Portable nucleic acid detection micro-fluidic chip, nucleic acid detection device and method

Abstract

The invention discloses a portable nucleic acid detection micro-fluidic chip, a nucleic acid detection device and a nucleic acid detection method, and belongs to the field of micro-fluidic chips, a detection module comprises a quantitative chamber, a micro-channel, a reaction chamber, a first connecting channel and a color developing module; the reaction chamber comprises an RPA reaction chamber in which an RPA amplification reagent is pre-embedded, a porous filter membrane and a CRISPR reaction chamber in which a CRISPR detection component is pre-embedded, and the RPA reaction chamber, the porous filter membrane and the CRISPR reaction chamber are sequentially positioned at the same position of the three adjacent chip layers; the aperture of the porous filter membrane is larger than the particle size of an RPA amplification product and smaller than the particle size of a core enzyme in a CRISPR detection component; two ends of the micro-channel are respectively communicated with the quantitative chamber and the RPA reaction chamber; two ends of the first connecting channel are respectively communicated with the CRISPR reaction chamber and the color developing module; the flow resistance of the first connecting channel is smaller than that of the micro-channel. According to the invention, the sequential reaction of RPA amplification and CRISPR cleavage can be completed without depending on artificial transfer of amplification products.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic chips, and more specifically, relates to a portable nucleic acid detection microfluidic chip, a nucleic acid detection device and a method. Background Art

[0002] With the rise of public health awareness, the demand for home molecular diagnostics continues to grow. Nucleic acid testing, particularly for screening for infections like human papillomavirus (HPV) and respiratory viruses, has become the preferred choice due to its high sensitivity and specificity. However, traditional qPCR technology has high requirements for instrumentation, operation, and environment, making it difficult to meet the "simple, low-cost, and instrument-free" needs of home users.

[0003] In recent years, the CRISPR-Cas system has demonstrated great potential for rapid on-site detection. Its high specificity stems from its combination of crRNA-mediated target recognition and Cas protein-mediated reporter probe cleavage. However, direct nucleic acid detection using the CRISPR system remains limited in sensitivity and often requires combination with isothermal amplification techniques such as recombinase polymerase amplification (RPA) to enhance the signal.

[0004] Most current RPA-CRISPR integration platforms are laboratory-grade solutions, which present the following challenges:

[0005] (1) The amplification and identification steps require multiple manual transfers, which can easily lead to contamination or operational errors;

[0006] (2) If the RPA product is co-located with the CRISPR system, premature shearing may lead to template depletion and inhibit the amplification reaction;

[0007] (3) Most of them use electronically controlled pumps / valves or complex structures, which are not suitable for resource-constrained scenarios and non-professional users.

[0008] To this end, the development of a portable microfluidic chip that combines sequential reaction control, high structural integration, and user-friendliness has become the key to promoting CRISPR on-site testing to home scenarios. In the patent document with application number 2024119465786, a portable microfluidic chip is proposed. When this chip is used for nucleic acid detection, it is necessary to pre-embed the RPA amplification reagent in the quantitative chamber, and at the same time pre-embed the CRISPR detection component in the reaction chamber. Since the RPA amplification is located in the shared quantitative chamber, the difficulty of designing multiple primers is increased. Moreover, after RPA amplification, it takes a certain amount of time before it is transferred to the CRISPR chamber. The time needs to be strictly controlled, and it is impossible to do a one-step method. In actual operation, the test results may be affected by time control errors. Summary of the Invention

[0009] In response to the defects of the existing technology and the need for improvement, the present invention provides a portable nucleic acid detection microfluidic chip, a nucleic acid detection device and a method, the purpose of which is to complete the temporal reaction of RPA amplification and CRISPR cleavage without relying on manual transfer of RPA amplification reaction products, thereby reducing the difficulty of detection and improving the accuracy of detection in home detection scenarios.

[0010] To achieve the above object, according to one aspect of the present invention, a portable nucleic acid detection microfluidic chip is provided, comprising: a chip body, and M detection modules arranged on the chip body;

[0011] The detection module includes: a quantitative chamber, a microchannel, a reaction chamber, N first connecting channels and N color development modules;

[0012] The quantitative chamber is provided with a sample addition port;

[0013] The reaction chamber includes an RPA reaction chamber, a porous filter membrane, and a CRISPR reaction chamber, which are located on three adjacent chip layers and overlap with each other on the chip layer. The RPA reaction chamber is pre-embedded with RPA amplification reagents; the CRISPR reaction chamber is pre-embedded with CRISPR detection components. The porous filter membrane is located between the RPA reaction chamber and the CRISPR reaction chamber, and its pore size is larger than the particle size of the RPA amplification product but smaller than the particle size of the core enzyme in the CRISPR detection component.

[0014] a microchannel, one end of which is connected to the bottom end of the quantitative chamber and the other end of which is connected to the RPA reaction chamber, and the connection point between the microchannel and the RPA reaction chamber is lower than the bottom end of the quantitative chamber;

[0015] The bottom ends of the N first connecting channels are all connected to the top of the CRISPR reaction chamber, and the top ends of the N first connecting channels are respectively connected to the top ends of the N color development modules; the flow resistance of the first connecting channels is less than the flow resistance of the microchannel;

[0016] The color development module is used to detect the products of the CRISPR cleavage reaction;

[0017] Wherein, M and N are both positive integers greater than or equal to 1.

[0018] In some optional embodiments, the color development module includes: a color development chamber located at the top, a substrate chamber located at the bottom, and a second connecting channel for connecting the color development chamber and the substrate chamber;

[0019] The color development chamber is connected to the top of the first connecting channel;

[0020] The substrate chamber is pre-buried with a colorimetric substrate for detecting the products of the CRISPR cleavage reaction;

[0021] An interception structure is provided in the first connection channel for intercepting unreleased microsphere probes and allowing the enzyme produced by the CRISPR cleavage reaction to pass through.

[0022] In some optional embodiments, the color development module includes a test strip for detecting products of the CRISPR cleavage reaction.

[0023] Furthermore, the color development module further comprises: a detection chamber for accommodating the test strip;

[0024] Water absorbing materials are respectively arranged at both ends of the detection chamber.

[0025] Furthermore, the quantitative chamber, the microchannel and the RPA reaction chamber are located on the same chip layer.

[0026] Furthermore, the quantitative chamber is in a "V" shape, and the vertex of the "V" shape is the bottom end of the quantitative chamber.

[0027] Furthermore, the porous filter membrane is a polycarbonate membrane.

[0028] Furthermore, N>1.

[0029] According to another aspect of the present invention, there is provided a nucleic acid detection device comprising: a base and K portable nucleic acid detection microfluidic chips;

[0030] K slots are provided on the base, and K portable nucleic acid detection microfluidic chips are fixed in the K slots respectively;

[0031] Among them, the portable nucleic acid detection microfluidic chip is the above-mentioned portable nucleic acid detection microfluidic chip provided by the present invention; K is a positive integer greater than or equal to 1.

[0032] According to another aspect of the present invention, a microfluidic detection method for non-medical diagnosis purposes based on the portable nucleic acid detection microfluidic chip is provided, comprising the following steps:

[0033] S1: Place the portable nucleic acid detection microfluidic chip upright, add the sample to be tested into the quantitative chamber of each detection module, and complete the sample quantification;

[0034] S2: Applying a positive force to the portable nucleic acid detection microfluidic chip causes the sample in each quantitative chamber to break through the microchannel and transfer to the RPA reaction chamber, where an RPA amplification reaction occurs. The RPA amplification product then diffuses to the CRISPR reaction chamber, where a CRISPR cleavage reaction occurs.

[0035] S3: After the sample in the chamber to be quantified is completely transferred to the reaction chamber and the CRISPR cleavage reaction is completed, the portable nucleic acid detection microfluidic chip is placed upside down so that the product of the CRISPR cleavage reaction flows to the color development module through the first connecting channel;

[0036] S4: After the preset time, the detection result of the color development module is read to complete the nucleic acid detection.

[0037] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

[0038] (1) The portable nucleic acid detection microfluidic chip provided by the present invention has a layered design for the reaction chamber. Specifically, the reaction chamber is designed as an RPA reaction chamber and a CRISPR reaction chamber located in different chip layers, with a porous filter membrane set between the two. Based on this design, after the sample quantification is completed, the sample can be transferred to the RPA reaction chamber, and the product of the RPA amplification reaction will automatically diffuse through the porous filter membrane into the CRISPR chamber to undergo a CRISPR cleavage reaction. Without the need to manually transfer the RPA amplification reaction product, the sequential reaction of RPA amplification and CRISPR detection can be completed, which effectively reduces the operational difficulty of nucleic acid detection and can improve the accuracy of nucleic acid detection.

[0039] (2) The portable nucleic acid detection microfluidic chip provided by the present invention, in its preferred embodiment, the color development module for detecting the CRISPR cleavage reaction product includes a color development chamber located at the top, a substrate chamber located at the bottom, and a second connecting channel for connecting the color development chamber and the substrate chamber. Based on this structural design, after the CRISPR cleavage reaction is completed, the chip is inverted, so that the product of the CRISPR cleavage reaction and the color development substrate in the substrate chamber can flow into the color development chamber to achieve detection. Compared with the test strip, the length of the color development module based on this structure is greatly shortened, and the entire chip structure is more compact.

[0040] (3) The portable nucleic acid detection microfluidic chip provided by the present invention, in its optional scheme, uses a test strip as a color development module, and in its further preferred scheme, is further provided with a detection chamber for preventing the test strip from transferring. Water-absorbing materials are respectively provided at both ends of the detection chamber, thereby improving the efficiency of transferring the reaction product in the reaction chamber or the enhancement reagent in the enhancement reagent chamber to the test strip.

[0041] (4) The portable nucleic acid detection microfluidic chip provided by the present invention has multiple color development modules set in the same detection module, and correspondingly has multiple first connection channels, so that different detections can be completed in parallel for the same sample, thereby improving detection efficiency.

[0042] (5) In the preferred embodiment of the portable nucleic acid detection microfluidic chip provided by the present invention, the quantitative chamber, microchannel and RPA reaction chamber are located on the same chip layer, which simplifies the layered structure design of the chip while ensuring the detection function.

[0043] (6) In the portable nucleic acid detection microfluidic chip provided by the present invention, in its preferred embodiment, the quantitative chamber is in a "V" shape, and the top of the "V" shape is the bottom of the quantitative chamber, which facilitates the quantification of samples and the transfer of samples in the quantitative chamber to the reaction chamber. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the structure of a portable nucleic acid detection microfluidic chip provided in Example 1 of the present invention;

[0045] Figure 2 A schematic structural diagram of the reaction chamber provided in Example 1 of the present invention;

[0046] Figure 3 Schematic diagram of the layered design of the portable nucleic acid detection microfluidic chip provided in Example 1 of the present invention;

[0047] Figure 4 A schematic diagram of the structure of a portable nucleic acid detection microfluidic chip provided in Example 2 of the present invention;

[0048] Figure 5 Schematic diagram of the layered design of the portable nucleic acid detection microfluidic chip provided in Example 2 of the present invention;

[0049] Figure 6 A schematic diagram of the interception structure provided in Example 2 of the present invention;

[0050] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0051] 1-Chip body;

[0052] 2-Detection module;

[0053] 21-quantification chamber;

[0054] 22-microchannel;

[0055] 23-reaction chamber; 231-RPA reaction chamber, 232-porous filter membrane, 233-CRISPR reaction chamber;

[0056] 24-first connecting channel;

[0057] 25-color development module; 251-test strip; 252-color development chamber, 253-second connecting channel, 254-substrate chamber;

[0058] 26-Interception structure. DETAILED DESCRIPTION

[0059] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0060] Those skilled in the art should understand that these embodiments are merely used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. It should be noted that, in the description of the present invention, the terms "upper," "lower," "left," "right," "inner," "outer," and the like indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is merely for ease of description and does not indicate or imply that the device or element must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, it should not be understood as limiting the present invention. In addition, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they can mean fixed connection, detachable connection, or integral connection; mechanical connection or electrical connection; direct connection, indirect connection through an intermediate medium, or internal communication between two components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances. The terms "first," "second," and the like (if any) in the present invention and the accompanying drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0061] To reduce the difficulty and complexity of fluid manipulation and improve the portability of microfluidic chips, the present invention provides a portable nucleic acid detection microfluidic chip, nucleic acid detection device, and method. By improving the flow channel and chamber structure of the microfluidic chip, the RPA amplification reaction products are automatically transferred by diffusion to the CRISPR reaction chamber for CRISPR cleavage. This allows the sequential reaction of RPA amplification and CRISPR cleavage to be completed without manual transfer of RPA amplification reaction products. This reduces the difficulty of detection and improves detection accuracy in home testing scenarios.

[0062] It is easy to understand that in the present invention, descriptions such as "top," "bottom," "high," and "low" are all relative to the chip in an upright position. In this position, the highest point of each structure is the top of the corresponding structure, and the lowest point of each structure is the bottom of the corresponding structure. "Forward force" and "reverse force" are relative to the top and bottom of the chip. A force from the top of the chip toward the bottom is a forward force, while a force from the bottom of the chip toward the top is a reverse force.

[0063] The following are examples.

[0064] Example 1:

[0065] A portable nucleic acid detection microfluidic chip, such as Figure 1 and Figure 2 As shown, it includes: a chip body 1, and five detection modules 2 arranged on the chip body 1;

[0066] The detection module 2 includes: a quantitative chamber 21, a microchannel 22, a reaction chamber 23, a first connecting channel 24 and a color development module 25;

[0067] The quantitative chamber 1 is provided with a sample addition port; optionally, in this embodiment, the quantitative chamber 1 is in a "V" shape, and the vertex of the "V" shape is the bottom end of the quantitative chamber, which facilitates the quantification of the sample and the transfer of the sample in the quantitative chamber to the reaction chamber;

[0068] like Figure 2 and Figure 3 As shown, the reaction chamber 23 includes an RPA reaction chamber 231, a porous filter membrane 232 and a CRISPR reaction chamber 233, which are located in three adjacent chip layers and overlap with each other on the chip layer; RPA amplification reagents (including primers, enzymes, buffers, etc.) are pre-embedded in the RPA reaction chamber 231; CRISPR detection components (such as Cas12a / crRNA / ssDNA reporter probes) are pre-embedded in the CRISPR reaction chamber 233; the porous filter membrane 232 is located between the RPA reaction chamber 231 and the CRISPR reaction chamber 233, and its pore size is larger than the particle size of the RPA amplification product and smaller than the particle size of the core enzyme in the CRISPR detection component; optionally, in this embodiment, the pore size of the porous filter membrane 232 is 10 to 50 nm, and the porous filter membrane is a polycarbonate membrane, thereby ensuring that the RPA amplification product is allowed to diffuse during the nucleic acid detection process, but prevents the circulation of macromolecules such as proteases, and ensures that the substances in the RPA reaction chamber are not prematurely sheared during amplification;

[0069] The microchannel 22 has one end connected to the bottom end of the quantitative chamber 21 and the other end connected to the RPA reaction chamber 231, and the connection point between the microchannel 22 and the RPA reaction chamber 231 is lower than the bottom end of the quantitative chamber 21;

[0070] The bottom end of the first connecting channel 24 is connected to the top of the CRISPR reaction chamber 233, and the top end of the first connecting channel 24 is connected to the top of the color development module 25; the flow resistance of the first connecting channel 24 is smaller than the flow resistance of the microchannel 22;

[0071] The color development module 25 is used to detect the products of the CRISPR cleavage reaction.

[0072] Optionally, in this embodiment, the color development module 25 includes a test strip 251 for detecting the product of the CRISPR cutting reaction, and in order to improve the efficiency of transferring the reaction product in the reaction chamber or the enhancement reagent in the enhancement reagent chamber to the test strip, in this embodiment, the color development module 25 also includes a detection chamber for accommodating the test strip; water-absorbing materials are respectively provided at both ends of the detection chamber.

[0073] The chip of this embodiment specifically includes multiple chip layers, and an optional layered design scheme is as follows: Figure 2 As shown, from left to right are: the chip bottom cover; the chip layer where the test strips are located; the chip layer where the water-absorbing material, the second connection channel, and the test strip connection hole are located; the chip layer where the CRISPR reaction chamber and the second connection channel are located; the chip layer where the porous filter membrane is located; the chip layer where the quantitative chamber, microchannel, and RPA reaction chamber are located; and the chip top cover. It is easy to understand that the chip is also equipped with air holes that connect each chamber to the atmosphere.

[0074] Based on the setting of the flow resistance relationship between the microchannel and the first connecting channel, in the layered design of this embodiment, the quantitative chamber, microchannel and RPA reaction chamber can be designed in the same chip layer. This design scheme simplifies the layered structure design of the chip while ensuring the detection function.

[0075] In practical applications, the portable nucleic acid detection microfluidic chip provided in this embodiment is used for nucleic acid detection in the following steps:

[0076] (1) Sample loading and quantification: Place the chip upright and add the sample (e.g., lysed sample solution) to the quantitative chamber. The sample is automatically quantified using the V-shaped structure, and the sample remains at the front end of the microchannel.

[0077] (2) Amplification and cleavage reaction: Apply positive force to the chip (such as manually or by gently squeezing the chip with a heating pad) to allow the sample to break through the microchannel and flow into the RPA reaction chamber, where RPA amplification is performed at 37-42°C to generate the target sequence; after the reaction, the amplified product can naturally diffuse through the porous filter membrane to the CRISPR reaction chamber, initiating the CRISPR cleavage reaction; in the CRISPR cleavage reaction, the amplified product and crRNA recognize and activate the Cas12a protein, thereby cleaving the ssDNA reporter probe and generating a color signal pre-reaction;

[0078] (3) Signal output: After the sample in the chamber to be quantified is completely transferred to the reaction chamber and the RPA amplification reaction and CRISPR cleavage reaction are completed, the chip is inverted. The reaction products in the CRISPR reaction chamber enter the detection chamber through the first connecting channel under the action of gravity and interact with the test strip to form a visible or semi-quantitative response;

[0079] (4) Reading the results: After a preset time (e.g., 5 minutes), observe the color development of the test line / quality control line on the test strip to complete the nucleic acid test result reading.

[0080] Based on the above detection steps, it can be seen that when the portable nucleic acid detection microfluidic chip provided in this embodiment is used for nucleic acid detection, the sample can be transferred to the RPA reaction chamber after the sample quantification is completed. The product of the RPA amplification reaction will automatically diffuse through the porous filter membrane into the CRISPR chamber to undergo a CRISPR cleavage reaction. Without the need to manually transfer the RPA amplification reaction product, the sequential reaction of RPA amplification and CRISPR detection can be completed, which effectively reduces the operational difficulty of nucleic acid detection and can improve the accuracy of nucleic acid detection.

[0081] It should be noted that, in actual applications, the number of detection modules provided on the same chip body can be flexibly set to other values ​​according to actual detection requirements, or only one detection module can be provided. In addition, in order to improve detection efficiency, in some other embodiments of the present invention, a plurality of color development modules (the specific number is set according to the detection requirements) can be provided in the same detection module. In this case, a plurality of first connection channels connected to these color development modules are correspondingly provided, and these first connection channels are all connected to the CRISPR reaction chamber. In this case, for the same sample, different detections can be completed in parallel, effectively improving detection efficiency.

[0082] Example 2:

[0083] A portable microfluidic chip for nucleic acid detection. Figure 4 and Figure 5 As shown, this embodiment is similar to the above-mentioned embodiment 1, except that, in this embodiment, the color development module includes: a color development chamber 252 located at the top, a substrate chamber 254 located at the bottom, and a second connecting channel 253 for connecting the color development chamber 252 and the substrate chamber 254;

[0084] The color development chamber 252 is connected to the top of the first connecting channel 24;

[0085] The substrate chamber 254 is pre-buried with a colorimetric substrate (such as TMB or ABTS) for detecting the products of the CRISPR cleavage reaction;

[0086] An interception structure 26 is provided in the first connection channel 24 for intercepting unreleased microsphere probes and allowing the enzyme produced by the CRISPR cleavage reaction to pass through; the interception structure 26 may be a micro-dam or slit structure, or other structures that can achieve corresponding functions, such as Figure 6As shown, when the nucleic acid sequence generated by the RPA amplification reaction will connect the microspheres and the corresponding enzyme, if the target nucleic acid is present in the sample, the target nucleic acid will be cut in the CRISPR cutting reaction, releasing the corresponding enzyme (such as HRP), and the released enzyme can pass through the interception structure to complete the subsequent color development reaction; on the contrary, if the target nucleic acid is not present in the sample, the connected nucleic acid will not be cut, HRP cannot be released, and cannot enter the color development chamber through the interception structure. At this time, no color development reaction occurs in the color development chamber, ensuring high specificity.

[0087] The portable nucleic acid detection microfluidic chip provided in this embodiment, when used for nucleic acid detection, has operating steps similar to those of the above-mentioned embodiment 1, except that, in the signal output step, after the chip is placed upside down, the product in the CRISPR reaction chamber will enter the color development chamber through the first connecting channel and the intercepting structure therein, and at the same time, the chromogenic substrate in the substrate chamber will enter the color development chamber through the second connecting channel and undergo a color development reaction with the product of the CRISPR cleavage reaction; accordingly, in the result reading step, the color development result is directly read from the color development chamber.

[0088] Figure 5 The figure shows an optional layered design scheme for the portable nucleic acid detection microfluidic chip provided by this embodiment. In this scheme, the entire color development module is located in the same chip layer, which further simplifies the layered design of the chip compared to Example 1. At the same time, in Example 1, in order to complete the corresponding detection, the test strip is longer, while in this embodiment, as long as the second connecting channel can achieve communication between the color development chamber and the substrate chamber, its length can be set shorter, even shorter than the first connecting channel, making the entire chip structure more compact and the detection sensitivity higher. This embodiment is suitable for closed reaction systems without test strips, and is suitable for low viral load scenarios or low-concentration targets that are difficult to distinguish with the naked eye.

[0089] In this embodiment, the specific implementation of the remaining structures can refer to the description in the above embodiment 1 and will not be repeated here.

[0090] Example 3:

[0091] A nucleic acid detection device, comprising: a base and K portable nucleic acid detection microfluidic chips;

[0092] K slots are provided on the base, and K portable nucleic acid detection microfluidic chips are fixed in the K slots respectively;

[0093] Among them, the portable nucleic acid detection microfluidic chip is the above-mentioned portable nucleic acid detection microfluidic chip provided by the present invention; K is a positive integer greater than or equal to 1.

[0094] The nucleic acid detection device provided in this embodiment integrates multiple portable nucleic acid detection microfluidic chips together through a base, which can further improve the detection channel.

[0095] Example 4:

[0096] A microfluidic detection method for non-medical diagnostic purposes is proposed in this embodiment based on the portable nucleic acid detection microfluidic chip provided in Example 1 or Example 2 above. This embodiment includes the following steps:

[0097] S1: Place the portable nucleic acid detection microfluidic chip upright, add the sample to be tested into the quantitative chamber of each detection module, and complete the sample quantification;

[0098] S2: Applying a positive force to the portable nucleic acid detection microfluidic chip causes the sample in each quantitative chamber to break through the microchannel and transfer to the RPA reaction chamber, where an RPA amplification reaction occurs. The RPA amplification product then diffuses to the CRISPR reaction chamber, where a CRISPR cleavage reaction occurs.

[0099] S3: After the sample in the chamber to be quantified is completely transferred to the reaction chamber and the CRISPR cleavage reaction is completed, the portable nucleic acid detection microfluidic chip is placed upside down so that the product of the CRISPR cleavage reaction flows to the color development module through the first connecting channel;

[0100] S4: After the preset time, the detection result of the color development module is read to complete the nucleic acid detection.

[0101] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A portable microfluidic chip for nucleic acid detection, characterized in that: include: A chip body, and M detection modules disposed on the chip body; The detection module includes: a quantitative chamber, a microchannel, a reaction chamber, N first connecting channels and N color development modules; The quantitative chamber is provided with a sample addition port; The reaction chamber includes an RPA reaction chamber, a porous filter membrane, and a CRISPR reaction chamber, which are located on three adjacent chip layers and overlap with each other on the chip layers; the RPA reaction chamber is pre-embedded with an RPA amplification reagent; the CRISPR reaction chamber is pre-embedded with a CRISPR detection component; the porous filter membrane is located between the RPA reaction chamber and the CRISPR reaction chamber, and its pore size is larger than the particle size of the RPA amplification product but smaller than the particle size of the core enzyme in the CRISPR detection component; One end of the microchannel is connected to the bottom end of the quantitative chamber, and the other end is connected to the RPA reaction chamber, and the connection point between the microchannel and the RPA reaction chamber is lower than the bottom end of the quantitative chamber; The bottom ends of the N first connecting channels are all connected to the top of the CRISPR reaction chamber, and the top ends of the N first connecting channels are respectively connected to the top ends of the N color development modules; the flow resistance of the first connecting channels is less than the flow resistance of the microchannel; The color development module is used to detect the product of the CRISPR cleavage reaction; Wherein, M and N are both positive integers greater than or equal to 1.

2. The portable nucleic acid detection microfluidic chip according to claim 1, characterized in that: The color development module includes: a color development chamber located at the top, a substrate chamber located at the bottom, and a second connecting channel for connecting the color development chamber and the substrate chamber; The color development chamber is connected to the top of the first connecting channel; The substrate chamber is pre-buried with a colorimetric substrate for detecting the product of the CRISPR cleavage reaction; The first connecting channel is provided with an interception structure for intercepting unreleased microsphere probes and allowing the enzyme produced by the CRISPR cleavage reaction to pass through.

3. The portable nucleic acid detection microfluidic chip according to claim 1, characterized in that: The color development module includes a test strip for detecting the products of the CRISPR cleavage reaction.

4. The portable nucleic acid detection microfluidic chip according to claim 3, characterized in that: The color development module further includes: a detection chamber for accommodating the test strip; Water absorbing materials are respectively provided at both ends of the detection chamber.

5. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 4, characterized in that: The quantitative chamber, the microchannel and the RPA reaction chamber are located on the same chip layer.

6. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 4, characterized in that: The quantitative chamber is in a "V" shape, and the top of the "V" shape is the bottom of the quantitative chamber.

7. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 4, characterized in that: The porous filter membrane is a polycarbonate membrane.

8. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 4, characterized in that: N>1。 9. A nucleic acid detection device, characterized in that: include: Base and K portable nucleic acid detection microfluidic chips; The base is provided with K slots, and the K portable nucleic acid detection microfluidic chips are respectively fixed in the K slots; Wherein, the portable nucleic acid detection microfluidic chip is the portable nucleic acid detection microfluidic chip according to any one of claims 1 to 8; K is a positive integer greater than or equal to 1.

10. A microfluidic detection method for non-medical diagnosis purposes based on the portable nucleic acid detection microfluidic chip according to any one of claims 1 to 8, characterized in that: The steps include: S1: Place the portable nucleic acid detection microfluidic chip upright, add the sample to be tested into the quantitative chamber of each detection module, and complete the sample quantification; S2: applying a positive force to the portable nucleic acid detection microfluidic chip, so that the sample in each quantitative chamber breaks through the microfluidic channel and is transferred to the RPA reaction chamber, where an RPA amplification reaction occurs, and the RPA amplification product diffuses to the CRISPR reaction chamber, where a CRISPR cleavage reaction occurs; S3: After the sample in the quantification chamber is completely transferred to the reaction chamber and the CRISPR cleavage reaction is completed, the portable nucleic acid detection microfluidic chip is placed upside down, so that the product of the CRISPR cleavage reaction flows to the color development module through the first connecting channel; S4: After a preset time, the detection result of the color development module is read to complete the nucleic acid detection.

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