with a step away from the center of rotation based on the arc and radius of the distribution channel. Under the action of centrifugal force, the liquid in the upstream chamber sequentially fills the corresponding first metering pool through the distribution channel. The step has an arc-shaped cross-section, and the radius r of the arc is related to the radial distance L of the step. The radius r of the arc satisfies: ； in, The correction factor is 2.2mm-2.3mm; L is the radial distance of the step; L0 is the standard distance, with a value of 2mm; L≥L0; The area of ​​the opening of the first metering pool located downstream of the step is not greater than the area of ​​the opening of the first metering pool located upstream of the step. The first metering pool is divided into several groups based on the steps. The first metering pools in the same group have the same depth, and the depth of the first metering pool in the upstream group is less than that in the downstream group. The radial distance L of the steps satisfies: 1mm≤L≤5mm.

2. The multiplex nucleic acid detection chip according to claim 1, characterized in that, A flow control valve is installed between the upstream chamber and the distribution channel.

3. The multiplex nucleic acid detection chip according to claim 1, characterized in that, There is a certain gap between the opening of the first metering cell and the depth of the first metering cell; the gap is related to the radial distance of the step.

4. The multiplex nucleic acid detection chip according to claim 1, characterized in that, The upstream chamber includes a sample addition cell, a lysis cell, and a mixing cell connected in sequence. The mixing tank is connected to the downstream chamber via a distribution channel.

5. The multiplex nucleic acid detection chip according to claim 4, characterized in that, The sample addition chamber includes: Sample dispensing port; Vent hole; The first-level groove is disposed close to the surface of the chip; The second-level groove is located away from the surface of the chip and has a sealing film at its bottom. The bottom surface of the first-level groove is connected to the second-level groove. The cross-sectional area of ​​the first-level groove is larger than that of the second-level groove. The sample inlet and the vent are connected to the bottom surface of the second-level groove. The sealing film seals the sample inlet and the vent.

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