Portable nucleic acid detection microfluidic chip, nucleic acid detection device and method

Abstract

This invention discloses a portable nucleic acid detection microfluidic chip, nucleic acid detection device, and method, belonging to the field of microfluidic chips. Its detection module includes: a quantitative chamber, a microchannel, a reaction chamber, a first connecting channel, and a colorimetric module. The reaction chamber includes an RPA reaction chamber pre-embedded with RPA amplification reagent, a porous filter membrane, and a CRISPR reaction chamber pre-embedded with CRISPR detection components, all three located sequentially at the same position on three adjacent chip layers. The pore size of the porous filter membrane 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 components. The two ends of the microchannel are connected to the quantitative chamber and the RPA reaction chamber, respectively. The two ends of the first connecting channel are connected to the CRISPR reaction chamber and the colorimetric module, respectively. The flow resistance of the first connecting channel is less than that of the microchannel. This invention enables the sequential reaction of RPA amplification and CRISPR cleavage without relying on manual transfer of amplification products.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chips, and more specifically, relates to portable nucleic acid detection microfluidic chips, nucleic acid detection devices, and methods. Background Technology

[0002] With increasing public health awareness, the demand for home-based molecular diagnostics continues to grow, especially in screening for human papillomavirus (HPV) and respiratory viruses, where nucleic acid testing has become the preferred choice due to its high sensitivity and specificity. However, traditional qPCR technology has high requirements for instruments, operation, and environment, making it difficult to meet the needs of home users for "convenience, low cost, and instrument-free" testing.

[0003] In recent years, the CRISPR-Cas system has shown great potential for rapid on-site detection, exhibiting good specificity through crRNA-mediated target recognition and Cas protein-mediated reporter probe cleavage. However, the sensitivity of directly using the CRISPR system to detect nucleic acids remains limited, and it usually needs to be combined with isothermal amplification techniques such as recombinase polymerase amplification (RPA) to enhance the signal.

[0004] Current RPA-CRISPR integration platforms are mostly laboratory-level solutions, which present the following challenges: (1) The amplification and identification steps require multiple manual transfers, which can easily lead to contamination or operational errors; (2) If RPA products are co-located with the CRISPR system, template depletion may occur due to premature shearing, which may inhibit the amplification reaction. (3) They often use electrically controlled pumps / valves or complex structures, which are not suitable for resource-constrained scenarios and non-professional users.

[0005] Therefore, developing a portable microfluidic chip that combines sequential reaction control, high structural integration, and user-friendliness is crucial for advancing CRISPR on-site detection into home scenarios. Patent application number 2024119465786 proposes a portable microfluidic chip for nucleic acid detection. However, this chip requires pre-embedding RPA amplification reagents in the quantitative chamber and CRISPR detection components in the reaction chamber. Since RPA amplification is located within a shared quantitative chamber, it increases the difficulty of designing multiple primers. Furthermore, after RPA amplification, a certain time is required before transferring the chip to the CRISPR chamber, and this time must be strictly controlled, making a one-step method impossible. In practical operation, errors in time control may affect the detection results. Summary of the Invention To address the shortcomings and improvement needs of existing technologies, this invention provides a portable nucleic acid detection microfluidic chip, nucleic acid detection device, and method. The purpose is to complete the sequential reaction of RPA amplification and CRISPR cutting without relying on manual transfer of RPA amplification reaction products, thereby reducing the difficulty of detection and improving detection accuracy in home testing scenarios.

[0006] To achieve the above objectives, according to one aspect of the present invention, a portable nucleic acid detection microfluidic chip is provided, comprising: a chip body, and a chip disposed on the chip body. M One detection module; The detection module includes: a quantitative chamber, a microchannel, and a reaction chamber. N The first connection channel and N One color display module; The quantitative chamber is equipped with a sample dispensing port; The reaction chamber includes an RPA reaction chamber, a porous membrane, and a CRISPR reaction chamber. These three chambers are located on three adjacent chip layers, and their positions on the chip layers overlap. The RPA reaction chamber contains RPA amplification reagents. The CRISPR reaction chamber contains CRISPR detection components. The porous 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. The microchannel is connected at one end to the bottom of the quantitative chamber and at the other end to the RPA reaction chamber, and the connection point between the microchannel and the RPA reaction chamber is lower than the bottom of the quantitative chamber. N The bottom of each of the first connection channels is connected to the top of the CRISPR reaction chamber. N The top of each of the first connection channels is respectively connected to N The tops of the color-developing modules are connected; the flow resistance of the first connecting channel is less than that of the microchannel; The colorimetric module is used to detect the products of the CRISPR cleavage reaction; in, M and N All are positive integers greater than or equal to 1.

[0007] In some alternative implementations, the color development module includes: a color development chamber at the top, a substrate chamber 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 contains a chromogenic substrate for detecting the products of the CRISPR cutting reaction; The first connection channel is equipped with an interception structure to trap unreleased microsphere probes while allowing enzymes generated by the CRISPR cleavage reaction to pass through.

[0008] In some alternative implementations, the colorimetric module includes a test strip for detecting the products of the CRISPR cleavage reaction.

[0009] Furthermore, the colorimetric module also includes a detection chamber for accommodating the test strip; Water-absorbing material is installed at both ends of the testing chamber.

[0010] Furthermore, the quantitative chamber, microchannel, and RPA reaction chamber are located on the same chip layer.

[0011] Furthermore, the quantitative chamber is V-shaped, with the apex of the V-shape being the bottom of the quantitative chamber.

[0012] Furthermore, the porous filter membrane is a polycarbonate membrane.

[0013] Furthermore, N >1.

[0014] According to another aspect of the present invention, a nucleic acid detection device is provided, comprising: a base and... K A portable microfluidic chip for nucleic acid detection; The base is equipped with K One slot, K Each portable nucleic acid detection microfluidic chip is fixed in K In each slot; Among them, the portable nucleic acid detection microfluidic chip is the portable nucleic acid detection microfluidic chip provided by the present invention; K It is a positive integer greater than or equal to 1.

[0015] According to another aspect of the present invention, a microfluidic detection method for non-medical diagnostic purposes based on the above-described portable nucleic acid detection microfluidic chip is provided, comprising the following steps: 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: Apply a positive force to the portable nucleic acid detection microfluidic chip, causing the samples in each quantitative chamber to break through the microchannels and transfer to the RPA reaction chamber, where the RPA amplification reaction occurs, and the RPA amplification products diffuse into the CRISPR reaction chamber, where the CRISPR cleavage reaction occurs. S3: After the sample in the quantitative chamber has been completely transferred to the reaction chamber and the CRISPR cleavage reaction has been completed, the portable nucleic acid detection microfluidic chip is placed upside down so that the product of the CRISPR cleavage reaction flows to the colorimetric module through the first connection channel. S4: After a preset time, read the detection results from the colorimetric module to complete the nucleic acid test.

[0016] In summary, the above-described technical solutions conceived in this invention can achieve the following beneficial effects: (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 between them. Based on this design, after sample quantification, the sample can be transferred to the RPA reaction chamber. The product of the RPA amplification reaction will automatically diffuse through the porous filter membrane into the CRISPR chamber to undergo the CRISPR cleavage reaction, without the need for manual transfer of the RPA amplification reaction product. This completes the sequential reaction of RPA amplification and CRISPR detection, effectively reducing the operational difficulty of nucleic acid detection and improving the accuracy of nucleic acid detection.

[0017] (2) In the preferred embodiment of the portable nucleic acid detection microfluidic chip provided by the present invention, the colorimetric module for detecting CRISPR cleavage reaction products includes a colorimetric chamber at the top, a substrate chamber at the bottom, and a second connecting channel for connecting the colorimetric chamber and the substrate chamber. Based on this structural design, after the CRISPR cleavage reaction is completed, the chip is inverted so that the CRISPR cleavage reaction products and the colorimetric substrate in the substrate chamber can flow into the colorimetric chamber to achieve detection. Compared with test strips, the length of the colorimetric module based on this structure is greatly shortened, and the entire chip structure is more compact.

[0018] (3) The portable nucleic acid detection microfluidic chip provided by the present invention uses a test strip as a color development module in its optional scheme, and in its further preferred scheme, a detection chamber for placing the test strip is also provided, and water-absorbing materials are respectively provided at both ends of the detection chamber, thereby improving the efficiency of the transfer of reaction products in the reaction chamber or enhancing reagents in the enhancing reagent chamber to the test strip.

[0019] (4) The portable nucleic acid detection microfluidic chip provided by the present invention has multiple color development modules in the same detection module and multiple first connection channels, so that different detections can be completed in parallel for the same sample, thereby improving detection efficiency.

[0020] (5) In the preferred embodiment of the portable nucleic acid detection microfluidic chip provided by the present invention, the quantitative chamber, the microchannel and the RPA reaction chamber are located on the same chip layer, which simplifies the layered structure design of the chip while ensuring the detection function.

[0021] (6) In the preferred embodiment of the portable nucleic acid detection microfluidic chip provided by the present invention, the quantitative chamber is V-shaped, and the apex of the V-shape is the bottom 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. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the portable nucleic acid detection microfluidic chip structure provided in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the structure of the reaction chamber provided in Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the layered design of the portable nucleic acid detection microfluidic chip provided in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the structure of the portable nucleic acid detection microfluidic chip provided in Embodiment 2 of the present invention; Figure 5 This is a schematic diagram of the layered design of the portable nucleic acid detection microfluidic chip provided in Embodiment 2 of the present invention; Figure 6 This is a schematic diagram of the interception structure provided in Embodiment 2 of the present invention; In all the accompanying drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1-Chip body; 2-Detection module; 21-Quantitative chamber; 22-Microchannel; 23-Reaction chamber; 231-RPA reaction chamber; 232-Porous filter membrane; 233-CRISPR reaction chamber; 24 - First connection channel; 25-Color development module; 251-Test strip; 252-Color development chamber; 253-Second connecting channel; 254-Substrate chamber; 26-Interception structure. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be 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 illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0024] 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, terms such as "upper," "lower," "left," "right," "inner," and "outer," 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, or be constructed and operated in a specific orientation; therefore, it should not be construed as a limitation of the present invention. Furthermore, it should be noted that in the description of the present invention, unless otherwise explicitly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; a mechanical connection or an electrical connection; a direct connection or an indirect connection through an intermediate medium; or a connection within two elements. Those skilled in the art can understand the specific meaning of the above terms in the present invention according to the specific circumstances. The terms "first," "second," etc. (if present), in the present invention and accompanying drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0025] To reduce the difficulty and complexity of fluid manipulation and improve the portability of microfluidic chips, this invention provides a portable nucleic acid detection microfluidic chip, nucleic acid detection device, and method. The flow channel and chamber structure of the microfluidic chip are improved, enabling the products of the RPA amplification reaction to automatically diffuse into the CRISPR reaction chamber for CRISPR cleavage. This completes the sequential reaction of RPA amplification and CRISPR cleavage without relying on manual transfer of RPA amplification products. In home testing scenarios, this reduces the difficulty of detection and improves detection accuracy.

[0026] It is easy to understand that in this invention, descriptions such as "top," "bottom," "high," and "low" are all relative to the chip when it is placed upright. When the chip is placed upright, the highest point of each structure is the top of that structure, and the lowest point of each structure is the bottom of that structure. "Forward force" and "reverse force" are relative to the top and bottom of the chip. The force from the top to the bottom of the chip is the forward force, and conversely, the force from the bottom to the top of the chip is the reverse force.

[0027] The following is an example.

[0028] Example 1: A portable microfluidic chip for nucleic acid detection, such as Figure 1 and Figure 2 As shown, it includes: a chip body 1, and five detection modules 2 disposed on the chip body 1; The detection module 2 includes: a quantitative chamber 21, a microchannel 22, a reaction chamber 23, a first connecting channel 24, and a colorimetric module 25; The quantitative chamber 21 is provided with a sample addition port; optionally, in this embodiment, the quantitative chamber 21 is V-shaped, and the apex of the V-shape is the bottom 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. 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. These three chambers are located on three adjacent chip layers, and their positions on the chip layers overlap. The RPA reaction chamber 231 contains pre-embedded RPA amplification reagents (including primers, enzymes, buffers, etc.). The CRISPR reaction chamber 233 contains pre-embedded CRISPR detection components (such as Cas12a / crRNA / ssDNA reporter probes). The porous filter membrane 232 is located between the RPA reaction chamber 231 and the CRISPR reaction chamber 233. 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. Optionally, in this embodiment, the pore size of the porous filter membrane 232 is 10~50nm, and the porous filter membrane is a polycarbonate membrane. This ensures that the RPA amplification product can diffuse during nucleic acid detection, but prevents the flow of large molecules such as proteases, ensuring that the substances in the RPA reaction chamber are not prematurely sheared during amplification. Microchannel 22, one end of which is connected to the bottom of quantitative chamber 21, and the other end of which is connected to RPA reaction chamber 231, and the connection point between microchannel 22 and RPA reaction chamber 231 is lower than the bottom of quantitative chamber 21. The bottom end of the first connecting channel 24 is connected to the top end of the CRISPR reaction chamber 233, and the top end of the first connecting channel 24 is connected to the top end of the color development module 25; the flow resistance of the first connecting channel 24 is less than the flow resistance of the microchannel 22. The colorimetric module 25 is used to detect the products of the CRISPR cleavage reaction.

[0029] Optionally, in this embodiment, the colorimetric module 25 includes a test strip 251 for detecting the products of the CRISPR cutting reaction. In order to improve the efficiency of the transfer of reaction products in the reaction chamber or enhancing reagents in the enhancing reagent chamber to the test strip, in this embodiment, the colorimetric module 25 also includes a detection chamber for accommodating the test strip; the two ends of the detection chamber are respectively provided with absorbent materials.

[0030] The chip in this embodiment specifically includes multiple chip layers, one of which is an optional layered design scheme as follows: Figure 2As shown, from left to right: the chip bottom cover; the chip layer containing the test strip; the chip layer containing the absorbent material, the second connection channel, and the test strip connection hole; the chip layer containing the CRISPR reaction chamber and the second connection channel; the chip layer containing the porous filter membrane; the chip layer containing the quantitative chamber, microchannel, and RPA reaction chamber; and the chip top cover. It's easy to understand that the chip also has vents that connect each chamber to the atmosphere.

[0031] Based on the flow resistance relationship between the microchannel and the first connecting channel, in the layered design of this embodiment, the quantitative chamber, the microchannel, and the RPA reaction chamber can be designed in the same chip layer. This design simplifies the layered structure design of the chip while ensuring the detection function.

[0032] In practical applications, the portable nucleic acid detection microfluidic chip provided in this embodiment is used for nucleic acid detection in the following steps: (1) Sample loading and quantification: Place the chip upright and add the sample (such as lysed sample solution) into the quantification chamber; the sample quantification is automatically completed with the help of the V-shaped structure, and the sample stays at the front end of the microchannel; (2) Amplification and cleavage reaction: Apply positive force to the chip (e.g., 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. RPA amplification is performed at 37~42°C to generate the target sequence. After the reaction, the amplification product can naturally diffuse through the porous filter membrane into the CRISPR reaction chamber to initiate the CRISPR cleavage reaction. In the CRISPR cleavage reaction, the amplification product recognizes and activates the Cas12a protein with crRNA, thereby cleaving the ssDNA reporter probe and generating a colorimetric signal pre-reaction. (3) Signal output: After the sample in the quantitative chamber is completely transferred to the reaction chamber and the RPA amplification reaction and CRISPR cutting reaction are completed, the chip is inverted. The reaction product in the CRISPR reaction chamber enters the detection chamber through the first connection channel under the action of gravity, and interacts with the test strip to form a visible or semi-quantitative response. (4) Result reading: After a preset time (e.g., 5 minutes), observe the color development of the test strip's detection line / control line to complete the reading of the nucleic acid test result.

[0033] Based on the above detection steps, it can be seen that when the portable nucleic acid detection microfluidic chip provided in this embodiment is applied to nucleic acid detection, the sample can be transferred to the RPA reaction chamber after sample quantification. The product of the RPA amplification reaction will automatically diffuse through the porous filter membrane into the CRISPR chamber to undergo CRISPR cleavage reaction, without the need for manual transfer of the RPA amplification reaction product. This completes the sequential reaction of RPA amplification and CRISPR detection, effectively reducing the operational difficulty of nucleic acid detection and improving the accuracy of nucleic acid detection.

[0034] It should be noted that in practical applications, the number of detection modules on the same chip can be flexibly set to other values ​​according to actual detection needs, or only one detection module may be set. Furthermore, to improve detection efficiency, in some other embodiments of the present invention, multiple color development modules (the specific number is set according to detection needs) can be set in the same detection module. In this case, multiple first connection channels connected to these color development modules are correspondingly set, and these first connection channels are all connected to the CRISPR reaction chamber. In this way, different detections can be performed in parallel for the same sample, effectively improving detection efficiency.

[0035] Example 2: A portable microfluidic chip for nucleic acid detection. For example... Figure 4 and Figure 5 As shown, this embodiment is similar to the above 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. The color development chamber 252 is connected to the top of the first connecting channel 24; The substrate chamber 254 is pre-embedded with a chromogenic substrate (such as TMB or ABTS) for detecting the products of the CRISPR cleavage reaction. The first connection channel 24 is provided with an interception structure 26 for trapping unreleased microsphere probes while allowing enzymes generated by the CRISPR cleavage reaction to pass through. The interception structure 26 can be a microdam or a slit structure, or other structures that can achieve the corresponding function, such as... Figure 6 As shown, when the nucleic acid sequence generated by the RPA amplification reaction is linked to the microsphere and the corresponding enzyme, if the target nucleic acid is present in the sample, it will be cleaved in the CRISPR cleavage reaction, releasing the corresponding enzyme (e.g., HRP). The released enzyme can then pass through the interception structure to complete the subsequent colorimetric reaction. Conversely, if the target nucleic acid is not present in the sample, the linked nucleic acid will not be cleaved, HRP cannot be released, and it cannot pass through the interception structure to enter the colorimetric chamber. In this case, no colorimetric reaction occurs in the colorimetric chamber, ensuring high specificity.

[0036] The portable nucleic acid detection microfluidic chip provided in this embodiment operates similarly to that in Embodiment 1 above when used for nucleic acid detection. The difference is that, in the signal output step, after the chip is inverted, the product in the CRISPR reaction chamber will enter the colorimetric chamber through the first connection channel and the interception structure therein. At the same time, the chromogenic substrate in the substrate chamber will enter the colorimetric chamber through the second connection channel and react with the product of the CRISPR cleavage reaction to produce a colorimetric reaction. Correspondingly, in the result reading step, the colorimetric result is directly read from the colorimetric chamber.

[0037] Figure 5 The illustration shows an optional layered design scheme for the portable nucleic acid detection microfluidic chip provided in this embodiment. In this scheme, the entire colorimetric module is located on the same chip layer, further simplifying the layered design of the chip compared to Embodiment 1. Furthermore, in Embodiment 1, the test strip is relatively long to complete the detection, while in this embodiment, the second connecting channel only needs to connect the colorimetric chamber and the substrate chamber; its length can be set shorter, even shorter than the first connecting channel. This results in a more compact chip structure and higher detection sensitivity. This embodiment is suitable for closed reaction systems without test strips and is adapted to low viral load scenarios or low-concentration targets that are difficult to distinguish with the naked eye.

[0038] In this embodiment, the specific implementation of the remaining structures can be referred to the description in Embodiment 1 above, and will not be repeated here.

[0039] Example 3: A nucleic acid testing device includes: a base and K A portable microfluidic chip for nucleic acid detection; The base is equipped with K One slot, K Each portable nucleic acid detection microfluidic chip is fixed in K In each slot; Among them, the portable nucleic acid detection microfluidic chip is the portable nucleic acid detection microfluidic chip provided by the present invention; K It is a positive integer greater than or equal to 1.

[0040] 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 throughput.

[0041] Example 4: A microfluidic detection method for non-medical diagnostic purposes is proposed in this embodiment, which is based on the portable nucleic acid detection microfluidic chip provided in Embodiment 1 or Embodiment 2 above. This embodiment includes the following steps: 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: Apply a positive force to the portable nucleic acid detection microfluidic chip, causing the samples in each quantitative chamber to break through the microchannels and transfer to the RPA reaction chamber, where the RPA amplification reaction occurs, and the RPA amplification products diffuse into the CRISPR reaction chamber, where the CRISPR cleavage reaction occurs. S3: After the sample in the quantitative chamber has been completely transferred to the reaction chamber and the CRISPR cleavage reaction has been completed, the portable nucleic acid detection microfluidic chip is placed upside down so that the product of the CRISPR cleavage reaction flows to the colorimetric module through the first connection channel. S4: After a preset time, read the detection results from the colorimetric module to complete the nucleic acid test.

[0042] Those skilled in the art will readily understand 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 within the scope of protection of the present invention.

Claims

1. A portable nucleic acid detection microfluidic chip, characterized in that, include: The chip body and the detection module are arranged on the chip body M The chip body and the detection module are arranged on the chip body The detection module includes: a quantitative chamber, a microchannel, and a reaction chamber. N The first connection channel and N One color display module; The quantitative chamber is equipped with a sample dispensing 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 in their positions on the chip layers. The RPA reaction chamber contains embedded RPA amplification reagents. The CRISPR reaction chamber contains embedded 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 components. The microchannel has one end connected to the bottom of the quantitative chamber and the other end connected to the RPA reaction chamber, and the connection point between the microchannel and the RPA reaction chamber is lower than the bottom of the quantitative chamber. The N The bottom end of each of the first connection channels is connected to the top end of the CRISPR reaction chamber. N The top ends of the first connection channels are respectively connected to the N The tops of the color-displaying modules are connected; the flow resistance of the first connecting channel is less than the flow resistance of the microchannel; The colorimetric module is used to detect the products of the CRISPR cleavage reaction; in, M and N All are positive integers greater than or equal to 1; the color development module includes: a color development chamber at the top, a substrate chamber 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 contains a chromogenic substrate for detecting the products of the CRISPR cleavage reaction. The first connection channel is provided with an interception structure to trap unreleased microsphere probes and allow enzymes generated by the CRISPR cleavage reaction to pass through.

2. The portable nucleic acid detection microfluidic chip as described in claim 1, characterized in that, The colorimetric module includes a test strip for detecting the products of the CRISPR cleavage reaction.

3. The portable nucleic acid detection microfluidic chip as described in claim 2, characterized in that, The colorimetric module further includes a detection chamber for accommodating the test strip; Water-absorbing material is provided at both ends of the detection chamber.

4. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 3, characterized in that, The quantitative chamber, the microchannel, and the RPA reaction chamber are located on the same chip layer.

5. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 3, characterized in that, The quantitative chamber is V-shaped, and the apex of the V-shape is the bottom of the quantitative chamber.

6. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 3, characterized in that, The porous filter membrane is a polycarbonate membrane.

7. The portable nucleic acid detection microfluidic chip according to any one of claims 1 to 3, characterized in that, N >1。 8. A nucleic acid detection device, characterized in that, include: base and K A portable microfluidic chip for nucleic acid detection; The base is provided with K Each slot, the K Each portable nucleic acid detection microfluidic chip is fixed to the above K In each slot; The portable nucleic acid detection microfluidic chip is the portable nucleic acid detection microfluidic chip according to any one of claims 1 to 7; K It is a positive integer greater than or equal to 1.

9. A microfluidic detection method for non-medical diagnostic purposes based on the portable nucleic acid detection microfluidic chip according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1: Place the portable nucleic acid detection microfluidic chip upright, add the sample to be detected into the quantitative chamber of each detection module, and complete the sample quantification; S2: Apply a positive force to the portable nucleic acid detection microfluidic chip, causing the samples in each quantitative chamber to break through the microchannel and transfer to the RPA reaction chamber, where an RPA amplification reaction occurs, and the RPA amplification products diffuse into the CRISPR reaction chamber, where a CRISPR cleavage reaction occurs. S3: After the sample in the quantitative chamber is completely transferred to the reaction chamber and the CRISPR cutting reaction is completed, the portable nucleic acid detection microfluidic chip is placed upside down so that the product of the CRISPR cutting reaction flows to the colorimetric module through the first connection channel. S4: After a preset time, read the detection result of the color development module to complete the nucleic acid detection.

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