Systems and Applications and Methods for Synthesizing Oligonucleotides

Abstract

The present invention relates to a system, application and method for synthesizing oligonucleotides. The method includes the steps of: importing a sequence to be synthesized into a control module; providing a chip including a microelectrode array having a plurality of pixel units; decomposing bases added in each synthesis step and determining pixel units to be opened; performing validity detection and marking on the pixel units to be opened; updating bases added in each synthesis step and determining valid pixel units to be opened; adjusting the synthesis voltage or current input to the chip according to the number of valid pixel units to be opened; and electrochemically synthesizing oligonucleotides in a working area corresponding to the opened valid pixel units. The present invention realizes the on-line real-time self-check of pixel units by introducing a plurality of through holes into the microelectrode array and respectively connecting each through hole to a control circuit to detect the conductivity of the through holes, thereby improving the recognition of valid pixel units and ensuring the synthesis efficiency and accuracy of oligonucleotides.

Description

Technical Field

[0001] The present invention relates to biosynthesis technology, and in particular to a system, application and method for high-throughput electrochemical synthesis of oligonucleotides. Background Art

[0002] Microelectrode arrays have a wide range of applications in the fields of biomedical research, neuroscience, medical diagnosis, etc. However, with the increase in the size and number of microelectrode arrays (e.g., reaching tens of thousands or millions), the manufacturing yield and uniformity of microelectrode arrays have become the main bottlenecks restricting their large-scale application.

[0003] To promote the application of microelectrode arrays in the synthesis of oligonucleotides, in addition to improving the microfabrication quality and quality control of microelectrode arrays, a method for quickly detecting the electrical conduction quality of microelectrode array units is also required, so as to be able to quickly detect the conductivity of microelectrode array units without introducing samples, and further reduce the impact of the failure of microelectrode array units on oligonucleotide synthesis. Summary of the Invention

[0004] The object of the present invention is to provide a method and system for electrochemical synthesis of oligonucleotides, which perform electrical conductivity self-check based on through-holes provided in a microelectrode array, thereby significantly improving the effectiveness and reliability in the oligonucleotide synthesis of the microelectrode array.

[0005] To achieve the above object, the present invention proposes a system for electrochemical synthesis of oligonucleotides, comprising:

[0006] A microelectrode array, including a plurality of pixel units, and each pixel unit includes two or more through-holes;

[0007] A self-check structure, including two or more transistor switches and a control circuit;

[0008] Wherein, each of the through-holes is electrically connected to a corresponding one of the transistor switches; and

[0009] The control circuit is connected to the transistor switches in pairs in a polling control manner.

[0010] According to one embodiment, the pixel unit includes a working electrode and a counter electrode surrounding the working electrode; wherein, the working electrode includes an array electrode structure and a ring electrode structure surrounding the array electrode structure.

[0011] According to one embodiment, the layer structure where the microelectrode array is located is a microelectromechanical system structure, and the layer structure where the control circuit is located is a complementary metal oxide semiconductor structure.

[0012] According to one embodiment, the transistor switch is a metal-oxide-semiconductor field-effect transistor switch.

[0013] According to one embodiment, the transistor switch is integrated into the system by a 180-nm complementary metal-oxide semiconductor process.

[0014] According to one embodiment, the microelectrode array is a microelectrode array within 88,000 pixel units.

[0015] According to one embodiment, the via holes are formed in the complementary metal-oxide semiconductor structure.

[0016] According to one embodiment, the control circuit further includes a pull-up resistor.

[0017] According to another aspect, there is provided an application of the above system for electrochemical synthesis of oligonucleotides.

[0018] According to still another aspect of the present invention, there is provided a method for electrochemical synthesis of oligonucleotides, comprising the steps of:

[0019] Importing the sequence to be synthesized into the control module;

[0020] Providing a chip including a microelectrode array, and the microelectrode array includes a plurality of pixel units;

[0021] Decomposing the bases added in each synthesis step and determining the pixel units to be opened;

[0022] Performing validity detection on the pixel units to be opened, and marking the detected valid pixel units and invalid pixel units;

[0023] Updating the bases added in each synthesis step and determining the valid pixel units to be opened;

[0024] Adjusting the synthesis voltage or current input to the chip according to the number of the valid pixel units to be opened;

[0025] Electrochemically synthesizing oligonucleotides in the working area corresponding to the opened valid pixel units.

[0026] According to one embodiment, the performing validity detection on the pixel units to be opened includes:

[0027] Setting N via holes in each of the pixel units to be opened;

[0028] Setting N transistor switches respectively connected to the N via holes;

[0029] Selecting any 2 of the N transistor switches and electrically connecting them to the control circuit, applying a detection voltage or current, and performing connectivity detection;

[0030] Wherein, N is a natural number greater than or equal to 2.

[0031] According to one embodiment, the validity detection of the pixel unit to be turned on and the marking of the detected valid pixel units include:

[0032] Performing N × (N - 1) / 2 times of connectivity detection on the pixel unit to be turned on; and

[0033] When there are no electrically connected paired vias in the pixel unit to be turned on, marking this pixel unit as an invalid pixel unit;

[0034] When there is at least one pair of electrically connected vias in the pixel unit to be turned on, marking this pixel unit as a valid pixel unit.

[0035] According to one embodiment, the microelectrode array includes a working electrode and a counter electrode surrounding the working electrode.

[0036] According to one embodiment, the working electrode includes one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures, and the surface of the working electrode has a protecting group.

[0037] According to one embodiment, electrochemically synthesizing oligonucleotides in the working area corresponding to the turned-on valid pixel units includes:

[0038] Introducing an electrochemical deprotection reagent into the working area;

[0039] Under the action of the synthesis voltage or current, deprotecting the surface of the working electrode in the working area;

[0040] Introducing raw materials for synthesizing oligonucleotides into the working area, and performing coupling, oxidation, and capping.

[0041] According to one embodiment, the raw materials include an electrochemical deprotection reagent, a coupling reagent, an oxidation reagent, and a capping reagent.

[0042] According to one embodiment, the electrochemical deprotection reagent includes p-phenol, p-benzoquinone, and an organic base;

[0043] The coupling reagent includes a base monomer and an acetonitrile solution of tetrazole;

[0044] The oxidation reagent includes iodine, water, and pyridine;

[0045] The capping reagent includes an acetonitrile solution of N-methylimidazole and acetic anhydride.

[0046] According to one embodiment, the transistor switch is integrated on the chip in the form of a complementary metal oxide semiconductor process or is included in a module of the control circuit.

[0047] According to one embodiment, the validity detection performs a one-by-one detection on each pixel unit through polling control.

[0048] The system according to the present invention includes a microelectrode array having a plurality of through holes, each through hole is respectively connected to a control circuit, thereby enabling the conduction condition of each through hole to be detected in real time, further improving the electrical conductivity of each electrode, and detecting the electrical conductivity of each electrode in real time online to ensure that the target sequence is correctly synthesized on an effective microelectrode array. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The following will describe specific embodiments with reference to the drawings. The scope of the present invention is not limited thereto, wherein:

[0050] Figure 1 FIG. shows a schematic structural diagram of a pixel unit in a microelectrode array according to an embodiment of the present invention;

[0051] Figure 2 FIG. shows a schematic structural diagram of a pixel unit in a microelectrode array according to another embodiment of the present invention;

[0052] Figure 3 FIG. shows a schematic structural diagram of the connection between a through hole and a control circuit of a pixel unit in a microelectrode array according to an embodiment of the present invention;

[0053] Figure 4 FIG. shows a schematic diagram of a self-check loop based on a through hole according to an embodiment;

[0054] Figure 5 FIG. shows a schematic diagram of a self-check loop based on a through hole according to another embodiment;

[0055] Figure 6 FIG. shows a schematic cross-sectional view of a layer structure of a chip according to the present invention;

[0056] Figure 7 FIG. shows a process flow chart of forming a MEMS structure in a chip according to the present invention;

[0057] Figure 8 FIG. shows a schematic structural diagram of a chip according to the present invention;

[0058] Figure 9 FIG. shows a complete flow chart of a method for synthesizing oligonucleotides according to the present invention;

[0059] Figures 10 to 12 FIG. shows a schematic diagram of a self-check result of a chip according to an embodiment;

[0060] Figure 13 Shows a flow chart of the synthesis steps of the method for synthesizing oligonucleotides according to the present invention.

[0061] Explanation of reference numerals:

[0062] 1 - via hole; 2 - working electrode; 3 - pixel region; 4 - pad region; 5 - counter electrode; 6 - normal pixel unit; 7 - defective pixel unit; 21 - array electrode structure; 22 - annular electrode structure; 23 - first dielectric structure; 24 - second dielectric structure; 40 - second substrate; 41 - insulating layer; 42 - via; 48 - second hole; 49 - third dielectric structure; 50 - first hole. Detailed implementation manners

[0063] In the prior art, a system for electrochemically synthesizing oligonucleotides usually inputs a synthesis voltage or current to a chip according to the need for synthesizing a target oligonucleotide, and evenly distributes the synthesis voltage or current to each opened pixel on the chip, in order to expect to synthesize the target oligonucleotide on each pixel.

[0064] However, in practical applications, there is a situation where a pixel fails during the synthesis process (usually caused by defects in the chip processing technology). Then, the actual voltage or current introduced into each effective pixel will be larger than the set value, which will cause side reactions such as depurination during the synthesis of oligonucleotides, and will cause misinsertion during insertion. For example, assuming that each pixel unit needs to obtain 0.1 μA of current for synthesizing the target oligonucleotide, and 5000 pixel units are opened in the current synthesis situation, the system will input 500 μA of current to the chip. If all pixel units are effective, the current on each pixel unit is 0.1 μA. However, if 500 out of these 5000 pixel units are defective, the current on each pixel unit is 0.11 μA. Thus, the actual current distributed to the pixel unit is greater than the expected value of 0.1 μA, which may lead to unexpected synthesis side reactions.

[0065] The present invention provides a system for synthesizing oligonucleotides. By introducing a self - inspection structure into the pixel unit, the effective state of the pixel can be understood in a timely manner, so that the synthesis voltage or current can be correctly allocated to the pixel unit, and the target sequence can be correctly synthesized on the normal pixel unit. For example, in the above example, if 500 out of these 5000 pixel units are defective, only 450 μA of current needs to be input to the chip, so that the current on each pixel unit remains 0.1 μA.

[0066] 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.

[0067] Among them, the electrode structure layer may include a microelectrode array, and the microelectrode array includes a plurality of pixel units, and each pixel unit includes two or more through holes. The above through holes are located at the bottom layer of the electrode structure layer and are used for electrical connection with the self-checking structure below.

[0068] Specifically, referring to Figure 1 and Figure 2 , according to one embodiment, the pixel unit of the microelectrode array may include an electrode structure unit and two or more through holes 1, for example, it may include two through holes, or three through holes, or four through holes. Further, the electrode structure unit may include a working electrode 2, a counter electrode 5 surrounding the working electrode 2, and a second dielectric structure 24 separating the working electrode 2 and the counter electrode 5.

[0069] According to one embodiment, as Figure 1 shown, the working electrode 2 includes a plurality of array electrode structures 21 and an annular electrode structure 22 surrounding the plurality of array electrode structures 21. Further, the electrode structure unit may further include a first dielectric structure 23 located between the array electrode structures 21 and between the array electrode structures 21 and the annular electrode structure 22.

[0070] According to another embodiment, as Figure 2 shown, the working electrode 2 is a single electrode structure. Other structures are the same as those of the Figure 1 shown embodiment.

[0071] In the above Figure 1 , the electrode structure unit includes an annular electrode structure 22, so that the array electrode structures 21 in the electrode structure unit are separated from the array electrode structures of other pixel units.

[0072] Further referring to Figure 3 , the through hole 1 is formed in the first dielectric layer where the first dielectric structure 23 is located in the electrode structure unit, and a conductive material is filled in the through hole 1. The conductive material may only fill the hole part of the through hole, or may fill both the hole part of the through hole and form a conductive material layer 11 on the lower surface of the first dielectric layer for electrical connection with the self-checking structure.

[0073] A first conductive layer, a second dielectric layer, and a second conductive layer are sequentially formed on the first dielectric layer. After patterning, the second dielectric layer and the second conductive layer have a hole structure. The first conductive layer exposed by the hole structure is used as the working electrode 2, the second conductive layer forms the counter electrode 5 after patterning, and the second dielectric layer forms the second dielectric structure 24 after patterning.

[0074] According to a specific embodiment, the working electrode 2 may be formed of a noble metal material, for example, it may be a Pt electrode. The counter electrode 5 may be formed of the same or different noble metal materials as the working electrode 2, and is preferably also a Pt electrode. The conductive material filled in the through hole 1 may be copper or aluminum.

[0075] In the system of the present invention, the self-checking structure may include two or more transistor switches and a control circuit. Among them, the number of transistor switches is the same as that of the through holes, and each through hole is connected to a corresponding transistor switch for detecting the conductivity of the electrode array.

[0076] In the system of the present invention, the transistor switch may be a Metal-Oxide-Semiconductor Field-Effect Transistor (MOSFET).

[0077] Specifically, in Figure 4 and Figure 5 In the specific embodiment shown, the control circuit may further include a pull-up resistor R for connecting to the first transistor switch, while the second transistor switch is grounded. Denote the potentials before and after the pull-up resistor as V0 and V t , so that the electrical connection of the through hole can be judged by the potential V t .

[0078] In Figure 4 and Figure 5 , the control circuit is configured to inject a detection voltage or current between a pair of through holes. When the electrical connection between the two through holes is conductive, a current forms a loop, and V t is at a low level, then the pixel unit where the electrode corresponding to the through hole is located at this time is marked as a valid pixel unit (as shown in Figure 4 ); when the electrical connection of any one through hole is not conductive, the current cannot form a loop, and V t is at a high level, then the pixel unit where the electrode corresponding to the through hole is located at this time is marked as a failed pixel unit (as shown in Figure 5 ); and the valid pixel units and failed pixel units are counted respectively. The control circuit connects the transistor switches and the through holes in pairs one by one in a polling control manner and performs conductivity detection.

[0079] That is to say, when the pixel unit includes N through holes (N is a natural number greater than or equal to 2), the self-checking structure correspondingly includes N transistor switches, that is, the number of through holes is equal to the number of transistor switches, and the N through holes are connected to the N transistor switches in one-to-one correspondence. The control circuit needs to perform N×(N - 1) / 2 connectivity detections to realize the conductivity detection of these N pairs of through hole - transistor switches.

[0080] In an embodiment of the present invention, the above-mentioned microelectrode array may be a Micro-Electro-Mechanical Systems (MEMS) structure. The transistor switch connected to the through-holes in the microelectrode array can be integrated into the system in the form of a Complementary Metal-Oxide-Semiconductor (CMOS) process or can be included in the module of the control circuit.

[0081] Figure 6 A specific embodiment is shown. The system for synthesizing oligonucleotides according to the present invention may include an MEMS structure and a CMOS structure, preferably consisting of an MEMS structure and a CMOS structure.

[0082] Specifically, the above-mentioned MEMS structure may include:

[0083] A base layer, usually the first dielectric layer, may also include a first substrate (such as a silicon substrate) and a first dielectric layer formed on the first substrate. The base layer may include a plurality of through-holes 1, and the first dielectric layer having a plurality of through-holes 1 is the first dielectric structure 23;

[0084] A first conductive layer located on the base layer, and the first conductive layer is patterned to form a working electrode 2;

[0085] A second dielectric layer formed on the working electrode 2 and separating the working electrodes 2, and the second dielectric layer is patterned to form a second dielectric structure 24;

[0086] A second conductive layer formed on the second dielectric structure 24, and the second conductive layer is patterned to form a counter electrode 5; and

[0087] A third dielectric layer formed on the counter electrode 5, and the third dielectric layer is patterned to form a third dielectric structure 49.

[0088] Among them, the multiple through-holes 1 are located below the working electrode and filled with a conductive metal material, such as copper or aluminum. Among them, the first dielectric layer, the second dielectric layer, and the third dielectric layer can be independently formed of a dielectric material, such as one or more of silicon oxide, silicon nitride, and aluminum oxide. The first dielectric layer and the second dielectric layer can be formed of conventional insulating materials respectively, and the materials of the two can be the same or different. For example, the first dielectric layer can be formed of silicon nitride, and the second dielectric layer can be formed of silicon oxide. The working electrode 2 and the counter electrode 5 can be formed of the same or different noble metal materials, such as Pt. Further, the first dielectric layer forms a hole structure in the region corresponding to the working electrode 2 to expose a part of the working electrode 2. For example, the first hole 50 formed by the window open process is used to realize the connection between different functional layers, or the second hole 48 formed by the pad open process is used to ensure good contact and conductive performance between the pad and the external lead or packaging material.

[0089] As Figure 7 shown, the manufacturing method of the above MEMS structure includes the following steps:

[0090] S110. Flatten the base layer;

[0091] S120. Etch the flattened base layer to form multiple through-holes;

[0092] S130. Electroplate the through-holes with a conductive metal material (such as copper);

[0093] S140. Perform chemical mechanical polishing (CMP) on the electroplated base layer;

[0094] S150. Deposit a first noble metal material on the base layer after chemical mechanical polishing and pattern it to form a working electrode;

[0095] S160. Deposit a first insulating material and a second noble metal material on the working electrode and the base layer in sequence and pattern them to form a counter electrode and a hole structure, while exposing a part of the working electrode;

[0096] S170. Deposit a second insulating material on the counter electrode and perform window opening process and pad opening process to expose a part of the working electrode and the counter electrode respectively.

[0097] Among them, the above-mentioned base layer can be a first dielectric layer, or include a first substrate (such as a silicon substrate) and a first dielectric layer formed on the first substrate. The first dielectric layer can be formed of a dielectric material, for example, it can be formed of one or more of silicon oxide, silicon nitride, and aluminum oxide. The above-mentioned first noble metal material and the second noble metal material can be the same or different, for example, it can be Pt. The above-mentioned first insulating material and the second insulating material can be the same or different, for example, the first insulating material can be silicon nitride, and the second insulating material can be silicon oxide.

[0098] Furthermore, the above-mentioned CMOS structure can include a second substrate (such as a silicon substrate) 40 and an insulating layer 41 formed on the second substrate 40, and a plurality of vias 42 are formed in the insulating layer 41. Further, the vias 42 are filled with a conductive material, and the conductive material can be copper or aluminum. The CMOS structure is combined with the MEMS structure by electrically connecting the conductive material in the vias 42 to the conductive metal material in the through hole 1 in the above-mentioned base layer, thereby completing the construction of the system for electrochemically synthesizing oligonucleotides according to the present invention.

[0099] According to a specific embodiment, see Figure 8 , the system for synthesizing oligonucleotides according to the present invention can be a chip. Among them, Figure 8 shows a pixel region 3 and a pad region 4. Specifically, the chip can be a chip including a microelectrode array of 88,000 pixel units. Each pixel contains two or more through holes, and these through holes are respectively used to connect to MOSFET switches in the control circuit, which can be realized by a 180nm CMOS process.

[0100] See Figure 9 , on the other hand, according to the present invention, a method for synthesizing oligonucleotides is provided, including the following steps:

[0101] S210. 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 can include parameters such as the temperature of the synthesis reaction, the addition time and addition amount of various synthesis reagents, and other reaction conditions (such as light conditions), ensuring that oligonucleotides are accurately synthesized in accordance with 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.

[0102] S220. Provide a chip including a microelectrode array and a self-checking structure, and the microelectrode array includes a plurality of pixel units. Each pixel unit includes a working electrode and a counter electrode surrounding the working electrode, and includes two or more through holes; wherein the working electrode includes an array electrode structure and an annular electrode structure surrounding the array electrode structure. The self-checking structure may include a transistor switch and a control circuit respectively connected to the above through holes. Two or more through holes and two or more transistor switches are arranged in each pixel unit, and the through holes and the transistor switches are connected in one-to-one correspondence.

[0103] S230. Decompose the bases added in each synthesis step and determine the pixel units to be opened. During the process of electrochemically synthesizing 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.

[0104] S240. Perform validity detection on the pixel units to be opened, and mark the detected valid pixel units and invalid pixel units.

[0105] Among them, this step includes: Select any 2 of the N transistor switches and connect them to the control circuit, and apply a constant current or voltage for conduction detection. Wherein, N is a natural number greater than or equal to 2. Specifically, when the electrical connection of the two through holes is conductive and the current forms a loop, the pixel unit where the electrode corresponding to the through hole is located at this time is marked as a valid pixel unit (as Figure 4 shown); when the electrical connection of any one through hole is not conductive and the current cannot form a loop, and V t is at a high level, the pixel unit where the electrode corresponding to the through hole is located at this time is marked as a failed pixel unit (as Figure 5 shown). This step also includes counting the valid pixel units and failed pixel units respectively, and feeding the counting results back to the control module for determining the magnitude of the current that the control circuit needs to apply.

[0106] Figure 10 shows the self-checking result of a chip according to an embodiment of the present invention, where the gray pixels in the boxes are the self-checking qualified pixels, and the red pixels are the self-checking unqualified pixels.

[0107] Figure 10 The "G" pattern within box A of

[0108] is a defect deliberately designed by the inventor (that is, the Pt working electrode for connecting to the via is not plated in the pixel), and B is a randomly detected defect. Therefore, the chip design can correctly detect the defects of the electrode structure.

[0108] For the detected defects other than the deliberately designed ones, the inventor performs Focused Ion Beam (FIB) tests on them (asFigure 11 and Figure 12 as shown). Among them, Figure 11 is Figure 10 an enlarged view of the area shown by box A in the Chinese side, including the failed pixel unit 7 and the normal pixel unit 6, where the dashed "G"-shaped pattern represents the failed pixel unit 7 without a Pt working electrode (as shown by the rectangular box in the pixel unit) designed specifically. Figure 12 The test results of show that the Pt working electrode is discontinuous at the through hole. Therefore, this chip design can detect defects in the processing of the microelectrode array.

[0109] S250. If all pixel units are effective, the control module determines the magnitude of the current or voltage that the control circuit needs to apply according to the number of effective pixel units determined in step S240 and the magnitude of the current or voltage expected to be obtained by each pixel. For example, if the number of effective pixel units determined in step S240 is 5000 and the current expected to be obtained by each pixel is 0.1 μA, then the current that the control circuit needs to apply to the system is 500 μA.

[0110] S260. Electrochemically synthesize oligonucleotides in the working area corresponding to the effective pixel units.

[0111] S270. If an ineffective pixel unit is detected, the control module deletes the sequence information to be synthesized on the marked ineffective pixel unit, updates the bases added in each synthesis step, and determines the effective pixel units that need to be opened.

[0112] S280. Adjust the input current or voltage of the chip according to the number of effective pixel units that need to be opened and the magnitude of the current or voltage expected to be obtained by each pixel. In this step, the number of determined effective pixel units has subtracted the number of ineffective pixel units, and the pre-applied current or voltage determined thereby meets the expectations, ensuring that the current or voltage allocated to each pixel unit meets the magnitude of the current or voltage required for the expected synthesis sequence, avoiding side reactions such as depurination and mis-incorporation of sequences introduced or inserted due to excessive current or voltage allocated to the remaining effective pixel units caused by some ineffective pixel units, thereby ensuring the correct synthesis of the required sequence on the effective pixels.

[0113] S290. Electrochemically synthesize oligonucleotides in the working area corresponding to the opened effective pixel units.

[0114] In the method of the present invention, the electrochemical synthesis of oligonucleotides includes introducing the synthetic raw materials for the electrochemical synthesis of oligonucleotides, and generating a localized acidic region on the surface of the electrode by means of an electrochemical reaction, 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 synthetic raw materials can include base reagents, deprotection reagents, coupling agents, oxidation reagents, and capping reagents.

[0115] Specifically, the working electrode for the electrochemical synthesis of oligonucleotides usually has a protecting group, dimethoxytrityl (DMT), pre-attached. For example, the electrode can be immersed in an anhydrous ethanol solution of 0.1 mM thiol for 12 hours, and then a chemical phosphorylation reagent and a coupling reagent are added to the electrode together. The phosphate group of the chemical phosphorylation reagent will couple with the hydroxyl group on the thiol, and then a monolayer film with a DMT protecting group is formed on the electrode. After that, the chip can be used as a carrier to synthesize oligonucleotides according to the phosphoramidite method.

[0116] See Figure 13 , the steps for the electrochemical synthesis of oligonucleotides according to the present invention can include: introducing the raw materials for the electrochemical synthesis of oligonucleotides into the working area; under the action of the input current, performing deprotection, coupling, oxidation, and capping on the surface of the working electrode in the working area.

[0117] The above-mentioned raw materials include an electrochemical deprotection reagent, a coupling reagent, an oxidation reagent, and a capping reagent.

[0118] Among them, the chemical phosphorylation reagent can be . The thiol can be HSCH2(CH2)9 CH2O(CH2)2O(CH2)2O(CH2)2OH.

[0119] The electrochemical deprotection reagent can include p-phenol, p-benzoquinone, and an organic base, preferably composed of p-phenol, p-benzoquinone, and an organic base. The organic base can be pyridine or 2,6-dimethylpyridine; particularly, it can be 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 an organic basic substance.

[0120] The coupling reagent can include an acetonitrile solution of a base monomer and tetrazole; the oxidation reagent can include iodine, water, and pyridine; and the capping reagent can include an acetonitrile solution of N-methylimidazole and acetic anhydride.

[0121] By using the electrochemical oligonucleotide synthesis method of the present invention, before introducing reagents for synthesis, the conductivity of the microelectrode array of the chip is first tested to eliminate invalid pixel units in the microelectrode array, and the failure of sequence synthesis is predicted in advance, so that the current can be correctly distributed to the effective pixel units, and then the required sequence can be correctly synthesized on the effective pixel units, which not only improves the synthesis efficiency of the oligonucleotide, but also improves the accuracy of the synthesized sequence.

Claims

1. A system for synthesizing oligonucleotides, characterized in that, Comprising: A microelectrode array including a plurality of pixel units, and each pixel unit includes an electrode structure unit and two or more through-holes; A self-checking structure including two or more transistor switches and a control circuit; Wherein, each of the through-holes is electrically connected to a corresponding one of the transistor switches; The electrode structure unit includes a first dielectric layer formed with the through-holes, and a first conductive layer, a second dielectric layer, and a second conductive layer sequentially located on the first dielectric layer, and the second dielectric layer and the second conductive layer have a hole structure such that the first conductive layer exposed by the hole structure serves as a working electrode; The through-holes are filled with a conductive material; and The control circuit is connected to the transistor switches pair by pair in a polling control manner.

2. The system according to claim 1, wherein, The pixel unit includes a working electrode and a counter electrode surrounding the working electrode; Wherein, the working electrode includes an array electrode structure and a ring electrode structure surrounding the array electrode structure, or is a single electrode structure.

3. The system according to claim 1, wherein The layer structure where the microelectrode array is located is a microelectromechanical system structure, and the layer structure where the control circuit is located is a complementary metal oxide semiconductor structure.

4. The system according to claim 1, wherein, The transistor switch is a metal-oxide-semiconductor field effect transistor switch.

5. The system according to claim 1, wherein The transistor switch is integrated into the system through a 180 nm complementary metal oxide semiconductor process.

6. The system according to any one of claims 1 to 5, wherein, The microelectrode array is a microelectrode array within 88,000 pixel units.

7. The system according to claim 3, wherein, The through-holes are formed in the complementary metal oxide semiconductor structure.

8. The system according to any one of claims 1 to 5, wherein, The control circuit further includes a pull-up resistor connected to the transistor switch.

9. Application of the system according to any one of claims 1 to 8 in electrochemically synthesizing oligonucleotides.

10. A method for synthesizing oligonucleotides, characterized in that, Including steps: Importing the sequence to be synthesized into a control module; Providing a chip including a microelectrode array, and the microelectrode array includes a plurality of pixel units; Decomposing the bases added in each synthesis step and determining the pixel units to be opened; Performing validity detection on the pixel units to be opened, and marking the detected valid pixel units and invalid pixel units; Updating the bases added in each synthesis step and determining the valid pixel units to be opened; Adjusting the synthesis voltage or current input to the chip according to the number of the valid pixel units to be opened; Electrochemically synthesizing oligonucleotides in the working area corresponding to the opened valid pixel units; Wherein, each pixel unit includes an electrode structure unit and two or more through-holes, the electrode structure unit includes a first dielectric layer formed with the through-holes, and a first conductive layer, a second dielectric layer, and a second conductive layer sequentially located on the first dielectric layer, and the second dielectric layer and the second conductive layer have a hole structure such that the first conductive layer exposed by the hole structure forms a working electrode; and The through-holes are filled with a conductive material; Wherein, the performing validity detection on the pixel units to be opened includes: Setting N through-holes in each of the pixel units to be opened; Setting N transistor switches respectively connected to the N through-holes; Select any two of the N transistor switches and electrically connect them to the control circuit, apply a detection voltage or current, and perform a connectivity detection; where N is a natural number greater than or equal to 2.

11. The method according to claim 10, wherein, Perform an effectiveness detection on the pixel unit to be turned on, and marking the detected effective pixel units includes: Perform N × (N - 1) / 2 times of connectivity detection on the pixel unit to be turned on; and When there are no electrically connected paired vias in the pixel unit to be turned on, mark this pixel unit as a failed pixel unit; When there is at least one pair of electrically connected vias in the pixel unit to be turned on, mark this pixel unit as an effective pixel unit.

12. The method according to claim 10 or 11, wherein, The microelectrode array includes a working electrode and a counter electrode surrounding the working electrode array.

13. The method according to claim 12, wherein, The working electrode includes one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures, and the surface of the working electrode has a protecting group.

14. The method according to claim 13, wherein, Electrochemically synthesizing oligonucleotides in the working area corresponding to the turned-on effective pixel unit includes: Introduce an electrochemical deprotection reagent into the working area; Under the action of the synthesis voltage or current, deprotect the surface of the working electrode in the working area; Introduce the raw materials for synthesizing oligonucleotides into the working area, and perform coupling, oxidation and capping.

15. The method according to claim 14, wherein, The raw materials include a coupling reagent, an oxidation reagent and a capping reagent.

16. The method according to claim 15, wherein, The electrochemical deprotection reagent includes p-phenol, p-benzoquinone and an organic base; The coupling reagent includes a base monomer and an acetonitrile solution of tetrazole; The oxidation reagent includes iodine, water and pyridine; The capping reagent includes an acetonitrile solution of N-methylimidazole and acetic anhydride.

17. The method according to claim 10, wherein The transistor switch is integrated on the chip in the manner of complementary metal oxide semiconductor process, or is included in the module of the control circuit.

18. The method according to claim 10, wherein, The effectiveness detection is performed on each pixel unit one by one through polling control.

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