Micro-fluidic chip, preparation method and detection method

Abstract

The invention provides a micro-fluidic chip, a preparation method and a detection method, and relates to the technical field of nucleic acid detection. The chip comprises a first substrate, wherein the first substrate is sequentially provided with an electric cracking area, an electric enrichment area and a detection area along the flowing direction of a sample to be detected; the electric cracking area is used for electric cracking of the to-be-detected sample; the electric enrichment area is used for enriching the to-be-detected sample flowing out of the electric cracking area; the detection area is used for detecting the to-be-detected sample flowing out of the electric enrichment area; and the electrode assembly is connected with the first substrate. The electric cracking area, the electric enrichment area and the detection area are integrated, the to-be-detected sample is driven by the electrode assembly to rapidly flow and sequentially flow through the electric cracking area and the electric enrichment area, effective substances are enriched in the electric enrichment area, loss is reduced, the detection concentration of the to-be-detected sample entering the detection area is guaranteed, and the requirement for instant detection is met; the rapidity, the sensitivity and the accuracy of the detection of the to-be-detected sample are improved.

Description

Technical Field

[0001] This application relates to the technical field of nucleic acid detection, and particularly relates to a microfluidic chip, a preparation method, and a detection method. Background Art

[0002] mRNA detection is a nucleic acid detection method that reveals gene expression patterns, identifies disease-related biomarkers, and guides personalized medicine or vaccine development by analyzing the expression level of messenger ribonucleic acid. For example, in individuals affected by radiation, the radiation dose is evaluated through mRNA in the blood for medical intervention.

[0003] In the prior art, the detection of mRNA mainly includes RT-PCR, RNA sequencing (RNA-seq), and gene chips (Microarray). These methods usually analyze the sample to be tested based on peripheral blood and are supplemented by chromosomal gene mutation detection to evaluate genomic instability.

[0004] However, the above-mentioned detection has a high cost and a long detection time. Moreover, as the detection time increases, mRNA will degrade rapidly, reducing the accuracy of the detection results. Summary of the Invention

[0005] This application provides a microfluidic chip, a preparation method, and a detection method to solve the problems of high detection cost, long detection time, and rapid degradation of mRNA with the increase of detection time, reducing the accuracy of detection results.

[0006] On the one hand, this application provides a microfluidic chip for nucleic acid detection, including: a first substrate, on which an electrolysis region, an electroenrichment region, and a detection region are sequentially arranged along the flow direction of the sample to be tested; the electrolysis region is used for electrolyzing the sample to be tested; the electroenrichment region is used for enriching the sample to be tested flowing out of the electrolysis region; the detection region is used for detecting the sample to be tested flowing out of the electroenrichment region; an electrode assembly, which is connected to the first substrate and is used to drive the sample to be tested to flow through the electrolysis region and the electroenrichment region in sequence and enter the detection cell for detection.

[0007] In a possible implementation manner, in the microfluidic chip of the embodiment of this application, at least two flow channels for the sample to be tested to flow are arranged on the first substrate, the two flow channels are arranged oppositely, and each flow channel communicates with the electrolysis region, the electroenrichment region, and the detection region.

[0008] In a possible implementation manner, in the microfluidic chip of the embodiment of this application, the flow channel includes a first flow channel located in the electrolysis region, and at least a part of the first flow channel bends towards the edge of the first substrate.

[0009] In one possible implementation, the microfluidic chip in the embodiment of the present application has an enrichment pool in the electrical enrichment zone, which is used to receive the sample to be tested flowing through the electrolysis zone. The enrichment pool has a waste liquid outlet and a sampling outlet, and the waste liquid outlet is located above the sampling outlet, and the sampling outlet is connected to the detection zone.

[0010] In one possible implementation, in the microfluidic chip in the embodiment of the present application, at least one bending portion is provided on the lower side of the enrichment tank to form a waste liquid outlet and a sampling outlet, and a sampling tube is provided between the sampling outlet and the enrichment tank, and the inner diameter of the sampling tube gradually decreases from the enrichment tank to the sampling outlet.

[0011] In one possible implementation, in the microfluidic chip in the embodiment of the present application, the electrode area includes an enrichment electrode area, the enrichment electrode area includes a positive electrode and a negative electrode, the upper part of the enrichment tank is connected to the positive electrode, and a connecting part is provided at the bottom of the enrichment tank, a plurality of grooves are arranged side by side on the connecting part, a membrane is provided in the groove, and the connecting part is connected or in contact with the negative electrode on the side away from the enrichment tank.

[0012] In a possible implementation, in the microfluidic chip in the embodiment of the present application, each flow channel is connected to at least one enrichment pool, and the enrichment pools are arranged in an interlaced manner; the detection area has at least one detection pool, and each detection pool is connected to the enrichment pool in a one-to-one correspondence.

[0013] In a possible implementation, in the microfluidic chip in the embodiment of the present application, the flow channel width of the electrolysis flow channel is 0.4 mm to 2.0 mm.

[0014] On the other hand, the present application also provides a method for preparing a microfluidic chip as in the above embodiment, including: providing a first substrate, the first substrate being sequentially provided with an electrolysis zone, an electro-enrichment zone and a detection zone along the flow direction of the sample to be tested; performing freeze-drying modification on the first substrate, including: dripping a reaction system mixture containing different primers into the detection zone; pre-cooling the first substrate for a preset time and a preset temperature, and drying and freeze-drying under preset vacuum conditions; providing an electrode assembly, the electrode assembly being connected to the first substrate, and the electrode assembly being used to drive the sample to be tested to flow through the electro-lysis zone and the electro-enrichment zone in sequence, and enter the detection pool for detection.

[0015] In addition, the present application also provides a method for detecting a sample to be tested, which is applied to a microfluidic chip such as the one in the above embodiment, including: passing the sample to be tested into the electrolysis zone; energizing the electrode assembly so that the electrode assembly drives the sample to be tested to pass through the electrolysis zone and the electrical enrichment zone in sequence and enter the detection zone; after the liquid in the detection zone reaches a preset value, the microfluidic chip is placed in a heating device for a preset reaction time to obtain a detection result.

[0016] A microfluidic chip, a preparation method and a detection method provided by the present application. The chip includes: a first substrate, and an electrocracking area, an electroenrichment area and a detection area are sequentially arranged on the first substrate along the flowing direction of the sample to be detected; the electrocracking area is used for electrocracking the sample to be detected; the electroenrichment area is used for enriching the sample to be detected flowing out of the electrocracking area; the detection area is used for detecting the sample to be detected flowing out of the electroenrichment area; an electrode assembly, which is connected to the first substrate and is used for driving the sample to be detected to flow through the electrocracking area and the electroenrichment area in sequence and enter the detection area for detection. The present application integrates the electrocracking area, the electroenrichment area and the detection area, and drives the sample to be detected to flow quickly through the electrode assembly, flow through the electrocracking area and the electroenrichment area in sequence, and quickly enrich the effective substances in the electroenrichment area, reduce the loss, ensure the detection concentration of the sample to be detected entering the detection area, meet the requirements of point-of-care testing, and improve the rapidity, sensitivity and accuracy of detecting the sample to be detected. Description of the Drawings

[0017] The drawings here are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present application, and are used together with the specification to explain the principles of the present application.

[0018] Figure 1 It is a schematic structural diagram of the microfluidic chip provided by the embodiment of the present application;

[0019] Figure 2 is Figure 1 a schematic structural diagram of another perspective of the microfluidic chip in ;

[0020] Figure 3 is Figure 1 a schematic structural diagram of the enrichment pool in ;

[0021] Figure 4 is Figure 1 a distribution diagram of the electric field intensity in the electrocracking area in ;

[0022] Figure 5 is Figure 1 a schematic diagram of the modification of the freeze-dried reagent of the microfluidic chip in ;

[0023] Figure 6 It is a verification experimental diagram of the microfluidic chip provided by the embodiment of the present application;

[0024] Figure 7 It is a verification experimental diagram of the microfluidic chip provided by the embodiment of the present application for different samples to be detected;

[0025] Figure 8 It is a functional verification experimental diagram of the electroenrichment of the microfluidic chip provided by the embodiment of the present application;

[0026] Figure 9 It is a module difference verification diagram of the electroenrichment area of the microfluidic chip provided by the embodiment of the present application;

[0027] Figure 10 It is a test verification diagram of the fluorescence detection sensitivity of the microfluidic chip provided by the embodiment of the present application;

[0028] Figure 11 It is an application verification experimental diagram of the microfluidic chip provided by the embodiment of the present application in clinical practice;

[0029] Figure 12 It is a flowchart of the preparation method of the microfluidic chip provided by the embodiment of the present application.

[0030] Explanation of reference numerals:

[0031] 100 - First substrate; 110 - Electrochemical cracking region; 111 - First flow channel; 112 - Sampling port; 114 - Bending section; 120 - Electrochemical enrichment region; 121 - Sampling tube; 122 - Enrichment cell; 123 - Bending part; 124 - Waste liquid outlet; 125 - Sampling outlet; 126 - Groove; 130 - Detection region; 131 - Detection cell; 140 - Flow channel; 200 - Second substrate; 210 - Electrode assembly; 211 - Enrichment electrode region; 212 - Positive electrode; 213 - Negative electrode.

[0032] Through the above - mentioned drawings, specific embodiments of the present application have been shown, and there will be more detailed descriptions hereinafter. These drawings and written descriptions are not intended to limit the scope of the concept of the present application in any way, but to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. Detailed description of the specific embodiments

[0033] Here, the exemplary embodiments will be described in detail, and their examples are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent the embodiments consistent with the present application. On the contrary, they are only examples of devices and methods consistent with some aspects of the present application.

[0034] It should be noted that in the description of the embodiments of the present application, the terms indicating the orientation or positional relationship such as "upper", "lower", "inner", "outer", etc. are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of description, rather than indicating or implying that the device or component must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be understood as a limitation to the embodiments of the present application.

[0035] In addition, it should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this application, the meaning of "a plurality" is two or more, unless otherwise specifically defined.

[0036] In this application, unless otherwise clearly specified and defined, terms such as "installed", "connected", "fixed", etc. shall be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or integrated; it may be a mechanical connection, an electrical connection, or capable of communicating with each other; it may be a direct connection, or indirectly connected through an intermediate medium, and may be the communication inside two components or the interaction relationship between two components. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0037] First, the relevant concepts or terms involved in this application are explained:

[0038] mRNA: It refers to messenger ribonucleic acid.

[0039] RT-PCR: It refers to an mRNA detection method that undergoes reverse transcription (RNA is reversed to cDNA) and PCR amplification (multiple cycles) through reverse transcription polymerase chain reaction for quantitative analysis.

[0040] RNA sequencing (RNA-seq): It refers to an mRNA detection method that unbiasedly sequences RNA molecules in a sample to be tested through transcriptome sequencing, covering known and unknown transcripts. Its principle includes RNA fragmentation, reverse transcription into a cDNA library, sequencing, and bioinformatics analysis.

[0041] Gene chip (Microarray): It refers to an mRNA detection method that, based on the principle of probe hybridization, immobilizes DNA probes or RNA probes with known sequences on a solid-phase carrier and detects the expression level of specific genes through fluorescence labeling.

[0042] mRNA detection reveals gene expression patterns, identifies disease-related biomarkers, and guides personalized medicine or vaccine development by analyzing the expression level of messenger RNA. For example, for an individual affected by radiation, the radiation dose is evaluated through the mRNA in the blood to enable medical intervention.

[0043] In the prior art, the detection of mRNA mainly includes RT-PCR, RNA sequencing (RNA-seq) and gene chips (Microarray). These methods usually analyze the sample to be tested based on peripheral blood, and are supplemented by chromosomal gene mutation detection to evaluate genomic instability.

[0044] However, the above detection methods have high costs (such as expensive equipment and reagent costs, and dependence on professional technicians), long detection times (usually about 72 hours), and as the detection time increases, mRNA will degrade rapidly, reducing the accuracy of the detection results.

[0045] In addition, in terms of the lysis and purification of the sample to be tested, it still relies on chemical lysis buffers, magnetic beads or silicon membrane technologies. These technologies usually require the sample to be tested to stay in a specific area for a long time to complete the reaction, making the process difficult to control. Moreover, in order to prevent the loss or degradation of the sample to be tested, mechanical disturbance needs to be minimized, which further prolongs the detection time and reduces the detection efficiency. Therefore, the chip detection in traditional technologies is more about simplifying the experimental process, rather than significantly improving the detection quality. At the same time, the use of multiple reagents may cause cross-reactions in the chip, affecting the accuracy of the detection results.

[0046] In view of this, the present application provides a microfluidic chip, a preparation method and a detection method. The chip includes: a first substrate, and an electro-lysis area, an electro-enrichment area and a detection area are sequentially arranged on the first substrate along the flow direction of the sample to be tested; the electro-lysis area is used for electro-lyzing the sample to be tested; the electro-enrichment area is used for enriching the sample to be tested flowing out of the electro-lysis channel; the detection area is used for detecting the sample to be tested flowing out of the electro-enrichment area; an electrode assembly, which is connected to the first substrate and is used to drive the sample to be tested to flow through the electro-lysis area and the electro-enrichment area in sequence and enter the detection area for detection. The present application integrates the electro-lysis area, the electro-enrichment area and the detection area, and drives the sample to be tested to flow rapidly through the electrode assembly, flow through the electro-lysis area and the electro-enrichment area in sequence, and rapidly enrich the effective substances in the electro-enrichment area, reducing the loss, ensuring the detection concentration of the sample to be tested entering the detection area, and improving the rapidity, sensitivity and accuracy of the detection of the sample to be tested.

[0047] The following combines Figures 1 to 12 with specific embodiments to elaborate on the microfluidic chip, preparation method and detection method involved. The embodiments can be combined arbitrarily with each other, and the present application does not limit this.

[0048] The technical solution of the present application is as follows:

[0049] On the one hand, the present application provides a microfluidic chip for nucleic acid detection, including: a first substrate 100, on which an electrolysis region 110, an electroenrichment region 120, and a detection region 130 are sequentially arranged along the flow direction of the sample to be tested; the electrolysis region 110 is used for electrolyzing the sample to be tested; the electroenrichment region 120 is used for enriching the sample to be tested flowing out of the electrolysis region 110; the detection region 130 is used for detecting the sample to be tested flowing out of the electroenrichment region 120; an electrode assembly 210, which is connected to the first substrate 100 and is used for driving the sample to be tested to flow through the electrolysis region 110 and the electroenrichment region 120 in sequence and enter the detection region 130 for detection.

[0050] The electrolysis region 110 is used for electrolyzing the sample to be tested, and the electrolysis region 110 has a first flow channel 111; the electroenrichment region 120 is used for enriching the sample to be tested flowing out of the first flow channel 111; the detection region 130 is used for detecting the sample to be tested flowing out of the electroenrichment region 120.

[0051] The electrode assembly 210 can be a plurality of metal electrode wires. The electrode assembly 210 is arranged at the bottom of the first substrate 100 and is used for generating an electric field to drive the sample to be tested to flow through the electrolysis region 110 and the electroenrichment region 120 in sequence and enter the detection cell 131 for detection.

[0052] An injection port 112 is arranged on the first substrate 100. The injection port 112 is communicated with the electrolysis region 110, and the electroenrichment region 120 is communicated with the electrolysis region 110. The electrolysis region 110 has a waste liquid outlet 124 and a sampling outlet 125. For the sample to be tested flowing from the electrolysis region 110 to the electroenrichment region 120, part of the sample to be tested flows out from the waste liquid outlet 124, and part of the sample to be tested flows out from the sampling outlet 125 to the detection region 130. The detection region 130 can be a fluorescence detection region. In addition, a liquid outlet is also arranged on the detection region 130. When the detection of the sample to be tested is completed, the sample to be tested can be discharged from the liquid outlet from the microfluidic chip.

[0053] At least two flow channels for the sample to be tested to flow are arranged on the first substrate 100, and the electrolysis region 110, the electroenrichment region 120, and the detection region 130 are communicated through the flow channels.

[0054] It should be noted that the electroenrichment region 120 can include a plurality of enrichment cells 122. Enrichment cells 122 are arranged on each flow channel 140. The number of enrichment cells 122 can be four, and two enrichment cells 122 are arranged on each flow channel 140. At least two waste liquid outlets 124 can be arranged on each enrichment cell 122 to enable the ineffective substances of the sample to be tested to flow out quickly, so as to accelerate the flow of the sample to be tested and improve the enrichment effect.

[0055] The detection area 130 has multiple detection cells 131, and each detection cell 131 corresponds to an enrichment cell 122 one by one, so that the above-mentioned microfluidic chip can detect four different targets simultaneously.

[0056] In addition, this application may further include a second substrate 200. The second substrate 200 is connected to the first substrate 100. An electrode assembly 210 is provided on the second substrate 200, and the electrode assembly 210 is connected to the first substrate 100.

[0057] It should be noted that the microfluidic chip can be fabricated by numerical control machining, and the first substrate 100 and the second substrate 200 can be obtained by cutting an acrylic plate.

[0058] The microfluidic chip includes a first substrate 100 and a second substrate 200, and the first substrate 100 and the second substrate 200 are arranged opposite to each other. The first substrate 100 and the second substrate 200 can be made of the same material and have the same size. Of course, the first substrate 100 and the second substrate 200 can be made of different materials and have different sizes. For example, the overall length of the microfluidic chip can be 90 mm, the width can be 25 mm, and the overall thickness can be 2 mm. Among them, the width of a single flow channel 140 in the electrocracking area 110 is 0.8 mm. There are four enrichment cells 122, and the length of the energized area of each enrichment cell 122 is 4 mm, the width is 2 mm, and the height is 0.3 mm.

[0059] The electrode assembly 210 includes four conductive members. Each enrichment cell 122 is arranged between two conductive members. One of the two conductive members is a positive electrode 212, and the other conductive member is a negative electrode 213. A plurality of grooves 126 are provided in the part of the enrichment cell 122 close to the negative electrode 213 for modifying and fixing the Nafion solution. A selection membrane is provided in the groove 126, and the selection membrane is an ion-selective membrane, so that the effective substances in the sample to be measured can pass through, thereby selectively enriching specific ions or molecules, such as nucleic acids. Modifying and fixing the Nafion solution is to stabilize the Nafion in the groove 126. The Nafion solution is a solution mainly composed of Nafion. Nafion is a perfluorosulfonic acid ion exchange resin, and its molecular structure includes a perfluorocarbon main chain and sulfonic acid groups connected to the main chain, and has ion exchange properties.

[0060] Specifically, 29 uniformly distributed rectangular blocks with a length of 1 mm, a width of 0.1 mm, and a height of 0.1 mm are provided on one side of the enrichment cell 122 close to the negative electrode 213 to form grooves 126 or channels for modifying and fixing the Nafion solution.

[0061] It should be noted that Nafion solution is a colloidal dispersion system containing perfluorosulfonic acid type ion exchange polymer. Its core component is a block copolymer composed of polytetrafluoroethylene main chain and sulfonic acid group side chain, which has a unique amphiphilic structure: the hydrophobic carbon fluorine main chain provides mechanical stability and chemical inertness.

[0062] The detection area 130 has four detection pools 131, and the present application does not limit the structure and size of the detection pools 131. Exemplarily, the detection pools 131 are circular, with a radius of 3 mm and a depth of 1 mm, so as to modify different reagents to react with corresponding targets.

[0063] In addition, the electrode assembly 210 can be arranged inside the second substrate 200 or on the surface of the second substrate 200 (e.g., the side of the second substrate 200 facing the first substrate 100 or the side of the second substrate 200 facing away from the first substrate 100), the second substrate 200 is arranged opposite to the first substrate 100, and the electrode assembly 210 includes a conductive member, which is a wire. In some embodiments, the electrode assembly 210 can also be arranged on the back of the first substrate 100, and the electrode assembly 210 is integrated on the first substrate 100.

[0064] The second substrate 200 is provided with an electrode groove, and the electrode assembly 210 is arranged in the electrode groove, and the width of the electrode groove is 0.8 mm and the depth is 1 mm. Among them, in the microfluidic chip in the embodiment of the present application, at least two flow channels 140 for the flow of the sample to be tested are arranged on the first substrate 100, and the two flow channels 140 are arranged oppositely, and each flow channel 140 connects the electrolysis area 110, the electro-enrichment area 120 and the detection area 130.

[0065] It should be noted that the first substrate 100 is the main body of the chip structure, integrating the electrolysis area 110, the electrical enrichment area 120 and the detection area 130. The second substrate 200 is arranged opposite to the first substrate 100 and is used to carry the electrode assembly 210 to drive the flow of the sample to be tested.

[0066] In addition, in the microfluidic chip of the embodiment of the present application, the flow channel 140 includes a first flow channel 111, which is located in the electrolysis area 110 and at least partially bent toward the edge of the first substrate 100. The sample to be tested is electrolyzed in the first flow channel 111.

[0067] For example, the first flow channel 111 has a plurality of continuous bending segments 114 , and the bending segments 114 are sequentially arranged in a plurality of “X” shapes.

[0068] In a possible implementation, for the microfluidic chip in the embodiments of the present application, the electro-enrichment region 120 has an enrichment pool 122. The enrichment pool 122 is used to receive the sample to be tested flowing through the electro-cracking region 110. The enrichment pool 122 has a waste liquid outlet 124 and a sampling outlet 125. The waste liquid outlet 124 is located above the sampling outlet 125, and the sampling outlet 125 is communicated with the detection pool 131.

[0069] In a possible implementation, for the microfluidic chip in the embodiments of the present application, at least one bending portion 123 is provided on the lower side of the enrichment pool 122 to form the waste liquid outlet 124 and the sampling outlet 125. A sampling tube 121 is provided between the sampling outlet 125 and the enrichment pool 122. The inner diameter of the sampling tube 121 gradually decreases from the enrichment pool 122 to the sampling outlet 125.

[0070] The enrichment pool 122 has a waste liquid outlet 124 and a sampling outlet 125. The sampling outlet 125 is communicated with the detection pool. The waste liquid outlet 124 is located above the sampling outlet 125. A Z-shaped or M-shaped fold angle is provided on the lower side of the enrichment pool 122. The waste liquid outlet 124 is formed above the Z-shaped part, and the sampling outlet 125 is formed below. The diameter of the sampling outlet 125 gradually decreases along the detection direction of the sample to be tested, that is, the diameter facing the detection pool is smaller than the diameter facing the enrichment pool 122.

[0071] The bending structure is a Z-shaped or M-shaped arranged fold angle. The upper fold angle part of the Z-shaped forms the waste liquid outlet 124, and the lower fold angle part forms the sampling outlet 125. The inner diameter of the waste liquid outlet 124 is larger than the maximum inner diameter of the sampling outlet 125. The waste liquid outlet 124 is located above the sampling outlet 125 in the direction of gravity, realizing the layered diversion of the waste liquid and the sample to be tested.

[0072] In a possible implementation, for the microfluidic chip in the embodiments of the present application, the electrode region 210 includes an enrichment electrode region 211. The enrichment electrode region 211 includes a positive electrode 212 and a negative electrode 213. The upper part of the enrichment pool 122 is connected to the positive electrode 212. A connecting portion is provided at the bottom of the enrichment pool 122. A plurality of grooves 126 are arranged side by side on the connecting portion. A selection membrane is provided in the grooves 126. The side of the connecting portion facing away from the enrichment pool 122 is connected or in contact with the negative electrode 213.

[0073] The bottom of the enrichment cell 122 is electrified. For example, the electrode assembly 210 of the second substrate 200 makes the enrichment cell 122 charged. The enrichment cell 122 is arranged on the first substrate 100, and the first substrate 100 and the second substrate 200 are arranged opposite to each other. The electrode region of the second substrate 200 is divided into a positive electrode and a negative electrode, so that the upper part of the enrichment cell 122 is the positive electrode and the lower part is the negative electrode. In the region of the enrichment cell 122 close to the negative electrode, a plurality of grooves 126 or channels are evenly distributed to modify the Nafion membrane to complete the selective permeation of ions, so as to enrich the useful detection substances in the sample to be detected.

[0074] The grooves 126 or channels are evenly distributed along the radial direction of the enrichment cell 122, and their shapes include at least one of strip, polygon or circle, and the depth matches the thickness of the Nafion membrane to achieve the directional modification of the ion channel. The positive electrode 212 and the negative electrode 213 are respectively attached to the upper and lower parts of the enrichment cell 122, and the electrode material is selected from conductive polymers or metal composite materials to optimize the electric field distribution and ion migration efficiency. It should be noted that the conductive part can be a gold electrode, and gold is used as the conducting wire.

[0075] In a possible implementation manner, in the microfluidic chip of the embodiment of the present application, at least one enrichment cell 122 is connected to each flow channel, and the enrichment cells 122 are arranged at intervals in a staggered manner; at least one detection cell 131 is provided, and at least two detection cells 131 are in one-to-one correspondence and communication with the enrichment cells 122.

[0076] For example, the number of enrichment cells 122 is four. Two enrichment cells 122 are arranged on one flow channel 140, and the other two enrichment cells 122 are arranged on another flow channel 140. The two enrichment cells 122 on the same flow channel 140 are arranged at intervals, and the two enrichment cells 122 in one row are located at the corresponding positions of the interval regions of the two enrichment cells 122 in the other row, and the two enrichment cells 122 in the other row are also located at the corresponding positions of the interval regions of the two enrichment cells 122 in one row.

[0077] In a possible implementation manner, in the microfluidic chip of the embodiment of the present application, the flow channel width of the first flow channel 111 is 0.4 mm to 2.0 mm. For example, the flow channel width of the first flow channel 111 is 0.8 mm. The width mainly affects the lysis efficiency by changing the electric field strength in the interval. By changing the width of the lysis flow channel of the chip for verification, five chips with different sizes of 0.4 mm, 0.8 mm, 1.2 mm, 1.6 mm, and 2.0 mm are designed. When the flow channel width of the first flow channel 111 is 0.8 mm, the cell lysis rate reaches the optimum. The width of the groove 126 is 0.05 mm to 0.15 mm, and the length of the groove 126 is 0.5 mm to 1.5 mm.

[0078] In some embodiments, the enrichment pool 122 is square or has a length of 2 mm to 6 mm, and the width of the enrichment pool 122 is 1 mm to 3 mm.

[0079] On the other hand, the present application also provides a method for preparing a microfluidic chip as in the above embodiments, including: S100. Provide a first substrate 100, and an electrolytic cracking region 110, an electro-enrichment region 120, and a detection region 130 are sequentially arranged on the first substrate 100 along the flow direction of the sample to be tested. S200. Perform freeze-drying modification on the first substrate 100, including: dropping a reaction system mixture containing different primers in the detection region 130; pre-cooling the first substrate 100 at a preset time and a preset temperature in sequence, and then drying and freeze-drying under a preset vacuum condition. S300. Provide an electrode assembly 210, the electrode assembly 210 is connected to the first substrate 100, and the electrode assembly 210 is used to drive the sample to be tested to flow through the electrolytic cracking region 110 and the electro-enrichment region 120 in sequence, and enter the detection cell 131 for detection.

[0080] In addition, a second substrate 200 can be provided, an electrode assembly 210 is arranged on the second substrate 200, the electrode assembly 210 is connected to the first substrate 100, and the electrode assembly 210 is used to drive the sample to be tested to flow through the electrolytic cracking region 110 and the electro-enrichment region 120 in sequence, and enter the detection cell 131 for detection.

[0081] S400. Connect the first substrate 100 and the second substrate 200. For example, when bonding the first substrate 100 and the second substrate 200, it includes: fixing wires on the second substrate 200 to form an electrode region; using UV glue to bond the first substrate 100 and the second substrate 200; coating glue along the edge of the substrate in batches, and making the glue penetrate and fill through pressure; completing the encapsulation after curing at room temperature.

[0082] The first substrate 100 and the second substrate 200 of the microfluidic chip can be made of acrylic plates and prepared by 3D printing, and the thickness of the acrylic plate can be 2 mm.

[0083] S200. Perform freeze-drying modification on the first substrate 100, and the specific modification steps are as follows:

[0084] S201: Clean, dry, and modify the acrylic plate, and drop 20 μL of the RT-LAMP reaction system mixture in the fluorescence detection region 130. The primers in the mixture dropped in different regions are different.

[0085] S202: Place the chip in a -20°C refrigerator for pre-cooling, and then place it in an -80°C refrigerator for pre-freezing for 2 hours;

[0086] S203: Take it out and place it in a freeze-dryer, set the temperature to -50°C, set the vacuum degree to 10 Pa, and keep it for more than six hours;

[0087] S204: Raise the temperature to 10 degrees, keep the vacuum degree unchanged, and continue for more than 4 hours;

[0088] S205: After confirming that the reagent is dry, gradually expose it to the vacuum state and fill it with nitrogen to prevent the freeze-dried reagent from getting moist;

[0089] S206: Take out the chip and quickly seal it, place it in a -20-degree refrigerator, and store it in a dry environment for a long time.

[0090] S400. Connect the processed first substrate 100 and the second substrate 200, and the connection method is bonding. The specific bonding steps are as follows:

[0091] S401: Fix the gold wire on the lower second substrate 200 (the material is an acrylic board), that is, an electrode assembly 210 is provided on the second substrate 200;

[0092] S402: Use uv glue to bond the complex structures inside the chip;

[0093] S403: After the bonding is completed, press the upper and lower plates forcefully. Each time, take 20 μL of acrylic glue and slowly trace along the edges of the first substrate 100 and the second substrate 200. The glue slowly enters the inside under the drive of the pressure difference until it is completely filled;

[0094] S404: Press at room temperature until it is firm to obtain the chip used in the experiment.

[0095] The present invention detects the blood of eight different people. Before the detection, each blood sample is processed with 3 different radiation doses, and the untreated one is used as a control group. Through the detection of the actual samples to be tested, the fast detection speed, high sensitivity and other characteristics of the present invention are further demonstrated. As Figure 11 shown, Figure 11 This is the application of the microfluidic chip provided by the embodiment of the present application in clinical practice (a is the physical diagram of the chip for detecting blood samples treated with different radiation irradiations; b is the detection results obtained by detecting eight different samples to be tested, each sample being processed in four different ways).

[0096] In addition, the present application also provides a method for detecting a sample to be tested, which is applied to the microfluidic chip in the above-mentioned embodiment, and includes: introducing the sample to be tested into the electrocracking area 110; energizing the electrode assembly 210 so that the electrode assembly 210 drives the sample to be tested to pass through the electrocracking area 110 and the electroenrichment area 120 in sequence and enter the detection area 130; after the liquid in the detection area 130 reaches a preset value, place the microfluidic chip in a heating device and react for a preset time to obtain the detection result.

[0097] The pretreated sample to be measured is introduced into the liquid inlet of the microfluidic chip; the second substrate 200 is electrically connected to the power supply, an electric field is applied to the electrode region to drive the sample to be measured to sequentially pass through the electro-cracking region 110 and the electro-concentration region 120, and finally enter the detection region 130; after the detection region 130 is filled with liquid, the second substrate 200 is disconnected from the power supply, and the microfluidic chip is placed in a constant temperature device at 65 °C for 30 minutes, and the result is output through the fluorescence signal of the detection region 130. The microfluidic chip, the external power supply, the heating device and the detection device jointly realize the detection of the sample to be measured. Taking the sample to be measured as a cell solution as an example, cell culture is required, which is specifically as follows:

[0098] S01: A549 cells are cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0099] S02: Place the incubator at 37 °C and 5% CO 2 Take out when the cell density reaches 80-90%. Gently aspirate the culture medium and wash twice with PBS.

[0100] S03: Add 0.25% Trypsin-EDTA, evenly cover the cell surface, and place it in an incubator at 37 °C for digestion for 2-3 min. Observe under a microscope that the cells detach from the bottom and stop digestion.

[0101] S04: Transfer the cell suspension to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. Add an appropriate amount of PBS and gently pipette to mix well for standby.

[0102] The electrode assembly 210 is connected to the DC power supply, the voltage is set according to 10v, 20v, 30v, 40v, 50v, and the power-on time increases sequentially according to 5s, 10s, 15s, 20s, 25s. After the power-on is completed, turn off the power supply. After the electro-concentration region 120 collects the cracking products, purification, purity measurement, reverse transcription and other operations are carried out for real-time fluorescence quantitative detection (qRT-PCR).

[0103] In addition, the principle and performance optimization of the chip electro-cracking design provided by the examples of the present application are as follows. As shown below, a fluid and electrode model are established in the corresponding software, and the model of the chip is imported into the software for simulation and adjustment.

[0104] Establish an Electric Currents (EC) physical field, select a two-dimensional structure, and the materials are selected as 1xPBS and gold electrodes. That is, a current field model is established in the simulation software to simulate the distribution and behavior of current in a specific structure. Both 1xPBS and gold electrodes are conductive media.

[0105] The overall potential and electric field distribution inside the chip are obtained through simulation. The electro-lysis region 110 of the microfluidic chip is used to study the lysis effect of the electric field and potential on cells, and the first flow channel 111 is the electro-lysis flow channel. The specific steps are as follows:

[0106] S11: Inject a 1xPBS solution containing cells (about 10 6 cells and about 200 PBS) at the inlet, with a flow rate of 120 (according to the above flow channel splitting simulation, the flow rate per region is 60 ).

[0107] S12: When the sample to be tested completely flows into the electro-lysis region 110, turn on the power supply voltage (30 v). After the liquid has passed through completely, collect the lysed sample to be tested and conduct subsequent tests.

[0108] S13: During the experiment, it was found that the width and length of the flow channel 140 jointly affect the performance of the electro-lysis region 110. The width mainly affects the lysis efficiency by changing the electric field strength in the region.

[0109] S14: Verification was carried out by changing the width of the chip lysis flow channel. Five chips with different sizes of 0.4 mm, 0.8 mm, 1.2 mm, 1.6 mm, and 2.0 mm were designed. Multiple experiments showed that in an environment where the electric field strength is higher than 350 v / cm, the cell lysis rate is similar and reaches the optimum.

[0110] S15: Finally, considering the lysis efficiency and the practicality of the experiment, the flow channel width of the first flow channel 111 of 0.8 mm has a better effect. Therefore, the scheme with a width of 0.8 mm was selected to design the final electro-lysis region 110.

[0111] S16: The length mainly affects the lysis efficiency through the energization time. Set the initial flow rates of the injection pump to 40 , 80 , 120 , 160 , 200 .

[0112] S17: Through experiments, it was found that the lysis efficiency can reach the optimum under the conditions of 80 , 120 . As Figure 6 shown,[[]] Figure 6Verification experiment diagram of the microfluidic chip provided by the embodiment of the present application. Among them, (a is the mortality rate of cells under different voltage lysis conditions photographed under a fluorescence microscope. Generally, green represents live cells stained with AM dye, and red represents dead cells stained with PI dye. It can be seen from Figure a that green are live cells and red are dead cells; b is a line graph of the mortality rate of electro-lysed cells under different voltages and times; c is the detection result obtained by real-time quantitative fluorescence detection (qPCR) of the products of electro-lysed cells under different voltages and times. The optimal condition is a voltage of 30V and an energization time of 10s, and the CT value under this condition is 16.79).

[0113] Figure 7 Verification experiment diagram of the microfluidic chip provided by the embodiment of the present application for different samples to be measured. Among them, the lysis effect of electro-lysis is compared with that of a commercial kit. (a is the result obtained by capillary electrophoresis of the products of electro-lysis and reagent lysis. The red area is the core detection area 130; b is a comparison of the core areas in the capillary electrophoresis results of electro-lysis and kit lysis; c is a differential calculation of the above core area, electro-lysis, and reagent lysis to obtain the corresponding area ratio; d is a qPCR detection of the products of electro-lysis and reagent lysis, and a comparison of the detection results of four target genes, BAX, CDKN1A, GADD45A, and GAPDH).

[0114] In addition, this embodiment also provides the principle and performance optimization of chip electro-concentration design. An object model is established in the software, and the model of the chip is imported into the software. Establish Laminar Flow (SPF) and Transport of Diluted Species (T physical fields, and establish a multi-physical field. Select a two-dimensional structure, and the material is selected as 1xPBS solution. Determine the approximate enrichment area and flow direction of negatively charged molecules in the chip through simulation. Use the electro-concentration area 120 of the microfluidic chip to study the enrichment effect of the electric field and flow rate on anions, specifically as follows:

[0115] The enrichment sites are mainly affected by the flow rate of the sample to be measured and the electric field strength. In this experiment, the same initial flow rate (optimal flow rate) is used, and the difference in enrichment sites is mainly explored by changing the magnitude of the electric field strength.

[0116] Five enrichment pools 122 with different sizes of 2cm×1cm, 1.2cm×0.6cm, 0.8cm×0.4cm, 0.4cm×0.2cm, and 0.2cm×0.1cm in length and width were prepared in the experiment for exploration.

[0117] This application uses 20 , 30 The initial flow rate is used to introduce the sample to be tested, which is a fluorescent sample to be tested. Five different magnitudes of electric field strength are provided: 50 , 100 , 150 , 200 , 250 . Through multiple experiments, it is found that the larger the width of the enrichment pool 122, the greater the electric field strength required to achieve stable enrichment. For the same enrichment size, if the electric field strength is too small, a stable enrichment band may not be formed. If the electric field strength is too large, enrichment sites will be formed prematurely, and a "dispersion" phenomenon will occur. As Figure 8 shown, Figure 8 is the functional verification experimental diagram of the electro-enrichment of the microfluidic chip provided by the embodiment of the present application (a shows the enrichment of the sample to be tested in the chip under different voltage and time conditions; b shows the comparison of the effects of chip electro-enrichment and commercial kit purification; c shows the enrichment effects of in-chip electro-enrichment and kit purification for different DNA concentrations; d shows that the enrichment results of the kit for different DNA concentrations have a linear correlation; e shows that the enrichment results of chip electro-enrichment for different DNA concentrations have a linear correlation).

[0118] Combined with the voltage conditions in electro-cracking, an enrichment pool 122 with a size of 0.4 cm × 0.2 cm was finally determined for subsequent experiments.

[0119] In the embodiments of the present application, the sensitivity of the detection area 130 (such as the fluorescence detection area) in the microfluidic chip is also provided. Standard plasmids used in the experiment. The four standard plasmids of BAX, CDKN1A, GADD45A, and GAPDH were respectively diluted to 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 . In the four-well plate, BAX, CDKN1A, GADD45A, GAPDH primers and LAMP reaction system reagents were modified from left to right.

[0120] The plasmid samples to be tested at each concentration were added dropwise in sequence according to the concentration gradient. The completed detection pool 131, such as the four-well plate, was placed on a water bath heating box.

[0121] After the reaction was completed, the well plate was taken out and placed in a photography studio for fluorescence photography. And the data of the photographed results were statistically analyzed, and it was found that the fluorescence intensities generated by the four target reactions all had a linear correlation with the concentration. Figure 9Verification diagram of module differences in the electro-enrichment region of the microfluidic chip provided by the embodiments of the present application (a is the difference comparison of the upper two channels, in which four targets of BAX, CADKN1A3, GADD45A, and GAPDH are detected and compared; b is the difference comparison of the lower two channels, in which four targets of BAX, CADKN1A3, GADD45A, and GAPDH are detected and compared).

[0122] The primer sequences of BAX, CDKN1A, GADD45A, and GAPDH are as follows in the table:

[0123] Table 1 shows the primer sequence of BAX

[0124]

[0125] Among them, Amplification represents the amplification type, primer represents the primer, Sequence (5'-3') represents the sequence (5'-3'), and PCR primers represent conventional polymerase chain reaction, which is used to amplify a specific region of the BAX gene in traditional PCR.

[0126] LAMP primers represent loop-mediated isothermal amplification, which is used in the LAMP technique to rapidly amplify the BAX gene under certain temperature conditions. Forward represents the forward primer, and Reverse represents the reverse primer.

[0127] Fip (Forward Inner Primer) includes the F2 region (binding to the target sequence) and the F1c region (initiating strand displacement), which is used to form a circular structure. Bip (Backward Inner Primer) contains the B1c region and the B2 region, which is used for the amplification of the reverse complementary strand.

[0128] F3 (Forward Outer Primer) has the same sequence as the forward primer of PCR and is used for initial amplification. B3 (Backward Outer Primer) has the same sequence as the reverse primer of PCR and is used for initial amplification. F1c is the complementary region of F1, and both are used to participate in the formation of the circular structure. F2 and B2 are used to bind to the target sequence and initiate strand displacement.

[0129] Loop F and Loop B are used to accelerate the reaction and promote the extension of the circular structure.

[0130] The LAMP primer contains 6 core primers (Fip / Bip / F3 / B3 / F2 / B2) and 2 loop primers (LoopF / LoopB), and can achieve the effect of efficient amplification by recognizing multiple regions of the target gene.

[0131] Table 2 shows the primer sequences of CDKN1A

[0132]

[0133] Table 3 shows the primer sequences of GADD45A

[0134]

[0135] Table 4 shows the primer sequences of GAPDH

[0136]

[0137] This example also provides a method using a microfluidic chip to detect fluorescence intensity. The specific operation steps are as follows:

[0138] S31: In this experiment, 1.2 mL of the sample to be tested was processed to detect mRNA biomarkers related to cell repair.

[0139] S32: Fix the chip on a plane, connect the injection pump and the DC power supply, and introduce the sample to be tested into the microfluidic chip at a speed of 120 per unit.

[0140] S33: When the sample to be tested enters the electro-lysis zone 110, turn on the DC power supply to form a high electric field strength, and rapidly electro-lyze the sample in the electro-lysis zone 110 to break the cell membrane and release the components inside the cell, including mRNA. To distinguish the characteristics of reagent lysis and electro-lysis after lysis, we used an enzyme-linked immunosorbent assay (ELISA) reader for detection. The results are as follows:

[0141] Table 5 shows the detection results of the ELISA reader

[0142]

[0143] Among them, well refers to the detection cell 131, and 260, 230, and 280 represent different wavelengths, with the unit of nm. Among them, 260 nm refers to the maximum absorption wavelength of nucleic acids, 230 nm refers to the absorption wavelength of carbohydrates, polypeptides, or certain organic pollutants, and 280 nm is mainly used to detect whether there is protein contamination in the sample. The absorbance ratios at these wavelengths (such as 260 / 280 and 260 / 230) are important indicators for evaluating the purity of nucleic acid samples. For example, the A260 / A280 ratio of pure DNA is usually about 1.8, and that of pure RNA is about 2.0; while the A260 / A230 ratio is usually between 2.0 and 2.2, and a value lower than this range may indicate carbohydrate or salt contamination.

[0144] S34: A small amount of bubbles generated during the electro-lysis process, due to their electrical neutrality, are confined to the area near the cathode and do not affect the flow of the lysate to the next functional area.

[0145] S35: The liquid sample to be tested after lysis is then guided to the electro-enrichment region 120. Under the combined action of the lateral driving force and the vertical enrichment force, mRNA molecules are concentrated to form an "ion flow", and are transported to the detection region 130 through the sampling tube 121.

[0146] S36: The entire enrichment process avoids bubble interference, ensures rapid enrichment of the mRNA target within 10 seconds, reaches a concentration multiple of about 10 times, and directly flows into the detection region 130 after the formation of the enrichment site.

[0147] S37: During the detection, the detection cell 131 in the detection region 130 will be pre-loaded with freeze-dried reagents, including reverse transcriptase, primers, LAMP buffer, and other components required for the reaction. The enriched mRNA is mixed with the freeze-dried reagents, and the chip is placed on a thermostatic water bath heating container for isothermal amplification.

[0148] S38: The width of the effective enrichment region of the sampling tube 121 is 0.2 mm. An area of about 30 is reserved in the detection region 130 to collect the enriched mRNA.

[0149] Thus, it significantly increases the initial concentration of mRNA in the reaction, and the loss rate of mRNA during the enrichment process is relatively low. The increase in this concentration greatly improves the overall sensitivity of the detection process, making this system particularly suitable for detecting low-abundance mRNA biomarkers in blood. As Figure 10 shown, Figure 10 This is the test verification diagram of the fluorescence detection sensitivity of the microfluidic chip provided by the embodiment of the present application (a is the detection of Tq values for different concentrations of BAX plasmids, and there is a linear correlation between the logarithm of the concentration and the Tq value; b is the detection of Tq values for different concentrations of CDKN1A plasmids, and there is a linear correlation between the logarithm of the concentration and the Tq value; is the detection of Tq values for different concentrations of GADD454A plasmids, and there is a linear correlation between the logarithm of the concentration and the Tq value; is the detection of Tq values for different concentrations of GAPDH plasmids, and there is a linear correlation between the logarithm of the concentration and the Tq value).

[0150] By combining a microfluidic chip based on electro-lysis, electro-enrichment, and chemical amplification methods, there is great potential to simplify the preparation and detection processes of the sample to be tested in the high-efficiency analysis of cancer biomarkers.

[0151] This application integrates the lysis, electro-enrichment, and nucleic acid amplification detection of the sample to be tested on a microfluidic chip. Compared with traditional methods, it has the advantages of being fast, sensitive, and accurate, and can be widely applied to point-of-care testing (POCT), especially in the fields of early cancer screening, radiation exposure assessment, and real-time disease monitoring. The microfluidic chip includes two parallel electro-lysis zones 110, and an accurate inlet shunt structure is used to ensure the dynamic matching of the lysis time of the sample to be tested and the initial flow rate of electro-enrichment. The lysed sample to be tested directly enters the enrichment zone, and the optimized channel design minimizes the flow rate difference between different enrichment channels, improving the enrichment efficiency and stability.

[0152] In addition, at the outlet of the enrichment zone, the target molecules in the enrichment band are collected to the greatest extent, thereby improving the detection sensitivity. As shown by the experimental results, this can enrich the target molecules by up to 15 times. The entire system can process up to 1.2 mL of the blood sample to be tested at a time, and can complete lysis and enrichment within 20 seconds. Subsequently, the enriched sample to be tested is transported to the LAMP amplification module in the detection zone 130 to achieve rapid and efficient nucleic acid detection throughout the process. It should be noted that the LAMP amplification module (Loop-mediated Isothermal Amplification Module) is the core operation unit of the loop-mediated isothermal amplification technology, and its full Chinese name is the loop-mediated isothermal amplification module. This module integrates reaction reagents, primer design, and detection systems to achieve efficient amplification of target DNA / RNA at a certain temperature (60 - 65 degrees), without relying on the thermal cycling equipment of traditional PCR.

[0153] Those skilled in the art will readily think of other embodiments of this application after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application, which follow the general principles of this application and include the common general knowledge or conventional technical means in this technical field not disclosed in this application. The specification and examples are only regarded as exemplary.

[0154] It should be understood that this application is not limited to the exact structure described above and shown in the drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A microfluidic chip for nucleic acid detection, characterized in that: include: A first matrix (100), wherein the first matrix (100) is provided with an electrolysis zone (110), an electro-enrichment zone (120), and a detection zone (130) in sequence along the flow direction of the sample to be tested; The electrolysis zone (110) is used for electrolysis of a sample to be tested; The electro-enrichment zone (120) is used to enrich the sample to be tested flowing out of the electro-lysis zone (110); The detection area (130) is used to detect the sample to be tested flowing out of the electrical enrichment area (120); An electrode assembly (210), the electrode assembly (210) being connected to the first substrate (100), the electrode assembly (210) being used to drive the sample to be tested to flow through the electrolysis zone (110) and the electro-enrichment zone (120) in sequence and enter the detection zone (130) for detection.

2. The microfluidic chip according to claim 1, characterized in that: At least two flow channels (140) for the flow of a sample to be tested are arranged on the first substrate (100), the two flow channels (140) are arranged opposite to each other, and each flow channel (140) is connected to the electrolysis area (110), the electro-enrichment area (120) and the detection area (130).

3. The microfluidic chip according to claim 2, characterized in that: The flow channel (140) comprises a first flow channel (111), the first flow channel (111) being located in the electrolysis zone (110), and the first flow channel (111) being at least partially bent towards an edge of the first substrate (100).

4. The microfluidic chip according to any one of claims 2 to 3, characterized in that: The electro-enrichment zone (120) has an enrichment pool (122), the enrichment pool (122) is used to receive the sample to be tested that flows through the electrolysis zone (110), the enrichment pool (122) has a waste liquid outlet (124) and a sampling outlet (125), the waste liquid outlet (124) is located above the sampling outlet (125), and the sampling outlet (125) is connected to the detection zone (130).

5. The microfluidic chip according to claim 4, characterized in that: At least one bent portion (123) is provided on the lower side of the enrichment tank (122) to form the waste liquid outlet (124) and the sampling outlet (125); a sampling tube (121) is provided between the sampling outlet (125) and the enrichment tank (122); and the inner diameter of the sampling tube (121) gradually decreases in a direction from the enrichment tank (122) to the sampling outlet (125).

6. The microfluidic chip according to claim 4, characterized in that: The electrode assembly (210) has an enrichment electrode area (211), the enrichment electrode area (211) includes a positive electrode (212) and a negative electrode (213), the upper part of the enrichment tank (122) is connected to the positive electrode (212), the bottom of the enrichment tank (122) is provided with a connection part, a plurality of grooves (126) are arranged side by side on the connection part, a selection membrane is arranged in the groove (126), and the connection part is connected or in contact with the negative electrode (213) on a side away from the enrichment tank (122).

7. The microfluidic chip according to claim 4, characterized in that: Each of the flow channels (140) is connected to at least one enrichment pool (122), and the enrichment pools (122) are arranged in an alternating manner; The detection area (130) has at least one detection pool (131), and each of the detection pools (131) is connected to the enrichment pool (122) in a one-to-one correspondence.

8. The microfluidic chip according to claim 3, characterized in that: The flow channel width of the first flow channel (111) is 0.4 mm to 2.0 mm.

9. A method for preparing a microfluidic chip, characterized in that: include: Providing a first matrix (100), wherein the first matrix (100) is provided with an electrolysis area (110), an electro-enrichment area (120), and a detection area (130) in sequence along a flow direction of a sample to be tested; The first matrix (100) is subjected to freeze-drying modification, comprising: dropping a reaction system mixture containing different primers into the detection area (130); precooling the first matrix (100) for a preset time and a preset temperature, and then drying and freeze-drying the first matrix (100) under preset vacuum conditions; An electrode assembly (210) is provided, the electrode assembly (210) being connected to the first substrate (100), and the electrode assembly (210) being used to drive the sample to be tested to flow through the electrolysis zone (110) and the electro-enrichment zone (120) in sequence and enter the detection zone (130) for detection.

10. A method for detecting a sample to be tested, applied to the microfluidic chip according to any one of claims 1 to 8, characterized in that: include: Passing the sample to be tested into the electrolysis zone (110); Powering the electrode assembly (210) so that the electrode assembly (210) drives the sample to be tested to sequentially pass through the electrolysis zone (110) and the electro-enrichment zone (120) and enter the detection zone (130); After the liquid in the detection area (130) reaches a preset value, the microfluidic chip is placed in a heating device to react for a preset time to obtain a detection result.

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