Constant-temperature home self-checking microfluidic detection device and application thereof

Abstract

The invention provides a constant-temperature household self-checking microfluidic detection device and application thereof, and belongs to the technical field of biochemical detection.The device comprises an upper cover, a thermal reaction bottom plate and a microfluidic reaction plate, and the upper cover is provided with a sample adding hole and a sample adding hole cover used for covering the sample adding hole; a water injection hole and a water injection hole plug matched with the water injection hole are arranged at the position close to the sample adding hole; a heating bin is arranged on the thermal reaction bottom plate, the heating bin comprises a thermal reaction tank A and a thermal reaction tank B, the thermal reaction tank A and the thermal reaction tank B are used for carrying out heating reaction of the heating bag and thermal slow-release reaction of the heat release bag respectively, and a heat exchange liquid flow port is formed between the thermal reaction tank A and the thermal reaction tank B; and water or thermal reaction liquid which is added through the water injection hole and used for thermal reaction is heated by the heating bag in the thermal reaction tank A and then flows to the thermal reaction tank B through the heat exchange liquid flow port. The device can thoroughly solve the technical problem that the device can only be used by an instrument, a power supply, a circuit board or a lithium battery. The method has the advantages of short time, low cost, rapidness, simplicity, safety and reliability.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to a constant-temperature home self-testing microfluidic detection device and applications thereof. Background Art

[0002] With the rapid development of nucleic acid molecular detection technology, it has been widely applied in numerous fields, including pathogen detection, genetics, forensic identification, environmental microbiology, and genetic breeding. Mainstream molecular detection methodologies include PCR, NGS, and constant-temperature detection techniques. PCR and NGS rely primarily on high-precision instrumentation, software interpretation, and bioinformatics support. Therefore, these technologies have become high-end research tools for hospitals, universities, research institutes, and other institutions with comprehensive laboratory facilities and facilities.

[0003] In the early 21st century, the emergence of molecular isothermal amplification methodologies opened up new avenues for molecular testing. This technology requires minimal, if any, instrumentation, significantly reducing the hardware investment and operational barriers to entry for molecular testing. This allowed molecular testing to truly enter the community and home testing market as a point-of-care (POCT) product and testing technology. Home testing eliminates the need for medical facilities, long journeys, and lengthy waits for doctors to take samples and perform tests. Instead, users can complete the entire process of sampling, testing, and obtaining results independently at home. Home testing offers numerous advantages, including convenience, visual and rapid results, strong privacy, contactlessness, environmental flexibility, and the ability to conserve medical resources. Therefore, home testing holds broad application prospects in numerous health areas, including epidemic prevention and control, private disease diagnosis, companion diagnostics, and daily health monitoring.

[0004] The invention patent with publication number CN115537320A discloses a fully enclosed nucleic acid rapid test paper detection microfluidic chip and its portable system. In the upper layer of the fully enclosed nucleic acid rapid test paper detection microfluidic chip, the amplification reaction liquid storage chamber is connected to the sample and lysate sampling hole through the first microchannel, the amplification reaction liquid storage chamber is connected to the amplification liquid sampling hole, and the amplification liquid sampling hole is correspondingly provided with an amplification liquid sampling hole plug; the middle hole of the sample and lysate sampling hole in the middle layer is connected to the sample and lysate sampling hole. The upper chamber of the liquid storage chamber is connected through the second microfluidic channel, the upper hole of the sample and lysate sampling hole is connected with the middle hole of the sample and lysate sampling hole to form the sample and lysate sampling hole, and the sample and lysate sampling hole is correspondingly provided with a reaction chamber sampling hole plug; the reaction chamber in the lower layer is connected with the lower chamber of the liquid storage chamber through the third microfluidic channel, the detection chamber is connected with the lower chamber of the liquid storage chamber and a nucleic acid rapid detection test paper is arranged inside, wherein the lower chamber of the liquid storage chamber is connected with the upper chamber of the liquid storage chamber to form the liquid storage chamber.

[0005] The invention with announcement number: CN113512490B discloses a self-driven microfluidic detection device and its use. The device includes: an upper cover, a bottom plate fixed under the upper cover, an interlayer arranged between the upper cover and the bottom plate, a sample loading hole arranged on the upper cover, a microfluidic guide component connected to the sample loading hole, a sample loading groove arranged on the interlayer and corresponding to the position of the microfluidic guide component, a functional chamber arranged on the interlayer and connected to the microfluidic guide component, and a chromatography test paper connected to the microfluidic guide component; the microfluidic guide component includes: a capillary guide channel, and an on-off connector connected to the capillary guide channel and controlling the connection and disconnection of the capillary guide channel; this device realizes precise control of the sample loading and detection process, improves detection accuracy, can be operated quickly and easily without professional training, and is safe and reliable; this device can detect a variety of samples and has a very wide range of applications.

[0006] The detection device in the prior art also has the following shortcomings: Existing detection devices all rely on external power sources or built-in lithium batteries to complete the constant temperature heating and amplification process. Detection requires the use of printed circuit boards for circuit control. This results in a highly complex production process, making mass production difficult and leading to high retail prices.

[0007] Existing detection devices often use photodetectors or electrochemical potential detection devices to collect and analyze the signals of amplified products, requiring sophisticated equipment. This significantly limits the widespread application of nucleic acid self-testing at home among non-professionals. Furthermore, since these self-tests require consumers to purchase the test device, this also significantly impacts customer repurchase rates and user experience.

[0008] While current home self-testing products based on lateral flow test strips (LFDs) do address the need for complex and precise photoelectric detectors, the testing process still relies on a power supply, circuit boards, or lithium batteries. This still significantly lags behind home tests based on antigen colloidal gold in terms of ease of use, low cost, and rapid response. Summary of the Invention

[0009] The present invention aims to provide a constant-temperature, home-based self-testing microfluidic detection device and its use, which can completely solve the technical problem of relying on instruments, power supplies, circuit boards, or lithium batteries for use. It has the advantages of being quick, low-cost, quick, simple, safe, and reliable.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is: A constant temperature home self-test microfluidic detection device, comprising an upper cover, a thermal reaction bottom plate and a microfluidic reaction plate. The upper cover is provided with a sample addition hole and a sample addition hole cover for covering the sample addition hole, and a water injection hole and a water injection hole plug matching the water injection hole are provided near the sample addition hole. A heating chamber is provided on the thermal reaction base plate. The heating chamber includes a thermal reaction pool A and a thermal reaction pool B, which respectively carry out the heating reaction of the heating pack and the thermal slow-release reaction of the heat-releasing pack. A heat exchange liquid flow port is provided between the thermal reaction pool A and the thermal reaction pool B. Water or thermal reaction liquid for thermal reaction added through the water injection hole is heated by the heating pack in the thermal reaction pool A and then flows to the thermal reaction pool B through the heat exchange liquid flow port. An air insulation area is provided around the heating chamber. The upper cover and the thermal reaction base plate are buckled together, and the microfluidic reaction plate is located between the upper cover and the thermal reaction base plate. A constant temperature amplification reaction chamber for constant temperature amplification is distributed on the microfluidic reaction plate. Wide groove siphon flow channels and product diffusion flow channels for liquid flow control of samples and amplification products are distributed at both ends of the constant temperature amplification reaction chamber.

[0011] Furthermore, the wide-slot iris suction flow channel is engaged with the narrow-slot iris suction flow channel of the upper cover.

[0012] Furthermore, the product diffusion channel is divided into two sections, namely an upstream buffer channel and a downstream diffusion channel. The amplification products flowing out of the constant temperature amplification reaction chamber flow into the diffusion channel through the buffer channel.

[0013] Among them, a product detection area is set downstream of the diffusion channel, and the product detection area has several lateral flow chromatography test strips. Each lateral flow chromatography test strip corresponds to a diffusion channel, and each test strip is embedded in a test strip limiting groove. The test strip has a test strip limiting baffle at the water absorption end to fix the test strip.

[0014] Furthermore, an observation window is provided on the upper cover.

[0015] Among them, a number of visual grids are set on the observation window, and each visual grid corresponds to a lateral flow chromatography test paper.

[0016] The observation window is covered with a transparent cover to seal and protect the product detection area.

[0017] Furthermore, the product detection zone is located downstream of the product diffusion channel, and the horizontal position of the product detection zone is lower than that of the product diffusion channel.

[0018] Furthermore, the constant-temperature amplification reaction chamber is located between the wide-slot siphon flow channel and the product diffusion channel. The chamber contains constant-temperature amplification reagent beads. Sample solution is added through the sample injection port and evenly distributed within the microfluidic flow channel. Once the constant-temperature amplification reagent beads dissolve, the amplification reaction can then proceed within the chamber.

[0019] The present invention also provides a constant temperature home self-test microfluidic detection device for use, which is used for influenza A, influenza B, adenovirus, and new coronavirus nucleic acid detection.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention mainly consists of an upper cover, a thermal reaction base plate, and a microfluidic reaction plate. The present invention adopts a microfluidic method to achieve precise and autonomous distribution of nucleic acid samples in the flow channel, and complete complex isothermal reactions and visual detection. The purpose of the present invention is to provide a microfluidic detection device and its use. The microfluidic flow channel automatic distribution technology is used to achieve multiple indicators such as sample addition and autonomous flow of samples, high-precision liquid separation, and automatic disintegration and melting of reaction spheres. The operation process is almost the same as that of the antigen colloidal gold mode, which is time-saving, low-cost, fast, simple, safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 It is a schematic diagram of the overall structure of the present invention.

[0023] Figure 2 It is an explosion schematic diagram of the present invention.

[0024] Figure 3 It is a schematic diagram of the overall structure of the upper cover of the present invention.

[0025] Figure 4 It is a schematic diagram of the overall structure of the thermal reaction base plate of the present invention.

[0026] Figure 5 It is a schematic diagram of the overall structure of the microfluidic reaction plate of the present invention.

[0027] Figure 6 It is a schematic diagram of the internal structure of the present invention.

[0028] Reference numerals: 100-top cover, 101-sample loading port, 102-water injection port, 103-narrow slot siphon flow channel, 104-observation window, 1041-visual grid, 200-thermal reaction bottom plate, 201-thermal reaction pool, 2011-thermal reaction pool A, 2012-thermal reaction pool B, 2013-heat exchange liquid flow port, 204-air insulation area, 300-microfluidic reaction plate, 301-constant temperature amplification reaction chamber, 302-wide slot siphon flow channel, 303-product diffusion channel, 3031-buffer channel, 3032-diffusion channel, 304-product detection area, 3041-test paper limiting groove, 3042-test paper limiting baffle, 400-sample loading port cover, 500-transparent cover. DETAILED DESCRIPTION

[0029] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0030] In the description of the embodiments of the present invention, it should be understood that the terms "length", "vertical", "horizontal", "top", "bottom", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the embodiments of the present invention.

[0031] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly specified.

[0032] In the embodiments of the present invention, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," "fixed," etc. should be understood in a broad sense. For example, they can refer to fixed connection, detachable connection, or integration; mechanical connection, electrical connection, or communication; direct connection or indirect connection through an intermediate medium; internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the embodiments of the present invention based on specific circumstances.

[0033] In the embodiments of the present invention, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Furthermore, a first feature being "above," "above," and "above" a second feature may include the first feature being directly above and obliquely above the second feature, or may simply mean that the first feature is higher in level than the second feature. A first feature being "below," "below," and "below" a second feature may include the first feature being directly above and obliquely above the second feature, or may simply mean that the first feature is lower in level than the second feature.

[0034] The disclosure below provides many different embodiments or examples for implementing different structures of the embodiments of the present invention. In order to simplify the disclosure of the embodiments of the present invention, the components and configurations of specific examples are described below. Of course, these are merely examples and are not intended to limit the embodiments of the present invention. In addition, the embodiments of the present invention may repeat reference numerals and / or reference letters in different examples. Such repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or configurations discussed.

[0035] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0036] See Figures 1-6 This embodiment discloses a constant temperature home self-test microfluidic detection device, comprising an upper cover 100, a thermal reaction base plate 200 and a microfluidic reaction plate 300. The upper cover 100 is provided with a sample addition hole 101 and a sample addition hole cover 400 for covering the sample addition hole 101. A water injection hole 102 and a water injection hole plug matching the water injection hole 102 are provided near the sample addition hole 101. Thermal reaction pools 201 are provided on the thermal reaction base plate 200. Thermal reaction pools 201 include thermal reaction pool A 2011 and thermal reaction pool B 2012, which respectively perform the heating reaction of the heating pack and the slow-release heat reaction of the heat-releasing pack. A heat exchange liquid flow port 2013 is provided between thermal reaction pools A 2011 and B 2012. Water or thermal reaction liquid for the thermal reaction, added through the water injection hole 102, is heated by the heating pack in thermal reaction pool A 2011 and then flows through the heat exchange liquid flow port 2013 to thermal reaction pool B 2012. An air insulation area 204 is provided around the thermal reaction pools 201. The upper cover 100 and the thermal reaction base plate 200 are buckled together, and the microfluidic reaction plate 300 is located between the upper cover 100 and the thermal reaction base plate 200. A constant temperature amplification reaction chamber 301 for constant temperature amplification is distributed on the microfluidic reaction plate 300. Wide groove siphon flow channels 302 and product diffusion channels 303 for liquid flow control of samples and amplification products are distributed at both ends of the constant temperature amplification reaction chamber 301.

[0037] Furthermore, the wide-slot iris suction flow channel 302 is engaged with the narrow-slot iris suction flow channel 103 of the upper cover 100 .

[0038] Furthermore, the product diffusion channel 303 is divided into two sections, namely an upstream buffer channel 3031 and a downstream diffusion channel 3032 . The amplification products flowing out of the constant temperature amplification reaction chamber 301 flow through the buffer channel 3031 to the diffusion channel 3032 .

[0039] Among them, a product detection area 304 is set downstream of the diffusion channel 3032. The product detection area 304 has several lateral flow chromatography test strips. Each lateral flow chromatography test strip corresponds to a diffusion channel 3032. Each test strip is embedded in a test strip limiting groove 3041. A test strip limiting baffle 3042 is provided at the water absorption end of the test strip to fix the test strip.

[0040] Furthermore, an observation window 104 is provided on the upper cover 100 .

[0041] A plurality of visual grids 105 are provided on the observation window 104 , and each visual grid 105 corresponds to a lateral flow chromatography test paper.

[0042] The observation window 104 is covered with a transparent cover plate 500 for sealing and protecting the product detection area 304 .

[0043] Furthermore, the product detection zone 304 is located downstream of the product diffusion channel 303 , and the horizontal position of the product detection zone 304 is lower than that of the product diffusion channel 303 .

[0044] Furthermore, the constant-temperature amplification reaction chamber 301 is located between the wide-slot siphon flow channel 302 and the product diffusion channel 303. The constant-temperature amplification reaction chamber contains constant-temperature amplification reagent beads. Sample solution is added through the sample injection port 101 and evenly distributed within the microfluidic flow channel. Once the constant-temperature amplification reagent beads are dissolved, an amplification reaction can then be performed within the constant-temperature amplification reaction chamber.

[0045] The present invention also provides a constant temperature home self-test microfluidic detection device for use, which is used for influenza A, influenza B, adenovirus, and new coronavirus nucleic acid detection.

[0046] This invention utilizes a microfluidic chip to achieve precise, autonomous distribution of nucleic acid sample solutions within the flow channel, as well as complex constant-temperature reactions and visual detection. Utilizing microfluidic channel automated distribution technology, it achieves multiple performance goals, including sample loading and autonomous flow, high-precision liquid separation, and automated disintegration and melting of reaction spheres. This approach offers the advantages of rapid, cost-effective, simple, safe, and reliable processing.

[0047] In the present invention, the thermal reaction pool 201 is composed of a thermal reaction pool A 2011 and a thermal reaction pool B 2012 ; a heat exchange liquid flow port 2013 is provided between the thermal reaction pool A 2011 and the thermal reaction pool B 2012 .

[0048] The narrow groove siphon suction flow channel 103 located on the upper cover 100, the wide groove siphon suction flow channel 302 located on the microfluidic reaction plate 300, the constant temperature amplification reaction chamber and the product diffusion flow channel 303 together constitute a microfluidic reaction assembly (microfluidic reaction plate 300).

[0049] In the present application, the sample loading hole 101 of the upper cover 100 is connected to the narrow slot siphon flow channel 103 to drain the added sample to the constant temperature amplification reaction chamber, and the sample loading hole cover 400 of the upper cover 100 is tightly combined with the sample loading hole 101 of the upper cover 100 to seal the sample loading hole 101; Preferably, the observation windows 104 of the upper cover 100 are 1-5 observation ports, covering the lateral flow chromatography test paper in the detection area, and each observation port formed corresponds to a lateral flow chromatography test paper for observing the chromatography results.

[0050] In the present application, the thermal reaction pool A2011 is connected to the water injection hole 102 of the upper cover 100, and the thermal reaction pool A2011 is connected to the thermal reaction pool B2012 at the bottom via a heat exchange liquid outlet 2013. Water or other liquids participating in the thermal reaction are heated by the heating pack of the thermal reaction pool A2011 and can flow to the thermal reaction pool B2012 to exchange heat with the heating pack of the thermal reaction pool B2012. The thermal reaction pool B2012 is embedded in the constant temperature amplification reaction chamber of the upper microfluidic reaction plate 300 to perform heat controlled release for the constant temperature amplification reaction.

[0051] The air insulation area 204 of the thermal reaction base plate 200 separates the thermal reaction pools 201A and B, reduces heat loss in the thermal reaction pools 201A and B, maintains a constant temperature in the detection area, and controls the temperature of the detection area to below 37°C.

[0052] The narrow slot siphon flow channel 103 is embedded in the wide slot siphon flow channel 302 and the constant temperature amplification reaction chamber 301, breaking the surface tension of the liquid, forming a liquid siphon force between the siphon channels that is greater than the internal adhesion of the liquid, thereby guiding the liquid flow. The product diffusion channel 303 is located downstream of the constant temperature amplification reaction chamber 301. The product diffusion channel 303 consists of a buffer channel 3031 and a diffusion channel 3032. The buffer channel 3031 is narrower than the diffusion channel 3032. The amplification product solution enters the buffer channel 3031 from the constant temperature amplification reaction chamber and flows toward the diffusion channel 3032 under capillary action. Upon reaching the diffusion channel 3032, the capillary action weakens because the diffusion channel 3032 is wider and horizontally lower than the buffer channel 3031, and the siphon force takes over, resulting in slow flow, thus preventing rapid surge of the product solution. As the product solution slowly flows, it diffuses to the product detection area 304. Each diffusion channel 3032 corresponds to a lateral flow chromatography test strip.

[0053] The narrow slot siphon suction flow channel of the preferred siphon suction flow channel is located on the inner side of the upper cover 100, and the slot spacing range is: 0.01-5mm; the wide slot siphon suction flow channel 302 of the siphon suction flow channel is located on the microfluidic reaction plate 300, and can be interlocked with the siphon suction flow channel located on the inner side of the upper cover 100, and the slot spacing range is: 0.02-10mm; the constant temperature amplification reaction chamber is located at the end of the wide slot siphon suction flow channel 302, and is interlocked with the end of the narrow slot siphon suction flow channel 103, and the volume range of the constant temperature amplification reaction chamber is: 10-100μL; the width range of the buffer channel 3031 of the product diffusion flow channel 303 is: 0.02-10mm, and the width range of the diffusion channel 3032 of the product diffusion flow channel 303 is: 0.01-10mm.

[0054] In order to facilitate those skilled in the art to further understand the present invention, the present invention is further described below with reference to specific implementation cases.

[0055] See Figure 1-Figure 5 , this embodiment discloses a constant temperature home self-test microfluidic detection device, comprising an upper cover 100, a thermal reaction base plate 200 and a microfluidic reaction plate 300; The system also includes a sample loading hole 101 provided on the upper cover 100 and a sample loading hole cover 400 covering the sample loading hole 101 to provide sealing protection for the sample loading hole 101. A water injection hole 102 is provided adjacent to the sample loading hole 101 for injecting water or heating reaction liquid into the thermal reaction base plate 200 engaged with the upper cover 100, and an independent water injection hole plug is provided for sealing the water injection hole 102.

[0056] The thermal reaction base plate 200 is provided with a thermal reaction pool 201, which includes a thermal reaction pool A and a thermal reaction pool B2012, which respectively perform a heating reaction of the heating pack and a heat-releasing pack heat-release reaction. A heat exchange liquid flow port 2013 is provided between the thermal reaction pool A and the thermal reaction pool B2012. Water or thermal reaction liquid for thermal reaction added through the water injection hole 102 is heated by the heating pack in the thermal reaction pool A and flows to the thermal reaction pool B2012 through the heat exchange liquid flow port 20132013. The heat-releasing pack performs a heat-releasing reaction. The air insulation area 204 provided around the thermal reaction pool 201 plays a role in maintaining a constant temperature.

[0057] The microfluidic reaction plate 300 is arranged between the upper cover 100 and the thermal reaction base plate 200. A constant temperature amplification reaction chamber 301 for constant temperature amplification is distributed on the microfluidic reaction plate 300. Wide groove siphon suction flow channels 302 and product diffusion flow channels 303 for liquid flow control of samples and amplification products are distributed at both ends of the constant temperature amplification reaction chamber 301.

[0058] The wide-groove siphon suction flow channel 302 is interlocked with the narrow-groove siphon suction flow channel 103 of the upper cover 100. The sample solution is added through the sample loading hole 101 and self-distributed to the constant temperature amplification reaction chamber 301 in the siphon flow channel. The constant temperature amplification reagent beads in the constant temperature amplification reaction chamber 301 automatically disintegrate and melt. The constant temperature amplification reaction chamber 301 is located above the thermal reaction pool 201. The constant temperature amplification reaction of the nucleic acid target sequence in the sample is completed under heating conditions. The reaction product flows to the product diffusion flow channel 303 along with the stop solution added from the sample loading hole 101. The product diffusion flow channel 303 is divided into two sections, namely the upstream buffer channel 3031 and the downstream diffusion channel 3032. The amplification product flowing out of the constant temperature amplification reaction chamber 301 flows to the diffusion channel 3032 through the buffer channel 3031.

[0059] A product detection zone 304 is provided downstream of the diffusion channel 3032. This zone includes several lateral flow chromatography test strips, each corresponding to a diffusion channel 3032. Each test strip is embedded in a test strip retaining groove 3041, and a test strip retaining baffle 3042 is provided at the water absorption end of the test strip to secure the test strip. The isothermal amplification product flows through the diffusion channel 3032 into the sample zone of the lateral flow chromatography test strip, undergoing specific hybridization and color development with the detection probe on the lateral flow chromatography test strip. The test results are ultimately observed through the observation window 104 of the upper cover 100, displayed on the lateral flow chromatography test strip. The observation window 104 includes several visual grilles 105, each corresponding to a lateral flow chromatography test strip. A transparent cover 500 seals and protects the product detection zone 304.

[0060] This application integrates nucleic acid sample guidance, nucleic acid amplification product distribution, and result display in a microfluidic reaction device. In actual operation, the flow distribution of nucleic acid samples and amplification products involved is completed by the microfluidic reaction assembly. The microfluidic reaction assembly is composed of functional components in multiple areas, including a narrow siphon suction flow channel 103 on the inner side of the upper cover 100, a wide siphon suction flow channel 302 located on the microfluidic counterplate, a product diffusion flow channel 303, and a constant temperature amplification reaction chamber.

[0061] The present invention can expand the microfluidic reaction component into a parallel multiple structure according to different detection requirements. Specifically as follows: the nucleic acid sample is added through the sample addition hole 101, and the sample addition hole 101 has a number of openings that are connected to the narrow slot siphon suction flow channel 103 according to different detection requirements. The nucleic acid sample solution enters the narrow slot siphon suction flow channel 103 through capillary force, and the narrow slot siphon suction flow channel 103 is embedded in the wide slot siphon suction flow channel 302. The nucleic acid sample solution forms a siphon effect in the mosaic siphon suction flow channel composed of the narrow slot siphon suction flow channel 103 and the wide slot siphon suction flow channel 302. The siphon force of the liquid is greater than the adhesion force inside the liquid, thereby guiding the nucleic acid sample solution to flow to the constant temperature amplification reaction chamber.

[0062] After the isothermal amplification reaction is completed, a stop solution is added through sample loading port 101. The stop solution flows into the isothermal amplification reaction chamber via a chimeric siphon flow channel consisting of a narrow-slot siphon flow channel 103 and a wide-slot siphon flow channel 302, terminating the isothermal amplification reaction. Simultaneously, the stop solution flows along with the product into the product diffusion channel 303 located downstream of the isothermal amplification reaction chamber. The isothermal amplification product first enters the buffer channel 3031 and, under capillary action, flows toward the diffusion channel 3032. Upon reaching the diffusion channel 3032, the product solution slowly diffuses into the product detection zone 304. The isothermal amplification product in each diffusion channel 3032 flows to the corresponding lateral flow chromatography test strip, where it undergoes specific hybridization with the detection probe and undergoes a colorimetric reaction.

[0063] The specific steps are as follows: Step 1: Place the microfluidic detection device on a horizontal table, remove the sample well cover 400, add the nucleic acid sample solution to the sample well 101, and then cover the sample well cover 400.

[0064] Step 2: Use a syringe to draw tap water or purified water, add it to the water injection hole 102, and seal the water injection hole 102 with a water injection hole plug. As the water enters, the heating reaction starts, and the heat released simultaneously triggers the isothermal amplification reaction in the isothermal amplification reaction chamber.

[0065] Step 3: After waiting for 12-15 minutes, open the sample well cover 400, add the stop solution to the sample well 101, and replace the sample well cover 400. As the stop solution is added, the product of the isothermal amplification reaction flows to the lateral flow chromatography test paper.

[0066] Step 4: After waiting for 4-6 minutes, the isothermal amplification reaction product completes the hybridization color reaction with the specific detection probe on the chromatography test paper. The detection result is observed from the observation window 104 through the transparent cover plate 500 and the result is interpreted.

[0067] The detection performance of the microfluidic detection device is further described below through detection applications.

[0068] The constant temperature home self-testing microfluidic detection device is used for influenza A, influenza B, adenovirus, and novel coronavirus nucleic acid detection.

[0069] Specific application 1: 1.Main test items include: Influenza A virus (Flu A) nucleic acid, influenza B virus (Flu B) nucleic acid, adenovirus (ADV) nucleic acid, novel coronavirus nucleic acid, internal reference gene (Human GAPDH).

[0070] 2. The required materials and specific methods are as follows: (1) Reagents: Pre-loaded in the thermostatic amplification reaction chamber are freeze-dried reagent beads for constant temperature detection of influenza A (Flu A), influenza B (Flu B), adenovirus (ADV), novel coronavirus, and internal reference gene (Human GAPDH).

[0071] The lateral flow chromatography test paper pre-loaded in the product detection area 304 is used to detect the isothermal amplification products of influenza A (Flu A), influenza B (Flu B), adenovirus (ADV), novel coronavirus, and internal reference gene (Human GAPDH) nucleic acid. The lateral flow chromatography test paper is coated with a specific detection probe for the item to be tested.

[0072] (2) Test samples: The test samples were prepared using national reference materials for influenza A virus nucleic acid detection reagents, national reference materials for influenza B virus nucleic acid detection reagents, national reference materials for respiratory adenovirus nucleic acid detection reagents, national reference materials for novel coronavirus nucleic acid detection reagents, and human genomic DNA quantitative standard substances.

[0073] (3) Amplification primers and detection probes: Design amplification primers for Flu A, Flu B, ADV, novel coronavirus, and Human GAPDH.

[0074] After primer synthesis, the primers were diluted to 100 μM using a 10 mM Tris-HCl (pH 8.5) solution prepared with DEPC water. The primers were then mixed in a ratio of D1:D2:RS1:RS2:R1:R2:F1:F2 = 1:1:2:2:4:4:4:4 (D1:D2:RS1:RS2:R1:R2:F1:F2, see Table 1 for details) to form a primer mix, where the 5' end of the F1 primer was labeled with FAM, and the 5' end of the F2 primer was labeled with Biotin.

[0075] Based on the selected amplification sequence, Flu A, Flu B, ADV, novel coronavirus, and Human GAPDH detection probes CP were designed and coated on the NC membrane of the lateral flow chromatography test paper.

[0076] The primers and probes are shown in Tables 1-5 below: Table 1: Table 2: Table 3: Table 4: Table 5: The components of the amplification reagent beads are shown in Table 6: Table 6: Nucleic acid releaser: 1-100mM Tris-HCl pH7.0-7.5, 0.1-1mM EDTA, 1-100mM KCl, 0.1-5mM DTT, 0.1‰-0.1% tetradecyltrimethylammonium oxalate.

[0077] Stop solution: 1-100mM Tris-HCl pH8.5-9.0, 50mM EDTA, 0.1‰-0.1 TritonX-100.

[0078] The detection method is as follows: Collect a throat swab from the subject and immerse the swab tip in a sample tube containing 0.2-0.5mL of nucleic acid release agent. Rotate the swab and press the bottom of the sample processing liquid tube (where the disposable sampler tip is immersed) with your hand. Gently squeeze this area at least five times, taking care not to squeeze out the liquid in the tube. After squeezing, let it sit at room temperature for 3-5 minutes. This is the sample to be tested.

[0079] Open the sample well cover 400 on a horizontal table, add 5-20 drops of sample into the sample well 101, and then close the well cover.

[0080] Use a 25mL syringe to draw 20mL of tap water and inject all the tap water into the water injection hole 102. Then plug the water injection hole plug.

[0081] After waiting for 15 minutes, open the sample well cover 400, add 0.5-2 mL of stop solution into the sample well 101, and close the sample well cover 400.

[0082] After 5 minutes, visually observe the microfluidic cartridge window and determine the results according to Table 7.

[0083] Table 7: result: The National Reference Material for Influenza A Virus Nucleic Acid Detection Reagent, the National Reference Material for Influenza B Virus Nucleic Acid Detection Reagent, the National Reference Material for Respiratory Adenovirus Nucleic Acid Detection Reagent, the National Reference Material for Novel Coronavirus Nucleic Acid Detection Reagent, and the Human Genomic DNA Quantification Standard were diluted with RNase-free deionized water to prepare concentrations of 1000 cps / swab, 500 cps / swab, 100 cps / swab, 50 cps / swab, and 10 cps / swab, respectively. Ten swabs of each concentration were prepared for each test item, and ten no-nucleic acid controls were used.

[0084] The specific results are shown in Table 8-13: Table 8: Table 9: Table 10: Table 11: Table 12: Table 13: When the sample concentration is 1000cps / swab, the detection rates of influenza A (Flu A), influenza B (Flu B), adenovirus (ADV), novel coronavirus, and internal reference gene (Human GAPDH) are all 100%; When the sample concentration is 500cps / swab, the detection rates of influenza A (Flu A), influenza B (Flu B), adenovirus (ADV), novel coronavirus, and internal reference gene (Human GAPDH) are all 100%; When the sample concentration is 100 cps / swab, the detection rates of influenza A (Flu A), influenza B (Flu B), adenovirus (ADV), novel coronavirus, and internal reference gene (Human GAPDH) are all 100%; When the sample concentration is 50cps / swab, the detection rate of influenza A (Flu A) is 90%, the detection rate of influenza B (Flu B) is 70%, the detection rate of adenovirus (ADV) is 70%, the detection rate of new coronavirus is 80%, and the detection rate of the internal reference gene (Human GAPDH) is 100%; When the sample concentration is 10cps / swab, the detection rate of influenza A (Flu A) is 20%, the detection rate of influenza B (Flu B) is 10%, the detection rate of adenovirus (ADV) is 0%, the detection rate of the new coronavirus is 10%, and the detection rate of the internal reference gene (Human GAPDH) is 3%.

[0085] No nucleic acid sample results were normal, all were negative.

[0086] in conclusion: Experimental results demonstrate that a thermostatic microfluidic cartridge can be used for nucleic acid detection of influenza A (Flu A), influenza B (Flu B), adenovirus (ADV), and the novel coronavirus. The cartridge can complete nucleic acid detection for the four viruses within 20 minutes of sample addition, with a sensitivity of at least 100 cps / swab.

[0087] Specific application 2: This application provides a constant temperature microfluidic cartridge for use in a method for detecting nucleic acids of hepatitis B virus DNA (HBV DNA) and hepatitis C virus RNA (HCV RNA).

[0088] (1) Test items: Hepatitis B virus DNA (HBV DNA), hepatitis C virus RNA (HCV RNA), and internal reference gene (Human GAPDH).

[0089] (2) Materials and methods Reagents: Hepatitis B virus (HBV), hepatitis C virus (HCV), and internal reference gene (Human GAPDH) nucleic acid constant temperature detection freeze-dried reagent beads pre-loaded in the constant temperature amplification reaction chamber 301 .

[0090] The lateral flow chromatography test paper pre-loaded in the product detection area 304304 is used to detect the isothermal amplification products of hepatitis B virus (HBV), hepatitis C virus (HCV), and internal reference gene (Human GAPDH) nucleic acid. The lateral flow chromatography test paper is coated with specific detection probes for the items to be tested.

[0091] Test sample: The national reference materials for hepatitis B virus nucleic acid were used as the test samples (negative reference materials: N1-N8; positive reference materials: P1-P9; sensitivity reference materials: 1000 IU / mL).

[0092] The national reference material for hepatitis C virus nucleic acid was used as the sample to be tested (positive and negative reference materials: N1-N10, P1-P10; minimum detection limit reference material: 8.0×106IU / vial).

[0093] Human genomic DNA quantitative standard (3.8×104cps / μL).

[0094] Amplification Primers and Detection Probes: Design isothermal amplification primers for HBV, HCV, and Human GAPDH. After primer synthesis, dilute the primers to 100 μM in 10 mM Tris-HCl (pH 8.5) in DEPC water. Mix the primers in a ratio of D1:D2:RS1:RS2:R1:R2:F1:F2 = 1:1:2:2:4:4:4:4 (see Tables 14-16 for details) to create a primer mix. The 5' end of the F1 primer is labeled with FAM, and the 5' end of the F2 primer is labeled with biotin.

[0095] Based on the selected amplified sequences, HBV, HCV, and Human GAPDH detection probes CP were designed and coated on the NC membrane of the lateral flow chromatography test paper.

[0096] The primers and probes are shown in Tables 14-16 below: Table 14: Table 15: Table 16: The components of the amplification reagent beads are shown in Table 17: Table 17: Nucleic acid releaser: 1-100mM Tris-HCl pH7.0-7.5, 0.1-1mM EDTA, 1-100mM KCl, 0.1-5mM DTT, 0.1‰-0.1% tetradecyltrimethylammonium oxalate.

[0097] Stop solution: 1-100mM Tris-HCl pH8.5-9.0, 50mM EDTA, 0.1‰-0.1 TritonX-100.

[0098] The detection method is as follows: Wash your hands with alcohol disinfectant, hand sanitizer or soap.

[0099] Massage from the base of the finger to the fingertip several times to make the fingertip congested, remove the needle cap of the disposable peripheral blood collection device, aim at the blood collection site on the fingertip, and press the needle of the peripheral blood collection device to puncture and collect blood.

[0100] Use a pipette to draw up 10-100 μL of blood and add it to a sample tube containing 0.1-0.5 mL of nucleic acid release agent. Gently squeeze the pipette 3-5 times to allow the sample to fully enter the sample processing solution tube, taking care not to squeeze out the liquid in the tube. After squeezing, let it sit at room temperature for 3-5 minutes. This is the sample to be tested.

[0101] Open the sample well cover 400 on a horizontal table, add 5-20 drops of sample into the sample well 101, and then close the well cover.

[0102] Use a 25mL syringe to draw 20mL of tap water and inject all the tap water into the water injection hole 102. Then plug the water injection hole plug.

[0103] After waiting for 15 minutes, open the sample well cover 400, add 0.5-2 mL of stop solution into the sample well 101, and close the sample well cover 400.

[0104] After 5 minutes, visually observe the microfluidic cartridge window and determine the results according to Table 7.

[0105] result: 100uL of hepatitis B virus nucleic acid positive reference materials P1-P8 and hepatitis B virus nucleic acid negative reference materials N1-N8 were respectively pipetted and added to the sample tube containing nucleic acid release agent for treatment; 100uL of hepatitis C virus nucleic acid positive reference materials P1-P10 and hepatitis C virus nucleic acid negative reference materials N1-N10 were respectively pipetted and added to the sample tube containing nucleic acid release agent for sample treatment; using HBV negative plasma, the hepatitis B virus nucleic acid sensitivity reference material was diluted to 100IU / mL and 30IU / mL, and 100uL was pipetted and added to the sample tube containing nucleic acid release agent for treatment, and 10 copies were processed for each; HC was used to For V-negative plasma, dilute the hepatitis C virus nucleic acid sensitivity reference substance to 100 IU / mL and 50 IU / mL, draw 100 uL with a pipette, and add it to the sample tube containing the nucleic acid release agent for processing, processing 10 copies each time; dilute the human genomic DNA quantitative standard with DNase- and RNase-free purified water to 10,000 cps / mL and 100 cps / mL, draw 100 uL with a pipette, and add it to the sample tube containing the nucleic acid release agent for processing, processing 10 copies at each concentration; use nucleic acid-free purified water as a pure negative control, draw 100 uL with a pipette, and add it to the sample processing tube containing the nucleic acid release agent for processing, processing a total of 10 copies.

[0106] The specific results are shown in Tables 18-23: Table 18: Table 19: Table 20: Table 21: Table 22: Table 23: Table 24: Table 25: Positive and negative detection rate: The present invention was used to test national reference materials for hepatitis B and hepatitis C virus nucleic acid. Finally, all positive reference materials were detected, and all negative reference materials were negative. This shows that the detection method provided in this example has good detection specificity.

[0107] Detection sensitivity: The present invention was used to detect hepatitis B and hepatitis C virus nucleic acid sensitivity test samples. All hepatitis B detection sensitivity reference samples of 50 IU / mL were detected, and all hepatitis C detection sensitivity reference samples of 30 IU / mL were detected. The experimental results show that the detection method provided in this embodiment has good detection sensitivity.

[0108] No nucleic acid sample results were normal, all were negative.

[0109] in conclusion: Experimental results show that a constant-temperature home self-testing microfluidic detection device can be applied to the nucleic acid detection method of hepatitis B virus DNA (HBV DNA) and hepatitis C virus RNA (HCV RNA), and the nucleic acid detection of the two viruses can be completed within 20 minutes after sample addition.

[0110] In summary, the present invention adopts the method of microfluidic chip to realize the precise and autonomous distribution of nucleic acid sample solution in the flow channel, and completes the complex constant temperature reaction and visual detection work without relying on instruments, power supplies, etc. Using almost the same operating procedures and detection time as the antigen colloidal gold mode, the detection performance comparable to PCR is achieved. It provides technical support for constant temperature amplification in multiple fields such as laboratory medicine, animal and plant testing, animal and plant molecular breeding, and pathogenic microorganism detection. Let the nucleic acid home detection mode, like the antigen colloidal gold mode, be short in time, low in cost, fast and simple, safe and reliable, so that nucleic acid home detection is heading towards the market and thousands of households.

[0111] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A constant temperature home self-test microfluidic detection device, comprising an upper cover, a thermal reaction base plate, and a microfluidic reaction plate, characterized in that: The upper cover is provided with a sample addition hole and a sample addition hole cover for covering the sample addition hole, and a water injection hole and a water injection hole plug matching the water injection hole are provided near the sample addition hole. A heating chamber is provided on the thermal reaction base plate. The heating chamber includes a thermal reaction pool A and a thermal reaction pool B, which respectively carry out the heating reaction of the heating pack and the thermal slow-release reaction of the heat-releasing pack. A heat exchange liquid flow port is provided between the thermal reaction pool A and the thermal reaction pool B. Water or thermal reaction liquid for thermal reaction added through the water injection hole is heated by the heating pack in the thermal reaction pool A and then flows to the thermal reaction pool B through the heat exchange liquid flow port. An air insulation area is provided around the heating chamber. The upper cover and the thermal reaction base plate are buckled together, and the microfluidic reaction plate is located between the upper cover and the thermal reaction base plate. A constant temperature amplification reaction chamber for constant temperature amplification is distributed on the microfluidic reaction plate. Wide groove siphon flow channels and product diffusion flow channels for liquid flow control of samples and amplification products are distributed at both ends of the constant temperature amplification reaction chamber.

2. The constant temperature home self-test microfluidic detection device according to claim 1, characterized in that: The wide-slot iris suction flow channel is engaged with the narrow-slot iris suction flow channel of the upper cover.

3. The constant temperature home self-test microfluidic detection device according to claim 1, characterized in that: The product diffusion channel is divided into two sections, namely the upstream buffer channel and the downstream diffusion channel. The amplification products flowing out of the constant temperature amplification reaction chamber flow to the diffusion channel through the buffer channel.

4. The constant temperature home self-test microfluidic detection device according to claim 3, characterized in that: A product detection area is set downstream of the diffusion channel. The product detection area has several lateral flow chromatography test strips. Each lateral flow chromatography test strip corresponds to a diffusion channel. Each test strip is embedded in a test strip limiting groove. A test strip limiting baffle is provided at the water absorption end of the test strip to fix the test strip.

5. The constant temperature home self-testing microfluidic detection device according to claim 4, characterized in that: An observation window is provided on the upper cover.

6. The constant temperature home self-testing microfluidic detection device according to claim 5, characterized in that: A plurality of visual grids are provided on the observation window, and each visual grid corresponds to a lateral flow chromatography test paper.

7. The constant temperature home self-test microfluidic detection device according to claim 6, characterized in that: The observation window is covered with a transparent cover to seal and protect the product detection area.

8. The constant temperature home self-testing microfluidic detection device according to claim 3, characterized in that: The product detection zone is located downstream of the product diffusion channel, and the horizontal position of the product detection zone is lower than the product diffusion channel.

9. A constant temperature home self-testing microfluidic detection device according to any one of claims 1 to 8, characterized in that: The constant temperature amplification reaction chamber is located between the wide groove siphon flow channel and the product diffusion flow channel; The constant temperature amplification reaction chamber contains constant temperature amplification reagent beads. The sample solution is added through the sample injection hole and evenly distributed to the constant temperature amplification reaction chamber in the microfluidic flow channel. After the constant temperature amplification reagent beads are dissolved, the amplification reaction can be carried out in the constant temperature amplification reaction chamber.

10. A use of a constant temperature home self-testing microfluidic detection device, characterized by: The constant temperature home self-test microfluidic detection device described in any one of claims 1-9 is used for influenza A, influenza B, adenovirus, and new coronavirus nucleic acid detection.

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