DNA synthesis chip

Abstract

The utility model relates to a DNA synthesis chip, which comprises a first substrate, a second substrate, a first electrode group and a second electrode group, wherein the first substrate is provided with a plurality of first grooves and a plurality of electrode groups which are distributed at intervals; the electrode group comprises a first electrode and a second electrode; each first groove is in contact with one first electrode and one second electrode; the second substrate is provided with a reagent injection channel; the first substrate and the second substrate are configured to be combined with each other; when the first substrate and the second substrate are combined, the reagent injection channel is communicated with the first grooves, and a runner communicated with each first groove is arranged between the first substrate and the second substrate; the first substrate is provided with a first surface facing the second substrate. According to the DNA synthesis chip disclosed by the embodiment of the invention, the difficulty of chip modification is reduced, the possibility of crosstalk is reduced, the synthesis amount is increased, and the chip can be repeatedly used, so that the synthesis cost is greatly reduced.

Description

Technical Field

[0001] The present utility model relates to the technical field of DNA synthesis, and more particularly to a DNA synthesis chip. Background Art

[0002] Synthetic biology is a new discipline based on multiple disciplines such as genetic engineering, systems biology, and computer engineering. Its application demands in the fields of medicine, energy, information, agriculture, etc. are increasing day by day. With broad investment prospects and great development potential, it has become a must-compete area in the scientific and technological strategic layouts of various countries. The essence of synthetic biology lies in design and creation. Whether it is "modifying existing natural biological systems" or "designing and building new biological components, devices, and systems", it is all based on DNA. As a key basic technology of synthetic biology, DNA synthesis is as important as the support of sequencing technology for genomics. The chemical synthesis method is the main method for directly synthesizing DNA. Based on the principle of solid-phase chemical synthesis, it consists of multiple reaction cycles. A single cycle mainly includes four steps: deprotection, coupling, capping, and oxidation. The protected end of the DNA strand on the synthesis carrier is cyclically deprotected, and then a new protected nucleotide monomer is linked, so that the DNA strand length increases by one.

[0003] Traditional oligonucleotide synthesis is generally called column synthesis. It is currently relatively mature and has a wide range of applications, but the synthesis throughput is limited. The electrochemical method is one of the currently relatively mature technical routes for high-throughput DNA synthesis. A commonly used electrochemical method is to integrate a high-density electrode array on a chip. Each electrode can be independently controlled. When an electrode is energized, it can produce acid, and under the action of the acid, the protecting group of the DNA monomer is removed, and then the DNA monomer is linked. The preparation process of the highly integrated chip used in this method is complex and cannot be reused, so the consumable cost is high, and the problem of crosstalk is likely to occur, reducing the synthesis accuracy. Summary of the Utility Model

[0004] The following is an overview of the subject matter described in detail in this article. This overview is not intended to limit the protection scope of this application.

[0005] The embodiments of the present application provide a DNA synthesis chip, which reduces the difficulty of chip modification, reduces the possibility of crosstalk, increases the synthesis amount, and the chip can be reused, greatly reducing the synthesis cost.

[0006] The embodiments of the present application provide a DNA synthesis chip, which comprises:

[0007] A first substrate, on which a plurality of first grooves and a plurality of electrode groups are distributed at intervals; each electrode group includes a first electrode and a second electrode; each first groove is in contact with a first electrode and a second electrode;

[0008] A second substrate having a reagent injection channel thereon;

[0009] The first substrate and the second substrate are configured to be capable of being combined with each other; when the first substrate and the second substrate are combined, the reagent injection channel communicates with the first groove, and there is a flow channel communicating with each of the first grooves between the first substrate and the second substrate; the first substrate has a first surface facing the second substrate.

[0010] In some embodiments of the present application, the first groove is located in the first substrate, the first groove is recessed towards the interior of the first substrate along a direction away from the first surface, and an opening of the first groove is located on the first surface.

[0011] In some embodiments of the present application, there is a retaining wall protruding in a direction towards the second substrate on the first surface; the first groove is located on the first surface and is enclosed by the retaining wall and the first surface.

[0012] In some embodiments of the present application, the electrode group satisfies any one or more of the following conditions:

[0013] 1) The first electrode is located on the inner wall of the first groove;

[0014] 2) The first electrode is located on the first surface and is in contact with the first groove;

[0015] 3) The second electrode is located on the inner wall of the first groove;

[0016] 4) The second electrode is located on the first surface and is in contact with the first groove.

[0017] In some embodiments of the present application, there are a plurality of second grooves spaced apart on the second substrate;

[0018] The second grooves are configured such that when the first substrate and the second substrate are combined, each of the second grooves communicates with one of the first grooves in a direction perpendicular to the first substrate.

[0019] In some embodiments of the present application, a positive projection of the first groove on the first substrate falls within a range of a positive projection of the second groove on the first substrate; or,

[0020] A positive projection of the second groove on the first substrate falls within a range of a positive projection of the first groove on the first substrate.

[0021] In some embodiments of the present application, the DNA synthesis chip further includes a first magnetic plate, or includes a first magnetic plate and a second magnetic plate;

[0022] The first magnetic plate is configured to be capable of binding to the side of the first substrate away from the first surface, and the orthographic projection of each of the first grooves on the first substrate falls within the orthographic projection of the first magnetic plate on the first substrate;

[0023] The second magnetic plate is configured to be capable of binding to the side of the second substrate away from the first surface, and the orthographic projection of each of the second grooves on the second substrate falls within the orthographic projection of the second magnetic plate on the second substrate.

[0024] In some embodiments of the present application, the distance between any two opposite sides of the opening of the first groove is 5 μm to 100 μm.

[0025] In some embodiments of the present application, the depth of the first groove is 2 μm to 50 μm.

[0026] In some embodiments of the present application, the height of the flow channel is 1 μm to 50 μm.

[0027] Other features and advantages of the present utility model will be described in the following specification, and, in part, will be obvious from the specification, or will be understood by implementing the present utility model. The objectives and other advantages of the present utility model can be achieved and obtained by the structures specifically pointed out in the specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings are used to provide a further understanding of the technical solutions of the present utility model, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present utility model, and do not constitute a limitation to the technical solutions of the present utility model.

[0029] Figure 1 is a schematic longitudinal sectional structure diagram of a DNA synthesis chip according to an exemplary embodiment of the present application;

[0030] Figure 2 is Figure 1 a schematic cross-sectional structure diagram of the DNA synthesis chip shown;

[0031] Figure 3 is a schematic longitudinal sectional structure diagram of another DNA synthesis chip according to an exemplary embodiment of the present application;

[0032] Figure 4 is a schematic longitudinal sectional structure diagram of yet another DNA synthesis chip according to an exemplary embodiment of the present application;

[0033] Figure 5 Schematic longitudinal sectional structure diagram of another DNA synthesis chip according to an exemplary embodiment of the present application;

[0034] Figure 6 Schematic partial longitudinal sectional structure diagram of a DNA synthesis chip with a TFT control unit adopting a 2T1C structure;

[0035] Figure 7 Pixel circuit diagram of a TFT control unit adopting a 2T1C structure;

[0036] Figure 8 Schematic reaction flow diagram of a DAN synthesis method according to an exemplary embodiment of the present application;

[0037] Figure 9 Schematic reaction flow diagram of an application method of the DNA synthesis method according to an exemplary embodiment of the present application in single-base mutation detection;

[0038] Figure 10 Schematic reaction flow diagram of an application method of the DNA synthesis method according to an exemplary embodiment of the present application in nucleic acid purification;

[0039] Figure 11 Schematic reaction flow diagram of an application method of the DNA synthesis method according to an exemplary embodiment of the present application in fluorescence detection.

[0040] The meanings of the reference symbols in the drawings are as follows:

[0041] 10 - First substrate; 11 - First groove; 111 - First surface; 12 - First electrode; 13 - Second electrode; 14 - Retaining wall; 20 - Second substrate; 21 - Reagent injection channel; 22 - Second groove; 30 - Flow channel; 40 - First magnetic plate; 50 - Second magnetic plate; 60 - Power supply; 70 - Liquid / gas path system; 81 - Gate insulating layer; 82 - Passivation layer; 100 - DNA carrier particles; 101 - Detection particles; 102 - Addressing sequence; 103 - Probe sequence; 104 - Target sequence; 105 - Purification particles; 106 - Target nucleic acid fragment; 107 - Fluorescent probe; T1 - First transistor; T2 - Second transistor; C - Capacitor; P1 - First source electrode; P2 - First drain electrode; P3 - Second source electrode; P4 - Second drain electrode; G1 - First gate electrode; G2 - Second gate electrode. Detailed implementation manners

[0042] To make the objectives, technical solutions, and advantages of the present application clearer and more understandable, the embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be noted that, without conflict, the embodiments and features in the embodiments of the present application can be combined arbitrarily with each other.

[0043] The embodiments in this application can be implemented in multiple different forms. It is easy for those of ordinary skill in the art to understand the fact that the implementation methods and content can be transformed into various forms without departing from the purpose and scope of this application. Therefore, this application should not be construed as being limited only to the content described in the following embodiments. Without conflict, the embodiments and features in the embodiments of this application can be combined arbitrarily with each other.

[0044] The drawing ratios in this application can be used as a reference in actual processes, but are not limited thereto. For example, the width-to-length ratio of the channel, the thickness and spacing of each film layer, and the width and spacing of each signal line can be adjusted according to actual needs. The number of pixels in the display substrate and the number of sub-pixels in each pixel are not limited to the numbers shown in the figures. The drawings described in this application are only schematic diagrams of the structure, and one embodiment of this application is not limited to the shapes or values shown in the drawings.

[0045] In this specification, for convenience, terms indicating orientation or positional relationships such as "middle", "upper", "lower", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. are used to describe the positional relationships of the components with reference to the drawings. This is only for the convenience of describing this specification and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation to this application. The positional relationships of the components are appropriately changed according to the directions describing each component. Therefore, it is not limited to the terms described in the specification and can be appropriately replaced according to the situation.

[0046] In this specification, unless otherwise clearly specified and limited, the terms "arranged" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected, indirectly connected through an intermediate member, or the internal communication of two components. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific situations.

[0047] In the description of this application, ordinal numbers such as "first" and "second" are set to avoid confusion of components, rather than to limit the quantity.

[0048] In traditional electrochemical synthesis, hydroxyl groups and linkers are modified on the surface of metal electrodes. Metal electrodes are usually stable inert metal materials. The modified groups are not easily combined and are prone to falling off, and the modified film layer is usually an insulating material (for example, glycosyl modification is used), resulting in unstable power-on caused by current blocking, thus increasing the possibility of crosstalk.

[0049] An embodiment of the present application provides a DNA synthesis chip. Figure 1 It is a schematic longitudinal sectional structure diagram of a DNA synthesis chip according to an exemplary embodiment of the present application; Figure 2 is Figure 1 A schematic cross-sectional structure diagram of the DNA synthesis chip shown.

[0050] As Figure 1 and Figure 2 As shown, the DNA synthesis chip includes: a first substrate 10 and a second substrate 20;

[0051] The first substrate 10 has a plurality of first grooves 11 and a plurality of electrode groups distributed at intervals; the first grooves 11 are configured to be able to accommodate DNA carrier particles 100 required for DNA synthesis; the electrode group includes a first electrode 12 and a second electrode 13; each first groove 11 is in contact with a first electrode 12 and a second electrode 13; the first electrode 12 and the second electrode 13 are configured to be connected to a power supply 60;

[0052] The second substrate 20 has a reagent injection channel 21;

[0053] The first substrate 10 and the second substrate 20 are configured to be able to be combined with each other; when the first substrate 10 and the second substrate 20 are combined, the reagent injection channel 21 communicates with the first grooves 11, and there is a flow channel 30 communicating with each first groove 11 between the first substrate 10 and the second substrate 20; the first substrate 10 has a first surface 111 facing the second substrate 20.

[0054] In the DNA synthesis chip of the embodiment of the present application, the electrodes do not need to carry modification groups, but the modification groups are modified on the DNA carrier particles. The modification technology of the particles is mature, and the diversity modification of the particle surface is easier to achieve, which can reduce the modification difficulty of the chip, and there will be no unstable power-on caused by current blockage, avoiding the occurrence of crosstalk problems.

[0055] Moreover, compared with planar modification, in the present application, the modification groups are modified on the DNA carrier particles, increasing the specific surface area of the reaction and improving the synthesis amount. In addition, when the modification groups are modified on the DNA carrier particles, the electrodes of the DNA synthesis chip do not need to carry modification groups, but the modification groups are modified on the DNA carrier particles. The modification technology of the particles can be reused, greatly reducing the synthesis cost. And the obtained DNA carrier particles carrying the target DNA fragment can be directly applied to single-base mutation detection, nucleic acid purification, fluorescence detection, etc. The DNA synthesis chip of the present application has a richer application scenario.

[0056] In some embodiments of the present application, the reagent injection channel is used to provide the reagents required for DNA electrochemical synthesis and an oxygen-free and water-free environment, and can be connected to the currently commonly used liquid / gas path system 70, which will not be elaborated here.

[0057] In some embodiments of the present application, the materials of the first substrate and the second substrate can independently be glass, silicon wafers, plastics, etc. Considering the price and the advantages of mass production, the materials of the first substrate and the second substrate can both be glass.

[0058] In some embodiments of the present application, the distance between any two opposite sides of the opening of the first groove can be 5 μm to 100 μm. For example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, or 100 μm.

[0059] For another example, the distance between any two opposite sides of the opening of the first groove can be 30 μm to 50 μm.

[0060] In some embodiments of the present application, the size of the opening of the first groove can be 5 μm to 100 μm.

[0061] In some embodiments of the present application, the depth of the first groove can be 2 μm to 50 μm. For example, it can be 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm.

[0062] If the slope angle of the first groove is small, the DNA carrier particles filled therein are likely to run out. To avoid crosstalk, when the second substrate is encapsulated, the distance between the first substrate and the second substrate can be smaller than the size of the DNA carrier particles.

[0063] In some embodiments of the present application, the particle size of the DNA carrier particles can be 50 nm to 30 μm. For example, it can be 10 μm to 30 μm, or 50 nm to 1 μm. The particle size of the DNA carrier particles should be smaller than the size of the first groove so that the first groove can accommodate the DNA carrier particles.

[0064] When the particle size of the DNA carrier particles is relatively large, for example, 10 μm to 30 μm, in order to avoid crosstalk, the height of the flow channel can be designed to be less than the particle size of the DNA carrier particles. In some embodiments of the present application, the height of the flow channel can be 1 μm to 50 μm. For example, it can be 1 μm, 2 μm, 4 μm, 6 μm, 8 μm, 10 μm, 12 μm, 14 μm, 165 μm, 18 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 55 μm.

[0065] Since the process of electrochemically synthesizing DNA requires multiple flushes and cleanings, the first groove with a smaller slope angle is more conducive to the exchange and cleaning of the solution. On the contrary, the first groove with a larger slope angle is not conducive to the exchange and cleaning of the solution.

[0066] In some embodiments of the present application, the slope angle of the first groove may not exceed 50°. For example, it can be 30°, 35°, 40°, 45° or 50°.

[0067] In some embodiments of the present application, as Figure 2 shown, a plurality of first grooves 11 are arranged in an array on the first substrate.

[0068] In some embodiments of the present application, as Figure 1 shown, the first groove 11 is located in the first substrate 10. The first groove 11 is recessed toward the inside of the first substrate 10 along a direction away from the first surface 111, and the opening of the first groove 11 is located on the first surface 111.

[0069] As Figure 1 shown, the first groove can be formed by the following method: First, an array pattern of a hard mask, for example, a circular array pattern, is formed on the first substrate by photolithography, and is etched using a reagent such as hydrofluoric acid to form a first groove recessed toward the inside of the first substrate; then the hard mask is washed away.

[0070] In some embodiments of the present application, the DNA carrier particles can be plastic particles, silica particles or magnetic beads.

[0071] In order to further increase the yield of DNA synthesis, the size of the DNA carrier particles can be reduced, such as 50 nm to 1 μm, and for another example, 500 nm. In this way, the specific surface area of the DNA carrier particles is further increased, but it is difficult to achieve nanoscale control of the distance between the first substrate and the second substrate (i.e., the height of the flow channel), thereby restricting the crosstalk of the particles. Therefore, magnetic beads can be selected as the DNA carrier particles, and the magnetic beads are bound in the first groove by a magnet, avoiding the crosstalk of the DNA carrier particles between multiple first grooves.

[0072] Figure 3The longitudinal sectional structure diagram of another DNA synthesis chip according to an exemplary embodiment of the present application. As Figure 3 shown, in some embodiments of the present application, the DNA synthesis chip further includes a first magnetic plate 40, and the first magnetic plate 40 is configured to be capable of binding to the side of the first substrate 10 away from the first surface 111, and the orthographic projection of each first groove 11 on the first substrate 10 falls within the range of the orthographic projection of the first magnetic plate 40 on the first substrate 10.

[0073] When using the DNA synthesis chip as Figure 3 shown for DNA synthesis, the modified magnetic bead solution can be added to the first grooves to make the magnetic beads evenly dispersed. Subsequently, the first substrate is placed on the first magnetic plate, and the magnetic beads are adsorbed on the first substrate under the action of magnetic force. The magnetic beads outside the first grooves will also fall into the first grooves under the action of diffusion force and magnetic force. In this process, it is not necessary to ensure that the number of magnetic beads in each first groove is the same, as long as there are magnetic beads in each first groove. To avoid crosstalk of magnetic beads, the first substrate is placed on the first magnetic plate during the reaction process to ensure that the magnetic beads all fall into the first grooves.

[0074] Figure 4 The longitudinal sectional structure diagram of yet another DNA synthesis chip according to an exemplary embodiment of the present application. As Figure 4 shown, in some embodiments of the present application, the second substrate 20 has a plurality of second grooves 22 distributed at intervals;

[0075] The second grooves 22 are configured such that when the first substrate 10 and the second substrate 20 are combined, each second groove 22 communicates with a first groove 11 in a direction perpendicular to the first substrate 10. For example, the positions of the second grooves 22 and the first grooves 11 can be vertically aligned one by one.

[0076] The particle size of the DNA carrier microparticles should be smaller than the size of the second grooves, so that the second grooves can accommodate the DNA carrier microparticles.

[0077] In some embodiments of the present application, as Figure 4 shown, the DNA synthesis chip further includes a first magnetic plate 40 and a second magnetic plate 50;

[0078] The first magnetic plate 40 is configured to be capable of binding to the side of the first substrate 10 away from the first surface 111, and the orthographic projection of each first groove 11 on the first substrate 10 falls within the range of the orthographic projection of the first magnetic plate 40 on the first substrate 10;

[0079] The second magnetic plate 50 is configured to be capable of being combined with the side of the second substrate 20 away from the first surface 111, and the orthographic projection of each second groove 22 on the second substrate 20 falls within the range of the orthographic projection of the second magnetic plate 50 on the second substrate 20.

[0080] To ensure the stability and correctness of the DNA synthesis reaction, the cleaning during the reaction process and the binding after liquid replacement need to be as sufficient as possible to avoid the influence of residual liquid on the next reaction. To ensure sufficient cleaning and binding, the first substrate and the second substrate can be combined with the first magnetic plate and the second magnetic plate respectively. First, the first magnetic plate under the first substrate is energized to adsorb the magnetic beads uniformly dispersed in the DNA synthesis chip into the first grooves. Subsequently, during cleaning and liquid replacement, the first magnetic plate and the second magnetic plate are alternately energized in sequence to move the magnetic beads up and down to ensure sufficient cleaning and binding, as Figure 4 shown. To avoid the crosstalk of magnetic beads when the second magnetic plate is energized, the second substrate can also be processed into a structure with a second groove array, and the positions of the first grooves and the second grooves are aligned to avoid the crosstalk of magnetic beads.

[0081] To further increase the flux of the electrochemical reaction, more and more methods now use TFT or CMOS as the control unit to control the opening and closing of each electrode. However, in this way, the first grooves cannot be directly processed inside the first substrate. Therefore, the control unit and the electrodes can be first processed on the first substrate, and the control unit can be located below the first electrode and the second electrode. Subsequently, a dam 14 can be formed on the first surface 111 where the first electrode and the second electrode are processed by using a curable stable material such as photoresist or PDMS, and a plurality of first grooves 11 are enclosed by the dam 14 and the first surface 111. Figure 5 It is a schematic longitudinal sectional structure diagram of another DNA synthesis chip according to an exemplary embodiment of the present application.

[0082] As Figure 5 shown, in some embodiments of the present application, there is a dam 14 protruding in the direction towards the second substrate 20 on the first surface 111; the first grooves 11 are located on the first surface 111 and are enclosed by the dam 14 and the first surface 111.

[0083] In some embodiments of the present application, the control unit can adopt the pixel circuit design commonly used in displays, such as the traditional 2T1C pixel circuit design.

[0084] Figure 6 It is a schematic partial longitudinal sectional structure diagram of a DNA synthesis chip with a TFT control unit adopting a 2T1C structure. As Figure 6As shown, a first groove 11 corresponds to a TFT control unit of a 2T1C structure, and the control unit is located below a first electrode 12 and a second electrode 13. A TFT control unit of a 2T1C structure includes a first transistor T1, a second transistor T2, and a capacitor C. The first transistor T1 includes a first source electrode P1, a first drain electrode P2, a first active layer C1 located between the first source electrode P1 and the first drain electrode P2, and a first gate electrode G1. There is a gate insulating layer between the first active layer C1 and the first gate electrode G1. The second transistor T2 includes a second source electrode P3, a second drain electrode P4, a second active layer C2 located between the second source electrode P3 and the second drain electrode P4, and a second gate electrode G2. There is a gate insulating layer 81 between the second active layer C2 and the second gate electrode G2. The pores in the TFT control unit of the 2T1C structure are filled with a passivation layer 82.

[0085] Figure 7 It is a pixel circuit diagram of a TFT control unit adopting a 2T1C structure. As Figure 7 shown, the first source electrode P1 of the first transistor T1 is connected to a bit line (Bit Line, BL) (for inputting a data signal V data ), the first drain electrode P2 of the first transistor T1 is connected to the capacitor C and the second gate electrode G2 of the second transistor T2, and the first gate electrode G1 of the first transistor T1 is connected to a word line (Word Line, WL) (for inputting a scan signal V scan ); the second source electrode P3 of the second transistor T2 is connected to the positive power supply (for example, V dd ), the second drain electrode P4 of the second transistor T2 is connected to the first electrode 12 (for example, the positive electrode) of an electrode group of the DNA synthesis chip, and the second electrode 13 (for example, the negative electrode) of the electrode group is grounded.

[0086] When performing a synthesis reaction using the DNA synthesis chip of the embodiment of the present application, by controlling the timing of the input data signal V data input through the bit line BL and the scan signal V scan input through the word line WL, the switching control of each positive electrode is realized, and thus high-throughput electrochemical reaction synthesis is realized.

[0087] In some embodiments of the present application, the first electrode is located on the inner wall of the first groove.

[0088] In some embodiments of the present application, the first electrode is located on the first surface and is in contact with the first groove.

[0089] In some embodiments of the present application, the first electrode is located on the inner wall of the first groove, and, the first electrode is located on the first surface and is in contact with the first groove.

[0090] In some embodiments of the present application, the second electrode is located on the inner wall of the first groove.

[0091] In some embodiments of the present application, the second electrode is located on the first surface and in contact with the first groove.

[0092] In some embodiments of the present application, the second electrode is located on the inner wall of the first groove, and the second electrode is located on the first surface and in contact with the first groove.

[0093] For example, as Figure 1 shown, the first electrode 12 is located on the inner wall of the first groove 11, and the second electrode 13 is located on the first surface 111 and in contact with the first groove 11.

[0094] For another example, as Figure 5 shown, the first electrode 12 is located on the first surface 111 and in contact with the first groove 11. Since a part of the first surface 111 is a part of the interior of the first groove 11, the first electrode 12 is also located on the inner wall of the first groove 11; the second electrode 13 is simultaneously located on the inner wall of the first groove 11 and on the first surface 111, and in contact with the first groove 11.

[0095] In some embodiments of the present application, the first electrode and the second electrode made of metal material can be formed by evaporation or sputtering processes, and are respectively used as the positive electrode and the negative electrode for DNA synthesis reaction.

[0096] In some embodiments of the present application, the orthographic projection of the first groove on the first substrate falls within the range of the orthographic projection of the second groove on the first substrate.

[0097] In some embodiments of the present application, the orthographic projection of the second groove on the first substrate falls within the range of the orthographic projection of the first groove on the first substrate.

[0098] The embodiments of the present application further provide a DNA synthesis method, which includes:

[0099] Modifying DNA carrier microparticles to obtain DNA carrier microparticles carrying a modifying group;

[0100] Injecting the DNA carrier microparticles carrying the modifying group and the reagents required for DNA synthesis into the first groove of the DNA synthesis chip as described above;

[0101] Connecting the first electrode and the second electrode of the DNA synthesis chip to a power supply, and performing DNA synthesis reaction in each of the first grooves.

[0102] The DNA synthesis method of the embodiments of the present application modifies the modifying group on the DNA carrier microparticles, so that there is no need to modify the modifying group on the metal electrode. The modification technology of microparticles is mature, and the diverse modification on the surface of microparticles is easier to achieve, which can reduce the modification difficulty of the chip, and there will be no unstable power-on caused by current blocking, avoiding the occurrence of crosstalk problems.

[0103] Moreover, compared with planar modification, the present application modifies the modifying group on the DNA carrier microparticles, increasing the specific surface area of the reaction and improving the synthesis amount. In addition, if the modifying group is modified on the DNA carrier microparticles, then the electrodes of the DNA synthesis chip do not need to carry the modifying group, but the modifying group is modified on the DNA carrier microparticles. The modification technology of microparticles can be reused, greatly reducing the synthesis cost. Moreover, the obtained DNA carrier microparticles carrying the target DNA fragment can be directly applied to single-base mutation detection, nucleic acid purification, fluorescence detection, etc. The DNA synthesis method of the present application has a richer application scenario.

[0104] In some embodiments of the present application, the particle size of the DNA carrier microparticles can be 50 nm to 30 μm. For example, the particle size of the DNA carrier microparticles is 10 μm to 30 μm, or 50 nm to 1 μm.

[0105] In some embodiments of the present application, the particle size of the DNA carrier microparticles is greater than the height of the flow channel.

[0106] In some embodiments of the present application, the material of the DNA carrier microparticles is not a metal material.

[0107] In some embodiments of the present application, the DNA carrier microparticles can be plastic microparticles, silica microparticles or magnetic beads.

[0108] In some embodiments of the present application, the DNA carrier microparticles can be magnetic beads;

[0109] The DNA synthesis method further includes:

[0110] When performing the DNA synthesis reaction, place the first substrate on the first magnetic plate and apply electricity to the first magnetic plate. The first magnetic plate adsorbs the magnetic beads in the first groove in the first groove.

[0111] In some embodiments of the present application, the DNA synthesis chip is the DNA synthesis chip as described above; the DNA carrier microparticles are magnetic beads;

[0112] The DNA synthesis method further includes:

[0113] When performing a DNA synthesis reaction, place the first substrate on the first magnetic plate and apply electricity to the first magnetic plate, which adsorbs the magnetic beads in the first groove within the first groove;

[0114] After completing the DNA synthesis reaction, perform washing and liquid replacement, and during the washing and liquid replacement process, alternately apply electricity to the first magnetic plate and the second magnetic plate, and the magnetic beads move between the first groove and the second groove.

[0115] In some embodiments of the present application, the modification of DNA carrier microparticles includes:

[0116] First, modify hydroxyl groups on the DNA carrier microparticles, then modify cleavable linkers, and subsequently modify hydroxyl protecting groups (e.g., DMT (dimethyl terephthalate), etc.).

[0117] In some embodiments of the present application, the modification of DNA carrier microparticles includes:

[0118] First, modify hydroxyl groups on the DNA carrier microparticles, then modify hydroxyl protecting groups (e.g., DMT (dimethyl terephthalate), etc.).

[0119] In practical applications, if the DNA fragment needs to be cut off from the DNA carrier microparticles for use after synthesizing the DNA fragment, a linker needs to be connected to the DNA carrier microparticles; if the DNA carrier microparticles connected with the DNA fragment can be directly used without cutting the DNA fragment off from the DNA carrier microparticles, then a linker does not need to be connected to the DNA carrier microparticles.

[0120] In some embodiments of the present application, the DNA synthesis reaction includes a deprotection reaction, a coupling reaction, a capping reaction, and an oxidation reaction. Among them, the deprotection reaction can be achieved by electro-generated acid.

[0121] In some embodiments of the present application, the DNA synthesis reaction consists of multiple reaction cycles, and a single cycle mainly includes a deprotection reaction, a coupling reaction, a capping reaction, and an oxidation reaction.

[0122] Figure 8 It is a schematic diagram of the reaction process of a DAN synthesis method according to an exemplary embodiment of the present application. As Figure 8 shown, in some embodiments of the present application, the DNA synthesis method includes:

[0123] First, hydroxyl groups are modified on the DNA carrier microparticles, and then cleavable linkers and hydroxyl protecting groups (such as DMT (dimethyl terephthalate), etc.) are modified; subsequently, the hydroxyl protecting groups are removed through a deprotection reaction (such as electrochemically induced acid deprotection) to expose the hydroxyl groups, and then nucleotides are linked through coupling reactions, capping reactions, and oxidation reactions.

[0124] In some embodiments of the present application, injecting the DNA carrier microparticles carrying the modification groups and the reagents required for DNA synthesis into the first grooves of the DNA synthesis chip as described above includes:

[0125] Injecting one or more of the DNA carrier microparticles into each of the first grooves;

[0126] Performing a DNA synthesis reaction in each of the first grooves includes: performing the same DNA synthesis reaction in the same first groove to obtain DNA with the same sequence in the same first groove.

[0127] In some embodiments of the present application, the DNA synthesis reaction includes:

[0128] According to the DNA sequence map to be synthesized, the corresponding electrodes are energized to perform deprotection reactions, coupling reactions, capping reactions, and oxidation reactions until the synthesis is completed; subsequently, the second substrate is removed, and the DNA carrier microparticles with the target DNA fragments rinsed out are collected by a test tube, enriched by centrifugation, and then the DNA carrier microparticles with the target DNA fragments are added to ammonia water to shear off the synthesized DNA fragments, and then the temperature is raised to evaporate the ammonia gas in the ammonia water, and the final solution obtained is the target DNA sequence group solution.

[0129] Compared with the traditional electrochemical synthesis method, the DNA synthesis method of the embodiments of the present application modifies the modification groups on the DNA carrier microparticles, reducing the possibility of electrochemically induced acid crosstalk, and also increasing the specific surface area of the DNA carrier microparticles, which is equivalent to increasing the reaction area of DNA synthesis, improving the yield, and increasing the concentration of the enriched DNA sequence group solution.

[0130] The embodiments of the present application also provide an application of the DNA synthesis method as described above in single-base mutation detection, and the application includes:

[0131] Synthesizing the detection microparticles required for the single-base mutation detection by using the DNA synthesis method as described above, and the detection microparticles are DNA carrier microparticles carrying an addressing sequence and a probe sequence;

[0132] Mixing the detection microparticles with the sample to be detected to perform single-base mutation detection.

[0133] In some embodiments of the present application, the mixing of the detection microparticles with the sample to be detected for single-base mutation detection includes:

[0134] Inject the sample to be detected into the first groove of the DNA synthesis chip, mix it with the detection microparticles synthesized in the first groove, and perform single-base mutation detection in the first groove.

[0135] Figure 9 It is a schematic diagram of the reaction process of an application method of the DNA synthesis method of the exemplary embodiment of the present application in single-base mutation detection.

[0136] Such as Figure 9 As shown, in some embodiments of the present application, the application of the DNA synthesis method as described above in single-base mutation detection includes:

[0137] Use the DNA synthesis method as described above to synthesize the detection microparticles 101 required for single-base mutation detection. The detection microparticles 101 are DNA vector microparticles 100 carrying an addressing sequence 102 and a probe sequence 103;

[0138] Inject the sample 110 to be detected containing the target sequence 104 into the first groove of the DNA synthesis chip, mix it with the detection microparticles 101 synthesized in the first groove, and perform in-situ single-base mutation detection in the first groove.

[0139] A currently common single-base mutation detection method needs to synthesize a variety of detection microparticles carrying addressing sequences and probe sequences, and then fill the detection microparticles into a micro-well array for detection. However, the single-base mutation detection method of the embodiments of the present application can not only directly synthesize a variety of detection microparticles carrying addressing sequences and probe sequences, but also directly perform in-situ detection after obtaining the detection microparticles, without the need to fill the detection microparticles into a micro-well array, simplifying the operation process of single-base mutation detection; moreover, in the in-situ synthesis and detection process, the DNA synthesis chip of the embodiments of the present application can be used both as a reaction device for DNA synthesis and as a detection device for single-base mutation detection, thus streamlining the instrument equipment required for detection and reducing the detection cost. In addition, the DNA synthesis chip of the embodiments of the present application can be reused, which can further reduce the cost of single-base mutation detection.

[0140] In some embodiments of the present application, the application of the DNA synthesis method as described above in single-base mutation detection includes:

[0141] Use the DNA synthesis method as described above to synthesize the detection microparticles required for single-base mutation detection in the first DNA synthesis chip. The detection microparticles are DNA vector microparticles carrying an addressing sequence and a probe sequence;

[0142] Take out the synthesized detection particles from the first DNA synthesis chip, inject the detection particles and the sample to be detected into the first groove of the second DNA synthesis chip, and perform single-base mutation detection in the first groove of the second DNA synthesis chip.

[0143] The embodiment of the present application also provides an application of the DNA synthesis method as described above in nucleic acid purification.

[0144] Figure 10 It is a schematic diagram of the reaction process of an application method of the DNA synthesis method in nucleic acid purification in an exemplary embodiment of the present application.

[0145] As Figure 10 shown, in some embodiments of the present application, the application of the DNA synthesis method as described above in nucleic acid purification includes:

[0146] Synthesize the purification particles 105 required for nucleic acid purification by using the DNA synthesis method as described above. The purification particles 105 are DNA carrier particles 100 carrying purification probe fragments (including addressing sequences 102 and probe sequences 103), and the purification probe fragments are complementary to the target nucleic acid fragment 106 to be purified.

[0147] Take out the purification particles from the DNA synthesis chip, mix them with the sample to be purified containing the target nucleic acid fragment. The target nucleic acid fragment in the sample to be purified is connected to the purification particles through base complementary pairing. Remove the remaining sample to be purified, and separate the target nucleic acid fragment from the purification particles to obtain the purified target nucleic acid fragment.

[0148] The embodiment of the present application also provides an application of the DNA synthesis method as described above in fluorescence detection. Figure 11 It is a schematic diagram of the reaction process of an application method of the DNA synthesis method in fluorescence detection in an exemplary embodiment of the present application.

[0149] As Figure 11 shown, the application of the DNA synthesis method as described above in fluorescence detection includes:

[0150] Synthesize the detection particles 101 required for fluorescence detection by using the DNA synthesis method as described above. The detection particles 101 are DNA carrier particles 100 carrying addressing sequences 102 and probe sequences 103, and the probe sequences are complementary to the bases at one end of the target nucleic acid fragment 106 to be detected.

[0151] Mix the detection particles 101 with the sample to be detected containing the target nucleic acid fragment 106 and the fluorescence probe 107 to obtain a mixed detection solution; the fluorescence probe 107 is complementary to the bases at the other end of the target nucleic acid fragment 106.

[0152] Drop the mixed detection solution onto the test strip. The detection particles and the fluorescent probe stop at the same position on the test strip, and the detection of the target nucleic acid fragment is achieved through fluorescence interpretation.

[0153] Although the embodiments disclosed in this application are as above, the content described is only the embodiments adopted for the convenience of understanding this application and is not used to limit this application. Any person skilled in the art within the scope of this application can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed in this application. However, the protection scope of this application shall still be subject to the scope defined by the appended claims.

Claims

1. A DNA synthesis chip, characterized in that: include: A first substrate having a plurality of first grooves and a plurality of electrode groups distributed at intervals; The electrode group includes a first electrode and a second electrode; Each of the first grooves is in contact with a first electrode and a second electrode; a second substrate having a reagent injection channel; The first substrate and the second substrate are configured to be able to be combined with each other; when the first substrate and the second substrate are combined, the reagent injection channel is connected to the first groove, and there is a flow channel connected to each of the first grooves between the first substrate and the second substrate; the first substrate has a first surface facing the second substrate.

2. The DNA synthesis chip according to claim 1, characterized in that: The first groove is located in the first substrate, the first groove is recessed toward the inside of the first substrate along a direction away from the first surface, and an opening of the first groove is located on the first surface.

3. The DNA synthesis chip according to claim 1, characterized in that: The first surface has a retaining wall protruding in a direction toward the second substrate; the first groove is located on the first surface and is surrounded by the retaining wall and the first surface.

4. The DNA synthesis chip according to claim 1, characterized in that: The electrode group satisfies any one or more of the following conditions: 1) The first electrode is located on the inner wall of the first groove; 2) The first electrode is located on the first surface and in contact with the first groove; 3) The second electrode is located on the inner wall of the first groove; 4) The second electrode is located on the first surface and contacts the first groove.

5. The DNA synthesis chip according to any one of claims 1 to 4, characterized in that: The second substrate has a plurality of second grooves distributed at intervals; The second grooves are configured such that, when the first substrate and the second substrate are combined, each of the second grooves is communicated with one of the first grooves in a direction perpendicular to the first substrate.

6. The DNA synthesis chip according to claim 5, characterized in that: The orthographic projection of the first groove on the first substrate falls within the range of the orthographic projection of the second groove on the first substrate; or, An orthographic projection of the second groove on the first substrate falls within a range of an orthographic projection of the first groove on the first substrate.

7. The DNA synthesis chip according to claim 5, characterized in that: Also includes a first magnetic plate, or includes a first magnetic plate and a second magnetic plate; The first magnetic plate is configured to be able to be combined with a side of the first substrate away from the first surface, and the orthographic projection of each of the first grooves on the first substrate falls within the range of the orthographic projection of the first magnetic plate on the first substrate; The second magnetic plate is configured to be capable of being combined with a side of the second substrate away from the first surface, and an orthographic projection of each of the second grooves on the second substrate falls within a range of an orthographic projection of the second magnetic plate on the second substrate.

8. The DNA synthesis chip according to any one of claims 1 to 4, characterized in that: A distance between any two opposite sides of the opening of the first groove is 5 μm to 100 μm.

9. The DNA synthesis chip according to any one of claims 1 to 4, characterized in that: The first groove has a depth of 2 μm to 50 μm.

10. The DNA synthesis chip according to any one of claims 1 to 4, characterized in that: The height of the flow channel is 1 μm to 50 μm.

Images