Microfluidic chip and nucleic acid enrichment and detection method based on gradient gel electrophoresis

Abstract

The application belongs to the field of nucleic acid detection, and particularly relates to a microfluidic chip based on gradient gel electrophoresis and a nucleic acid enrichment detection method. The method comprises the following steps: obtaining a lysate mixture of a sample to be detected, injecting a second concentration gel into a second perfusion port and waiting for the gel to solidify, injecting a first concentration gel into a first perfusion port 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 a cathode incubation hole, adding a buffer, adding the buffer into an anode incubation hole; and applying a voltage for a set time length, so that nucleic acid molecules in the sample to be detected move in the electrophoresis gel, and specific nucleic acid molecules are enriched in specific regions, wherein the specific regions are located on both sides of the junction between the first-concentration gel and the second-concentration gel of the electrophoresis gel. In the application, the design of the microfluidic chip and the setting of the gradient gel enable the target nucleic acid to be successfully enriched in the electrophoresis process.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection, and more specifically, to a microfluidic chip based on gradient gel electrophoresis and a method and system for nucleic acid enrichment detection. Background Technology

[0002] Microfluidic chip technology was proposed in the early 1990s and belongs to the rapidly developing high-tech and cutting-edge field of science and technology. Microfluidics is a technology that manipulates the volume of fluid from nanoliters to picoliters in micron-scale structures. Its purpose is to miniaturize, chip-scale, integrate and increase the throughput of analytical equipment, which is 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 for nucleic acid extraction include techniques based on centrifuge columns and magnetic beads. Centrifuge column methods, relying on high-speed centrifuges, are difficult to adapt to miniaturized systems, making them incompatible with portable diagnostic devices. Magnetic bead methods require multi-chamber structures to complete their purification and elution steps, which greatly increases the complexity of the system. Multi-chamber structures also lead to large system size and high manufacturing costs. Even methods that bypass the elution step still require complex control systems, resulting in increased instrument size and operating costs. Electrophoresis uses an electric field to move negatively charged nucleic acids towards the positive electrode. Compared to traditional DNA extraction, electrophoresis has significant advantages, enabling rapid separation and enrichment of nucleic acid fragments in a short time. For biomolecules, although their charge-to-mass ratios are similar, their sizes differ, such as proteins and nucleic acids, gel electrophoresis is more suitable. On a gel electrophoresis platform, small molecules move freely in the gel network with less resistance, while large molecules are hindered and migrate more slowly. However, traditional gel electrophoresis consumes a large amount of sample, and samples (nucleic acids and proteins) are often precious and scarce. In addition, conventional gel electrophoresis generates cumulative Joule heat under high voltage, which may lead to gel dissolution.

[0004] In the nucleic acid extraction step, existing technologies that are similar to those in this application include:

[0005] The article "Excisome Extraction 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 nanoporous membranes and agarose gel electrophoresis, and formed a vertical channel for sample introduction and an enrichment cavity channel for enriching exosomes by injecting 1% concentration of agarose.

[0006] Patent CN116139950A discloses a microfluidic system for rapid extraction of miRNA. From one side to the other, the microfluidic chip comprises a first electrode region, a first hydrogel region, a lysis chamber region, a separation hydrogel region, an elution chamber region, a second hydrogel region, and a second electrode region. The first electrode region includes a first electrode and a first electrode fluid channel; the second electrode region 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 sequentially adjacent to each other. This patent achieves rapid nucleic acid extraction by dividing the microfluidic chip into multiple distinct regions and applying different AC or DC voltages when cells are in different regions.

[0007] However, current technologies mainly focus on nucleic acid extraction, and also include microfluidic chips based on the isothermal amplification detection principle. There are no microfluidic chips that can integrate nucleic acid amplification and detection. The reason for this is that current microfluidic chips are designed for nucleic acid extraction in terms of nucleic acid amplification, and there are obstacles to their integration with nucleic acid detection.

[0008] Highly miniaturized microfluidic technology enables the integration of nucleic acid extraction, amplification, and signal detection onto a single chip. This reduces operational complexity, making microfluidic chips a powerful tool for point-of-care testing (POCT) development. Microfluidics provides a cost-effective approach to molecular diagnostics and also offers new opportunities for the point-of-care 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. By designing a new flow channel, nucleic acid enrichment and extraction are achieved, and a suitable electrophoresis voltage is determined in the microfluidic chip scenario. At the same time, nucleic acid extraction, nucleic acid amplification and nucleic acid detection are integrated into a single detection microfluidic chip.

[0010] This application (first aspect) discloses a microfluidic chip for gradient gel electrophoresis, including a substrate having an anode incubation hole, a cathode incubation hole, a microchannel, a first infusion port, and a second infusion port. The microchannel serves as a channel for gel electrophoresis, and the first infusion port and the second infusion port are connected to the side of the microchannel.

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

[0012] Using any of the microfluidic chips described above, wherein the cathode incubation port of the microfluidic chip is connected to the negative electrode of electrophoresis and the anode incubation port is connected to the positive electrode of electrophoresis, the method of use includes the following steps:

[0013] Obtain the lysis buffer mixture of the sample to be tested.

[0014] A second concentration of gel was injected into the second infusion port near the positive electrode of the electrophoresis apparatus, and the gel was allowed to solidify.

[0015] A first concentration gel is injected into the first infusion port near the negative electrode to connect the low-concentration gel with the second concentration gel. After the gel solidifies, an electrophoretic gel is obtained. The first concentration is less than the second concentration.

[0016] Add the lysis buffer mixture to the cathode incubation well and add buffer solution, and add buffer solution to the anode incubation well;

[0017] After nucleic acid molecules in the sample are moved in the electrophoresis gel by applying a set voltage for a set time, specific nucleic acid molecules are enriched in a specific region, which is located on both sides of the interface between the first concentration gel and the second concentration gel of the electrophoresis gel.

[0018] Furthermore, the specific area is 1-5 mm on each side of the boundary;

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

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

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

[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, a second concentration of gel is injected by pushing out one side of the venting blade, waiting for the gel to solidify, and then pushing it back in, so that the interface between the first and second concentration gels is perpendicular.

[0024] Furthermore, the method also includes:

[0025] Nucleic acid amplification is performed by adding a reaction system to the specific region.

[0026] The pathogen species were obtained by heating the region where nucleic acid amplification was completed and then performing nucleic acid detection.

[0027] Optionally, the gel in the specific area can be cut and removed, and the removed gel can be subjected to nucleic acid amplification and nucleic acid detection to obtain the pathogen species.

[0028] Furthermore, 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;

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

[0030] Optionally, the gel is an agarose gel, with the first concentration being 0.8% and the second concentration being 3%.

[0031] Optionally, the set voltage and 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 duration is 15min.

[0033] Furthermore, the specific steps for obtaining the lysis solution mixture are as follows:

[0034] A lysozyme solution was prepared by dissolving buffered lysozyme powder in lysozyme buffer. The lysozyme solution had the following components and concentrations: 1 mg / mL lysozyme, 20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; and 1.2% Triton X-100.

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

[0036] Add 0.5 μL of 20 mg / mL proteinase K to the first lysis buffer mixture and incubate for 5 minutes to obtain the lysis buffer mixture.

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

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

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

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

[0041] Acquisition unit: used to acquire the lysis buffer mixture of the sample to be tested.

[0042] Gradient gel preparation unit: used to inject a second concentration of gel into a second inlet near the positive electrode of electrophoresis and wait for the gel to solidify, and to inject a first concentration of gel into a first inlet near the negative electrode of electrophoresis so that the low concentration gel is connected with the second concentration gel and wait for the gel to solidify to obtain an electrophoretic gel, wherein the first concentration is less than the second concentration.

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

[0044] Electrophoretic enrichment unit: used to enrich specific nucleic acid molecules in a specific region after the nucleic acid molecules in the sample move in the electrophoresis gel when energized with a set voltage for a set time. The specific region is located on both sides of the interface between 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 microfluidic chip for extraction and detection, which 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 microfluidic chip design and gradient gel setup in this application enable the target nucleic acid to be successfully enriched during electrophoresis, and then amplified and extracted, which is simple and efficient.

[0048] (3) The detection method based on the microfluidic chip of this application can be applied to the detection of a variety of respiratory pathogens and has a very broad application prospect. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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 effort.

[0050] Figure 1 This is a schematic diagram of the structure of the microfluidic chip provided in the first aspect of the present invention;

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

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

[0053] Figure 4This is a schematic diagram illustrating the various steps of nucleic acid extraction, nucleic acid amplification, and nucleic acid detection based on a microfluidic chip, as provided in the embodiments of the present invention.

[0054] Figure 5 This is a schematic diagram illustrating the principle of low-density gel and high-density gel distribution for nucleic acid extraction using a pre-filled gel electrophoresis microfluidic chip, as provided in an embodiment of the present invention.

[0055] Figure 6 This is a schematic diagram of charge distribution and target region during nucleic acid extraction using a pre-filled gel electrophoresis microfluidic chip, provided by an embodiment of the present invention.

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

[0057] Figure 8 This is a schematic diagram illustrating the enrichment and amplification effects of nucleic acids enriched in different regions of a microchannel, as provided in an embodiment of the present invention.

[0058] Figure 9 This is a schematic diagram illustrating the effect of proteinase K concentration on amplification, provided in an embodiment of the present invention.

[0059] Figure 10 A diagram showing the principle of enrichment and the correspondence between the enrichment region of a microchannel is provided for an embodiment of the present invention.

[0060] Figure 11 This is a schematic diagram illustrating the detection effect of a microfluidic chip on several viruses, as provided in an embodiment of the present invention.

[0061] Figure 12 This is a schematic diagram of a microfluidic nucleic acid enrichment and detection system based on gradient gel electrophoresis, provided in an embodiment of the present invention. Detailed Implementation

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

[0063] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as S101, S102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

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

[0065] The proposed solution selected agar electrophoresis as the method for nucleic acid extraction and detection in microfluidic chips. To successfully miniaturize agar electrophoresis, the following methods were employed:

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

[0067] This method places the sample containing pathogens along with the gel in an electric field. The electric field breaks down cells and forces the charged contents into the gel. As long-chain biomolecules, nucleic acids exhibit significantly different electromigration rates compared to other biomolecules within the gel, resulting in distinct positions. Commonly used nucleic acid electrophoresis gel materials can perform the above-mentioned nucleic acid screening. However, since the gel containing the target nucleic acid needs to be added to the amplification system along with the nucleic acid, this places demands on the gel's physicochemical properties. Agarose is preferred as the 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, which are completely independent of reagent addition and only require temperature control. Thus, the gel acts as a solid for nucleic acid screening during extraction and dissolves in the reaction solution as a liquid during amplification. In preparation, molten agarose is injected into a prepared thin channel using a syringe. The liquid agarose forms a column due to surface tension, which can be freely moved and repositioned by gravity or pressure. After repositioning, it solidifies into a usable, fixed columnar gel. The solidified gel can be preserved for a long time without deterioration or shrinkage, and remains in close contact with the sidewalls, provided it is sealed and immersed in a solution. Figure 3 The diagram shown illustrates the principle of nucleic acid extraction via gel electrophoresis in this section.

[0068] (2) Designing a density gradient to stop nucleic acid migration: The microstructure of the gel can filter electromigration molecules based on their size. To achieve better separation, a gel density gradient is designed, such as... Figure 3 As shown. The gel density in the cell-contact region is lower, facilitating nucleic acid entry and blocking uncharged macromolecules in the solution. The concentration of low-density agarose gel is preferably 0.5-1%, which allows nucleic acid molecules to pass through normally while effectively blocking cell debris and larger protein molecules. The gel density at the ends of the gel, away from the sample, should be higher to hinder nucleic acid entry 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 hinders the migration of target nucleic acids as much as possible without hindering the passage of nucleic acid debris and charged small molecules, thus achieving the purpose of screening and purification. In this way, nucleic acids of different lengths within a certain range can migrate into the lower concentration gel and remain at the density abrupt change position of the gel after migrating a certain distance. The gel with density gradient can achieve the purpose of enriching nucleic acid fragments of the target length at the preset density abrupt change position. After the electrophoresis step, the density abrupt change region can be directly cut to obtain agarose blocks enriched with nucleic acid fragments from the sample, which can be added to the pre-prepared amplification system for melting and amplification.

[0069] (3) Optimization of Electrophoresis Buffer and Field Strength: The buffer is used to prepare the gel and is mixed with the sample before being added to the sample cell. Unlike conventional electrophoresis, the field strength is greater during cell electrolysis, and the pH of the solution near the cells should also be higher. The strong electric field and alkaline environment promote the rupture of the phospholipid membrane structure and release of the nucleic acid-containing substances. In combination with conditions that maintain nucleic acid stability, a TAE solution with a pH of 7.5-8.0 is preferred 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 clean proteins and other substances. Based on this, 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 be selected from common components in quantitative amplification solutions, such as Tris, Tween-20, DMSO, etc., and amplification tests should be performed to ensure that the amplification efficiency is not affected. The electrophoresis device is powered by a platinum electrode with one end in direct contact with the solutions on both sides and the other end leading to an external power source. Since there is no need for precise resolution of nucleic acid bands, and the electrode distance in miniaturized devices is relatively short, a relatively small applied voltage is sufficient to achieve the required electric field strength. In actual experiments, the electric field strength should be greater than 30 V / cm. A higher field strength is beneficial for accelerating membrane structure disruption and promoting nucleic acid migration, thereby speeding up nucleic acid purification.

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

[0071] (5) Stability test: Electrophoretic nucleic acid extraction was performed using samples containing several known components of the same kind in different proportions and repeated to compare the extraction stability of the platform under different sample input conditions. Combined with fluorescence amplification, parameters of the accuracy of sample component reduction were obtained.

[0072] 2. Gel-pathogen nucleic acid mixture multisite quantitative detection technology:

[0073] (1) Input of gel-pathogen nucleic acid mixture into amplification system: After nucleic acid extraction, the gel containing the nucleic acid to be tested is directly input into the pre-prepared and packaged amplification system for nucleic acid amplification. Since the aforementioned density gradient gel has a localization enrichment effect on nucleic acids, the target nucleic acid will be trapped in the density mutation region. Therefore, only the gel at the density mutation position is cut off, directly mixed with the pre-prepared amplification system, and then the agarose gel is heated to melt, which completes 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; the fixed position can be removed. The entire volume of the cut-off nucleic acid-gel mixture contains more nucleic acid to be tested but also contains more agarose, which increases the volume of the amplon solution. By comparing the amplification efficiency of different input volumes, the optimal amplification input amount is obtained. A reference dye of a fixed concentration is premixed in the amplification system as a calibration basis.

[0074] (2) Selection of target sites and primer design: 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 almost no impact on PCR. Choosing appropriate reaction systems and temperatures minimizes the influence of gel and electrophoresis buffer on amplification to ensure efficiency and accuracy. Adding appropriate concentrations of surfactants and small-molecule proteins (such as fetal bovine serum albumin) can further reduce the influence of agarose on amplification. For primers with different sequences, the corresponding annealing temperatures and concentrations are optimized, and fluorescence changes during the amplification process are used for detection.

[0076] (4) Specificity and sensitivity tests: Further gel electrophoresis and sequencing analysis were performed on the amplification products from the amplification detection stage to understand the sequence characteristics of the products and thus determine the specificity of the amplification detection. Samples containing target nucleic acids at different concentrations were input, extracted, and detected, and the detection limits of the detection platform for different pathogens were statistically analyzed.

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

[0078] (1) Microfluidic chip gel electrophoresis module: Two structures containing semi-cylindrical cavities are cast using PDMS material and a positive mold, and then bonded together 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 internal cylindrical cavity has a diameter between 5mm and 1cm, ensuring that the soft flow channel does not easily collapse and facilitating the stable formation of a liquid column of molten agarose. Agarose of different densities is injected in stages and solidifies. After maintaining a low temperature to allow the agarose to undergo a phase transition and form a gel, electrophoresis buffer is filled into the cavity connected to the positive electrode, reducing subsequent experimental procedures and preventing the agarose gel from shrinking. The sample input cavity is left empty. Before extraction, the entire outer surface of the 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 introduced into the negative electrode connection area. Because the two electrodes in the chip are close together, the required electric field strength can be obtained efficiently without high-voltage DC during electrophoresis. The electrophoresis time should be sufficient to migrate the nucleic acids to the vicinity of the gel density gradient interface. The transparent properties of PDMS also facilitate the mixing of nucleic acid dyes into the sample for real-time fluorescence observation during electrophoresis. Figure 5 Nucleic acid extraction was performed using a pre-filled gel electrophoresis microfluidic chip.

[0079] (2) Real-time quantitative amplification module: The structural diagram and operation procedure of this module are as follows: Figure 12As shown. Since the final position of nucleic acid migration in the gel is relatively fixed, in actual operation, the gel a few millimeters before and after the density interface is directly cut off as a template and placed into the pre-prepared amplification system in the tube for amplification. Since the size of the electrophoresis microfluidic chip is fixed and PDMS itself is soft and easy to cut, a track with double-layer transparent blades can be prefabricated to fix the gel cutting process. The blade material is preferably a perforated rigid plastic sheet to facilitate 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 pre-placed and placed in a modular disposable reaction chamber. The reaction chamber is pre-filled with amplification reaction solution as needed, and its surface sealing film is in close contact with the chip. After cutting the gel, the blade directly pushes out the agarose gel portion, destroys the sealing film of the reaction chamber, and places it into the reaction solution. The temperature during the amplification process will directly melt the low-melting-point agarose and release nucleic acids. After this, the concentration of agarose mixed in the solution will be insufficient to solidify or hinder the amplification reaction. The released pathogen nucleic acid will bind to specific primers, amplify and generate a fluorescent signal, which can be observed. Figure 12 );

[0080] (3) External power supply, temperature control, and fluorescence system: The electrophoresis and amplification processes described above require a DC power input and a temperature control system to function. The power supply system uses a rechargeable lithium battery for direct power supply. Because the current and power requirement are very small during electrophoresis, a 2Ah capacity lithium battery is sufficient for multiple electrophoresis extractions. The microchip is directly connected to a reaction chamber. The total volume of the amplification reaction is small, and each amplification only takes about 1-1.5 hours. Therefore, the power requirement for temperature control is also low. It can be achieved using a programmed microcontroller with a small heating pad. Temperature control is also powered by the aforementioned lithium battery. The bottom of the rigid plate used to cut the electrophoresis module directly contacts the probe containing the microlens to amplify the fluorescence signal in the reaction tube. The probe is sealed on all sides and its diameter is no larger than the cylinder of the well plate. This allows it to occupy the cavity cut out inside the electrophoresis module under the protection of the cutting plate. The probe can be used with a fluorescence microscope for precise quantitative detection at the end of the reaction, or it can be directly integrated with a programmable fluorescence excitation detection device for automatic detection. For integrated fluorescence detection equipment, a temperature control microcontroller is required. During the amplification extension stage, the sample is irradiated with the wavelength corresponding to the dye (e.g., 480nm for SYBR, achieved via a white LED and color filter). Signal acquisition then occurs through an emission filter within the probe (e.g., 520nm for SYBR) and a post-sensor connected by an optical cable. These signals are recorded on a data acquisition card and storage medium, and a fluorescence curve is automatically generated by software at the end of the experiment. When simultaneously detecting multiple samples and various pathogens, 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 parallel detection needs.

[0081] (4) Clinical evaluation and application of the detection platform: The agarose gel electrophoresis nucleic acid extraction and amplification system will be packaged into a microfluidic chip and combined with an external control system to form a complete rapid respiratory pathogen detection platform. In cooperation with relevant provincial and municipal institutions, such as grassroots disease control centers, samples will be collected, and existing chemical extraction methods and quantitative real-time PCR will be used as references to determine the sensitivity and accuracy of this detection platform. Clinical evaluation of the platform application and products will be conducted, and qualified products will be promoted and tested. The technical roadmap is as follows: Figure 12 This is the technology roadmap for this project.

[0082] Figure 1 This is a chip structure diagram of a microfluidic chip for respiratory pathogen detection provided in an embodiment of the present invention. The microfluidic chip includes: a cathode incubation hole (1), a microchannel (2), a first infusion port (3), an exhaust port (4), a second infusion port (5), and an anode incubation hole (6).

[0083] Considering that electrophoresis is the electrolysis of water and the liquid will decrease, this application designs cathode incubation wells and anode incubation wells with larger volumes, so that no liquid needs to be added during the electrolysis process. The cathode incubation wells are designed to be larger than the anode incubation wells because the cathode incubation wells need to add both electrophoresis buffer and sample, while the anode incubation wells only need to add electrophoresis buffer.

[0084] I. Chip Design and Fabrication

[0085] The gel electrophoresis microfluidic chip developed in this study uses a PC sheet with dimensions of approximately 35mm*15mm as the substrate for the microfluidic channels. The microfluidic channels are designed based on this substrate, and the channel design is as follows: Figure 1 As shown, Figure 2 The figure shown is a top view and a schematic diagram of the size of the microfluidic chip of this application. Figure 3 The diagram shown is a schematic front view of the microfluidic chip of this application;

[0086] In some embodiments, the microfluidic chip is 3D printed.

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

[0088] In some embodiments, the microchannel consists of a horizontal channel with dimensions of 30mm*1mm*depth mm and two infusion ports with dimensions of 5mm*1mm*depth mm and vent holes with dimensions of 3mm*1mm*depth mm connected perpendicularly to the horizontal channel.

[0089] The microchannel has buffer loading areas on both sides, namely the cathode incubation well and the anode incubation well;

[0090] In the figure, the two incubation holes and the DC channel have different depths. The incubation hole depth is 2mm and the DC channel depth is 1mm. A slope was designed.

[0091] Among them, the first injection port (3), the exhaust port (4), and the second injection port (5) can all be connected to the DC channel on either side of the DC channel;

[0092] The DC channel is connected to the cathode incubation hole and the anode incubation hole on both sides, respectively.

[0093] During 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 includes a cover plate, which is used to cover the microchannels during use.

[0096] II. Instructions for Use

[0097] This application also provides a procedure for nucleic acid extraction, nucleic acid amplification, and nucleic acid detection using the microfluidic chip designed in this application, as follows: Figure 4 As shown,

[0098] 2.1 Nucleic acid extraction includes:

[0099] Step 1: Preparation of agarose gel ( Figure 4 (Left side)

[0100] Agarose powder from Biowest (Spain) and 0.5×TAE solution from Sangon Biotech (China) were heated to 95°C until dissolved to prepare 3% (w / v) and 0.8% (w / v) agarose gels. Transparent tape was then applied to the surface of the microfluidic chip, ensuring that the vent and infusion ports were not covered to avoid obstructing gas escape and sample flow.

[0101] Then, draw 20 μL of 3% agarose gel and inject it through the second infusion port. During injection, ensure that the 3% agarose gel does not exceed the vent hole.

[0102] After the 3% agarose gel solidifies, take 20 μL of 0.8% agarose gel and inject it into the channel through the first infusion port to form a continuous structure with the 3% agarose gel. Figure 9 (as shown in E);

[0103] In some embodiments, after infusing 3% agarose gel, a baffle is placed in the microchannel. After the 3% agarose gel solidifies, the baffle is removed. Then, 20 μL 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.

[0104] In some embodiments, after infusing 3% agarose gel, a pull-out blade located on the side of the vent near the anode incubation port is pushed out in the microchannel. After the 3% agarose gel solidifies, the blade is pushed back, and then 20 μL 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: Pyrolysis process ( Figure 4 (A right side)

[0106] Lysozyme powder (Sangon Biotech, China) was dissolved in lysozyme buffer (Sangon Biotech, China) to prepare a lysozyme solution with the following concentrations: lysozyme 1 mg / mL, 20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; 1.2% Triton X-100. 20 μL of the sample was mixed with 5 μL of the 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 lysis buffer mixture, and the mixture was incubated for another 5 minutes.

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

[0109] Step 3: Electrophoresis

[0110] The depths of the sample inlet and the DC channel in the diagram are different; the sample inlet depth is 2mm, and the DC channel depth is 1mm. A ramp was designed (the ramp is shown in the diagram). Figure 3 (As shown in the front view of A), the condensation function is implemented;

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

[0112] In some embodiments, the division of regions A, B, C, and D corresponding to the microchannels of the two concentration gels is as follows: Figure 7 A, Figure 7 As shown in Figure B, the design of the sample loading port and microfluidic channel combined with gradient agarose in this application results in different lengths of nucleic acid fragments enriched in different regions. Figure 7 As shown in Figure A, the microchannels of the microfluidic chip are divided into four regions: Region A, Region B, Region C, and Region D. Figure 7 B).

[0113] The microfluidic chip of this application is divided into regions as follows: the microchannel from the cathode incubation hole to the anode incubation hole is approximately divided into 4 segments, namely region A, region B, region C, and region D.

[0114] Region A is the area from the junction of the cathode incubation hole and the microchannel to the side of the first injection port that is biased towards the anode.

[0115] Area B corresponds to the first injection port near the anode side starting from the side of the vent hole near the anode side;

[0116] Region C corresponds to a small section extending from the side of the vent hole closest to the anode to the side of the second injection port closest to the anode.

[0117] Region D corresponds to the endpoint of region C, extending to the junction of the microchannel and the anode incubation hole.

[0118] Assuming all nucleic acid clusters are negatively charged, after electrophoresis, smaller nucleic acid molecules move faster in low-concentration agarose gels. While their movement speed decreases upon entering high-concentration agarose gels, it remains relatively fast. Therefore, from... Figure 9 As can be seen from A, the nucleic acid enriched in region D is the smallest. As the length of the nucleic acid molecule increases, the probability of the nucleic acid molecule reaching region D decreases. Therefore, the nucleic acid molecules in region D are mainly small in length (<2500bp).

[0119] For nucleic acid molecules with a length of approximately 15000 bp, they are mainly enriched in region B. Figure 9 A),

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

[0121] This is because high concentrations of agarose gel hinder the movement of long nucleic acid molecules, thus causing nucleic acids to accumulate in fixed regions.

[0122] Figure 8 A shows the amplification effect of the enriched nucleic acids: Figure 8 A. The Agilent nucleic acid analyzer was used to measure the fragment distribution and length of nucleic acids in each region. The x-axis represents nucleic acid length, and the y-axis represents nucleic acid content. Figure 8 B. Total nucleic acid concentration in each region on the left Y-axis, and the number of amplifiable fragments on the right Y-axis; (A) Distribution of bacterial DNA extracted from the four regions; (B) DNA concentration (left Y-axis) and amplifiable fragments (right Y-axis) in the four regions;

[0123] In some embodiments, we also tested the distribution of DNA in four regions when the length of the DNA fragments detected by electrophoresis was fixed. Since the DNA fragment lengths were fixed, the length of DNA appearing in each region was necessarily fixed. However, according to... Figure 7 As shown in Figure C, for any fixed length of DNA, the enrichment is highest in regions B and C, further proving that the microchannels in the microfluidic chip designed in this application can achieve the effect of nucleic acid enrichment. Figure 7 (as shown in C); Figure 7 D shows the E. coli DNA extracted using the kit, which was 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, the suitable electrophoresis voltage for E. coli on a microfluidic chip was determined experimentally to be 70V for 15 minutes. Since the genome length of E. coli is 4.7 million base pairs, this voltage is also applicable to the nucleic acids of other pathogens with genome lengths within this range.

[0125] 2.2. Nucleic acid amplification

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

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

[0128] Loop-mediated isothermal amplification (LAMP) is an isothermal amplification assay that utilizes the strand substitution activity of Bst DNA polymerase from Bacillus stearothermophilus to efficiently and stably amplify any target nucleic acid using at least four primers (F3, B3, FIP, and BIP). LAMP technology has been successfully used to detect a wide range 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 electrophoresis, the pull-out blades located at the first and second filling ports are extended to truncate the target region. Reagents added during amplification are then added to the target region (regions B and C), and the region is heated to 65°C for amplification before detection. Figure 4 (as shown in B).

[0130] In some embodiments, after electrophoresis is completed, according to Figure 7 A, Figure 7 As shown in B, regions B and C are divided and extracted, and then amplified in the tube using RCA technology.

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

[0132] Figure 9 The diagram illustrates the effects of proteinase K concentration, lysozyme concentration, and electrophoresis buffer type and concentration on amplification, as provided in this embodiment of the invention. The overall purpose is to demonstrate the purification effect of agarose gel, proving that the low protein content in the target region will not affect amplification.

[0133] The problem of nucleic acid enrichment in microfluidic chips

[0134] (2) Problems in detecting the fragmentation of microfluidic chips

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

[0136] In some embodiments, an external cutting device is used according to Figure 7 As shown in Figure B, the target regions of regions B and C are agarose gels.

[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 performed directly in the target area of ​​the microfluidic chip;

[0139] In some embodiments, the target region 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 gradient gel electrophoresis-based microchannel nucleic acid enrichment detection method proposed in this application were used to detect pathogens, and the detection rates of the corresponding fecA gene, glpQ gene, and ply gene in Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae, Phlegm, and negative bacteria were measured (e.g., Figure 11 (As shown).

[0141] The widespread transmission of respiratory pathogens has placed higher demands on the integration of rapid, on-site detection. Microfluidic-based point-of-care testing (POCT) systems offer an effective solution. We have developed a compact, integrated microfluidic system that efficiently handles complex samples and a variety of pathogens. By using LAMP amplification technology combined with a small fluorescence detection device, the system provides rapid and convenient results. Furthermore, the platform requires no specialized personnel, has low production costs, and requires minimal sample volume. Due to its low dependence on large laboratory equipment, the system can be applied to various environments outside of traditional laboratories. It fills the gap in the current market for highly integrated respiratory pathogen detection platforms suitable for use outside of laboratory settings.

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

[0143] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between apparatuses or units through some interfaces, and may be electrical, mechanical, or other forms.

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

[0145] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

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

Claims

1. A method for nucleic acid enrichment detection based on a gradient gel electrophoresis microfluidic chip for non-disease diagnostic purposes, characterized in that, The microfluidic chip includes a substrate on which are constructed a cathode incubation port, a microchannel, a first infusion port, a second infusion port, an anode incubation port, and an exhaust port. The microchannel serves as a channel for gel electrophoresis, and the first and second infusion ports are connected to the sides of the microchannel. The cathode and anode incubation ports are located on both sides of the microchannel and connected to it. The cathode and anode incubation ports have the same depth, which is greater than the depth of the microchannel. The anode incubation port is connected to the positive electrode, and the cathode incubation port is connected to the negative electrode. The exhaust port is located between the first and second infusion ports and is connected to the side of the microchannel. The enrichment detection method includes the following steps: 1) Obtain the lysis buffer mixture of the sample to be tested; 2) Using the microfluidic chip, a second concentration gel is injected into the second infusion port near the positive electrode of electrophoresis. After the gel solidifies, a first concentration gel is injected into the first infusion port near the negative electrode to connect the first concentration gel with the second concentration gel. After the gel solidifies, an electrophoretic gel is obtained. The first concentration is less than the second concentration, forming an electrophoretic gel with a continuous structure in which the concentration increases from the first infusion port to the second infusion port. 3) Add the lysis buffer mixture to the cathode incubation well, and add buffer solution to both the cathode and anode incubation wells; 4) After the set voltage is applied for a set time, nucleic acid molecules in the sample to be tested move in the electrophoresis gel and nucleic acid molecules of a specific length are enriched in the enrichment region, which is located on both sides of the interface between the first concentration gel and the second concentration gel of the electrophoresis gel. 5) Add the reaction system to the enriched region to amplify nucleic acid; 6) Nucleic acid detection is performed on the regions where nucleic acid amplification has been completed to obtain the pathogen species.

2. The nucleic acid enrichment detection method according to claim 1, characterized in that, The enrichment region is 1-5 mm to each side of the boundary between the first concentration gel and the second concentration gel.

3. The nucleic acid enrichment detection method according to claim 1, characterized in that, The microfluidic chip also includes an external baffle, the size of which is the same as the width of the microchannel; in step 2), the baffle is first placed in the middle of the vent hole, and then a second concentration gel is injected through the second infusion port. After the second concentration gel solidifies, the baffle is removed, and a first concentration gel is injected through the first infusion port, so that the interface between the first concentration gel and the second concentration gel is perpendicular.

4. The nucleic acid enrichment detection method according to claim 1, characterized in that, At least one side of the vent hole has a built-in push-pull blade; in step 2), the blade on one side of the vent hole is pushed out, and then the second concentration gel is injected through the second injection port. After the second concentration gel solidifies, the blade is pushed back, and the first concentration gel is injected through the first injection port, so that the interface between the first concentration gel and the second concentration gel is perpendicular.

5. The nucleic acid enrichment detection method according to claim 3 or 4, characterized in that, The microfluidic chip also includes a cover for an exhaust port.

6. The nucleic acid enrichment detection method according to claim 1, characterized in that, The first and second infusion ports have built-in push-pull blades. The push-pull blade of the first infusion port is located on the side of the first infusion port near the cathode incubation hole, and the push-pull blade of the second infusion port is located on the side of the second infusion port near the anode incubation hole. In step 5), the gel in the enrichment area is cut and removed using the push-pull blades. The removed gel is then subjected to nucleic acid amplification and nucleic acid detection to obtain the pathogen type.

7. The nucleic acid enrichment detection method according to claim 1, characterized in that, The first and second concentrations of the gel are determined based on the nucleic acid length of the target pathogen for respiratory pathogen detection.

8. The nucleic acid enrichment detection method according to claim 7, characterized in that, The first concentration is 0.5-1%, and the second concentration is 3-3.5%.

9. The nucleic acid enrichment detection method according to claim 1, characterized in that, The set voltage and set duration are determined based on the nucleic acid length of the target pathogen for respiratory pathogen detection.

10. The nucleic acid enrichment detection method according to claim 9, characterized in that, The set voltage is 70V and the set duration is 15min.

11. The nucleic acid enrichment detection method according to claim 1, characterized in that, After adding buffer solution to the cathode and anode incubation wells, the liquid level is lower than or equal to the gel height in the microchannels.

12. The nucleic acid enrichment detection method according to claim 1, characterized in that, The specific steps for obtaining the lysis solution mixture in step 1) are as follows: Lysozyme powder was dissolved in lysozyme buffer to prepare a lysozyme solution. The lysozyme solution had the following components and concentrations: 1 mg / mL lysozyme, 20 mM Tris·Cl, pH 8.0; and 2 mM sodium EDTA. 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. After incubation at 37 °C for a certain period of time, the first lysis buffer mixture is obtained. Add 0.5 µL of 20 mg / mL proteinase K to the first lysis buffer mixture and incubate for 5 minutes to obtain the lysis buffer mixture.

13. The nucleic acid enrichment detection method according to claim 1, characterized in that, The nucleic acid amplification is performed by qPCR, LAMP, RPA, or RCA.

14. The nucleic acid enrichment detection method according to claim 1, characterized in that, The sample to be tested contains multiple pathogens, the nucleic acid amplification reaction system contains primers for multiple pathogens, and the nucleic acid detection detects multiple pathogens simultaneously.

15. The nucleic acid enrichment detection method according to claim 1, characterized in that, The substrate is made of PMMA, PDMS, or PC.

16. The nucleic acid enrichment detection method according to claim 1, characterized in that, The depth of the anode incubation hole and the cathode incubation hole is greater than the depth of the microchannel, and a slope is formed at the connection with the microchannel.

17. The nucleic acid enrichment detection method according to claim 16, characterized in that, The slope angle is 15°~45°.

18. The nucleic acid enrichment detection method according to claim 1, characterized in that, The microfluidic chip has a length of 20-50 mm, a width of 10-20 mm, and a height of 3-8 mm.

19. The nucleic acid enrichment detection method according to claim 18, characterized in that, The microfluidic chip has a length of 35mm, a width of 15mm, and a height of 5mm; the microchannel has a width of 1mm, a length of 30mm, and a depth of 1mm.

20. The nucleic acid enrichment detection method according to claim 19, characterized in that, The depth of the anode incubation hole and the cathode incubation hole is 2 mm.

21. The nucleic acid enrichment detection method according to claim 20, characterized in that, The width of the first and second injection ports and the microchannel connection port is 1 mm.

22. The nucleic acid enrichment detection method according to claim 21, characterized in that, The lengths of both the first and second injection ports are 3-5 mm.

23. The nucleic acid enrichment detection method according to claim 22, characterized in that, The ends of both the first and second injection ports are circular, and the diameter of the circle is greater than or equal to 1 mm.

24. The nucleic acid enrichment detection method according to claim 1, characterized in that, The chip has power terminals on both sides, which are used to connect to the positive and negative terminals of the electrophoresis power supply, respectively.

25. The application of the nucleic acid enrichment detection method according to any one of claims 1 to 24 in the detection of pathogens for non-disease diagnostic purposes.

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