System, application and method for synthesizing oligonucleotides

Abstract

The invention relates to a system for synthesizing oligonucleotides, application and a method. The method comprises the following steps: importing a fitted sequence into a control module; providing a chip comprising a microelectrode array comprising a plurality of pixel units; decomposing basic groups added in each synthesis step, and determining pixel units to be opened; carrying out validity detection and marking on the pixel units to be opened; updating basic groups added in each synthesis step, and determining effective pixel units needing to be opened; according to the number of the effective pixel units needing to be opened, the synthesized voltage or current input into the chip is adjusted; and carrying out electrochemical synthesis on oligonucleotides in the working areas corresponding to the opened effective pixel units. According to the invention, a plurality of through holes are introduced into the microelectrode array, and each through hole is respectively connected with the control circuit to detect the conductivity of the through hole, so that the online real-time self-inspection of the pixel unit is realized, the recognition of the effective pixel unit is improved, and the synthesis efficiency and accuracy of oligonucleotide are ensured.

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 are widely used in biomedical research, neuroscience, and medical diagnosis. However, as the size and number of microelectrode arrays increase (e.g., reaching tens of thousands or millions), the manufacturing yield and uniformity of microelectrode arrays become the main bottleneck restricting their large-scale application.

[0003] In order to promote the application of microelectrode arrays in oligonucleotide synthesis, in addition to improving the micromachining quality and quality control of microelectrode arrays, a method is also needed to quickly detect the conductivity quality of microelectrode array units, so as to achieve rapid conductivity detection of microelectrode array units without introducing samples, thereby reducing the impact of microelectrode array unit failure on oligonucleotide synthesis. Summary of the invention

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

[0005] In order to achieve the above object, the present invention provides a system for electrochemically synthesizing oligonucleotides, comprising: A microelectrode array, comprising a plurality of pixel units, and each pixel unit comprises two or more through holes; A self-test 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; and The control circuit is connected to the transistor switches pair by pair in a polling control manner.

[0006] 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.

[0007] According to one embodiment, the layer structure where the microelectrode array is located is a micro-electromechanical system structure, and the layer structure where the control circuit is located is a complementary metal oxide semiconductor structure.

[0008] According to one embodiment, the transistor switch is a metal-oxide-semiconductor field-effect transistor switch.

[0009] According to one embodiment, the transistor switch is integrated into the system using a 180 nm complementary metal oxide semiconductor process.

[0010] According to one embodiment, the microelectrode array is a microelectrode array within 88,000 pixel units.

[0011] According to one embodiment, the via is formed in the complementary metal oxide semiconductor structure.

[0012] According to one embodiment, the control circuit further comprises a pull-up resistor.

[0013] According to another aspect, there is provided use of the above system for electrochemical synthesis of oligonucleotides.

[0014] According to another aspect of the present invention, there is provided a method for electrochemically synthesizing oligonucleotides, comprising the steps of: Import the fitted sequence into the control module; Providing a chip including a microelectrode array, wherein the microelectrode array includes a plurality of pixel units; Decompose the bases added at each synthesis step and determine the pixel cells to be opened; Performing validity detection on the pixel units to be turned on, and marking the detected valid pixel units and invalid pixel units; Update the bases added in each synthesis step and determine the valid pixel units that need to be turned on; According to the number of effective pixel units that need to be turned on, adjusting the composite voltage or current input to the chip; Oligonucleotides are electrochemically synthesized in the working area corresponding to the turned-on effective pixel unit.

[0015] According to one embodiment, the performing validity detection on the pixel unit to be turned on includes: Setting N through holes in each pixel unit that needs to be opened; Setting N transistor switches, connected to the N through holes respectively; Select any two of the N transistor switches to be electrically connected to the control circuit, apply a detection voltage or current, and perform a connectivity test; Wherein, N is a natural number greater than or equal to 2.

[0016] According to one embodiment, performing validity detection on the pixel unit to be turned on and marking the detected valid pixel unit includes: Performing N × (N-1) / 2 connectivity checks on the pixel units to be opened; and When there are no electrically connected pairs of through holes in the pixel unit to be opened, marking the pixel unit as a failed pixel unit; When there is at least one pair of electrically connected through holes in the pixel unit to be opened, the pixel unit is marked as a valid pixel unit.

[0017] According to one embodiment, the microelectrode array includes a working electrode and a counter electrode surrounding the working electrode.

[0018] According to one embodiment, the working electrode comprises one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures, and a surface of the working electrode has a protective group.

[0019] According to one embodiment, electrochemically synthesizing oligonucleotides in a working area corresponding to an opened effective pixel unit includes: introducing an electrochemical deprotection reagent into the working area; Under the action of the synthetic voltage or current, deprotecting the surface of the working electrode in the working area; The raw materials for synthesizing oligonucleotides are introduced into the working area for coupling, oxidation and capping.

[0020] According to one embodiment, the raw materials include an electrochemical deprotection reagent, a coupling reagent, an oxidation reagent, and a capping reagent.

[0021] According to one embodiment, 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 includes iodine, water and pyridine; The capping reagent includes an acetonitrile solution of N-methylimidazole and acetic anhydride.

[0022] According to one embodiment, the transistor switch is integrated on the chip in a complementary metal oxide semiconductor process, or is included in a module of the control circuit.

[0023] According to one embodiment, the validity detection is performed by detecting each pixel unit one by one through patrol control.

[0024] The system according to the present invention includes a microelectrode array having a plurality of through holes, each of which is connected to a control circuit, thereby making it possible to detect the conduction status of each through hole in real time, thereby improving the electrical conductivity of each electrode, and detecting the electrical conductivity of each electrode online in real time to ensure that the target sequence is correctly synthesized on the effective microelectrode array. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The specific embodiments will be described below with reference to the accompanying drawings, but the scope of the present invention is not limited thereto, wherein: Figure 1 A schematic diagram of the structure of a pixel unit in a microelectrode array according to an embodiment of the present invention is shown; Figure 2 A schematic diagram showing the structure of a pixel unit in a microelectrode array according to another embodiment of the present invention is shown; Figure 3 A schematic diagram showing the structure of the connection between the through hole of the pixel unit and the control circuit in the microelectrode array according to one embodiment of the present invention; Figure 4 A schematic diagram of a through-hole based self-test loop according to one embodiment is shown; Figure 5 A schematic diagram of a through-hole based self-test loop according to another embodiment is shown; Figure 6 A schematic cross-sectional view of the layer structure of a chip according to the present invention is shown; Figure 7 A process flow chart of forming a MEMS structure in a chip according to the present invention is shown; Figure 8 A schematic diagram of the chip structure according to the present invention is shown; Fig. 9 A complete flow chart of the method for synthesizing oligonucleotides according to the present invention is shown; Figures 10 to 12 A schematic diagram of a chip self-test result according to an embodiment is shown; Fig.13 A flow chart showing the synthesis steps of the method for synthesizing oligonucleotides according to the present invention is shown.

[0026] Description of reference numerals: 1-through hole; 2-working electrode; 3-pixel area; 4-pad area; 5-counter electrode; 6-normal pixel unit; 7-failed 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 DESCRIPTION

[0027] In the prior art, electrochemical oligonucleotide synthesis systems usually input a synthesis voltage or current into the chip according to the needs of synthesizing the target oligonucleotide, and evenly distribute the synthesis voltage or current to each open pixel of the chip in the hope of synthesizing the target oligonucleotide on each pixel.

[0028] However, in actual applications, there are cases where pixels fail during the synthesis process (usually due to defects in the chip processing technology), then the voltage or current actually introduced to each effective pixel will be larger than the set value, which will cause side reactions such as depurination during the synthesis of the oligonucleotide and cause insertion errors. For example, assuming that the synthesis of the target oligonucleotide requires each pixel unit to obtain a current of 0.1μA, when 5000 pixel units are turned on in the current synthesis state, the system will input 500μA of current to the chip. If all pixel units are valid, the current on each pixel unit is 0.1μA, but if 500 of the 5000 pixel units fail, the current on each pixel unit is 0.11μA, so the current actually allocated to the pixel unit is greater than the expected value of 0.1μA, which may cause undesirable synthesis side reactions.

[0029] The present invention provides a system for synthesizing oligonucleotides, which can timely understand the effective state of the pixel by introducing a self-checking structure in the pixel unit, so that the synthesis voltage or current can be correctly allocated to the pixel unit, so that the target sequence can be correctly synthesized on the normal pixel unit. For example, in the above example, if 500 pixel units out of the 5000 pixel units fail, only 450μA current needs to be input to the chip, so that the current on each pixel unit remains at 0.1μA.

[0030] According to one aspect, the present invention provides a system for synthesizing oligonucleotides, which may mainly include an electrode structure layer and a control circuit layer.

[0031] The electrode structure layer may include a microelectrode array, and the microelectrode array includes a plurality of pixel units, each pixel unit includes two or more through holes. The through holes are located at the bottom layer of the electrode structure layer and are used to electrically connect to the self-test structure below.

[0032] Specifically, see 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, 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.

[0033] According to one embodiment, Figure 1 As 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 also 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.

[0034] According to another embodiment, Figure 2 As shown, the working electrode 2 is a separate electrode structure. Figure 1 The embodiments shown are identical.

[0035] In the above Figure 1 In the embodiment, the electrode structure unit includes a ring-shaped electrode structure 22, so that the array electrode structure 21 in the electrode structure unit is separated from the array electrode structures of other pixel units.

[0036] See also Figure 3 A 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 portion of the through hole, or may fill the hole portion of the through hole, and may also form a conductive material layer 11 on the lower surface of the first dielectric layer for electrical connection with the self-test structure.

[0037] 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 a working electrode 2. The second conductive layer is patterned to form a counter electrode 5. The second dielectric layer is patterned to form a second dielectric structure 24.

[0038] According to a specific embodiment, the working electrode 2 may be formed of a noble metal material, such as a Pt electrode. The counter electrode 5 may be formed of the same or different noble metal material as the working electrode 2, preferably also a Pt electrode. The conductive material filled in the through hole 1 may be copper or aluminum.

[0039] In the system of the present invention, the self-test structure may include two or more transistor switches and a control circuit, wherein the number of transistor switches is the same as the number of through holes, and each through hole is connected to a corresponding transistor switch for conductivity detection of the electrode array.

[0040] In the system of the present invention, the transistor switch may be a Metal-Oxide-Semiconductor Field-Effect Transistor (MOSFET).

[0041] Specifically, in Figure 4 and Figure 5 In the specific embodiment shown, the control circuit may further include a pull-up resistor R, which is connected to the first transistor switch, and the second transistor switch is grounded. The potentials before and after the pull-up resistor are denoted as V0 and V t , so that the potential V tTo determine whether the electrical connection of the through hole is effective.

[0042] exist Figure 4 and Figure 5 In the embodiment, the control circuit is configured to inject a detection voltage or current between the paired through holes. When the electrical connection between the two through holes is turned on, the current forms a loop, V t is low level, then the pixel unit where the electrode corresponding to the through hole is located is marked as a valid pixel unit (such as Figure 4 As shown); when the electrical connection of any through hole is not conductive, the current cannot form a loop, V t is high level, then the pixel unit where the electrode corresponding to the through hole is located is marked as a failed pixel unit (such as Figure 5 The control circuit connects the transistor switches and the through holes one by one in a round-robin control manner and performs a conductivity test.

[0043] That is, when the pixel unit includes N through holes (N is a natural number greater than or equal to 2), the self-test structure includes N transistor switches accordingly, that is, the through holes have the same number as the transistor switches, and the N through holes are connected to the N transistor switches in a one-to-one correspondence. The control circuit needs to perform N× (N-1) / 2 connectivity tests to achieve the conductivity test of these N pairs of through holes-transistor switches.

[0044] In one embodiment of the present invention, the above-mentioned microelectrode array can be a micro-electro-mechanical system (MEMS) structure, and the transistor switches 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, and can also be included in a module of the control circuit.

[0045] Figure 6 A specific embodiment is shown, and the system for synthesizing oligonucleotides according to the present invention may include a MEMS structure and a CMOS structure, and preferably consists of a MEMS structure and a CMOS structure.

[0046] Specifically, the above-mentioned MEMS structure may include: The base layer is usually a first dielectric layer, and 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. The first dielectric layer having a plurality of through holes 1 is the first dielectric structure 23. A first conductive layer located on the base layer, wherein the first conductive layer is patterned to form a working electrode 2; A second dielectric layer formed on the working electrode 2 and separating the working electrode 2, wherein the second dielectric layer is patterned to form a second dielectric structure 24; A second conductive layer is formed on the second dielectric structure 24, wherein the second conductive layer is patterned to form a counter electrode 5; and A third dielectric layer is formed on the counter electrode 5 , and the third dielectric layer is patterned to form a third dielectric structure 49 .

[0047] Wherein, the above-mentioned multiple through holes 1 are located below the working electrode, and are filled with conductive metal materials, such as copper or aluminum. Wherein, the above-mentioned first dielectric layer, the second dielectric layer and the third dielectric layer can be independently formed of dielectric materials, for example, they can be formed of one or more of silicon oxide, silicon nitride and aluminum oxide. The above-mentioned 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 above-mentioned working electrode 2 and the counter electrode 5 can be formed of the same or different precious metal materials, for example, they can be formed of Pt. Further, the first dielectric layer forms a hole structure in the area corresponding to the working electrode 2 to expose a part of the working electrode 2, such as a first hole 50 formed by a window opening process to achieve connection between different functional layers, or a second hole 48 formed by a pad opening process to ensure good contact and conductivity between the pad and the external lead or packaging material.

[0048] like Figure 7 As shown, the preparation method of the above-mentioned MEMS structure includes the following steps: S110. The base layer is planarized; S120. The planarized base layer is etched to form a plurality of through holes; S130. Plating the through hole with a conductive metal material (eg, copper); S140. Performing chemical mechanical polishing (CMP) on the electroplated substrate layer; S150. Depositing a first noble metal material on the base layer after the metal chemical mechanical polishing, and patterning to form a working electrode; S160. Depositing a first insulating material and a second noble metal material on the working electrode and the substrate layer in sequence, and patterning them to form a counter electrode and a hole structure, while exposing a portion of the working electrode; S170. Depositing a second insulating material on the counter electrode, and performing a window opening process and a pad opening process to expose a portion of the working electrode and the counter electrode, respectively.

[0049] The base layer may be a first dielectric layer, or include a first substrate (e.g., a silicon substrate) and a first dielectric layer formed on the first substrate. The first dielectric layer may be formed of a dielectric material, for example, one or more of silicon oxide, silicon nitride, and aluminum oxide. The first noble metal material and the second noble metal material may be the same or different, for example, they may be Pt. The first insulating material and the second insulating material may be the same or different, for example, the first insulating material may be silicon nitride, and the second insulating material may be silicon oxide.

[0050] Further, the CMOS structure may include a second substrate (e.g., 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 may 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.

[0051] According to a specific embodiment, see Figure 8 , the system for synthesizing oligonucleotides according to the present invention may be a chip. Figure 8 The pixel area 3 and the pad area 4 are shown. Specifically, the chip may be a chip of a microelectrode array including 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 180nm CMOS process.

[0052] See also Fig. 9 According to another aspect of the present invention, there is provided a method for synthesizing oligonucleotides, comprising the following steps: S210. Import the fitted sequence into the control module. After receiving the fitted sequence, the control module will parse and process the sequence and 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 lighting conditions) to ensure that oligonucleotides are accurately synthesized according to the predetermined sequence at different positions of the chip. At the same time, the control module can also monitor various parameters and states during the synthesis process in real time, and warn and handle possible abnormal situations to ensure the efficiency and quality of the synthesis.

[0053] S220. A chip including a microelectrode array and a self-test structure is provided, wherein 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 a ring electrode structure surrounding the array electrode structure. The self-test structure may include a transistor switch and a control circuit respectively connected to the through holes. Two or more through holes and two or more transistor switches are provided in each pixel unit, and the through holes and the transistor switches are connected one-to-one.

[0054] S230. Decomposition of bases added at each synthesis step and determination of pixel cells to be turned on. During the electrochemical synthesis of oligonucleotides using a chip, the synthesis is performed step by step, base by base. Each synthesis step only adds a specific base, such as adenine (A), guanine (G), cytosine (C), or thymine (T).

[0055] S240. Perform validity detection on the pixel units to be turned on, and mark the detected valid pixel units and invalid pixel units.

[0056] The step includes: selecting any two of the N transistor switches to connect to the control circuit, and applying a constant current or voltage to perform a conductivity test. N is a natural number greater than or equal to 2. Specifically, when the electrical connection between the two through holes is turned on, the current forms a loop, and the pixel unit where the electrode corresponding to the through hole is located is marked as a valid pixel unit (such as Figure 4 As shown); when the electrical connection of any through hole is not conductive, the current cannot form a loop, V t is high level, then the pixel unit where the electrode corresponding to the through hole is located is marked as a failed pixel unit (such as Figure 5 This step also includes counting the effective pixel units and the failed pixel units respectively, and feeding back the counting results to the control module to determine the current size that the control circuit needs to apply.

[0057] Fig.10 The self-test result of a chip according to an embodiment of the present invention is shown, wherein the gray pixels in the box are pixels that pass the self-test, and the red pixels are pixels that fail the self-test.

[0058] Fig.10 The "G" pattern in the box A is a defect designed by the inventor (i.e., the Pt working electrode for connecting to the via hole is not plated in the pixel), and B is a defect detected randomly. Therefore, the chip design can correctly detect the defects of the electrode structure.

[0059] For the defects detected beyond human design, the inventors conducted focused ion beam (FIB) tests (such as Fig.11 and Fig.12 ). Among them, Fig.11 for Fig.10 The enlarged view of the area shown in the box A includes the failed pixel unit 7 and the normal pixel unit 6, wherein the dotted line "G"-shaped pattern represents the failed pixel unit 7 that is intentionally designed without a Pt working electrode (as shown in the rectangular box in the pixel unit). Fig.12 The test results showed that the Pt working electrode was discontinuous at the through-hole. Therefore, this chip design is capable of detecting defects in microelectrode array processing.

[0060] S250. If all pixel units are valid, the control module determines the magnitude of the current or voltage that the control circuit needs to apply according to the number of valid pixel units determined in step S240 and the magnitude of the current or voltage that each pixel expects to obtain. For example, if the number of valid pixel units determined in step S240 is 5000 and the current that each pixel expects to obtain is 0.1 μA, the current that the control circuit needs to apply to the system is 500 μA.

[0061] S260. Electrochemically synthesizing oligonucleotides in the working area corresponding to the effective pixel unit.

[0062] S270. If an invalid pixel unit is detected, the control module deletes the sequence information to be synthesized on the marked invalid pixel unit, updates the base added in each synthesis step, and determines the valid pixel unit that needs to be opened.

[0063] S280. According to the number of effective pixel units to be opened and the magnitude of the current or voltage expected to be obtained by each pixel, the input current or voltage of the chip is adjusted. In this step, the number of effective pixel units determined has been subtracted from the number of invalid pixel units, and the pre-applied current or voltage determined thereby meets expectations, so as to ensure that the current or voltage allocated to each pixel unit meets the magnitude of the current or voltage required for the expected synthesis sequence, and avoid the situation where the current or voltage allocated to the remaining effective pixel units is too large due to the invalidity of some pixel units, resulting in side reactions such as depurination and the introduction or insertion of the wrong sequence, thereby ensuring that the required sequence is correctly synthesized on the effective pixels.

[0064] S290. Electrochemically synthesizing oligonucleotides in the working area corresponding to the turned-on effective pixel unit.

[0065] In the method of the present invention, the electrochemical synthesis of oligonucleotides includes introducing the synthesis raw materials of the electrochemical synthesis of oligonucleotides, and using the electrochemical reaction to generate a localized acidic region on the electrode surface, thereby avoiding the need to add a strong acid in the deprotection step in the chemical synthesis method. The electrochemical synthesis of oligonucleotides can adopt the phosphoramidite triester method, which can generally include four steps of deprotection, coupling, oxidation and capping. In this method, the synthesis raw materials may include a base reagent, a deprotection reagent, a coupling agent, an oxidizing reagent and a capping reagent.

[0066] Specifically, the working electrode for electrochemical synthesis of oligonucleotides is usually pre-protected with dimethoxytrityl (DMT), for example, the electrode can be immersed in a 0.1mM thiol anhydrous ethanol solution for 12 hours, and then a chemical phosphorylation reagent and a coupling reagent are added to the electrode. The phosphate group of the chemical phosphorylation reagent will couple with the hydroxyl group on the thiol, thereby forming a monolayer with a DMT protective group on the electrode. After that, the chip can be used as a carrier to synthesize oligonucleotides according to the phosphoramidite tripeptide method.

[0067] See also Fig.13 According to the present invention, the steps of electrochemically synthesizing oligonucleotides may include: introducing the raw materials for electrochemically synthesizing oligonucleotides into the working area; and performing deprotection, coupling, oxidation and capping on the surface of the working electrode within the working area under the action of the input current.

[0068] The raw materials include electrochemical deprotection reagents, coupling reagents, oxidation reagents and capping reagents.

[0069] Among them, the chemical phosphorylation reagent can be The thiol may be HSCH2(CH2)9CH2O(CH2)2O(CH2)2O(CH2)2OH.

[0070] The electrochemical deprotection reagent may include p-phenol, p-benzoquinone and an organic base, preferably consisting of p-phenol, p-benzoquinone and an organic base, wherein the organic base may be pyridine or 2,6-lutidine; in particular, it may 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; and p-benzoquinone can be reduced on the counter electrode to generate an organic alkaline substance.

[0071] The coupling reagent may include a base monomer and tetrazole in acetonitrile; the oxidizing reagent may include iodine, water and pyridine; and the capping reagent may include N-methylimidazole and acetic anhydride in acetonitrile.

[0072] 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 include: A microelectrode array, comprising a plurality of pixel units, and each pixel unit comprises two or more through holes; A self-test 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; 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 comprises 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 separate electrode structure.

3. The system according to claim 1, wherein: The layer structure where the microelectrode array is located is a micro-electromechanical 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 switches are integrated into the system using 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 via is formed in a complementary metal oxide semiconductor structure.

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

9. Use of the system according to any one of claims 1 to 8 for electrochemical synthesis of oligonucleotides.

10. 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 pixel units; Decompose the bases added at each synthesis step and determine the pixel cells to be opened; Performing validity detection on the pixel units to be turned on, and marking the detected valid pixel units and invalid pixel units; Update the bases added in each synthesis step and determine the valid pixel units that need to be turned on; According to the number of effective pixel units that need to be turned on, adjusting the composite voltage or current input to the chip; Oligonucleotides are electrochemically synthesized in the working area corresponding to the turned-on effective pixel unit.

11. The method according to claim 10, wherein: The performing validity detection on the pixel unit to be turned on includes: Setting N through holes in each pixel unit that needs to be opened; Setting N transistor switches, connected to the N through holes respectively; Select any two of the N transistor switches to be electrically connected to the control circuit, apply a detection voltage or current, and perform a connectivity test; Wherein, N is a natural number greater than or equal to 2.

12. The method according to claim 11, wherein: Performing validity detection on the pixel unit to be turned on and marking the detected valid pixel unit includes: Performing N × (N-1) / 2 connectivity checks on the pixel units to be opened; and When there are no electrically connected pairs of through holes in the pixel unit to be opened, marking the pixel unit as a failed pixel unit; When there is at least one pair of electrically connected through holes in the pixel unit to be opened, the pixel unit is marked as a valid pixel unit.

13. The method according to any one of claims 10 to 12, wherein: The microelectrode array includes a working electrode and a counter electrode surrounding the working electrode array.

14. The method according to claim 13, wherein: The working electrode comprises one or more array electrode structures and a ring electrode structure surrounding the one or more array electrode structures, and a surface of the working electrode has a protective group.

15. The method according to claim 14, wherein: The electrochemically synthesized oligonucleotides in the working area corresponding to the opened effective pixel unit include: introducing an electrochemical deprotection reagent into the working area; Under the action of the synthetic voltage or current, deprotecting the surface of the working electrode in the working area; The raw materials for synthesizing oligonucleotides are introduced into the working area for coupling, oxidation and capping.

16. The method according to claim 15, wherein: The raw materials include coupling reagents, oxidizing reagents and capping reagents.

17. The method according to claim 16, 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 includes iodine, water and pyridine; The capping reagent includes an acetonitrile solution of N-methylimidazole and acetic anhydride.

18. The method according to claim 11, wherein: The transistor switch is integrated on the chip in a complementary metal oxide semiconductor process, or is included in a module of the control circuit.

19. The method according to claim 10, wherein: The effectiveness detection is performed by detecting each pixel unit one by one through patrol control.

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