System for synthesizing oligonucleotides and application and method thereof

Abstract

The invention relates to a system for synthesizing oligonucleotides as well as application and a method thereof. The method comprises the following steps: importing a fitted sequence into a control module; providing a chip comprising a microelectrode array, wherein the microelectrode array comprises a plurality of electrodes and dielectric structures between the electrodes; carrying out surface modification on the dielectric structure; and carrying out electrochemical synthesis on the oligonucleotide by taking the surface-modified dielectric structure as a synthesis region. According to the invention, the surface-modified dielectric structure is used as a synthesis region, so that the difficulty of surface modification can be reduced, and the influence of the electrochemical characteristic change of the synthesis electrode on the synthesis effect in the oligonucleotide synthesis process can be avoided, thereby improving the synthesis load and the synthesis effect.

Description

Technical Field

[0001] The present invention relates to biosynthesis technology, and particularly to a system for synthesizing oligonucleotides, and its applications and methods. Background Art

[0002] Microelectrode arrays have extensive applications in fields such as biomedical research, neuroscience, and medical diagnosis, especially for high-throughput electrochemical synthesis of oligonucleotides. A microelectrode array typically includes a working electrode and a counter electrode. Usually, the working electrode serves as the synthesis region, that is, synthesis sites are generated on the working electrode through chemical treatment; protons generated by the electrochemical reaction deprotect the sites on the working electrode. The limitation of this method is that since the synthesis region is directly on the working electrode, as the synthesis process progresses, the electrochemical characteristics of the working electrode will change accordingly, which will affect the synthesis efficiency and accuracy of oligonucleotides, and this effect is particularly significant for the synthesis of long sequences. Summary of the Invention

[0003] The object of the present invention is to provide a method and system for synthesizing oligonucleotides, which use a dielectric structure disposed within a microelectrode array after surface modification as the synthesis region, thereby significantly reducing the process difficulty and improving the effectiveness and reliability of the oligonucleotide synthesis process.

[0004] According to one aspect, the present invention provides a system for synthesizing oligonucleotides, including: A microelectrode array, including one or more pixel units, and each of the pixel units includes a plurality of electrodes and a dielectric structure located between the electrodes; and A control circuit; Wherein, the dielectric structure has a surface modification layer containing a linker molecule with a protecting group, so that the dielectric structure can be used as the synthesis region for electrochemically synthesizing oligonucleotides.

[0005] According to one embodiment, the surface modification layer includes an organosilane layer containing epoxy groups covalently bonded to the surface of the dielectric structure, a surface modification structure bonded to the organosilane layer, and a linker molecule with a protecting group grafted onto the surface modification structure.

[0006] According to one embodiment, in each of the pixel units, the microelectrode array includes a working electrode and a counter electrode surrounding the working electrode; and the working electrode includes one or more array electrode structures and an annular electrode structure surrounding the one or more array electrode structures.

[0007] According to one embodiment, the synthesis region is constituted by the dielectric structure located between the plurality of array electrode structures, between the plurality of array electrode structures and the annular electrode structure, and between the annular electrode structure and the counter electrode.

[0008] According to one embodiment, the layer structure where the microelectrode array is located includes a first conductive layer, a dielectric layer formed on the first conductive layer, and a second conductive layer formed on the dielectric layer; Wherein, the dielectric layer is patterned to form a dielectric structure with a first through-hole structure, so that a part of the first conductive layer exposed by the first through-hole structure is used as a working electrode; The second conductive layer is patterned to have a second through-hole structure that exposes the dielectric structure and the working electrode, and a counter electrode; and The exposed dielectric structure is surface-modified to have the surface-modified layer.

[0009] According to one embodiment, the layer structure where the microelectrode array is located includes a first conductive layer, and a dielectric layer formed on the first conductive layer; Wherein, the first conductive layer includes a working electrode, a counter electrode surrounding the working electrode, and a first sub-dielectric structure located between the working electrode and the counter electrode; The dielectric layer located in the area of the working electrode is patterned to form a second sub-dielectric structure with a through-hole structure, so that a part of the first conductive layer exposed through the through-hole structure is used as a working electrode; and The first sub-dielectric structure and the second sub-dielectric structure are surface-modified to have the surface-modified layer.

[0010] According to another aspect of the present invention, there is provided a system for synthesizing oligonucleotides, including a microelectrode array and a control circuit, wherein, The layer structure where the microelectrode array is located includes a dielectric layer having a plurality of first through-hole structures arranged at intervals, and an electrode array structure; The surface of the dielectric layer has a surface-modified layer containing a linker molecule with a protecting group, so that the dielectric layer serves as a synthesis region for synthesizing oligonucleotides.

[0011] According to one embodiment, each electrode in the electrode array structure includes a first part filling the corresponding first through-hole in the dielectric layer, a second part formed on the upper surface of the dielectric layer and communicating with the first part, and a third part formed on the lower surface of the dielectric layer and communicating with the first part.

[0012] According to one embodiment, the control circuit includes a driving board, and the driving board is electrically connected to the electrode array structure through a welding structure or a conductive adhesive.

[0013] According to one embodiment, the system further includes a carrier board located between the welding structure or the conductive adhesive and the driving board; Among them, the carrier board has a plurality of second through-hole structures arranged at intervals, and a conductive structure filling the second through-hole structures, which is used to electrically connect the welding structure or conductive adhesive to the driving board.

[0014] According to another aspect of the present invention, there is provided an application of the above system in medium-throughput electrochemical synthesis of oligonucleotides.

[0015] According to another aspect of the present invention, a method for synthesizing oligonucleotides is proposed, including the steps of: Importing the sequence to be synthesized into the control module; Providing a chip including a microelectrode array, and the microelectrode array includes a plurality of electrodes and a dielectric structure located between the electrodes; Performing surface modification on the dielectric structure so that the surface of the dielectric structure has a surface modification layer; Using the surface-modified dielectric structure as a synthesis region to electrochemically synthesize oligonucleotides.

[0016] According to one embodiment, the dielectric structure is formed of a dielectric material selected from silicon oxides and silicon nitrides.

[0017] According to one embodiment, the surface modification of the dielectric structure includes: Forming an organosilane layer containing epoxy groups on the surface of the dielectric structure; Reacting an imine polymer with the organosilane layer to form an amino group to form a surface modification structure; Grafting a linking molecule containing a protecting group on the surface modification structure.

[0018] According to one embodiment, the organosilane layer containing epoxy groups is formed of epoxypropoxy trialkoxysilane. Among them, the organosilane layer containing epoxy groups is formed of an epoxy silane selected from epoxypropoxy trimethoxysilane and epoxypropoxy triethoxysilane. And, the organosilane layer containing epoxy groups is bonded to the surface of the dielectric structure through a covalent bond to form a monolayer.

[0019] According to one embodiment, the imine polymer is selected from polyalkyleneimine and polyamidoamine polymer. Preferably, the polyalkyleneimine is selected from polymethyleneimine, polyethyleneimine, and polypropyleneimine. The polyamidoamine polymer is a dendritic polyamidoamine polymer having an alkylene diamine core, wherein the alkylene diamine is selected from methanediamine, ethanediamine, propanediamine, and butanediamine.

[0020] According to one embodiment, the linking molecule containing a protecting group is a linking molecule containing 4,4'-dimethoxytriphenylmethyl.

[0021] According to one embodiment, the microelectrode array includes a working electrode and a counter electrode surrounding the working electrode; and the working electrode includes one or more array electrode structures and an annular electrode structure surrounding the one or more array electrode structures. Preferably, the ratio of the surface area of the annular electrode structure to the total surface area of the one or more array electrode structures is 0.1 to 10.

[0022] According to one embodiment, in the one or more array electrode structures, each array electrode structure has a microporous structure at the top.

[0023] According to one embodiment, the synthesis region includes a dielectric structure located between the plurality of array electrode structures, between the plurality of array electrode structures and the annular electrode structure, and between the annular electrode structure and the counter electrode.

[0024] According to one embodiment, electrochemically synthesizing oligonucleotides in the synthesis region with the surface-modified dielectric structure includes: Introducing an electrochemical deprotection reagent into the synthesis region; Under the action of an input voltage or current, deprotection is performed on the surface of the dielectric structure in the synthesis region; Introducing raw materials for synthesizing oligonucleotides into the synthesis region, and performing coupling, oxidation, and capping.

[0025] According to one embodiment, the raw materials include a coupling reagent, an oxidation reagent, and a capping reagent.

[0026] According to one embodiment, the electrochemical deprotection reagent includes p-phenol, p-benzoquinone, and an organic base; The coupling reagent includes an acetonitrile solution of a base monomer and tetrazole; The oxidation reagent includes iodine, water, and pyridine; and / or The capping reagent includes an acetonitrile solution of N-methylimidazole and acetic anhydride.

[0027] The system according to the present invention includes a surface-modified dielectric structure as a synthesis region, thereby reducing the difficulty of surface modification compared with electrodes, and reducing the influence of electrode characteristic changes on the oligonucleotide synthesis process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following will describe specific embodiments with reference to the drawings. The scope of the present invention is not limited thereto, where: Figure 1 Shows a schematic structural diagram of a pixel unit in a microelectrode array according to one embodiment of the present invention; Figure 2 Shows a schematic structural diagram of a surface modification layer of a dielectric structure according to one embodiment of the present invention; Figure 3 Shows a schematic structural diagram of the surface modification layer of the dielectric structure according to another embodiment of the present invention; Figure 4 Shows a longitudinal sectional schematic diagram of the layer structure where the electrode structure is located according to an embodiment; Figure 5 Shows the formation of Figure 4 The process flow chart of the shown electrode structure; Figure 6 Shows a longitudinal sectional schematic diagram of the layer structure where the electrode structure is located according to another embodiment; Figure 7 Shows the formation of Figure 6 The process flow chart of the shown electrode structure; Figure 8 Shows the physical diagram of the chip according to the present invention; Figure 9 Shows Figure 8 The enlarged structural diagram of area A of the shown chip; Figure 10 Shows a longitudinal sectional schematic diagram of the system according to yet another embodiment; Figure 11 Shows the complete flow chart of the method for electrochemically synthesizing oligonucleotides according to the present invention; Figure 12 Shows the flow chart of the synthesis steps of the method for electrochemically synthesizing oligonucleotides according to the present invention; Figure 13 Shows the schematic diagram of the pixel structure of the model for evaluating the influence of the electrode structure on the proton distribution according to the present invention; Figure 14 Shows the proton concentration distribution curve along the Figure 13 dotted line in the case of the surface area ratio of different ring electrode structures and array electrode structures; Figure 15 And Figure 16 Shows the influence of the electrode structure without a ring working electrode on the deprotection performance; Figure 17 And Figure 18 Shows the influence of the electrode structure with a ring working electrode on the deprotection performance.

[0029] Explanation of reference numerals: 1 - counter electrode; 2 - working electrode; 3 - first sub - dielectric structure; 4 - second sub - dielectric structure; 5 - dielectric layer; 7 - carrier plate; 8 - driving plate; 21 - array electrode structure; 22 - ring electrode structure; 51 - the second part of the electrode; 52 - the first part of the electrode; 54 - the third part of the electrode; 61 - liquid inlet; 62 - liquid outlet; 63 - flow cell; 64 - welding structure; 65 - conductive structure. Detailed implementation manners

[0030] In the prior art, a system for electrochemically synthesizing oligonucleotides generally includes a working electrode with a surface modification as a synthesis region. Therefore, the performance of the electrode has a direct impact on the progress and effect of the synthesis. If the electrode fails or the electrode characteristics change during the synthesis process, it will affect the synthesis effect, especially significantly affecting the synthesis of long sequences.

[0031] The present invention provides a system for synthesizing oligonucleotides. After surface modification of the dielectric structure of the microelectrode array in the system, it is used as a synthesis region, thereby reducing the difficulty of the surface modification process and improving the stability of the synthesis process.

[0032] According to one aspect, the present invention provides a system for synthesizing oligonucleotides, which mainly includes an electrode structure layer and a control circuit layer.

[0033] Among them, the electrode structure layer may include a microelectrode array, and the microelectrode array includes a plurality of pixel units. Each pixel unit includes a plurality of electrodes and a dielectric structure located between the electrodes.

[0034] Specifically, referring to Figure 1 , according to one embodiment, the pixel unit of the microelectrode array may include an electrode structure. The electrode structure may include a counter electrode 1 and a working electrode 2. The working electrode 2 may include one or more array electrode structures 21 and a ring electrode structure 22 surrounding the one or more array electrode structures 21, and the counter electrode 1 may surround the ring electrode structure 22. Among the one or more array electrode structures 21, the top of each array electrode structure has a microporous structure.

[0035] According to a specific embodiment, the above-mentioned working electrode 2 may be formed of a noble metal material, for example, it may be a Pt electrode. The counter electrode 1 may be formed of the same or different noble metal materials as the working electrode 2, and preferably is also a Pt electrode.

[0036] Furthermore, there is a first sub-dielectric structure 3 between the array electrode structures 21, between the array electrode structure 21 and the ring electrode structure 22, and there is a second sub-dielectric structure 4 between the ring electrode structure 22 and the counter electrode 1.

[0037] In the system of the present invention, the above-mentioned dielectric structure may be formed of a dielectric material selected from silicon oxides and silicon nitrides, and is preferably formed of silicon dioxide.

[0038] Further, the above dielectric structure has a surface modification layer after surface modification, so that the dielectric structure can be used as a synthesis region for synthesizing oligonucleotides. Specifically, the dielectric structures located between multiple array electrode structures, between the multiple array electrode structures and the annular electrode structure, and between the annular electrode structure and the counter electrode constitute the above synthesis region.

[0039] Under the action of a certain voltage or current, protons are generated near the working electrode, and these protons diffuse into the synthesis region for the electrochemical synthesis process of oligonucleotides. Since the working electrode includes an array electrode structure and an annular electrode structure surrounding the array electrode structure, protons are more likely to be evenly distributed in the synthesis region, thereby improving the synthesis loading and synthesis effect of oligonucleotides.

[0040] According to a specific embodiment, the ratio of the surface area of the above annular electrode structure to the total surface area of one or more array electrode structures is 0.1 to 10, which is beneficial to ensuring the spatial uniformity of the proton concentration. According to a preferred embodiment, the surface area ratio can be 0.5 to 9, preferably 1 to 8.5, such as 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 8.3.

[0041] In the system according to the present invention, the dielectric structure used as the synthesis region has a surface modification layer. The surface modification layer includes a silane layer bonded to the surface of the dielectric structure, a surface modification structure bonded to the silane layer, and a linking molecule containing a protecting group grafted onto the surface modification structure.

[0042] According to an embodiment, the above silane layer can be a silane layer containing epoxy groups. Preferably, the silane layer containing epoxy groups can be formed by epoxypropoxy trialkoxysilane, such as formed by an epoxy silane selected from epoxypropoxy trimethoxysilane and epoxypropoxy triethoxysilane. And, the silane layer containing epoxy groups is bonded to the surface of the dielectric structure by a covalent bond to form a monolayer.

[0043] According to an embodiment, the above surface modification structure can be formed by an amino reaction of an imine polymer with the above silane layer bonded to the surface of the dielectric structure, especially a monolayer formed by covalently bonding to the surface of the dielectric structure. The imine polymer can be selected from polyalkyleneimine and polyamidoamine polymer (Polyamidoamine, PAMAM).

[0044] Preferably, refer to Figure 2, the above-mentioned polyalkyleneimine may be selected from polymethyleneimine, polyethyleneimine, and polypropyleneimine, and may be, for example, 3-(2,3-epoxypropoxy)propyltrimethoxysilane. Further, the imine polymer may have polyalkyleneimine side chains. The number average molecular weight (Mn) of the imine polymer may be from 20,000 to 30,000, and may be, for example, 25,000.

[0045] See Figure 3 , the above-mentioned polyamidoamine polymer may be a dendritic polyamidoamine polymer having an alkylene diamine core. Among them, the alkylene diamine may be selected from methylenediamine, ethylenediamine, propylenediamine, and butylenediamine. Preferably, the polyamidoamine polymer may be a polyamidoamine polymer having an ethylenediamine core, such as a polyethylamidoamine polymer having an ethylenediamine core. Further, the dendritic structure of the polyamidoamine polymer may have an algebra of 0 to 7.0 (i.e., the number of branching layers of the dendritic structure), and a number average molecular weight (Mw) of 500 to 120,000. For example, the PAMAM may be generation 0 (MW 516.8), generation 1.0 (MW 1429.85), generation 2.0 (MW 28824.81), generation 3.0 (MW 6908.83), generation 4.0 (MW 14214.17), generation 5.0 (MW 28824.81), generation 6.0 (MW 58048), or generation 7.0 (MW 116493), etc.

[0046] According to one embodiment, the above-mentioned linking molecule containing a protecting group is a linking molecule containing 4,4'-dimethoxytrityl (DMT). The linking molecule may be a Universal Linker Molecule (ULM).

[0047] See further Figure 2 and Figure 3 , according to a specific embodiment, on the surface of a silicon dioxide (SiO 2 ), or silicon nitride (SiN x ) dielectric structure, 3-(2,3-epoxypropoxy)propyltrimethoxysilane is covalently bonded through a silicon-oxygen bond (-Si-O-) or a silicon-nitrogen bond (-Si-N-) to form a monomolecular organosilane layer; then, through the reaction of the epoxy functional group with the amino group of the imine polymer, a surface modification structure is formed; the linking molecule containing the DMT protecting group is grafted onto the surface of the surface modification structure through the formation of an amide group (-CO-NH-) to form a synthesis site.

[0048] In one embodiment of the present invention, the above-mentioned control circuit may be integrated into the system in a Complementary Metal-Oxide-Semiconductor (CMOS) process manner.

[0049] Figure 4 Shows a single pixel unit of a system for synthesizing oligonucleotides according to an embodiment of the present invention, wherein the layer structure where the electrode structure is located may have a bilayer structure. Specifically, the electrode structure may include a counter electrode 1 and a working electrode, as well as a dielectric structure. The dielectric structure may include a first sub-dielectric structure 3 located on the working electrode, and a second sub-dielectric structure 4 located between the counter electrode 1 and the working electrode. According to a specific embodiment, the first sub-dielectric structure 3 may have a through-hole array structure, and the second sub-dielectric structure 4 may be an annular structure. Through the through-hole array structure, the exposed conductive structure serves as the working electrode. According to a specific embodiment, the working electrode may include an array electrode structure 21 and an annular electrode structure 22 surrounding the array electrode structure 21.

[0050] The above-mentioned working electrode may be formed of a noble metal material, for example, it may be a Pt electrode. The counter electrode 1 may be formed of the same or different noble metal materials as the working electrode 2, and preferably is also a Pt electrode.

[0051] The above-mentioned dielectric structure may be formed of a dielectric material selected from silicon oxide and silicon nitride, and is preferably formed of silicon dioxide. The above-mentioned dielectric structure has a surface modification layer after surface modification, so that the dielectric structure can be used as a synthesis region for synthesizing oligonucleotides. Specifically, the first sub-dielectric structure 3 located on the working electrode 2 and the second sub-dielectric structure 4 located between the working electrode 2 and the counter electrode 1 together constitute the synthesis region. The surface modification layer here has a three-layer structure composed of an organosilane layer, a surface modification structure, and a linking molecule containing a protecting group. Among them, the organosilane layer, the surface modification structure, and the linking molecule containing a protecting group are respectively as described above.

[0052] See Figure 5 , the layer structure where the above-mentioned electrode structure is located is formed by the following method: S101. Form a conductive layer; S102. Pattern the formed conductive layer to form an electrode structure including a counter electrode and a working electrode, and a first via structure separating the counter electrode and the working electrode; S103. Deposit a dielectric layer on the electrode structure formed in the above step S102; S104. Pattern the above-mentioned dielectric layer so that a first sub-dielectric structure having a second via structure is formed on the working electrode, and a second sub-dielectric structure filling the first via structure.

[0053] The system manufacturing process according to this embodiment is simple, the surface modification of the synthesis region is less difficult, and the proton concentration is evenly distributed in the synthesis space, which is beneficial to the synthesis of target oligonucleotides.

[0054] Figure 6A single pixel unit of a system for synthesizing oligonucleotides according to another embodiment of the present invention is shown. The layer structure where the electrode structure is located may have a three-layer structure, namely, a first conductive layer, a second conductive layer, and a dielectric layer located between the first conductive layer and the second conductive layer. On the other hand, the electrode structure may include a working electrode region and a counter electrode region. In particular, the counter electrode region surrounds the working electrode region. Specifically, the working electrode region is located in the central part of the first conductive layer, and a dielectric structure 3 with a through-hole structure is formed on the working electrode region, so that the first conductive layer exposed by the through-hole structure serves as the working electrode 2. The counter electrode region includes the first conductive layer and the second conductive layer and the dielectric layer located between the first conductive layer and the second conductive layer, where the second conductive layer in this region serves as the counter electrode 1.

[0055] The above-mentioned working electrode may be formed of a noble metal material, for example, it may be a Pt electrode. The counter electrode 1 may be formed of the same or different noble metal materials as the working electrode 2, and preferably is also a Pt electrode.

[0056] The above-mentioned dielectric structure may be formed of a dielectric material selected from silicon oxides and silicon nitrides, and is preferably formed of silicon dioxide. The above-mentioned dielectric structure has a surface modification layer after surface modification, so that the dielectric structure can be used as a synthesis region for synthesizing oligonucleotides. The surface modification layer here has a three-layer structure composed of an organosilane layer, a surface modification structure, and a linking molecule containing a protecting group. Among them, the organosilane layer, the surface modification structure, and the linking molecule containing a protecting group are respectively described as above.

[0057] See Figure 7 , the layer structure where the above-mentioned electrode structure is located is formed by the following method: S201. Form a first conductive layer and pattern the formed first conductive layer; S202. Form a dielectric layer on the first conductive layer; S203. Form a second conductive layer on the dielectric layer; S204. Pattern the formed second conductive layer to form a counter electrode; S205. Pattern the dielectric layer exposed by the above-mentioned second conductive layer to form a dielectric structure with a through-hole structure on the working electrode region of the first conductive layer, and make the exposed first conductive layer form a working electrode including one or more array electrode structures and an annular electrode structure surrounding the one or more array electrode structures.

[0058] The system according to this embodiment includes an electrode structure with a three-layer structure, which has a better-defined synthesis region and can provide more stable synthesis performance.

[0059] The above system can be used for high-throughput (e.g., with a throughput of 10,000 to 1,000,000) electrochemical synthesis of oligonucleotides.

[0060] Figures 8 to 10 Figure 4 shows a system for synthesizing oligonucleotides according to another embodiment of the present invention. Among them, Figure 8 is a schematic external view of the system, including a chip connected to a carrier plate. Figure 9 is Figure 8 a partial enlarged view of region A in the chip of FIG. 4, which shows a microelectrode array and a dielectric structure.

[0061] Further, referring to Figure 10 FIG. 5, the system according to this embodiment may include a chip, a flow cell 63, a liquid inlet 61, and a liquid outlet 62. Further, the chip may include a microelectrode array and a control circuit. The layer structure where the microelectrode array is located includes a dielectric layer 5 having a plurality of first through-hole structures arranged at intervals, and an electrode array structure.

[0062] Each electrode in the above electrode array structure includes a first portion 52 filling the corresponding first through-hole in the dielectric layer, a second portion 51 formed on the upper surface of the dielectric layer 5 and communicating with the first portion 52, and a third portion 54 formed on the lower surface of the dielectric layer 5 and communicating with the first portion 52. As Figure 9 shown in FIG. 6, the second portion 51 of the electrode presents an approximate annular structure, which is beneficial to the diffusion of generated protons and keeps the protons evenly distributed in the synthesis region; its surface has a surface modification layer of a linking molecule containing a protecting group, so that the dielectric layer is used as a synthesis region for synthesizing oligonucleotides. Among them, the surface modification layer includes a silane layer bonded to the surface of the dielectric layer, a surface modification structure bonded to the silane layer, and a linking molecule containing a protecting group grafted on the surface modification structure. Among them, the silane layer, the surface modification structure, and the linking molecule containing a protecting group are respectively described as above.

[0063] The above control circuit may include a driving board 8, and the driving board 8 may be electrically connected to the electrode array structure through a welding structure 64, especially electrically connected to the third portion 54 of the electrode array structure. Specifically, the welding structure 64 may be a solder ball or a conductive adhesive. According to another embodiment, the driving board may also be electrically connected to the electrode array structure through a conductive adhesive.

[0064] The above chip may further include a carrier plate 7 located between the welding structure 64 or the conductive adhesive and the driving board 8, so as to ensure that the welding structure 64 or the conductive adhesive is more stably electrically connected to the driving board 8. Among them, the carrier plate 7 has a plurality of second through-hole structures arranged at intervals, and a conductive structure 65 filling the second through-hole structures, which is used to better electrically connect the welding structure 64 or the conductive adhesive to the driving board 8.

[0065] The chip in this embodiment includes a first via structure and a second via structure, which are usually formed by Through Glass Via (TGV) technology. The control circuit uses a driving board, and the chip and the driving board are connected through solder balls or conductive adhesives. The manufacturing process is simple and can be used for medium-throughput synthesis of oligonucleotides.

[0066] According to another aspect of the present invention, there is provided an application of the above chip for synthesizing oligonucleotides, particularly for medium-throughput (e.g., 1000 - 10000 throughput) electrochemical synthesis of oligonucleotides.

[0067] See Figure 11 , according to another aspect of the present invention, there is provided a method for synthesizing oligonucleotides, including the following steps.

[0068] S301. Import the sequence to be synthesized into the control module. After receiving the sequence to be synthesized, the control module will analyze and process the sequence to generate a series of control instructions to coordinate the work of each synthesis unit on the chip. These control instructions may include parameters such as the temperature of the synthesis reaction, the addition time and amount of various synthesis reagents, and other reaction conditions (such as light conditions), to ensure that oligonucleotides are accurately synthesized according to the predetermined sequence at different positions on the chip. At the same time, the control module can also monitor various parameters and states during the synthesis process in real time, and give early warnings and handle possible abnormal situations to ensure the efficiency and quality of the synthesis.

[0069] Decompose the bases added in each synthesis step. In the process of electrochemical synthesis of oligonucleotides using the chip, the synthesis is carried out step by step for each base. Only one specific base, such as adenine (A), guanine (G), cytosine (C), or thymine (T), is added in each synthesis step.

[0070] S302. Provide a chip including a microelectrode array and a control circuit, and the microelectrode array includes a plurality of electrodes and a dielectric structure located between the electrodes. Among them, the microelectrode array includes a plurality of pixel units, and the electrodes in each pixel unit include one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures.

[0071] Among them, the ratio of the surface area of the above ring electrode structure to the total surface area of the one or more array electrode structures is 0.1 - 10, preferably 0.5 - 9, more preferably 0.8 - 8.5, such as 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 8.3.

[0072] S303. Modify the surface of the dielectric structure so that the surface of the dielectric structure has a surface modification layer.

[0073] See Figure 2 , the above surface modification step may include: First, an organosilane containing an epoxy group, preferably an epoxy silane (e.g., 3-(2,3-epoxypropoxy)propyltrimethoxysilane), is used to form a monolayer on the surface of the dielectric structure (e.g., SiN x or SiO 2 ) through covalent bonding of silanol groups; Then, through the reaction of the epoxy group of the organosilane layer with the iminoalkylidene group (or -NH-CH 2 =CH 2 ) on the imine polymer (e.g., polyethyleneimine or PAMAM), a surface modification structure is formed; Finally, a universal linker molecule containing a DMT protecting group is grafted onto the surface of the imine polymer of the surface modification structure to form the final synthesis site.

[0074] S304. Electrochemically synthesize oligonucleotides using the surface-modified dielectric structure as the synthesis region.

[0075] In the method of the present invention, the electrochemical synthesis of oligonucleotides includes introducing the synthesis raw materials for the electrochemical synthesis of oligonucleotides, and generating a localized acidic region on the surface of the electrode by using an electrochemical reaction. The protons generated on the surface of the electrode diffuse into the synthesis region (i.e., the surface of the dielectric structure with the surface modification layer), avoiding the need to add strong acids in the deprotection step in chemical synthesis. The electrochemical synthesis of oligonucleotides can adopt the phosphoramidite method, which generally includes four steps: deprotection, coupling, oxidation, and capping. In this method, the synthesis raw materials may include base reagents, deprotection reagents, coupling agents, oxidation reagents, and capping reagents.

[0076] Specifically, the surface of the dielectric structure for the electrochemical synthesis of oligonucleotides is usually pre-coated with a protecting group dimethoxytrityl (DMT). For example, the dielectric structure can be immersed in a 10 mM epoxy silane solution for 16 to 24 hours, then soaked in a 1.5% polyethyleneimine aqueous solution for 16 to 24 hours, and finally a solution of the linker molecule with DMT is added to the dielectric structure and incubated for 6 hours, thereby forming a monolayer film with a DMT protecting group on the dielectric structure. After that, the chip can be used as a carrier to synthesize oligonucleotides according to the phosphoramidite method.

[0077] See Figure 12 , the steps for the electrochemical synthesis of oligonucleotides according to the present invention may include: introducing the raw materials for the electrochemical synthesis of oligonucleotides into the synthesis region of the chip; under the action of an input current or voltage, performing deprotection, coupling, oxidation, and capping on the surface of the dielectric material in the synthesis region.

[0078] The above raw materials include an electrochemical deprotection reagent, a coupling reagent, an oxidation reagent, and a capping reagent.

[0079] The electrochemical deprotection reagent may include p - phenol and p - benzoquinone, especially 25 mM p - phenol, 25 mM p - benzoquinone, and 5 mM pyridine. Among them, p - phenol can be oxidized on the working electrode to generate hydrogen ions; while p - benzoquinone can be reduced on the counter electrode to generate organic basic substances.

[0080] The coupling reagent may include a base monomer and an acetonitrile solution of tetrazole; the oxidation reagent may include iodine water and pyridine; and the capping reagent may include an acetonitrile solution of N - methylimidazole and acetic anhydride.

[0081] According to the method for electrochemically synthesizing oligonucleotides of the present invention, by using a dielectric structure as the synthesis region, compared with the surface modification of the modified electrode, the surface modification difficulty of the dielectric structure is reduced and it is easy to achieve. On the other hand, by combining the array electrode structure and the ring electrode structure, the distribution of protons in the pixel unit region is more uniform, and then the required sequence can be correctly synthesized on the pixel unit, which not only improves the synthesis efficiency of oligonucleotides but also improves the correct rate of the synthesized sequence.

[0082] Performance evaluation Influence of electrode structure on proton distribution See Figure 13 , a model as shown in the figure was designed to verify the influence effect of the electrode structure on proton generation and diffusion. In this model, the proton distribution in the middle pixel was mainly investigated; and the influence of different ratios of the surface area of the ring electrode structure to the total surface area of the array electrode structure (represented by the ring structure / array structure in the figure) on the proton concentration (i.e., hydrogen ion distribution) was studied.

[0083] Figure 14 The normalized concentration distribution curves of protons along the dotted line direction are shown when the ratio of the surface area of the ring electrode structure to the total surface area of the array electrode structure is 0 (i.e., without the ring electrode structure), 0.92, 1.35, and 1.78 respectively. It can be seen from the curves in the figure that: the electrode structure represented by curve 1 has no peripheral ring electrode structure, and protons are mainly distributed in the middle region of the pixel; the electrode structures represented by curves 2 to 4 include a ring electrode structure and an array electrode structure, and the surface area ratios are 0.92, 1.35, and 1.78 respectively. Protons can better cover the pixel, and when the surface area ratio of the ring electrode structure to the array electrode structure reaches 1.78, the distribution of protons in the entire pixel region is more uniform.

[0084] Influence of electrode structure on oligonucleotide synthesis effect Figure 15A two - layer electrode structure is shown, including a counter electrode (Pt) 1, an array electrode structure (Pt) 21, a first sub - dielectric structure 3 located between the array electrode structures 21, and a second sub - dielectric structure 4 located between the array electrode structure 21 and the counter electrode 1. Among them, the silica synthesis region is circled by circle C1, and the array electrode structure region is circled by circle C2. In this structure, there are 37 circular Pt working electrodes, and the rest is the silica synthesis region, without a ring - shaped electrode structure.

[0085] By immersing the SiO 2 synthesis region in a 10 mM epoxy silane (i.e., glycidoxytrimethoxysilane) solution for 16 to 24 hours, then soaking it in a 1.5% aqueous solution of polyethyleneimine for 16 to 24 hours, and finally immersing the synthesis region in an equimolar mixed solution of a DMT - protected phosphoramidite biotin reagent solution (60 mM) and 5 - Ethylthio - 1H - Tetrazole (ETT). By using the method of electrochemical deprotection, after removing the DMT protecting group, streptavidin with a Cy5 fluorescent group is then attached. Directly observing the proton diffusion and deprotection situation, the results are as Figure 16 shown. The bright areas in the figure are the successfully deprotected regions, which can be used for subsequent oligonucleotide synthesis steps. It can be seen from Figure 16 that only the dielectric structure within the middle working electrode region is effectively deprotected and becomes an effective synthesis region, that is, this electrode structure cannot deprotect the entire synthesis region.

[0086] Figure 17 A two - layer electrode structure is shown, including a counter electrode 1, an array electrode structure 21, a ring - shaped electrode structure 22, a first sub - dielectric structure 3 between the array electrode structures 21 and between the array electrode structure 21 and the ring - shaped electrode structure 22, and a second sub - dielectric structure 4 between the ring - shaped electrode structure 22 and the counter electrode 1. In this structure, there are 37 circular Pt working electrodes and ring - shaped working electrodes, and the synthesis region includes the silica region circled by circle C1 and the second sub - dielectric structure 4 which is also made of silica material on the periphery.

[0087] By immersing the SiO 2 synthesis region in a 10 mM epoxy silane (i.e., glycidoxytrimethoxysilane) solution for 16 to 24 hours, then soaking it in a 1.5% aqueous solution of polyethyleneimine for 16 to 24 hours, and finally immersing the synthesis region in an equimolar mixed solution of a DMT - protected phosphoramidite biotin reagent solution (60 mM) and 5 - Ethylthio - 1H - Tetrazole. By using the method of electrochemical deprotection, after removing the DMT protecting group, streptavidin with a Cy5 fluorescent group is then attached. Directly observing the proton diffusion and deprotection situation, the results are as Figure 18As shown. The bright areas in the figure are the successfully deprotected areas, which can be used for subsequent oligonucleotide synthesis steps. From Figure 18 It can be seen that both the second sub-dielectric structure 4 on the periphery and the first sub-dielectric structure 3 in the center are effectively deprotected and become effective synthesis areas, that is, this electrode structure can deprotect the entire synthesis area.

Claims

1. A system for synthesizing oligonucleotides, characterized in that include: A microelectrode array, comprising one or more pixel units, wherein each of the pixel units comprises a plurality of electrodes and a dielectric structure located between the electrodes; and Control circuit; The dielectric structure has a surface modification layer of a linker molecule containing a protective group, so that the dielectric structure can be used as a synthesis region for electrochemically synthesizing oligonucleotides.

2. The system according to claim 1, wherein: The surface modification layer comprises an organic silane layer containing epoxy groups covalently bonded to the surface of the dielectric structure, a surface modification structure bonded to the organic silane layer, and a linking molecule containing a protective group grafted onto the surface modification structure.

3. The system according to claim 2, wherein: The organosilane layer is a monomolecular layer bonded to the surface of the dielectric structure through siloxy covalent bonds; The surface modification structure includes an imine polymer bonded to the organosilane layer, wherein the imine polymer is selected from polyalkylene imine and polyamidoamine polymer.

4. The system according to claim 3, wherein: The organosilane layer containing epoxy groups is formed of epoxy silane selected from glycidoxytrimethoxysilane and glycidoxytriethoxysilane; The polyalkylene imine is selected from polymethylene imine, polyethylene imine and polypropylene imine; The polyamidoamine polymer is a dendritic polyamidoamine polymer having an alkylene diamine core, wherein the alkylene diamine is selected from methylenediamine, ethylenediamine, propylenediamine and butylenediamine; and / or The linking molecule containing a protecting group is a linking molecule containing a 4,4′-dimethoxytrityl group.

5. The system according to claim 1, wherein: In each of the pixel units, the microelectrode array includes a working electrode and a counter electrode surrounding the working electrode; and The working electrode includes one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures.

6. The system according to claim 3, wherein: The ratio of the surface area of ​​the annular electrode structure to the total surface area of ​​the one or more array electrode structures is 0.1-10.

7. The system according to claim 5, wherein: Among the one or more array electrode structures, a top of each array electrode structure has a microporous structure.

8. The system according to claim 5, wherein: The synthesis area is composed of the dielectric structure located between the plurality of array electrode structures, between the plurality of array electrode structures and the ring electrode structure, and between the ring electrode structure and the counter electrode.

9. The system according to claim 1, wherein: The layer structure where the microelectrode array is located includes a first conductive layer, a dielectric layer formed on the first conductive layer, and a second conductive layer formed on the dielectric layer; The dielectric layer is patterned to form a dielectric structure having a first through-hole structure, so that a portion of the first conductive layer exposed by the first through-hole structure is used as a working electrode; The second conductive layer is patterned to have a second through-hole structure exposing the dielectric structure and the working electrode, and a counter electrode; and The exposed dielectric structure is surface-modified to have the surface modification layer.

10. The system according to claim 1, wherein: The layer structure where the microelectrode array is located includes a first conductive layer and a dielectric layer formed on the first conductive layer; Wherein, the first conductive layer includes a working electrode, a counter electrode surrounding the working electrode, and a first sub-dielectric structure located between the working electrode and the counter electrode; The dielectric layer located on the region of the working electrode is patterned to form a second sub-dielectric structure having a through-hole structure, so that a portion of the first conductive layer exposed through the through-hole structure serves as a working electrode; and The first sub-dielectric structure and the second sub-dielectric structure are surface-modified to have the surface-modified layer.

11. The system according to claim 9 or 10, wherein: The layer structure where the control circuit is located is a complementary metal oxide semiconductor structure.

12. Use of the system according to any one of claims 1 to 11 for high-throughput electrochemical synthesis of oligonucleotides.

13. A system for synthesizing oligonucleotides, comprising a microelectrode array and a control circuit, characterized in that: The layer structure where the microelectrode array is located includes a dielectric layer having a plurality of first through-hole structures arranged at intervals, and an electrode array structure; The surface of the dielectric layer has a surface modification layer of attachment molecules containing protective groups, so that the dielectric layer is used as a synthesis region for synthesizing oligonucleotides.

14. The system according to claim 13, wherein: The surface modification layer comprises an organic silane layer containing epoxy groups covalently bonded to the surface of the dielectric layer, a surface modification structure bonded to the organic silane layer, and a linking molecule containing a protective group grafted onto the surface modification structure.

15. The system of claim 14, wherein: The organosilane layer is a monomolecular layer bonded to the surface of the dielectric layer by covalent bonds; The surface modification structure includes an imine polymer bonded to the organosilane layer, wherein the imine polymer is selected from polyalkylene imine and polyamidoamine polymer.

16. The system of claim 15, wherein: The organosilane layer containing epoxy groups is formed of epoxy silane selected from glycidoxytrimethoxysilane and glycidoxytriethoxysilane; The polyalkylene imine is selected from polymethylene imine, polyethylene imine and polypropylene imine; The polyamidoamine polymer is a dendritic polyamidoamine polymer having an alkylene diamine core, wherein the alkylene diamine is selected from methylenediamine, ethylenediamine, propylenediamine and butylenediamine; and / or The linking molecule containing a protecting group is a linking molecule containing a 4,4′-dimethoxytrityl group.

17. The system of claim 13, wherein: Each electrode in the electrode array structure includes a first portion filling the corresponding first through hole in the dielectric layer, a second portion formed on the upper surface of the dielectric layer and connected to the first portion, and a third portion formed on the lower surface of the dielectric layer and connected to the first portion.

18. The system of claim 13, wherein: The control circuit includes a driving board, and the driving board is electrically connected to the electrode array structure through a welding structure or a conductive adhesive.

19. The system of claim 18, further comprising a carrier plate located between the welding structure or conductive glue and the driving board; in, The carrier board has a plurality of second through-hole structures arranged at intervals, and a conductive structure filling the second through-hole structures, which is used to electrically connect the welding structure or the conductive glue with the driving board.

20. Use of the system according to any one of claims 13 to 19 for medium-throughput electrochemical synthesis of oligonucleotides.

21. A method for synthesizing oligonucleotides, characterized in that: Includes steps: Import the fitted sequence into the control module; Providing a chip including a microelectrode array, wherein the microelectrode array includes a plurality of electrodes and a dielectric structure located between the electrodes; Performing surface modification on the dielectric structure so that the surface of the dielectric structure has a surface modification layer; Oligonucleotides are electrochemically synthesized using the surface-modified dielectric structure as a synthesis region.

22. The method according to claim 1, wherein: The dielectric structure is formed of a dielectric material selected from silicon oxide and silicon nitride.

23. The method according to claim 22, wherein: The surface modification of the dielectric structure comprises: forming an organosilane layer containing epoxy groups on the surface of the dielectric structure; Using an imine polymer to react with the organosilane layer to form a surface modified structure; A linking molecule containing a protecting group is grafted onto the surface modification structure.

24. The method according to claim 23, wherein: The organosilane layer containing epoxy groups is formed of glycidoxytrialkoxysilane; The imine polymer is selected from polyalkylene imine and polyamidoamine polymers; and The linking molecule containing a protecting group is a linking molecule containing a 4,4′-dimethoxytrityl group.

25. The method according to claim 23, wherein: The epoxy group-containing organosilane layer is formed of epoxysilane selected from glycidoxytrimethoxysilane and glycidoxytriethoxysilane.

26. The method of claim 23, wherein: The organic silane layer containing epoxy groups is bonded to the surface of the dielectric structure through covalent bonds to form a monomolecular layer.

27. The method according to claim 24, wherein: The polyalkylene imine is selected from polymethylene imine, polyethylene imine and polypropylene imine; and / or The polyamidoamine polymer is a dendritic polyamidoamine polymer having an alkylene diamine core, wherein the alkylene diamine is selected from methylenediamine, ethylenediamine, propylenediamine and butylenediamine.

28. The method according to any one of claims 21 to 27, wherein: The microelectrode array includes a working electrode and a counter electrode surrounding the working electrode; and The working electrode includes one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures.

29. The method according to claim 28, wherein: The ratio of the surface area of ​​the annular electrode structure to the total surface area of ​​the one or more array electrode structures is 0.1-10.

30. The method of claim 28, wherein: Among the one or more array electrode structures, a top of each array electrode structure has a microporous structure.

31. The method of claim 28, wherein: The synthesis region includes dielectric structures located between the plurality of array electrode structures, between the one or more array electrode structures and the ring electrode structure, and between the ring electrode structure and the counter electrode.

32. The method of claim 21, wherein: The electrochemical synthesis of oligonucleotides using the surface-modified dielectric structure as the synthesis region comprises: introducing an electrochemical deprotection reagent into the synthesis area; Under the action of input voltage or current, deprotecting the surface of the dielectric structure in the synthesis area; The starting materials for synthesizing oligonucleotides are introduced into the synthesis area, coupled, oxidized and capped.

33. The method of claim 32, wherein: The raw materials include coupling reagents, oxidizing reagents and capping reagents.

34. The method of claim 33, wherein: The electrochemical deprotection reagent comprises p-phenol, p-benzoquinone and an organic base; The coupling reagent includes an acetonitrile solution of a base monomer and tetrazole; The oxidizing agent comprises iodine, water and pyridine; and / or The capping reagent includes an acetonitrile solution of N-methylimidazole and acetic anhydride.

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