Nucleic acid detection microfluidic chip

Abstract

This invention relates to the field of molecular diagnostics technology and discloses a nucleic acid detection microfluidic chip, comprising a sample processing layer, a reaction layer, an electrode layer, and a first sealing layer. The sample processing layer has a sample shunt chamber containing several sequentially connected sample storage chambers. The reaction layer is located below the sample processing layer and has several reaction chambers, each corresponding to and connected to a sample storage chamber. The reaction layer has a first pump valve chamber and a gas channel, while the sample processing layer has a second pump valve chamber containing pumps and valves. The electrode layer is located below the reaction layer. The first sealing layer is located above the sample processing layer and has an opening located above a lysis chamber and a washing chamber. In this invention, the sample fills each sample storage chamber uniformly and synchronously under capillary action. Driven by the pumps and valves, the sample is synchronously transferred to the reaction chamber, ensuring consistent liquid volume distributed to each reaction chamber.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, specifically to a microfluidic chip for nucleic acid detection. Background Technology

[0002] Microfluidic chip technology integrates the basic operational units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, onto a single chip, automating the entire analysis process. It is an ideal approach for achieving rapid on-site molecular diagnostics.

[0003] In existing technologies, to achieve multiplex detection, multiple reaction chambers are usually set on a microfluidic chip, and the nucleic acid solution to be tested is transferred to the reaction chamber one by one through a transfer device. During the transfer process, there is a problem of uneven sample distribution in each reaction chamber. Inaccurate distribution will seriously affect the comparability of detection results from different channels. More seriously, the asynchronous flow of liquids during the distribution process can easily lead to aerosol crosstalk between different chambers, causing fatal cross-contamination and rendering the multiplex detection results meaningless. Summary of the Invention

[0004] This invention provides a microfluidic chip for nucleic acid detection, which solves the problem in the prior art that the nucleic acid solution to be tested is difficult to be uniformly and synchronously distributed into multiple parallel reaction chambers.

[0005] This invention provides a nucleic acid detection microfluidic chip, comprising: The sample processing layer has several reagent chambers and sample splitting chambers connected in sequence. The reagent chambers are used to extract nucleic acid from the sample, and the sample splitting chambers have several sample storage chambers connected in sequence. The reaction layer is located below the sample processing layer. The reaction layer has several reaction chambers, which are connected to the sample storage chambers one by one. The reaction layer has a first pump valve chamber and a gas channel, which connect the first pump valve chamber and the reaction chamber. The sample processing layer has a second pump valve chamber, which is connected to the first pump valve chamber. Pump valves are installed in the first pump valve chamber and the second pump valve chamber. An electrode layer is disposed below the reaction layer. Several reaction regions are disposed on the electrode layer, and each reaction region is connected to the reaction chamber in a one-to-one correspondence. A first sealing layer is disposed above the sample processing layer. The first sealing layer has an opening located above the remaining reagent chambers, excluding those adjacent to the sample shunt chamber.

[0006] Beneficial effects: In this invention, after the sample is processed in the reagent chamber, it is driven into the sample distribution chamber by squeezing the first sealing layer. Under capillary action, the sample uniformly and synchronously fills each sample storage chamber. Driven by the pump valve, the sample is synchronously transferred to the reaction chamber, ensuring that the amount of liquid distributed to each reaction chamber is consistent. This not only guarantees that the start time of all parallel reactions is completely consistent, improving the comparability and accuracy of the data, but also eliminates cross-contamination between chambers due to asynchronous liquid flow from a physical structure perspective. The design of multiple independent reaction chambers allows for multiple detections of a single sample, with results mutually corroborating each other or covering multiple targets, greatly improving the throughput and reliability of the detection.

[0007] In one alternative implementation, the upper dimension of the sample storage cavity is larger than the lower dimension.

[0008] Beneficial effects: The present invention sets the sample storage chamber into a conical structure with a larger top and a smaller bottom. The longitudinal cross-section of each storage chamber is trapezoidal, and its width decreases uniformly from top to bottom, so that liquid can quickly fill the sample storage chamber through capillary force and Laplace pressure difference.

[0009] In one optional implementation, the sample shunt cavity further includes a shunt channel, and several sample temporary storage cavities are arranged in a straight line, with the shunt channel connecting adjacent sample temporary storage cavities.

[0010] Beneficial effects: Since the straight-line distance between two points is the shortest, the sample storage cavities are arranged in a straight line, which allows the sample to be quickly diverted to each storage cavity.

[0011] In one optional embodiment, the reaction layer is provided with lyophilized reagent chambers and transfer channels. The number of lyophilized reagent chambers is the same as that of the reaction chambers. The transfer channels connect the reaction chambers and the lyophilized reagent chambers. The lyophilized reagent chambers are connected to the sample storage chambers in a one-to-one correspondence. The starting end of the transfer channels is located at the bottom of the lyophilized reagent chambers.

[0012] Beneficial effects: The present invention sets the starting end of the transfer channel at the bottom of the lyophilized reagent chamber, which facilitates the complete transfer of liquid in the transfer channel to the reaction chamber.

[0013] In one alternative embodiment, at least one mixing chamber is provided in the transfer channel.

[0014] Beneficial effects: The mixing chamber of this invention can fully mix the sample and lyophilized reagent during the transfer process, thereby improving the detection effect.

[0015] In one alternative implementation, the gas passage is equidistant from the first pump valve chamber to each reaction chamber.

[0016] Beneficial effects: Since the path length of the gas channel from the first pump valve chamber to the reaction chamber is consistent, the present invention can provide the same driving force to each group of reaction chambers, so that the liquid transfer time is consistent.

[0017] In one optional embodiment, the reagent chamber includes a magnetic bead chamber, a lysis chamber, a washing chamber, and an elution chamber connected in sequence. The bottom of the elution chamber is connected to the sample splitting chamber. A waste liquid chamber is provided in the sample splitting chamber, and the waste liquid chamber is connected to the sample temporary storage chambers located at both ends.

[0018] Beneficial effects: The present invention connects the bottom of the elution chamber to the sample splitting chamber, which facilitates the transfer of all samples in the elution chamber to the sample splitting chamber; the waste liquid chamber can be set up so that excess samples can flow into the waste liquid chamber for collection through the splitting channel, ensuring that each sample storage chamber can be filled with samples.

[0019] In one alternative embodiment, the pyrolysis chamber and the cleaning chamber are provided with sealing covers, which have a communicating state that connects adjacent chambers and a blocking state that isolates adjacent chambers.

[0020] Beneficial effects: The sealing cap of this invention can seal the reagents pre-filled in the chamber to prevent leakage or contamination, and it can also be used to prevent the transfer of reagents between adjacent chambers during transportation.

[0021] In one optional embodiment, the sealing cap includes a cap body, a handle, and partitions. The cap body is movably disposed in the pyrolysis chamber and the cleaning chamber. The handle is disposed on the top of the cap body. The two partitions are disposed parallel to each other at the bottom of the cap body, forming a connecting channel between the two partitions. The connecting channel has a communicating state that connects adjacent chambers and a blocking state that isolates adjacent chambers.

[0022] Beneficial effects: The present invention provides a handle on the sealing cap for easy operation by the user, and the partition at the bottom of the cap can be used to prevent the transfer of reagents between adjacent chambers during transportation.

[0023] In one optional embodiment, the nucleic acid detection microfluidic chip further includes a second sealing layer disposed between the sample processing layer and the reaction layer. The second sealing layer has a first opening communicating between the reaction chamber and the sample storage chamber, and a second opening communicating between the first pump valve chamber and the second pump valve chamber. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a three-dimensional structural schematic diagram of a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 2 This is an exploded structural diagram of a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 3 This is a top view schematic diagram of the sample processing layer in a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 4 This is a perspective view of the sample processing layer in a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 5 This is a schematic diagram and a perspective view of the sealing cap in a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 6 This is a schematic diagram of the reaction layer in a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 7 This is a schematic diagram of the electrode layer in a nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 8 This is a three-dimensional structural schematic diagram of another nucleic acid detection microfluidic chip according to an embodiment of the present invention; Figure 9 This is an exploded structural diagram of another nucleic acid detection microfluidic chip according to an embodiment of the present invention.

[0026] Explanation of reference numerals in the attached figures: 1. Sample processing layer; 11. Magnetic bead chamber; 111. First channel; 12. Lysis chamber; 121. Second channel; 13. Washing chamber; 131. First washing chamber; 132. Second washing chamber; 133. Third channel; 134. Fourth channel; 14. Elution chamber; 141. Fifth channel; 142. Sixth channel; 15. Sample splitting chamber; 151. Sample storage chamber; 152. Splitting channel; 153. Waste liquid chamber; 16. Second pump valve chamber; 17. Pump valve; 18. Sealing cap; 181. Cap body; 182. Handle; 183. Partition; 184. Connecting channel; 2. Reaction layer; 21. Reaction chamber; 22. First pump / valve chamber; 23. Gas channel; 231. First gas channel; 232. Second gas channel; 233. Third gas channel; 234. Fourth gas channel; 24. Lyophilized reagent chamber; 25. Transfer channel; 251. Mixing chamber; 3. Electrode layer; 30. Reaction region; 31. Working electrode; 32. Counter electrode; 33. Reference electrode; 34. Electrode lead-out terminal; 4. First sealing layer; 5. Second sealing layer. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] In related technologies, most microfluidic chips still have the following limitations in realizing the "sample in, result out" process for nucleic acid detection. First, many chips are single-function and fail to integrate the front-end nucleic acid extraction step, resulting in a fragmented detection process and cumbersome operation. Second, the fluid drive of many chips relies on complex external fluid control equipment, increasing the complexity and cost of the system and hindering portability. Finally, most chips are designed only for optical detection, limiting their application in detection methods that require electrochemical detection and are more suitable for resource-constrained scenarios.

[0029] The following is combined with Figures 1 to 9 The following describes embodiments of the present invention.

[0030] According to embodiments of the present invention, such as Figures 1 to 9 As shown, a nucleic acid detection microfluidic chip is provided, including a sample processing layer 1, a reaction layer 2, an electrode layer 3, and a first sealing layer 4. The sample processing layer 1 has a plurality of reagent chambers and a sample distribution chamber 15 connected in sequence. The reagent chambers are used to extract nucleic acid from the sample. The sample distribution chamber 15 contains a plurality of sample storage chambers 151 connected in sequence. The reaction layer 2 is located below the sample processing layer 1 and has a plurality of reaction chambers 21, which are connected to the sample storage chambers 151 in a one-to-one correspondence. The reaction layer 2 also has a first pump valve chamber 22 and a gas channel 23. Gas channel 23 connects the first pump valve chamber 22 and the reaction chamber 21. The sample processing layer 1 is provided with a second pump valve chamber 16. The first pump valve chamber 22 and the second pump valve chamber 16 are connected. Pump valves 17 are installed in the first pump valve chamber 22 and the second pump valve chamber 16. Electrode layer 3 is provided below the reaction layer 2. Several reaction areas 30 are provided on the electrode layer 3. The reaction areas 30 are connected to the reaction chamber 21 in a one-to-one correspondence. The first sealing layer 4 is provided above the sample processing layer 1. The first sealing layer 4 is provided with an opening. The opening is located above the other reagent chambers except those adjacent to the sample diversion chamber 15.

[0031] Specifically, in this embodiment, sample processing layer 1 is used for nucleic acid extraction and uniform distribution of samples, reaction layer 2 is used for the "one-pot" reaction (amplification, transcription and detection) of the target to be tested, and electrode layer 3 is used for electrochemical signal output.

[0032] like Figures 1 to 4As shown, the shape of the sample processing layer 1 is not specifically limited in this embodiment. For example, the projection of the sample processing layer 1 in this embodiment is rectangular. The top surface of the sample processing layer 1 is recessed inward and has multiple reagent chambers and a sample diversion chamber 15. In this embodiment, the reagent chambers are connected in sequence. The reagent chamber at the first end is used to add the sample to be processed, and the reagent chamber at the last end is connected to the sample diversion chamber 15. The reagent chamber at the last end is covered with a first sealing layer 4. The remaining reagent chambers and the second pump valve chamber 16 are open. After the nucleic acid is extracted in the reagent chamber, the sample enters the sample diversion chamber 15.

[0033] In this embodiment, as Figure 3 and Figure 4 As shown, the sample diversion cavity 15 contains multiple sample storage cavities 151, with the bottom of each sample storage cavity 151 penetrating the sample processing layer 1. The number of sample storage cavities 151 corresponds to the expected number of targets to be detected and can be increased or decreased according to actual needs.

[0034] In this embodiment, as Figure 1 , Figure 2 and Figure 6 As shown, the reaction layer 2 is located below the sample processing layer 1. Multiple reaction chambers 21 are provided on the reaction layer 2, the number of which is the same as the number of sample storage chambers 151, and their positions correspond one-to-one. The top of each reaction chamber 21 is connected to the bottom of the sample storage chamber 151. The reaction layer 2 also has a first pump valve chamber 22, which is connected to the reaction chambers 21 via multiple gas channels 23. The sample processing layer 1 has a second pump valve chamber 16, which is connected to the first pump valve chamber 22. Pump valves 17 are installed in both the first and second pump valve chambers 22 and 16. Driven by the pump valves 17, the sample is transferred from the sample storage chamber 151 to the reaction chamber 21. In this embodiment, the pump valve 17 is not specifically limited; for example, it can be a rubber cap, silicone cap, etc. The pump valve 17 is initially in a compressed state. When in use, the pump valve 17 rebounds, and the air in the gas channel 23 flows back towards the pump valve 17, making the reaction chamber 21 negative pressure, thereby transferring the sample from the sample storage chamber 151 to the reaction chamber 21.

[0035] like Figure 3 As shown, the number of sample distribution chambers 15 is not specifically limited in this embodiment. For example, this embodiment sets 8 sample distribution chambers 15, which can evenly divide the sample into 8 portions, and the liquid volume of each sample storage chamber 151 is 30 μL. Figure 2 and Figure 6 As shown, there are 8 sample storage chambers 151 and 8 reaction chambers 21 in the reaction layer 2. The 8 sample storage chambers 151 and the 8 reaction chambers 21 are vertically corresponding to each other.

[0036] In this embodiment, the reaction chamber 21 extends through the reaction layer 2. For example... Figure 2 and Figure 7 As shown, the reaction region 30 of the electrode layer 3 is located at the bottom of the reaction chamber 21 and is used for biochemical reactions and detection of electrochemical signals after the reaction.

[0037] In this embodiment, as Figure 2 As shown, the first sealing layer 4 is disposed above the sample processing layer 1, and the first sealing layer 4 is made of a flexible membrane. In this embodiment, the material of the first sealing layer 4 is not specifically limited; for example, PE, PDMS, TPU, etc., can be used. After nucleic acid extraction is completed, the sample can be transferred into the sample splitting chamber 15 by pressing the first sealing layer 4 above the reagent chamber adjacent to the sample splitting chamber 15, causing the air in the reagent chamber to be squeezed.

[0038] In this embodiment, as Figure 6 As shown, four sets of detection branches are distributed on both sides of the reaction layer 2, with the gas channel 23 as the boundary. The paths and shapes of the gas channels 23 in the detection branches are mirror images of each other with the gas channel 23 as the axis of symmetry. After the pump valve 17, which is in the pressed state, is released, the sample in the sample storage chamber 151 will be transferred to the reaction chamber 21 under the action of gas driving force. Corresponding to the eight reaction chambers 21, eight electrodes are provided on the electrode layer 3. The electrodes can be gold electrodes, carbon electrodes, glassy carbon electrodes, etc. The reaction areas 30 of the electrodes correspond vertically to the reaction chambers 21 one by one. After the solution transferred to the reaction chamber 21 reacts, the electrochemical signal is detected by the electrode layer 3.

[0039] In this invention, after the sample is processed in the reagent chamber, it is driven into the sample distribution chamber 15 by squeezing the first sealing layer 4. Under capillary action, the sample uniformly and synchronously fills each sample storage chamber 151. Driven by the pump valve 17, the sample is synchronously transferred to the reaction chamber 21, ensuring that the liquid volume distributed to each reaction chamber 21 is consistent. This not only ensures that the start time of all parallel reactions is completely consistent, improving the comparability and accuracy of the data, but also eliminates cross-contamination between chambers due to asynchronous liquid flow from a physical structure perspective. The design of multiple independent reaction chambers 21 allows for multiple detections of a single sample, with results mutually corroborating each other or covering multiple targets, greatly improving the throughput and reliability of the detection.

[0040] In one embodiment, such as Figure 4 As shown, the upper dimension of the sample storage cavity 151 is larger than the lower dimension.

[0041] Specifically, in this embodiment, the sample storage cavity 151 is designed as a conical structure with a larger top and a smaller bottom. The longitudinal cross-section of each sample storage cavity 151 is trapezoidal, and its width decreases uniformly from one end to the other, allowing the liquid to quickly fill the sample storage cavity 151 through capillary force and Laplace pressure difference. The surface interface of the sample storage cavity 151 is hydrophilic. The channel surface can be modified by plasma treatment, hydrophilic polymer coating (such as PEG, PVA), silanization, or chemical vapor deposition to form a hydrophilic gradient distribution, increasing the hydrophilicity from the large opening at the top to the small opening at the bottom of the sample storage cavity 151, ensuring that the liquid can fill the sample storage cavity 151 more quickly.

[0042] In this embodiment, since the bottom diameter of the sample storage chamber 151 is small, it can generate an upward capillary force, so the liquid has the ability to be retained, thereby ensuring that the sample is evenly distributed in each sample storage chamber 151 and is synchronously and equally transferred to the freeze-drying reaction chamber 21 by the pump valve 17.

[0043] In this embodiment, the bottom opening diameter D2 of the sample temporary storage cavity 151 is subject to the following constraints:

[0044] Where h is the height of the sample storage cavity 151, ρ is the liquid density, γ is the surface tension, θ is the contact angle, and g represents the gravitational acceleration.

[0045] The present invention sets the sample storage chamber 151 into a conical structure with a larger top and a smaller bottom. The longitudinal cross-section of each storage chamber is trapezoidal, and its width decreases uniformly from top to bottom, so that liquid can quickly fill the sample storage chamber 151 through capillary force and Laplace pressure difference.

[0046] In one embodiment, such as Figure 3 As shown, the sample diversion cavity 15 also includes a diversion channel 152, and several sample temporary storage cavities 151 are arranged in a straight line. The diversion channel 152 connects adjacent sample temporary storage cavities 151.

[0047] Specifically, in this embodiment, the arrangement of the sample storage cavity 151 is consistent with the arrangement of the reagent cavity and parallel to the long side of the sample processing layer 1.

[0048] In this embodiment, the number of reaction chambers 21 corresponds to the number of detection targets and can be increased or decreased according to actual conditions. Multiple reaction chambers 21 are arranged in a straight line, consistent with the arrangement direction of the sample storage chambers 151.

[0049] Because the straight-line distance between two points is the shortest, the sample storage cavities 151 are arranged in a straight line, which allows the sample to be quickly diverted to each storage cavity.

[0050] In one embodiment, such as Figure 6As shown, the reaction layer 2 is provided with a lyophilized reagent chamber 24 and a transfer channel 25. The number of lyophilized reagent chambers 24 is the same as that of the reaction chambers 21. The transfer channel 25 connects the reaction chambers 21 and the lyophilized reagent chambers 24. The lyophilized reagent chambers 24 are connected to the sample storage chambers 151 in a one-to-one correspondence. The starting end of the transfer channel 25 is located at the bottom of the lyophilized reagent chambers 24.

[0051] Specifically, in this embodiment, the reaction layer 2 is provided with multiple lyophilized reagent chambers 24 and multiple transfer channels 25. The lyophilized reagent chambers 24 and the reaction chambers 21 are connected through the transfer channels 25. The number of lyophilized reagent chambers 24 corresponds to the number of detection targets and can be increased or decreased according to actual needs. The multiple lyophilized reagent chambers 24 are arranged in a straight line, parallel to the long side of the reaction layer 2. Each reaction chamber 21 corresponds one-to-one with a lyophilized reagent chamber 24. The lyophilized reagent chambers 24 are vertically aligned with the small opening at the bottom of the sample storage chamber 151. Driven by the pump valve 17, the sample is transferred from the sample storage chamber 151 to the lyophilized reagent chamber 24 and mixed with the pre-loaded reagents.

[0052] In this embodiment, the reagents pre-loaded in the lyophilized reagent chamber 24 are not specifically limited. For example, the pre-loaded reagents in this embodiment may be one-pot reagents for amplification-transcription-CRISPR detection or reaction reagents involving other detection methods.

[0053] In this embodiment, the freeze-drying reagent chamber 24 penetrates the reaction layer 2, the starting end of the transfer channel 25 is located at the bottom of the freeze-drying reagent chamber 24, extends in an arc shape to the upper surface of the reaction layer 2, and the end is connected to the reaction chamber 21.

[0054] The present invention sets the starting end of the transfer channel 25 at the bottom of the freeze-drying reagent chamber 24, so that all the liquid in the transfer channel 25 can be transferred to the reaction chamber 21.

[0055] In one embodiment, such as Figure 6 As shown, at least one mixing chamber 251 is provided in the transfer channel 25.

[0056] Specifically, in this embodiment, corresponding to the eight sample storage chambers 151, the reaction layer 2 is provided with eight lyophilized reagent chambers 24 and eight transfer channels 25. The eight sample storage chambers 151 and eight lyophilized reagent chambers 24 are vertically aligned. Two hexagonal mixing chambers 251 are provided within the eight transfer channels 25. The lyophilized reagent chambers 24 are pre-encapsulated with dry powder of the RPA-transcription-CRISPR / Cas13a homogeneous electrochemical one-pot detection system.

[0057] In this embodiment, at least one mixing chamber 251 is provided in the portion of the transfer channel 25 located on the upper surface of the reaction layer 2. The mixing chamber 251 can be polygonal, circular, elliptical, etc., and is used to thoroughly mix the mixture of sample and lyophilized reagent during the transfer process.

[0058] In this embodiment, after the pump valve 17 is released, the sample in the sample storage chamber 151 is transferred from the small opening of the sample storage chamber 151 to the lyophilization reagent chamber 24 under the action of gas driving force, and mixed with the pre-placed one-pot lyophilization reagent. It continues to be transferred and mixed through the mixing chamber 251 in the transfer channel 25, and finally transferred to the reaction chamber 21. Since the starting end of the transfer channel 25 is on the lower surface of the reaction layer 2, i.e., the bottom of the lyophilization reagent chamber 24, all the reagent can be transferred. Furthermore, since the end of the transfer channel 25 is on the upper surface of the reaction chamber 21, the transferred reagent will not be transferred again through the gas channel 23.

[0059] The present invention provides a mixing chamber 251 to thoroughly mix the sample and lyophilized reagent mixture during the transfer process, thereby improving the detection effect.

[0060] In one embodiment, such as Figure 6 As shown, the gas passage 23 is equidistant from the first pump valve chamber 22 to each reaction chamber 21.

[0061] Specifically, in this embodiment, the first pump valve chamber 22 is located in the middle of the reaction layer 2. The first pump valve chamber 22 is connected to various branch gas channels through gas channels 23. The branch gas channels include a first gas channel 231, a second gas channel 232, a third gas channel 233, and a fourth gas channel 234. Each branch gas channel is connected to a reaction chamber 21 at its end. The branch gas channels are distributed mirror images on both sides with gas channel 23 as the dividing line. Furthermore, the distance from the first pump valve chamber 22 to each gas channel 23 is consistent, ensuring that the driving force on the liquid in each parallel path is consistent.

[0062] Because the path length of the gas channel 23 from the first pump valve chamber 22 to the reaction chamber 21 is consistent, the present invention can provide the same driving force to each group of reaction chambers 21, so that the liquid transfer time is consistent.

[0063] In one embodiment, such as Figure 3 As shown, the reagent chamber includes a magnetic bead chamber 11, a lysis chamber 12, a washing chamber 13, and an elution chamber 14 connected in sequence. The bottom of the elution chamber 14 is connected to the sample splitting chamber 15. A waste liquid chamber 153 is provided in the sample splitting chamber 15. The waste liquid chamber 153 is connected to the sample temporary storage chambers 151 located at both ends.

[0064] Specifically, the reagent chamber includes a magnetic bead chamber 11, a lysis chamber 12, a washing chamber 13, and an elution chamber 14. The washing chamber 13 includes a first washing chamber 131 connected to the lysis chamber 12, and a second washing chamber 132 connected to the elution chamber 14, with the second washing chamber 132 connected to the first washing chamber 131. The elution chamber 14 extends through the sample processing layer 1. An opening is provided above the lysis chamber 12 and the washing chamber 13 corresponding to the first sealing layer 4.

[0065] In this embodiment, as Figures 2 to 4 As shown, the magnetic bead chamber 11 and the pyrolysis chamber 12 are connected through a first channel 111; the pyrolysis chamber 12 and the first cleaning chamber 131 are connected through a second channel 121; the first cleaning chamber 131 and the second cleaning chamber 132 are connected through a third channel 133; and the second cleaning chamber 132 and the elution chamber 14 are connected through a fourth channel 134. The first channel 111, the second channel 121, the third channel 133, and the fourth channel 134 are arranged in a straight line for magnetic bead transfer.

[0066] In this embodiment, the magnetic bead chamber 11 is pre-loaded with magnetic beads used for nucleic acid extraction; the lysis chamber 12 is pre-loaded with lysis buffer for lysing pathogens to release nucleic acids, and also serves as a sample loading chamber for adding samples; the first washing chamber 131 and the second washing chamber 132 are pre-loaded with washing buffer for washing the nucleic acid-enriched magnetic beads; and the elution chamber 14 is pre-loaded with elution buffer for eluting nucleic acids from the magnetic beads. The reagents pre-loaded in these chambers include, but are not limited to, the above-mentioned reagents. The number of chambers is not specifically limited; chambers can be added or removed if necessary.

[0067] In this embodiment, the magnetic beads can be superparamagnetic particles made of iron oxide such as magnetite, with their surface modified with active groups such as carboxyl groups. Under high-salt, low-pH conditions, the magnetic bead surface becomes positively charged and binds to negatively charged nucleic acids through electrostatic attraction and hydrogen bonds. An external magnetic field can cause the nucleic acid-bound magnetic beads to aggregate, achieving the separation of nucleic acids from impurities. During elution, the low-salt, high-pH buffer solution disrupts the electrostatic and hydrogen bond interactions, causing the nucleic acids to desorb from the magnetic beads.

[0068] In this embodiment, after the sample is added to the lysis chamber 12, a magnet is used to guide the magnetic beads in the magnetic bead chamber 11 through the first channel 111, the second channel 121, the third channel 133, and the fourth channel 134 into the elution chamber 14 to complete the nucleic acid extraction and purification process. Finally, nucleic acid elution is performed to obtain the nucleic acid of the pathogen. After the nucleic acid extraction is completed, the eluent in the elution chamber 14 can be transferred to the sample splitting chamber 15 through the fifth channel 141 by pressing the first sealing layer 4 above the elution chamber 14.

[0069] In this embodiment, as Figure 4As shown, the elution chamber 14 and the sample splitting chamber 15 are connected by a fifth channel 141. The fifth channel 141 is used to transfer the sample eluent from the elution chamber 14 to the sample splitting chamber 15. The starting end of the fifth channel 141 is at the bottom of the elution chamber 14, and it connects the lower surface of the elution chamber 14 and the upper surface of the sample splitting chamber 15 through a sixth channel 142 that vertically penetrates the sample processing layer 1. The fifth channel 141 is used to transfer the sample eluent from the elution chamber 14 to the sample splitting chamber 15. The vertically arranged sixth channel 142 facilitates the complete delivery of the sample from the bottom of the elution chamber 14 to the sample splitting chamber 15.

[0070] In this embodiment, after nucleic acid extraction is completed, the first sealing layer 4 above the reagent chamber elution chamber 14 adjacent to the sample splitting chamber 15 can be pressed to squeeze the air in the reagent chamber 14 and drive the eluent in the elution chamber 14 to be transferred to the sample splitting chamber 15 through the sixth channel 142 and the fifth channel 141.

[0071] In this embodiment, a waste liquid chamber 153 is also provided in the sample diversion chamber 15. The two ends of the waste liquid chamber 153 and the diversion channel 152 are connected, and excess sample can flow into the waste liquid chamber 153 through the diversion channel 152.

[0072] In this embodiment, the waste liquid chamber 153 is pre-filled with absorbent material. The absorbent material is not specifically limited in this embodiment; for example, a sponge, absorbent pad, or absorbent paper can be used. The extracted nucleic acid sample is dispersed into each sample storage chamber 151 through the capillary action of the diversion channel 152. Excess sample is absorbed by the absorbent material in the waste liquid chamber 153 through both ends of the diversion channel 152, ensuring that each sample storage chamber 151 is filled with sample.

[0073] The present invention connects the bottom of the elution chamber 14 to the sample distribution chamber 15, which facilitates the transfer of all samples in the elution chamber 14 to the sample distribution chamber 15; a waste liquid chamber 153 is provided so that excess samples can flow into the waste liquid chamber 153 for collection through the distribution channel 152, ensuring that each sample storage chamber 151 can be filled with samples; an absorbent pad is pre-installed in the waste liquid chamber 153 to absorb excess samples, ensuring that there are basically no sample residues in the distribution channel 152.

[0074] In one embodiment, such as Figure 1 and Figure 2 As shown, the pyrolysis chamber 12 and the cleaning chamber 13 are provided with sealing covers 18. The sealing covers 18 have a communicating state that connects adjacent chambers and a blocking state that isolates adjacent chambers.

[0075] Specifically, in this embodiment, the lysis chamber 12, the first washing chamber 131, and the second washing chamber 132 are each provided with a sealing cap 18. The sealing cap 18 is used to seal the reagents pre-filled in the chambers to prevent leakage or contamination, and also to prevent the reagents in the lysis chamber 12, the first washing chamber 131, the second washing chamber 132, and the elution chamber 14 from transferring to each other through the first channel 111, the second channel 121, the third channel 133, and the fourth channel 134 during transportation.

[0076] For example, during transportation, when several chambers do not need to be connected, the sealing cover 18 is adjusted to an isolation state, preventing communication between the chambers. When magnetic beads need to be transferred between chambers, the sealing cover 18 is adjusted to a communication state, allowing communication between the chambers.

[0077] The present invention provides a sealing cap 18 to seal the reagents pre-filled in the chamber to prevent leakage or contamination, and also to prevent the transfer of reagents between adjacent chambers during transportation.

[0078] In one embodiment, such as Figure 5 As shown, the sealing cover 18 includes a cover body 181, a handle 182, and a partition 183. The cover body 181 is movably disposed in the pyrolysis chamber 12 and the cleaning chamber 13. The handle 182 is disposed on the top of the cover body 181. The two partitions 183 are disposed parallel to each other at the bottom of the cover body 181. A connecting channel 184 is formed between the two partitions 183. The connecting channel 184 has a communicating state that connects adjacent chambers and a blocking state that isolates adjacent chambers.

[0079] Specifically, such as Figures 1 to 5 As shown, in this embodiment, the shapes of the magnetic bead cavity 11, the first cleaning chamber 131 and the second cleaning chamber 132 of the lysis chamber 12 are not specifically limited. For example, in this embodiment, the magnetic bead cavity 11, the first cleaning chamber 131 and the second cleaning chamber 132 of the lysis chamber 12 and the elution chamber 14 are circular. The diameter of the cover 181 matches the diameter of the lysis chamber 12, the first cleaning chamber 131 and the second cleaning chamber 132. A handle 182 is provided on the top of the cover 181 for rotating the sealing cover 18 and for easy opening when adding samples to the lysis chamber 12.

[0080] In this embodiment, as Figure 5As shown, the bottom of the cover 181 has two partitions 183, which are parallel to the handle 182. A connecting channel 184 is formed between the two partitions 183. The width of the connecting channel 184 is the same as the width of the first channel 111 to the fifth channel 141. When adjacent chambers do not need to be connected, the handle 182 can be rotated so that the handle 182 is not parallel to the long side of the sample processing layer 1. At this time, the connecting channel 184 between the partitions 183 is not on the same straight line as the first channel 111, the second channel 121, the third channel 133, or the fourth channel 134, so the adjacent chambers cannot be connected. When magnetic beads need to be transferred between chambers, the handle 182 is rotated so that it is parallel to the long side of the sample processing layer 1. At this time, the connecting channel 184 between the partitions 183 is on the same straight line as the first channel 111, the second channel 121, the third channel 133, and the fourth channel 134, so the chambers can be connected to each other.

[0081] In some other embodiments, the magnetic bead cavity 11, the pyrolysis cavity 12, the first cleaning cavity 131, the second cleaning cavity 132, and the elution cavity 14 may also be square in shape, and the cover 181 is correspondingly square. When adjusting the state of the sealing cover 18, the state can be adjusted by removing the sealing cover 18, turning it 90°, and then installing it into the corresponding cavity.

[0082] The present invention provides a handle 182 on the sealing cover 18 to facilitate the user's rotation operation, and the partition 183 at the bottom of the cover can be used to prevent the transfer of reagents between adjacent chambers during transportation.

[0083] In one embodiment, such as Figure 2 As shown, the nucleic acid detection microfluidic chip also includes a second sealing layer 5, which is disposed between the sample processing layer 1 and the reaction layer 2. The second sealing layer 5 is provided with a first opening that connects the reaction chamber 21 and the sample storage chamber 151, and a second opening that connects the first pump valve chamber 22 and the second pump valve chamber 16.

[0084] Specifically, in this embodiment, the second sealing layer 5 is disposed between the sample processing layer 1 and the reaction layer 2, and is a non-flexible membrane, the material of which can be PC, PMMA, etc. The second sealing layer 5 is provided with a first opening connecting the reaction chamber 21 and the sample storage chamber 151, and a second opening connecting the first pump valve chamber 22 and the second pump valve chamber 16 at the corresponding position.

[0085] In this embodiment, when the reaction layer 2 is provided with a lyophilized reagent chamber 24, the first opening connects the lyophilized reagent chamber 24 with the sample storage chamber 151.

[0086] In this embodiment, as Figure 7As shown, electrode layer 3 is located below reaction layer 2, and multiple reaction regions 30 are provided on electrode layer 3. Each reaction region 30 corresponds vertically to a reaction chamber 21. The reaction region 30 includes a three-electrode system: working electrode 31, counter electrode 32, and reference electrode 33.

[0087] In this embodiment, the material of the working electrode 31 is not specifically limited. For example, the material of the working electrode 31 can be carbon, gold, silver, or platinum. The material of the counter electrode 32 is not specifically limited. For example, the counter electrode 32 can be carbon, gold, silver, or platinum. The material of the reference electrode 33 is not specifically limited. For example, the reference electrode 33 can be silver / silver chloride. The working electrode 31, counter electrode 32, and reference electrode 33 each correspond to an electrode lead 34, and the electrode lead 34 can be made of silver.

[0088] More specifically, in this embodiment, the working electrode 31 is made of gold, the counter electrode 32 is made of carbon, the reference electrode 33 is made of silver / silver chloride, and the electrode lead 34 is made of silver.

[0089] The nucleic acid detection process of the nucleic acid detection microfluidic chip provided in this invention is as follows: When in use, open the sealing cover 18 of the lysis chamber 12, place the collected saliva, serum, nasopharyngeal swab preservation solution and other samples into the lysis chamber 12, close the sealing cover 18 and rotate all the sealing covers 18 so that the sealing covers 18 are in a connected state.

[0090] A magnet is placed above the magnetic bead cavity 11 and moved along the first channel 111 to transfer the magnetic beads into the lysis cavity 12. The magnet is then removed to release the magnetic beads. After the reaction is complete, the magnet is placed above the lysis cavity 12 again and moved along the second channel 121 to transfer the magnetic beads into the first washing cavity 131. The magnet is then removed to release the magnetic beads. After the reaction is complete, the magnetic beads are transferred to the second washing cavity 132 along the same trajectory for a second washing. After washing, the beads are transferred to the elution cavity 14, and the magnet is removed to complete nucleic acid elution. After the reaction is complete, the magnetic beads are transferred back to the second washing cavity 132 using the magnet. A mechanical rod is used to press the first sealing layer 4 above the elution cavity 14 to transfer the eluent along the sixth channel 142 and the fifth channel 141 into the sample splitting cavity 15, thus completing the uniform distribution of the sample.

[0091] After the distribution is completed, the pump valve 17, which is in the pressed state, is released to complete the mixing and homogenization of the sample and the pre-filled one-pot dry powder reagent in the lyophilized reagent chamber 24, and then the sample is transferred to the reaction chamber 21 for one-pot reaction. After the reaction is completed, electrochemical signal detection is performed to obtain the peak current values ​​of 8 channels to determine whether the target to be tested exists in the sample.

[0092] The nucleic acid detection microfluidic chip in this invention, through the design of sample shunt chamber 15 and synchronous press pump valve 17, can solve the problem of sample equalization and synchronous start-up in multi-channel microfluidic detection. Combined with integrated electrochemical detection electrode and one-pot lyophilized reagent, it realizes fully integrated, high-throughput, and highly reliable on-site rapid detection of multiple pathogens.

[0093] This invention integrates nucleic acid extraction and multiplex detection by placing the sample reaction layer 2 and the processing layer in the same chip. Users can complete the detection by simply adding the original sample and performing a simple operation, which greatly reduces the professional skill requirements for operators.

[0094] The conical sample storage chamber 151 is designed to allow liquid to fill it uniformly and synchronously under capillary action and Laplace pressure differential, ensuring a high degree of consistency in the liquid volume distributed to each reaction chamber 21. Synchronous transfer of liquid within all channels is achieved by mechanically squeezing the first sealing layer 4. This not only guarantees that the start times of all parallel reactions are completely consistent, improving data comparability and accuracy, but also physically eliminates cross-contamination between chambers caused by asynchronous liquid flow.

[0095] The setup of multiple independent detection channels enables multiple detections of a single sample, with results corroborating each other or covering multiple targets, greatly improving the throughput and reliability of the detection.

[0096] This invention is not only applicable to "one-pot" nucleic acid detection, but its core fluid control and distribution structure is also applicable to other biochemical analysis processes that require precise distribution and synchronous reaction, providing a universal technical platform for the development of various multiplex detection products.

[0097] In another embodiment, such as Figure 8 and Figure 9 As shown, there are two sample splitting chambers 15. Each sample splitting chamber 15 is equidistant from the edge of the long side of the sample processing layer 1, and each sample splitting chamber 15 includes a set of eight sample storage chambers 151. Below each set of sample storage chambers 151 are a set of eight lyophilized reagent chambers 24, and each sample storage chamber 151 and lyophilized reagent chamber 24 are vertically and one-to-one corresponding. The elution chamber 14 is equidistant from the two sample splitting chambers 15, so that the nucleic acid extract can be evenly distributed into the two sample splitting chambers 15. The two sets of lyophilized reagent chambers 24 correspond one-to-one with a set of transfer channels 25 and a reaction chamber 21, and each transfer channel 25 is provided with two mixing chambers 251. The two sets of eight reaction chambers 21 are vertically corresponding to the reaction areas 30 in the two sets of electrode layers 3. The sample processing layer 1 is provided with two second pump valve chambers 16, each with two pre-installed pump valves 17.

[0098] The nucleic acid detection process of the microfluidic chip in this embodiment is the same as that in the above embodiment. The difference is that the two pump valves 17 need to be activated simultaneously to detect 16 targets at the same time. The rest of the process will not be described here.

[0099] In some other embodiments, the top surface of the sample processing layer 1 may also be square, and the number of sample splitting chambers 15 is set to four. Each sample splitting chamber 15 is equidistant from the edge of the sample processing layer 1, and the elution chamber 14 is located in the center of the sample processing layer 1, so that the distance from the elution chamber 14 to the four sample splitting chambers 15 is equidistant, and the eluent in the elution chamber 14 can be evenly distributed to the four sample splitting chambers 15. Each sample splitting chamber 15 includes a set of eight sample storage chambers 151, and each set of sample storage chambers 151 corresponds to a set of eight lyophilized reagent chambers 24 below it. Similarly, each sample storage chamber 151 and the lyophilized reagent chamber 24 are vertically corresponding one-to-one. The four sets of lyophilized reagent chambers 24 correspond one-to-one to a set of transfer channels 25 and reaction chambers 21, and each transfer channel 25 is provided with two mixing chambers 251. The four sets of eight reaction chambers 21 are vertically corresponding to the reaction regions 30 in the four sets of electrode layers 3. The sample processing layer 1 is provided with four second pump valve chambers 16, and four pump valves 17 are pre-installed in each chamber. This setting can further increase the number of simultaneous detections.

[0100] The nucleic acid detection process of the microfluidic chip in this embodiment is the same as that in the above embodiment. The difference is that the four pump valves 17 need to be activated simultaneously to detect 32 targets at the same time. The rest of the process will not be described in detail here.

[0101] Although embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations all fall within the scope defined by the appended claims.

Claims

1. A microfluidic chip for nucleic acid detection, characterized in that, include: The sample processing layer (1) is provided with a plurality of reagent chambers and sample diversion chambers (15) connected in sequence. The reagent chambers are used to extract nucleic acid from the sample. The sample diversion chambers (15) are provided with a plurality of sample storage chambers (151) connected in sequence. A reaction layer (2) is disposed below the sample processing layer (1). The reaction layer (2) is provided with a plurality of reaction chambers (21). The reaction chambers (21) are connected to the sample storage chambers (151) one by one. The reaction layer (2) is provided with a first pump valve chamber (22) and a gas channel (23). The gas channel (23) connects the first pump valve chamber (22) and the reaction chambers (21). The sample processing layer (1) is provided with a second pump valve chamber (16). The first pump valve chamber (22) and the second pump valve chamber (16) are connected. Pump valves (17) are installed in the first pump valve chamber (22) and the second pump valve chamber (16). An electrode layer (3) is disposed below the reaction layer (2). A plurality of reaction regions (30) are disposed on the electrode layer (3). The reaction regions (30) are connected to the reaction chamber (21) one by one. A first sealing layer (4) is disposed above the sample processing layer (1). An opening is provided on the first sealing layer (4), and the opening is located above the remaining reagent chambers excluding those adjacent to the sample diversion chamber (15).

2. The nucleic acid detection microfluidic chip according to claim 1, characterized in that, The upper dimension of the sample storage cavity (151) is larger than the lower dimension.

3. The nucleic acid detection microfluidic chip according to claim 1 or 2, characterized in that, The sample shunt cavity (15) further includes: The diversion channel (152) and several sample storage cavities (151) are arranged in a straight line, and the diversion channel (152) connects adjacent sample storage cavities (151).

4. The nucleic acid detection microfluidic chip according to claim 1, characterized in that, The reaction layer (2) is provided with a lyophilized reagent chamber (24) and a transfer channel (25). The number of lyophilized reagent chambers (24) is the same as that of the reaction chambers (21). The transfer channel (25) connects the reaction chambers (21) and the lyophilized reagent chambers (24). The lyophilized reagent chambers (24) and the sample storage chambers (151) are connected in a one-to-one correspondence. The starting end of the transfer channel (25) is located at the bottom of the lyophilized reagent chambers (24).

5. The nucleic acid detection microfluidic chip according to claim 4, characterized in that, At least one mixing chamber (251) is provided in the transfer channel (25).

6. The nucleic acid detection microfluidic chip according to claim 1, characterized in that, The gas passage (23) is equidistant from the first pump valve chamber (22) to each of the reaction chambers (21).

7. The nucleic acid detection microfluidic chip according to claim 1, characterized in that, The reagent chamber includes a magnetic bead chamber (11), a lysis chamber (12), a washing chamber (13), and an elution chamber (14) connected in sequence. The bottom of the elution chamber (14) is connected to the sample splitting chamber (15). A waste liquid chamber (153) is provided in the sample splitting chamber (15). The waste liquid chamber (153) is connected to the sample temporary storage chamber (151) located at both ends.

8. The nucleic acid detection microfluidic chip according to claim 7, characterized in that, The pyrolysis chamber (12) and the cleaning chamber (13) are provided with sealing covers (18), which have a communicating state that connects adjacent chambers and a blocking state that isolates adjacent chambers.

9. The nucleic acid detection microfluidic chip according to claim 8, characterized in that, The sealing cap (18) includes: A cover (181) is movably disposed in the pyrolysis chamber (12) and the cleaning chamber (13); A handle (182) is disposed on the top of the cover (181); Partition (183), two partitions (183) are arranged in parallel at the bottom of the cover (181), and a connecting channel (184) is formed between the two partitions (183). The connecting channel (184) has a connected state that connects adjacent chambers and a blocking state that separates adjacent chambers.

10. The nucleic acid detection microfluidic chip according to claim 1, characterized in that, Also includes: The second sealing layer (5) is disposed between the sample processing layer (1) and the reaction layer (2). The second sealing layer (5) is provided with a first opening that connects the reaction chamber (21) and the sample storage chamber (151), and a second opening that connects the first pump valve chamber (22) and the second pump valve chamber (16).

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