Microfluidic chip and method for recovering nucleic acids of different fragment sizes

Abstract

The application discloses a micro-fluidic chip and a method for recovering nucleic acids with different fragment sizes. The method can realize recovery of nucleic acids with different fragment sizes by repeatedly washing and recovering magnetic beads through the micro-fluidic chip. The method can improve the overall nucleic acid recovery effect by adjusting the adding concentration of the magnetic beads and PEG salt and the washing times, and can distinguish and control the nucleic acid length, and all the nucleic acids with different fragment lengths can be obtained by continuously adjusting the concentration. Moreover, the method can realize small volume, high throughput and automation.

Description

Technical Field

[0001] This article generally relates to the field of biotechnology, with a particular focus on a microfluidic chip and a method for recovering nucleic acids of different sizes. Background Technology

[0002] Nucleic acid molecules are the primary targets for processing in biology and genetic engineering, and they mainly include two types: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The backbone of nucleic acid molecules has a chain-like structure (straight-chain, branched-chain, or circular-chain). Based on the molecular weight of nucleic acid fragments, electrophoresis can be used to distinguish and recover target nucleic acid fragments. Electrophoresis is also one of the most commonly used methods for nucleic acid separation, purification, and enrichment.

[0003] Currently, existing methods for recovering nucleic acids from agarose gels typically involve first cutting the target fragment from the agarose gel electrophoresis plate, then adding a sol solution and dissolving the gel in a constant-temperature bath. The dissolved liquid then reversibly adsorbs and binds to the target fragment via a selective adsorption mechanism. After washing, the purified nucleic acid is obtained by elution. However, these existing methods are cumbersome, time-consuming, labor-intensive, and only recover a limited range of fragments, making them unsuitable for preparing large quantities of samples and hindering high-throughput processing. Summary of the Invention

[0004] Based on this, this application provides a microfluidic chip, comprising:

[0005] substrate;

[0006] Two or more processing zones are located on the substrate and are sequentially connected by channels, the channels containing valves for controlling fluid flow.

[0007] The processing area includes two or more inlet channels and one or more outlet channels, and the processing area also includes a magnetic control device capable of controlling the changes in the magnetic field in the processing area;

[0008] The opening and closing of the valve, two or more inlet channels, and one or more outlet channels are controlled by external signals.

[0009] On the other hand, this application also provides a method for recovering nucleic acids of different fragment sizes, including:

[0010] The sample solution is mixed with magnetic beads and then added to the microfluidic chip described herein. The mixed solution flows sequentially through each processing zone in the microfluidic chip, and the final waste liquid is discharged through the outlet channel of the last processing zone.

[0011] The mixed solution undergoes the following treatment steps in each treatment zone:

[0012] 1) Magnetic bead adsorption: Magnetic beads are pre-stored in each processing zone or magnetic bead stock solution is added through the first inlet channel. Depending on the concentration of magnetic beads and salt, nucleic acids of different fragment sizes are adsorbed onto the magnetic beads.

[0013] 2) Magnetic bead aggregation: The magnetic field of the processing zone is changed by the magnetic control device, causing the magnetic beads to aggregate on the outer wall of the processing zone. At this time, the valve is opened to allow the solution with the magnetic beads removed to flow into the next processing zone for further processing.

[0014] 3) Magnetic bead cleaning: Close the valve, remove the magnetic field, add cleaning fluid from the second inlet channel, so that the magnetic beads are resuspended in the cleaning fluid. After thorough cleaning, reapply the magnetic field so that the magnetic beads gather again on the outer wall of the processing area.

[0015] 4) Nucleic acid elution: The washing solution is discharged from the outlet channel, and the elution solution is added from the second inlet channel. The magnetic field is removed, so that the magnetic beads are resuspended in the elution solution. After sufficient elution, the magnetic field is reapplied, so that the magnetic beads are re-aggregated on the outer wall of the processing area. The elution solution containing nucleic acid fragments is discharged and recycled through the outlet channel.

[0016] On the other hand, this application also provides the use of the microfluidic chips described herein in gene sequencing, nucleic acid detection, molecular diagnostics, biological sample analysis and / or environmental analysis.

[0017] Traditional magnetic bead recovery processes require manual operation, which is time-consuming, labor-intensive, and prone to significant human error. This application utilizes microfluidic chips to repeatedly wash and recover magnetic beads, enabling the recovery of nucleic acids of different fragment sizes. By adjusting the concentration of magnetic beads and PEG salt and the number of washing cycles, this application improves the overall nucleic acid recovery efficiency and allows for the differentiation and control of nucleic acid lengths. Nucleic acid fragments of all lengths can be recovered by continuously adjusting the concentration.

[0018] The beneficial technical effects of this application include:

[0019] 1) Nucleic acid sorting can be achieved in microfluidic chips, enabling small volume, high throughput, and automation.

[0020] 2) The groove structure of the processing zone helps to retain more magnetic beads, reduce liquid residue, and improve the purification effect.

[0021] 3) The spherical protrusions in the processing area can increase the turbulence effect but reduce nucleic acid damage; at the same time, they can promote the redispersion of magnetic beads without causing magnetic bead residue.

[0022] 4) Multiple washing is possible, and nucleic acids from all fragments can be obtained by continuously adjusting the concentration.

[0023] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the solutions described in the description and the accompanying drawings. Attached Figure Description

[0024] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0025] Figure 1 This is a flowchart of the traditional magnetic bead sorting process.

[0026] Figure 2 This is a top view of the microfluidic chip in one embodiment of this application.

[0027] Figure 3 This is a diagram illustrating the structural optimization of the processing area in one embodiment of this application.

[0028] Figure 4 This is a side view of the processing area structure in one embodiment of this application.

[0029] Figure 5 This is a top view of the microfluidic chip in another embodiment of this application. Detailed Implementation

[0030] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood as equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0031] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0032] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0033] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0034] Traditional magnetic bead sorting processes require manual operation. An example of this process is... Figure 1 As shown: Adjust the concentrations of magnetic beads and PEG salt in the mixed solution according to the desired nucleic acid length. First, allow the magnetic beads to adsorb larger fragments. After removing the supernatant, wash the magnetic beads with 80% ethanol, then elute the nucleic acids with an aqueous solution. Smaller fragments in the supernatant are adsorbed by fresh magnetic beads. After removing the supernatant, wash the magnetic beads with 80% ethanol, then elute the nucleic acids with an aqueous solution. Repeat this process multiple times to separate nucleic acids of different sizes. This process requires continuous manual operation, which is tedious and time-consuming, and prone to human error. The magnetic beads are adsorbed by a magnet on the outside of the tube, which can easily leave liquid residue, affecting the experimental results.

[0035] In one aspect, this application provides a microfluidic chip, comprising:

[0036] substrate;

[0037] Two or more processing zones are located on a substrate and are connected sequentially by channels, which contain valves for controlling fluid flow.

[0038] The processing area includes two or more inlet channels and one or more outlet channels, and also includes a magnetic control device that can control the changes in the magnetic field within the processing area.

[0039] The opening and closing of the valve, two or more inlet channels, and one or more outlet channels are controlled by external signals.

[0040] In some embodiments, the magnetic control device is an electromagnet or a permanent magnet whose raising and lowering is controlled by an electrical circuit. The magnetic control device can be located on the sidewall surface outside the processing area or above the outside of the processing area. In some embodiments, the magnetic control device is located above the outside of the processing area or integrated into the upper surface structure of the processing area. In some embodiments, the magnetic control device is an electromagnet. When the electromagnet is energized, it provides a magnetic field to attract the magnetic beads. When the electromagnet is de-energized, the magnetic field disappears, and the magnetic beads can disperse again within the processing area. In some embodiments, the magnetic control device is a permanent magnet that is mechanically lowered or raised. When the permanent magnet is mechanically lowered, it provides a magnetic field to attract the magnetic beads. When the permanent magnet is mechanically raised, the magnetic field disappears, and the magnetic beads can disperse again within the processing area. Depending on the location of the magnetic control device, the magnetic beads can be gathered onto the sidewall or upper surface of the processing area.

[0041] In some embodiments, the upper surface of the treatment zone further includes one or more inwardly facing grooves. In some embodiments, the grooves are sloping. In some embodiments, the grooves are located near the outlet of the channel in the treatment zone, forming a gradually increasing slope, and the magnet is also located at this outlet. When a magnetic field is provided, the magnetic beads are attracted to the upper wall of the chamber by the magnet and the flow velocity, and the grooves at this location make it easier for the liquid to be retained, while the liquid can flow down in both directions due to gravity, thereby reducing liquid residue.

[0042] In some embodiments, the upper and / or lower surfaces of the processing zone further include one or more protrusions. These protrusions can increase the turbulence effect of the liquid flow in the processing zone, increase the shear force of the liquid flow, and facilitate better mixing of the solution. In some embodiments, the lower surface of the processing zone is an elastic film structure that can be deformed under external control. In some embodiments, the lower surface of the processing zone is an elastic film structure that can generate spherical protrusions under external control. In some embodiments, the protrusions are spherical. The spherical shape of the protrusions minimizes damage to nucleic acids and improves mixing efficiency. Furthermore, the spherical protrusions also promote the redispersion of magnetic beads without causing magnetic bead residue.

[0043] In some implementations, the processing zone also includes a temperature control device and / or a vibration device. Both the temperature control device and the vibration device are controlled by external signals to change the temperature of the processing zone and generate vibrations to promote the mixing and reaction of the components in the mixed solution.

[0044] In some implementations, external control is circuit-based. In other implementations, external control is achieved by directly controlling the input signal via an external signal line, or indirectly through active addressing.

[0045] In some embodiments, the substrate may contain two or more processing zones, which may be numbered sequentially from front to back. In some embodiments, the processing zones are sequentially classified into primary processing zones, secondary processing zones, and so on, up to Nth-level processing zones, based on their position on the substrate, where N represents the order of the processing zones on the substrate. In some embodiments, the substrate may contain two processing zones, namely a primary processing zone and a secondary processing zone. In some embodiments, the substrate may contain three processing zones, namely a primary processing zone, a secondary processing zone, and a tertiary processing zone. In some embodiments, the substrate may contain four processing zones, namely a primary processing zone, a secondary processing zone, a tertiary processing zone, and a quaternary processing zone. In some embodiments, the substrate may contain N processing zones, namely a primary processing zone, a secondary processing zone, a tertiary processing zone, and so on, up to Nth-level processing zones. In some embodiments, the primary processing zone further includes an air inlet allowing gas to enter, and the Nth-level processing zone further includes an air outlet allowing gas to exit. The air inlet and outlet are used to introduce and extract air into the processing zones, using the airflow to dry the assembled magnetic beads.

[0046] In some implementations, the microfluidic chip described herein consists of two layers: the upper surface of the lower layer has recessed channels formed by etching or casting, and the upper layer has a sloping recess in the processing area, with the entrance accessible by conventional external means for different solutions to enter.

[0047] On the other hand, this application also provides a method for recovering nucleic acids of different fragment sizes, including:

[0048] The sample solution is mixed with magnetic beads and then added to the microfluidic chip described herein. The mixed solution flows sequentially through each processing zone in the microfluidic chip, and the final waste liquid is discharged through the outlet channel of the last processing zone.

[0049] The mixed solution undergoes the following treatment steps in each treatment zone:

[0050] 1) Magnetic bead adsorption: Magnetic beads are pre-stored in each processing zone or magnetic bead stock solution is added through the first inlet channel. Depending on the concentration of magnetic beads and salt, nucleic acids of different fragment sizes are adsorbed onto the magnetic beads.

[0051] 2) Magnetic bead aggregation: The magnetic field of the processing zone is changed by a magnetic control device, causing the magnetic beads to aggregate on the outer wall of the processing zone. At this time, the valve is opened to allow the solution with the magnetic beads removed to flow into the next processing zone for further processing.

[0052] 3) Cleaning the magnetic beads: Close the valve, remove the magnetic field, add cleaning fluid through the second inlet channel, so that the magnetic beads are resuspended in the cleaning fluid. After thorough cleaning, reapply the magnetic field so that the magnetic beads gather again on the outer wall of the processing area.

[0053] 4) Nucleic acid elution: The washing solution is discharged from the outlet channel, and the elution solution is added from the second inlet channel. The magnetic field is removed, so that the magnetic beads are resuspended in the elution solution. After sufficient elution, the magnetic field is reapplied, so that the magnetic beads are re-aggregated on the outer wall of the processing area. The elution solution containing nucleic acid fragments is discharged and recycled through the outlet channel.

[0054] In some embodiments, in step 2), magnetic beads are gathered on the sidewall or upper surface of the processing zone using a magnetic control device. In some embodiments, in step 2), magnetic beads are gathered in a groove above the processing zone using a magnetic control device. In some embodiments, in step 2), under the combined action of magnetic force and liquid flow, the magnetic beads can be gathered in a sloping groove on the upper surface of the processing zone near the outlet, while the liquid can flow down in both directions under gravity, thereby reducing liquid residue.

[0055] In some implementations, in steps 1), 3), and / or 4), the deformation of the elastic film on the lower surface of the processing zone is controlled by an external signal to form spherical protrusions, thereby mixing the solution in the processing zone. One or more spherical protrusions can increase the turbulence effect of the liquid flow in the processing zone, increase the shear force of the liquid flow, and help the solution mix better. The spherical shape of the protrusions can minimize damage to nucleic acids and improve mixing efficiency. On the other hand, spherical protrusions can also promote the redispersibility of magnetic beads without causing magnetic bead residue.

[0056] In some implementations, in steps 1), 3), and / or 4), the temperature and / or vibration of the solution in the processing zone are controlled by an external signal-controlled temperature control device and / or vibration device. This better promotes the mixing and reaction of the components in the mixed solution.

[0057] In some implementations, step 3) and / or step 4) further includes a step of drying the magnetic beads: while keeping the magnetic beads aggregated, controlling the opening and closing of the valve, and venting air from the inlet and / or drawing air from the outlet to dry the magnetic beads.

[0058] In some embodiments, the magnetic bead washing step may be repeated once or more until satisfactory pure nucleic acid fragments are obtained. In some embodiments, the washing buffer is approximately 75%-85% (v / v) ethanol. In some embodiments, the washing buffer is approximately 80% (v / v) ethanol. In some embodiments, other types of washing buffers may also be used as needed.

[0059] In some implementations, the eluent is enzyme-free water or tris EDTA.

[0060] In some implementations, an additional resuspension and adsorption step is included between steps 3) and 4): the magnetic beads are resuspended with elution buffer and the nucleic acid fragments are eluted, and then a salt solution containing PEG high salt ions is passed through, so that some nucleic acids are re-adsorbed onto the magnetic beads. A magnetic field is reapplied to make the magnetic beads aggregate, and the nucleic acids remaining in the solution enter the next processing zone for further processing. The nucleic acids adsorbed on the magnetic beads are then processed by repeating steps 3) and 4).

[0061] In some implementations, in step 1), the size of the nucleic acids adsorbed onto the magnetic beads is controlled by controlling the concentration of PEG salt in the solution, thereby controlling the size of the nucleic acids recovered in each processing zone.

[0062] In some implementations, the substrate may contain only two processing zones to achieve the recovery of nucleic acids of various fragment sizes. First, in the primary processing zone, a high concentration of magnetic beads and PEG salt is used to adsorb large nucleic acid fragments onto the beads, while only small fragments remain in solution for further purification and recovery in the secondary processing zone. The magnetic beads adsorbed with large nucleic acid fragments are then eluted, and the magnetic beads and PEG salt are diluted to a specific concentration, allowing slightly smaller fragments to remain in solution for further purification and recovery in the secondary processing zone. Larger fragments remain in the primary processing zone for subsequent processing. Through this cyclical process and the adjustment of the magnetic bead and PEG salt concentrations, the recovery of nucleic acids of various fragment sizes can be achieved.

[0063] On the other hand, this application also provides the use of the microfluidic chips described herein in gene sequencing, nucleic acid detection, molecular diagnostics, biological sample analysis and / or environmental analysis.

[0064] This application utilizes a microfluidic chip for magnetic bead sorting. By continuously adjusting the concentration of the magnetic bead stock solution, a supernatant solution gradually obtains fragments ranging from small to large. These fragments are then subjected to magnetic bead adsorption, washing, and elution within the solution to obtain nucleic acids of varying sizes, achieving zoned recovery. The processing zone features magnetic fields and grooves on the chip surface, resulting in high magnetic bead retention and minimal liquid residue during supernatant removal. Temperature and vibration modules can be added to the processing zone as needed to better promote mixing and reactions of the components in the mixed solution.

[0065] The microfluidic chip and method for recovering nucleic acids of different sizes described in this paper can be applied to a variety of scenarios. For example, in CAR library construction, the lengths of nucleic acid fragments vary considerably, and existing methods lack effective control over fragment lengths. Furthermore, the impact of fragment lengths during subsequent library construction is not considered, resulting in a lack of length optimization systems. The microfluidic chip presented in this application can effectively distinguish and control the length of nucleic acid fragments, which is beneficial for providing higher-quality CAR libraries.

[0066] For example, the microfluidic chip of this application can be used as an optimization system for evaluating the effect of magnetic beads, and can evaluate and analyze the performance of the developed magnetic beads, which is beneficial to the development and optimization of magnetic beads.

[0067] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0068] This application includes and contemplates combinations of features known to those skilled in the art. The embodiments and features disclosed in this application can also be combined with any conventional features to form a unique inventive scheme as defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme as defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0069] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0070] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0071] Example

[0072] Example 1.

[0073] This embodiment illustrates an improvement to the processing region in a microfluidic chip. In one implementation, the improvement is as follows: Figure 3 As shown in the figure, the upper layer of the chip gradually deepens in the processing area. The sample mixture flows from top to bottom in the left image, corresponding to the liquid flowing from left to right in the upper cross-section of the right image. The upper layer of the chip is not at a uniform height; it is higher near the magnetic outlet, forming a groove that allows the magnetic beads to aggregate better, and the liquid flows around it with less residue. The upper and lower surfaces have multiple circular protrusions, which enhance the mixing effect of the solution in the processing area, as shown in the lower right cross-section.

[0074] In another implementation, an improvement to the circular protrusion is as follows: Figure 4 As shown, the bottom of the chip can adopt an elastic thin film structure. This design can increase the capacity of the processing area, and the bottom can perform operations such as mixing the liquid in the processing area by applying force.

[0075] Example 2.

[0076] Nucleic acids of different fragment sizes are obtained through multi-stage processing.

[0077] 1) Sample distribution and adsorption

[0078] Primary Processing Zone: The sample and 0.2× (diluted 5 times) magnetic beads (adjustable according to the size of the recovered fragments and needs) are mixed externally and introduced into the primary processing zone through the inlet (the number can be increased as needed, and control units can be used to control different solutions separately). A magnetic force is applied above the primary processing zone (either by energizing an electromagnet or mechanically lowering a permanent magnet). Magnetic beads with large nucleic acid fragments are magnetically concentrated in the upper layer of the primary processing zone, while small and medium-sized nucleic acid fragments remain in the solution and pass through the primary processing zone into the secondary processing zone.

[0079] Secondary processing zone: Pre-stored magnetic beads or added magnetic bead stock solution through the secondary processing zone inlet, "diluting" the magnetic beads to a concentration of 0.2× (adjustable according to the size of the recovered fragments and requirements) by the nucleic acid solution containing small and medium-sized fragments flowing out of the primary processing zone. A magnetic force is applied above the secondary processing zone (either by energizing an electromagnet or mechanically lowering a permanent magnet). Magnetic beads with medium-sized nucleic acid fragments are magnetically concentrated in the upper layer of the secondary processing zone, while small nucleic acid fragments remain in solution and pass through the secondary processing zone into the tertiary processing zone.

[0080] Level 3-N processing zones: Multiple processing zones may be involved as needed. Magnetic beads are pre-stored in the final processing zone, or magnetic bead stock solution is added through an inlet. This "dilutes" the magnetic beads to a concentration of 0.8× (a relatively high concentration) in the nucleic acid solution containing the smallest fragments flowing out from the previous processing zone. A magnetic force is applied above this processing zone (either by energizing an electromagnet or mechanically lowering a permanent magnet). The magnetic beads adsorbed with small nucleic acid fragments are magnetically concentrated in the upper layer of this processing zone. Since the solution has undergone several rounds of magnetic bead adsorption, the concentration is already low, and the demand for low-fragment solutions is not significant. The supernatant from this zone can be discharged as waste liquid without the need for further magnetic bead addition.

[0081] 2. Cleaning

[0082] The cleaning solution (80% ethanol) is introduced into each treatment zone through the cleaning / elution inlet (the number of inlets can be increased and control units can be added to control different solutions as needed). The magnetism is removed in each zone (the electromagnet is de-energized or the permanent magnet is mechanically raised), and the magnetic beads are suspended by gravity and the shear force of the cleaning solution. Magnetism is then applied again above that zone (the electromagnet is energized or the permanent magnet is mechanically lowered), and the waste liquid flows away from the cleaning outlet of each zone. The cleaning process can be repeated once or multiple times.

[0083] To ensure minimal liquid residue in the cavity, the valves in the channels between each stage can be opened while maintaining the magnetic force, and the magnetic beads can be further dried in the main channel by ventilation or evacuation.

[0084] 3. Washing

[0085] The blocking valves in the channels between each processing zone are closed. The elution solution (enzyme-free water or tris EDTA) is introduced into each processing zone from the washing / elution inlet (the number can be increased and control units can be used to control different solutions separately). The magnetic beads are suspended by gravity and the shear force of the washing solution. A magnetic force is applied again above this area of ​​the chip (the electromagnet is energized to open or the permanent magnet is lowered mechanically). The solution with different fragment lengths flows out from the washing outlet of each processing zone (a dedicated outlet can be used as needed) to recover nucleic acids of different fragment sizes.

[0086] Example 3.

[0087] Unlike Example 2, which involves initial allocation followed by cleaning and recycling in each processing zone, this example uses only two processing zones (e.g., ...). Figure 5 As shown in the image, magnetic beads of different sizes are obtained through multiple elution and washing processes. The specific steps are described below:

[0088] The sample is mixed with 0.8× concentration magnetic beads (adjustable according to the size of the recovered fragments and as needed, with the aim of retaining only small fragments in the solution) outside the chip and enters through the inlet of the primary processing zone (the number can be increased as needed, and control units can be used to control different solutions separately). A magnetic force is applied above the primary processing zone (either by energizing an electromagnet or by mechanically lowering a permanent magnet). Magnetic beads with large nucleic acid fragments adsorbed are magnetically gathered in the upper layer of the primary processing zone, while small nucleic acid fragments in the solution pass through the primary processing zone and enter the secondary processing zone.

[0089] Secondary processing zone: Pre-stored magnetic beads or added magnetic bead stock solution through the secondary processing zone inlet, "dilute" the magnetic beads to a certain concentration with the nucleic acid solution containing small and medium-sized fragments flowing out of the primary processing zone (this concentration can be adjusted according to the size of the fragments to be recovered and the needs, the purpose of which is to recover nucleic acids). Magnetic force is applied above the secondary processing zone (electromagnet is turned on or a permanent magnet is lowered mechanically), and the magnetic beads with small nucleic acid fragments adsorbed are magnetically gathered on the upper layer of the chip.

[0090] The cleaning solution (80% ethanol) is introduced into the secondary treatment zone from the cleaning / elution inlet of the secondary treatment zone (the number of inlets can be increased and control units can be added to control different solutions as needed). The magnetism in the secondary zone is removed (the electromagnet is de-energized or the permanent magnet is mechanically raised). The magnetic beads are suspended by gravity and the shear force of the cleaning solution. Magnetism is then applied again above the secondary treatment zone (the electromagnet is energized or the permanent magnet is mechanically lowered). The waste liquid flows away from the secondary cleaning outlet. To ensure minimal liquid residue in the chamber, the magnetic beads can be further dried by ventilation or evacuation while maintaining the magnetism. The cleaning step can be repeated once or multiple times.

[0091] Enzyme-free water or tris EDTA is introduced into the secondary processing zone through the washing / elution inlet (the number of inlets can be increased and control units can be used to control different solutions as needed). Magnetic beads are suspended by gravity and the shear force of the washing solution. Magnetic force is applied again above the secondary processing zone (the electromagnet is energized or the permanent magnet is lowered mechanically). The solution with short fragment lengths flows out from the secondary washing outlet (a dedicated outlet can be used as needed) for the recovery of nucleic acid fragments.

[0092] This completes the small fragment collection.

[0093] In the primary processing zone, washing is performed in a similar manner, and nucleic acids are eluted from the magnetic beads using enzyme-free water or Tris EDTA. At this point, a salt solution containing high levels of PEG ions is introduced, causing the nucleic acids to re-adsorb onto the magnetic beads. Adjusting the volume and concentration of the salt solution allows the magnetic beads to adsorb large fragments while leaving medium-sized fragments in the solution. A magnetic force is applied above the primary processing zone, causing the magnetic beads with large nucleic acid fragments to accumulate in the upper layer of the primary processing zone, while the medium-sized nucleic acid fragments remain in the solution and pass through the primary processing zone into the secondary processing zone. Medium-sized fragments are recovered and eluted using a similar method. To further improve fragment purity, after each elution, a solution of magnetic beads is introduced for re-selection and discharged with the waste liquid, and a fresh solution of magnetic beads is introduced for the next elution.

Claims

1. A microfluidic chip, characterized in that, include: substrate; Two or more processing zones are located on the substrate and are sequentially connected by channels, the channels containing valves for controlling fluid flow. The processing area includes two or more inlet channels and one or more outlet channels, and the processing area also includes a magnetic control device capable of controlling the changes in the magnetic field in the processing area; The upper surface of the processing area also includes one or more inward grooves, which are slopes with gradually increasing height in the direction of fluid flow. Magnetic beads are gathered in the grooves above the processing area by a magnetic control device. The upper and / or lower surface of the processing area also includes one or more protrusions, which are spherical protrusions. The lower surface of the processing area is an elastic film structure, which can deform under external control to form spherical protrusions. The opening and closing of the valve, two or more inlet channels, and one or more outlet channels are controlled by external signals.

2. The microfluidic chip according to claim 1, characterized in that, The magnetic control device is an electromagnet or a permanent magnet whose raising and lowering are controlled by a circuit; and The magnetic control device is located above the outside of the processing area, or integrated into the upper surface structure of the processing area.

3. The microfluidic chip according to claim 2, characterized in that, The processing area also includes a temperature control device and / or a vibration device.

4. The microfluidic chip according to claim 3, characterized in that, The processing areas are divided into primary processing areas, secondary processing areas, and N-level processing areas according to their positions on the substrate. The primary processing area also includes an air inlet that allows gas to enter, and the N-level processing area also includes an air outlet that allows gas to exit. Here, N represents the order of the processing areas on the substrate.

5. A method for recovering nucleic acids of different fragment sizes, comprising: The sample solution is mixed with magnetic beads and then added to the microfluidic chip of claim 4. The mixed solution flows sequentially through each processing zone in the microfluidic chip, and the final waste liquid is discharged through the outlet channel of the last processing zone. The mixed solution undergoes the following treatment steps in each treatment zone: 1) Magnetic bead adsorption: Depending on the concentration of the magnetic beads and the salt concentration, nucleic acids of different sizes are adsorbed onto the magnetic beads; 2) Magnetic bead aggregation: The magnetic field of the processing zone is changed by the magnetic control device, causing the magnetic beads to aggregate on the outer wall of the processing zone. At this time, the valve is opened to allow the solution with the magnetic beads removed to flow into the next processing zone for further processing. 3) Magnetic bead cleaning: Close the valve, remove the magnetic field, add cleaning fluid from the second inlet channel, so that the magnetic beads are resuspended in the cleaning fluid. After thorough cleaning, reapply the magnetic field so that the magnetic beads gather again on the outer wall of the processing area. 4) Nucleic acid elution: The washing solution is discharged from the outlet channel, and the elution solution is added from the second inlet channel. The magnetic field is removed, so that the magnetic beads are resuspended in the elution solution. After sufficient elution, the magnetic field is reapplied, so that the magnetic beads are re-aggregated on the outer wall of the processing area. The elution solution containing nucleic acid fragments is discharged and recycled through the outlet channel.

6. The method according to claim 5, characterized in that, In steps 1), 3), and / or 4), the deformation of the elastic film on the lower surface of the treatment zone is controlled by an external signal to form spherical protrusions, thereby mixing the solution in the treatment zone; and / or In steps 1), 3), and / or 4), the temperature and / or vibration state of the solution in the processing zone are controlled by external signals through the temperature control device and / or the vibration device in the processing zone.

7. The method according to claim 5, characterized in that, In step 3) and / or step 4), the step of drying the magnetic beads is further included: while keeping the magnetic beads aggregated, the opening and closing of the valve is controlled, and air is introduced from the air inlet and / or drawn from the air outlet to dry the magnetic beads.

8. The method according to claim 5, characterized in that, This also includes repeating step 3) once or more.

9. The method according to claim 5, characterized in that, The washing solution is 75%-85% (v / v) ethanol, and the elution solution is enzyme-free water or tris EDTA.

10. The method according to claim 5, characterized in that, Between steps 3) and 4), there is an additional resuspension and adsorption step: the magnetic beads are resuspended by elution buffer and the nucleic acid fragments are eluted, and then a salt solution containing PEG high salt ions is passed through, so that some nucleic acids are re-adsorbed onto the magnetic beads. A magnetic field is reapplied to make the magnetic beads aggregate. The nucleic acids remaining in the solution enter the next processing area for further processing. The nucleic acids adsorbed on the magnetic beads are repeated in steps 3) and 4).

11. The method according to any one of claims 5-10, characterized in that, In step 1), the size of the nucleic acids adsorbed onto the magnetic beads is controlled by controlling the concentration of PEG salt in the solution, thereby controlling the size of the nucleic acids recovered in each processing zone.

12. Use of the microfluidic chip according to any one of claims 1-4 in biological sample analysis or environmental analysis.

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