Micro-fluidic chip based on gradient gel electrophoresis and nucleic acid enrichment detection method

Abstract

The invention belongs to the field of nucleic acid detection, and particularly relates to a micro-fluidic chip based on gradient gel electrophoresis and a nucleic acid enrichment detection method. The method comprises the following steps: acquiring a lysis solution mixture of a sample to be detected, injecting second-concentration gel into a second filling port to wait for the gel to be solidified, injecting first-concentration gel into a first filling port to connect low-concentration gel with the second-concentration gel to wait for the gel to be solidified so as to obtain electrophoresis gel, and the first concentration is smaller than the second concentration; adding the lysate mixture into a cathode incubation hole, adding a buffer solution, and adding a buffer solution into an anode incubation hole; specific nucleic acid molecules are enriched to specific areas after nucleic acid molecules in a to-be-detected sample move in the electrophoresis gel after the to-be-detected sample is electrified at a set voltage for a set time length, and the specific areas are positioned on two sides of a junction of the first-concentration gel and the second-concentration gel of the electrophoresis gel. The design of the micro-fluidic chip is matched with the setting of the gradient gel, so that target nucleic acid is smoothly enriched in the electrophoresis process.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection, and more particularly, to a microfluidic chip based on gradient gel electrophoresis, a nucleic acid enrichment detection method, and a system. Background Art

[0002] Microfluidic chip technology was proposed in the early 1990s of the 20th century and belongs to the currently rapidly developing high-tech and cutting-edge technology fields. Microfluidic technology is a technology for manipulating fluids in the nanoliter to picoliter volume scale in a micron-scale structure, aiming to miniaturize, chipize, integrate, and high-throughput analyze equipment, namely the so-called "lab-on-a-chip".

[0003] The general process of pathogen detection includes: nucleic acid extraction, nucleic acid amplification, and nucleic acid detection. The main methods of nucleic acid extraction include technologies based on centrifugal columns and magnetic beads. The centrifugal column method is difficult to adapt to a miniaturized system due to its dependence on a high-speed centrifuge, which is incompatible with portable diagnostic devices. The magnetic bead method requires a multi-chamber structure to complete its purification and elution steps, which greatly increases the complexity of the system. The multi-chamber structure also results in a large system volume and high manufacturing costs. Even those methods that bypass the elution step still require a complex control system, resulting in an increase in the instrument volume and operating costs. Electrophoretic separation uses an electric field to move negatively charged nucleic acids towards the positive electrode. Compared with traditional DNA extraction, electrophoresis has obvious advantages and can quickly separate and enrich nucleic acid fragments in a short time. For biological macromolecules, although their charge-to-mass ratios are similar, their sizes are different, such as proteins and nucleic acids, and gel electrophoresis is more suitable. On the gel electrophoresis platform, small molecules move freely in the gel network with less resistance, while large molecules are hindered and have a slower migration speed. However, the sample consumption of traditional gel electrophoresis is relatively large, and samples (nucleic acids and proteins) are often precious and scarce. In addition, traditional gel electrophoresis generates cumulative Joule heat at high voltages, which may cause the gel to dissolve.

[0004] In the step of nucleic acid extraction, the prior art relatively close to the present application includes:

[0005] The article "Extraction of Exosomes by Gel Electrophoresis Microfluidic Chip and Determination of miRNA-21 in Human Plasma Exosomes" published by Luo Dan et al. discloses a method for extracting and determining miRNA-21 using a gel electrophoresis microfluidic chip. They independently designed and prepared a microfluidic chip based on a nanoporous film and agarose gel electrophoresis, and formed a vertical channel for sample injection and an enrichment chamber channel for enriching exosomes by injecting 1% concentration of agarose.

[0006] The invention patent with document number CN116139950A discloses a microfluidic system for rapid extraction of miRNA, which includes, from one side to the other side of the microfluidic chip, a first electrode area, a first hydrogel area, a lysis chamber area, a separation hydrogel area, an elution chamber area, a second hydrogel area, and a second electrode area; the first electrode area includes a first electrode and a first electrode fluid channel; the second electrode area includes a second electrode and a second electrode fluid channel; the first electrode, the first electrode fluid channel, the second electrode fluid channel, and the second electrode are adjacent to each other in sequence; the patent divides the microfluidic chip into multiple different areas, and applies different AC voltages or DC voltages when cells are in different areas, thereby achieving rapid extraction of nucleic acids.

[0007] However, the current existing technologies are mainly for the extraction of nucleic acids. In addition, they also include microfluidic chips that can achieve amplification based on the principle of constant temperature amplification detection. There is no microfluidic chip that can integrate nucleic acid amplification detection. The reason for this phenomenon is that the current microfluidic chips are designed for nucleic acid extraction in nucleic acid amplification, and there are obstacles to their integrated combination with nucleic acid detection.

[0008] Highly miniaturized microfluidics technology is able to integrate nucleic acid extraction, amplification, and signal detection on a single chip. This reduces the complexity of operation and makes microfluidics chips a powerful tool for the development of point-of-care testing (POCT). Microfluidics provides a cost-effective method for molecular diagnostics, while also providing new opportunities for the immediate detection of infectious pathogens outside of hospitals and laboratories. Summary of the invention

[0009] In view of the above problems, the present invention provides a microfluidic nucleic acid enrichment and detection method based on gradient gel electrophoresis, which realizes nucleic acid enrichment and nucleic acid extraction by designing a new flow channel, and determines the appropriate electrophoresis voltage in the microfluidic chip scenario, while integrating nucleic acid extraction, nucleic acid amplification, and nucleic acid detection into an integrated detection microfluidic chip.

[0010] The present application (first aspect) discloses a microfluidic chip for gradient gel electrophoresis, comprising a substrate on which are constructed an anode incubation hole, a cathode incubation hole, a microfluidic channel, a first perfusion port, and a second perfusion port. The microfluidic channel is a channel for gel electrophoresis, and the first perfusion port and the second perfusion port are connected to the side of the microfluidic channel.

[0011] The second aspect of the present application discloses a microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis, comprising:

[0012] Using any of the above-mentioned microfluidic chips, the cathode incubation hole of the microfluidic chip is connected to the negative electrode of electrophoresis, and the anode incubation hole is connected to the positive electrode of electrophoresis, and the use method comprises the following steps:

[0013] Obtain the lysate mixture of the sample to be tested,

[0014] Inject the second concentration gel into the second infusion port near the electrophoresis positive electrode and wait for the gel to solidify.

[0015] Injecting a first concentration gel into a first infusion port near the negative electrode of the electrode to connect the low concentration gel with the second concentration gel and wait for the gel to solidify to obtain an electrophoresis gel, wherein the first concentration is less than the second concentration;

[0016] Add the lysate mixture to the cathode incubation wells and add buffer, and add buffer to the anode incubation wells;

[0017] After the nucleic acid molecules in the sample to be tested move in the electrophoresis gel at a set voltage and for a set time, specific nucleic acid molecules are enriched in a specific area, and the specific area is located on both sides of the junction of the first concentration gel and the second concentration gel of the electrophoresis gel.

[0018] Furthermore, the specific area is 1 to 5 mm on both sides of the junction;

[0019] Optionally, the specific area is the area between the first filling port and the second filling port;

[0020] Optionally, the specific area is an area between the left boundary of the first filling port and the right boundary of the second filling port;

[0021] Optionally, a second concentration gel is injected and a baffle is placed in the middle of the exhaust port and then pulled out after the gel solidifies, so that the interface between the first concentration gel and the second concentration gel is vertical;

[0022] Optionally, the width of the specific area corresponds to the width of the exhaust port, and the specific area is obtained by pushing out the push-pull blades on both sides of the exhaust port;

[0023] Optionally, after injecting the gel of the second concentration, the blade on one side of the exhaust port is pushed out and then pushed back after waiting for the gel to solidify, so that the interface between the gel of the first concentration and the gel of the second concentration is vertical.

[0024] Furthermore, the method further comprises:

[0025] Adding a reaction system to the specific region to perform nucleic acid amplification;

[0026] After heating the area where nucleic acid amplification has been completed, nucleic acid detection is performed to obtain the type of pathogen;

[0027] Optionally, the gel in the specific area is cut and removed, and nucleic acid amplification and nucleic acid detection are performed on the removed gel to obtain the type of pathogens.

[0028] Further, the method further comprises: the first concentration and the second concentration are determined according to the nucleic acid length of the target pathogen of the respiratory pathogen detection;

[0029] Optionally, the first concentration is 0.5-1%, and the second concentration is 3-3.5%;

[0030] Optionally, the gel is agarose gel, the first concentration is 0.8%, and the second concentration is 3%;

[0031] Optionally, the set voltage and the set duration are determined based on the nucleic acid of the target pathogen for respiratory pathogen detection;

[0032] Optionally, the set voltage is 70V and the set time is 15 minutes.

[0033] Further, the specific steps of obtaining the lysate mixture are:

[0034] The buffered lysozyme powder was dissolved in the lysozyme buffer to prepare a lysozyme solution, wherein the concentration of the components of the lysozyme solution was as follows: 1 mg / mL lysozyme, 20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; 1.2% Triton X-100;

[0035] Take 20 μL of the sample to be tested and mix it with 5 μL of lysozyme solution in a test tube, incubate at 37°C for 5 minutes to obtain the first lysate mixture.

[0036] 0.5 μL of 20 mg / mL proteinase K was added to the first lysate mixture and incubated for 5 minutes to obtain the lysate mixture.

[0037] Furthermore, the nucleic acid amplification is any one or more of the following: qPCR, LAMP, RCA;

[0038] Optionally, the sample to be tested contains multiple virus bodies, and the nucleic acid amplification is performed using qPCR. The reaction system of the nucleic acid amplification contains primers for multiple pathogens, and the nucleic acid detection simultaneously detects the multiple pathogens contained in the sample to be tested.

[0039] The third aspect of the present application discloses an application of a microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis in respiratory virus detection.

[0040] A microfluidic nucleic acid enrichment detection system based on gradient gel electrophoresis, the system comprising:

[0041] Acquisition unit: used to obtain the lysate mixture of the sample to be tested,

[0042] Gradient gel preparation unit: used for injecting a second concentration gel into a second infusion port near the electrophoresis positive electrode and waiting for the gel to solidify, injecting a first concentration gel into a first infusion port near the negative electrode to connect the low concentration gel with the second concentration gel and waiting for the gel to solidify to obtain the electrophoresis gel, wherein the first concentration is less than the second concentration;

[0043] Sample loading unit: add the lysate mixture to the cathode incubation well and add buffer; add buffer to the anode incubation well;

[0044] Electrophoresis enrichment unit: used to energize at a set voltage and for a set time, so that the nucleic acid molecules in the sample to be tested move in the electrophoresis gel and specific nucleic acid molecules are enriched in a specific area, and the specific area is located on both sides of the junction of the first concentration gel and the second concentration gel of the electrophoresis gel.

[0045] This application has the following beneficial effects:

[0046] (1) This application designs an integrated extraction and detection microfluidic chip that completes pathogen lysis, nucleic acid enrichment, and nucleic acid amplification on a single chip, thereby increasing the integration of the microfluidic chip and improving detection efficiency;

[0047] (2) The design of the microfluidic chip in the present application, combined with the setting of the gradient gel, can smoothly enrich the target nucleic acid during the electrophoresis process, and amplify and extract it, which is simple and efficient.

[0048] (3) The detection method based on the microfluidic chip of the present application can be applied to the detection of a variety of respiratory pathogens, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0050] Figure 1 is a schematic structural diagram of a microfluidic chip provided by the first aspect of an embodiment of the present invention;

[0051] Figure 2 is a schematic diagram of a top view and size of a microfluidic chip provided by an embodiment of the present invention;

[0052] Figure 3 Schematic diagram of a front view (A) and a right view (B) of a microfluidic chip provided in an embodiment of the present invention;

[0053] Figure 4It is a schematic diagram of various steps of nucleic acid extraction, nucleic acid amplification, and nucleic acid detection based on a microfluidic chip provided in an embodiment of the present invention;

[0054] Figure 5 It is a schematic diagram of the principle of low-density gel and high-density gel distribution for nucleic acid extraction using a pre-filled gel electrophoresis microfluidic chip provided by an embodiment of the present invention;

[0055] Figure 6 It is a schematic diagram of charge distribution and target area when a pre-filled gel electrophoresis microfluidic chip is used for nucleic acid extraction provided by an embodiment of the present invention;

[0056] Figure 7 A schematic diagram of a microfluidic chip and its corresponding area division provided in an embodiment of the present invention;

[0057] Figure 8 A schematic diagram of the enrichment effect and amplification effect of nucleic acids enriched in different regions of a microfluidic channel provided in an embodiment of the present invention;

[0058] Fig. 9 A schematic diagram of the effect of proteinase K concentration on amplification provided in an embodiment of the present invention;

[0059] Fig.10 A diagram of an enrichment principle provided by an embodiment of the present invention and a corresponding diagram of a microchannel enrichment area;

[0060] Fig.11 A schematic diagram of the detection effect of a microfluidic chip on several viruses provided by an embodiment of the present invention.

[0061] Fig.12 A schematic diagram of a microfluidic nucleic acid enrichment detection system based on gradient gel electrophoresis provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0062] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0063] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The sequence numbers of the operations, such as S101, S102, etc., are only used to distinguish between different operations, and the sequence numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0064] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0065] The scheme selected agar electrophoresis technology as the method for nucleic acid extraction and detection on microfluidic chips. In order to smoothly realize the miniaturization of agar electrophoresis technology, the following methods were adopted:

[0066] (1) Preparation of agarose gel column by perfusion method:

[0067] This method places the sample containing pathogens and gel in an electric field together, breaks the cells by the electric field and drives the charged contents therein into the gel. As a biological macromolecule with a long chain structure, the electrical migration speed of nucleic acids in the gel will be significantly different from that of other biological molecules, thereby showing a position difference in the gel. Commonly used nucleic acid electrophoresis gel materials can complete the above-mentioned screening of nucleic acids, but since the gel containing the nucleic acid to be tested needs to be put into the next amplification system together with the nucleic acid therein, this puts forward requirements for the physicochemical properties of the gel. Agarose is preferably used as a separation gel material. This is not only because agarose is a mature nucleic acid gel electrophoresis material, but also because it has the ability to repeatedly undergo solid-liquid phase transitions, and it does not rely on the addition of reagents during the phase transition process, and only requires temperature control to complete. As a result, the gel is used as a solid to screen nucleic acids during extraction, and can be dissolved in the reaction solution in liquid form during amplification. During preparation, it is only necessary to inject molten agarose into the prepared fine pipe with a syringe, and the liquid agarose will form a liquid column by surface tension. The liquid column can move freely to adjust the position by gravity or pressure, and solidify after adjustment to become a usable and fixed columnar gel. The solidified gel can be stored for a long time without deterioration or shrinkage if it is sealed and immersed in a solution, and can remain tightly attached to the side walls. Figure 3 Shown is a schematic diagram of the principle of nucleic acid extraction by gel electrophoresis in this section.

[0068] (2) Designing density gradient cutoff for nucleic acid migration: The microstructure of the gel can screen the molecules that migrate based on their size. In order to achieve better separation, the gel density gradient is designed, such as Figure 3 As shown. The gel density in the area in contact with the cells is low, which is conducive to the entry of nucleic acids and blocks the uncharged macromolecules in the solution. The concentration of low-density agarose gel is preferably 0.5-1%, so that it can not only pass through nucleic acid molecules normally, but also effectively block impurities such as cell debris and larger protein molecules. The density of the gel at the end of the gel away from the sample should be higher, and the entry of nucleic acids is blocked without affecting the passage of charged small molecules through the gel into the solution. The concentration of high-density agarose gel is preferably 3-3.5%, which blocks the migration of target nucleic acids as much as possible, while not blocking the passage of nucleic acid fragments and charged small molecules, so as to achieve the purpose of screening and purification. In this way, nucleic acids of different lengths within a certain range can migrate into gels with lower concentrations and stay at the density mutation position of the gel after migrating a certain distance. The gel with a density gradient can achieve the purpose of enriching nucleic acid fragments of the target length at the preset density mutation position. After the electrophoresis step is completed, the density mutation area is directly cut to obtain an agarose block enriched with nucleic acid fragments in the sample, and it can be put into a prefabricated amplification system for melting and amplification.

[0069] (3) Optimize electrophoresis buffer and applied field strength: The buffer is used to prepare the gel, mix with the sample, and add it to the sample pool. Unlike conventional electrophoresis, the field strength during the electrolysis of cells is relatively large, and the pH value of the solution near the cells should also be relatively high. The strong electric field plus the alkaline environment promotes the rupture of the phospholipid membrane structure and the release of the nucleic acid contained therein. Combined with the conditions for maintaining the stability of nucleic acids, a TAE solution with a pH of 7.5-8.0 is preferably used as the basic electrophoresis buffer. The buffer for nucleic acid electrophoresis extraction should also have a certain concentration of surfactant (such as 1% Tween-20) to play a role in washing proteins and other substances. On this basis, the appropriate buffer ratio is determined by comparing the cell lysis efficiency and nucleic acid stability in different buffers. The components of the buffer should also select common components in quantitative amplification solutions, such as Tris, Tween-20, DMSO, etc., and amplification tests should be performed to ensure that they do not affect the amplification efficiency. The electrophoresis device is powered by a platinum electrode with one end directly in contact with the solutions on both sides and the other end leading to an external power supply. Since there is no need to accurately distinguish nucleic acid bands and the distance between electrodes in miniaturized devices is short, the applied voltage does not need to be very large to achieve the required field strength. In actual experiments, the electric field strength should be greater than 30V / cm. A higher field strength is conducive to accelerating the fragmentation of membrane structures and accelerating the migration of nucleic acids, thereby accelerating nucleic acid purification.

[0070] (4) Detection of nucleic acid extraction efficiency and purity: By inputting a variety of known standard samples (including human cells, bacteria, viruses, etc.) and performing nucleic acid extraction under different parameters (represented by gel density, buffer concentration, electric field strength), the required time and extraction results (including final nucleic acid concentration, protein and other impurity concentrations, and the impact on subsequent amplification, etc.) are compared and analyzed uniformly, and the optimal condition combination is selected through comprehensive comparison. Corresponding standard samples are set for different application scenarios, and by exploring parameter combinations, a variety of optimal solutions suitable for different types of pathogens (such as bacteria and viruses) are given.

[0071] (5) Stability test: Electrophoresis nucleic acid extraction was performed using samples containing several known similar components in different composition ratios and repeated. The extraction stability of the platform under different sample input conditions was compared. Combined with fluorescence amplification, parameters for the accuracy of sample component restoration were obtained.

[0072] 2. Gel--Pathogen Nucleic Acid Mixture Multi-site Quantitative Detection Technology:

[0073] (1) Gel-pathogen nucleic acid mixture is input into the amplification system: After the nucleic acid extraction is completed, the gel containing the nucleic acid to be tested is directly put into the pre-prepared and packaged amplification system for nucleic acid amplification. Since the aforementioned density gradient gel has a localized enrichment effect on nucleic acids, the target nucleic acid will be trapped in the density mutation area, so only the gel at the density mutation position is cut off and directly mixed with the pre-prepared amplification system, and then the agarose gel is heated to melt to complete the preparation process of the amplification solution. Because the density mutation position is fixed in the agarose gel block, the gel cutting operation does not require auxiliary means such as fluorescence, and the fixed position can be cut off. The entire volume of the nucleic acid-gel mixture cut off has more nucleic acid to be tested but also contains more agarose, which expands the volume of the amplification solution. By comparing the amplification efficiency of different input volumes, the optimal amplification input amount is obtained. A fixed concentration of reference dye is pre-mixed in the amplification system as a calibration basis.

[0074] (2) Select target sites and design primers: Eight important respiratory pathogens (influenza A virus, influenza B virus, novel coronavirus, respiratory syncytial virus, human rhinovirus, adenovirus, Haemophilus influenzae and Streptococcus pneumoniae) were selected as detection targets. Specific primers were designed and synthesized by searching for specific gene fragments contained in each of the above pathogens.

[0075] (3) Optimization of amplification reaction conditions: Low melting point agarose remains in a liquid state throughout the PCR process and has little effect on PCR. Select a suitable reaction system and temperature to minimize the effect of gel and electrophoresis buffer on amplification to ensure efficiency and accuracy. Adding appropriate concentrations of surfactants and small molecule proteins (such as bovine serum albumin) can further reduce the effect of agarose on amplification. For primers with different sequences, optimize the corresponding annealing temperature and concentration, and perform detection based on the fluorescence changes during the amplification process.

[0076] (4) Detection specificity and sensitivity test: Further gel electrophoresis and sequencing analysis are performed on the amplification products in the amplification detection stage to understand the sequence characteristics of the products and then determine the specificity of the amplification detection. Samples with different concentrations of target nucleic acid are input and extracted and detected, and the detection limit of the detection platform for different pathogens is calculated.

[0077] 3. Develop a chip that integrates electrophoresis nucleic acid extraction and quantitative amplification detection:

[0078] (1) Microfluidic chip gel electrophoresis module: Two structures containing semi-cylindrical cavities are cast by combining PDMS material with a positive mold and bonded to form a microfluidic chip. Openings are reserved at corresponding positions to place electrodes and pour molten agarose, such as Figure 5 As shown. The diameter of the internal cylindrical cavity is between 5mm and 1cm, which not only ensures that the soft flow channel is not easy to collapse, but also facilitates the molten agarose to form a liquid column stably. Agarose of different densities is injected and solidified in batches. After keeping the temperature low to make the agarose phase change to form a gel, the electrophoresis buffer is filled into the cavity connected to the positive electrode of the electrode to reduce the subsequent experimental operation content and prevent the agarose gel from shrinking. The sample input cavity is vacant. Before being used for extraction, the outside of the entire chip should be sterilized and sealed with a sealing film to prevent contamination and liquid loss. When extracting nucleic acids by electrophoresis, the seal is broken and the sample is mixed with the electrophoresis buffer and then passed into the negative electrode connection area of ​​the electrode. Because the two electrodes in the chip are close to each other, the required electric field strength can be efficiently obtained without high voltage direct current during electrophoresis. The electrophoresis time should be sufficient to migrate the nucleic acid to the vicinity of the gel density gradient interface. The transparent property of PDMS is also conducive to mixing nucleic acid dyes into the sample for real-time fluorescence observation of electrophoresis. Figure 5 Use pre-filled gel electrophoresis microfluidic chip for nucleic acid extraction;

[0079] (2) Real-time quantitative amplification module: The structure and operation flow of this module are as follows: Fig.12As shown. Since the final position of nucleic acid migration in the gel is relatively fixed, in actual operation, the gel several millimeters before and after the gel density interface is directly cut off as a template, and then put into the pre-prepared amplification system in the tube for amplification. Since the size of the electrophoresis microfluidic chip is fixed and the PDMS itself is soft and easy to cut, a track with a double-layer transparent blade can be prefabricated to fix the gel cutting process. The blade material is preferably a hard plastic sheet with holes to facilitate the internal gel melting and fluorescence detection. On the other side of the chip corresponding to the track position, a fixed temperature control and fluorescence detection device is preset, and a modular disposable reaction chamber is placed. The reaction chamber is pre-filled with amplification reaction liquid as needed, and its surface sealing film is in close contact with the chip. After cutting the gel, the blade directly pushes out part of the agarose gel, destroys the sealing film of the reaction chamber, and puts it into the reaction liquid. The temperature during the amplification process will directly melt the low-melting point agarose and release the nucleic acid. After that, the concentration of agarose mixed in the solution will not be enough to solidify or hinder the amplification reaction. The released pathogen nucleic acid will bind to the specific primer, amplify to produce a fluorescent signal and be observed, ( Fig.12 );

[0080] (3) External power supply, temperature control, and fluorescence system: The above electrophoresis and amplification processes require direct current input and temperature control systems to function. The power supply system uses a rechargeable lithium battery for direct power supply. Because the current during electrophoresis is very small and the power demand is low, the capacity of an ordinary lithium battery of 2Ah can meet the needs of multiple electrophoresis extractions. The microchip is directly connected to a reaction chamber. The total volume of the amplification reaction is small and one amplification only takes about 1-1.5 hours. Therefore, the temperature control power requirement is also low. It can be achieved using a programmed single-chip microcomputer with a small heating paste. The temperature control is also powered by the above lithium battery. The bottom of the hard plate used to cut the electrophoresis module directly contacts the probe containing a microlens, which is used to amplify the fluorescence signal in the reaction tube. The probe is sealed on all sides and has a diameter no larger than the orifice cylinder, so that it can occupy the cavity cut inside the electrophoresis module under the protection of the cutting plate. The probe can be used with a fluorescence microscope to take pictures and perform precise quantitative detection at the end of the reaction, or it can be directly integrated with a programmed fluorescence excitation detection device for automatic detection. For integrated fluorescence detection equipment, it is necessary to link a temperature-controlled microcontroller to irradiate with the wavelength corresponding to the dye (such as SYBR corresponding to a wavelength of 480nm, achieved by white light LED plus a filter) during the extension phase of amplification, and then collect signals through the emission light filter in the probe (such as SYBR corresponding to 520nm) and the post-photosensitive element connected by an optical cable. These signals are recorded in the data acquisition card and storage medium, and the fluorescence curve is automatically generated by the software at the end of the experiment. When multiple samples and multiple pathogens are tested in parallel, multiple electrophoresis channels and amplification tubes need to be connected in parallel. For this requirement, a power supply, temperature control, and fluorescence detection system with power and space redundancy can be designed to meet the needs of parallel detection.

[0081] (4) Clinical evaluation and application of the detection platform: Encapsulate the agarose gel electrophoresis nucleic acid extraction and amplification system into a microfluidic chip, and combine it with an external control system to form a complete rapid detection platform for respiratory pathogens. Cooperate with relevant provincial and municipal institutions such as grassroots disease control to collect samples, use existing chemical extraction processes and fluorescent quantitative PCR as a reference to determine the sensitivity and accuracy of this detection platform. Conduct clinical evaluation of platform applications and products, and promote and trial qualified products. The technical roadmap is as follows: Fig.12 This is the technical roadmap for this project.

[0082] Figure 1 The present invention provides a chip structure diagram of a microfluidic chip for respiratory pathogen detection, wherein the microfluidic chip comprises: a cathode incubation hole (1), a microfluidic channel (2), a first perfusion port (3), an exhaust port (4), a second perfusion port (5), and an anode incubation hole (6);

[0083] Taking into account that electrophoresis is the electrolysis of water and the amount of liquid will be reduced, the present application designs cathode incubation holes and anode incubation holes with larger volumes, so that there is no need to replenish liquid during the electrolysis process. Among them, the cathode incubation hole is designed to be larger than the anode incubation hole because the cathode incubation hole not only needs to add electrophoresis buffer but also needs to add sample, while the anode incubation hole only needs to add electrophoresis buffer.

[0084] 1. Chip design and preparation

[0085] The gel electrophoresis microfluidic chip developed by this research institute uses a PC sheet with a size of about 35mm*15mm as the substrate of the microfluidic channel. The microfluidic channel is designed based on the substrate. The channel design is as follows Figure 1 As shown, Figure 2 Shown is a schematic diagram of the top view and size of the microfluidic chip of the present application, Figure 3 Shown is a schematic diagram of a front view of the microfluidic chip of the present application;

[0086] In some embodiments, the microfluidic chip is obtained by 3D printing.

[0087] In some embodiments, the microfluidic chip is directly milled by a computer-controlled milling machine.

[0088] In some embodiments, the microchannel is composed of a horizontal channel with a size of 30mm*1mm*depth mm and two infusion ports with a size of 5mm*1mm*depth mm and an exhaust hole with a size of 3mm*1mm*depth mm connected vertically to the horizontal channel.

[0089] The two sides of the microfluidic channel are buffer loading areas, which are the cathode incubation hole and the anode incubation hole respectively;

[0090] Among them, the depths of the two incubation holes and the direct flow channel in the figure are different, the depth of the incubation hole is 2mm, the depth of the direct flow channel is 1mm, and a slope is designed;

[0091] The first filling port (3), the exhaust port (4) and the second filling port (5) can be connected to the direct current channel at any side of the direct current channel;

[0092] The two sides of the direct current channel are respectively connected to the cathode incubation hole and the anode incubation hole;

[0093] When performing electrophoresis, the anode incubation hole is connected to the positive electrode; the cathode incubation hole is connected to the negative electrode;

[0094] In some embodiments, the shape design of the cathode incubation hole and the anode incubation hole can be changed as needed to ensure that the capacity of the incubation hole meets the capacity requirements for adding samples and buffer solutions.

[0095] In some embodiments, the microfluidic chip further comprises a cover sheet, which covers the microfluidic channel during use.

[0096] 2. Usage

[0097] The present application also provides a method for using the microfluidic chip designed in the present application to extract nucleic acid, amplify nucleic acid, and detect nucleic acid. Figure 4 As shown,

[0098] 2.1 Nucleic acid extraction includes:

[0099] Step 1: Prepare agarose gel ( Figure 4 A Left side)

[0100] Agarose powder from Biowest, Spain, and 0.5×TAE solution from Sangon Biotech, China, were heated at 95°C until dissolved to prepare 3% (w / v) and 0.8% (w / v) agarose gels. Transparent tape was applied to the surface of the microfluidic chip, but it was ensured that the exhaust port and the injection port were not covered to avoid obstruction of gas discharge and sample flow.

[0101] Then, draw 20 μl of 3% agarose gel and inject it into the channel through the second injection port. During the injection process, make sure that the 3% agarose gel does not exceed the vent hole.

[0102] After the 3% agarose gel solidifies, 20 μl of 0.8% agarose gel is drawn and injected into the channel through the first infusion port to form a continuous structure with the 3% agarose gel ( Fig. 9 E);

[0103] In some embodiments, a baffle is set in the microfluidic channel after the 3% agarose gel is injected, and the baffle is removed after the 3% agarose gel solidifies; then 20 microliters of 0.8% agarose gel is drawn and injected into the channel through the first injection port to form a continuous structure with the 3% agarose gel.

[0104] In some embodiments, after infusing 3% agarose gel, in the microfluidic channel, the pull-out blade located on the side of the exhaust hole close to the anode incubation hole is pushed out, and the blade is pushed back after the 3% agarose gel solidifies, and then 20 microliters of 0.8% agarose gel is aspirated and injected into the channel through the first infusion port to form a continuous structure with the 3% agarose gel.

[0105] Step 2: Cracking process ( Figure 4 A right side)

[0106] Lysozyme solution was prepared by dissolving buffered lysozyme powder (Sangon Biotech, China) in lysozyme buffer (Sangon Biotech, China), the component concentrations of which were as follows: lysozyme 1 mg / mL, 20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; 1.2% Triton X-100. 20 μL of sample was mixed with 5 μL of lysozyme solution in a test tube and incubated at 37°C for 5 minutes.

[0107] Then, 0.5 μL of 20 mg / mL proteinase K (Beyotime, China) was added to the lysate mixture and incubated for another 5 minutes.

[0108] Alternatively, chip lysis can be used. The basic steps of chip lysis are the same as those of tube lysis, except that the incubation time of proteinase K on the chip needs to be extended to 10 minutes. The amount of reagent can be adjusted proportionally according to the sample volume. After lysis is completed, add the lysate mixture to the cathode incubation well of the chip, and then add 0.5×TAE buffer, with a total of 80μL. Add 60μL TAE buffer to the anode well.

[0109] Step 3: Electrophoresis

[0110] In the figure, the depths of the sample well and the direct current channel are different. The sample well depth is 2mm, and the direct current channel depth is 1mm. A slope is designed (the slope is as shown in Figure 3 A front view), achieving the concentration function;

[0111] like Figure 5 As shown, after the gradient gel constructed in step 1 of the present application and the buffer solution added in step 2, the buffer solution and the solid phase structure on the chip are as follows Figure 5As shown in the figure, after the power is turned on, the uncharged and positively charged molecules are trapped in the buffer solution, and the negatively charged molecules enter the agarose gel. When the nucleic acid molecules are large, due to the high concentration of the high-density gel, the large molecules move slowly and are retained in the high-density gel ( Figure 6 )

[0112] In some embodiments, the regions A, B, C, and D corresponding to the microfluidic channels of the two concentrations of gel are divided as follows: Figure 7 A. Figure 7 As shown in B, it can be seen that the design of the sample addition port, microfluidic channel and gradient agarose in the present application makes the lengths of nucleic acid fragments enriched in different areas different. Figure 7 As shown in A, the microchannel of the microfluidic chip is divided into four areas: area A, area B, area C, and area D ( Figure 7 B).

[0113] The area division of the microfluidic chip of the present application is expressed as follows: the microchannel is approximately divided into 4 sections from the cathode incubation hole to the anode incubation hole, which are area A, area B, area C, and area D in sequence;

[0114] Region A is a section from the junction of the cathode incubation hole and the microfluidic channel to the first perfusion port on the side biased toward the anode;

[0115] Area B corresponds to the first injection port, starting from the side close to the anode and ending at the exhaust hole, close to the anode;

[0116] Area C corresponds to a small area starting from the side of the exhaust hole close to the anode to the second injection port and extending to the side close to the anode;

[0117] The D region corresponds to the end point of the C region to the junction of the microfluidic channel and the anode incubation hole.

[0118] Assuming that all nucleic acid groups are negatively charged, when the electrophoresis is electrified, small nucleic acid molecules move faster in low-concentration agarose gel. After entering high-concentration agarose gel, although the movement speed decreases, it is still relatively fast. Therefore, Fig. 9 As can be seen in A, the size of the nucleic acid enriched in the D region is the smallest. As the length of the nucleic acid molecule increases, the probability of the nucleic acid molecule reaching the D region decreases. Therefore, the nucleic acid molecules in the D region are mainly small in length (<2500bp);

[0119] For nucleic acid molecules with a length of about 15,000 bp, they are mainly enriched in the B region ( Fig. 9 A),

[0120] For nucleic acid molecules with a length of about 7000 bp, they are mainly enriched in the C region ( Fig. 9 A),

[0121] This is because high concentration of agarose gel will hinder the movement of long-fragment nucleic acid molecules, causing the nucleic acids to be enriched in fixed areas.

[0122] Figure 8 A shows the amplification effect of the enriched nucleic acid: Figure 8 A. The fragment distribution and length of nucleic acid in each region are measured using the Agilent Nucleic Acid Analyzer. The horizontal axis is the nucleic acid length, and the vertical axis is the nucleic acid content. Figure 8 B The left Y axis is the total nucleic acid concentration in each area, and the right Y axis is the number of fragments that can be amplified; (A) Distribution of bacterial DNA extracted from four areas; (B) DNA concentration (left Y axis) and amplifiable fragments (right Y axis) in four areas;

[0123] In some embodiments, we also tested the distribution of DNA in four regions when the length of the DNA fragment detected by electrophoresis was fixed. Since the length of the DNA fragment was fixed, the length of the DNA in each region must be fixed. Figure 7 As shown in C, for any fixed length of DNA, the enrichment in regions B and C is the highest, which further proves that the microchannel in the microfluidic chip designed in this application can achieve the effect of nucleic acid enrichment ( Figure 7 C); Figure 7 D shows the E. coli DNA extracted using the kit and then quantified by dPCR using 10 7 ,10 6 ,10 5 ,10 4 ,10 3 ,10 2 ,10 1 The detection limit of the test chip for the concentration of copise / ul;

[0124] In one embodiment, it is experimentally determined that the electrophoresis voltage suitable for Escherichia coli on a microfluidic chip is 70V for 15 minutes. Since the genome length of Escherichia coli is 4.7 million base pairs, this voltage is applicable to other pathogenic nucleic acids with genome lengths within this range.

[0125] Nucleic Acid Amplification

[0126] In some embodiments, in situ amplification is employed.

[0127] In some embodiments, qPCR+LAMP amplification is used; because the detection limit of LAMP amplification is high and the false positive rate is high, only qualitative judgment can be made, while qPCR can be used for quantitative judgment;

[0128] Loop-mediated isothermal amplification (LAMP) is an isothermal amplification test that utilizes the strand displacement activity of Bst DNA polymerase from Bacillus stearothermophilus to efficiently and robustly amplify any target nucleic acid using at least four primers (F3, B3, FIP, and BIP). The LAMP technique has been successfully used to detect a variety of infectious agents in humans, animals, and plants. This technique has also been used to detect a large number of plant viruses.

[0129] In some embodiments, after the electrophoresis is completed, the pull-out blades located at the first infusion port and the second infusion port are pushed out to cut off the target area, and the reagents added during amplification are added to the target area (area B and area C), and the amplification is heated to 65°C and then detected ( Figure 4 B).

[0130] In some embodiments, after electrophoresis is completed, according to Figure 7 A. Figure 7 The area division shown in B is performed, and area B and area C are cut out and taken out, and amplified in the tube using RCA technology.

[0131] In some embodiments, multiple pathogens are detected simultaneously, in which case in situ amplification is employed.

[0132] Fig. 9 The diagram shows the effect of proteinase K concentration on amplification provided by the embodiment of the present invention, the effect of lysozyme concentration on amplification, and the effect of electrophoresis buffer type and concentration on amplification. The overall purpose is to demonstrate the purification effect of agarose gel and prove that the protein content in the target area is low and will not affect amplification.

[0133] Problems with nucleic acid enrichment on microfluidic chips

[0134] (2) Problems with lysis detection on microfluidic chips

[0135] In some embodiments, the push-pull blade disposed on the side of the first perfusion hole close to the anode incubation hole is pushed out to cut the target area (i.e., the specific area).

[0136] In some embodiments, an external cutting device is used according to Figure 7 Shown in B is an agarose gel of the target regions of region B and region C.

[0137] There are two detection methods: qPCR and LAMP;

[0138] In some embodiments, after the push-pull blade cuts off the target area, nucleic acid amplification and nucleic acid detection are directly performed in the target area of ​​the microfluidic chip;

[0139] In some embodiments, the target area of ​​the microfluidic chip is cut and moved into an external container (such as an EP tube) for external amplification and detection.

[0140] In some embodiments, the microfluidic chip and the microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis proposed in the present application were used to detect pathogens, and the detection rates of fecA gene, glpQ gene, and ply gene corresponding to Escherichia coli (Ecoli), Streptococcus pneumoniae (S.pneumoniae), Haemophilus influenzae (Hi), Phlegm (sputum), and negative were measured (e.g. Fig.11 shown).

[0141] The large-scale spread of respiratory pathogens has put forward higher requirements for the integration level of rapid on-site detection. Microfluidics-based POCT systems provide an effective solution. We have developed a compact, integrated microfluidic system that effectively handles complex samples and multiple pathogens. By using LAMP amplification technology combined with a small fluorescence detection device, the system can obtain results quickly and conveniently. In addition, the platform does not require professional personnel, has low production costs, and requires minimal sample volume. Due to the low dependence on large laboratory equipment, the system can be applied to various environments outside traditional laboratories. It fills the gap in the current market for a highly integrated respiratory pathogen detection platform suitable for use outside the laboratory environment.

[0142] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0143] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0144] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0145] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0146] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. It should be understood by those skilled in the art that various modifications and combinations may be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A microfluidic chip for gradient gel electrophoresis, comprising a substrate, on which a cathode incubation hole, a microchannel, a first perfusion port, a second perfusion port, and an anode incubation hole are constructed, characterized in that: The microfluidic channel is a channel for gel electrophoresis, and the first perfusion port and the second perfusion port are connected to the side of the microfluidic channel.

2. The microfluidic chip for gradient gel electrophoresis according to claim 1, characterized in that: The microfluidic chip further comprises an exhaust hole, which is located between the first infusion port and the second infusion port and communicates with the side of the microchannel; Optionally, the material of the substrate is any one or more of the following: PMMA, PDMS, PC; Optionally, the depth of the anode incubation hole and the cathode incubation hole is greater than the depth of the microfluidic channel, and a slope is formed at the connection with the microfluidic channel; Optionally, the slope angle is 15° to 45°; Optionally, at least one side of the exhaust hole is provided with a push-pull blade; Optionally, at least one side of the first filling port and the second filling port is provided with a push-pull blade; Optionally, the push-pull blade of the first perfusion port is arranged on a side of the first perfusion port close to the cathode incubation hole, and the push-pull blade of the second perfusion port is arranged on a side of the second perfusion port close to the anode incubation hole. Optionally, the microfluidic chip further comprises an upper cover with an exhaust hole; Optionally, the microfluidic chip further comprises an external baffle, the size of the baffle being the same as the width of the microchannel; Optionally, the length of the microfluidic chip is 20 to 50 mm, the width is 10 to 20 mm, and the height is 3 to 8 mm; Optionally, the microfluidic chip has a length of 35 mm, a width of 15 mm, and a height of 5 mm; the microchannel has a width of 1 mm, a length of 30 mm, and a depth of 1 mm; Optionally, the depth of the anode incubation hole and the cathode incubation hole is 2 mm; Optionally, the width of the first perfusion port, the second perfusion port and the microchannel communication port is 1 mm; Optionally, the length of the first perfusion port and the second perfusion port is 3 to 5 mm; Optionally, the terminals of the first filling port and the second filling port are circular, and the diameter of the circle is greater than or equal to 1 mm; Optionally, power connection holes are provided on both sides of the chip, which are respectively used to connect the positive electrode and the negative electrode of the electrophoresis power supply.

3. A microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis, characterized in that: The microfluidic chip according to any one of claims 1 to 2 is used, wherein the cathode incubation hole of the microfluidic chip is connected to the negative electrode of electrophoresis, and the anode incubation hole is connected to the positive electrode of electrophoresis, and the method comprises the following steps: Obtain the lysate mixture of the sample to be tested, Injecting a second concentration gel into a second infusion port near the electrophoresis positive electrode and waiting for the gel to solidify, injecting a first concentration gel into a first infusion port near the negative electrode to connect the low concentration gel with the second concentration gel and waiting for the gel to solidify to obtain an electrophoresis gel, wherein the first concentration is less than the second concentration; adding the lysate mixture into the cathode incubation well, and adding a buffer, and adding a buffer into the anode incubation well; After the nucleic acid molecules in the sample to be tested move in the electrophoresis gel at a set voltage and for a set time, the nucleic acid molecules of a specific length are enriched in the enrichment area, which is located on both sides of the junction of the first concentration gel and the second concentration gel of the electrophoresis gel.

4. The microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis according to claim 3, characterized in that: The enrichment area is 1 to 5 mm on both sides of the junction; Optionally, the enrichment area is the area between the first perfusion port and the second perfusion port; Optionally, the enrichment region is a region between the left boundary of the first perfusion port and the right boundary of the second perfusion port; Optionally, a second concentration gel is injected and a baffle is placed in the middle of the exhaust port and then pulled out after the gel solidifies, so that the interface between the first concentration gel and the second concentration gel is vertical; Optionally, after injecting the gel of the second concentration, the blade on one side of the exhaust port is pushed out and then pushed back after waiting for the gel to solidify, so that the interface between the gel of the first concentration and the gel of the second concentration is vertical.

5. The microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis according to claim 3, characterized in that: The method further comprises: Adding a reaction system to the enrichment region to perform nucleic acid amplification; After heating the area where nucleic acid amplification has been completed, nucleic acid detection is performed to obtain the type of pathogen; Optionally, the gel in the enrichment area is cut and removed, and nucleic acid amplification and nucleic acid detection are performed on the removed gel to obtain the pathogen type.

6. The microfluidic nucleic acid enrichment and detection method based on gradient gel electrophoresis according to claim 3, characterized in that: The method further includes: the first concentration and the second concentration are determined based on the nucleic acid length of the target pathogen for respiratory pathogen detection; Optionally, the first concentration is 0.5-1%, and the second concentration is 3-3.5%; Optionally, the gel is agarose gel, the first concentration is 0.8%, and the second concentration is 3%; Optionally, the set voltage and the set duration are determined based on the nucleic acid of the target pathogen of respiratory pathogen detection; Optionally, the set voltage is 70V and the set time is 15 minutes.

7. The microfluidic nucleic acid enrichment and detection method based on gradient gel electrophoresis according to claim 3, characterized in that: After adding buffer into the cathode incubation well and the anode incubation well, the liquid level is consistent with or slightly lower than the gel height in the microfluidic channel; Optionally, the specific steps of obtaining the lysate mixture are: The buffered lysozyme powder was dissolved in the lysozyme buffer to prepare a lysozyme solution, wherein the concentration of the components of the lysozyme solution was: 1 mg / mL lysozyme, 20 mM Tris·Cl, pH 8.0; 2 mM EDTA sodium; 1.2% Triton X-100; Take 20 μL of the sample to be tested and mix it with 5 μL of lysozyme solution in a test tube or in a cathode incubation chamber, and incubate at 37°C for a certain period of time to obtain the first lysate mixture. 0.5 μL of 20 mg / mL proteinase K was added to the first lysate mixture and incubated for 5 minutes to obtain the lysate mixture.

8. The microfluidic nucleic acid enrichment and detection method based on gradient gel electrophoresis according to claim 4, characterized in that: The nucleic acid amplification is any one or more of the following: qPCR, LAMP, RPA, RCA; optionally, the sample to be tested contains multiple pathogens, qPCR is used to perform the nucleic acid amplification, the reaction system of the nucleic acid amplification contains primers for multiple pathogens, and the nucleic acid detection simultaneously detects the multiple pathogens contained in the sample to be tested.

9. Use of the microfluidic nucleic acid enrichment detection method based on gradient gel electrophoresis according to any one of claims 3 to 8 in the detection of respiratory pathogens.

10. A microfluidic nucleic acid enrichment detection system based on gradient gel electrophoresis, characterized in that: The system comprises: Acquisition unit: used to obtain the lysate mixture of the sample to be tested, Gradient gel preparation unit: used for injecting a second concentration gel into a second infusion port near the electrophoresis positive electrode and waiting for the gel to solidify, injecting a first concentration gel into a first infusion port near the negative electrode to connect the low concentration gel with the second concentration gel and waiting for the gel to solidify to obtain the electrophoresis gel, wherein the first concentration is less than the second concentration; Sample loading unit: add the lysate mixture to the cathode incubation well and add buffer; add buffer to the anode incubation well; Electrophoresis enrichment unit: used to enrich nucleic acid molecules of a specific length in the sample to be tested in the electrophoresis gel after the nucleic acid molecules move in the electrophoresis gel at a set voltage and for a set time. The enrichment area is located on both sides of the junction of the first concentration gel and the second concentration gel of the electrophoresis gel.

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