Electrowetting chip for high-throughput gene synthesis and sample adding card box thereof

Abstract

The invention belongs to the technical field of digital micro-fluidic chips, and provides an electrowetting chip for high-throughput gene synthesis and a sample adding card box thereof.The electrowetting chip comprises ITO glass, a sample adding device and the electrowetting chip for high-throughput gene synthesis, the bottom of the ITO glass is fixedly connected with a dielectric layer through a flow cavity, the sample adding device is arranged at the top of the ITO glass, and the sample adding device is arranged at the bottom of the ITO glass; the sample adding device is provided with an operation hole group, the ITO glass is provided with a plurality of sample outlet holes, and the operation hole group is communicated with the flowing chamber through the plurality of sample outlet holes. The electrowetting chip for high-throughput gene synthesis comprises a PCB (Printed Circuit Board) substrate, wherein a conductive component is arranged in the PCB substrate; the chip substrate is arranged at the top of the PCB substrate, a plurality of electrode layers are arranged at the top of the chip substrate, and the plurality of electrode layers are electrically connected with the conductive assembly; and the dielectric layer is laid above the plurality of electrode layers. The synthesis precision and the synthesis flux of gene synthesis can be improved, and meanwhile, the synthesis cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of digital microfluidic chips, and in particular relates to an electrowetting chip for high-throughput gene synthesis and a sample loading card box thereof. Background Art

[0002] Electrowetting-on-dielectric (EWOD) is a new type of microfluidic manipulation technology, which is widely used in digital microfluidics (DMF) systems. It has many advantages that traditional microfluidics do not have, such as no need for external pumps and valve drives, flexible droplet manipulation, and high degree of integration, especially in the biomedical field. The biochip based on EWOD has programmable path planning, which can accurately manipulate microdroplets to move, split, merge, etc. in the range of microliters to nanoliters, and realize the pipetting, mixing, and separation of various biological reagents in biochemical experiments, simplifying the operation process, improving efficiency, and reducing reagent consumption. At the same time, the digital microfluidics (DMF) system based on the EWOD chip has the advantages of small size and high integration, and is easy to integrate with the thermal cycle system and fluorescence detection module, which is very suitable for gene synthesis and detection.

[0003] In the field of gene synthesis, the mainstream gene synthesis method on the market is still solid-phase column synthesis. Compared with traditional gene synthesis methods, the synthesis method based on gene chips has the advantages of low reagent consumption, low synthesis cost, and high synthesis flux. With the continuous development of digital microfluidic chips, the application of dielectric wetting technology in the field of gene synthesis is becoming more and more extensive. However, the digital microfluidic chips currently used for gene synthesis still have problems such as low synthesis flux, high synthesis cost, and complex manufacturing process. Summary of the invention

[0004] The purpose of the present invention is to provide an electrowetting chip for high-throughput gene synthesis and a sample loading card box thereof to solve the above-mentioned problems and achieve the purpose of improving synthesis accuracy and synthesis flux while reducing synthesis cost.

[0005] To achieve the above object, the present invention provides the following scheme: an electrowetting chip for high-throughput gene synthesis, comprising:

[0006] A PCB substrate, wherein a conductive component is arranged in the PCB substrate;

[0007] A chip substrate, wherein the chip substrate is arranged on the top of the PCB substrate, and a plurality of electrode layers are arranged on the top of the chip substrate, and the plurality of electrode layers are electrically connected to the conductive components;

[0008] A dielectric layer is laid on top of the plurality of electrode layers.

[0009] Preferably, the conductive component includes a plurality of top metal contacts arranged on the top of the PCB substrate and a plurality of bottom metal contacts arranged on the bottom of the PCB substrate, a plurality of internal metal wires are arranged in the PCB substrate, a plurality of the top metal contacts are electrically connected to a plurality of the bottom metal contacts through a plurality of the internal metal wires, a plurality of the bottom metal contacts are electrically connected to an external drive and controller, and a plurality of the top metal contacts are electrically connected to a plurality of the electrode layers.

[0010] Preferably, a first hydrophobic layer is provided on a side of the dielectric layer away from the electrode layer.

[0011] Preferably, the plurality of electrode layers are arranged on the top of the chip substrate, and the plurality of electrode layers are divided into path electrodes, sample inlet region electrodes, reaction region electrodes, sample outlet region electrodes and waste liquid region electrodes.

[0012] An electrowetting chip sample loading card box for high-throughput gene synthesis comprises an ITO glass, a sample loading device and the electrowetting chip for high-throughput gene synthesis, wherein the bottom of the ITO glass is fixedly connected to a dielectric layer via a flow chamber, the sample loading device is arranged on the top of the ITO glass, an operation hole group is arranged on the sample loading device, a plurality of sample outlet holes are opened on the ITO glass, and the operation hole group is connected to the flow chamber via the plurality of sample outlet holes.

[0013] Preferably, the operation hole group includes a sampling hole, a sampling liquid storage tank, a filling medium loading tank and a waste liquid tank, the sampling hole is located above the sampling area electrode, the sampling liquid storage tank is located above the sampling area electrode, the waste liquid tank is located above the waste liquid area electrode, and the filling medium loading tank is used to fill the medium into the flow chamber.

[0014] Preferably, the bottom surface of the ITO glass is a conductive surface, the conductive surface is provided with a second hydrophobic layer, and the second hydrophobic layer is connected to the flow chamber.

[0015] Preferably, the conductive surface of the ITO glass is further modified with a plurality of gene sites, and the plurality of gene sites are located above the reaction area electrodes.

[0016] Preferably, the bottom edge of the ITO glass and the top edge of the chip substrate are connected by tape, the flow chamber is formed between the ITO glass, the chip substrate and the tape, and the first hydrophobic layer and the second hydrophobic layer are respectively located at the bottom and top of the flow chamber.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention can improve the flux of gene synthesis, enhance the synthesis efficiency, and reduce the synthesis cost by providing an electrowetting chip for high-throughput gene synthesis and a sample loading card box thereof. The raw material for gene synthesis is oligo pools, and gene synthesis of any number of fluxes can be performed simultaneously, such as 96-flux gene synthesis at the same time, which is an order of magnitude higher than the traditional synthesis method, and the synthesis price of a single base is much lower than the traditional synthesis method. The driving circuit of the digital microfluidic chip is designed on a PCB substrate and combined with the chip electrodes prepared by the MEMS process. Without reducing the number of driving electrodes, the accuracy and flatness of the microelectrode are guaranteed, and the processing cost and manufacturing difficulty are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is a schematic diagram of the structure of the chip of the present invention;

[0020] Figure 2 It is a schematic diagram of the structure of the chip and the sample adding device of the present invention;

[0021] Figure 3 It is an isometric view of the chip and the sample loading device of the present invention;

[0022] Figure 4 for Figure 3 A partial cross-sectional view of

[0023] Figure 5 The bottom view of the PCB substrate;

[0024] Figure 6 It is a schematic diagram of the internal structure of the PCB substrate;

[0025] Figure 7 It is a schematic diagram of the structure of the interconnection between the PCB substrate and the chip substrate;

[0026] Figure 8 It is a schematic diagram of the liquid storage tank structure of the sample adding device;

[0027] Fig. 9 It is a schematic diagram of the waste liquid tank structure of the sample adding device;

[0028] Fig.10 It is a schematic diagram of the sampling port structure of the sampling device;

[0029] Fig.11 Schematic diagram of different electrode areas of the digital microfluidic chip;

[0030] Fig.12 Schematic diagram of droplet actuation in digital microfluidic chip.

[0031] Among them, 1. PCB substrate; 2. chip substrate; 3. substrate metal wire layer; 4. electrode layer; 5. dielectric layer; 6. first hydrophobic layer; 7. PCB top metal contact; 8. tape; 9. chamber filling medium; 10. ITO glass; 11. gene site; 12. sample loading device; 13. second hydrophobic layer; 14. pipette; 1-a. thermal conductive copper sheet at the bottom of PCB substrate; 4-a. path electrode; 4-b. sample injection area electrode; 4-c. reaction area electrode; 4-d. sample output area electrode; 4-e. waste liquid area electrode; 7-a. PCB top metal contact; 7-b. PCB bottom metal contact; 7-c. PCB internal metal wire; 12-a. sampling hole; 12-b. sample injection reservoir; 12-c. filling medium loading tank; 12-d. waste liquid tank. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Embodiment 1:

[0035] Reference Figure 1 , Figure 6 and Figure 7 The present invention provides an electrowetting chip for high-throughput gene synthesis, comprising:

[0036] A PCB substrate 1, wherein a conductive component is arranged in the PCB substrate 1;

[0037] A chip substrate 2, the chip substrate 2 is arranged on the top of the PCB substrate 1, a plurality of electrode layers 4 are arranged on the top of the chip substrate 2, and the plurality of electrode layers 4 are electrically connected to the conductive components;

[0038] The dielectric layer 5 is laid on the electrode layers 4 .

[0039] The main function of the conductive component is to conduct the voltage to the electrode layers 4. The main function of the dielectric layer 5 is to insulate the electrode layers 4 from the droplets, acting as a capacitor in dielectric wetting, allowing charges to accumulate therein to produce a dielectric wetting effect, causing the contact angle of the droplets to change.

[0040] According to a further optimization scheme, the chip substrate 2 material includes a silicon substrate, a glass substrate, and a polymer substrate.

[0041] According to a further optimization scheme, the material of the dielectric layer 5 can be silicon nitride, aluminum oxide, gallium nitride, SU-8, PDMS, or parylene.

[0042] In a further optimized solution, the chip substrate 2 and the PCB substrate 1 are connected, and the connection method can be welding, ejector connection, adapter connection, etc.

[0043] A further optimized solution is provided, wherein the conductive components include a plurality of top metal contacts 7-a arranged on the top of the PCB substrate and a plurality of bottom metal contacts 7-b arranged on the bottom of the PCB substrate 1, a plurality of internal metal wires 7-c are arranged inside the PCB substrate 1, a plurality of top metal contacts 7-a are electrically connected to a plurality of bottom metal contacts 7-b through a plurality of internal metal wires 7-c, a plurality of bottom metal contacts 7-b are electrically connected to an external drive and controller, and a plurality of top metal contacts 7-a are electrically connected to a plurality of electrode layers 4, respectively.

[0044] According to a further optimization scheme, a plurality of electrode layers 3 are provided in the chip substrate 2 , and the plurality of electrode layers 3 are used to electrically connect a plurality of top metal contacts 7 - a and a plurality of electrode layers 4 .

[0045] According to a further optimization scheme, materials of the electrode layer 3 and the internal metal wire 7 - c include gold, copper, aluminum, titanium, platinum, etc.

[0046] In a further optimized solution, a first hydrophobic layer 6 is provided on a side of the dielectric layer 5 away from the electrode layer 4 .

[0047] The first hydrophobic layer 6 is prepared on the dielectric layer 5 to provide the high contact angle required for EWOD to drive the droplets, so that the droplets are hydrophobic, which is a necessary condition for contact angle modulation of the dielectric wetting effect.

[0048] According to a further optimization scheme, the material of the first hydrophobic layer 6 can be amorphous fluororesin such as Teflon AF, CYTOP, and FluoroPel.

[0049] According to a further optimization scheme, a PCB bottom thermal conductive copper sheet 1-a is provided at the bottom center of the PCB substrate 1. The main function of the PCB bottom thermal conductive copper sheet 1-a is to improve the thermal conductivity of the electrowetting chip for high-throughput gene synthesis of the present invention.

[0050] According to a further optimization scheme, a plurality of electrode layers 4 are arranged on the top of the chip substrate 2, and the plurality of electrode layers 4 are divided into path electrodes 4-a, sample injection area electrodes 4-b, reaction area electrodes 4-c, sample output area electrodes 4-d and waste liquid area electrodes 4-e.

[0051] Embodiment 2:

[0052] refer to Figure 2-Figure 5 and Figure 8-Figure 10 The present invention also provides an electrowetting chip sample loading card box for high-throughput gene synthesis, including an ITO glass 10, a sample loading device 12 and the above-mentioned electrowetting chip for high-throughput gene synthesis, the bottom of the ITO glass 10 is fixedly connected to the dielectric layer 5 through a flow chamber, the sample loading device 12 is arranged on the top of the ITO glass 10, an operation hole group is arranged on the sample loading device 12, a plurality of sample outlet holes are opened on the ITO glass 10, and the operation hole group is connected to the flow chamber through the plurality of sample outlet holes.

[0053] The main function of the flow chamber is to keep a certain distance between the ITO glass 10 and the chip substrate 2, and to store the chamber filling medium. The flow chamber is also filled with a gene splicing solution.

[0054] A further optimized solution is provided, in which the operation hole group includes a sampling hole 12-a, a sampling liquid storage tank 12-b, a filling medium loading tank 12-c and a waste liquid tank 12-d. The sampling hole 12-a is located above the sample outlet area electrode 4-d, the sampling liquid storage tank 12-b is located above the sample injection area electrode 4-b, the waste liquid tank 12-d is located above the waste liquid area electrode 4-e, and the filling medium loading tank 12-c is used to fill the medium into the flow chamber.

[0055] The loading device 12 can be provided with a plurality of injection liquid storage tanks 12-b according to the operation requirements, such as Figure 4As shown, three groups of sample injection storage tanks 12-b are provided to improve the use efficiency of the sample loading card box of the present invention, which helps to achieve the effect of being able to simultaneously carry out gene synthesis work of any flux number. The sample injection storage tank 12-b is located above the sample injection area electrode 4-b, and is used to preload samples, store reagents, oligo pools and cleaning solutions required for the reaction, and split and transport the reagents to the reaction area electrode 4-c through the sample injection area electrode 4-b. The waste liquid tank 12-d is located above the waste liquid area electrode 4-e, and is used to collect the reagents and cleaning solutions after the reaction. The sampling hole 12-a is located above the sample outlet electrode 4-d, aligned with the sample outlet hole on the ITO glass 10, and the product is extracted through the ITO glass 10 and the sampling hole 12-a of the sample loading device 12. The filling medium loading slot 12-c is located at the four corners of the sample loading device 12, and is used to inject the chamber filling medium 9 into the flow chamber, and to make the chamber filling medium 9 expel air, completely infiltrate the flow chamber, and avoid reaction failure and air pollution caused by reagent volatilization. Preferably, the chamber filling medium 9 may be dimethyl silicone oil, hexadecane, dodecane, dodecamethylpentasiloxane or the like.

[0056] According to a further optimized solution, the bottom surface of the ITO glass 10 is a conductive surface, a second hydrophobic layer 13 is provided on the conductive surface, and the second hydrophobic layer 13 is connected to the flow chamber.

[0057] According to a further optimized solution, the conductive surface of the ITO glass 10 is plated with carmine tin oxide, and the resistance value is 1 to 100 ohms.

[0058] According to a further optimization scheme, when the chip of the present invention is used, the conductive surface of the ITO glass is grounded at the same time, forming a circuit together with the electrode layer 4 and the droplets.

[0059] According to a further optimization scheme, the conductive surface of the ITO glass 10 is further modified with a plurality of gene sites 11, and the plurality of gene sites 11 are located above the reaction area electrode 4-c.

[0060] The main function of gene locus 11 is to capture the target gene in oligo pools.

[0061] In a further optimization scheme, the shapes of the electrode layers 4 on the chip substrate 2 are not fixed, and their sizes and arrangements can be adjusted according to needs. According to different functions, the electrodes can be divided into path electrodes 4-a, sample injection area electrodes 4-b, reaction area electrodes 4-c, sample output area electrodes 4-d and waste liquid area electrodes 4-e. The path electrode 4-a is the unit pixel electrode of the chip. The path electrode 4-a is a regular polygon and is arranged in a rectangular manner between other electrode areas to connect different electrode areas for transporting droplets to various functional areas. The sampling area electrode 4-b is located on one side of the electrode area. The number of the sampling area electrodes 4-b can be adjusted according to the number of reaction reagents. The reaction area electrode 4-c is located in the center of the chip and occupies the main electrode area. The reaction area electrode 4-c is located directly below the gene site 11 on the ITO glass 10. When the oligo pools move to the reaction area electrode 4-c, the gene site 11 can be completely covered to perform a capture reaction. The sample outlet area electrode 4-d is adjacent to the reaction area electrode 4-c. Above the sample outlet area electrode 4-d is the sampling hole 12-a of the ITO glass 10 and the sample adding device 12. The number of the sample outlet area electrodes 4-d is the same as that of the reaction area electrodes 4-c. When the biochemical reaction is completed, the product will move to the sample outlet area electrode 4-d, located below the sampling hole 12-a, and can be extracted from above by the pipette 14. Each sample outlet area corresponds to a sample outlet to avoid cross contamination between different products.

[0062] According to a further optimization scheme, the bottom edge of the ITO glass 10 is connected to the top edge of the chip substrate 2 by means of a tape 8, a flow chamber is formed between the ITO glass 10, the chip substrate 2 and the tape 8, and the first hydrophobic layer 6 and the second hydrophobic layer 13 are respectively located at the bottom and top of the flow chamber.

[0063] According to the size and spacing of the electrode layer 4, the thickness of the tape 8 for controlling the spacing between the ITO glass 10 and the chip substrate 2 can be less than 0.1 mm. The material of the tape 8 can be PET, PI, OCA, or UV glue.

[0064] In a further optimization scheme, the material of the sample adding device 12 can be PC, PMMA, PDMS. The sample adding device 12 is connected to the ITO glass 10 by bonding or bonding. The bonding material can be double-sided tape, photosensitive resin, chemical bonding, and the bonding method can be high-temperature pressing, laser bonding, ultrasonic bonding, chemical modification, or plasma bonding.

[0065] Embodiment three:

[0066] like Fig.11As shown, the driving of the electrowetting chip applies an electrical signal. When driving the droplet, a certain voltage is applied to the electrode layer 4. Under the action of electrowetting, the contact angle of the droplet above the electrode will decrease. At this time, the droplet is in a hydrophilic state and is located above the driving electrode; at this time, the electrode is closed and the adjacent electrode is opened, and the droplet returns to a hydrophobic state. At this time, the adjacent electrode is in an activated state, and the electrowetting effect generated makes its surface hydrophilic. At this time, the droplet will be captured by the hydrophilic area and move to the adjacent electrode area, thereby completing a step of the droplet movement.

[0067] Based on the above embodiment, a method for using an electrowetting chip loading card box for high-throughput gene synthesis is as follows: pre-load the synthetic raw material oligo pools, the reagents required for the reaction, and the cleaning buffer in the sample injection reservoir 12-b; add the filling chamber filling medium 9 to the flow chamber to completely exhaust the air; start the control program to control the oligo pools to split into droplets and transmit them to each reaction area electrode, give a certain incubation temperature, so that the target gene of the gene site 11 modified on the ITO glass 10 captures enough target genes; after the capture is completed, the oligo at the reaction area electrode 4-c is placed in the reaction area electrode 4-c. The pools solution is moved to the waste liquid area electrode 4-e; the cleaning buffer solution is controlled to be distributed to each reaction area electrode 4-c, and after cleaning the reaction area, it is moved to the waste liquid area electrode 4-e, and repeated 3 times; the reagents required for the reaction (enzymes, buffer solutions) are controlled to be distributed to each reaction area electrode 4-c, and a temperature control cycle is started (not shown in the figure) to allow the target gene captured by the modification point to fall off and be synthesized; after the synthesis is completed, the product at the reaction area electrode 4-c is moved to the sample area electrode 4-d, and the product is transferred and collected through the upper pipette 14.

[0068] To further optimize the solution, the temperature control cycle is a common setting, so its working principle and process will not be described in detail.

[0069] Embodiment 4:

[0070] As a further optimization solution, the manufacturing method of the PCB substrate 1 can be manufactured through standard PCB processing.

[0071] As a further optimization scheme, the MEMS process of the electrode layer 4 can be lift-off, in which the pattern on the mask is transferred to the photoresist of the chip substrate 2 through photolithography and development, and then the metal image is covered on the chip substrate 2 through a coating process, thereby obtaining the metal electrode pattern.

[0072] In a further optimization scheme, the MEMS process of the dielectric layer 5 can be to deposit the dielectric layer material onto the electrode layer 4 by vapor deposition or atomic layer film process, and its thickness can be 10-500nm depending on the material and process. When using photoresist or polymer material as the dielectric layer 5, it can be prepared by high-speed spin coating, and its thickness is usually controlled to be around 1um.

[0073] In a further optimization scheme, the material of the first hydrophobic layer 6 can be amorphous fluororesin, and its manufacturing process can be high-speed spin coating. During spin coating, the amorphous fluororesin is diluted to 0.5% to 2%, and the dilution solvent is an electronic fluoride liquid, such as FC-40 or FC-75. About 100ul of the diluted solution is dropped on the surface of the chip substrate 2 and evenly spread on the chip. The thickness of the hydrophobic layer 6 finally formed is in the range of 10 to 1000nm.

[0074] The preparation process of the second hydrophobic layer 13 is the same as that of the first hydrophobic layer 6 .

[0075] Finally, the chip substrate 2 with the electrode pattern and the dielectric layer 5 is connected to the PCB substrate 1, so that the electrowetting chip for high-throughput gene synthesis in Example 1 is obtained.

[0076] Embodiment five:

[0077] In the second embodiment, the preparation process of the gene array formed by a plurality of gene sites 11 can be: prepare a second hydrophobic layer 13 on the ITO glass 10, cover the second hydrophobic layer 13 with a mask with an array pattern, and plasma-treat the ITO glass 10 covered with the mask, remove the second hydrophobic layer 13 exposed at the array through-hole, and obtain micropores with an array pattern on the surface of the second hydrophobic layer 13. This array area has no hydrophobic layer and is a local hydrophilic point. The substrate of the local hydrophilic point is the conductive surface of the ITO glass 10. After the plasma treatment, the chemical groups on the surface of the ITO glass 10 are activated, and the intermediate groups can be covalently connected to the substrate through chemical bonds. Finally, the target gene of the target gene is connected to the intermediate group by incubation, and the gene array on the ITO glass 10 is obtained. According to the different target genes incubated and connected to the modified points, different gene fragments can be captured and synthesized under the condition of using the same oligo pool.

[0078] For further optimization, the mask material can be glass, silicon, PMMA, PC, PI, PEEK, and the intermediate group can be streptavidin, biotin, azide, or silane coupling agent.

[0079] In the description of the present invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0080] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An electrowetting chip for high-throughput gene synthesis, characterized in that: include: A PCB substrate (1), wherein a conductive component is arranged inside the PCB substrate (1); A chip substrate (2), the chip substrate (2) being arranged on the top of the PCB substrate (1), a plurality of electrode layers (4) being arranged on the top of the chip substrate (2), and the plurality of electrode layers (4) being electrically connected to the conductive components; A dielectric layer (5), wherein the dielectric layer (5) is laid on top of the plurality of electrode layers (4).

2. The electrowetting chip for high-throughput gene synthesis according to claim 1, characterized in that: The conductive component comprises a plurality of top metal contacts (7-a) arranged on the top of the PCB substrate and a plurality of bottom metal contacts (7-b) arranged on the bottom of the PCB substrate (1); a plurality of internal metal wires (7-c) are arranged in the PCB substrate (1); the plurality of top metal contacts (7-a) are electrically connected to the plurality of bottom metal contacts (7-b) through the plurality of internal metal wires (7-c), the plurality of bottom metal contacts (7-b) are electrically connected to an external drive and controller, and the plurality of top metal contacts (7-a) are electrically connected to the plurality of electrode layers (4).

3. The electrowetting chip for high-throughput gene synthesis according to claim 2, characterized in that: A first hydrophobic layer (6) is provided on a side of the dielectric layer (5) away from the electrode layer (4).

4. The electrowetting chip for high-throughput gene synthesis according to claim 3, characterized in that: The plurality of electrode layers (4) are arranged on the top of the chip substrate (2), and the plurality of electrode layers (4) are divided into path electrodes (4-a), sample injection area electrodes (4-b), reaction area electrodes (4-c), sample output area electrodes (4-d) and waste liquid area electrodes (4-e).

5. An electrowetting chip loading card box for high-throughput gene synthesis, characterized in that: The invention comprises an ITO glass (10), a sample loading device (12) and an electrowetting chip for high-throughput gene synthesis as described in claims 1 to 4, wherein the bottom of the ITO glass (10) is fixedly connected to a dielectric layer (5) via a flow chamber, the sample loading device (12) is arranged on the top of the ITO glass (10), an operation hole group is arranged on the sample loading device (12), a plurality of sample outlet holes are opened on the ITO glass (10), and the operation hole group is connected to the flow chamber via the plurality of sample outlet holes.

6. The electrowetting chip loading card box for high-throughput gene synthesis according to claim 5, characterized in that: The operation hole group includes a sampling hole (12-a), a sample injection liquid storage tank (12-b), a filling medium loading tank (12-c) and a waste liquid tank (12-d), wherein the sampling hole (12-a) is located above the sample output area electrode (4-d), the sample injection liquid storage tank (12-b) is located above the sample injection area electrode (4-b), the waste liquid tank (12-d) is located above the waste liquid area electrode (4-e), and the filling medium loading tank (12-c) is used to fill the medium into the flow chamber.

7. The electrowetting chip loading card box for high-throughput gene synthesis according to claim 6, characterized in that: The bottom surface of the ITO glass (10) is a conductive surface, the conductive surface is provided with a second hydrophobic layer (13), and the second hydrophobic layer (13) is connected to the flow chamber.

8. The electrowetting chip loading card box for high-throughput gene synthesis according to claim 7, characterized in that: The conductive surface of the ITO glass (10) is also modified with a plurality of gene sites (11), and the plurality of gene sites (11) are located above the reaction area electrode (4-c).

9. The electrowetting chip loading card box for high-throughput gene synthesis according to claim 7, characterized in that: The bottom edge of the ITO glass (10) and the top edge of the chip substrate (2) are connected via an adhesive tape (8); the flow chamber is formed between the ITO glass (10), the chip substrate (2) and the adhesive tape (8); the first hydrophobic layer (6) and the second hydrophobic layer (13) are respectively located at the bottom and top of the flow chamber.

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