Nucleic acid extraction and enrichment system based on digital microfluidic technology

Abstract

The invention discloses a nucleic acid extraction and enrichment system. The system comprises a mixing and splitting module, a cleaning and eluting module, a liquid drop control module, a pressure control module and an extrusion module. The nucleic acid extraction and enrichment system can complete 10-20 times of nucleic acid enrichment, solves the problem of false negative in nucleic acid detection caused by lower virus concentration at the initial stage of infection, can reduce the influence of personnel operation on the result, reduces aerosol pollution, and also can reduce the reagent dosage.

Description

Technical Field

[0001] The invention discloses a nucleic acid extraction and enrichment system. Background Art

[0002] The detection of biological samples first requires the extraction and enrichment of biological molecules in the samples, such as nucleic acids and proteins. However, the current extraction and enrichment technologies rely on laboratories and complex analytical instruments, and can only be completed by professionals after multiple operation steps, which makes the analysis of the test results time-consuming. For example, nucleic acid extraction generally requires four steps: lysis, binding, washing, and elution, plus subsequent nucleic acid molecule hybridization, polymerase chain reaction (PCR), and biochip detection steps, making the entire detection process time-consuming.

[0003] Digital microfluidics (DMF) is a technology for manipulating single droplets. Its principle is to use the electrowetting (EWOD) effect on the medium. It does not require complex microfluidic channels, nor pumps or valves to generate droplets from the chip reservoir and perform basic operations such as moving, splitting, and merging. It can realize droplet manipulation in air environments and oils, so it is also suitable for volatile or easily oxidized reagents. Therefore, it is widely used in chip laboratories and instant detection technology (POCT, Point-of-Care Testing) and other fields.

[0004] In view of the fact that the prior art is unable to solve the problems of false negatives that are prone to occur in the early stage of epidemiological infection due to low nucleic acid concentration, a long time of infection required to detect nucleic acid, aerosol contamination, and large errors in manual nucleic acid extraction, the purpose of the present invention is to provide a nucleic acid extraction and enrichment system with EWOD chip as the core, which can complete automatic extraction and enrichment of nucleic acid in a relatively short time and can realize automatic extraction of nucleic acid under sealed conditions. Summary of the invention

[0005] Based on the above purpose, the present invention first provides a nucleic acid extraction and enrichment system, the system comprising: a mixing and lysis module, a cleaning and elution module, a droplet control module, a pressure control module and an extrusion module, wherein The mixing and lysis module is used to mix the working reagent with the sample to be detected, lyse the protein, release the nucleic acid and capture the released nucleic acid with magnetic beads. The mixing and lysis module includes an injection slot, a mixing and lysis slot, a gear stirring mechanism, a mixing and heating device, and a pre-packaged sealed form of protease, magnetic beads with adsorption effect on nucleic acid and lysing agent; the pre-packaged sealed form of protease, magnetic beads with adsorption effect on nucleic acid and lysing agent is communicated with the injection slot of the mixing and lysis module through a slot hole set at the bottom of the placement position, and the injection slot is connected with the mixing and lysis slot, so as to mix and lyse the protease, magnetic beads with adsorption effect on nucleic acid and lysing agent with the added sample to be detected and then transmit them to the digital microfluidic chip; The cleaning and elution module is used to clean and elute the captured nucleic acid, and includes a flow channel layer, a digital microfluidic chip, and a pre-packaged sealed form of a cleaning solution and an eluent. The upper surface of the flow channel layer is provided with a complete reagent injection area for placing proteases, magnetic beads with adsorption effects on nucleic acids, lysing agents, and pre-packaged sealed forms of cleaning solutions and eluents. The proteases, magnetic beads with adsorption effects on nucleic acids, and lysing agents are mixed with the added sample to be tested and lysed, and then transferred to the digital microfluidic chip through the magnetic bead transfer channel of the flow channel layer. The cleaning solution and eluent are squeezed by the pressure control module and directly transferred to the digital microfluidic chip for cleaning and elution; the digital microfluidic chip is used to complete nucleic acid extraction The digital microfluidic chip includes, from bottom to top, a printed circuit board, a dielectric layer, a first hydrophobic layer, a spacer, a water-absorbing layer, a second hydrophobic layer, and a conductive layer, wherein the printed circuit board is provided with upper and lower plate electrodes, a heating electrode, a liquid separation electrode, a waste liquid suction electrode, and a tail liquid suction connection electrode for connecting the printed circuit board and the conductive layer into a loop, and passing the eluent that has been extracted and enriched from the card box into the test container through the tail liquid suction flow channel; The droplet control module is used to control the size of droplets, the direction of movement of magnetic beads, and the combination and dissociation of magnetic beads and working reagents during nucleic acid extraction and enrichment, and includes a magnetic permeability structure with switchable magnetic strength, wherein the magnetic permeability structure with switchable magnetic strength includes a weak magnetic strength for assisting the driving of magnetic beads, and a strong magnetic strength for gathering magnetic beads and separating them from reagents; The pressure control structure includes positive pressure and negative pressure devices for driving and transmitting liquid, and a gas valve plug for controlling the liquid to enter the mixing and pyrolysis tank from the injection tank and enter the chip from the mixing and pyrolysis tank, and a release plug for controlling the liquid to enter the digital microfluidic chip from the flow channel layer; The extrusion module includes a mechanical arm, microcolumns and steel balls arranged on the upper surface of a pre-packaged flow channel layer for placing working reagents. The extrusion module is used to extrude the unsealed and released protease, magnetic beads with adsorption effect on nucleic acid and lysing agent in the pre-packaged sealed form to enter the mixed lysis module, and the cleaning liquid and eluent directly enter the digital microfluidic chip, and after the lysis is completed, the release plug is squeezed to allow the liquid to enter the digital microfluidic chip from the flow channel layer, and then the air valve plug is squeezed to make the mixed lysis tank relatively sealed.

[0006] In a preferred embodiment, the extrusion module comprises one robot arm and extrudes sequentially or comprises a plurality of robot arms and extrudes in parallel.

[0007] In a specific embodiment of the present invention, the flow channel layer and the digital microfluidic chip are placed in a card box, and the card box includes a card box cover, a flow channel layer and a digital microfluidic chip from top to bottom. A slot is provided at the bottom of the reagent injection area on the upper surface of the flow channel layer to communicate with the injection slot of the flow channel layer, and the injection slot is connected to the mixing and lysis slot to transfer the working reagent transferred from the reagent injection area and the added sample to be detected to the digital microfluidic chip. The working reagents share a slot hole to enter the sample feeding slot; the flow channel layer is also provided with an air valve slot and a release slot, and the release slot and the air valve slot are respectively connected to the mixed lysis slot through two flow channels, and the release slot is provided with a release plug and a release hole; the air valve slot is in an open state before and during mixed lysis, for releasing the positive pressure released outward after the pre-packaged sealed form is unsealed and driving the working reagent to enter the mixed lysis slot from the sample feeding slot, and the release slot is also connected to the mixed lysis slot, and a release plug is provided thereon to pierce the sealing film that blocks the magnetic bead transfer flow channel and the digital microfluidic chip, so that the release plug pierces the sealing film after the mixed lysis is completed to allow the working reagent to enter the digital microfluidic chip, and then the slot hole of the air valve slot is plugged, and the mixed lysis slot is relatively sealed. Under the positive pressure of the pressure control structure, the liquid in the mixed lysis slot can only enter the chip unidirectionally from the mixed lysis slot through the magnetic bead transfer flow channel.

[0008] In a specific embodiment of the present invention, the flow channel layer includes a top film, a flow channel layer body and a bottom film, thereby forming a closed flow channel space for transporting relevant reagents and magnetic beads.

[0009] In a specific embodiment of the present invention, the first common flow channel shared by the protease, magnetic beads, and lysate (the protease / magnetic beads / lysate common flow channel), the second common flow channel shared by the magnetic beads and lysate (the magnetic beads / lysate common flow channel), and the lysate flow channel share a common outlet to enter the injection tank.

[0010] In a specific embodiment of the present invention, the reagent is prepackaged in a blister, and the size of the blister can be set to 80 μL-1000 μL according to the required amount of the prepackaged reagent. The blister is connected to the cartridge by being attached to the corresponding position on the upper surface of the flow channel layer.

[0011] In a preferred embodiment, the reagents include protease, lysing agent, eluent, cleaning agent, and magnetic beads with adsorption effect on nucleic acid. In a specific embodiment of the present invention, the protease is proteinase K.

[0012] In a specific embodiment of the present invention, the magnetic beads having an adsorption effect on nucleic acids may be magnetic beads with silanol chemically modified on the surface.

[0013] In a specific embodiment of the present invention, a robotic arm can be used to squeeze the pre-packaged form of the reagent, and the microcolumns on the flow channel layer will puncture the pre-package to release the reagent into the corresponding slot and merge into the mixed lysis tank. In specific applications, the five liquids and microcolumns are specifically divided into three types: ① Proteinase K, there is a flat-headed microcolumn with a height of 0.4mm under the magnetic bead bubble; ② There is no microcolumn at the bottom of the lysate bubble, and there is a 3.67mm groove where the lysate bubble is attached to the flow channel layer. When working, the robotic arm first presses the steel ball with a diameter of about 2mm on the lysate bubble, and then squeezes the bubble to allow the working liquid to enter the flow channel; ③ There are pointed microcolumns with a height of 0.4~0.6mm under the eluent and cleaning liquid bubbles.

[0014] In a preferred embodiment, the gear stirring mechanism is used to drive the mixing and stirring in the mixing and lysing tank, and the gear promotes protein lysis by alternating forward and reverse rotations.

[0015] In a preferred embodiment of the present invention, the heating electrodes provided in the digital microfluidic chip include a pyrolysis heating electrode for heating the mixed pyrolysis module and an elution heating electrode for heating the cleaning elution module, wherein the pyrolysis heating electrode heats the mixed pyrolysis tank through a copper block under the mixed pyrolysis tank. In a specific embodiment of the present invention, the heating temperature is between 60 and 70°C.

[0016] In a preferred embodiment of the present invention, the switchable magnetic strength of the magnetic guide structure is divided into a weak magnetic strength for driving the magnetic beads to move in the liquid but not to separate from the reagent liquid and a strong magnetic strength for driving the magnetic beads to gather and separate from the reagent liquid. In the process of the magnetic beads adsorbed with nucleic acid being transferred from the flow channel layer to the digital microfluidic chip, the magnetic guide structure will give a strong magnetic effect, so that the waste liquid is completely transferred, and the magnetic beads adsorbed with nucleic acid are retained. Afterwards, under the action of the electric field force and magnetic field generated by the digital microfluidic chip, various types of liquids are separated, cleaned and eluted. The strong magnetism generated by the magnetic field plays a role in separating or mixing the magnetic beads with the specific liquid in this process, and the weak magnetism plays a role in dispersing the magnetic beads in the specific liquid in this process.

[0017] In a preferred embodiment of the present invention, the dielectric layer of the digital microfluidic chip is a 2 μm polyester film.

[0018] In a preferred embodiment of the present invention, the conductive layer is an indium tin oxide coating with glass as the substrate, an indium tin oxide coating with a flexible film as the substrate, or an indium tin oxide coating with the lower surface of the flow channel layer as the substrate. In a specific embodiment, the flexible film can be 9795R single-sided adhesive (Funna Thin Film Materials (Shanghai) Co., Ltd.).

[0019] In a preferred embodiment of the present invention, the first hydrophobic layer and the second hydrophobic layer are processed by a resin solution with a solid content of 3%, a spin coating parameter of 400 rpm for 18 s, 2000 rpm for 60 s, and a drying process of 120°C for 40 min.

[0020] In a preferred embodiment of the present invention, the liquid separation electrode includes a small electrode and a large electrode, the length and width of the small electrode are both 2 mm, and the length of the large electrode is 10 mm and the width is 2 mm. Based on the space separated by the spacer with a thickness of 0.2~0.25mm, the volume of each droplet of the small electrode is 0.8 μL~1.0 μL, which can achieve precise control of the minimum droplet of 0.8~1 μL, and the large electrode can achieve precise movement and liquid separation of 4~5μL droplets.

[0021] In a more preferred embodiment, a droplet feedback mechanism is provided in the small electrode and the large electrode, and whether there are droplets on the electrode can be determined by detecting the change in capacitance. When the number of small electrodes with droplets does not reach a threshold, the mechanical arm of the extrusion module, the positive pressure control module and / or the droplet control module drive the droplet to move; when the number of small electrodes with droplets reaches a threshold, the mechanical arm of the extrusion module and / or the droplet control module stop driving the droplet to move or the negative pressure control structure drives the droplet to move.

[0022] The threshold value of the present invention corresponds to the number of droplets that need to enter. In a specific embodiment of the present invention, the threshold value of the cleaning solution is set to 10 (10 small electrodes), and the threshold value of the elution solution is set to 15 (15 small electrodes).

[0023] In a specific embodiment of the present invention, only a small part of the large electrode has droplets, that is, the entire electrode displays droplets; when there are droplets on the small electrode, only this grid has droplet feedback, and when there are droplets on two small electrodes, these two grids have droplet feedback. Therefore, the area in the middle of the chip that needs to accurately control the amount of liquid entering is all small electrodes.

[0024] In a specific embodiment of the present invention, after the mixed lysis is completed, when the robotic arm squeezes the release plug and the air valve plug, the strong magnet is turned on. At this time, if no droplets enter the large electrode and / or the small electrode, there is no droplet feedback from the electrodes on the digital microfluidic chip. At this time, the pressure control structure starts the positive pressure pump to operate at a low speed, and the positive pressure drives the mixed solution into the large electrode and / or the small electrode, and the strong magnet gathers the entering magnetic beads. At this time, there is droplet feedback on the electrode near the absorbent paper, and the positive pressure pump operates with a certain duty cycle (the positive pressure pump operates intermittently to prevent excessive pressure and liquid from covering the PCB), the waste liquid in the mixed lysis tank is sucked away by the absorbent layer, and then the strong magnet drags the magnetic beads to mix with the cleaning solution to enter the cleaning and elution program; if there is still no droplet feedback from the electrode near the absorbent paper on the chip after the pressure control structure starts the positive pressure low-speed operation, the positive pressure is increased to high-speed operation until droplet feedback is generated.

[0025] In another specific embodiment of the present invention, in the cleaning and elution procedure, after the mixed droplets of magnetic beads and cleaning solution enter the digital microfluidic chip, if the small electrode of the chip has reached the threshold value (10 or 15 grids), the robotic arm stops squeezing, and the number of droplets entering the digital microfluidic chip can be controlled to 10 or 15 μL, and the droplets are driven by the electric field to perform the cleaning and elution procedures in the chip; if the droplets in the small electrode are less than 10 or 15 grids, the robotic arm continues to squeeze the bubble until it is full of 10 or 15 grids and enters the cleaning and elution procedures. Before the elution procedure, the cleaned magnetic beads will be dragged to the elution heating zone by a strong magnet, and the elution step will be performed after the residual cleaning solution on the surface of the magnetic beads is evaporated.

[0026] In another specific embodiment of the present invention, after the elution procedure is completed, the strong magnetic drags the magnetic beads out of the elution zone, and the droplets move to the tail aspiration point under the drive of the electric field. At this time, the logic judges whether the tail aspiration connection electrode has 9μL (the 3*3 nine-square grid formed by the tail aspiration connection electrode and the other 8 small electrodes around it is covered with droplets). If it does not reach 9μL, the electric field continues to drive the eluent into the elution zone, otherwise, the negative pressure drive is started to complete the extraction and enrichment of the eluent from the tail aspiration hole facing the electrode through the tail aspiration flow channel from the card box into the container for inspection. In a specific embodiment, the container for inspection is an EP tube.

[0027] In a preferred embodiment, the upper and lower plate electrodes are connected to connect the PCB circuit board and ITO to form a loop, so as to realize the change of positive and negative poles of the upper and lower plates when alternating current is supplied.

[0028] The waste liquid absorbing electrode can assist in making the waste liquid on the chip more easily absorbed by the water absorbing layer.

[0029] In a preferred embodiment, a bonding layer for providing buffering and protection is provided between the flow channel layer and the digital microfluidic chip.

[0030] In a preferred embodiment, the water-absorbing layer is used to absorb the lysis waste liquid of the mixed lysis tank and the cleaning waste liquid of the cleaning step, and the residual cleaning waste liquid after absorption by the water-absorbing layer is removed by heating; in a specific embodiment of the present invention, the water-absorbing layer is absorbent paper.

[0031] In yet another preferred embodiment, the interior of the digital microfluidic chip is an air phase.

[0032] In a preferred embodiment of the pressure control module of the present invention, a positive pressure input port and a negative pressure input port are provided, which can be connected to a positive pressure device and a negative pressure device respectively, for example, a positive pressure pump can be provided, a positive pressure is formed by a positive pressure pump stirring paddle, and the positive pressure is transmitted through a positive pressure pump connecting pipe, and a negative pressure pump can be provided, a negative pressure pump can be provided, a negative pressure is formed by a negative pressure pump stirring paddle, and the negative pressure is transmitted through a negative pressure pump connecting pipe. The positive pressure device is used to send proteinase K, magnetic beads, and lysate into a mixed lysate tank, and the lysate waste liquid is sent into a positive pressure transmission system of a water-absorbing layer via a magnetic bead transfer channel, and the negative pressure device is used to suck the elution tail liquid out of the cartridge into a container in a container for inspection.

[0033] In another preferred embodiment, an air valve plug and a release plug are also provided on the upper surface of the flow channel layer. The release plug is provided on the release groove to puncture the sealing film that blocks the magnetic bead transfer flow channel and the digital microfluidic chip, so as to puncture the sealing film after the mixed lysis is completed so that the mixed lysis liquid enters the digital microfluidic chip. The air valve plug is connected to the mixed lysis tank through the flow channel, and is in an open state before the mixed lysis is completed, and is used to release the positive pressure generated by the pre-packaged reagent after being punctured, and / or drive the liquid from the injection slot into the mixed lysis tank. After the positive pressure is released, the air valve plug is tightened by applying pressure by the mechanical arm, and the mixed lysis tank is relatively sealed; in a specific embodiment of the present invention, the sealing film is set to tin foil, and the release plug can be squeezed by the mechanical arm to puncture the sealing film to connect the flow channel layer and the digital microfluidic chip.

[0034] The steps of a specific embodiment of nucleic acid extraction and enrichment based on digital microfluidics technology of the present invention are as follows: (1) Injection step: The sample to be tested is added to the injection tank through a pipette, and the blister package containing proteinase K, magnetic beads, and lysis solution is pressurized by a robotic arm, and enters the injection tank along the common flow channel through the slot under the blister; by inputting positive pressure into the flow channel, the mixed solution is driven from the injection tank into the mixing lysis tank.

[0035] (2) Mixed lysis step: The lysis heating electrode on the PCB starts to heat up, and the mixed lysis tank is heated by the copper block with silicone grease on both the upper and lower surfaces; at this time, the gear starts to rotate (forward and reverse rotation are performed alternately to make the lysis more complete), and the lysis step is performed. In this process, the viral nucleic acid will be released under the action of proteinase K and the lysis solution, and at the same time bind to the magnetic beads (the surface of the magnetic beads is chemically modified (silicon hydroxyl magnetic beads), which can bind to nucleic acids). After the lysis is completed, the gear stops rotating, the PCB stops heating, and the robot moves to the release plug for squeezing, pierces the tin foil with the puncture plug, and then squeezes to the air valve plug to seal the mixed lysis tank. At the same time, positive pressure is input; to prevent the magnetic beads from sinking to the bottom, the gear also starts to rotate; the mixed liquid in the mixed lysis tank is evenly transferred from the bottom hole of the mixed lysis tank through the magnetic bead transfer channel from the release hole and the magnetic bead injection hole on the chip into the chip. At the same time, the strong magnet is turned on, and the magnetic beads in the mixed lysis tank flow out together with the liquid in the mixed lysis tank. The magnetic beads are adsorbed to the magnetic bead injection hole on the digital microfluidic chip under the action of the strong magnet and enter the digital microfluidic chip; the liquid in the mixed lysis tank flows out under the action of positive pressure and is absorbed by the absorbent paper on the digital microfluidic chip.

[0036] (3) Cleaning steps: The robot squeezes the cleaning solution bubble, and after the bubble is punctured, it enters the chip through the slot set at the bottom of the reagent placement area (if manual liquid addition is used), the robot cooperates with the droplet feedback system on the chip, and the cleaning solution is driven by the electric field force generated by the digital microfluidic chip. A fixed volume of cleaning solution set by the threshold is injected. The magnetic beads are mixed with the cleaning solution under the action of a strong magnet, and are first moved back and forth on the chip for cleaning under the assistance of a weak magnet and the action of an electric field; then the weak magnet is removed, and the magnetic beads and the cleaning solution mixture are cleaned on the chip under the action of the electric field, and finally moved to the waste liquid absorption electrode under the action of the electric field. The strong magnet is turned on, and the waste liquid is absorbed under the action of the electric field and the drainage of thin absorbent paper, and this is repeated three times; the magnetic beads that have completed three washes are dragged to the elution area by the strong magnet and heated to evaporate the residual cleaning solution on the surface of the magnetic beads.

[0037] (4) Elution step: The reagent eluent is pre-packaged and squeezed by the robotic arm. After the pre-package is punctured, it enters the chip through the slot set at the bottom of the placement area (if manual liquid addition is used, it can be added manually). The robotic arm and the droplet feedback system on the chip cooperate, and the eluent is driven by the electric field force generated by the digital microfluidic chip to inject a fixed volume of eluent set by the threshold. The magnetic beads are mixed with the eluent under the action of a strong magnet, and are first moved back and forth on the chip under the assistance of a weak magnet and the action of an electric field for elution; then the weak magnet is removed, and the magnetic beads and eluent mixture are eluted on the chip under the action of the electric field. Finally, the strong magnetic action separates the magnetic beads from the eluent, and the eluent is transferred to the enrichment sample receiving port at the tail of the cartridge under the action of the electric field. During this process, the chip droplet feedback will also cooperate with the pump to suck all the liquid in the tail liquid suction area into the EP tube for PCR or leave the droplets on the chip for on-chip PCR amplification.

[0038] In the present invention, the chip used for cleaning and elution in the process of nucleic acid extraction / purification uses a digital microfluidic chip (EWOD chip). Through the design of the present invention, the EWOD chip can operate microliter-level or even smaller droplets, so the use of magnetic beads, cleaning solution and elution solution can be reduced, wherein the magnetic beads are reduced to 1 / 10, and the same nucleic acid extraction and enrichment effect can be achieved; the cleaning solution is 1 / 20 (30 / 600) of the existing one; the elution solution is reduced by 1 / 20~3 / 20 (5 / 100~15 / 100), while achieving nucleic acid extraction and enrichment, it can also save the amount of reagents; at the same time, the card box is small and portable, and can be used in POCT with a small supporting instrument (length * width * height: 367 mm * 318 mm * 280 mm), which greatly reduces the site restrictions for nucleic acid extraction. This is of great significance in responding to nucleic acid detection such as epidemics / HIV in the early stages of infection.

[0039] The card box of the present invention can store various reagents, perform various mixing, heating and other reactions as needed. The card box of the present invention is used for lysis, and the various reagents for nucleic acid extraction are not directly stored in the card box, but are stored in blisters / vesicles. The blisters are connected to the card box by pasting, and enter the microchannel under the squeezing of the mechanical arm, so as to perform automatic lysis, cleaning and elution of nucleic acid extraction / purification. This has the advantage that the amount of reagents can be increased / decreased as needed, and the test process can be modified without manual operation. At the same time, the card box and chip involved in the present invention are also in a sealed state, which can avoid aerosol contamination and sample cross-contamination. In addition, the nucleic acid extraction reagent in the blister can be replaced with different extraction reagents at any time according to the application scenario, and can be applied to the extraction of more sudden disease nucleic acids, with great flexibility.

[0040] The present invention uses a thinner 2 μm PET (polyester film) dielectric layer on the digital microfluidic chip, and optimizes the processing technology of the first hydrophobic layer on the upper surface of the dielectric layer on the PCB and the second hydrophobic layer spin-coated on the lower surface of the ITO glass. After optimization, the hydrophobic layer increases wear resistance and better adhesion to ITO / PCB. Compared with the existing solution of using 5-10 μm PI as the dielectric layer and imported hydrophobic Teflon / Hyflon as the hydrophobic layer, it reduces the cost while improving the driving performance and reducing the probability of electrolysis. The reason is that the thinner dielectric layer can increase the electric field strength provided by the PCB, and the good drying process of the hydrophobic layer can make the hydrophobic layer play the role of both the dielectric layer and the hydrophobic layer. Moreover, the overall thinner chip is more suitable for the effectiveness of the magnetic field force of the magnetic guide structure.

[0041] The present invention has a simple design in the microfluidic channel, which reduces the processing cost; wherein, the first common flow channel (protease / magnetic beads / lysate common flow channel) shared by proteinase K, magnetic beads, and lysate, the second common flow channel (magnetic beads / lysate common flow channel) shared by magnetic beads and lysate, and the lysate flow channel share an outlet to enter the injection tank; the design can reduce the residue of liquid (proteinase K, magnetic beads) in the flow channel; the flow channel function is simple to implement, and only the top film and the bottom film need to be pasted, which saves costs. The present invention can reduce the loss of magnetic beads by repeated flushing through the unique design of the shared flow channel, and then only hydrophobic modification is performed on the magnetic bead transfer flow channel, gears, and mixed lysis tank to reduce magnetic bead adsorption, so as to ensure the minimum residue of magnetic beads. Such modification can save the use of hydrophobic reagents while also achieving the purpose of reducing magnetic bead residue and improving enrichment efficiency.

[0042] The present invention only needs to configure one robotic arm for the extrusion of blisters of different sizes (80 μL-1000 μL), air valve plugs and release plugs, which reduces the complexity of the design; in addition, different extrusion sequences and extrusion methods are designed for different types of extruded objects so that they can be integrated on one robotic arm to complete the extrusion of multiple extruded objects. The extrusion method and sequence are specifically as follows: small blisters such as proteases and magnetic beads are extruded once / multiple times to make them completely extruded; for oversized blisters such as lysate blisters, steel balls can be set on the blister placement area, and the steel balls on the blister placement area are first squeezed, and then the main part of the blister is squeezed. Such an extrusion method and design can reduce the problem of liquid leakage from the pasting due to the excessive size of the blister and multiple squeezing; the release plug and the air valve plug are designed to be of suitable size with the contact surface of the robotic arm for extrusion, which is a one-time extrusion; the extrusion of the cleaning solution and the eluent can be extended to the robotic arm to cooperate with the droplet feedback on the chip for extrusion. Such a robotic arm design greatly reduces the fixed cost and maintenance cost of the instrument.

[0043] The chip design of the card box of the present invention can realize the precise segmentation of droplets. In combination with the overall instrument design containing the card box of the present invention, it can be expanded to a robotic arm and a corresponding logic circuit design to complete complex logic judgment and droplet feedback; and through the logic circuit design, the positive pressure pump and the droplet feedback on the chip are used to realize the release of liquid in the mixed lysis tank and the transfer of magnetic beads, which can complete the maximum transfer of magnetic beads and the suction of waste liquid.

[0044] The device of the present invention can also achieve automatic conversion between strong and weak magnetic fields through the configuration of strong magnetic fields and weak magnetic fields through the control of mechanical structure and software sequence. Such a setting can reduce the residual magnetic beads when strong magnetic fields are needed to drag the magnetic beads; and the auxiliary effect of weak magnetic fields during cleaning and elution can make cleaning and elution more complete, greatly promoting the extraction and enrichment of nucleic acids.

[0045] The present invention has an air phase inside the digital microfluidic chip instead of an oil-water dual phase, which saves costs and has higher efficiency when heating and evaporating the cleaning liquid remaining on the surface of the magnetic beads after cleaning, thereby improving the extraction efficiency of nucleic acids.

[0046] In summary, the designs of all the above chips, card boxes, software and mechanical structures can achieve better nucleic acid extraction and enrichment. Compared with the traditional 96-well plate type fully automatic nucleic acid extractor, it can achieve 10~20 times nucleic acid enrichment, thus largely solving the problem of false negative nucleic acid detection caused by low virus concentration in the early stage of infection; and the fully enclosed nucleic acid start-up extraction can reduce the impact of human operation on the results and avoid aerosol contamination; in addition, the amount of cleaning fluid and elution fluid is reduced, which can save costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a main mechanical structure diagram of the automatic nucleic acid extraction and enrichment instrument of the present invention, and the used mechanical arm, pump, cartridge, magnetic field, and stepper motor are marked in detail; wherein 1 is an EP tube, 34 is a magnetic guide mechanism, 35 is a stepper motor for controlling the magnetic field, 11 is a flow channel layer, 21 is a cartridge cover, 70 is a negative pressure control device, 31 is a mechanical arm, 33 is a stepper motor for controlling the mechanical arm, 32 is a lysis tank gear drive mechanism, and 69 is a positive pressure control device.

[0048] Figure 2 This is an exploded view of the digital microfluidic chip, cartridge and EP tube assembly used in the entire nucleic acid extraction process used in the present invention, with the main components of the chip (PCB, ITO spacer, etc.) and the main components of the cartridge (consisting of the flow channel layer and the cartridge cover) marked in detail.

[0049] Figure 3 It is a front plan view of the card box used in the present invention ( Figure 3 A), reverse view ( Figure 3 B) and oblique side view ( Figure 3 C) provides more detailed annotations of the features of the cartridge other than the flow channel.

[0050] Figure 4 The front side of the flow channel layer used in the present invention ( Figure 4 A), back ( Figure 4 C), side ( Figure 4 B) Detailed annotation of each flow channel, which can provide a clearer understanding of the droplet flow direction, pump intake and suction processes during nucleic acid extraction and enrichment.

[0051] Figure 5 Exploded diagram of the digital microfluidic chip used in the present invention ( Figure 5 C), side view ( Figure 5 A) and front view ( Figure 5 B), where 3 is a dielectric layer, which is 2μm PET, and can provide a strong electric field; 4 is a hydrophobic layer, which is formed by spin coating a resin solution with a solid content of 3% of a hydrophobic reagent, and its spin coating process and drying process are optimized; the thinner dielectric layer 3 is matched with the optimized hydrophobic layer 4 to achieve a low electrolysis rate and good drive for the cleaning liquid and the eluent; 6 is absorbent paper, which is used to absorb working waste liquids such as lysate during nucleic acid extraction; 5 is a spacer, which is used to isolate the space between the upper plate and the lower plate for droplet movement, and has a thickness of 0.2~0.25 mm; 7 is ITO glass, which is coated with indium tin oxide and a hydrophobic layer 4 on the lower surface. In addition, there are connection structures such as a magnetic bead injection hole 71 on the chip, a cleaning liquid injection hole 72 on the chip, and a tail liquid suction hole 74 on the chip eluent injection hole 73.

[0052] Figure 6 The PCB array design plane used in the present invention, wherein Figure 6 A is the PCB front array diagram, Figure 6 B is a PCB front array diagram showing the heating electrode and the electrode connection pins. Combined with the liquid separation electrode, lysis heating electrode, elution heating electrode, large electrode and small electrode in the diagram, we can have a clearer understanding of the functions completed by the chip. Among them, 211 is the lysis heating electrode, which is mainly used for heating the nucleic acid lysis process; 212 is the elution zone heating electrode, which is mainly used for evaporation and elution heating of the residual cleaning liquid; 213 is the upper and lower plate connection electrode. During the chip manufacturing process, conductive foam will be pasted on it to connect the upper and lower plates of the chip (PCB and ITO) to form a loop, which can change the positive and negative poles of the upper and lower plates; 214 is a small electrode, whose length and width are both 2 mm, the chip spacer 5 height is 0.2~0.25 mm, and the volume of each droplet is 0.8 μL~1.0μL, which can achieve precise control of the minimum droplet of 0.8~1.0 μL, and 215 is a large electrode, which is 10 mm long and 2 mm wide, so it can achieve precise movement and liquid separation of 4~5 μL droplets. 216 is a liquid separation electrode, which can cooperate with the sequence and the extrusion of the robot arm to complete the precise segmentation of the droplets when the cleaning liquid and the elution liquid enter the chip; 217 is a waste liquid suction electrode, and its auxiliary function can make the waste liquid on the chip easier to be sucked away by the absorbent paper; 218 is a tail liquid suction connection electrode, and the nine-square grid formed by it and other small electrodes around it is covered with droplets, which is the condition for turning on the negative pressure pump. The eluent that has completed extraction and enrichment can enter the EP tube 1 through the tail liquid suction hole 74 directly opposite to the electrode via the tail liquid suction channel 46.

[0053] Figure 7 Detailed drawing of absorbent paper used in the present invention, wherein 611 is thin absorbent paper ( Figure 7 B), with a thickness of 0.2~0.25mm, mainly plays a drainage role, 612 is thick absorbent paper ( Figure 7 A), with a thickness of 1.8 mm, mainly serves to absorb waste liquid during nucleic acid extraction. Figure 7 C is the absorbent paper actually used in the chip, which is assembled from thin absorbent paper and thick absorbent paper.

[0054] Figure 8 The spacer used in the present invention has double-sided adhesive on its upper and lower surfaces, which can adhere the PCB and ITO together to form a double-board digital microfluidic chip.

[0055] Fig. 9 The top film and bottom film used in the present invention are respectively adhered to the upper surface and the lower surface of the flow channel layer to form various microchannels in the nucleic acid extraction process. Fig. 9 B is the top membrane, Fig. 9 A and Fig. 9 D are the oblique side view and front view of the top film attached to the upper surface of the flow channel layer; Fig. 9 C is the base film, Fig. 9 FIG. E is a front view of the bottom film attached to the lower surface of the flow channel layer.

[0056] Fig.10 The release plug 19 used in the present invention, wherein 191 is a puncture needle and 192 is a silicone plug, can play a buffering role when the robot arm 31 is squeezed. Fig.10 A is the front view of the release plug. Fig.10 B is a front cross-sectional view of the release plug.

[0057] Fig.11 The specific position of the release plug used in the present invention in the flow channel layer is described in detail, wherein Fig.11 A is the position of the release plug on the front of the flow channel layer. Fig.11 B and Fig.11 C are the position of the release plug in the cross section of the flow channel layer and the enlarged view of the position, Fig.11 D and Fig.11 E respectively represent the position of the release plug on the oblique side of the flow channel layer and an enlarged view of the position.

[0058] Fig.12 A detailed description of the gears used in the present invention to make the lysis more complete and the magnetic beads sink to the bottom during the lysis and magnetic bead transfer. Fig.12 A and Fig.12 B are the position diagram of the gear on the front of the flow channel layer and the enlarged diagram of the position, Fig.12 C and an enlarged view of the gear's location in the cross section of the flow channel layer.

[0059] Fig.13 This is a front view of a copper block that conducts heat during the cracking step of the present invention ( Fig.13 A), top view ( Fig.13 B) and side view ( Fig.13 C), which can perfectly mesh with the mixed cracking tank, and the thermal conductivity of the silicone grease on the upper and lower surfaces of the copper block can achieve good temperature control of the cracking when the PCB is heated.

[0060] Fig.14 The design diagram of the magnetic guide structure of the present invention, wherein the area of ​​341 is 6.14*6.07cm, which is a strong magnet, and the magnetic force is defined as 10 in the present invention; the area of ​​342 is 6.14*3.78cm, which is a weak magnet, and the magnetic force is defined as 6.2 in the present invention. Among them, A is the right side view of the magnetic guide structure, B is the front view of the magnetic guide structure, C is the left side view of the magnetic guide structure, and D is the right oblique side view of the magnetic guide structure.

[0061] Fig.15The positive pressure pump and negative pressure pump used in the present invention are equivalent to the specific position side view of the cartridge ( Fig.15 A) and top view ( Fig.15 B), wherein 69 is a positive pressure pump, including 691 is a positive pressure pump stirring paddle for forming positive pressure, and 692 is a positive pressure pump connecting pipe; 70 is a negative pressure pump, including 701 is a negative pressure pump stirring paddle for forming negative pressure, and 692 is a negative pressure pump connecting pipe.

[0062] Fig.16 Detailed description of the robot arm relative to the bubble cap, release plug and air valve plug that need to be squeezed, wherein 31 is the robot arm that squeezes, and 33 is the stepping motor that controls the robot arm.

[0063] Fig.17 Detailed description of the instrument housing of the nucleic acid extractor of the present invention, wherein 78 is the instrument door switch; 79 is the instrument injection door; 80 is the reset button for one-key reset in case of failure; 81 is a USB connection line for connecting to a computer operating the instrument; and 82 is the instrument power line.

[0064] Fig.18 The figure is a full flow chart of the automatic nucleic acid extraction of the present invention.

[0065] Fig.19 The figure is a flow chart of the cleaning solution and the eluting solution entering the chip, the instrument robot 31 and the droplet feedback logic judgment flow chart.

[0066] Fig. 20 The chip, negative pressure pump, and magnetic field cooperate with the logic judgment flow chart when aspirating liquid at the tail.

[0067] Fig.21 This is a diagram showing the effect of magnetic beads under the action of magnetic field of the present invention; Fig.21 When A is weak magnetic assistance, the magnetic beads are scattered in the washing solution or elution solution; Fig.21 B is the state where the magnetic beads are scattered in the washing solution or elution solution after the weak magnetic assistance is cancelled; Fig.21 C is the state where the magnetic beads are separated from the elution solution under the action of a strong magnet. DETAILED DESCRIPTION

[0068] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0069] In conjunction with the drawings of the present invention, in a specific embodiment of a nucleic acid extraction and enrichment system based on digital microfluidics technology provided by the present invention, the system includes: a mixing and lysis module, a cleaning and elution module, a droplet control module, a pressure control module and an extrusion module.

[0070] The mixing and lysis module is used to mix the working reagent with the sample to be detected and lyse the protein, release the nucleic acid and capture the released nucleic acid with magnetic beads. The mixing and lysis module includes a sample injection slot 12, a mixing and lysis slot 13, a gear stirring mechanism 14, a mixing and heating device 10, and a pre-packaged sealed form of a protease, magnetic beads with an adsorption effect on nucleic acid and a lysis agent; The extrusion module includes a robotic arm 31, and a microcolumn (or steel ball) 68 arranged on the upper surface of the flow channel layer in a pre-packaged form for placing the working reagent. The extrusion module is used to squeeze the blister of the pre-packaged working reagent to open the seal and release the working reagent into the mixing and lysis module, and squeeze the release plug 19 after the lysis is completed to allow the liquid to enter the digital microfluidic chip from the flow channel layer, and then squeeze the air valve plug 20 to seal the mixing and lysis tank 13, and squeeze the cleaning liquid and the elution liquid directly into the chip for the cleaning and elution steps.

[0071] The cleaning and elution module is used to clean and elute the captured nucleic acid, and includes a flow channel layer 11 and a digital microfluidic chip (composed of a PCB 2, a dielectric layer 3, a hydrophobic layer 4, a spacer 5, a blotting paper 6, and an ITO 7), as well as a pre-packaged sealed form of a cleaning solution and an eluting solution. A reagent injection area is provided on the upper surface of the flow channel layer 11 to place a pre-packaged sealed working reagent. After being squeezed by the pressure control module, the lysis working solution in the mixed lysis tank 13 is transferred to the digital microfluidic chip, the magnetic beads adsorbed with nucleic acid are retained by the strong magnet, and the waste liquid is absorbed by the blotting paper; the digital microfluidic chip is used to complete the cleaning step and the elution step in the process of nucleic acid extraction and enrichment, and to provide heating for the lysis and elution steps. The digital microfluidic chip 2 is automatically From bottom to top, it includes: a printed circuit board (PCB) 2, a dielectric layer 3, a first hydrophobic layer 4, a spacer, a water-absorbing layer 6, a second hydrophobic layer 4, and a conductive layer 7; wherein the printed circuit board 2 is provided with upper and lower plate connecting electrodes 213 that connect the printed circuit board and the conductive layer into a loop, a lysis heating electrode 211, an elution heating electrode 212, a liquid separation electrode 216, a waste liquid suction electrode 217, and a tail liquid suction connection electrode 218 that transfers the extracted and enriched eluate from the card box into the test container through the tail liquid suction flow channel.

[0072] In the specific implementation scheme, the flow channel layer 11 and the digital microfluidic chip 2 are placed in the card box, and the card box includes the following components in the longitudinal direction: a card box cover 21, a flow channel layer 11, a bonding layer 8, a digital microfluidic chip (composed of a PCB 2, a dielectric layer 3, a hydrophobic layer 4, a spacer layer 5, absorbent paper 6, and ITO 7). The components are stacked and attached from top to bottom and are fixed by the card box cover. The card box cover 21 is provided with a gear 14 for driving mixed cracking and stirring. The flow channel layer 11 and the digital microfluidic chip are responsible for forming the working liquid into a microfluidic and transporting it to the working groove defined by the flow channel layer or the position controlled by the digital microfluidic chip. The digital microfluidic chip is composed of a PCB 2, a dielectric layer 3, a hydrophobic layer 4, a spacer 5 for isolating the space between the upper plate and the lower plate for droplet movement, an absorbent paper 6 for absorbing working waste liquid, a hydrophobic layer 4, and ITO 7 from bottom to top.

[0073] The flow channel layer 11 is provided with a sample injection slot 12 for adding samples, a mixed lysis slot 13 for mixed lysis, a flow channel layer placement area for pasting proteinase K 22, magnetic beads 23, lysis solution 24, cleaning solution 25 and eluent 26 in pre-packaged form, a release slot 15, an air valve plug slot 16, a positive pressure pump connection port 29, a negative pressure pump connection port 30, an EP tube connection port 28 and various flow channels for forming microfluids (such as a negative pressure pump flow channel 45, etc.); in addition, a top film positioning hole 27 and a card box positioning hole 67 are also provided to facilitate the assembly of the film and the card box (facilitating the precise combination of the flow channel layer and the card box cover to form a card box).

[0074] The specific design of the digital microfluidic chip can be found in Figure 5 and Figure 6, wherein 211 is a lysis heating electrode, which is used to provide the heat source required for the lysis step in the nucleic acid extraction process, and cooperates with the copper block and silicone grease to heat the mixed lysis tank of the flow channel layer; 212 is an elution heating electrode, which is used to remove the cleaning solution residue on the surface of the magnetic beads after the cleaning step and provide the temperature required for eluting nucleic acids; 213 is a connection electrode for the upper and lower plates (upper plate ITO, lower plate PCB), and conductive foam will be pasted on these two electrodes during the chip manufacturing process to connect the upper and lower plates (pasting conductive foam is a preferred embodiment of the present invention, aiming to obtain higher driving ability, and droplets can also be driven without conductive foam), realizing the diversity of power supply methods (the positive and negative poles of the upper and lower plates can be changed to achieve better driving of droplets); 21 4 is a small electrode, which can be used with 216 liquid separation electrode to accurately divide μL-level droplets (5~15μL cleaning solution and elution solution); 215 is a large electrode, which is filled with 4~5μL liquid. Therefore, to manipulate a 10μL droplet, it only needs to occupy 2 large electrodes, and such a design can reduce electrode interfaces and reduce instrument costs; 217 is a waste liquid suction electrode. When sucking away the waste liquid in the nucleic acid extraction process, such a design combined with the shape of 611 thin absorbent paper can make the waste liquid more fully absorbed by the absorbent paper; 218 is a tail liquid suction electrode. When the surrounding nine-square grid small electrodes are filled, the negative pressure pump will start, and the extracted liquid will enter the relevant flow channel of the flow channel layer from the tail liquid suction hole 74 on the ITO through this electrode, and enter the EP tube.

[0075] The proteinase K 22, magnetic beads 23, lysate 24, cleaning solution 25 and eluent 26 are placed in the flow channel layer placement area in the form of blisters, and the blisters are pierced by external force during operation to make the corresponding proteinase K 22, magnetic beads 23, lysate 24, cleaning solution 25 and eluent 26 flow out and enter the microfluidic channel through the slots. After the sample is added to the injection slot 12 by a pipette and the injection plug 18 is covered, the reagents pre-stored in the blister enter the microfluidic channel in a certain order under the combined action of the extrusion of the mechanical arm and the microcolumn 68 on the flow channel layer, completing the process of nucleic acid extraction. During the period when the robot arm squeezes the bubble, in order to avoid excessive pressure in the mixing and cracking tank 13, the flow channel layer is equipped with an air valve plug and a mixing and cracking tank connecting hole 64, a sampling slot and a mixing and cracking tank connecting groove channel 43 and an air valve plug groove 16, so that the positive pressure generated by the bubble extrusion and the pressure of the positive pressure pump 69 allow the liquid to enter the mixing and cracking tank 13 from the sampling slot 12, and then be discharged from the air valve plug and the mixing and cracking tank connecting hole 64 through the sampling slot and the mixing and cracking tank connecting groove channel 43 from the air valve plug groove 16. Therefore, the air valve plug 20 is in an unplugged state at this time.

[0076] In the technical solution of the present invention, the strong magnetic field strength (intensity 1) in the droplet control module refers to the magnetic field strength that can make the magnetic beads quickly converge to one point. When used in the magnetic bead transfer step, the magnetic beads are gathered at the strong magnetic point from the mixed lysis tank waste liquid so that the magnetic beads can be collected; and the magnetic beads are dragged on the chip magnetic bead chip to perform the cleaning and elution steps and the magnetic beads and liquid separation steps. The weak magnetic field (intensity 2) can pull the magnetic beads to move, but does not bring the magnetic beads together, but can disperse the magnetic beads in the liquid, so it can be used to pull the dispersed magnetic beads for mixing, washing, and elution assistance. The absolute strength of the magnetic field is related to the size and material of the magnetic beads, and is also related to the medium (material, thickness, distance, etc.) between the magnet and the flow channel. Under normal circumstances, the weak magnetic field (intensity 2) used is between 50% and 70% of the strong magnetic field (intensity 1). In a specific embodiment of the present invention, in the magnetic permeability structure with switchable magnetic strength, the area of ​​the magnet for providing strong magnetic strength is 6.14 cm*6.07 cm, and the area of ​​the magnet for providing weak magnetic strength is 6.14 cm*3.78 cm.

[0077] In the technical solution of the present invention, the dielectric layer on the digital microfluidic chip is PET, and its thickness is 2 μm. The optimized formula of the hydrophobic layer is: using domestic hydrophobic reagent 3% T6 (resin solution), the spin coating parameters are 400 rpm 18 s, 2000 rpm60 s, and the drying process is 120 ℃, 40 min. In the technical solution of the present invention, the heating method used to remove the residual cleaning liquid in the digital microfluidic chip is PCB electrode heating 212, and the heating temperature is between 60 and 70 ℃; the heating method used in the cracking step is PCB electrode heating 211, thermal conductive silicone grease and copper block 10 direct heating, and the heating temperature is between 60 and 70 ℃, wherein the copper block is located at the card slot of the mixed cracking tank 13, and the inner surface of the copper block 10 in contact with the mixed cracking tank 13 and the inner surface of the copper block 10 in contact with the PCB 2 is coated with thermal conductive silicone grease.

[0078] In the technical solution of the present invention, the thickness of the spacer is 0.2~0.25 mm, and a square electrode area and a rectangular electrode area are provided on the PCB circuit board. In the square electrode area, the length and width of each square electrode are 2 mm, and the droplet diameter generated and controlled is 0.8~1.0 μL. In the rectangular electrode area, the length of each rectangular electrode is 10 mm and the width is 2 mm, and the droplet diameter generated and controlled is 4~5 μL. The rectangular electrode is convenient for controlling droplets of about 10 μL in the cleaning and elution steps, and the rectangular electrodes are connected together by copper coating on the surface, which can also save electrode interfaces and reduce costs; the square electrode plays an important role in the separation of the cleaning solution 25 and the elution solution 26.

[0079] In the technical solution of the present invention, the power supply of the electrode is alternating current. The specific circuit uses a 24V switching power supply as the main power supply of the instrument, outputs a relatively small voltage value after being processed by the power management module, and converts the low voltage into a high voltage through a transformer after voltage stabilization. The high voltage is provided by the high-voltage shift register HV507PG-G (hereinafter referred to as the high-voltage chip), and the 64 output pins of the high-voltage chip are connected to the electrodes respectively. When a certain electrode needs to be operated with direct current, the control end inputs the corresponding control signal to the high-voltage chip, turns on the corresponding pin to output a high voltage, and turns on the MOS tube inside the chip at the same time, so that the ITO is connected to the ground, and the operation of direct current power supply to the electrode is realized; when a certain electrode needs to be operated with alternating current, then similarly, the above-mentioned operation of direct current power supply is performed first; after a period of time, the MOS tube inside the chip is controlled to be turned on, so that the electrode is connected to the ground through the chip pin, and the output pin connected to the ITO is turned on at the same time, and a high level is output, which is equivalent to an operation of alternating power supply between an electrode and the ITO, and the operation of alternating current power supply to the electrode is realized. By setting the time interval of each voltage flip, the frequency adjustable function of alternating current power supply can be achieved.

[0080] The specific steps of extracting and enriching nucleic acids of the present invention are: Injection step: After the sample is added to the injection slot 12, the mechanical arm 31 controlled by the stepper motor squeezes the proteinase K 22 into the flow channel from the proteinase K injection hole 52, and enters the injection slot 12 from the injection slot hole 62 along the proteinase K / magnetic bead / lysate common flow channel (first common flow channel) 37; the magnetic beads 23 enter the flow channel from the magnetic bead injection hole 53, and enter the injection slot 12 from the injection slot hole 62 along the magnetic bead / lysate common flow channel (second common flow channel 38) and the proteinase K / magnetic bead / lysate common flow channel; the lysate 24 enters the flow channel from the lysate injection hole 56, and enters the injection slot 12 along the lysate flow channel 39, the magnetic bead / lysate common flow channel 38 and the proteinase K / Magnetic beads / lysate shared flow channel 37 enters the sampling slot 12 from the sampling slot hole 62; at the same time, the positive pressure pump 69 is turned on, and the gas passes through the positive pressure pump connecting pipe 691, the positive pressure pump connecting port 29, from the positive pressure pump flow channel 66 to the positive pressure pump and the sampling slot connecting flow channel 44 (with a certain buffering effect), and enters the sampling slot 12 through the positive pressure pump air inlet hole 65 to form a positive pressure, and the mixed liquid flows through the sampling slot bottom hole 58 through the sampling slot and the mixing lysis slot connecting slot flow channel 43, from the sampling slot into the mixing lysis slot hole 63, and enters the mixing lysis slot 13.

[0081] Mixed lysis step: After the lysate 24 completely enters the mixed lysis slot hole 63 from the injection slot and enters the mixed lysis slot 13, the lysis heating electrode 211 on the PCB 2 starts to heat, and the mixed lysis slot 13 is heated through the copper block 10 with silicone grease on both the upper and lower surfaces; at this time, the gear 14 also starts to rotate (forward and reverse rotation are performed alternately to make the lysis more complete), and the lysis step is performed. In this process, the viral nucleic acid will be released under the action of proteinase K 22 and the lysis solution 24, and at the same time bind to the magnetic beads 23 (the surface of the magnetic beads is chemically modified (silicon hydroxyl magnetic beads), which can bind to nucleic acids).

[0082] After 5 minutes of lysis, the gear 14 stops rotating, the PCB 2 stops heating, the mechanical arm 31 moves to the valve plug 20 for squeezing, and then moves to the release plug 19 for squeezing, piercing the tin foil 77. At the same time, the positive pressure pump 69 starts to operate according to a certain logic judgment. To prevent the magnetic beads from sinking to the bottom, the gear 14 also starts to rotate. The mixed liquid in the mixed lysis tank 13 is uniformly transferred from the mixed lysis tank 13 from the bottom hole 59 of the mixed lysis tank through the magnetic bead transfer channel 36 from the release hole 60 and the magnetic bead injection hole 71 on the chip into the chip. At the same time, the strong magnet 341 in the magnetic field 34 is turned on, and the magnetic beads 23 in the mixed lysis tank 13 flow out together with the liquid in the mixed lysis tank 13, wherein the magnetic beads 23 are adsorbed to the magnetic bead injection hole 71 on the chip under the action of the strong magnet 341; the liquid in the mixed lysis tank 13 flows out under the action of positive pressure and is sucked away by the absorbent paper 6 on the digital microfluidic chip.

[0083] Cleaning step: The cleaning liquid 25 enters the digital microfluidic chip from the cleaning liquid injection hole 54 of the bubble cap through the cleaning liquid inlet channel 40 (lower), the upper and lower connecting holes 57 of the cleaning liquid injection channel and the cleaning liquid injection channel 47 (upper) from the cleaning liquid injection hole 50 and the cleaning liquid injection hole 72 on the chip. With the cooperation of the mechanical arm and the chip droplet feedback, the cleaning liquid entering the chip occupies 10 grids (i.e., 8~10 μL); then, under the action of a strong magnet, the magnetic bead 23 moves to the small electrode 214 in the middle of the chip to mix with the cleaning solution 25, and first moves repeatedly between the large electrode 215 and the small electrode 214 in the digital microfluidic chip with the assistance of a weak magnet 342 and the action of an electric field, and then the weak magnet 342 is turned off, and continues to move between the large electrode 215 and the small electrode 214 under the action of the electric field for cleaning, and the working waste liquid formed after the cleaning is completed moves to the waste liquid absorption electrode 217 under the action of the electric field, and is absorbed by the absorbent paper 6, and this is repeated 3 times.

[0084] Elution step: The washed magnetic beads 23 are moved to the elution heating electrode 212 of the digital microfluidic chip under the action of the strong magnet 341, and the residual cleaning solution 25 on the magnetic beads 23 is removed by heating. Then, the eluent 26 passes through the eluent inlet channel 41 (lower), the upper and lower connecting holes 83 of the eluent inlet channel and the eluent inlet channel 48 (upper) from the bubble cap eluent injection hole 55, and enters the digital microfluidic chip from the eluent injection hole 49 and the eluent injection hole 73 on the chip. With the cooperation of the mechanical arm and the chip droplet feedback, the eluent 26 entering the chip occupies 10 to 15 grids (i.e., 8 to 15 grids) set by the threshold value of the small electrode 214. μL); after that, under the action of the strong magnet, the magnetic beads 23 move to the small electrode 214 to mix with the eluent 26, and first elute between the small electrode 214 and the elution heating electrode 212 with the assistance of the weak magnet 342 and the action of the electric field, then the weak magnet 342 is turned off, and the magnetic beads 23 continue to move back and forth between the small electrode 214 in the middle of the chip and the elution heating electrode 212 under the action of the electric field to elute the nucleic acid enriched on the magnetic beads 23; after the elution is completed, the mixed solution first moves to the tail liquid suction connection electrode 218, and after occupying the 9 electrodes there, the strong magnet 341 is turned on, and the magnetic beads 23 are moved to the waste liquid suction electrode 217 to separate from the eluent 26 enriched with nucleic acid. At this time, the negative pressure pump 70 is turned on, and the negative pressure pump flow channel 45 and the EP tube 1 connected thereto form a negative pressure, and the eluent 26 is sucked into the EP tube 1 from the EP tube connection port 28 through the tail liquid suction hole 74 and the tail liquid suction flow channel 46.

[0085] Preparation Example 1. Modification of the magnetic bead transfer channel 36, the mixing and lysis tank 13 and the gear 14 In order to reduce and minimize the loss of magnetic beads in the flow channel body during the lysis step and magnetic bead transfer, it is necessary to perform hydrophobic modification on the relevant parts of the flow channel body. The specific steps are: use a pipette to take an appropriate amount of hydrophobic reagent to the magnetic bead transfer flow channel 36, and then use a small brush to evenly apply the solution; after completion, use the same method to add an appropriate amount of hydrophobic reagent to the surface of the mixed lysis tank 13 and the surface of the gear 14, and then put it in an oven at 80°C for 960 min to dry, and wait for the hydrophobic reagent to complete vitrification.

[0086] 2. Assembly of flow channel layer and chip: Step 1: The formation of the microchannel of the flow channel layer 11 and the top part need to be pasted with the bottom film and the top film respectively. The specific operation is: tear off the white protective layer of the bottom film adapted to the flow channel layer, and use the micro-pillars 68 and the release plug 19 to position and paste it inside the flow channel layer, and then use the hand to flatten the entire bottom glue and then use a small shovel to squeeze out the air near the flow channel to prevent the solution from overflowing.

[0087] Step 2: The reagents for nucleic acid extraction need to be pasted in sequence on the front of the flow channel layer (see Figure 3). The specific operation is: tear off the back protective layer of each blister and stick it to the corresponding placement area (see Figure 3 ), and then use a small shovel to press the edge of the bubble cap to expel the air and prevent liquid leakage from the bubble cap.

[0088] Step 3: Before the cracking step is completed, the release plug 19 should be sealed, and it needs to be sealed with tin foil 77, double-sided tape, etc. The specific operation is: stick the double-sided tape on the release hole, tear off the protective film and lay the cut one on the pressure-sensitive adhesive, and then use a small shovel to press the tin foil 77 and the pressure-sensitive adhesive to expel the air (be careful not to damage the tin foil).

[0089] Step 4: The copper block 10 of the cracking step of the present invention is heated, and silicone grease needs to be applied between the copper block 10 and the chip, and between the copper block 10 and the mixed cracking tank to improve the thermal conductivity. The specific operation is: use a needleless syringe to absorb a certain amount of silicone grease, evenly apply an appropriate amount of thermal conductive silicone grease on the inner side of the copper block 10, install it in the card slot on the back of the mixed cracking tank, and then use the same method to take an appropriate amount of thermal conductive silicone grease and apply it on the back of the copper block 10, and then place it at the cracking heating electrode 211.

[0090] Step 5: Installation of the digital microfluidic chip and the buffer pad of the flow channel layer 11: Take the chip with good quality inspection, adjust the absorbent paper, take the rubber pad 8 with a thickness of 1.2mm and place it on the chip ITO 7, align the holes of the rubber pad 8 with the holes of ITO 7, and then buckle the chip and the rubber pad 8 into the flow channel layer 11 according to the slot holes, and use tape to stick the two sides of the chip to the chip and the flow channel layer 11. The position where the tape is pasted should not cover the bottom electrode of the chip.

[0091] Step 6: Assemble the cartridge cover 21, chip, flow channel layer 11 and gear 14 into a complete nucleic acid extraction cartridge. The specific operation is: put the gear into the mixed lysis tank 13, then buckle the cartridge cover 21 onto the flow channel layer 11 according to the cartridge positioning column 76, and put the injection plug 18, release plug 19 and valve plug cover 20 in the corresponding holes 12, 15, 16. Finally, take a clean 200 μLEP tube 1 and install it in the cartridge EP tube connection port 28.

[0092] Application examples: The present invention mainly provides a system for automatically extracting and enriching nucleic acids, which will be described in detail below by taking the extraction of syncytial pseudovirus as an example.

[0093] (1) Sample addition: Remove the injection plug 18, take 200 μL of the oscillated and mixed throat swab (mixed with an appropriate amount of syncytial pseudovirus and put into the throat swab) and add it to the injection slot, and plug it tightly with the rubber stopper; also insert the air valve plug 20 into the air valve plug slot 16, and the release plug 19 into the release plug slot 15 (both do not need to be tightly closed).

[0094] (2) System reading and software use: Open the digital microfluidic nucleic acid extraction system control software, enter the [operation interface], check [mainboard and driver board connection], [device connection], and display the green light. Click [Read], select the full process sequence, click [Write], and wait for the log record to display [Ask Auto-Run], which means the writing is complete. Open the lift door, insert the card box, and wait for the software to display that the card is inserted correctly, the temperature control self-test is successful, and the high-voltage self-test is successful.

[0095] (3) Nucleic acid extraction and enrichment: Click [Run] in the software to start the whole process. After the whole process is completed (about 30-40 minutes), remove the 200 μL EP tube 1 and take the corresponding volume of liquid in the tube for the next step of amplification.

[0096] (4) Nucleic acid result detection: According to the instruction manual of Respiratory Syncytial Virus Type A / B Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method) of Daan Gene, version number: 003, prepare the amplification reagent and perform nucleic acid amplification.

[0097] Comparative Example. Daan Gene Smart32 Automatic Nucleic Acid Extraction Syncytial Pseudovirus (5000 Copies / mL) Turn on the UV light of the nucleic acid extractor Smart32. After sterilization for about 30 minutes, add the syncytial nucleic acid extraction reagent and test samples to the 96-well plate according to the instructions of the nucleic acid extraction kit (the amount of eluent used is 100μL~200μL), set the program of the instrument Smart32, put in the 96-well plate, and press the start button to start nucleic acid extraction.

[0098] The application example is a nucleic acid extraction and enrichment scheme of the present invention, which can achieve that after adding the sample to the injection slot, no artificial sterilization is required (the factory cartridge is sterilized and free of nuclease), and a pure enriched sample that can be directly PCR amplified can be obtained after about 30 to 40 minutes; the control example 2 is a commercial instrument nucleic acid extraction scheme, which can achieve that after adding reagents and samples, a sample that can be PCR amplified can be obtained after about 1 hour of extraction. After the extraction is completed and tested, EWOD (application example) can complete nucleic acid enrichment compared with Smart32 (control example).

[0099] Table 1. Comparison of reagent addition amounts between Smart32 and EWOD nucleic acid extractors

[0100] *150μL magnetic beads are obtained by diluting 20μL magnetic beads in equal proportions, and the original amount of magnetic beads is the same.

[0101] Table 2. Comparison of Ct values ​​between EWOD automatic washing and elution and smart32

[0102] The digital microfluidic nucleic acid extractor and Smart32 were used to extract 5000 copies / mL of syncytial pseudovirus. The Ct value of the digital microfluidic nucleic acid extractor was 28.07, and the Ct value of Smart32 was 31.05. The Ct value of the digital microfluidic nucleic acid extractor used in the present invention was advanced by about 3 cycles, that is, the nucleic acid was enriched by 8 times. Compared with the control example, the nucleic acid extraction and enrichment of the present invention is short in time, the reagent dosage is small, and the nucleic acid concentration is higher.

Claims

1. A nucleic acid extraction and enrichment system based on digital microfluidics technology, characterized in that: The system includes: a mixing and cracking module, a cleaning and elution module, a droplet control module, a pressure control module, and an extrusion module, wherein The mixing and lysis module is used to mix the working reagent with the sample to be detected, lyse the protein, release the nucleic acid and capture the released nucleic acid with magnetic beads. The mixing and lysis module includes an injection slot, a mixing and lysis slot, a gear stirring mechanism, a mixing and heating device, and a pre-packaged sealed form of protease, magnetic beads with adsorption effect on nucleic acid and lysing agent; the pre-packaged sealed form of protease, magnetic beads with adsorption effect on nucleic acid and lysing agent is communicated with the injection slot of the mixing and lysis module through a slot hole set at the bottom of the placement position, and the injection slot is connected with the mixing and lysis slot, so as to mix and lyse the protease, magnetic beads with adsorption effect on nucleic acid and lysing agent with the added sample to be detected and then transmit them to the digital microfluidic chip; The cleaning and elution module is used to clean and elute the captured nucleic acid, and includes a flow channel layer, a digital microfluidic chip, and a pre-packaged sealed form of a cleaning solution and an eluent. A reagent injection area is provided on the upper surface of the flow channel layer for placing proteases, magnetic beads with an adsorption effect on nucleic acids, a lysing agent, and a pre-packaged sealed form of a cleaning solution and an eluent; the proteases, magnetic beads with an adsorption effect on nucleic acids, and a lysing agent are mixed with the added sample to be detected and lysed, and then transferred to the digital microfluidic chip through the magnetic bead transfer flow channel of the flow channel layer; the cleaning solution and the pre-packaged sealed form of the eluent are directly transferred to the digital microfluidic chip after being squeezed by the pressure control module. The digital microfluidic chip is used for cleaning and elution; the digital microfluidic chip is used to complete the cleaning step and the elution step in the process of nucleic acid extraction and enrichment and provide heating for the lysis and elution steps, and the digital microfluidic chip comprises from bottom to top: a printed circuit board, a dielectric layer, a first hydrophobic layer, a spacer, a water-absorbing layer, a second hydrophobic layer, and a conductive layer; wherein the printed circuit board is provided with upper and lower plate connecting electrodes, a heating electrode, a liquid separation electrode, a waste liquid suction electrode, and a tail liquid suction connection electrode for connecting the printed circuit board and the conductive layer into a loop, and the eluent after extraction and enrichment is passed from the card box to the test container through the tail liquid suction flow channel; The droplet control module is used to control the size of droplets, the direction of movement of magnetic beads, and the combination and dissociation of magnetic beads and working reagents during nucleic acid extraction and enrichment, and includes a magnetic permeability structure with switchable magnetic strength, wherein the magnetic permeability structure with switchable magnetic strength includes a weak magnetic strength for assisting the driving of magnetic beads, and a strong magnetic strength for gathering magnetic beads and separating them from reagents; The pressure control structure includes positive and negative pressure devices for driving and transmitting liquid, and a gas valve plug for controlling the liquid to enter the mixing and lysis tank from the injection tank, from the mixing and lysis tank to the digital microfluidic chip, and a release plug for controlling the liquid to enter the digital microfluidic chip from the flow channel layer; The extrusion module includes a mechanical arm, micro-columns and steel balls arranged on the upper surface of a pre-packaged flow channel layer for placing working reagents; the extrusion module is used to extrude the unsealed and released protease, magnetic beads with adsorption effect on nucleic acid and lysing agent of the pre-packaged sealed form to enter the mixed lysis module, and the cleaning liquid and eluent directly enter the digital microfluidic chip, and extrude the release plug after the lysis is completed to allow the liquid to enter the digital microfluidic chip from the flow channel layer, and then extrude the air valve plug to seal the mixed lysis tank.

2. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: The heating electrodes provided in the digital microfluidic chip include a pyrolysis heating electrode for heating the mixed pyrolysis module and an elution heating electrode for heating the cleaning and elution module, wherein the pyrolysis heating electrode heats the mixed pyrolysis tank through a copper block under the mixed pyrolysis tank.

3. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: The dielectric layer of the digital microfluidic chip is a 2 μm polyester film.

4. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: The conductive layer is an indium tin oxide coating with glass as a substrate, an indium tin oxide coating with a flexible film as a substrate, or an indium tin oxide coating with the lower surface of the flow channel layer as a substrate.

5. According to the nucleic acid extraction and enrichment system according to claim 1, the processing method of the first hydrophobic layer and the second hydrophobic layer is a resin solution with a solid content of 3%, the spin coating parameters are 400 rpm 18 s, 2000 rpm 60 s, and the drying process is 120 ° C, 40 min.

6. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: The electrodes include a small electrode and a large electrode. The length and width of the small electrode are both 2 mm, and the length of the large electrode is 10 mm and the width is 2 mm.

7. The nucleic acid extraction and enrichment system according to claim 6, characterized in that: A droplet feedback mechanism is set on the electrodes on the digital microfluidic chip. When the number of small electrodes with droplets does not reach a threshold, the mechanical arm of the extrusion module, the positive pressure control module and / or the droplet control module drive the droplets to move; when the number of small electrodes with droplets reaches a threshold, the mechanical arm of the extrusion module and / or the droplet control module drive the droplets to move or the negative pressure control structure drives the droplets to move.

8. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: A bonding layer for providing buffering and protection is arranged between the flow channel layer and the digital microfluidic chip.

9. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: The interior of the digital microfluidic chip is an air phase.

10. The nucleic acid extraction and enrichment system according to claim 1, characterized in that: The extrusion module includes one robot arm and extrudes sequentially or multiple robot arms and extrudes in parallel.

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