Biosynthesis chip, biosynthesis device and synthesis method
By setting up isolated first and second synthesis chambers on the biosynthesis chip and performing quality inspection after each round of cycle operations, the problem of low synthesis accuracy of existing DNA monomer synthesis technology is solved, and a higher synthesis accuracy is achieved.
Patent Information
- Application Number
- CN202510213707.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-05-30
AI Technical Summary
The synthesis accuracy of existing DNA monomer synthesis technology is low, and how to improve the synthesis accuracy has become an urgent problem for technicians.
A biosynthesis chip is designed, including an isolated first synthesis chamber and a second synthesis chamber, and perform multiple rounds of cycle operations simultaneously under the same synthesis conditions, and the formed monomer is quality-checked through the second synthesis chamber after each cycle operation to judge the synthesis quality of the first synthesis chamber.
The synthesis quality is judged through the quality inspection results, which improves the synthesis accuracy of DNA fragments, makes up for the shortcomings in the existing technology, and has broad application prospects.
Smart Images
Figure CN120054370A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biosynthesis, and in particular to a biosynthetic chip, a biosynthetic device and a synthesis method. Background Art
[0002] Synthetic Biology is a new and interdisciplinary subject that can use gene modules and engineering means to assemble and build a living system that does not exist in nature. The essence of synthetic biology lies in design and creation, and its core basis is deoxyribonucleic acid (DNA).
[0003] However, the current DNA monomer synthesis technology has the problem of low synthesis accuracy rate. How to improve the synthesis accuracy rate of DNA monomer synthesis technology has become a technical problem to be solved urgently by those skilled in the art. Summary of the Invention
[0004] In order to solve at least one of the above problems, the first embodiment of the present invention provides a biosynthetic chip, which includes a first region and a second region, wherein,
[0005] The first region includes a first synthesis chamber, and the first synthesis chamber includes a plurality of first electrode units for synthesizing deoxyribonucleic acid fragments of a first length;
[0006] The second region includes a second synthesis chamber isolated from the first synthesis chamber, and the second synthesis chamber includes the same number of second electrode units as the first length;
[0007] The first synthesis chamber and the second synthesis chamber perform multiple rounds of cyclic operations synchronously under the same synthesis conditions. Each round of cyclic operation completes monomer synthesis at a specified position by controlling the voltages of the first electrode units and the second electrode units; the second synthesis chamber is used to perform quality inspection operations on the formed monomers after each round of cyclic operation.
[0008] For example, in the biosynthetic chip provided in some embodiments of the present application, the biosynthetic chip further includes a first sample inlet and a first sample outlet communicating with the first synthesis chamber, and a second sample inlet and a second sample outlet communicating with the second synthesis chamber;
[0009] The first sample inlet is isolated from the second sample inlet and the second sample outlet respectively, and the second sample inlet is isolated from the first sample inlet and the first sample outlet respectively.
[0010] For example, in the biosynthetic chip provided in some embodiments of the present application, the area of the first electrode unit is less than or equal to the area of the second electrode unit;
[0011] The first electrode units are arranged in an array along the row direction and / or the column direction, and the area of each first electrode unit is less than or equal to 1 mm 2 , and the distance between adjacent first electrode units is greater than or equal to 50 μm;
[0012] The second electrode units are arranged in an array along the row direction and / or the column direction, and the area of each second electrode unit is greater than or equal to 1 mm 2 , and less than or equal to 100 mm 2 , and the distance between adjacent second electrode units is greater than or equal to 1 mm.
[0013] For example, in the biosynthetic chip provided by some embodiments of the present application, the same synthesis conditions include the same current and the same energization time applied to the first electrode units and the second electrode units, and the dosage of the reagent for monomer synthesis.
[0014] For example, in the biosynthetic chip provided by some embodiments of the present application, the ratio of the dosage of the reagent for monomer synthesis applied to the first electrode units to the area of the first electrode units is the same as the ratio of the dosage of the reagent for monomer synthesis applied to the second electrode units to the area of the second electrode units.
[0015] For example, in the biosynthetic chip provided by some embodiments of the present application, the first length is greater than or equal to 20 bp.
[0016] The second embodiment of the present invention provides a biosynthetic device, including the biosynthetic chip described in the first embodiment.
[0017] The third embodiment of the present invention provides a synthesis method applied to the biosynthetic device described in the second embodiment, including:
[0018] Initializing a first operation quantity and a second operation quantity, where the first operation quantity is the number of cycles of the deoxyribonucleic acid fragments in the biosynthetic chip, the number of cycles is the length of the deoxyribonucleic acid fragments, and the second operation quantity is the number of cycles of the monomer synthesis that has been completed;
[0019] Judging the second operation quantity according to the first operation quantity, and exiting the synthesis method when the second operation quantity is greater than or equal to the first operation quantity;
[0020] According to the second operation quantity, synchronously perform monomer synthesis on a plurality of first electrode units at specified positions in the first synthesis chamber of the biosynthetic chip and the second electrode units corresponding to the second operation quantity in the second synthesis chamber under the same synthesis conditions;
[0021] Using a detection reagent to detect the monomers formed in this round of cyclic operation in the second synthesis chamber and generate a detection result;
[0022] Update the second operation quantity according to the detection result, and jump to judging the second operation quantity according to the first operation quantity.
[0023] For example, in the synthesis method provided by some embodiments of the present application,
[0024] The detecting the monomers formed in the current cycle operation in the second synthesis chamber by using a detection reagent and generating a detection result further includes:
[0025] Injecting a fluorescence detection reagent corresponding to the monomers formed in the current cycle operation into the second synthesis chamber for detection operation;
[0026] Performing a cleaning operation on the second synthesis chamber by using a cleaning reagent;
[0027] Detecting the second synthesis chamber by using a fluorescence microscope, if fluorescence is detected, generating a first detection result, otherwise generating a second detection result;
[0028] The updating the second operation quantity according to the detection result further includes: when the detection result is the first detection result, adding 1 to the second operation quantity, and when the detection result is the second detection result, keeping the second operation quantity unchanged.
[0029] For example, in the synthesis method provided by some embodiments of the present application, the performing monomer synthesis on a plurality of first electrode units at designated positions in the first synthesis chamber of the biosynthetic chip and second electrode units corresponding to the second operation quantity in the second synthesis chamber synchronously under the same synthesis conditions according to the second operation quantity further includes:
[0030] Performing a cleaning operation on the first synthesis chamber and the second synthesis chamber respectively by using a cleaning reagent;
[0031] Injecting a deprotecting reagent into the first synthesis chamber and the second synthesis chamber respectively, and controlling the current and energization time of a plurality of first electrode units at designated positions in the first synthesis chamber and second electrode units corresponding to the second operation quantity in the second synthesis chamber;
[0032] Injecting monomer reagents and active agents required for the current cycle operation into the first synthesis chamber and the second synthesis chamber respectively for monomer synthesis;
[0033] Injecting a blocking agent into the first synthesis chamber and the second synthesis chamber respectively for blocking operation;
[0034] Injecting an oxidant into the first synthesis chamber and the second synthesis chamber respectively for oxidation operation;
[0035] Clean the first synthesis chamber and the second synthesis chamber respectively using a cleaning reagent.
[0036] The fourth embodiment of the present invention provides a computer-readable storage medium, on which a computer program is stored. When the program is executed by a processor, the method described in the third embodiment is implemented.
[0037] The fifth embodiment of the present invention provides a computer device, including a memory, a processor, and a computer program stored on the memory and executable on the processor. When the processor executes the program, the method described in the third embodiment is implemented.
[0038] The beneficial effects of the present invention are as follows:
[0039] In view of the existing problems, the present invention provides a biosynthetic chip, a biosynthetic device, and a synthesis method. By setting up isolated first and second synthesis chambers, monomer synthesis operations are synchronously carried out under the same synthesis conditions. After completing one round of monomer synthesis operations, it is determined whether each monomer synthesized in the first synthesis chamber in this round of monomer synthesis operations is correct according to the quality inspection result of the monomers synthesized in the second synthesis chamber. Thus, the problems existing in the prior art are made up for, the synthesis accuracy rate of the biosynthetic chip is effectively improved, and it has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0041] Figure 1 Schematic diagram showing the synthesis sequence length distribution of deoxyribonucleic acid fragments of the same length;
[0042] Figure 2 Schematic diagram showing the structure of the biosynthetic chip according to an embodiment of the present invention;
[0043] Figure 3 Flowchart showing the synthesis method according to an embodiment of the present invention;
[0044] Figure 4 Block diagram showing the structure of the biosynthetic device according to an embodiment of the present invention;
[0045] Figure 5 Schematic diagram showing the structure of the biosynthetic chip after monomer synthesis according to an embodiment of the present invention;
[0046] Figure 6Schematic structural diagram of the second synthesis chamber after fluorescence detection according to an embodiment of the present invention;
[0047] Figure 7 Schematic structural diagram of a computer device according to another embodiment of the present invention. Detailed implementation manners
[0048] To describe the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.
[0049] Synthetic biology is a new discipline based on multiple disciplines such as genetic engineering, systems biology, and computer engineering. Its application demands in fields such as medicine, energy, information, and agriculture are increasing day by day, with broad investment prospects and huge development potential. Synthetic biology combines the knowledge and technologies of multiple disciplines such as biology, engineering, informatics, and chemistry, and is a new interdisciplinary research field. The core goal of synthetic biology is to design and construct new biological parts (such as enzymes, genetic circuits, cells, etc.), biological systems, and biological machines, or to redesign existing natural biological systems so that they can perform some specific and useful functions.
[0050] According to the technical principle, the mainstream DNA monomer synthesis technologies can be divided into four generations. The first-generation DNA monomer synthesis technology uses the phosphoramidite triester synthesis method, generally referred to as column synthesis; the second-generation DNA monomer synthesis technology is based on chips, including inkjet method, photochemical method, and electrochemical method; the third-generation DNA monomer synthesis technology is the ultra-high-throughput synthesis technology, and its core feature lies in the combination of semiconductor and electrochemical method, with high throughput and low cost; the current latest-generation DNA monomer synthesis technology is the enzymatic synthesis technology, mainly including microarray method, in vivo DNA monomer synthesis method in yeast, and ligation-mediated DNA synthesis method. However, since this technology is not yet mature, it has not been commercialized. Among the above DNA monomer synthesis technologies, the second-generation DNA monomer synthesis technology has entered the stage of domestic substitution, and the third-generation ultra-high-throughput synthesis technology has also entered a period of rapid growth.
[0051] Synthesizing nucleic acid fragments on a chip is an advanced technology in synthetic biology, which usually involves using microfluidic chips or solid-phase synthesis technologies to parallelly synthesize a large number of short DNA fragments. These technologies can significantly improve the efficiency of DNA synthesis, reduce costs, and accelerate the gene manufacturing and detection processes.
[0052] Microfluidic chip technology is a key technology used for synthesizing nucleic acid fragments on a chip. By performing DNA synthesis on a microfluidic chip, high-throughput production of DNA fragments can be achieved. Microfluidic chips can create tiny fluid channels and reaction chambers, which can be used to synthesize DNA fragments and precisely control the reaction conditions through an integrated fluid control system.
[0053] DNA in-situ synthesis chips connect nucleotide molecules one by one in sequence at specific positions. Since a large number of oligonucleotide sequences can be synthesized on a relatively small area, this technology can produce chips arranged with high-density nucleic acid sequences in large quantities. For example, the chemical synthesis reaction of oligonucleotides in chip synthesis is completed on a carrier that modifies the chip. According to the predetermined base sequence, the corresponding four phosphoramidite synthesis monomers (i.e., adenine A, thymine T, guanine G, and cytosine C) and other chemical reagents are sequentially added to the solid-phase carrier that has been pre-modified on the surface through a liquid path system (for example, a system including microchannels) to complete the synthesis of the specified oligonucleotide sequence. During chip synthesis, the synthesis volume of a single oligonucleotide is small, which can greatly reduce the consumption of reagents and achieve the purpose of low-cost synthesis.
[0054] For example, using an electric current for DNA synthesis is a technology based on electrochemical principles. This method usually involves directly synthesizing a DNA sequence on the surface of an electrode and controlling the chemical reaction process through an electric current to add nucleotides one by one. As Figure 1 shown, it is a schematic diagram of the length distribution for detecting the finally synthesized DNA sequence when biosynthesizing deoxyribonucleic acid fragments of the same length, for example, when synthesizing a 64bp length. Among them, the x-axis is the length of the synthesized DNA sequence, and the y-axis is the number of synthesized DNA sequences. As shown, the length with the largest proportion in the finally synthesized DNA sequence is 64bp. At the same time, there are also cases where the lengths are 63bp / 62bp / 61bp / 60bp / 59bp, that is, one or more bases are lost in the synthesized DNA sequence, affecting the synthesis accuracy rate of the DNA sequence.
[0055] In view of the above situation, through a large amount of research and experiments, the inventors proposed that according to the electrochemical technology principle of using an electric current for DNA synthesis, considering that only the target base raw materials are contained in the reaction reagents in each step of synthesis, and the type (ATCG) of bases will not be incorrect during the synthesis process, the problem resulting in a reduction in the DNA sequence length is that in each round of synthesis reaction, the reaction reagents and the electrifying time will affect the synthesis effect. For example, insufficient electrifying time - poor acid production effect - incomplete de-capping - this round of synthesis reaction is invalid - base loss.
[0056] Based on the above problems and the reasons causing these problems, as Figure 2As shown, an embodiment of the present invention provides a biosynthetic chip 100, which includes a first region A1 and a second region A2. Among them,
[0057] the first region A1 includes a first synthesis chamber 10, and the first synthesis chamber 10 includes a plurality of first electrode units 11 for synthesizing deoxyribonucleic acid fragments of a first length;
[0058] the second region A2 includes a second synthesis chamber 20 isolated from the first synthesis chamber 10, and the second synthesis chamber 20 includes a number of second electrode units 21 equal to the number of the first length;
[0059] the first synthesis chamber 10 and the second synthesis chamber 20 perform multiple rounds of cyclic operations synchronously under the same synthesis conditions. Each round of cyclic operation completes monomer synthesis at a specified position by controlling the voltages of the first electrode units 11 and the second electrode units 21; the second synthesis chamber 20 is used to perform quality inspection operations on the formed monomers after each round of cyclic operation.
[0060] In this embodiment, in view of the situation of base loss in monomer synthesis based on the electrochemical principle, by providing a first synthesis chamber and a second synthesis chamber that are isolated from each other on the biosynthetic chip, that is, the first synthesis chamber and the second synthesis chamber are independently provided and there is no transfer channel, and they do not affect each other during the synthesis reaction. Specifically, the first synthesis chamber includes a first electrode unit for synthesizing DNA fragments. For example, the length of the DNA fragment is 64 bp. The first synthesis chamber includes a plurality of electrode units and modification structures provided on the substrate for providing the monomer synthesis voltage. The second synthesis chamber also includes electrode units and modification structures provided on the substrate for providing the monomer synthesis voltage, and the number of the second electrode units in the second synthesis chamber is the same as the number of the lengths of the DNA fragments. That is, the length of the DNA fragment to be synthesized in the first synthesis chamber is 64 bp, and 64 rounds of cyclic operations are required. The second synthesis chamber includes 64 second electrode units. Under the same synthesis conditions, the monomer synthesis operations in the first synthesis chamber and the second synthesis chamber are carried out synchronously. That is, on the basis of the monomer synthesis in the first synthesis chamber, the same monomer synthesis is carried out on one of the second electrode units in the second synthesis chamber at the same time. The monomer synthesis on the second electrode unit is used as a control group for the monomer synthesis on the first electrode unit. After the monomer synthesis operation, by judging the quality of the monomers synthesized on the second electrode unit, the synthesis quality of the first electrode unit can be judged. For example, by performing fluorescence detection on the second electrode unit, when fluorescence is detected, it indicates that this round of synthesis operation is effective and the next round of T base synthesis can continue. When no fluorescence is detected, it indicates that this round of synthesis operation is invalid and the current round of A base synthesis operation needs to be repeated until fluorescence is detected by performing fluorescence detection on the second electrode unit. That is, by performing quality inspection on the monomers synthesized in the second synthesis chamber, the effective synthesis of the DNA fragment is ensured, thereby improving the synthesis accuracy rate of the DNA fragment.
[0061] In an alternative embodiment, the biosynthetic chip further includes a first sample inlet and a first sample outlet communicating with the first synthesis chamber, and a second sample inlet and a second sample outlet communicating with the second synthesis chamber; the first sample inlet is isolated from the second sample inlet and the second sample outlet respectively, and the second sample inlet is isolated from the first sample inlet and the first sample outlet respectively.
[0062] In this embodiment, considering the injection and discharge of reagents through the sample inlet and sample outlet of the synthesis chamber, to further improve the operational independence of the first synthesis chamber and the second synthesis chamber, the first sample inlet of the first synthesis chamber is isolated from the second sample inlet and the second sample outlet of the second synthesis chamber, that is, the reagents injected into the second sample inlet of the second synthesis chamber and the reagents discharged from the second sample outlet will not affect the first synthesis chamber; similarly, the second sample inlet of the second synthesis chamber is isolated from the first sample inlet and the first sample outlet of the first synthesis chamber, that is, the reagents injected into the first sample inlet of the first synthesis chamber and the reagents discharged from the first sample outlet will not affect the second synthesis chamber, thereby further improving the mutual isolation between the first synthesis chamber and the second synthesis chamber.
[0063] To further illustrate the specific implementation manner of this embodiment, a round of synthesis operation and quality inspection operation are used for illustration. As Figure 4 shown, a biosynthesis device provided by the present application includes a Figure 2 biosynthesis chip as shown, and a control unit and a microfluidic unit for example. The control unit can be configured to provide an electrical signal to the biosynthesis chip, for example, provide an electrical signal for monomer synthesis to the electrode unit. The microfluidic unit includes pipes and pumps for controlling the entry or exit of reagents into or out of the synthesis chamber, thereby controlling the synthesis of DNA fragments. Figure 2 The biosynthesis chip shown includes two regions arranged side by side on a substrate. The first region A1 and the second region A2 do not overlap. The first synthesis chamber and the second synthesis chamber are formed by grooving on the two regions respectively. The first synthesis chamber is used to form DNA fragments and includes a plurality of first electrode units. The length of the DNA fragment is 64 bp and 64 rounds of synthesis cycle operations are required. The second synthesis chamber includes 64 second electrode units. When each round of synthesis operation is performed in the first synthesis chamber, one second electrode unit in the second synthesis chamber synchronously performs a synthesis operation to form a control group for the monomer synthesis on the first electrode unit in the first synthesis chamber. As Figure 3 shown, the control unit of the biosynthesis device includes the following steps:
[0064] In the first step, initialize the first operation quantity and the second operation quantity. The first operation quantity is the number of cycle operations of the deoxyribonucleic acid fragment of the biosynthesis chip. The number of cycle operations is the length of the deoxyribonucleic acid fragment. The second operation quantity is the number of cycle operations of the completed monomer synthesis.
[0065] In this embodiment, the controller of the biosynthesis device defines two variables for judging whether to perform a cycle operation and initializes them before starting the synthesis operation. Specifically, the number of cycle operations of the DNA fragment is used as the initial value of the first operation quantity, and the second operation quantity for identifying the number of cycle operations of the completed monomer synthesis. The initial value of the second operation quantity is 0.
[0066] In the second step, determine the second operation quantity according to the first operation quantity, and exit the synthesis method when the second operation quantity is greater than or equal to the first operation quantity.
[0067] In this embodiment, the first operation quantity representing the number of cycles of DNA fragment operation and the second quantity of the number of cycles of monomer synthesis completed are used as the judgment for whether to continue the cyclic synthesis operation. Specifically, when the second operation quantity is less than the first operation quantity, it indicates that there are still monomers to be synthesized, and the synthesis operation continues; when the second operation quantity is greater than or equal to the first operation quantity, it indicates that there are no monomers to be synthesized, the DNA fragment has been synthesized, and it does not enter the cyclic synthesis operation.
[0068] In the third step, according to the second operation quantity, synchronously perform monomer synthesis on a plurality of first electrode units at specified positions in the first synthesis chamber of the biosynthetic chip and the second electrode units corresponding to the second operation quantity in the second synthesis chamber under the same synthesis conditions.
[0069] In this embodiment, on the basis of performing monomer synthesis in the first synthesis chamber, the same monomer synthesis is simultaneously performed on a second electrode unit in the second synthesis chamber, and the monomer synthesis on the second electrode unit is used as a control group for the monomer synthesis on the first electrode unit.
[0070] Specifically, as Figure 2 shown, in the first round of cyclic operation, synthesize base A, control the voltage and energization time applied to the first electrode units at each specified position in the first synthesis chamber, control the voltage and energization time applied to the first second electrode unit in the second synthesis chamber under the same conditions, and perform the synthesis operation by injecting sufficient reagents for monomer synthesis into the sample inlet of the first synthesis chamber and the sample inlet of the second synthesis chamber respectively.
[0071] Considering the reliability of the second electrode unit for quality inspection, in an optional embodiment, the area of the first electrode unit is less than or equal to the area of the second electrode unit;
[0072] The first electrode units are arranged in an array along the row direction and / or the column direction, and the area of each first electrode unit is less than or equal to 1 mm 2 , and the spacing L1 between adjacent first electrode units is greater than or equal to 50 μm;
[0073] The second electrode units are arranged in an array along the row direction and / or the column direction, and the area of each second electrode unit is greater than or equal to 1 mm 2 , and less than or equal to 100 mm 2 , and the spacing L2 between adjacent second electrode units is greater than or equal to 1 mm.
[0074] In this embodiment, the area of the second electrode unit is greater than or equal to a first preset size. For example, the area of the second electrode unit is greater than or equal to 1 mm 2 , so as to ensure the stability and reliability of the fluorescence reaction during subsequent fluorescence detection. Compared with the sizes of the bases on the synthesized DNA fragment, the area of the first electrode unit in the first synthesis chamber is less than or equal to the area of the second electrode unit in the second synthesis chamber. As Figure 2 shown, the first electrode units in the first synthesis chamber are arranged in an array, and the second electrode units in the second synthesis chamber are arranged in an array. The present application does not specifically limit the shape of the electrode unit, which can be circular, rectangular, or polygonal. When the electrode unit is circular, the distance between adjacent two electrode units is greater than or equal to the radius of the electrode unit; when the electrode unit is rectangular or polygonal, the distance between adjacent two electrode units is greater than or equal to half of the width of the electrode unit.
[0075] Considering further meeting the sufficient reagents for monomer synthesis, in an optional embodiment, the ratio of the amount of reagent used for monomer synthesis applied to the first electrode unit to the area of the first electrode unit is the same as the ratio of the amount of reagent used for monomer synthesis applied to the second electrode unit to the area of the second electrode unit.
[0076] In this embodiment, according to the area sum of the first electrode units corresponding to the number of A bases in the first round of cyclic operation in the first synthesis chamber, the sufficient reagents required are determined, and according to the area of one second electrode unit in the second synthesis chamber, the sufficient reagents required are determined. The two satisfy the same ratio of electrode unit area to reagent, thereby further ensuring monomer synthesis under the same conditions.
[0077] It should be noted that the present application does not specifically limit the length of the DNA fragment, which can be any length greater than or equal to 20 bp. By detecting the synthesized bases after each round of cyclic operation, the synthesis effectiveness and synthesis accuracy rate are improved. Considering that when the synthesis accuracy rate of the DNA fragment is the product of the accuracy rates of each round of cyclic operation, the shorter the length of the DNA fragment, the higher the synthesis accuracy rate, and vice versa.
[0078] In a specific example, the cyclic operation includes:
[0079] First, use a cleaning reagent to perform cleaning operations on the first synthesis chamber and the second synthesis chamber respectively.
[0080] In this embodiment, the cleaning reagent is acetonitrile. Acetonitrile is respectively injected into the first synthesis chamber and the second synthesis chamber for cleaning operations. After discharging the cleaning agent, argon gas is used to dry the first synthesis chamber and the second synthesis chamber.
[0081] Second, inject the deprotection reagent into the first synthesis chamber and the second synthesis chamber respectively, and control the current and energization time of a plurality of first electrode units at a specified position in the first synthesis chamber and the second electrode units corresponding to the second operation quantity in the second synthesis chamber.
[0082] In this embodiment, the deprotection reagent is an acetonitrile solution containing hydroquinone, etc. Inject the deprotection reagent into the first synthesis chamber and the second synthesis chamber respectively, energize the first electrode units to be synthesized in this round of cyclic operation in the first synthesis chamber and the first second electrode unit in the second synthesis chamber for 60 s, then use the cleaning reagent to clean the first synthesis chamber and the second synthesis chamber, and after discharging the cleaning agent, use argon to dry the first synthesis chamber and the second synthesis chamber.
[0083] Third, inject the monomer reagent and the activating agent required for this round of cyclic operation into the first synthesis chamber and the second synthesis chamber respectively at the same time for monomer synthesis.
[0084] In this embodiment, inject the A base monomer of this round of cyclic operation and tetrazole as the activator into the first synthesis chamber and the second synthesis chamber respectively and simultaneously, wait for 120 s and then discharge, and use argon to dry the first synthesis chamber and the second synthesis chamber.
[0085] Fourth, inject the capping agent into the first synthesis chamber and the second synthesis chamber respectively for capping operation.
[0086] In this embodiment, the capping reagents are Cap A and Cap B. Inject the Cap A and Cap B reagents into the first synthesis chamber and the second synthesis chamber respectively at the same time, wait for 35 s and then discharge, and use argon to dry the first synthesis chamber and the second synthesis chamber.
[0087] Fifth, inject the oxidizing agent into the first synthesis chamber and the second synthesis chamber respectively for oxidation operation.
[0088] In this embodiment, the oxidizing agent is a tetrahydrofuran solution of iodine. Inject the oxidizing agent into the first synthesis chamber and the second synthesis chamber respectively, wait for 30 s and then discharge, and use argon to dry the first synthesis chamber and the second synthesis chamber.
[0089] Sixth, use the cleaning reagent to clean the first synthesis chamber and the second synthesis chamber respectively.
[0090] In this embodiment, inject acetonitrile into the first synthesis chamber and the second synthesis chamber again for cleaning operation, and after discharging the cleaning agent, use argon to dry the first synthesis chamber and the second synthesis chamber.
[0091] Fourth step, use the detection reagent to detect the monomer formed in this round of cyclic operation in the second synthesis chamber and generate a detection result;
[0092] In this embodiment, after the monomer synthesis operation, the synthesis quality of the first electrode unit is judged by judging the quality of the monomer synthesized by the second electrode unit. Specifically, in this embodiment, fluorescence detection is performed on the second electrode unit. When fluorescence is detected, it indicates that the current synthesis operation is effective and the next round of T base synthesis can continue. When no fluorescence is detected, it indicates that the current synthesis operation is ineffective and the current round of A base synthesis operation needs to be repeated until fluorescence detection is performed on the second electrode unit and fluorescence is detected. That is, the quality inspection of the monomer synthesized in the second synthesis chamber is performed to ensure the effective synthesis of the DNA fragment, thereby improving the synthesis accuracy of the DNA fragment.
[0093] In a specific example, as Figure 5 shown, the detection operation includes:
[0094] First, the fluorescence detection reagent corresponding to the monomer formed in the current cycle operation is injected into the second synthesis chamber for detection operation.
[0095] In this embodiment, the fluorescent T base 31 paired with the A base is used for fluorescence detection with the first second electrode unit in the second synthesis chamber.
[0096] Second, a cleaning reagent is used to clean the second synthesis chamber.
[0097] In this embodiment, acetonitrile is injected into the second synthesis chamber for cleaning operation. After discharging the cleaning agent, argon is used to dry the second synthesis chamber.
[0098] Third, a fluorescence microscope is used to detect the second synthesis chamber. If fluorescence is detected, a first detection result is generated; otherwise, a second detection result is generated.
[0099] In this embodiment, as Figure 5 shown, when fluorescence 32 is captured during detection using a fluorescence microscope, it indicates that the current monomer synthesis is effective and a first detection result is generated; otherwise, it indicates that the current monomer synthesis is ineffective and a second detection result is generated.
[0100] Fifth step, update the second operation quantity according to the detection result, and jump to judging the second operation quantity according to the first operation quantity.
[0101] In this embodiment, when the detection result is the first detection result that the current monomer synthesis is effective, the second operation quantity is incremented by 1, indicating that the next round of base T synthesis can continue; when the detection result is the second detection result that the current monomer synthesis is ineffective, the second operation quantity remains unchanged, indicating that the current round of base A synthesis is repeated until fluorescence 32 is captured under the fluorescence microscope.
[0102] So far, a round of synthesis operation and quality inspection operation are completed. In this embodiment, by setting the isolated first synthesis chamber and the second synthesis chamber, the monomer synthesis operations are synchronously performed under the same synthesis conditions. After a round of monomer synthesis operation is completed, it is determined whether each monomer synthesized in the first synthesis chamber in this round of monomer synthesis operation is correct based on the quality inspection result of the monomers synthesized in the second synthesis chamber. When the second operation quantity is equal to the first operation quantity, as Figure 6 shown, all the second electrode units 21 of the second synthesis chamber 20 have completed monomer synthesis, and fluorescence detection is performed with the supporting base 31 to form corresponding fluorescence 32, indicating that the synthesis of the DNA fragment is effective, realizing the quality inspection of the monomer synthesis with a length of 64bp of the DNA fragment, and effectively improving the synthesis accuracy rate.
[0103] It should be noted that the structure of the biosynthetic chip of the present application is not specifically limited and can be various structures currently in use. For example, it is a chip formed by mating an upper substrate and a lower substrate. The first synthesis chamber and the second synthesis chamber can be arranged with side-by-side slots, or can be arranged in other ways, with the design criterion that the two are independent and isolated from each other, which will not be elaborated here.
[0104] Based on the biosynthetic chip of the above embodiment, an embodiment of the present application further provides a biosynthetic device, including the above biosynthetic chip.
[0105] In this embodiment, as Figure 4 shown, the biosynthetic device includes a biosynthetic chip, a control unit, and a microfluidic unit. The control unit can be configured to provide an electrical signal to the biosynthetic chip, such as providing an electrical signal for monomer synthesis to the electrode unit. The microfluidic unit includes pipelines and pumps for controlling the entry or outflow of reagents into or out of the synthesis chamber, thereby controlling the synthesis of the DNA fragment. In this embodiment, the first synthesis chamber and the second synthesis chamber are formed in two regions respectively. The first synthesis chamber is used to form the DNA fragment and includes a plurality of first electrode units. The second synthesis chamber includes the second electrode units with the number of DNA fragment lengths. When each round of synthesis operation is performed in the first synthesis chamber, a second electrode unit in the second synthesis chamber synchronously performs the synthesis operation to form a control group for the monomer synthesis on the first electrode unit in the first synthesis chamber, and the monomers synthesized by the second electrode are detected. By judging the quality of the monomers synthesized by the second electrode unit, the synthesis quality of the first electrode unit is judged. When it is detected that this round of synthesis operation is effective, the synthesis of the next round of base is continued. When it is detected that this round of synthesis operation is ineffective, this round of base synthesis operation needs to be repeated until the second electrode unit is detected and the monomer synthesis is effective. For the specific implementation manners of this embodiment, refer to the foregoing embodiments and will not be elaborated here.
[0106] As Figure 3As shown in the figure, an embodiment of the present application further provides a synthesis method, including:
[0107] Initialize the first operation quantity and the second operation quantity. The first operation quantity is the number of cycles of the deoxyribonucleic acid fragments on the biosynthetic chip, and the number of cycles is the length of the deoxyribonucleic acid fragments. The second operation quantity is the number of cycles of monomer synthesis that has been completed.
[0108] Judge the second operation quantity according to the first operation quantity. When the second operation quantity is greater than or equal to the first operation quantity, exit the synthesis method.
[0109] According to the second operation quantity, synchronously perform monomer synthesis on a plurality of first electrode units at a specified position in the first synthesis chamber of the biosynthetic chip and the second electrode units corresponding to the second operation quantity in the second synthesis chamber under the same synthesis conditions.
[0110] Use a detection reagent to detect the monomers formed in this round of cyclic operation in the second synthesis chamber and generate a detection result.
[0111] Update the second operation quantity according to the detection result, and jump to judge the second operation quantity according to the first operation quantity.
[0112] In this embodiment, by setting up isolated first and second synthesis chambers, monomer synthesis operations are synchronously performed under the same synthesis conditions. After completing one round of monomer synthesis operations, it is judged whether each monomer synthesized in this round of monomer synthesis operations in the first synthesis chamber is correct according to the quality inspection result of the monomers synthesized in the second synthesis chamber, thereby making up for the problems existing in the prior art and effectively improving the synthesis accuracy rate of the biosynthetic chip. For the specific implementation manners of this embodiment, refer to the foregoing embodiments and will not be elaborated here.
[0113] In an optional embodiment, the step of using a detection reagent to detect the monomers formed in this round of cyclic operation in the second synthesis chamber and generate a detection result further includes: injecting a fluorescence detection reagent corresponding to the monomers formed in this round of cyclic operation into the second synthesis chamber for detection operations; using a cleaning reagent to perform cleaning operations on the second synthesis chamber; using a fluorescence microscope to detect the second synthesis chamber. If fluorescence is detected, generate a first detection result, otherwise generate a second detection result.
[0114] The step of updating the second operation quantity according to the detection result further includes: adding 1 to the second operation quantity when the detection result is the first detection result, and keeping the second operation quantity unchanged when the detection result is the second detection result.
[0115] In this embodiment, by performing fluorescence detection on the monomers synthesized in the second synthesis chamber to determine whether the synthesis of each monomer in the first synthesis chamber in this round of cyclic operation is effective, the synthesis accuracy rate of DNA fragments can be improved. For the specific implementation manners of this embodiment, refer to the foregoing embodiments and will not be elaborated herein.
[0116] Another embodiment of the present invention provides a computer-readable storage medium, on which a computer program is stored, and when the program is executed by a processor, a synthesis method based on a biological synthesis device is implemented.
[0117] In practical applications, the computer-readable storage medium may adopt any combination of one or more computer-readable media. The computer-readable medium may be a computer-readable signal medium or a computer-readable storage medium. The computer-readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the above. More specific examples (a non-exhaustive list) of the computer-readable storage medium include: an electrical connection having one or more wires, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the above. In this embodiment, the computer-readable storage medium may be any tangible medium that contains or stores a program, and the program can be used by or in combination with an instruction execution system, apparatus, or device.
[0118] The computer-readable signal medium may include a data signal propagated in a baseband or as a part of a carrier wave, in which computer-readable program code is carried. Such a propagated data signal may take various forms, including but not limited to electromagnetic signals, optical signals, or any suitable combination of the above. The computer-readable signal medium may also be any computer-readable medium other than the computer-readable storage medium, and the computer-readable medium can send, propagate, or transmit a program for use by or in combination with an instruction execution system, apparatus, or device.
[0119] The program code included on the computer-readable medium can be transmitted by any appropriate medium, including but not limited to wireless, wire, optical cable, RF, etc., or any suitable combination of the above.
[0120] Computer program code for performing the operations of the present invention may be written in one or more programming languages or combinations thereof. The programming languages include object-oriented programming languages such as Java, Smalltalk, C++, and also include conventional procedural programming languages such as the "C" language or similar programming languages. The program code may be executed entirely on the user's computer, partially on the user's computer, executed as a stand-alone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In the case of a remote computer, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or may be connected to an external computer (e.g., through the Internet using an Internet service provider).
[0121] As Figure 7 shown, a schematic structural diagram of a computer device provided by another embodiment of the present invention is shown. Figure 7 The computer device T12 shown is only an example and should not impose any limitations on the functions and usage scope of the embodiments of the present invention.
[0122] As Figure 7 shown, the computer device T12 is presented in the form of a general-purpose computing device. The components of the computer device T12 may include, but are not limited to: one or more processors or processing units T16, a system memory T28, and a bus T18 connecting different system components (including the system memory T28 and the processing unit T16).
[0123] The bus T18 represents one or more of several types of bus architectures, including a memory bus or memory controller, a peripheral bus, a graphics acceleration port, a processor, or a local bus using any of the various bus architectures. For example, these architectures include, but are not limited to, an Industry Standard Architecture (ISA) bus, a Micro Channel Architecture (MAC) bus, an Enhanced ISA bus, a Video Electronics Standards Association (VESA) local bus, and a Peripheral Component Interconnect (PCI) bus.
[0124] The computer device T12 typically includes a variety of computer system-readable media. These media can be any available media accessible by the computer device T12, including volatile and non-volatile media, removable and non-removable media.
[0125] System memory T28 can include computer system readable media in the form of volatile memory, such as random access memory (RAM) T30 and / or cache memory T32. The computer device T12 can further include other removable / non-removable, volatile / non-volatile computer system storage media. By way of example only, a storage system T34 can be used for reading and writing on non-removable, non-volatile magnetic media ( Figure 7 not shown, typically referred to as a "hard disk drive"). Although Figure 7 not shown in the figure, a disk drive for reading and writing on removable non-volatile disks (such as "floppy disks") and an optical disk drive for reading and writing on removable non-volatile optical disks (such as CD-ROM, DVD-ROM or other optical media) can be provided. In these cases, each drive can be connected to the bus T18 through one or more data media interfaces. The memory T28 can include at least one program product having a set (e.g., at least one) of program modules configured to perform the functions of the embodiments of the present invention.
[0126] A program / utility T40 having a set (at least one) of program modules T42 can be stored, for example, in the memory T28. Such program modules T42 include, but are not limited to, an operating system, one or more application programs, other program modules, and program data. Each or some combination of these examples may include an implementation of a network environment. The program modules T42 generally perform the functions and / or methods in the embodiments described in the present invention.
[0127] The computer device T12 can also communicate with one or more external devices T14 (such as a keyboard, a pointing device, a display T24, etc.), and can also communicate with one or more devices that enable a user to interact with the computer device T12, and / or communicate with any device that enables the computer device T12 to communicate with one or more other computing devices (such as a network card, a modem, etc.). Such communication can be carried out through an input / output (I / O) interface T22. Moreover, the computer device T12 can also communicate with one or more networks (such as a local area network (LAN), a wide area network (WAN), and / or a public network, such as the Internet) through a network adapter T20. As Figure 7 shown, the network adapter T20 communicates with other modules of the computer device T12 through the bus T18. It should be understood that although Figure 7 not shown in the figure, other hardware and / or software modules can be used in combination with the computer device T12, including but not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data backup storage systems, etc.
[0128] The processor unit T16 executes various functional applications and data processing by running the programs stored in the system memory T28, for example, implementing a synthesis method based on a biosynthesis device provided by the embodiments of the present invention.
[0129] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.
Claims
1. A biosynthesis chip, characterized in that: It includes a first area and a second area, wherein: The first region includes a first synthesis chamber, which includes a plurality of first electrode units for synthesizing DNA fragments of a first length; The second region includes a second synthesis chamber isolated from the first synthesis chamber, and the second synthesis chamber includes a number of second electrode units of the first length; The first synthesis chamber and the second synthesis chamber synchronously perform multiple rounds of cycle operation under the same synthesis conditions, and each round of cycle operation completes monomer synthesis at a specified position by controlling the voltage of the first electrode unit and the second electrode unit; The second synthesis chamber is used to perform quality inspection on the formed monomers after each round of cycle operation.
2. The biosynthesis chip according to claim 1, characterized in that: The biosynthesis chip further comprises a first sample inlet and a first sample outlet connected to the first synthesis chamber, and a second sample inlet and a second sample outlet connected to the second synthesis chamber; The first injection port is isolated from the second injection port and the second outlet, respectively, and the second injection port is isolated from the first injection port and the first outlet, respectively.
3. The biosynthesis chip according to claim 1, characterized in that: The area of the first electrode unit is less than or equal to the area of the second electrode unit; The first electrode units are arranged in an array along the row direction and / or the column direction, and the area of each first electrode unit is less than or equal to 1mm 2 , a spacing between adjacent first electrode units is greater than or equal to 50 μm; The second electrode units are arranged in an array along the row direction and / or the column direction, and the area of each second electrode unit is greater than or equal to 1 mm 2 , and less than or equal to 100mm 2 , the spacing between adjacent second electrode units is greater than or equal to 1 mm.
4. The biosynthesis chip according to claim 3, characterized in that: The same synthesis conditions include the same current loaded on the first electrode unit and the second electrode unit and the same power-on time, as well as the amount of reagents used for monomer synthesis.
5. The biosynthesis chip according to claim 4, characterized in that: The ratio of the amount of reagent used for monomer synthesis of the first electrode unit to the area of the first electrode unit is the same as the ratio of the amount of reagent used for monomer synthesis of the second electrode unit to the area of the second electrode unit.
6. The biosynthesis chip according to any one of claims 1 to 5, characterized in that: The first length is greater than or equal to 20 bp.
7. A biosynthesis device, characterized in that: Comprising the biosynthesis chip as described in any one of claims 1-6.
8. A synthesis method applied to the biosynthesis device according to claim 7, characterized in that: include: Initializing a first operation number and a second operation number, wherein the first operation number is the number of cyclic operation rounds of the deoxyribonucleic acid fragment of the biosynthesis chip, the number of cyclic operation rounds is the length of the deoxyribonucleic acid fragment, and the second operation number is the number of cyclic operation rounds of completed monomer synthesis; Determining the second operation number according to the first operation number, and exiting the synthesis method when the second operation number is greater than or equal to the first operation number; According to the second operation quantity, under the same synthesis conditions, a plurality of first electrode units at designated positions in the first synthesis chamber of the biosynthesis chip and second electrode units corresponding to the second operation quantity in the second synthesis chamber are simultaneously synthesized into monomers; Using a detection reagent to detect the monomers formed in the current cycle operation in the second synthesis chamber and generate a detection result; The second operation quantity is updated according to the detection result, and the process jumps to determining the second operation quantity according to the first operation quantity.
9. The synthesis method according to claim 8, characterized in that The step of using a detection reagent to detect the monomers formed in the current cycle operation in the second synthesis chamber and generating a detection result further includes: Injecting the fluorescent detection reagent corresponding to the monomer formed in this round of cyclic operation into the second synthesis chamber for detection operation; Using a cleaning reagent to clean the second synthesis chamber; Using a fluorescence microscope to detect the second synthesis chamber, if fluorescence is detected, a first detection result is generated, otherwise a second detection result is generated; Updating the second operation quantity according to the detection result further includes: when the detection result is the first detection result, the second operation quantity is increased by 1, and when the detection result is the second detection result, the second operation quantity remains unchanged.
10. The synthesis method according to claim 8, characterized in that The step of synchronously performing monomer synthesis on a plurality of first electrode units at designated positions in the first synthesis chamber of the biosynthesis chip and a second electrode unit corresponding to the second operation quantity in the second synthesis chamber under the same synthesis conditions according to the second operation quantity further comprises: Using a cleaning reagent to clean the first synthesis chamber and the second synthesis chamber respectively; Injecting a deprotection reagent into the first synthesis chamber and the second synthesis chamber respectively, and controlling the current and power-on time of a plurality of first electrode units at designated positions in the first synthesis chamber and a second electrode unit corresponding to the second operation number in the second synthesis chamber; The monomer reagent and the active agent required for the synthesis of this round of cycle operation are simultaneously injected into the first synthesis chamber and the second synthesis chamber respectively to perform monomer synthesis; Injecting a sealing agent into the first synthesis chamber and the second synthesis chamber respectively to perform a sealing operation; Injecting an oxidant into the first synthesis chamber and the second synthesis chamber respectively to perform an oxidation operation; The first synthesis chamber and the second synthesis chamber are cleaned respectively using a cleaning reagent.
11. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the program is executed by a processor, the method according to any one of claims 8 to 10 is implemented.
12. A computer device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the program, the method according to any one of claims 8 to 10 is implemented.