Nucleic acid multiple detection micro-fluidic chip

Abstract

According to the nucleic acid multiple detection micro-fluidic chip provided by the utility model, a multi-turn roundabout fluid channel is arranged between a reaction cavity and a detection cavity to realize fluid communication, and the aperture of the fluid channel is smaller than that of a sample injection flow channel of the reaction cavity, so that a sample can flow into the reaction cavity from a sample adding area under the action of gravity; and after being pressurized by the simple pressure applying device, the sample can smoothly enter the detection cavity from the reaction cavity through the fluid channel, so that the controllable transfer of the sample in different cavities is realized, and the backflow of the sample can be effectively prevented. The micro-fluidic chip is simpler in structure, only the exhaust channel and the waterproof breathable film need to be arranged in the detection cavity, the reaction cavity does not need to be provided with the exhaust channel, samples can be converted among different cavities only by means of gravity or a simple pressure applying device on the chip, extra driving equipment is not needed, and the cost is reduced. The structure and the operation process are greatly simplified on the basis of ensuring the detection precision, so that the device can adapt to more simple detection environments, is low in cost and integrated, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The utility model relates to the field of microfluidic control, and in particular to a nucleic acid multiple detection microfluidic chip. 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 for site, personnel and equipment in the detection process.

[0003] Existing microfluidic detection chips have realized the integration of multiple steps such as nucleic acid amplification and nucleic acid detection on a small chip, quickly completing the "sample in - result out" detection process. Since microfluidic chips are free from the dependence on precision pipetting devices and professionals, they have great potential in the field of instant detection.

[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 to achieve accurate detection, which is called nucleic acid multiplex detection. Nucleic acid multiplex detection requires the sample to be tested to be controllably transferred in multiple reaction chambers, and can prevent cross-influence caused by sample reflux. 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 and injection pumps, has a strong driving force, but needs to be 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 siphon action liquid flow driving method does not require equipment, the driving force is small and there is a problem of volatilization during the injection process.

[0006] In order to realize multiple nucleic acid detection, the inventor has applied for invention patent 2023117199558, disclosing a microfluidic chip that can realize multiple nucleic acid detection without relying on external force. A reaction chamber and a detection chamber are arranged on the microfluidic chip. Both the reaction chamber and the detection chamber need to be provided with exhaust channels and waterproof breathable membranes, and a valve block is added between the reaction chamber and the detection chamber. When the valve block is solid, the reaction chamber and the detection chamber cannot be fluidically connected; when the valve block is liquid, the detection chamber is fluidically connected to the reaction chamber. The temperature-increasing phase change performance of the valve block and the characteristics of the waterproof breathable membrane, combined with the principle of internal and external balance of atmospheric pressure, cleverly realize the controllable transfer of the samples to be tested in different chambers.

[0007] However, the setting of the valve block adds trouble to the preparation process of the microfluidic chip, and the heating temperature needs to be controlled to control the state of the valve block, which adds extra steps to the operation process. Therefore, it is urgent to find a nucleic acid multiple detection microfluidic chip with a simpler structure, more convenient operation, lower instrument requirements and lower manufacturing cost. Utility Model Content

[0008] In order to solve the above problems, the utility model provides a nucleic acid multiple detection microfluidic chip, which realizes fluid communication by setting a multi-turn circuitous fluid channel between the reaction chamber and the detection chamber, and the aperture of the fluid channel is smaller than the aperture of the reaction chamber injection flow 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, and then after being pressurized by a simple pressure 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 the sample from flowing back. The microfluidic chip has a simpler structure, and only needs to set an exhaust channel and a waterproof breathable membrane in the detection chamber, and the reaction chamber does not need to set an exhaust channel. The sample can be transferred in different chambers only by gravity or a simple pressure device on the chip, without the need for additional driving equipment. On the basis of ensuring the detection precision, the structure and operation process are greatly simplified, so that it can adapt to more simple detection environments, low-cost integration, and suitable for large-scale promotion and application.

[0009] On the one hand, the utility model provides a nucleic acid multiplex detection microfluidic chip, the chip comprising a sample addition area, a reaction chamber for sample pretreatment and a detection chamber for detecting samples; an injection channel is provided between the sample addition area and the reaction chamber; the reaction chamber and the detection chamber are fluidically connected via a fluid channel; the aperture of the fluid channel is smaller than the injection channel; under the action of gravity, the sample can flow from the sample addition area into the reaction chamber, but will not flow into the fluid channel.

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

[0011] During use, the microfluidic chip needs to be kept in a vertical state with the injection port facing upward. The following descriptions of the positional relationship between the various flow channels and chambers in the microfluidic chip are based on the microfluidic chip being kept in a vertical state. In some methods, the microfluidic chip can be inserted into a matching detection device, so that the microfluidic chip is kept vertically placed with the injection port facing upward, and the detection device can provide a heat source to heat specific positions of the microfluidic chip (such as reaction chambers, valve blocks, etc.); in some methods, the detection device can also detect the fluorescent substance produced after the sample reaction in the microfluidic chip and read the detection results.

[0012] Since the gas pressure in the system is balanced, the samples in the sample loading area do not require an external driving device and can be autonomously transferred to the reaction chamber under gravity alone. This process is easy to achieve. Once the sample enters the sample loading area, it will flow downward on its own and enter the reaction chamber through the injection channel.

[0013] The main channel of the microfluidic chip and the injection channel of the reaction chamber have large apertures, which 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, so that after the sample fills the reaction chamber, it will be blocked when continuing to move forward, 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.

[0014] Furthermore, the detection chamber is provided with an exhaust channel, and a waterproof breathable membrane is provided at the outlet of the exhaust channel to control the connection state between the detection chamber and the external atmosphere; the reaction chamber is not provided with an exhaust channel.

[0015] The setting of the exhaust channel and the waterproof breathable membrane of the detection cavity can realize the discharge of the gas in the reaction cavity and the detection cavity 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, so that the entire system is in a closed state and the liquid in the system stops 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 moistened by the exhaust channel, the system in the chip is in a closed state, so that the sample in the detection cavity stops flowing and no backflow occurs. In some methods, the waterproof breathable membrane is made of polymer material.

[0016] Existing nucleic acid multiplex detection microfluidic chips usually require a valve to be set between the reaction chamber and the detection chamber for isolation, so both the reaction chamber and the detection chamber need to be provided with an exhaust channel and a waterproof breathable membrane respectively.

[0017] In the chip provided by the utility model, the reaction chamber does not need to be provided with an exhaust channel, and the multi-turn and small-diameter fluid channel can act as a valve to temporarily block the flow of liquid between the reaction chamber and the detection chamber, but the reaction chamber and the detection chamber can always be ventilated, so 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 provided with an exhaust channel and a waterproof breathable membrane. Such a design makes the structure simpler, the cost lower, and the operation more convenient, while also ensuring the accuracy of the test results.

[0018] Furthermore, the fluid channel comprises a sample inlet section, a middle section and a sample outlet section, the middle section is arched upward, and the position of the middle section is higher than the sample inlet section and the sample outlet section.

[0019] The middle section of the fluid channel is arched 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.

[0020] Furthermore, the fluid channel is located between the reaction chamber and the detection chamber, and the middle section of the fluid channel extends toward the upper end of the reaction chamber.

[0021] The middle section of the fluid channel extends toward the upper end of the reaction chamber, thereby extending the length of the fluid channel and further increasing the difficulty for the sample in the reaction chamber to enter the fluid channel without additional pressure.

[0022] Furthermore, the fluid channel is a multi-turn circuitous flow channel and is provided with at least one bent structure.

[0023] Arranging multiple bent structures in the fluid channel can extend the length of the fluid channel as much as possible, maximizing the difficulty for the sample in the reaction chamber to enter the fluid channel without additional pressure, while providing a guarantee for all samples in the reaction chamber to remain in the reaction chamber.

[0024] Furthermore, the inlet of the fluid channel is located at the top of the reaction chamber; the outlet of the injection channel is located at the bottom 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.

[0025] The outlet of the injection flow channel is equivalent to the injection port of the reaction chamber, and the inlet of the fluid channel is equivalent to the outlet of the reaction chamber. The utility model is also designed for the positions of the injection port and the outlet of the reaction chamber. The injection port is at the bottom of the reaction chamber, and the outlet is at the top of the reaction chamber. The purpose of this is to ensure that when the sample enters the reaction chamber, it can be exhausted to the outlet smoothly to maintain the air pressure balance. At the same time, because the outlet is at the top of the reaction chamber, it is more difficult for the sample to flow out, so that the sample stays in the reaction chamber more stably.

[0026] The outlet of the fluid channel is equivalent to the injection port of the detection chamber, and the inlet of the exhaust channel is equivalent to the exhaust port of the detection chamber. The injection port 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 exhausted to the exhaust port smoothly when entering the detection chamber to maintain air pressure balance. Under the pressure of 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 full, the sample can also enter the exhaust channel and contact the waterproof breathable membrane, causing the waterproof breathable membrane to lose its breathability, making the chip as a whole in a closed state, and the sample stops flowing, so that it is completely retained in the detection chamber for detection reaction and completion of the detection of the target nucleic acid.

[0027] Furthermore, the detection chamber is located higher than the reaction chamber, and the bottom of the detection chamber is not lower than the top of the reaction chamber.

[0028] The position of the detection chamber is higher than the reaction chamber, which increases the difficulty for the sample in the reaction chamber to enter the detection chamber without additional pressure, and helps to prevent the sample in the reaction chamber from flowing into the detection chamber in advance.

[0029] Furthermore, it also includes a main channel, the injection channel is in fluid communication with the branch port of the main channel, the injection channel is an arcuate channel that arches upward, the inlet and outlet of the injection channel are respectively located at the low points on both sides of the arcuate channel, and the inlet is higher than the outlet.

[0030] Furthermore, the highest point of the arc-shaped flow channel is not lower than the top of the reaction chamber.

[0031] The arc-shaped 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, it is difficult for the sample to return from the injection flow channel to the main flow channel after entering the reaction chamber because the injection port of the reaction chamber is located at the lowest point of the injection flow channel. The sample cannot overcome the gravity and the pressure caused by the upward arched arc flow channel to return to the main flow channel and can only remain in the reaction chamber, thereby preventing sample reflux in the reaction chamber.

[0032] 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, the sample cannot flow into the fluid channel, and 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 stably remain in the reaction chamber for reaction.

[0033] 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.

[0034] It is understandable that the apertures of the injection channel and the fluid channel can be successfully prepared into a microfluidic chip, and the fluid channel can play a certain role in preventing liquid from easily flowing in due to the small aperture under the action of gravity and surface tension. The limitation on the aperture here does not mean that the aperture cannot be used if it exceeds or is smaller than the aperture. It is just a preferred implementation. For example, the aperture of the injection channel reaches 801μm, 805μm, 810μm, 820μm, 850μm, 900μm, 399μm, 395μm, 390μm, 385μm, 380μm, etc., or the aperture 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.

[0035] Furthermore, 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.

[0036] The purpose of setting the pressure zone is that after the sample completes the amplification reaction in the reaction chamber, the sample needs to enter the detection chamber from the reaction chamber. 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. Therefore, the pressure zone needs to provide sufficient pressure to drive the sample to be tested from the reaction chamber into the detection chamber.

[0037] It is understandable that the pressure area can be set at any position of the chip, such as in the middle of the chip, at the bottom of the chip, on the side of the chip, in the sample adding area of ​​the chip, in the detection area of ​​the chip, etc. It can even be set on the tube cover, as long as the air bag in the pressure area is pressed to provide pressure for the liquid in the reaction chamber, so that it can smoothly enter the detection area.

[0038] In some embodiments, the pressure zone is preferably set at the upper left or upper right of the chip, such as one side of the chip sample loading area, etc., because if the pressure zone is set on the sample loading area or the tube cover, although it can also provide a certain pressure, due to the limited volume of the sample loading area or the upper end of the tube cover, the airflow pressure provided is also 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 in advance. Therefore, the pressure zone is preferably set at the upper left or upper right of the microfluidic chip, and it should also reach a specific volume (specifically, it can be calculated according to the size of the 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.

[0039] The airbag in the pressure application area may be pressed manually or by using a matching detection device to provide a specific force to press the airbag.

[0040] In some embodiments, the pressure-applying area may be connected to the sample pool via a flow diversion channel, and airflow may be discharged from the flow diversion channel to the sample pool into the microfluidic chip by pressing.

[0041] Furthermore, the microfluidic chip also includes a sample adding area, which includes 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 microfluidic chip.

[0042] In some embodiments, the reaction chamber has a circular cross-section, and the circular cross-section can accommodate more samples in the reaction chamber.

[0043] In some embodiments, the cross-section of the detection cavity is in the shape of a water drop, and the exhaust port is located at the top of the water drop.

[0044] 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 gather upward, so that it is easier to enter the exhaust channel.

[0045] It is understandable that the solid reagents built into the reaction chamber and the detection chamber can be flexibly arranged according to needs, and different chambers can be equipped with different reaction reagents, such as the reaction chamber can be equipped with amplification primers of the target, and the detection chamber can be equipped with detection probes of different targets.

[0046] In some embodiments, the reaction chamber can serve as an ERA amplification chamber, and the detection chamber can serve as an ERA detection chamber.

[0047] In some embodiments, the reaction chamber can be used as an ERA reaction chamber, and the detection chamber can be used as a CRISPR detection chamber.

[0048] In some embodiments, the reaction chamber can be used as an ERA reaction chamber, and the detection chamber can be used as an Ago detection chamber.

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

[0050] In some embodiments, the microfluidic chip is used for multiple detection of (DNA and / or RNA) nucleic acids, and the reaction chamber is used for nucleic acid amplification. A nucleic acid amplification reagent needs to be placed in the reaction chamber in advance, and 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 acidsequence-based amplification), HDA (helicase-dependent amplification), etc.

[0051] In some embodiments, the solid detection reagent specifically includes a detection primer probe and / or a detection premix solution, and the detection premix solution includes an enzyme and a buffer solution required for the detection.

[0052] In some embodiments, the chip body structure uses a material with low cost, easy processing performance and good biocompatibility, such as PMMA (polymethyl methacrylate), PP (Polypropylene), PC (Polycarbonate) and other materials as a substrate, and directly uses laser etching technology to make channels and reaction grooves, or etches the structure on the PMMA substrate and then uses PDMS to cast the mold.

[0053] The method for multiple nucleic acid detection using the microfluidic chip provided by the utility model comprises the following steps:

[0054] (1) Insert the microfluidic chip vertically into the detection device;

[0055] (2) 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;

[0056] (3) The sample to be tested undergoes nucleic acid amplification in the reaction chamber;

[0057] (4) squeezing the pressure area to allow the sample to be tested after nucleic acid amplification to enter the detection chamber for reaction;

[0058] (5) Read the test results.

[0059] The nucleic acid multiple detection microfluidic chip provided by the utility model has a cleverly designed structure of the sample injection channel, fluid channel, chamber and exhaust channel. Without an external drive, the sample can enter the reaction chamber by its own gravity 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, thereby realizing the controllable transfer of samples in different chambers without the occurrence of sample reflux. The main beneficial effects are as follows:

[0060] 1. No external driving device is required, and the sample can be autonomously transferred to the reaction chamber only under gravity drive;

[0061] 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. The sample cannot enter under the action of gravity alone. As a result, after the sample fills the reaction chamber, it will be blocked when continuing to move forward, and it is difficult to flow out of the fluid channel at the top of the reaction chamber. All of the sample remains in the reaction chamber to complete the nucleic acid amplification reaction;

[0062] 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 gravity and the pressure brought by the injection channel to return to the main channel, and all remain in the reaction chamber to prevent sample reflux;

[0063] 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 exhaust channel and a waterproof breathable membrane, which has a simpler structure and lower cost.

[0064] 5. A simple pressure device is designed. When the sample completes nucleic acid amplification in the reaction chamber, pressing the pressure device can make the sample enter the detection chamber from the reaction chamber, realizing the controllable transfer of samples in different chambers;

[0065] 6. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a back view of the nucleic acid multiplex detection microfluidic chip in Example 1;

[0067] Figure 2 It is a front view of the nucleic acid multiplex detection microfluidic chip in Example 1;

[0068] Figure 3 A three-dimensional image of the nucleic acid multiplex detection microfluidic chip in Example 1 (back side facing upward);

[0069] Figure 4 It is a three-dimensional diagram of the nucleic acid multiplex detection microfluidic chip in Example 1;

[0070] Figure 5 This is a schematic diagram of the detection unit structure of the nucleic acid multiple detection microfluidic chip in Example 1;

[0071] Figure 6 It is a back view of the simple nucleic acid multiple detection microfluidic chip in Example 2;

[0072] Figure 7 It is a front view of the simple nucleic acid multiple detection microfluidic chip in Example 2;

[0073] Figure 8 This is a three-dimensional diagram of the simple nucleic acid multiplex detection microfluidic chip in Example 2. DETAILED DESCRIPTION

[0074] The preferred embodiments of the present invention are described in further detail below in conjunction with the accompanying drawings. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention. The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0075] In the description of the present utility model, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present utility model and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present utility model. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present utility model, unless otherwise specified, "multiple" means two or more.

[0076] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood by specific circumstances.

[0077] Example 1: Structure of gravity-driven microfluidic chip

[0078] The structure of the nucleic acid multiplex detection microfluidic chip provided in this embodiment is as follows Figures 1 to 5 As shown, Figure 1 This is the back view of the nucleic acid multiplex detection microfluidic chip. Figure 2 This is a front view of a nucleic acid multiplex detection microfluidic chip; Figure 3 A three-dimensional image of a nucleic acid multiplex detection microfluidic chip (back side facing upward); Figure 4 A three-dimensional diagram of a nucleic acid multiplex detection microfluidic chip; Figure 5 Schematic diagram of the detection unit structure.

[0079] like Figures 1 to 4 As shown, the nucleic acid multiple detection microfluidic chip 28 (hereinafter referred to as chip 28) provided in this embodiment includes a sample adding area 1 and a detection area 2. The sample adding area 1 includes a sample inlet 4, a sample pool 3 and a tube cover (not shown in the figure). The sample pool 3 is used to hold the sample to be tested. When the sample is added to the chip 28, it first enters the sample pool 3, then enters the main channel 6 under the action of gravity, and then flows into the detection area 2.

[0080] The detection area 2 can be provided with any number 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 detection units 7 are preferably provided, which are arranged in two columns, and each column is provided with five 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.

[0081] During use, the chip 28 needs to be kept in a vertical state, with the injection port 4 placed upward. When the positional relationship between the flow channels and chambers in the chip 28 is involved in this embodiment, the description is based on the chip 28 being kept in a vertical state. Since the air pressure in the system is balanced, 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 achieve. Once the sample enters the sample adding area 1, it will flow downward by itself and enter the reaction chamber 8 through the injection flow channel 17. The main flow channel 6 of the chip 28 and the injection flow 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 small aperture. Only under the action of gravity, the liquid cannot enter and flow smoothly, so that after the sample fills the reaction chamber 8, it will be blocked when continuing to advance, and it is 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 enter the detection chamber 9 through the fluid channel 19 under pressure to ensure accurate detection. The channel width of the main channel 6 and multiple sample injection channels 17 in the chip 28 is 400μm to 800μm. In this embodiment, the width of the main channel 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.

[0082] 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, which is used to control the connection state 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 make the gas in the reaction chamber 8 and the detection chamber 9 be discharged 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 will lose its breathability, so that the entire system is in a closed state and the liquid in the system stops flowing. Therefore, the waterproof breathable membrane 13 can help the sample in the detection chamber 9 to establish internal and external pressure balance, and can regulate the flow direction of the liquid. 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, so that the sample in the detection chamber 9 stops flowing and no backflow occurs. The waterproof breathable membrane is made of polymer material, such as high-grade synthetic rubber, with good sealing performance, no adhesive and residue, and no reaction with the reaction reagent or detection reagent. In this embodiment, the waterproof breathable membrane is preferably made of material (manufacturer: Membrane; item number: VET022H60). The chip 28 can also be covered with a plastic sheet 42 at the waterproof breathable membrane position. There is a gap between the plastic sheet 42 and the chip 28, which can not only connect the waterproof breathable membrane 13 with the outside gas, but also prevent the waterproof breathable membrane 13 from falling off, thereby protecting the waterproof breathable membrane 13.

[0083] In the chip 28, the reaction chamber 8 does not need to be provided with an exhaust channel, and the multi-turn and small-diameter fluid channel 19 can act as a valve to temporarily block the flow of liquid between the reaction chamber 8 and the detection chamber 9, but the reaction chamber 8 and the detection chamber 9 can always be ventilated, so the reaction chamber 8 and the detection chamber 9 can share the exhaust channel 11 located on the detection chamber 9 for exhaust, and the reaction chamber 8 does not need to be provided with an exhaust channel 11 and a waterproof breathable membrane 13. Such a design makes the structure simpler, the cost lower, and the operation more convenient, while also ensuring the accuracy of the test results.

[0084] like Figure 5The fluid channel 19 includes a sample injection section 14, a middle section 15 and a sample output section 16, and the middle section 15 is arched upward, and the position of the middle section 15 is higher than the sample injection section 14 and the sample output 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 the sample in the reaction chamber 8 to remain 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, so that the length of the fluid channel 19 is extended, which increases 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, and is provided with at least one bent structure 21. The fluid channel 19 is provided with a plurality of bent structures 21, which can extend the length of the fluid channel 19 as much as possible, and increase the difficulty of the sample in the reaction chamber 8 entering the fluid channel 19 without additional pressure, but provide a guarantee for the sample in the reaction chamber 8 to remain in the reaction chamber 8. The inlet 22 of the fluid channel 19 is located at the top 10 of the reaction chamber; the outlet 23 of the injection channel 17 is located at the bottom 24 of the reaction chamber; the outlet 25 of the fluid channel 19 is located at the bottom 26 of the detection chamber; and the inlet 27 of the exhaust channel 20 is located at the top 29 of the detection chamber.

[0085] The outlet 23 of the injection channel 17 is equivalent to the injection port 30 of the reaction chamber 8, and the inlet 22 of the fluid channel 19 is equivalent to the outlet 31 of the reaction chamber 8. The injection port 30 is at the bottom of the reaction chamber 8, and the outlet 31 is at the top 10 of the reaction chamber. The purpose of this is to ensure that when the sample enters the reaction chamber 8, it can be exhausted to the outlet 31 smoothly to maintain the air pressure balance. At the same time, since the outlet 31 is at the top of the reaction chamber 8, it is more difficult for the sample to flow out, so that the sample stays in the reaction chamber 8 more stably. The outlet 25 of the fluid channel 19 is equivalent to the injection port 32 of the detection chamber 9, and the inlet 27 of the exhaust channel 11 is equivalent to the exhaust port 33 of the detection chamber 9. The injection port 32 is at the bottom 26 of the detection chamber 9, and the exhaust port 33 is at the top 29 of the detection chamber 9. The purpose of this is to ensure that when the sample enters the detection chamber 9, it can be exhausted to the exhaust port 33 smoothly to maintain the air pressure balance. Under the pressure of the pressure-applying area 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 chip 28 as a whole in a closed state, and the sample stops flowing, thereby being completely retained in the detection chamber 9 for detection reaction and completion of the detection of the target nucleic acid.

[0086] The detection chamber 9 is located 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.

[0087] like 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 arc channel 36. The inlet 37 and the outlet 38 of the sample inlet channel 17 are respectively located at the low points on both sides of the arc channel 36, and the inlet 37 is higher than the outlet 38; the highest point 39 of the arc channel 36 is not lower than the top 10 of the reaction chamber. The arc channel design of the sample inlet channel 17 can make it easier for the sample to enter the sample inlet channel 17 from the branch port 35 and then enter the reaction chamber 8 through the sample inlet channel 17. However, it is difficult for the sample to return to the main channel 6 from the sample inlet channel 17 after entering the reaction chamber 8, 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 gravity and the pressure brought by the upwardly arched arc channel 36 to return to the main channel 6, and can only remain in the reaction chamber 8, thereby preventing the sample from flowing back in the reaction chamber 8.

[0088] 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 in 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 and small-aperture flow channel, the sample cannot flow into the fluid channel 19, and the sample is also difficult 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 all the samples will stably remain in the reaction chamber 8 for reaction.

[0089] like Figure 2The pressure area 34 of the chip 28 is located on one side of the sample adding 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 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 according to the size of the chip 28 and the required pressure, so as to set the airbag 40 of a suitable volume), so that the pressing process provides a 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 the airbag 40 can be pressed by a matching detection device to provide a specific force. The pressure application area 34 may be connected to the sample pool 3 via the branch channel 41 , and by pressing, airflow is discharged from the branch channel 41 to the sample pool 3 and into the chip 28 .

[0090] like Figure 5 As shown, 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 in the shape of a water drop, and the exhaust port 33 is located at the top of the water drop. The cross section of the detection chamber 9 is in the shape of a water drop, which helps the gas or liquid in the detection chamber 9 to converge upward, so that it is 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, and different chambers can be built 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.

[0091] A nucleic acid amplification reagent needs to be placed in the reaction chamber 8 in advance. 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 acidsequence-based amplification), HDA (helicase-dependent amplification), etc.

[0092] The detection chamber 9 is used for the 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 the detection.

[0093] The main structure of the chip 28 provided in this embodiment adopts a material with low cost, easy processing performance and good biocompatibility chip, such as PMMA (polymethyl methacrylate), PP (Polypropylene), PC (Polycarbonate) and other materials as a substrate, and directly uses laser etching technology to make channels and reaction grooves, or etches the structure on the PMMA substrate and then uses PDMS to cast.

[0094] The chip 28 provided in this embodiment cleverly designs the structures of the sample injection 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-applying device 34, thereby realizing the controllable transfer of samples between different chambers without causing sample reflux.

[0095] Example 2: Simple nucleic acid multiplex detection microfluidic chip

[0096] This embodiment provides a simplified version of a nucleic acid multiplex detection microfluidic chip, such as Figures 6 to 8 As shown, Figure 6 This is the back view of a simple nucleic acid multiplex detection microfluidic chip. Figure 7 This is a front view of a simple nucleic acid multiplex detection microfluidic chip. Figure 8 A three-dimensional image of a simple nucleic acid multiplex detection microfluidic chip.

[0097] The difference between the simple nucleic acid multiple detection microfluidic chip provided in this embodiment and the nucleic acid multiple detection microfluidic chip in embodiment 1 is that only a single row of detection units 7 are designed, and the detection units 7 are placed vertically, the reaction chamber 8 is located below, the detection chamber 9 is located directly above the reaction chamber 8, and a fluid channel 19 is arranged between the reaction chamber 8 and the detection chamber 9; the sample pool 3 is located on one side of the detection unit 7, and 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, it is squeezed by the pressure area 34 ( Figure 6 ) can make the sample smoothly enter the detection chamber 9, and enter the exhaust channel 11 of the detection chamber 9, contact the waterproof breathable membrane 13, so that the whole system is closed, the sample stops flowing, and the detection is completed in the detection chamber 9.

[0098] Example 3: Method for using a nucleic acid multiplex detection microfluidic chip

[0099] 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 takes multiplex detection based on enzymatic isothermal amplification technology as an example to illustrate the use of the chip 28:

[0100] 1) The chip 28 is vertically inserted into the detection device;

[0101] 2) Transfer: The sample to be tested containing the lysate is introduced into the chip 28 from the injection port 4, and the sample flows into the reaction chamber 8 under the action of gravity, and the tube cover is tightened to seal the chip;

[0102] 3) The sample to be tested undergoes nucleic acid amplification in the reaction chamber 8;

[0103] 4) After the nucleic acid amplification reaction is completed, the pressure application area 34 is squeezed to allow the sample to be tested after the nucleic acid amplification to enter the detection chamber 9 for reaction;

[0104] 3) Read the results: After the reaction is completed, the fluorescence signal is read by the device to obtain the test results.

[0105] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0106] The above is only a specific implementation mode of the utility model, and its description is relatively specific and detailed, but the protection scope of the utility model is not limited to this. Any technician familiar with the technical field can easily think of changes or replacements without departing from the concept of the utility model, which belong to the protection scope of the utility model. Therefore, the protection scope of the utility model patent shall be based on the protection scope of the claims. Although the utility model is disclosed as above, the utility model is not limited to this. For example, it can be expanded according to its scope of application in the field of microfluidics. Any technical personnel in this field can make various changes and modifications without departing from the spirit and scope of the utility model, so the protection scope of the utility model shall be based on the scope defined by the claims.

Claims

1. A nucleic acid multiplex detection microfluidic chip, characterized in that: It includes a sample adding area, a reaction chamber for sample pretreatment and a detection chamber for sample detection; an injection channel is provided between the sample adding area and the reaction chamber; the reaction chamber and the detection chamber are fluidically connected via a fluid channel; the aperture of the fluid channel is smaller than the injection channel; under the action of gravity, the sample can flow from the sample adding area into the reaction chamber, but will not flow into the fluid channel.

2. The nucleic acid multiplex detection microfluidic chip according to claim 1, characterized in that: The detection chamber is provided with an exhaust channel, and a waterproof breathable membrane is provided at the outlet of the exhaust channel to control the connection state between the detection chamber and the outside atmosphere; the reaction chamber is not provided with an exhaust channel.

3. The nucleic acid multiplex detection microfluidic chip according to claim 2, characterized in that: The fluid channel comprises a sample injection section, a middle section and a sample output section. The middle section is arched upwards and is located higher than the sample injection section and the sample output section.

4. The nucleic acid multiplex detection microfluidic chip according to claim 3, characterized in that: The fluid channel is located between the reaction chamber and the detection chamber, and the middle section of the fluid channel extends toward the upper end of the reaction chamber.

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

6. The nucleic acid multiplex detection microfluidic chip according to claim 5, characterized in that: The inlet of the fluid channel is located at the top of the reaction chamber; the outlet of the injection channel is located at the bottom 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.

7. The nucleic acid multiplex detection microfluidic chip according to claim 6, characterized in that: The detection chamber is located higher than the reaction chamber, and the bottom of the detection chamber is not lower than the top of the reaction chamber.

8. The nucleic acid multiplex detection microfluidic chip according to claim 7, 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.

9. The nucleic acid multiplex detection microfluidic chip according to claim 8, characterized in that: The highest point of the arc-shaped flow channel is not lower than the top of the reaction chamber.

10. The nucleic acid multiplex detection microfluidic chip according to claim 9, 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.

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