Multi-target micro-fluidic chip with novel liquid shunting mode

Abstract

The invention relates to the technical field of nucleic acid detection, and discloses a multi-target micro-fluidic chip with a novel liquid distribution mode, which comprises a reaction layer, a sealing layer and a liquid distribution layer, the reaction layer is provided with a plurality of amplification reaction cavities, sample loading buffer bins and test paper storage grooves; a vertically arranged sample inlet, a plurality of sample shunting channels and a plurality of sample vertical runners are arranged in the liquid separation layer; the sample inlet and the sample vertical runners upwards and downwards penetrate through the top surface and the bottom surface of the liquid separation layer respectively; the sample vertical flow channels, the sample shunting channels and the amplification reaction cavity are correspondingly arranged in position, the sample shunting channels are respectively communicated with the sample inlet, the sample shunting channels are the same in size and are arranged at intervals along a conical surface, the sample vertical flow channels are also the same in size, and the sample vertical flow channels are communicated with the amplification reaction cavity. The sample can be completely equally divided, and a plurality of parallel reactions are promoted to be simultaneously carried out; and as many samples enter the amplification reaction cavity as possible, so that the liquid loss is reduced, and the detection accuracy can be further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a multi-target microfluidic chip with a novel liquid diversion mode. Background Art

[0002] Pathogen detection technologies include traditional detection technologies such as smear microscopy, isolation culture and biochemical reaction, and tissue cell culture, as well as commonly used clinical methods such as antigen-antibody detection and nucleic acid detection. Traditional detection technologies are time-consuming and not suitable for rapid screening of pathogens. Compared with antigen-antibody detection, nucleic acid detection has the advantages of high sensitivity and high specificity, so it is of great significance for early diagnosis and screening of asymptomatic infections.

[0003] The main limitation of nucleic acid detection technology in point-of-care testing is that it relies on professional operation and professional instruments and equipment. However, in the primary healthcare system, nucleic acid detection technology is difficult to popularize due to the lack of professional molecular biology laboratories. In order to meet the needs of rapid and large-scale screening at the grassroots level, a low-cost, portable, instrument-free microfluidic chip suitable for real-time detection is urgently needed.

[0004] The microfluidic technology currently used for multi-target detection aims to achieve simultaneous detection and analysis of multiple biomolecules through integration and miniaturization, in order to improve detection efficiency and accuracy. However, this technology faces significant challenges in practical applications, especially in the precise distribution of liquid samples. On the one hand, when the chip design allows for complete equal distribution of liquid, it is usually limited by the number of targets that can be detected. This is because most liquid distribution is based on a 1-in-2 flow channel, so the number of targets must be 2. n (n≥1). On the other hand, the current microfluidic chip design has the diversion channel and the amplification reaction chamber on the same plane, which cannot ensure that the liquid does not flow back or remain in the channel, thus increasing the liquid loss.

[0005] Therefore, it is urgent to develop a multi-target microfluidic chip with a new liquid diversion method to get rid of the limitation on the number of detection targets and effectively reduce liquid loss. Summary of the invention

[0006] The object of the present invention is to provide a multi-target microfluidic chip with a novel liquid diversion method, which can get rid of the limitation on the number of detection targets and effectively reduce liquid loss.

[0007] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0008] A multi-target microfluidic chip with a novel liquid diversion method, comprising:

[0009] The top surface of the reaction layer is inwardly recessed and has a plurality of amplification reaction chambers and a plurality of sample loading buffer chambers, and also has a plurality of test paper storage slots; the plurality of amplification reaction chambers, the plurality of sample loading buffer chambers and the plurality of test paper storage slots are arranged in a one-to-one corresponding position, and the amplification reaction chambers are connected to the corresponding sample loading buffer chambers;

[0010] A sealing layer, arranged on the reaction layer;

[0011] The liquid separation layer is installed at one end of the sealing layer and protrudes from the top surface of the sealing layer. The liquid separation layer cooperates with the sealing layer to seal the reaction layer. The liquid separation layer is provided with a vertically arranged sample inlet, a plurality of sample diversion channels and a plurality of sample vertical flow channels. The sample inlet penetrates the top surface of the liquid separation layer upward, and the sample vertical flow channel penetrates the bottom surface of the liquid separation layer downward. The plurality of sample diversion channels are radially distributed, and the top end of the sample diversion channel is connected to the sample inlet, and the bottom end is connected to the sample vertical flow channel; wherein,

[0012] The plurality of sample vertical flow channels, the plurality of sample diversion channels and the plurality of amplification reaction chambers are arranged in corresponding positions one by one, the plurality of sample diversion channels have the same size and are arranged at intervals along a conical surface, the plurality of sample vertical flow channels have the same size, the conical surface is arranged coaxially with the injection port, and the amplification reaction chamber is vertically opposite to and connected to the corresponding sample vertical flow channel.

[0013] Furthermore, the amplification reaction chamber and the sample vertical flow channel are arranged at a vertical distance.

[0014] Furthermore, the amplification reaction chamber is connected to the sample loading buffer chamber through a flow resistance channel.

[0015] Furthermore, the flow resistance channel includes a first U-shaped tube and two second U-shaped tubes respectively connected to both ends of the first U-shaped tube, and the opening direction of the second U-shaped tube is opposite to the opening direction of the first U-shaped tube.

[0016] Furthermore, it also includes a capillary wall, one end of which enters the sample loading buffer chamber, and the other end of which is connected to the test paper in the test paper storage groove.

[0017] Furthermore, the bottom wall of the sample loading buffer chamber is lower than the bottom wall of the flow resistance channel.

[0018] Furthermore, it also includes a base, which is installed at the bottom of the reaction layer. A heating module storage slot is provided in the base. The heating module storage slot is used to store a heating module, and the heating module is used to heat the amplification reaction chamber.

[0019] Furthermore, the base is slidably connected to the reaction layer.

[0020] Furthermore, the liquid separation layer is provided with a diluent bubble placement hole, a plurality of diluent diversion channels arranged radially, and a plurality of diluent vertical flow channels corresponding to and connected with the bottom ends of the plurality of diluent diversion channels one by one; the diluent bubble placement hole and the injection port are arranged at a distance in the horizontal direction; the diluent bubble placement hole is used to accommodate diluent bubbles; the top ends of the plurality of diluent diversion channels are respectively connected to the diluent bubble placement hole, and a plurality of diluent cavities are provided on the reaction layer, and the diluent cavities vertically correspond to and are connected with the diluent vertical flow channels one by one.

[0021] Furthermore, the sealing layer is provided with result display windows, and the number of the result display windows is the same as the number of the amplification reaction chambers.

[0022] The technical solution of the present invention has the following advantages:

[0023] 1. The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention has a vertically arranged sample inlet, a plurality of sample diversion channels and a plurality of sample vertical flow channels, the sample inlet penetrates the top surface of the liquid diversion layer upward, the sample vertical flow channel penetrates the bottom surface of the liquid diversion layer downward, and the plurality of sample diversion channels are radially distributed, the top of the sample diversion channel is connected to the sample inlet, and the bottom is connected to the sample vertical flow channel; the plurality of sample vertical flow channels, the plurality of sample diversion channels and the plurality of amplification reaction chambers are arranged in a one-to-one position corresponding to each other, the plurality of sample diversion channels have the same size and are arranged at intervals along a conical surface, and the plurality of sample vertical flow channels have the same size. The conical surface is coaxially arranged with the injection port, and the amplification reaction chamber is vertically opposite to and connected with the corresponding sample vertical flow channel. Therefore, the sample and the diluent can be evenly divided into the corresponding amplification reaction chamber to achieve multi-target detection. In addition, since a sample vertical flow channel is also arranged below the sample diversion channel, and the liquid separation layer protrudes from the top surface of the sealing plate, the vertical flow path of the sample and the diluent can be increased, so that the sample and the diluent have a greater flow speed under the action of their own gravity, and under the action of inertia, as much as possible enter the amplification reaction chamber, thereby reducing liquid loss, ensuring the concentration of the sample and the diluent, and thus improving the detection accuracy. Furthermore, using a multi-target microfluidic chip with a novel liquid diversion method, the sample only needs to be lysed at room temperature, and then added to the amplification reaction chamber through the sample inlet for amplification reaction. The amplification product is diluted by the diluent and enters the sample loading buffer chamber and then reaches the test paper in the test paper storage tank. The test results are conveniently displayed by the test paper. Therefore, low-cost, portable and real-time detection can be achieved. It does not need to rely on professional operation and professional instruments and equipment, and can meet the detection needs of grassroots rapid and large-scale screening. In the form of radially arranged sample diversion channels, it gets rid of the limitation on the number of detection targets.

[0024] 2. The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention has a vertical spacing between the amplification reaction chamber and the sample vertical flow channel. This can reduce the risk of liquid in the amplification reaction chamber entering the sample vertical flow channel, further reduce the possibility of liquid loss, and increase detection accuracy.

[0025] 3. The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention comprises a flow resistance channel including a first U-shaped tube and two second U-shaped tubes respectively connected to the two ends of the first U-shaped tube, and the opening direction of the second U-shaped tube is opposite to the opening direction of the first U-shaped tube. Such a flow resistance channel can, on the one hand, prevent the sample added to the amplification reaction chamber from directly flowing into the sample loading buffer chamber before or during the amplification reaction, and on the other hand, can further mix the amplification product and the diluent before the test paper detection.

[0026] 4. In the multi-target microfluidic chip with a novel liquid diversion method provided by the present invention, the bottom wall of the loading buffer chamber is lower than the bottom wall of the flow resistance channel, so that a portion of the diluted amplification products can be stored to prevent a large amount of diluted amplification products from directly flowing to the test paper, resulting in inaccurate test results.

[0027] 5. The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention has a base that is slidably connected to the reaction layer, and the heating module can be easily removed for repeated use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 Schematic diagram of the three-dimensional combination of the multi-target microfluidic chip in Example 1 of the present invention;

[0030] Figure 2 This is a perspective schematic diagram of a three-dimensional exploded view of a multi-target microfluidic chip in Example 1 of the present invention;

[0031] Figure 3 Schematic diagram of a top view of a reaction layer of a multi-target microfluidic chip in Example 1 of the present invention;

[0032] Figure 4 This is a perspective schematic diagram of a reaction layer of a multi-target microfluidic chip in Example 1 of the present invention;

[0033] Figure 5 It is a perspective schematic diagram of the liquid separation layer of the multi-target microfluidic chip in Example 1 of the present invention;

[0034] Figure 6 Schematic perspective view of the base of the multi-target microfluidic chip in Example 1 of the present invention;

[0035] Figure 7 Schematic diagram of the three-dimensional assembly of the multi-target microfluidic chip in the second embodiment of the present invention;

[0036] Figure 8 It is a perspective schematic diagram of a three-dimensional exploded view of a multi-target microfluidic chip in Embodiment 2 of the present invention;

[0037] Fig. 9 It is a perspective schematic diagram of the base of the multi-target microfluidic chip in the second embodiment of the present invention;

[0038] Fig.10 Schematic diagram of the three-dimensional assembly of the multi-target microfluidic chip in the third embodiment of the present invention;

[0039] Fig.11 This is a perspective schematic diagram of a three-dimensional exploded view of a multi-target microfluidic chip according to Embodiment 3 of the present invention;

[0040] Fig.12 This is a schematic top view of the reaction layer of the multi-target microfluidic chip of Example 3 of the present invention;

[0041] Fig.13 This is a perspective schematic diagram of a reaction layer of a multi-target microfluidic chip according to Embodiment 3 of the present invention;

[0042] Fig.14 This is a perspective schematic diagram of the liquid separation layer of the multi-target microfluidic chip according to Example 3 of the present invention;

[0043] Fig.15 Schematic diagram of the three-dimensional assembly of the multi-target microfluidic chip in the fourth embodiment of the present invention;

[0044] Fig.16 Schematic diagram of a three-dimensional exploded perspective view of a multi-target microfluidic chip in Embodiment 4 of the present invention;

[0045] Fig.17 Schematic diagram of a top view of a reaction layer of a multi-target microfluidic chip in Embodiment 4 of the present invention;

[0046] Fig.18 This is a perspective schematic diagram of a reaction layer of a multi-target microfluidic chip in Example 4 of the present invention;

[0047] Fig.19 It is a perspective schematic diagram of the liquid separation layer of the multi-target microfluidic chip in the fourth embodiment of the present invention;

[0048] Fig. 20 This is a schematic diagram of the structure of the test paper in an embodiment of the present invention;

[0049] Fig.21 This is a schematic diagram of the structure of another test paper in an embodiment of the present invention;

[0050] Fig. 22 Schematic diagram of the structure of another test paper in an embodiment of the present invention.

[0051] Description of reference numerals:

[0052] 1. Liquid separation layer; 11. Sample injection port; 12. Sample diversion channel; 13. Sample vertical flow channel; 14. Sealing cover; 15. Dilution vesicle; 16. Dilution diversion channel; 17. Dilution vertical flow channel; 2. Sealing layer; 21. Result display window; 22. First fixed component; 3. Reaction layer; 31. Second fixed component; 32. Amplification reaction chamber; 33. Flow resistance channel; 34. Sample loading buffer chamber; 35. Capillary wall; 36. Test paper storage slot; 37. Slide; 38. Dilution chamber; 4. Base; 41. Sliding block; 42. Heating module storage slot; 43. Wire clamp slot; 5. Test paper; 51. Bottom plate; 52. Sample pad; 53. Binding pad; 54. NC membrane; 55. Water-absorbing pad. DETAILED DESCRIPTION

[0053] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] In the description of the present invention, it should be noted that the terms "upper", "lower", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0055] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0056] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0057] like Figures 1 to 22 As shown, the present invention provides a multi-target microfluidic chip with a novel liquid diversion method for nucleic acid detection (hereinafter referred to as a multi-target microfluidic chip), the multi-target microfluidic chip comprises a liquid separation layer 1, a sealing layer 2, a reaction layer 3, and a base 4. The liquid separation layer 1 is used to evenly divide the sample and the diluent. The reaction layer 3 is used to amplify and detect the target to be detected.

[0058] The top surface of the reaction layer 3 is inwardly recessed to form a plurality of amplification reaction chambers 32 and a plurality of sample loading buffer chambers 34. The reaction layer 3 also has a plurality of chambers for storing test strips 5 (such as Figure 20 to Figure 22 The plurality of amplification reaction chambers 32, the plurality of sample loading buffer chambers 34 and the plurality of test paper storage slots 36 are arranged in a one-to-one correspondence, and the amplification reaction chambers 32 are connected to the corresponding sample loading buffer chambers 34.

[0059] The sealing layer 2 is installed on the reaction layer 3 , and the liquid separation layer 1 is installed at one end of the sealing layer 2 . The sealing layer 2 and the liquid separation layer 1 cooperate to seal the reaction layer 3 .

[0060] The liquid separation layer 1 is provided with a vertically arranged sample inlet 11, a plurality of sample shunt channels 12 and a plurality of sample vertical flow channels 13. The sample inlet 11 penetrates upwardly through the top surface of the liquid separation layer 1. The sample vertical flow channel 13 penetrates downwardly through the bottom surface of the liquid separation layer 1, and the plurality of sample shunt channels 12 are radially arranged and respectively connected to the same sample inlet 11, the top of the sample shunt channel 12 is connected to the sample inlet 11, and the bottom is connected to the sample vertical flow channel 13. Among them, the plurality of sample vertical flow channels 13, the plurality of sample shunt channels 12 and the plurality of amplification reaction chambers 32 below the sample inlet 11 are arranged in a position corresponding to each other. The plurality of sample shunt channels 12 have the same size and are arranged at intervals along a conical surface, the plurality of sample vertical flow channels 13 also have the same size, the conical surface is arranged coaxially with the sample inlet 11, and the amplification reaction chamber 32 is directly opposite to and vertically connected to the sample vertical flow channel 13.

[0061] In the present invention, since a sample inlet 11, a plurality of sample diversion channels 12 and a plurality of sample vertical flow channels 13 are provided on the liquid separation layer 1, the plurality of sample vertical flow channels 13, the plurality of sample diversion channels 12 and the plurality of amplification reaction chambers 32 are arranged in corresponding positions one by one, the plurality of sample diversion channels 12 are respectively connected to the same sample inlet 11, and the plurality of sample diversion channels 12 have the same size and are arranged at intervals along a conical surface, the plurality of sample vertical flow channels 13 have the same size, and the amplification reaction chamber 32 is directly opposite to and vertically connected to the sample vertical flow channel 13, so that the sample and the diluent can be evenly divided into the corresponding amplification reaction chamber 32, so that simultaneous amplification of multiple targets can be achieved. In addition, the sample inlet 11 is vertically arranged, and a sample vertical flow channel 13 is also arranged below the sample diversion channel 12. The liquid separation layer 1 also protrudes from the top surface of the sealing layer 2, thereby increasing the vertical flow path of the sample and the diluent, so that the sample and the diluent have a greater flow velocity under the action of their own gravity, and under the action of inertia, enter the amplification reaction chamber 32 as much as possible, reduce liquid loss, and ensure that the amount of sample and diluent entering multiple amplification reaction chambers 32 is consistent, thereby ultimately improving the accuracy of nucleic acid detection.

[0062] Furthermore, the amplification reaction chamber 32 is directly opposite to and connected to the sample vertical flow channel 13 , and a vertical spacing is set between the amplification reaction chamber 32 and the sample vertical flow channel 13 , thereby reducing the risk of the sample and diluent entering the amplification reaction chamber 32 flowing back into the sample vertical flow channel 13 .

[0063] In order to simplify the operation steps, the diluent is built into the multi-target microfluidic chip, and a tin foil membrane is provided at the bottom of the diluent vesicle 15 storing the diluent, and the vesicle material is thin soft plastic. The diluent vesicle 15 is squeezed downward to break through the tin foil membrane to release the diluent. Specifically, the diluent and the sample have their own flow paths, that is, a diluent vesicle placement hole, a plurality of diluent shunt channels 16 and a plurality of diluent vertical flow channels 17 are also provided on the liquid separation layer 1. The diluent vesicle placement hole penetrates the top surface of the liquid separation layer 1 upward. The diluent vertical flow channel 17 penetrates the bottom surface of the liquid separation layer 1 downward, and the plurality of diluent shunt channels 16 are radially arranged and respectively connected to the same diluent vesicle placement hole, and the top of the diluent shunt channel 16 is connected to the diluent vesicle placement hole, and the bottom is connected to the diluent vertical flow channel 17. Correspondingly, a plurality of diluent chambers 38 are provided in the aforementioned reaction layer 3, and the plurality of diluent chambers 38 are the same in number and connected to the plurality of amplification reaction chambers 32 in one-to-one correspondence. The plurality of diluent shunt channels 16, the plurality of diluent vertical flow channels 17, and the plurality of diluent chambers 38 below the diluent vesicle placement hole are arranged in one-to-one correspondence. The plurality of diluent shunt channels 16 have the same size and are arranged at intervals along a conical surface, and the plurality of diluent vertical flow channels 17 have the same size. The conical surface is arranged coaxially with the diluent vesicle placement hole, and the diluent chamber 38 is vertically opposite to and connected to the diluent vertical flow channel 17. In this way, the sample can be evenly divided through the injection port 11, the sample shunt channel 12, and the sample vertical flow channel 13 and then enter the amplification reaction chamber 32, and the diluent can be evenly divided through the diluent vesicle placement hole, the diluent shunt channel 16, and the diluent vertical flow channel 17 and then enter the diluent chamber 38, so as to realize multi-target detection of equal distribution of samples and diluents.

[0064] In order to prevent foreign matter from entering the injection port 11, the injection port 11 is sealed by a sealing cover 14. Specifically, the sealing cover 14 is threadedly connected to the injection port 11. Of course, it can also be sealed in the form of a sealing film.

[0065] In order to facilitate viewing of the test results, a result display window 21 is provided on the sealing layer 2, and the result display window 21 corresponds to the color development part on the test paper 5. In order to facilitate the fixed connection between the sealing layer 2 and the reaction layer 3, a first fixing component 22 (such as a column / card slot) is provided on the sealing layer 2, and a second fixing component 31 (corresponding to a card slot / column) is provided on the reaction layer. The sealing layer 2 and the reaction layer 3 are fixed together by the cooperation of the first fixing component 22 and the second fixing component 31.

[0066] The base 4 is connected to the reaction layer 3 in the form of a sliding connection. Specifically, a slider 41 is provided on the base 4, and a slide groove 37 is provided on the reaction layer 3, and the slider 41 slides along the slide groove 37. The base 4 and the reaction layer 3 are set in a sliding connection because a heating module storage groove 42 for storing the heating module is provided in the base 4. By sliding the base 4 open, the heating module in the heating module storage groove 42 can be taken out, so that it can be separated from the entire device and used for the next detection reaction, thereby realizing the reuse of the heating module. The heating module is used to provide temperature incubation for the amplification reaction.

[0067] Embodiment 1

[0068] like Figures 1 to 6 , Figure 20 to Figure 22 As shown, the number of the sample shunt channel 12, the sample vertical flow channel 13 and the amplification reaction chamber 32 under the injection port 11, the diluent shunt channel 16 and the diluent vertical flow channel 17 under the diluent vesicle placement hole, and the result display window 21 are all three. The angle between two adjacent sample shunt channels 12 is 120°, and the angle between adjacent diluent shunt channels 16 is 120°. The angle between the extension direction of the sample shunt channel 12 and the bottom surface of the injection port 11 (that is, the horizontal plane) is 35° to 55°, preferably 45°. The angle between the extension direction of the diluent shunt channel 16 and the bottom surface of the diluent vesicle placement hole (that is, the horizontal plane) is 35° to 55°, preferably 45°. The height of the sample vertical flow channel is 0.55mm to 0.65mm, preferably 0.6mm. Since three sample diversion channels 12, three sample vertical flow channels 13, three diluent diversion channels 16 and three diluent vertical flow channels 17 are provided respectively, the sample added to the amplification reaction chamber from the injection port 11 and the diluent released from the diluent vesicle 15 can be divided into three parts, thereby simplifying the number of liquid addition operations.

[0069] The height of the amplification reaction chamber 32 is 1 mm, and the RPA reaction volume (reaction volume refers to the space occupied by substances reacting and generating substances under certain conditions) is 10 μL. Each amplification reaction chamber 32 is connected to a flow resistance channel 33 downstream, and the flow resistance channel 33 can prevent the sample from flowing directly downstream after entering the amplification reaction chamber 32, thereby improving the uniformity of the diluent and the amplification product. Specifically, the flow resistance channel 33 includes a first U-shaped tube, and two second U-shaped tubes respectively connected to the two ends of the first U-shaped tube, and the opening direction of the second U-shaped tube is opposite to the opening direction of the first U-shaped tube. Such a flow resistance channel can, on the one hand, prevent the sample added to the amplification reaction chamber from directly flowing into the sample loading buffer chamber 34 before or during amplification, and on the other hand, the amplification product and the diluent can be further mixed before the test paper detection.

[0070] A sample loading buffer chamber 34 is connected downstream of each flow resistance channel 33. A capillary wall 35 is provided on the side wall of each sample loading buffer chamber 34 facing the test paper storage slot 36. The capillary wall 35 is composed of a plurality of vertical thin sheets spaced 0.2 mm apart to generate capillary force, thereby transporting the liquid to the sample loading end of the test paper 5 stored in the test paper storage slot 36. One end of the capillary wall 35 enters the sample loading buffer chamber 34, and the other end is connected to the sample loading end of the test paper 5.

[0071] The base 4 is provided with a heating module storage slot 42 directly below the amplification reaction chamber 32. The heating module is specifically an electric heating plate, and a wire clamping slot 43 of the electric heating plate is provided on the side wall of the base 4 for external power supply. The electric heating plate is pre-adhered to the bottom of the heating module storage slot 42 by double-sided tape. The bottom of the reaction layer 3 fits the electric heating plate to ensure that the electric heating plate can directly contact the bottom of the amplification reaction chamber 32 to fully supply heat. The electric heating plate relies on resistance heating to convert electrical energy into thermal energy.

[0072] The following describes the process of nucleic acid detection using the multi-target microfluidic chip in Example 1:

[0073] Place the saliva and nasopharyngeal swab samples into a soft bottle containing lysis solution for room temperature lysis reaction.

[0074] Unscrew the sealing cover 14 and place the multi-target microfluidic chip horizontally;

[0075] Place the mouth of the soft bottle downward in the injection port 11, the mouth of the soft bottle is sealed and connected to the injection port 11, squeeze the body of the soft bottle, and evenly distribute the sample into the amplification reaction chamber 32, discard the soft bottle, and cover it with a sealing cap 14;

[0076] Turn on the power of the electric heating plate to heat, wait for the amplification reaction to end, and then turn off the power;

[0077] The diluent vesicle 15 is squeezed to evenly distribute the diluent into the diluent chamber 38, and then flows into the amplification reaction chamber 32 and then flows along the flow resistance channel 33 to the loading buffer chamber 34. The continuously accumulated liquid passes through the capillary wall 35 to reach the loading end of the test paper 5 stored in the test paper storage groove 36. The loading detection process is completed through the capillary force of the capillary wall 35 and the test paper 5, and finally the detection result is read through the result display window 21;

[0078] After the detection is completed, the reaction layer 3 is pushed away from the base 4, and the remaining parts except the base 4 are discarded. The base 4 and the internal heating module can be reused.

[0079] Embodiment 2

[0080] like Figures 7 to 9 , Figure 20 to Figure 22As shown, the difference between the second embodiment and the first embodiment is that the heating module is not an electric heating sheet but a hand warmer. The hand warmer contains iron powder, which can generate a large amount of heat through chemical reaction when in contact with air.

[0081] The following describes the process of nucleic acid detection using the multi-target microfluidic chip in Example 2:

[0082] Place the sample saliva and nasopharyngeal swab into a soft bottle containing lysis solution for room temperature lysis reaction.

[0083] Take the hand warmer bag out of the sealed bag and place it in the heating module storage slot 42 in the base 4;

[0084] Unscrew the sealing cover 14 and place the multi-target microfluidic chip horizontally;

[0085] Place the mouth of the soft bottle downward in the injection port 11, the mouth of the soft bottle is sealed and connected to the injection port 11, squeeze the body of the soft bottle, and evenly distribute the reagent into the amplification reaction chamber 32, discard the soft bottle, and cover it with a sealing cap 14;

[0086] After waiting for the amplification reaction to end, the diluent vesicle 15 is squeezed to evenly distribute the diluent into the diluent chamber 38, and then the diluent enters the amplification reaction chamber 32 and flows along the flow resistance channel 33 to the loading buffer chamber 34. The continuously accumulated liquid passes through the capillary wall 35 to reach the loading end of the test paper 5 stored in the test paper storage tank 36. The loading detection process is completed through the capillary force of the capillary wall 35 and the test paper 5, and finally the detection result is read through the result display window 21;

[0087] After testing is complete, the entire device is discarded.

[0088] Embodiment 3

[0089] like Figures 10 to 14 As shown, the difference between Example 3 and Example 1 is that the number of the sample diversion channel 12, the sample vertical flow channel 13 and the amplification reaction chamber 32 under the injection port 11, the diluent vesicle placement hole under the diluent vesicle placement hole, the diluent diversion channel 16 and the diluent vertical flow channel 17, and the result display window 21 are all four.

[0090] The process of performing nucleic acid detection using the multi-target microfluidic chip in Example 3 is the same as that in Example 1 and will not be repeated here.

[0091] Embodiment 4

[0092] like Figures 15 to 19As shown, the difference between Example 4 and Example 1 is that the number of the sample diversion channel 12, the sample vertical flow channel 13, the amplification reaction chamber 32, the diluent chamber 38, the flow resistance channel 33, the sample loading buffer chamber 34, the capillary wall 35 and the test paper storage slot 36 below the injection port 11 is five, and the diluent is also divided into five portions.

[0093] The process of using the multi-target microfluidic chip in Example 4 to perform nucleic acid detection is the same as that in Example 1 and will not be repeated here.

[0094] Embodiment 5

[0095] This embodiment provides a nucleic acid detection method for Example 1 (such as Figures 1 to 6 As shown) or Example 2 (as Figures 7 to 9 The multi-target microfluidic chip provided in the embodiment of the present invention is used for nucleic acid detection, and the method comprises the following steps:

[0096] Constructing a three-target RPA reaction system: Each RPA reaction system includes amplification primers and probes of a target, reaction buffer and amplification functional enzymes. The dry powders of the RPA reaction system are respectively placed in three amplification reaction chambers 32, and the sample vertical flow channel 13 in the liquid separation layer 1 is aligned with the amplification reaction chamber 32. In the RPA system, the 5' end of the probe is modified with FITC, and the 5' end of the anti-primer is modified with biotin.

[0097] Construct a lateral flow chromatography test paper detection system: Lateral flow chromatography test paper displays the test results through the principle of gold nanoparticle aggregation and color development. Fig. 20 As shown, the lateral flow chromatography test paper 5 is composed of a bottom plate 51, a sample pad 52, a conjugate pad 53, an NC membrane 54 and a water absorbent pad 55, wherein a T band and a C band are marked on the NC membrane 54, streptavidin is fixed on the T band, rabbit anti-mouse secondary antibody is fixed on the C band, and the nanogold particles are labeled with anti-FITC antibodies.

[0098] Add the sample to be tested for amplification reaction: add the sample to be tested into the lysis solution soft bottle for lysis at room temperature, squeeze the soft bottle, and transfer the sample after lysis to the amplification reaction chamber 32 for RPA amplification reaction. At this time, the amplification product is a double-stranded DNA labeled with biotin and a double-stranded DNA labeled with FITC.

[0099] Release the diluent of the test paper for chromatographic detection: squeeze the diluent vesicle 15 to release the diluent into the diluent chamber 38. After the diluent enters the amplification reaction chamber 32, it flows along the flow resistance channel 33 with the amplification product into the sample loading buffer chamber 34, and finally reaches the sample pad 52 of the test paper 5 through the capillary wall 35, completing the sample loading detection process. When the sample is positive, the biotin end of the double-stranded DNA is intercepted by the streptavidin of the T band, and the FITC end of the double-stranded DNA intercepts the nano-gold particles labeled with the anti-FITC antibody, causing the T band to display red. Correspondingly, when the sample is negative, no color is displayed because the nano-gold particles cannot be intercepted.

[0100] In this embodiment, the RPA reaction volume ranges from 5 μL to 50 μL.

[0101] In this embodiment, the reaction time range of the sample to be tested and the RPA reaction system is 5 min-40 min.

[0102] In this embodiment, the total magnesium acetate concentration in each RPA amplification system ranges from 12 mM to 40 mM.

[0103] In this embodiment, the concentration range of each target primer in each RPA amplification system is 150nM-600nM, the concentration range of the probe is 50nM-150nM, the ratio of primer 1, primer 2, and probe can be freely adjusted, but the concentration range of oligonucleotides in a single RPA reaction system should be maintained at 750nM-2000nM.

[0104] In this embodiment, the reaction temperature range of the sample to be tested and the RPA reaction system is 45°C-42°C.

[0105] In this embodiment, the volume of the sample solution of the lateral flow chromatography test paper ranges from 100 μL to 200 μL.

[0106] Embodiment 6

[0107] This embodiment provides a nucleic acid detection method for Example 1 (such as Figures 1 to 6 As shown) or Example 2 (as Figures 7 to 9 The multi-target microfluidic chip provided in the embodiment of the present invention is used for nucleic acid detection, and the method comprises the following steps:

[0108] Constructing a three-target RPA reaction system: Each RPA reaction system includes amplification primers and probes of two targets, reaction buffer and amplification functional enzymes. The dry powders of the RPA reaction system are respectively built into three amplification reaction chambers 32, and the sample vertical flow channel 13 in the liquid separation layer 1 is aligned with the amplification reaction chamber 32. In the RPA system, one target probe is modified with FITC at the 5' end, and the anti-primer is modified with biotin at the 5' end; the other target probe is modified with FITC at the 5' end, and the anti-primer is modified with digoxigenin at the 5' end.

[0109] Construct a lateral flow chromatography test paper detection system: Lateral flow chromatography test paper displays the test results through the principle of gold nanoparticle aggregation and color development. Fig.21 As shown, the lateral flow chromatography test paper 5 is composed of a bottom plate 51, a sample pad 52, a conjugate pad 53, an NC membrane 54 and a water absorbent pad 55, wherein two T bands and one C band are marked on the NC membrane, streptavidin and anti-digoxigenin antibody are fixed on the two T bands respectively, rabbit anti-mouse secondary antibody is fixed on the C band, and the nanogold particles are labeled with anti-FITC antibody.

[0110] Add the sample to be tested for amplification reaction: add the sample to be tested into the lysis solution soft bottle for lysis at room temperature, squeeze the soft bottle to transfer the lysed sample to the amplification reaction chamber 32 for RPA amplification reaction. At this time, the amplification product of one target is a double-stranded DNA labeled with biotin and a double-stranded DNA labeled with FITC; the amplification product of the other target is a double-stranded DNA labeled with digoxigenin and a double-stranded DNA labeled with FITC.

[0111] Release the diluent of the test paper for chromatographic detection: squeeze the diluent vesicle 15, release the diluent into the diluent chamber 38, the diluent flows into the amplification reaction chamber 32, and then flows along the flow resistance channel 33 with the amplification product into the sample loading buffer chamber 34, and finally reaches the sample pad 52 of the test paper 5 through the capillary wall 35, completing the sample loading detection process. When the sample is positive, the biotin / digoxigenin end of the double-stranded DNA is intercepted by the streptavidin / anti-digoxigenin antibody of the T band, and the FITC end of the double-stranded DNA intercepts the nano-gold particles labeled with the anti-FITC antibody, causing the T band to display red. Correspondingly, when the sample is negative, no color is displayed because the nano-gold particles cannot be intercepted.

[0112] In this embodiment, the total RPA reaction volume ranges from 5 μL to 50 μL.

[0113] In this embodiment, the reaction time range of the sample to be tested and the RPA reaction system is 5 min-40 min.

[0114] In this embodiment, the total magnesium acetate concentration in each RPA amplification system ranges from 12 mM to 40 mM.

[0115] In this embodiment, the concentration range of each target primer in each RPA amplification system is 150nM-600nM, the concentration range of the probe is 50nM-150nM, the ratio of primer 1, primer 2, and probe can be freely adjusted, but the concentration range of oligonucleotides in a single RPA reaction system should be maintained at 750nM-2000nM.

[0116] In this embodiment, the reaction temperature range of the sample to be tested and the RPA reaction system is 45°C-42°C.

[0117] In this embodiment, the volume of the sample solution of the lateral flow chromatography test paper ranges from 100 μL to 200 μL.

[0118] Embodiment 7

[0119] This embodiment provides another nucleic acid detection method for Example 1 (such as Figures 1 to 6 As shown) or Example 2 (as Figures 7 to 9 The multi-target microfluidic chip provided in the embodiment of the present invention is used for nucleic acid detection, and the method comprises the following steps:

[0120] Constructing a nine-target RPA reaction system: Each RPA reaction system includes amplification primers and probes of three targets, reaction buffer and amplification functional enzymes. The dry powders of the RPA reaction system are respectively placed in three amplification reaction chambers 32, and the sample vertical flow channel 13 in the liquid separation layer 1 is aligned with the amplification reaction chamber 32. The 5' ends of the probes of the three targets in each RPA system are respectively modified with FITC, and the anti-primers 5' are respectively modified with biotin, rhodamine and digoxigenin.

[0121] Construct a lateral flow chromatography test paper detection system: Lateral flow chromatography test paper displays the test results through the principle of gold nanoparticle aggregation and color development. Fig. 22 As shown, the lateral flow chromatography test paper 5 is composed of a bottom plate 51, a sample pad 52, a conjugate pad 53, an NC membrane 54 and a water absorbent pad 55, wherein three T bands and one C band are marked on the NC membrane 54, streptavidin, anti-rhodamine antibody and anti-digoxigenin antibody are fixed on the T band respectively, rabbit anti-mouse secondary antibody is fixed on the C band, and the nanogold particles are labeled with anti-FITC antibody.

[0122] Add the sample to be tested for amplification reaction: add the sample to be tested into the lysis solution soft bottle for room temperature lysis, squeeze the soft bottle, and transfer the sample after lysis to the amplification reaction chamber 32 for RPA amplification reaction. At this time, the amplification product in each RPA amplification system is a double-stranded DNA labeled with biotin / rhodamine / digoxigenin and a double-stranded DNA labeled with FITC.

[0123] Release the test paper diluent for chromatographic detection: squeeze the diluent vesicle 15, release the diluent into the diluent chamber 38, the diluent flows into the amplification reaction chamber 32, and then flows along the flow resistance channel 33 with the amplification product into the sample loading buffer chamber 34, and finally reaches the sample pad of the test paper 5 through the capillary wall 35, completing the sample loading detection process. When the sample is positive, the biotin / rhodamine / digoxigenin end of the double-stranded DNA is intercepted by the streptavidin / anti-rhodamine antibody / anti-digoxigenin antibody of the T band, and the FITC end of the double-stranded DNA intercepts the nano-gold particles labeled with the anti-FITC antibody, causing the T band to display red. Correspondingly, when the sample is negative, no color is displayed because the nano-gold particles cannot be intercepted.

[0124] In this embodiment, the RPA reaction volume ranges from 5 μL to 50 μL.

[0125] In this embodiment, the total magnesium acetate concentration in each RPA amplification system ranges from 12 mM to 40 mM.

[0126] In this embodiment, the concentration range of each target primer in each RPA amplification system is 150nM-600nM, the concentration range of the probe is 50nM-150nM, the ratio of primer 1, primer 2, and probe can be freely adjusted, but the concentration range of oligonucleotides in a single RPA reaction system should be maintained at 750nM-2000nM.

[0127] In this embodiment, the reaction time range of the sample to be tested and the RPA reaction system is 5 min-40 min.

[0128] In this embodiment, the reaction temperature range of the sample to be tested and the RPA reaction system is 45°C-42°C.

[0129] In this embodiment, the volume of the sample solution of the lateral flow chromatography test paper ranges from 100 μL to 200 μL.

[0130] The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention can completely evenly divide samples and diluents, promote multiple parallel reactions at the same time, and allow as many samples and diluents as possible to enter the amplification reaction chamber 32, thereby reducing liquid loss and improving detection accuracy.

[0131] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A multi-target microfluidic chip with a novel liquid diversion method, characterized in that: The multi-target microfluidic chip comprises: The reaction layer (3) has a top surface that is inwardly recessed and is provided with a plurality of amplification reaction chambers (32), a plurality of sample loading buffer chambers (34), and a plurality of test paper storage slots (36); the plurality of amplification reaction chambers (32), the plurality of sample loading buffer chambers (34), and the plurality of test paper storage slots (36) are arranged in a one-to-one corresponding position, and the amplification reaction chambers (32) are connected to the corresponding sample loading buffer chambers (34); A sealing layer (2) is arranged on the reaction layer (3); The liquid separation layer (1) is installed at one end of the sealing layer (2) and protrudes from the top surface of the sealing layer (2). The liquid separation layer (1) cooperates with the sealing layer (2) to seal the reaction layer (3). A vertically arranged sample inlet (11), a plurality of sample diversion channels (12) and a plurality of sample vertical flow channels (13) are provided in the liquid separation layer (1). The sample inlet (11) penetrates the top surface of the liquid separation layer (1) upward, and the sample vertical flow channel (13) penetrates the bottom surface of the liquid separation layer (1) downward. The plurality of sample diversion channels (12) are radially distributed. The top end of the sample diversion channel (12) is connected to the sample inlet (11), and the bottom end is connected to the sample vertical flow channel (13); wherein, The plurality of sample vertical flow channels (13), the plurality of sample shunt channels (12) and the plurality of amplification reaction chambers (32) are arranged in a corresponding position. The plurality of sample shunt channels (12) have the same size and are arranged at intervals along a conical surface. The plurality of sample vertical flow channels (13) also have the same size. The conical surface is arranged coaxially with the sample inlet (11). The amplification reaction chamber (32) is vertically opposite to and connected to the corresponding sample vertical flow channel (13).

2. The multi-target microfluidic chip with a novel liquid diversion method according to claim 1, characterized in that: The amplification reaction chamber (32) and the sample vertical flow channel (13) are arranged at a vertical distance.

3. The multi-target microfluidic chip with a novel liquid diversion method according to claim 1, characterized in that: The amplification reaction chamber (32) is connected to the sample loading buffer chamber (34) via a flow resistance channel (33).

4. The multi-target microfluidic chip with a novel liquid diversion method according to claim 3, characterized in that: The flow resistance channel (33) comprises a first U-shaped tube and two second U-shaped tubes respectively connected to the two ends of the first U-shaped tube, and the opening direction of the second U-shaped tube is opposite to the opening direction of the first U-shaped tube.

5. The multi-target microfluidic chip with a novel liquid diversion method according to claim 3 or 4, characterized in that: It also includes a capillary wall (35), one end of which enters the sample loading buffer chamber (34) and the other end of which is connected to the test paper (5) in the test paper storage groove (36).

6. The multi-target microfluidic chip with a novel liquid diversion method according to claim 3, characterized in that: The bottom wall of the sample loading buffer chamber (34) is lower than the bottom wall of the flow resistance channel (33).

7. The multi-target microfluidic chip with a novel liquid diversion method according to any one of claims 1 to 4 or 6, characterized in that: It also includes a base (4) installed at the bottom of the reaction layer (3), and a heating module storage groove (42) is provided in the base (4). The heating module storage groove (42) is used to store a heating module, and the heating module is used to heat the amplification reaction chamber (32).

8. The multi-target microfluidic chip with a novel liquid diversion method according to claim 7, characterized in that: The base (4) is slidably connected to the reaction layer (3).

9. The multi-target microfluidic chip with a novel liquid diversion method according to claim 1, characterized in that: The liquid separation layer (1) is also provided with a diluent vesicle placement hole, a plurality of diluent shunt channels (16) arranged in a radial shape, and a plurality of diluent vertical flow channels (17) corresponding to and connected with the bottom ends of the plurality of diluent shunt channels (16); the diluent vesicle placement hole and the sample inlet (11) are arranged at intervals in the horizontal direction; the diluent vesicle placement hole is used to receive the diluent vesicle (15); the top ends of the plurality of diluent shunt channels (16) are respectively connected with the bottom ends of the diluent vesicle placement hole; the reaction layer (3) is provided with a plurality of diluent cavities (38), and the diluent cavities (38) are vertically opposite to and connected with the diluent vertical flow channels (17).

10. The multi-target microfluidic chip with a novel liquid diversion method according to claim 1, characterized in that: The sealing layer (2) is provided with result display windows (21), and the number of the result display windows (21) is the same as the number of the amplification reaction chambers (32).

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