An EWOD-based end-to-end nucleic acid detection chip and its applications

Abstract

The present invention discloses a full-process nucleic acid detection chip based on EWOD and its application, the chip comprises: SiO 2 Support plate, SiO 2 glass, a dielectric layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer and an ITO glass layer; a magnetic bead nucleic acid extraction zone and a dual-temperature zone nucleic acid amplification zone are formed between the first and second Teflon hydrophobic layers; the magnetic bead nucleic acid extraction zone includes: a lysis chamber, a first washing chamber, a second washing chamber and an elution chamber; the dual-temperature zone nucleic acid amplification zone includes: a low-temperature constant temperature zone and a high-temperature constant temperature zone; SiO 2 The glass is provided with interdigital electrodes and EWOD driving electrodes, and the droplets circulate and move in the low-temperature constant temperature zone and the high-temperature constant temperature zone to achieve amplification. The present invention integrates electrolysis, magnetic bead nucleic acid extraction, EWOD droplet drive and dual-temperature zone constant temperature control, has the advantages of short detection time, low cost and small size, and has good application prospects in the field of PCR detection.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chips, and more specifically to an EWOD-based full-process nucleic acid detection chip and applications thereof. Background Art

[0002] Medical full-process nucleic acid detection chip technology is developing rapidly, integrating multiple steps such as sample processing, nucleic acid extraction, amplification and detection, and can realize the integration and automation of nucleic acid detection. Loop-mediated isothermal amplification (LAMP) and PCR are usually used for pathogen detection. Among them, PCR technology is called the "gold standard" for nucleic acid detection due to its high sensitivity, good characteristics, and excellent quantitative ability. The existing PCR temperature control method mainly relies on traditional thermal cyclers to complete DNA amplification reactions by accurately controlling the temperature changes of heating modules and cooling systems. Commonly used technologies include semiconductor temperature control (Peltier effect) and thermocouple sensors. Although these technologies have been widely used in actual operations, there are still some shortcomings. First, the temperature control accuracy is limited. Due to the delay in temperature conduction, it is difficult to achieve rapid temperature rise and fall, especially in experiments requiring high reaction speeds. Second, the entire reaction process takes a long time because the sample needs to be heated and cooled step by step, which prolongs the experimental cycle and reduces efficiency (usually more than 60 minutes); third, the equipment is large in size and high in energy consumption. Traditional thermal cyclers are not only bulky but also consume a lot of power, which is not suitable for portable operation or use in resource-constrained environments. In addition, in order to meet the full-process testing needs, the existing full-process nucleic acid detection chips have a variety of structural designs and external control components, complex equipment structures, huge equipment volumes, and high costs, such as the GeneXpert product of Cepei. Therefore, it is urgent to develop a low-cost, ultra-fast, and full-process nucleic acid detection chip to meet the needs of rapid on-site testing.

[0003] Electrowetting on dielectrics (EWOD) is an electrically controlled surface tension driving method that can achieve rapid drive of droplets. It has been applied to the field of PCR in recent years. It changes the wetting characteristics of the dielectric membrane and the surface liquid by applying an electric potential on the microelectrode array under the dielectric membrane, and can drive droplets accurately and quickly. A typical EWOD device usually adopts a three-layer structure, that is, the controlled droplets are sandwiched between the upper and lower plates. The lower plate is composed of a substrate, a microelectrode array, a dielectric layer and a hydrophobic layer. The hydrophobic layer can ensure the smoothness and stability of the droplet movement through the layer. The filling material between the upper and lower plates can be air or silicone oil. Silicone oil can be used as a lubricant to reduce the driving damping of the droplets, reduce the driving voltage, and reduce the evaporation of the droplets. However, the traditional EWOD chip is prepared using PCB technology, MEMS technology or flexible circuit technology. It is expensive and the electrode thickness is relatively thick (minimum about 35 microns), which is not conducive to the movement of droplets and is not suitable for use as a disposable consumable. Therefore, it is urgent to develop a low-cost EWOD chip for PCR detection. Summary of the invention

[0004] The purpose of the present invention is to provide a full-process nucleic acid detection chip based on EWOD and its application, so as to solve the problems of long detection time, complex chip structure, high cost, large equipment size, etc. in the full-process nucleic acid detection of the prior art.

[0005] To solve the above problems, the present invention adopts the following technical solutions:

[0006] According to a first aspect of the present invention, a full-process nucleic acid detection chip based on EWOD is provided, wherein the full-process nucleic acid detection chip comprises: SiO 2 Support plate, SiO 2 glass, a dielectric layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer and an ITO glass layer; a magnetic bead nucleic acid extraction zone and a dual-temperature zone nucleic acid amplification zone are formed between the first Teflon hydrophobic layer and the second Teflon hydrophobic layer; the magnetic bead nucleic acid extraction zone includes: a lysis chamber, a first washing chamber, a second washing chamber and an elution chamber; the dual-temperature zone nucleic acid amplification zone includes: a low-temperature constant temperature zone and a high-temperature constant temperature zone; the SiO 2 An interdigitated electrode and an EWOD driving electrode are provided on the glass. The interdigitated electrode is aligned with the lysis chamber to realize the electric lysis of the bacterial sample. The EWOD driving electrode is aligned with the elution chamber and the dual-temperature zone nucleic acid amplification zone. By controlling the power supply of the EWOD driving electrode to make its surface hydrophilic, the droplets circulate in the low-temperature constant temperature zone and the high-temperature constant temperature zone to realize amplification.

[0007] Preferably, the interdigital electrodes and the EWOD driving electrodes are both printed on SiO 2It is printed on glass, and the paste used is conductive silver paste, conductive gold paste, or conductive platinum paste.

[0008] Preferably, the thickness of the interdigital electrodes and the EWOD driving electrodes is 5-10 μm.

[0009] It should be understood that the electrode prepared by the screen printing process is thinner. According to a preferred embodiment of the present invention, the prepared electrode is about 6 μm thick. Compared with the EWOD driving electrodes prepared by other processes (thickness is at least 35 μm, or even up to 50 μm), the electrode prepared by screen printing is thinner, which is more conducive to the movement of droplets, reduces the driving voltage, and can avoid the influence of bubbles generated by reagent electrolysis on the movement of droplets to a greater extent.

[0010] The interdigital electrodes and EWOD driving electrodes are prepared by multi-layer screen printing. The screen printing plate uses a 300-mesh or 380-mesh screen, and the slurry used is conductive silver slurry, conductive gold slurry, or conductive platinum slurry. Compared with the traditional PCB board process or flexible circuit preparation process for processing EWOD driving electrodes, the electrodes prepared by multi-layer screen printing are lower in cost and convenient for disposable use as consumables. It has obvious advantages in cost-effectiveness, preparation speed and flexibility. At the same time, the multi-layer screen printing process is used to prepare EWOD driving electrodes and electrode leads, which can also avoid the interference of the leads on droplet drive when the electrode leads and the EWOD driving electrodes are on the same layer.

[0011] Preferably, the support layer has a thickness of 0.2-0.5 mm, which can better provide space for the droplets.

[0012] Preferably, the lysis chamber, the first washing chamber, the second washing chamber, the elution chamber, the low-temperature constant temperature zone and the high-temperature constant temperature zone are all filled with mineral oil to ensure that the magnetic beads can move freely between the chambers.

[0013] Preferably, in the multi-layer screen printing process, the screen printing plate uses a 300-mesh or 380-mesh screen.

[0014] Preferably, by changing the structure of the screen printing plate, the parallel design of multiple nucleic acid detection pathways is achieved, thereby achieving simultaneous detection of multiple nucleic acids.

[0015] According to a second aspect of the present invention, there is provided an application of an EWOD-based full-process nucleic acid detection chip as described above in full-process nucleic acid detection, comprising the following steps: S1, adding a test sample to a lysis chamber, and electrolyzing the bacteria by applying a voltage of a certain frequency to the interdigitated electrodes; S2, after the electrolysis is completed, controlling the magnetic beads to enter the lysis chamber by a magnetic rod, mixing by moving the magnetic rod, and then controlling the magnetic rod to move the magnetic beads so that they pass through the first washing chamber and the second washing chamber in sequence; S3, connecting the EWOD driving electrode to a power source, adding a nucleic acid extract to the elution chamber, controlling the magnetic rod to move the magnetic beads to the elution chamber for elution, and then controlling the magnetic rod again to move the magnetic beads back to the second washing chamber, and then controlling the EWOD driving electrodes to be energized respectively, and moving the reaction reagents in the elution chamber to a high-temperature constant temperature zone for pre-denaturation for 30 to 60 seconds. After completion, by controlling the power on and off of the EWOD driving electrodes, the droplets are circulated in the high-temperature constant temperature zone and the low-temperature constant temperature zone to achieve nucleic acid amplification; S4, nucleic acid detection is performed by fluorescent photography.

[0016] Preferably, the interdigitated electrodes are connected to a DC voltage of 10 to 30 V or an AC voltage of 10 to 30 V, which can release the nucleic acid in the cells within 1 minute.

[0017] Preferably, by controlling the power on and off of the EWOD driving electrode, the movement and residence of the droplets are controlled so that in each cycle the droplets stay in the high temperature constant temperature zone for 3 seconds and in the low temperature zone for 1 second, and move between the high temperature constant temperature zone and the low temperature constant temperature zone for several cycles, and the entire amplification time does not exceed 7 minutes.

[0018] The existing EWOD technology for nucleic acid detection usually adopts PCB, flexible circuit or MEMS technology, which has high processing cost, while the present invention adopts multi-layer screen printing of conductive silver paste to prepare EWOD driving electrodes, which has lower cost. In addition, the present invention also creatively adopts screen printing technology to prepare on-chip electrolysis electrodes, and integrates a magnetic bead nucleic acid extraction module by modifying the surface hydrophilic sites. Afterwards, the present invention integrates a dual-temperature zone heating module to combine it with the EWOD droplet drive technology, so that it can realize the ultra-fast PCR amplification link. Through the combination of the above technologies, the chip can complete the whole process of nucleic acid detection in a low-cost, multi-channel and portable manner.

[0019] One of the inventive points of the present invention is to prepare on-chip interdigitated electrodes and use electrolysis technology to lyse cells. The principle is to generate a transmembrane potential by applying an electric field. When the potential reaches a certain level, the cell membrane ruptures, thereby releasing the cell contents. The interdigitated width is 50-200 μm, and the interdigitated spacing is 50-200 μm. The electrode voltage is 10-30V DC or 10-30V AC. Compared with other lysis methods, electrolysis is fast and efficient, and can release nucleic acids in cells within 1 minute. It is easy to operate, does not require complex equipment or additional chemical reagents; and has strong controllability. By adjusting the intensity and action time of the electric field, the degree of cell lysis can be accurately controlled to obtain the desired lysis effect. The present invention uses screen printing to prepare a low-cost conductive electrode, and the material of the electrode is a screen printing paste with good conductive properties to reduce the partial pressure on the electrode and enhance the lysis efficiency at the same voltage. The printing material of the electrode is a highly conductive printing paste such as conductive silver paste, conductive gold paste or platinum paste.

[0020] Another inventive point of the present invention is that a magnetic bead method nucleic acid extraction module is integrated on the EWOD chip. The module consists of a lysis chamber, a first washing chamber, a second washing chamber and an elution chamber. The surfaces of the lysis chamber, the first washing chamber and the second washing chamber are etched to expose SiO 2 The surface of the module is hydrophilic and is used to fix the reagent sample and the washing liquid. The chambers are filled with mineral oil to ensure that the magnetic beads can move freely between the chambers. The module is characterized by the modification of the hydrophilic sites to firmly fix the droplets and prevent the droplets from being dragged when the magnetic beads move, which may cause crosstalk between the chambers.

[0021] Another inventive point of the present invention is that a dual-zone constant temperature heating block is used as a heating method, and a low temperature constant temperature zone of 60°C and a high temperature constant temperature zone of 98°C are set respectively. Compared with the semiconductor temperature control method, the dual-zone method consumes less power and takes less time, avoids the temperature rise / fall process of the temperature control module, and can realize an ultra-fast and portable PCR process.

[0022] Another inventive point of the present invention is that the amplification reagent is driven by EWOD, and the surface is rendered hydrophilic by electrification, thereby better fixing the amplification reagent in the elution chamber, and the reagent can also move and react in different temperature ranges. The driving method of EWOD can accurately control the path and morphology of the droplets, and is easy to integrate with other technologies on the chip. In addition, the operation of the droplets is carried out in a closed environment, reducing exogenous pollution.

[0023] During the creation of the present invention, the inventors discovered that a larger driving voltage causes the reagent to electrolyze and produce more bubbles, which will have a greater impact on the movement of droplets. The present invention creatively uses a very thin dielectric layer and a hydrophobic layer, and adopts conductive silver paste and other slurries with better conductivity; it uses multi-layer screen printing and layers the electrodes and leads, which can prevent the hydrophilicity caused by the conduction of the leads and the hydrophilicity after the electrodes are energized from interfering with each other, resulting in the droplets being unable to move smoothly.

[0024] Compared with the prior art, the superiority of the present invention mainly lies in the ultra-fast full process, and the entire process of sample lysis, nucleic acid extraction and nucleic acid amplification can be completed within 10 minutes. Through dual-temperature zone heating and low power consumption, the droplets can quickly reach the predetermined temperature, and the thinner electrode layer, dielectric layer and hydrophobic layer reduce the driving voltage, reduce reagent electrolysis, and quickly drive the droplets at the same time, so that the amplification time is controlled within 7 minutes. The conventional full-process nucleic acid detection chip commonly used for EWOD chips often takes about 30 minutes. The present invention reduces costs through low-cost screen printing technology, and can integrate the entire process of electrolysis, elution, extraction and amplification into one chip.

[0025] In summary, the present invention integrates electrolysis technology, magnetic bead nucleic acid extraction technology, EWOD droplet drive technology and dual-temperature zone constant temperature control technology. Electrolysis can complete the release of cell nucleic acid within 1 minute, and the magnetic beads can be controlled by magnetic rods to complete the extraction of nucleic acid within 2 minutes. The rapid movement of droplets driven by EWOD in the dual-temperature interval can complete 40 cycles of PCR within 7 minutes, thereby achieving full-process automated detection of nucleic acids within 10 minutes, exceeding the conventional PCR detection volume and heating rate, and reducing the detection time. Therefore, such a full-process nucleic acid detection chip based on EWOD provided by the present invention has the advantages of short detection time, low cost, and small size, and has good application prospects in the field of PCR detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the entire process of a full-process nucleic acid detection chip provided according to a preferred embodiment of the present invention;

[0027] Figure 2 Yes Figure 1 An exploded view of the full-process nucleic acid detection chip is shown;

[0028] Figure 3 Yes Figure 1 The schematic diagram of the structure of the electrolysis and nucleic acid extraction area of ​​the full-process nucleic acid detection chip is shown;

[0029] Figure 4 Yes Figure 1 The schematic diagram of the nucleic acid amplification region structure of the full-process nucleic acid detection chip is shown;

[0030] Figure 5 This is the electron microscope image of the thickness of the conductive silver paste electrode in the electrolysis zone;

[0031] Figure 6 It is a schematic diagram of contact angles of hydrophobic and hydrophilic regions;

[0032] Figure 7 This is a comparison chart of live and dead cells of E. coli electrolysis. The brighter green in the yellow circle in the upper picture is live bacteria, and the red in the lower picture is dead bacteria.

[0033] Figure 8 These are negative and positive control images before and after amplification; the upper image is negative and the lower image is positive. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0035] Example 1 Preparation of a full-process nucleic acid detection chip based on EWOD According to the present invention, an ultra-fast detection chip for performing the full process of electrolysis, elution, extraction and amplification in an EWOD microfluidic chip is provided. The ultra-fast full-process detection is completed in 10 minutes, and the dual-temperature zone amplification is completed in a very short time by controlling the rapid movement of tiny droplets through electrodes, which exceeds the conventional PCR detection volume and heating rate, thereby reducing the detection time.

[0036] Combination Figure 1 , Figure 2 As shown, a full-process nucleic acid detection chip based on EWOD is provided according to a preferred embodiment of the present invention. The specific structure is described in detail as follows:

[0037] Specific reference Figure 2 The full-process nucleic acid detection chip includes the following components arranged from bottom to top: SiO 2 Support plate 1, SiO 2 Glass 2 , PET dielectric layer 3 , first Teflon hydrophobic layer 4 , support layer 5 , second Teflon hydrophobic layer 6 and ITO glass layer 7 .

[0038] See also Figure 1A magnetic bead nucleic acid extraction zone 10 and a dual-temperature zone nucleic acid amplification zone 20 are formed between the first Teflon hydrophobic layer 4 and the second Teflon hydrophobic layer 6; the magnetic bead nucleic acid extraction zone 10 includes: a lysis chamber 11, a first washing chamber 12, a second washing chamber 13 and an elution chamber 14; the dual-temperature zone nucleic acid amplification zone 20 includes: a low-temperature constant temperature zone 21 and a high-temperature constant temperature zone 22.

[0039] Combination Figure 2 , Figure 3 As shown, SiO 2 The glass 2 is provided with an interdigitated electrode 30 and an EWOD driving electrode 40. The interdigitated electrode 30 is aligned with the lysis chamber 11 to realize the electrolysis of the bacterial sample. The EWOD driving electrode 40 is aligned with the elution chamber 14 and the dual-temperature zone nucleic acid amplification zone 20. By controlling the power supply of the EWOD driving electrode 40 to make its surface hydrophilic, the droplets circulate in the low-temperature constant temperature zone 21 and the high-temperature constant temperature zone 22 to realize amplification.

[0040] Specifically, combined Figure 1 to Figure 4 As shown, the chip bottom uses 2 to 3 mm SiO 2 The support plate 1 is used as a support layer. 2 Grooves of specific shapes are carved on the support layer 1 to facilitate the movement of the magnetic rod 50 and the heating of the heating blocks (60°C constant temperature heating block 71, 98°C constant temperature heating block 72); SiO 2 A thin SiO layer with a thickness of 0.4 to 0.5 mm is used on the support layer 1. 2 Glass 2; in SiO 2 The glass 2 is printed with conductive silver paste using a multi-layer screen printing process to print interdigital electrodes 30 and EWOD driving electrodes 40; a 2μm thick PET dielectric layer 3 is sprayed on the electrode, and then Teflon is sprayed on it as the first Teflon hydrophobic layer 4; a 100-400μm thick PET chamber is used as the support layer 5 on the first Teflon hydrophobic layer 4; the top layer uses 1.1mm thick ITO glass as a sealing layer, and the side of the ITO glass in contact with the support layer is also sprayed with Teflon as the second Teflon hydrophobic layer 6; different glass and support layers are bonded by high temperature resistant non-fluorescent UV glue. In addition, a spring pin connection method is used to make the spring pin contact with the external electrode to conduct the circuit.

[0041] According to the present invention, Figure 3 As shown, in the magnetic bead nucleic acid extraction area 10 and the dual-temperature zone nucleic acid amplification area 20, an etching process is used to etch away the hydrophobic layer and the PET dielectric layer to expose the hydrophilic SiO 2 Therefore, the area where the droplet is located is hydrophilic, and the outside is hydrophobic. The difference in contact angles ensures that the sample to be tested, the eluent, and the extract are fixed in the hydrophilic area and will not move. Figure 5As shown, the contact angle of the hydrophilic area after hydrophilic treatment is 17.9°, and the contact angle of the hydrophobic area is 117.5°.

[0042] According to the present invention, the most important structural layer is the electrode layer printed by a multi-layer screen printing process. The thinner the layer is, the better, so as to facilitate the smooth movement of the droplets. Figure 6 As shown, according to a preferred embodiment of the present invention, the thickness of the electrode prepared by screen printing is 6 μm, which is much smaller than other preparation methods, is more conducive to droplet driving and reduces driving voltage, and can greatly reduce reagent electrolysis.

[0043] According to a preferred embodiment of the present invention, each layer in the above structure is indispensable, and the upper and lower Teflon hydrophobic layers are necessary conditions, because the interface driven by the EWOD droplet must be a hydrophobic interface, and conventional EWOD chip preparation has two hydrophobic layers. The support layer 5 only uses PET material to provide a chamber with a thickness of 0.2 to 0.5 mm. Other materials, such as PC, can also be used to form the chamber required for the movement of silicone oil and reaction reagents. In addition, the dielectric layer is also very important. According to a preferred embodiment of the present invention, the thickness of the dielectric layer is 2 μm. According to the research of the present invention, it is found that if the dielectric layer is too thick and the driving voltage is too high, the hydrolysis and bubble problems will be more serious; on the contrary, if the dielectric layer is too thin, it is easy to cause electrical breakdown, causing the chip to fail. Therefore, the dielectric layer is preferably selected to be 2 to 3 μm thick, and the material is not limited to PET (polyethylene terephthalate), but can also be PI (polyimide).

[0044] Example 2 Application of a full-process nucleic acid detection chip in a full-process nucleic acid detection According to this embodiment, an application of a full-process nucleic acid detection chip based on EWOD as shown in Example 1 in a full-process nucleic acid detection is provided, comprising the following steps:

[0045] 1) First, add the test sample (such as bacteria such as Escherichia coli or Bacillus, saliva from the respiratory tract, throat swab collection, etc.) into the lysis chamber 11, and lyse the test sample by applying a certain frequency voltage (AC voltage 30V, frequency 100,000 Hz) to the interdigital electrode 30. The entire lysis time does not exceed 1 minute. SYTO 9 is used to stain the bacteria before and after lysis, showing live and dead bacteria in green; PI staining agent stains dead bacteria in red. The staining results are shown in Figure 1. Figure 7 As shown, it shows that electrolysis can effectively lyse cells.

[0046] 2)Reference Figure 3 After the electrolysis is completed, the magnetic rod 50 controls the magnetic beads 60 to enter the lysis chamber 11, and the magnetic rod 50 is moved to mix, and then the magnetic rod 50 is controlled to move to the bottom of the elution chamber 14, so that the magnetic beads pass through the silicone oil and enter the first washing chamber 12. Repeat the above process to allow the magnetic beads to enter the second washing chamber 13.

[0047] 3) The EWOD driving electrode 40 below the elution chamber 14 is powered on, and the extracting solution is added to the elution chamber 14, and the magnetic bar 50 is controlled to move the magnetic beads 60 to the elution chamber 14 for elution, and then the magnetic bar 50 is controlled again to move the magnetic beads 60 back to the second washing chamber 13. Then the EWOD driving electrodes 40 are controlled to be powered on respectively, and the 4 μL reaction reagent in the elution chamber is moved to the 98°C temperature zone for 30-60s of pre-denaturation. After completion, the droplets are circulated in the high temperature constant temperature zone 22 (98°C) and the low temperature constant temperature zone 21 (60°C) by controlling the power on and off of the EWOD driving electrodes. In each cycle, the droplets stay in the high temperature constant temperature zone 22 for 3s and stay in the low temperature constant temperature zone 21 for 1s. Under this condition, the droplets move between the high temperature constant temperature zone 22 and the low temperature constant temperature zone 21 for 40 cycles, and the entire amplification time does not exceed 7min. The moving time of the droplets between adjacent electrodes is set to 500ms-1000ms to ensure that the droplets can move stably. The fastest moving time of the droplets between the electrodes of the chip of the present invention can reach 100ms.

[0048] 4) Fluorescence photography was used to detect the negative and positive control groups (for example, Bacillus, FAM channel). After 40 cycles, the negative and positive fluorescence intensity controls were as follows: Figure 8 As shown in the figure, the fluorescence intensity is significantly enhanced. The entire ultra-fast process takes no more than 10 minutes.

[0049] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A full-process nucleic acid detection chip based on EWOD, characterized in that: The full-process nucleic acid detection chip includes: a SiO2 support plate, a SiO2 glass, a dielectric layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer and an ITO glass layer arranged in sequence from bottom to top; A magnetic bead nucleic acid extraction zone and a dual-temperature zone nucleic acid amplification zone are formed between the first Teflon hydrophobic layer and the second Teflon hydrophobic layer; The magnetic bead nucleic acid extraction area includes: a lysis chamber, a first washing chamber, a second washing chamber and an elution chamber arranged in sequence; The dual-temperature zone nucleic acid amplification zone includes: a low-temperature constant temperature zone and a high-temperature constant temperature zone; The SiO2 glass is provided with interdigital electrodes and EWOD driving electrodes, the interdigital electrodes are aligned with the lysis chamber to realize the electric lysis of the bacterial sample, the EWOD driving electrodes are aligned with the elution chamber and the dual-temperature zone nucleic acid amplification zone, and the surface of the EWOD driving electrodes is made hydrophilic by controlling the power supply, and the droplets circulate and move in the low-temperature constant temperature zone and the high-temperature constant temperature zone to realize amplification; Among them, the interdigital electrodes and EWOD driving electrodes are printed on SiO2 glass by multi-layer screen printing. In the magnetic bead nucleic acid extraction area and the dual-temperature zone nucleic acid amplification area, an etching process is used to etch away the hydrophobic layer and the dielectric layer to expose the hydrophilic SiO2. The thickness of the dielectric layer is 2~3μm.

2. The full-process nucleic acid detection chip according to claim 1, characterized in that: The interdigital electrodes and the EWOD driving electrodes are printed on SiO2 glass by multi-layer screen printing, and the paste used is conductive silver paste, conductive gold paste, or conductive platinum paste.

3. The full-process nucleic acid detection chip according to claim 1, characterized in that: The thickness of the interdigital electrodes and the EWOD driving electrodes is 5-10 μm.

4. The full-process nucleic acid detection chip according to claim 1, characterized in that: The support layer has a thickness of 0.2-0.5 mm, providing storage space for droplets.

5. The full-process nucleic acid detection chip according to claim 1, characterized in that: The lysis chamber, the first washing chamber, the second washing chamber, the elution chamber, the low-temperature constant temperature zone and the high-temperature constant temperature zone are all filled with mineral oil to ensure that the magnetic beads can move freely between the chambers.

6. The full-process nucleic acid detection chip according to claim 1, characterized in that: In the multi-layer screen printing process, the screen printing plate uses a 300-mesh or 380-mesh screen.

7. The full-process nucleic acid detection chip according to claim 6, characterized in that: By changing the structure of the screen printing plate, the parallel design of multiple nucleic acid detection pathways can be achieved, thereby realizing the simultaneous detection of multiple nucleic acids.

8. An application of the full-process nucleic acid detection chip based on EWOD as claimed in any one of claims 1 to 7 in full-process nucleic acid detection, characterized in that: The following steps are involved: S1, adding the test sample into the lysis chamber, and applying a voltage of a certain frequency to the interdigital electrodes to electrolyze the bacteria; S2, after the electrolysis is completed, the magnetic beads are controlled by the magnetic rod to enter the lysis chamber, and the magnetic rod is moved to mix, and then the magnetic rod is controlled to move the magnetic beads to pass through the first washing chamber and the second washing chamber in sequence; S3, connect the EWOD driving electrode to the power supply, add the nucleic acid extract solution into the elution chamber, control the magnetic bar to move the magnetic beads to the elution chamber for elution, and then control the magnetic bar again to move the magnetic beads back to the second washing chamber, and then control the EWOD driving electrode to energize, move the reaction reagent in the elution chamber to the high temperature constant temperature zone, and perform pre-denaturation for 30 to 60 seconds. After completion, control the power on and off of the EWOD driving electrode to make the droplets circulate between the high temperature constant temperature zone and the low temperature constant temperature zone to achieve nucleic acid amplification; S4, nucleic acid detection is performed using fluorescent photography.

9. The use according to claim 8, characterized in that Connecting the interdigital electrodes to a DC voltage of 10-30V or an AC voltage of 10-30V can release nucleic acids in cells within 1 minute.

10. The use according to claim 8, characterized in that By controlling the power on and off of the EWOD driving electrode, the movement and residence of the droplets are controlled so that in each cycle the droplets stay in the high temperature constant temperature zone for 3 seconds and in the low temperature zone for 1 second. The droplets move between the high temperature constant temperature zone and the low temperature constant temperature zone for several cycles, and the entire amplification time does not exceed 7 minutes.

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