A microfluidic chip, its fabrication method and detection method

Abstract

This application provides a microfluidic chip, its preparation method, and a detection method, relating to the field of nucleic acid detection technology. The chip includes: a first substrate, on which an electrolytic region, an electroenrichment region, and a detection region are sequentially disposed along the flow direction of the sample to be tested; the electrolytic region is used for electrolytically breaking down the sample to be tested; the electroenrichment region is used for enriching the sample to be tested flowing out of the electrolytic region; the detection region is used for detecting the sample to be tested flowing out of the electroenrichment region; and an electrode assembly connected to the first substrate. This application integrates the electrolytic region, the electroenrichment region, and the detection region. The electrode assembly drives the sample to flow rapidly, sequentially passing through the electrolytic region and the electroenrichment region, enriching effective substances in the electroenrichment region, reducing losses, ensuring the detection concentration of the sample entering the detection region, meeting the needs of instantaneous detection, and improving the speed, sensitivity, and accuracy of sample detection.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection technology, and in particular to a microfluidic chip, its preparation method, and its inspection method. Background Technology

[0002] mRNA detection is a nucleic acid testing method that analyzes the expression levels of messenger ribonucleic acid (MRNA) to reveal gene expression patterns, identify disease-related biomarkers, and guide personalized medicine or vaccine development. For example, in individuals affected by radiation, mRNA in the blood can be used to assess the radiation dose in order to facilitate medical intervention.

[0003] In existing technologies, mRNA detection mainly includes RT-PCR, RNA sequencing (RNA-seq), and microarray. These methods are usually based on peripheral blood samples for analysis and are supplemented by chromosomal gene mutation detection to assess genomic instability.

[0004] However, the above-mentioned tests are costly and time-consuming. Furthermore, as the testing time increases, the mRNA degrades rapidly, reducing the accuracy of the test 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 as the detection time increases, which reduces the accuracy of the detection results.

[0006] On one hand, this application provides a microfluidic chip for nucleic acid detection, comprising: a first substrate, wherein the first substrate is sequentially provided with an electrolytic region, an electroenrichment region and a detection region along the flow direction of the sample to be tested; the electrolytic region is used for electrolytically dissolving the sample to be tested; the electroenrichment region is used for enriching the sample to be tested flowing out of the electrolytic region; the detection region is used for detecting the sample to be tested flowing out of the electroenrichment region; and an electrode assembly connected to the first substrate, wherein the electrode assembly is used to drive the sample to be tested to flow sequentially through the electrolytic region and the electroenrichment region and enter the detection cell for detection.

[0007] In one possible implementation, the microfluidic chip in this application embodiment has at least two channels on a first substrate for supplying the flow of the sample to be tested. The two channels are arranged opposite to each other, and each channel connects the electrolytic region, the electroenrichment region, and the detection region.

[0008] In one possible implementation, the microfluidic chip in this application embodiment has a flow channel including a first flow channel located in the electrolytic region, and the first flow channel is at least partially bent toward the edge of the first substrate.

[0009] In one possible implementation, the microfluidic chip in this embodiment has an enrichment cell in the electro-enrichment region. The enrichment cell is used to receive the sample to be tested flowing through the electrolytic region. The enrichment cell has a waste liquid outlet and a sampling outlet. The waste liquid outlet is located above the sampling outlet, and the sampling outlet is connected to the detection region.

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

[0011] In one possible implementation, the microfluidic chip in this application embodiment has an electrode region including an enrichment electrode region, which includes a positive electrode and a negative electrode. The upper part of the enrichment cell is connected to the positive electrode, and the bottom of the enrichment cell is provided with a connection part. Multiple grooves are arranged side by side on the connection part, and a membrane is disposed in the grooves. The side of the connection part away from the enrichment cell is connected to or in contact with the negative electrode.

[0012] In one possible implementation, the microfluidic chip in this application embodiment has at least one enrichment cell connected to each flow channel, and the enrichment cells are arranged alternately at intervals; the detection area has at least one detection cell, and each detection cell is connected to the enrichment cell in a one-to-one correspondence.

[0013] In one possible implementation, the microfluidic chip in this application embodiment has an electrolytic flow channel width of 0.4 mm to 2.0 mm.

[0014] On the other hand, this application also provides a method for fabricating a microfluidic chip as described in the above embodiments, comprising: providing a first substrate, wherein the first substrate is sequentially provided with an electrolytic region, an electro-enrichment region, and a detection region along the flow direction of the sample to be tested; performing freeze-drying modification on the first substrate, including: adding a reaction system mixture containing different primers to the detection region; pre-cooling the first substrate sequentially for a preset time and a preset temperature, and then drying and freeze-drying it under preset vacuum conditions; providing an electrode assembly, wherein the electrode assembly is connected to the first substrate, and the electrode assembly is used to drive the sample to be tested to sequentially flow through the electrolytic region and the electro-enrichment region and enter the detection cell for detection.

[0015] In addition, this application also provides a method for detecting a sample to be tested, applied to a microfluidic chip as described in the above embodiments, comprising: passing the sample to be tested into an electrolytic region; energizing an electrode assembly to drive the sample to be tested sequentially through an electrolytic region and an electroaccumulation region, and into a detection region; after the liquid in the detection region reaches a preset value, placing the microfluidic chip in a heating device to react for a preset time to obtain a detection result.

[0016] This application provides a microfluidic chip, its fabrication method, and a detection method. The chip includes: a first substrate, on which an electrolytic region, an electroenrichment region, and a detection region are sequentially disposed along the flow direction of the sample to be tested; the electrolytic region is used for electrolytically breaking down the sample to be tested; the electroenrichment region is used for enriching the sample to be tested flowing out of the electrolytic region; the detection region is used for detecting the sample to be tested flowing out of the electroenrichment region; and an electrode assembly connected to the first substrate, which drives the sample to be tested to flow sequentially through the electrolytic region and the electroenrichment region before entering the detection region for detection. This application integrates the electrolytic region, the electroenrichment region, and the detection region. The electrode assembly drives the sample to flow rapidly, sequentially through the electrolytic region and the electroenrichment region, and rapidly enriches the effective substance in the electroenrichment region, reducing losses and ensuring the detection concentration of the sample to be tested entering the detection region. This meets the needs of real-time detection and improves the speed, sensitivity, and accuracy of sample detection. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0018] Figure 1 This is a schematic diagram of the structure of the microfluidic chip provided in the embodiments of this application;

[0019] Figure 2 for Figure 1 Another structural diagram of a microfluidic chip;

[0020] Figure 3 for Figure 1 Schematic diagram of the medium enrichment tank;

[0021] Figure 4 for Figure 1 Electric field intensity distribution diagram of the electrolytic cracking region;

[0022] Figure 5 for Figure 1 A schematic diagram of the lyophilized reagent modification of a microfluidic chip;

[0023] Figure 6 A verification experiment diagram of the microfluidic chip provided in the embodiments of this application;

[0024] Figure 7 The diagram shows the verification experiments of the microfluidic chip provided in the embodiments of this application on different test samples;

[0025] Figure 8 Experimental diagram for functional verification of electrical enrichment of microfluidic chip provided in the embodiments of this application;

[0026] Figure 9 A module difference verification diagram of the electrically enriched region of the microfluidic chip provided in the embodiments of this application;

[0027] Figure 10 The graph shows the test and verification of the fluorescence detection sensitivity of the microfluidic chip provided in the embodiments of this application.

[0028] Figure 11 The diagram shows the clinical application verification experiment of the microfluidic chip provided in the embodiments of this application;

[0029] Figure 12 A flowchart illustrating the fabrication method of the microfluidic chip provided in this application embodiment.

[0030] Explanation of reference numerals in the attached figures:

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

[0032] The accompanying drawings illustrate specific embodiments of this application, which will be described in more detail below. These drawings and descriptions are not intended to limit the scope of the concept in any way, but rather to illustrate the concept of this application to those skilled in the art through reference to particular embodiments. Detailed Implementation

[0033] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application.

[0034] It should be noted that in the description of the embodiments of this application, the terms "upper", "lower", "inner", "outer" and other terms indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of description, and are not intended to indicate or imply that the device or component must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the embodiments of this application.

[0035] Furthermore, it should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0036] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "fixation," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, an electrical connection, or a connection that allows communication between them; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication between two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

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

[0038] mRNA refers to messenger ribonucleic acid.

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

[0040] RNA sequencing (RNA-seq) is a method for detecting mRNA. It uses transcriptome sequencing to perform unbiased sequencing of RNA molecules in the sample, covering both known and unknown transcripts. Its principles include RNA fragmentation, reverse transcription into a cDNA library, sequencing, and bioinformatics analysis.

[0041] Microarray: refers to an mRNA detection method based on the principle of probe hybridization, in which DNA or RNA probes with known sequences are immobilized on a solid support, and the expression level of a specific gene is detected by fluorescent labeling.

[0042] mRNA testing analyzes the expression levels of messenger RNA to reveal gene expression patterns, identify disease-related biomarkers, and guide personalized medicine or vaccine development. For example, in individuals affected by radiation, mRNA in the blood can be used to assess the radiation dose in order to facilitate medical intervention.

[0043] In existing technologies, mRNA detection mainly includes RT-PCR, RNA sequencing (RNA-seq), and microarray. These methods are usually based on peripheral blood samples for analysis and are supplemented by chromosomal gene mutation detection to assess genomic instability.

[0044] However, the above-mentioned detection methods are costly (e.g., expensive equipment and reagents, reliance on professional technicians), take a long time (usually about 72 hours), and as the detection time increases, mRNA degrades rapidly, reducing the accuracy of the test results.

[0045] Furthermore, the lysis and purification of analytes still rely on chemical lysis buffers, magnetic beads, or silica membranes. These techniques typically require the analyte to remain in a specific area for an extended period to complete the reaction, making process control difficult. Moreover, to prevent analyte loss or degradation, mechanical disturbances must be minimized, further prolonging detection time and reducing efficiency. Therefore, traditional chip-based detection techniques primarily simplify experimental procedures rather than significantly improving detection quality. Additionally, the use of multiple reagents can lead to cross-reactions within the chip, affecting the accuracy of the results.

[0046] In view of this, this application provides a microfluidic chip, a fabrication method, and a detection method. The chip includes: a first substrate, on which an electrolytic region, an electroenrichment region, and a detection region are sequentially disposed along the flow direction of the sample to be tested; the electrolytic region is used for electrolytically breaking down the sample to be tested; the electroenrichment region is used for enriching the sample to be tested flowing out of the electrolytic flow channel; the detection region is used for detecting the sample to be tested flowing out of the electroenrichment region; and an electrode assembly connected to the first substrate, which is used to drive the sample to be tested to flow sequentially through the electrolytic region and the electroenrichment region, and then enter the detection region for detection. This application integrates the electrolytic region, the electroenrichment region, and the detection region. The electrode assembly drives the sample to flow rapidly, sequentially through the electrolytic region and the electroenrichment region, and rapidly enriches the effective substance in the electroenrichment region, reducing losses and ensuring the detection concentration of the sample to be tested entering the detection region, thereby improving the speed, sensitivity, and accuracy of the detection of the sample to be tested.

[0047] The following is combined Figures 1 to 12 The present application describes the microfluidic chip, preparation method and detection method involved through specific embodiments. The embodiments can be combined arbitrarily, and this application does not limit them.

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

[0049] On one hand, this application provides a microfluidic chip for nucleic acid detection, comprising: a first substrate 100, wherein the first substrate 100 is sequentially provided with an electrolytic region 110, an electroenrichment region 120 and a detection region 130 along the flow direction of the sample to be tested; the electrolytic region 110 is used for electrolytically dissecting the sample to be tested; the electroenrichment region 120 is used for enriching the sample to be tested flowing out of the electrolytic region 110; the detection region 130 is used for detecting the sample to be tested flowing out of the electroenrichment region 120; and an electrode assembly 210, which is connected to the first substrate 100 and is used to drive the sample to be tested to flow sequentially through the electrolytic region 110 and the electroenrichment region 120 and enter the detection region 130 for detection.

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

[0051] The electrode assembly 210 can be multiple metal electrode wires. The electrode assembly 210 is disposed at the bottom of the first substrate 100. The electrode assembly 210 is used to generate an electric field to drive the sample to be tested to flow sequentially through the electrolytic region 110 and the electro-enrichment region 120 and enter the detection cell 131 for detection.

[0052] A sample inlet 112 is provided on the first substrate 100, which is connected to the electrolysis zone 110, and the electroenrichment zone 120 is also connected to the electrolysis zone 110. The electrolysis zone 110 has a waste liquid outlet 124 and a sampling outlet 125. The sample to be tested flowing from the electrolysis zone 110 to the electroenrichment zone 120 has a portion flowing out from the waste liquid outlet 124 and a portion flowing out from the sampling outlet 125 to the detection zone 130. The detection zone 130 can be a fluorescence detection zone. In addition, the detection zone 130 is also provided with a liquid outlet, so that the sample can be easily discharged from the microfluidic chip after the detection is completed.

[0053] At least two flow channels are provided on the first substrate 100 for the flow of the sample to be tested, and the flow channels connect the electrolytic region 110, the electro-enrichment region 120 and the detection region 130.

[0054] It should be noted that the electro-enrichment zone 120 may include multiple enrichment cells 122, and each flow channel 140 is provided with an enrichment cell 122. The number of enrichment cells 122 can be four, with two enrichment cells 122 provided on each flow channel 140. Each enrichment cell 122 may be provided with at least two waste liquid outlets 124 to allow the ineffective substances of the sample to flow out quickly, thereby accelerating the flow of the sample and improving the enrichment effect.

[0055] The detection area 130 has multiple detection pools 131, each of which corresponds to an enrichment pool 122, enabling the microfluidic chip to detect four different targets simultaneously.

[0056] Furthermore, this application may also include a second substrate 200 connected to the first substrate 100, and an electrode assembly 210 disposed on the second substrate 200, the electrode assembly 210 being connected to the first substrate 100.

[0057] It should be noted that microfluidic chips can be manufactured using CNC machining, and the first substrate 100 and the second substrate 200 can be obtained by cutting acrylic sheets.

[0058] The microfluidic chip includes a first substrate 100 and a second substrate 200, which 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 dimensions; however, they can also be made of different materials and have different dimensions. For example, the overall length of the microfluidic chip can be 90 mm, the width 25 mm, and the overall thickness 2 mm. The width of a single flow channel 140 in the electrolytic region 110 is 0.8 mm, and there are four enrichment cells 122. Each enrichment cell 122 has an energized area with a length of 4 mm, a width of 2 mm, and a height of 0.3 mm.

[0059] Electrode assembly 210 includes four conductive elements. Each enrichment cell 122 is disposed between two conductive elements, one of which is a positive electrode 212, and the other is a negative electrode 213. The portion of the enrichment cell 122 near the negative electrode 213 has multiple grooves 126 for modifying and immobilizing the Nafion solution. A selective membrane, specifically an ion-selective membrane, is disposed within the grooves 126 to allow the passage of active substances in the analyte, thereby selectively enriching specific ions or molecules, such as nucleic acids. Modifying and immobilizing the Nafion solution stabilizes the Nafion within the grooves 126. The Nafion solution is a solution with Nafion as its main component. Nafion is a perfluorosulfonic acid ion exchange resin whose molecular structure contains a perfluorocarbon backbone and sulfonic acid groups attached to the backbone, exhibiting ion exchange properties.

[0060] Specifically, the enrichment cell 122 has 29 uniformly distributed rectangular blocks with a length of 1 mm, a width of 0.1 mm, and a height of 0.1 mm on the side near 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 a perfluorosulfonic acid-type ion exchange polymer. Its core component is a block copolymer composed of a polytetrafluoroethylene backbone and sulfonic acid side chains, possessing a unique amphiphilic structure: the hydrophobic fluorocarbon backbone provides mechanical stability and chemical inertness.

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

[0063] Furthermore, the electrode assembly 210 may be disposed inside the second substrate 200 or on the surface of the second substrate 200 (e.g., on the side of the second substrate 200 facing the first substrate 100 or on the side of the second substrate 200 away from the first substrate 100). The second substrate 200 is disposed opposite to the first substrate 100. The electrode assembly 210 includes a conductive element, which is a wire. In some embodiments, the electrode assembly 210 may also be disposed on the back of the first substrate 100, integrating the electrode assembly 210 onto the first substrate 100.

[0064] The second substrate 200 is provided with an electrode groove, and the electrode assembly 210 is disposed in the electrode groove. The electrode groove has a width of 0.8 mm and a depth of 1 mm. In the microfluidic chip of this application embodiment, the first substrate 100 is provided with at least two flow channels 140 for the flow of the sample to be tested. The two flow channels 140 are arranged opposite to each other, and each flow channel 140 connects the electrolysis region 110, the electroenrichment region 120 and the detection region 130.

[0065] It should be noted that the first substrate 100 is the main body of the chip structure, integrating the electrolytic region 110, the electro-enrichment region 120 and the detection region 130. The second substrate 200 is disposed opposite to the first substrate 100 and is used to support the electrode assembly 210 to drive the flow of the sample to be tested.

[0066] Furthermore, in the microfluidic chip of this embodiment, the flow channel 140 includes a first flow channel 111, which is located in the electrolytic region 110, and the first flow channel 111 is at least partially bent toward the edge of the first substrate 100. The sample to be tested undergoes electrolytic degradation within the first flow channel 111.

[0067] For example, the first flow channel 111 has multiple continuous bends 114, and each bend 114 is arranged in a series of Z-shaped configurations.

[0068] In one possible implementation, the microfluidic chip in this embodiment has an electro-enrichment region 120 with an enrichment cell 122 for receiving the sample to be tested flowing through the electrolytic region 110. The enrichment cell 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 cell 131.

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

[0070] The enrichment tank 122 has a waste liquid outlet 124 and a sampling outlet 125. The sampling outlet 125 is connected to the detection tank. The waste liquid outlet 124 is located above the sampling outlet 125. The lower side of the enrichment tank 122 is provided with a Z-shaped or M-shaped bend. The waste liquid outlet 124 is formed above the Z-shape, and the sampling outlet 125 is formed below it. The diameter of the sampling outlet 125 decreases sequentially along the detection direction of the sample to be tested, that is, the diameter facing the detection tank is smaller than the diameter facing the enrichment tank 122.

[0071] The bending structure consists of Z-shaped or M-shaped bends, with the upper bend of the Z-shape forming the waste liquid outlet 124 and the lower bend forming 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, achieving stratified flow guidance between the waste liquid and the sample to be tested.

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

[0073] The bottom of the enrichment cell 122 is energized, for example, by the electrode assembly 210 of the second substrate 200. The enrichment cell 122 is disposed on the first substrate 100, which is positioned opposite to the second substrate 200. The electrode area 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 near the negative electrode, multiple grooves 126 or channels are uniformly distributed to modify the Nafion membrane to achieve selective ion permeation, thereby enriching the useful detection substances in the sample.

[0074] Grooves 126 or channels are uniformly distributed radially along the enrichment cell 122, and their shapes include at least one of strip, polygon, or circle, with depth matching the thickness of the Nafion film to achieve directional modification of ion channels. Positive electrode 212 and negative electrode 213 are respectively attached to the upper and lower parts of the enrichment cell 122, and the electrode materials are selected from conductive polymers or metal composites to optimize electric field distribution and ion migration efficiency. It should be noted that the conductive element can be a gold electrode, using gold as the conductive wire.

[0075] In one possible implementation, the microfluidic chip in this embodiment of the application has at least one enrichment pool 122 connected to each flow channel, and the enrichment pools 122 are arranged alternately at intervals; at least one detection pool 131 is provided, and at least two detection pools 131 are connected to the enrichment pools 122 in a one-to-one correspondence.

[0076] For example, there are four enrichment pools 122. Two enrichment pools 122 are set on one flow channel 140, and the other two enrichment pools 122 are set on another flow channel 140. The two enrichment pools 122 in the same flow channel 140 are arranged at intervals. The two enrichment pools 122 in one row are located at the position corresponding to the interval area of ​​the two enrichment pools 122 in the other row. The two enrichment pools 122 in the other row are also located at the position corresponding to the interval area of ​​the two enrichment pools 122 in the first row.

[0077] In one possible implementation, the microfluidic chip in this embodiment has a channel width of 0.4 mm to 2.0 mm for the first channel 111. For example, the channel width of the first channel 111 is 0.8 mm. The width mainly affects the lysis efficiency by changing the electric field strength of the region. Verification was conducted by changing the width of the lysis channel of the chip. Five different chip sizes (0.4 mm, 0.8 mm, 1.2 mm, 1.6 mm, and 2.0 mm) were designed, and the cell lysis rate was optimal when the channel width of the first channel 111 was 0.8 mm. 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 tank 122 is square or has a length of 2mm to 6mm and a width of 1mm to 3mm.

[0079] On the other hand, this application also provides a method for fabricating a microfluidic chip as described in the above embodiments, comprising: S100, providing a first substrate 100, wherein the first substrate 100 is sequentially provided with an electrolytic region 110, an electro-enrichment region 120, and a detection region 130 along the flow direction of the sample to be tested. S200, performing lyophilization modification on the first substrate 100, comprising: adding a reaction system mixture containing different primers to the detection region 130; precooling the first substrate 100 sequentially for a preset time and a preset temperature, and then drying and lyophilizing it under a preset vacuum condition. S300, providing an electrode assembly 210, wherein 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 sequentially flow through the electrolytic region 110 and the electro-enrichment region 120, and enter the detection cell 131 for detection.

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

[0081] S400, Connect the first substrate 100 and the second substrate 200. For example, bonding the first substrate 100 and the second substrate 200 includes: fixing wires on the second substrate 200 to form an electrode area; bonding the first substrate 100 and the second substrate 200 with UV adhesive; applying adhesive in multiple layers along the edge of the substrate, and filling the adhesive by applying pressure; and 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 sheets and fabricated by 3D printing. The thickness of the acrylic sheet can be 2 mm.

[0083] S200. The first substrate 100 is freeze-dried and modified. The specific modification steps are as follows:

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

[0085] S202: Place the chip in a -20 degree freezer for pre-cooling, and then place it in a -80 degree freezer for 2 hours to pre-freeze;

[0086] S203: Remove and place in a freeze dryer, set the temperature to -50 degrees Celsius and the vacuum degree to 10 Pa, for more than six hours;

[0087] S204: Raise the temperature to 10 degrees Celsius while maintaining the vacuum level for more than 4 hours;

[0088] S205: After confirming that the reagent is dry, gradually bring it into a vacuum state and fill it with nitrogen to prevent the freeze-dried reagent from becoming damp;

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

[0090] S400. Connect the processed first substrate 100 to the second substrate 200 by adhesive bonding. The specific adhesive bonding steps are as follows:

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

[0092] S402: Uses UV adhesive to bond complex structures within the chip;

[0093] S403: After bonding is complete, press the upper and lower plates together firmly. Take 20μL of acrylic glue each time and slowly run it along the edge of the first substrate 100 and the second substrate 200. Driven by the pressure difference, the glue slowly enters the interior until it is completely filled.

[0094] S404: Press firmly at room temperature to obtain the chip used in the experiment.

[0095] This invention tested the blood of eight different individuals. Before testing, each blood sample was treated with three different radiation doses. An untreated sample served as a control group. The testing of actual samples further demonstrated the invention's fast detection speed and high sensitivity. Figure 11 As shown, Figure 11 The application of the microfluidic chip provided in this application in clinical practice (a is a physical image of the chip for detecting blood treated with different radiation; b is the detection results obtained after eight different test samples, each of which was processed in four different ways).

[0096] Furthermore, this application also provides a method for detecting a sample to be tested, applied to a microfluidic chip as described in the above embodiments, comprising: passing the sample to be tested into the electrolysis region 110; energizing the electrode assembly 210 so that the electrode assembly 210 drives the sample to be tested to pass sequentially through the electrolysis region 110 and the electroenrichment region 120 and enter the detection region 130; after the liquid in the detection region 130 reaches a preset value, placing the microfluidic chip in a heating device to react for a preset time to obtain the detection result.

[0097] The pretreated sample is introduced into the inlet of the microfluidic chip. The second substrate 200 is electrically connected to the power supply, applying an electric field to the electrode area, driving the sample through the electrolysis zone 110 and the electroaccumulation zone 120 sequentially, and finally into the detection zone 130. After the detection zone 130 is filled with liquid, the second substrate 200 is disconnected from the power supply, and the microfluidic chip is placed in a 65°C constant temperature device for 30 minutes. The result is output through the fluorescence signal in the detection zone 130. The microfluidic chip, external power supply, heating device, and detection equipment work together to detect the sample. Taking a cell solution as an example, cell culture is required, as detailed below:

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

[0099] S02: Place in an incubator at 37 degrees Celsius with 5% CO2 until the cell density reaches 80-90%. Gently aspirate the culture medium and wash twice with PBS.

[0100] S03: Add 0.25% Trypsin-EDTA to evenly cover the cell surface, and incubate at 37 degrees Celsius for 2-3 minutes. Observe under a microscope as the cells detach from the bottom, then 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, gently pipette to mix, and set aside.

[0102] Electrode assembly 210 is connected to a DC power supply, with voltages set to 10V, 20V, 30V, 40V, and 50V, and energizing times increasing sequentially from 5s to 25s. The power is then turned off after energizing is complete. The electro-enrichment region 120 collects the lysis products, purifies them, measures their purity, performs reverse transcription, and then performs real-time quantitative PCR (qRT-PCR).

[0103] Furthermore, this application provides examples of chip electrolytic cracking design principles and performance optimization. As shown below, fluid and electrode models are established in the corresponding software, and the chip model is imported into the software for simulation and adjustment.

[0104] An Electric Currents (EC) physical field was established, using a two-dimensional structure and 1xPBS and gold electrodes as materials. Specifically, a current field model was created in the simulation software to simulate the distribution and behavior of current within the specific structure, with both 1xPBS and gold electrodes serving as conductive media.

[0105] The overall potential and electric field distribution inside the chip were obtained through simulation. The effect of electric field and potential on cell lysis was studied using the electrolysis region 110 of the microfluidic chip; the first flow channel 111 is the electrolysis flow channel. The specific steps are as follows:

[0106] S11: Inject 1xPBS solution containing cells (approximately 10 cells) at the inlet. 6 1, PBS approximately 200 ), flow rate 120 (Based on the above flow channel splitting simulation, the flow velocity in each region is 60) ).

[0107] S12: The sample to be tested flows completely into the electrolysis zone 110, the power supply voltage is turned on (30V), and after the liquid has been introduced, the pyrolyzed sample is collected and subsequent detection is performed.

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

[0109] S14: By changing the width of the chip lysis channel, five different chip sizes of 0.4mm, 0.8mm, 1.2mm, 1.6mm, and 2.0mm were designed. Multiple experiments showed that the cell lysis rate was similar and reached the optimal level under an electric field strength higher than 350V / cm.

[0110] S15: Ultimately, considering both the pyrolysis efficiency and the practicality of the experiment, a flow channel width of 0.8 mm for the first flow channel 111 yielded better results. Therefore, a 0.8 mm width scheme was chosen to design the final electrolytic pyrolysis zone 110.

[0111] S16: The length mainly affects the pyrolysis efficiency through the energizing time. The initial flow rate of the injection pump is set to 40. 80 120 160 200 .

[0112] S17: Experiments have shown that at 80 120 Under these conditions, the pyrolysis efficiency can reach its optimal level. For example... Figure 6 As shown, Figure 6The following are experimental verification diagrams of the microfluidic chip provided in the embodiments of this application. (a) shows the cell mortality rate under different voltage lysis conditions as captured by a fluorescence microscope. Generally, green represents live cells stained with AM dye, and red represents dead cells stained with PI dye. As can be seen from Figure a, green represents live cells and red represents dead cells. (b) is a line graph showing the cell mortality rate of electrolyzed cells under different voltage and time conditions. (c) shows the detection results obtained by real-time quantitative fluorescence detection (qPCR) of the products of electrolyzed cells under different voltage and time conditions. The optimal conditions are 30V voltage and 10s energizing time, under which the CT value is 16.79).

[0113] Figure 7 The following diagrams illustrate the validation experiments of the microfluidic chip provided in this application for different test samples. The electrolysis effect is compared with that of commercially available reagent kits (a) shows the results of capillary electrophoresis of the electrolysis and reagent lysis products, where the red area is the core detection region 130; b) compares the core regions in the capillary electrophoresis results of electrolysis and reagent lysis; c) calculates the area ratio of the core region, electrolysis, and reagent lysis; d) shows the qPCR detection of the electrolysis and reagent lysis products and compares the detection results of four targets: BAX, CDKN1A, GADD45A, and GAPDH.

[0114] Furthermore, this embodiment also provides the principles and performance optimization of chip electro-enrichment design. An object model is created in the software, and the chip model is imported into the software. Laminar Flow (SPF) and Transport of Diluted Species (T physics fields) are established, and a multiphysics field is also established. A two-dimensional structure is selected, and 1xPBS solution is chosen as the material. Simulation is used to determine the approximate enrichment region and flow direction of negatively charged molecules within the chip. The effect of electric field and flow rate on the enrichment of anions is studied using the electro-enrichment region 120 of the microfluidic chip, as detailed below:

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

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

[0117] This application is respectively in 20 30 An initial flow rate is used to introduce the sample, which is a fluorescent sample. Five different electric field intensities are provided: 50... 100 150 200 250 Multiple experiments revealed that the wider the enrichment cell 122, the greater the electric field strength required to achieve stable enrichment. For the same enrichment size, too low an electric field strength may prevent the formation of stable enriched bands, while too high an electric field strength will cause premature formation of enrichment sites and be accompanied by a "diffuse" phenomenon. Figure 8 As shown, Figure 8 The functional verification experiment diagrams of the microfluidic chip electro-enrichment provided in the embodiments of this application are as follows: (a) the enrichment of the sample in the chip under different voltage and time conditions; b) the comparison of the effect of chip electro-enrichment and purification by commercial kits; c) the enrichment effect of in-chip electro-enrichment and kit purification for different DNA concentrations; d) the linear correlation between the enrichment results of the kit for different DNA concentrations; e) the linear correlation between the enrichment results of chip electro-enrichment for different DNA concentrations.

[0118] Based on the voltage conditions in electrolysis, an enrichment cell 122 with dimensions of 0.4 cm × 0.2 cm was finally determined for subsequent experiments.

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

[0120] Plasmid samples of varying concentrations were added sequentially according to the concentration gradient. The completed detection chamber 131, such as a four-well plate, was then placed on a water bath.

[0121] After the reaction was complete, the plate was removed and placed in a photography studio for fluorescence imaging. Statistical analysis of the imaging results revealed a linear correlation between the fluorescence intensity of the four target reactions and their concentrations. Figure 9The following diagrams illustrate the module differences in the electrically enriched region of the microfluidic chip provided in this application embodiment: (a) shows the difference comparison between the two channels in the upper half, in which four targets, BAX, CADKN1A3, GADD45A, and GAPDH, are detected and compared; (b) shows the difference comparison between the two channels in the lower half, in which four targets, BAX, CADKN1A3, GADD45A, and GAPDH, are detected and compared).

[0122] The primer sequences for BAX, CDKN1A, GADD45A, and GAPDH are shown in the table below:

[0123] Table 1 shows the BAX primer sequences.

[0124]

[0125] Here, Amplification indicates the amplification type, primer indicates the primer, Sequence (5'-3') indicates the sequence (5'-3'), and PCR primers indicate conventional polymerase chain reaction, used for conventional PCR amplification of specific regions of the BAX gene.

[0126] LAMP primers refer to loop-mediated isothermal amplification, used in LAMP technology to rapidly amplify the BAX gene under specific temperature conditions. Forward indicates the forward primer, and Reverse indicates the reverse primer.

[0127] The Fip (Forward Inner Primer) includes the F2 region (for binding to the target sequence) and the F1c region (for initiator strand replacement), used to form a loop structure. The Bip (Backward Inner Primer) contains the B1c and B2 regions, used for amplification of the reverse complementary strand.

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

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

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

[0131] Table 2 shows the CDKN1A primer sequences.

[0132]

[0133] Table 3 shows the primer sequences for GADD45A.

[0134]

[0135] Table 4 shows the GAPDH primer sequences.

[0136]

[0137] This example also provides a method for detecting fluorescence intensity using a microfluidic chip, with the specific steps as follows:

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

[0139] S32: Fix the chip on the plane, connect the syringe pump and DC power supply, and inject the sample at 120... The speed of introducing microfluidic chips.

[0140] S33: When the sample enters the electrolysis zone 110, the DC power supply is turned on to create a high electric field, achieving rapid electrolysis of the sample within the zone, disrupting the cell membrane and releasing intracellular components, including mRNA. To distinguish the characteristics of reagent lysis and electrolysis after completion, we used a microplate reader for detection. The results are as follows:

[0141] Table 5 shows the results of the enzyme-linked immunosorbent assay (ELISA) test.

[0142]

[0143] Here, "well" refers to detection cell 131, and 260, 230, and 280 represent different wavelengths, measured in nm. 260nm refers to the maximum absorption wavelength of nucleic acids, 230nm refers to the absorption wavelength of carbohydrates, peptides, or certain organic contaminants, and 280nm primarily detects protein contamination in the sample. The absorbance ratios of these wavelengths (such as 260 / 280 and 260 / 230) are important indicators for assessing the purity of nucleic acid samples. For example, the A260 / A280 ratio for pure DNA is typically around 1.8, and for pure RNA around 2.0; while the A260 / A230 ratio is usually between 2.0 and 2.2. A ratio below this range may indicate carbohydrate or salt contamination.

[0144] S34: The small number of bubbles generated during the electrolytic cracking process are electrically neutral and are limited to the area near the cathode, and will not affect the flow of cracking products to the next functional area.

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

[0146] S36: The entire enrichment process avoids bubble interference, ensuring that the mRNA target is rapidly enriched within 10 seconds, reaching a concentration factor of approximately 10 times, and flows directly into the detection zone 130 after the enrichment site is formed.

[0147] S37: During the detection process, the detection chamber 131 of the detection area 130 is pre-loaded with lyophilized reagents, including reverse transcriptase, primers, LAMP buffer, and other components required for the reaction. The enriched mRNA is mixed with the lyophilized reagents, and the chip is placed on a constant-temperature water bath for isothermal amplification.

[0148] S38: The width of the effective enrichment area of ​​sampling tube 121 is 0.2 mm. Approximately 30 mm is reserved in the detection area 130. The enriched mRNA is collected from the region.

[0149] This significantly increases the initial concentration of mRNA in the reaction, while minimizing mRNA loss during enrichment. This increased concentration dramatically improves the overall sensitivity of the detection process, making this system particularly suitable for detecting low-abundance mRNA biomarkers in blood. Figure 10 As shown, Figure 10 The following graphs illustrate the test and verification of the fluorescence detection sensitivity of the microfluidic chip provided in the embodiments of this application: (a) Tq values ​​were detected for different concentrations of BAX plasmid, and a linear correlation was found between the logarithm of the concentration and the Tq value; (b) Tq values ​​were detected for different concentrations of CDKN1A plasmid, and a linear correlation was found between the logarithm of the concentration and the Tq value; (c) Tq values ​​were detected for different concentrations of GADD454A plasmid, and a linear correlation was found between the logarithm of the concentration and the Tq value; (d) Tq values ​​were detected for different concentrations of GAPDH plasmid, and a linear correlation was found between the logarithm of the concentration and the Tq value).

[0150] By combining microfluidic chips based on electrolysis, electroenrichment, and chemical amplification methods, there is a great potential to simplify the sample preparation and detection process in the efficient analysis of cancer biomarkers.

[0151] This application integrates sample lysis, electroaccumulation, and nucleic acid amplification detection onto a microfluidic chip. Compared to traditional methods, it offers advantages such as speed, sensitivity, and accuracy, and can be widely applied to point-of-care testing (POCT), particularly in areas such as early cancer screening, radiation exposure assessment, and real-time disease monitoring. The microfluidic chip contains two parallel electrolysis zones 110, and a precise inlet shunt structure ensures dynamic matching between the sample lysis time and the initial flow rate of electroaccumulation. The lysed sample directly enters the enrichment zone, and the optimized flow channel design minimizes the flow rate differences between different enrichment channels, improving enrichment efficiency and stability.

[0152] Furthermore, at the exit of the enrichment zone, target molecules in the enriched bands are collected to the maximum extent, thereby improving detection sensitivity. Experimental results show that this can achieve a target molecule enrichment factor of up to 15 times. The entire system can process up to 1.2 mL of blood sample at a time, completing lysis and enrichment within 20 seconds. The enriched sample is then transferred to the LAMP amplification module in the detection zone 130, achieving rapid and efficient nucleic acid detection throughout the entire process. It should be noted that the LAMP amplification module (Loop-mediated Isothermal Amplification Module) is the core operating unit of loop-mediated isothermal amplification technology. This module integrates reaction reagents, primer design, and a detection system to achieve efficient amplification of target DNA / RNA at a specific temperature (60-65 degrees Celsius), eliminating the need for traditional PCR thermal cycling equipment.

[0153] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only.

[0154] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying 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: The first substrate (100) is provided with an electrolytic region (110), an electro-enrichment region (120) and a detection region (130) in sequence along the flow direction of the sample to be tested. The electrolytic cracking region (110) is used for electrolytic cracking of the sample to be tested; The electro-enrichment region (120) is used to enrich the test sample flowing out from the electro-pyrolysis region (110); The detection zone (130) is used to detect the sample to be tested flowing out from the electro-enrichment zone (120), and the detection zone (130) has a detection cell (131) for pre-loading lyophilized reagents; Electrode assembly (210) is connected to the first substrate (100). The electrode assembly (210) is used to generate an electric field to drive the sample to be tested to flow sequentially through the electrolytic region (110) and the electro-enrichment region (120) and enter the detection region (130) for detection. The enrichment region (120) has an enrichment cell (122), and the electrode assembly (210) has 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 cell (122) is connected to the positive electrode (212). The bottom of the enrichment cell (122) is provided with a connecting part. Multiple grooves (126) are arranged side by side on the connecting part. A selective membrane is provided in the groove (126). The selective membrane is a Nafion membrane. The side of the connecting part away from the enrichment cell (122) is connected to or in contact with the negative electrode (213). The enrichment cell (122) is used to receive the sample to be tested flowing through the electrolytic cracking zone (110). The enrichment cell (122) has a waste liquid outlet (124) and a sampling outlet (125). The waste liquid outlet (124) and the sampling outlet (125) are located on opposite sides of the liquid inlet side of the enrichment cell (122) and on the upper side of the plurality of grooves (126). The waste liquid outlet (124) is located above the sampling outlet (125) in the direction of gravity. The sampling outlet (125) is connected to the detection zone (130).

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

3. The microfluidic chip according to claim 2, characterized in that, The flow channel (140) includes a first flow channel (111) located in the electrolytic region (110), and the first flow channel (111) is at least partially bent toward the edge of the first substrate (100).

4. The microfluidic chip according to claim 2 or 3, characterized in that, At least one bend (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). The inner diameter of the sampling tube (121) gradually decreases from the enrichment tank (122) to the sampling outlet (125).

5. The microfluidic chip according to claim 2 or 3, 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 alternately at intervals; The detection area (130) has at least one detection pool (131), and each detection pool (131) is connected to the enrichment pool (122) in a one-to-one correspondence.

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

7. A method for fabricating a microfluidic chip, used to fabricate the microfluidic chip according to any one of claims 1-6, characterized in that, include: A first substrate (100) is provided, wherein an electrolytic region (110), an electro-enrichment region (120) and a detection region (130) are sequentially provided along the flow direction of the sample to be tested, and the electro-enrichment region (120) has an enrichment cell (122). The first substrate (100) is freeze-dried, including: adding a mixture of reaction systems containing different primers to the detection area (130); pre-cooling the first substrate (100) for a preset time and a preset temperature, and then drying and freeze-drying it under a preset vacuum condition; An electrode assembly (210) is provided, which is connected to the first substrate (100). The electrode assembly (210) is used to generate an electric field to drive the sample to be tested to flow sequentially through the electrolytic region (110) and the electro-enrichment region (120) and enter the detection region (130) for detection.

8. A method for detecting a sample, applied to the microfluidic chip according to any one of claims 1-6, characterized in that, include: The sample to be tested is introduced into the electrolytic region (110). The electrode assembly (210) is energized so that the electrode assembly (210) drives the sample to be tested to pass sequentially through the electrolytic region (110) and the electroenrichment region (120) and enter the detection region (130). After the liquid in the detection area (130) reaches a preset value, the microfluidic chip is placed in a heating device and reacted for a preset time to obtain the detection result.

Images