An enhanced micro-mixing device for nucleic acid detection and a control method thereof

Abstract

The application provides an enhanced micro-mixing device for nucleic acid detection and a control method thereof. The device is sequentially connected from top to bottom with an upper plate, a reinforced mixing channel, a medium layer, an electrode layer and a lower plate. The reinforced mixing channel comprises a first groove for connecting a liquid storage area on the electrode layer to a mixing reaction area, a second groove for connecting a recovery area on the electrode layer to the mixing reaction area, and a micro-column array arranged at the intersection of each groove. The electrode layer is divided into a liquid storage area, a droplet generation area, a mixing reaction area and a recovery area. The electrode layer comprises a plurality of first electrodes for storing droplets, a plurality of second electrodes for pinching off droplets, a plurality of third electrodes for mixing droplets to generate reactions, a plurality of fourth electrodes for recovering droplets after reactions, and a plurality of fifth electrodes for transmitting droplets between areas. The device drives droplet generation, transmission and mixing based on the EWOD technology, and enhances flow field disturbance through the micro-column array to improve mixing efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, and in particular to an enhanced micro-mixing device for nucleic acid detection and a control method thereof. BACKGROUND

[0002] Microfluidic analysis systems have the characteristics of miniaturization, integration and portability. Microfluidic chips are one of the most important parts of microfluidic analysis systems, and have received extensive attention in the fields of nucleic acid detection, protein analysis and nanomaterial synthesis. Micro-mixers are the core part of microfluidic chips, and micro-droplet mixing is an important step to realize biochemical reactions. Micro-mixers can meet various detection needs and provide technical support for high-throughput, low-cost and portable microfluidic analysis systems.

[0003] In traditional microfluidic systems, the manipulation of liquid usually relies on the characteristics of continuous flow, which leads to slow molecular diffusion at the microscale and makes it difficult to achieve efficient mixing. Based on this, micro-mixers have developed various strategies, including passive mixers through the design of serpentine, spiral and contraction-expansion structures. However, these traditional designs still have certain limitations in handling diversified droplets, achieving efficient mixing at low flow rates, and meeting complex reaction conditions.

[0004] Digital microfluidics (DMF) technology is a microscale droplet manipulation technology that controls the movement and mixing of liquids through external forces such as electric field, magnetic field or surface tension. It belongs to active mixers and controls droplets through electrowetting or dielectric, achieving independent and programmable control of liquids in various micro-reaction units, providing unprecedented flexibility and automation for various biochemical operations such as sample distribution, dilution, mixing, reaction and detection. However, since the DMF system usually uses discrete droplets as working units, it is still challenging to achieve efficient micro-mixing due to limitations in droplet generation, merging, splitting and transmission efficiency.

[0005] In summary, both types of micro-mixers have obvious defects. Therefore, how to provide a low-cost and simple-to-assemble micro-mixer to achieve efficient mixing of micro-droplets in the pipeline is a problem that engineers in the field need to solve. SUMMARY

[0006] The technical problem to be solved by the present application is to provide an enhanced micro-mixing device for nucleic acid detection.

[0007] Another technical problem to be solved by the present application is to provide a control method for the enhanced micro-mixing device for nucleic acid detection.

[0008] The technical solution adopted by the present application is:

[0009] An enhanced micro-mixing device for nucleic acid detection, comprising an upper electrode plate 1, a reinforced mixing channel 2, a medium layer 3, an electrode layer 4 and a lower electrode plate 5 connected in sequence from top to bottom, wherein,

[0010] The reinforced mixing channel 2 comprises a first groove 21 for connecting the liquid storage area on the electrode layer 4 to the mixing reaction area (liquid storage area, droplet generation area, mixing reaction area), a second groove 22 for connecting the recovery area on the electrode layer 4 to the mixing reaction area, and a micro-column array 23 arranged at the intersection of each groove;

[0011] The electrode layer 4 is divided into a liquid storage area, a droplet generation area, a mixing reaction area and a recovery area; the electrode layer 4 comprises a plurality of first electrodes for storing droplets, a second electrode for pinching droplets, a third electrode for mixing and reacting droplets, a fourth electrode for recovering droplets after reaction, and a fifth electrode for transmitting droplets between areas; the second electrode is composed of sub-electrodes of different shapes, including two crescent electrodes 421 and one spindle electrode 422; the crescent electrodes 421 are arranged on the upper and lower sides of the spindle electrode 422 to jointly realize the function of droplet generation; the crescent electrodes 421 are used to preliminarily guide the flow direction of droplets, and the spindle electrode 422 is used to pinch the neck of the droplet through the electric field to realize precise pinching; the third electrode is composed of sub-electrodes of different shapes, including four ring segment electrodes 431 and one circular electrode 432, which jointly realize the function of enhanced mixing of droplets; the ring segment electrodes 431 are used to guide the rotation trajectory of droplets, and the circular electrode 432 acts as a mixing center to enhance the disturbance of the vortex; the fifth electrode 45 comprises a plurality of independent transmission electrodes for connecting the liquid storage area and the droplet generation area, the droplet generation area and the mixing reaction area, and the mixing reaction area and the recovery area; the first electrode is arranged in the liquid storage area, the second electrode is arranged in the droplet generation area, the third electrode is arranged in the mixing reaction area, and the fourth electrode is arranged in the recovery area; the fifth electrode acts as a droplet transmission electrode and is arranged between the areas; the first electrode 41 is arranged at the center of the liquid storage area and connected to the droplet generation area through the fifth electrode 45; the spindle electrode 422 of the second electrode is located in the middle of the droplet generation area and adjacent to the fifth electrode 45; the crescent electrode 421 of the second electrode is located at the edge of the droplet generation area; the ring segment electrode 431 of the third electrode is located at the periphery of the mixing area and surrounds the circular electrode 432 of the third electrode; the circular electrode 432 of the third electrode is located at the center of the mixing reaction area and aligned with the micro-column array 23; the fourth electrode 44 is located at the center of the recovery area and connected to the mixing reaction area through the fifth electrode 45.

[0012] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the enhanced mixing channel 2 is located between the upper electrode plate 1 and the medium layer 3, and the enhanced mixing channel 2 and the medium layer 3 are connected with the electrode layer 4 by bonding technology (including direct bonding, indirect bonding, etc.); the first groove 21 and the second groove 22 of the enhanced mixing channel 2 are aligned with the corresponding area of the electrode layer and the electrode, and the droplet is driven into the first groove 21 by the electrode and combined with the micro-column array 23 for enhanced mixing.

[0013] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the micro-column array 23 is composed of a plurality of micro-columns, and the micro-columns are fixed with blades (the blades can be one layer, two layers, three layers, etc.).

[0014] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the shape of the blades on the micro-columns is spiral, paddle or anchor.

[0015] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the height of the micro-column array 23 is 100 μm, 500 μm, 1000 μm, 2000 μm, 3000 μm or more.

[0016] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the diameter of a single micro-column in the micro-column array 23 is 10 μm, 20 μm, 30 μm, 40 μm, 50 μm or more.

[0017] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the material of the electrode layer 4 is gold, copper, aluminum or indium tin oxide.

[0018] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein each group of the first electrode is a rectangular electrode.

[0019] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein each group of the fourth electrode is a square electrode.

[0020] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the enhanced mixing channel 2 is located between the upper electrode plate 1 and the medium layer 3, and the material of the enhanced mixing channel 2 is a hydrophobic polymer (for example, polydimethylsiloxane).

[0021] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the groove wall thickness of the enhanced mixing channel 2 is 10 μm, 15 μm, 20 μm, 25 μm, 30 μm or more.

[0022] Preferably, the enhanced micro-mixing device for nucleic acid detection is provided, wherein the groove depth of the enhanced mixing channel 2 is 100 μm, 500 μm, 1000 μm, 2000 μm, 3000 μm or more.

[0023] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the material of the medium layer 3 is a material with high dielectric constant and good hydrophobic property, which can be bonded with polymer (e.g. parylene, polytetrafluoroethylene, silicon dioxide, polydimethylsiloxane, etc.), and the medium layer 3 is coated on the electrode layer 4.

[0024] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the thickness of the medium layer 3 is 10 μm, 15 μm, 30 μm, 50 μm or more.

[0025] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the upper electrode plate comprises a substrate 11, a conductive layer 12 and a hydrophobic layer 13 fixedly connected in sequence from top to bottom, and the material of the substrate 11 is glass.

[0026] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the substrate 11 of the upper electrode plate is provided with an opening, a first hole 111 for adding liquid to the liquid storage area, and a second hole 112 for recovering liquid droplets from the recovery area.

[0027] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the first hole and the second hole are square or circular.

[0028] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the material of the conductive layer 12 is indium tin oxide.

[0029] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the material of the hydrophobic layer 13 is Teflon, polydimethylsiloxane or polytetrafluoroethylene.

[0030] Preferably, in the enhanced micro-mixing device for nucleic acid detection, the material of the lower electrode plate is a glass plate or a printed circuit board.

[0031] The control method of the enhanced micro-mixing device for nucleic acid detection is as follows:

[0032] (1) The first liquid and the second liquid to be mixed are respectively placed in the corresponding first holes in the upper electrode plate, the first liquid is a nucleic acid sample to be detected (such as blood, saliva), the second liquid is a nucleic acid amplification reagent (such as a lysis solution, a primer mixture), and the level of the first electrode is switched from low to high, and the liquid is sucked to the liquid storage area under the action of the electro-wetting force;

[0033] (2) The first liquid and the second liquid are switched by the high-low level of the second electrode, and the large droplets in the liquid storage area are pinched off to generate small droplets with precise volume;

[0034] (3) The two droplets of precise volume generated are transported to the reaction area by the fifth electrode and smoothly merged, the third electrode is switched between high and low levels in turn, the droplets start to rotate under the action of the electric field, and the droplets rotate through the micro-pillar array to strengthen the mixing effect, the micro-pillar array induces internal turbulence of the droplets through the blade structure, increases the interface contact area, accelerates diffusion mixing, and thus improves the mixing efficiency.

[0035] (4) After the mixing is completed, the droplets are transported to the recovery area by the fifth electrode and recovered through the second hole.

[0036] Preferably, the control method adopts a power supply voltage of 150V, 100V, 50V, 25V, 15V or smaller.

[0037] The beneficial effects of the present application are:

[0038] The enhanced micro-mixing device for nucleic acid detection combines microfluidic technology and electrowetting-on-dielectric (EWOD) technology, integrates the structure, arranges the electrode layer and the enhanced mixing channel in three dimensions, the electrode layer (EWOD control layer) is located below the medium layer, the enhanced mixing channel (passive microfluidic layer) is located above the medium layer, and the three are integrated through bonding technology; the electrowetting controls the movement of the droplets, accurately generates and transports the droplets to the mixing area; after the droplets enter the groove, they are further disturbed by the passive disturbance of the micro-pillar array, thereby further improving the mixing efficiency, so that the device has an enhanced mixing effect and has the characteristics of miniaturization and automation; the control method of the device switches the voltage applied to the electrode, generates and transports the droplets to the mixing area, and then the ring segment-shaped electrode and the circular electrode of the third electrode drive the droplets to rotate through high and low voltage switching, the droplets pass through the micro-pillar array 23 during rotation, the blade structure induces vortex, and a double synergistic mixing mode of "electric field driving + fluid disturbance" is realized. Specifically:

[0039] (1) Automatic control, full-automatic droplet generation, transportation, mixing and recovery are realized through programmed voltage switching, without the need for external peristaltic pumps, and the volume of the microfluidic device is reduced.

[0040] (2) The three-dimensional microfluidic structure is combined with the electrowetting microfluidic chip to enhance the mixing of small droplets and improve the mixing efficiency.

[0041] (3) The device combines electrowetting droplet control with three-dimensional microfluidic channels, can automatically generate droplets, and only needs a small voltage to realize the double-mode mixing control of "electric field driving + passive mixing" of the droplets, saving manpower and resource cost. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1It is a schematic diagram of the upper plate of the enhanced micro-mixing device for nucleic acid detection.

[0043] Figure 2 It is a schematic diagram of the enhanced mixing channel of the enhanced micro-mixing device for nucleic acid detection.

[0044] Figure 3 It is a micro-column array detail view of the enhanced micro-mixing device for nucleic acid detection, and (a), (b), (c) are three kinds of blade shapes.

[0045] Figure 4 It is a schematic diagram of the electrode layer of the enhanced micro-mixing device for nucleic acid detection.

[0046] Figure 5 It is a schematic diagram of the simulated flow channel of the enhanced micro-mixing device for nucleic acid detection.

[0047] Figure 6 It is a schematic diagram of the overall layered view of the enhanced micro-mixing device for nucleic acid detection. DETAILED DESCRIPTION

[0048] In order to further illustrate the present application, the technical solutions are described clearly and completely in combination with the following embodiments.

[0049] Embodiment 1

[0050] As shown in the description, the enhanced micro-mixing device for nucleic acid detection comprises, from top to bottom, an upper plate 1, an enhanced mixing channel 2, a medium layer 3, an electrode layer 4 and a lower plate 5 connected in sequence, wherein, Figures 1-6 The upper plate comprises, from top to bottom, a substrate 11, a conductive layer 12 and a hydrophobic layer 13 fixedly connected in sequence, the material of the substrate 11 is glass, the material of the conductive layer 12 is indium tin oxide, and the material of the hydrophobic layer 13 is polydimethylsiloxane PDMS, the substrate 11 of the upper plate is provided with a circular opening, a first hole 111 is used for adding liquid to the liquid storage area, and a second hole 112 is used for recovering liquid drops from the recovery area.

[0051]

[0052] ​The reinforced mixing channel 2 is located between the upper plate 1 and the medium layer 3, the material of the reinforced mixing channel 2 is a hydrophobic polymer (for example, polydimethylsiloxane PDMS), the reinforced mixing channel 2 and the medium layer 3 are connected with the electrode layer 4 by a bonding technology, which can be direct bonding or indirect bonding; the reinforced mixing channel 2 includes a first groove 21 for connecting the liquid storage area, the droplet generation area and the mixing reaction area on the electrode layer 4, a second groove 22 for connecting the recovery area to the mixing reaction area on the electrode layer 4, and a micro column array 23 arranged at the intersection of each groove; the groove wall thickness of the reinforced mixing channel 2 is 10 μm (which can also be 15 μm, 20 μm, 25 μm, 30 μm or more), and the groove depth is 100 μm (which can also be 500 μm, 1000 μm, 2000 μm, 3000 μm or more); the height of the micro column array 23 is 100 μm (which can also be 500 μm, 1000 μm, 2000 μm, 3000 μm or more), and the diameter of a single micro column in the micro column array 23 is 10 μm (which can also be 20 μm, 30 μm, 40 μm, 50 μm or more); the first groove 21 and the second groove 22 of the reinforced mixing channel 2 are aligned with the corresponding area of the electrode layer and the electrode, and the droplets are driven into the first groove 21 by the electrode and combined with the micro column array 23 to enhance mixing, the micro column array 23 is composed of a plurality of micro columns, and a blade is fixed on the micro column, the shape of the blade on the micro column is spiral 231, paddle 232 or anchor 233;

[0053] The material of the medium layer 3 is a material (for example, polydimethylsiloxane PDMS) with high dielectric constant and good hydrophobic performance, which can be bonded with the polymer, and the medium layer 3 is coated on the electrode layer 4; the thickness of the medium layer is 10 μm (which can also be 15 μm, 30 μm, 50 μm or more);

[0054] The material of the electrode layer 4 is gold (also can be copper, aluminum or indium tin oxide), and the electrode layer 4 is divided into a liquid storage area I, a droplet generation area II, a mixing reaction area III and a recovery area IV; the electrode layer 4 includes a plurality of first electrodes 41 for storing droplets, a second electrode for pinching off droplets, a third electrode for mixing and reacting droplets, a fourth electrode 44 for recovering droplets after reaction, and a fifth electrode 45 for transmitting droplets between areas; the second electrode is composed of sub-electrodes of different shapes, including two crescent electrodes 421 and a spindle electrode 422, the crescent electrodes 421 are arranged on the upper and lower sides of the spindle electrode 422 to achieve the function of droplet generation, wherein the crescent electrodes 421 are used to preliminarily guide the flow direction of the droplets, and the spindle electrode 422 is used to pinch off the droplet neck through the electric field to achieve precise pinching off; the third electrode is composed of sub-electrodes of different shapes, including four ring segment electrodes 431 and a circular electrode 432, which work together to achieve the function of enhanced mixing of droplets, wherein the ring segment electrodes 431 are used to guide the rotation trajectory of the droplets, and the circular electrode 432 is used as a mixing center to enhance the disturbance of the vortex; the fifth electrode 45 includes a plurality of independent transmission electrodes, which are respectively used to connect the liquid storage area and the droplet generation area, the droplet generation area and the mixing reaction area, and the mixing reaction area and the recovery area; the first electrode is arranged in the liquid storage area, the second electrode is arranged in the droplet generation area, the third electrode is arranged in the mixing reaction area, and the fourth electrode is arranged in the recovery area; the fifth electrode is used as a droplet transmission electrode and is arranged between the areas; the first electrode 41 is a rectangular electrode arranged in the center of the liquid storage area and connected to the droplet generation area through the fifth electrode 45; the spindle electrode 422 of the second electrode is located in the middle of the droplet generation area and adjacent to the fifth electrode 45; the crescent electrode 421 of the second electrode is located at the edge of the droplet generation area; the ring segment electrode 431 of the third electrode is located at the periphery of the mixing area and surrounds the circular electrode 432 of the third electrode; the circular electrode 432 of the third electrode is located in the center of the mixing reaction area and aligned with the micro pillar array 23; the fourth electrode 44 is a square electrode located in the center of the recovery area and connected to the mixing reaction area through the fifth electrode 45.

[0055] The material of the lower plate is a glass plate.

[0056] The processing technology of the above-mentioned enhanced mixing channel 2 can adopt micro-channel casting processing technology or two-photon micro-nano 3D printing technology, etc. The processing technology of the above-mentioned dielectric layer 3 can adopt spin coating, magnetron sputtering, atomic layer deposition or vapor deposition, etc. The processing technology of the above-mentioned electrode layer 4 can adopt etching, magnetron sputtering or thermal evaporation, etc.

[0057] The control method for the enhanced micro-mixing device for nucleic acid detection described above allows the device to be operatively connected to a computer. Electrode layer 4 integrates a control circuit, which, when connected to the computer, enables programmed voltage drive. Power is supplied via computer control, using a power supply voltage of 150V (or 100V, 50V, 25V, 15V, or lower), achieving automated mixing operation. The specific steps are as follows:

[0058] (1) Place the first liquid 6 and the second liquid 7 to be mixed into the corresponding first wells 111 in the upper electrode plate. The first liquid is the nucleic acid sample to be tested (such as blood or saliva), and the second liquid is the nucleic acid amplification reagent (such as lysis buffer or primer mixture). Under program control, the voltage level of the first electrode switches from low to high. Figure 5 As shown, under the action of electrowetting force, the first liquid 6 and the first liquid 7 are drawn into the storage area, and the first electrode 41 stops operating.

[0059] (2) Precise droplets must be generated in the droplet generation region:

[0060] (a) The spindle-shaped electrode 422 and the crescent-shaped electrode 421 of the second electrode switch from low level to high level, and the second electrode becomes a hydrophilic surface, so that the first liquid 6 and the second liquid 7 flow into the spindle-shaped electrode 422 and the crescent-shaped electrode 421 and the fifth electrode 45 connected before and after the second electrode.

[0061] (b) After the first liquid 6 and the second liquid 7 flow into the spindle-shaped electrode 422 and the crescent-shaped electrode 421 of the second electrode, the spindle-shaped electrode 422 switches to a high level, while the crescent-shaped electrode 421 remains at a low level, making the spindle-shaped electrode 422 hydrophobic and the crescent-shaped electrode 421 still hydrophilic. At this time, the liquid flowing into the second electrode is squeezed into the shape of the crescent-shaped electrode 421.

[0062] (c) After the liquid flowing into the second electrode is squeezed into the shape of the crescent-shaped electrode 421, both the spindle-shaped electrode 422 and the crescent-shaped electrode 421 switch to a low level, and the second electrode becomes a hydrophobic surface, thereby pinching off the large droplets in the liquid storage area to generate small droplets of precise volume. The droplets remain on the fifth electrode 45, completing the precise generation of droplets. The generated droplet volume is 0.1-10 μL.

[0063] (3) After the generation of the precise droplet, the fifth electrode 5 is switched from low level to high level, and the droplet is transported to the ring segment electrode 431 and the circular electrode 432 of the third electrode, and the two droplets meet in the reaction area, the four ring segment electrodes 431 of the third electrode are switched from high level to low level in sequence, and the circular electrode 432 keeps low level, and the droplets are rotated and mixed under the driving of the electrowetting force, the droplets pass through the micro pillar array 23 while rotating, the micro pillar array and the blades thereof are used to increase the vortex disturbance to the droplets to form a turbulent flow to increase the interface contact area, accelerate diffusion mixing, thereby improving the mixing efficiency and enhancing the droplet mixing;

[0064] (4) After the mixing is completed, the ring segment electrode 431 and the circular electrode 432 of the third electrode of the reaction area are switched to the level, the droplet is transported to the fourth electrode 44 of the droplet recovery area through the fifth electrode 45, and finally the droplet is recovered through the second hole 112.

[0065] In summary, the enhanced micro mixing device for nucleic acid detection disclosed in the application combines the traditional micro flow channel on the electrowetting microfluidic chip, generates droplets through the EWOD technology, and performs distribution and high-throughput transmission and mixing of the droplets; meanwhile, the droplets are further mixed through the special designed micro flow channel, so that the high-efficiency mixing and reaction effect of the droplets are realized.

[0066] The above-described embodiments only describe the preferred embodiments of the application, and do not limit the scope of the application, and various deformations and improvements of the technical solutions of the application made by the ordinary engineering technicians in the art without departing from the design spirit of the application shall fall within the protection scope of the claims of the application.

Claims

1. An enhanced micro-mixing device for nucleic acid detection, characterized by: The electrode layer is connected to the upper plate, the reinforced mixing channel, the medium layer and the lower plate in sequence from top to bottom. The reinforced mixing channel comprises a first groove for connecting the liquid storage area on the electrode layer to the mixing reaction area, a second groove for connecting the recovery area on the electrode layer to the mixing reaction area, and a micro column array arranged at the intersection of the grooves. The electrode layer comprises a plurality of first electrodes for storing droplets, a second electrode for pinching droplets, a third electrode for mixing and reacting droplets, a fourth electrode for recovering reacted droplets, and a fifth electrode for transmitting droplets between the areas.

2. The enhanced micro-mixing device for nucleic acid detection of claim 1, wherein: The first electrode is arranged in the liquid storage area, the second electrode is arranged in the droplet generation area, the third electrode is arranged in the mixing reaction area, and the fourth electrode is arranged in the recovery area.

3. The enhanced micro-mixing device for nucleic acid detection of claim 1, wherein: The fifth electrode is arranged between the areas as a droplet transmission electrode.

4. The enhanced micro-mixing device for nucleic acid detection of claim 3, wherein: The first electrode is arranged in the center of the liquid storage area and connected to the droplet generation area through the fifth electrode.

5. The enhanced micro-mixing device for nucleic acid detection of claim 1, wherein: The spindle-shaped electrode of the second electrode is arranged in the middle of the droplet generation area adjacent to the fifth electrode.

6. The enhanced micro-mixing device for nucleic acid detection of claim 1, wherein: The crescent-shaped electrode of the second electrode is arranged at the edge of the droplet generation area.

7. The enhanced micro-mixing device for nucleic acid detection of claim 1, wherein: The ring segment-shaped electrode of the third electrode is arranged at the periphery of the mixing area surrounding the circular electrode of the third electrode. The circular electrode of the third electrode is arranged in the center of the mixing reaction area and aligned with the micro column array. The fourth electrode is arranged in the center of the recovery area and connected to the mixing reaction area through the fifth electrode. The reinforced mixing channel is arranged between the upper plate and the medium layer. The reinforced mixing channel and the medium layer are connected to the electrode layer through bonding technology. The first groove and the second groove of the reinforced mixing channel are aligned with the corresponding area of the electrode layer and the electrode. The micro column array comprises a plurality of micro columns. The shape of the micro column is spiral, paddle or anchor. The height of the micro column array is not less than 100 μm. The diameter of a single micro column in the micro column array is not less than 10 μm. The material of the electrode layer is gold, copper, aluminum or indium tin oxide. Each group of the first electrode is a rectangular electrode. Each group of the fourth electrode is a square electrode. The material of the reinforced mixing channel is a hydrophobic polymer. The groove wall thickness of the reinforced mixing channel is not less than 10 μm. The groove depth of the reinforced mixing channel is not less than 100 μm. The material of the medium layer is a material with high dielectric constant and good hydrophobic property that can be bonded with the polymer. The medium layer is coated on the electrode layer. The thickness of the medium layer is not less than 10 μm.

8. The enhanced micro-mixing device for nucleic acid detection of claim 1, wherein: The upper electrode plate comprises a substrate, a conductive layer and a hydrophobic layer fixedly connected in sequence from top to bottom, the material of the substrate is glass, the substrate of the upper electrode plate is provided with an opening, a first hole is used for adding liquid to the liquid storage area, and a second hole is used for recovering liquid drops from the recovery area; the material of the conductive layer is indium tin oxide; the material of the hydrophobic layer is Teflon, polydimethylsiloxane or polytetrafluoroethylene.

9. The enhanced microfluidic device for nucleic acid detection of claim 1, wherein: The material of the lower electrode plate is a glass plate or a printed circuit board.

10. The method for controlling the enhanced micro-mixing device for nucleic acid detection according to any one of claims 1 to 9, characterized in that: The specific steps are as follows: (1) the first liquid and the second liquid to be mixed are respectively placed in the corresponding first holes in the upper electrode plate, the first liquid is a nucleic acid sample to be detected, the second liquid is a nucleic acid amplification reagent, the level of the first electrode is switched from low to high, and the liquid is sucked to the liquid storage area under the action of the electro-wetting force; (2) the first liquid and the second liquid are switched by the high-low level of the second electrode, the large liquid drop of the liquid storage area is pinched off to generate a small liquid drop with precise volume; (3) the two small liquid drops with precise volume generated are transported to the reaction area by the fifth electrode and smoothly merged, the third electrode is switched in sequence from high to low, the liquid drop starts to rotate under the action of the electric field, and the liquid drop rotates while passing through the micro-column array to strengthen the mixing effect; (4) after the mixing is completed, the liquid drop is transported to the recovery area by the fifth electrode and recovered through the second hole.

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