Multi-target microfluidic chip with a novel liquid splitting method

Abstract

The present invention relates to the technical field of nucleic acid detection, and discloses a multi-target microfluidic chip with a novel liquid diversion method, which includes a reaction layer, a sealing layer, and a liquid separation layer; the reaction layer is provided with a plurality of amplification reaction chambers, a sample loading buffer chamber, and a test strip storage groove; a vertically arranged sample inlet, a plurality of sample diversion channels, and a plurality of sample vertical channels are arranged in the liquid separation layer, and the sample inlet and the sample vertical channels penetrate the top surface and the bottom surface of the liquid separation layer upward and downward respectively; the sample vertical channels, the sample diversion channels, and the amplification reaction chambers are arranged in corresponding positions, and the plurality of sample diversion channels are respectively communicated with the sample inlet, the sizes of the plurality of sample diversion channels are the same and are arranged at intervals along a conical surface, the sizes of the plurality of sample vertical channels are also the same, and the sample vertical channels are communicated with the amplification reaction chambers. It can evenly divide the samples completely, promote the simultaneous progress of multiple parallel reactions; and enable as many samples as possible to enter the amplification reaction chambers, reduce liquid loss, and thus improve the detection accuracy.

Description

Technical Field

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

[0002] Pathogenic microorganism detection technologies include traditional detection techniques such as smear microscopy, isolation culture and biochemical reactions, and tissue cell culture, as well as commonly used clinical methods such as antigen-antibody detection and nucleic acid detection. Traditional detection techniques 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 infected persons.

[0003] The limitation of nucleic acid detection technology in point-of-care testing mainly lies in its dependence on professional personnel operation and professional instrument equipment. However, in the primary healthcare system, due to the lack of professional molecular biology laboratories, nucleic acid detection technology is difficult to popularize. In order to meet the detection needs such as rapid and large-scale screening at the grassroots level, there is an urgent need for a microfluidic chip with low cost, portability, instrument-free dependence, and suitable for real-time detection.

[0004] Currently, the microfluidic technology for multi-target detection aims to achieve simultaneous detection and analysis of multiple biomolecules through integration and miniaturization means, in order to improve the 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 complete liquid equalization, it is usually limited by the number of detection targets. This is because most liquid equalization is based on the 1-to-2 splitting of the flow channel, so the number of targets must be 2 n (n≥1). On the other hand, the current splitting flow channels and amplification reaction chambers in the microfluidic chip design are on the same plane, which cannot ensure that the liquid does not flow back or stay in the flow channel, thus resulting in an increase in liquid loss.

[0005] Therefore, there is an urgent need to develop a multi-target microfluidic chip with a novel liquid splitting method to get rid of the limitation of the number of detection targets and effectively reduce liquid loss. Summary of the Invention

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

[0007] 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 splitting method, comprising:

[0009] The reaction layer has a top surface that is recessed inward to form a plurality of amplification reaction chambers, a plurality of loading buffer chambers, and a plurality of test strip storage grooves; the plurality of amplification reaction chambers, the plurality of loading buffer chambers, and the plurality of test strip storage grooves are arranged in one-to-one correspondence in position, and the amplification reaction chambers communicate with the corresponding loading buffer chambers;

[0010] The sealing layer is installed 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 and the sealing layer cooperate 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 upward through the top surface of the liquid separation layer, and the sample vertical flow channels penetrate downward through the bottom surface of the liquid separation layer. The plurality of sample diversion channels are radially distributed. The top end of the sample diversion channel communicates with the sample inlet, and the bottom end communicates with 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 one-to-one correspondence in position. 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 also have the same size. The conical surface is coaxial with the sample inlet, and the amplification reaction chambers are vertically aligned with and communicate with the corresponding sample vertical flow channels.

[0013] Further, a vertical distance is set between the amplification reaction chamber and the sample vertical flow channel.

[0014] Further, the amplification reaction chamber and the loading buffer chamber are communicated through a flow resistance channel.

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

[0016] Further, it further includes a capillary wall. One end of the capillary wall enters the loading buffer chamber, and the other end is connected to the test strip in the test strip storage groove.

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

[0018] Further, it further includes a base installed at the bottom of the reaction layer. A heating module storage groove is provided in the base for storing a heating module, and the heating module is used to heat the amplification reaction chamber.

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

[0020] Furthermore, a diluent vesicle placement hole, a plurality of diluent diversion channels arranged radially, and a plurality of diluent vertical flow channels corresponding to and communicating with the bottom ends of the plurality of diluent diversion channels are formed in the liquid separation layer; the diluent vesicle placement hole and the sample inlet are spaced in the horizontal direction; the diluent vesicle placement hole is used for receiving diluent vesicles; the top ends of the plurality of diluent diversion channels communicate with the diluent vesicle placement hole respectively, a plurality of diluent chambers are formed in the reaction layer, and the diluent chambers vertically face and communicate with the diluent vertical flow channels one by one.

[0021] Furthermore, a result display window is provided on the sealing layer, and the number of the result display windows is the same as that 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. Since a vertically arranged sample inlet, a plurality of sample diversion channels and a plurality of sample vertical flow channels are formed in the liquid separation layer, the sample inlet penetrates upward through the top surface of the liquid separation layer, the sample vertical flow channels penetrate downward through the bottom surface of the liquid separation layer, the plurality of sample diversion channels are radially distributed, the top ends of the sample diversion channels communicate with the sample inlet, and the bottom ends communicate with the sample vertical flow channels; the plurality of sample vertical flow channels, the plurality of sample diversion channels and the plurality of amplification reaction chambers are arranged corresponding to each other in position, the sizes of the plurality of sample diversion channels are the same and are arranged at intervals along a conical surface, the sizes of the plurality of sample vertical flow channels are also the same, the conical surface and the sample inlet are coaxially arranged, and the amplification reaction chambers vertically face and communicate with the corresponding sample vertical flow channels. Therefore, the sample and the diluent can be evenly divided into the corresponding amplification reaction chambers to realize multi-target detection. In addition, since a sample vertical flow channel is further 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 velocity under the action of their own gravity, and under the action of inertia, as much as possible enters the amplification reaction chamber, reducing liquid loss, ensuring the concentration of the sample and the diluent, and further improving the detection accuracy. Moreover, when using the multi-target microfluidic chip with a novel liquid diversion method, only the sample needs to be lysed at room temperature and then added into the amplification reaction chamber through the sample inlet for amplification reaction. The amplification product is diluted by the diluent and then enters the loading buffer chamber and then reaches the test strip in the test strip storage groove, and the test result is conveniently displayed by the test strip. Therefore, low-cost, portable and real-time detection can be realized, and it does not need to rely on the operation of professionals and professional instrument equipment, can meet the detection requirements such as rapid and large-scale screening at the grass-roots level, and in the form of radially arranged sample diversion channels, 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. In this way, the risk of the liquid in the amplification reaction chamber entering the sample vertical flow channel can be reduced, the possibility of liquid loss can be further reduced, and the detection accuracy can be increased.

[0025] 3. The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention has a flow resistance channel including a first U-shaped tube and two second U-shaped tubes respectively connected to both ends of the first U-shaped tube. The opening directions of the second U-shaped tubes are 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 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 strip detection.

[0026] 4. The multi-target microfluidic chip with a novel liquid diversion method provided by the present invention has the bottom wall of the loading buffer chamber lower than the bottom wall of the flow resistance channel, so as to store a part of the diluted amplification product and prevent a large amount of the diluted amplification product from directly flowing towards the test strip, resulting in inaccurate test results.

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

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is a three-dimensional combined schematic diagram of the multi-target microfluidic chip in Embodiment 1 of the present invention;

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

[0031] Figure 3 It is a top view schematic diagram of the reaction layer of the multi-target microfluidic chip in Embodiment 1 of the present invention;

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

[0034] Figure 6 Perspective schematic diagram of the base of the multi-target microfluidic chip in Embodiment 1 of the present invention;

[0035] Figure 7 Three-dimensional combined schematic diagram of the multi-target microfluidic chip in Embodiment 2 of the present invention;

[0036] Figure 8 Three-dimensional exploded perspective schematic diagram of the multi-target microfluidic chip in Embodiment 2 of the present invention;

[0037] Figure 9 Perspective schematic diagram of the base of the multi-target microfluidic chip in Embodiment 2 of the present invention;

[0038] Figure 10 Three-dimensional combined schematic diagram of the multi-target microfluidic chip in Embodiment 3 of the present invention;

[0039] Figure 11 Three-dimensional exploded perspective schematic diagram of the multi-target microfluidic chip in Embodiment 3 of the present invention;

[0040] Figure 12 Top view schematic diagram of the reaction layer of the multi-target microfluidic chip in Embodiment 3 of the present invention;

[0041] Figure 13 Three-dimensional perspective schematic diagram of the reaction layer of the multi-target microfluidic chip in Embodiment 3 of the present invention;

[0042] Figure 14 Perspective schematic diagram of the liquid separation layer of the multi-target microfluidic chip in Embodiment 3 of the present invention;

[0043] Figure 15 Three-dimensional combined schematic diagram of the multi-target microfluidic chip in Embodiment 4 of the present invention;

[0044] Figure 16 Three-dimensional exploded perspective schematic diagram of the multi-target microfluidic chip in Embodiment 4 of the present invention;

[0045] Figure 17 Top view schematic diagram of the reaction layer of the multi-target microfluidic chip in Embodiment 4 of the present invention;

[0046] Figure 18 Three-dimensional perspective schematic diagram of the reaction layer of the multi-target microfluidic chip in Embodiment 4 of the present invention;

[0047] Figure 19 Perspective schematic diagram of the liquid separation layer of the multi-target microfluidic chip in Embodiment 4 of the present invention;

[0048] Figure 20 Schematic diagram of the structure of the test strip in the embodiment of the present invention;

[0049] Figure 21 It is a schematic structural diagram of another test strip in the embodiment of the present invention;

[0050] Figure 22 It is a schematic structural diagram of yet another test strip in the embodiment of the present invention.

[0051] Explanation of reference numerals:

[0052] 1. Liquid separation layer; 11. Sampling port; 12. Sample shunt channel; 13. Sample vertical flow channel; 14. Sealing cover; 15. Diluent vesicle; 16. Diluent shunt channel; 17. Diluent vertical flow channel; 2. Sealing layer; 21. Result display window; 22. First fixing component; 3. Reaction layer; 31. Second fixing component; 32. Amplification reaction cavity; 33. Flow resistance channel; 34. Loading buffer chamber; 35. Capillary wall; 36. Test strip storage groove; 37. Slide groove; 38. Diluent chamber; 4. Base; 41. Slide block; 42. Heating module storage groove; 43. Wire card slot; 5. Test strip; 51. Bottom plate; 52. Sample pad; 53. Conjugate pad; 54. NC membrane; 55. Absorbent pad. Detailed implementation manners

[0053] Next, the technical solutions of the present invention will be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0054] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is 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 should not be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed 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 directly connected or indirectly connected through an intermediate medium, and it can be the internal communication of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0056] In addition, the technical features involved in 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] As Figures 1 to 22 shown, the present invention provides a multi-target microfluidic chip with a novel liquid splitting method for nucleic acid detection (hereinafter referred to as the multi-target microfluidic chip). The multi-target microfluidic chip includes a liquid splitting layer 1, a sealing layer 2, a reaction layer 3, and a base 4. The liquid splitting layer 1 is used to evenly divide the sample and the diluent. The reaction layer 3 is used for amplifying and detecting the target to be measured.

[0058] The top surface of the reaction layer 3 is recessed inward to form a plurality of amplification reaction chambers 32 and a plurality of loading buffer chambers 34. The reaction layer 3 is also provided with a plurality of test strip storage grooves 36 for storing test strips 5 (as Figures 20 to 22 shown). The plurality of amplification reaction chambers 32, the plurality of loading buffer chambers 34, and the plurality of test strip storage grooves 36 are arranged in one-to-one correspondence, and the amplification reaction chambers 32 communicate with the corresponding loading buffer chambers 34.

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

[0060] The liquid splitting layer 1 is provided with a vertically arranged sample inlet 11, a plurality of sample splitting channels 12, and a plurality of sample vertical channels 13. The sample inlet 11 penetrates upward through the top surface of the liquid splitting layer 1. The sample vertical channels 13 penetrate downward through the bottom surface of the liquid splitting layer 1. The plurality of sample splitting channels 12 are radially arranged and respectively communicate with the same sample inlet 11. The top end of the sample splitting channel 12 communicates with the sample inlet 11, and the bottom end communicates with the sample vertical channel 13. Among them, the plurality of sample vertical channels 13, the plurality of sample splitting channels 12, and the plurality of amplification reaction chambers 32 below the sample inlet 11 are arranged in one-to-one correspondence. The sizes of the plurality of sample splitting channels 12 are the same and are arranged at intervals along a conical surface. The sizes of the plurality of sample vertical channels 13 are also the same. The conical surface is coaxial with the sample inlet 11, and the amplification reaction chambers 32 are directly opposite to and vertically communicate with the sample vertical channels 13.

[0061] In the present invention, since a sample inlet 11, a plurality of sample diversion channels 12 and a plurality of sample vertical channels 13 are provided on the liquid separation layer 1, the plurality of sample vertical channels 13, the plurality of sample diversion channels 12 and the plurality of amplification reaction chambers 32 are arranged in one-to-one corresponding positions. The plurality of sample diversion channels 12 are respectively connected to the same sample inlet 11, and the sizes of the plurality of sample diversion channels 12 are the same and are arranged at intervals along a conical surface. The sizes of the plurality of sample vertical channels 13 are also the same. The amplification reaction chambers 32 are directly opposite and vertically communicate with the sample vertical channels 13. Therefore, the sample and the diluent can be evenly divided into the corresponding amplification reaction chambers 32 so as to enable simultaneous amplification of multiple targets. In addition, the sample inlet 11 is vertically arranged, and a sample vertical channel 13 is further provided below the sample diversion channel 12. The liquid separation layer 1 also protrudes from the top surface of the sealing layer 2. Therefore, the vertical flow path of the sample and the diluent can be increased, 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, as much as possible enters the amplification reaction chambers 32, reducing liquid loss, ensuring that the amounts of the sample and the diluent entering the plurality of amplification reaction chambers 32 are consistent, and ultimately improving the accuracy of nucleic acid detection.

[0062] Further, the amplification reaction chambers 32 are directly opposite and communicate with the sample vertical channels 13, and the vertical distance between the amplification reaction chambers 32 and the sample vertical channels 13 is set. In this way, the risk that the sample and the diluent entering the amplification reaction chambers 32 flow back into the sample vertical channels 13 can be reduced.

[0063] To simplify the operation steps, the diluent is built into the multi-target microfluidic chip. A tin foil film is provided at the bottom of the diluent vesicle 15 storing the diluent, and the vesicle material is thin soft plastic. By squeezing the diluent vesicle 15 downward, the tin foil film can be broken through to release the diluent. Specifically, the diluent and the sample have their respective flow paths. That is, a diluent vesicle placement hole, a plurality of diluent diversion channels 16, and a plurality of diluent vertical channels 17 are also provided on the liquid separation layer 1. The diluent vesicle placement hole penetrates upward through the top surface of the liquid separation layer 1. The diluent vertical channels 17 penetrate downward through the bottom surface of the liquid separation layer 1. The plurality of diluent diversion channels 16 are arranged radially and are respectively connected to the same diluent vesicle placement hole. The top end of the diluent diversion channel 16 is connected to the diluent vesicle placement hole, and the bottom end is connected to the diluent vertical channel 17. Correspondingly, a plurality of diluent chambers 38 are provided in the reaction layer 3 described above. The number of the plurality of diluent chambers 38 is the same as that of the plurality of amplification reaction chambers 32 and they are connected in a one-to-one corresponding position. The plurality of diluent diversion channels 16, the plurality of diluent vertical channels 17, and the plurality of diluent chambers 38 below the diluent vesicle placement hole are arranged in a one-to-one corresponding position. The sizes of the plurality of diluent diversion channels 16 are the same and they are arranged at intervals along a conical surface. The sizes of the plurality of diluent vertical channels 17 are also the same. This conical surface is arranged coaxially with the diluent vesicle placement hole. The diluent chamber 38 is vertically opposite to and connected to the diluent vertical channel 17. In this way, the sample can be evenly divided through the sample inlet 11, the sample diversion channel 12, and the sample vertical channel 13 and then enter the amplification reaction chamber 32. The diluent is evenly divided through the diluent vesicle placement hole, the diluent diversion channel 16, and the diluent vertical channel 17 and then enters the diluent chamber 38, so as to realize the multi-target detection of the evenly divided sample and diluent.

[0064] To prevent foreign objects from entering the sample inlet 11, the sample inlet 11 is sealed by a sealing cover 14. Specifically, the sealing cover 14 is threadedly connected to the sample inlet 11. Of course, it can also be sealed in the form of a sealing film.

[0065] To facilitate viewing the detection results, a result display window 21 is provided on the sealing layer 2. The result display window 21 corresponds to the color-developing part on the test strip 5. To facilitate the fixed connection between the sealing layer 2 and the reaction layer 3, a first fixing component 22 (such as a column / groove) is provided on the sealing layer 2, and a second fixing component 31 (corresponding to a groove / column) is provided on the reaction layer. The sealing layer 2 and the reaction layer 3 are fixed together through the cooperation of the first fixing component 22 and the second fixing component 31.

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

[0067] Example 1

[0068] As Figures 1 to 6 , Figures 20 to 22 shown, the number of the sample shunt channels 12, the sample vertical channels 13, and the amplification reaction chambers 32 under the sample inlet 11, the number of the diluent shunt channels 16 and the diluent vertical channels 17 under the diluent vesicle placement holes, and the number of the result display windows 21 are all three. The included angle between two adjacent sample shunt channels 12 is 120°, and the included angle between two adjacent diluent shunt channels 16 is 120°. The extending direction of the sample shunt channel 12 and the bottom surface of the sample inlet 11 (i.e., the horizontal plane) form an angle of 35° to 55°, preferably 45°. The extending direction of the diluent shunt channel 16 and the bottom surface of the diluent vesicle placement hole (i.e., the horizontal plane) form an angle of 35° to 55°, preferably 45°. The height of the sample vertical channel is 0.55 mm to 0.65 mm, preferably 0.6 mm. Since there are three sample shunt channels 12, three sample vertical channels 13, three diluent shunt channels 16, and three diluent vertical channels 17 respectively, the samples added to the amplification reaction chamber from the sample inlet 11 and the diluent released from the diluent vesicles 15 can be evenly divided into three parts, simplifying the number of liquid addition operation times.

[0069] The height of the amplification reaction chamber 32 is 1 mm, and the RPA reaction volume (the reaction volume refers to the space occupied by substances when they react and generate substances under certain conditions) is 10 μL. A flow resistance channel 33 is connected to the downstream of each amplification reaction chamber 32. The flow resistance channel 33 can prevent the sample from flowing directly downstream after entering the amplification reaction chamber 32, improving the uniformity effect 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. The opening directions of the second U-shaped tubes are 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 flowing directly into the loading buffer chamber 34 before or during the amplification, and on the other hand, can further mix the amplification product and the diluent before the test strip detection.

[0070] A sampling buffer bin 34 is connected downstream of each flow resistance channel 33. A capillary wall 35 is provided on the side wall of each sampling buffer bin 34 facing the test strip storage slot 36. The capillary wall 35 is composed of a plurality of vertical small flakes with a spacing of 0.2 mm to generate capillary power, thereby transporting the liquid to the sampling end of the test strip 5 stored in the test strip storage slot 36. One end of the capillary wall 35 enters the sampling buffer bin 34, and the other end is connected to the sampling end of the test strip 5.

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

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

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

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

[0075] Place the mouth of the soft bottle downward into the sampling port 11. The mouth of the soft bottle is sealed to the sampling port 11. Squeeze the body of the soft bottle to evenly distribute the sample into the amplification reaction chamber 32, discard the soft bottle, and cover the sealing cap 14;

[0076] Connect the power supply of the electric heating sheet for heating, wait for the amplification reaction to end, and then turn off the power supply;

[0077] Squeeze the dilution liquid vesicle 15 to evenly distribute the dilution liquid into the dilution liquid chamber 38, then it flows into the amplification reaction chamber 32 and flows upward along the flow resistance channel 33 to the sampling buffer bin 34. The continuously accumulating liquid reaches the sampling end of the test strip 5 stored in the test strip storage slot 36 through the capillary wall 35, and the sampling and detection process is completed through the capillary power of the capillary wall 35 and the test strip 5. Finally, read the detection result through the result display window 21;

[0078] After the detection is completed, push the reaction layer 3 away from the base 4 and discard the rest except the base 4. The base 4 and the internal heating module can be reused.

[0079] Example 2

[0080] As Figures 7 to 9 、 Figures 20 to 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, and after the iron powder contacts the air, a large amount of heat can be generated through a chemical reaction.

[0081] The following introduces the process of nucleic acid detection using the multi-target microfluidic chip in the second embodiment:

[0082] Put the sample saliva and nasopharyngeal swab into a soft bottle storing lysis buffer first, and carry out a lysis reaction at room temperature.

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

[0084] Unscrew the sealed cap 14 and place the multi-target microfluidic chip horizontally;

[0085] Place the mouth of the soft bottle downward into the sample inlet 11. The mouth of the soft bottle is hermetically connected to the sample inlet 11. Squeeze the body of the soft bottle, and the reagent is evenly distributed into the amplification reaction chamber 32. Discard the soft bottle and cover the sealed cap 14;

[0086] Wait for the amplification reaction to end. Squeeze the diluent vesicle 15, evenly distribute the diluent into the diluent chamber 38, and after entering the amplification reaction chamber 32, flow upward along the flow resistance channel 33 to the loading buffer chamber 34. The continuously accumulating liquid reaches the loading end of the test strip 5 stored in the test strip storage slot 36 through the capillary wall 35, and the loading and detection process is completed through the capillary action of the capillary wall 35 and the test strip 5. Finally, read the detection result through the result display window 21;

[0087] After the detection is completed, discard the entire device.

[0088] The third embodiment

[0089] As Figures 10 to 14 shown, the difference between the third embodiment and the first embodiment is that: the number of the sample shunt channels 12, sample vertical flow channels 13 and amplification reaction chambers 32 under the sample inlet 11, the diluent vesicle placement holes under the diluent vesicle placement holes, diluent shunt channels 16 and diluent vertical flow channels 17, and the result display window 21 are all four.

[0090] The process of nucleic acid detection using the multi-target microfluidic chip in the third embodiment is the same as that in the first embodiment, and will not be elaborated here.

[0091] The fourth embodiment

[0092] As Figures 15 to 19As shown in the figure, the difference between Example 4 and Example 1 lies in that: the numbers of the sample shunt channel 12, the sample vertical flow channel 13, the amplification reaction chamber 32, the diluent chamber 38, the flow resistance channel 33, the loading buffer chamber 34, the capillary wall 35 and the test strip storage groove 36 below the sample inlet 11 are all five, and the diluent is also divided into five equal parts.

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

[0094] Example 5

[0095] This example provides a nucleic acid detection method for performing nucleic acid detection on the multi-target microfluidic chip provided in Example 1 (as Figures 1 to 6 shown) or Example 2 (as Figures 7 to 9 shown). The method includes the following steps:

[0096] Construct a triple-target RPA reaction system: Each RPA reaction system includes amplification primers and probes for one target, reaction buffer, and amplification functional enzymes. The dry powder of the RPA reaction system is 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. Among them, the 5' end of the probe in the RPA system is modified with FITC, and the 5' end of the reverse primer is modified with biotin.

[0097] Construct a lateral flow chromatography test strip detection system: The lateral flow chromatography test strip shows the detection result through the principle of color development by the aggregation of gold nanoparticles. As Figure 20 shown, the lateral flow chromatography test strip 5 is composed of a bottom plate 51, a sample pad 52, a conjugate pad 53, an NC membrane 54 and an absorbent pad 55. Among them, 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 gold nanoparticles are labeled with anti-FITC antibody.

[0098] Add the sample to be detected for amplification reaction: Add the sample to be detected 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 is a double-stranded DNA labeled with biotin and a double-stranded DNA labeled with FITC.

[0099] Release the test strip diluent 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 strip 5 through the capillary wall 35, completing the sample loading and detection process. When the sample is positive, the biotin end of the double-stranded DNA is intercepted by the streptavidin on the T band, and the FITC end of the double-stranded DNA intercepts the gold nanoparticles labeled with anti-FITC antibody, resulting in the T band showing red. Correspondingly, when the sample is negative, no color is shown because the gold nanoparticles 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 between the above-mentioned sample to be tested and the RPA reaction system is 5 min to 40 min.

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

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

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

[0105] In this embodiment, the sample loading liquid volume range of the lateral flow chromatographic test strip is 100 μL to 200 μL.

[0106] Example Six

[0107] This embodiment provides a nucleic acid detection method for nucleic acid detection of the multi-target microfluidic chip provided in Example 1 (as Figures 1 to 6 shown) or Example 2 (as Figures 7 to 9 shown). The method includes the following steps:

[0108] Construct a three-target RPA reaction system: Each RPA reaction system includes amplification primers and probes for 2 targets, reaction buffer, and amplification functional enzymes. The dry powder of the RPA reaction system is respectively built 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. Among them, the 5' end of one target probe in the RPA system is modified with FITC, and the 5' end of the reverse primer is modified with biotin; the 5' end of the other target probe is modified with FITC, and the 5' end of the reverse primer is modified with digoxin.

[0109] Construct a lateral flow chromatographic test strip detection system: The lateral flow chromatographic test strip shows the test result based on the principle of color development by the aggregation of gold nanoparticles. As Figure 21 shown, the lateral flow chromatographic test strip 5 is composed of a base plate 51, a sample pad 52, a conjugate pad 53, an NC membrane 54, and an absorbent pad 55. Two T bands and one C band are marked on the NC membrane. Streptavidin and anti-digoxin antibody are respectively immobilized on the two T bands, and rabbit anti-mouse secondary antibody is immobilized on the C band. The gold nanoparticles are labeled with anti-FITC antibody.

[0110] Add the sample to be detected for amplification reaction: Add the sample to be detected into the lysis buffer soft bottle for lysis at room temperature, and 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 one labeled with FITC; the amplification product of the other target is a double-stranded DNA labeled with digoxin and one labeled with FITC.

[0111] Release the test strip diluent for chromatographic detection: Squeeze the diluent vesicle 15 to 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 loading buffer chamber 34, and finally reaches the sample pad 52 of the test strip 5 through the capillary wall 35 to complete the loading and detection process. When the sample is positive, the biotin / digoxin end of the double-stranded DNA is intercepted by the streptavidin / anti-digoxin antibody on the T band, and the FITC end of the double-stranded DNA intercepts the gold nanoparticles labeled with anti-FITC antibody, resulting in the T band showing red. Correspondingly, when the sample is negative, there is no color display because the gold nanoparticles cannot be intercepted.

[0112] In this embodiment, the total reaction volume range of RPA is 5 μL - 50 μL.

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

[0114] In this embodiment, the total magnesium acetate concentration range in each RPA amplification system is 12 mM - 40 mM.

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

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

[0117] In this embodiment, the volume range of the sample loading solution of the lateral flow chromatography test strip is 100 μL - 200 μL.

[0118] Example VII

[0119] This embodiment provides another nucleic acid detection method for nucleic acid detection of the multi-target microfluidic chip provided in Example 1 (as Figures 1 to 6 shown) or Example 2 (as Figures 7 to 9 shown). The method includes the following steps:

[0120] Construct a nine-target RPA reaction system: Each RPA reaction system includes amplification primers and probes for three targets, a reaction buffer, and an amplification functional enzyme. The dry powder of the RPA reaction system is respectively placed inside 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. Among them, the 5' ends of the probes for the three targets in each RPA system are respectively modified with FITC, and the 5' ends of the reverse primers are respectively modified with biotin, rhodamine, and digoxin.

[0121] Construct a lateral flow chromatography test strip detection system: The lateral flow chromatography test strip shows the detection result through the principle of color development by the aggregation of gold nanoparticles. As Figure 22 shown, the lateral flow chromatography test strip 5 is composed of a bottom plate 51, a sample pad 52, a binding pad 53, an NC membrane 54, and a water absorption pad 55. Among them, three T bands and one C band are marked on the NC membrane 54. Streptavidin, anti-rhodamine antibody, and anti-digoxin antibody are respectively fixed on the T bands, and rabbit anti-mouse secondary antibody is fixed on the C band. The gold nanoparticles are labeled with anti-FITC antibody.

[0122] Add the sample to be detected for amplification reaction: Add the sample to be detected 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 in each RPA amplification system is a double-stranded DNA labeled with biotin / rhodamine / digoxin and a double-stranded DNA labeled with FITC.

[0123] Release the test strip diluent for chromatography detection: Squeeze the diluent vesicle 15 to 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 strip 5 through the capillary wall 35 to complete the sample loading detection process. When the sample is positive, the biotin / rhodamine / digoxin end of the double-stranded DNA is intercepted by the streptavidin / anti-rhodamine antibody / anti-digoxin antibody on the T band, and the FITC end of the double-stranded DNA intercepts the gold nanoparticles labeled with anti-FITC antibody, resulting in the T band showing red. Correspondingly, when the sample is negative, there is no color display because the gold nanoparticles 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 of each target primer in each RPA amplification system ranges from 150 nM to 600 nM, and the probe concentration ranges from 50 nM to 150 nM. The ratio of primer 1, primer 2, and the probe can be freely adjusted, but the oligonucleotide concentration in a single RPA reaction system should be maintained within the range of 750 nM to 2000 nM.

[0127] In this embodiment, the reaction time between the above-mentioned sample to be tested and the RPA reaction system ranges from 5 min to 40 min.

[0128] In this embodiment, the reaction temperature between the above-mentioned sample to be tested and the RPA reaction system ranges from 45 °C to 42 °C.

[0129] In this embodiment, the volume of the loading solution for the lateral flow chromatography strip ranges from 100 μL to 200 μL.

[0130] The multi-target microfluidic chip with a novel liquid splitting method provided by the present invention can evenly distribute the sample and the diluent completely, promote multiple parallel reactions to proceed simultaneously, and enable as much sample and diluent as possible to enter the amplification reaction chamber 32, reducing liquid loss, thereby improving the detection accuracy.

[0131] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill 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 enumerate all the implementation manners here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A multi-target microfluidic chip with a novel liquid splitting method, characterized in that, The multi-target microfluidic chip includes: A reaction layer (3) with a plurality of amplification reaction chambers (32), a plurality of sample loading buffer chambers (34), and a plurality of test strip storage grooves (36) recessed inward on the top surface; the plurality of amplification reaction chambers (32), the plurality of sample loading buffer chambers (34), and the plurality of test strip storage grooves (36) are arranged in one-to-one correspondence, and the amplification reaction chamber (32) communicates with the corresponding sample loading buffer chamber (34); A sealing layer (2) installed on the reaction layer (3); A liquid separation layer (1) installed at one end of the sealing layer (2) and protruding above the top surface of the sealing layer (2), the liquid separation layer (1) and the sealing layer (2) cooperate to seal the reaction layer (3), a vertically arranged sample injection port (11), a plurality of sample diversion channels (12), and a plurality of sample vertical channels (13) are provided in the liquid separation layer (1), the sample injection port (11) penetrates upward through the top surface of the liquid separation layer (1), the sample vertical channels (13) penetrate downward through the bottom surface of the liquid separation layer (1), the plurality of sample diversion channels (12) are radially distributed, the top end of the sample diversion channel (12) communicates with the sample injection port (11), and the bottom end communicates with the sample vertical channel (13); wherein, The plurality of sample vertical channels (13), the plurality of sample diversion channels (12), and the plurality of amplification reaction chambers (32) are arranged in one-to-one correspondence, 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 channels (13) also have the same size, the conical surface is coaxial with the sample injection port (11), and the amplification reaction chamber (32) is vertically aligned with and communicates with the corresponding sample vertical channel (13).

2. The multi-target microfluidic chip with a novel liquid splitting method according to claim 1, wherein The amplification reaction chamber (32) is vertically spaced from the sample vertical channel (13).

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

4. The multi-target microfluidic chip with a novel liquid splitting method according to claim 3, wherein, The flow resistance channel (33) 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 directions of the second U-shaped tubes are opposite to the opening direction of the first U-shaped tube.

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

6. The multi-target microfluidic chip with a novel liquid splitting 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 splitting method according to any one of claims 1-4 or 6, characterized in that, It further includes a base (4) installed at the bottom of the reaction layer (3), 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 splitting method according to claim 7, wherein, The base (4) is slidably connected to the reaction layer (3).

9. The multi-target microfluidic chip with a novel liquid splitting method according to claim 1, characterized in that, A dilution liquid vesicle placement hole, a plurality of dilution liquid diversion channels (16) arranged radially, and a plurality of dilution liquid vertical flow channels (17) corresponding to and communicating with the bottom ends of the plurality of dilution liquid diversion channels (16) are further formed in the liquid separation layer (1); the dilution liquid vesicle placement hole and the sample inlet (11) are spaced in the horizontal direction; the dilution liquid vesicle placement hole is used for receiving a dilution liquid vesicle (15); the top ends of the plurality of dilution liquid diversion channels (16) are respectively communicated with the bottom end of the dilution liquid vesicle placement hole, a plurality of dilution liquid cavities (38) are formed in the reaction layer (3), and the dilution liquid cavities (38) are vertically and respectively opposite to and communicated with the dilution liquid vertical flow channels (17).

10. The multi-target microfluidic chip with a novel liquid splitting method according to claim 1, characterized in that, A result display window (21) is provided on the sealing layer (2), 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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