Double-cavity centrifugal micro-fluidic chip having reaction cavity and test cavity and used for nucleic acid test, and test method

Abstract

Provided are a double-cavity micro-fluidic chip having a reaction cavity and a test cavity and used for nucleic acid test, and a test method thereof. A fluid channel is provided between the reaction cavity and the test cavity, and only one exhaust channel is provided. The combination of the fluid channel and the exhaust channel can automatically provide resistance for a sample entering the test cavity from the reaction cavity, thereby achieving a controllable transfer of the sample between the reaction cavity and the test cavity. A gravity micro-fluidic chip and a centrifugal micro-fluidic chip are designed. The gravity micro-fluidic chip has a multi-turning circuitous fluid channel of which the diameter is smaller than that of a sample injection channel, such that under the action of gravity, the sample can flow into a reaction cavity from a sample adding region but cannot flow into the fluid channel, and then under pressurization by a simple pressurization device, the sample can enter a test cavity from the reaction cavity. The centrifugal micro-fluidic chip provides an exhaust channel branched from a fluid channel, and makes the exhaust direction opposite to a rotary centrifugal direction and the position of an exhaust opening higher than that of the fluid channel; therefore, by combining the principle of internal and external balance of the atmospheric pressure and the centrifugal driving performance, the centrifugal micro-fluidic chip effectively controls the flowing of a gas and a liquid in reaction cavities and test cavities, so as to effectively control the amount of the sample entering each cavity, so that multiple nucleic acid tests can be completed simply by means of one or two times of centrifugation. In addition, for a chip which only has a reaction cavity, an exhaust channel is connected to a liquid storage cavity, and a gravity-driving chip and an asymmetric pressure-driving chip are designed, thereby effectively avoiding aerosol pollution.

Description

A dual-chamber nucleic acid detection centrifugal microfluidic chip with a reaction chamber and a detection chamber and a detection method Technical Field

[0001] The present invention relates to the field of microfluidic control, and in particular to a centrifugal microfluidic chip for nucleic acid detection and a detection method. Background Art

[0002] Microfluidic chip technology, also known as chip laboratory, can integrate or basically integrate basic operations such as sample preparation, biological and chemical reactions, separation and detection involved in fields such as biology and chemistry on a chip of a few square centimeters. It is used to complete different biological or chemical reaction processes and analyze their products. This technology can greatly reduce the requirements of the detection process for space, personnel and equipment.

[0003] Existing microfluidic detection chips have integrated multiple steps, including nucleic acid amplification and detection, into a compact chip, rapidly completing the "sample in - result out" testing process. Because microfluidic chips eliminate the need for precise pipetting devices and specialized personnel, they hold enormous potential for point-of-care testing.

[0004] Isothermal amplification has gained increasing attention in the field of instant testing due to its fast reaction speed and low requirements for instruments and equipment. However, due to the complexity of the samples to be tested, such as blood, sputum, urine, tissue, etc., it is usually necessary to react with detection reagents to amplify the signal after isothermal amplification in order to achieve accurate detection, which is called nucleic acid multiplex detection. In order to achieve nucleic acid multiplex detection, a reaction chamber and a detection chamber must be provided in the microfluidic chip. The reaction chamber is used for amplification reaction, and the detection chamber is used for signal amplification. The sample to be tested needs to be controllably transferred between the reaction chamber and the detection chamber, and cross-influence caused by sample reflux must be prevented. Most existing microfluidic chips only have one reaction chamber for nucleic acid amplification and detection, which makes it difficult to achieve nucleic acid multiplex detection.

[0005] At the same time, existing microfluidic chips generally require external pressure devices, centrifugal force, and siphon action as driving forces. The use of external pressure devices, such as negative pressure vacuum pumps, injection pumps, etc., has a strong driving force, but needs to be externally connected to the chip, making the detection platform structure complex. Using centrifugal force as a drive has the problem of dependence on electricity and centrifugal equipment. Although the liquid flow drive method of siphon action does not require equipment, the driving force is relatively small, and there is a problem of volatilization during the sampling process. In order to realize multiple nucleic acid detection, there is currently also a method of controlling multiple detections through an external pressure supply device, and its operation process is complicated and has high requirements. For example, the patent with publication number CN113736643 provides a microfluidic chip that can realize multiple nucleic acid detection, but it requires an external pressure supply device for sampling, and a hydrophobic valve and a membrane valve are specially provided between the reaction amplification chamber and the detection chamber. When the external pressure provides positive pressure, the membrane valve and the hydrophobic valve are closed, and when the positive pressure is released, the membrane valve and the hydrophobic valve are opened. At the same time, an electromagnetic valve is required to control the external pressure supply device. In addition, the microfluidic chip structure is complex and the cost is high, which greatly limits the use of microfluidic chips in resource-limited environments.

[0006] Centrifugal microfluidic chips drive the flow of liquids by rotating the chip, enabling the manipulation of liquids at the submillimeter scale using centrifugal force. Compared to traditional microfluidic chips, the entire device is more compact and simpler to handle, leading to its increasing application in diagnostics. In a centrifugal microfluidic chip, microchannels are distributed radially along a circular disc. As the chip rotates, the test liquid is moved along multiple microchannels toward the various reaction cells under the action of centrifugal force, achieving distribution and sample addition of the test liquid.

[0007] Current diagnostic technologies typically require multiple indicators to determine whether a patient has a disease. For example, during clinical testing, the same patient sample needs to be screened for multiple respiratory pathogens, and these indicators vary depending on individual conditions. High-throughput centrifugal microfluidic chips also suffer from short, inaccurate control processes and complex structures. When testing multiple indicators simultaneously, the chambers in the centrifugal microfluidic chip are prone to uneven sample injection volumes, and the sample may enter other chambers during centrifugation or reflux after centrifugation, leading to mutual interference and contamination. As a result, existing centrifugal microfluidic chips can only perform simultaneous amplification of a single sample or a few detection indicators. For example, the nucleic acid multiplex detection microfluidic chip provided by patent publication number CN220166182U has low detection throughput and is difficult to complete sample and multi-indicator pathogen screening in a short period of time. When faced with the need to amplify multiple samples and multiple indicators, it not only fails to provide efficient and rapid amplification, but also faces high chip replacement costs.

[0008] CN115684014A provides a microfluidic chip for centrifugation. Although it can also achieve high-throughput detection of multiple indicators, its structure and operation process are relatively complex. Ventilation holes are set on the bottom plate and cover plate for ventilation of the entire chip. The sample enters the corresponding chamber through centrifugation. Since it is difficult to control the amount of sample entering the chamber during each centrifugation, a transition pool is required to be set up, and the volume of the transition pool is used to measure the sample amount. Therefore, it requires three stages of centrifugation to allow the liquid to reach the target location for detection (first stage centrifugation to reach the transition pool, second stage centrifugation to reach the reaction chamber, and third stage centrifugation to reach the detection chamber). Moreover, since the centrifugal speed of the latter stage must be significantly higher than that of the previous stage to allow the liquid to enter the next chamber, the three-stage centrifugation inevitably requires higher centrifugal force centrifugation, which is not only more complicated to operate, but also the pre-embedded freeze-dried or vitrified reagents in the chamber are easily precipitated by high-speed centrifugation when they are not fully dissolved, resulting in reaction failure and seriously affecting the detection results. At the same time, the chip has many holes, and the flow channels and holes are very small, which makes it difficult to control the work tolerance, resulting in easy liquid misalignment and loss of control during sample addition.

[0009] Therefore, there is an urgent need to find a nucleic acid multiplex detection microfluidic chip with a simpler structure, more convenient operation, lower instrument requirements, and lower manufacturing costs. At the same time, for centrifugal nucleic acid multiplex detection microfluidic chips, it is hoped that only one or two centrifugations can be required to accurately direct samples into the corresponding chamber, and the amount of sample entering each chamber can be effectively controlled to prevent sample backflow or leakage into other chambers, completely solving the problems of mutual interference and contamination. Moreover, it is possible to perform high-throughput testing of multiple samples and multiple detection items, while ensuring the accuracy of the test results.

[0010] Summary of the Invention

[0011] To solve the above problems, the present invention provides a nucleic acid detection microfluidic chip and a detection method thereof, including a nucleic acid multiple detection microfluidic chip. The nucleic acid multiple detection microfluidic chip is configured by setting a fluid channel between the reaction chamber and the detection chamber, and making the reaction chamber and the detection chamber share an exhaust channel. The combination of the fluid channel and the exhaust channel can automatically provide resistance for the sample in the reaction chamber to enter the detection chamber, thereby realizing the controllable transfer of the sample between the reaction chamber and the detection chamber, and can greatly simplify the structure and operation process while ensuring the detection precision, thereby designing a gravity microfluidic chip and a centrifugal microfluidic chip.

[0012] The gravity microfluidic chip has a multi-turn winding fluid channel and the aperture of the fluid channel is smaller than the aperture of the reaction chamber injection channel, so that the sample can flow from the sample addition area into the reaction chamber under the action of gravity, but will not flow into the fluid channel. After being pressurized by a simple pressure-applying device, the sample can smoothly enter the detection chamber from the reaction chamber through the fluid channel, realizing the controllable transfer of samples in different chambers and effectively preventing sample backflow. Moreover, the structure is simple. Only an exhaust channel and a waterproof breathable membrane need to be set in the detection chamber. No exhaust channel is required in the reaction chamber. The sample can be converted between different chambers by gravity or a simple pressure-applying device on the chip, without the need for additional driving equipment.

[0013] The centrifugal microfluidic chip leads out an exhaust channel on the fluid channel between the reaction chamber and the detection chamber, and makes the exhaust direction of the exhaust channel opposite to the rotating centrifugal direction, and the exhaust port is located higher than the fluid channel. Combined with the principle of internal and external balance of atmospheric pressure and centrifugal driving performance, it can effectively control the flow of gas and liquid in the reaction chamber and the detection chamber, and can effectively control the amount of samples entering each chamber. Only one or two centrifugations are required to complete multiple nucleic acid tests. After entering each chamber, the test samples can be stably retained and reacted, effectively preventing sample backflow and eliminating mutual interference or contamination problems. At the same time, by setting one or more concentric circle sub-chips outside the chip body, high-throughput detection of more samples and more detection items can be achieved, thereby improving detection efficiency.

[0014] At the same time, the present invention also provides a microfluidic chip for nucleic acid typing detection. The chip is only equipped with a reaction chamber but no detection chamber. Multiple reaction chambers are connected to the liquid storage chamber, and the exhaust channel of the reaction chamber is connected to the liquid storage chamber. A gravity typing chip and an asymmetric pressure typing chip are designed to effectively avoid aerosol contamination.

[0015] In one aspect, the present invention provides a nucleic acid detection microfluidic chip, wherein the chip is provided with a reaction chamber and a detection chamber; the reaction chamber is used to amplify a target nucleic acid in a sample to obtain an amplified product; the detection chamber is used to detect the target nucleic acid in the amplified product;

[0016] The reaction chamber and the detection chamber are connected via a fluid channel, and the fluid channel is used to achieve liquid or gas flow between the reaction chamber and the detection chamber;

[0017] The reaction chamber and the detection chamber share an exhaust channel, and the exhaust channel is used to control the flow of gas or liquid in the reaction chamber or the detection chamber;

[0018] The exhaust channel is connected to the fluid channel or the detection chamber; the combination of the fluid channel and the exhaust channel can automatically provide resistance for the sample in the reaction chamber to enter the detection chamber.

[0019] In some embodiments, the microfluidic chip provided by the present invention is mainly used for multiplex detection of nucleic acids, and the multiplex detection of nucleic acids means that the nucleic acids need to be detected in two steps. In the first step, the sample enters the reaction chamber, and the target nucleic acid in the sample is amplified in the reaction chamber; in the second step, the amplification is completed, and the amplified product enters the detection chamber. Therefore, the microfluidic chip must meet the controllable transfer of samples in the reaction chamber and the detection chamber, and can prevent the cross-influence caused by the backflow of the sample. The controllable transfer of the sample in the reaction chamber and the detection chamber means that when the sample enters the reaction chamber, all the samples to be tested enter the reaction chamber smoothly, and all remain in the reaction chamber for amplification reaction. After the amplification reaction is completed, the amplified product flows out of the reaction chamber, enters the detection chamber through the fluid channel, and all remain in the detection chamber to complete the detection reaction until the final successful detection.

[0020] Existing nucleic acid multiplex detection microfluidic chips usually require a valve to be set between the reaction chamber and the detection chamber to block the sample from entering the detection chamber when entering the reaction chamber.

[0021] In some methods, a valve block can be added between the reaction chamber and the detection chamber to control the transfer of samples between the reaction chamber and the detection chamber. When the valve block (paraffin) is solid, the reaction chamber and the detection chamber cannot be fluidically connected; when the valve block is liquid, the detection chamber and the reaction chamber are fluidically connected. Utilizing the temperature-increasing phase change properties of the valve block and the characteristics of the waterproof and breathable membrane, combined with the principle of internal and external atmospheric pressure balance, the controlled transfer of the sample to be tested between different chambers can be cleverly achieved. When using this method, both the reaction chamber and the detection chamber require exhaust channels and waterproof and breathable membranes. The installation of the valve block also adds complexity to the preparation process of the microfluidic chip, and the heating temperature must be controlled to control the state of the valve block, which adds additional steps to the operation process.

[0022] The combination of the fluid channel and the exhaust channel described in the present invention refers to the combination of the design of various characteristics of the fluid channel (quantity, length, shape, position height, aperture, etc.) and the design of various characteristics of the exhaust channel (quantity, distance from the detection cavity, position, design of the exhaust port, orientation of the exhaust channel, aperture, etc.).

[0023] The present invention automatically provides resistance for the sample in the reaction chamber by improving the structure and positional relationship of the fluid channel and the exhaust channel, thereby cleverly achieving controllable transfer of the sample between the reaction chamber and the detection chamber.

[0024] When the reaction chamber and detection chamber share a common exhaust channel, the structure and location of the exhaust channel primarily include the distance between the exhaust channel and the detection chamber, or the orientation of the exhaust channel. It is understood that the distance between the exhaust channel and the detection chamber is directly related to the resistance to sample entry into the detection chamber. Only when the exhaust channel is connected to the top of the detection chamber, when liquid flows from the reaction chamber to the detection chamber and enters the detection chamber from the bottom of the detection chamber, will the exhaust direction also be from the bottom of the detection chamber to the top of the detection chamber and then discharged out of the exhaust channel. The exhaust direction is consistent with the liquid flow direction, thus minimizing the resistance to liquid sample entering the detection chamber. However, when the exhaust channel is connected to other parts of the detection chamber, or is not connected to the detection chamber but to the fluid channel, the exhausted gas will be discharged from the bottom of the detection chamber to the top, then turn back and pass through the liquid before reaching the exhaust channel. In other words, part of the exhaust process is opposite to the direction of liquid flow. This process of turning back and passing through the liquid will significantly increase the resistance to sample entry into the detection chamber. Therefore, changing the position of the exhaust channel will directly affect the effectiveness of the controlled transfer of the sample between the reaction chamber and the detection chamber.

[0025] Improving the structure of the fluid channel between the reaction chamber and the detection chamber can directly affect the resistance of the sample from the reaction chamber into the detection chamber. For example, the longer the fluid channel, the more complex the structure (the more winding or folded structures), the higher the height of the fluid channel than the reaction chamber, the smaller the aperture, etc., the more difficult it is for the sample in the reaction chamber to enter the detection chamber. This allows the fluid channel to automatically provide resistance to the sample in the reaction chamber, making it difficult for the sample in the reaction chamber to automatically enter the detection chamber. The sample must be subjected to a certain pressure (a simple pressure device on the chip) or centrifuged before entering the detection chamber.

[0026] The gravity microfluidic chip provided by the present invention is equipped with multiple groups of detection units, each group of detection units is arranged in multiple levels on the chip body, and each group of detection units is equipped with a reaction chamber and a detection chamber. It can be applied to the simultaneous detection of multiple different target analytes, such as the detection of multiple different nucleic acid targets, and each group of detection units can realize multiple nucleic acid detection. Of course, it is understandable that the gravity microfluidic chip is not limited to multiple nucleic acid detection, but is also suitable for the detection of other types of molecules such as proteins and antibodies. The reaction chamber can be used for sample pretreatment, and the detection chamber is used for sample detection.

[0027] Furthermore, the chip includes a gravity microfluidic chip and / or a centrifugal microfluidic chip; when the chip is a gravity microfluidic chip, the fluid channel can automatically provide resistance for the sample in the reaction chamber, making it difficult for the sample in the reaction chamber to automatically enter the detection chamber.

[0028] A gravity microfluidic chip refers to a chip in which the sample can directly reach the reaction chamber from the sample area under the action of gravity without providing any external driving force. For example, when used for multiple nucleic acid detection, the sample can directly reach the reaction chamber from the sample area under the action of gravity. However, when the sample enters the reaction chamber, the fluid channel needs to act as a valve to prevent the sample from entering the detection chamber at the same time. By setting the structure of the fluid channel, the fluid channel can provide resistance for the sample to enter the detection chamber from the reaction chamber, thereby acting as an automatic valve. The structural setting of the fluid channel mainly includes setting the length, structure, aperture, height, inner wall roughness of the fluid channel, etc. to provide resistance for the sample to enter the detection chamber from the reaction chamber.

[0029] In some methods, the fluid channel can automatically provide resistance for the sample to enter the detection chamber from the reaction chamber by designing a combination of one, two, three, or four of the length, structure, aperture, height, etc. of the fluid channel. Some preferred methods can also be selected to increase the controllability of the resistance provided by the fluid channel and improve the effect of the fluid channel acting as a valve.

[0030] Furthermore, the length of the fluid channel is longer than the straight-line distance between the reaction chamber and the detection chamber.

[0031] It is understandable that when the length of the fluid channel is lengthened, the resistance of the sample from the reaction chamber through the fluid channel to the detection chamber will also be greater. There are many ways to lengthen the length of the fluid channel, such as increasing the degree of curvature, repeated bending, multiple detours on the path passed, or extending in different directions, or increasing the height span of the local or multiple points in the fluid channel, etc., which can make the length of the fluid channel longer and increase the resistance of the sample from the reaction chamber to the detection chamber. These methods can be used alone, or any type can be selected for combination. According to the required resistance size, certain screening and optimization experiments can be carried out to find a more preferred and suitable fluid channel design method, such as increasing the degree of curvature while increasing the number of repeated bendings, such as increasing the degree of curvature while increasing the height span, etc., to try to improve the controllability of the resistance provided by the fluid channel and improve the effect of the fluid channel acting as a valve.

[0032] Furthermore, the fluid channel is a folding, spiral or multi-turn circuitous flow channel, and the folding or multi-turn circuitous flow channel is provided with at least one bent structure.

[0033] When the fluid channel is designed to have a structure containing multiple folds, spirals, or multiple turns, the length of the fluid channel can be extended as much as possible, thereby maximizing the difficulty for the sample in the reaction chamber to enter the fluid channel without additional pressure, while ensuring that all the samples in the reaction chamber remain in the reaction chamber.

[0034] Furthermore, the fluid channel extends toward the upper end of the reaction chamber or the upper end of the detection chamber or directly above the fluid channel.

[0035] It is understandable that extending the fluid channel in any direction will lengthen the fluid channel or increase the degree of tortuosity and folding, thereby increasing the difficulty of the sample from the reaction chamber through the fluid channel into the detection chamber without additional pressure.

[0036] Furthermore, the position of the detection chamber is higher than the reaction chamber, or the position of the sample inlet of the detection chamber is higher than the position of the sample outlet of the reaction chamber, or the bottom of the detection chamber is not lower than the top of the reaction chamber.

[0037] During use, the gravity microfluidic chip needs to be kept in a vertical state with the sample inlet facing upwards. The following descriptions of the positional relationship between the various flow channels and chambers in the gravity microfluidic chip are based on the situation where the gravity microfluidic chip is kept in a vertical state. In some ways, the gravity microfluidic chip can be inserted into a matching detection device so that the gravity microfluidic chip is kept vertically with the sample inlet facing upwards, and the heat source can be provided by the detection device to heat specific positions of the gravity microfluidic chip (such as the reaction chamber, valve block, etc.); in some ways, the fluorescent substance produced after the sample reaction in the gravity microfluidic chip can also be detected by the detection device to read the detection results.

[0038] In some embodiments, the positional relationship of the gravity microfluidic chip described herein can refer to the height relationship of the various chambers when the chip is held in a vertical position. Of course, this does not mean that the fluid channels of the gravity microfluidic chip can only provide resistance to the flow of samples from the reaction chamber into the detection chamber when the chip is held in a vertical position. In fact, the gravity microfluidic chip can provide resistance to the flow of samples from the reaction chamber into the detection chamber regardless of whether the chip is held in a horizontal, tilted, or vertical position.

[0039] It is understandable that when the gravity microfluidic chip remains in a vertical state, the position of the detection chamber is higher than the reaction chamber, and it is more difficult for the sample to enter the detection chamber from the reaction chamber under the action of gravity, and it is impossible to enter the detection chamber from the reaction chamber under the action of gravity.

[0040] In some methods, the sample flows out from the sample outlet of the reaction chamber, passes through the fluid channel, and then enters the sample inlet of the detection chamber. Therefore, the position of the sample inlet of the detection chamber is higher than the position of the sample outlet of the reaction chamber, which will also make it more difficult for the sample to enter the detection chamber from the reaction chamber under the action of gravity, and it is impossible for the sample to enter the detection chamber from the reaction chamber under the action of gravity.

[0041] Furthermore, at least one point in the fluid channel is higher than the reaction chamber.

[0042] In some methods, when the gravity microfluidic chip is maintained in a vertical state, there is a section of flow channel or a point in the fluid channel that is higher than the reaction chamber, which can also make it more difficult for the sample to enter the detection chamber from the reaction chamber under the action of gravity, and it is impossible to enter the detection chamber from the reaction chamber under the action of gravity.

[0043] In some embodiments, the fluid channel includes a flow passage or a point higher than the sample outlet of the reaction chamber.

[0044] Furthermore, the fluid channel includes at least one arched flow channel, which includes an inlet section, a middle section and an outlet section. The middle section is arched upward, and the position of the middle section is higher than the inlet section and the outlet section, and at least the position of the middle section is higher than the reaction chamber.

[0045] The middle section of the fluid channel arches upward, which increases the difficulty for the sample in the reaction chamber to enter the fluid channel without additional pressure, and helps to keep all the samples in the reaction chamber in the reaction chamber to complete the nucleic acid amplification reaction.

[0046] Furthermore, it also includes a sample adding area; a sample injection channel is provided between the sample adding area and the reaction chamber; and the aperture of the fluid channel is smaller than that of the sample injection channel.

[0047] In some embodiments, the aperture of the fluid channel is smaller than that of the sample inlet channel, so that the sample can flow from the sample addition area into the reaction chamber under the action of gravity but will not flow into the fluid channel.

[0048] Since the air pressure in the system is balanced, the sample in the sample addition area does not require an external drive device and can be autonomously transferred to the reaction chamber under gravity alone. Moreover, this process is very easy to implement. Once the sample enters the sample addition area, it will flow downward on its own and enter the reaction chamber through the sample inlet channel. The main channel of the gravity microfluidic chip and the sample inlet channel of the reaction chamber have large apertures, which can enable the liquid to flow smoothly under gravity. However, the aperture of the fluid channel is significantly smaller than these channels. The fluid channel is a multi-turn, circuitous channel with a small aperture. Under the action of gravity alone, the liquid cannot enter and flow smoothly. As a result, after the sample fills the reaction chamber, it will be blocked when continuing to advance, and it will be difficult to flow out of the fluid channel at the top of the reaction chamber. All of it will remain in the reaction chamber to complete the nucleic acid amplification reaction. After the nucleic acid amplification reaction is completed, it can enter the detection chamber through the fluid channel under pressure, thereby ensuring accurate detection.

[0049] In some methods, the pore size of the fluid channel does not have to be completely uniform. Different pore sizes can be designed in different parts of the fluid channel to increase the resistance of the liquid to impact, sudden turns, etc. in the fluid channel, thereby increasing the difficulty of the sample entering the fluid channel.

[0050] In some embodiments, the fluid channel provided by the present invention effectively combines the above-mentioned several designs, and the design points include the following: 1. The aperture of the fluid channel is smaller than the sample inlet channel; 2. The fluid channel is a multi-turn circuitous channel with at least one bend-shaped structure; 3. The fluid channel includes an arched flow channel, and the arched flow channel includes an inlet section, an intermediate section and a sample outlet section. The intermediate section is arched upward, and the position of the intermediate section is higher than the inlet section and the sample outlet section, and at least the position of the intermediate section is higher than the reaction chamber; 4. The position of the detection chamber is higher than the reaction chamber, the position of the sample inlet of the detection chamber is higher than the position of the sample outlet of the reaction chamber, and the bottom of the detection chamber is not lower than the top of the reaction chamber; and any one, two, three, or four of these methods are used in combination to achieve a better automatic valve effect, thereby accurately realizing the controllable transfer of samples between the reaction chamber and the detection chamber.

[0051] In some embodiments, the pore size of the injection channel is 400 μm to 800 μm, and the pore size of the fluid channel is 50 to 300 μm.

[0052] It is understandable that the aperture of the injection channel and the fluid channel can be such that a gravity microfluidic chip can be successfully prepared, and the fluid channel can play a certain role in preventing liquid from flowing in easily under the action of gravity and surface tension due to its small aperture. The limitation on the pore size here does not mean that it cannot be used if it exceeds or is smaller than the pore size. It is just a preferred embodiment. For example, the pore size of the injection channel reaches 801μm, 805μm, 810μm, 820μm, 850μm, 900μm, 399μm, 395μm, 390μm, 385μm, 380μm, etc., or the pore size of the fluid channel reaches 301μm, 302μm, 305μm, 310μm, 320μm, 350μm, 390μm, 49μm, 48μm, 46μm, 41μm, 40μm, 35μm, etc., which can still be achieved under the appropriate chip size.

[0053] Furthermore, it also includes a main channel, the sampling channel is connected to the branch port of the main channel, the sampling channel is an upward arched arc channel, the inlet and outlet of the sampling channel are respectively located at the low points on both sides of the arc channel, and the inlet is higher than the outlet; the highest point of the arc channel is not lower than the top of the reaction chamber.

[0054] The curved flow channel design of the injection flow channel can make it easier for the sample to enter the injection flow channel from the branch port and then enter the reaction chamber through the injection flow channel. However, after entering the reaction chamber, it is difficult for the sample to return to the main flow channel from the injection flow channel because the injection port of the reaction chamber is located at the lowest point of the injection flow channel. The sample cannot overcome the effect of gravity and the pressure caused by the upward arched curved flow channel to return to the main flow channel, and can only remain in the reaction chamber, thereby preventing sample backflow in the reaction chamber.

[0055] After a specific volume of the sample to be tested is added from the sample adding area, the lid of the sample adding area is closed, and the sample enters the reaction chamber of each detection unit from the main channel under the action of gravity. The sample inlet of the reaction chamber is at a low position, which can make the sample to be tested flow into the reaction chamber quickly and gradually fill it, but it will not flow out of the reaction chamber from the sample outlet at this time, because the sample outlet is at the top of the reaction chamber, and the fluid channel is a multi-turn, small-aperture flow channel, and the sample cannot flow into the fluid channel. At the same time, it is also difficult for the sample to return to the main channel from the sample inlet channel, because the reaction chamber sample inlet is located at the lowest point of the sample inlet channel, and the highest point of the sample inlet channel is higher than the top of the reaction chamber. The sample cannot return to the main channel under the action of gravity, so all the samples will remain stably in the reaction chamber for reaction.

[0056] Furthermore, the exhaust channel is connected to the detection chamber.

[0057] The location of the exhaust channel directly affects the controlled transfer of samples between the reaction chamber and the detection chamber. This is primarily due to the difficulty of this controlled transfer due to the distance between the exhaust channel and the detection chamber. If the exhaust channel is too close to the reaction chamber and too far from the detection chamber, transferring the sample into the detection chamber will be significantly more difficult. The sample may even wet the waterproof breathable membrane due to premature entry into the exhaust channel, rendering it impermeable, completely sealing the interior of the microfluidic chip and preventing liquid flow. Therefore, the location of this exhaust channel, shared by the reaction and detection chambers, is critical. It must be kept ventilated as much as possible during the sample's entry into the reaction chamber, ensuring that ventilation is quickly restored even if occlusion occurs during the process.

[0058] In some approaches, for gravity-based microfluidic chips, the exhaust channel can be directly located within the detection chamber. Consequently, the fluid channels within the chip have a smaller aperture and are more circuitous, providing sufficient resistance to entry into the detection chamber. Furthermore, the exhaust channel's location within the detection chamber makes it easier to control sample flow, preventing the system from closing due to sample entry. Once the sample enters the detection chamber, it flows into the exhaust channel, sealing the system and preventing it from exiting the detection chamber. This facilitates controlled sample transfer between the reaction chamber and the detection chamber.

[0059] Furthermore, a waterproof and breathable membrane is provided at the outlet of the exhaust passage of the detection cavity to control the communication state between the detection cavity and the outside atmosphere.

[0060] The exhaust channel and the waterproof breathable membrane of the detection cavity enable the gas in the reaction cavity and the detection cavity to be discharged to the outside through the waterproof breathable membrane, but the liquid cannot be discharged through the waterproof breathable membrane. When the exhaust channel is filled with liquid, the waterproof breathable membrane will lose its breathability, causing the entire system to be in a closed state and the liquid in the system to stop flowing. Therefore, the waterproof breathable membrane can help the sample in the detection cavity to establish internal and external pressure balance and can regulate the direction of liquid flow. When the detection cavity is filled with liquid and the waterproof breathable membrane is wetted by the exhaust channel, the system in the chip is in a closed state, thereby stopping the flow of the sample in the detection cavity and preventing backflow. In some embodiments, the waterproof breathable membrane is made of polymer material.

[0061] Existing nucleic acid multiplex detection microfluidic chips usually require a valve to be set between the reaction chamber and the detection chamber for isolation. Therefore, both the reaction chamber and the detection chamber need to be equipped with an exhaust channel and a waterproof breathable membrane respectively.

[0062] In the gravity microfluidic chip provided by the present invention, the reaction chamber does not need to be equipped with an exhaust channel. The multi-turn, circuitous, and relatively small-aperture fluid channel can act as a valve to temporarily block the flow of liquid between the reaction chamber and the detection chamber. However, ventilation can always be maintained between the reaction chamber and the detection chamber. Therefore, the reaction chamber and the detection chamber can share the exhaust channel located on the detection chamber for exhaust, and the reaction chamber does not need to be equipped with a separate exhaust channel and waterproof breathable membrane. This design makes the structure simpler, lowers the cost, and is more convenient to operate, while also ensuring the accuracy of the test results.

[0063] Furthermore, the outlet of the sample inlet channel is located at the bottom of the reaction chamber; the inlet of the fluid channel is located at the top of the reaction chamber; the outlet of the fluid channel is located at the bottom of the detection chamber; and the inlet of the exhaust channel is located at the top of the detection chamber.

[0064] The outlet of the injection channel is equivalent to the sample inlet of the reaction chamber, and the inlet of the fluid channel is equivalent to the sample outlet of the reaction chamber. The gravity microfluidic chip is also designed with the position of the sample inlet and sample outlet of the reaction chamber in mind. The sample inlet is located at the bottom of the reaction chamber, and the sample outlet is located at the top of the reaction chamber. This is to ensure that when the sample enters the reaction chamber, it can be exhausted to the sample outlet smoothly, maintaining air pressure balance. At the same time, since the sample outlet is at the top of the reaction chamber, it is more difficult for the sample to flow out, so that the sample stays more stably in the reaction chamber.

[0065] The outlet of the fluid channel is equivalent to the sample inlet of the detection chamber, and the inlet of the exhaust channel is equivalent to the exhaust port of the detection chamber. The sample inlet is at the bottom of the detection chamber, and the exhaust port is at the top of the detection chamber. The purpose of this is to ensure that the sample can be smoothly exhausted to the exhaust port when entering the detection chamber to maintain air pressure balance. Under the action of the pressure in the pressure zone, the sample in the reaction chamber enters the detection chamber from the bottom of the detection chamber through the fluid channel. Since the sample volume is usually relatively full, the sample can also enter the exhaust channel and contact the waterproof breathable membrane, causing the waterproof breathable membrane to lose its breathable function, making the entire gravity microfluidic chip in a closed state, and the sample stops flowing, thereby being completely retained in the detection chamber for detection reaction and completion of the detection of the target nucleic acid.

[0066] Furthermore, it also includes a pressure area, which is located on the chip or on a tube cover used to seal the chip sampling area, and is used to increase the pressure of the liquid in the reaction chamber so that the liquid in the reaction chamber flows into the detection chamber through the fluid channel.

[0067] Furthermore, it also includes a pressure area, which is located on the chip or on a tube cover used to seal the chip sampling area, and is used to increase the pressure of the liquid in the reaction chamber so that the liquid in the reaction chamber flows into the detection chamber through the fluid channel.

[0068] The purpose of the pressure zone is to ensure that, after the sample completes amplification and other reactions in the reaction chamber, it must fully enter the detection chamber. This requires overcoming the pressure of both the atmospheric pressure and the pressure of the multi-turn, narrow-aperture fluidic channel. Therefore, the pressure zone must provide sufficient pressure to propel the sample from the reaction chamber into the detection chamber.

[0069] It is understandable that the pressure area can be set at any position of the chip, such as the middle of the chip, the bottom of the chip, the side of the chip, the sample addition area of ​​the chip, the detection area of ​​the chip, etc., and can even be set on the tube cover. As long as the air bag in the pressure area is pressed, pressure can be provided for the liquid in the reaction chamber to allow it to enter the detection area smoothly.

[0070] In some embodiments, the pressure zone is preferably set in the upper left or upper right of the chip, such as on one side of the chip sample loading area, etc., because if the pressure zone is set on the sample loading area or on the tube cover, although it can also provide a certain pressure, the volume of the sample loading area or the upper end of the tube cover is limited, resulting in the airflow pressure provided being very limited, and the pressing operation is inconvenient. When tightening the tube cover, it is easy to accidentally touch the airbag on the tube cover, causing the airbag to release the airflow prematurely. Therefore, the pressure zone is preferably set in the upper left or upper right of the gravity microfluidic chip, and should also reach a specific volume (specifically, it can be calculated based on the size of the gravity microfluidic chip and the required pressure, so as to set an airbag of appropriate volume), so that the pressing process provides more accurate airflow pressure to drive the sample to be tested from the reaction chamber into the detection chamber.

[0071] The airbag in the pressure area can be pressed manually or by using a matching detection device to provide a specific force to press the airbag.

[0072] In some embodiments, the pressure-applying area may be connected to the sample pool via a branch channel, and airflow may be discharged from the branch channel to the sample pool and into the interior of the gravity microfluidic chip by pressing.

[0073] Furthermore, the gravity microfluidic chip further comprises a sample adding area, which comprises a sample pool and a tube cover. The sample pool is used to accommodate the sample to be tested, and the tube cover is used to seal the gravity microfluidic chip.

[0074] In some embodiments, the reaction chamber of the gravity microfluidic chip has a circular cross-section, and the circular cross-section can accommodate more samples in the reaction chamber.

[0075] In some embodiments, the cross-section of the detection cavity of the gravity microfluidic chip is in the shape of a water droplet, and the exhaust port is located at the top of the water droplet.

[0076] The cross section of the detection cavity is in the shape of a water drop, which helps the gas or liquid in the detection cavity to converge upward, thereby making it easier for it to enter the exhaust channel.

[0077] The gravity microfluidic chip provided by the present invention, through the ingenious design of the structure of the sample inlet flow channel, fluid channel, chamber and exhaust channel, does not require external drive. The sample can use its own gravity to enter the reaction chamber and will not enter the detection chamber. After the amplification reaction in the reaction chamber is completed, it can enter the detection chamber under a simple pressure device, realizing the controllable transfer of samples between different chambers without sample backflow.

[0078] On the other hand, when the chip is a centrifugal microfluidic chip, the exhaust channel is connected to the fluid channel, so that the combination of the exhaust channel and the fluid channel can automatically provide resistance for the sample in the reaction chamber.

[0079] The exhaust channel is connected to the fluid channel, which significantly increases the difficulty of the sample entering the detection chamber. In the absence of an external driving force, it is difficult to make the sample enter the detection chamber by simply setting a simple pressure device on the chip. However, this method is particularly suitable for centrifugal microfluidic chips. The centrifugal force provided by the centrifuge can promote the smooth entry of the sample into the detection chamber, and the magnitude of the centrifugal force can be controlled by the centrifugal speed. The centrifugal speed required for this method is not high and can be completely controlled within the medium-speed centrifugation range, which will not affect the amplification and detection of the target nucleic acid in the sample.

[0080] Furthermore, the chip includes a chip body with a rotation center, the reaction chamber, detection chamber, fluid channel and exhaust channel are located in the chip body, the rotation center is used to connect to the centrifugal drive device, thereby driving the centrifugal microfluidic chip to centrifuge; the exhaust channel includes an inlet and an outlet, the inlet is the connection between the fluid channel and the exhaust channel, and the outlet is the other end of the exhaust channel; the outlet is closer to the rotation center than the inlet, so that the exhaust direction of the exhaust channel is opposite to the rotation centrifugal direction or at least partially opposite.

[0081] The centrifugal microfluidic chip provided by the present invention has multiple groups of detection units on the chip body, each group of detection units is provided with a reaction chamber and a detection chamber, and can be applied to the simultaneous detection of multiple different target analytes, such as the detection of multiple different nucleic acid targets, and each group of detection units can realize multiple nucleic acid detection. Of course, it is understandable that the centrifugal microfluidic chip of the present application is not limited to multiple nucleic acid detection, but is also suitable for other types of molecular detection such as proteins and antibodies. The reaction chamber can be used for sample nucleic acid amplification, and the detection chamber is used for sample detection.

[0082] During use, the centrifugal microfluidic chip needs to be kept horizontal, with the sample port facing upward. In some embodiments, the centrifugal microfluidic chip can be inserted into a matching detection device, with the chip placed horizontally with the sample port facing upward. The rotation center is fastened and fixed to the detection device's centrifugal shaft, allowing the shaft to drive the centrifugal microfluidic chip for centrifugal rotation. In some embodiments, the detection device can provide a heat source for heating the reaction chamber of the centrifugal microfluidic chip. In some embodiments, the detection device can also detect fluorescent substances produced after sample reaction in the centrifugal microfluidic chip and read the detection results.

[0083] Because centrifugal microfluidic chips require centrifugation to control the flow of liquid, they cannot have exhaust channels in each reaction chamber and detection chamber. This is because if each chamber is equipped with an exhaust channel, it is difficult to control the flow of samples during centrifugation. The sample may directly pass through the reaction chamber and reach the detection chamber under the action of centrifugation, affecting the amplification and detection results. Therefore, existing centrifugal microfluidic chips usually only have air holes on the chip floor or cover. Such a setting is not suitable for multiple nucleic acid detection chips because multiple chambers are required. It is difficult to exhaust the rear chamber during centrifugation, and a greater centrifugal force is required to push the sample in. High-speed centrifugation can easily cause the freeze-dried reagents pre-placed in each chamber to not dissolve fully, resulting in precipitation, which seriously affects the accuracy of the test results.

[0084] The centrifugal microfluidic chip provided by the present invention is provided with an exhaust channel on the fluid channel connecting the reaction chamber and the detection chamber in each detection unit, so that the reaction chamber and the detection chamber share the same exhaust channel, and the structure of the exhaust channel is improved. Combined with the performance of centrifugation, the reaction chamber and the detection chamber can be smoothly exhausted when they need to be exhausted, and can be smoothly closed to prevent interference when they need to be closed for reaction. The amount of sample entering the reaction chamber and the detection chamber can also be effectively controlled. Multiple nucleic acid detection can be completed by one or two centrifugations, and the problem of leakage or sample reflux can be perfectly avoided. After the sample to be tested enters the corresponding chamber, it can be stably retained in the chamber without reflux and will not flow into other chambers, thereby ensuring that each step of the reaction can be completed smoothly, providing a guarantee for the accurate detection of the centrifugal microfluidic chip.

[0085] The fluid channel connecting the reaction chamber and the detection chamber refers to a channel connecting the outlet of the reaction chamber and the inlet of the detection chamber.

[0086] Furthermore, the exhaust channel includes an inlet and an outlet, the inlet is the connection between the fluid channel and the exhaust channel, and the outlet is the other end of the exhaust channel; the outlet is closer to the rotation center than the inlet, so that the exhaust direction of the exhaust channel is opposite to or at least partially opposite to the centrifugal direction of rotation.

[0087] When the centrifugal microfluidic chip is centrifuged, the flow direction of the fluid sample under the action of centrifugation is emitted from the center of the circle to the outside. In the centrifugal microfluidic chip provided by the present invention, the exhaust channel is led out from the fluid channel between the reaction chamber and the detection chamber and approaches the rotation center, so that the exhaust direction is opposite to the centrifugal direction. During centrifugation, the fluid sample will not enter the exhaust channel, but will accurately enter the corresponding chamber along the centrifugal direction.

[0088] In some embodiments, the exhaust channel is led out from the middle of the fluid channel or toward a position close to the detection chamber, and the initial section of the exhaust channel is an arc-shaped channel, and the angle between the arc-shaped channel and the fluid channel close to the reaction chamber is less than 90 degrees.

[0089] In some embodiments, the exhaust channel can be led out slightly closer to the detection chamber. Because the exhaust channel is closer to the detection chamber, the pressure that the sample needs to overcome to enter the detection chamber during centrifugation can be reduced, making it easier for the sample to enter the detection chamber during centrifugation.

[0090] When the exhaust channel is led out from the fluid channel, the smaller the angle between the initial section and the fluid channel close to the reaction chamber, the less likely the fluid will enter the exhaust channel. However, this angle cannot be too small, otherwise it may cause exhaust difficulties. It is preferred to use an arc-shaped channel at one end for transition, and the angle between the arc-shaped channel and the fluid channel close to the reaction chamber is less than 90 degrees.

[0091] In some embodiments, the exhaust passage has an inner diameter smaller than that of the fluid passage.

[0092] Furthermore, there are two ways to set the outlet of the exhaust channel. One is to connect with the outside world, and a waterproof breathable membrane is provided at the outlet connected with the outside world; the other is to connect with the main channel of the chip body.

[0093] Furthermore, there are two ways to set the outlet of the exhaust channel. One is to connect with the outside world, and a waterproof breathable membrane is provided at the outlet connected with the outside world; the other is to connect with the main channel of the chip body.

[0094] When the first setting method is used, only one centrifugation is required to complete the multiple nucleic acid detection; when the second setting method is used, only two centrifugations are required to complete the multiple nucleic acid detection.

[0095] The first configuration involves exhausting the sample to the outside. The exhaust port is covered with a waterproof, breathable membrane. This membrane is permeable to air but impermeable to water, allowing gases to escape from the reaction or detection chamber while preventing liquids from passing through. This membrane helps balance the internal and external pressures of the sample within the reaction or detection chamber, regulating the flow of liquid to prevent leakage or backflow. In some configurations, the membrane is made of a polymer material.

[0096] The flow process of the test sample in the centrifugal microfluidic chip mainly consists of two steps: the first step is that the test sample enters the reaction chamber through the main channel under the action of injection pressure for reaction; the second step is that after the reaction is completed, the test sample flows out of the reaction chamber through low-speed centrifugation and enters the detection chamber for reaction and detection.

[0097] When the first setting is adopted, the sample is injected from the injection port into the main channel through a pipette or syringe, and under the action of the injection pressure, it flows from the main channel into each reaction chamber, and then the injection port is sealed. In the first step, when the sample enters the reaction chamber, the gas in the reaction chamber needs to be discharged outward through the exhaust channel. However, since the detection chamber is not provided with an exhaust channel, it is difficult for the sample to flow in. At the same time, since the exhaust direction of the exhaust channel is opposite to the centrifugal direction and is higher than the fluid channel, the inner diameter of the exhaust channel is smaller than the inner diameter of the fluid channel. Therefore, the sample can only reach the intersection of the fluid channel and the exhaust channel in the first step and cannot continue to move forward, thereby controlling the volume of the sample entering the reaction chamber. At this time, since the injection port is sealed, that is, only the exhaust channel of the entire microfluidic chip is connected to the atmosphere, and the rest of the parts are in a sealed state, the pressure inside and outside is balanced, and the sample in the reaction chamber can no longer flow from the reaction chamber to other areas, because flowing out of the reaction chamber will inevitably require a large pressure to break the established internal and external pressure balance. Therefore, all the samples will remain in the reaction chamber for reaction, and leakage or backflow is impossible.

[0098] In the second step, after the sample completes its reaction in the reaction chamber, it undergoes low-speed centrifugation (e.g., 100-1000 rpm). This disrupts the initial pressure equilibrium, causing the sample to flow out of the reaction chamber under the action of centrifugal force. At this point, because the exhaust direction of the exhaust channel is opposite to the centrifugal direction, the sample does not enter the exhaust channel and flows entirely into the detection chamber, while the gas in the detection chamber is discharged through the exhaust channel. At this point, the pressure inside the detection chamber also reaches equilibrium, preventing the sample from flowing out and remaining in the detection chamber for reaction and detection.

[0099] Therefore, when the first setting method is adopted, under the joint action of the exhaust channel and the fluid flow channel, the internal and external pressure balance of the sample in the reaction chamber or detection chamber can be established, and the direction of liquid flow can be regulated to prevent leakage or backflow of the sample in the reaction chamber or detection chamber. It can also effectively control the amount of sample entering the reaction chamber or detection chamber, and multiple nucleic acid detection can be completed with only one low-speed centrifugation.

[0100] The second setting is to exhaust air to the main channel. Neither the reaction chamber nor the detection chamber needs to be connected to the outside atmosphere. The exhaust channel is directly connected to the main channel, and the air pressure inside the reaction chamber or detection chamber is discharged back to the main channel. This is equivalent to discharging the air pressure in the liquid-filled part to the unfilled part through the exhaust channel, which plays a role in controlling the flow of liquid. Moreover, after exhausting air to the main channel through the exhaust channel, the air pressure in the chip reaches equilibrium. The sample entering the reaction chamber or detection chamber is also difficult to flow out of the reaction chamber and reflux is difficult to occur, forming a closed and circulating environment. Compared with the first setting, since it is not connected to the outside world, it can effectively avoid aerosol contamination, making the detection results more accurate and reliable. At the same time, the second setting eliminates the steps of preparing air holes and covering with a waterproof breathable membrane, making the preparation process simpler and easier, and lowering the cost.

[0101] The process includes three steps: the first step is that the sample enters the main channel from the sample injection port; the second step is low-speed centrifugation to allow the sample to enter the reaction chamber from the main channel for reaction; the third step is that after the reaction is completed, the sample flows out of the reaction chamber through medium-speed centrifugation and enters the detection chamber for reaction and detection. Unlike the first setting method, when using the second setting method, it is also necessary to control the flow of the sample by controlling the centrifugal rate. The sample is allowed to enter the reaction chamber from the main channel through low-speed centrifugation (such as 100-1000 rpm), and then the sample is allowed to enter the detection chamber from the reaction chamber through medium-speed centrifugation (such as 1000-3000 rpm). The method of controlling the flow of samples in different chambers by controlling the centrifugal rate is simpler and easier to operate, and the entire process is more controllable and the test results are more accurate.

[0102] In the first step, the sample is injected into the main channel from the injection port through a pipette or syringe. After filling the serpentine main channel, the sample will flow to the final waste liquid pool, the injection will stop, and the injection port will be sealed. This process is equivalent to the process of the sample filling the main channel, and it is also the process of the gas filled with liquid being discharged to the unfilled part of the main channel through the exhaust channel. Because the inner diameter of the branch channel connecting the main channel to the reaction chamber is small and there is a large difference in the inner diameter of the main channel, the sample cannot flow into the reaction chamber. Moreover, it is difficult to discharge the gas in the reaction chamber from the exhaust channel back to the main channel by relying solely on injection pressure, so the sample will first stay in the main channel.

[0103] In the second step, after the sample has completely filled the main channel, it is centrifuged clockwise at a low speed. Centrifugal force forces the sample within the main channel into the reaction chamber. However, the detection chamber lacks a separate exhaust channel, creating a nearly sealed enclosure. Under low-speed centrifugation, the sample struggles to overcome the gas pressure within the detection chamber and is unable to flow into the detection chamber. Furthermore, because the exhaust channel's direction of exhaust is opposite to the centrifugal direction and the exhaust channel is higher than the fluid, the sample can only reach the intersection of the fluid channel and the exhaust channel during the second step before being unable to move forward. This effectively controls the volume of sample entering the reaction chamber. During the low-speed centrifugation process, the sample enters the reaction chamber while the gas within the reaction chamber is simultaneously exhausted back into the main channel, further facilitating the flow of sample from the main channel into the reaction chamber. After the low-speed centrifugation, the sample remains in the reaction chamber for the amplification reaction. In this sealed state, pressure equilibrium is achieved inside and outside the reaction chamber, preventing the sample from flowing out of the reaction chamber to other areas. Exiting the reaction chamber would require a significant pressure increase to disrupt the established pressure balance. Therefore, the sample remains entirely within the reaction chamber, preventing leakage or backflow.

[0104] In the third step, after the sample completes the amplification reaction in the reaction chamber, the centrifugal speed is increased. After medium-speed centrifugation, the initial pressure balance is broken, driving the sample from the reaction chamber to the detection chamber. At this time, since the exhaust direction of the exhaust channel is opposite to the centrifugal direction and the inner diameter of the exhaust channel is smaller, the sample will not enter the exhaust channel and will all flow into the detection chamber. Since the entire system is in a closed state, the liquid in the detection chamber will not reflux, and thus it can be used stably for detection.

[0105] Therefore, the second setting adopts two-stage centrifugation to complete multiple nucleic acid detection. The centrifugation rates of the first and second stages can differ greatly, the control is stable, and high-speed centrifugation (such as 3000-10000 rpm) is not required, so as to avoid the precipitation of pre-embedded freeze-dried or vitrified reagents due to insufficient dissolution caused by high-speed centrifugation, thereby causing reaction failure and affecting the test results.

[0106] Furthermore, when the centrifugal microfluidic chip is placed horizontally, the outlet of the exhaust channel is higher than the inlet. When the outlet of the exhaust channel is connected to the outside world, an air permeable cavity is also provided at the outlet of the exhaust channel, and the volume of the air permeable cavity is not less than that of the reaction chamber.

[0107] The outlet of the exhaust channel is located higher than the inlet, which can further increase the difficulty of the fluid sample entering the exhaust channel, thereby helping to control the flow of liquid in the chip and facilitating the exhaust of gas from the reaction chamber and detection chamber.

[0108] The provision of the vent cavity can be used to help exhaust the reaction cavity and the detection cavity, making it easier for the sample to enter the reaction cavity, and also facilitates the transfer of gas in the detection cavity from the detection cavity to the vent cavity during the centrifugation process.

[0109] Furthermore, the chip body is circular; when the outlet of the exhaust channel is connected to the outside world, the main channel is arranged in a wave-like manner; when the outlet of the exhaust channel is connected to the main channel, the main channel is arranged in a serpentine manner; the main channel is bent into a ring, and the chip body, the ring and the rotation center are coaxially arranged.

[0110] In some methods, the main flow channel adopts a serpentine (wavy) flow channel with a large curvature and evenly spaced intervals, which can help maintain the air pressure balance in the chip, smoothly control the flow of liquid, and help prevent the various detection units from directly interfering with each other.

[0111] In some embodiments, when the outlet of the exhaust channel is connected to the main channel, the main channel is arranged in a serpentine shape. The serpentine shape makes the main channel more curved and longer, so that the samples are first stably retained in the main channel.

[0112] Furthermore, the serpentine main channel includes U-shaped channels arranged at equal distances along a circular ring, and the bottom end of the U-shaped channel is connected to the reaction chamber; when the outlet of the exhaust channel is connected to the main channel, the connected part is the top end of the U-shaped channel.

[0113] In some embodiments, the bottom end of the U-shaped flow channel is provided with a branch channel connected to the inlet of the reaction chamber, and the top end of the U-shaped flow channel is connected to the exhaust channel. The left and right sides of the U-shaped flow channel are longer, and the top end is closer to the center of rotation than the bottom end, so that the bottom and top ends of the U-shaped flow channel are farther apart. When the exhaust channel exhausts toward the top end of the U-shaped flow channel, it will not immediately affect the air pressure balance at the bottom end, which helps the sample to flow smoothly into the reaction chamber.

[0114] The U-shaped flow channels are evenly arranged, and the bottom end of each U-shaped flow channel is connected to a reaction chamber. Therefore, during centrifugation, the sample in each U-shaped flow channel will enter the corresponding reaction chamber under the action of centrifugal force. This is equivalent to the U-shaped flow channel evenly distributing the sample in advance, which can help control the volume of the sample entering the reaction chamber.

[0115] Furthermore, the main channel is further provided with a sample addition port. When the outlet of the exhaust channel is connected to the main channel, the sample addition port is located at the starting end of the main channel. A waste liquid pool is further provided at the end of the main channel.

[0116] When the exhaust channel adopts the first setting mode, the main channel is a connected circular closed loop, and only one opening needs to be set in the main channel for sample injection.

[0117] The exhaust channel adopts the second setting method, that is, when the sampling port is located at the starting end of the main channel, the two ends of the main channel are not connected, and the sampling port and the waste liquid pool are respectively set at both ends, where the sampling port is located at the starting end of the main channel and the waste liquid pool is located at the end of the main channel.

[0118] The waste liquid reservoir serves as a reservoir for excess sample, which flows into the waste liquid reservoir as the sample fills the main channel. In the second method, because the injection pressure is insufficient to force the sample into the reaction chamber, the excess sample begins to flow into the waste liquid reservoir after the sample has filled the main channel. This serves as a reminder that the sample volume has been sufficient and that addition can be stopped. Therefore, the waste liquid reservoir also aids in sample quantification.

[0119] In some embodiments, the sample loading port of the centrifugal microfluidic chip is covered with a transparent film to provide a seal. Sample loading is performed by puncturing the transparent film with a syringe. After the sample fills the main channel, the sample loading port is sealed with the transparent film again.

[0120] Furthermore, it also includes one or more sub-chips, which are concentric ring chips located outside the chip body. The sub-chip is also provided with a main channel, a reaction chamber and a detection chamber; when the chip body rotates centrifugally, the sub-chip can rotate centrifugally along with the chip body.

[0121] The centrifugal microfluidic chip provided by the present invention decomposes the traditional single centrifugal microfluidic chip into a chip body and a sub-chip. The chip body and the sub-chip operate independently, are compact, and easy to operate. They can perform multi-index detection of multiple samples simultaneously, greatly improving detection efficiency and meeting the needs of fast, convenient, and low-cost high-throughput diagnosis. It has important application value in the fields of clinical diagnosis or rapid on-site screening.

[0122] The chip body and the sub-chip can be directly molded and manufactured in one chip and directly connected into one body, or they can be manufactured separately and fixed in use by means of snap fastening or the like. In some embodiments, in order to make the centrifugation process more stable and controllable, the chip body and the sub-chip are integrally molded and cannot be disassembled.

[0123] In some methods, the sub-chip, the chip body and the entire microfluidic chip are all in the same plane. The sub-chip is located outside the chip body and has a larger diameter, so more groups of detection units can be set up. At the same time, the sub-chip can also be provided with a sample addition port and a waste liquid pool to independently perform multiple nucleic acid detections. It can detect samples different from the chip body, realize simultaneous detection of multiple samples, and can detect more indicators at the same time (each detection unit can detect a nucleic acid target), significantly increasing the detection throughput and improving the detection efficiency.

[0124] In some methods, the number of sub-chips and the number of detection units in each sub-chip or chip can be flexibly set according to the number of samples to be tested and the index detection requirements, and the reaction chamber of each detection unit and the solid reagent built into the detection chamber can be flexibly set according to actual needs. For example, amplification primers for different respiratory targets can be built into different reaction chambers, and corresponding detection reagents can be built into corresponding detection chambers, so that multiple detection of respiratory pathogens can be flexibly achieved.

[0125] Furthermore, the rotation center is a slot, and the slot enables the centrifugal microfluidic chip to be clamped on the rotation axis of the centrifugal drive device, thereby performing centrifugal rotation.

[0126] In some embodiments, the slot is a circular slot with a protrusion, and the protrusion is in the shape of a triangle, trapezoid, etc. The rotating shaft on the detection device also has a fixed groove that matches the size of the slot, so that the centrifugal microfluidic chip is fixed on the rotating shaft through the slot and rotates centrifugally with the rotating shaft.

[0127] In some embodiments, the cross-section of the reaction chamber is circular.

[0128] The cross-sectional shape of the reaction chamber of the centrifugal microfluidic chip is set to be circular, so that the sample to be tested reacts and is discharged in the reaction chamber without leaving any dead corners, and it is not easy to leave residues during the inflow and outflow processes.

[0129] The cross-section of the detection chamber of the centrifugal microfluidic chip is approximately semicircular and larger than the reaction chamber, which can ensure that all samples in the reaction chamber enter the detection chamber.

[0130] It can be understood that, whether it is a gravity microfluidic chip or a centrifugal microfluidic chip, the solid reagents built into the reaction chamber and the detection chamber can be flexibly set according to needs, and different chambers can have different reaction reagents built into them, such as amplification primers for the target built into the reaction chamber and detection probes for different targets built into the detection chamber.

[0131] In another aspect, the present invention provides a nucleic acid detection method, characterized in that the detection is performed using the nucleic acid multiplex detection microfluidic chip as described above. When the nucleic acid multiplex detection microfluidic chip is a centrifugal microfluidic chip, the detection method is divided into two types:

[0132] A. When the outlet of the exhaust duct is connected to the outside world, the detection method includes the following steps:

[0133] (1) Place the centrifugal microfluidic chip horizontally and add the sample to be tested from the sample injection port;

[0134] (2) The sample to be tested enters the reaction chamber of the centrifugal microfluidic chip from the sample loading area and is incubated at a temperature for nucleic acid amplification;

[0135] (3) clamping the slot of the centrifugal microfluidic chip onto the rotating shaft of the detection device;

[0136] (4) centrifugation to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction;

[0137] (5) Read the test results;

[0138] B. When the outlet of the exhaust channel is connected to the main channel, the detection method includes the following steps:

[0139] (a) Place the centrifugal microfluidic chip horizontally and add the sample to be tested through the sample injection port;

[0140] (b) clamping the card slot of the centrifugal microfluidic chip onto the rotating shaft of the detection device;

[0141] (c) low-speed centrifugation, where the sample to be tested enters the reaction chamber of the centrifugal microfluidic chip from the sample loading area for nucleic acid amplification;

[0142] (d) centrifuging at a moderate speed to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction;

[0143] (e) reading the test results;

[0144] When the nucleic acid multiplex detection microfluidic chip is a gravity microfluidic chip, the detection method comprises the following steps:

[0145] (I) Insert the microfluidic chip vertically into the detection device;

[0146] (II) The sample enters the microfluidic chip from the sample inlet and flows into the reaction chamber under the action of gravity, and the tube is capped;

[0147] (III) nucleic acid amplification of the sample to be tested in the reaction chamber;

[0148] (IV) squeezing the pressure zone to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction;

[0149] (V) Read the test results

[0150] In some embodiments, in step (2), (c) or (III), the nucleic acid amplification is isothermal amplification, and a heating device is required to provide the reaction chamber with a suitable temperature required for isothermal amplification (such as 30-65° C.).

[0151] On the other hand, the present invention provides a microfluidic chip, which is a nucleic acid typing detection chip. Unlike the aforementioned nucleic acid multiplex detection microfluidic chip, it is only provided with a reaction chamber but no detection chamber, and is more suitable for nucleic acid typing detection. It can be used to perform nucleic acid detection of different targets on pre-processed samples. However, in some embodiments, the nucleic acid typing detection chip is suitable for both genotyping detection and nucleic acid multiplex detection. When used for nucleic acid multiplex detection, the liquid storage chamber is used for target nucleic acid cleavage, and amplification and detection reagents for different nucleic acids need to be pre-placed in the reaction chamber; when used for nucleic acid typing detection, the liquid storage chamber is used for target nucleic acid amplification, and nucleic acid amplification reagents need to be pre-placed in the liquid storage chamber, and detection reagents for different target nucleic acids need to be pre-placed in the reaction chamber.

[0152] The nucleic acid typing detection chip includes a liquid storage chamber for adding or storing reagents and a reaction chamber for amplification or detection; an injection channel is provided between the liquid storage chamber and the reaction chamber to facilitate the flow of liquid from the liquid storage chamber to the reaction chamber under the action of gravity; the reaction chamber is also provided with an exhaust channel, which is ultimately connected to the liquid storage chamber to discharge the gas therein back to the liquid storage chamber.

[0153] Existing microfluidic chips generally have certain drawbacks, such as complex chip structures, prone to flow channel blockage, and reliance on power devices and centrifugal devices to achieve directional movement of liquids, resulting in a dependence on power and centrifugal devices. Furthermore, existing microfluidic chips often require vent holes connected to the outside world to ensure proper flow of liquid within the chip, which complicates the chip structure and requires prevention of leakage.

[0154] The nucleic acid typing detection chip provided by the present invention is provided with a liquid storage chamber and a plurality of reaction chambers from top to bottom, and the chambers are connected by flow channels. When there is a sample in the liquid storage chamber, the sample can enter the main channel under the action of gravity, and is radially connected to the multiple reaction chambers by the end of the main channel; the reaction chamber is provided with an injection flow channel and an exhaust channel, the injection flow channel is designed to have a larger aperture, which is convenient for the liquid to flow from the liquid storage chamber to the reaction chamber under the action of gravity, and the exhaust channel is a multi-turn circuitous flow channel with a smaller aperture, which is finally connected to the liquid storage chamber. After the sample enters the reaction chamber, the gas in the cavity can flow back to the liquid storage chamber through the exhaust channel, and no gas exchange occurs with the outside world, thereby avoiding aerosol contamination. Due to the air pressure balance in the system, it is difficult for the sample entering the reaction chamber to flow out of the reaction chamber, and it is difficult for reflux to occur, forming a circulating closed environment. Therefore, the sample in the liquid storage chamber does not need an external driving device, and can be autonomously transferred to the reaction chamber only under gravity drive. The chip greatly simplifies the structure and operation process while ensuring detection precision. It has a fast reaction speed and a closed reaction to avoid aerosol contamination. It can be expanded to the rapid and visual detection of nucleic acids of other pathogens such as respiratory and reproductive tract samples, and is suitable for large-scale promotion and application.

[0155] During use, the nucleic acid typing detection chip needs to be kept upright with the sample port facing upward. In some methods, the nucleic acid typing detection chip can be inserted into a matching detection device, with the microfluidic chip kept upright with the sample port facing upward. The detection device can then provide a heat source to heat specific locations on the nucleic acid typing detection chip (such as the reaction chamber, valve block, etc.). In some methods, the detection device can also detect the fluorescent substance produced by the sample reaction in the nucleic acid typing detection chip and read the test results.

[0156] Furthermore, the reaction chamber is further provided with an inlet channel, the cross-sectional area of ​​which is larger than the cross-sectional area of ​​the exhaust channel, so that the sample can flow into the reaction chamber from the inlet channel but cannot flow out of the reaction chamber from the exhaust channel.

[0157] Furthermore, the aperture of the injection channel is larger than the aperture of the exhaust flow channel.

[0158] In some embodiments, the pore size of the injection channel is greater than 400 μm.

[0159] In some embodiments, the pore size of the injection channel is 400-800 μm.

[0160] The apertures of the main channel of the nucleic acid typing detection chip and the reaction chamber's sample flow channel, as well as other channels used for sample flow, are designed to ensure smooth liquid flow. However, the aperture of the reaction chamber's exhaust channel is significantly smaller than these channels. The exhaust channel is a multi-turn, small-aperture flow channel that only allows for ventilation but not for smooth liquid drainage. As a result, after the sample fills the reaction chamber, further advancement is hindered, making it difficult to exit the exhaust channel and remain in the reaction chamber to complete the reaction and be accurately detected.

[0161] Furthermore, the pore size of the exhaust channel is less than 300 μm.

[0162] In some embodiments, the pore size of the exhaust channel is 50 to 300 μm.

[0163] Furthermore, the exhaust passage is a multi-turn circuitous flow passage and is provided with at least one bent structure.

[0164] Providing multiple bent structures in the exhaust channel can extend the length of the exhaust channel as much as possible, so that there are more pipes to store the exhausted gas, preventing the situation where there are many reaction chambers but only one liquid storage chamber, which is difficult to fully accommodate all the gases and causes the gas pressure to increase.

[0165] In some embodiments, the exhaust channel is further provided with a small cavity at a bend for accommodating the excess gas discharged.

[0166] In some embodiments, the exhaust passage has at least one passage that bends back and forth.

[0167] In some embodiments, before the exhaust channel is connected to the liquid storage chamber, an exhaust chamber may be provided to store the exhausted excess gas, which then returns to the liquid storage chamber after the exhaust chamber is filled.

[0168] Furthermore, the number of the reaction chamber is at least one, and each of the reaction chambers is connected to the main channel fluid of the microfluidic chip through an injection channel.

[0169] Nucleic acid typing detection chips are equipped with multiple reaction chambers, suitable for multiplexed detection of different target nucleic acids or detection of different genotypes. Of course, it is understood that nucleic acid typing detection chips are not limited to genotyping detection or multiplexed nucleic acid detection, but are also suitable for detection of other molecular types such as proteins and antibodies. The liquid storage chamber can be used for sample pretreatment, and the reaction chamber is used for sample detection.

[0170] In some embodiments, the number of the reaction chambers is 2 to 10, and the reaction chambers can be arranged in any manner, such as horizontally, vertically, or radially at the end of the main channel in any other manner. Multiple bifurcations can also be set at the lower end of the main channel for the arrangement of the reaction chambers.

[0171] Furthermore, the reaction chamber is provided with an injection port and an exhaust port, the injection port is in fluid communication with the injection channel, and the exhaust port is in gas communication with the exhaust channel; the exhaust port is positioned higher than the injection port.

[0172] Furthermore, the injection port is located at a lower position of the reaction chamber, and the exhaust port is located at an upper position of the reaction chamber.

[0173] The present invention also designs the positions of the sample inlet and exhaust port of the reaction chamber. The sample inlet is located at the lower position of the reaction chamber, and the exhaust port is located at the upper position of the reaction chamber. The purpose of this is to ensure that the sample can be smoothly exhausted upward after entering the reaction chamber, and at the same time, it can stay more stably in the reaction chamber to prevent backflow.

[0174] After the sample to be tested completes a reaction that is conducive to detection in the reaction chamber (such as reacting with the detection reagent to amplify the signal, etc.), the result is detected directly in the reaction chamber. One side of the reaction chamber is prepared with a transparent film. The detection instrument can detect fluorescent substances and other substances generated by the reaction through the film and read the test results.

[0175] Furthermore, the liquid storage chamber is provided with a liquid outlet and an air inlet, the liquid outlet is in fluid communication with the main channel, and the air inlet is in gas communication with the exhaust channel of the reaction chamber; the air inlet is positioned higher than the liquid outlet.

[0176] The position of the air inlet in the liquid storage chamber is higher than the liquid outlet, which can enable all samples in the liquid storage chamber to be discharged smoothly and prevent interference from the incoming gas.

[0177] Furthermore, a valve block can be added between the liquid storage chamber and the reaction chamber to control the state of fluid communication between the liquid storage chamber and the reaction chamber; the valve block has two forms, solid and liquid; when the valve block is solid, the liquid storage chamber and the reaction chamber cannot be fluidically connected; when the valve block is liquid, the liquid storage chamber and the reaction chamber are fluidically connected.

[0178] Furthermore, when the valve block is solid, it is located in the main channel connecting the liquid storage chamber and the reaction chamber, and is used to prevent the sample to be tested in the liquid storage chamber from flowing into the reaction chamber; when the valve block is liquid, it will flow into the exhaust channel of the reaction chamber; the valve block is paraffin or lipid material, which is solid at room temperature and melts into liquid after heating; the density of the valve block is less than that of water.

[0179] Because microfluidic chips are equipped with multiple reaction chambers and the sample inlets of the reaction chambers are gathered together, sample backflow is very likely to occur, causing the sample to overflow from one reaction chamber and enter another reaction chamber, resulting in cross-influence. Therefore, it is very important to prevent sample backflow and ensure the accuracy of the test results.

[0180] The nucleic acid typing detection chip provides an exhaust channel in the reaction chamber and a valve block between the liquid storage chamber and the reaction chamber. The valve block's temperature-induced phase change properties allow the liquid storage chamber and reaction chamber to be blocked and connected. When the valve block melts, the flow channels between the liquid storage chamber and each reaction chamber are connected, allowing the sample in the liquid storage chamber to flow into each reaction chamber. The melted valve block, with its low density, floats above the sample, flowing into the reaction chamber along with the sample and condensing near the exhaust port of the reaction chamber, blocking the exhaust channel (since the sample is quantitative, it will at least reach the exhaust port of the reaction chamber). This also helps the sample to be tested remain more stably within the reaction chamber after entering it, making it less likely to reflux and flow into other chambers. This ensures that each step of the reaction is completed smoothly until accurate detection is achieved.

[0181] It can be seen that the valve block can serve multiple purposes, such as controlling the connection state between the liquid storage chamber and the reaction chamber, preventing the sample from flowing out of the reaction chamber, etc.

[0182] In some embodiments, the lipid substances include n-hexadecane, n-octadecane, paraffin, stearic acid, palmitic acid, etc.

[0183] In some embodiments, the valve block is paraffin.

[0184] Paraffin wax can achieve phase change at a specific temperature threshold between 40 and 90°C.

[0185] Furthermore, when using pre-processed samples for nucleic acid testing for different purposes, there is no need to place a lysis solution in the liquid storage chamber, and there is no need to place a valve block between the liquid storage chamber and the reaction chamber. The liquid storage chamber and the reaction chamber are in a state of fluid communication, and under the action of gravity, the liquid storage chamber reagent directly enters different reaction chambers.

[0186] In some embodiments, the cross-section of the reaction chamber is in the shape of a water droplet, and the exhaust port is located at the top of the water droplet.

[0187] The cross-section of the reaction chamber is in the shape of a water droplet, which helps the liquid paraffin to gather upward, making it easier to condense near the exhaust port.

[0188] It is understandable that the solid reagents built into the liquid storage chamber and the reaction chamber can be flexibly set according to needs. Different chambers can have different reaction reagents built into them. For example, the liquid storage chamber can have amplification primers for the target built into it, and each reaction chamber can have detection probes for different genotypes built into it, so as to realize gene typing detection.

[0189] In some embodiments, the sample loading area further includes a tube cover, which is used to seal the microfluidic chip.

[0190] The nucleic acid typing detection chip provided by the present invention includes a gravity typing chip and an asymmetric pressure typing chip. The tube cover of the gravity typing chip can be prepared in the form of a plug, or the cover surface of the tube cover can be concave. When the tube cover is tightened to seal the microfluidic chip, a certain downward pressure can be provided, prompting the sample near the sample injection port to flow completely into the liquid storage chamber, and the amplification product in the liquid storage chamber is placed in a relatively high-pressure state.

[0191] The gravity typing chip means that the sample can automatically move from the liquid storage chamber to the reaction chamber directly under gravity drive.

[0192] The asymmetric pressure parting chip is similar to the gravity parting chip, except that its tube cover can apply pressure to the chip after the chip is sealed, forming an instantaneous asymmetric pressure. Driven by the instantaneous asymmetric air pressure, the sample can quickly flow from the injection channel into the reaction chamber, but cannot flow out of the reaction chamber through the exhaust channel.

[0193] After the chip is sealed, the tube cover of the asymmetric pressure typing chip can also apply pressure to the chip, generating asymmetric pressure inside the chip, prompting the sample to flow from the liquid storage chamber through the injection channel into the reaction chamber.

[0194] Existing microfluidic chips require continuous external force to achieve directional flow, placing high demands on instrumentation and drive stability. The asymmetric pressure-sensing chip provided by the present invention achieves this by eliminating the need for any external force to drive the liquid flow, relying solely on the instantaneous asymmetric pressure generated by twisting or pressing the tube cap.

[0195] In the asymmetric pressure typing chip, a certain pressure is applied to the sealing system by twisting or pressing the tube cap. The reaction chamber is equipped with an inlet channel and an exhaust channel. The exhaust channel is a multi-turn, circuitous channel with a smaller aperture. Because the inlet channel aperture is larger than the exhaust channel aperture, an instantaneous asymmetric pressure is generated to drive liquid movement. As the sample flows into the reaction chamber through the inlet channel, the gas inside the reaction chamber is connected to the liquid storage chamber through the exhaust channel. At this time, the air pressure in the microfluidic chip is balanced, and the sample entering the reaction chamber is difficult to flow out of the reaction chamber, which reduces the risk of backflow. This creates a closed and circulating environment, effectively preventing aerosol contamination.

[0196] The main channel of the microfluidic chip and the injection channel of the reaction chamber are channels for sample flow, and their aperture settings can enable the liquid to flow smoothly.

[0197] The cross-sectional area of ​​the exhaust channel is smaller than that of the sample inlet channel. This small exhaust channel only allows for ventilation, but not for smooth liquid drainage. When a certain pressure is applied, a transient asymmetric pressure is generated. Once the sample fills the reaction chamber, further flow is blocked, making it difficult to exit the exhaust channel. Consequently, the sample remains in the reaction chamber, completing the reaction and enabling accurate detection.

[0198] The tube cover is used to seal the microfluidic chip and provide instantaneous pressure. The tube cover can be manufactured using a screw-down mechanism or a piston embedded in the cover surface. When the tube cover seals the microfluidic chip, or after the chip is sealed, twisting or pressing the tube cover will apply a certain amount of air pressure to the chip. Because the aperture of the inlet channel is significantly larger than that of the exhaust channel, and the exhaust channel is a multi-turn, circuitous channel with a smaller aperture, this creates an instantaneous asymmetric pressure, promoting the transfer of liquid from the liquid storage chamber to the reaction chamber.

[0199] Furthermore, after the chip is sealed, the tube cover can apply pressure to the chip by twisting or pressing the tube cover.

[0200] Furthermore, the cap is equipped with a pushable airbag at the top. After sealing the chip, pushing the airbag downward applies pressure to the chip, creating an asymmetric pressure inside the chip and encouraging the sample to flow from the liquid reservoir into the reaction chamber. The airbag ensures that the cap remains sealed to the chip, even during movement.

[0201] In some embodiments, the spiral tube cover is detachably connected to the liquid storage chamber, and while closing the system, it provides air pressure to the chip system to drive the liquid in the liquid storage chamber to transfer to the reaction chamber.

[0202] The method for detecting using the gravity typing chip provided by the present invention comprises the following steps:

[0203] (1) Place the sample to be tested containing the lysate into the gravity typing chip from the sample injection port and cover the tube with the cap;

[0204] (2) Insert the microfluidic chip vertically into the detection device;

[0205] (3) The sample to be tested is lysed or nucleic acid amplified in the liquid storage chamber;

[0206] (4) Heating to change the valve block from solid to liquid;

[0207] (5) The sample to be tested is driven by gravity into each reaction chamber for reaction;

[0208] (6) Read the test results.

[0209] In step (3), the nucleic acid amplification is isothermal amplification, and a heating device is required to provide the liquid storage chamber with a suitable temperature required for isothermal amplification (such as 20 to 65° C.).

[0210] In step (4), the detection device needs to heat the valve block (paraffin) (eg, 40-90° C.) to reach its phase change temperature and melt it into liquid.

[0211] The gravity typing chip provided by the present invention, through the ingenious design of flow channel connections and chamber positions, does not require external drive, allowing the liquid to achieve the amplification and detection reaction of the sample to be tested step by step and independently according to its own gravity. The entire process is simple to operate, has a fast reaction speed, and can effectively achieve high-throughput detection of pathogens. In addition, the amplification / detection reagents of the microfluidic chip are all dry reagents, which can realize the storage and transportation of the chip at room temperature, avoiding the limitations of cold chain transportation and storage at -20°C.

[0212] The method for detecting using the asymmetric pressure typing chip provided by the present invention comprises the following steps:

[0213] (1) Place the sample to be tested containing the lysate into the asymmetric pressure typing chip from the sample inlet and close the tube cap;

[0214] (2) vertically inserting the asymmetric pressure parting chip into the detection device;

[0215] (3) The sample to be tested is lysed or nucleic acid amplified in the liquid storage chamber;

[0216] (4) Heating to change the valve block from solid to liquid;

[0217] (5) Press the air bag on the tube cover, and the sample to be tested is driven into each reaction chamber by asymmetric pressure to react;

[0218] (6) Read the test results.

[0219] In step (3), the nucleic acid amplification is isothermal amplification, and a heating device is required to provide the liquid storage chamber with a suitable temperature required for isothermal amplification (such as 20 to 65° C.).

[0220] In step (4), the detection device needs to heat the valve block (paraffin) (eg, 40-90° C.) to reach its phase change temperature and melt it into liquid.

[0221] The asymmetric pressure typing chip provided by the present invention cleverly designs the flow channel connection and chamber position, applies pressure to the closed system by screwing the tube cap, and at the same time, because the sample inlet flow channel is significantly larger than the exhaust channel, instantaneous asymmetric pressure is generated, thereby realizing the sequential and independent amplification and detection reactions of the sample to be tested. The entire process is not only simple to operate and has a fast reaction speed, but the chip is designed with a fully enclosed structure to effectively avoid aerosol contamination, and can effectively achieve high-throughput detection of pathogens. In addition, the amplification / detection reagents of the microfluidic chip are all dry reagents, which can realize the storage and transportation of the chip at room temperature, avoiding the limitations of cold chain transportation and storage at -20°C.

[0222] The nucleic acid multiplex detection microfluidic chip provided by the present invention has the following beneficial effects:

[0223] 1. On the basis of ensuring the detection precision, the structure and operation process are greatly simplified, the operation is more convenient, the cost is lower, and it is suitable for large-scale promotion and application;

[0224] 2. Provides a gravity microfluidic chip, which has the following beneficial effects:

[0225] (1) No external driving device is required; the sample can be autonomously transferred to the reaction chamber under gravity alone;

[0226] (2) A fluid channel is designed between the reaction chamber and the detection chamber. The fluid channel is a multi-turn, small-diameter, long, and high-positioned flow channel. Under the action of gravity alone, the sample cannot enter. As a result, after the sample fills the reaction chamber, it will be blocked when it continues to move forward, making it difficult to flow out of the fluid channel at the top of the reaction chamber. All of the sample will remain in the reaction chamber to complete the nucleic acid amplification reaction;

[0227] (3) The structure of the injection channel is designed so that the injection port of the reaction chamber is located at the lowest point of the injection channel, so that the sample cannot overcome the effect of gravity and the pressure brought by the injection channel to return to the main channel, and all of it remains in the reaction chamber to prevent sample backflow;

[0228] (4) The reaction chamber does not need to be equipped with an exhaust channel. The multi-turn and small-diameter fluid channel can serve as a valve to temporarily block the flow of liquid between the reaction chamber and the detection chamber, and can also serve as a temporary exhaust channel for the reaction chamber. The reaction chamber does not need to be equipped with an additional exhaust channel and a waterproof and breathable membrane, which makes the structure simpler and the cost lower.

[0229] (5) A simple pressure device is designed. When the sample completes nucleic acid amplification in the reaction chamber, pressing the pressure device can move the sample from the reaction chamber into the detection chamber, realizing the controllable transfer of the sample between different chambers; thus, it can adapt to more simple detection environments, be low-cost and integrated, and be suitable for large-scale promotion and application.

[0230] 3. Provides a centrifugal microfluidic chip, which has the following beneficial effects:

[0231] (1) By introducing an exhaust channel into the fluid channel between the reaction chamber and the detection chamber, and making the exhaust direction of the exhaust channel opposite to the centrifugal direction and positioned higher than the fluid channel, the flow of gas and liquid in the reaction chamber and the detection chamber can be effectively controlled, and the amount of sample entering the reaction chamber can be effectively controlled. In addition, only one or two low-speed plus medium-speed centrifugations are required to complete multiple nucleic acid detection, which makes the operation more convenient and the test results more accurate.

[0232] (2) Two exhaust channel modes are set up. The first is connected to the outside atmosphere, and the second is to directly discharge the gas back to the main channel. Both exhaust modes can well realize multiple nucleic acid detection;

[0233] (3) In particular, the second type can avoid aerosol contamination because it does not need to be connected to the outside world, and the manufacturing process is more convenient and the cost is lower. After the sample enters each chamber, it can be stably retained and reacted, effectively preventing sample backflow, eliminating mutual interference or contamination problems, and providing a guarantee for accurate detection.

[0234] (4) One or more concentric circle sub-chips are set outside the chip body, which can realize high-throughput detection of more samples and more detection indicators, and improve detection efficiency; it has a simple structure, small size, and convenient operation, which can meet the needs of rapid detection anytime, anywhere, and adapt to more simple detection environments, truly achieving low-cost integration and suitable for large-scale production applications.

[0235] The nucleic acid typing detection microfluidic chip provided by the present invention has the following beneficial effects:

[0236] (1) The sample in the liquid storage chamber can be autonomously transferred to the reaction chamber under gravity drive or asymmetric pressure drive without the need for an external driving device;

[0237] (2) The reaction chamber does not need to be connected to the outside atmosphere. The exhaust channel of the reaction chamber is directly connected to the liquid storage chamber, and the atmosphere pressed out of the reaction chamber is discharged back to the liquid storage chamber. At this time, the air pressure in the microfluidic chip is balanced, and the sample entering the reaction chamber is difficult to flow out of the reaction chamber, and reflux is difficult to occur, forming a closed circulation environment;

[0238] (3) The structure and operation process are greatly simplified while ensuring the detection precision. It is not only easy to operate and has a fast response speed, but also the chip adopts a fully enclosed structure design to effectively avoid aerosol contamination. It can be extended to the rapid and visual detection of nucleic acids of other pathogens such as respiratory and reproductive tract samples, and is suitable for large-scale promotion and application.

[0239] Detailed description

[0240] In the description of the present invention, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second" and the like are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, features defined as "first", "second" and the like may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0241] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "connected" and "connection" should be understood in a broad sense. For example, they can refer to fixed connection, detachable connection, or integral connection; mechanical connection or electrical connection; direct connection or indirect connection through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0242] sample

[0243] In the present invention, the sample or specimen to be tested includes a biological fluid. The initial state of the sample can be liquid, solid, or semi-solid. The solid or semi-solid sample can be converted into a liquid sample by any appropriate method, such as mixing, crushing, maceration, incubation, dissolution, enzymatic hydrolysis, etc., and then poured into a collection chamber. The sample is then tested for the presence of the analyte using a test element. The sample can be obtained from the human body, animals, plants, or nature. Samples obtained from the human body can include, for example, liquid samples such as blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, and gastric juice; solid or semi-solid samples such as feces, hair, keratin, tartar, and nails. Samples obtained from plants can include, for example, solid samples such as roots, stems, and leaves; liquid or semi-solid samples such as tissue fluid and cell fluid prepared from roots, stems, and leaves. Samples obtained from nature can include, for example, liquid samples such as rainwater, river water, seawater, and groundwater; and solid or semi-solid samples such as soil, rock, ore, and petroleum.

[0244] Nucleic Acids

[0245] The term "nucleic acid" includes any compound and / or substance that can be incorporated into an oligonucleotide chain. Exemplary nucleic acids used in accordance with the present application include, but are not limited to, DNA, and RNA includes messenger RNA (mRNA), hybrids thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like.

[0246] As used herein, the term "nucleic acid" includes one or more of the following types: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base or a modified purine or pyrimidine base (including abasic sites). The term "nucleic acid," as used herein, also includes covalently bonded polymers of ribonucleosides or deoxyribonucleosides, typically through phosphodiester bonds between subunits, but in some cases through phosphorothioate, methylphosphonate, and the like. "Nucleic acid" includes single-stranded and double-stranded DNA and single-stranded and double-stranded RNA. Exemplary nucleic acids include, but are not limited to, gDNA; hnRNA; mRNA; rRNA, tRNA, microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and small sequential RNA (stRNA), and any combination thereof.

[0247] microfluidic chip

[0248] Microfluidic chip analysis uses chips as the operating platform, grounded in analytical chemistry, backed by microelectromechanical processing technology, and characterized by microchannel networks. Currently, its primary application targets life sciences. The goal is to integrate the entire laboratory function, including sampling, dilution, reagent addition, reaction, separation, and detection, onto a microchip, allowing for multiple uses. The device's primary characteristic is that its effective fluid-containing structures (channels, reaction chambers, and other functional components) are micrometer-scale in at least one dimension. This micrometer-scale structure enables fluids to exhibit and generate unique properties distinct from those observed at the macroscale. Gene chips and protein chips are simply hybridization chips with zero microfluidic flow rates and very limited functionality. These chips are specialized types of microfluidic chips, which offer a wider range of types, functions, and applications. They enable the development of analytical systems such as biocomputers, gene and protein sequencing, mass spectrometry, and chromatography, forming a crucial technological foundation for systems biology, particularly systems genetics.

[0249] The microfluidic chip provided by the present invention can use materials with low cost, easy processing performance and good biocompatibility chip materials such as PMMA (polymethyl methacrylate), PP (polypropylene), PC (polycarbonate) as the substrate, and directly use laser etching technology to make channels and reaction grooves, or etch structures on the PMMA substrate and then use PDMS mold to make it.

[0250] Nucleic acid multiplex detection microfluidic chip

[0251] The nucleic acid multiple detection microfluidic chip provided by the present invention is composed of any number of detection units, each of which can be used to detect one or more target nucleic acids. Therefore, the chip is mainly used to detect target nucleic acids in samples. In fact, it can be understood that the microfluidic chip provided by the present invention can not only be used for nucleic acid detection, but also can be used for the detection of any other target substances in samples. For example, the reaction chamber can be used for the pretreatment of target substances in samples, and the detection chamber can be used for the detection of target substances in samples.

[0252] In some embodiments, the nucleic acid multiplex detection microfluidic chip provided by the present invention is, for example, a gravity microfluidic chip or a centrifugal microfluidic chip.

[0253] In some cases, a gravity microfluidic chip refers to a chip in which a sample can be moved directly from a sample area to a specific chamber under the action of gravity without providing any external driving force. For example, when used for multiple nucleic acid detection, the sample can be moved directly from a sample area to a reaction chamber under the action of gravity.

[0254] In some methods, a centrifugal microfluidic chip means that the sample needs to be driven by centrifugal force to reach a specific chamber. For example, when the sample is in the sample area, it can enter the reaction chamber under the drive of centrifugal force; when in the reaction chamber, it can enter the detection chamber under the drive of centrifugal force.

[0255] In some embodiments, the centrifugal microfluidic chip can be a chip in which the sample enters the reaction chamber under the action of gravity and then enters the detection chamber under the drive of centrifugal force. In this case, the centrifugal microfluidic chip is also a type of gravity microfluidic chip.

[0256] Nucleic acid typing detection microfluidic chip

[0257] A nucleic acid typing detection microfluidic chip refers to a microfluidic chip that can simultaneously detect multiple genotypes of the same target nucleic acid, or can simultaneously detect multiple target nucleic acids of the same sample.

[0258] The nucleic acid typing detection microfluidic chips provided by the present invention, such as gravity typing chips, asymmetric pressure typing chips, etc., do not require any external force to drive when the liquid flows therein, and can be achieved only by the instantaneous asymmetric pressure generated by screwing or pressing the tube cover itself. The gravity typing chip refers to a chip in which the sample can automatically reach the reaction chamber directly from the liquid storage chamber under gravity drive, but cannot flow out of the reaction chamber from the exhaust channel. The asymmetric pressure typing chip is similar to the gravity typing chip, but the difference is that its tube cover can apply pressure to the chip after the chip is sealed, forming an instantaneous asymmetric pressure, so that the sample can quickly flow into the reaction chamber from the injection channel under the action of the instantaneous asymmetric air pressure drive, but cannot flow out of the reaction chamber from the exhaust channel.

[0259] Nucleic acid multiplex detection

[0260] The nucleic acid multiplex detection described in the present invention means that nucleic acid needs to be detected in two steps. After the target nucleic acid in the sample is amplified, it cannot be directly detected due to unclear signal or other reasons, and a second step of signal amplification is required before detection.

[0261] Therefore, in order to meet the needs of multiple nucleic acid detection, each detection unit in the chip needs to contain a reaction chamber and a detection chamber, so as to be able to control the two steps of nucleic acid detection separately, and must meet the controllable transfer of samples between the reaction chamber and the detection chamber, and prevent cross-influence caused by sample reflux.

[0262] reaction chamber

[0263] The nucleic acid detection microfluidic chip provided by the present invention includes any number of detection units, and each detection unit includes a reaction chamber and a detection chamber.

[0264] When used for multiple nucleic acid detection, when the reaction chamber is used to amplify the target nucleic acid in the sample, a nucleic acid amplification reagent must be pre-placed in the reaction chamber; the nucleic acid amplification reagent can be a liquid reagent or a solid reagent, preferably a solid amplification reagent that is freeze-dried or dried. The nucleic acid amplification reagent is a common variable temperature or isothermal nucleic acid amplification reagent, including but not limited to ERA (Enzymatic Recombinase Amplification), RPA (Recombinase Polymerase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), NASBA (Nuclear acid sequence-based amplification), HDA (helicase-dependent amplification), TMA, SAT, RCR, etc. The form of the solid amplification reagent includes but is not limited to spherical, powdered, sheet or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0265] In some embodiments, the reaction chamber can be used as an amplification chamber or a reaction chamber, such as an ERA amplification chamber, an ERA reaction chamber, an RPA amplification chamber, an RPA reaction chamber, and the like.

[0266] In some methods, the reaction chamber is not limited to amplifying the target nucleic acid in the sample. When the microfluidic chip is used to detect any other target substances in the sample, such as proteins, chemical molecules, etc., the reaction chamber can be pre-set with sample pretreatment reagents, including sample extraction, purification, derivatization, signal amplification and other reagents.

[0267] In some embodiments, the reaction chamber can also be used for sample lysis processing, serving as a lysis reaction chamber, and the detection chamber is used for amplification and detection of target nucleic acids in the sample after lysis, serving as an amplification reaction chamber.

[0268] Detection cavity

[0269] When used for multiple nucleic acid detection, the reaction chamber is used for amplifying the target nucleic acid, and the reaction chamber contains a nucleic acid amplification reagent, while the detection chamber is used for detecting the target nucleic acid, and the detection chamber contains a nucleic acid detection reagent.

[0270] In some methods, when used for multiple nucleic acid detection, detection reagents need to be pre-placed in the detection chamber; the detection reagents can be liquid reagents or solid reagents, preferably solid detection reagents that are freeze-dried or dried, including but not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0271] In some embodiments, the detection reagent includes a detection primer probe and / or a detection premix, and the detection premix includes an enzyme and a buffer required for the detection.

[0272] In some embodiments, the detection reagents include but are not limited to various detection systems established by ERA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, PCR, hybridization probe technology, CRISPR, Ago, RNase H, etc.

[0273] In some embodiments, the detection chamber can be used as a nucleic acid detection chamber, for example, the detection chamber can be used as an ERA detection chamber, a CRISPR detection chamber, an Ago detection chamber, an RNase H detection chamber, and the like.

[0274] In some methods, the detection cavity is not only used for the detection of target nucleic acids in samples. When the microfluidic chip is used to detect any other target substances in the sample, such as proteins, chemical molecules, etc., the detection reagents of the target substances can be pre-set in the detection cavity, including antibodies, antigens, fluorescent markers, chemiluminescent reagents, microspheres and other reagents.

[0275] Fluid Channel

[0276] The reaction chamber and the detection chamber are connected by a fluid channel, which can be used for the circulation of liquid or gas, so that the liquid or gas in the reaction chamber is transferred to the detection chamber, and the liquid or gas in the detection chamber is also transferred to the reaction chamber.

[0277] In some embodiments, the fluid channel can be of any shape, such as a straight line or an arc, and the aperture within the fluid channel can be continuously variable, from wide to thin or from thin to wide, etc., according to actual needs. Alternatively, the aperture of the fluid channel can be changed to a point where liquid cannot pass through, while gas can still flow, depending on actual needs, such as allowing only gas to pass through and not liquid.

[0278] In some methods, for gravity microfluidic chips, the fluid channel needs to act as a valve when connecting the reaction chamber and the detection chamber. The sample cannot enter the detection chamber when entering the reaction chamber, and can only enter the detection chamber under pressure. At this time, the fluid channel needs to be set as a fluid channel with a valve effect as needed, such as reducing the aperture, extending the fluid channel, raising the position of the fluid channel, setting it in an arch shape, setting multiple tortuous structures, etc. to increase the resistance to entering the detection chamber.

[0279] In some methods, for gravity microfluidic chips, it is also possible to consider setting the exhaust channel on the fluid channel, and not setting the exhaust channel in the detection chamber. In this case, the resistance of the sample entering the detection chamber can be increased, thereby achieving controllable transfer of the sample between the reaction chamber and the detection chamber.

[0280] In some approaches, for centrifugal microfluidic chips, the fluid channel can also function as a valve when connecting the reaction chamber and the detection chamber. By placing the exhaust channel on the fluid channel, the detection chamber has no exhaust channel, and the sample cannot enter directly, requiring centrifugal force to enter the detection chamber.

[0281] In some methods, for centrifugal microfluidic chips, the exhaust channel can also be set to have a smaller aperture, an extended fluid channel, an elevated fluid channel position, an arched setting, multiple tortuous structures, etc. to prevent the sample from entering the detection cavity, thereby acting as a valve.

[0282] exhaust duct

[0283] The exhaust channel is used to balance the pressure within the microfluidic chip. For example, it can exhaust the gas in the reaction chamber so that the sample can enter the reaction chamber smoothly, and exhaust the gas in the detection chamber so that the sample can enter the detection chamber. Of course, the exhaust channel is not only for gas flow. In some cases, such as when the sample overflows, it may also enter the exhaust channel.

[0284] In existing nucleic acid detection microfluidic chips, in order to achieve controllable conversion of samples between the reaction chamber and the detection chamber, exhaust channels must usually be set in the reaction chamber and the detection chamber respectively, so that the reaction chamber and the detection chamber can be smoothly exhausted and the sample can flow in smoothly.

[0285] In the microfluidic chip provided by the present invention, the reaction chamber and the detection chamber share an exhaust channel, that is, this exhaust channel can help exhaust both the reaction chamber and the detection chamber, thereby controlling the flow of liquid samples in the reaction chamber and the detection chamber.

[0286] The connection between the exhaust channel and the outside world is covered with a waterproof and breathable membrane. This membrane blocks the passage of liquids but not gases, thus enabling exhaust. However, if the membrane gets wet, it blocks the vent holes, rendering it unable to vent, turning the microfluidic chip into a fully enclosed system, making exhaust impossible and the liquid stagnating within the system.

[0287] In some embodiments, this shared exhaust channel can be located anywhere within the detection unit, as long as it facilitates smooth exhaust of the reaction and detection chambers. Of course, the location of the exhaust channel directly affects the effectiveness of the controlled transfer of the sample between the reaction and detection chambers. For example, the distance between the exhaust channel and the detection chamber is directly related to the resistance to sample entry into the detection chamber. Only when the exhaust channel is connected to the top of the detection chamber, when liquid flows from the reaction chamber to the detection chamber and enters the detection chamber from the bottom, the exhaust direction is also from the bottom of the detection chamber to the top of the detection chamber and then discharged out of the exhaust channel. The exhaust direction is consistent with the liquid flow direction, thus minimizing the resistance to the liquid sample entering the detection chamber. However, when the exhaust channel is connected to other parts of the detection chamber, or is not connected to the detection chamber but to the fluid channel, the exhausted gas will be discharged from the bottom of the detection chamber to the top, then turn back and pass through the liquid before reaching the exhaust channel. In other words, part of the exhaust process is opposite to the direction of liquid flow. This process of turning back and passing through the liquid significantly increases the resistance to sample entry into the detection chamber. Therefore, changing the location of the exhaust channel can directly affect the effectiveness of the controlled transfer of the sample between the reaction and detection chambers. For example, if the exhaust channel is too close to the reaction chamber and too far away from the detection chamber, it will significantly increase the difficulty of transferring the sample into the detection chamber. For another example, if the exhaust channel is set directly on the reaction chamber or the injection channel, not only will it be too difficult to transfer the sample into the detection chamber, but the sample will also enter the exhaust channel prematurely after entering the reaction chamber and wet the waterproof breathable membrane, making it no longer breathable. The interior of the microfluidic chip is completely closed, and the liquid can no longer flow. Therefore, the location of the exhaust channel shared by the reaction chamber and the detection chamber is very critical. It must be kept ventilated as much as possible when the sample enters the reaction chamber, and it must not be closed. Even if it is closed during the process, ventilation can be quickly restored.

[0288] In some embodiments, for a centrifugal microfluidic chip, an exhaust channel can be provided in the fluid channel, slightly away from the detection chamber. This is because even if the distance from the detection chamber makes it more difficult for the sample to enter the detection chamber, controlling the centrifugal force can still provide greater power, thereby prompting the sample to smoothly enter the detection chamber from the reaction chamber. On the contrary, if the exhaust channel is too close to the detection chamber, or is directly located on the detection chamber, it is impossible to distinguish the force required for the sample to enter the reaction chamber or the detection chamber by controlling the centrifugal speed, resulting in failure of the controllable transfer of the sample between the reaction chamber and the detection chamber. Therefore, in the present invention, the exhaust channel of the centrifugal microfluidic chip is preferably provided in the fluid channel, and can be located near the reaction chamber or the detection chamber on the fluid channel, or in the middle, and can be adjusted arbitrarily according to needs.

[0289] In some approaches, for gravity-based microfluidic chips, the exhaust channel can be directly located within the detection chamber. Consequently, the fluid channels within the chip have a smaller aperture and are more circuitous, providing sufficient resistance to entry into the detection chamber. Furthermore, the exhaust channel's location within the detection chamber makes it easier to control sample flow, preventing the system from closing due to sample entry. Once the sample enters the detection chamber, it flows into the exhaust channel, sealing the system and preventing it from exiting the detection chamber. This facilitates controlled sample transfer between the reaction chamber and the detection chamber.

[0290] Combination of fluid channels and exhaust channels

[0291] The combination of the fluid channel and the exhaust channel described in the present invention refers to the combination of the design of various characteristics of the fluid channel (quantity, length, shape, position height, aperture, etc.) and the design of various characteristics of the exhaust channel (quantity, distance from the detection cavity, position, design of the exhaust port, orientation of the exhaust channel, aperture, etc.).

[0292] In some embodiments, the aperture of the fluid channel is smaller than the aperture of the sample inlet channel, and the exhaust channel is connected to the fluid channel. In some embodiments, the exhaust channel can be connected to any position in the fluid channel, such as the middle of the fluid channel, a position in the fluid channel close to the reaction chamber, or a position in the fluid channel closer to the detection chamber.

[0293] In some embodiments, the exhaust channel can also be directly connected to the fluid channel. In this case, in order to make the sample enter the detection chamber from the reaction chamber, it is necessary to provide greater power or pressure, or even an additional external driving force. It is understandable that the exhaust channel can be set at any position in the fluid channel, but setting it at different positions in the fluid channel will also affect the resistance of the sample entering the detection chamber. For example, when the exhaust channel is set in the fluid channel, if it is too close to the reaction chamber or too far away from the detection chamber, the difficulty of transferring the sample into the detection chamber will be significantly increased, and it may even be difficult to enter the detection chamber. If the exhaust channel is too close to the detection chamber, or is directly set on the detection chamber, the resistance of the sample entering the detection chamber will be reduced, and the sample may also enter the detection chamber when entering the reaction chamber, making it difficult to achieve controllable transfer of the sample between the reaction chamber and the detection chamber. Therefore, the specific position of the exhaust channel can be selected according to the actual product situation and the size of the force that can be provided by the pressure-applying device of the chip or the centrifugal force.

[0294] In some embodiments, the fluid channel is a tortuous channel, and the exhaust channel is connected to the fluid channel.

[0295] In some embodiments, the fluid channel is a spiral flow channel, and the exhaust channel is connected to the detection chamber. In some embodiments, the exhaust channel can be connected to any position of the detection chamber, such as the top of the detection chamber, the bottom of the detection chamber, or the side of the detection chamber.

[0296] In some embodiments, the fluid channel is a multi-fold channel, the exhaust channel is connected to the fluid channel, and the aperture of the fluid channel is smaller than the aperture of the injection channel, and the exhaust channel outlet is connected to the main channel.

[0297] In some embodiments, the fluid channel is a tortuous flow channel, including multiple curved structures, similar to the shape of the "Great Wall". The exhaust channel is connected to the fluid channel, and the aperture of the fluid channel is smaller than the aperture of the injection channel. The outlet of the exhaust channel is connected to the main channel.

[0298] In some embodiments, the fluid channel is a tortuous channel including multiple bends and turns, the exhaust channel is connected to the top of the detection chamber, and the aperture of the fluid channel is smaller than the aperture of the injection channel (gravity microfluidic chip).

[0299] In some embodiments, the exhaust channel is connected to the fluid channel, and the outlet of the exhaust channel is connected to the main flow channel or the external atmosphere (centrifugal microfluidic chip).

[0300] Controllable transfer between reaction chamber and detection chamber

[0301] The controllable transfer between the reaction chamber and the detection chamber described in the present invention means that the sample can enter the reaction chamber according to a pre-conceived plan. When the sample is expected to flow out of the reaction chamber, the sample will flow out of the reaction chamber smoothly, enter the detection chamber through the fluid channel, and remain in the detection chamber to complete the detection reaction until the final successful detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0302] FIG1 is a back view of the gravity microfluidic chip in Example 1;

[0303] FIG2 is a front view of the gravity microfluidic chip in Example 1;

[0304] FIG3 is a perspective view of the gravity microfluidic chip in Example 1 (with the back side facing upward);

[0305] FIG4 is a perspective view of the gravity microfluidic chip in Example 1;

[0306] FIG5 is a schematic structural diagram of a detection unit of the gravity microfluidic chip in Example 1;

[0307] FIG6 is a back view of the simple gravity microfluidic chip in Example 2;

[0308] FIG7 is a front view of the simple gravity microfluidic chip in Example 2;

[0309] FIG8 is a perspective view of the simple gravity microfluidic chip in Example 2;

[0310] FIG9 is a front view of the centrifugal microfluidic chip in Example 4 (the first exhaust channel, including the core plate body and the sub-chip);

[0311] FIG10 is a back view of the centrifugal microfluidic chip in Example 4 (the first exhaust channel, including the core plate body and the sub-chip);

[0312] FIG11 is a perspective view of the centrifugal microfluidic chip in Example 4 (the first exhaust channel, including the core plate body and the sub-chip);

[0313] FIG12 is a schematic diagram of the structure of the detection unit in the centrifugal microfluidic chip in Example 4 (the first exhaust channel);

[0314] FIG13 is a front view of the centrifugal microfluidic chip in Example 5 (the first exhaust channel, without the sub-chip);

[0315] FIG14 is a back view of the centrifugal microfluidic chip in Example 5 (the first exhaust channel, without a sub-chip);

[0316] FIG15 is a perspective view of the centrifugal microfluidic chip in Example 5 (the first exhaust channel, without a sub-chip);

[0317] FIG16 is a schematic diagram of the structure of the detection unit in the centrifugal microfluidic chip in Example 5 (the first exhaust channel, without a sub-chip);

[0318] FIG17 is a front view of the centrifugal microfluidic chip in Example 6 (second exhaust channel);

[0319] FIG18 is a back view of the centrifugal microfluidic chip in Example 6 (second exhaust channel);

[0320] FIG19 is a perspective view of the centrifugal microfluidic chip in Example 6 (the second exhaust channel);

[0321] FIG20 is a schematic diagram of the structure of the detection unit in the centrifugal microfluidic chip in Example 6 (the second exhaust channel).

[0322] FIG21 is a front view of the gravity typing chip with four reaction chambers in Example 9;

[0323] FIG22 is a back view of the gravity-partitioning chip with four reaction chambers in Example 9;

[0324] FIG23 is a top view of the gravity-partitioning chip with four reaction chambers in Example 9;

[0325] FIG24 is a schematic diagram of the structure of the sample loading area of ​​the gravity typing chip with four reaction chambers in Example 9;

[0326] FIG25 is a schematic cross-sectional view of the sample loading area of ​​the gravity typing chip with four reaction chambers in Example 9;

[0327] FIG26 is a schematic diagram of the detection area structure of the gravity typing chip with four reaction chambers in Example 9;

[0328] FIG27 is a structural diagram of a gravity typing chip with six reaction chambers arranged in a radioactive manner in Example 9;

[0329] FIG28 is a structural diagram of a gravity typing chip with four reaction chambers arranged in a radial manner in Example 9;

[0330] FIG29 is a structural diagram of a gravity typing chip with four reaction chambers arranged horizontally in Example 9.

[0331] FIG30 is a diagram showing the tube cover structure of the asymmetric pressure parting chip in Example 13;

[0332] FIG31 is a cross-sectional structural diagram of the tube cover of the asymmetric pressure parting chip in Example 13;

[0333] FIG32 is a front view of the asymmetric pressure splitting chip with four reaction chambers in Example 13;

[0334] FIG33 is a back view of the asymmetric pressure-partitioning chip with four reaction chambers in Example 13;

[0335] FIG34 is a top view of the asymmetric pressure-partitioning chip with four reaction chambers in Example 13;

[0336] FIG35 is a schematic diagram of the sample loading area structure of the asymmetric pressure typing chip with four reaction chambers in Example 13;

[0337] FIG36 is a schematic cross-sectional view of the sample loading area of ​​the asymmetric pressure typing chip with four reaction chambers in Example 13;

[0338] FIG37 is a schematic diagram of the detection area structure of the asymmetric pressure typing chip with four reaction chambers in Example 13;

[0339] FIG38 is a structural diagram of the asymmetric pressure typing chip with six reaction chambers arranged radially in Example 13;

[0340] FIG39 is a structural diagram of an asymmetric pressure-type chip with four reaction chambers arranged radially in Example 13;

[0341] FIG40 is a structural diagram of an asymmetric pressure typing chip with four reaction chambers arranged laterally in Example 13. DETAILED DESCRIPTION

[0342] The preferred embodiments of the present invention are described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and do not limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0343] Example 1: Structure of gravity microfluidic chip

[0344] The structure of the nucleic acid multiple detection microfluidic chip provided in this embodiment is shown in Figures 1 to 5, wherein Figure 1 is a back view of the nucleic acid multiple detection microfluidic chip, and Figure 2 is a front view of the nucleic acid multiple detection microfluidic chip; Figure 3 is a three-dimensional view of the nucleic acid multiple detection microfluidic chip (back facing up); Figure 4 is a three-dimensional view of the nucleic acid multiple detection microfluidic chip; and Figure 5 is a schematic diagram of the detection unit structure.

[0345] As shown in Figures 1 to 4, the nucleic acid multiplex detection microfluidic chip 28 (hereinafter referred to as chip 28) provided in this embodiment includes a sample loading area 1 and a detection area 2. The sample loading area 1 includes a sample inlet 4, a sample reservoir 3, and a tube cap (not shown). The sample reservoir 3 is used to hold the sample to be tested. When the sample is added to the chip 28, it first enters the sample reservoir 3, then enters the main flow channel 6 under the action of gravity, and then flows into the detection area 2.

[0346] The detection area 2 can be provided with any number of groups of detection units 7 according to the needs of the project, so as to realize the simultaneous detection of multiple targets. In this embodiment, ten groups of detection units 7 are preferably provided, which are arranged in two columns, and each column is provided with five groups of detection units 7. Each group of detection units 7 includes a reaction chamber 8 and a detection chamber 9 connected to the reaction chamber 8. The reaction chamber 8 is connected to the main channel 6 through the injection channel 17, and the reaction chamber 8 and the detection chamber 9 are fluidically connected through a fluid channel 19. The aperture of the fluid channel 19 is smaller than the injection channel 17; under the action of gravity, the sample can flow from the sample addition area 1 into the reaction chamber 8, but will not flow into the fluid channel 19.

[0347] During use, the chip 28 needs to be kept in a vertical state with the injection port 4 facing upward. When the positional relationship of the various flow channels and chambers in the chip 28 is involved in this embodiment, the description is based on the case where the chip 28 is kept in a vertical state. Since the air pressure is balanced in the system, the sample in the sample adding area 1 does not require an external driving device and can be autonomously transferred to the reaction chamber 8 only under gravity drive, and this process is easy to implement. Once the sample enters the sample adding area 1, it will flow downward by itself and enter the reaction chamber 8 through the injection channel 17. The main channel 6 of the chip 28 and the injection channel 17 of the reaction chamber 8 have a large aperture, which can enable the liquid to flow smoothly under gravity drive. However, the aperture of the fluid channel 19 is significantly smaller than these channels. The fluid channel 19 is a multi-turn, circuitous channel with a relatively small aperture. Under the action of gravity alone, liquid cannot enter and flow smoothly. As a result, after the sample fills the reaction chamber 8, it will be blocked when continuing to advance, and it will be difficult to flow out of the fluid channel 19 at the top 10 of the reaction chamber. All of it will remain in the reaction chamber 8 to complete the nucleic acid amplification reaction. After the nucleic acid amplification reaction is completed, it can only enter the detection chamber 9 through the fluid channel 19 under pressure, thereby ensuring accurate detection. The channel width of the main channel 6 and the multiple sample injection channels 17 in the chip 28 is 400μm to 800μm. In this embodiment, the main channel width is preferably 400-600μm, and the depth is 400-600μm. The channel width of the fluid channel is 50-300μm. In this embodiment, the width of the fluid channel is preferably 100μm.

[0348] The detection chamber 9 is provided with an exhaust channel 11, and a waterproof breathable membrane 13 is provided at the outlet 12 of the exhaust channel 11 to control the connection between the detection chamber 9 and the outside atmosphere; the reaction chamber 8 is not provided with an exhaust channel 11. The exhaust channel 11 and the waterproof breathable membrane 13 of the detection chamber 9 can discharge the gas in the reaction chamber 8 and the detection chamber 9 to the outside through the waterproof breathable membrane 13, but the liquid cannot be discharged through the waterproof breathable membrane 13. When the exhaust channel 11 is filled with liquid, the waterproof breathable membrane 13 loses its breathability, causing the entire system to be in a closed state and the liquid in the system to stop flowing. Therefore, the waterproof breathable membrane 13 can help the sample in the detection chamber 9 establish internal and external pressure balance and can regulate the direction of liquid flow. When the detection chamber 9 is filled with liquid and the waterproof breathable membrane 13 is wetted by the exhaust channel 11, the system in the chip 28 is in a closed state, thereby stopping the sample in the detection chamber 9 from flowing and preventing backflow. The waterproof, breathable membrane is made of a polymer material, such as high-grade synthetic rubber, which offers excellent sealing properties, is free of adhesive and residue, and does not react with reaction or detection reagents. In this embodiment, the waterproof, breathable membrane is preferably made of material (Manufacturer: Membrane; Part Number: VET022H60). Chip 28 can also be covered with a plastic sheet 42 in place of the waterproof, breathable membrane. A gap exists between plastic sheet 42 and chip 28, allowing the waterproof, breathable membrane 13 to communicate with the outside air while preventing it from falling off, thus protecting the membrane 13.

[0349] In chip 28, reaction chamber 8 does not require an exhaust channel. Instead, the multi-turn, narrow-aperture fluid channel 19 acts as a valve to temporarily block the flow of liquid between reaction chamber 8 and detection chamber 9. However, ventilation between the two chambers is always maintained. Therefore, both chambers 8 and 9 can share exhaust channel 11 located on detection chamber 9 for exhaust, eliminating the need for a separate exhaust channel 11 and waterproof, breathable membrane 13 in reaction chamber 8. This design simplifies the structure, reduces costs, and facilitates operation, while also ensuring the accuracy of test results.

[0350] As shown in Figure 5, the fluid channel 19 includes an injection section 14, a middle section 15, and a sample outlet section 16. The middle section 15 is arched upward, and the position of the middle section 15 is higher than the injection section 14 and the sample outlet section 16. The middle section 15 of the fluid channel 19 is arched upward, which increases the difficulty for the sample in the reaction chamber 8 to enter the fluid channel 19 without additional pressure, and helps to keep all the samples in the reaction chamber 8 in the reaction chamber 8 to complete the nucleic acid amplification reaction. The fluid channel 19 is located between the reaction chamber 8 and the detection chamber 9, and the middle section 15 of the fluid channel 19 extends toward the upper end 18 of the reaction chamber 8, extending the length of the fluid channel 19, and increasing the difficulty for the sample in the reaction chamber 8 to enter the fluid channel 19 without additional pressure. The fluid channel 19 is a multi-turn circuitous flow channel with at least one bend-shaped structure 21. The multiple curved structures 21 provided in fluid channel 19 maximize the length of fluid channel 19, making it difficult for the sample in reaction chamber 8 to enter fluid channel 19 without additional pressure, while ensuring that the sample in reaction chamber 8 remains completely within reaction chamber 8. The inlet 22 of fluid channel 19 is located at the top 10 of the reaction chamber; the outlet 23 of injection channel 17 is located at the bottom 24 of the reaction chamber; the outlet 25 of fluid channel 19 is located at the bottom 26 of the detection chamber; and the inlet 27 of exhaust channel 20 is located at the top 29 of the detection chamber.

[0351] The outlet 23 of the sample inlet channel 17 corresponds to the sample inlet 30 of the reaction chamber 8, and the inlet 22 of the fluid channel 19 corresponds to the sample outlet 31 of the reaction chamber 8. The sample inlet 30 is located at the bottom of the reaction chamber 8, and the sample outlet 31 is located at the top 10 of the reaction chamber. This is done to ensure that the sample can be smoothly exhausted to the sample outlet 31 when entering the reaction chamber 8, maintaining air pressure balance. At the same time, since the sample outlet 31 is located at the top of the reaction chamber 8, it is more difficult for the sample to flow out, allowing the sample to remain more stably in the reaction chamber 8. The outlet 25 of the fluid channel 19 corresponds to the sample inlet 32 ​​of the detection chamber 9, and the inlet 27 of the exhaust channel 11 corresponds to the exhaust outlet 33 of the detection chamber 9. The sample inlet 32 ​​is located at the bottom 26 of the detection chamber 9, and the exhaust outlet 33 is located at the top 29 of the detection chamber 9. This is done to ensure that the sample can be smoothly exhausted to the exhaust outlet 33 when entering the detection chamber 9, maintaining air pressure balance. Under the pressure of the pressure zone 34, the sample in the reaction chamber 8 enters the detection chamber 9 from the bottom 26 of the detection chamber through the fluid channel 19. Since the sample volume is usually relatively full, the sample can also enter the exhaust channel 11 and contact the waterproof breathable membrane 13, causing the waterproof breathable membrane 13 to lose its breathability, making the entire chip 28 in a closed state, and the sample stops flowing, thereby being completely retained in the detection chamber 9 for detection reaction and completing the detection of the target nucleic acid.

[0352] The detection chamber 9 is positioned higher than the reaction chamber 8, and the bottom 26 of the detection chamber 9 is not lower than the top 10 of the reaction chamber 8, which increases the difficulty for the sample in the reaction chamber 8 to enter the detection chamber 9 without additional pressure, and helps prevent the sample in the reaction chamber 8 from flowing into the detection chamber 9 prematurely.

[0353] As shown in Figure 5, the sample inlet channel 17 of the chip 28 is in fluid communication with the branch port 35 of the main channel 6. The sample inlet channel 17 is an upwardly arched curved channel 36. The inlet 37 and outlet 38 of the sample inlet channel 17 are located at the lowest points on either side of the curved channel 36, with the inlet 37 being higher than the outlet 38. The highest point 39 of the curved channel 36 is no lower than the top 10 of the reaction chamber. The curved channel design of the sample inlet channel 17 allows the sample to enter the sample inlet channel 17 more easily from the branch port 35 and then enter the reaction chamber 8 through the sample inlet channel 17. However, after entering the reaction chamber 8, the sample has difficulty returning from the sample inlet channel 17 to the main channel 6 because the sample inlet 30 of the reaction chamber 8 is located at the lowest point of the sample inlet channel 17. The sample cannot overcome the effects of gravity and the pressure caused by the upwardly arched curved channel 36 to return to the main channel 6. Instead, the sample remains entirely in the reaction chamber 8, thereby preventing sample backflow within the reaction chamber 8.

[0354] After a specific volume of the sample to be tested is added from the sample adding area 1, the tube cover of the sample adding area 1 is covered, and the sample enters the reaction chamber 8 of each detection unit 7 from the main channel 6 under the action of gravity. The sample inlet 30 of the reaction chamber 8 is at a low position, which can make the sample to be tested flow into the reaction chamber 8 quickly and gradually fill it, but it will not flow out of the reaction chamber 8 from the sample outlet 31 at this time, because the sample outlet 31 is at the top 10 of the reaction chamber 8, and the fluid channel 19 is a multi-turn, small-aperture flow channel, the sample cannot flow into the fluid channel 19, and it is also difficult for the sample to return to the main channel 5 from the sample inlet channel 17, because the sample inlet 30 of the reaction chamber 8 is located at the lowest point of the sample inlet channel 17, and the highest point of the sample inlet channel 17 is higher than the top 10 of the reaction chamber 8. The sample cannot return to the main channel 6 under the action of gravity, so the sample will all remain stably in the reaction chamber 8 for reaction.

[0355] As shown in Figure 2, the pressure area 34 of the chip 28 is located on one side of the sample addition area 1, and is used to increase the pressure of the liquid in the reaction chamber 8, so that the liquid in the reaction chamber 8 flows into the detection chamber 8 through the fluid channel 19. The purpose of setting the pressure area 34 is that after the sample completes the amplification reaction and other reactions in the reaction chamber 8, the sample needs to enter the detection chamber 9 from the reaction chamber 8. At this time, it is necessary to overcome a certain air pressure and the pressure brought by the multi-turn and small-aperture fluid channel 19. The pressure area 34 needs to be set at the upper left or upper right of the chip 28, and it should also reach a specific volume (specifically, it can be calculated based on the size of the chip 28 and the required pressure, so as to set an airbag 40 of a suitable volume), so that the pressing process provides more accurate airflow pressure to drive the sample to be tested from the reaction chamber 8 into the detection chamber 9. Pressing the airbag 40 in the pressure area 35 can be done manually, or by using a matching detection device to provide a specific force to press the airbag 40. The pressure area 34 can be connected to the sample pool 3 through the branch channel 41 , and by pressing, the air flow is discharged from the branch channel 41 to the sample pool 3 and into the interior of the chip 28 .

[0356] As shown in Figure 5, the cross-section of the reaction chamber 8 is circular, and the circular cross-section setting allows the reaction chamber 8 to accommodate more samples. The cross-section of the detection chamber 9 is teardrop-shaped, and the exhaust port 33 is located at the top of the teardrop. The teardrop-shaped cross-section of the detection chamber 9 helps the gas or liquid in the detection chamber 9 to converge upward, making it easier to enter the exhaust channel 11. The solid reagents built into the reaction chamber 8 and the detection chamber 9 can be flexibly set as needed. Different chambers can be equipped with different reaction reagents, such as the reaction chamber 8 has built-in amplification primers for the target, and the detection chamber 9 has built-in detection probes for different targets.

[0357] A nucleic acid amplification reagent needs to be placed in advance in the reaction chamber 8. The nucleic acid amplification reagent is a solid amplification reagent that is freeze-dried or dried. The nucleic acid amplification reagent is a common variable temperature or isothermal nucleic acid amplification reagent, including but not limited to ERA (Enzymatic Recombinase Amplification), RPA (Recombinase Polymerase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), NASBA (Nuclear acid sequence-based amplification), HDA (helicase-dependent amplification), etc.

[0358] The detection chamber 9 is used for fluorescence detection reaction between the amplified sample and the detection reagent, and the detection reagent includes but is not limited to ERA (Enzymatic Recombinase Amplification), RPA (Recombinase Polymerase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), and various probe detection systems based on Ago, RNase H, etc. The solid detection reagent specifically includes a detection primer probe and / or a detection premix, and the detection premix includes the enzyme and buffer required for detection.

[0359] The main structure of the chip 28 provided in this embodiment uses a chip material with low cost, easy processing performance and good biocompatibility, such as PMMA (polymethyl methacrylate), PP (polypropylene), PC (polycarbonate), etc. as the substrate, and the channels and reaction grooves are directly made by laser etching technology, or the structure is etched on the PMMA substrate and then molded with PDMS.

[0360] The chip 28 provided in this embodiment cleverly designs the structures of the sample inlet channel, fluid channel, chamber and exhaust channel. Without the need for external drive, the sample can use its own gravity to enter the reaction chamber 8 and will not enter the detection chamber 9. After the amplification reaction in the reaction chamber 8 is completed, it can enter the detection chamber 9 under the action of a simple pressure device 34, thereby realizing the controllable transfer of samples between different chambers without the occurrence of sample reflux.

[0361] Example 2: Simple gravity microfluidic chip

[0362] This embodiment provides a simplified version of a nucleic acid multiplex detection microfluidic chip, as shown in Figures 6 to 8, wherein Figure 6 is a back view of the simplified nucleic acid multiplex detection microfluidic chip, Figure 7 is a front view of the simplified nucleic acid multiplex detection microfluidic chip, and Figure 8 is a stereoscopic view of the simplified nucleic acid multiplex detection microfluidic chip.

[0363] The difference between the simple nucleic acid multiple detection microfluidic chip provided in this embodiment and the nucleic acid multiple detection microfluidic chip in Example 1 is that only a single row of detection units 7 is designed, and the detection units 7 are placed vertically, the reaction chamber 8 is located below, and the detection chamber 9 is located directly above the reaction chamber 8, and a fluid channel 19 is set between the reaction chamber 8 and the detection chamber 9; the sample pool 3 is located on one side of the detection unit 7. After the sample enters the sample pool, it will directly flow into the reaction chamber 8 under the action of gravity. Due to the small aperture of the fluid channel 19 and the circuitous multi-turn design, the sample cannot enter the reaction chamber 8 under the action of gravity. After the amplification reaction is completed, the sample is squeezed through the pressure area 34 (Figure 6) located on the side of the sample addition area 1 to allow the sample to smoothly enter the detection chamber 9 and enter the exhaust channel 11 of the detection chamber 9, contacting the waterproof and breathable membrane 13, thereby closing the entire system, stopping the sample flow, and completing the detection in the detection chamber 9.

[0364] Example 3: Method for using gravity microfluidic chip

[0365] The nucleic acid multiplex detection microfluidic chip 28 provided in Example 1 or Example 2 can be applied to a variety of nucleic acid detection methods. This embodiment uses multiplex detection based on enzymatic isothermal amplification technology as an example to illustrate the use of the chip 28:

[0366] 1) The chip 28 is vertically inserted into the detection device;

[0367] 2) Transfer: The sample to be tested containing the lysate is introduced into the chip 28 from the injection port 4. The sample flows into the reaction chamber 8 under the action of gravity. The cap is tightened to seal the chip.

[0368] 3) The sample to be tested undergoes nucleic acid amplification in the reaction chamber 8;

[0369] 4) After the nucleic acid amplification reaction is completed, the pressure area 34 is squeezed to allow the sample to be tested after the nucleic acid amplification to enter the detection chamber 9 for reaction;

[0370] 3) Read the results: After the reaction is completed, the fluorescence signal is read by the device to obtain the test results.

[0371] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. 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.

[0372] Example 4: Centrifugal microfluidic chip with the first configuration for the exhaust channel

[0373] The structure of the centrifugal microfluidic chip for multiple nucleic acid detection provided in this embodiment is shown in Figures 9 to 12, wherein Figure 9 is a front view of the centrifugal microfluidic chip (the first exhaust channel), and Figure 10 is a back view of the centrifugal microfluidic chip (the first exhaust channel); Figure 11 is a three-dimensional view of the centrifugal microfluidic chip (the first exhaust channel); and Figure 12 is a schematic diagram of the detection unit structure in the centrifugal microfluidic chip (the first exhaust channel).

[0374] As shown in Figures 9 to 11, the centrifugal microfluidic chip 43 for multiple nucleic acid detection provided in this embodiment includes a chip body 45 with a rotation center 44, and a sub-chip 69 arranged on the outside of the chip body 45. Multiple groups of detection units 8 can be set on the chip body 45 and the sub-chip 69 according to project needs or according to the size of the chip, so as to realize the simultaneous detection of multiple targets. Each group of detection units 50 is provided with a reaction chamber 46 and a detection chamber 47. The reaction chamber 46 is used for sample nucleic acid amplification, and the detection chamber 47 is used for sample detection. Therefore, each group of detection units can realize multiple nucleic acid detection. When the chip body 45 is centrifuged, the sub-chip 69 can be centrifuged along with the chip body 45. The chip body 45 and the sub-chip 69 are both provided with a main channel 58, and the main channel 58 is also provided with a sample port 59 (including the first sample port 171 in the chip body 45 and the second sample port 172 in the sub-chip 27). The main channel 58 is a connected circular closed loop, and the sample port 59 is an opening for sampling provided in the circular closed loop of the main channel 58. The main channel 58 is arranged in a wave-like manner and bent into a circular ring. The chip body 45, the circular ring and the rotation center 44 are coaxially arranged. The reaction chamber 46 and the detection chamber 47 are connected by a fluid channel 48. The fluid channel 48 is also provided with an exhaust channel 49, which is drawn out from a position approximately in the middle of the fluid channel 48. The exhaust channel 49 is used to control the flow of gas in the reaction chamber 46 or the detection chamber 47; the rotation center 44 is used to connect to the centrifugal drive device, thereby driving the centrifugal microfluidic chip 43 to centrifuge.

[0375] During use, the centrifugal microfluidic chip 43 needs to be kept in a horizontal state and placed in the matching detection device (centrifugal drive device), with the sample port 51 placed horizontally facing upward, and the rotation center 44 is fastened and fixed to the rotating shaft used for centrifugation of the detection device, so that the rotating shaft can drive the centrifugal microfluidic chip 43 to rotate centrifugally; at the same time, the detection device can also provide a heat source for the reaction chamber 46 of the centrifugal microfluidic chip 43 for heating; the detection device can also detect the fluorescent substance produced after the sample reaction in the centrifugal microfluidic chip 43 and read the test results.

[0376] The chip body 45 is circular, and the sub-chip 69 is located on the concentric circle outside the chip body 45. The chip body 45 and the sub-chip 69 can be directly integrally formed and cannot be disassembled. Of course, if necessary, sub-chips can be further arranged on the outside of the sub-chip 69. The sub-chip 69, the chip body 45 and the entire microfluidic chip 43 are all in the same plane. The sub-chip 69 is located outside the chip body 45 and has a larger diameter. More groups of detection units 50 can be set up. The sub-chip 69 and the chip body 45 can independently perform multiple nucleic acid detections, and can detect samples different from the chip body 45, realize the simultaneous detection of multiple samples, and can detect more indicators at the same time (each detection unit can detect a nucleic acid target), significantly increasing the detection flux and improving the detection efficiency. The number of sub-chips 69 and the number of detection units 50 in each sub-chip 69 or chip body 45 can be flexibly set according to the number of samples to be tested and the index detection requirements, and the solid reagents built into the reaction chamber 46 and the detection chamber 47 of each detection unit 50 can be flexibly set according to actual needs. For example, amplification primers for different respiratory targets can be built into different reaction chambers 46, and corresponding detection reagents can be built into the corresponding detection chambers 47, so that multiple detection of respiratory pathogens can be flexibly achieved.

[0377] As shown in Figures 9 to 12, an exhaust channel 49 is provided on the fluid channel 48 connecting the reaction chamber 46 and the detection chamber 47 in each detection unit 50 of the centrifugal microfluidic chip 43, so that the reaction chamber 46 and the detection chamber 47 share the same exhaust channel 49. The fluid channel 48 is a channel that connects the outlet 51 of the reaction chamber 46 with the inlet 52 of the detection chamber 47. The exhaust channel 49 includes an inlet 53 and an outlet 54. The inlet 53 is the connection point between the fluid channel 48 and the exhaust channel 49, and the outlet 54 is the other end of the exhaust channel 49. The outlet 54 is closer to the rotation center 44 than the inlet 53, so that the exhaust direction of the exhaust channel 49 is opposite, or at least partially opposite, to the centrifugal direction of rotation. When the centrifugal microfluidic chip 43 is centrifuged, the flow direction of the fluid sample under the action of centrifugation is emitted from the center of the circle to the outside. At this time, since the exhaust channel 49 is led out from the fluid channel 48 between the reaction chamber 46 and the detection chamber 47 and approaches the rotation center 44, the exhaust direction is opposite to the centrifugal direction. It is difficult for the fluid sample to enter the exhaust channel 49 during centrifugation, but it enters the corresponding chamber accurately along the centrifugal direction.

[0378] Preferably, as shown in Figure 12, when the centrifugal microfluidic chip 43 is placed horizontally, the outlet 54 of the exhaust channel 49 is higher than the inlet 53. Such a design can further increase the difficulty of the fluid sample entering the exhaust channel 49, thereby helping to control the flow of liquid in the chip and preventing the sample from entering the exhaust channel 49. At the same time, it can also facilitate the discharge of gas from the reaction chamber 46 and the detection chamber 47, so that the centrifugal microfluidic chip 43 can perform multiple nucleic acid detection more accurately.

[0379] As shown in Figure 12, exhaust channel 49 extends from the middle of the fluid channel or toward the detection chamber 47. The initial section of the exhaust channel 49 is an arcuate channel 55. The angle 56 between the arcuate channel 55 and the fluid channel near the reaction chamber 46 is less than 90 degrees. Exhaust channel 49 can be slightly offset toward the detection chamber 47. This offset reduces the pressure required for the sample to enter the detection chamber 47 during centrifugation, making it easier for the sample to enter the detection chamber 47 during centrifugation. When exhaust channel 49 extends from fluid channel 48, the smaller the angle 56 between the initial section and the fluid channel near the reaction chamber 46, the less likely the fluid will enter exhaust channel 49. However, this angle 56 should not be too small, as this may lead to exhaust difficulties. Preferably, an arcuate channel 55 is used as a transition point, and the angle between the arcuate channel 55 and the fluid channel near the reaction chamber 46 is less than 90 degrees. Preferably, the inner diameter of exhaust channel 49 is smaller than the inner diameter of fluid channel 48, further preventing the sample from entering exhaust channel 49 during centrifugation.

[0380] As shown in Figure 12, the outlet 54 of the exhaust channel 49 is connected to the outside world, and the outlet 54 connected to the outside world is covered with a waterproof breathable membrane 57. The waterproof breathable membrane 57 is a membrane that is breathable but not water-permeable. It can achieve the discharge of gas from the reaction chamber 46 or the detection chamber 47, but liquid cannot pass through. The waterproof breathable membrane 57 can help the sample in the reaction chamber 46 or the detection chamber 47 to establish internal and external pressure balance, and can regulate the direction of liquid flow so that the sample in the reaction chamber 46 or the detection chamber 47 will not leak or flow back. The waterproof breathable membrane 57 can be made of polymer materials, such as high-grade synthetic glue, with good sealing properties, no adhesive backing and residue, and does not react with reaction reagents or detection reagents. In this embodiment, the waterproof breathable membrane is preferably made of material (manufacturer: Membrane; Article No.: VET022H60). When this setting is adopted, the centrifugal microfluidic chip 43 only needs one centrifugation to complete multiple nucleic acid detection. Preferably, a ventilation cavity 72 is further provided at the outlet 54 of the exhaust channel 49 . The volume of the ventilation cavity 72 is not less than that of the reaction chamber 46 and is used to help exhaust the reaction chamber 46 so that the sample can enter the reaction chamber 46 more easily.

[0381] The flow process of the sample to be tested in the centrifugal microfluidic chip 43 mainly includes two steps: the first step is that the sample to be tested enters the reaction chamber 46 through the main channel 58 under the action of injection pressure for reaction; the second step is that after the reaction is completed, the sample to be tested flows out of the reaction chamber 46 through low-speed centrifugation and enters the detection chamber 47 for reaction and detection.

[0382] The sample is injected through the sample injection port 59 into the wavy main channel 58 via a pipette or syringe, and under the action of the injection pressure, flows from the main channel 58 into each reaction chamber 46, and then the sample injection port 59 is sealed. In the first step, when the sample enters the reaction chamber 46, the gas in the reaction chamber 46 needs to be discharged outward through the exhaust channel 49. However, since the detection chamber 47 is not provided with an exhaust channel 49, it is difficult for the sample to flow in. At the same time, since the exhaust direction of the exhaust channel 49 is opposite to the centrifugal direction and is higher than the fluid channel 48, and the inner diameter of the exhaust channel 49 is smaller than the inner diameter of the fluid channel 48, the sample can only reach the intersection of the fluid channel 48 and the exhaust channel 49 (the inlet 53 of the exhaust channel 49) at most in the first step and cannot continue to move forward, thereby controlling the volume of the sample entering the reaction chamber 46. At this point, since the sample inlet 59 is sealed, that is, only the exhaust channel 49 of the entire centrifugal microfluidic chip 43 is connected to the atmosphere, and the rest of the chip is sealed, the internal and external pressures are balanced, and the sample in the reaction chamber 46 cannot flow out of the reaction chamber 46 to other areas. Because flowing out of the reaction chamber 46 would inevitably require a greater pressure to break the established internal and external pressure balance, the sample will remain in the reaction chamber 46 to react, and leakage or backflow is impossible. In the second step, after the sample completes the reaction in the reaction chamber 46, it is centrifuged at a low speed (e.g., 100-1000 rpm, preferably 200-800 rpm, more preferably 500-600 rpm) to break the initial pressure balance. The sample flows out of the reaction chamber 46 under the action of centrifugal force. At this time, since the exhaust direction of the exhaust channel 49 is opposite to the centrifugal direction, the sample will not enter the exhaust channel 49 and will flow entirely into the detection chamber 47. The gas in the detection chamber 47 is then discharged through the exhaust channel 49. At this time, the pressure in the detection chamber 47 is also balanced, so that the sample in the detection chamber 47 can no longer flow out of the detection chamber 47 and all of the sample remains in the detection chamber 47 for reaction and detection.

[0383] Therefore, when this setting is adopted, under the joint action of the exhaust channel 49 and the fluid flow channel 48, the internal and external pressure balance of the sample in the reaction chamber 46 or the detection chamber 47 can be established, and the direction of liquid flow can be regulated so that the sample in the reaction chamber 46 or the detection chamber 47 will not leak or flow back. It can also effectively control the amount of sample entering the reaction chamber 46 or the detection chamber 47, and multiple nucleic acid detection can be completed with only one low-speed centrifugation.

[0384] Preferably, the main flow channel 58 is a wavy flow channel with a large curvature and evenly spaced, which can help maintain the air pressure balance in the chip 43, smoothly control the flow of liquid, and help prevent the various detection units 50 from directly interfering with each other.

[0385] The number of sub-chips 69, the number of reaction chambers 46 and detection chambers 47 provided on each sub-chip 69, and the built-in solid reagents can be flexibly set as needed. Preferably, the number of sub-chips 69 can be set to 2 to 5, and the number of amplification chambers 46 and detection chambers 47 can be set to 4 to 80. In this embodiment, the number of sub-chips 69 is 1, the chip body 45 is provided with 34 reaction chambers 46 and detection chambers 47, and the sub-chip 69 is provided with 66 reaction chambers 46 and detection chambers 47.

[0386] The surface of the sample port 59 of the centrifugal microfluidic chip 43 is covered with a transparent film for sealing. When adding sample, the transparent film is pierced by a syringe to add sample. After the sample fills the main channel 58, the surface of the sample port 59 needs to be covered with a transparent film for sealing.

[0387] As shown in Figures 9 to 12, the rotation center 44 is a slot 70. The slot 70 can be clamped on the rotating shaft of the detection device to perform centrifugal rotation, thereby driving the entire chip to perform centrifugal rotation. Preferably, the slot 70 is a circular slot with a protrusion 71. The shape of the protrusion 71 is triangular, trapezoidal, etc. The rotating shaft on the detection device also has a fixing groove that can match the size of the slot 70. Therefore, the centrifugal microfluidic chip 43 is fixed to the rotating shaft through the slot 70 and performs centrifugal rotation along with the rotating shaft.

[0388] Preferably, the cross-section of reaction chamber 46 is circular, ensuring that the sample reacts and exits the reaction chamber 46 without leaving any dead corners and preventing residual flow both in and out. The cross-section of detection chamber 5 is approximately semicircular and larger than reaction chamber 46, ensuring that all sample in reaction chamber 46 enters detection chamber 47.

[0389] In some embodiments, the reaction chamber 46 is used for amplifying the target nucleic acid, and the reaction chamber 46 has a built-in nucleic acid amplification reagent, and the detection chamber 47 is used for detecting the target nucleic acid, and the detection chamber 47 has a built-in nucleic acid detection reagent; in some embodiments, the reaction chamber 46 can be used as an ERA amplification chamber, and the detection chamber 47 can be used as an ERA detection chamber; in some embodiments, the reaction chamber 46 can be used as an ERA reaction chamber, and the detection chamber 47 can be used as a CRISPR detection chamber; in some embodiments, the reaction chamber 46 can be used as an ERA reaction chamber, and the detection chamber 47 can be used as an Ago detection chamber; in some embodiments, the reaction chamber 46 can be used as an ERA reaction chamber, and the detection chamber 47 can be used as an RNase H detection chamber; in some embodiments, the amplification reaction includes but is not limited to EPA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, RCR, etc., and the form of the solid amplification reagent includes but is not limited to spherical, powdered, sheet-like or block preparations in the form of freeze-dried, dried, air-dried, etc. In some methods, the detection reagents include but are not limited to various detection systems established by ERA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, PCR, hybridization probe technology, CRISPR, Ago, RNase H, etc., and the detection reagents are solid detection reagents, including but not limited to spherical, powdered, sheet or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0390] When the sample to be tested is added to the sample addition port 59 through a pipetting device (not shown in the figure), the sample to be tested can be evenly diverted to the branch channel 61 along the main channel 58, reach the reaction chamber 46, mix with the solid amplification reagent therein, and at a certain temperature, an amplification reaction occurs; while the amplification reaction is completed, the rotating shaft in the detection device rotates clockwise, driving the centrifugal microfluidic chip 43 to perform centrifugal motion, and the liquid in the reaction chamber 46 is transferred to the detection chamber 47, and a fluorescence detection reaction occurs. After the experiment is completed, the results can be observed with the naked eye through the perspective window of the detection device, or an RGB sensor can be set in the detection device for automatic identification and automatic judgment of the results. Preferably, an LED lamp or a laser lamp is set in the detection device to illuminate the detection chamber 47 by releasing blue light, thereby improving the color development effect of the detection chamber 47 (when observed by the naked eye as green, it is judged that positive amplification has occurred, and when observed by the naked eye as dark orange, it is judged as negative). The above are all existing technologies and will not be repeated.

[0391] Preferably, a tapered microchannel structure is used between the main channel 58, the branch channel 61 (including the fluid channel 48), and the air-permeable channel 49 to prevent uneven sample distribution caused by excessively rapid sample droplets. The width of the main channel 58 ranges from 500 μm to 1000 μm, and the depth ranges from 500 μm to 1000 μm. The width of the branch channel 61 ranges from 400 μm to 800 μm, and the depth ranges from 150 μm to 300 μm. The width of the air-permeable channel 49 ranges from 50 μm to 500 μm, and the depth ranges from 50 μm to 350 μm. In this embodiment, the widths of the main channel 58, the branch channel 61, and the air-permeable channel 49 are 800 μm, 500 μm, and 300 μm, respectively, and the depths are 500 μm, 200 μm, and 100 μm, respectively.

[0392] The centrifugal microfluidic chip 43 provided in this embodiment is made of low-cost, easy-to-process, and biocompatible chip materials, such as polymethyl methacrylate (PMMA), polycarbonate (PC), and polypropylene (PP). A waterproof, breathable membrane is bonded to the chip body, forming a sealed space that effectively prevents liquid leakage. Depending on the detection requirements, the reaction chamber 46 is filled with a fixed amplification reagent for nucleic acid amplification, and the detection chamber 47 is filled with a solid detection substance for detection, such as a lyophilized pellet, lyophilized powder, or a quality control internal standard reagent.

[0393] The centrifugal microfluidic chip 43 is designed with multiple chips (chip body 45 and sub-chip 69). Each chip operates independently and is combined with an active drive mode. While accurately controlling the re-dissolution and mixing process of samples and reagents, it has the characteristics of ultra-high throughput and can be used for multi-sample and multi-index detection (such as detecting multiple different respiratory pathogens screening in multiple patient samples).

[0394] Example 5: Centrifugal microfluidic chip with the first configuration for the exhaust channel (without a sub-chip)

[0395] The structure of the centrifugal microfluidic chip for multiple nucleic acid detection provided in this embodiment is shown in Figures 13 to 16, wherein Figure 13 is a front view of the centrifugal microfluidic chip (the first exhaust channel), and Figure 14 is a back view of the centrifugal microfluidic chip (the first exhaust channel); Figure 15 is a three-dimensional view of the centrifugal microfluidic chip (the first exhaust channel); and Figure 16 is a schematic diagram of the detection unit structure in the centrifugal microfluidic chip (the first exhaust channel).

[0396] As shown in FIG. 13 to FIG. 16 , the centrifugal microfluidic chip 43 for multiplex nucleic acid detection provided in this embodiment is different from the chip in Example 4 in that it only contains a chip body 45 and does not contain a sub-chip 69 .

[0397] The centrifugal microfluidic chip 43 provided in this embodiment is combined with a centrifugal drive method. While accurately controlling the re-dissolution and mixing process of the sample and reagent, it has the characteristics of ultra-high throughput and can be used for single sample multi-index detection (such as detecting multiple different respiratory pathogens screening in a single patient sample).

[0398] Example 6: Nucleic Acid Multiple Detection Centrifugal Microfluidic Chip with the Second Exhaust Channel Configuration

[0399] The structure of the centrifugal microfluidic chip for multiple nucleic acid detection provided in this embodiment is shown in Figures 17 to 20, wherein Figure 17 is a front view of the centrifugal microfluidic chip (the second exhaust channel); Figure 18 is a back view of the centrifugal microfluidic chip (the second exhaust channel); Figure 19 is a three-dimensional view of the centrifugal microfluidic chip (the second exhaust channel); and Figure 20 is a schematic diagram of the detection unit structure in the centrifugal microfluidic chip (the second exhaust channel).

[0400] As shown in Figures 17 to 20, the centrifugal microfluidic chip 43 for multiple nucleic acid detection provided in this embodiment includes a chip body 45 with a rotation center 44. The chip body 45 is provided with a reaction chamber 46 for sample nucleic acid amplification and a detection chamber 47 for sample detection; the reaction chamber 46 and the detection chamber 47 are connected by a fluid channel 48, and the fluid channel 48 is also provided with an exhaust channel 49, which is led out from approximately the middle of the fluid channel 48. The exhaust channel 49 is used to control the flow of gas in the reaction chamber 46 or the detection chamber 47; the rotation center 44 is used to connect to a centrifugal drive device to drive the centrifugal microfluidic chip 43 to centrifuge.

[0401] The centrifugal microfluidic chip 43 can be provided with any number of detection units 50 according to project requirements or chip size, thereby realizing simultaneous detection of multiple targets. Each detection unit 50 is provided with a reaction chamber 46 and a detection chamber 47, which can be applied to the detection of a variety of different target analytes (such as a variety of different nucleic acid targets), and each detection unit can realize multiple nucleic acid detection. During use, the centrifugal microfluidic chip 43 needs to be kept in a horizontal state and placed in a matching detection device (centrifugal drive device), with the sample injection port 51 facing upward and placed horizontally. The rotation center 44 is fastened and fixed to the rotating shaft of the detection device for centrifugation, so that the rotating shaft can drive the centrifugal microfluidic chip 43 to rotate centrifugally; at the same time, the detection device can also provide a heat source for heating the reaction chamber 46 of the centrifugal microfluidic chip 43; the detection device can also detect the fluorescent substance produced after the sample reaction in the centrifugal microfluidic chip 43 and read the test results.

[0402] The fluid channel 48 connecting the reaction chamber 46 and the detection chamber 47 refers to the channel connecting the outlet 51 of the reaction chamber 46 and the inlet 52 of the detection chamber 47. The exhaust channel 49 includes an inlet 53 and an outlet 54. The inlet 53 is the connection between the fluid channel 48 and the exhaust channel 49, and the outlet 54 is the other end of the exhaust channel 49. The outlet 54 is closer to the rotation center 44 than the inlet 53, so that the exhaust direction of the exhaust channel 49 is opposite to, or at least partially opposite to, the centrifugal direction of rotation. When the centrifugal microfluidic chip 43 is centrifuged, the fluid sample flows outward from the center of the circle under the action of centrifugation. At this time, because the exhaust channel 49 is drawn from the fluid channel 48 between the reaction chamber 46 and the detection chamber 47 and approaches the rotation center 44, the exhaust direction is opposite to the centrifugal direction. Therefore, it is difficult for the fluid sample to enter the exhaust channel 49 during centrifugation, but it accurately enters the corresponding chamber along the centrifugal direction.

[0403] As shown in Figure 20, exhaust channel 49 extends from the middle of the fluid channel or toward the detection chamber 47. The initial section of the exhaust channel is an arc-shaped channel 55. The angle 56 between the arc-shaped channel 55 and the fluid channel near the reaction chamber 46 is less than 90 degrees. Exhaust channel 49 can be slightly offset toward the detection chamber 47 because this position reduces the pressure required for the sample to enter the detection chamber 47 during centrifugation, making it easier for the sample to enter the detection chamber 47 during centrifugation. When exhaust channel 49 extends from fluid channel 48, the smaller the angle 56 between the initial section and the fluid channel near the reaction chamber 46, the less likely the fluid will enter exhaust channel 48. However, this angle 56 should not be too small, otherwise exhaust may become difficult. Preferably, a single-end arc-shaped channel 55 is used for transition, and the angle between the arc-shaped channel 55 and the fluid channel near the reaction chamber 46 is less than 90 degrees. Preferably, the inner diameter of the exhaust channel 49 is smaller than the inner diameter of the fluid channel 48 (as shown in FIG. 20 ), which can further prevent the sample from entering the exhaust channel 49 during centrifugation.

[0404] The outlet 54 of the exhaust channel 7 is connected to the main channel 58 of the chip body 45, and exhaust is discharged to the main channel 58 (see Figures 9 to 12). When this setting is adopted, only two centrifugations are required to complete the multiple nucleic acid detection. Neither the reaction chamber 46 nor the detection chamber 47 needs to be connected to the outside atmosphere. The exhaust channel 49 is directly connected to the main channel 58, and the atmosphere pressed out of the reaction chamber 46 or the detection chamber 47 is discharged back to the main channel 58, which is equivalent to discharging the air pressure of the liquid-filled part to the unfilled part through the exhaust channel 49, thereby controlling the flow of liquid. Moreover, after exhausting the gas to the main channel 58 through the exhaust channel 49, the air pressure in the chip 43 is balanced, and the sample entering the reaction chamber 46 or the detection chamber 47 is difficult to flow out of the reaction chamber 46, and it is difficult for backflow to occur, forming a circulating closed environment. Moreover, compared with the setting mode provided in Example 4 or 5 in which the exhaust channel outlet 54 is connected to the outside world, since it is not connected to the outside world, it can effectively avoid aerosol contamination, making the detection results more accurate and reliable. At the same time, since the steps of preparing air holes and covering the waterproof breathable membrane 57 are omitted, the preparation process is simpler and easier, and the cost is lower.

[0405] When this setting is used, the process of the flow and detection of the sample to be tested in the centrifugal microfluidic chip 43 includes three steps: the first step is that the sample enters the main channel 58 from the sample injection port 59; the second step is low-speed centrifugation to allow the sample to enter the reaction chamber 46 from the main channel 58 for reaction; and the third step is that after the reaction is completed, the sample flows out of the reaction chamber 46 through medium-speed centrifugation and enters the detection chamber 47 for reaction and detection. Unlike the first setting, when the second setting is used, it is also necessary to control the flow of the sample by controlling the centrifugal rate. The sample is allowed to enter the reaction chamber 5 from the main channel 16 through low-speed centrifugation (such as 100-1000 rpm), and then enter the detection chamber 47 from the reaction chamber 46 through medium-speed centrifugation (such as 1000-3000 rpm). The method of controlling the flow of samples in different chambers by controlling the centrifugal rate is simpler and easier to operate, and the entire process is more controllable, and the detection results are more accurate.

[0406] As shown in Figures 17 to 20, when this setting is adopted, in the first step, the sample is injected from the sample port 59 into the main channel 58 through a pipette or syringe. After filling the serpentine main channel 58, the sample will flow to the final waste liquid pool 60, the injection will stop, and the sample port 59 will be sealed. This process is equivalent to the process of the sample filling the main channel 58, and it is also the process of the gas filled with liquid being discharged to the unfilled main channel 58 through the exhaust channel 7. Because the inner diameter of the branch channel 61 connecting the main channel 58 to the reaction chamber 46 is smaller, there is a large difference with the inner diameter of the main channel 58, so the sample cannot flow into the reaction chamber 46. Moreover, it is difficult to discharge the gas in the reaction chamber 46 back to the main channel 58 from the exhaust channel 49 by relying solely on injection pressure, so the sample will first stay in the main channel 58. In the second step, after the sample completely fills the main channel 58, it is centrifuged instantaneously in a clockwise low speed (e.g., 100 to 1000 rpm, preferably 500 rpm in this embodiment). The sample in the main channel 58 enters the reaction chamber 46 under the action of centrifugal force. However, since the detection chamber 47 does not have a separate exhaust channel 49 and is almost an enclosed sealed structure, it is difficult for the sample to overcome the gas pressure in the detection chamber 47 under low-speed centrifugation, and therefore cannot flow into the detection chamber 47. At the same time, since the exhaust direction of the exhaust channel 49 is opposite to the centrifugal direction, and the exhaust channel 49 is higher than the fluid channel 48, the sample can only reach the intersection of the fluid channel 48 and the exhaust channel 49 at most in the second step and cannot continue to move forward, thereby controlling the volume of the sample entering the reaction chamber 46. During the low-speed centrifugation process, the sample enters the reaction chamber 46, and at the same time, the gas in the reaction chamber 46 is discharged back to the main channel 58, which can also help the sample in the main channel 58 flow into the reaction chamber 46. After low-speed centrifugation, the sample remains in reaction chamber 46 for amplification reaction. In a sealed state, pressure equilibrium is achieved inside and outside the reaction chamber 46. The sample in reaction chamber 46 cannot flow out of reaction chamber 46 to other areas because flowing out of reaction chamber 46 would require a greater pressure to break the established internal and external pressure equilibrium. Therefore, the sample remains entirely in reaction chamber 46 for reaction, and leakage or backflow is impossible. In the third step, after the sample completes the amplification reaction in reaction chamber 46, the centrifuge speed is increased. After a moderate-speed centrifugation (e.g., 1000-3000 rpm, preferably 2000 rpm in this embodiment), the initial pressure equilibrium is broken, driving the sample from reaction chamber 46 to detection chamber 47. At this time, because the exhaust direction of exhaust channel 49 is opposite to the centrifugal direction and the inner diameter of exhaust channel 49 is smaller, the sample does not enter exhaust channel 49 and flows entirely into detection chamber 47. Since the entire system is in a closed state, the liquid in detection chamber 47 does not reflux, thus being stable for detection.

[0407] Therefore, this setting adopts two-stage centrifugation to complete multiple nucleic acid detection. The centrifugation rates of the first and second stages can differ greatly, the control is stable, and high-speed centrifugation (such as 3000-10000 rpm) is not required, so as to avoid the precipitation of pre-embedded freeze-dried or vitrified reagents due to insufficient dissolution caused by high-speed centrifugation, thereby causing reaction failure and affecting the test results. In addition, there is no need to connect to the outside world, thus avoiding aerosol contamination, and it is more convenient to manufacture and has lower cost.

[0408] The chip body 45 is circular, and the main flow channel 58 is arranged in a serpentine shape and curved into a ring. The chip body 45 and the ring are coaxially arranged with the rotation center 44. The main flow channel 58 adopts a serpentine flow channel with a large curvature and evenly spaced intervals. It helps maintain the air pressure balance within the chip 43, smoothly controls the flow of liquid, and helps prevent direct interference between the individual detection units 50.

[0409] As shown in Figures 17 to 20, when the outlet 54 of the exhaust channel 49 is connected to the main channel 58, the main channel 58 is arranged in a serpentine shape. This serpentine shape makes the main channel 58 more curved and longer, allowing the sample to be initially and stably retained in the main channel 58. The serpentine main channel 58 includes U-shaped channels 62 arranged equidistantly along the annular ring. The bottom end 63 of the U-shaped channel 62 is connected to the reaction chamber 46. When the outlet 54 of the exhaust channel 49 is connected to the main channel 58, the connection is at the top end 64 of the U-shaped channel 62.

[0410] Preferably, the bottom end 63 of the U-shaped channel 62 is provided with a branch channel 61 connected to the reaction chamber entrance, and the top end 64 of the U-shaped channel 62 is connected to the exhaust channel 49. The left and right sides of the U-shaped channel 62 are longer, and the top end 64 is closer to the rotation center 44 than the bottom end 63. This results in a greater distance between the bottom end 63 and the top end 64 of the U-shaped channel 62. When the exhaust channel 49 exhausts the top end 64 of the U-shaped channel 62, it does not immediately affect the pressure balance at the bottom end 63, thus facilitating a smooth flow of the sample into the reaction chamber 46. As shown in Figures 17 to 19, the U-shaped channels 62 are evenly arranged, with the bottom end 63 of each U-shaped channel 62 connected to a reaction chamber 46. Therefore, during centrifugation, the sample in each U-shaped channel 62 will enter the corresponding reaction chamber 46 under the action of centrifugal force. This is equivalent to the U-shaped channel 62 evenly distributing the sample in advance, which can help control the volume of the sample entering the reaction chamber 46.

[0411] Preferably, a tapered microchannel structure is used between the main channel 58, the branch channel 61 (including the fluid channel 48), and the air-permeable channel 49 to prevent uneven sample distribution caused by excessively rapid sample droplets. The width of the main channel 58 ranges from 500 μm to 1000 μm, and the depth ranges from 500 μm to 1000 μm. The width of the branch channel 61 ranges from 400 μm to 800 μm, and the depth ranges from 200 μm to 400 μm. The width of the air-permeable channel 49 ranges from 50 μm to 500 μm, and the depth ranges from 100 μm to 300 μm. In this embodiment, the widths of the main channel 58, the branch channel 61, and the air-permeable channel 49 are 800 μm, 400 μm, and 200 μm, respectively, and the depths are 500 μm, 400 μm, and 200 μm, respectively.

[0412] The main channel 58 is provided with a sample addition port 59 (including the first sample addition port 171 in the chip body 45 and the second sample addition port 172 in the sub-chip 69). As shown in Figures 17 to 20, the sample addition port 59 is located at the starting end 65 of the main channel 58. The end 66 of the main channel 58 is also provided with a waste liquid pool 60 (including the first waste liquid pool 181 in the chip body 45 and the second waste liquid pool 182 in the sub-chip 69). The two ends of the main channel 58 are not connected. The waste liquid pool 60 has a liquid storage function and is used to receive excess sample. When the sample fills the main channel 58, the excess sample will enter the waste liquid pool 60. Since the injection pressure is not enough to allow the sample to enter the reaction chamber 46, after the sample fills the main channel 58, the excess sample will begin to enter the waste liquid pool 60. At this time, it is equivalent to prompting that the sample addition amount is sufficient and the sample addition can be stopped. Therefore, the waste liquid pool 60 also has the effect of helping the sample quantification.

[0413] The surface of the sample port 59 of the centrifugal microfluidic chip 43 is covered with a transparent film for sealing. When adding sample, the transparent film is pierced by a syringe to add sample. After the sample fills the main channel 58, the surface of the sample port 59 needs to be covered with a transparent film for sealing.

[0414] As shown in Figures 17 to 20, the centrifugal microfluidic chip 43 is provided with a sub-chip 69 outside the chip body 45. The sub-chip 69 is a concentric ring chip located outside the chip body 45. The sub-chip 69 is also provided with a main channel 58, a reaction chamber 46 and a detection chamber 47. When the chip body 45 is centrifugally rotated, the sub-chip 69 can be centrifugally rotated along with the chip body 45. The sub-chip 69 is a concentric ring chip located outside the chip body 45. Of course, if necessary, more sub-chips can be set outside the sub-chip 69. The chip body 45 and the sub-chip 69 can be directly molded and manufactured in one chip 43 and cannot be disassembled. The sub-chip 69, the chip body 45 and the entire microfluidic chip 43 are all in the same plane. The sub-chip 69 is located outside the chip body 45 and has a larger diameter, so more groups of detection units 50 can be set up. At the same time, the sub-chip 69 can also be provided with a sample addition port 59 and a waste liquid pool 60 to independently perform multiple nucleic acid detections. It can detect samples different from the chip body 45, realize simultaneous detection of multiple samples, and can detect more indicators at the same time (each detection unit 50 can detect a nucleic acid target), significantly increasing the detection throughput and improving the detection efficiency.

[0415] As shown in Figures 17 to 20, the rotation center 44 is a slot 70. The slot 70 can be clamped on the rotating shaft of the detection device to perform centrifugal rotation, thereby driving the entire chip to perform centrifugal rotation. Preferably, the slot 70 is a circular slot with a protrusion 71. The protrusion 71 is shaped like a triangle, trapezoid, etc. The rotating shaft on the detection device also has a fixing groove that can match the size of the slot 70. Therefore, the centrifugal microfluidic chip 43 is fixed to the rotating shaft through the slot 70 and performs centrifugal rotation along with the rotating shaft.

[0416] Preferably, the cross-section of reaction chamber 46 is circular, ensuring that the sample reacts and exits the reaction chamber 46 without leaving any dead corners and preventing residual flow both in and out. The cross-section of detection chamber 47 is approximately semicircular and larger than reaction chamber 46, ensuring that all sample in reaction chamber 46 enters detection chamber 47.

[0417] In some embodiments, the reaction chamber 46 is used for amplifying the target nucleic acid, and the reaction chamber 46 contains a nucleic acid amplification reagent. The detection chamber 47 is used for detecting the target nucleic acid, and the detection chamber 47 contains a nucleic acid detection reagent.

[0418] The centrifugal microfluidic chip 43 provided in this embodiment is designed with multiple chips (chip body 35 and sub-chip 69). Each chip operates independently and is combined with an active drive mode. While accurately controlling the re-dissolution and mixing process of samples and reagents, it has the characteristics of ultra-high throughput and can be used for multi-sample and multi-index detection (such as detecting multiple different respiratory pathogens in multiple patient samples).

[0419] Example 7: Method for using a centrifugal microfluidic chip for multiplex nucleic acid detection

[0420] 1. Examples 4 and 5 provide methods for using a centrifugal microfluidic chip (the first configuration, where the exhaust channel is connected to the outside world)

[0421] The centrifugal microfluidic chips provided in Examples 4 and 5 can be applied to multiple nucleic acid detection, comprising the following steps:

[0422] Step 1: Add sample and re-dissolve

[0423] The pipette is used to draw different test samples and inject them into the chip body 45 and the sub-chip 69 through the first and second sample injection holes 171 and 172, respectively. Under the action of the exhaust channel 49, the liquid is evenly distributed along the main channel 58 in the chip body 45 and the sub-chip 69 to the branch channel 61 and enters the reaction chamber 46, redissolving the solid amplification reagent in the reaction chamber 46 and remaining in the reaction chamber 46 due to the balance of air pressure.

[0424] Step 2: Amplification reaction

[0425] Place the mixed centrifugal microfluidic chip 43 parallel to the center of the detection device, with the slot 70 fixed to the rotating shaft of the detection device. The chip is then heated, and the nucleic acid in the sample undergoes an amplification reaction at a certain temperature. The amplification reaction includes, but is not limited to, isothermal amplification and PCR.

[0426] Step 3: Detection reaction

[0427] After step 2 is completed, the centrifugal device is started. At this time, the centrifugal microfluidic chip 43 rotates in a clockwise direction (at a speed of 800 rpm). The amplification product in the reaction chamber 46 flows into the corresponding detection chamber 47 under the action of centrifugal force, and after mixing with the detection reagent, a detection reaction occurs. The detection reaction includes but is not limited to a variety of detection systems established by hybridization probe technology, CRISPR, Ago, RNaseH, etc.; during the transfer of the sample to be tested from the reaction chamber 46 to the detection chamber 47, the gas in the detection chamber 47 will enter the outside through the exhaust channel 49;

[0428] Step 4: Data reading

[0429] After step 3 is completed, the fluorescence intensity on the detection channel of the microfluidic chip is read by the optical device of the detection device to obtain the detection result.

[0430] 2. Example 6 provides a method for using a centrifugal microfluidic chip (second configuration, where the exhaust channel is connected to the main channel)

[0431] The centrifugal microfluidic chip provided in Example 6 can be applied to multiple nucleic acid detection, comprising the following steps:

[0432] Step 1: Add sample and re-dissolve

[0433] The pipette is used to add different test samples to the first and second sample wells 171 and 172, respectively. Driven by the air pressure of the pipette, the test sample is evenly distributed along the main channel 58, and the excess liquid enters the waste liquid reservoir 60. When the main channel 58 is completely filled, the sample port is sealed with a transparent film. Due to the difference in the pore size between the main channel 58 and the branch channel 61, the liquid cannot enter the reaction chamber 46.

[0434] Step 2: Amplification

[0435] The sealed centrifugal microfluidic chip 43 is placed parallel to the centrifugal drive device of the detection device and centrifuged clockwise at a low speed (500 / min). The sample to be tested is distributed to the reaction chamber 46 through the main channel 58. At this time, the chip heats up, and the nucleic acid in the sample to be tested undergoes an amplification reaction at a certain temperature.

[0436] Step 3: Detection reaction

[0437] After step 2 is completed, the centrifugal drive device of the detection device is restarted and the centrifugal speed is increased (to 1500 rpm). Driven by the centrifugal force, the sample to be tested is transferred from the reaction chamber 46 to the corresponding detection chamber 47; after the amplification product is mixed with the detection reagent, a detection reaction will occur at a certain temperature. The detection reaction includes but is not limited to a variety of detection systems established by hybridization probe technology, CRISPR, Ago, RNaseH, etc.

[0438] Step 4: Data reading

[0439] After step 3 is completed, the fluorescence intensity on the detection channel of the centrifugal microfluidic chip 43 is read by the optical device of the detection device to obtain the detection result.

[0440] According to the detection requirements, the fixed amplification reagent for nucleic acid amplification in the reaction chamber 46 and the detection substance for detection in the detection chamber 47 include freeze-dried balls, freeze-dried powder or quality control internal standard reagent.

[0441] Both the amplification reaction and the detection reaction need to be performed at a certain temperature. Therefore, a heating device is required in step 2 to provide a suitable temperature for the centrifugal microfluidic chip 43 . For example, an isothermal amplification reaction needs to provide a reaction temperature of 30-65° C.

[0442] Example 8: Verification of the detection effect of the centrifugal microfluidic chip for multiplex nucleic acid detection

[0443] 1. Using Example 4 (Figures 9 to 12) to provide a centrifugal microfluidic chip (the first setting mode, the exhaust channel is connected to the outside world) for genotyping detection

[0444] Blood specimens from patients with suspected respiratory infections were tested for nucleic acid detection of respiratory pathogens (influenza A, influenza B, respiratory syncytial virus, adenovirus, human rhinovirus, Mycoplasma pneumoniae, human metapneumovirus, and parainfluenza virus) using ERA-CRISPR technology (enzymatic recombinase amplification combined with CRISPR detection). This was compared and validated using a commercially available qPCR assay. Oral swabs were lysed using a sample release agent, and the resulting lysate served as a template. Using the same template loading, samples were amplified using a microfluidic chip device and qPCR. The ERA-CRISPR assay uses lyophilized ERA microspheres preloaded with specific amplification primers in the amplification chamber of the chip device, and lyophilized microspheres preloaded with a specific CRISPR detection system in the detection chamber. The samples were tested for the presence of nucleic acid from the corresponding pathogens. The results are shown in Table 1.

[0445] Table 1. Centrifugal microfluidic chip detection performance verification list (first setting method)

[0446] As shown in Table 1, under the same conditions, the detection results of the centrifugal microfluidic chip device of Example 1 are consistent with the results of the qPCR detection method, but it is more convenient and quick to use, has a lower overall cost, and is more suitable for on-site detection in the field and special environments.

[0447] 2. Using the centrifugal microfluidic chip provided in Example 6 (the second configuration, where the exhaust channel is connected to the main channel) for genotyping detection

[0448] This embodiment uses the centrifugal microfluidic chip of Example 6 (Figures 17 to 20) for typing detection, and uses ERA-CRISPR detection technology (enzymatic recombination amplification combined with CRISPR detection technology) to perform respiratory pathogen nucleic acid detection (influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human rhinovirus, Mycoplasma pneumoniae, human metapneumovirus, parainfluenza virus) on blood specimens suspected of respiratory tract infection, and uses qPCR detection method (commercial reagent) for comparative verification. The oral swab is subjected to nucleic acid cleavage using a sample release agent, and the resulting cleavage product is used as a template. The same template loading amount is used, and the sample is amplified and detected using a microfluidic chip device and a qPCR detection method, respectively. The ERA-CRISPR detection technology pre-loads ERA freeze-dried microspheres containing specific amplification primers in the amplification chamber of the chip device, and the detection chamber is pre-loaded with freeze-dried microspheres containing a specific CRISPR detection system. Determine whether the sample contains the corresponding pathogenic microorganism nucleic acid, and the results are shown in Table 2.

[0449] Table 2. Centrifugal microfluidic chip detection performance verification list (second setting method)

[0450] As shown in Table 2, when other conditions are the same, the detection results of the centrifugal microfluidic chip device of Example 3 are consistent with the results of the qPCR detection method, but it is more convenient and quick to use, has a lower overall cost, and is more suitable for on-site detection in the field and special environments.

[0451] At the same time, this embodiment also conducted hundreds of detection experiments to comprehensively compare the detection accuracy of the centrifugal microfluidic chip of Example 4 (first setting) and Example 6 (second setting). It was found that the accuracy of the centrifugal microfluidic chip with the first setting reached 98.1%, and the accuracy of the centrifugal microfluidic chip with the second setting reached 99.9%. The reason may be that the second setting can completely avoid aerosol contamination, and therefore has higher accuracy.

[0452] Example 9: Structure of Gravity Typing Chip

[0453] The structure of the gravity typing chip provided in this embodiment is shown in Figures 21 to 29, wherein Figure 21 is a front view of the gravity typing chip with four reaction chambers; Figure 22 is a back view of the gravity typing chip with four reaction chambers; Figure 23 is a top view of the gravity typing chip with four reaction chambers; Figure 24 is a schematic structural diagram of the sample addition area of ​​the gravity typing chip with four reaction chambers; Figure 25 is a schematic cross-sectional structural diagram of the sample addition area of ​​the gravity typing chip with four reaction chambers; Figure 26 is a schematic structural diagram of the detection area of ​​the gravity typing chip with four reaction chambers; Figure 27 is a structural diagram of the gravity typing chip with six reaction chambers arranged radially; Figure 28 is a structural diagram of the gravity typing chip with four reaction chambers arranged radially; and Figure 29 is a structural diagram of the gravity typing chip with four reaction chambers arranged horizontally.

[0454] As shown in Figures 21 to 26, the gravity typing chip 201 provided in this embodiment includes a sample addition area 202 and a detection area 203. The sample addition area 202 includes a liquid storage chamber 204, which is used to accommodate the sample to be tested or perform pre-processing on the sample (such as amplification of the target nucleic acid or lysis of the sample). There is only one liquid storage chamber 204, which is cylindrical. When the sample is added to the microfluidic chip 201, it will basically stay in the liquid storage chamber 204 first. The microfluidic chip 201 can also be equipped with a tube cover for sealing the microfluidic chip 201. The tube cover can be in the form of a plug. When the tube cover is tightened downward, the plug can provide downward pressure, prompting the sample near the sample addition port 206 to flow completely into the liquid storage chamber 204, and the sample in the liquid storage chamber 204 is in a high-pressure state. The channels in the microfluidic chip 201 include a main channel 207 and multiple branch channels 208 . The channel width is 400 μm to 800 μm. In this embodiment, the width of the main channel 207 and the branch channels 208 is preferably 400 to 600 μm, and the depth is 400 to 600 μm.

[0455] The detection area 203 can be provided with any number of reaction chambers 209 according to the needs of the project typing test, for sample processing and detection (such as for multiple typing tests for the same target nucleic acid, or multiple tests for different target nucleic acids). The gravity-driven microfluidic chip 201 shown in FIG21 is provided with six reaction chambers 209, which are arranged in a multi-cavity central radial arrangement from one end of the main channel 207. The reaction chamber 209 is provided with an exhaust channel 210, which is connected to the liquid storage chamber 204. Such a design can make the reaction chamber 209 of the microfluidic chip 201 unnecessary to be connected to the outside atmosphere. The exhaust channel 210 of the reaction chamber 209 is directly connected to the liquid storage chamber 204, and the atmosphere pressed out of the reaction chamber 209 is discharged back to the liquid storage chamber 204. At this time, the air pressure in the microfluidic chip 201 is balanced, and the sample entering the reaction chamber 209 is difficult to flow out of the reaction chamber 209, and reflux is difficult to occur. When the liquid flows in the microfluidic chip 201 , no external force is required to drive it, and the flow is achieved solely by the gravity of the liquid itself.

[0456] As shown in Figure 26, the reaction chamber 209 is also provided with an inlet channel 211. The exhaust channel 210 has a smaller aperture than the inlet channel 211, and the exhaust channel utilizes a multi-turn, circuitous flow path with a smaller aperture. This allows the sample to flow into the reaction chamber 209 from the inlet channel 211, but prevents it from flowing out of the reaction chamber 209 from the exhaust channel 210. The channels for sample flow, such as the main channel 207 of the microfluidic chip 201 and the inlet channel 211 of the reaction chamber 209, have relatively wide apertures, enabling smooth liquid flow. However, the aperture of the exhaust channel 210 of the reaction chamber 209 is significantly smaller than these channels. The small aperture of the exhaust channel 210 allows only ventilation, but not smooth liquid drainage. Consequently, after the sample fills the reaction chamber 209, further advancement is hindered, making it difficult for the sample to flow out of the exhaust channel 210 of the reaction chamber 209, and the sample will remain in the reaction chamber 209 to complete the reaction and be accurately detected. The pore size of the exhaust channel 210 is less than 300 μm, so that it can have a certain barrier effect on liquid. Preferably, the pore size of the exhaust channel 210 is 50 to 300 μm. In this embodiment, the pore size of the exhaust channel 210 is 100 μm.

[0457] As shown in Figures 21 to 26, the exhaust channel 210 is provided with at least one bent shape 212. The bent shape 212 can extend the length of the exhaust channel 210 as much as possible, so that there are more pipes to store the exhausted gas, and prevent the situation where there are many reaction chambers 209 and only one liquid storage chamber 204, which makes it difficult to fully accommodate all the gases, resulting in an increase in the air pressure in the microfluidic chip 201. At the same time, the exhaust channel 210 is also provided with a small cavity 214 at the bend 213 of the bent shape 212, which is used to temporarily accommodate the excess gas discharged. Preferably, the bent shape 212 provided on the exhaust channel 210 is a channel that bends back and forth. As shown in Figure 24, before the exhaust channel 210 is connected to the liquid storage chamber 204, an exhaust cavity 223 can also be provided to store the excess gas discharged, and the exhaust cavity 223 will continue to return to the liquid storage chamber 204 after it is filled.

[0458] The microfluidic chip 201 can be provided with multiple reaction chambers 209, each of which is fluidically connected to the main channel 207 of the microfluidic chip 201 via a sample inlet channel 211, to facilitate detection of different target nucleic acids or different genotypes. The microfluidic chip 201 shown in FIG27 has six reaction chambers 209, while the microfluidic chip 1 shown in FIG21 to FIG26, 28, and 29 has four reaction chambers 209. The number of reaction chambers 209 can be selected based on the actual detection requirements of the project. In the microfluidic chip 201, the reaction chambers 209 can be arranged in any arrangement, such as horizontally, vertically, or radially arranged in any other manner at the end 215 of the main channel 207. A plurality of bifurcations 216 can also be provided at the lower end of the main channel 207 for arranging the reaction chambers 209. The reaction chambers 209 of the microfluidic chip 201 shown in Figures 27 and 28 are arranged in a radial manner, and the reaction chambers 209 of the microfluidic chip 201 shown in Figure 29 are arranged in a horizontal transverse manner; the reaction chambers 209 of the microfluidic chip 201 shown in Figures 21 to 26 are arranged with two bifurcations 216, and two reaction chambers 209 are arranged transversely at each bifurcation.

[0459] The reaction chamber 209 is provided with an injection port 217 and an exhaust port 218. The injection port 217 is in fluid communication with the injection channel 211, and the exhaust port 218 is in gas communication with the exhaust channel 210. The exhaust port 218 is located higher than the injection port 217. As shown in Figure 25, the injection port 217 is located at the lower part of the reaction chamber 209, and the exhaust port 218 is located at the upper part of the reaction chamber 209. The purpose of doing so is to ensure that the sample can be smoothly exhausted upward after entering the reaction chamber 209, and at the same time, it can also stay more stably in the reaction chamber 209 to prevent backflow. After the sample to be tested completes a reaction that is conducive to detection in the reaction chamber 209 (such as reacting with a detection reagent to amplify the signal, etc.), the result is detected directly in the reaction chamber 209. One side of the reaction chamber 209 is made of a transparent film. The detection instrument can detect substances such as fluorescence generated by the reaction through the film and read the test results.

[0460] As shown in Figure 25 , the liquid storage chamber 204 is provided with a liquid outlet 219 and an air inlet 220. The liquid outlet 219 is in fluid communication with the main channel 207, while the air inlet 220 is in gaseous communication with the exhaust channel 210 of the reaction chamber 207. The air inlet 220 is positioned higher than the liquid outlet 219. The position of the air inlet 220 in the liquid storage chamber 204, which is higher than the liquid outlet 219, allows the entire sample in the liquid storage chamber 204 to be discharged smoothly, preventing interference from incoming gas.

[0461] As shown in Figure 26, a valve block cavity 222 is provided between the liquid storage chamber 204 and the reaction chamber 209 for placing a solid valve block 221 for controlling the state of fluid communication between the liquid storage chamber 204 and the reaction chamber 209; the valve block 221 has two forms, solid and liquid; when the valve block 221 is solid, the liquid storage chamber 204 and the reaction chamber 209 cannot be fluidically connected; when the valve block 221 is liquid, the liquid storage chamber 204 and the reaction chamber 209 are fluidically connected. Meanwhile, when valve block 221 is solid, it is located within main channel 207 connecting liquid reservoir 204 and reaction chamber 209, preventing the sample to be tested from liquid reservoir 204 from flowing into reaction chamber 209. When valve block 221 is liquid, the sample flows toward exhaust channel 210 of reaction chamber 209. Valve block 221 is paraffin or a lipid (such as n-octadecane, stearic acid, palmitic acid, or other lipids), which is solid at room temperature and melts into a liquid state upon heating. Valve block 221 must have a density less than that of water. When valve block 221 is liquid, it can float above the sample to be tested, facilitating condensation at exhaust port 218 at the top of reaction chamber 209. If valve block 221 has a density greater than that of water, when valve block 221 is liquid and flows into reaction chamber 209 along with the sample, it is likely to accumulate at inlet 217 of reaction chamber 209, causing blockage. Therefore, valve block 221 must have a density less than that of water. In this embodiment, paraffin is preferably used as the valve block 221. Paraffin can achieve phase change at a specific temperature threshold between 40 and 90°C. In this embodiment, paraffin (manufacturer: Sigma-Aldrich, model: 411663) undergoes phase change at about 65°C.

[0462] Because the microfluidic chip 201 has multiple reaction chambers 209 and their sample inlets 217 are collectively connected, sample backflow is prone to occur, causing the sample to overflow from one reaction chamber 209 and enter another, resulting in cross-effects. Therefore, preventing sample backflow is crucial to ensure the accuracy of test results. By providing an exhaust channel 211 within each reaction chamber 209 and a valve block 221 between the liquid storage chamber 204 and the reaction chamber 209, the temperature-dependent phase change properties of the valve block 221 are utilized to achieve blocking and connecting the liquid storage chamber 204 and the reaction chamber 209. After the valve block 221 heats up and melts, the flow channels between the liquid storage chamber 204 and each reaction chamber 209 are connected, and the sample in the liquid storage chamber 204 flows into each reaction chamber 209. The melted valve block 221 has a low density and will float above the sample, flowing into the reaction chamber 209 together with the sample, and condensing near the exhaust port 218 of the reaction chamber 209, blocking the exhaust channel 210 (because the sample is quantitative, it can at least reach the exhaust port 218 of the reaction chamber 209). It also helps to make the sample to be tested more stably remain in the reaction chamber 209 after entering the reaction chamber 209, making it difficult to reflux and not flow into other chambers, thereby ensuring that each step of the reaction can be completed smoothly until accurate detection is finally achieved.

[0463] Preferably, the cross-section of the reaction chamber 209 is in the shape of a water drop ( FIG. 26 ), and the exhaust port 218 is located at the top of the water drop, thereby helping the liquid paraffin to gather upward, making it easier to condense near the exhaust port 218 .

[0464] The solid reagents contained in the liquid reservoir 204 and reaction chamber 209 can be flexibly arranged as needed, with different chambers containing different reaction reagents. For example, when the microfluidic chip 201 provided in this embodiment is used for genotyping detection of a target nucleic acid, the liquid reservoir 204 is used for amplification of the target nucleic acid and contains nucleic acid amplification reagents; while the reaction chamber 209 is used for detecting different genotypes of the target nucleic acid and contains detection reagents for different genotypes. The liquid storage chamber 204 is used for amplifying the target nucleic acid. A nucleic acid amplification reagent must be placed in the liquid storage chamber 204 in advance. The reagent used for lysis is added from the sample injection port 206 during sample detection. The nucleic acid amplification reagent is a solid amplification reagent that is freeze-dried or dried, including but not limited to freeze-dried, dried, air-dried, or other forms of spheres, powders, tablets, or blocks. The amplification reactions involved include but are not limited to RPA (Recombinase Polymerase Amplification), ERA (Enzymatic Recombinase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), NASBA (Nuclear acid sequence-based amplification), HDA (helicase-dependent amplification), TMA (transcription mediated amplification), SAT (Simultaneous Amplification and Testing, real-time fluorescence nucleic acid constant temperature amplification detection technology) etc.The reaction chamber 209 is used for fluorescence detection reaction between the amplified sample and the detection reagent. The detection reagent needs to be placed in advance in the reaction chamber 209. The detection reagent is a solid detection reagent, including but not limited to spherical, powdered, sheet or block preparations in the form of freeze-dried, dried, air-dried, etc. The detection reactions involved include but are not limited to those based on RPA (Recombinase Polymerase Amplification), ERA (Enzymatic Recombinase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), NASBA (Nuclear acid sequence-based amplification), HDA (helicase-dependent amplification), TMA (transcription mediated amplification), SAT (Simultaneous Amplification and Testing, real-time fluorescence nucleic acid constant temperature amplification detection technology), hybridization probe technology, CRISPR, Ago, RNaseH and other established probe detection systems. The detection results vary according to actual needs, including but not limited to fluorescence detection reactions, test strip visualization detection methods. When the microfluidic chip 201 provided in this embodiment is used for multiple detections of target nucleic acids, the liquid storage chamber 204 is used to add sample nucleic acid lysis products, and the reaction chamber 209 needs to pre-place solid detection reagents for the amplification and detection of different nucleic acids, specifically including detection primer probes and detection premixes, and the detection premixes include enzymes and buffers required for detection.

[0465] The chip body structure of the microfluidic chip 201 uses a chip material with low cost, easy processing performance and good biocompatibility, such as PMMA (polymethyl methacrylate), PP (polypropylene), PC (polycarbonate), etc. as the substrate. Channels and reaction grooves are directly made using laser etching technology, or structures are etched on the PMMA substrate and then molded with PDMS.

[0466] During use, the microfluidic chip 201 must be kept upright with the sample port 206 facing upward. During use, the microfluidic chip 201 can be inserted into a matching detection device, positioned vertically with the sample port 206 facing upward. The detection device can then provide a heat source to heat specific locations on the microfluidic chip 201 (e.g., the reaction chamber 209, the valve block 221, etc.). The detection device can also detect fluorescent substances generated by sample reactions in the microfluidic chip 1 and read the test results.

[0467] The microfluidic chip 201 provided in this embodiment, through ingenious design of flow channel connections and chamber placement, eliminates the need for external drivers, allowing liquids to independently and sequentially amplify and detect samples based on their own gravity. This process is simple to operate, offers rapid reaction times, and effectively enables high-throughput pathogen detection. Furthermore, the amplification and detection reagents in this microfluidic chip are all dry reagents, enabling the chip to be stored and transported at room temperature, avoiding the limitations of cold chain transportation and -20°C storage.

[0468] Example 10: Method for using gravity typing chip

[0469] The gravity-driven microfluidic chip provided in Example 9 can be applied to a variety of nucleic acid detection methods.

[0470] Method 1: Using genotyping detection based on enzymatic isothermal amplification technology as an example, the use of the microfluidic chip device is described:

[0471] 1) Transfer: The sample to be tested containing the lysate is introduced into the liquid storage chamber 204 of the microfluidic chip from the sample injection port 206. The solid amplification reagent in the liquid storage chamber 204 is re-dissolved by the sample to be tested. At this time, the tube cap is tightened to completely seal the chip;

[0472] 2) Reaction: The chip is heated. After the temperature reaches a certain level, an amplification reaction occurs in the liquid storage chamber 204. The amplification reaction includes but is not limited to a constant temperature amplification reaction and a PCR reaction. While the amplification reaction is proceeding, the valve block 221 separating the liquid storage chamber 204 and the reaction chamber 209 is also continuously melting. After the valve block 221 is completely melted, the air pressure in the system is balanced, and the amplification product moves toward the reaction chamber 209 under the action of gravity.

[0473] 3) Read the results: After the reaction is completed, the fluorescence signal is read by the device to obtain the test results.

[0474] Method 2: Using enzymatic isothermal amplification technology for multiplex detection as an example, the use of the microfluidic chip device is described:

[0475] 1) Transfer: The sample to be tested containing the lysate is introduced into the liquid storage chamber 204 of the microfluidic chip from the sample injection port 206, and the tube cap is tightened to seal the chip.

[0476] 2) Reaction: The chip is heated. When the temperature reaches a certain level, the valve block 221 between the liquid storage chamber 204 and the detection chamber 209 continues to melt. After the valve block 221 is completely melted, the air pressure in the system is balanced, and the sample lysis products move to the reaction chamber 209 under the action of gravity. At the same time, the reagents in the reaction chamber 209 dissolve and an amplification reaction occurs, which includes but is not limited to a constant temperature amplification reaction and a PCR reaction.

[0477] 3) Read the results: After the reaction is completed, the fluorescence signal is read by the device to obtain the test results.

[0478] Example 11: Verification of Gravity Typing Chip Typing Detection Results

[0479] This embodiment uses the gravity-driven microfluidic chip of Example 9 (Figure 21) for typing detection, and uses ERA-CRISPR detection technology (enzymatic recombination amplification combined with CRISPR detection technology) to perform malarial parasite nucleic acid detection (Plasmodium Spp., Plasmodium F., Plasmodium V., Plasmodium K.) on blood samples of people suspected of being infected with malarial parasites, and uses qPCR detection method (commercial reagent) for comparative verification. The blood sample is subjected to nucleic acid cleavage using a sample release agent, and the resulting cleavage product is used as a template. The same template loading amount is used, and the sample is amplified and detected using a gravity-driven microfluidic chip device and a qPCR detection method, respectively. The ERA-CRISPR detection technology pre-loads ERA freeze-dried microspheres containing specific amplification primers in the liquid storage chamber of the chip device, and the reaction chamber is pre-loaded with freeze-dried microspheres containing a specific CRISPR detection system. Determine whether the sample contains the corresponding pathogenic microorganism nucleic acid, and the results are shown in Table 3.

[0480] Table 3. Gravity typing chip detection performance verification results

[0481] As shown in Table 3, under the same other conditions, the detection results of the gravity typing chip device of Example 9 are consistent with the results of the qPCR detection method, but it is more convenient and faster to use and is more suitable for on-site detection in the wild and special environments.

[0482] Example 12: Verification of Multiple Detection Effects Using Gravity Typing Chips

[0483] This example utilizes the gravity-driven microfluidic chip device of Example 9 ( FIG. 26 ) for multiplex testing. ERA (enzymatic recombinant amplification) detection technology is used to test nucleic acid in shrimp samples suspected of infection for white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), acute hepatopancreatic necrosis disease (AHPND / EMS), shrimp iridovirus (SHIV), and enterocytosis (EHP). Quantitative quantitative PCR (qPCR) detection (using commercial reagents) is also used for comparative validation. Crushed shrimp meat samples are subjected to nucleic acid lysis using a sample release agent. The resulting lysate serves as a template. Using the same template loading amount, samples are amplified using both the gravity-driven microfluidic chip and qPCR detection methods. The ERA detection technology uses freeze-dried microspheres pre-loaded with specific amplification primers and detection probes, containing the ERA amplification detection system, in the reaction chamber of the gravity-driven microfluidic chip device. After the reaction, the results were read to determine whether the sample contained the corresponding pathogenic microorganism nucleic acid. The results are shown in Table 4.

[0484] Table 4. Gravity typing chip detection performance verification list

[0485] As shown in Table 4, under the same conditions, the detection results of the gravity-driven microfluidic chip device of Example 9 are consistent with the results of the qPCR detection method. It is more convenient and quick to use and is more suitable for on-site detection in the wild and special environments.

[0486] Example 13: Asymmetric pressure-type chip structure

[0487] The structure of the asymmetric pressure-driven microfluidic chip provided in this embodiment is shown in Figures 30 to 40, wherein Figure 30 is a tube cover structure of the asymmetric pressure-driven microfluidic chip; Figure 31 is a cross-sectional view of the tube cover structure of the asymmetric pressure-driven microfluidic chip; Figure 32 is a front view of the asymmetric pressure-driven microfluidic chip with four reaction chambers; Figure 33 is a back view of the asymmetric pressure-driven microfluidic chip with four reaction chambers; Figure 34 is a top view of the asymmetric pressure-driven microfluidic chip with four reaction chambers; and Figure 35 is a diagram of the asymmetric pressure driven microfluidic chip with four reaction chambers. Schematic diagram of the structure of the sample loading area of ​​the asymmetric pressure-driven microfluidic chip; Figure 36 is a schematic diagram of the cross-sectional structure of the sample loading area of ​​the asymmetric pressure-driven microfluidic chip with four reaction chambers; Figure 37 is a schematic diagram of the structure of the detection area of ​​the asymmetric pressure-driven microfluidic chip with four reaction chambers; Figure 38 is a structural diagram of the asymmetric pressure-driven microfluidic chip with six reaction chambers arranged radially; Figure 39 is a structural diagram of the asymmetric pressure-driven microfluidic chip with four reaction chambers arranged radially; Figure 40 is a structural diagram of the asymmetric pressure-driven microfluidic chip with four reaction chambers arranged horizontally.

[0488] As shown in Figures 30 to 37, the asymmetric pressure-driven microfluidic chip 301 provided in this embodiment includes a sample loading area 302 and a detection area 303. The sample loading area 302 includes a liquid reservoir 304 and a tube cover 305. The liquid reservoir 304 is used to accommodate the sample to be tested or perform pre-processing on the sample (such as amplification of the target nucleic acid or sample lysis, etc.). There is only one cylindrical liquid reservoir 304. When the sample is added to the microfluidic chip 301, it will basically stay in the liquid reservoir 304 first. The tube cover 305 seals the microfluidic chip 301 and can apply pressure to the microfluidic chip 301 by pressing or twisting. Tube cap 305 can be in the form of a plug or a screw. When tightened, it not only seals the chip but also provides instantaneous downward pressure, forcing the sample near injection port 306 to flow completely into liquid reservoir 304. Because exhaust channel 310 is a multi-turn, circuitous flow channel with a smaller aperture and a smaller aperture than injection channel 311, the pressure applied by twisting or pressing tube cap 305 creates an instantaneous asymmetric pressure, causing the sample in liquid reservoir 304 to transfer through the flow channel to detection area 303. The channels within microfluidic chip 301 include a main channel 307 and multiple branch channels 308.

[0489] As shown in Figures 30 and 31, the top of the tube cover 305 provided in this embodiment is provided with a pushable airbag 324. After sealing the microfluidic chip 301, the airbag 324 is pushed downward from the top of the tube cover 305 to a position close to the tube cover's spiral 325. This can apply pressure to the chip, creating an asymmetric pressure within the chip, prompting the sample to flow from the liquid reservoir 304 into the reaction chamber 309. The tube cover 305 can also be detachably connected to the liquid reservoir 304, providing air pressure to the chip system while sealing the system, driving the liquid in the liquid reservoir 304 to transfer to the reaction chamber 309. The channels for sample flow, such as the main channel 307 of the microfluidic chip 301 and the sample inlet channel 311 of the reaction chamber 309, have apertures that enable smooth liquid flow.

[0490] The detection area 303 can be provided with any number of reaction chambers 309 according to the needs of the project typing test, for sample processing and detection (such as for multiple typing tests for the same target nucleic acid, or multiple tests for different target nucleic acids). The asymmetric pressure-driven microfluidic chip 301 shown in FIG37 is provided with six reaction chambers 309, which are arranged in a multi-cavity central radial arrangement from one end of the main channel 307. The reaction chamber 309 is provided with an exhaust channel 310, which is connected to the liquid storage chamber 304. Such a design can make the reaction chamber 309 of the microfluidic chip 301 unnecessary to be connected to the outside atmosphere. The exhaust channel 310 of the reaction chamber 309 is directly connected to the liquid storage chamber 304, and the atmosphere pressed out of the reaction chamber 309 is discharged back to the liquid storage chamber 304. At this time, the air pressure in the microfluidic chip 301 is balanced, and the sample entering the reaction chamber 309 is difficult to flow out of the reaction chamber 309, and reflux is difficult to occur.

[0491] As shown in Figure 37, reaction chamber 309 also has an inlet channel 311. The aperture of inlet channel 311 is significantly larger than that of exhaust channel 310. The exhaust channel uses a multi-turn, circuitous flow path with a smaller aperture. When the cap 305 is twisted or pressed, an instantaneous asymmetric pressure is generated, allowing the sample to flow into the reaction chamber 309 through inlet channel 311 but preventing it from flowing out of the reaction chamber 309 through exhaust channel 310. The channels for sample flow, such as the main channel 307 of the microfluidic chip 301 and the inlet channel 311 of the reaction chamber 309, have wide apertures to ensure smooth liquid flow. However, the aperture of the exhaust channel 310 of the reaction chamber 309 is significantly smaller than these flow channels. The aperture of the exhaust channel 310 is small, which can only achieve ventilation, and the drainage is not smooth. As a result, after the sample fills the reaction chamber 309, it will be blocked when continuing to move forward, and it will be difficult to flow out of the exhaust channel 310 of the reaction chamber 309. The sample will remain in the reaction chamber 309 to complete the reaction and be accurately detected.

[0492] As shown in Figures 30 to 37, the exhaust channel 310 is provided with at least one bent shape 312. The bent shape 312 can extend the length of the exhaust channel 310 as much as possible, so that there are more pipes to store the exhausted gas, and prevent the situation where there are many reaction chambers 309 and only one liquid storage chamber 304, which makes it difficult to fully accommodate all the gases, resulting in an increase in the air pressure in the microfluidic chip 301. At the same time, the exhaust channel 310 is also provided with a small cavity 314 at the bend 313 of the bent shape 312, which is used to temporarily accommodate the excess exhausted gas. Preferably, the bent shape 312 provided on the exhaust channel 310 is a channel that bends back and forth. As shown in Figure 34, before the exhaust channel 310 is connected to the liquid storage chamber 304, an exhaust cavity 323 can also be provided to store the exhausted excess gas, which will continue to return to the liquid storage chamber 304 after the exhaust cavity 323 is filled.

[0493] The microfluidic chip 301 can be provided with multiple reaction chambers 309, each of which is fluidically connected to the main channel 307 of the microfluidic chip 301 via a sample injection channel 311, so as to be suitable for detecting different target nucleic acids or different genotypes. The microfluidic chip 301 shown in Figures 30 and 37 has six reaction chambers 309, while the microfluidic chip 301 shown in Figures 31 to 36, 38, and 40 has four reaction chambers 309. The number of reaction chambers 309 can be selected based on the actual detection needs of the project. In the microfluidic chip 301, the reaction chambers 309 can be arranged in any arrangement, such as horizontally, vertically, or radially arranged in any other manner at the end 315 of the main channel 307. A plurality of bifurcations 316 can also be provided at the lower end of the main channel 307 for arranging the reaction chambers 309. The reaction chambers 309 of the microfluidic chip 301 shown in Figures 37 and 38 are arranged in a radial form, and the reaction chambers 309 of the microfluidic chip 301 shown in Figures 30 and 40 are arranged in a horizontal transverse form; the reaction chambers 309 of the microfluidic chip 301 shown in Figures 31 to 36 are arranged with two bifurcations 316, and two reaction chambers 309 are arranged transversely at each bifurcation.

[0494] The reaction chamber 309 is provided with an injection port 317 and an exhaust port 318. The injection port 317 is in fluid communication with the injection channel 311, and the exhaust port 318 is in gas communication with the exhaust channel 310. The exhaust port 318 is located higher than the injection port 317. As shown in Figure 35, the injection port 317 is located at the lower part of the reaction chamber 309, and the exhaust port 318 is located at the upper part of the reaction chamber 309. The purpose of doing this is to ensure that the sample can be smoothly exhausted upward after entering the reaction chamber 309, and at the same time, it can stay more stably in the reaction chamber 309 to prevent backflow. After the sample to be tested completes a reaction that is conducive to detection in the reaction chamber 309 (such as reacting with a detection reagent to amplify the signal, etc.), the result is detected directly in the reaction chamber 309. One side of the reaction chamber 309 is made of a transparent film. The detection instrument can detect substances such as fluorescence generated by the reaction through the film and read the test results.

[0495] As shown in Figure 36 , the liquid storage chamber 304 is provided with a liquid outlet 319 and an air inlet 320. The liquid outlet 319 is in fluid communication with the main channel 307, while the air inlet 320 is in gas communication with the exhaust channel 310 of the reaction chamber 307. The air inlet 320 is positioned higher than the liquid outlet 319. The position of the air inlet 320 in the liquid storage chamber 304, which is higher than the liquid outlet 319, allows the entire sample in the liquid storage chamber 304 to be discharged smoothly, preventing interference from incoming gas.

[0496] As shown in Figure 37, a valve block cavity 322 is provided between the liquid storage chamber 304 and the reaction chamber 309 for placing a solid valve block 321 for controlling the state of fluid communication between the liquid storage chamber 304 and the reaction chamber 309; the valve block 321 has two forms, solid and liquid; when the valve block 321 is solid, the liquid storage chamber 304 and the reaction chamber 309 cannot be fluidically connected; when the valve block 321 is liquid, the liquid storage chamber 304 and the reaction chamber 309 are fluidically connected. Meanwhile, when the valve block 321 is solid, it is located within the main channel 307 connecting the liquid storage chamber 304 and the reaction chamber 309, preventing the sample to be tested in the liquid storage chamber 304 from flowing into the reaction chamber 309. When the valve block 321 is liquid, the sample flows into the exhaust channel 310 of the reaction chamber 309. The valve block 321 is a paraffin wax or a lipid (such as n-octadecane, stearic acid, palmitic acid, etc.), which is solid at room temperature and melts into a liquid state upon heating. The density of the valve block 321 must be less than that of water. When the valve block 321 is converted to a liquid state, it can always float above the sample to be tested, facilitating condensation at the exhaust port 318 at the top of the reaction chamber 309. If the density of the valve block 321 is greater than that of water, when the valve block 321 is converted to a liquid state and flows into the reaction chamber 309 along with the sample, it is likely to accumulate at the inlet 317 of the reaction chamber 309, causing blockage. Therefore, the density of the valve block 321 must be less than that of water. In this embodiment, paraffin is preferably used as the valve block 321. Paraffin can achieve phase change at a specific temperature threshold between 40 and 90°C. In this embodiment, paraffin (manufacturer: Sigma-Aldrich, model: 411663) undergoes phase change at about 65°C.

[0497] Because the microfluidic chip 301 has multiple reaction chambers 309, and their inlets 317 are collectively connected, sample backflow is prone to occur, causing the sample to overflow from one reaction chamber 9 and enter another reaction chamber 309, resulting in cross-effects. Therefore, preventing sample backflow is crucial to ensure the accuracy of test results. By providing an exhaust channel 310 in each reaction chamber 309 and a valve block 321 between the liquid storage chamber 304 and the reaction chamber 309, the temperature-dependent phase change properties of the valve block 321 are utilized to achieve blocking and connecting the liquid storage chamber 304 and the reaction chamber 309. After the valve block 321 heats up and melts, the flow channels between the liquid storage chamber 304 and each reaction chamber 309 are connected, and the sample in the liquid storage chamber 304 flows into each reaction chamber 309. The melted valve block 321 has a low density and will float above the sample, flowing into the reaction chamber 309 together with the sample, and condensing near the exhaust port 318 of the reaction chamber 309, blocking the exhaust channel 310 (because the sample is quantitative, it can at least reach the exhaust port 318 of the reaction chamber 309). This also helps to ensure that the sample to be tested can remain more stably in the reaction chamber 309 after entering the reaction chamber 309, making it difficult for backflow to occur and will not flow into other chambers, thereby ensuring that each step of the reaction can be completed smoothly until accurate detection is finally achieved.

[0498] Preferably, the cross-section of the reaction chamber 309 is in the shape of a water drop ( FIG. 36 ), and the exhaust port 318 is located at the top of the water drop, thereby helping the liquid paraffin to gather upward, making it easier to condense near the exhaust port 18 .

[0499] The solid reagents contained in the liquid reservoir 304 and reaction chamber 309 can be flexibly arranged as needed, with different chambers containing different reaction reagents. When the microfluidic chip 301 provided in this embodiment is used for genotyping of a target nucleic acid, the liquid reservoir 304 is used for amplification of the target nucleic acid and contains nucleic acid amplification reagents; the reaction chamber 309 is used for detecting different genotypes of the target nucleic acid and contains detection reagents for different genotypes. The liquid storage chamber 304 is used for amplifying the target nucleic acid. A nucleic acid amplification reagent must be pre-placed in the liquid storage chamber 304, and the reagent used for lysis is added from the sample injection port 306 during sample detection. The nucleic acid amplification reagent is a solid amplification reagent that is freeze-dried or dried, including but not limited to freeze-dried, dried, air-dried, or other forms of spheres, powders, tablets, or blocks. The amplification reactions involved include but are not limited to RPA (Recombinase Polymerase Amplification), ERA (Enzymatic Recombinase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), NASBA (Nuclear acid sequence-based amplification), HDA (helicase-dependent amplification), TMA (transcription mediated amplification), etc.Reaction chamber 309 is used to perform a fluorescence detection reaction between the amplified sample and the detection reagent. The detection reagent must be pre-placed in the reaction chamber 309. The detection reagent is a solid detection reagent, including but not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, oven-dried or air-dried. The detection reactions involved include but are not limited to various probe detection systems based on RPA (Recombinase Polymerase Amplification), ERA (Enzymatic Recombinase Amplification), LAMP (Loop-mediated Isothermal Amplification), NEAR (Nicking enzyme-Assisted Reaction), NASBA (Nuclear acid sequence-based amplification), HDA (helicase-dependent amplification), TMA (transcription mediated amplification), hybridization probe technology, CRISPR, Ago, RNaseH, etc. Detection results vary depending on actual needs, including but not limited to fluorescence detection reactions and test strip visualization detection methods. When the microfluidic chip 301 provided in this embodiment is used for multiplex detection of target nucleic acids, the liquid reservoir 304 is used to add sample nucleic acid lysis products, and the reaction chamber 309 is pre-placed with solid detection reagents for amplification and detection of different nucleic acids, specifically including detection primer probes and detection premixes. The detection premixes include the enzymes and buffers required for detection.

[0500] The chip body structure of the microfluidic chip 301 uses a chip material with low cost, easy processing performance and good biocompatibility, such as PMMA (polymethyl methacrylate), PP (polypropylene), PC (polycarbonate), etc. as the substrate. Channels and reaction grooves are directly produced by laser etching technology, or structures are etched on the PMMA substrate and then molded with PDMS.

[0501] During use, the microfluidic chip 301 must be kept upright with the sample port 306 facing upward. During use, the microfluidic chip 301 can be inserted into a matching detection device, positioned vertically with the sample port 306 facing upward. The detection device can then provide a heat source to heat specific locations on the microfluidic chip 301 (e.g., the reaction chamber 309, the valve block 321, etc.). The detection device can also detect fluorescent substances produced by sample reactions in the microfluidic chip 301 and read the test results.

[0502] The microfluidic chip 301 provided in this embodiment, through ingenious design of flow channel connections and chamber positions, eliminates the need for external drivers. This allows the liquid to independently and sequentially amplify and detect the sample under test based on its own asymmetric pressure. This process is simple to operate, offers rapid reaction times, and effectively enables high-throughput pathogen detection. Furthermore, the amplification and detection reagents in this microfluidic chip are all dry reagents, enabling the chip to be stored and transported at room temperature, avoiding the limitations of cold chain transportation and -20°C storage.

[0503] Example 14: Method for using an asymmetric pressure-partitioning chip

[0504] The asymmetric pressure typing chip provided in Example 13 can be applied to a variety of nucleic acid detection methods.

[0505] Method 1: Using genotyping detection based on enzymatic isothermal amplification technology as an example, the use of the microfluidic chip device is described:

[0506] 1) Transfer: The sample to be tested containing the lysate is added to the liquid storage chamber 304 of the microfluidic chip from the sample injection port 306. At this time, the tube cap 305 is tightened to seal the chip. The sample enters the bottom of the liquid storage chamber 304 and dissolves the built-in solid amplification reagent.

[0507] 2) Reaction: The chip is heated. After the temperature reaches a certain level, an amplification reaction occurs in the liquid storage chamber 304. The amplification reaction includes but is not limited to a constant temperature amplification reaction and a PCR reaction. While the amplification reaction is proceeding, the valve block 321, which blocks the liquid storage chamber 304 and the detection chamber 309, is also continuously melting. After the valve block 321 is completely melted, pressing or twisting the tube cap 305 drives the amplification product to move toward the reaction chamber 309 containing the solid detection reagent under the action of instantaneous asymmetric pressure, thereby achieving air pressure balance in the system. At the same time, the reagent in the reaction chamber 309 dissolves and the detection reaction proceeds.

[0508] 3) Read the results: After the reaction is completed, the fluorescence signal is read by the device to obtain the test results.

[0509] Method 2: Using enzymatic isothermal amplification technology for multiplex detection as an example, the use of the microfluidic chip device is described:

[0510] 2) Transfer: Add the sample to be tested containing the lysate into the liquid storage chamber 304 of the microfluidic chip from the sample injection port 306 and tighten the tube cap 305 to seal the chip.

[0511] 2) Reaction: The chip is heated. When the temperature reaches a certain level, the valve block 321, which blocks the liquid storage chamber 304 and the detection chamber 309, continues to melt. After the valve block 321 is completely melted, the sample liquid treated in the liquid storage chamber 304 is driven by the instantaneous asymmetric pressure of pressing or twisting the tube cover 305. The sample liquid moves to the reaction chamber 309 containing the solid amplification reagent, thereby achieving pressure balance in the system. At the same time, the reagent in the reaction chamber 309 dissolves and an amplification reaction occurs. The amplification reaction includes but is not limited to isothermal amplification reaction and PCR reaction.

[0512] 3) Read the results: After the reaction is completed, the fluorescence signal is read by the device to obtain the test results.

[0513] Example 15: Verification of the typing detection effect of asymmetric pressure-driven microfluidic chip

[0514] This example uses the asymmetric pressure-driven microfluidic chip of Example 13 (Figure 31) for typing detection, and uses ERA-CRISPR detection technology (enzymatic recombination amplification combined with CRISPR detection technology) to detect grass carp reovirus (GCRV) nucleic acid (GCRV, type I GCRV, type II GCRV, type III GCRV) in tissue specimens suspected of being infected with grass carp hemorrhagic disease, and uses qPCR detection method (commercial reagent) for comparative verification. The diseased fish specimens are lysed using a sample release agent to release nucleic acids, and the resulting lysate is used as a template. Using the same template loading amount, the samples are amplified and detected using an asymmetric pressure-driven microfluidic chip device and a qPCR detection method, respectively. The ERA-CRISPR detection technology pre-loads ERA freeze-dried microspheres containing specific amplification primers in the liquid storage chamber of the chip device, and the reaction chamber is pre-loaded with freeze-dried microspheres containing a specific CRISPR detection system. The sample is determined to determine whether it contains the corresponding pathogenic microorganism nucleic acid, and the results are shown in Table 5.

[0515] Table 5. Detection performance verification results of typing microfluidic chip

[0516] As shown in Table 5, when other conditions are the same, the detection results of the asymmetric pressure-driven microfluidic chip device of Example 13 are consistent with the results of the qPCR detection method, but it is more convenient and faster to use and is more suitable for on-site detection in the wild and special environments.

[0517] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. 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.

[0518] Although the present invention is disclosed above, it is not limited thereto. The present invention may be expanded according to its application scope in the field of microfluidics. Any person skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the claims.

Claims

1. A nucleic acid detection microfluidic chip, characterized in that: The chip is provided with a reaction chamber and a detection chamber; the reaction chamber is used for amplifying the target nucleic acid in the sample. obtaining amplification products; The detection chamber is used to detect the target nucleic acid in the amplification product; The reaction chamber and the detection chamber are connected via a fluid channel, and the fluid channel is used to achieve liquid or gas flow between the reaction chamber and the detection chamber; The reaction chamber and the detection chamber share an exhaust channel, and the exhaust channel is used to control the flow of gas or liquid in the reaction chamber or the detection chamber; The exhaust channel is connected to the fluid channel or the detection chamber; the combination of the fluid channel and the exhaust channel can automatically provide resistance for the sample in the reaction chamber to enter the detection chamber.

2. The nucleic acid detection microfluidic chip according to claim 1, characterized in that: The chip includes a gravity microfluidic chip and / or a centrifugal microfluidic chip; when the chip is a gravity microfluidic chip, the fluid channel can automatically provide resistance for the sample in the reaction chamber, making it difficult for the sample in the reaction chamber to automatically enter the detection chamber.

3. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: The length of the fluid channel is longer than the straight-line distance between the reaction chamber and the detection chamber.

4. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: The fluid channel is a folding or spiral or multi-turn circuitous flow channel, and the folding or multi-turn circuitous flow channel is provided with at least one bent structure.

5. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: The fluid channel extends toward the upper end of the reaction chamber or the upper end of the detection chamber or directly toward the top of the fluid channel.

6. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: The position of the detection chamber is higher than that of the reaction chamber, or the position of the sample inlet of the detection chamber is higher than that of the sample outlet of the reaction chamber, or the bottom of the detection chamber is not lower than the top of the reaction chamber.

7. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: At least one point of the fluid channel is higher than the reaction chamber.

8. The nucleic acid detection microfluidic chip according to claim 7, characterized in that: The fluid channel includes at least one arched flow channel, which includes an inlet section, a middle section and an outlet section. The middle section is arched upward, and the position of the middle section is higher than the inlet section and the outlet section, and at least the position of the middle section is higher than the reaction chamber.

9. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: It also includes a sample adding area; a sample injection channel is arranged between the sample adding area and the reaction chamber; and the aperture of the fluid channel is smaller than that of the sample injection channel.

10. The nucleic acid detection microfluidic chip according to claim 9, characterized in that: It also includes a main channel, the injection channel is fluidly connected to the branch port of the main channel, the injection channel is an upwardly arched arc channel, the inlet and outlet of the injection channel are respectively located at the low points on both sides of the arc channel, and the inlet is higher than the outlet; the highest point of the arc channel is not lower than the top of the reaction chamber.

11. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: The exhaust channel is connected to the detection chamber.

12. The nucleic acid detection microfluidic chip according to claim 11, characterized in that: A waterproof and breathable membrane is provided at the outlet of the exhaust passage of the detection cavity, which is used to control the connection state between the detection cavity and the outside atmosphere.

13. The nucleic acid detection microfluidic chip according to claim 12, characterized in that: The outlet of the injection channel is located at the bottom of the reaction chamber; the inlet of the fluid channel is located at the top of the reaction chamber; the outlet of the fluid channel is located at the bottom of the detection chamber; and the inlet of the exhaust channel is located at the top of the detection chamber.

14. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: It also includes a pressure application area, which is located on the chip or on a tube cover used to seal the chip sampling area, and is used to increase the pressure of the liquid in the reaction chamber so that the liquid in the reaction chamber flows into the detection chamber through the fluid channel.

15. The nucleic acid detection microfluidic chip according to claim 2, characterized in that: When the chip is a centrifugal microfluidic chip, the exhaust channel is connected to the fluid channel, so that the combination of the exhaust channel and the fluid channel can automatically provide resistance for the sample in the reaction chamber.

16. The nucleic acid detection microfluidic chip according to claim 15, characterized in that: The chip includes a chip body with a rotation center, the reaction chamber, the detection chamber, the fluid channel and the exhaust channel are located in the chip body, the rotation center is used to connect with the centrifugal driving device, so as to drive the centrifugal microfluidic chip to centrifuge; the exhaust channel includes an inlet and an outlet, the inlet is the connection between the fluid channel and the exhaust channel, and the outlet is the other end of the exhaust channel; the outlet is closer to the rotation center than the inlet, so that the exhaust direction of the exhaust channel is opposite to or at least partially opposite to the rotation centrifugal direction.

17. The nucleic acid detection microfluidic chip according to claim 16, characterized in that: There are two ways to set the outlet of the exhaust channel. One is to connect with the outside world, and a waterproof breathable membrane is provided at the outlet connected with the outside world; the other is to connect with the main flow channel of the chip body.

18. The nucleic acid detection microfluidic chip according to claim 17, characterized in that: When the centrifugal microfluidic chip is placed horizontally, the outlet of the exhaust channel is higher than the inlet. When the outlet of the exhaust channel is connected to the outside, a gas permeable cavity is also provided at the outlet of the exhaust channel, and the volume of the gas permeable cavity is not less than the reaction chamber.

19. The nucleic acid detection microfluidic chip according to claim 17, characterized in that: The chip body is circular; when the outlet of the exhaust channel is connected to the outside, the main channel is arranged in a wave-like manner; when the outlet of the exhaust channel is connected to the main channel, the main channel is arranged in a serpentine manner; the main channel is bent into a circular ring, and the chip body, the circular ring and the rotation center are coaxially arranged.

20. The nucleic acid detection microfluidic chip according to claim 19, characterized in that: The serpentine main flow channel includes U-shaped flow channels arranged at equal distances along a circular ring, and the bottom end of the U-shaped flow channel is connected to the reaction chamber; when the outlet of the exhaust channel is connected to the main flow channel, the connected part is the top end of the U-shaped flow channel.

21. The nucleic acid detection microfluidic chip according to claim 17, characterized in that: The main channel is also provided with a sample adding port. When the outlet of the exhaust channel is connected with the main channel, the sample adding port is located at the starting end of the main channel. A waste liquid pool is also provided at the end of the main channel.

22. The nucleic acid detection microfluidic chip according to claim 16, characterized in that: It also includes one or more sub-chips, which are concentric ring chips located outside the chip body. The sub-chip is also provided with a main channel, a reaction chamber and a detection chamber; when the chip body rotates centrifugally, the sub-chip can rotate centrifugally along with the chip body.

23. The nucleic acid detection microfluidic chip according to claim 16, characterized in that: The rotation center is a slot, and the slot enables the centrifugal microfluidic chip to be stuck on the rotation axis of the centrifugal drive device, thereby performing centrifugal rotation.

24. A method for nucleic acid detection, characterized in that: The nucleic acid detection microfluidic chip according to any one of claims 1 to 23 is used for detection. When the nucleic acid detection microfluidic chip is a centrifugal microfluidic chip, the detection method is divided into two types: A. When the outlet of the exhaust passage is connected to the outside, the detection method includes the following steps: (1) Place the centrifugal microfluidic chip horizontally and add the sample to be tested from the sample injection port; (2) The sample to be tested enters the reaction chamber of the centrifugal microfluidic chip from the sample loading area and is incubated at a temperature to perform nucleic acid amplification; (3) clamping the card slot of the centrifugal microfluidic chip on the rotating shaft of the detection device; (4) centrifuging to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction; (5) Read the test results; B. When the outlet of the exhaust channel is connected to the main channel, the detection method includes the following steps: (a) Place the centrifugal microfluidic chip horizontally and add the sample to be tested from the sample injection port; (b) clamping the card slot of the centrifugal microfluidic chip on the rotating shaft of the detection device; (c) low-speed centrifugation, where the sample to be tested enters the reaction chamber of the centrifugal microfluidic chip from the sample loading area for nucleic acid amplification; (d) centrifuging at a moderate speed to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction; (e) reading the test results; When the nucleic acid detection microfluidic chip is a gravity microfluidic chip, the detection method comprises the following steps: (I) vertically inserting the microfluidic chip into the detection device; (II) The sample enters the microfluidic chip from the sample inlet and flows into the reaction chamber under the action of gravity, and the tube is covered with a cap; (III) the sample to be tested undergoes nucleic acid amplification in the reaction chamber; (IV) squeezing the pressure application area to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction; (V) Read the test results.

25. A microfluidic chip, characterized in that: It includes a liquid storage chamber for adding or storing reagents and a reaction chamber for amplification or detection; an injection channel is provided between the liquid storage chamber and the reaction chamber to facilitate the liquid to flow from the liquid storage chamber to the reaction chamber under the action of gravity; the reaction chamber is also provided with an exhaust channel, which is finally connected to the liquid storage chamber to discharge the gas therein back to the liquid storage chamber.

26. The microfluidic chip according to claim 25, characterized in that: The reaction chamber is also provided with an injection channel, the cross-sectional area of ​​which is larger than the cross-sectional area of ​​the exhaust channel, so that the sample can flow into the reaction chamber from the injection channel but cannot flow out of the reaction chamber from the exhaust channel.

27. The microfluidic chip according to claim 26, characterized in that: The aperture of the injection channel is larger than the aperture of the exhaust flow channel.

28. The microfluidic chip according to claim 27, characterized in that: The exhaust passage is a multi-turn circuitous flow passage and is provided with at least one bent structure.

29. The microfluidic chip according to claim 25, characterized in that: The number of the reaction chamber is at least one, and each of the reaction chambers is connected to the main channel fluid of the microfluidic chip through an injection channel.

30. The microfluidic chip according to claim 29, characterized in that: The reaction chamber is provided with an injection port and an exhaust port, wherein the injection port is in fluid communication with an injection channel, and the exhaust port is in gas communication with an exhaust channel; and the exhaust port is located higher than the injection port.

31. The microfluidic chip according to claim 30, characterized in that: The injection port is located at the lower part of the reaction chamber, and the exhaust port is located at the upper part of the reaction chamber.

32. The microfluidic chip according to claim 31, characterized in that: The liquid storage chamber is provided with a liquid outlet and an air inlet, the liquid outlet is in fluid communication with the main channel, and the air inlet is in gas communication with the exhaust channel of the reaction chamber; the air inlet is located higher than the liquid outlet.

33. The microfluidic chip according to claim 25, characterized in that: A valve block may be added between the liquid storage chamber and the reaction chamber to control the state of fluid communication between the liquid storage chamber and the reaction chamber; the valve block has two forms, solid and liquid; when the valve block is solid, the liquid storage chamber and the reaction chamber cannot be fluidically connected; when the valve block is liquid, the liquid storage chamber and the reaction chamber are fluidically connected.

34. The microfluidic chip according to claim 33, characterized in that: When the valve block is solid, it is located in the main flow channel connecting the liquid storage chamber and the reaction chamber, and is used to prevent the sample to be tested in the liquid storage chamber from flowing into the reaction chamber; when the valve block is liquid, it will flow to the exhaust channel of the reaction chamber; the valve block is paraffin or lipid material, which is solid at room temperature and melts into liquid after heating; the density of the valve block is less than that of water.

35. The microfluidic chip according to claim 34, characterized in that: After the chip is sealed, the tube cover can also apply pressure to the chip to generate asymmetric pressure in the chip, thereby prompting the sample to flow from the liquid storage cavity into the reaction cavity through the injection channel.

36. The microfluidic chip according to claim 35, characterized in that: After the tube cover is closed, pressure can be applied to the chip by twisting or pressing the tube cover.

37. The microfluidic chip according to claim 36, characterized in that: A pushable air bag is provided on the top of the tube cover. After the chip is closed, the air bag is pushed downward to apply pressure to the inside of the chip.

Images