CN115253953A

CN115253953A - Nucleic acid synthesis system

Info

Publication number: CN115253953A
Application number: CN202211195209.9A
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: 2022-09-29
Publication date: 2022-11-01
Also published as: CN219784710U

Abstract

The invention discloses a nucleic acid synthesis system, which comprises: the system comprises an industrial personal computer, a PLC (programmable logic controller), a multi-path pre-column liquid path, an anti-mixing valve, a synthesis column, a circulating valve, a circulating liquid path and a liquid discharge path; each pre-column liquid path comprises an inlet valve and an infusion pump, the inlet valve is connected with the infusion pump, and the infusion pump is connected with an anti-mixing valve; the anti-mixing valve is connected with the synthetic column; the synthetic column is connected with a circulating valve, one outlet of the circulating valve is connected with a circulating liquid path, and the other outlet of the circulating valve is connected with a liquid discharge path; the outlet of the circulating liquid path is connected with the inlet valve of a certain path of the front liquid path of the column; obtaining feedback pressure data through a pressure sensor in front of the column; obtaining ultraviolet absorption data through an ultraviolet detector; the industrial personal computer obtains control commands for each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve by utilizing a synthetic control algorithm according to the feedback pressure data and the ultraviolet absorption data, so that the flow of the delivered liquid is more accurate, and the industrial personal computer has the advantages of high stability, high automation degree and the like.

Description

Nucleic acid synthesis system

Technical Field

The invention belongs to the technical field of nucleic acid synthesis, and particularly relates to a nucleic acid synthesis system.

Background

With the rapid development of oligonucleotide chain synthesis technology, synthesis cost is continuously reduced, and synthesis length and precision are continuously improved, so that large-scale DNA and RNA synthesis starting from oligonucleotide chains becomes possible, and a nucleic acid synthesis system is provided.

However, the current nucleic acid synthesis systems have drawbacks including: delivery of reagents is not accurate enough, waste of reaction reagents is severe, equipment failure rate is high, airtightness is not good enough, price is high, and maintenance period is too long.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects of the conventional nucleic acid synthesis system, the invention provides the nucleic acid synthesis system which has the advantages of more accurate flow of the conveyed liquid, high stability, low failure rate, high automation degree and the like.

The technical scheme is as follows: a nucleic acid synthesis system, comprising: the system comprises an industrial personal computer, a PLC (programmable logic controller), a multi-path pre-column liquid path, an anti-mixing valve, a synthetic column, a circulating valve, a circulating liquid path and a liquid discharge path;

each pre-column liquid path 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 an anti-mixing valve; the outlet of the anti-mixing valve is connected with the column inlet of the synthesis 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 drainage liquid path; the outlet of the circulating liquid path is connected with the inlet valve of a certain path of pre-column liquid path;

each inlet of the anti-mixing valve is provided with a pre-column pressure sensor for obtaining feedback pressure data of a corresponding pre-column liquid path;

an ultraviolet detector is arranged at an outlet of the circulating valve and used for monitoring ultraviolet absorption data of the substance flowing out of the circulating valve;

the industrial personal computer adopts a synthetic control algorithm to obtain control commands for each inlet valve, each infusion pump, each anti-mixing valve and each circulation valve according to feedback pressure data and ultraviolet absorption data of a liquid path in front of the column;

the PLC is connected with the ultraviolet detector and is used for acquiring ultraviolet absorption data monitored by the ultraviolet detector; the PLC is connected with each pressure sensor in front of the column and used for acquiring feedback pressure data of each pressure sensor in front of the column corresponding to a liquid path in front of the column, is electrically connected with the industrial personal computer and used for uploading the feedback pressure data and ultraviolet absorption data to the industrial personal computer and receiving a control command issued by the industrial personal computer, and is connected with each inlet valve, each infusion pump, an anti-mixing valve and a circulating valve and used for correspondingly controlling each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve based on the control command issued by the industrial personal computer;

the synthesis control algorithm comprises the following steps:

based on the nucleic acid synthesis flow, preset liquid path parameters and feedback pressure data of each pre-column liquid path, control commands for each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve are obtained, and nucleic acid synthesis is carried out according to the nucleic acid synthesis flow under the condition that the preset liquid path parameters are met; the nucleic acid synthesis process comprises five nucleic acid synthesis stages of deprotection, activation, coupling, oxidation and capping; the liquid path parameters include: maximum allowable flow rate, pre-column pressure maximum, pre-column target pressure, target monomer or reagent volume, on-off state of inlet valve, flow rate of infusion pump, run time of infusion pump, on-off state of anti-mix valve, on-off state of circulation valve, and mode state of circulation valve; the mode states of the circulation valve include a circulation mode and a liquid discharge mode; when the circulating valve is in a circulating mode, a passage is formed between the circulating valve and the circulating liquid path, and a circuit is broken between the circulating valve and the liquid discharge liquid path; when the circulating valve is in a liquid discharge mode, the circulating valve is in an open circuit with the circulating liquid path, and a passage is formed between the circulating valve and the liquid discharge liquid path;

optimizing preset liquid path parameters based on the ultraviolet absorption data and the feedback pressure data of the liquid paths in front of each column, and obtaining control commands for each inlet valve, each infusion pump, each anti-mixing valve and each circulation valve according to the optimized liquid path parameters; the method specifically comprises the following steps:

presetting deprotection peak integration conditions for a deprotection nucleic acid synthesis stage, judging whether ultraviolet absorption data in the stage meet the deprotection peak integration conditions, if not, adjusting the flow rate of an infusion pump on a liquid path in front of a relevant column to the maximum allowable flow rate, and not limiting the operation time of the infusion pump until the ultraviolet absorption data meet the deprotection peak integration conditions;

if the ultraviolet absorption data at this stage meet the integral condition of deprotection peak, the following steps are carried out one by one:

judging whether the peak-off time of the deprotection peak exceeds the peak-off time threshold of the deprotection peak, and if so, optimizing the flow rate of the infusion pump according to the formula (1); if not, maintaining the preset liquid path parameters;

（1）

in the formula (I), the compound is shown in the specification,

in order to optimize the flow rate of the infusion pump,

the flow rate of the infusion pump in the preset fluid path parameters,

in order to increase the coefficient for the flow rate,

is the peak-off time threshold of the deprotection peak,

for the current threshingThe peak-off time of the protection peak;

judging whether the rising time of the deprotection peak is larger than a rising time threshold value or not, and if so, optimizing the flow rate of the infusion pump according to the formula (2); if not, maintaining the preset liquid path parameters;

（2）

in the formula (I), the compound is shown in the specification,

is the threshold value of the rise time of the deprotection peak,

is the rise time of the current deprotected peak;

judging whether the falling time of the deprotection peak is larger than a falling time threshold value or not, and if so, optimizing the flow rate of the infusion pump according to the formula (3); if not, maintaining the preset liquid path parameters;

（3）

in the formula (I), the compound is shown in the specification,

is the threshold value for the fall time of the deprotected peak,

is the fall time of the current deprotected peak;

judging whether the feedback pressure data of the liquid path in front of the column exceeds the maximum value of the pressure in front of the column, if so, optimizing the flow rate of the infusion pump according to the formula (4), and if not, optimizing the flow rate of the infusion pump according to the formula (5);

（4）

in the formula (I), the compound is shown in the specification,

in order to reduce the coefficient for the flow rate,

in order to feed back the pressure data at the present time,

maximum value of pressure before column;

（5）

and for the activation stage, the coupling stage, the oxidation stage and the cap nucleic acid synthesis stage, judging whether the feedback pressure data of the liquid path in front of the column exceeds the maximum value of the pressure in front of the column, if so, optimizing the flow rate of the infusion pump according to the formula (4), and if not, optimizing the flow rate of the infusion pump according to the formula (5).

Further, the obtaining of control commands for each inlet valve, each infusion pump, each anti-mixing valve, and each circulation valve based on the nucleic acid synthesis flow, preset fluid path parameters, and feedback pressure data of each pre-column fluid path specifically includes:

for any inlet valve, obtaining a control command of a switch state according to the nucleic acid synthesis stage;

for any infusion pump, obtaining a control command of the flow rate of the infusion pump through an incremental PID algorithm according to feedback pressure data of a corresponding pre-column liquid path and a corresponding pre-column target pressure; obtaining a control command of the operation time of the infusion pump according to the accumulated volume algorithm;

for the anti-mixing valve, a control command of a switch state is obtained according to the nucleic acid synthesis stage;

for the circulating valve, according to the nucleic acid synthesis stage, a control command of a switch state and a control command of a mode state are obtained.

Further, the obtaining of the control command of the flow rate of the infusion pump through the incremental PID algorithm includes:

（6）

（7）

（8）

wherein:

is composed of

The flow rate at a time is controlled in increments,

is a coefficient of proportionality that is,

in order to be the integral coefficient of the light,

in order to be the differential coefficient,

is composed of

The difference between the time-of-day feedback pressure data and the corresponding pre-column target pressure,

is composed of

The feedback pressure data at the time of day,

is composed of

The corresponding pre-column target pressure at the time,

is composed of

The flow rate control value of the infusion pump at the time,

is composed of

is composed of

Feeding back the difference between the pressure data and the corresponding target pressure before the column at any moment;

is composed of

A flow rate control value of the time infusion pump; the flow rate control value of the infusion pump constitutes a control command for the flow rate of the infusion pump.

Further, the obtaining of the control command of the infusion pump running time according to the cumulative volume algorithm specifically includes:

according to equation (9), calculate

Cumulative volume of time of day

：

（9）

In the formula (I), the compound is shown in the specification,

is composed of

The cumulative volume of the time of day,

is composed of

The flow rate of the infusion pump at the time,

to represent

At the moment of time, the time of day,

to represent

Time of day;

judgment of

Cumulative volume of time of day

Whether the volume of the target monomer or reagent is reached, and if so, obtaining a control command that the running time of the infusion pump reaches; otherwise, obtaining a control command of prolonging the operation time of the infusion pump.

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

the valve group structure comprises: the liquid inlet is in one-to-one correspondence with the air cylinder assemblies; a plurality of holes and a common channel are arranged in the valve base, each hole is communicated with the common channel, the positions of the liquid inlets on the valve base are in one-to-one correspondence with the positions of the holes in the valve base, so that the common channel is communicated with the liquid inlets, and the liquid outlets are positioned at the tail end of the common channel;

each air cylinder assembly consists of a diaphragm, a shell seat, a movable rod, a shell, a spring, a retainer ring, a piston and a pneumatic connector; the shell seat is fixed with the shell, the movable rod extends downwards into the shell seat and is fixed with the diaphragm, 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 the check ring;

a space for the piston to move downwards is formed 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 formed 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 an 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;

the PLC controller controls the on-off state of the inlet valve by controlling whether the external electromagnetic valve is ventilated.

Furthermore, the anti-mixing valve and the circulating valve are both valve group structures.

Furthermore, the outlet of each infusion pump is connected with a flow limiting valve device;

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 inside 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 a sealing gasket of the flow limiting valve; the vertical end of the T-shaped block is sleeved with the flow limiting valve spring, 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 is reserved between the vertical end of the T-shaped block and the pressing block; the pressing block is fixed inside the valve body of the flow limiting valve through a jackscrew;

the liquid inlet of the flow limiting valve and the liquid outlet of the flow limiting valve are both arranged on the valve body of the flow limiting valve, a liquid inlet channel is arranged between the liquid inlet of the flow limiting valve and the sealing gasket of the flow limiting valve, and a liquid outlet channel is arranged between the liquid outlet of the flow limiting valve and the sealing gasket of the flow limiting valve;

when the infusion pump works, a monomer or a reagent is pressed into the liquid inlet of the restriction valve, when the fluid pressure overcomes the friction force between the sealing gasket of the restriction valve and the valve body of the restriction valve and the compression force of the spring of the restriction valve, the spring of the restriction valve is in a compressed state, the sealing gasket of the restriction valve moves upwards, the liquid inlet channel is a passage, the liquid outlet channel is a passage, and the monomer or the reagent in the liquid inlet channel flows out of the liquid outlet of the restriction valve through the liquid outlet channel;

when the infusion pump does not work, the spring of the flow limiting valve is in a free state, and the sealing gasket of the flow limiting valve blocks the liquid inlet channel and the liquid outlet channel.

Further, the synthesis column comprises: the device comprises a pressure solvent inlet, an exhaust port, a column casing, a lower end cover assembly, a piston assembly, an upper end cover guide sleeve and an auxiliary cylinder assembly; the auxiliary cylinder assembly is fixed on the upper cover plate assembly;

the lower end cover component is fixed at the bottom of the column casing; the upper cover plate component is fixed on the upper part of the column casing, a central hole is arranged on the upper cover plate component, and the upper end cover guide sleeve is arranged in the central hole; the pressure solvent inlet and the emptying port are correspondingly arranged at two ends of the upper cover plate component;

the piston assembly comprises a piston main body and a guide rod; the piston main body is arranged in the column barrel; the piston main body comprises a piston guide ring, a piston sieve plate and a piston distributor; the guide rod sequentially penetrates through the guide sleeve of the upper end cover and the auxiliary cylinder assembly upwards, and a limiting plate is arranged at the top of the guide rod; the piston sieve plate is fixed at the lower part of the piston distributor, the piston guide ring is concentrically fixed with the upper part of the piston distributor, and the guide rod is downwards fixed at the upper part of the piston distributor; a circle of groove is formed at the joint of the piston guide ring and the piston distributor, the lower end of the piston guide ring and the upper end of the piston distributor;

the auxiliary cylinder assembly includes: the device comprises a guide seat, a cylinder body, a cylinder cover, a piston plate, a cylinder body guide sleeve, a first joint, a first speed regulating valve, a second speed regulating valve and a second joint;

a cylinder body guide sleeve is arranged in the guide seat, and the lower end of the guide seat is hermetically connected with the upper end cover assembly;

the upper end of the guide seat is provided with a piston plate, and the upper end of the guide seat and the piston plate are both positioned in the cylinder body;

a first cavity is formed among the cylinder body, the guide seat and the lower part of the piston plate, a first joint is arranged on the outer side of the cylinder body, one end of the first joint is communicated with the first cavity, and the other end of the first joint is connected with a first speed regulating valve;

the cylinder cover penetrates through the guide rod and is fixed on the upper portion of the cylinder body, a second cavity is formed among the cylinder body, the upper portion of the piston plate and the lower portion of the cylinder cover, a second connector is installed on the upper portion of the cylinder cover, one end of the second connector is communicated with the second cavity, and the other end of the second connector is connected with a second speed regulating valve.

Further, the device also comprises a column mounting device; the device column device is a flushing valve;

the inlet of the flushing valve is connected with the other outlet of the anti-mixing valve; one outlet of the flushing valve is connected with the pressure solvent inlet of the synthesis column; the other outlet of the flushing valve is connected with the inlet of the liquid drainage circuit;

the flushing valve is connected with the PLC;

the PLC controller controls the flushing valve according to a control command for the flushing valve issued by the industrial personal computer;

according to the nucleic acid synthesis stage, the control command for the flushing valve comprises a control command of an on-off state and a control command of a flushing valve mode state; the flush valve mode state includes: a column packing mode and a washing mode; when the flushing valve is in the column filling 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 the flush mode, one outlet of the flush valve is closed and the other outlet of the flush valve is open.

Further, still include inert gas protection device, inert gas protection device includes: a plurality of protective gas circuits;

each protection gas circuit comprises a gas source distributor, a protection pressure regulating valve and a protection pressure sensor;

the inlet of the protective pressure regulating valve is used for obtaining inert gas, the outlet of the protective 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 into the monomer bottle/reagent bottle;

the protective pressure sensor is arranged on a pipeline between the outlet of the protective pressure regulating valve and the monomer bottle/reagent bottle;

the protection pressure sensor and the protection pressure regulating valve are both connected with a PLC (programmable logic controller), and the PLC controls the protection pressure regulating valve according to a control command for the protection pressure regulating valve, which is issued by an industrial personal computer;

the control command for protecting the pressure regulating valve is a control command for protecting the flow rate of the pressure regulating valve obtained by an incremental PID algorithm according to the feedback pressure data of the protection pressure sensor on the same liquid path and the corresponding target protection pressure.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages:

(1) The invention controls each infusion pump and each valve through a control algorithm, so that the flow of the liquid delivered in the nucleic acid synthesis process is more accurate, the consumption of the reaction reagent is reduced, and the automation degree is high;

(2) According to the invention, technological parameters in the nucleic acid synthesis process are optimized through a control algorithm, so that the accuracy of the flow of the conveyed liquid is greatly improved, and the nucleic acid synthesis efficiency is improved;

(3) By adopting the valve group structure, the volume of a liquid path is reduced, the consumption of reagents is saved, the pressure resistance of the membrane in each valve is improved to 20bar, and the pressure resistance stability of a nucleic acid synthesis system is greatly improved;

(4) According to the invention, the flow limiting valve device is adopted, so that the fluid driven by the micro-positive pressure gas is prevented from entering the system, the precision of reagent conveying capacity is further improved, the material proportioning accuracy is ensured, and the purity of a target product is greatly improved;

(5) The invention adopts the synthetic column structure with a new structure, ensures uniform distribution in the column and improves the nucleic acid synthesis efficiency.

Drawings

FIG. 1 is a schematic view of a valve block configuration;

FIG. 2 is a schematic view of a flow-limiting valve assembly;

FIG. 3 is a schematic view of a synthesis column structure;

FIG. 4 is a schematic view of the piston assembly of the synthetic column;

FIG. 5 is a schematic view of the auxiliary cylinder assembly of the synthetic column;

FIG. 6 is a schematic view of a state in which the piston screen deck is disassembled;

fig. 7 is a schematic view of another state when the piston sieve plate is disassembled;

FIG. 8 is a schematic perspective view of a nucleic acid synthesis system;

FIG. 9 is a schematic diagram showing a schematic front structure of a nucleic acid synthesis system;

FIG. 10 is a process flow diagram of a nucleic acid synthesis system;

FIG. 11 is a schematic view of an inert gas blanket;

FIG. 12 is a schematic representation of the deprotection peak.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings and the embodiment.

Example 1:

the present example discloses a nucleic acid synthesis system comprising: industrial control computer, PLC controller, multichannel pre-column liquid way, anti-mixing valve, synthetic column, circulating valve, circulating liquid way and flowing back liquid way.

Each pre-column liquid path comprises an inlet valve and an infusion pump, 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 the circulating valve, one outlet of the circulating valve is connected with the inlet of the circulating liquid path, and the other outlet of the circulating valve is connected with the inlet of the liquid drainage liquid path; the outlet of the circulating liquid path is connected with the inlet valve of a certain path of the front liquid path of the column; each inlet of the anti-mixing valve is provided with a pre-column pressure sensor for obtaining feedback pressure data of a corresponding pre-column liquid path; an ultraviolet detector is arranged at an outlet of the circulating valve and is used for monitoring ultraviolet absorption data of the substance flowing out of the circulating valve.

The industrial personal computer adopts a synthetic control algorithm to obtain control commands for each inlet valve, each infusion pump, each anti-mixing valve and each circulating valve according to feedback pressure data and ultraviolet absorption data of the liquid path in front of the column.

The PLC is connected with the ultraviolet detector, each column front pressure sensor, the industrial personal computer, the inlet valves, the infusion pumps, the anti-mixing valves and the circulating valves, and is used for correspondingly controlling the inlet valves, the infusion pumps, the anti-mixing valves and the circulating valves based on control commands issued by the industrial personal computer.

The synthesis control algorithm of the embodiment comprises the following steps:

based on the nucleic acid synthesis flow, preset liquid path parameters and feedback pressure data of each pre-column liquid path, control commands for each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve are obtained, and nucleic acid synthesis is carried out according to the nucleic acid synthesis flow under the condition that the preset liquid path parameters are met; the nucleic acid synthesis process comprises five nucleic acid synthesis stages of deprotection, activation, coupling, oxidation and capping; the liquid path parameters include: maximum allowable flow rate, pre-column pressure maximum, pre-column target pressure, target monomer or reagent volume, on-off state of inlet valve, flow rate of infusion pump, run time of infusion pump, on-off state of anti-mix valve, on-off state of circulation valve, and mode state of circulation valve; the mode states of the circulating valve include a circulation mode and a liquid discharge mode; when the circulating valve is in a circulating mode, a passage is formed between the circulating valve and the circulating liquid path, and a broken circuit is formed between the circulating valve and the liquid discharge path; when the circulating valve is in the liquid discharge mode, the circulating valve is disconnected with the circulating liquid path, and a passage is formed between the circulating valve and the liquid discharge liquid path.

（1）

in the formula (I), the compound is shown in the specification,

in order to optimize the flow rate of the infusion pump,

the flow rate of the infusion pump in the preset fluid path parameters,

in order to increase the coefficient for the flow rate,

is the peak-off time threshold of the deprotection peak,

the peak-off time of the current deprotection peak;

（2）

in the formula (I), the compound is shown in the specification,

is the threshold value of the rise time of the deprotected peak,

is the rise time of the current deprotected peak;

（3）

in the formula (I), the compound is shown in the specification,

is the threshold value for the fall time of the deprotected peak,

is the fall time of the current deprotected peak;

（4）

in the formula (I), the compound is shown in the specification,

in order to reduce the coefficient for the flow rate,

in order to feed back the pressure data at the present time,

is the maximum value of the pressure before the column;

（5）

and (3) judging whether the feedback pressure data of the liquid path in front of the column exceeds the maximum value of the pressure in front of the column in the activation stage, the coupling stage, the oxidation stage and the capping nucleic acid synthesis stage, if so, optimizing the flow rate of the infusion pump according to the formula (4), and if not, optimizing the flow rate of the infusion pump according to the formula (5).

The nucleic acid synthesis system of the embodiment has the advantages of more accurate liquid flow rate for conveying, less consumption of reaction reagents, high automation degree, high nucleic acid synthesis efficiency, capability of being used for mmol-level nucleic acid synthesis and the like.

Example 2:

Each pre-column liquid path comprises an inlet valve and an infusion pump, 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 an 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 the circulating valve, one outlet of the circulating valve is connected with the inlet of the circulating liquid path, and the other outlet of the circulating valve is connected with the inlet of the liquid drainage liquid path; the outlet of the circulating liquid path is connected with the inlet valve of a certain path of the front liquid path of the column; each inlet of the anti-mixing valve is provided with a pre-column pressure sensor for obtaining feedback pressure data of a corresponding pre-column liquid path; an ultraviolet detector is arranged at an outlet of the circulating valve and is used for monitoring ultraviolet absorption data of the substance flowing out of the circulating valve.

The industrial personal computer adopts a synthesis control algorithm to obtain control commands for each inlet valve, each infusion pump, each anti-mixing valve and each circulation valve according to feedback pressure data and ultraviolet absorption data of the liquid path in front of the column;

the PLC is connected with the ultraviolet detector, connected with each pressure sensor in front of the column, used for acquiring ultraviolet absorption data monitored by the ultraviolet detector and feedback pressure data of a liquid path in front of the column corresponding to each pressure sensor in front of the column, electrically connected with the industrial personal computer, used for uploading the feedback pressure data and the ultraviolet absorption data to the industrial personal computer, receiving a control command issued by the industrial personal computer, connected with each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve, and used for correspondingly controlling each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve based on the control command issued by the industrial personal computer.

based on the nucleic acid synthesis flow, preset liquid path parameters and feedback pressure data of each pre-column pressure sensor, control commands for each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve are obtained, and nucleic acid synthesis is sequentially carried out under the condition that the liquid path parameters of each pre-column liquid path meet the preset liquid path parameters; the nucleic acid synthesis process comprises five nucleic acid synthesis stages of deprotection, activation, coupling, oxidation and capping; the liquid path parameters include: maximum allowable flow rate, pre-column pressure maximum, pre-column target pressure, target monomer or reagent volume, open and close state of inlet valve, flow rate of infusion pump, running time of infusion pump, open and close state of anti-mix valve, open and close state of circulating valve, and mode state of circulating valve; the mode states of the circulating valve include a circulation mode and a liquid discharge mode; when the circulating valve is in a circulating mode, a passage is formed between the circulating valve and the circulating liquid path, and a circuit is broken between the circulating valve and the liquid discharge liquid path; when the circulating valve is in the liquid discharge mode, the circulating valve is disconnected from the circulating liquid path, and a passage is formed between the circulating valve and the liquid discharge path. The method specifically comprises the following steps:

for any infusion pump, obtaining a control command of the flow rate of the infusion pump through an incremental PID algorithm according to the feedback pressure data of the corresponding pre-column liquid path and the corresponding pre-column target pressure; the incremental PID algorithm is represented as:

（6）

（7）

（8）

wherein:

is composed of

The flow rate at a time is controlled in increments,

is a coefficient of proportionality that is,

in order to be the integral coefficient of the light,

in order to be the differential coefficient,

is composed of

is composed of

The feedback pressure data at the time of day,

is composed of

The corresponding pre-column target pressure at the time,

is composed of

The flow rate control value of the infusion pump at the time,

is composed of

is composed of

is composed of

A flow rate control value of the infusion pump at that time; the flow rate control value of the infusion pump constitutes a control command of the flow rate of the infusion pump.

For any infusion pump, the calculation is made according to equation (9)

Cumulative volume of time of day

：

（9）

In the formula (I), the compound is shown in the specification,

is composed of

The cumulative volume at a time of day,

is composed of

The flow rate of the infusion pump at the time,

represent

At the time of day, the user may,

represent

Time of day;

judgment of

Cumulative volume of time of day

Whether the target monomer or reagent volume is reached or not, and if so, obtaining a control command that the running time of the infusion pump reaches; otherwise, obtaining a control command of prolonging the operation time of the infusion pump. .

For the anti-mixing valve, a control command of an on-off state is obtained according to the nucleic acid synthesis stage;

if the ultraviolet absorption data at this stage meet the integral condition of deprotection peak, one by one:

（1）

in the formula (I), the compound is shown in the specification,

in order to optimize the flow rate of the infusion pump,

the flow rate of the infusion pump in the preset fluid path parameters,

in order to increase the coefficient for the flow rate,

is the peak-off time threshold of the deprotected peak,

the peak-off time of the current deprotection peak;

judging whether the rising time of the deprotection peak is greater than a rising time threshold value or not, and if so, optimizing the flow rate of the infusion pump according to the formula (2); if not, maintaining the preset liquid path parameters;

（2）

in the formula (I), the compound is shown in the specification,

is the threshold value of the rise time of the deprotected peak,

is the rise time of the current deprotection peak;

（3）

in the formula (I), the compound is shown in the specification,

is the threshold value of the fall time of the deprotected peak,

is the fall time of the current deprotected peak;

（4）

in the formula (I), the compound is shown in the specification,

in order to reduce the coefficient for the flow rate,

for the purpose of the current feedback pressure data,

maximum value of pressure before column;

（5）

Example 3:

to address the situation where the sanitary-grade structure cannot withstand higher pressures during use, the present embodiment replaces the inlet valve referred to in embodiment 1 or embodiment 2 with an inlet valve having a valve block structure.

As shown in fig. 1, it mainly includes: the liquid inlet valve 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, the liquid inlets 30 correspond to the cylinder assemblies one by one, namely, one liquid inlet 30 corresponds to one cylinder assembly; a plurality of holes and a common channel are arranged in the valve base 32, 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 at the arrangement positions of the valve base, so that the common channel is communicated with the liquid inlets, and the liquid outlets 31 are positioned at the tail end of the common channel.

Each cylinder assembly is composed of a diaphragm 33, a compression ring 34, a shell seat 35, a movable rod 36, a shell 37, a spring 38, a retainer ring 39, a piston 40, a sealing ring 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 pressing ring 34, and the movable rod extends upwards and is fixedly connected with the piston 40 through the retaining ring 39. The lower end of the movable rod 36 is movably connected with the shell seat 35, that is, 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 seal ring 41 and the pneumatic connector 42 are all arranged in the housing 37, specifically, a space for the piston 40 to move downwards exists between the lower end of the piston 40 and the housing seat, the bottom of the spring 38 is fixed on the upper surface of the housing seat, the upper portion of the spring 38 is sleeved on the outer side of 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 pressing the spring. The sealing ring 41 is sleeved on the upper end of the piston 40, and a space for air to enter is arranged between the upper end of the piston 40 and the pneumatic connector 42. The pressing ring 34 and the seal ring 41 mainly perform a sealing function. The pneumatic connector is connected with an external air source. The external air source is controlled by an external electromagnetic valve.

When the external air source is not ventilated, the spring 38 is in a free state, the check ring 39 pulls the movable rod 36, the diaphragm 33 is away from the bottom plane of the hole in the valve base, and the corresponding liquid inlet is in an open state. When an 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.

Fluid enters through the liquid inlet 30, the diaphragm 33 is opened, and flows out through the liquid outlet 31 through the common channel, the liquid inlet 30 is not opened for 2 or more states at the same time, the whole flow path can flow reversely, and the liquid outlet can be used as a liquid inlet.

The diaphragm 33 of the embodiment is a double-layer diaphragm (PTFE + EPDM), the pressure resistance of the diaphragm of the current diaphragm valve is not more than 6bar, but the structure of the embodiment can make the pressure resistance of the diaphragm valve greater than 20bar, thereby effectively solving the problem that the sanitary-grade structure cannot withstand higher pressure in the application process.

The external electromagnetic valve can be connected with the PLC, and the industrial personal computer controls the opening and closing state of the inlet valve according to the nucleic acid synthesis stage, so that a control command of the ventilation state of the external electromagnetic valve is issued by the industrial personal computer, and the PLC controls the external electromagnetic valve according to the received control command of the ventilation state of the external electromagnetic valve, so that the inlet valve is opened or closed.

Example 4:

in this embodiment, on the basis of embodiment 3, 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 3.

Example 5:

since the monomers and reagents used in the nucleic acid synthesis process have high hydrophilicity and need to be isolated from the moisture in the air by applying inert gas to maintain micro-positive pressure, and since the liquid path of the whole pilot nucleic acid synthesis system is a through structure, in order to prevent the micro-positive pressure gas from driving fluid to enter the system, which causes the abnormal pushing amount of the monomers and reagents, the present embodiment connects a flow limiting valve device at the outlet of each infusion pump on the basis of embodiment 1 or embodiment 2 or embodiment 3 or embodiment 4.

As shown in fig. 2, the restriction valve device of the present embodiment includes a restriction valve body 43, a first restriction valve liquid inlet 44, a second restriction valve liquid inlet 45, a restriction valve liquid outlet 46, a restriction valve sealing gasket 47, a T-shaped block 48, a restriction valve spring 49, a pressing block 50, and a jackscrew 51; wherein, the sealing gasket 47 of the flow limiting valve is positioned in the valve body 43 of the flow limiting valve and is in interference fit with the valve body 43 of the flow limiting valve,

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 49 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 a jackscrew 51; the T-block 48 of this embodiment serves to prevent over-compression of the constrictor valve spring 49 while also avoiding over-displacement of the constrictor valve gasket 47.

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

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

When the infusion pump does not work, the fluid pressure (not more than 0.35 bar) in the liquid inlet of any one restriction valve is smaller than the friction force between the restriction valve sealing gasket 47 and the restriction valve body 43 and the elastic force of the restriction valve spring 49, the elastic force of the restriction valve spring 49 drives the restriction valve sealing gasket 47 to reset, at the moment, the three liquid passages are plugged by the restriction valve sealing gasket 47 again, and the interruption of the flow path is realized.

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

The restriction valve device of this embodiment can prevent the inert gas pressure in monomer bottle and the reagent bottle, and the synthetic post of driving fluid infiltration can improve the precision of reagent delivery volume greatly, ensures that the material ratio is accurate, makes the target product purity good.

Example 6;

in this embodiment, on the basis of any of the above embodiments, the synthesis column concerned is replaced with a synthesis column of a new structure, and the synthesis column of the new structure mainly includes: the device comprises a synthetic column body, a column inlet, a column outlet, an in-column piston positioned in the synthetic column body, a pressure solvent inlet and an evacuation port; the column body of the synthetic column is divided into a column height adjusting area and a reaction area by the piston in the column, the column height adjusting area is arranged above the piston in the column, and the reaction area is arranged below the 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 column body of the synthesis column, and the column inlet is positioned at the upper end of the column body of the synthesis column; the pressure solvent inlet and the emptying port are respectively arranged at two ends of the column body of the synthesis column, and particularly, the pressure solvent inlet and the emptying port are communicated with the column height adjusting area.

In order to realize rapid column loading, namely the height of the reaction zone can be rapidly adjusted according to the process requirement; in this embodiment, a flush valve is added, and the quick column mounting is realized through the flush valve and a front end liquid path thereof. Specifically, the inlet of the flushing valve is connected with the other outlet of the anti-mixing valve; one outlet of the flushing valve is connected with the pressure solvent inlet of the synthesis column; the other outlet of the flushing valve is connected with the inlet of the liquid drainage circuit.

The flushing valve is connected with a PLC (programmable logic controller), and the PLC controls the flushing valve according to a control command for the flushing valve, which is issued by the industrial personal computer. According to the nucleic acid synthesis stage, the control command for the flushing valve comprises a control command of an on-off state and a control command of a flushing valve mode state; the flush valve mode state includes: a column packing mode and a washing mode; when the flushing valve is in the column filling 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 the flush mode, one outlet of the flush valve is closed and the other outlet of the flush valve is open.

When the column is required to be packed, the pressure solvent sequentially enters the column height adjusting area through the flushing valve and the pressure solvent inlet, the piston in the column is forced to move downwards, the height adjustment of the reaction area is realized, in the column packing process, the emptying port is connected with any external waste liquid barrel, the emptying port is opened firstly, the gas in the column height adjusting area is emptied, and in the real column packing process, the emptying port is closed.

Example 7:

this embodiment provides a device for quickly removing a column based on embodiment 6, and the device for quickly removing a column includes a pressure sensor for removing a column, a pressure regulating valve for removing a column, a related fluid path, and related components. The outlet of the column disassembling pressure regulating valve is connected with the column outlet of the synthetic column; the column detaching pressure sensor is arranged at an outlet of the column detaching pressure regulating valve.

The column dismounting pressure regulating valve and the column dismounting pressure sensor are both connected with a PLC (programmable logic controller), and the PLC controls the column dismounting pressure regulating valve according to a control command for the column dismounting pressure regulating valve, which is issued by an industrial personal computer; according to the nucleic acid synthesis stage, the control command for the column-disassembling pressure regulating valve comprises a control command of an on-off state.

When the column needs to be disassembled, the emptying port is opened, inert gas enters the outlet of the column through the column disassembling pressure regulating valve, the pressure in the reaction zone is increased, the piston in the column faces upwards, and the height of the reaction zone is regulated.

Example 8:

in the whole nucleic acid synthesis process, the monomer bottle and the reagent bottle need to be pressurized and protected by inert gas (argon or nitrogen) so as to achieve the purpose of isolating air. Therefore, this embodiment adds an inert gas protection device to any of the above embodiments, and the inert gas protection device includes: and (4) multiple protective gas circuits.

Each protection gas circuit comprises a gas source distributor, a protection pressure regulating valve and a protection pressure sensor; the inlet of the protective pressure regulating valve is used for obtaining inert gas, the outlet of the protective 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 into the monomer bottle/reagent bottle; and the protective pressure sensor is arranged on a pipeline between the outlet of the protective pressure regulating valve and the monomer bottle/reagent bottle.

The protection pressure sensor and the protection pressure regulating valve are both connected with a PLC (programmable logic controller), and the PLC controls the protection pressure regulating valve according to a control command for the protection pressure regulating valve, which is sent by an industrial personal computer; and the control command for protecting the pressure regulating valve is a control command for protecting the flow rate of the pressure regulating valve obtained by an incremental PID algorithm according to the feedback pressure data of the protection pressure sensor on the same liquid path and the corresponding target protection pressure.

Example 9:

in order to improve the synthesis efficiency, the synthesis column may be replaced by another synthesis column with a new structure, as shown in fig. 3, the synthesis column with the new structure mainly comprises: the device comprises a column cylinder 52, a lower end cover 53, a lower end cover sieve plate 54, an E-shaped sealing ring 55, a piston assembly 56, an upper end cover 57, an upper end cover sealing ring 58, an upper end cover guide sleeve 59, a frame assembly 60, a rotating shaft 61, a connecting plate 62 and an auxiliary cylinder assembly 63.

The connection condition among all parts is as follows:

the column casing 52 is fixed with a connecting plate 62 on each side, a rotating shaft 61 is mounted on each side of the upper portion of the frame assembly 60, and each connecting plate 62 is fixed with the corresponding rotating shaft 61 through bolts, so that the column casing 52 is arranged on the frame assembly 60. A turntable 67 is also mounted on any one of the shafts 61, and a latch 68 is mounted on the upper portion of the frame assembly 60 near the turntable. In this embodiment, the cylinder 52 can be turned over by the rotating shaft 61, so that the composition in the cylinder 52 can be taken out conveniently.

When the synthesis column needs to be turned over, the plug 68 is pulled out of the turntable 67, the column barrel 52 is rotated to enable the whole synthesis column to rotate along the axis of the rotating shaft 61, the rotating angles are 45 degrees, 90 degrees, 135 degrees and 180 degrees, and the plug is inserted into the rotating disc to position the column barrel 52 at a certain angle after the plug is rotated to a certain angle.

The lower end cover sieve plate 54 is clamped on the lower end cover 53 through an E-shaped sealing ring 55 to form a whole and then is fixed at the bottom of the column casing 52 through bolt connection.

When the lower end cover screen 54 needs to be removed, the fixing bolts on the lower end cover 53 are removed, the lower end cover 53, the lower end cover screen 54 and the E-shaped seal ring 55 are taken out integrally from the lower end of the column casing 52, and the E-shaped seal ring 55 is pulled open to take out the lower end cover screen 54.

The upper end cover 57 is fixed on the upper part of the column casing 52 through bolts, an upper end cover sealing ring 58 is arranged between the upper end cover 57 and the upper part of the column casing 52, a central hole is arranged on the upper end cover 57, an upper end cover guide sleeve 59 is arranged in the central hole, two valves are arranged on the upper part of the upper end cover 57, the two valves respectively correspond to a pressure solvent inlet 64 and an exhaust port 65 and are used for opening the pressure solvent inlet 64 and the exhaust port 65, and when the column is installed, the pressure solvent inlet 64 is connected with a column installing device. By pumping pressure solvent, the constant pressure in the column is ensured, and the piston assembly is always attached to the column bed.

As shown in fig. 4, the piston assembly 56 includes a piston main body and a guide rod 561. A piston main body is formed by a piston guide ring 562, a first piston seal ring 563, a piston sieve plate 564, a second piston seal ring 565, a third piston seal ring 567, a fourth piston seal ring 568, a fifth piston seal ring 569 and a piston distributor 5610, and a guide rod 561 is fixed on the piston main body; wherein, the piston body is arranged in the column cylinder 52, the guide rod 561 upwards sequentially passes through the holes at the centers of the upper end cover guide sleeve 59 and the auxiliary cylinder assembly 63, and the top of the guide rod 561 is provided with a limit plate 66. The auxiliary cylinder assembly 63 is bolted to the upper end cap 57 through a guide rod 561. Piston screen plate 564 is bolted to the lower portion of piston distributor 5610 with a third piston seal 567 therebetween, second piston seal 565 is seated in a groove around piston screen plate 564, piston guide ring 562 is bolted to the upper portion of piston distributor 5610, fourth piston seal 568 is seated between piston guide ring 562 and

piston distributor

5610, and 2 fourth piston seals 568 are seated in grooves around piston guide ring 562. The guide rod 561 is fixed to an upper portion of the piston distributor 5610 by a bolt, and a fifth piston packing 569 is installed between the guide rod and the piston distributor.

In the present embodiment, a ring of grooves 5611 is formed at the junction of the piston guide ring 562 and the piston distributor 5610, the lower end of the piston guide ring 562, and the upper end of the piston distributor 5610, and the grooves 5611 divide the piston into two layers. With this arrangement, the piston body need not be completely disengaged from the spar 52 when it is desired to disassemble the piston screen 564. The problem of current piston sieve often can't dismantle, and waste time and energy when dismantling is solved.

In this embodiment, a positioning conical surface 5612 is provided between the piston guide ring 562 and the piston distributor 5610 to ensure that the piston guide ring 562 and the piston distributor 5610 are concentric and do not leak. The embodiment ensures even distribution by adding the piston distributor, so that the synthesis column with the structure is convenient to disassemble and assemble.

In the present embodiment, by providing the auxiliary cylinder assembly 63 on the top of the composite column, as shown in fig. 5, the auxiliary cylinder assembly 63 includes a guide seat 631, a first cylinder seal 632, a cylinder 633, a cylinder cover 634, a second cylinder seal 635, a third cylinder seal 636, a piston plate 637, a fourth cylinder seal 638, a cylinder guide sleeve 639, a first joint 6310, a first speed control valve 6311, a second speed control valve 6312, a fifth cylinder seal 6313, a sixth cylinder seal 6314, a second joint 6315, and a seventh cylinder seal 6316.

A cylinder guide sleeve 639 is installed in the guide seat 631, a seventh cylinder packing 6316 is installed between the lower end of the guide seat 631 and the upper end cap 57, a piston plate 637, a second cylinder packing 635 and a third cylinder packing 636 are installed at the upper end of the guide seat 631, and a cylinder 633 is installed outside the guide seat 631, in other words, the upper end of the guide seat 631 and the piston plate 637 are both placed inside the cylinder 633, and the connected portions thereof are respectively installed with a first cylinder packing 632 and a fifth cylinder packing 6313. The cylinder cover 634 penetrates through the guide rod 561 and is fixed to the upper portion of the cylinder body 633 through bolts, a sixth cylinder body sealing ring 6314 is installed between the cylinder cover 634 and the cylinder body 633, a second cavity is formed among the cylinder body 633, the upper portion of the piston plate 637 and the lower portion of the cylinder cover 634, a second joint 6315 is installed on the upper portion of the cylinder cover 634, one end of the second joint 6315 is communicated with the second cavity, and the other end of the second joint 6315 is connected with a second speed regulating valve 6312. A first cavity is formed in front of the lower portion of the cylinder 633, the guide seat 631 and the piston plate 637, a first joint 6310 is installed on the outer side of the cylinder 633, one end of the first joint 6310 is communicated with the first cavity, and the other end of the first joint is connected with a first speed regulating valve 6311.

The auxiliary cylinder assembly of the present embodiment is used to assist in loading the piston assembly into the cylinder or pressing the piston out of the cylinder.

When the piston sieve plate needs to be disassembled, the second speed regulating valve 6312 is kept in an open state, and compressed air (or liquid may be pumped) is introduced from the second speed regulating valve 6312 through the second joint 6315, so that the first cylinder seal 632, the cylinder 633, the cylinder cover 634, the fourth cylinder seal 638, the sixth cylinder seal 6314, the second speed regulating valve 6312, the second joint 6315, the first speed regulating valve 6311, and the first joint 6310 move upward as a whole, and the state shown in fig. 6 is reached. The pressure solvent inlet 64 is opened, the evacuation port 65 is closed, pressure solvent is pumped from the pressure solvent inlet 64, the piston assembly 56 is pushed to move downwards, the pressure solvent is stopped being pumped after the lower surface of the limiting plate 66 is attached to the upper surface of the cylinder cover 634, the piston assembly 56 stops moving downwards, the state of the piston assembly 56 is just as shown in fig. 7, and the groove of the piston assembly 56 is exposed out of the lower end surface of the column cylinder 52. The plunger screen plate 564 is bolted down and the plunger screen plate 564 is removed.

When it is desired to install the piston assembly 56 into the barrel, the piston will typically need to be removed for the first installation or for significant maintenance; the column body is integrally rotated by 180 degrees by the column barrel, the lower end cover 53, the lower end cover sieve plate 54, the E-shaped sealing ring 55 and the limiting plate 66 are ensured to be in a detached state, and the lower surface of the cylinder cover 634 is attached to the upper surface of the piston plate 637. The guide rod 561 of the piston assembly 56 is passed down through the central bore of the barrel cylinder 52, upper end cap 57 and auxiliary cylinder assembly 63 with the guide rod 561 and the fourth cylinder seal 638 in a sealed condition. The stopper plate 66 is fitted to a position corresponding to the guide rod 561. The second speed regulation valve port is kept in an open state, compressed air is introduced from the second speed regulation valve 6312 through the second joint 6315 (or liquid can be pumped in), so that the first cylinder sealing ring 632, the cylinder 633, the cylinder cover 634, the fourth cylinder sealing ring 638, the sixth cylinder sealing ring 6314, the second speed regulation valve 6312, the second joint 6315, the first speed regulation valve 6311 and the first joint 6310 move up integrally, the cylinder cover 634 pushes the limit plate 66 and the piston assembly 56 to move integrally, the piston assembly 56 enters the cylinder 52, and the first piston sealing ring 563 is in a sealed state. Rotating the whole column body back to a normal working state, opening the pressure solvent inlet 64, opening the evacuation port 65, pumping the pressure solvent from the pressure solvent inlet 64 until the evacuation port 65 continuously exhausts liquid and no gas is exhausted, stopping pumping the pressure solvent, closing the evacuation port 65, pumping the solvent in the column from the pressure solvent inlet 64, forming negative pressure in the column, sucking the piston assembly 56 into the column barrel 52, and completing the installation of the piston assembly 56.

Example 10:

the nucleic acid synthesis system of the present embodiment mainly includes: the device comprises an industrial personal computer, a PLC (programmable logic controller), a cabinet body 1, a single inlet valve 2, a first reagent inlet valve 3, a second reagent inlet valve 4, a third infusion pump 5, a second infusion pump 6, a first infusion pump 7, a second pressure sensor 8, a first pressure sensor 9, a three-way joint 10, an anti-mixing valve 11, a synthetic column 12, a third pressure sensor 13, a conductivity detector 14, a circulating valve 15, a flushing valve 16, an ultraviolet detector 17, a back pressure valve 18, a waste discharge valve 19, a single bottle 20, a first pressure regulating valve 21, a fourth pressure sensor 22, a first air source distributor 23, a reagent bottle 24, a fifth pressure sensor 25, a second pressure regulating valve 26, a third pressure regulating valve 27, a sixth pressure sensor 28 and a second air source distributor 29. The inlet of the monomer inlet valve 2 is connected with the monomer bottle 20, and the inlet of the first reagent inlet valve 3 and the inlet of the second reagent inlet valve 4 are respectively connected with different reagent bottles 24.

As shown in fig. 8 and 9, an industrial personal computer, a PLC controller, a single inlet valve 2, a first reagent inlet valve 3, a second reagent inlet valve 4, a waste discharge valve 19, a third infusion pump 5, a second infusion pump 6, a first infusion pump 7, a second pressure sensor 8, a first pressure sensor 9, a tee joint 10, a third pressure sensor 13, a conductivity detector 14, a circulating valve 15, an anti-mixing valve 11, a flushing valve 16, an ultraviolet detector 17 and a back pressure valve 18 are all arranged on the cabinet 1. The single bottle 20 and the reagent bottle 24 are placed below the cabinet 1, and the single bottle 20 and the reagent bottle 24 can be fixed below the cabinet 1 by a tray.

The air pipe components are arranged on the left side and the lower side of the cabinet body 1, and comprise a pressure sensor four 22, a pressure sensor five 25, a pressure sensor six 28, a pressure regulating valve one 21, a pressure regulating valve two 26, a pressure regulating valve three 27, an air source distributor one 23 and an air source distributor two 29, wherein the pressure sensor four 22, the pressure sensor five 25, the pressure sensor six 28, the pressure regulating valve one 21, the pressure regulating valve two 26 and the pressure regulating valve three 27 are arranged on 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 body 1. The synthetic column 12 is fixed on the left side of the cabinet 1 through a clamp.

The embodiment can also arrange chuck joints at the left and right sides of the cabinet body 1, and when a large amount of solvent is used, the large amount of solvent can be connected with an external vulcanization hose 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 synthesis 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. 10, the three inlet valves of the present embodiment respectively correspond to three pre-column liquid paths, and the first pre-column liquid path is composed of a single inlet valve 2, a liquid delivery pump three 5 and a pressure sensor two 8; the outlet of the single inlet valve 2 is connected with the inlet of the infusion pump three 5 through a liquid pipeline, the outlet of the infusion pump three 5 is connected with the inlet of the anti-mixing valve 11 through a liquid pipeline, and the pressure sensor two 8 is arranged on the liquid pipeline connecting the infusion pump three 5 and the anti-mixing valve 11. When the fluid is conveyed by using the liquid path in front of the first column, the second pressure sensor 8 is used for monitoring feedback pressure data in front of the synthetic column 12 and uploading the monitored feedback pressure data to the industrial personal computer, and the industrial personal computer judges whether to perform high-pressure alarm or not, so that hardware damage and carrier damage in the synthetic column 12 caused by overpressure can be prevented.

The front liquid path of the second column consists of a reagent inlet valve I3, an infusion pump II 6 and a pressure sensor I9; 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 path consists of a reagent inlet valve II 4 and a liquid 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 a liquid path in front of a second column is used for conveying fluid or a liquid path in front of a third column is used for conveying fluid, a first pressure sensor 9 is used for monitoring feedback pressure data in front of a synthesis column 12 and uploading the monitored feedback pressure data to an industrial personal computer, and the industrial personal computer judges whether high-pressure alarm is carried out or not, so that hardware damage and carrier damage in the synthesis column 12 caused by overpressure can be prevented. The three pre-column liquid paths are connected with the synthetic 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 synthetic column 12 through a liquid pipeline, a column outlet of the synthetic column 12 is connected with an inlet of the circulating valve 15 through a liquid pipeline, and one outlet of the circulating 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 a backpressure valve 18 through a liquid pipeline, and the outlet of the backpressure valve 18 is connected with a waste discharge valve 19 through a liquid pipeline to realize liquid discharge; the ultraviolet detector 17 is arranged on a liquid pipeline connecting the other outlet of the circulating valve 15 and the backpressure valve 18, in other words, the backpressure valve 18 is positioned at the tail end of the ultraviolet detector 17; the ultraviolet detector 17 is provided to obtain ultraviolet absorption data of the material after the reaction of the chemical reagent and upload the ultraviolet absorption data to the industrial personal computer, and the industrial personal computer monitors the reaction conditions and reaction results of the material at different process stages according to the ultraviolet absorption data. The purpose of the back pressure valve 18 is to provide the synthesis system of this embodiment with an appropriate back pressure, to prevent bubbles from being generated in the front end liquid path and affecting the detection data, and to keep the reaction in the column sufficient. The liquid circulation and drainage functions of the synthesis system of this embodiment will now be further described with reference to fig. 3.

The circulating valve 15 of the present embodiment is controlled by an industrial personal computer to circulate or discharge liquid. When the circulating valve 15 is used for liquid circulation, a liquid circulation passage is formed by the reagent inlet valve II 4, the infusion pump I7, the pressure sensor I9, the anti-mixing valve 11, the synthesis column 12, the pressure sensor III 13, the conductivity detector 14, the circulating valve 15 and a plurality of liquid pipelines; the first infusion pump 7 drives the liquid to complete the circulation process inside the liquid circulation path.

The liquid discharge passage of the embodiment is formed by sequentially connecting a reagent inlet valve I3, a liquid delivery pump II 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 liquid delivery pump 6 drives the liquid in the liquid discharge passage to discharge the liquid from the waste valve 19.

The synthesis column 12 of this example has the structure disclosed in example 6 or example 9.

The flush valve 16 of the present embodiment has a rapid column packing function, an inlet of the flush valve 16 is connected to the other outlet of the mix preventing valve 11, an 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 an inlet of the back pressure valve 18. When the column loading is needed, the evacuation port of the synthesis column 12 is closed, and the liquid flows into the synthesis column 12 through the reagent inlet valve I3, the infusion pump II 6, the pressure sensor I9, the anti-mixing valve 11 and the flushing valve 16 through the pipeline, so that the column loading is completed.

The pressure sensor six 28 and the pressure regulating valve three 27 of the embodiment 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 a gas source into the synthesis column 12, and the pressure sensor six 28 is arranged on a gas pipeline of the third pressure regulating valve 27 connected with the synthesis column 12 and is used for regulating according to the pressure required by the synthesis column 12. When the column needs to be disassembled, the evacuation port of the synthesis column 12 is opened, the outlet of the third pressure regulating valve 27 is connected with the column outlet of the synthesis column 12, and the pressure in the synthesis column is regulated.

During the whole reaction process, the monomer bottle 20 and the reagent bottle 24 need to be protected by pressurization with inert gas (argon or nitrogen) to achieve the purpose of air isolation. As shown in fig. 11, an inert gas protection device is composed of a pressure sensor four 22, a pressure sensor five 25, a pressure regulating valve one 21, a pressure regulating valve two 26, a gas source distributor one 23 and a gas source distributor two 29; an inert gas source is distributed to a gas source distributor I23 and a gas source distributor II 29 through a four-way joint, and the gas source distributor I23 and the gas source distributor II 29 are respectively used for distributing inert gas into the monomer bottle 20 and the reagent bottle 24; the pressure sensor four 22 is arranged on a gas pipeline of the pressure regulating valve one 21 connected with the gas source distributor one 23, the pressure sensor four 22 is arranged for acquiring pressure data in the monomer bottle 20 in real time, and the pressure regulating valve one 21 is arranged for regulating the pressure in the monomer bottle 20 according to process requirements. The pressure sensor five 25 is arranged on a gas pipeline connected with the pressure regulating valve two 26 and the gas source distributor two 29, and the pressure sensor five 25 is arranged for acquiring 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.

The gas pipeline and the liquid pipeline adopted in the embodiment can adopt a tetrafluoride pipe or a PEEK pipe.

The nucleic acid synthesis scheme of this example was:

the first process is as follows: trichloroacetic acid enters a synthesis column 12 through a reagent inlet valve I3, a liquid delivery pump II 6 and an anti-mixing valve 11 in sequence to be deprotected (Deblocking); after the reaction is finished, the acetonitrile enters a reagent inlet valve I3 to wash the pipeline, flows out of the synthesis column 12, and is discharged through a pressure sensor III 13, a conductivity detector 14, a circulating valve 15, an ultraviolet detector 17, a back pressure valve 18 and a waste discharge valve 19 in sequence, so that pipeline washing and liquid discharge are finished.

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

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

The second process is called Activation. Prior to coupling, the monomer is mixed with tetrazole and enters the synthesis column 12, where tetrazole provides a proton to the N atom of the diisopropylamine group on the 3' phosphate, and the protonated diisopropylamine is a good free group to form a reactive intermediate, phosphoramidite tetrazole, with tetrazole.

The third process: controlling the circulating valve 15 to be in a liquid circulating state, starting the infusion pump I7, setting the flow rate, and enabling the monomers and the tetrazole in the synthesis column 12 to flow into the infusion pump I7 through the three-way joint 10, the pressure sensor I9, the anti-mixing valve 11, the synthesis column 12, the pressure sensor III 13, the conductivity detector 14, the circulating valve 15 and the reagent inlet valve II 4 to form a closed-loop circulating loop; after the reaction is finished, controlling the circulating valve 15 to be in a liquid discharge state, enabling the acetonitrile to sequentially pass through the monomer inlet valve 2, the infusion pump III 5, the pressure sensor II 8, the anti-mixing valve 11, the synthetic column 12, the pressure sensor III 13, the conductivity detector 14, the circulating valve 15 and the reagent inlet valve II 4, flow into the infusion pump I7, the three-way joint 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 then discharging, and finishing pipeline flushing.

The process is called as follows: coupling (Coupling). The tetrazole phosphoramidite undergoes a nucleophilic reaction with the 5' hydroxyl group of the nucleotide to which CPG is linked when colliding with the tetrazole, coupling occurs and the tetrazole is removed, and the synthesized oligonucleotide chain is extended by one.

The process four is as follows: and the oxidant enters the synthesis column 12 through the reagent inlet valve II 4, the infusion pump I7, the tee joint 10, the pressure sensor I9 and the anti-mixing valve 11, after the reaction is finished, the acetonitrile enters through the flow path, and is discharged through the pressure sensor III 13, the conductivity detector 14, the circulating 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 as follows: oxidation (Oxidation). The newly added nucleotide after the coupling reaction is connected with the oligonucleotide chain on the CPG through a phosphorous ester bond (phosphorous is trivalent), the phosphorous ester bond is unstable and is easy to be hydrolyzed by acid and alkali, so that the trivalent phosphorous is required to be oxidized into pentavalent phosphorous through the oxidation process.

And a fifth process: the capping agent A enters the three-way joint 10 through the first reagent inlet valve 3 and the second infusion pump 6, and the capping agent B enters the three-way joint 10 through the second reagent inlet valve 4 and the first infusion pump 7; after the two reagents enter the three-way joint 10, the two reagents sequentially pass through the first pressure sensor 9 and the anti-mixing valve 11 to enter the synthetic column 12 to complete reaction, acetonitrile enters 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 pipeline flushing.

The process is called as follows: caps (Capping). To prevent the unreacted 5' hydroxyl group attached to CPG from being extended in subsequent cycles, it is necessary to block the coupling reaction after it has proceeded sufficiently.

By repeating the above-mentioned processes one to five, the oligonucleotide chain can be extended to a desired length. In order to improve the automation degree of a test nucleic acid synthesis system in the embodiment, improve the synthesis efficiency and improve the accuracy of liquid flow rate, a synthesis control algorithm is arranged in the industrial personal computer in the embodiment, the industrial personal computer is electrically connected with a PLC (programmable logic 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 are all connected with the PLC, 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 feedback pressure data and ultraviolet absorption data monitored by the PLC, the PLC uploads the received feedback pressure data and ultraviolet absorption data to the industrial personal computer, and the synthesis control commands of each pump and each valve are obtained through calculation and analysis by the synthesis control algorithm arranged in the industrial personal computer; the PLC controller is connected to each pump and each valve, and controls the operation of each pump and each valve in accordance with a control command for each pump and each valve.

Specifically, the PLC controls the flow rate of the pump through a Modbus; the PLC controls the valve to be switched on and off through the switching value; the PLC acquires 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 through the Modbus.

The synthesis control algorithm of the present embodiment mainly includes:

sending control signals for each pump and each valve to a PLC (programmable logic controller) based on preset process parameters and data of each pressure sensor to realize nucleic acid synthesis; the method comprises the following steps:

based on the nucleic acid synthesis flow, preset fluid path parameters, and feedback pressure data of each pressure sensor, control commands to each inlet valve, to each infusion pump, to the anti-mix valve 11, to the circulation valve 15, to the back pressure valve 18, and to the waste discharge valve 19 are obtained for the purpose of: and (3) carrying out nucleic acid synthesis according to preset liquid path parameters in sequence. The fluid path parameters herein include, but are not limited to: maximum allowable flow rate, pre-column pressure maximum, pre-column target pressure, target monomer or reagent volume, inlet valve on-off state, infusion pump flow rate, infusion pump run time, anti-mix valve 11 on-off state, circulation valve 15 on-off state, and circulation valve 15 mode state; at different nucleic acid synthesis stages, the liquid path parameters corresponding to the same device are different. The mode states of the circulation valve 15 include a circulation mode and a liquid discharge mode.

Now, the control process of each pump and each valve by the PLC controller based on the control command of the industrial personal computer will be described with reference to the nucleic acid synthesis process.

In the deprotection stage, the preset liquid path parameters mainly include: liquid path parameters of a deprotection reaction stage and liquid path parameters of a deprotection cleaning stage; the liquid path parameters of the deprotection reaction stage mainly comprise: the input reagent name, the opening and closing state of the reagent inlet valve I3 is open, the opening and closing state of the anti-mixing valve 11 is open, the preset flow rate of the infusion pump II 6, the preset running time of the infusion pump II 6 and the opening and closing state of the synthesis column 12 are open; the liquid path parameters of the deprotection cleaning stage mainly comprise: the inputted reagent name, the opening and closing state of the reagent inlet valve I3 is open, the opening and closing state of the mixing prevention valve 11 is open, the preset flow rate of the infusion pump II 6, the preset running time of the infusion pump II 6, the opening and closing state of the circulating valve 15 is open, the mode state of the circulating valve 15 is a liquid discharge mode, the percentage preset value of the back pressure valve 18, and the opening state of the waste discharge valve 19 is open.

In the activation stage, the preset fluid path parameters mainly include: the input reagent name, the opening state of the single inlet valve 2 is open, the opening state of the mixing prevention valve 11 is open, the opening state of the reagent inlet valve two 4 is open, the preset flow rate of the infusion pump three 5, the preset running time of the infusion pump three 5, the preset flow rate of the infusion pump one 7 and the preset running time of the infusion pump one 7.

In the coupling stage, the preset fluid path parameters mainly include: the liquid path parameters of the coupling reaction stage and the liquid path parameters of the coupling cleaning stage; the liquid path parameters of the coupling reaction stage mainly comprise: the inputted reagent name, the on/off state of the circulation valve 15 is open, the mode state of the circulation valve 15 is circulation mode, the preset flow rate of the infusion pump one 7, the preset operation time of the infusion pump one 7, the on/off state of the mix preventing valve 11 is open, and the open state of the reagent inlet valve two 4 is open. The liquid path parameters of the coupling cleaning stage mainly comprise: the inputted reagent name, the on-off state of the circulation valve 15 is open, the mode state of the circulation valve 15 is a drain mode, the open state of the individual inlet valve 2 is open, the on-off state of the flush valve 16 is open, the mode of the flush valve 16 is a flush mode, the percentage preset value of the back pressure valve 18, the on-off state of the waste discharge valve 19 is open, the preset flow rate of the infusion pump three 5, the preset operation time of the infusion pump three 5, the preset flow rate of the infusion pump one 7, and the preset operation time of the infusion pump one 7.

In the oxidation stage, the preset liquid path parameters mainly include: liquid path parameters of an oxidation reaction stage and liquid path parameters of an oxidation cleaning stage; the liquid path parameters of the oxidation reaction stage mainly comprise: the input reagent name, the preset flow rate of the infusion pump I7, the preset running time of the infusion pump I7, the opening and closing state of the reagent inlet valve II 4 is open, and the opening and closing state of the anti-mixing valve 11 is open. The liquid path parameters of the oxidation cleaning stage mainly comprise: the input reagent name, the preset flow rate of the infusion pump I7, the preset running time of the infusion pump I7, the on-off state of the reagent inlet valve II 4 is open, the on-off state of the anti-mixing valve 11 is open, the on-off state of the circulating valve 15 is open, the mode state of the circulating valve 15 is a liquid discharge mode, the percentage preset value of the back pressure valve 18 and the on-off state of the waste discharge valve 19 are open.

In the capping stage, the preset liquid path parameters mainly include: liquid path parameters of a cap reaction stage and liquid path parameters of a cap cleaning stage; the liquid path parameters of the cap reaction stage mainly comprise: the input reagent name, the switch state of the first reagent inlet valve 3 is open, the switch state of the second reagent inlet valve 4 is open, the switch state of the anti-mixing valve 11 is open, the preset flow rate of the second infusion pump 6, the preset running time of the second infusion pump 6, the preset flow rate of the first infusion pump 7 and the preset running time of the first infusion pump 7. The liquid path parameters of the cap cleaning stage mainly comprise: the on-off state of the circulating valve 15 is open, the mode state of the circulating valve 15 is a liquid discharge mode, the percentage preset value of the back pressure valve 18, the on-off state of the waste discharge valve 19 is open, the on-off state of the reagent inlet valve I3 is open, the on-off state of the reagent inlet valve II 4 is open, the on-off state of the anti-mixing valve 11 is open, the preset flow rate of the infusion pump II 6, the preset running time of the infusion pump II 6, the preset flow rate of the infusion pump I7 and the preset running time of the infusion pump I7.

How to control the rotation speed and the operation time of the infusion pump to reach the preset flow rate and the preset operation time according to the feedback pressure data of each pressure sensor is further described.

Taking the infusion pump three 5 as an example, the operation mode of the infusion pump three 5 is divided into a flow rate mode and a pressure mode, and when the flow rate mode is in the flow rate mode, the flow rate of the output liquid of the infusion pump three 5 is controlled to the target flow rate of the output liquid, that is, the rotation speed control value of the infusion pump three 5 is obtained according to the pre-calibrated flow rate-rotation speed ratio of the infusion pump three 5. The default mode of the infusion pump three 5 in the embodiment is a pressure mode, and when the infusion pump three 5 is in the pressure mode, a control command of the flow rate of the infusion pump three 5 is calculated through an incremental PID algorithm according to feedback pressure data of the pressure sensor two 8 and preset target pressure before the column.

（6）

（7）

（8）

Wherein:

is composed of

The flow rate at a time is controlled in increments,

is a coefficient of proportionality that is,

in order to be the coefficient of integration,

in order to be the differential coefficient,

is composed of

is composed of

The feedback pressure data of the time of day,

is composed of

The corresponding pre-column target pressure at that time,

is composed of

The flow rate control value of the infusion pump at the time,

is composed of

is composed of

Feeding back the difference between the pressure data and the corresponding target pressure in front of the column at any moment;

is composed of

The operation time of the infusion pump three 5 is divided into a time mode and a volume mode, the operation time in the time mode is a fixed preset target operation time, the operation time in the volume mode is determined according to the accumulated volume calculation, and the operation time is reached when the accumulated volume reaches the target volume. The operating time of the infusion pump three 5 of the present embodiment defaults to the operating time in volume mode.

According to equation (9), calculate

Cumulative volume of time of day

：

（9）

In the formula (I), the compound is shown in the specification,

is composed of

The cumulative volume at a time of day,

is composed of

The flow rate of the infusion pump at the time,

represent

At the moment of time, the time of day,

represent

Time of day;

judgment of

Cumulative volume of time of day

Whether the target monomer or reagent volume is reached or not, and if so, obtaining a control command that the running time of the infusion pump reaches; otherwise, obtaining a control command of prolonging the running time of the infusion pump.

The industrial personal computer controls the back pressure valve 18 into a percentage mode and a pressure mode, and in the percentage mode, a target percentage value is directly output to the PLC according to a preset target percentage value, and the PLC controls the back pressure valve 18; in the pressure mode, a percentage control value of the back pressure valve 18 is calculated by using the incremental PID algorithm of the back pressure valve through the feedback pressure data of the third pressure sensor 13 and a preset target pressure, so that the pressure behind the synthetic column is controlled.

The back pressure valve incremental PID algorithm is expressed as:

（10）

（11）

（12）

wherein:

is composed of

The back pressure valve percentage at that time controls the increment,

is a coefficient of proportionality that is,

in order to be the integral coefficient of the light,

in order to be the differential coefficient,

is composed of

The difference between the current feedback pressure data at the time and the preset target pressure,

is composed of

The current feedback pressure data at the time of day,

is composed of

The preset target pressure at the time of day,

is composed of

The percentage control value of the back pressure valve at the moment,

is composed of

The percentage control value of the back-pressure valve at the moment,

is composed of

The difference between the current feedback pressure data at that time and the preset target pressure,

is composed of

The difference between the current feedback pressure data at the moment and the preset target pressure.

The synthesis control algorithm of the present embodiment further includes:

optimizing preset liquid path parameters based on the ultraviolet absorption data and the feedback pressure data of the pressure sensors in front of each column, and mainly obtaining control commands for each infusion pump according to the optimized liquid path parameters; the method specifically comprises the following steps:

presetting deprotection peak integration conditions for a deprotection nucleic acid synthesis stage, judging whether ultraviolet absorption data in the stage meet the deprotection peak integration conditions, if not, adjusting the flow rate of an infusion pump on a related liquid path to the maximum allowable flow rate, and not limiting the operation time of the infusion pump until the ultraviolet absorption data meet the deprotection peak integration conditions; the second infusion pump 6 will now be described as an example. When the flow rate is not satisfied, the flow rate of the infusion pump II 6 is increased to the maximum allowable flow rate, the operation is carried out for 10-30 minutes, and if no deprotection peak still exists, the deprotection reagent is considered to be abnormal, namely trichloroacetic acid is abnormal; if the deprotection peak is immediately finished just before appearing, the second infusion pump 6 is operated at the maximum allowable rotating speed, and the operation time is increased by 3-20 minutes on the basis of 10-30 minutes.

As shown in FIG. 12, the number in the figure indicates a deprotection peak, starting from "deprotection" and ending at "deprotection washing". The beginning of the peak indicates that the uv absorption data began to rise, and the end of the peak indicates that the uv absorption data dropped to baseline (bottom-most) stability. From the deprotection stage, the time until the ultraviolet absorption data rises is the peak time; the time from the drop in uv absorption data to baseline stabilization (end of peak) to the end of deprotection phase is the end of peak wait time.

Ideally, the deprotection regime is short peak off time and short peak end wait time. As shown in fig. 12, the short peak-off time and peak-end latency (essentially 0) are "good" deprotection processes.

judging whether the peak-off time of the deprotection peak exceeds the peak-off time threshold of the deprotection peak, and if so, optimizing the flow rate of the infusion pump according to the formula (1); if not, maintaining the preset liquid path parameters, because the shorter the peak output time of the deprotection peak is, the better.

（1）

In the formula (I), the compound is shown in the specification,

in order to optimize the flow rate of the infusion pump,

the flow rate of the infusion pump in the preset fluid path parameters,

in order to increase the coefficient for the flow rate,

is the peak-off time threshold of the deprotected peak,

the peak-off time of the current deprotection peak;

judging whether the rising time of the deprotection peak is larger than a rising time threshold value or not, and if so, optimizing the flow rate of the infusion pump according to the formula (2); if not, the preset liquid path parameters are maintained, because the shorter the rising time of the deprotection peak is, the better.

（2）

In the formula (I), the compound is shown in the specification,

is the threshold value of the rise time of the deprotection peak,

is the rise time of the current deprotected peak;

judging whether the falling time of the deprotection peak is larger than a falling time threshold value or not, and if so, optimizing the flow rate of the infusion pump according to the formula (3); if the peak value is not greater than the preset threshold value, maintaining the preset liquid path parameters, wherein the shorter the falling time of the deprotection peak is, the better the falling time is;

（3）

in the formula (I), the compound is shown in the specification,

is the threshold value of the fall time of the deprotected peak,

is the fall time of the current deprotected peak;

judging whether the feedback pressure data of the pressure sensor in front of the column exceeds the maximum value of the pressure in front of the column, if so, optimizing the flow rate of the infusion pump according to the formula (4), and if not, optimizing the flow rate of the infusion pump according to the formula (5);

（4）

in the formula (I), the compound is shown in the specification,

in order to reduce the coefficient for the flow rate,

for the purpose of the current feedback pressure data,

maximum value of pressure before column;

（5）

in the embodiment, after the deprotection peak reaction is finished, judging whether the waiting time length exceeds a preset waiting time length or not, if so, reducing the deprotection finishing waiting time, wherein the new finishing waiting time = the old finishing waiting time-the exceeded waiting time; if not, no optimization is done, since the shorter the deprotection end latency, the better.

And (3) judging whether the feedback pressure data of the pressure sensor in front of the column exceeds the maximum value of the pressure in front of the column in the activation stage, the coupling stage, the oxidation stage and the capping nucleic acid synthesis stage, if so, optimizing the flow rate of the infusion pump according to the formula (4), and if not, optimizing the flow rate of the infusion pump according to the formula (5).

Claims

1. A nucleic acid synthesis system, comprising: the method comprises the following steps: the system comprises an industrial personal computer, a PLC (programmable logic controller), a multi-path pre-column liquid path, an anti-mixing valve, a synthesis column, a circulating valve, a circulating liquid path and a liquid discharge path;

each pre-column liquid path 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 an anti-mixing valve; the outlet of the anti-mixing valve is connected with the column inlet of the synthesis column; the column outlet of the synthesis column is connected with the inlet of the circulating valve, one outlet of the circulating valve is connected with the inlet of the circulating liquid path, and the other outlet of the circulating valve is connected with the inlet of the liquid drainage liquid path; the outlet of the circulating liquid path is connected with the inlet valve of a certain path of pre-column liquid path;

the industrial personal computer adopts a synthetic control algorithm to obtain control commands for each inlet valve, each infusion pump, each anti-mixing valve and each circulation valve according to feedback pressure data and ultraviolet absorption data of the liquid path in front of the column;

the PLC is connected with the ultraviolet detector and is used for acquiring ultraviolet absorption data monitored by the ultraviolet detector; the PLC is connected with each pre-column pressure sensor and used for acquiring feedback pressure data of each pre-column pressure sensor corresponding to a pre-column liquid path, is electrically connected with an industrial personal computer and used for uploading the feedback pressure data and ultraviolet absorption data to the industrial personal computer and receiving a control command issued by the industrial personal computer, is connected with each inlet valve, each infusion pump, an anti-mixing valve and a circulating valve and used for correspondingly controlling each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve based on the control command issued by the industrial personal computer;

the synthesis control algorithm comprises the following steps:

based on the nucleic acid synthesis flow, preset liquid path parameters and feedback pressure data of each pre-column liquid path, control commands for each inlet valve, each infusion pump, the anti-mixing valve and the circulating valve are obtained, and nucleic acid synthesis is carried out according to the nucleic acid synthesis flow under the condition that the preset liquid path parameters are met; the nucleic acid synthesis process comprises five nucleic acid synthesis stages of deprotection, activation, coupling, oxidation and capping; the liquid path parameters include: maximum allowable flow rate, pre-column pressure maximum, pre-column target pressure, target monomer or reagent volume, open and close state of inlet valve, flow rate of infusion pump, running time of infusion pump, open and close state of anti-mix valve, open and close state of circulating valve, and mode state of circulating valve; the mode states of the circulation valve include a circulation mode and a liquid discharge mode; when the circulating valve is in a circulating mode, a passage is formed between the circulating valve and the circulating liquid path, and a circuit is broken between the circulating valve and the liquid discharge liquid path; when the circulating valve is in a liquid discharge mode, the circulating valve is disconnected with the circulating liquid path, and a passage is formed between the circulating valve and the liquid discharge path;

（1）

in the formula (I), the compound is shown in the specification,

is superior toThe flow rate of the infusion pump after the infusion is changed,

the flow rate of the infusion pump in the preset fluid path parameters,

in order to increase the coefficient for the flow rate,

is the peak-off time threshold of the deprotected peak,

the peak-off time of the current deprotection peak;

（2）

in the formula (I), the compound is shown in the specification,

is the threshold value of the rise time of the deprotection peak,

is the rise time of the current deprotection peak;

（3）

in the formula (I), the compound is shown in the specification,

is the threshold value for the fall time of the deprotected peak,

is the fall time of the current deprotected peak;

（4）

in the formula (I), the compound is shown in the specification,

in order to reduce the coefficient for the flow rate,

for the purpose of the current feedback pressure data,

is the maximum value of the pressure before the column;

（5）

2. A nucleic acid synthesis system according to claim 1, wherein: the method is characterized in that control commands for each inlet valve, each infusion pump, each anti-mixing valve and each circulation valve are obtained based on a nucleic acid synthesis flow, preset liquid path parameters and feedback pressure data of each pre-column liquid path, and the method specifically comprises the following steps:

3. A nucleic acid synthesis system according to claim 2, wherein: the control command of the flow rate of the infusion pump is obtained through an incremental PID algorithm, and the control command comprises the following steps:

（6）

（7）

（8）

wherein:

is composed of

The flow rate at a time is controlled in increments,

is a coefficient of proportionality that is,

in order to be the coefficient of integration,

in order to be the differential coefficient,

is composed of

is composed of

The feedback pressure data at the time of day,

is composed of

The corresponding pre-column target pressure at the time,

is composed of

The flow rate control value of the infusion pump at the time,

is composed of

is composed of

is composed of

A flow rate control value of the time infusion pump;

the flow rate control value of the infusion pump constitutes a control command for the flow rate of the infusion pump.

4. A nucleic acid synthesis system according to claim 2, wherein: the obtaining of the control command of the infusion pump running time according to the cumulative volume algorithm specifically comprises:

according to equation (9), calculate

Cumulative volume of time of day

：

（9）

In the formula (I), the compound is shown in the specification,

is composed of

The cumulative volume at a time of day,

is composed of

The flow rate of the infusion pump at the time,

to represent

At the moment of time, the time of day,

to represent

Time of day;

judgment of

Cumulative volume of time of day

Whether the target monomer or reagent volume is reached or not, and if so, obtaining a control command that the running time of the infusion pump reaches; otherwise, obtaining a control command of prolonging the operation time of the infusion pump.

5. A nucleic acid synthesis system according to claim 1, wherein: the inlet valve is of a valve bank structure;

each air cylinder assembly consists of a diaphragm, a shell seat, a movable rod, a shell, a spring, a retainer 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 diaphragm, 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 the check ring;

6. A nucleic acid synthesis system according to claim 5, wherein: the anti-mixing valve and the circulating valve are both valve group structures.

7. A nucleic acid synthesis system according to claim 1, wherein: the outlet of each infusion pump is connected with a flow limiting valve device;

8. A nucleic acid synthesis system according to claim 1, wherein: the synthesis column comprises: the device comprises a pressure solvent inlet, an evacuation port, a column casing, a lower end cover assembly, a piston assembly, an upper end cover guide sleeve and an auxiliary cylinder assembly; the auxiliary cylinder assembly is fixed on the upper cover plate assembly;

9. A nucleic acid synthesis system according to claim 8, wherein: the device also comprises a column mounting device; the device column device is a flushing valve;

the flushing valve is connected with the PLC;

the PLC controls the flushing valve according to a control command for the flushing valve sent by the industrial personal computer;

10. A nucleic acid synthesis system according to claim 1, wherein: the nucleic acid synthesis system further comprises an inert gas protection device comprising: a plurality of protective gas circuits;

the protection pressure sensor and the protection pressure regulating valve are both connected with a PLC (programmable logic controller), and the PLC controls the protection pressure regulating valve according to a control command for the protection pressure regulating valve, which is sent by an industrial personal computer;