CN219784710U

CN219784710U - Nucleic acid synthesis system and flow limiting valve device

Info

Publication number: CN219784710U
Application number: CN202321158270.6U
Authority: CN
Inventors: 王祥林; 刘根水; 王凯; 王正涛; 刘杰; 郑洲; 张中春
Original assignee: Jiangsu Hanbon Science and Technology Co Ltd
Current assignee: Jiangsu Hanbon Science and Technology Co Ltd
Priority date: 2022-09-29
Filing date: 2023-05-15
Publication date: 2023-10-03
Anticipated expiration: 2033-05-15
Also published as: CN115253953A

Abstract

The utility model discloses a nucleic acid synthesis system, comprising: the device comprises a multipath pre-column liquid path, an anti-mixing valve, a synthetic column, a circulating valve, a circulating liquid path and a liquid discharge liquid path; each liquid path in front of the column comprises an inlet valve and an infusion pump, wherein the inlet of the inlet valve is connected with a monomer bottle or a reagent bottle, the outlet of the inlet valve is connected with the inlet of the infusion pump, and the outlet of the infusion pump is connected with the inlet of the anti-mixing valve; the outlet of the anti-mixing valve is connected with the column inlet of the synthetic column; the column outlet of the synthetic column is connected with the inlet of a circulating valve, one outlet of the circulating valve is connected with the inlet of a circulating liquid path, and the other outlet of the circulating valve is connected with the inlet of a liquid draining path; the outlet of the circulating liquid path is connected with an inlet valve of a liquid path in front of a certain path of column; a flow limiting valve device is connected to the outlet of each infusion pump; the nucleic acid synthesis system has the advantage of more accurate reagent delivery; the utility model discloses a flow limiting valve device, which enables reagent delivery to be more accurate.

Description

Nucleic acid synthesis system and flow limiting valve device

Technical Field

The utility model belongs to the technical field of nucleic acid synthesis, and particularly relates to a nucleic acid synthesis system and a flow limiting valve device.

Background

With the rapid development of oligonucleotide chain synthesis technology, synthesis cost is continuously reduced, synthesis length and precision are continuously improved, so that large-scale DNA and RNA synthesis taking an oligonucleotide chain as a starting material is possible, and a nucleic acid synthesis system is developed.

However, the current nucleic acid synthesis systems suffer from drawbacks including: reagent delivery is not accurate enough; one reason for the inaccurate reagent delivery is that: the monomer and the reagent used in the nucleic acid synthesis process have higher hydrophilicity, and the reagent is required to be isolated from moisture in the air by applying inert gas to keep micro-positive pressure.

Disclosure of Invention

The utility model aims to: in order to solve the problem that the micro-positive pressure gas drives a reagent to enter the system to cause abnormal pushing amount of monomers and reagents, the utility model provides a nucleic acid synthesis system and a flow limiting valve device, so that the flow of the conveyed liquid is more accurate.

The technical scheme is as follows: a nucleic acid synthesis system, comprising: the device comprises a multipath pre-column liquid path, an anti-mixing valve, a synthetic column, a circulating valve, a circulating liquid path and a liquid discharge liquid path;

each liquid path in front of the column comprises an inlet valve and an infusion pump, wherein the inlet of the inlet valve is connected with a monomer bottle or a reagent bottle, the outlet of the inlet valve is connected with the inlet of the infusion pump, and the outlet of the infusion pump is connected with the inlet of the anti-mixing valve; the outlet of the anti-mixing valve is connected with the column inlet of the synthetic column; the column outlet of the synthetic column is connected with the inlet of a circulating valve, one outlet of the circulating valve is connected with the inlet of a circulating liquid path, and the other outlet of the circulating valve is connected with the inlet of a liquid draining liquid path; the outlet of the circulating liquid path is connected with an inlet valve of a liquid path in front of a certain path of column;

a flow limiting valve device is connected to the outlet of each infusion pump;

the flow limiting valve device comprises a flow limiting valve body, a flow limiting valve liquid inlet, a flow limiting valve liquid outlet, a flow limiting valve sealing gasket, a T-shaped block, a flow limiting valve spring, a pressing block and a jackscrew;

the flow limiting valve sealing gasket is positioned in the flow limiting valve body and is in interference fit with the flow limiting valve body; the transverse end of the T-shaped block is fixed with the sealing gasket of the flow limiting valve; the flow limiting valve spring is sleeved on the vertical end of the T-shaped block, one end of the flow limiting valve spring is fixed on the transverse end of the T-shaped block, the other end of the flow limiting valve spring is fixedly connected with the pressing block, and a space exists between the vertical end of the T-shaped block and the pressing block; the pressing block is fixed in the valve body of the flow-limiting valve through a jackscrew;

the liquid inlet of the limiting valve and the liquid outlet of the limiting valve are both arranged on the valve body of the limiting valve, a liquid inlet channel is arranged between the liquid inlet of the limiting valve and the sealing pad of the limiting valve, and a liquid outlet channel is arranged between the liquid outlet of the limiting valve and the sealing pad of the limiting valve.

Further, the inlet valve is of a valve group structure;

the valve group structure includes: the device comprises a plurality of liquid inlets, a liquid outlet, a valve base and a plurality of cylinder assemblies, wherein the plurality of cylinder assemblies are arranged on the valve base, and the liquid inlets correspond to the cylinder assemblies one by one; the valve base is internally provided with a plurality of holes and a common channel, each hole is communicated with the common channel, the liquid inlets are in one-to-one correspondence with the arrangement positions of the holes in the valve base at the arrangement positions of the valve base, so that the common channel is communicated with each liquid inlet, and the liquid outlets are positioned at the tail ends of the common channel;

each air cylinder assembly consists of a diaphragm, a shell seat, a movable rod, a shell, a spring, a check ring, a piston and a pneumatic connector; the shell seat is fixed with the shell, the movable rod extends downwards into the shell seat, the lower end of the movable rod is fixed with the membrane, and the movable rod moves up and down in the shell seat; the movable rod extends upwards and is fixedly connected with the piston through a check ring;

a space for the piston to move downwards is reserved between the lower end of the piston and the shell seat, and the bottom of the spring is fixed on the upper surface of the shell seat; the upper part of the spring is sleeved outside the lower end of the piston and is fixedly connected with a limiting ring on the outer side of the lower end of the piston; a space for air to enter is reserved between the upper end of the piston and the pneumatic connector; the pneumatic connector is connected with an external air source;

when the external air source is not ventilated, the spring is in a free state, the diaphragm leaves the bottom plane of the hole in the valve base, and the corresponding liquid inlet is in an open state;

when the external air source is ventilated, the spring is in a compressed state, the diaphragm is pressed on the bottom plane of the hole in the valve base, and the corresponding liquid inlet is in a closed state.

Further, the anti-mixing valve is of a valve group structure.

Further, the circulating valve is of a valve group structure.

Furthermore, the nucleic acid synthesis system also comprises a multi-path protection gas path;

each path of protection gas circuit comprises a gas source distributor and a protection pressure regulating valve;

the inlet of the protection pressure regulating valve is used for acquiring inert gas, the outlet of the protection pressure regulating valve is connected with the inlet of the gas source distributor, and the outlet of the gas source distributor distributes the inert gas to the monomer bottle/the reagent bottle.

Further, the nucleic acid synthesis system further comprises a flush valve; the inlet of the flushing valve is connected with the other outlet of the anti-mixing valve; an outlet of the flushing valve is connected with a pressure solvent inlet of the synthetic column; the other outlet of the flushing valve is connected with the inlet of the liquid draining path. The flush valve has two modes, including: a column loading mode and a flushing mode; when the flushing valve is in the column loading mode, one outlet of the flushing valve is opened, and the other outlet of the flushing valve is closed; when the flush valve is in flush mode, one outlet of the flush valve is closed and the other outlet of the flush valve is open.

The utility model also discloses a flow limiting valve device, which comprises a flow limiting valve body, a flow limiting valve liquid inlet, a flow limiting valve liquid outlet, a flow limiting valve sealing gasket, a T-shaped block, a flow limiting valve spring, a pressing block and a jackscrew;

The beneficial effects are that: compared with the prior art, the utility model has the following advantages:

(1) According to the utility model, by adopting the flow limiting valve device, the reagent is prevented from being driven into the system by micro-positive pressure gas, the accuracy of the reagent conveying amount is further improved, the material proportioning is ensured to be accurate, and the purity of a target product is greatly improved;

(2) The utility model adopts the valve group structure, reduces the volume of the liquid path, saves the consumption of reagents, improves the pressure resistance of the membrane in each valve to 20bar, and greatly improves the pressure resistance stability of the nucleic acid synthesis system.

Drawings

FIG. 1 is a schematic perspective view of a nucleic acid synthesis system;

FIG. 2 is a schematic diagram of a forward structure of a nucleic acid synthesis system;

FIG. 3 is a block diagram of a nucleic acid synthesis system;

FIG. 4 is a schematic view of an inert gas shield apparatus;

FIG. 5 is a schematic diagram of a flow restrictor valve device;

fig. 6 is a schematic diagram of a valve block structure.

Detailed Description

The technical scheme of the utility model is further described with reference to the accompanying drawings and the embodiments.

Example 1:

as shown in fig. 1, 2 and 3, the nucleic acid synthesis system of the present embodiment mainly includes: the device comprises a PLC controller, a cabinet body 1, a monomer inlet valve 2, a reagent inlet valve 3, a reagent inlet valve 4, an infusion pump 5, an infusion pump 6, an infusion pump 7, a pressure sensor 8, a pressure sensor 9, a three-way joint 10, an anti-mixing valve 11, a synthesis column 12, a pressure sensor 13, a conductivity detector 14, a circulation valve 15, a flushing valve 16, an ultraviolet detector 17, a back pressure valve 18, a waste discharge valve 19, a monomer bottle 20, a pressure regulating valve 21, a pressure sensor 22, an air source distributor 23, a reagent bottle 24, a pressure sensor 25, a pressure regulating valve 26, a pressure regulating valve 27, a pressure sensor 28 and an air source distributor 29. The inlet of the monomer inlet valve 2 is connected with a monomer bottle 20, and the inlet of the reagent inlet valve I3 and the inlet of the reagent inlet valve II 4 are respectively connected with different reagent bottles 24.

The PLC controller, the single inlet valve 2, the reagent inlet valve I3, the reagent inlet valve II 4, the waste discharge valve 19, the infusion pump III 5, the infusion pump II 6, the infusion pump I7, the pressure sensor II 8, the pressure sensor I9, the three-way joint 10, the pressure sensor III 13, the conductivity detector 14, the circulation valve 15, the mixing prevention valve 11, the flushing valve 16, the ultraviolet detector 17 and the back pressure valve 18 are all arranged on the cabinet body 1. The monomer bottle 20 and the reagent bottle 24 are arranged below the cabinet body 1, and the monomer bottle 20 and the reagent bottle 24 can be fixed below the cabinet body 1 by adopting a tray.

The air pipe component is arranged at the left side and the lower side of the cabinet body 1, wherein the air pipe component comprises a pressure sensor IV 22, a pressure sensor V25, a pressure sensor VI 28, a pressure regulating valve I21, a pressure regulating valve II 26, a pressure regulating valve III 27, an air source distributor I23 and an air source distributor II 29, and specifically, the pressure sensor IV 22, the pressure sensor V25, the pressure sensor VI 28, the pressure regulating valve I21, the pressure regulating valve II 26 and the pressure regulating valve III 27 are arranged at the left side of the cabinet body 1; the first air source distributor 23 and the second air source distributor 29 are arranged below the cabinet 1. The synthetic column 12 is fixed to the left side of the cabinet 1 by a clip.

In this embodiment, chuck joints may be further disposed on the left and right sides of the cabinet body 1, and when a large amount of solvent is used, the chuck joints may be connected to an external vulcanization hose, so as to complete the access and efficient connection of the large amount of solvent. The cabinet body 1 of the embodiment is designed as an explosion-proof cabinet; the synthetic column 12 can be configured with various specifications according to the process requirements; the number of lines for the inlet valve and the waste valve may be reduced or increased depending on the process requirements.

As shown in fig. 3, the three inlet valves in this embodiment correspond to three pre-column liquid paths respectively, and the first pre-column liquid path is composed of a single inlet valve 2, a third infusion pump 5 and a second pressure sensor 8; the outlet of the monomer inlet valve 2 is connected with the inlet of the infusion pump III 5 through a liquid pipeline, the outlet of the infusion pump III 5 is connected with one inlet of the anti-mixing valve 11 through a liquid pipeline, and the pressure sensor II 8 is arranged on the liquid pipeline connecting the infusion pump III 5 with the anti-mixing valve 11. When the first pre-column liquid path is used for conveying fluid, the second pressure sensor 8 is used for monitoring feedback pressure data before the synthetic column 12 and uploading the monitored feedback pressure data to the PLC, the PLC can upload the data to the industrial personal computer, and the industrial personal computer judges whether to perform high-pressure alarm or not, so that overpressure can be prevented, and hardware damage and carrier damage in the synthetic column 12 can be prevented.

The second pre-column liquid route consists of a first reagent inlet valve 3, a second infusion pump 6 and a first pressure sensor 9; the outlet of the reagent inlet valve I3 is connected with the inlet of the infusion pump II 6 through a liquid pipeline, the outlet of the infusion pump II 6 is connected with the three-way joint 10 through a liquid pipeline, and the outlet of the three-way joint 10 is connected with the other inlet of the anti-mixing valve 11 through a liquid pipeline; the first pressure sensor 9 is arranged on a liquid pipeline connecting the three-way joint 10 and the anti-mixing valve 11.

The third pre-column liquid route consists of a reagent inlet valve II 4 and an infusion pump I7; the outlet of the reagent inlet valve II 4 is connected with the inlet of the infusion pump I7, and the outlet of the infusion pump I7 is connected with the three-way joint 10 through a liquid pipeline. When the second pre-column liquid path is used for conveying fluid or the third pre-column liquid path is used for conveying fluid, the first pressure sensor 9 is used for monitoring feedback pressure data before the synthetic column 12 and uploading the monitored feedback pressure data to the PLC, the PLC can upload the data to the industrial personal computer, and the industrial personal computer can judge whether high-pressure alarm is carried out or not, so that overpressure can be prevented, and hardware damage and carrier damage in the synthetic column 12 can be caused. The three pre-column liquid paths are connected with the synthesis column 12 through the anti-mixing valve 11, namely, one outlet of the anti-mixing valve 11 is connected with a column inlet of the synthesis column 12 through a liquid pipeline, the column outlet of the synthesis column 12 is connected with an inlet of the circulation valve 15 through a liquid pipeline, and one outlet of the circulation valve 15 is connected with an inlet of the reagent inlet valve II 4 through a liquid pipeline to form liquid circulation; the other outlet of the circulating valve 15 is connected with the inlet of the back pressure valve 18 through a liquid pipeline, and the outlet of the back pressure valve 18 is connected with the waste discharge valve 19 through a liquid pipeline to realize liquid discharge; the ultraviolet detector 17 is disposed on a liquid line connecting the other outlet of the circulation valve 15 and the back pressure valve 18, in other words, the back pressure valve 18 is located at the tail end of the ultraviolet detector 17; the ultraviolet detector 17 is configured to acquire ultraviolet absorption data of a substance after a chemical reagent is reacted, and upload the ultraviolet absorption data to the PLC controller, where the PLC controller may upload the data to the industrial personal computer, and the industrial personal computer monitors the substance reaction conditions and reaction results in different process stages according to the ultraviolet absorption data. The back pressure valve 18 is provided to provide an appropriate back pressure to the synthesis system of the present embodiment, prevent the front-end liquid path from generating bubbles and affecting the detection data, and keep the in-column reaction sufficient.

The liquid circulation and drainage functions of the synthesis system of this embodiment will now be further described with reference to fig. 3.

When the circulation valve 15 is used for liquid circulation, a liquid circulation passage is formed by a reagent inlet valve II 4, an infusion pump I7, a pressure sensor I9, an anti-mixing valve 11, a synthesis column 12, a pressure sensor III 13, a conductivity detector 14, the circulation valve 15 and a plurality of liquid pipelines; the infusion pump 7 drives the liquid to complete the circulation process in the liquid circulation path.

The liquid discharge path of the embodiment is formed by sequentially connecting a first reagent inlet valve 3, a second infusion pump 6, an anti-mixing valve 11, a synthetic column 12, a conductivity detector 14, a circulating valve 15, an ultraviolet detector 17, a back pressure valve 18 and a waste discharge valve 19 through liquid pipelines; the second infusion pump 6 drives the liquid in the liquid discharge passage to be discharged from the waste valve 19.

The synthesis column 12 of the present embodiment may adopt the following structure, mainly including: the device comprises a synthetic column body, a column inlet, a column outlet, a column inner piston positioned in the synthetic column body, a pressure solvent inlet and an emptying port; the column piston in the column divides the synthetic column body into a column height adjusting area and a reaction area, the column height adjusting area is arranged above the column piston in the column, and the reaction area is arranged below the column piston in the column; the column inlet and the column outlet are communicated with the reaction zone, the column outlet is positioned at the lower end of the synthetic column body, and the column inlet is positioned at the upper end of the synthetic column body; the pressure solvent inlet and the emptying port are respectively arranged at two ends of the synthetic column body, and specifically, the pressure solvent inlet and the emptying port are communicated with the column height adjusting area.

The flushing valve 16 of the embodiment has a rapid column-loading function, and the rapid column-loading is realized through the flushing valve 16 and the front liquid path thereof, namely, the height of the reaction zone can be rapidly adjusted according to the process requirement. Specifically, the inlet of the flush valve 16 is connected to the other outlet of the anti-mixing valve 11, one outlet of the flush valve 16 is connected to the pressurized solvent inlet of the synthesis column 12, and the other outlet of the flush valve 16 is connected to the inlet of the back pressure valve 18. When the column is required to be assembled, the emptying port of the synthetic column 12 is closed, and liquid flows into the synthetic column 12 through the first reagent inlet valve 3, the second infusion pump 6, the first pressure sensor 9, the anti-mixing valve 11 and the flushing valve 16 by pipelines, so that the column is assembled. When the column is required to be assembled, the pressure solvent sequentially passes through the flushing valve and the pressure solvent inlet to enter the column height adjusting region, the piston in the column is forced to downwards, the height adjustment of the reaction region is realized, in the column assembling process, the emptying port is connected with an external arbitrary waste liquid barrel, the emptying port is firstly opened, the gas in the column height adjusting region is emptied, and in the real column assembling process, the emptying port is closed.

The pressure sensor six 28 and the pressure regulating valve three 27 form a quick column dismounting device, and have a quick column dismounting function; the outlet of the third pressure regulating valve 27 is connected with the column outlet of the synthesis column 12, and is used for conveying an air source into the synthesis column 12, and the air source is regulated according to the pressure required by the synthesis column 12, and the sixth pressure sensor 28 is arranged on an air pipeline connected with the synthesis column 12 by the third pressure regulating valve 27. When the column is required to be disassembled, an evacuation port of the synthetic column 12 is opened, and an outlet of the third pressure regulating valve 27 is connected with a column outlet of the synthetic column 12 to regulate the pressure in the synthetic column 12. When the column is required to be disassembled, the emptying port is opened, inert gas enters the outlet of the column through the column disassembly pressure regulating valve, the pressure in the reaction zone is increased, the piston in the column is upward, and the height of the reaction zone is regulated. Throughout the reaction, the monomer bottles 20 and reagent bottles 24 need to be pressurized with an inert gas (argon or nitrogen) for air isolation. As shown in fig. 4, an inert gas protection device is formed by a pressure sensor IV 22, a pressure sensor V25, a pressure regulating valve I21, a pressure regulating valve II 26, a gas source distributor I23 and a gas source distributor II 29; the inert gas source is distributed to a first gas source distributor 23 and a second gas source distributor 29 through four-way connectors, and the first gas source distributor 23 and the second gas source distributor 29 are respectively used for distributing inert gas to the monomer bottles 20 and the reagent bottles 24; the fourth pressure sensor 22 is arranged on a gas pipeline connected with the first pressure regulating valve 21 and the first gas source distributor 23, the fourth pressure sensor 22 is arranged to acquire pressure data in the monomer bottle 20 in real time, and the first pressure regulating valve 21 is arranged to regulate the pressure in the monomer bottle 20 according to process requirements. The fifth pressure sensor 25 is arranged on a gas pipeline connected with the second pressure regulating valve 26 and the second gas source distributor 29, and the purpose of the fifth pressure sensor 25 is to acquire the pressure data in the reagent bottle 24 in real time; the second pressure regulating valve 26 is used for regulating the pressure in the reagent bottle 24 according to the process requirement.

In this embodiment, a restrictor valve device is connected to the outlet of each infusion pump. The flow limiting valve device can prevent inert gas pressure in the monomer bottle and the reagent bottle, drive fluid to permeate into the synthetic column, greatly improve the accuracy of reagent conveying amount, ensure accurate material proportioning and ensure that the target product has good purity.

As shown in fig. 5, the restrictor valve device of the present embodiment includes a restrictor valve body 43, a first restrictor valve inlet 44, a second restrictor valve inlet 45, a restrictor valve outlet 46, a restrictor valve gasket 47, a T-block 48, a restrictor valve spring 49, a pressing block 50, and a jackscrew 51; the flow limiting valve sealing gasket 47 is positioned inside the flow limiting valve body 43 and is in interference fit with the flow limiting valve body 43, the transverse end of the T-shaped block 48 is fixed with the flow limiting valve sealing gasket 47, the flow limiting valve spring 49 is sleeved on the vertical end of the T-shaped block 48, one end of the flow limiting valve spring 49 is fixed on the transverse end of the T-shaped block 48, the other end of the flow limiting valve spring is fixedly connected with the pressing block 50, a space exists between the vertical end of the T-shaped block 48 and the pressing block 50, and the pressing block 50 is fixed inside the flow limiting valve body 43 through the jackscrew 51; the T-block 48 of this embodiment serves to prevent excessive compression of the restrictor spring 49 while also avoiding excessive displacement of the restrictor gasket 47.

The first restrictor inlet 44 and the second restrictor inlet 45 of the present embodiment are both disposed at the lower end of the restrictor valve body 43, specifically, the first restrictor inlet 44 is disposed at the lower left side of the restrictor valve body 43, the second restrictor inlet 45 is disposed directly below the restrictor valve body 43, and the restrictor outlet 46 is disposed at the upper right side of the restrictor valve body 43, so that three liquid passages are formed inside the restrictor valve body 43, including a first inlet liquid passage, a second inlet liquid passage and a liquid outlet passage, and are collected inside the restrictor valve body 43, in the present embodiment, the three liquid passages are collected at the center of the restrictor valve body 43, and the restrictor seal 47 is disposed at the center of the restrictor valve body 43 where the three liquid passages are collected, i.e., where the restrictor seal 47 is disposed, in the present embodiment. When the restrictor spring 49 is in a free state, the restrictor gasket 47 blocks the three liquid passages, so that the fluid in the first/second liquid inlet passages cannot enter the liquid outlet passage.

When the infusion pump works, a single body or a reagent is pressed into any one of the liquid inlets of the limiting valves, when the fluid pressure (up to 27.5 Mpa) overcomes the friction force between the limiting valve sealing gasket 47 and the limiting valve body 43 and the compression force of the limiting valve spring 49, the limiting valve sealing gasket 47 moves upwards, the limiting valve spring 49 is compressed through the T-shaped block 48, the limiting valve spring 49 is in a compressed state, the limiting valve sealing gasket 47 moves upwards, and at the moment, any one of the liquid inlets of the limiting valves is communicated with the liquid outlet 46 of the limiting valve, and the input of fluid is completed.

When the infusion pump does not work, the fluid pressure (not more than 0.35 bar) in any one of the flow limiting valve liquid inlets is smaller than the friction force between the flow limiting valve sealing gasket 47 and the flow limiting valve body 43 and the elastic force of the flow limiting valve spring 49, the elastic force of the flow limiting valve spring 49 drives the flow limiting valve sealing gasket 47 to reset, and at the moment, the flow limiting valve sealing gasket 47 blocks three liquid passages again to realize interruption of the flow paths.

And in the infusion pump III, the outlet of the infusion pump is connected with the liquid inlet of any one of the flow limiting valves, and the liquid outlet of the flow limiting valve is connected with one inlet of the anti-mixing valve through a liquid pipeline.

The gas and liquid lines used in this embodiment may be tetrafluoro or PEEK.

The nucleic acid synthesis procedure of this example is:

process one: trichloroacetic acid sequentially enters a synthesis column 12 through a first reagent inlet valve 3, a second infusion pump 6 and an anti-mixing valve 11 to be deprotected (deblocked); after the reaction is completed, acetonitrile enters the first reagent inlet valve 3 to flush the pipeline, flows out of the synthesis column 12, and then sequentially passes through the third pressure sensor 13, the conductivity detector 14, the circulation valve 15, the ultraviolet detector 17, the back pressure valve 18 and the waste discharge valve 19 to be discharged, so that the pipeline flushing and liquid discharging are completed.

Deprotection (Deblocking) is the first step of synthesis: DMT on the CPG-linked nucleoside was removed by trichloroacetic acid to expose the 5' hydroxyl group for the next coupling step.

And a second process: the monomer sequentially passes through a monomer inlet valve 2, a third infusion pump 5 and a second pressure sensor 8 to enter an anti-mixing valve 11; tetrazole sequentially enters an anti-mixing valve 11 through a reagent inlet valve II 4, an infusion pump I7 and a pressure sensor I9; the two solvents are mixed in an anti-mixing valve 11 and then enter a synthesis column 12.

Process two is called Activation (Activation). The monomer is mixed with the tetrazole prior to coupling into the synthesis column 12, where the tetrazole provides a proton to the N atom of the diisopropylamine group on the 3' phosphate, and the protonated diisopropylamine is a good free radical to form a phosphoramidite tetrazole reactive intermediate with the tetrazole.

And a third process: the circulation valve 15 is controlled to a liquid circulation state, the infusion pump I7 is started, the flow rate is set, and the monomers and tetrazoles in the synthetic column 12 flow into the infusion pump I7 through the three-way joint 10, the pressure sensor I9, the anti-mixing valve 11, the synthetic column 12, the pressure sensor III 13, the conductivity detector 14, the circulation valve 15 and the reagent inlet valve II 4 to form a closed loop circulation circuit; after the reaction is finished, the circulation valve 15 is controlled to be in a liquid discharge state, acetonitrile sequentially passes through the monomer inlet valve 2, the infusion pump III 5, the pressure sensor II 8, the anti-mixing valve 11, the synthesis column 12, the pressure sensor III 13, the conductivity detector 14, the circulation valve 15 and the reagent inlet valve II 4, flows into the infusion pump I7, the three-way connector 10, the pressure sensor I9, the anti-mixing valve 11, the flushing valve 16, the ultraviolet detector 17, the back pressure valve 18 and the waste discharge valve 19 and is discharged, and the pipeline flushing is finished.

The process is called: coupling (Coupling). When phosphoramidite tetrazole collides with a nucleotide connected with CPG, nucleophilic reaction is carried out on the phosphoramidite tetrazole and 5' hydroxyl, coupling is carried out, tetrazole is removed, and the synthesized oligonucleotide chain is prolonged by one.

And a process IV: the oxidant enters the synthesis column 12 through the reagent inlet valve II 4, the infusion pump I7, the three-way joint 10, the pressure sensor I9 and the mixing prevention valve 11, after the reaction is finished, acetonitrile enters through the flow path, and is discharged through the pressure sensor III 13, the conductivity detector 14, the circulation valve 15, the ultraviolet detector 17, the back pressure valve 18 and the waste discharge valve 19, so that the pipeline flushing is finished.

The process is called: oxidation (Oxidation). The newly added nucleotide is connected with the oligonucleotide chain on CPG through a phosphorous ester bond (phosphorus is trivalent), and the phosphorous ester bond is unstable and easy to be subjected to acid and alkaline hydrolysis, so that the trivalent phosphorus is required to be oxidized into pentavalent phosphorus through the oxidation process.

And a fifth process: the capping agent A enters the three-way joint 10 from the first reagent inlet valve 3 and the second infusion pump 6, and meanwhile, the capping agent B enters the three-way joint 10 from the second reagent inlet valve 4 and the first infusion pump 7; after two reagents enter the three-way joint 10, the two reagents enter the synthesis column 12 through the first pressure sensor 9 and the mixing prevention valve 11 in sequence to complete the reaction, acetonitrile enters the synthesis column through the flow path, and is discharged through the third pressure sensor 13, the conductivity detector 14, the circulating valve 15, the ultraviolet detector 17, the back pressure valve 18 and the waste discharge valve 19 to complete the pipeline flushing.

The process is called: capping (Capping). For preventing the unreacted 5' -hydroxyl group attached to CPG from being prolonged in the subsequent cycle, it is necessary to block it after the coupling reaction is sufficiently progressed.

The above-mentioned process one to process five are circulated, and the extension of the oligonucleotide chain to a desired length is completed.

In order to improve the automation degree of the nucleic acid synthesis system and the synthesis efficiency and the accuracy of the flow rate of the transported liquid, the pressure sensor II 8, the pressure sensor I9, the pressure sensor III 13, the ultraviolet detector 17, the pressure sensor IV 22, the pressure sensor V25 and the pressure sensor VI 28 are all connected with a PLC controller, the pressure sensor II 8, the pressure sensor I9, the pressure sensor III 13, the ultraviolet detector 17, the pressure sensor IV 22, the pressure sensor V25 and the pressure sensor VI 28 all upload the feedback pressure data and the ultraviolet absorption data monitored by the pressure sensor II, the pressure sensor V25 and the pressure sensor VI 28 to the PLC controller, and the PLC controller can selectively upload the received feedback pressure data and the ultraviolet absorption data to the industrial personal computer for calculation and analysis. In this embodiment, a PLC controller may be connected to each pump and each valve to control the operation of each pump and each valve. The present embodiment may not employ a PLC controller, and the functions of the nucleic acid synthesis system of the present embodiment may not be affected by the absence of the PLC controller.

Specifically, the PLC collects data of the second pressure sensor 8, the first pressure sensor 9, the third pressure sensor 13, the ultraviolet detector 17, the fourth pressure sensor 22, the fifth pressure sensor 25, and the sixth pressure sensor 28 via Modbus.

Example 2:

there is no sanitary-grade diaphragm valve capable of withstanding 15bar pressure in the market at present, and in order to solve the situation that the sanitary-grade structure cannot withstand higher pressure during application, the inlet valve involved in the embodiment 1 is replaced with an inlet valve having a valve group structure.

As shown in fig. 6, it mainly includes: the liquid inlet device comprises a plurality of liquid inlets 30, a liquid outlet 31, a valve base 32 and a plurality of cylinder assemblies, wherein the plurality of cylinder assemblies are arranged on the valve base 32, and the liquid inlets 30 are in one-to-one correspondence with the cylinder assemblies, namely one liquid inlet 30 corresponds to one cylinder assembly; the valve base 32 is internally provided with a plurality of holes and a common channel, each hole is communicated with the common channel, the liquid inlets 30 are in one-to-one correspondence with the arrangement positions of the holes in the valve base 32, so that the common channel is communicated with each liquid inlet, and the liquid outlets 31 are positioned at the tail ends of the common channel.

Each cylinder assembly consists of a diaphragm 33, a compression ring 34, a housing seat 35, a movable rod 36, a housing 37, a spring 38, a retainer ring 39, a piston 40, a seal 41 and a pneumatic connector 42. The shell seat 35 is fixed with the shell 37, the movable rod 36 extends downwards into the shell seat 35, the lower end of the movable rod 36 is fixed with the diaphragm 33 through the compression ring 34, and the movable rod extends upwards to be fixedly connected with the piston 40 through the check ring 39. The lower end of the movable rod 36 is movably connected with the shell seat 35, i.e. the movable rod 36 can move up and down in the shell seat 35.

The spring 38, the retainer ring 39, the piston 40, the sealing ring 41 and the pneumatic connector 42 are all arranged in the shell 37, specifically, a space for the piston 40 to move downwards exists between the lower end of the piston 40 and the shell seat, the bottom of the spring 38 is fixed on the upper surface of the shell seat, the upper part of the spring 38 is sleeved outside the lower end of the piston 40, the spring 38 is fixed with a limiting ring on the outer side of the lower end of the piston 40, and the limiting ring is used for limiting the spring, limiting the upward moving distance of the piston and applying pressure to the spring. The sealing ring 41 is sleeved on the upper end of the piston 40, and a space for air to enter is reserved between the upper end of the piston 40 and the pneumatic connector 42. The compression ring 34 and the seal ring 41 mainly play a sealing role. The pneumatic connector is connected with an external air source. The external air source is controlled by an external solenoid valve.

When the external air source is not ventilated, the spring 38 is in a free state, the movable rod 36 is pulled by the check ring 39, the diaphragm 33 is separated from the bottom plane of the hole inside the valve base, and the corresponding liquid inlet is in an open state. When the external air source is ventilated, the air pressure drives the piston 40 to compress the spring 38, the spring 38 is in a compressed state, the movable rod 36 presses the diaphragm 33 on the bottom plane of the hole in the valve base, and the corresponding liquid inlet is in a closed state, so that the flow path is closed.

The fluid enters through the liquid inlet 30, the membrane 33 is opened, and flows out through the liquid outlet 31 through the common channel, 2 or more states of opening the liquid inlet 30 simultaneously do not exist, the whole flow path can be reversed, and the liquid outlet can be used as the liquid inlet.

The diaphragm 33 in this embodiment is a double-layer diaphragm (ptfe+epdm), and the pressure resistance of the diaphragm valve diaphragm is not greater than 6bar at present, but the pressure resistance of the diaphragm valve diaphragm can be greater than 20bar by the structure in this embodiment, so that the problem that the sanitary structure cannot withstand higher pressure in the application process is effectively solved.

Example 3:

in this embodiment, on the basis of embodiment 2, the anti-mixing valve is replaced with an anti-mixing valve having a valve block structure, and the circulation valve is replaced with a circulation valve having a valve block structure. The valve block structure of this embodiment is the same as that disclosed in embodiment 2.

Claims

1. A nucleic acid synthesis system, characterized in that: comprising the following steps: the device comprises a multipath pre-column liquid path, an anti-mixing valve, a synthetic column, a circulating valve, a circulating liquid path and a liquid discharge liquid path;

a flow limiting valve device is connected to the outlet of each infusion pump;

2. The nucleic acid synthesis system of claim 1, wherein: the inlet valve is of a valve group structure;

3. The nucleic acid synthesis system of claim 2, wherein: the anti-mixing valve is of a valve group structure.

4. The nucleic acid synthesis system of claim 2, wherein: the circulating valve is of a valve group structure.

5. The nucleic acid synthesis system of claim 1, wherein: the nucleic acid synthesis system also comprises a multi-path protection gas path;

6. The nucleic acid synthesis system of claim 1, wherein: the nucleic acid synthesis system further comprises a flush valve; the inlet of the flushing valve is connected with the other outlet of the anti-mixing valve; an outlet of the flushing valve is connected with a pressure solvent inlet of the synthetic column; the other outlet of the flushing valve is connected with the inlet of the liquid draining path;

the flush valve has two modes, including: a column loading mode and a flushing mode; when the flushing valve is in the column loading mode, one outlet of the flushing valve is opened, and the other outlet of the flushing valve is closed; when the flush valve is in flush mode, one outlet of the flush valve is closed and the other outlet of the flush valve is open.

7. A restrictor valve device, characterized by: the flow limiting valve device comprises a flow limiting valve body, a flow limiting valve liquid inlet, a flow limiting valve liquid outlet, a flow limiting valve sealing gasket, a T-shaped block, a flow limiting valve spring, a pressing block and a jackscrew;