Fully enclosed chip based on gravity drive and micro-NAA-LFD integration and method thereof

Abstract

The present invention discloses a fully enclosed chip and method based on gravity drive and micro-NAA-LFD integration. The replenisher liquid storage chamber accommodates replenisher liquid, and a replenisher liquid sampling port is provided at the upper end; the reaction liquid storage chamber temporarily accommodates nucleic acid amplification reaction liquid and the sample solution to be tested, and a reaction liquid sampling port is provided at the upper end; the nucleic acid amplification chamber accommodates nucleic acid amplification reaction liquid and the sample solution to be tested for nucleic acid amplification reaction; the test strip detection chamber is used for test strip detection, and a lateral chromatography test strip is placed in advance; the replenisher liquid storage chamber and the reaction liquid storage chamber are connected by a channel to form a first annular circulation path; each two of the reaction liquid storage chamber, the nucleic acid amplification chamber and the test strip detection chamber are connected by a channel to form a second annular circulation path. The present invention can realize integrated nucleic acid amplification and test strip detection of trace nucleic acid amplification liquid, and only a trace amount of nucleic acid amplification reagent is needed to meet the use requirements of the test strip, thereby reducing the detection cost.

Description

Technical Field

[0001] The present invention discloses a gravity-driven and micro-NAA-LFD integrated fully enclosed chip for nucleic acid detection, which relates to an integrated chip combining nucleic acid amplification (NAA), lateral flow dipstick (LFD) and microfluidics technology, and belongs to the field of microbiology and biochemical analysis. Background Art

[0002] Nucleic acid amplification (NAA) is a general term for a broad category of methods, including conventional PCR, real-time fluorescence PCR, and a range of isothermal nucleic acid amplification techniques. Since the polymerase chain reaction (PCR) was proposed by Mullis et al. in 1985, new nucleic acid amplification techniques have been continuously developed as more and more enzyme tools have been discovered. Currently, nucleic acid amplification technology has been widely used in the field of nucleic acid detection.

[0003] Lateral flow dipsticks (LFDs) utilize the principles of immunochromatography, using capillary action to force the test sample to flow laterally through a matrix, allowing antigens to bind to antibodies and visualizing the test results. LFDs typically consist of a sample pad, a conjugate pad coated with colloidal gold or microsphere-labeled antibodies, and a filter membrane containing a T-line coated with the antigen and a C-line coated with a control antibody. Lateral flow dipsticks utilize a large-pore microporous filter membrane (NC membrane, nitrocellulose membrane) as a carrier, with specific antigens or antibodies immobilized on the NC membrane. When the test sample is applied to the sample pad at one end of the strip, it migrates laterally through capillary action, reacting specifically with the colloidal gold or microsphere-labeled reagent on the conjugate pad. The sample then migrates to the NC membrane, where it is captured by the antigens or antibodies immobilized on the membrane surface and accumulates on the test strip. Visual observation of the light reflection signal density from the markers (colloidal gold or latex particles) on the NC membrane surface provides intuitive colorimetric results. Unbound markers flow through the test strip and into the absorbent pad, achieving automatic separation. This technology is simple to operate and provides rapid test results, effectively replacing time-consuming immunoassays and instrumental testing in the laboratory. It offers the advantages of speed, simplicity, single-sample testing, and affordability. It is now widely used in medical testing, food quality monitoring, environmental monitoring, and other fields.

[0004] Whether it is a dye-based or probe-based nucleic acid amplification, it needs to rely on fluorescence emission and monitoring modules to reflect the amplification situation. The addition of lateral flow test strips has freed nucleic acid amplification from bulky and expensive fluorescence modules. The combination of NAA and LFD technologies has promoted the development of on-site testing in a more convenient and low-cost direction. However, the current NAA-LFD technology still has some shortcomings. For example, NAA amplification will produce a large number of amplicons, which can easily cause aerosol contamination in the process of opening the lid and taking the amplification product to the test strip, resulting in false positive results in subsequent tests; the process of taking liquid also increases the complexity of the operation, and requires the use of consumables such as droppers or pipette tips; the infiltration of the test strip requires more nucleic acid amplification reagents (about 50-100 microliters), which is more expensive. Summary of the Invention

[0005] To address the problems in the background technology, the present invention proposes a fully enclosed chip based on gravity drive and micro-NAA-LFD, and a corresponding detection method, which can realize integrated nucleic acid amplification and test strip detection of trace nucleic acid amplification liquid. The chip and detection method only require a trace amount of nucleic acid amplification reagent to meet the use requirements of the test strip, reducing the cost of detection. In addition, it also unties the microfluidic chip and the detection reagent, and simplifies the operating procedures of the NAA-LFD nucleic acid detection method.

[0006] The technical solutions of the present invention are as follows:

[0007] 1. A fully enclosed chip based on gravity drive and micro-NAA-LFD:

[0008] The chip comprises an L-shaped replenishing liquid storage chamber for accommodating replenishing liquid, and a replenishing liquid sampling port is provided at the upper end of the replenishing liquid storage chamber and is connected to the outside of the chip, and the replenishing liquid sampling port is used to add replenishing liquid;

[0009] The chip includes a reaction liquid storage chamber for temporarily storing nucleic acid amplification reaction solutions and test sample solutions. A reaction liquid injection port, connected to the chip's exterior, is located at the upper end of the reaction liquid storage chamber for adding reaction liquid. The injection port can be positioned freely on the front, back, or top of the chip.

[0010] It includes a nucleic acid amplification chamber for containing nucleic acid amplification reaction solution and a sample solution to be tested for nucleic acid amplification reaction;

[0011] It includes a test strip detection cavity for test strip detection, in which a lateral flow test strip is pre-placed;

[0012] The replenishing liquid storage chamber and the reaction liquid storage chamber are connected through a channel inside the chip, forming a first annular circulation path that is approximately rectangular inside the chip; the first circulation path located on the left side allows the replenishing liquid to flow in the replenishing liquid storage chamber.

[0013] Each of the reaction liquid storage chamber, the nucleic acid amplification chamber, and the test strip detection chamber is connected by a channel inside the chip, forming a second annular circulation path inside the chip; the second circulation path located on the right side allows the reaction liquid to flow from the reaction liquid storage chamber to the nucleic acid amplification chamber and then to the test strip detection chamber, and the replenisher to flow from the replenisher storage chamber to the nucleic acid amplification chamber and then to the test strip detection chamber, thereby realizing the integration of nucleic acid amplification and test strip detection.

[0014] These annular circulation paths automatically balance the air pressure inside the chip, and the liquid flows inside the chip by relying on the flipping / rotation of the chip and its own gravity, without the need for external equipment to provide power.

[0015] The "L" shape of the replenishing liquid storage chamber includes a vertical part and a horizontal part, wherein the vertical part serves as a flow channel for the replenishing liquid, the upper end of the vertical part is connected to the replenishing liquid injection port, and the horizontal part is used to store the replenishing liquid.

[0016] The side wall at the lower end of the reaction liquid storage chamber is provided with an arc-shaped inner concave surface, and the arc-shaped design can facilitate the reaction liquid to flow into the nucleic acid amplification chamber below.

[0017] The bottom surface of the nucleic acid amplification chamber adopts an arc-shaped concave surface, and the arc-shaped design can reduce the residual liquid at this point.

[0018] In the initial state, the reaction liquid storage chamber and the test strip detection chamber are located as a whole at the bottom of the replenisher liquid storage chamber and above the nucleic acid amplification chamber. The reaction liquid storage chamber is located between the top of the replenisher liquid storage chamber and the test strip detection chamber. The top of the reaction liquid storage chamber is level with the top of the replenisher liquid storage chamber, the nucleic acid amplification chamber is level with the bottom of the replenisher liquid storage chamber, and the test strip detection chamber is level with the reaction liquid storage chamber.

[0019] The side of the nucleic acid amplification chamber close to the reaction liquid storage chamber is connected to the reaction liquid storage chamber through an arc-shaped liquid channel, the other side of the nucleic acid amplification chamber is connected to the side of the test strip detection chamber away from the reaction liquid storage chamber through a liquid channel, and the side of the test strip detection chamber close to the reaction liquid storage chamber is connected to the reaction liquid storage chamber or the reaction liquid injection port through a gas channel; the bottom of the supplementary liquid storage chamber is connected to the middle of the arc-shaped channel between the nucleic acid amplification chamber and the reaction liquid storage chamber through a horizontal capillary liquid channel, and the top of the supplementary liquid storage chamber is connected to the reaction liquid storage chamber through a horizontal gas channel.

[0020] The liquid channel between the nucleic acid amplification chamber and the test strip detection chamber is configured in an inverted "L" shape, so that the vertical portion of the inverted "L"-shaped liquid channel can prevent the reaction liquid from flowing into the test strip detection chamber in advance before amplification.

[0021] The volume of the reaction liquid storage chamber is smaller than the volume of the supplementary liquid storage chamber.

[0022] The chip is connected to the outside world only at the supplementary liquid sampling port and the reaction liquid sampling port. When connected to the outside world, it is only used for sampling. When the supplementary liquid sampling port and the reaction liquid sampling port are closed, the chip becomes a fully closed chip.

[0023] 2. Method for using a gravity-driven and micro-NAA-LFD integrated fully enclosed chip:

[0024] S1. Place the lateral flow test strip, front-up and sample pad facing outward, into the bottom of the test chamber. Use tape to seal the entire front of the chip, leaving an appropriate sample port for later use.

[0025] When in use, the chip is initially placed horizontally or vertically. Specifically, when the sample port is located on the front or back of the chip, the chip is placed horizontally; when the sample port is located on the top of the chip, the chip is placed vertically.

[0026] Add 15-35 μL of nucleic acid amplification reaction solution to the supplementary liquid injection port of the reaction solution storage chamber. The nucleic acid amplification reaction solution can be prepared as needed. The marker used should match the test strip. The nucleic acid amplification method and detection target should be matched and selected accordingly.

[0027] Add a supplementary solution with a volume greater than that of the nucleic acid amplification reaction solution to the reaction solution injection port of the supplementary solution storage chamber, and the total amount of the supplementary solution and the nucleic acid amplification reaction solution added meets the working conditions of the test strip (50-100 μL);

[0028] The supplementing solution can be clean, uncontaminated PBS buffer, Tris-EDTA buffer or pure water, and an appropriate surfactant can be added as needed.

[0029] After adding the sample, seal the two sample ports of the supplementary solution and the reaction solution with tape;

[0030] S2. Flip the chip over or not. The liquid in the supplemental solution storage chamber and the reaction solution storage chamber will flow downward due to gravity. If the sample injection port is on the back or front of the chip, flip the chip over after injection so that the chip's base surface is perpendicular to the ground. If the sample injection port is on the top of the chip, flipping is not necessary.

[0031] The replenishing liquid flows along the vertical part of the "L"-shaped replenishing liquid storage chamber and is stored in the horizontal part, and the reaction liquid flows along the arc-shaped liquid channel from the reaction liquid storage chamber to the nucleic acid amplification chamber;

[0032] S3, then the temperature control device outside the nucleic acid amplification chamber is used to heat the nucleic acid amplification chamber to perform the nucleic acid amplification reaction; the reaction temperature is adjusted to the optimal temperature for the nucleic acid amplification method used. After the amplification reaction is completed, the reaction amplification solution is obtained in the nucleic acid amplification chamber;

[0033] S4. Flip the chip. Specifically, rotate the chip 90° clockwise about an axis perpendicular to the reference plane. The liquid in the replenisher liquid storage chamber and the nucleic acid amplification chamber now flows downward due to gravity, ultimately flowing into the lowest point of the flipped test strip detection chamber, i.e., the side of the test strip detection chamber that is away from the reaction liquid storage chamber. The replenisher liquid continuously impacts or pushes the nucleic acid amplification liquid into the test strip detection chamber, mixing with it to form a mixed liquid. The mixed liquid then moves laterally from the sample pad toward the absorbent pad of the test strip through capillary action. Finally, the test result is interpreted based on the color development of the C and T lines on the test strip.

[0034] In response to the problems and shortcomings of the prior art, the present invention proposes a fully enclosed chip based on gravity-driven and micro-NAA-LFD integration, which achieves the integration of nucleic acid amplification and test strip detection. This chip has several unique advantages: First, it is equipped with two chambers for holding a replenisher and a nucleic acid amplification reagent, respectively, separated by a capillary valve. Due to the introduction of a large volume of replenisher, the micro-NAA-LFD of the present invention greatly reduces the amount of nucleic acid amplification reagent required compared to existing large-volume NAA-LFD methods, thereby reducing detection costs. Second, compared to nucleic acid detection chips with pre-embedded detection reagents, the chip of the present invention has a sample injection port connected to the outside world, allowing detection reagents to be added on-the-spot without the need for pre-embedded chambers. The nucleic acid amplification method and the components of the detection reagents are also not restricted, and users can add them according to the detection target. Third, the flow of liquid in the chip is driven by the liquid's own gravity, eliminating the need for external equipment such as centrifuges and pumps. Fourth, the chip integrates nucleic acid amplification and test strip detection. After amplification, the chip is rotated, and the amplification liquid automatically flows into the test strip detection chamber, simplifying the detection operation process. Fifth, after the sample and supplement are added, the two sample ports are sealed with tape or other means, ensuring the entire chip system is airtight, preventing aerosols from being released into the surrounding environment and preventing false positives in subsequent tests. Sixth, the ventilation channels not only balance the air pressure inside the chip, but also allow liquids to flow smoothly within the closed environment.

[0035] The beneficial effects of the present invention are:

[0036] First, the chip and method of the present invention enable integrated micro-NAA for LFD nucleic acid detection. The chip features two chambers for a replenisher and a nucleic acid amplification reagent, separated by a capillary valve. By introducing a large volume of replenisher, the micro-NAA of the present invention significantly reduces the amount of nucleic acid amplification reagent required compared to existing large-volume NAA-LFD methods, thereby lowering detection costs.

[0037] Second, to complete the test, the user only needs to perform two simple steps. First, add a certain amount of supplemental solution and reaction solution to the sample injection ports of the supplemental solution storage chamber and the reaction solution storage chamber, respectively, and then seal them. Second, after the nucleic acid amplification is completed, rotate the chip, and the amplification solution automatically flows into the test strip detection chamber. The entire test process eliminates the need for opening the lid, pipetting, and manual mixing, simplifying the test process. The second rotation step can also be equipped with a simple instrument to automatically complete the operation after amplification. This way, the operator only needs to add the sample in one step.

[0038] Third, compared to nucleic acid detection chips that require pre-embedded detection reagents, the present chip has a separate nucleic acid reaction chamber connected to the outside world. Detection reagents can be added on demand, without pre-embedded reagents in the chamber. There are no restrictions on the nucleic acid amplification method or the composition of the detection reagents; users can add them based on the detection target.

[0039] Fourth, the flow of liquid in this chip is driven by the liquid's own gravity, and the flow direction is precisely controlled by the chamber and flow channel, without the need for external equipment such as centrifuges and pumps.

[0040] 5. After the test sample and supplementary liquid are added, the two sample addition ports are sealed with tape. The entire chip system is airtight, and aerosols cannot be released into the surrounding environment, avoiding false positives in subsequent test results.

[0041] 6. The ventilation channel can not only balance the air pressure inside the chip, but also allow the liquid to flow smoothly in a closed environment.

[0042] The chip of the present invention has a simple structure and low cost. Compared with the existing methods, the chip and method of the present invention are reagent-saving and simple to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the chip structure of the present invention. 1. Reference surface; 2. Supplementary liquid storage chamber; 3. Supplementary liquid sample port; 4. Reaction liquid storage chamber; 5. Reaction liquid sample port; 6. Nucleic acid amplification chamber; 7. Test strip detection chamber; 81, 82, 83. Liquid channels; 91, 92. Gas channels. This diagram uses sample ports 3 and 5 located at the top of the chip as an example. In practice, the locations of the sample ports are not limited and can be located at the top, front, or back of the storage chamber.

[0044] Figure 2yes Figure 1 The capillary valve structure formed by the replenishing liquid storage chamber 2 and the liquid channel 81 in the chip is shown.

[0045] Figure 3 yes Figure 1 The schematic diagram shows a static state of the chip after the supplementary solution and the reaction solution flow into the supplementary solution storage chamber 2 and the nucleic acid amplification chamber 6 respectively under the action of gravity. The dark grey part represents the flowing solution.

[0046] Figure 4 yes Figure 3 The chip shown is a static schematic diagram after being rotated 90° clockwise with the direction perpendicular to the reference plane as the axis, after the supplementary liquid and the amplification liquid flow into the test strip detection cavity 7. The dark gray part is the solution flowing into the test strip detection cavity 7.

[0047] Figure 5 These are three test results of the nucleic acid chromatography test strip of Example 1: a) positive result, b) negative result, and c) invalid test result.

[0048] Figure 6 These are five test results of the dual-index nucleic acid chromatography test strip of Example 2: a) both targets 1 and 2 are positive, b) target 1 is positive and target 2 is negative, c) target 2 is positive and target 1 is negative, c) both targets 1 and 2 are negative, and c) an invalid test result. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the accompanying drawings and specific implementations.

[0050] like Figure 1 As shown, the chip contains four independent chambers: a replenisher fluid storage chamber 2, a reaction fluid storage chamber 4, a nucleic acid amplification chamber 6, and a test strip detection chamber 7, along with channels 81, 82, 83, 91, and 92 connecting these chambers. The replenisher fluid storage chamber 2 has a sample injection port 3. The reaction fluid storage chamber 4 has a sample injection port 5. The location of the sample injection port is not restricted and can be located on the top, front, or back of the storage chamber.

[0051] The chip comprises an L-shaped replenisher liquid storage chamber 2 for accommodating replenisher liquid. A replenisher liquid sampling port 3 is provided at the upper end of the replenisher liquid storage chamber 2, connected to the outside of the chip. The replenisher liquid sampling port 3 is used to add replenisher liquid. The location of the replenisher liquid sampling port is not restricted and can be set on the front, back, or top of the chip. The L-shaped replenisher liquid storage chamber 2 comprises a vertical portion and a horizontal portion. The vertical portion serves as a flow path for the replenisher liquid, with the upper end of the vertical portion connected to the replenisher liquid sampling port 3, and the horizontal portion is used to store the replenisher liquid.

[0052] The chip includes a reaction liquid storage chamber 4 for temporarily storing nucleic acid amplification reaction liquid and the sample solution to be tested. A reaction liquid sampling port 5 is provided at the upper end of the reaction liquid storage chamber 4, connected to the outside of the chip, for adding reaction liquid. The position of the reaction liquid sampling port 5 is not restricted and can be set on the front, back, or top of the chip. The lower end sidewall of the reaction liquid storage chamber 4 is provided with an arc-shaped inner concave surface. The arc design facilitates the flow of reaction liquid into the nucleic acid amplification chamber 6 below.

[0053] The apparatus comprises a nucleic acid amplification chamber 6 for accommodating nucleic acid amplification reaction liquid and a sample solution for nucleic acid amplification. The bottom of the chamber 6 is an arc-shaped concave surface, which reduces liquid residue. A temperature control device for heating is provided on the outside of the chamber 6.

[0054] It includes a test strip detection chamber 7 for test strip detection, in which a lateral flow test strip is pre-placed;

[0055] The replenisher liquid storage chamber 2 and the reaction liquid storage chamber 4 are connected through a channel inside the chip, forming a first annular circulation path located inside the chip that is approximately rectangular; each two of the reaction liquid storage chamber 4, the nucleic acid amplification chamber 6 and the test strip detection chamber 7 are connected through a channel inside the chip, forming a second annular circulation path located inside the chip.

[0056] like Figure 1 As shown, the specific position of each chamber is described with the chip's reference plane 1 perpendicular to the ground. The replenisher liquid storage chamber 2 is located on the lower left side of the chip. The reaction liquid storage chamber 4 is located on the upper right side of the replenisher liquid storage chamber 2, with its highest point aligned with the highest point of the replenisher liquid storage chamber 2. The nucleic acid amplification chamber 6 is located on the lower right side of the reaction liquid storage chamber 4. The added reaction liquid flows from the reaction liquid storage chamber 4 along the arc-shaped liquid channel 82 to be stored in the nucleic acid amplification chamber 6. The chip is rotated 90° clockwise with the direction perpendicular to the reference plane 1 as the axis. At this point, the replenisher liquid storage chamber 2 and the nucleic acid amplification chamber 6 are located on the upper left side of the test strip detection chamber 7. The liquid in the replenisher liquid storage chamber 2 and the nucleic acid amplification chamber 6 flows downward due to gravity and eventually flows into the lowest point of the test strip detection chamber 7. The chambers can also be arranged in a left-right mirror-symmetrical layout, as long as the liquid inlets of the replenisher liquid storage chamber 2 and the test strip detection chamber 7 are located on different sides of the nucleic acid amplification chamber 6.

[0057] The replenisher liquid storage chamber 2 and the reaction liquid storage chamber 4 are connected by three internal channels 81, 82, and 91, forming a roughly rectangular circulation path within the chip. The reaction liquid storage chamber 4, the nucleic acid amplification chamber 6, and the test strip detection chamber 7 are each connected by channels 82, 83, and 92 within the chip, forming a single internal circulation path. These two circulation paths automatically balance the internal pressure of the chip, allowing liquid to flow through the chip by gravity, without the need for external power.

[0058] The nucleic acid amplification chamber 6 is close to the left side of the reaction liquid storage chamber 4 and is connected to the reaction liquid storage chamber 4 through an arc-shaped liquid channel 82. The nucleic acid amplification chamber 6 is far from the right side of the reaction liquid storage chamber 4 and is connected to the test strip detection chamber 7 is far from the right side of the reaction liquid storage chamber 4 through a liquid channel 83. The test strip detection chamber 7 is close to the left side of the reaction liquid storage chamber 4 and is connected to the reaction liquid storage chamber 4 or the reaction liquid sampling port 5 through a gas channel 92, thereby forming a circulation path to maintain air pressure balance.

[0059] The liquid channel between the nucleic acid amplification chamber 6 and the test strip detection chamber 7 is configured in an inverted "L" shape, so that the vertical portion of the inverted "L"-shaped liquid channel can prevent the reaction liquid from flowing into the test strip detection chamber 7 before amplification.

[0060] The horizontal portion of the L-shaped bottom of the replenisher liquid storage chamber 2 intersects and connects with the middle of the arcuate channel between the nucleic acid amplification chamber 6 and the reaction liquid storage chamber 4 via a horizontal capillary liquid channel 81. Furthermore, the top of the replenisher liquid storage chamber 2 is connected to the reaction liquid storage chamber 4 via a horizontal gas channel 91. This forms a circulation path between the chambers.

[0061] The capillary liquid channel connecting the replenisher liquid storage chamber 2 with the arcuate channel between the nucleic acid amplification chamber 6 and the reaction liquid storage chamber 4 on the right side is particularly important. The left side of this narrow capillary liquid channel and the right side of the larger replenisher liquid storage chamber 2 form a capillary valve structure, which intercepts the reaction liquid flowing in from the intersection and achieves separation between the replenisher liquid and the reaction liquid when the chip is in the vertical position.

[0062] The circulation path on the left ensures the flow of the replenishing liquid in the replenishing liquid storage chamber 2. The circulation path on the right ensures the flow of the reaction liquid from the reaction liquid storage chamber 4 to the nucleic acid amplification chamber 6 and then to the test strip detection chamber 7, and the flow of the replenishing liquid from the replenishing liquid storage chamber 2 to the nucleic acid amplification chamber 6 and then to the test strip detection chamber 7, thereby realizing the integration of nucleic acid amplification and test strip detection. Figure 2 yes Figure 1The large replenisher liquid storage chamber 2 and the narrow liquid channel 81 in the chip form a capillary valve structure, which intercepts the reaction liquid flowing from the liquid channel 82 and separates the replenisher liquid from the reaction liquid. The curved portions of the liquid channels 82 and 83 are designed to facilitate the flow of liquid.

[0063] The volumes of the replenisher storage chamber 2, the reaction solution storage chamber 4, and the nucleic acid amplification chamber 6 can be adjusted according to the amount of replenisher and reaction solution added. The total volume of the replenisher and reaction solution should meet the working conditions of the test strip, 50-100 μL, as much as possible.

[0064] The chip is connected to the outside world only at the supplementary liquid sampling port 3 and the reaction liquid sampling port 5. When connected to the outside world, it is only used for sampling. When the supplementary liquid sampling port 3 and the reaction liquid sampling port 5 are blocked, the chip becomes a fully closed chip.

[0065] The working process of the above chip of the present invention is as follows:

[0066] The flow of liquid within the chip is precisely controlled by the various chambers and channels. If the sample port is on the back or front of the chip, flip the chip over after loading the sample so that the chip's reference surface 1 is perpendicular to the ground. If the sample port is on the top of the chip, flipping is not necessary.

[0067] A large amount of lower-cost replenisher is added to replenisher storage chamber 2 through replenisher injection port 3, while a small amount of higher-cost nucleic acid amplification reaction solution and test sample solution is added to reaction solution storage chamber 4 through reaction solution injection port 5. After the additions, the liquids in replenisher storage chamber 2 and reaction solution storage chamber 4 flow downward due to gravity, with the replenisher flowing along the L-shaped vertical portion of replenisher storage chamber 2 to the bottom horizontal portion where it is stored. The reaction solution then flows along the curved liquid channel from reaction solution storage chamber 4 to the nucleic acid amplification chamber 6 where it is stored for amplification.

[0068] After the amplification reaction is complete, the chip is rotated 90° clockwise about the axis perpendicular to the reference plane 1. At this point, the liquid in the replenisher storage chamber 2 and the nucleic acid amplification chamber 6 flows downward due to gravity, ultimately flowing into the lowest point on the right side of the test strip detection chamber 7.

[0069] When a large amount of nucleic acid amplification liquid is added, the nucleic acid amplification liquid fills the entire nucleic acid amplification chamber 6. Due to the large gravity and the absence of surface tension, the nucleic acid amplification liquid first flows from the nucleic acid amplification chamber 6 into the test strip detection chamber 7. The nucleic acid amplification liquid first accumulates on the right side of the test strip detection chamber 7, and the replenishing liquid then flows in from the replenishing liquid storage chamber 2. Subsequently, the nucleic acid amplification liquid is mixed by the replenishing liquid due to the continuous impact of the larger volume of replenishing liquid, and is finally slowly absorbed into the test strip by capillary action.

[0070] When the amount of nucleic acid amplification liquid added is small, the nucleic acid amplification liquid does not fill the nucleic acid amplification chamber 6. Due to the small gravity and the presence of surface tension (when the amount of nucleic acid amplification liquid is small, the nucleic acid amplification chamber is not filled, so there is no liquid in the liquid channel 83 on the right side of the nucleic acid amplification chamber 6. Therefore, after rotation, the liquid needs to overcome capillary force to enter the liquid channel 83; when the amount of nucleic acid amplification liquid is large, there is already liquid in the liquid channel 83 on the right side of the nucleic acid amplification chamber 6 before rotation. Therefore, after rotation, there is no need to overcome capillary force), the nucleic acid amplification liquid cannot flow into the test strip detection chamber 7 through the liquid channel on its own. At this time, the nucleic acid amplification liquid is pushed by the replenishing liquid to flow into the test strip detection chamber 7 (after rotation, the replenishing liquid is above the nucleic acid amplification liquid, and its downward flow will naturally push the nucleic acid amplification liquid below to flow together). The replenishing liquid pushes and impacts the nucleic acid amplification liquid, thereby mixing the two, and finally causing the liquid to be slowly absorbed into the test strip by capillary action.

[0071] Figure 3 and Figure 4 The diagrams show the static state of the liquid after it flows under the action of gravity. If the sample injection port is on the back or front of the chip, flip the chip after injection so that the reference surface of the chip is perpendicular to the ground. If the sample injection port is on the top of the chip, there is no need to flip it. The replenishing liquid flows along the vertical part of the "L"-shaped replenishing liquid storage chamber 2 to be stored in the horizontal part, and the reaction liquid flows along the arc-shaped liquid channel 81 from the reaction liquid storage chamber 4 to be stored in the nucleic acid amplification chamber 6 (such as Figure 3 After the chip is rotated 90° clockwise with the direction perpendicular to the reference plane 1 as the axis, the supplementary liquid flows along the liquid channels 81, 82, and 83 into the lowest point of the test strip detection cavity 7, and the amplification liquid flows along the liquid channel 83 into the lowest point of the test strip detection cavity 7.

[0072] The setting of the replenishing liquid storage chamber 2 and the capillary liquid channel and the presence of the replenishing liquid not only make it possible to use trace amounts of NAA in combination with LFD, reducing the use of amplification reagents; they also promote the flow of trace amounts of nucleic acid amplification liquid in the process of flowing to the test strip detection chamber 7, mix it with the nucleic acid amplification liquid; and take away the nucleic acid amplification liquid remaining in the chamber and channel.

[0073] The specific detection implementation process of the present invention is described below:

[0074] 1. Placement of test strips and addition of supplementary solution and reaction solution

[0075] Place the lateral flow test strip with the front side facing up and the sample pad facing outward into the test strip detection chamber 7. Seal the front of the chip with tape for later use. When in use, the chip is placed horizontally or vertically in the initial state. Specifically, when the sample port is located on the front or back of the chip, the chip is placed horizontally; when the sample port is located on the top of the chip, the chip is placed vertically. Add 15-35μL of nucleic acid amplification reaction solution to the sample port 5 of the reaction liquid storage chamber 4. The nucleic acid amplification reaction solution is prepared by itself as needed, and the marker used should match the test strip. Add supplementary liquid to the sample port 3 of the supplementary liquid storage chamber 2. The total amount of supplementary liquid and reaction liquid added should try to meet the working conditions of the test strip (50-100μL). The supplementary liquid can be clean, uncontaminated PBS buffer, Tris-EDTA buffer or pure water, and appropriate surfactants can be added as needed. After adding the sample, seal the two sample ports 3 and 5 with tape.

[0076] 2. Nucleic acid amplification reaction

[0077] If the sample loading port is on the back or front of the chip, flip the chip over after loading the sample so that the reference surface 1 of the chip is perpendicular to the ground. If the sample loading port is on the top of the chip, there is no need to flip it over. At this time, the liquid in the replenishing liquid storage chamber 2 and the reaction liquid storage chamber 4 flows downward due to gravity. The replenishing liquid flows along the vertical part of the "L"-shaped replenishing liquid storage chamber 2 to and is stored in the horizontal part. The reaction liquid flows along the arc-shaped liquid channel 81 from the reaction liquid storage chamber 4 to and is stored in the nucleic acid amplification chamber 6. The nucleic acid amplification chamber 6 is heated by the temperature control device on the outside of the nucleic acid amplification chamber 6 to perform a nucleic acid amplification reaction. The reaction temperature is adjusted to the optimal temperature of the nucleic acid amplification method used.

[0078] 3. Lateral flow test strip detection of amplified products

[0079] After the amplification reaction is completed, the reaction amplification liquid is obtained in the nucleic acid amplification chamber 6. The chip is rotated 90° clockwise with the axis perpendicular to the reference plane 1. At this point, the liquid in the replenisher storage chamber 2 and the nucleic acid amplification chamber 6 flows downward due to gravity, eventually flowing into the lowest point of the test strip detection chamber 7. The replenisher liquid continuously impacts the amplification liquid and mixes with it. The mixed liquid moves laterally from the sample pad to the absorbent pad through capillary action. Finally, the test result is interpreted based on the color development of the C and T lines on the test strip.

[0080] Implementation examples of the present invention:

[0081] 1. PCR amplification and single-indicator lateral flow test strip detection of African swine fever virus

[0082] The structure of the chip used in this embodiment is as follows Figure 1 The chip is made of polymethyl methacrylate (PMMA) sheet with a thickness of 4 mm. Figure 1The structure shown (the dimensions are subject to the text description below).

[0083] The chip contains a replenishing liquid storage chamber 2, a reaction liquid storage chamber 4, a nucleic acid amplification chamber 6 and a test strip detection chamber 7, as well as channels 81, 82, 83, 91, and 92 connecting these chambers. There is a sample addition port 3 on the top of the replenishing liquid storage chamber 2. There is a sample addition port 5 on the top of the reaction liquid storage chamber 4. The replenishing liquid storage chamber 2 is located on the lower left side of the chip. The reaction liquid storage chamber 4 is located on the upper right side of the replenishing liquid storage chamber 2, and its highest point is level with the highest point of the replenishing liquid storage chamber 2. The nucleic acid amplification chamber 6 is located on the lower right side of the reaction liquid storage chamber 4 and is roughly level with the replenishing liquid storage chamber 2. The test strip detection chamber 7 is located above the nucleic acid amplification chamber 6 and to the right of the reaction liquid storage chamber. The chambers can also be arranged in a left-right mirror-symmetrical layout, as long as the liquid inlets of the replenishing liquid storage chamber 2 and the test strip detection chamber 7 are located on different sides of the nucleic acid amplification chamber 6.

[0084] The vertical portion of the L-shaped replenisher liquid storage chamber 2 is 4 mm wide and 23 mm long, while the horizontal portion is 3 mm wide and 28 mm long. The replenisher liquid injection port 3 is 3 mm wide and 4 mm long. The reaction liquid storage chamber 4 is 3 mm wide and 11 mm long. The reaction liquid injection port 5 is 3 mm wide and 4 mm long. The arc-shaped nucleic acid amplification chamber 6 has a chord length of 7.5 mm. The rectangular test strip detection chamber 7 is 4 mm wide and 65 mm long. All of these chambers are 2 mm deep. Liquid channel 81 has a cross-sectional dimension of 0.3 x 0.3 mm. Together with the replenisher liquid storage chamber 2, which has a cross-sectional dimension of 2 x 3 mm, it forms a capillary valve structure, trapping the reaction liquid flowing in from liquid channel 82 and achieving separation between the replenisher liquid and the reaction liquid. The cross-sectional dimension of liquid channel 82 is 0.5 x 0.5 mm. If it is too thin, the reaction liquid will not flow smoothly, while if it is too thick, a large amount of reaction liquid will remain. The cross-sectional dimension of liquid channel 83 is 0.5 x 0.5 mm. Gas channels 91 and 92 are 0.3 mm wide and 0.3 mm deep. The length and shape of each channel are determined based on actual needs. The rounded corners of liquid channels 82 and 83 facilitate liquid flow within the channels. To ensure faster and smoother solution flow within the chip, a surfactant can be added to the replenisher and reaction solutions.

[0085] This example takes the detection of African swine fever virus as an example, and uses the P72 gene in the African swine fever virus as the target to perform PCR amplification and lateral flow test strip detection.

[0086] Place the lateral flow test strip with the front side facing up and the sample pad facing outward into the test strip detection chamber 7. The test line of the test strip is coated with avidin (SA), and the latex microspheres are labeled with anti-FAM antibodies. The nucleic acid amplification products containing both Biotin and FAM labels are detected by the sandwich method. Seal the front of the chip with PMMA tape. In the initial state, the chip is placed vertically. Add 25 μL of nucleic acid amplification reaction solution to the sample port 5 of the reaction solution storage chamber 4. Add 50 μL of pure water to the sample port 3 of the supplementary solution storage chamber 2. After adding the sample, seal the two sample ports 3 and 5 with PMMA tape.

[0087] The PCR reaction solution system is as follows:

[0088]

[0089]

[0090] Flip the chip so that its reference surface 1 is perpendicular to the ground. At this point, the liquid in the replenisher liquid storage chamber 2 and the reaction liquid storage chamber 4 flows downward due to gravity. Pure water flows along the vertical portion of the L-shaped replenisher liquid storage chamber 2 to be stored in the horizontal portion. The reaction liquid flows along the curved liquid channel 81 from the reaction liquid storage chamber 4 to be stored in the nucleic acid amplification chamber 6. An external temperature control device is used to heat the outside of the nucleic acid amplification chamber 6. The configured temperature control device needs to be able to cycle the temperature between 50°C and 95°C.

[0091] After the amplification reaction is completed, the reaction amplification liquid is obtained in the nucleic acid amplification chamber 6. The chip is rotated 90° clockwise with the direction perpendicular to the reference plane 1 as the axis. At this time, the liquid in the replenishing liquid storage chamber 2 and the nucleic acid amplification chamber 6 flows downward due to gravity and eventually flows into the lowest point of the test strip detection chamber 7. The replenishing liquid continuously impacts the amplification liquid and mixes with it. The mixed liquid moves laterally from the sample pad to the absorbent pad through capillary action. Finally, the test results are interpreted based on the color development of the C line and T line on the test strip. If both the C line and the T line are colored, it means that the tested sample contains the P72 gene ( Figure 5 a). If the C line is colored and the T line is not colored, it means that the sample does not contain the P72 gene or the content is lower than the detection limit of the test strip ( Figure 5 b). If both the C line and the T line are colored, it means that the operation is wrong or the test strip has deteriorated and is invalid. In this case, the problem should be eliminated and the test should be repeated ( Figure 5 c).

[0092] 2. Dual LAMP amplification and dual-index test strip detection of hepatitis B virus (HBV) and hepatitis A virus (HCV)

[0093] The structure of this embodiment is as follows Figure 1 As shown, the chip structure and processing method are the same as those in Example 1.

[0094] Among hepatotropic viruses, hepatitis B virus (HBV) and hepatitis A virus (HCV) are the most contagious. This embodiment takes the detection of HBV and HCV as an example, and performs dual LAMP amplification and dual-indicator test strip detection.

[0095] Place the lateral flow test strip with the front side facing up and the sample pad facing outward into the test strip detection chamber 7. The detection line T1 of the dual-index test strip is coated with anti-FITC antibody, and the detection line T2 is coated with anti-Digoxin antibody. Some latex microspheres are labeled with streptavidin, and some latex microspheres are labeled with anti-TAMRA antibody. The nucleic acid amplification products containing the corresponding labels are detected by the sandwich method. Seal the front of the chip with PMMA tape. In the initial state, the chip is placed vertically. Add 20 μL of nucleic acid amplification reaction solution to the sample port 5 of the reaction solution storage chamber 4. Add 70 μL of Tris-EDTA buffer to the sample port 3 of the supplementary liquid storage chamber 2. After adding the sample, seal the two sample ports 3 and 5 with PMMA tape.

[0096] The LAMP reaction solution system is as follows:

[0097]

[0098]

[0099] Flip the chip so that its reference surface 1 is perpendicular to the ground. At this point, the liquid in the replenisher liquid storage chamber 2 and the reaction liquid storage chamber 4 flows downward due to gravity. The replenisher liquid flows along the vertical portion of the L-shaped replenisher liquid storage chamber 2 to the horizontal portion, where it is stored. The reaction liquid flows along the curved liquid channel 81 from the reaction liquid storage chamber 4 to the nucleic acid amplification chamber 6, where it is stored. An external temperature control device is used to heat the outside of the nucleic acid amplification chamber 6 to approximately 65°C. The reaction is continued for 30 minutes.

[0100] After the amplification reaction is completed, the reaction amplification liquid is obtained in the nucleic acid amplification chamber 6. The chip is rotated 90° clockwise with the direction perpendicular to the reference plane 1 as the axis. At this time, the liquid in the replenishing liquid storage chamber 2 and the nucleic acid amplification chamber 6 flows downward due to gravity, and eventually flows into the lowest point of the test strip detection chamber 7. The replenishing liquid continuously impacts the amplification liquid and mixes with it. The mixed liquid moves laterally from the sample pad to the absorbent pad through capillary action. Finally, the test results are interpreted based on the color development of the C line, T1 line and T2 line on the test strip. If the C line, T1 line and T2 line are all colored, it means that the test is valid and the tested sample contains both HBV and HCV viruses ( Figure 6 a). If the C line and T1 line are colored, and the T2 line is not colored, it means that the test is valid and the sample contains HBV but not HCV ( Figure 6 b). If the C line and T2 line are colored, and the T1 line is not colored, it means that the test is valid and the sample contains HCV but not HBV ( Figure 6 c). If the C line is colored and the T1 and T2 lines are not colored, it means that the test is valid and the sample does not contain HBV and HCV ( Figure 6 d). If the C line does not show color, it means that the operation is wrong or the test strip has deteriorated and is invalid. In this case, the problem should be eliminated and the test should be repeated ( Figure 6 e).

Claims

1. A fully enclosed chip based on gravity drive and micro-NAA-LFD, characterized by: The invention comprises an L-shaped supplementary liquid storage chamber (2) for accommodating the supplementary liquid, wherein the upper end of the supplementary liquid storage chamber (2) is provided with a supplementary liquid injection port (3) connected to the outside of the chip, and the supplementary liquid injection port (3) is used for adding the supplementary liquid; The chip comprises a reaction liquid storage chamber (4) for temporarily accommodating nucleic acid amplification reaction liquid and a sample solution to be tested. The upper end of the reaction liquid storage chamber (4) is provided with a reaction liquid injection port (5) connected to the outside of the chip. The reaction liquid injection port (5) is used to inject the reaction liquid. It includes a nucleic acid amplification chamber (6) for accommodating a nucleic acid amplification reaction solution and a sample solution to be tested to perform a nucleic acid amplification reaction; It includes a test strip detection cavity (7) for test strip detection, in which a lateral flow test strip is pre-placed; The supplementary liquid storage chamber (2) and the reaction liquid storage chamber (4) are connected via a channel inside the chip, forming a first annular circulation path inside the chip; Each two of the reaction liquid storage chamber (4), the nucleic acid amplification chamber (6) and the test strip detection chamber (7) are connected through a channel inside the chip, forming a second annular circulation path inside the chip; In the initial state, the reaction liquid storage chamber (4) and the test strip detection chamber (7) are both located at the bottom of the replenishing liquid storage chamber (2) and above the nucleic acid amplification chamber (6), the reaction liquid storage chamber (4) is located between the top of the replenishing liquid storage chamber (2) and the test strip detection chamber (7), the top of the reaction liquid storage chamber (4) is flush with the top of the replenishing liquid storage chamber (2), the nucleic acid amplification chamber (6) is flush with the bottom of the replenishing liquid storage chamber (2), and the test strip detection chamber (7) is flush with the reaction liquid storage chamber (4); The side of the nucleic acid amplification chamber (6) close to the reaction liquid storage chamber (4) is connected to the reaction liquid storage chamber (4) through an arc-shaped liquid channel, the other side of the nucleic acid amplification chamber (6) is connected to the side of the test strip detection chamber (7) away from the reaction liquid storage chamber (4) through a liquid channel, and the side of the test strip detection chamber (7) close to the reaction liquid storage chamber (4) is connected to the reaction liquid storage chamber (4) or the reaction liquid injection port (5) through a gas channel; the bottom of the supplementary liquid storage chamber (2) is connected to the middle of the arc-shaped channel between the nucleic acid amplification chamber (6) and the reaction liquid storage chamber (4) through a horizontal capillary liquid channel, and the top of the supplementary liquid storage chamber (2) is connected to the reaction liquid storage chamber (4) through a horizontal gas channel; The liquid channel between the nucleic acid amplification chamber (6) and the test strip detection chamber (7) is configured in an inverted "L" shape.

2. The fully enclosed chip based on gravity drive and micro-NAA-LFD integration according to claim 1, characterized in that: The "L" shape of the replenishing liquid storage chamber (2) includes a vertical portion and a horizontal portion, wherein the vertical portion serves as a flow channel for the replenishing liquid, the upper end of the vertical portion is connected to the replenishing liquid injection port (3), and the horizontal portion is used to store the replenishing liquid.

3. The fully enclosed chip based on gravity drive and micro-NAA-LFD integration according to claim 1, characterized in that: The lower side wall of the reaction liquid storage chamber (4) is provided with an arc-shaped inner concave surface.

4. The fully enclosed chip based on gravity drive and micro-NAA-LFD integration according to claim 1, characterized in that: The bottom surface of the nucleic acid amplification chamber (6) is an arc-shaped concave surface.

5. The fully enclosed chip based on gravity drive and micro-NAA-LFD integration according to claim 1, characterized in that: The volume of the reaction liquid storage chamber (4) is smaller than the volume of the supplementary liquid storage chamber (2).

6. The fully enclosed chip based on gravity drive and micro-NAA-LFD integration according to claim 1, characterized in that: The chip is connected to the outside world only at the supplementary liquid sampling port (3) and the reaction liquid sampling port (5). When the supplementary liquid sampling port (3) and the reaction liquid sampling port (5) are closed, the chip becomes a fully closed chip.

7. A method for using the gravity-driven and micro-NAA-LFD integrated fully enclosed chip as described in any one of claims 1 to 6, characterized in that: S1. Place the lateral flow test strip with the front side facing upward and the sample pad facing outward into the bottom of the test strip detection chamber (7), add 15-35 μL of nucleic acid amplification reaction solution to the supplementary solution injection port (3) of the reaction solution storage chamber (4), and add a supplementary solution with a volume greater than that of the nucleic acid amplification reaction solution to the reaction solution injection port (5) of the supplementary solution storage chamber (2). The total amount of the supplementary solution and the nucleic acid amplification reaction solution added should meet the working conditions of the test strip; after adding the sample, seal the two injection ports of the supplementary solution injection port (3) and the reaction solution injection port (5) with tape; S2. The "L" shape of the replenishing liquid storage chamber (2) includes a vertical portion and a horizontal portion; the chip is flipped or not flipped, and the liquid in the replenishing liquid storage chamber (2) and the reaction liquid storage chamber (4) flows downward due to gravity: the replenishing liquid flows along the vertical portion of the "L"-shaped replenishing liquid storage chamber (2) to be stored in the horizontal portion, and the reaction liquid flows along the arc-shaped liquid channel from the reaction liquid storage chamber (4) to the nucleic acid amplification chamber (6); S3, then the nucleic acid amplification chamber (6) is heated by a temperature control device outside the nucleic acid amplification chamber (6) to perform a nucleic acid amplification reaction; after the amplification reaction is completed, a reaction amplification solution is obtained in the nucleic acid amplification chamber (6); S4. The chip is flipped over. At this time, the liquid in the replenishing liquid storage chamber (2) and the nucleic acid amplification chamber (6) flows downward due to gravity and eventually flows into the lowest point of the flipped test strip detection chamber (7). The replenishing liquid continuously impacts or pushes the nucleic acid amplification liquid into the test strip detection chamber (7) and mixes with it to form a mixed liquid. The mixed liquid moves laterally from the sample pad to the absorbent pad of the test strip by capillary action. Finally, the test result is interpreted according to the color development of the test strip.

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