Multi-target microfluidic chip having novel liquid distribution method

Abstract

The present application relates to the technical field of nucleic acid testing. Disclosed is a multi-target microfluidic chip having a novel liquid distribution method, the multi-target microfluidic chip comprising a reaction layer, a sealing layer and a liquid distribution layer, wherein the reaction layer is provided with a plurality of amplification reaction chambers, a plurality of sample loading buffer reservoirs and a plurality of test strip storage slots; a vertically arranged sample inlet, a plurality of sample distribution channels and a plurality of sample vertical flow channels are provided within the liquid distribution layer, the sample inlet and the sample vertical flow channels vertically running through the top surface and the bottom surface of the liquid distribution layer; and the sample vertical flow channels, the sample distribution channels and the amplification reaction chambers are correspondingly positioned, the plurality of sample distribution channels are all in communication with the sample inlet, the plurality of sample distribution channels are of the same size and are arranged at intervals along a conical surface, and the plurality of sample vertical flow channels are also of the same size and are in communication with the amplification reaction chambers. Such a configuration allows a sample to be completely and equally distributed, enables multiple parallel reactions to happen simultaneously, and maximizes the amount of the sample entering the amplification reaction chambers to reduce liquid loss, thereby improving the testing accuracy.

Description

Multi-target microfluidic chip with novel liquid distribution mode

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202411274893.9, filed on September 11, 2024, and entitled "Multi-target microfluidic chip with novel liquid distribution mode", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of nucleic acid detection technology, in particular to a multi-target microfluidic chip with novel liquid distribution mode. BACKGROUND

[0004] Pathogenic microorganism detection technology includes traditional detection technologies such as smear microscopy, isolation culture and biochemical reaction, and tissue cell culture, as well as clinical commonly used methods such as antigen-antibody detection and nucleic acid detection. Traditional detection technologies take a long time, and are not suitable for rapid screening of pathogens. Compared with antigen-antibody detection, nucleic acid detection has the advantages of high sensitivity and high specificity, and therefore has important significance for early diagnosis and screening of asymptomatic infected persons.

[0005] The main limitation of the application of nucleic acid detection technology in instant detection is the dependence on professional operation and professional instrument equipment. However, in the primary medical system, due to the lack of professional molecular biology laboratory, nucleic acid detection technology is difficult to popularize. In order to meet the detection needs of rapid and large-scale screening at the grassroots level, there is an urgent need for a low-cost, portable, instrument-independent, and real-time detection suitable microfluidic chip.

[0006] Current microfluidic technology for multi-target detection aims to realize the simultaneous detection and analysis of multiple biological molecules through integration and miniaturization, in order to improve detection efficiency and accuracy. However, this technology faces significant challenges in practical application, especially in the accurate distribution of liquid samples. On the one hand, when the chip design allows the liquid to be completely evenly divided, it is usually limited by the number of detection targets. This is because most of the liquid division is based on the flow channel 1 / 2, so the number of targets must be 2 n (n≥1). On the other hand, the current microfluidic chip design divides the flow channel and the amplification reaction cavity in the same plane, which cannot guarantee that the liquid does not backflow or stagnate in the flow channel, thus increasing the liquid loss.

[0007] Therefore, there is an urgent need to develop a multi-target microfluidic chip with novel liquid distribution mode to get rid of the limitation of the number of detection targets and effectively reduce the liquid loss. SUMMARY

[0008] The application aims to provide a multi-target microfluidic chip with a new liquid distribution mode, which can break through the limitation of detecting target quantity and effectively reduce liquid loss.

[0009] To solve the above technical problems, the technical scheme of the application is as follows:

[0010] A multi-target microfluidic chip with a new liquid distribution mode, comprising:

[0011] A reaction layer, the top surface of which is recessed to form a plurality of amplification reaction cavities and a plurality of sample loading buffer compartments, and a plurality of test paper storage slots; the plurality of amplification reaction cavities, the plurality of sample loading buffer compartments and the plurality of test paper storage slots are arranged one-to-one in correspondence with each other, and the amplification reaction cavities are connected to the corresponding sample loading buffer compartments.

[0012] A sealing layer arranged on the reaction layer;

[0013] A liquid distribution layer arranged at one end of the sealing layer and protruding from the top surface of the sealing layer, the liquid distribution layer and the sealing layer cooperating to seal the reaction layer, the liquid distribution layer being provided with a vertical sample inlet, a plurality of sample distribution channels and a plurality of sample vertical flow channels, the sample inlet penetrating the top surface of the liquid distribution layer upward, the sample vertical flow channels penetrating the bottom surface of the liquid distribution layer downward, the plurality of sample distribution channels being distributed radially, the top ends of the sample distribution channels being connected to the sample inlet, and the bottom ends of the sample distribution channels being connected to the sample vertical flow channels; wherein,

[0014] The plurality of sample vertical flow channels, the plurality of sample distribution channels and the plurality of amplification reaction cavities are arranged one-to-one in correspondence with each other, the plurality of sample distribution channels have the same size and are arranged along a conical surface, the plurality of sample vertical flow channels also have the same size, the conical surface is coaxially arranged with the sample inlet, and the amplification reaction cavities vertically face and are connected to the corresponding sample vertical flow channels.

[0015] Optionally, the amplification reaction cavities and the sample vertical flow channels are arranged vertically at a distance.

[0016] Optionally, the amplification reaction cavities and the sample loading buffer compartments are connected through a flow resistance channel.

[0017] Optionally, the flow resistance channel comprises a first U-shaped tube and two second U-shaped tubes connected to the two ends of the first U-shaped tube respectively, and the opening direction of the second U-shaped tube is opposite to that of the first U-shaped tube.

[0018] Optionally, it further comprises a capillary wall, one end of which enters the sample loading buffer compartment, and the other end is connected to the test paper in the test paper storage slot.

[0019] Optionally, the bottom wall of the sample loading buffer bin is lower than the bottom wall of the flow resistance channel.

[0020] Optionally, a base is arranged at the bottom of the reaction layer, and a heating module storage groove is arranged in the base, the heating module storage groove is used for storing a heating module, and the heating module is used for heating the amplification reaction cavity.

[0021] Optionally, the base is in sliding connection with the reaction layer.

[0022] Optionally, a dilution liquid vesicle placing hole, a plurality of dilution liquid shunt channels arranged in a radial manner, and a plurality of dilution liquid vertical flow channels corresponding to the bottom ends of the dilution liquid shunt channels are arranged on the separation layer; the dilution liquid vesicle placing hole and the sample inlet are arranged at a horizontal distance; the dilution liquid vesicle placing hole is used for accommodating a dilution liquid vesicle; the top ends of the dilution liquid shunt channels are respectively connected to the dilution liquid vesicle placing hole; a plurality of dilution liquid cavities are arranged on the reaction layer, and the dilution liquid cavities are vertically and vertically correspond to and connected to the dilution liquid vertical flow channels.

[0023] Optionally, a result display window is arranged on the sealing layer, and the number of the result display windows is the same as the number of the amplification reaction cavities.

[0024] The technical scheme has the following advantages:

[0025] 1.The multi-target microfluidic chip with a novel liquid distribution mode provided by the application, wherein a vertical sample inlet, a plurality of sample distribution channels and a plurality of sample vertical flow channels are arranged on a liquid distribution layer; the sample inlet penetrates the top surface of the liquid distribution layer; the sample vertical flow channels penetrate the bottom surface of the liquid distribution layer; the sample distribution channels are radially distributed; the top end of the sample distribution channel is connected to the sample inlet, and the bottom end is connected to the sample vertical flow channel; the plurality of sample vertical flow channels, the plurality of sample distribution channels and the plurality of amplification reaction cavities are arranged in one-to-one correspondence; the plurality of sample distribution channels have the same size and are arranged 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 cavities vertically face and communicate with the corresponding sample vertical flow channels, so that the sample and the diluent can be evenly distributed into the corresponding amplification reaction cavities, so as to realize multi-target detection; in addition, the sample vertical flow channel is arranged below the sample distribution channel, and the liquid distribution layer protrudes from the top surface of the sealing plate, so as to increase the vertical flow path of the sample and the diluent, increase the flow speed of the sample and the diluent under the action of their own gravity, and under the action of inertia, as much as possible, the sample and the diluent enter the amplification reaction cavity, reducing liquid loss, ensuring the concentration of the sample and the diluent, and further improving the detection accuracy. Furthermore, the multi-target microfluidic chip with a novel liquid distribution mode only needs to add the sample into the amplification reaction cavity through the sample inlet after the sample is lysed at room temperature, and then the amplification reaction is carried out; the amplification product is diluted by the diluent, and then enters the test paper storage tank through the sample buffer warehouse and the test paper, and the detection result is displayed on the test paper, so as to realize low-cost, portable and real-time detection, and the professional operation and professional equipment are not required, which can meet the detection needs of rapid and large-scale screening at the grassroots level, and the number of detection targets is not limited by the radially arranged sample distribution channels.

[0026] 2.The multi-target microfluidic chip with a novel liquid distribution mode provided by the application, wherein the amplification reaction cavities and the sample vertical flow channels are vertically spaced apart, so as to reduce the risk of liquid in the amplification reaction cavities entering the sample vertical flow channels, further reduce the possibility of liquid loss, and increase the detection accuracy.

[0027] 3.The multi-target microfluidic chip with a novel liquid distribution mode provided by the application, wherein the flow resistance channel comprises a first U-shaped tube and two second U-shaped tubes connected to the two ends of the first U-shaped tube, respectively; and the opening direction of the second U-shaped tube is opposite to the opening direction of the first U-shaped tube; the flow resistance channel can prevent the sample added into the amplification reaction cavity from directly flowing into the sample buffer warehouse before or during the amplification reaction, and can further mix the amplification product and the diluent before the test paper detection.

[0028] 4. The multi-target microfluidic chip with a new liquid distribution mode provided in the application, the bottom wall of the sample buffer bin is 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 diluted amplification product from directly flowing to the test paper, resulting in inaccurate detection results.

[0029] 5. The multi-target microfluidic chip with a new liquid distribution mode provided in the application, the base and the reaction layer are slidingly connected, the heating module can be conveniently taken out, so as to be reused. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0031] Fig. 1 is a perspective combined schematic diagram of the multi-target microfluidic chip in the embodiment one of the application;

[0032] Fig. 2 is a perspective exploded schematic diagram of the multi-target microfluidic chip in the embodiment one of the application;

[0033] Fig. 3 is a top view schematic diagram of the reaction layer of the multi-target microfluidic chip in the embodiment one of the application;

[0034] Fig. 4 is a perspective schematic diagram of the reaction layer of the multi-target microfluidic chip in the embodiment one of the application;

[0035] Fig. 5 is a perspective schematic diagram of the distribution layer of the multi-target microfluidic chip in the embodiment one of the application;

[0036] Fig. 6 is a perspective schematic diagram of the base of the multi-target microfluidic chip in the embodiment one of the application;

[0037] Fig. 7 is a perspective combined schematic diagram of the multi-target microfluidic chip in the embodiment two of the application;

[0038] Fig. 8 is a perspective exploded schematic diagram of the multi-target microfluidic chip in the embodiment two of the application;

[0039] Fig. 9 is a perspective schematic diagram of the base of the multi-target microfluidic chip in the embodiment two of the application;

[0040] Fig. 10 is a perspective combined schematic diagram of the multi-target microfluidic chip in the embodiment three of the application;

[0041] Fig. 11 is a perspective exploded schematic diagram of the multi-target microfluidic chip in the embodiment three of the application;

[0042] Fig. 12 is a top view of the reaction layer of the multi-target microfluidic chip of Example 3 of the present application;

[0043] Fig. 13 is a perspective view of the reaction layer of the multi-target microfluidic chip of Example 3 of the present application;

[0044] Fig. 14 is a perspective view of the distribution layer of the multi-target microfluidic chip of Example 3 of the present application;

[0045] Fig. 15 is a perspective view of the multi-target microfluidic chip of Example 4 of the present application;

[0046] Fig. 16 is a perspective exploded view of the multi-target microfluidic chip of Example 4 of the present application;

[0047] Fig. 17 is a top view of the reaction layer of the multi-target microfluidic chip of Example 4 of the present application;

[0048] Fig. 18 is a perspective view of the reaction layer of the multi-target microfluidic chip of Example 4 of the present application;

[0049] Fig. 19 is a perspective view of the distribution layer of the multi-target microfluidic chip of Example 4 of the present application;

[0050] Fig. 20 is a structural view of a test paper of the present application;

[0051] Fig. 21 is a structural view of another test paper of the present application;

[0052] Fig. 22 is a structural view of still another test paper of the present application.

[0053] Legend of reference numerals: 1, distribution layer; 11, sample inlet; 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 assembly; 3, reaction layer; 31, second fixing assembly; 32, amplification reaction cavity; 33, flow resistance channel; 34, sample loading buffer bin; 35, capillary wall; 36, test paper storage groove; 37, sliding groove; 38, diluent cavity; 4, base; 41, sliding block; 42, heating module storage groove; 43, wire clamping groove; 5, test paper; 51, bottom plate; 52, sample pad; 53, conjugate pad; 54, NC membrane; 55, water absorption pad. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are some of the embodiments of the present application, but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts are within the scope of protection of the present application.

[0055] In the description of the present application, it should be noted that the terms "upper", "lower", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first" and "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0056] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0057] In addition, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as there is no conflict between them.

[0058] As shown in FIGS. 1-22, the present application provides a multi-target microfluidic chip with a new liquid diversion method for nucleic acid detection (hereinafter referred to as multi-target microfluidic chip), which comprises a distribution layer 1, a sealing layer 2, a reaction layer 3, and a base 4. The distribution layer 1 is used to divide the sample and the diluent. The reaction layer 3 is used to amplify and detect the target to be tested.

[0059] The top surface of the reaction layer 3 is inwardly recessed to form a plurality of amplification reaction cavities 32 and a plurality of sample loading buffer compartments 34. The reaction layer 3 is also provided with a plurality of test paper storage grooves 36 for storing test papers 5 (as shown in FIGS. 20-22). The plurality of amplification reaction cavities 32, the plurality of sample loading buffer compartments 34 and the plurality of test paper storage grooves 36 are arranged one-to-one in position, and the amplification reaction cavities 32 are in communication with the corresponding sample loading buffer compartments 34.

[0060] The sealing layer 2 is arranged on the reaction layer 3, and the distribution layer 1 is arranged at one end of the sealing layer 2. The sealing layer 2 and the distribution layer 1 cooperate to seal the reaction layer 3.

[0061] The sample injection port 11 vertically penetrates the top surface of the sample distribution layer 1. The sample vertical flow channels 13 vertically penetrate the bottom surface of the sample distribution layer 1. The sample diversion channels 12 are radially arranged and respectively communicate with the same sample injection port 11. The top ends of the sample diversion channels 12 communicate with the sample injection port 11, and the bottom ends of the sample diversion channels 12 communicate with the sample vertical flow channels 13. The sample vertical flow channels 13, the sample diversion channels 12 and the amplification reaction cavities 32 are arranged in one-to-one correspondence. The sample diversion channels 12 have the same size and are arranged along a conical surface. The sample vertical flow channels 13 also have the same size. The conical surface is coaxial with the sample injection port 11. The amplification reaction cavities 32 vertically communicate with the sample vertical flow channels 13.

[0062] In the present application, the sample injection port 11, the sample diversion channels 12 and the sample vertical flow channels 13 are arranged on the sample distribution layer 1. The sample vertical flow channels 13, the sample diversion channels 12 and the amplification reaction cavities 32 are arranged in one-to-one correspondence. The sample diversion channels 12 respectively communicate with the same sample injection port 11. The sample diversion channels 12 have the same size and are arranged along a conical surface. The sample vertical flow channels 13 also have the same size. The amplification reaction cavities 32 vertically communicate with the sample vertical flow channels 13. Therefore, the sample and the diluent can be evenly distributed into the corresponding amplification reaction cavities 32, so that the simultaneous amplification of multiple targets can be realized. In addition, the sample injection port 11 is vertically arranged, and the sample vertical flow channels 13 are arranged below the sample diversion channels 12. The sample distribution layer 1 protrudes from the top surface of the sealing layer 2. Therefore, the vertical flow path of the sample and the diluent can be increased. The sample and the diluent have a greater flow speed under the action of their own gravity. Under the action of inertia, the sample and the diluent can enter the amplification reaction cavities 32 as much as possible, reducing liquid loss, ensuring that the amount of the sample and the diluent entering the amplification reaction cavities 32 is consistent, and finally improving the accuracy of nucleic acid detection.

[0063] Optionally, the amplification reaction cavities 32 vertically communicate with the sample vertical flow channels 13, and the amplification reaction cavities 32 and the sample vertical flow channels 13 are vertically spaced. In this way, the risk of the sample and the diluent flowing back into the sample vertical flow channels 13 from the amplification reaction cavities 32 can be reduced.

[0064] In order to simplify the operation steps, the diluent is built-in on the multi-target microfluidic chip. The bottom of the diluent vesicle 15 storing the diluent is provided with a tin foil film, and the material of the vesicle is thin and soft plastic. The diluent vesicle 15 is squeezed downward to break the tin foil film and 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 distribution layer 1. The diluent vesicle placement hole penetrates through the top surface of the distribution layer 1 upward. The diluent vertical flow channel 17 penetrates through the bottom surface of the distribution layer 1 downward, and the plurality of diluent shunt channels 16 are arranged radially and respectively communicate with the same diluent vesicle placement hole. The top end of the diluent shunt channel 16 communicates with the diluent vesicle placement hole, and the bottom end communicates with the diluent vertical flow channel 17. Correspondingly, a plurality of diluent cavities 38 are provided in the reaction layer 3 as described above, and the plurality of diluent cavities 38 are in one-to-one position correspondence with the plurality of amplification reaction cavities 32. The plurality of diluent shunt channels 16 below the diluent vesicle placement hole, the plurality of diluent vertical flow channels 17 and the plurality of diluent cavities 38 are arranged in one-to-one position correspondence. The plurality of diluent shunt channels 16 are the same size and are arranged along a conical surface, and the plurality of diluent vertical flow channels 17 are also the same size. The conical surface is coaxially arranged with the diluent vesicle placement hole, and the diluent cavity 38 vertically faces and communicates with the diluent vertical flow channel 17. In this way, the sample can be evenly divided into the amplification reaction cavity 32 through the sample inlet 11, the sample shunt channel 12 and the sample vertical flow channel 13, and the diluent can be evenly divided into the diluent cavity 38 through the diluent vesicle placement hole, the diluent shunt channel 16 and the diluent vertical flow channel 17, so as to realize multi-target detection of sample and diluent.

[0065] In order to avoid foreign matter entering the sample inlet 11, the sample inlet 11 is sealed by the sealing cover 14. Specifically, the sealing cover 14 is threadedly connected with the sample inlet 11. Of course, it can also be sealed in the form of a sealing film.

[0066] In order to facilitate viewing of the detection results, the sealing layer 2 is provided with a result display window 21 corresponding to the color development part on the test paper 5. In order to facilitate the fixed connection of the sealing layer 2 and the reaction layer 3, the sealing layer 2 is provided with a first fixing component 22 (such as a column / slot), and the reaction layer is provided with a second fixing component 31 (corresponding to a slot / column). 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.

[0067] The base 4 is connected with the reaction layer 3 in a sliding connection mode. Specifically, the base 4 is provided with a sliding block 41, and the reaction layer 3 is provided with a sliding groove 37, and the sliding block 41 slides along the sliding groove 37. The base 4 and the reaction layer 3 are arranged in a sliding connection mode because the base 4 is provided with a heating module storage groove 42 for storing a heating module. The heating module in the heating module storage groove 42 can be taken out by sliding the base 4, so that the heating module is separated from the whole device and is 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.

[0068] Embodiment one

[0069] As shown in FIGS. 1-6, 20-22, the number of sample diversion channels 12 under the sample inlet 11, sample vertical flow channels 13 and amplification reaction cavities 32, dilution liquid diversion channels 16 under the dilution liquid vesicle placement hole and dilution liquid vertical flow channels 17, and the number of result display windows 21 are all three. The included angle between two adjacent sample diversion channels 12 is 120°, and the included angle between two adjacent dilution liquid diversion channels 16 is 120°. The included angle between the extension direction of the sample diversion channel 12 and the bottom surface (i.e. the horizontal surface) of the sample inlet 11 is 35°-55°, preferably 45°. The included angle between the extension direction of the dilution liquid diversion channel 16 and the bottom surface (i.e. the horizontal surface) of the dilution liquid vesicle placement hole is 35°-55°, preferably 45°. The height of the sample vertical flow channel is 0.55-0.65 mm, preferably 0.6 mm. Since three sample diversion channels 12, three sample vertical flow channels 13, three dilution liquid diversion channels 16 and three dilution liquid vertical flow channels 17 are respectively arranged, the sample added into the amplification reaction cavity from the sample inlet 11 and the dilution liquid released from the dilution liquid vesicle 15 can be divided into three parts, thereby simplifying the number of liquid addition operations.

[0070] The height of the amplification reaction cavity 32 is 1 mm, and the RPA reaction volume (the reaction volume refers to the space occupied by the generated substances under certain conditions) is 10 μL. Each amplification reaction cavity 32 is connected with a flow resistance channel 33 downstream, and the flow resistance channel 33 can prevent the sample from flowing directly to the downstream after entering the amplification reaction cavity 32, thereby improving the uniformity of the dilution liquid and the amplification product. Specifically, the flow resistance channel 33 includes a first U-shaped tube and two second U-shaped tubes connected with the two ends of the first U-shaped tube, respectively, and the opening direction of the second U-shaped tube is opposite to that of the first U-shaped tube. Such a flow resistance channel can prevent the sample added into the amplification reaction cavity from flowing directly into the sample buffer bin 34 before or during the amplification, and can further mix the amplification product and the dilution liquid before the test paper detection.

[0071] The downstream of each flow resistance channel 33 is connected with an upper sample buffer bin 34. The sidewall of each upper sample buffer bin 34 is provided with a capillary wall 35 towards the test paper storage groove 36. The capillary wall 35 is composed of a plurality of vertical small pieces with a spacing of 0.2 mm to generate capillary power to transport liquid to the upper sample end of the test paper 5 stored in the test paper storage groove 36. One end of the capillary wall 35 enters the upper sample buffer bin 34, and the other end is connected with the upper sample end of the test paper 5.

[0072] The base 4 is provided with a heating module storage groove 42 directly below the amplification reaction cavity 32. The heating module is specifically an electric heating sheet, and the sidewall of the base 4 is provided with an electric wire slot 43 of the electric heating sheet for external power supply. The electric heating sheet is previously adhered to the bottom of the heating module storage groove 42 by double-sided adhesive. The bottom of the reaction layer 3 is in close contact with the electric heating sheet, which ensures that the electric heating sheet can directly contact the bottom of the amplification reaction cavity 32 to provide sufficient heat. The electric heating sheet relies on resistance heating to convert electrical energy into heat energy.

[0073] The process of nucleic acid detection using the multi-target microfluidic chip in Example 1 is described below:

[0074] The saliva and nasopharyngeal swab samples are first placed in a soft bottle containing a lysis solution for a normal temperature lysis reaction.

[0075] The sealing cover 14 is twisted off, and the multi-target microfluidic chip is placed horizontally;

[0076] The bottle opening of the soft bottle is placed downward in the sample inlet 11, and the bottle opening of the soft bottle is sealingly connected with the sample inlet 11. The bottle body of the soft bottle is squeezed, the sample is evenly divided into the amplification reaction cavities 32, the soft bottle is discarded, and the sealing cover 14 is covered;

[0077] The power supply of the electric heating sheet is turned on for heating, and the power supply is turned off after the amplification reaction is completed;

[0078] The dilution liquid vesicle 15 is squeezed, the dilution liquid is evenly divided into the dilution liquid cavity 38, and then flows into the amplification reaction cavity 32, flows along the flow resistance channel 33 to the upper sample buffer bin 34, the liquid accumulated continuously reaches the upper sample end of the test paper 5 stored in the test paper storage groove 36 through the capillary wall 35, the sample loading detection process is completed through the capillary power of the capillary wall 35 and the test paper 5, and finally the detection result is read through the result display window 21;

[0079] 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, and the base 4 and the internal heating module can be repeatedly used.

[0080] Example Two

[0081] As shown in FIGS. 7-9 and 20-22, 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 a chemical reaction after contacting with air.

[0082] The process of using the multi-target microfluidic chip in the second embodiment for nucleic acid detection is described below:

[0083] The sample saliva or nasopharyngeal swab is first placed in a soft bottle containing a lysis solution for a normal temperature lysis reaction.

[0084] The hand warmer is taken out of the sealed bag and placed in the heating module storage slot 42 in the base 4;

[0085] The sealed cap 14 is twisted off, and the multi-target microfluidic chip is placed horizontally;

[0086] The soft bottle is placed with its mouth facing down in the sample inlet 11, and the mouth of the soft bottle is sealingly connected to the sample inlet 11. The bottle body of the soft bottle is squeezed to divide the reagent into the amplification reaction cavity 32. The soft bottle is discarded, and the sealed cap 14 is covered.

[0087] After waiting for the amplification reaction to end, the diluent vesicle 15 is squeezed to divide the diluent into the diluent cavity 38 and into the amplification reaction cavity 32, and then along the flow resistance channel 33 to the sample loading buffer bin 34. The continuously accumulated liquid passes through the capillary wall 35 to the sample paper 5 in the sample paper storage slot 36, and the sample loading detection process is completed through the capillary power of the capillary wall 35 and the sample paper 5. Finally, the detection result is read through the result display window 21.

[0088] After the detection is completed, the entire device is discarded.

[0089] Embodiment Three

[0090] As shown in FIGS. 10-14, the difference between the third embodiment and the first embodiment is that the number of sample diversion channels 12 under the sample inlet 11, sample vertical flow channels 13, amplification reaction cavities 32, diluent vesicle placement holes under the diluent vesicle placement holes, diluent diversion channels 16, diluent vertical flow channels 17, and result display windows 21 is four.

[0091] The process of using the multi-target microfluidic chip in the third embodiment for nucleic acid detection is the same as that in the first embodiment, which is not repeated here.

[0092] Embodiment Four

[0093] As shown in FIGS. 15-19, the difference between Example Four and Example One is that the number of sample shunt channels 12, sample vertical flow channels 13, amplification reaction cavities 32, diluent cavities 38, flow resistance channels 33, sample loading buffer reservoirs 34, capillary walls 35, and test paper storage slots 36 under the sample inlet 11 are all five, and the diluent is also divided into five portions.

[0094] The process of nucleic acid detection using the multi-target microfluidic chip in Example Four is the same as that in Example One, which will not be described here.

[0095] Example Five

[0096] The present embodiment provides a nucleic acid detection method for the multi-target microfluidic chip provided in Example 1 (as shown in FIGS. 1-6) or Example 2 (as shown in FIGS. 7-9), which comprises the following steps:

[0097] Constructing a three-target RPA reaction system: each RPA reaction system includes amplification primers and probes for one target, reaction buffer, and amplification enzymes. Dry powder of the RPA reaction system is inserted into each of the three amplification reaction cavities 32, and the sample vertical flow channels 13 in the separation layer 1 are aligned with the amplification reaction cavities 32. The probe in the RPA system is modified with FITC at the 5' end, and the reverse primer is modified with biotin at the 5' end.

[0098] Constructing a lateral flow chromatographic test paper detection system: the lateral flow chromatographic test paper displays the detection results by the principle of nano-gold particle aggregation color development. As shown in FIG. 20, the lateral flow chromatographic 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 absorption pad 55. The NC membrane 54 is marked with a T band and a C band. The T band is fixed with streptavidin, and the C band is fixed with a rabbit anti-mouse secondary antibody. The nano-gold particles are labeled with an anti-FITC antibody.

[0099] Adding the sample to be detected for amplification reaction: the sample to be detected is added to the lysate soft bottle for room temperature lysis, the soft bottle is squeezed, and the lysed sample is transferred to the amplification reaction cavity 32 for RPA amplification reaction. At this time, the amplification product is a double-stranded DNA labeled with biotin and FITC.

[0100] Releasing the test paper diluent for chromatographic detection: the diluent is released into the diluent cavity 38 by squeezing the diluent vesicle 15. The diluent enters the amplification reaction cavity 32, carrying the amplification product along the flow resistance channel 33 into the sample loading buffer reservoir 34, and finally through the capillary wall 35 to the sample pad 52 of the test paper 5, 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 is intercepted by the nano-gold particles labeled with the anti-FITC antibody, resulting in red color display on the T band. Correspondingly, when the sample is negative, no color is displayed due to the inability to intercept the nano-gold particles.

[0101] In this embodiment, the RPA reaction volume ranges from 5 μL to 50 μL.

[0102] In this embodiment, the reaction time of the above-mentioned sample to be tested and the RPA reaction system ranges from 5 min to 40 min.

[0103] In this embodiment, the total magnesium acetate concentration in each RPA amplification system ranges from 12 mM to 40 mM.

[0104] In this embodiment, the concentration of each target primer in each RPA amplification system ranges from 150 nM to 600 nM, and the concentration of the probe 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 kept within the range of 750 nM to 2000 nM.

[0105] In this embodiment, the reaction temperature of the above-mentioned sample to be tested and the RPA reaction system ranges from 45°C to 42°C.

[0106] In this embodiment, the sample liquid volume of the lateral flow chromatographic test paper ranges from 100 μL to 200 μL.

[0107] Embodiment Six

[0108] The present embodiment provides a nucleic acid detection method for nucleic acid detection of the multi-target microfluidic chip provided in Embodiment 1 (as shown in Figures 1 to 6) or Embodiment 2 (as shown in Figures 7 to 9), which comprises the following steps:

[0109] A three-target RPA reaction system is constructed: each RPA reaction system includes amplification primers and probes for two targets, reaction buffer, and amplification functional enzymes. The dry powder of the RPA reaction system is respectively built into three amplification reaction cavities 32, and the sample vertical flow channel 13 in the separation layer 1 is aligned with the amplification reaction cavities 32. Among them, one target probe in the RPA system is modified with FITC at the 5' end, and the reverse primer is modified with biotin at the 5' end; another target probe is modified with FITC at the 5' end, and the reverse primer is modified with digoxin at the 5' end.

[0110] A lateral flow chromatographic test paper detection system is constructed: the lateral flow chromatographic test paper displays the detection results through the principle of nano-gold particle aggregation color development. As shown in Figure 21, the lateral flow chromatographic 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 absorption pad 55. The NC membrane is marked with two T bands and one C band. The two T bands are respectively fixed with streptavidin and anti-digoxin antibody, and the C band is fixed with rabbit anti-mouse secondary antibody. The nano-gold particles are labeled with anti-FITC antibody.

[0111] Adding the sample to be detected for amplification reaction: the sample to be detected is added to the soft bottle of lysis solution for lysis at room temperature, and the soft bottle is squeezed to transfer the lysed sample to the amplification reaction cavity 32 for RPA amplification reaction. At this time, the amplification product of one target is a double-stranded DNA labeled with biotin and FITC; the amplification product of the other target is a double-stranded DNA labeled with digoxin and FITC.

[0112] Releasing the test paper diluent for chromatographic detection: the diluent capsule 15 is squeezed to release the diluent into the diluent cavity 38, and the diluent flows into the amplification reaction cavity 32 with the amplification product, flows into the sample buffer bin 34 along the flow resistance channel 33, and finally passes through the capillary wall 35 to reach the sample pad 52 of the test paper 5, completing the sample loading 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 is intercepted by the nanogold particles labeled with anti-FITC antibody, resulting in a red color on the T band. Correspondingly, when the sample is negative, no color is displayed due to the inability to intercept the nanogold particles.

[0113] In this embodiment, the total reaction volume of RPA is in the range of 5 μL-50 μL.

[0114] In this embodiment, the reaction time of the above-mentioned sample to be detected and the RPA reaction system is in the range of 5 min-40 min.

[0115] In this embodiment, the total magnesium acetate concentration in each RPA amplification system is in the range of 12 mM-40 mM.

[0116] In this embodiment, the concentration of each target primer in each RPA amplification system is in the range of 150 nM-600 nM, and the concentration of the probe is in the range of 50 nM-150 nM. The ratio of primer 1, primer 2, and probe can be freely adjusted, but the concentration of oligonucleotides in a single RPA reaction system should be in the range of 750 nM-2000 nM.

[0117] In this embodiment, the reaction temperature of the above-mentioned sample to be detected and the RPA reaction system is in the range of 45°C-42°C.

[0118] In this embodiment, the volume of the sample loading liquid of the lateral flow chromatographic test paper is in the range of 100 μL-200 μL.

[0119] Embodiment Seven

[0120] This embodiment provides another nucleic acid detection method for nucleic acid detection of the multi-target microfluidic chip provided in Embodiment 1 (as shown in FIGS. 1-6) or Embodiment 2 (as shown in FIGS. 7-9), which comprises the following steps:

[0121] The nine-target RPA reaction system is constructed: each RPA reaction system includes the amplification primers and probes of three targets, reaction buffer and amplification functional enzyme, the dry powder of the RPA reaction system is respectively built into three amplification reaction cavities 32, and the sample vertical flow channel 13 in the separation layer 1 is aligned with the amplification reaction cavities 32. The 5' end of the probe of each target in each RPA system is modified with FITC, and the 5' end of the reverse primer is modified with biotin, rhodamine and digoxin respectively.

[0122] The lateral flow chromatography test paper detection system is constructed: the lateral flow chromatography test paper displays the detection results by the principle of nano-gold particle aggregation color development. As shown in FIG. 22, 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 absorption pad 55, wherein the NC membrane 54 is marked with three T bands and one C band, the T bands are respectively fixed with streptavidin, anti-rhodamine antibody and anti-digoxin antibody, and the C band is fixed with rabbit anti-mouse secondary antibody, and the nano-gold particles are labeled with anti-FITC antibody.

[0123] The sample to be detected is added for amplification reaction: the sample to be detected is added to the soft bottle of lysis solution for room temperature lysis, the soft bottle is squeezed, and the lysis completed sample is transferred to the amplification reaction cavity 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.

[0124] The test paper diluent is released for chromatographic detection: the diluent vesicle 15 is squeezed to release the diluent into the diluent cavity 38, and the diluent flows into the amplification reaction cavity 32 with the amplification product, flows into the sample buffer bin 34 along the flow resistance channel 33, and finally reaches the sample pad of the test paper 5 through the capillary wall 35, completing the sample 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 of the T band, the FITC end of the double-stranded DNA is intercepted by the nano-gold particles labeled with anti-FITC antibody, resulting in red display of the T band. Correspondingly, when the sample is negative, no color is displayed due to the inability to intercept the nano-gold particles.

[0125] In this embodiment, the RPA reaction volume ranges from 5 μL to 50 μL.

[0126] In this embodiment, the total magnesium acetate concentration in each RPA amplification system ranges from 12 mM to 40 mM.

[0127] In this embodiment, the concentration of each target primer in each RPA amplification system ranges from 150 nM to 600 nM, the concentration of the probe ranges from 50 nM to 150 nM, and the ratio of primer 1, primer 2 and probe can be freely adjusted, but the oligonucleotide concentration in a single RPA reaction system should be kept within the range of 750 nM to 2000 nM.

[0128] In the present embodiment, the reaction time of the above-mentioned sample to be tested and the RPA reaction system ranges from 5 min to 40 min.

[0129] In the present embodiment, the reaction temperature of the above-mentioned sample to be tested and the RPA reaction system ranges from 45°C to 42°C.

[0130] In the present embodiment, the sample liquid volume of the lateral flow chromatography test paper ranges from 100 μL to 200 μL.

[0131] The multi-target microfluidic chip with a novel liquid shunting mode provided by the present application can completely and evenly divide the sample and the diluent, promote multiple parallel reactions to be performed simultaneously, and enable as many samples and diluents as possible to enter the amplification reaction cavity 32, thereby reducing liquid loss and improving detection accuracy.

[0132] Obviously, the above embodiments are merely examples for clear illustration, rather than limitation on the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, it is not necessary and impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A multi-target microfluidic chip with a novel liquid splitting mode, characterized in that, The multi-target microfluidic chip comprises: a reaction layer (3) having a plurality of amplification reaction cavities (32), a plurality of sample loading buffer storages (34) and a plurality of test paper storages (36) on the top surface; the amplification reaction cavities (32), the sample loading buffer storages (34) and the test paper storages (36) are arranged one by one in a position corresponding manner, and the amplification reaction cavities (32) are connected to the corresponding sample loading buffer storages (34); a sealing layer (2) arranged on the reaction layer (3); a liquid distribution layer (1) arranged at one end of the sealing layer (2) and protruding from the top surface of the sealing layer (2), the liquid distribution layer (1) and the sealing layer (2) cooperate to seal the reaction layer (3), the liquid distribution layer (1) is provided with a vertical sample inlet (11), a plurality of sample branch channels (12) and a plurality of sample vertical flow channels (13), the sample inlet (11) penetrates the top surface of the liquid distribution layer (1), the sample vertical flow channels (13) penetrate the bottom surface of the liquid distribution layer (1), the sample branch channels (12) are distributed in a radial manner, the top ends of the sample branch channels (12) are connected to the sample inlet (11), and the bottom ends of the sample branch channels (12) are connected to the sample vertical flow channels (13); wherein the sample vertical flow channels (13), the sample branch channels (12) and the amplification reaction cavities (32) are arranged one by one in a position corresponding manner, the sample branch channels (12) have the same size and are arranged along a conical surface, the sample vertical flow channels (13) also have the same size, the conical surface is coaxially arranged with the sample inlet (11), and the amplification reaction cavities (32) vertically face and are connected to the corresponding sample vertical flow channels (13).

2. The multi-target microfluidic chip with a novel liquid splitting mode according to claim 1, characterized in that, The amplification reaction cavities (32) and the sample vertical flow channels (13) are vertically spaced. 3.The multi-target microfluidic chip with a novel liquid splitting mode according to claim 1, wherein, The amplification reaction cavities (32) and the sample loading buffer storages (34) are connected through flow resistance channels (33).

4. The multi-target microfluidic chip with a novel liquid splitting mode according to claim 3, characterized in that, The flow resistance channels (33) comprise a first U-shaped tube and two second U-shaped tubes connected to the two ends of the first U-shaped tube, respectively, 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 splitting mode according to claim 3 or 4, characterized in that, A capillary wall (35) is further arranged, one end of the capillary wall (35) enters the sample loading buffer storage (34), and the other end of the capillary wall (35) is connected to the test paper (5) in the test paper storage (36).

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

7. The multi-target microfluidic chip with a novel liquid splitting mode according to any one of claims 1-4, 6, characterized in that, A base (4) is further arranged at the bottom of the reaction layer (3), the base (4) is provided with a heating module storage groove (42) for storing a heating module, and the heating module is used for heating the amplification reaction cavities (32).

8. The multi-target microfluidic chip with a novel liquid splitting mode according to claim 7, characterized in that, The base (4) and the reaction layer (3) are connected in a sliding manner.

9. The multi-target microfluidic chip with a novel liquid splitting mode according to claim 1, characterized in that, The liquid separation layer (1) is further provided with a diluent vesicle placing hole, a plurality of diluent shunt channels (16) arranged in a radial manner, and a plurality of diluent vertical flow channels (17) corresponding to the bottom ends of the plurality of diluent shunt channels (16) one by one and in communication; the diluent vesicle placing hole and the sample inlet (11) are arranged at a distance in the horizontal direction; the diluent vesicle placing hole is used for accommodating a diluent vesicle (15); the top ends of the plurality of diluent shunt channels (16) are respectively in communication with the bottom ends of the diluent vesicle placing hole, and the reaction layer (3) is provided with a plurality of diluent cavities (38), the diluent cavities (38) vertically correspond to and are in communication with the diluent vertical flow channels (17) one by one.

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

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