WO2023116712A1 - High-throughput nucleic acid synthesis chip and use method thereof - Google Patents

Abstract

A chip for nucleic acid synthesis. The chip comprises a plurality of synthesis units; each synthesis unit comprises a plurality of three-dimensional layers; the first layer of the plurality of three-dimensional layers is a synthesis layer of a short nucleic acid sequence, and the second layer to the last layer are splicing layers of a nucleic acid sequence, wherein a plurality of synthesis reaction points exist in the first layer to synthesize a plurality of short nucleic acid sequences, and splicing reaction points exist on the second layer to the last layer so as to orderly splice the shorter nucleic acid sequences synthesized on the previous layer into longer nucleic acid sequences until a nucleic acid sequence having a complete length is generated.

Description

A high-throughput nucleic acid synthesis chip and its application method

cross reference

This application claims the benefit of Chinese patent application 202111559747.7 filed on December 20, 2021, which is incorporated herein by reference in its entirety.

technical field

The present disclosure relates to the fields of biopharmaceuticals and biosynthesis, specifically a chip for nucleic acid synthesis, its use method, related equipment and system.

Background technique

Biological drugs are in a stage of rapid development, such as antibody drugs, protein preparations, nucleic acid drugs, immune cell drugs, gene therapy drugs, etc. have been put into use on a large scale. In order to meet the needs of different indications, various types of new drugs are under large-scale research and development. The research and development of various types of drugs requires the synthesis of artificially designed genes and nucleic acid sequences for genetic engineering of target biological carriers (such as CHO cells, insect cells, immune cells, etc.). As a key process, the synthesis of artificially designed nucleic acid sequences is in great demand in the development and production of various types of biopharmaceuticals. At present, there are synthesis methods using traditional synthesis equipment and manual synthesis methods for the synthesis of artificially designed nucleic acid sequences.

Traditional nucleic acid sequence synthesis methods and equipment are difficult to meet the rapidly growing demand for industrial gene synthesis. In traditional synthesis equipment and methods, only 1 to 2 nucleic acid sequence samples can be synthesized at a time, and the synthesis efficiency of multiple samples is low. If the parallel synthesis of multiple samples is required to meet the requirements of high-throughput screening, a corresponding number of traditional equipment needs to be arranged linearly, and the equipment purchase cost is high. At the same time, in order to install a large amount of equipment, it is necessary to match a large area of the site, which greatly increases the investment in fixed assets. Secondly, although traditional synthesis equipment can effectively synthesize nucleic acid sequences with a length of tens of nucleotides, the R&D and production of biopharmaceuticals at this stage require the synthesis of hundreds of nucleotides, thousands of nucleotides, or even tens of thousands of nucleotides. A nucleic acid sequence of nucleotides in length. The short nucleic acid sequence produced based on traditional equipment requires a complex splicing process, with high material and time costs. At the same time, errors generated by the traditional orifice plate operation method require additional error correction operations. Third, the volume of the reaction chamber used in traditional synthesis equipment is large. After each single nucleic acid binding, a large amount of unreacted residual nucleic acid needs to be discharged, resulting in a large amount of expensive reagent waste; at the same time, the cleaning process for removing protection at the nucleic acid binding site In the process, a large proportion of reagents is wasted, and the waste of large-capacity reagents greatly increases the cost of synthesis. Moreover, traditional synthesis equipment is also inefficient in terms of time. The synthesis of about 20-50 bases takes several hours, and the synthesis of a single long sequence takes several days, thus greatly increasing the cycle of new drug development and screening.

The current manual operation method can satisfy the synthesis of a single/small number of sequences, but it cannot realize the preparation of multiple samples in parallel from the mechanism, and cannot realize the synthesis and screening of a large number of different configurations in the process of new drug development.

There is a need in the art for economical and efficient devices and methods capable of high-throughput parallel synthesis of a large number of different nucleic acid sequences.

Contents of the invention

The present disclosure provides a chip for high-throughput nucleic acid synthesis, which adopts a design similar to an integrated circuit and a three-dimensional three-dimensional parallel synthesis method, so as to realize industrial-level large-scale gene synthesis. The chip can synthesize hundreds to tens of thousands of nucleic acid sequences up to 10kb in parallel, meeting the requirements of high-throughput parallel comparison and screening. The integrated chip adopts an effective miniaturized reaction system, which can reduce the amount of reagents used in the synthesis process by orders of magnitude and shorten the reaction cycle by orders of magnitude.

The chip of the present disclosure can realize the synthesis of a single long-sequence sample, starting with a single nucleic acid, synthesizing gene sequences at the level of hundreds to 10 KB, and synthesizing hundreds to thousands of gene sequences at the level of KB to 10 KB in parallel, satisfying high-throughput Side-by-side comparison of screening requirements. Since the disclosed chip adopts an effective miniaturized reaction system, the amount of reagents used in the synthesis process can be reduced by orders of magnitude, and the reaction cycle can be shortened by orders of magnitude. Moreover, due to the use of a new three-dimensional parallel synthesis method different from the traditional synthesis method, the synthesis efficiency is greatly improved. Reduce the cost of gene synthesis by orders of magnitude through a multidimensional approach, including:

--Reduce the number of equipment by orders of magnitude, and reduce the total investment of equipment in high-throughput scenarios by orders of magnitude;

--Reduce the footprint of the facility by an order of magnitude, and cancel the need for large-scale temperature and humidity control of the facility by traditional equipment;

-- The amount of reagent used in the order of magnitude reduction process;

--Industrialized large-scale production of chip consumables to ensure the cost of consumables within a reasonable range;

In addition, the chip of the present disclosure realizes the closed and intelligent operation of the whole operation process by adopting integrated circuit design, realizes the data collection of the whole process, realizes the controllability and monitoring of the fully automatic process, and realizes Quick configuration and intelligent splicing.

In one aspect, the present disclosure provides a chip for nucleic acid synthesis, the chip includes a plurality of synthesis units, each synthesis unit includes a plurality of three-dimensional layers, wherein the first layer of the plurality of three-dimensional layers is short The synthesis layer of nucleic acid sequences, the second to last layer is the splicing layer of nucleic acid sequences, wherein there are multiple synthesis reaction points in the first layer to synthesize multiple short nucleic acid sequences, and there are splicing reactions in the second to last layer Click to sequentially splice the shorter nucleic acid sequences synthesized in the previous layer into longer nucleic acid sequences until a full-length nucleic acid sequence is produced. Preferably, one or more of the following are adjustable: the number of layers of synthesis units, the number of synthesis units, the number of synthesis reaction points in each synthesis unit of the first layer, the second layer to the last layer The number of splicing reaction sites in each synthetic unit of . For example, depending on the number of target nucleic acids desired, the number of synthetic units can be as high as 10,000.

In some embodiments, for the first layer, each synthesis unit comprises one or more first layer reagent entry channels, one or more first layer reagent distribution channels, a plurality of synthesis reaction sites, and access to the second layer. transmission channel, where:

The reagent inlet channel of the first layer is used to send reagents into the chip,

The first-layer reagent distribution channels are used to distribute the incoming reagents to the synthesis units of the first layer,

The plurality of synthesis reaction sites form an array of short nucleic acid synthesis, and each synthesis reaction site includes a synthesis reaction chamber for short nucleic acid sequence synthesis.

In some embodiments, each synthesis reaction site of each synthesis unit further comprises a plurality of microchannels for delivering reagents to the synthesis reaction chamber, each microchannel corresponding to a different reagent delivery, so that the Different reagents in the unit are sent to the synthesis reaction chamber of the synthesis reaction site respectively.

In some embodiments, the plurality of microchannels comprises at least 8 microchannels, which respectively correspond to A nucleotide solution, G nucleotide solution, T/U nucleotide solution, C nucleotide solution, cleaning solution, error correcting enzyme solution, guard site removal reagent, and stock solution, optionally also containing microchannels for delivery of initial microparticle activation reagents.

In some embodiments, each synthesis reaction point of each synthesis unit also includes a reagent concentration and transmission channel, and the reagent concentration and transmission channel is arranged between the plurality of microchannels and the synthesis reaction chamber, with The different reagents delivered by the plurality of microchannels are concentrated and transported into the synthesis reaction chamber.

In some embodiments, the starting particle is a starting particle that has been implanted in the synthesis reaction chamber, or is delivered into the synthesis reaction chamber through a microchannel.

In some embodiments, the synthesis reaction chamber has a starting particle fixed electrode with an opposite charge to the starting particle to hold the starting particle in place.

In some embodiments, the starting particle is charged with the same polarity as the nucleic acid molecule.

In some embodiments, the plurality of microchannels are configured in a star configuration or a linear configuration. For example, according to the arrangement of the microchannels, the channel configuration of each reaction point in the first layer of the synthesis unit is a star configuration or a linear configuration.

In some embodiments, when the plurality of microchannels are configured in a linear configuration, the microchannels used to deliver the cleaning solution into the synthesis reaction chamber are located at the outermost ends of the linear configuration.

In some embodiments, the number of synthesis reaction points in each synthesis unit of the first layer is 4, 8, 16, 32, 64, 128 or more reaction points, thereby synthesizing 4 sequences in parallel, 8 sequences, 16 sequences, 32 sequences, 64 sequences, 96 sequences, 128 or more nucleic acid sequences.

In some embodiments, for the second layer to the last layer, each synthesis unit includes a reagent entry channel, a reagent distribution channel, a splicing reaction chamber and a transfer channel to the next layer corresponding to each layer.

In some embodiments, each layer of the synthesis unit further comprises a waste liquid drainage channel.

In some embodiments, each channel is configured with a corresponding microscale micro-actuated valve, such as a microscale diaphragm valve or a microscale comb actuator valve.

In some embodiments, the synthesis reaction chamber and the stitching reaction chamber are provided with microscale pumps, such as microscale diaphragm pumps or microscale gear pumps, to facilitate the pumping of liquids.

In some embodiments, the reagent entry channels of each layer contain transfer pumps to facilitate the pumping of reagents entering the channels into the reaction chamber, and/or to pump excess reagents and reaction waste into the waste discharge channels .

In some embodiments, an electrode assembly comprising a plurality of electrodes arranged in a linear arrangement, each capable of being charged with the same or opposite polarity as the nucleic acid molecule, is disposed along the walls of the synthesis reaction chamber and the stitching reaction chamber.

In some embodiments, each synthesis unit is also linked to a PCR chamber that amplifies said full length nucleic acid sequence.

In some embodiments, the synthesis unit is fabricated on silicon-based, glass-based or polymer material-based material substrates.

In some embodiments, the chip is prepared by using a wafer, and the cross section of the chip is in a circular or rectangular configuration.

In some embodiments, the plurality of electrodes can be turned on independently, so that the electrodes at corresponding positions can be turned on independently based on the length of the nucleic acid sequence during the synthesis process.

In some embodiments, the micro-sized comb actuator valve includes a valve chamber, a valve element, and a drive structure for driving the valve element, wherein the drive structure includes a comb-shaped electrostatic driver and a comb-shaped electrostatic drive connected to the comb-shaped electrostatic drive. a push rod between the driver and the valve element, the push rod is configured to drive the valve element to leave or enter the valve cavity under the action of the comb-shaped electrostatic driver to open or close the micro-sized comb shape actuator valve.

In some embodiments, the comb-like electrostatic drive includes a first comb portion and a second comb portion, the push rod is fixedly connected to the first comb portion, and wherein the first comb A portion is movable towards or away from the second comb portion to drive the valve element out of or into the valve chamber via the push rod.

In some embodiments, the valve element is configured as a valve plate.

In one aspect, the present disclosure provides a method for parallel high-throughput nucleic acid synthesis using a chip as disclosed herein, comprising:

Multiple short nucleic acid sequences are synthesized in parallel at multiple synthesis reaction points on the first layer, and the shorter nucleic acid sequences synthesized on the previous layer are sequentially spliced into longer nucleic acid sequences at the splicing reaction points from the second layer to the last layer, until A full-length nucleic acid sequence is produced, and the last layer of each synthetic unit produces a full-length nucleic acid sequence.

In some embodiments, the method includes:

On the first layer:

(a) distributing the reagents to the respective synthesis units of the first layer through the first layer of reagent inlet channels and the first layer of reagent distribution channels;

(b) immobilizing the starting particle in a synthesis reaction chamber and pumping a starting particle activation reagent to activate the first activation site;

(c) pumping in a solution of the first nucleotide for incorporation of the first nucleotide;

(d) pumping cleaning fluid to pump excess reagent and reaction waste into the waste discharge channel;

(e) pumping in the protection site stripping agent to expose the binding site of the nucleotide, pumping in the cleaning solution again to discharge the excess protection site stripping agent;

(f) pumping into the solution of the next nucleotide to carry out the combination of the next nucleotide;

(g) repeating steps (d)-(f) until the length of the short nucleic acid sequence reaches the program setting value;

(h) cleave the synthesized short nucleic acid sequence from the starting particle and send it to the next layer of transport channel; and

On the second to last layer:

(i) receiving the first short nucleic acid sequence and fixing it in the splicing reaction chamber;

(j) receiving the second short nucleic acid sequence, splicing the first short nucleic acid sequence with the second short nucleic acid sequence in the presence of reagents required for splicing;

(k) pumping cleaning solution to pump excess reagent and reaction waste into the waste discharge channel;

(l) Steps (j)-(k) are repeated until the sequence length at this layer reaches the program setting value, and then the spliced sequence is sent to the transmission channel of the next layer.

In some embodiments, during the synthesis process of the first layer, the electrode assembly configured on the wall of the synthesis reaction chamber applies a charge of opposite polarity to the nucleic acid molecule, thereby maintaining the high linearity of the nucleic acid sequence and simultaneously exposing the binding site at the tail end point for the incorporation of a new nucleotide molecule at the end of an existing sequence.

In some embodiments, between steps (g) and (h), excess reagents and reactions in the synthesis process are removed from the synthesis reaction chamber by applying charges of the same or opposite polarity to the nucleic acid molecules through the electrode assembly configured on the wall of the synthesis reaction chamber. Waste liquid is pumped into the waste liquid discharge channel.

In some embodiments, the synthetic short nucleic acid sequence is cleaved from the starting particle in step (h) by pumping in the starting particle cutting enzyme.

In some embodiments, delivery of reagents required for splicing occurs before or after receipt of the second short nucleic acid sequence.

In some embodiments, during the splicing process from the second layer to the last layer, by applying a sequential alternating electric field to the electrode assembly arranged on the wall of the splicing reaction chamber, the received short nucleic acid sequence is driven to reciprocate so as to restore the linear conformation type.

In some embodiments, the method further includes performing PCR amplification on the full-length nucleic acid sequence after finishing the last layer.

In one aspect, the present disclosure provides a chip operation device for operating the chip disclosed herein or implementing the method disclosed herein, which includes a box, and the box is provided with: a chip operation platform for implementing the chip Accurate positioning during operation; the microtube array working head is used to supply reagents to the reagent inlet channel of the chip; and the working head guide rail is used to realize the up and down movement of the microtube array working head.

In some embodiments, the chip operation platform is arranged on a platform transmission guide rail, so that the chip operation platform can move in a horizontal plane in a horizontal direction or a longitudinal direction on the platform transmission guide rail.

In some embodiments, the box includes a temperature control element and/or a humidity control element to provide a required temperature and/or humidity environment for nucleic acid synthesis.

In some embodiments, the chip manipulation device further comprises an array of micropumps and/or temperature-controlled reagent storage tanks.

In some embodiments, the micropump array uses micropumps for reagent delivery, and optionally prepares an independent reagent channel for each reagent.

In one aspect, the present disclosure provides a system comprising:

The chip disclosed in this paper;

A nucleic acid sequence determination module for each synthesis reaction point in each synthesis unit; a splicing reaction sequence order designation module; a switch control module for each valve; an electrode electric field control module; a synthesis reaction point number designation module; a splicing reaction point number designation module.

Additional and/or other aspects and advantages of the disclosure will be set forth in the description which follows, or may be obvious from the description, or may be learned by practice of the disclosure. Various technical features of the present disclosure can be combined arbitrarily as long as they are not contradictory to each other.

Description of drawings

The above-mentioned features and advantages of the present disclosure, other features and advantages, and the manner of achieving them will become more apparent with reference to the following detailed description of specific embodiments of the present disclosure in conjunction with the accompanying drawings. In the attached picture:

Fig. 1 is a schematic diagram of a chip in a circular configuration according to an embodiment of the present disclosure, wherein each grid represents a three-dimensional synthesis unit.

Fig. 2 is a schematic diagram of a chip in a rectangular configuration according to an embodiment of the present disclosure, wherein each grid represents a three-dimensional synthesis unit.

FIG. 3 is an isometric view of a schematic structure of a single synthesis unit according to an embodiment of the present disclosure.

4 is a top view of a schematic structure of a single synthesis unit according to an embodiment of the present disclosure, wherein each star-shaped structure represents a reagent entry microchannel for a synthesis reaction site.

5 is a side view of a basic hierarchical architecture of a single synthesis unit according to one embodiment of the present disclosure, which shows the basic hierarchical architecture of a single synthesis unit in which a plurality of synthesis reaction sites of the first layer and The splicing reaction points from the second layer to the Nth layer and the final PCR amplification reaction chamber.

Fig. 6 is a schematic flow diagram of nucleic acid synthesis in a three-dimensional synthesis unit according to an embodiment of the present disclosure, including parallel synthesis of shorter nucleic acid sequences in the first layer, and layer-by-layer splicing of nucleic acid sequences in the second to Nth layers , and finally PCR amplification of the complete nucleic acid sequence.

7 is an axonometric view of the synthesis reaction site array of the first layer of the synthesis unit according to an embodiment of the present disclosure, wherein each star-shaped structure represents a reagent entry microchannel of a synthesis reaction site.

FIG. 8 is a top view of the synthesis reaction site array of the first layer of the synthesis unit according to an embodiment of the present disclosure, wherein each star-shaped structure represents a reagent entering a microchannel of a synthesis reaction site.

Fig. 9 is the basic pipeline configuration of the synthesis reaction point of the first layer of the synthesis unit according to an embodiment of the present disclosure. As an example, it includes 8 separate reagents entering the microchannel and the main channel for short nucleic acid sequence synthesis reaction. Reaction chamber and transfer channel to the second layer.

10 is a front view of the first layer of the synthesis unit according to one embodiment of the present disclosure, wherein the cleaning liquid channel is arranged at the end of the linear structure.

FIG. 11 is a top view of the first layer of the synthesis unit according to an embodiment of the present disclosure, wherein the cleaning liquid channel is arranged at the end of the linear structure.

12 is a detailed view of a synthesis reaction chamber of a reaction point of the first layer of the synthesis unit according to an embodiment of the present disclosure, wherein the reagents entering the microchannel from various reagents enter the reaction chamber through the transfer pump after collection chamber, and linearized electrodes are arranged on the walls of the reaction chamber.

FIG. 13 is a composition flowchart of the first layer of the composition unit according to one embodiment of the present disclosure.

Figure 14 is a schematic diagram of driving + linearizing electrodes on the walls of a reaction chamber to maintain nucleic acids in a linear configuration, according to one embodiment of the present disclosure.

Fig. 15 is a schematic diagram of the ligation of initiator particles (also known as initiator particles) and nucleic acid sequences in a synthesis reaction chamber according to an embodiment of the present disclosure.

16 is a schematic diagram of a micro-sized diaphragm pump employed in a chip according to one embodiment of the present disclosure.

17 is a schematic diagram of a microscale comb driver valve employed in a chip according to one embodiment of the present disclosure.

Fig. 18 is a structural diagram of splicing reaction chambers of the second layer to the Nth layer of the synthesis unit according to an embodiment of the present disclosure.

FIG. 19 is a flowchart of the splicing reaction of the second layer to the Nth layer of the synthesis unit according to an embodiment of the present disclosure.

FIG. 20 is a chip operating device for operating a chip of the present disclosure according to one embodiment of the present disclosure.

Detailed ways

The present disclosure will be described below with reference to the accompanying drawings, which show several embodiments of the disclosure. However, it should be understood that the present disclosure can be presented in many different ways, and is not limited to the embodiments described below; in fact, the embodiments described below are intended to make the disclosure of the present disclosure more complete and contribute to this Those skilled in the art fully explain the protection scope of the present disclosure. It should also be understood that the embodiments disclosed herein can be combined in various ways to provide even more additional embodiments.

For purposes of description, the terms "upper", "lower", "left", "right", "vertical", "horizontal", "top", "bottom", "transverse", "lengthwise" and their Derivatives are all relative to the orientation in the drawings of the present disclosure. However, it should be understood that the present disclosure may employ various alternative modifications unless expressly stated to the contrary. For example, if the device in the figures is turned over, features described as "below" other features would then be oriented "above" the other features. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the relative spatial relationships interpreted accordingly.

The singular forms "a", "the" and "the" used in the specification include plural forms unless clearly stated otherwise. The terms "comprising", "comprising" and "comprising" are used in the specification to indicate the presence of claimed features but not to exclude the presence of one or more other features. The term "and/or" used in the specification includes any and all combinations of one or more of the related listed items.

The disclosure relates to a chip for nucleic acid synthesis, which adopts a design similar to an integrated circuit and a three-dimensional three-dimensional parallel synthesis method, so as to realize large-scale gene synthesis at an industrial level. The chip may contain multiple synthesis units, and each synthesis unit may contain multiple three-dimensional layers to synthesize different artificially designed nucleic acid sequences. The first layer of the plurality of three-dimensional layers may be a synthesis layer of short nucleic acid sequences, and the second to last layers may be spliced layers of nucleic acid sequences. Multiple synthesis reaction sites can exist in the first layer to synthesize multiple short nucleic acid sequences in parallel. There may be splicing reaction points in the second layer to the last layer to orderly splice the shorter nucleic acid sequences synthesized in the previous layer into longer nucleic acid sequences until a full-length nucleic acid sequence is generated.

Referring to FIG. 1 , there is shown a configuration of a circular chip according to an embodiment of the present disclosure, which contains n synthesis units. In some embodiments, circular chips can be fabricated directly using small-diameter wafers, such as 3-4 inch diameter wafers. Circular chips can be consumed from a single wafer. Fig. 2 shows the configuration of a rectangular chip according to an embodiment of the present disclosure, which contains n synthesis units. In some embodiments, rectangular chips can be fabricated using, for example, 8-12 inch diameter wafers, wherein a single wafer can be diced into multiple rectangular chip consumables. In some embodiments, the chip may include a substrate made of silicon base, glass base, polymer material base, etc., and the synthesis unit may be processed on the substrate.

Referring to FIGS. 3 to 5 , there are respectively shown an axonometric view, a top view and a side view of the main structure of a single synthesis unit according to an embodiment of the present disclosure.

As shown more clearly in FIG. 5 , a single synthesis unit according to an embodiment of the present disclosure may include multiple three-dimensional layers, such as a first layer, a second layer, . . . , an Nth layer. In some embodiments, each of the plurality of steric layers can include one or more synthesis reagent entry channels, one or more synthesis reagent distribution channels, and one or more synthesis reaction sites. Each of the plurality of three-dimensional layers may also include one or more synthetic waste liquid discharge channels for discharging synthetic waste liquid or excess reagents.

As shown in Figure 5, the entry channel of the first layer of synthetic reagents can exemplarily include 8 channels, and the 8 channels can correspond to the A nucleotide solution channel, the G nucleotide solution channel, and the T/U nucleoside channel respectively. Acid solution channel (according to whether DNA or RNA is to be synthesized), C nucleotide solution channel, cleaning solution channel, error correction enzyme solution channel, protection site stripping agent channel and stock solution channel. The stock solution channel can be used to deliver some reagents according to specific needs, such as initial microparticle activation reagent or initial microparticle shearing enzyme solution, etc. In some embodiments, the entry channels of the synthetic reagents from the second layer to the Nth layer may also include 8 channels, and the 8 channels may correspond to the A nucleotide solution channel, the G nucleotide solution channel, the T/U Nucleotide solution channel (according to whether DNA or RNA is to be synthesized), C nucleotide solution channel, cleaning solution channel, error correction enzyme solution channel, protection site stripping agent channel and stock solution channel. However, the present disclosure is not limited thereto, and the first layer to the Nth layer synthesis reagent inlet channels may also include other numbers of channels.

As shown in Figure 5, the first layer of each synthesis unit can contain multiple synthesis reaction points (forming a short gene sequence synthesis array), and the number of synthesis reaction points can be adjusted according to needs, such as 4, 8, 16 , 32, 64, 96, 128 and so on. The optimal number of other synthetic reaction sites can be selected based on the process. In some embodiments, the number of synthesis reaction sites in the first layer of each synthesis unit may be 2 ⁿ , the number of splicing reaction sites in the second layer may be 2 ^n-1 , and the number of splicing reaction sites in the third layer It can be 2 ^n-2 , etc., decreasing in turn.

In some embodiments, A, G, T/U, and C nucleotide solutions, protection site remover, and chip Pipeline cleaning solvent, error correction enzyme solution, etc.; then through the distribution channel of the first layer of synthetic reagents, A, G, T/U, C nucleotide solutions, protection site removal agent, chip internal pipeline cleaning solvent, Error-correcting enzyme solution etc. are distributed in each synthesis reaction point of the first layer of each synthesis unit, and make it enter the each synthesis reaction point of the first layer through the microchannel (for example 8) corresponding to each kind of reagent respectively Synthesis of short nucleic acid sequences is performed in the synthesis reaction chamber.

Then, the short nucleic acid sequence synthesized in the first layer enters the second layer, and splicing of the short nucleic acid sequence is performed in the presence of reagents entered from the synthesis reagent entry channel and the synthesis reagent distribution channel of the second layer. Similarly, the splicing of shorter nucleic acid sequences from the previous layer is continuously performed from the third layer to the last layer, and the desired complete nucleic acid sequence is generated in the last layer, which is then entered into the complete sequence PCR chamber to combine the synthesized High-fold amplification of the complete nucleic acid sequence. In some preferred embodiments, the length of the short nucleic acid sequence synthesized in the first layer may be about 20-50 nucleotides in length. The synthesis of short nucleic acid sequences within this length range can effectively guarantee the accuracy and accuracy of the synthesis, and at the same time greatly increase the synthesis speed, and quickly provide large doses of high-accuracy spliced fusion samples for the subsequent splicing layer. The specific synthesis sequence of each reaction point in each synthesis unit can be set by a software program.

Referring to FIG. 6 , it shows the parallel synthesis of short nucleic acid sequences in the first layer of the synthesis unit, the layer-by-layer splicing of nucleic acid sequences in the second layer to the Nth layer, and the final complete nucleic acid sequence according to an embodiment of the present disclosure. The process of PCR amplification.

Before the synthesis reaction starts, starting particles are provided to the synthesis reaction chamber for initiating the incorporation of the first nucleotide of each synthesis reaction site. The starting particles may be the starting particles that have been implanted into the synthesis reaction chamber when the chip is prepared, or the starting particles delivered into the synthesis reaction chamber through corresponding channels before starting the synthesis.

In some embodiments, the starting particle is immobilized in the synthesis reaction chamber by the starting particle fixed electrode, and the starting particle fixed electrode has an opposite charge to the starting particle to keep the position of the starting particle relatively fixed.

After the first nucleotide is synthesized in parallel at each synthesis reaction point of the first layer, all synthesis reaction points enter the sequence incorporation of the next nucleotide in parallel, and the next synthesis reaction point is pumped into each synthesis reaction point accordingly nucleotide solution. The sequence synthesized at each synthesis reaction point may be the same or different, and the program preferably sets which nucleotide should be used in each step of extension of the sequence of the synthesis reaction point. In the synthesis reaction site, after each step of elongation of the nucleotide sequence, the excess nucleotide solution is washed away by sending a cleaning solvent into the chip through the corresponding channel (optionally, at the same time, the protection site is washed away). Removing agent solution), and then add the protection site removing agent solution and the solution of the next nucleotide to carry out the next step of nucleic acid sequence extension.

In some embodiments, after the synthesis of the short nucleic acid sequence in the first layer is completed, the short nucleic acid sequence is cut from the starting particle by adding a cutting enzyme solution of the starting particle. In some embodiments, these short nucleic acid sequences enter the second layer through the transmission channel leading to the second layer after leaving the reaction chamber. Preferably, the sequence of the synthesized short nucleic acid sequences entering the second layer is set by a program. After entering the second layer, the short nucleic acid sequence that first enters (also called the first short nucleic acid sequence) is fixed in the splicing reaction chamber (for example, fixed in the splicing reaction chamber with a fixed electrode), and then another short nucleic acid sequence (also known as the second short nucleic acid sequence) into the splicing reaction chamber. In some embodiments, reagents such as synthetase solution, cleavage enzyme solution, error correction enzyme solution, chip internal channel cleaning solution are fed into the channel through the synthesis reagent entry channel of the second layer; The solution, the error correction enzyme solution, the chip internal channel cleaning solution, etc. are distributed to each splicing reaction point, and then enter the splicing reaction chamber for the splicing reaction to occur. The delivery of the synthetic agent can be preceded or followed by the delivery of another short nucleic acid sequence. Preferably, the correctness of the splicing points can also be checked by an error correction enzyme. Using error-correcting enzymes in parallel within each synthetic unit can greatly reduce the overall time of the high-throughput synthetic process while facilitating the design of related error-correcting enzymes.

In some embodiments, similar to the second layer, in the third to N layers (i.e., all splicing layers), the synthetic reagents entering the channel through each layer are sent into the synthetic unit into the synthetic enzyme solution, the cleavage enzyme solution, the error correction Enzyme solution, chip internal channel cleaning solution and other reagents; synthetic enzyme solution, error correction enzyme solution, chip internal channel cleaning solution, etc. are distributed to the splicing reaction points of each layer through the synthetic array reagent distribution channel of each layer, and enter the splicing reaction chamber to generate splice reaction.

In some embodiments, one splicing reaction is performed in one splicing reaction site of one splicing layer, ie, only one splicing of two shorter nucleic acid sequences is performed. In some embodiments, multiple stitching reactions are performed in one stitching reaction site of one stitching layer. For example, after the splicing of the two short nucleic acid sequences is completed, continue to send in the third short nucleic acid sequence for splicing with the spliced sequence of the former, and then send in the fourth short nucleic acid sequence..., so that the incoming Shorter nucleic acid sequences are spliced into longer sequences in 3-fold, 4-fold or more.

In some embodiments, after all the splicing reactions at the splicing reaction site are completed, excess enzyme solution is washed away by sending a cleaning solvent into the chip through the corresponding channel. In some embodiments, the spliced sequence generated by the splicing reaction site is sent to the next layer, preferably, the sequence of the spliced sequence entering the next layer is set by the program.

Referring to FIG. 7 and FIG. 8 , there are shown an isometric view and a top view, respectively, of a first layer of a synthesis unit according to an embodiment of the present disclosure. On the first layer of the chip, the number of synthesis units can be set according to the number of target nucleic acids to be synthesized. For example, if 10,000 target nucleic acids are to be synthesized, 10,000 synthesis units can be set. In addition, according to the length of the target nucleic acid and the convenience of the process, 2, 4, 8, 16, 32, 64, 256 or other synthetic process settings can be configured in the first layer of the synthesis unit synthesis reaction points. According to the length of the target nucleic acid and the convenience of the process, N layers can also be configured for each synthesis unit, including the synthesis reaction layer of the first layer and the splicing reaction layer of the 2-N layer, and N can be, for example, 3-50 and wherein any integer value of . The target nucleic acids of different synthetic units may be the same or different, and the target nucleic acids may be DNA or RNA. In some embodiments, the short nucleic acid fragments with a length of 20 to 50 nucleic acids are all synthesized in the synthesis units of the first layer.

As disclosed above, configuring a large number of parallel synthesis units, the advantages brought by the new architecture include:

1) A micron-scale synthesis unit is formed inside the chip, which can reduce the amount of reagents used in the process by an order of magnitude compared with traditional synthesis equipment; at the same time, the micron-scale reaction system can effectively optimize the reaction size and greatly improve the efficiency of the synthesis reaction process , greatly shorten the cycle of single nucleic acid binding and residual reagent cleaning;

2) Short fragments can effectively control the error rate in the synthesis process, and effectively control the correct rate of the entire sequence through segmented control;

3) Parallel synthesis of a large number of synthetic units can form high-throughput gene sequence (splicing) basic materials in a relatively short period of time;

4) In different stages of synthesis, different reagents can be pumped in the same channel, which can reduce the complexity of the spatial structure inside the unit.

Referring to FIG. 9 , a structure of a reaction site of a first layer of a synthesis unit according to an embodiment of the present disclosure is shown. The structure includes 8 microchannels, which are used as pumping pipelines for various reagents. The eight microchannels can correspond to A nucleotide solution channel, G nucleotide solution channel, T/U nucleotide solution channel, C nucleotide solution channel, cleaning solution channel, error correction enzyme solution channel, Guard site stripper channel and stock solution channel. The stock solution channel is used as a spare channel to enable the introduction of any solution required for the synthesis reaction. Nucleotide protection sites can use traditional DMT protection groups or other customized groups; correspondingly, protection site removal agents can use common reagents, and other customized protection site removal agents can also be used. Selection and preparation of various reagents are well known to those skilled in the art. The structure can also include: reagent concentration and transmission channels, which are used to concentrate and transport various reagents sent through the 8 microchannels and send them into the main reaction chamber (also called "synthesis reaction chamber") ; a synthesis waste liquid discharge channel, which is used to discharge excess reagents from the main reaction chamber; and a transfer channel leading to the second layer, which is used to transfer the short nucleic acid sequence synthesized in the main reaction chamber to the second layer in the splicing chamber. In some embodiments, the transport channel to the second layer may be located below the main reaction chamber of the first layer. Preferably, each channel can be configured with a corresponding valve, for example, a micron-scale micro valve.

In the embodiment shown in FIG. 9 , the eight microchannels may be distributed along the circumference around the reagent concentration and delivery channel, and may be in fluid communication with the reagent concentration and delivery channel located at the center of the circumference. Therefore, the structure shown in FIG. 9 may be referred to as a star arrangement or a star configuration.

Referring to FIG. 10 , another structure of a synthesis reaction site of the first layer of the synthesis unit according to an embodiment of the present disclosure is shown. In the structure shown in FIG. 10 , the 8 microchannels included in the synthesis reaction point are distributed along a straight line, so it is in a straight line configuration. Preferably, in order to ensure a more effective cleaning effect between different nucleotides, the cleaning solution channel for feeding the cleaning solution can be arranged at the outermost end of the linear configuration.

Referring to FIG. 11 , there is shown another structure of a synthesis reaction site of the first layer of the synthesis unit according to an embodiment of the present disclosure. As shown in FIG. 11 , in this structure, each nucleotide solution channel is arranged in a square shape, while other reagent channels are arranged in a straight line. Therefore, the structure shown in FIG. 11 is at least partially in a straight line configuration. Likewise, in order to ensure a more effective cleaning effect between different nucleotides, the cleaning solution channel for feeding the cleaning solution can be arranged at the outermost end of the linear configuration.

In order to meet the requirements of different synthesis processes, both the above-mentioned star-shaped and straight-line configurations can increase or decrease the number of solution channels, for example, they can contain 6, 7, 8, 9, 10 or more solution channels.

Similar to the structure shown in Figure 9, the structures shown in Figure 10 and Figure 11 may also include: reagent concentration and transmission channels, which are used to concentrate and transport various reagents sent through 8 microchannels and send them into a main reaction chamber; a synthesis waste discharge channel for removing excess reagents from the main reaction chamber; and a transfer channel to the second layer for transferring short nucleic acid sequences synthesized in the main reaction chamber into the splicing chamber on the second floor. In some embodiments, the transport channel to the second layer may be located below the main reaction chamber of the first layer.

In addition, in some embodiments, as shown in Figure 10 and Figure 11, there may also be an electrophoresis section in the channel after the main reaction chamber or the synthesis reaction chamber, which is used to pass the synthesized nucleic acid sequence and other nucleotides through Electrophoresis separates, thereby discharging other nucleotides into the synthesis waste discharge channel.

Although various different configurations of synthesis reaction sites according to the present disclosure are shown above with reference to FIGS. 9-11 , other configurations of synthesis reaction sites may also be adopted by those skilled in the art.

Referring to FIG. 12 , a more detailed structure of one synthesis reaction site of the first layer of the synthesis unit according to one embodiment of the present disclosure is shown. In this structure, the reagents sent through each microchannel are first collected in the reagent collection chamber, and then the collected reagents are sent into the main reaction chamber through the reagent delivery channel. A transfer pump may be installed in the reagent transfer channel for pumping or aspirating the reagent entering the channel into the main reaction chamber. The transfer pump can also form sufficient transfer force to transfer excess reagents and waste liquid in the synthesis process to the synthesis waste liquid discharge channel. Nucleotide reagents pumped into the main reaction chamber can be combined with the starting particle (incorporation of the first nucleotide), or incorporated into the end of the nucleic acid sequence bound on the starting particle (subsequent nucleoside Acid incorporation), so that the nucleic acid sequence is gradually extended. Preferably, the main reaction chamber is micron-scale, so that the binding efficiency between nucleotides can be effectively improved, and the amount of reagents used can be greatly reduced.

In some embodiments, the main reaction chamber may include a starting particle immobilization electrode to immobilize the starting particles. The starting particle-immobilized electrode may be disposed on the outer wall of the main reaction chamber. The starting particle immobilized electrode is charged with a polarity opposite to that of the starting particle. By using the fixed electrode to apply sufficient electric field strength, the stability of the initial particle can be guaranteed during the nucleotide binding process and can be kept at a relatively fixed position. In some embodiments, the starting particle and the nucleic acid molecule have the same polarity of charge, thereby helping the synthetic sequence to maintain a better linear spatial structure, exposing the binding point at the end of the sequence, and facilitating the binding of the next nucleotide ; In addition, it can be guaranteed that during the whole synthesis process, the initial particles and the nucleic acid molecules connected thereto are in a relatively stable binding position due to the action of the fixed electrodes of the initial particles with opposite polar charges.

In some embodiments, the upper and lower sides of the main reaction chamber may also be provided with electrode assemblies for driving and linearizing nucleic acid sequences. The electrode assembly may include a plurality of electrodes distributed along a straight line. The plurality of electrodes may be arranged on upper and lower sides of an outer wall of the main reaction chamber, thereby forming an electrode array. The plurality of electrodes may be turned on independently. During the synthesis process, based on the length of the nucleic acid sequence, the electrodes at the corresponding positions can be turned on, so as to maintain the high linearity of the nucleic acid sequence and at the same time expose the binding site at the end, so that the next nucleotide molecule is at the end of the existing nucleic acid sequence to combine. The electrode assembly is charged with the polarity opposite to that of the nucleic acid molecule during synthesis.

Referring to FIG. 14 , there is shown a schematic diagram of using an electrode assembly to maintain a linear configuration of a nucleic acid sequence according to an embodiment of the present disclosure. During the synthesis process, by adjusting the pH value of the solution or connecting groups on the nucleotides, the nucleotide units in the nucleic acid sequence can be charged with a specified polarity, and then the electrode assembly is set to be charged with the opposite polarity. As a result, a continuous electrostatic field attraction to the nucleic acid sequence is generated, thereby maintaining a stable linear configuration of the nucleic acid sequence. Maintaining a stable linear configuration of the nucleic acid sequence can effectively expose the binding site at the tail end of the nucleic acid sequence, facilitating the binding of new nucleotides.

Referring to FIG. 15 , there is shown the ligation of starting particles and nucleic acid sequences in a reaction chamber according to one embodiment of the present disclosure. The starting particles are linked to nucleic acid sequences via linking bases. The position where the first nucleotide binds to the ligation base is called the initiation ligation site.

After completing each step of elongation of the nucleic acid sequence (that is, after a single nucleotide is combined), the electrode cooperates with the transfer pump to transfer the remaining nucleotide solution in the main reaction chamber to the synthesis waste liquid discharge channel.

After the sequence synthesis at the reaction point is completed and before the synthesis sequence is cut off from the initial particle, the electrode array of the electrode assembly applies charges of the same polarity and opposite polarity to the nucleic acid according to the set time sequence to generate the set electrophoretic effect, so as to Transfer the remaining nucleotides, sequence fragments and reaction waste liquid during the synthesis process to the synthesis waste liquid discharge channel. During this process, a set electric field strength is also maintained in the main reaction chamber via the starting particle fixed electrode, so as to keep the starting particle and the synthesis sequence at a fixed position.

After the synthesized sequence is cleaved from the starting particle, the electrode array of the electrode assembly applies charges of the same or opposite polarity to the nucleic acid molecule step by step, and transports the synthesized sequence to the transport channel of the second layer. Preferably, the delivery pump maintains a set pumping pressure during this process.

As mentioned above, in some embodiments, each channel of the synthesis reaction site can be configured with a corresponding valve. For example, the synthesis reaction site can have a reagent channel valve to start the pumping of the corresponding reagent; a waste liquid discharge channel valve to start the discharge of the waste liquid; a starting particle channel valve to start the pumping of the starting particle; the initial particle activation reagent channel valve to start the pumping of the initial particle activation reagent; and so on.

Referring to FIG. 13 , a method of using a valve in combination with a first layer synthesis process according to an embodiment of the present disclosure is shown. In some embodiments, the starting particle channel valve is opened to pump the starting particle into the synthesis reaction chamber, and fixed electrode is used to immobilize it, and then the starting particle activation reagent channel valve is opened to activate the starting particle Reagents are pumped into the synthesis reaction chamber to activate the first binding site. In some embodiments, for a specific nucleotide to be combined, the nucleotide solution channel valve is opened to pump the nucleotide solution into the reaction chamber to carry out the binding reaction, and then open the pipeline in the chip Cleaning the valve of the solvent channel to pump the cleaning solvent into the reaction chamber, draining the excess nucleotide solution through washing, and then opening the valve of the guard site stripping agent channel to send the guard site stripping agent into In the reaction chamber, the binding site on the nucleotide is exposed, ready for the next nucleotide binding. In some embodiments, the valve of the pipeline cleaning solvent channel in the chip is opened again to pump the cleaning solvent into the reaction chamber, and the excess protection site removing agent is discharged through cleaning.

In some embodiments, after the short nucleic acid sequence is synthesized, sequence fragments and other impurities are separated from the synthesized nucleic acid by applying an electric field with the electrode assembly, transported to the waste liquid discharge channel and discharged through the waste liquid discharge channel. In some embodiments, the starting particle cleaving enzyme reagent channel valve is further pumped to pump the starting particle cleaving enzyme reagent into the synthesis reaction chamber to catalyze the disengagement of the synthesis sequence from the starting particle.

Referring to FIG. 16 , there is shown a transfer pump that may be used in each channel and reaction chamber according to one embodiment of the present disclosure. The transfer pump is exemplified as a micro-sized diaphragm pump, but other types of transfer pumps can be used, such as a micro-sized gear pump or other types of micro pumps that meet the requirements. In some embodiments, a microscale diaphragm pump can include a pump body, a membrane, and a plurality of electrodes for driving the membrane. An accommodating cavity for accommodating solvent is arranged between the pump body and the membrane. The pump body may be configured to include a plurality of grooves distributed along the length of the pump body, thereby forming a multi-stage structure. The plurality of grooves can cooperate with the film to form a plurality of accommodation cavities. The plurality of electrodes may be respectively arranged on the left and right sides of each groove or accommodating cavity. When in use, the solvent first enters the first chamber of the diaphragm pump; then, the electrodes located on the left and right sides of the first chamber are activated to drive the membrane toward the pump body, thereby driving the solvent in the first chamber to the The second receiving chamber after the receiving chamber. Afterwards, the electrodes located on the left and right sides of the second chamber and subsequent chambers are sequentially activated to drive the membrane toward the pump body, thereby gradually driving the solvent out of the last chamber.

Referring to Figure 17, there is shown a micro-sized comb driver valve that may be used in each channel according to one embodiment of the present disclosure. The comb driver valve may include a valve chamber, a valve element, and a driving structure for driving the valve element, wherein the valve element can enter and leave the valve chamber under the action of the driving structure, thereby opening or closing the comb driver valve. In some embodiments, the valve element can be configured in the form of a valve plate. The drive structure may include a comb-shaped electrostatic drive and a push rod connected between the comb-shaped electrostatic drive and the valve element. The electrostatic comb drive may comprise a first comb portion and a second comb portion, wherein the push rod may be fixedly connected to the first comb portion. The first comb portion is movable towards or away from the second comb portion to drive the valve element out of or into the valve chamber via a push rod connected to the first comb portion to open or close the comb actuator valve. In some embodiments, the valve cavity, valve element, comb electrostatic actuator, and push rod of the comb actuator valve can be fabricated by micromachining. By adopting this comb-like electrostatic driver structure, the driving force of the valve can be amplified. However, it should be understood that other microscopic valves that meet requirements can also be used. For example, the microscopic diaphragm pump shown in FIG. 16 can be simplified into a single-stage structure to form a microscopic diaphragm valve.

Referring to FIGS. 18 and 19 , the structures of the splicing reaction chambers and the corresponding splicing reaction processes of the second layer and subsequent layers are shown respectively according to an embodiment of the present disclosure. Similar to the synthesis reaction chamber of the first layer, driving electrodes are arranged on the upper and lower sides of the outer wall of the splicing reaction chamber, and the shorter nucleic acid sequence (called the first shorter nucleic acid sequence) of the upper layer enters the splicing reaction chamber Finally, the electrodes are driven to realize sequentially alternating electric fields, and the first short nucleic acid sequence that enters is driven to reciprocate, so that the first short nucleic acid sequence returns to a linear configuration; then the second short nucleic acid sequence enters the splicing reaction chamber, And in a similar manner, the second shorter nucleic acid sequence is also restored to a linear configuration; then the first and second shorter nucleic acid sequences are spliced using reagents. The enzymatic reagents required for the reaction are delivered via the reagent delivery channel, which may be performed before or after the introduction of the second shorter nucleic acid sequence. Optionally, multiple splicing can be performed in the splicing reaction chamber. After the splicing between the first and second shorter nucleic acid sequences is completed, a third shorter nucleic acid sequence can be similarly added to further splice the sequence obtained by splicing the first and second shorter nucleic acid sequences. This step can be performed iteratively.

Referring to FIG. 20 , there is shown a chip operating device for operating the three-dimensional chip of the present disclosure according to an embodiment of the present disclosure. In order to ensure the effective execution of the chip process flow, special operating equipment is required. The operating equipment may include, but is not limited to, one or more of the following components:

1) Cabinet: The cabinet may include temperature control elements and/or humidity control elements to provide a suitable temperature and/or humidity environment for nucleic acid synthesis. In the PCR amplification process, the temperature control components in the box can provide suitable heating and cooling curves to achieve the required high temperature and low temperature control;

2) Chip operation platform: it can be set in the box to realize the accurate positioning of the chip during operation, ensure the effective alignment of the liquid interface on the chip and the liquid interface on the microtube array working head, and ensure the reagents during operation. effective supply; the chip operation platform can be set on the platform transmission guide rail, so that the chip operation platform can move in a horizontal plane along the horizontal or vertical direction on the platform transmission guide rail;

3) Microtube array working head: it is used to supply reagents to the reagent entry channel of the chip, so that sufficient reagent supply can be guaranteed after being connected to the chip;

4) Working head guide rail: it is used to realize the up and down movement of the microtube array working head;

5) Micro-pump array: each reagent is prepared with an independent reagent channel. Micropumps are used for reagent transmission, and each channel maintains an appropriate back pressure to ensure that the reagents in each channel of the chip are effectively filled;

6) Temperature-controlled reagent storage tank: it is used to store various reagents to be used under a controlled temperature.

Beneficial effects of the present disclosure:

The present disclosure achieves the following benefits:

1) The high-throughput parallel synthesis of nucleic acid sequence arrays can realize the parallel delivery of hundreds to tens of thousands of different synthetic sequences, and realize industrial-level large-scale nucleic acid synthesis;

2) Through multi-level parallel operation, the operation cycle is greatly shortened to further meet the needs of high-throughput screening;

3) The use of miniature reaction chambers can effectively shorten the reaction time of the reaction system;

4) The use of micro-reaction chambers can effectively reduce the amount of reagents used in the synthesis process and optimize the cost of the synthesis process;

5) It is easy to integrate with large-scale automation systems (such as robotic arrays, pipetting units, etc.).

The invention also relates to the following embodiments:

1. A high-throughput gene synthesis chip basic structure and synthesis method, including gene synthesis chip operating equipment and a synthesis chip, characterized in that: the synthesis chip is on a material substrate such as a silicon base/glass base/polymer material base (Including but not limited to silicon-based, glass-based, polymer-based substrate materials) processing synthesis unit arrays, and synthesizing different artificially designed gene sequences in each synthesis unit;

In a single synthesis unit, build a three-dimensional multi-level synthesis system. On the first level, multiple reaction points synthesize short gene sequences in parallel, such as synthesizing 4 sequences, 8 sequences, 16 sequences, and 32 sequences at the same time , 64 sequences, 96 sequences, 128 sequences, (based on the process, select the optimal number of other synthesis points), the length of each short sequence is 20 to 50 nucleic acid lengths, starting from the second layer, and 2 from the upper layer Combination points (or) 4 synthesis points (or) 8 synthesis points, (based on the process, select the optimal number of synthesis points in the previous layer), the generated shorter sequences are spliced into longer sequences, from the second layer At the last layer, the shorter sequences synthesized in the previous layer are spliced into longer sequences in an orderly manner according to the program setting, until the full-length sequence is spliced. Determine the order in which the shorter genes of the previous layer enter the reaction chamber of this layer;

The number of layers N is set to different values, and the number of synthesis points is set to effectively adjust the total length of the sequence; each synthesis unit is connected to a PCR chamber for amplifying the complete sequence;

The basic hierarchical structure of the single synthesis unit: including the first layer of synthesis array reagent entry channel, the first layer of synthesis array reagent distribution channel, the first layer of short gene synthesis unit array, the second to N synthesis layer (splicing) units, the second To N synthesis layer (stitching) unit synthesis reagent entry channel, second to N layer synthesis array reagent distribution channel and complete sequence PCR amplification chamber;

The first layer of the synthesis chip can be configured with 2 (or) 4 (or) 8 (or) 16 (or) 32 (or) 64 (or) 256 or other synthetic process set quantity The short sequence synthesis unit can synthesize the same or different gene sequences in different synthesis units, synthesize short gene fragments of 20 to 50 nucleic acid lengths in the synthesis unit of the first layer, and configure a large number of parallel synthesis units at the same time;

The basic pipeline structure of the single synthesis unit on the first layer includes a main reaction chamber, the left front side of the main reaction chamber is provided with a reagent transmission channel, and the end of the reagent transmission channel is provided with various types of reagent pumping pipelines, The right rear side of the main reaction chamber is provided with a synthesis waste discharge channel, the bottom of the main reaction chamber is provided with a second transmission channel, and the lower end of the second transmission channel is provided with a second synthesis (splicing) chamber , the various types of reagent pumping pipelines include A nucleic acid solution, cleaning solution channel tube, T nucleic acid solution, stock solution channel, C nucleic acid solution, error correction enzyme solution, G nucleic acid solution, and protection site removal agent;

The configuration of the first layer of synthesis unit: including a main reaction chamber, the lower left side of the main reaction chamber is provided with a small fixed electrode, and the right side of the main reaction chamber is symmetrically provided with driving + linearization electrodes , the left side of the main reaction chamber is provided with initial binding particles, the upper left side of the main reaction chamber is provided with a reagent delivery channel, the reagent delivery channel is provided with a delivery pump, and the reagent delivery channel is The other end is provided with a reagent collection cavity, and the right side of the main reaction chamber is provided with a synthesis waste liquid transmission channel valve body;

The synthesis (splicing) cavity structure: includes a synthesis (splicing) cavity, the left end of the synthesis (splicing) cavity is provided with a reagent transmission channel, and the right end of the synthesis (splicing) cavity is provided with a synthetic waste liquid channel , the reagent transmission channel is provided with a reagent transmission channel valve body, the synthetic waste liquid channel is provided with a synthetic waste liquid transmission channel valve body, and the upper side of the synthesis (splicing) cavity is evenly provided with an upper stage The gene sequence enters the channel, and the upper and lower sides of the synthesis (splicing) cavity are provided with driving electrodes, and the middle of the lower side of the synthesis (splicing) cavity is provided with a transmission channel to the next level;

The gene synthesis chip operation equipment: includes a temperature control and humidity control chamber, the inner lower side of the temperature control and humidity control chamber is provided with a chip operation platform, and the lower side of the chip operation platform is symmetrically provided with a platform drive A guide rail, the end of the chip operation platform is provided with a gene synthesis chip, the inner upper right side of the temperature control and humidity control chamber is provided with a working head guide rail, and the lower left end of the working head guide rail is provided with a microtube array working head , the upper right side of the temperature control and humidity control chamber is provided with a micropump array, and the lower right side of the temperature control and humidity control chamber is provided with a temperature control reagent storage tank.

2. A high-throughput gene synthesis chip infrastructure and synthesis method according to embodiment 1, characterized in that: the synthesis chip can adopt a circular or rectangular configuration:

Circular configuration: the circular configuration can directly use small-diameter wafers, such as 3-4 inch diameter wafers, and a single wafer is a consumable;

Rectangular configuration: Wafers with a diameter of 8 to 12 inches can be used, and a single wafer can cut multiple consumables.

3. A high-throughput gene synthesis chip basic structure and synthesis method according to embodiment 1, characterized in that: the synthesis unit adopts a three-dimensional parallel synthesis method;

The first layer of synthetic array reagents enters the channel: A, G, T, C nucleic acid solutions, protection site removal agents, chip internal pipeline cleaning solvents, error correction enzyme solutions, etc. are sent into the unit;

The channel for distributing reagents for the first-layer synthesis array: distribute A, G, T, and C nucleic acid solutions, protective site removal agents, cleaning solvents for pipelines in the chip, error-correcting enzyme solutions, etc., to each short-sequence synthesis in the first layer within the unit;

The first layer of short gene synthesis unit array: configure 4 (or) 8 (or) 16 (or) 32 (or) 64 (or) 128 (or) other process-set quantity synthesis units, Batch synthesis of short gene sequences with a length of 20 to 50 nucleic acids, and the synthesis sequence in each unit is set by the synthesis software;

The second to N synthesis layer (splicing) unit: splicing the shorter sequence imported by the previous layer into a longer sequence by 2 times (or) 4 times (or) 8 times, and checking the correctness of the splicing point ;

The second to N synthesis layer (splicing) unit synthesis reagents enter the channel: send reagents such as synthetase solution, shear enzyme solution, error correction enzyme solution, chip internal pipeline cleaning solution into the unit;

The second to N layer synthetic array reagent distribution channel: distribute the synthetase solution, the error correction enzyme solution, the cleaning solution of the internal pipeline of the chip, etc. to the synthesis (splicing) sites of each layer;

The complete sequence PCR amplification chamber: perform high-fold replication of the synthesized complete sequence.

4. A high-throughput gene synthesis chip infrastructure and synthesis method according to embodiment 1, characterized in that: micron-scale synthesis units are formed inside the synthesis 1.

5. A high-throughput gene synthesis chip basic structure and synthesis method according to embodiment 1, characterized in that: the basic pipeline structure of the single synthesis unit on the first layer can adopt a linear configuration.

6. A high-throughput gene synthesis chip basic structure and synthesis method according to embodiment 1, characterized in that: the driving + linearization electrodes maintain the excellent linear configuration of the gene sequence.

7. A high-throughput gene synthesis chip basic structure and synthesis method according to embodiment 1, characterized in that: micro-sized diaphragm pumps; micro-sized gear pumps; other types of microscopic pumps that meet the needs are used in the synthesis chip body; the micro-sized diaphragm valve and micro-sized comb driver valve are used in the synthesis chip, and the driving force of the valve is amplified through the multi-tooth comb driver structure; other micro valve bodies that meet the requirements.

8. A high-throughput gene synthesis chip basic structure and synthesis method according to embodiment 1, characterized in that: the synthesis chip and synthesis process can realize: chemical synthesis method; enzymatic synthesis method; For the protection site of nucleic acid, traditional DMT protection group or other customized groups can be used. The protection site removal agent can use common reagents, and other customized protection site removal agents can also be used.

Exemplary embodiments of the present disclosure are described above with reference to the accompanying drawings. However, those skilled in the art should appreciate that the present disclosure is not limited to the specific structures disclosed. Various changes and modifications can be made to the exemplary embodiments of the present disclosure without departing from the spirit and scope of the present disclosure. All such variations and modifications are included within the scope of protection defined by the claims of the present disclosure.

Claims (39)

A chip for nucleic acid synthesis, the chip comprises a plurality of synthesis units, each synthesis unit comprises a plurality of three-dimensional layers, wherein the first layer of the plurality of three-dimensional layers is a synthesis layer of short nucleic acid sequences, and the second Layer to last layer is the splicing layer of nucleic acid sequences, wherein there are multiple synthesis reaction points in the first layer to synthesize multiple short nucleic acid sequences, and there are splicing reaction points in the second layer to the last layer to synthesize the previous layer. The orderly splicing of shorter nucleic acid sequences into longer nucleic acid sequences until the full length nucleic acid sequence is produced. The chip of claim 1, wherein for the first layer, each synthesis unit comprises one or more first layer reagent entry channels, one or more first layer reagent distribution channels, a plurality of synthesis reaction sites, and access to the second layer transmission channel, wherein: the first layer of reagents into the channel is used to send reagents into the chip, The first-layer reagent distribution channels are used to distribute the incoming reagents to the synthesis units of the first layer, The plurality of synthesis reaction sites form an array of short nucleic acid synthesis, and each synthesis reaction site includes a synthesis reaction chamber for short nucleic acid sequence synthesis. The chip of claim 1 or 2, wherein each synthesis reaction point of each synthesis unit further comprises a plurality of microchannels for delivering reagents to the synthesis reaction chamber, each microchannel corresponding to a different reagent delivery, thereby distributing Different reagents to the synthesis unit are fed into the synthesis reaction chambers of the synthesis reaction site respectively. The chip of claim 3, wherein said plurality of microchannels comprises at least 8 microchannels, which correspond respectively to A nucleotide solution, G nucleotide solution, T/U nucleotide solution, C nucleotide solution, Delivery of wash solutions, error correcting enzyme solutions, guard site removal reagents and stock solutions, optionally the plurality of microchannels further comprises microchannels for delivery of initial microparticle activation reagents. The chip according to claim 3 or 4, wherein each synthesis reaction point of each synthesis unit also includes a reagent concentration and transmission channel, and the reagent concentration and transmission channel is arranged between the plurality of microchannels and the synthesis reaction chamber between, for pooling the different reagents delivered by the plurality of microchannels and transporting them into the synthesis reaction chamber. 4. The chip of claim 4, wherein the starting particles are starting particles that have been implanted in the synthesis reaction chamber, or are delivered into the synthesis reaction chamber through microchannels. 6. The chip of claim 4 or 6, wherein said synthesis reaction chamber has a starting particle immobilized electrode with an opposite charge to the starting particle to maintain the position of the starting particle. 6. The chip of claim 6 or 7, wherein said starting particle has a charge of the same polarity as the nucleic acid molecule. The chip of any one of claims 3 to 5, wherein the plurality of microchannels are configured in a star configuration or a linear configuration. 9. The chip of claim 9, wherein when the plurality of microchannels are configured in a linear configuration, the microchannels for delivering the washing liquid into the synthesis reaction chamber are located at the outermost ends of the linear configuration. The chip of any one of the preceding claims, wherein one or more selected from the following is adjustable: the number of layers of synthesis units, the number of synthesis units, the number of synthesis reaction points in each synthesis unit of the first layer number, the number of splicing reaction points in each synthesis unit from the second layer to the last layer. The chip according to any one of the preceding claims, the number of synthesis reaction points in each synthesis unit of the first layer is 4, 8, 16, 32, 64, 128 or more reaction points, thereby synthesizing 4 in parallel sequences, 8 sequences, 16 sequences, 32 sequences, 64 sequences, 96 sequences, 128 or more nucleic acid sequences. The chip according to any one of the preceding claims, wherein for the second layer to the last layer, each synthesis unit comprises respectively a reagent entry channel, a reagent distribution channel, a splicing reaction chamber and a channel leading to the next layer corresponding to each layer. transmission channel. A chip according to any one of the preceding claims, wherein each layer of the synthesis unit further comprises a waste liquid drainage channel. A chip according to any one of the preceding claims, wherein each channel is provided with a corresponding microscale micro-actuated valve, such as a micro-sized diaphragm valve or a micro-sized comb actuator valve. The chip of any one of the preceding claims 2-15, wherein the synthesis reaction chamber and the stitching reaction chamber are provided with a microscale pump to facilitate the pumping of the liquid, said microscale pump being, for example, a microscale diaphragm pump or a microscale gear Pump. The chip according to any one of the preceding claims 2-16, wherein the reagent inlet channel of each layer comprises a transfer pump to facilitate the pumping of the reagent entering the channel into the reaction chamber, and/or the excess reagent and waste after the reaction The liquid is pumped into the waste discharge channel. The chip of any one of the preceding claims 2-17, wherein along the walls of the synthesis reaction chamber and the stitching reaction chamber, an electrode assembly comprising a plurality of electrodes arranged linearly, each of said electrodes capable of being applied to nucleic acid molecules charges of the same or opposite polarity. The chip of claim 18, wherein the plurality of electrodes can be turned on independently, so that the electrodes at corresponding positions can be turned on independently based on the length of the nucleic acid sequence during the synthesis process. A chip according to any one of the preceding claims, wherein each synthesis unit is also connected to a PCR chamber for amplifying said full length nucleic acid sequence. The chip according to any one of the preceding claims, wherein the synthesis unit is fabricated on a silicon-based, glass-based or polymer material-based material substrate. A chip according to any one of the preceding claims, wherein the chip is fabricated using a wafer and the cross-section of the chip is of circular or rectangular configuration. The chip according to claim 15, wherein said micro-sized comb actuator valve comprises a valve chamber, a valve element, and a driving structure for driving the valve element, wherein said driving structure comprises a comb electrostatic driver and is connected to said A push rod between the comb-shaped electrostatic driver and the valve element, the push rod is configured to drive the valve element to leave or enter the valve chamber under the action of the comb-shaped electrostatic driver to open or close the Micro-sized comb actuator valves. The chip of claim 23, wherein the comb-shaped electrostatic actuator includes a first comb portion and a second comb portion, the push rod is fixedly connected to the first comb portion, and wherein the The first comb portion is movable towards or away from the second comb portion to drive the valve element out of or into the valve cavity via the push rod. The chip of claim 23 or 24, wherein the valve element is configured as a valve plate. A method for performing parallel high-throughput nucleic acid synthesis using the chip according to any one of claims 1-25, comprising: synthesizing multiple short nucleic acid sequences in parallel at multiple synthesis reaction points on the first layer, and synthesizing multiple short nucleic acid sequences at the second layer to The splicing reaction point of the last layer sequentially splices the shorter nucleic acid sequences synthesized in the previous layer into longer nucleic acid sequences until a full-length nucleic acid sequence is produced, and the last layer of each synthesis unit produces a full-length nucleic acid sequence . The method of claim 26, wherein said method comprises: On the first layer: (a) distributing the reagents to the respective synthesis units of the first layer through the first layer of reagent inlet channels and the first layer of reagent distribution channels; (b) immobilizing the starting particle in a synthesis reaction chamber and pumping a starting particle activation reagent to activate the first activation site; (c) pumping in a solution of the first nucleotide for incorporation of the first nucleotide; (d) pumping cleaning fluid to pump excess reagent and reaction waste into the waste discharge channel; (e) pumping in the protection site stripping agent to expose the binding site of the nucleotide, pumping in the cleaning solution again to discharge the excess protection site stripping agent; (f) pumping into the solution of the next nucleotide to carry out the combination of the next nucleotide; (g) repeating steps (d)-(f) until the length of the short nucleic acid sequence reaches the program setting value; (h) cleave the synthesized short nucleic acid sequence from the starting particle and send it to the next layer of transport channel; and On the second to last layer: (i) receiving the first short nucleic acid sequence and fixing it in the splicing reaction chamber; (j) receiving the second short nucleic acid sequence, splicing the first short nucleic acid sequence with the second short nucleic acid sequence in the presence of reagents required for splicing; (k) pumping cleaning solution to pump excess reagent and reaction waste into the waste discharge channel; (l) Steps (j)-(k) are repeated until the sequence length at this layer reaches the program setting value, and then the spliced sequence is sent to the transmission channel of the next layer. The method of claim 26, wherein during the synthesis process of the first layer, the electrode assembly arranged on the wall of the synthesis reaction chamber is applied with a charge of opposite polarity to that of the nucleic acid molecule, so that the high linearity of the nucleic acid sequence is maintained and the tail is exposed simultaneously. The binding site at the terminal end, so that the new nucleotide molecule can bind at the end of the existing sequence. The method of claim 27, wherein between steps (g) and (h), by making the electrode assembly arranged on the wall of the synthesis reaction chamber apply an electric charge of the same or opposite polarity as that of the nucleic acid molecule, the excess in the synthesis process Reagent and reaction waste is pumped into the waste drain. 27. The method of any one of claims 27-29, wherein in step (h) the synthetic short nucleic acid sequence is cleaved from the starting particle by pumping in the starting particle cutting enzyme. 30. The method of any one of claims 27-30, wherein the introduction of reagents required for splicing occurs before or after receiving the second short nucleic acid sequence. The method according to any one of claims 27-31, wherein during the splicing process from the second layer to the last layer, the received short nucleic acid sequence is driven by applying a sequential alternating electric field to the electrodes arranged on the wall of the splicing reaction chamber The reciprocating motion restores the linear configuration. The method of any one of claims 26-32, further comprising performing PCR amplification of the full-length nucleic acid sequence after finishing the last layer. A chip operation device for operating the chip according to any one of claims 1-25 or implementing the method according to any one of claims 26-33, comprising a box, the box is provided with: The chip operation platform is used to realize the accurate positioning of the chip during operation; a microtube array head for supplying reagents to the reagent inlet channels of the chip; and The working head guide rail is used to realize the up and down movement of the microtube array working head. The chip handling apparatus according to claim 34, wherein the chip handling platform is arranged on a platform driving rail, so that the chip handling platform can move in a horizontal plane in a horizontal direction or a longitudinal direction on the platform driving rail. The chip handling device according to claim 34 or 35, wherein the box includes a temperature control element and/or a humidity control element to provide a required temperature and/or humidity environment for nucleic acid synthesis. 34. The chip handling device of claim 34 or 35, further comprising an array of micropumps and/or temperature-controlled reagent storage tanks. 37. The device-on-a-chip of claim 37, wherein said micropump array employs micropumps for reagent delivery, optionally with a separate reagent channel for each reagent. A system comprising: The chip of any one of claims 1-25; A nucleic acid sequence determination module for each synthesis reaction point in each synthesis unit; a splicing reaction sequence specification module; a switch control module for each valve; an electrode electric field control module; And optionally, synthesizing a reaction site number designation module and a splicing reaction site number designation module.

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