Solid-phase carrier microfluidic gene synthesis system and method

Abstract

This invention discloses a solid-phase carrier microfluidic gene synthesis system and method. The solid-phase carrier microfluidic gene synthesis system includes a substrate, microfluidic tubing, and a microfluidic valve assembly. Because an inlet is provided at the inlet of the main tubing, buffer solution can be injected into the main tubing through the inlet for cleaning before introducing various reagents or solid-phase carriers, avoiding cross-contamination. For various desired synthesis units, only the corresponding solid-phase carrier and synthesis reagent need to be selected and injected into the main tubing in sequence through their respective inlets to carry out the synthesis reaction, thus realizing the preparation of various synthesis units. Furthermore, the collection of various synthesis units is achieved by switching between the channels using the microfluidic valve assembly.

Description

Technical Field

[0001] This invention relates to the field of biofluidics, and in particular to a solid-phase carrier microfluidic gene synthesis system and method. Background Technology

[0002] As a core tool for achieving gene editing, gene synthesis technology has developed rapidly and given rise to a variety of oligonucleotide synthesis schemes based on solid-phase carriers and phosphorous amide chemical methods.

[0003] Currently, gene synthesis technologies generally suffer from the inability to independently control or collect solid-phase carriers, as well as the problem of cross-contamination or residues between reagents. Summary of the Invention

[0004] In view of this, the first objective of the present invention is to provide a solid-phase carrier microfluidic gene synthesis system, which aims to achieve carrier capture and independent gene synthesis, avoiding the problems of cross-contamination or residue between reagents.

[0005] The second objective of this invention is to provide a microfluidic gene synthesis method using a solid-phase carrier.

[0006] To achieve the first objective mentioned above, the present invention provides the following solution:

[0007] A solid-phase carrier microfluidic gene synthesis system, comprising:

[0008] substrate;

[0009] A microfluidic pipeline mounted on the substrate, the microfluidic pipeline including a main pipeline and a carrier trap, the main pipeline having multiple inlet ports at its inlet end and a drain port at its outlet end, the carrier trap being installed inside the main pipeline for capturing solid carriers;

[0010] A microfluidic valve assembly mounted on the substrate is used to switch the flow direction of fluid in the main pipeline to collect waste liquid or the solid carrier after the synthesis reaction.

[0011] In one specific implementation, the carrier trap includes a first trapping block and a second trapping block;

[0012] The first capture block and the second capture block are respectively installed in the main pipeline, and a capture gap is formed between the first capture block and the second capture block for capturing a single solid carrier. The gap formed between the first capture block and the second capture block and the pipe wall of the main pipeline adjacent to them allows the solid carrier to pass through.

[0013] In another specific implementation, along the direction in which the solid carrier flows through the carrier trap, the first trapping block and the second trapping block are tilted toward each other to trap the solid carrier.

[0014] In another specific implementation, both the first capture block and the second capture block are arc-shaped blocks.

[0015] In another specific implementation, the input port includes a buffer inlet / outlet, a carrier input port, and a synthetic reagent input port;

[0016] The discharge port includes a carrier collection port and a waste liquid discharge port.

[0017] In another specific implementation, the input port includes a buffer inlet / outlet, a carrier input port, and a synthetic reagent input port;

[0018] The discharge port includes a carrier collection port and a waste liquid discharge port.

[0019] In another specific implementation, the main road includes:

[0020] The input tube has a buffer inlet located at the inlet end, and the synthetic reagent inlet and the carrier inlet are sequentially spaced along the direction from the inlet end to the outlet end of the input tube.

[0021] An upstream pipe and a downstream pipe are respectively connected to the outlet end of the input pipe at their inlet ends. The carrier collection port is located at the outlet end of the upstream pipe, and the waste liquid discharge port is located at the outlet end of the downstream pipe.

[0022] A carrier tube is connected at both ends to the upstream pipe and the downstream pipe, respectively. The carrier trap is installed inside the carrier tube, with its first port connected to the upstream pipe and its second port connected to the downstream pipe.

[0023] When the solid support enters the support trap from the first communication port, the support trap is able to capture the solid support.

[0024] When the buffer solution enters the carrier trap from the second connection port, the carrier trap is able to flush the solid-phase carrier out of the carrier trap.

[0025] In another specific implementation, the carrier trap includes a first trapping block and a second trapping block;

[0026] The first capture block and the second capture block are respectively installed inside the carrier tube, and a capture gap is formed between the first capture block and the second capture block for capturing a single solid carrier. The gap formed between the first capture block and the second capture block and the tube wall of the carrier tube adjacent to them allows the solid carrier to pass through.

[0027] In another specific implementation, along the direction in which the solid carrier flows through the carrier trap, the first trapping block and the second trapping block are tilted toward each other to trap the solid carrier.

[0028] In another specific implementation, both the first capture block and the second capture block are arc-shaped blocks.

[0029] In another specific implementation, the microfluidic valve assembly includes:

[0030] An upstream pipeline control valve is used to control the on / off state of the upstream pipeline and the carrier pipeline;

[0031] Downstream pipeline control valve, used to control the on / off state of the downstream pipeline and the carrier pipeline;

[0032] A carrier collection port control valve is used to control the connection and disconnection between the carrier collection port and the upstream pipeline;

[0033] A waste liquid discharge outlet control valve is used to control the connection and disconnection between the waste liquid discharge outlet and the downstream pipeline.

[0034] In another specific implementation, the microfluidic valve assembly further includes a synthetic reagent inlet control valve, which is configured to correspond one-to-one with the synthetic reagent inlet and is used to control the on / off state of each synthetic reagent inlet and the main pipeline.

[0035] and / or

[0036] The microfluidic valve assembly also includes a buffer inlet control valve for controlling the connection and disconnection between the buffer inlet and the main pipeline;

[0037] and / or

[0038] The microfluidic valve assembly also includes a synthesis station control valve for controlling the connection and disconnection between the two ends of the carrier trap and the upstream and downstream pipes;

[0039] and / or

[0040] The microfluidic valve assembly also includes a carrier inlet control valve for controlling the connection and disconnection between the carrier inlet and the inlet tube.

[0041] In another specific implementation, the microfluidic valve assembly is located above the microfluidic pipeline, and each control valve of the microfluidic valve assembly includes a valve body and a valve tube. The inlet of the valve tube is used to introduce positive pressure, and the outlet of the valve tube is connected to the valve body. The valve body is located at the controllable on / off position of the main pipeline and deforms under the positive pressure input by the valve tube.

[0042] When the control valve is closed, positive pressure is introduced into the valve pipe, causing the valve body to deform downwards to block the controllable on / off position of the main pipeline.

[0043] In another specific embodiment, the ratio of the length of the valve body along the flow direction of the main pipeline to the width of the main pipeline is greater than or equal to 1.5;

[0044] The ratio of the width of the valve body perpendicular to the flow direction of the main pipeline to the width of the main pipeline is greater than or equal to 3.

[0045] In another specific embodiment, the ratio of the width of the valve pipe projected onto the main pipeline to the width of the main pipeline is less than or equal to 0.25.

[0046] The various embodiments of the present invention can be combined arbitrarily as needed, and the resulting embodiments are also within the scope of the present invention and are part of the specific implementation of the present invention.

[0047] To achieve the second objective mentioned above, the present invention provides the following solution:

[0048] A microfluidic gene synthesis method using a solid-phase carrier includes:

[0049] Step S1: Provide a solid-phase carrier microfluidic gene synthesis system as described in any one of the above;

[0050] Step S2: Determine whether the solid-phase carrier microfluidic gene synthesis system needs cleaning. If yes, proceed to step S3; otherwise, proceed to step S4.

[0051] Step S3: Control the microfluidic valve assembly to open the waste liquid outlet and input buffer solution into the buffer solution inlet so that the buffer solution flows through the main pipeline and is discharged from the waste liquid outlet. After cleaning is completed, proceed to step S4.

[0052] Step S4: Input solid carrier into the solid carrier inlet, the solid carrier flows along the main pipeline and is captured by the carrier catcher;

[0053] Step S5: Input the buffer solution into the buffer solution inlet. The buffer solution flows along the main pipeline to clean the main pipeline and the carrier trap, and discharge excess solid carrier.

[0054] Step S6: Input the corresponding synthetic reagent into the corresponding synthetic reagent input port according to the required synthetic unit. The synthetic reagent flows along the main pipeline and passes through the carrier trap to soak the solid support captured by the carrier trap, thereby completing the synthetic reaction.

[0055] Step S7: Control the microfluidic valve assembly to close the waste liquid outlet and open the carrier collection port, and input the buffer solution into the buffer solution inlet. The buffer solution flows along the main pipeline to discharge the solid-phase carrier that has completed the synthesis reaction, which has been captured by the carrier trap, to the carrier collection port.

[0056] In one specific implementation, step S8 is further included between step S6 and step S7: determining whether all types of synthetic reagents required for the synthetic unit to be prepared have participated in the synthetic reaction; if yes, proceed to step S5; if no, proceed to step S7.

[0057] In another specific implementation, when the carrier collection port is opened, the microfluidic valve assembly is controlled to connect the downstream pipe and the input pipe, and disconnect the upstream pipe and the input pipe;

[0058] When the waste liquid outlet is opened, the microfluidic valve assembly is controlled to disconnect the downstream pipe from the input pipe and connect the upstream pipe to the input pipe.

[0059] In another specific implementation, step S7 is followed by step S9: controlling the microfluidic valve assembly to open the waste liquid outlet and close the carrier collection port, inputting the buffer solution into the buffer solution inlet, the buffer solution flowing along the main pipeline to clean the main pipeline and the carrier trap, and discharging from the waste liquid outlet.

[0060] The solid-phase carrier microfluidic gene synthesis system provided by this invention allows for the cleaning of the main pipeline by injecting buffer solution into the inlet before introducing various reagents or solid-phase carriers, thus avoiding cross-contamination. For each desired synthesis unit, only the corresponding solid-phase carrier and synthesis reagent need to be selected and injected into the main pipeline in sequence through different inlets to carry out the synthesis reaction, thereby realizing the preparation of various synthesis units. Furthermore, the collection of various synthesis units is achieved by switching between different channels using a microfluidic valve assembly.

[0061] Furthermore, in this invention, a carrier trap is installed in the main pipeline, which can capture solid carriers, thereby enabling independent control and collection of the solid carriers. In addition, the carrier trap, in conjunction with a microfluidic valve assembly, can achieve the selective recovery of combined solid carriers, solving the technical problem in the prior art that it is impossible to control individual solid carriers. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0063] Figure 1 This is a schematic diagram of the front view of the microfluidic pipeline provided by the present invention;

[0064] Figure 2 This is a schematic diagram of the front view of the microfluidic valve assembly provided by the present invention.

[0065] Figure 3 This is a schematic diagram of the main structure of the solid-phase carrier microfluidic gene synthesis system provided by the present invention.

[0066] Figure 4 A three-dimensional structural schematic diagram of the solid-phase carrier microfluidic gene synthesis system without a substrate provided by the present invention;

[0067] Figure 5 A three-dimensional structural schematic diagram of the solid-phase carrier microfluidic gene synthesis system provided by the present invention;

[0068] Figure 6 This is a schematic diagram of the valve body and main pipeline provided by the present invention;

[0069] Figure 7 A partial structural schematic diagram of the carrier tube provided by the present invention;

[0070] Figure 8 A structural schematic diagram of the valve tube dimensions provided by the present invention;

[0071] Figure 9 This is a front view structural diagram of the valve body dimensions provided by the present invention. Detailed Implementation

[0072] The following will refer to the appendices in the embodiments of the present invention. Figures 1-9The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0073] In the description of this invention, it should be understood that the terms "upper," "lower," "top surface," "bottom surface," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the indicated position or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations of the invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0074] Combination Figures 1-5 As shown, this invention provides a solid-phase carrier microfluidic gene synthesis system to achieve carrier capture and independent gene synthesis, avoiding cross-contamination or residue between reagents. It is understood that the system provided by this invention is not limited to gene synthesis, but can also be used for the synthesis of cells, bacteria, or droplets.

[0075] Specifically, the solid-phase carrier microfluidic gene synthesis system includes a substrate 13, a microfluidic pipeline 11, and a microfluidic valve assembly 12.

[0076] Both the microfluidic tubing 11 and the microfluidic valve assembly 12 are fabricated using soft photolithography or photolithography processes, and are made of perfluoropolyether compatible with various synthetic reagents. It should be noted that the materials of the microfluidic tubing 11 and the microfluidic valve assembly 12 are not limited, and can also be made of flexible materials, such as PDMS (polydimethylsiloxane).

[0077] Both the microfluidic pipeline 11 and the microfluidic valve assembly 12 are mounted on the substrate 13. Specifically, the microfluidic pipeline 11 and the microfluidic valve assembly 12 are plasma-bonded to the substrate 13.

[0078] The microfluidic pipeline 11 includes a main pipeline 1101 and a carrier trap 1102. The inlet end of the main pipeline 1101 is provided with multiple inlet ports, and the outlet end of the main pipeline 1101 is provided with a drain port. The specific number of inlet ports and the number of drain ports can be set according to the needs.

[0079] The carrier catcher 1102 is installed inside the main pipeline 1101 to catch solid carriers.

[0080] The microfluidic valve assembly 12 is used to switch the flow direction of the main pipeline 1101 to collect waste liquid or solid carrier after the synthesis reaction is completed.

[0081] The solid-phase carrier microfluidic gene synthesis system provided by this invention allows for the cleaning of the main pipeline 1101 by injecting buffer solution into the input port before introducing various reagents or solid-phase carriers, thus avoiding cross-contamination. For each desired synthesis unit, only the corresponding solid-phase carrier and synthesis reagent need to be selected and injected into the main pipeline 1101 sequentially through different input ports to carry out the synthesis reaction, thereby realizing the preparation of various synthesis units. Furthermore, the collection of various synthesis units is achieved by switching between different channels using the microfluidic valve assembly 12.

[0082] Understandably, in this article, the synthetic unit specifically refers to a nucleic acid molecule, and different synthetic units refer to nucleic acid molecules with different sequences, such as the AGCT sequence, the ACCT sequence, etc.

[0083] In some embodiments, the present invention specifically discloses an input port including a buffer input port 1104, a carrier input port 110, and a synthetic reagent input port 1105. The number of synthetic reagent input ports 1105 is not limited and can be set according to specific needs.

[0084] In this embodiment, four synthesis reagent inlets 1105 are used: adenine synthesis reagent inlet 1105a, thymine synthesis reagent inlet 1105b, cytosine synthesis reagent inlet 1105c, and guanine synthesis reagent inlet 1105d. These inlets are used to input adenine, thymine, cytosine, and guanine synthesis reagents, respectively. Using separate inlets for different reagents avoids contamination caused by mixing them. It should be noted that the synthesis reagents are not limited to the four types of reactive bases; they can also include various modified bases or dibases.

[0085] The discharge port includes a carrier collection port 1106 and a waste liquid discharge port 1107. That is, the carrier collection port 1106 and the waste liquid discharge port 1107 are set separately to facilitate the separate collection of solid carrier and waste liquid.

[0086] The solid-phase carrier microfluidic gene synthesis system provided by this invention features a buffer inlet 1104 at the input end of the main pipeline 1101. This allows for the injection of buffer solution into the main pipeline 1101 before introducing various reagents or solid-phase carriers, preventing cross-contamination. For each desired synthesis unit, only the corresponding solid-phase carrier and synthesis reagent need to be selected and injected into the main pipeline 1101 sequentially through their respective carrier inlet 1103 and reagent inlet 1105 to initiate the synthesis reaction, thus achieving the preparation of various synthesis units. Furthermore, the microfluidic valve assembly 12 switches between channels, enabling the collection of synthesis units and controlling the flow direction of the solid-phase carrier according to different carriers, thereby achieving selective recovery of combined solid-phase carriers.

[0087] In some embodiments, the main pipeline 1101 includes an input tube 1101a, an upstream tube 1101b, a downstream tube 1101c, and a carrier tube 1101d. A buffer inlet 1104 is disposed at the inlet end of the input tube 1101a. A synthetic reagent inlet 1105 and a carrier inlet 1103 are sequentially and spaced apart on the input tube 1101a along the direction from the inlet end to the outlet end.

[0088] The inlet ends of the upstream pipe 1101b and the downstream pipe 1101c are respectively connected to the outlet end of the input pipe 1101a. The carrier collection port 1106 is located at the outlet end of the upstream pipe 1101b, and the waste liquid discharge port 1107 is located at the outlet end of the downstream pipe 1101c.

[0089] The two ends of the carrier tube 1101d are connected to the upstream tube 1101b and the downstream tube 1101c, respectively. The carrier trap 1102 is installed inside the carrier tube 1101d, and the first port of the carrier trap 1102 is connected to the upstream tube 1101b, and the second port of the carrier trap 1102 is connected to the downstream tube 1101c.

[0090] The number of carrier tubes 1101d is unlimited and can be set according to throughput requirements.

[0091] When the solid-phase carrier enters the carrier trap 1102 from the first connection port, the carrier trap 1102 can trap the solid-phase carrier; when the buffer solution enters the carrier trap 1102 from the second connection port, the carrier trap 1102 can flush the solid-phase carrier out of the carrier trap 1102.

[0092] It should be noted that the carrier capture device 1102 captures only one solid carrier at a time, so as to achieve independent capture of the solid carrier.

[0093] Furthermore, such as Figure 7As shown, the present invention discloses a carrier trap 1102 including a first trapping block 1102-1 and a second trapping block 1102-2. The first trapping block 1102-1 and the second trapping block 1102-2 are respectively installed in a carrier tube 1101d, and a trapping gap for trapping a single solid carrier is formed between the first trapping block 1102-1 and the second trapping block 1102-2. The gap formed between the first trapping block 1102-1 and the second trapping block 1102-2 and the tube wall of the carrier tube 1101d that are adjacent to each other allows the solid carrier to pass through.

[0094] To facilitate the capture of solid carriers, this invention discloses that, along the direction in which the solid carrier flows through the carrier capture device 1102, the surfaces of the first capture block 1102-1 and the second capture block 1102-2 used to capture the solid carrier are inclined towards each other. It should be noted that the surfaces of the first capture block 1102-1 and the second capture block 1102-2 used to capture the solid carrier refer to the surfaces of the first capture block 1102-1 and the second capture block 1102-2 that form the capture gap.

[0095] Furthermore, both the first capture block 1102-1 and the second capture block 1102-2 are curved blocks. It should be noted that the first capture block 1102-1 and the second capture block 1102-2 can also be set to other shapes of blocks, such as rectangular blocks, and the capture surface of the rectangular block can be set to a curved surface.

[0096] Specifically, this invention discloses that the solid carrier is a glass microsphere with controllable pore size. It should be noted that the solid carrier being a glass microsphere with controllable pore size is only one specific embodiment of this invention. In practical applications, other carriers that can be captured can also be used as the solid carrier.

[0097] In some embodiments, the microfluidic valve assembly 12 includes an upstream pipeline control valve 1201, a downstream pipeline control valve 1202, a carrier collection port control valve 1203, and a waste liquid discharge port control valve 1204.

[0098] Specifically, the upstream pipeline control valve 1201, the downstream pipeline control valve 1202, the carrier collection port control valve 1203, and the waste liquid discharge port control valve 1204 are located above the main pipeline 1101.

[0099] The upstream pipeline control valve 1201 is used to control the opening and closing of the upstream pipe 1101b and the carrier pipe 1101d. Specifically, the upstream control valve is installed on the upstream pipe 1101b and is located near the inlet end of the carrier pipe 1101d.

[0100] The downstream pipeline control valve 1202 is used to control the opening and closing of the downstream pipeline 1101c and the carrier pipeline 1101d. Specifically, the upstream control valve is installed on the downstream pipeline 1101c and is located near the outlet end of the carrier pipeline 1101d.

[0101] It should be noted that a three-way valve can also be installed at the position where the input pipe 1101a connects to the upstream pipe 1101b and the downstream pipe 1101c, and the three-way valve can replace the upstream pipe control valve 1201 and the downstream pipe control valve 1202.

[0102] The carrier collection port control valve 1203 is used to control the opening and closing of the carrier collection port 1106 and the upstream pipe 1101b, and the waste liquid discharge port control valve 1204 is used to control the opening and closing of the waste liquid discharge port 1107 and the downstream pipe 1101c.

[0103] When it is necessary to clean the main pipeline 1101 or to input the solid carrier and synthesis reagents for the synthesis reaction, the upstream pipeline control valve 1201 and the waste liquid discharge outlet control valve 1204 are opened, and the downstream pipeline control valve 1202 and the carrier collection port control valve 1203 are closed, so that the buffer solution or synthesis reagents flow along the input pipe 1101a, the upstream pipe 1101b and the carrier pipe 1101d, and the cleaned buffer solution, excess synthesis reagents or excess solid carriers are discharged from the waste liquid discharge outlet 1107.

[0104] When it is necessary to discharge the solid support after the synthesis reaction is completed, the upstream pipeline control valve 1201 and the waste liquid discharge outlet control valve 1204 are closed, and the downstream pipeline control valve 1202 and the support collection port control valve 1203 are opened, so that the buffer solution flows along the input pipe 1101a, the downstream pipe 1101c and the support pipe 1101d, and the solid support after the synthesis reaction is discharged from the support collection port 1106.

[0105] In some embodiments, the microfluidic valve assembly 12 further includes a synthetic reagent inlet control valve 1205, which is configured one-to-one with the synthetic reagent inlet 1105, and is used to control the on / off state of each synthetic reagent inlet 1105 and the main pipeline 1101. The synthetic reagent inlet control valve 1205 facilitates the control of the opening and closing of the synthetic reagent inlet 1105.

[0106] Since the synthetic reagent inlet control valve 1205 is configured in a one-to-one correspondence with the synthetic reagent inlet 1105, taking the number of synthetic reagent inlets 1105 as four (adenine synthetic reagent inlet 1105a, thymine synthetic reagent inlet 1105b, cytosine synthetic reagent inlet 1105c, and guanine synthetic reagent inlet 1105d) as an example, then correspondingly, the number of synthetic reagent inlet control valves 1205 is also four (adenine synthetic reagent inlet control valve 1205a, thymine synthetic reagent inlet control valve 1205b, cytosine synthetic reagent inlet control valve 1205c, and guanine synthetic reagent inlet control valve 1205d). It should be noted that the synthetic reagent inlet control valves 1205 are not limited to a one-to-one correspondence with the synthetic reagent inlet 1105; one synthetic reagent inlet 1105 can also correspond to two or more synthetic reagent inlet control valves 1205, etc.

[0107] To facilitate control of the buffer inlet 1104, the present invention discloses that the microfluidic valve assembly 12 further includes a buffer inlet control valve 1206, which is used to control the opening and closing of the buffer inlet 1104 and the main pipeline 1101.

[0108] Furthermore, the present invention also discloses that the microfluidic valve assembly 12 further includes a synthesis station control valve 1207, used to control the connection and disconnection of the two ends of the carrier trap 1102 with the upstream pipe 1101b and the downstream pipe 1101c.

[0109] Specifically, the synthesis station control valve 1207 is set in a one-to-one correspondence with the carrier tube 1101d.

[0110] In some embodiments, the microfluidic valve assembly 12 is located above the microfluidic pipeline 11, and each control valve of the microfluidic valve assembly 12 includes a valve body 1209 and a valve tube 1210. The inlet of the valve tube 1210 is used to introduce positive pressure, and the outlet of the valve tube 1210 is connected to the valve body 1209. The valve body 1209 is located at the controllable on / off position of the main pipeline 1101 and deforms under the positive pressure input by the valve tube 1210. Specifically, the valve body 1209 is made of a deformable membrane. When the control valve is closed, the positive pressure introduced by the valve tube 1210 causes the valve body 1209 to deform downward to block the controllable on / off position of the main pipeline 1101.

[0111] Furthermore, the cross-section of the main pipeline 1101 at the controllable on / off position is semi-circular, which, combined with the deformation and downward pressure of the valve body 1209, better blocks the pipeline 1101, such as... Figure 6 As shown.

[0112] Of course, a protruding structure can also be set at the position of the flow channel to be controlled in the main pipeline 1101, which can be adapted to the deformation and downward pressure of the valve body 1209 to achieve blocking.

[0113] Furthermore, a thin film can be placed at the junction of the main pipeline 1101 and the valve body 1209 to separate them. When positive pressure is introduced into the valve body 1209, the film deforms in the direction of the main pipeline 1101 until the main pipeline 1101 is blocked.

[0114] It should be noted that the control valve can also be an electrically controlled valve, etc.

[0115] In some embodiments, such as Figure 8 As shown, the ratio of the width L1 of the valve pipe 1210 projected onto the main pipeline 1101 to the width d of the main pipeline 1101 is less than or equal to 0.25, so as to avoid the valve pipe 1210 located directly above the main pipeline 1101 deforming downward and pressing onto the main pipeline 1101 when the positive pressure introduced into the valve pipe 1210 is too large, thus affecting the main pipeline 1101.

[0116] It should be noted that the above range values ​​are only one specific embodiment of the present invention. In practical applications, the above range values ​​can be adjusted as needed.

[0117] In some embodiments, such as Figure 9 As shown, the ratio of the length of the valve body 1209 along the flow direction of the main pipeline 1101 to the width of the main pipeline 1101 is greater than or equal to 1.5, and the ratio of the width of the valve body 1209 perpendicular to the flow direction of the main pipeline 1101 to the width of the main pipeline 1101 is greater than or equal to 3, in order to avoid the situation where the valve body 1209 fails to completely cut off the main pipeline 1101.

[0118] It should be noted that the above range values ​​are only one specific embodiment of the present invention. In practical applications, the above range values ​​can be adjusted as needed.

[0119] A second aspect of this invention provides a microfluidic gene synthesis method using a solid-phase carrier, comprising:

[0120] Step S1: Provide a solid-phase carrier microfluidic gene synthesis system as described above.

[0121] It should be noted that, in order to facilitate the operation of the microfluidic valve group 12, the microfluidic valve group 12 is initialized, which means closing all the control valves in the microfluidic valve group 12.

[0122] Step S2: Determine whether the solid-phase carrier microfluidic gene synthesis system needs cleaning. If yes, proceed to step S3; otherwise, proceed to step S4.

[0123] It should be noted that when using a solid-phase carrier microfluidic gene synthesis system, cleaning can be performed by default to avoid impurities from affecting the reaction of the synthesis reagents.

[0124] Step S3: Control the microfluidic valve assembly 12 to open the waste liquid outlet 1107 and input the buffer solution into the buffer solution inlet 1104 so that the buffer solution flows through the main pipeline 1101 and is discharged from the waste liquid outlet 1107. After cleaning is completed, proceed to step S4.

[0125] Specifically, controlling the microfluidic valve assembly 12 to open the waste liquid outlet 1107 is equivalent to opening the waste liquid outlet control valve 1204.

[0126] Step S4: Input solid carrier into solid carrier inlet 1103. Solid carrier flows along main pipeline 1101 and is captured by carrier capture device 1102.

[0127] Multiple carrier traps 1102 are installed on carrier tubes 1101d, and each one corresponds to a carrier tube 1101d.

[0128] Step S5: Input buffer solution into buffer inlet 1104. The buffer solution flows along main pipeline 1101 to clean main pipeline 1101 and carrier trap 1102 and discharge excess solid carrier.

[0129] By cleaning, excess solid support is removed, avoiding the need for multiple solid supports to be piled together for synthesis.

[0130] This enables the preparation of the same type of synthetic unit or at least two types of synthetic units within the same solid-phase carrier microfluidic gene synthesis system.

[0131] When preparing the same type of synthetic unit, only one synthetic station control valve 1207 on the carrier tube 1101d can be opened, or two or more synthetic station control valves 1207 on the carrier tube 1101d can be opened to prepare two or more synthetic units at one time.

[0132] When preparing different types of synthetic units, at least one synthetic station control valve 1207 on a carrier tube 1101d can be opened first to prepare one type of synthetic unit. Then, when preparing the next different synthetic unit, the synthetic station control valve 1207 for the previous synthetic unit is closed, and at least one other synthetic station control valve 1207 on a carrier tube 1101d is opened to prepare the required synthetic unit. This process is repeated sequentially to achieve the simultaneous preparation of two or more synthetic units.

[0133] Step S6: Input the corresponding synthesis reagent into the corresponding synthesis reagent input port 1105 according to the required synthesis unit. The synthesis reagent flows along the main pipeline 1101 and flows through the carrier trap 1102 to soak the solid carrier trapped by the carrier trap 1102, thus completing the synthesis reaction.

[0134] Understandably, when the required synthetic unit needs multiple synthetic reagents, the order in which these reagents are input should be in the order actually required.

[0135] Step S7: Control the microfluidic valve assembly 12 to close the waste liquid outlet 1107 and open the carrier collection port 1106, and input the buffer solution into the buffer inlet 1104. The buffer solution flows along the main pipeline 1101 to discharge the solid carrier that has completed the synthesis reaction, which has been captured by the carrier trap 1102, to the carrier collection port 1106.

[0136] Specifically, when the carrier collection port 1106 is opened, the microfluidic valve assembly 12 is controlled to connect the downstream tube 1101c and the input tube 1101a, and disconnect the upstream tube 1101b and the input tube 1101a, so that the buffer can discharge the solid-phase carrier that has completed the synthesis reaction on the carrier trap 1102 to the carrier collection port 1106 in the order of input tube 1101a, downstream tube 1101c, and carrier tube 1101d.

[0137] When the waste liquid outlet 1107 is opened, the microfluidic valve assembly 12 is controlled to disconnect the downstream pipe 1101c from the input pipe 1101a and connect the upstream pipe 1101b to the input pipe 1101a.

[0138] In some embodiments, step S8 is further included between step S6 and step S7: determining whether all types of synthetic reagents required for the synthetic unit to be prepared have participated in the synthetic reaction; if yes, proceed to step S5; if no, proceed to step S7.

[0139] It should be noted that whether all types of synthetic reagents required for the synthetic unit to be prepared have participated in the synthetic reaction includes: the types of synthetic reagents required to prepare one type of synthetic unit, and the types of synthetic reagents required to prepare two or more types of synthetic units.

[0140] In some embodiments, the present invention further includes step S9 after step S7: controlling the microfluidic valve assembly 12 to open the waste liquid outlet 1107 and close the carrier collection port 1106, inputting buffer solution into the buffer solution inlet 1104, the buffer solution flowing along the main pipeline 1101 to clean the main pipeline 1101 and the carrier trap 1102, and discharging from the waste liquid outlet 1107.

[0141] Step S9 cleans the entire solid-phase carrier microfluidic gene synthesis system, preventing residual substances from the synthesis reaction from affecting the next use of the system.

[0142] Furthermore, the present invention discloses that in the above steps, the solid-phase carrier microfluidic gene synthesis system is opened at each point that needs to be connected through the corresponding control valves of the microfluidic valve group 12, while the corresponding control valves at other locations are closed.

[0143] Understandably, or perhaps it is possible, that all points requiring conduction in the solid-phase carrier microfluidic gene synthesis system are opened through the corresponding control valves of the microfluidic valve group 12, while only the inputs that are not needed are closed.

[0144] Example 1

[0145] This embodiment uses adenine synthesis reagent inlet 1105a, thymine synthesis reagent inlet 1105b, cytosine synthesis reagent inlet 1105c, and guanine synthesis reagent inlet 1105d as examples, and adenine synthesis reagent inlet control valve 1205a, thymine synthesis reagent inlet control valve 1205b, cytosine synthesis reagent inlet control valve 1205c, and guanine synthesis reagent inlet control valve 1205d as examples, to illustrate the use of only adenine synthesis reagent.

[0146] The solid-phase carrier microfluidic gene synthesis system includes the following steps:

[0147] Step S1: Close all microfluidic valve groups 12 to complete the initialization of microfluidic valve groups 12;

[0148] Step S2: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204, so that the buffer solution is introduced from the inlet under positive pressure, flows along the main pipeline 1101 and is cleaned, and the cleaned waste liquid is discharged from the waste liquid outlet 1107;

[0149] Step S3: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204 to stop the buffer input and complete the initialization of the main pipeline 1101;

[0150] Step S4: Open the carrier inlet control valve 1208, the upstream pipeline control valve 1201, the synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and the waste liquid outlet control valve 1204, so that the solid carrier is input through the carrier inlet 1103 under positive pressure, flows along the pipeline and is captured by the carrier capture devices 1102a, 1102b, 1102c, 1102d, 1102e, 1102f, 1102g, 1102h, 1102i and 1102j;

[0151] Step S5: Close the carrier inlet control valve 1208, upstream pipeline control valve 1201, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204 to stop the solid carrier input;

[0152] Step S6: Open the buffer inlet control valve 1206, the upstream pipeline control valve 1201, the synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and the waste liquid outlet control valve 1204, so that the buffer solution is introduced from the inlet under positive pressure, flows along the pipeline and completes the carrier cleaning, while the excess carrier sample is discharged from the waste liquid outlet 1107;

[0153] Step S7: Close the buffer inlet control valve 1206, the upstream pipeline control valve 1201, the synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and the waste liquid outlet control valve 1204 to stop the buffer input;

[0154] Step S8: Open the adenine synthesis reagent inlet control valve 1205a, upstream pipeline control valve 1201, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204, so that the adenine synthesis reagent is input through the adenine synthesis reagent inlet 1105a under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier, and the overflow reagent is discharged from the waste liquid outlet 1107;

[0155] Step S9: Close the adenine synthesis reagent inlet control valve 1205a, upstream pipeline control valve 1201, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204 to stop the input of adenine synthesis reagent and complete the soaking of the solid carrier to achieve the synthesis reaction;

[0156] Step S10: Open the buffer inlet control valve 1206, the upstream pipeline control valve 1201, the synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and the waste liquid outlet control valve 1204, so that the buffer solution is introduced from the inlet under positive pressure, flows along the main pipeline 1101 and cleans the solid phase carrier, and at the same time, the residual adenine synthesis reagent is discharged from the waste liquid outlet 1107;

[0157] Step S11: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning;

[0158] Step S12: Open the buffer inlet control valve 1206, downstream pipeline control valve 1202, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and carrier collection port control valve 1203, so that the buffer solution is input through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and collects the synthesized solid carrier through the carrier collection port 1106;

[0159] Step S13: Close the buffer inlet control valve 1206, downstream pipeline control valve 1202, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and carrier collection port control valve 1203 to stop the buffer input and complete the solid carrier collection;

[0160] Step S14: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204, so that the buffer solution is input through the buffer solution inlet 1104 under positive pressure, flows along the main pipeline 1101 and performs post-synthesis cleaning, and the cleaning waste liquid is discharged through the waste liquid outlet 1107;

[0161] Step S15: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a, 1207b, 1207c, 1207d, 1207e, 1207f, 1207g, 1207h, 1207i, 1207j and waste liquid outlet control valve 1204 to stop buffer input and complete system recovery.

[0162] It should be noted that this embodiment is based on the use of only adenine synthesis reagent. When only thymine synthesis reagent, cytosine synthesis reagent, or guanine synthesis reagent is used, the steps are similar to those described above, except that the input port of the corresponding thymine synthesis reagent, cytosine synthesis reagent, or guanine synthesis reagent is replaced.

[0163] Example 2

[0164] This embodiment uses the synthesis of the target sequence AGCT (A represents adenine, G represents guanine, C represents cytosine, and T represents thymine) and the synthesis reaction carried out on the carrier trap 1102a as an example for illustration.

[0165] The solid-phase carrier microfluidic gene synthesis system includes the following steps:

[0166] Step S1: Close all microfluidic valve groups 12 to complete the initialization of microfluidic valve groups 12;

[0167] Step S2: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is input through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and is cleaned by the carrier trap 1102a, and the cleaned waste liquid is discharged from the waste liquid outlet 1107.

[0168] Step S3: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the buffer input and complete the cleaning and initialization.

[0169] Step S4: Open the carrier inlet control valve 1208, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the solid carrier is input through the carrier inlet 1103 under positive pressure, flows along the main pipeline 1101 and is captured by the carrier capture device 1102a.

[0170] Step S5: Close the carrier inlet control valve 1208, the upstream pipeline control valve 1201, the synthesis station control valve 1207a, and the waste liquid outlet control valve 1204 to stop the solid carrier input;

[0171] Step S6: Open the buffer inlet control valve 1206, the upstream pipeline control valve 1201, the synthesis station control valve 1207a and the waste liquid outlet control valve 1204, so that the buffer solution is input from the inlet under positive pressure, flows along the main pipeline 1101 and completes the carrier cleaning, while the excess solid carrier is discharged from the waste liquid outlet 1107.

[0172] Step S7: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the buffer input;

[0173] Step S8: Open the adenine synthesis reagent inlet control valve 1205a, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the adenine synthesis reagent is input through the adenine synthesis reagent inlet 1105a under positive pressure, flows along the main pipeline 1101 and completes the soaking of the capture and fixation carrier, and the overflow reagent is discharged from the waste liquid outlet 1107.

[0174] Step S9: Close the adenine synthesis reagent inlet control valve 1205a, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the input of adenine synthesis reagent and complete the soaking of the capture solid carrier to realize the synthesis reaction.

[0175] Step S10: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is input from the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time discharges the residual adenine synthesis reagent from the waste liquid outlet 1107.

[0176] Step S11: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0177] Step S12: Open the guanine synthesis reagent inlet control valve 1205d, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the guanine synthesis reagent is input through the guanine synthesis reagent inlet 1105d under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0178] Step S13: Close the guanine synthesis reagent inlet control valve 1205d, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the input of guanine synthesis reagent and complete the soaking of the capture solid carrier to realize the synthesis reaction.

[0179] Step S14: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is input from the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time discharges the residual guanine synthesis reagent from the waste liquid outlet 1107.

[0180] Step S15: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0181] Step S16: Open the cytosine synthesis reagent inlet control valve 1205c, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the cytosine synthesis reagent is input through the reagent inlet under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0182] Step S17: Close the cytosine synthesis reagent input control valve 1205c, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid discharge control valve 1204 to stop the input of cytosine synthesis reagent and complete the soaking of the capture solid support to realize the synthesis reaction.

[0183] Step S18: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is introduced through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time, the residual cytosine synthesis reagent is discharged from the waste liquid outlet 1107.

[0184] Step S19: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0185] Step S20: Open the thymine synthesis reagent inlet control valve 1205b, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the thymine synthesis reagent is input through the thymine synthesis reagent inlet 1105b under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0186] Step S21: Close the thymine synthesis reagent inlet control valve 1205b, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the input of thymine synthesis reagent and complete the soaking of the capture solid carrier to realize the synthesis reaction.

[0187] Step S22: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is input through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time, the residual thymine synthesis reagent is discharged from the waste liquid outlet 1107.

[0188] Step S23: Close the buffer inlet control valve 1206, the upstream pipeline control valve 1201, the synthesis station control valve 1207a and the waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0189] Step S24: Open the buffer inlet control valve 1206, downstream pipeline control valve 1202, synthesis station control valve 1207a and carrier collection port control valve 1203, so that the buffer solution is input from the inlet under positive pressure, flows along the main pipeline 1101 and the synthesized solid carrier is collected from the carrier collection port 1106.

[0190] Step S25: Close the buffer inlet control valve 1206, downstream pipeline control valve 1202, synthesis station control valve 1207a and carrier collection port control valve 1203 to stop the buffer input and complete the solid carrier collection step;

[0191] Step S26: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is input from the inlet under positive pressure, flows along the main pipeline 1101 and is cleaned, and the cleaned waste liquid is discharged from the waste liquid outlet 1107.

[0192] Step S27: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop buffer input and complete system recovery.

[0193] It should be noted that when other gene sequences need to be prepared, the corresponding synthetic reagents can be replaced according to the above steps.

[0194] The solid-phase carrier microfluidic gene synthesis system provided by the present invention can not only realize the synthesis of one gene sequence on one carrier trap 1102, but also realize the synthesis of two or more different gene sequences on two or more carrier traps 1102 respectively.

[0195] Example 3

[0196] This embodiment takes the synthesis of the AGCT gene sequence on vector trap 1102a and the synthesis of ACCT on vector trap 1102b as an example.

[0197] The solid-phase carrier microfluidic gene synthesis system includes the following steps:

[0198] Step S1: Close all microfluidic valve groups 12 to complete the initialization of microfluidic valve groups 12;

[0199] Step S2: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a and 1207b, and waste liquid outlet control valve 1204, so that the buffer solution is input through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the carrier traps 1102a and 1102b, and the cleaned waste liquid is discharged from the waste liquid outlet 1107;

[0200] Step S3: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the buffer input and complete the cleaning and initialization.

[0201] Step S4: Open the carrier inlet control valve 1208, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the solid carrier is input through the carrier inlet 1103 under positive pressure, flows along the main pipeline 1101 and is captured by the carrier traps 1102a and 1102b;

[0202] Step S5: Close the carrier inlet control valve 1208, the upstream pipeline control valve 1201, the synthesis station control valves 1207a and 1207b, and the waste liquid outlet control valve 1204 to stop the solid carrier input;

[0203] Step S6: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the buffer solution is input from the inlet under positive pressure, flows along the main pipeline 1101 and completes the capture and cleaning of the fixed carrier, while the excess solid carrier is discharged from the waste liquid outlet 1107.

[0204] Step S7: Close the buffer inlet control valve 1206, the upstream pipeline control valve 1201, the synthesis station control valves 1207a and 1207b, and the waste liquid outlet control valve 1204 to stop the buffer input;

[0205] Step S8: Open the adenine synthesis reagent inlet control valve 1205a, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the adenine synthesis reagent is input through the adenine synthesis reagent inlet 1105a under positive pressure, flows along the main pipeline 1101 and completes the soaking of the carrier for capturing and fixing, and the overflowing reagent is discharged from the waste liquid outlet 1107.

[0206] Step S9: Close the adenine synthesis reagent inlet control valve 1205a, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the input of adenine synthesis reagent and complete the soaking of the capture solid carrier to realize the synthesis reaction.

[0207] Step S10: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the buffer solution is input through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time, the residual adenine synthesis reagent is discharged from the waste liquid outlet 1107.

[0208] Step S11: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0209] Step S12: Open the guanine synthesis reagent inlet control valve 1205d, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the guanine synthesis reagent is input through the guanine synthesis reagent inlet 1105d under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0210] Step S13: Close the guanine synthesis reagent inlet control valve 1205d, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the input of guanine synthesis reagent and complete the soaking of the capture solid carrier to realize the synthesis reaction.

[0211] Step S14: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204, so that the buffer solution is input from the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time discharges the residual guanine synthesis reagent from the waste liquid outlet 1107.

[0212] Step S15: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207a and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0213] Step S16: Open the cytosine synthesis reagent inlet control valve 1205c, upstream pipeline control valve 1201, synthesis station control valve 1207b and waste liquid outlet control valve 1204, so that the cytosine synthesis reagent is input through the reagent inlet under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0214] Step S17: Close the cytosine synthesis reagent input control valve 1205c, upstream pipeline control valve 1201, synthesis station control valve 1207b and waste liquid discharge control valve 1204 to stop the input of cytosine synthesis reagent and complete the soaking of the capture solid support to realize the synthesis reaction.

[0215] Step S18: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207b and waste liquid outlet control valve 1204, so that the buffer solution is introduced through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the carrier, and at the same time, the residual cytosine synthesis reagent is discharged from the waste liquid outlet 1107.

[0216] Step S19: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valve 1207b and waste liquid outlet control valve 1204 to stop the buffer input and complete the carrier cleaning step;

[0217] Step S20: Open the cytosine synthesis reagent inlet control valve 1205c, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the cytosine synthesis reagent is input through the reagent inlet under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0218] Step S21: Close the cytosine synthesis reagent inlet control valve 1205c, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the input of cytosine synthesis reagent and complete the soaking of the capture solid support to realize the synthesis reaction.

[0219] Step S22: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the buffer solution is introduced through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the carrier, and at the same time, the residual cytosine synthesis reagent is discharged through the waste liquid outlet 1107.

[0220] Step S23: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the buffer input and complete the carrier cleaning step;

[0221] Step S24: Open the thymine synthesis reagent inlet control valve 1205b, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the thymine synthesis reagent is input through the thymine synthesis reagent inlet 1105b under positive pressure, flows along the main pipeline 1101 and completes the soaking of the solid carrier. The overflowing reagent will be discharged from the waste liquid outlet 1107.

[0222] Step S25: Close the thymine synthesis reagent inlet control valve 1205b, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the input of thymine synthesis reagent and complete the soaking of the capture solid carrier to realize the synthesis reaction.

[0223] Step S26: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the buffer solution is input through the buffer inlet 1104 under positive pressure, flows along the main pipeline 1101 and cleans the solid carrier, and at the same time, the residual thymine synthesis reagent is discharged from the waste liquid outlet 1107.

[0224] Step S27: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204 to stop the buffer input and complete the solid carrier cleaning step.

[0225] Step S28: Open the buffer inlet control valve 1206, downstream pipeline control valve 1202, synthesis station control valves 1207a and 1207b and carrier collection port control valve 1203, so that the buffer solution is input from the inlet under positive pressure, flows along the main pipeline 1101 and the synthesized solid carrier is collected from the carrier collection port 1106.

[0226] Step S29: Close the buffer inlet control valve 1206, downstream pipeline control valve 1202, synthesis station control valves 1207a and 1207b and carrier collection port control valve 1203 to stop the buffer input and complete the solid carrier collection step.

[0227] Step S30: Open the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a and 1207b and waste liquid outlet control valve 1204, so that the buffer solution is input from the inlet under positive pressure, flows along the main pipeline 1101 and is cleaned, and the cleaned waste liquid is discharged from the waste liquid outlet 1107;

[0228] Step S31: Close the buffer inlet control valve 1206, upstream pipeline control valve 1201, downstream pipeline control valve 1202, synthesis station control valves 1207a and 1207b, and waste liquid outlet control valve 1204 to stop buffer input and complete system recovery.

[0229] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0230] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and inventive features disclosed herein.

[0231] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0232] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to any specific implementation. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A solid-phase carrier microfluidic gene synthesis system, characterized in that, include: substrate; A microfluidic pipeline mounted on the substrate, the microfluidic pipeline including a main pipeline and a carrier trap, the main pipeline having multiple inlet ports at its inlet end and a drain port at its outlet end, the carrier trap being installed inside the main pipeline for capturing solid carriers; A microfluidic valve assembly mounted on the substrate is used to switch the flow direction of the fluid in the main pipeline to collect waste liquid or the solid carrier after the synthesis reaction is completed; The carrier capture device includes a first capture block and a second capture block; The first capture block and the second capture block are respectively installed in the main pipeline, and a capture gap is formed between the first capture block and the second capture block for capturing a single solid carrier. The gap formed between the first capture block and the second capture block and the pipe wall of the main pipeline adjacent to them allows the solid carrier to pass through. Along the direction in which the solid carrier flows through the carrier trap, the first trapping block and the second trapping block are tilted toward each other to trap the solid carrier.

2. The solid-phase carrier microfluidic gene synthesis system according to claim 1, characterized in that, Both the first capture block and the second capture block are arc-shaped blocks.

3. The solid-phase carrier microfluidic gene synthesis system according to claim 1, characterized in that, The input ports include a buffer solution input port, a carrier input port, and a synthetic reagent input port; The discharge port includes a carrier collection port and a waste liquid discharge port.

4. The solid-phase carrier microfluidic gene synthesis system according to claim 3, characterized in that, The main pipeline includes: The input tube has a buffer inlet located at the inlet end, and the synthetic reagent inlet and the carrier inlet are sequentially spaced along the direction from the inlet end to the outlet end of the input tube. An upstream pipe and a downstream pipe, whose inlets are respectively connected to the outlet of the input pipe, have a carrier collection port located at the outlet of the upstream pipe and a waste liquid discharge port located at the outlet of the downstream pipe. A carrier tube is connected at both ends to the upstream pipe and the downstream pipe, respectively. The carrier trap is installed inside the carrier tube, with its first port connected to the upstream pipe and its second port connected to the downstream pipe. When the solid carrier enters the carrier trap from the first communication port, the carrier trap is able to capture the solid carrier. When the buffer solution enters the carrier trap from the second connection port, the carrier trap is able to flush the solid-phase carrier out of the carrier trap.

5. The solid-phase carrier microfluidic gene synthesis system according to claim 4, characterized in that, The microfluidic valve assembly includes: An upstream pipeline control valve is used to control the on / off state of the upstream pipeline and the carrier pipeline; Downstream pipeline control valve, used to control the on / off state of the downstream pipeline and the carrier pipeline; A carrier collection port control valve is used to control the connection and disconnection between the carrier collection port and the upstream pipe; A waste liquid discharge outlet control valve is used to control the connection and disconnection between the waste liquid discharge outlet and the downstream pipe.

6. The solid-phase carrier microfluidic gene synthesis system according to claim 5, characterized in that, The microfluidic valve assembly also includes a synthetic reagent inlet control valve, which is configured to correspond one-to-one with the synthetic reagent inlet and is used to control the connection and disconnection between each synthetic reagent inlet and the main pipeline. and / or The microfluidic valve assembly also includes a buffer inlet control valve for controlling the connection and disconnection between the buffer inlet and the main pipeline; and / or The microfluidic valve assembly also includes a synthesis station control valve for controlling the connection and disconnection between the two ends of the carrier trap and the upstream and downstream pipes; and / or The microfluidic valve assembly also includes a carrier inlet control valve for controlling the connection and disconnection between the carrier inlet and the inlet tube.

7. The solid-phase carrier microfluidic gene synthesis system according to claim 6, characterized in that, The microfluidic valve assembly is located above the microfluidic pipeline, and each control valve of the microfluidic valve assembly includes a valve body and a valve tube. The inlet of the valve tube is used to introduce positive pressure, and the outlet of the valve tube is connected to the valve body. The valve body is located at the position to be controlled for the main pipeline and deforms under the positive pressure input by the valve tube. When the control valve is closed, positive pressure is introduced into the valve pipe, causing the valve body to deform downwards to block the controllable on / off position of the main pipeline.

8. The solid-phase carrier microfluidic gene synthesis system according to claim 7, characterized in that, The ratio of the length of the valve body along the flow direction of the main pipeline to the width of the main pipeline is greater than or equal to 1.5; The ratio of the width of the valve body perpendicular to the flow direction of the main pipeline to the width of the main pipeline is greater than or equal to 3.

9. The solid-phase carrier microfluidic gene synthesis system according to claim 7, characterized in that, The ratio of the width of the valve pipe projected onto the main pipeline to the width of the main pipeline is less than or equal to 0.

25.

10. A microfluidic gene synthesis method using a solid-phase carrier, characterized in that, include: Step S1: Provide a solid-phase carrier microfluidic gene synthesis system as described in any one of claims 3-9; Step S2: Determine whether the solid-phase carrier microfluidic gene synthesis system needs cleaning. If yes, proceed to step S3; otherwise, proceed to step S4. Step S3: Control the microfluidic valve assembly to open the waste liquid outlet and input buffer solution into the buffer solution inlet so that the buffer solution flows through the main pipeline and is discharged from the waste liquid outlet. After cleaning is completed, proceed to step S4. Step S4: Input a solid carrier into the carrier inlet, and the solid carrier flows along the main pipeline and is captured by the carrier catcher; Step S5: Input the buffer solution into the buffer solution inlet. The buffer solution flows along the main pipeline to clean the main pipeline and the carrier trap, and discharge excess solid carrier. Step S6: Input the corresponding synthetic reagent into the corresponding synthetic reagent input port according to the required synthetic unit. The synthetic reagent flows along the main pipeline and passes through the carrier trap to soak the solid support captured by the carrier trap, thereby completing the synthetic reaction. Step S7: Control the microfluidic valve assembly to close the waste liquid outlet and open the carrier collection port, and input the buffer solution into the buffer solution inlet. The buffer solution flows along the main pipeline to discharge the solid-phase carrier that has completed the synthesis reaction, which has been captured by the carrier trap, to the carrier collection port.

11. The solid-phase carrier microfluidic gene synthesis method according to claim 10, characterized in that, Between step S6 and step S7, there is also step S8: determining whether all types of synthetic reagents required for the synthetic unit to be prepared have participated in the synthetic reaction. If yes, proceed to step S7; otherwise, proceed to step S5.

12. The solid-phase carrier microfluidic gene synthesis method according to claim 10, characterized in that, The main pipeline includes an input pipe and an upstream pipe and a downstream pipe whose inlet ends are respectively connected to the outlet end of the input pipe. The buffer solution inlet is located at the inlet end of the input pipe. The synthetic reagent inlet and the carrier inlet are sequentially and spaced apart on the input pipe along the direction from the inlet end to the outlet end. The carrier collection port is located at the outlet end of the upstream pipe, and the waste liquid outlet is located at the outlet end of the downstream pipe. When the carrier collection port is opened, the microfluidic valve assembly is controlled to connect the downstream pipe and the input pipe, and disconnect the upstream pipe and the input pipe. When the waste liquid outlet is opened, the microfluidic valve assembly is controlled to disconnect the downstream pipe from the input pipe and connect the upstream pipe to the input pipe.

13. The solid-phase carrier microfluidic gene synthesis method according to claim 10, characterized in that, Step S7 is followed by step S9: controlling the microfluidic valve assembly to open the waste liquid outlet and close the carrier collection port, inputting the buffer solution into the buffer solution inlet, the buffer solution flowing along the main pipeline to clean the main pipeline and the carrier trap, and then discharging from the waste liquid outlet.

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