Digital microfluidic chip and automated DNA synthesis system

Abstract

The application provides a digital microfluidic chip and an automatic DNA synthesis system, the digital microfluidic chip is sequentially composed of an upper electrode plate, droplets and a lower electrode plate from top to bottom, wherein the upper electrode plate comprises a first substrate layer, a conductive layer and a hydrophobic layer, the lower electrode plate comprises a hydrophobic layer, a dielectric layer, a conductive layer and a second substrate layer, the automatic DNA synthesis system utilizes the high-throughput droplet parallel control capacity of the digital microfluidic chip, is based on the principle of subphosphoramidite DNA chemical synthesis method, is combined with specific chip electrode structure design, realizes automatic circulation distribution control of various synthesis reagents in the form of discrete micro-droplets on the microchip, so that miniaturization, integration, automation and intelligentization of high-throughput DNA synthesis equipment are realized, the reagent system of DNA chemical synthesis and enzyme method is compatible, high-throughput and low-cost DNA synthesis can be realized, and high-throughput parallel expansion capacity is possessed.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to a digital microfluidic chip and an automated DNA synthesis system. Background Technology

[0002] With the rapid development of DNA sequencing ("reading") and editing ("modifying") technologies, the demand for DNA synthesis ("writing") technologies is becoming increasingly urgent. Artificial DNA synthesis plays a crucial role in synthetic biology, including fields such as biomedicine, biosensing, molecular breeding, and information storage. High-throughput, low-cost DNA synthesis technology is a common key, and its core lies in high-throughput automated oligonucleotide synthesis equipment.

[0003] Currently, mature high-throughput DNA synthesis instruments mainly include three types: photochemical, electrochemical, and inkjet printing. All of them require a synthesis chip in conjunction with a bulky peripheral reagent dispensing system, resulting in a complex overall instrument structure and high cost. In addition, photochemical synthesizers require the use of special photosensitive monomer reagents; electrochemical synthesizers require continuous adjustment of the distance between the upper and lower electrodes of the chip and are affected by hydrogen ion crosstalk; while inkjet printing synthesizers suffer from problems such as printhead clogging and unstable jetting.

[0004] Digital microfluidic chips are miniaturized platforms capable of high-throughput distribution, movement, storage, mixing, reaction, and analysis of tiny droplets. This technology is primarily applied in biochemical analysis, enabling miniaturization, chip-based design, and portability of conventional biochemical laboratories. It reduces the consumption of expensive analytical samples and makes various analytical and detection functions more accurate, rapid, and efficient, demonstrating enormous development potential. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a digital microfluidic chip.

[0006] Another technical problem to be solved by the present invention is to provide an automated DNA synthesis system based on the above-mentioned digital microfluidic chip.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A digital microfluidic chip comprises, from top to bottom, an upper electrode, a droplet (6), and a lower electrode. The upper electrode includes a first substrate layer (1), a conductive layer (2), and a hydrophobic layer (3). The lower electrode includes a hydrophobic layer (3), a dielectric layer (4), a conductive layer (2), and a second substrate layer (5).

[0009] The conductive layer (2) includes a plurality of liquid storage electrodes (201), a plurality of driving electrodes (202), a plurality of reaction electrodes (203), and at least one waste liquid electrode (204). The plurality of liquid storage electrodes (201) are connected to the plurality of driving electrodes (202), the plurality of driving electrodes (202) are connected to the plurality of reaction electrodes (203), and the plurality of driving electrodes (202) are connected to at least one waste liquid electrode (204). The material is transported from the plurality of liquid storage electrodes (201) to the reaction electrodes (203) through the plurality of driving electrodes (202) to complete mixing and splitting. The waste liquid generated after the mixing and splitting of the material is discharged by the reaction electrodes (203). The liquid is delivered to at least one waste liquid electrode (204) through the driving electrode (202), and the cyclic reaction is achieved by alternating on and off of the electrodes; the upper electrode plate is provided with several strictly sealed liquid inlet interfaces (101) and liquid outlet interfaces (102). One end of the liquid inlet interface (101) is connected to the upper electrode plate, and the other end is connected to a silicone tube for providing materials to the digital microfluidic chip. The liquid inlet interface (101) is located above the storage electrode (201), and the liquid outlet interface (102) is located above the waste liquid electrode (204). There is a certain distance between the liquid outlet interface (102) and the waste liquid electrode (204).

[0010] The shapes of the electrodes described above are at least triangular, square, rectangular, pentagonal, hexagonal or other polygonal, and are arranged in a regular or irregular array.

[0011] Preferably, in the above-mentioned digital microfluidic chip, the liquid inlet interface (101) and the liquid storage electrode (201) correspond one-to-one, and each or two waste liquid electrodes (204) share one liquid outlet interface (102).

[0012] Preferably, the liquid inlet interface (101) and liquid outlet interface (102) of the above-mentioned digital microfluidic chip are made of polyetheretherketone (PEEK), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), or ethylene tetrafluoroethylene (ETFE).

[0013] Preferably, in the above-mentioned digital microfluidic chip, the inlet liquid path interface (101) and the outlet liquid path interface (102) are provided with silicone tubes and Luer connectors. One end of the Luer connector at the inlet liquid path interface (101) is connected to the first substrate layer (1) of the microfluidic chip, and the other end is connected to the silicone tube. The silicone tube provides materials to the digital microfluidic chip through the Luer connector. One end of the Luer connector at the outlet liquid path interface (102) is connected to the second substrate layer (5) of the microfluidic chip, and the other end is connected to the silicone tube. The silicone tube discharges waste liquid from the digital microfluidic chip through the Luer connector. During the recycling process, the waste liquid on the waste liquid electrode (204) can be connected to the outlet liquid path interface (102) through external equipment such as pumps, pipettes or syringes using the Luer connector, so that the waste liquid enters the outlet liquid path interface (102).

[0014] Preferably, in the above-mentioned digital microfluidic chip, the silicone tube is oil-sealed after the material conveying process is completed.

[0015] Preferably, in the above-mentioned digital microfluidic chip, the first base layer (1) of the upper electrode plate is made of glass, silicon wafer or flexible material, the conductive layer (2) is made of ITO or chromium gold conductive material, the thickness of the conductive layer is 50-1000nm, and the hydrophobic layer (3) is made of fluorinated cyclic polymers (Cytop) such as polydimethylsiloxane (PDMS) and Teflon material (polytetrafluoroethylene, Teflon).

[0016] Preferably, in the above-mentioned digital microfluidic chip, the hydrophobic layer (3) of the lower electrode plate is made of fluorinated cyclic polymers (Cytop) such as polydimethylsiloxane (PDMS) and Teflon (polytetrafluoroethylene), and the thickness of the hydrophobic layer is 0.01-20μm. The dielectric layer (4) is made of SU-8, polydimethylsiloxane (PDMS) or aluminum oxide, and the thickness of the dielectric layer is 0.2-15μm. The conductive layer (2) is made of ITO conductive material and the thickness of the conductive layer is 50-1000nm. The second substrate layer (5) is made of glass, silicon wafer or flexible material.

[0017] Preferably, in the above-mentioned digital microfluidic chip, the upper electrode plate is prepared by the following method:

[0018] 1) A conductive layer (2) is obtained on the substrate layer (1) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition;

[0019] 2) A hydrophobic layer (3) is prepared on the conductive layer (2) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition.

[0020] Preferably, in the above-mentioned digital microfluidic chip, the lower electrode plate is prepared by the following method:

[0021] 1) Electrodes are patterned on the second substrate layer (5) using photolithography, a metal thin film is grown, and the resist is removed to obtain a conductive layer (2);

[0022] 2) A dielectric layer (4) is prepared on the conductive layer (2) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition;

[0023] 3) A hydrophobic layer (3) is prepared on the dielectric layer (4) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition.

[0024] Preferably, in the above-mentioned digital microfluidic chip, the photolithography process employs ultraviolet light exposure or electron beam exposure.

[0025] Preferably, in the above-mentioned digital microfluidic chip, the upper and lower electrodes are arranged in parallel and sealed to provide a low-oxygen and low-water environment for DNA synthesis, and the distance between the upper and lower electrodes can provide space for liquid flow, and both the upper and lower electrodes are in contact with the liquid.

[0026] An automated DNA synthesis system includes the aforementioned digital microfluidic chip, as well as a microcontroller, a high-voltage control circuit, and a power drive module. The microcontroller is electrically connected to the high-voltage control circuit, and the high-voltage control circuit is electrically connected to both the power drive module and the digital microfluidic chip. The digital microfluidic chip is also electrically connected to the power drive module.

[0027] Preferably, in the above-mentioned automated DNA synthesis system, the control parameters of the digital microfluidic chip are obtained by creating the electrode power-up sequence for the user through a user interface program, and the control parameters are output to a high-voltage control circuit. The high-voltage control circuit is used to convert the control parameters into a level signal and send it to the digital microfluidic chip. The power drive module is used to provide a driving voltage to the digital microfluidic chip when the controlled terminal of the high-voltage control circuit is closed, so as to drive the droplets on the digital microfluidic chip to move according to the path specified by the user. The microcontroller stores and analyzes the signal and then transmits it to the user interface.

[0028] The specific steps of the DNA synthesis method using the above-mentioned automated DNA synthesis system are as follows:

[0029] (1) The reaction electrode (203) of the digital microfluidic chip is modified with biphenylamine cross-linked gold nanoparticles (AuNPs). The reaction electrode is modified by spin coating process, and then 6-mercapto-1-hexanol (MCH) solution is modified on the biphenylamine cross-linked gold nanoparticle layer by spin coating process.

[0030] (2) A self-assembled monolayer with terminal hydroxyl groups is formed on the modified reaction electrode. The active phosphoramide nucleotide monomer is introduced into the liquid inlet interface (101) and transported to the reaction electrode (203) by the storage electrode (201) of the digital microfluidic chip through the driving electrode (202) for DNA single-strand synthesis.

[0031] (3) Repeatedly and randomly introduce active phosphoramide nucleotide monomers until a complete DNA sequence is obtained;

[0032] (4) After completing one round of synthesis, the waste liquid generated is transported from the reaction electrode (203) to the waste liquid electrode (204) through the driving electrode.

[0033] Beneficial effects:

[0034] The aforementioned digital microfluidic chip utilizes the high-throughput droplet parallel manipulation capability of digital microfluidic chips. Based on the principle of phosphorous amide DNA chemical synthesis and combined with a specific chip electrode structure design, it realizes the automated cyclic distribution control of various synthetic reagents in the form of discrete microdroplets on the microchip. This enables the miniaturization, integration, automation, and intelligence of high-throughput DNA synthesis equipment, which is compatible with DNA chemical synthesis and enzymatic synthesis reagent systems. It can achieve high-throughput, low-cost DNA synthesis and has high-throughput parallel scalability. Attached Figure Description

[0035] Figure 1 This is a structural block diagram of the automated DNA synthesis system described in this invention.

[0036] Figure 2 This is a schematic diagram of the structure of the digital microfluidic chip described in this invention.

[0037] Figure 3 This is a schematic diagram of the conductive layer of the digital microfluidic chip described in this invention.

[0038] Figure 4 This is a schematic diagram of the fluid connection interface of the digital microfluidic chip described in this invention.

[0039] Figure 5 This is a schematic diagram of the electrode arrangement of the digital microfluidic chip described in this invention.

[0040] In the diagram: 20 - User interface program; 30 - Microcontroller; 40 - High voltage control circuit.

[0041] 50 - Digital microfluidic chip; 60 - Power drive module; 60 1 - First substrate layer

[0042] 2-Conductive layer; 3-Hydrophobic layer; 4-Dielectric layer; 5-Second substrate layer; 6-Droplet

[0043] 101 - Liquid inlet interface; 102 - Liquid outlet interface

[0044] 201-Storage electrode; 202-Driving electrode; 203-Reaction electrode; 204-Waste electrode

[0045] 701 - Basic Unit; 702 - Submodule; 703 - Parallel Synthesis Platform Detailed Implementation

[0046] The digital microfluidic chip and automated DNA synthesis system of the present invention will be described below with reference to the embodiments and accompanying drawings.

[0047] Example 1

[0048] like Figure 1 As shown, the automated DNA synthesis system includes a microcontroller 30, a high-voltage control circuit 40, a digital microfluidic chip 50, and a power drive module 60. The microcontroller 30 is electrically connected to the high-voltage control circuit 40, which is electrically connected to both the digital microfluidic chip 50 and the power drive module 60. The digital microfluidic chip 50 is also electrically connected to the power drive module 60.

[0049] The automated DNA synthesis system obtains the control parameters of the digital microfluidic chip by creating the electrode power-up sequence for the user through the user interface program 20, and outputs the control parameters to the high-voltage control circuit. Specifically, the user interface first receives the user's operation on the specified electrode, and then transmits the instruction to the microcontroller through serial communication (UART) between the microcontroller and the host computer. The microcontroller then performs bit operations on the corresponding ports according to the received data, and controls the output of different ports to output high and low levels, thereby controlling the relay. The high-voltage control circuit is used to convert the control parameters into level signals and send them to the digital microfluidic chip. The power drive module provides driving voltage to the digital microfluidic chip when the controlled terminal of the high-voltage control circuit is closed, so as to drive the droplets on the digital microfluidic chip to move according to the user-specified path. The microcontroller stores and analyzes the signal and then transmits it to the user interface.

[0050] like Figure 2-4 As shown, the aforementioned digital microfluidic chip consists of an upper electrode, a droplet 6, and a lower electrode from top to bottom. The upper electrode includes a first substrate layer 1, a conductive layer 2, and a hydrophobic layer 3. The lower electrode includes a hydrophobic layer 3, a dielectric layer 4, a conductive layer 2, and a second substrate layer 5.

[0051] The conductive layer 2 includes a plurality of liquid storage electrodes 201, a plurality of driving electrodes 202, a plurality of reaction electrodes 203, and at least one waste liquid electrode 204. Each electrode is square in shape. The liquid storage electrodes 201 are connected to the driving electrodes 202, the driving electrodes 202 are connected to the reaction electrodes 203, and the driving electrodes 202 are connected to at least one waste liquid electrode 204. Material is transported from the liquid storage electrodes 201 to the reaction electrodes 203 through the driving electrodes 202 to complete mixing and splitting. The waste liquid generated after mixing and splitting is discharged from the reaction electrodes 203. 3. The liquid is delivered to at least one waste liquid electrode 204 via the driving electrode 202, and a cyclic reaction is achieved by alternating on and off of the electrodes. The upper electrode plate is provided with several tightly sealed inlet liquid path interfaces 101 and outlet liquid path interfaces 102. One end of each inlet liquid path interface 101 is connected to the upper electrode plate, and the other end is connected to a silicone tube used to provide materials to the digital microfluidic chip. The inlet liquid path interface 101 is located above the storage electrode 201, and the outlet liquid path interface 102 is located above the waste liquid electrode 204. There is a certain distance between the outlet liquid path interface 102 and the waste liquid electrode 204.

[0052] The upper electrode plate has a first base layer 1 made of glass, a conductive layer 2 made of ITO conductive material with a thickness of 300nm, and a hydrophobic layer 3 made of Teflon material with a thickness of 300nm. The dimensions of the upper electrode plate are 25×30×1.1mm.

[0053] The second base layer 5 of the lower electrode plate is made of glass, the conductive layer 2 is made of chromium gold conductive material with a thickness of 300nm, the dielectric layer 4 is made of SU-8 material with a thickness of 10μm, and the hydrophobic layer 3 is made of Teflon material with a thickness of 300nm. The dimensions of the lower electrode plate are 25×30×1.1mm.

[0054] The fabrication process of the aforementioned digital microfluidic chip is as follows:

[0055] (1) Making the upper electrode plate

[0056] Cleaning: First, ultrasonically clean the 1.1mm thick glass in acetone for 15 minutes, then in ethanol for 15 minutes, and finally in deionized water for 15 minutes. The ultrasonic power was 40W and the ultrasonic temperature was 30℃. After cleaning, the glass was dried with a nitrogen gun and then dried in an oven at 125℃ for 30 minutes.

[0057] Electrode layer preparation: A 300 nm ITO thin film was grown on the dried glass surface using magnetron sputtering.

[0058] Activation: The glass with an ITO thin film grown on it was placed in an oxygen plasma cleaner for 2 minutes at a power of 300W.

[0059] Preparation of hydrophobic layer: The activated glass surface with an ITO film grown on it was spin-coated with a Teflon-AF 1600 layer. The Teflon solid was dissolved in fluorinated oil FC 40 to prepare the solution with a concentration of 0.5% to 2%. The spin-coating speed was set to 500 rpm for 10 seconds at low speed and 4000 rpm for 30 seconds at high speed. After spin-coating, the solution was placed on a hot plate at 150°C for 25 minutes to dry, thus completing the fabrication of the upper electrode plate.

[0060] (2) Fabrication of the lower electrode plate

[0061] Electrode Design: The electrode shape was designed using AutoCAD 2023 drawing software, and then the electrodes were fabricated using photolithography. The lower electrode plate has 8 liquid storage electrodes 201, 29 driving electrodes 202, 1 reaction electrode 203, 18 waste liquid electrodes 204, and 58 pin electrodes. The 8 liquid storage electrodes 201, 29 driving electrodes 202, 1 reaction electrode 203, and 18 waste liquid electrodes 204 are connected to the pin electrodes one by one.

[0062] Cleaning: The glass was first ultrasonically cleaned in acetone for 15 minutes, then ultrasonically cleaned in ethanol for 15 minutes, and finally ultrasonically cleaned in deionized water for 15 minutes. The ultrasonic power was 40W and the ultrasonic temperature was 30℃.

[0063] The cleaned glass substrate was dried with a nitrogen gun and placed in an oven at 125°C for 30 minutes.

[0064] The electrode layer is fabricated using standard photolithography processes:

[0065] Chromium / Gold plating: After the dried glass substrate is cooled to room temperature, 20 nm chromium and 100 nm gold are grown by magnetron sputtering.

[0066] Sputtering: Place the sputtered glass substrate on a spin coater, drop in an appropriate amount of AZ6130 photoresist, set the speed to low speed 600 rpm for 10 s, and high speed 3000 rpm for 30 s.

[0067] Pre-baking: Place the glass with photoresist spin-coated on it on a hot plate at 100°C and pre-bake for 1 minute.

[0068] Exposure: The pre-baked glass is then exposed to ultraviolet light using a mask at a dose of 100 mJ / cm². 2 .

[0069] Development: Develop the glass that has been exposed above for 1 minute. After development, rinse with deionized water and dry with a nitrogen gun.

[0070] Post-baking: Place the developed glass on a hot plate at 120°C and bake for 15 minutes.

[0071] Etching: The glass that has been post-baked is subjected to ion beam etching for 2 minutes according to the etching rate.

[0072] Photoresist removal: Immerse the etched glass in acetone to remove excess photoresist.

[0073] Fabrication of the dielectric layer:

[0074] Spin coat: Place the photolithographically completed glass on a spin coater, drop an appropriate amount of SU-8 2015 photoresist, set the speed to low speed of 500 rpm for 10 seconds, and high speed of 4000 rpm for 60 seconds.

[0075] Pre-baking: Place the glass after the above homogenization process on a hot plate at 65°C and bake for 10 minutes, then raise the temperature to 95°C and bake for 15 minutes.

[0076] Exposure: The glass after the pre-baking process is subjected to ultraviolet generalized exposure at a dose of 420 mJ / cm². 2 .

[0077] Post-baking: Place the glass that has been exposed above on a hot plate at 65°C and bake for 15 minutes, then raise the temperature to 95°C and bake for 20 minutes.

[0078] Preparation of the hydrophobic layer:

[0079] After the baked glass slide is cooled to room temperature, a layer of Teflon-AF 1600 is grown by spin coating. The Teflon solid is dissolved in fluorinated oil FC 40 to prepare the solution with a concentration of 0.5% to 2%. The spin coating speed is set to 500 rpm for 10 seconds at low speed and 4000 rpm for 30 seconds at high speed. After spin coating, the glass slide is placed on a hot plate at 150°C for 15 minutes to dry, thus completing the fabrication of the lower electrode plate.

[0080] (3) Fabrication of hydraulic connectors

[0081] like Figure 4 As shown, the upper plate of the digital microfluidic chip includes 8 liquid inlet interfaces 101 and 9 liquid outlet interfaces 102. The 8 liquid inlet interfaces 101 correspond one-to-one with the liquid storage electrode, and the 9 liquid outlet interfaces 102 are related to the waste liquid electrode in that two waste liquid electrodes share one liquid outlet interface.

[0082] The diameter of the liquid inlet interface is 0.5 mm, and the diameter of the liquid outlet interface is 0.75 mm.

[0083] Drill holes at the locations corresponding to the liquid storage electrode, with a diameter of 0.5 mm and a depth of 1.1 mm. Drill holes at the locations corresponding to the waste liquid electrode, with a diameter of 0.75 mm and a depth of 1.1 mm. Clean the drilled areas with anhydrous ethanol.

[0084] Apply some sealing silicone to the Luer connector of the appropriate size and install it in the corresponding position.

[0085] Connect the sealing silicone tube to the other end of the installed Luer connector.

[0086] Example 2

[0087] Using the aforementioned automated DNA synthesis system, oligonucleotide chains are synthesized on a digital microfluidic chip using a chemical method based on the principle of solid-phase synthesis, as detailed below:

[0088] Dissolve 1g of DMT-dT phosphorus amide monomer in 20-25ml of acetonitrile solution, mix thoroughly and shake in a centrifuge tube, and prepare DMT-dA, DMT-dC, and DMT-dG phosphorus amide monomer solutions in the same manner.

[0089] Power was applied to the liquid storage electrode on the digital microfluidic chip, and 15 μL of DMT-dT phosphorus amide monomer solution, 15 μL of DMT-dA phosphorus amide monomer solution, 15 μL of DMT-dC phosphorus amide monomer solution, 15 μL of DMT-dG phosphorus amide monomer solution, 50 μL of acetonitrile, 20 μL of trichloroacetic acid (TCA) deprotection reagent, 20 μL of capping reagent A / B, and 20 μL of iodine oxidant were introduced in sequence.

[0090] First, the deprotection reagent is delivered to the reaction electrode via a driving electrode to complete the deprotection reaction. Acetonitrile is then introduced for washing. Subsequently, the corresponding reservoir electrodes for DMT-dT phosphorus amide monomer solutions, DMT-dA phosphorus amide monomer solutions, DMT-dC phosphorus amide monomer solutions, and DMT-dG phosphorus amide monomer solutions are sequentially delivered to the reaction electrode via the driving electrode for coupling reactions. The phosphorus amide nucleotide monomer with the dimethoxytriphenylmethyl (DMT) protecting group reacts with the free hydroxyl group to form a phosphite bond. Capping reagent A is introduced to acetylate the unreacted 5' hydroxyl group. The residual acidic substances of capping reagent A are neutralized by capping reagent B, which provides a mild environment. Iodine solution is then introduced to further oxidize the DMT group into a more stable phosphate bond, removing the DMT group and exposing a reaction site for the next round of DNA synthesis. Acetonitrile is used for washing after each reaction, and the resulting waste liquid is driven to the waste liquid electrode by the driving electrode.

[0091] Example 3

[0092] The conventional method for amino modification of reaction electrodes can be divided into the following steps:

[0093] Step 1: Prepare a 0.5% to 2% NaOH solution, immerse the reaction electrode in it, stir magnetically at 300 rpm for 15 min, remove it and heat it on a hot plate at 130°C for 10 min.

[0094] Step 2: Soak in a piranha solution, which is prepared by mixing 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 (or 7:3), heating at 70-80℃ for 1 hour, then removing and rinsing with deionized water and drying with nitrogen.

[0095] Step 3: Aminoation modification of the electrode is carried out by reacting 3-aminopropyltriethoxysilane (APTS) with hydroxyl groups. A mixed solution of APTS and anhydrous ethanol at a volume ratio of 1:19 is prepared. The reaction electrode is immersed in the solution and magnetically stirred at 300 rpm for 1 hour. This is the process of the silane coupling agent reacting with the hydroxyl groups on the capillary surface. After removal, the electrode is cleaned with ethanol and deionized water respectively, and heated at 120°C for 30 minutes on a hot plate with a glass slide.

[0096] Step 4: Modify the electrode surface with a base monomer and immerse the reaction electrode in the prepared phosphoramide monomer solution (the first base monomer in the synthesis sequence) for 12 hours.

[0097] Example 4

[0098] Based on Example 3, gold nanoparticles were used for surface modification. Bisaniline cross-linked gold nanoparticles (AuNPs) were spin-coated onto the reaction electrode at room temperature. Then, 6-mercapto-1-hexanol (MCH) was modified onto the bisaniline cross-linked gold nanoparticle layer using a spin-coating process.

[0099] Example 5

[0100] like Figure 5 As shown, the structure designed above in this invention can be regarded as a basic unit 701. Two basic units can be combined as needed to construct a new sub-module 702. Furthermore, it can be expanded as needed in two directions (n ​​= 2, 4, 6...2k) to construct a higher throughput parallel synthesis platform 703, thereby realizing the infinite expansion of the digital microfluidic chip in two directions and thus realizing high throughput parallel synthesis.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A digital microfluidics chip, characterized by: The structure consists of an upper electrode plate, a droplet (6), and a lower electrode plate, arranged from top to bottom. The upper electrode plate includes a first substrate layer (1), a conductive layer (2), and a hydrophobic layer (3). The lower electrode plate includes a hydrophobic layer (3), a dielectric layer (4), a conductive layer (2), and a second substrate layer (5). The conductive layer (2) includes several liquid storage electrodes (201), several driving electrodes (202), several reaction electrodes (203), and at least one waste liquid electrode (204). The liquid storage electrodes (201) are connected to the driving electrodes (202), the driving electrodes (202) are connected to the reaction electrodes (203), and the driving electrodes (202) are connected to at least one waste liquid electrode (204). The upper electrode plate is provided with several tightly sealed liquid inlet interfaces (101) and liquid outlet interfaces (102). One end of the liquid inlet interface (101) is connected to the upper electrode plate. The other end is connected to a silicone tube for providing materials to a digital microfluidic chip. The liquid inlet interface (101) is located above the storage electrode (201), and the liquid outlet interface (102) is located above the waste electrode (204). There is a certain distance between the liquid outlet interface (102) and the waste electrode (204). The reaction electrode (203) is modified with bis-aniline cross-linked gold nanoparticles. The reaction electrode (203) is modified by spin coating. Then, 6-mercapto-1-hexanol solution is modified on the bis-aniline cross-linked gold nanoparticle layer by spin coating. A self-assembled monolayer with terminal hydroxyl groups is formed on the modified reaction electrode (203). The liquid inlet interface (101) is used to introduce active phosphoramide nucleotide monomers, which are transported to the reaction electrode (203) by the storage electrode (201) through the driving electrode (202) for DNA single-strand synthesis.

2. The digital microfluidics chip of claim 1, wherein: The liquid inlet interface (101) and the liquid storage electrode (201) are in one-to-one correspondence, and each or two waste liquid electrodes (204) share one liquid outlet interface (102).

3. The digital microfluidics chip of claim 1, wherein: The liquid inlet interface (101) and liquid outlet interface (102) are made of polyetheretherketone, polypropylene, polyethylene, polytetrafluoroethylene, fluorinated ethylene propylene, or ethylene tetrafluoroethylene.

4. The digital microfluidic chip according to any one of claims 1-3, characterized in that: The inlet liquid path interface (101) and outlet liquid path interface (102) are provided with silicone tubes and Luer connectors. One end of the Luer connector at the inlet liquid path interface (101) is connected to the first substrate layer (1) of the microfluidic chip, and the other end is connected to the silicone tube. The silicone tube provides materials to the digital microfluidic chip through the Luer connector. One end of the Luer connector at the outlet liquid path interface (102) is connected to the second substrate layer (5) of the microfluidic chip, and the other end is connected to the silicone tube. The silicone tube discharges waste liquid from the digital microfluidic chip through the Luer connector. During the recycling process, the waste liquid on the waste liquid electrode (204) is connected to the outlet liquid path interface (102) through a pump, pipette or syringe using the Luer connector, so that the waste liquid enters the outlet liquid path interface (102).

5. The digital microfluidic chip according to claim 1, characterized in that: The first base layer (1) of the upper electrode plate is made of glass, silicon wafer or flexible material, the conductive layer (2) is made of ITO or chromium gold conductive material, and the thickness of the conductive layer is 50-1000nm; the dielectric layer (4) of the lower electrode plate is made of SU-8, polydimethylsiloxane or aluminum oxide, and the thickness of the dielectric layer is 0.2-15μm, the conductive layer (2) is made of ITO conductive material, and the thickness of the conductive layer is 50-1000nm; the second base layer (5) is made of glass, silicon wafer or flexible material; the hydrophobic layer (3) of both the upper electrode plate and the lower electrode plate is made of fluorinated cyclic polymer, and the thickness of the hydrophobic layer is 0.01-20μm.

6. The digital microfluidic chip according to claim 1, characterized in that: The upper electrode plate is prepared by the following method: 1) A conductive layer (2) is obtained on the substrate layer (1) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition; 2) A hydrophobic layer (3) is prepared on the conductive layer (2) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition. The lower electrode plate is prepared by the following method: 1) The electrode is patterned on the second substrate layer (5) using photolithography, a metal thin film is grown, and the resist is removed to obtain the conductive layer (2). 2) A dielectric layer (4) is prepared on the conductive layer (2) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition. 3) A hydrophobic layer (3) is prepared on the dielectric layer (4) by spin coating, vapor deposition, magnetron sputtering or atomic layer deposition.

7. The digital microfluidic chip according to claim 1 or 6, characterized in that: The upper and lower electrodes are arranged in parallel and sealed to provide a low-oxygen and low-water environment for DNA synthesis. The distance between the upper and lower electrodes provides space for liquid flow, and both the upper and lower electrodes are in contact with the liquid.

8. An automated DNA synthesis system, comprising the digital microfluidic chip as described in any one of claims 1-7, a microcontroller, a high-voltage control circuit, and a power drive module, wherein the microcontroller is electrically connected to the high-voltage control circuit, the high-voltage control circuit is electrically connected to the power drive module and the digital microfluidic chip, and the digital microfluidic chip is electrically connected to the power drive module.

9. The automated DNA synthesis system according to claim 8, characterized in that: The user interface program creates an electrode power-up sequence for the user to obtain the control parameters of the digital microfluidic chip, and outputs the control parameters to the high-voltage control circuit. The high-voltage control circuit converts the control parameters into a level signal and sends it to the digital microfluidic chip. The power drive module provides a driving voltage to the digital microfluidic chip when the controlled terminal of the high-voltage control circuit is closed, so as to drive the droplets on the digital microfluidic chip to move along the path specified by the user. The microcontroller stores and analyzes the signal and then transmits it to the user interface.

10. A DNA synthesis method using the automated DNA synthesis system of claim 8 or 9, characterized in that: The specific steps are as follows: (1) The reaction electrode (203) of the digital microfluidic chip is modified with bisaniline cross-linked gold nanoparticles. The reaction electrode is modified by spin coating. Then, 6-mercapto-1-hexanol solution is modified on the bisaniline cross-linked gold nanoparticle layer by spin coating. (2) A self-assembled monolayer with terminal hydroxyl groups is formed on the modified reaction electrode. The active phosphoramide nucleotide monomer is introduced into the liquid inlet interface (101) and transported to the reaction electrode (203) by the storage electrode (201) of the digital microfluidic chip through the driving electrode (202) for DNA single-strand synthesis. (3) Repeatedly and randomly introduce active phosphoramide nucleotide monomers until a complete DNA sequence is obtained; (4) After one round of synthesis is completed, the waste liquid generated is transported from the reaction electrode (203) to the waste liquid electrode (204) through the driving electrode.

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