Multi-polypeptide synchronous reciprocating synthesis method and multi-channel synthesis device

Abstract

According to the multi-polypeptide synchronous reciprocating synthesis method and the multi-channel synthesis device, a DMF liquid storage bottle, a deprotection liquid storage bottle and a plurality of amino acid liquid storage bottles are arranged in liquid storage bottles and are communicated with a feeding port of a multi-channel injection pump, the multi-channel injection pump is communicated with a multi-channel switching valve, at least two reciprocating reaction modules are arranged, and the reciprocating reaction modules are communicated with the feeding port of the multi-channel injection pump. The waste liquid bottle and the reciprocating reaction module are communicated with the multi-channel switching valve, and the reciprocating reaction module is communicated with the waste liquid bottle. Reciprocating synthesis is adopted, a very small amount of reagent is in reciprocating contact with resin, and full utilization is achieved; and reciprocating reaction channels are allowed to be added, so that simultaneous reciprocating synthesis of multiple channels can be realized without mutual influence, and trace peptide synthesis can be performed at the same time. According to the method and the device thereof, the chemical synthesis mode is green, time is saved, the use amount of the resin can be reduced to 20 mg, the use amount of DMF is only 12-18 ml for connection of single amino acid, the time consumed by connection of one amino acid can be shortened from traditional 40 min to 5 min, and the time is shortened by 87.5%.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide synthesis, and in particular relates to a method for synchronously synthesizing multiple polypeptides and a multi-channel synthesis device. Background Art

[0002] Peptides are a class of biologically active molecules formed by amino acids connected by peptide bonds. They are widely present in organisms and participate in various physiological processes, such as cell growth, neural regulation, in vivo transport and hormone regulation. The development of peptide drugs, vaccines and diagnostic reagents has become a hot spot in biomedical research. Peptide drugs are highly stable and have low side effects. They are developed to treat a variety of diseases, such as anti-tumor, cancer cell targeting vectors, diabetes, osteoporosis, etc. With the development of science and technology, more and more peptide drugs have been approved for marketing, and the market size of peptide drugs in my country has also steadily increased.

[0003] The chemical synthesis method of peptides is divided into liquid phase synthesis and solid phase synthesis. However, the liquid phase synthesis method usually requires a large amount of reagents and solvents, and the reaction steps are complicated and the purification process is cumbersome. Solid phase synthesis is currently the most commonly used method for peptide synthesis. This method connects the C-terminus (carboxyl end) of the amino acid to an insoluble resin, and gradually adds DMF washing, deprotection solution to remove the Fmoc group, DMF washing, and amino acid activation solution coupling to extend the peptide chain.

[0004] The research and development of peptide drugs is a very long process. Nowadays, most of the research on peptide synthesis is carried out in traditional reaction vessels such as chromatography columns and reactors. Usually, a resin solid phase carrier is placed in it, and various reagents are added to the reaction vessel, and nitrogen bubbling or stirring is used to mix and react fully. After a certain period of time, the resin is retained by the filter plate at the port of the container, and the waste liquid is discharged. When using chromatography columns, reactors, etc. as reactors, a large amount of resin raw materials is required. When too little raw material is used, a large amount of resin sticks to the wall due to the wall hanging phenomenon caused by the resin solid support and the reaction vessel, resulting in very limited collection of products. Therefore, a small amount of raw materials is not realistic. The peptide synthesis method based on traditional reactors has problems such as long reaction cycle, large amount of reagents, and low synthesis efficiency. In the early synthesis process of new drug development, a large amount of raw materials and time are consumed, and it is difficult to meet the needs of high throughput and high efficiency. Summary of the invention

[0005] The present invention provides a method for synchronously synthesizing multiple polypeptides and a multi-channel synthesis device, which can effectively solve the above problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present application provides a multi-channel synthesis device for synchronously synthesizing multiple polypeptides, including a multi-channel injection pump, a multi-channel switching valve, a liquid storage bottle, a waste liquid bottle, and a reciprocating reaction control module. The liquid storage bottle is provided with a DMF liquid storage bottle, a deprotection liquid storage bottle, and several amino acid liquid storage bottles, which are respectively communicated with the feed inlet of the multi-channel injection pump. The discharge outlet of the multi-channel injection pump is communicated with the liquid inlet of the multi-channel switching valve. There are at least 2 reciprocating reaction control modules. The waste liquid bottle and the reciprocating reaction control module are respectively communicated with the liquid outlet of the multi-channel switching valve, and the reciprocating reaction control module is communicated with the waste liquid bottle.

[0008] As a further improvement, the reciprocating reaction control module is provided with a buffer bottle, a first two-position three-way solenoid valve, a retention sand core, a microfluidic chip, and a reciprocating injection pump. The retention sand core is provided with a first retention sand core and a second retention sand core. The buffer bottle, the first two-position three-way solenoid valve, the first retention sand core, the microfluidic chip, the second retention sand core, and the reciprocating injection pump are sequentially communicated. The upper end of the buffer bottle is communicated with the waste liquid bottle. The lower end of the buffer bottle is a sharp mouth, and the bottom of the sharp mouth is communicated with the first two-position three-way solenoid valve through a pipeline.

[0009] As a further improvement, a second two-position three-way solenoid valve is provided at the end of the second retention sand core and is communicated with the reciprocating injection pump.

[0010] As a further improvement, the pipe orifice of the pipeline connecting the buffer bottle and the waste liquid bottle is located in the middle and lower part of the buffer bottle and is arranged against the wall. The lower end of the buffer bottle is a sharp mouth, and the pipeline connecting the buffer bottle and the first two-position three-way solenoid valve extends into the bottom of the sharp mouth of the buffer bottle.

[0011] As a further improvement, the first retention sand core is a filter rod, and the second retention sand core is a filter ball.

[0012] As a further improvement, the multi-channel injection pump is reserved with an air inlet.

[0013] In a second aspect, the present application provides a method for synchronously synthesizing multiple polypeptides, including the following steps:

[0014] (1) Preliminary preparation:

[0015] Load the configured N,N-dimethylformamide (DMF), deprotection liquid, and each amino acid activation liquid into the DMF liquid storage bottle, the deprotection liquid storage bottle, and each amino acid liquid storage bottle; and swell the resin with Fmoc protection group with dichloromethane (DCM), and then configure it into a suspension with DMF and suck it into the microfluidic chip by a reciprocating injection pump.

[0016] (2) Washing step:

[0017] Control a multi-channel injection pump, a multi-channel switching valve, a first two-position three-way solenoid valve, a second two-position three-way solenoid valve, and a reciprocating injection pump, and cooperate with a DMF liquid storage bottle, a buffer bottle, and a waste liquid bottle to realize the washing of the connected pipeline and the microfluidic chip;

[0018] (3) Deprotection step:

[0019] 1) Deprotection preparation:

[0020] Control the multi-channel injection pump and the multi-channel switching valve to inject the deprotection liquid storage bottle into the buffer bottle;

[0021] 2) Deprotection:

[0022] Control the first two-position three-way solenoid valve, the reciprocating injection pump, and the second two-position three-way solenoid valve to inject the deprotection liquid in the buffer bottle into the microfluidic chip, and set the system reciprocating time and speed parameters. The reciprocating injection pump performs a reciprocating displacement so that the liquid passes through the microfluidic chip back and forth;

[0023] When the set reciprocating time ends, control the second two-position three-way solenoid valve and the reciprocating injection pump to discharge the reagent in the microfluidic chip into the waste liquid bottle;

[0024] (4) Coupling step:

[0025] 1) Coupling preparation:

[0026] Complete the washing according to the (2) washing step; then control the multi-channel injection pump and the multi-channel switching valve to inject the corresponding amino acid activation solution into the buffer bottle;

[0027] 2) Coupling:

[0028] Control the first two-position three-way solenoid valve, the reciprocating injection pump, and the second two-position three-way solenoid valve to inject the amino acid activation solution in the buffer bottle into the microfluidic chip, and set the system reciprocating time and speed parameters. The reciprocating injection pump performs a reciprocating displacement so that the liquid passes through the microfluidic chip back and forth;

[0029] When the set reciprocating time ends, control the second two-position three-way solenoid valve and the reciprocating injection pump to discharge the reagent in the microfluidic chip into the waste liquid bottle;

[0030] Thus, the coupling of one amino acid is completed. Subsequently, repeat steps (1)-(3) to sequentially connect the other amino acids of the polypeptide to be synthesized.

[0031] As a further improvement, a method for synchronous synthesis of multiple polypeptides specifically includes the following steps:

[0032] (1) Preliminary preparation:

[0033] Load the configured N,N-dimethylformamide (DMF), deprotection solution, and each amino acid activation solution into the DMF storage bottle, deprotection solution storage bottle, and each amino acid storage bottle; swell the resin with Fmoc protection group with dichloromethane (DCM), and then configure it with DMF into a suspension and suck it into the second retention sand core by a reciprocating injection pump. The second two-position three-way solenoid valve switches the passage to the air end, the reciprocating injection pump sucks a certain amount of air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, and the piston of the reciprocating injection pump advances, the resin is retained by the sand core, and the reagent is discharged to the waste liquid bottle.

[0034] (2) Washing step:

[0035] 1) The multi-channel injection pump switches the channel to suck DMF from the DMF storage bottle, then the multi-channel injection pump switches the channel to be connected to the multi-channel switching valve, the multi-channel switching valve switches the channel to the buffer bottle, and the multi-channel injection pump injects DMF into the buffer bottle; the multi-channel injection pump switches the channel to the air end to suck air, and then switches to be connected to the multi-channel switching valve, and discharges the remaining solvent in the pipeline to the buffer bottle through the air.

[0036] 2) The first two-position three-way solenoid valve switches to the buffer bottle end, the reciprocating injection pump completely sucks the liquid in the buffer bottle and forms a solid-liquid mixture with the resin to enter the retention sand core; the second two-position three-way solenoid valve switches the passage to the air end, the piston of the reciprocating injection pump advances to the reciprocating stroke, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, the system sets the reciprocating time and speed parameters, and the reciprocating injection pump performs reciprocating displacement, so that the liquid carrying the resin passes back and forth through the microfluidic chip.

[0037] 3) When the set reciprocating time ends, the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating injection pump sucks air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, and the piston of the reciprocating injection pump advances to discharge the reagent to the waste liquid bottle.

[0038] (3) Deprotection step:

[0039] 1) Deprotection preparation:

[0040] The multi-channel injection pump switches the channel to suck the deprotection solution from the deprotection solution storage bottle, then the multi-channel injection pump switches the channel to be connected to the multi-channel switching valve, the multi-channel switching valve switches the channel to the buffer bottle, and the multi-channel injection pump injects the deprotection solution into the buffer bottle; the multi-channel injection pump switches the channel to the air end to suck air, and then switches to be connected to the multi-channel switching valve, and discharges the remaining solvent in the pipeline to the buffer bottle through the air.

[0041] 2) Deprotection:

[0042] The first two-position three-way solenoid valve is switched to the buffer bottle end, and the reciprocating syringe pump completely aspirates the liquid in the buffer bottle; the second two-position three-way solenoid valve switches the passage to the air end, the piston of the reciprocating syringe pump advances to the reciprocating stroke, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, the system sets the reciprocating time and speed parameters, and the reciprocating syringe pump performs reciprocating displacement, so that the liquid carrying the resin passes back and forth through the microfluidic chip.

[0043] When the set reciprocating time ends, the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating syringe pump aspirates a certain amount of air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, and the piston of the reciprocating syringe pump advances to discharge the reagent to the waste liquid bottle.

[0044] (4) Coupling step:

[0045] 1) Coupling preparation:

[0046] After completing the washing according to the washing step in (2), the multi-channel syringe pump switches the channel to aspirate the corresponding amino acid activation solution from the amino acid solution storage bottle, and then the multi-channel syringe pump switches the channel to be connected to the multi-channel switching valve, the multi-channel switching valve switches the channel to the buffer bottle, and the multi-channel syringe pump injects the amino acid activation solution into the buffer bottle; the multi-channel syringe pump switches the channel to the air end to aspirate air, and then switches to be connected to the multi-channel switching valve to discharge the remaining amino acid activation solution in the pipeline to the buffer bottle through the air.

[0047] 2) Coupling:

[0048] The first two-position three-way solenoid valve is switched to the buffer bottle end, and the reciprocating syringe pump completely aspirates the amino acid activation solution in the buffer bottle; the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating syringe pump advances to the reciprocating stroke, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, the system sets the reciprocating time and speed parameters, and the reciprocating syringe pump performs reciprocating displacement, and the resin solid-liquid mixture passes back and forth through the microfluidic chip;

[0049] When the set reciprocating time ends, the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating syringe pump aspirates air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, and the piston of the reciprocating syringe pump advances to discharge the reagent to the waste liquid bottle;

[0050] At this point, the coupling of one amino acid is completed, and the subsequent steps (1)-(3) are repeated to sequentially connect the other amino acids of the polypeptide to be synthesized.

[0051] As a further improvement, before the deprotection preparation, the following steps are also included: the multi-channel injection pump switches channels to suck a certain amount of deprotection solution from the deprotection solution storage bottle, then the multi-channel injection pump switches channels to be connected to the multi-channel switching valve, the multi-channel switching valve switches channels to the waste liquid bottle, the multi-channel injection pump discharges the deprotection solution, then the multi-channel injection pump switches channels to the air port, sucks a certain amount of air and then switches to be connected to the multi-channel switching valve, and uses the air to discharge the remaining deprotection solution in the pipeline to complete the flushing of the pump chamber of the multi-channel injection pump with the deprotection solution.

[0052] As a further improvement, the washing step is repeated 2 - 3 times.

[0053] As a further improvement, the multi-channel switching valve is connected to a plurality of reciprocating reaction control modules composed of a buffer bottle, a two-way three-way solenoid valve, a retention sand core, a microfluidic chip and a reciprocating injection pump to complete the coupling steps of the same or different polypeptides.

[0054] As a further improvement, the different reciprocating reaction control modules synthesize polypeptides synchronously.

[0055] The speed of the reciprocating injection pump is 200 - 400 ul / s. If the flow rate is too small, the liquid cannot achieve the solid-liquid circulation. If the flow rate is too large, the pressure will increase, which may cause the bursting of the pipeline interface.

[0056] During the deprotection and coupling, the reciprocating stroke of the reciprocating injection pump is 5 ml. The reciprocating stroke is determined according to the liquid volume. Generally, on the basis of the injected reagent volume of xml, +2 ml is added. If the injection displacement is too small, the solid and liquid cannot fully circulate in the microfluidic chip channel, resulting in the microfluidic chip not reaching the usage effect. If the stroke is too large, unnecessary waiting time will be increased.

[0057] The length of the pipeline connecting the first two-way three-way solenoid valve to the waste liquid bottle is 2 - 3 times the reciprocating stroke of the reciprocating injection pump.

[0058] The beneficial effects of the present invention are as follows: Different from the mechanical stirring or bubbling of traditional reactors, the present invention adopts a reciprocating synthesis method to realize the reciprocating contact of extremely small amounts of reagents with the resin, achieving full utilization. And it can allow the addition of reciprocating reaction control modules to increase the reaction channels, and realize the simultaneous multi-channel reciprocating synthesis without mutual influence, and carry out the synthesis of trace peptides at the same time.

[0059] The method and its device feature a green chemical synthesis method: Based on the microfluidic microfluidic chip using a reciprocating synthesis process, it can complete polypeptide synthesis under the condition of using a very small amount of reagents, saving resources and protecting the environment; the resin dosage can be reduced to 20 mg, and for the connection of a single amino acid, the DMF usage is only 12 ml - 18 ml. The multi-channel synchronous reciprocating synthesis saves time: The reciprocating synthesis process design in this technology allows several channels to be synthesized simultaneously because each channel does not interfere with each other. By simply stacking the devices, the reaction modules can be increased. Shorten the synthesis time: Utilizing the advantages of the microfluidic chip, it can greatly enhance the mixing effect within a small space. The time consumed for the connection of one amino acid can be shortened from the traditional 40 minutes to 5 minutes, a time reduction of 87.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0061] Figure 1 FIG. is a schematic structural diagram of a multi-channel synthesis device for synchronous reciprocating synthesis of multiple polypeptides provided by an embodiment of the present invention.

[0062] Figure 2 FIG. is a schematic structural diagram of a microfluidic chip of a multi-channel synthesis device for synchronous reciprocating synthesis of multiple polypeptides provided by an embodiment of the present invention.

[0063] Figure 3 FIG. is a mass spectrometry diagram of a polypeptide obtained by a method for synchronous reciprocating synthesis of multiple polypeptides provided by Embodiment 1 of the present invention.

[0064] Figure 4 FIG. is a high performance liquid chromatography diagram of a polypeptide obtained by a method for synchronous reciprocating synthesis of multiple polypeptides provided by Embodiment 1 of the present invention.

[0065] DESCRIPTION OF THE REFERENCE NUMERALS:

[0066] 1 - multi-channel injection pump; 2 - multi-channel switching valve; 3 - waste liquid bottle; 4 - DMF storage bottle; 5 - deprotection liquid storage bottle; 6, 7, 8, 9 - amino acid storage bottles; 10, 17 - buffer bottles; 11, 18 - first two-position three-way solenoid valves; 12, 19 - second two-position three-way solenoid valves; 13, 21 - first retention sand core; 14, 22 - second retention sand core; 15, 23 - microfluidic chips; 151 - inlet and outlet; 152 - internal channels; 1521 - circulation channels; 1522 - direct current channels; 16, 20 - reciprocating injection pumps. Detailed implementation mode

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the present invention to be protected, but merely represents the selected embodiments of the present invention.

[0068] In the description of the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality" means two or more, unless otherwise specifically defined.

[0069] Continuous flow chemistry refers to carrying out chemical reactions in a continuously flowing fluid, also known as a continuous process. The specific operation is to use a peristaltic pump to inject two or more materials into a tubular, packed bed, or microchannel microfluidic chip through a mixing device at an appropriate flow rate to participate in the reaction, and then collect the reaction solution at the end outlet or enter the next process. However, continuously introducing the feed liquid will cause the raw material liquid to be discharged as waste liquid before complete reaction, resulting in waste of raw materials.

[0070] Microfluidic technology is a technology for processing and manipulating fluids at the micron scale. Microfluidic chips have a high specific surface area and good mass transfer performance, which can significantly improve the reaction rate and efficiency. Applying microfluidic technology to polypeptide synthesis can significantly improve the synthesis efficiency, reduce the consumption of reagents and solvents, and achieve automated synthesis. Microfluidic chips achieve the synthesis of polypeptides by manipulating fluids in channels at the micron scale. The existing polypeptide synthesis mainly uses rotary shaking or vibration stirring, which requires the participation of a large amount of raw materials and is not conducive to the requirements of trace amounts and rapidity for new drug development.

[0071] In response to this, the present invention aims to develop a multi-channel polypeptide automated reciprocating synthesis method and device for new drug development. Through microfluidic technology, the raw materials are reduced from the gram level in traditional research and development to the milligram level. Through reciprocating synthesis, the amount of raw materials used is greatly reduced, the synthesis efficiency is greatly improved, and several different peptides are allowed to complete their respective reactions synchronously through reciprocation, achieving the microscale synthesis of different polypeptides in a short time.

[0072] Refer to Figure 1 、 2As shown in the figure, a multi-channel synthesis device for synchronous synthesis of multiple polypeptides includes a multi-channel injection pump 1, a multi-channel switching valve 2, a liquid storage bottle, a waste liquid bottle 3, and a reciprocating reaction control module. The liquid storage bottle is provided with a DMF liquid storage bottle 4, a deprotection liquid storage bottle 5, and several amino acid liquid storage bottles (6, 7, 8, 9), which are respectively communicated with the feed inlet of the multi-channel injection pump 1. The discharge port of the multi-channel injection pump 1 is communicated with the liquid inlet of the multi-channel switching valve 2. There are at least 2 reciprocating reaction control modules. The waste liquid bottle 3 and the reciprocating reaction control module are respectively communicated with the liquid outlet of the multi-channel switching valve 2, and the reciprocating reaction control module is communicated with the waste liquid bottle 3.

[0073] Through modularization, the construction cost of the multi-channel synthesis device is reduced as much as possible to achieve low-cost multi-channel synthesis.

[0074] By using the multi-channel switching valve 2, a cleaning port is reserved to discharge the cleaning waste liquid. While the reaction module is reciprocating, with the addition of the multi-channel switching valve 2, the pump chamber can be effectively cleaned and the rinsing effect can be achieved, promoting a more effective reaction in the experiment.

[0075] Since different polypeptides use different amino acids, the liquid storage bottle functions to store different amino acids and reagents. At the same time, the system device combines a liquid distribution injection pump and a multi-channel switching valve 2 to inject the required amino acids into the buffer bottles 10 corresponding to the respective channels. Configuring buffer bottles 10 for different channels can avoid the correlation of pipeline air pressure between the liquid distribution injection pump and the reciprocating injection pump 16 (without a buffer bottle, the two pumps will form a passage through the multi-channel switching valve 2, and when injecting liquid, the rate differences of the two pumps need to be extra noted, otherwise it will cause a pressure difference and damage the pipeline). The system is more simple and has low requirements.

[0076] The microfluidic chip 15 is provided with an inlet and outlet 151 and an internal channel 152. The inlet and outlet 151 are arranged at both ends of the internal channel 152. The internal channel 152 is provided with a circulation channel 1521 and a direct current channel 1522. The circulation channel 1521 and the direct current channel 1522 are connected alternately, and the direct current channel 1522 is relatively arranged on the circulation channel 1521.

[0077] The resin and the reaction solution are continuously split and recombined through the internal channel 152 to enhance the mixing effect.

[0078] Further, the reciprocating reaction control module is provided with a buffer bottle 10, a first two-position three-way solenoid valve 11, a retention sand core, a microfluidic chip 15 and a reciprocating syringe pump 16. The retention sand core is provided with a first retention sand core 13 and a second retention sand core 14. The buffer bottle 10, the first two-position three-way solenoid valve 11, the first retention sand core 13, the microfluidic chip 15, the second retention sand core 14 and the reciprocating syringe pump 16 are connected in sequence. The upper end of the buffer bottle 10 is connected to the waste liquid bottle 3. The lower end of the buffer bottle 10 is a pointed end, and the bottom of the pointed end is connected to the first two-position three-way solenoid valve 11 through a pipeline.

[0079] In addition to the internal channels, the microfluidic chip 15 is provided with two connection ports, which are used as the inlet and outlet respectively, and are connected to the first retention sand core and the second retention sand core respectively.

[0080] The reciprocating reaction control module of another channel is provided with a buffer bottle 17, a first two-position three-way solenoid valve 18, a first retention sand core 21, a microfluidic chip 23, a second retention sand core 22 and a reciprocating syringe pump 20.

[0081] During the reciprocating process, both ends of the microfluidic chip 15 are respectively connected to the waste liquid bottle 3 and the reciprocating syringe pump 16. In order to prevent the liquid from entering the pump chamber of the reciprocating syringe pump 16, a sand core filter ball with a certain volume is customized, and the solid-liquid mixture is stored in the filter ball.

[0082] Through the first two-position three-way solenoid valve 11, the reaction pipeline connected to the microfluidic chip 15 is divided into two. After the reciprocating syringe pump 16 sucks the reagent in the buffer bottle 10, it is switched to the waste liquid path. At this time, the liquid of the reciprocating reaction no longer flows back into the buffer bottle 10 repeatedly, and the buffer bottle 10 is in an idle state and can be used for re-distribution of the liquid.

[0083] The buffer bottle 10 is designed with a pointed end at the lower end, and the pipeline extends to the bottom end, ensuring that a small amount of reaction liquid can be completely pumped by the reciprocating pump; the pipeline for injecting into the buffer bottle 10 is designed to be closely attached to the middle and lower part of the inner wall of the bottle (if it extends to the bottom end, if there are bubbles during injection, the bursting of the bubbles will cause liquid splashing, and if it is placed at the bottle mouth, the falling of the liquid flow will also cause liquid splashing).

[0084] Further, a second two-position three-way solenoid valve 12 is provided at the end of the second retention sand core 14 and is connected to the reciprocating syringe pump 16.

[0085] By adding a second two-position three-way switching valve, connecting it to the reciprocating syringe pump 16 and the air end, the displacement inside the reciprocating syringe pump 16 is additionally amplified to realize the discharge of the reaction liquid.

[0086] Further, the pipe opening of the pipe connecting the buffer bottle 10 and the waste liquid bottle 3 is located in the middle and lower part of the buffer bottle 10 and is arranged against the wall. The lower end of the buffer bottle 10 is a pointed mouth, and the pipe connecting the buffer bottle 10 and the first two-way three-way solenoid valve 11 extends into the bottom of the pointed mouth of the buffer bottle 10.

[0087] Further, the first intercepting sand core 13 is a filter rod, and the second intercepting sand core 14 is a filter ball.

[0088] An intercepting sand core filter is adopted for solid-liquid separation. By customizing the sand core filter ball and filter rod, the resin in the reciprocating process is intercepted in front of the pump chamber, realizing the persistent use of high-precision equipment. One end is a filter ball, and the filtration at the other side (the waste liquid bottle 3 end) is a filter rod. Compared with the filter ball, it is easier to completely discharge the liquid and reduce the residue of the reaction liquid.

[0089] Further, the multi-channel injection pump 1 is provided with an air inlet.

[0090] Since the liquid distribution injection pump injects the corresponding reaction liquid into the corresponding buffer bottle 10 through the multi-channel switching valve 2, there is still residual reaction liquid in the pipeline between the liquid distribution injection pump and the multi-channel switching valve 2. The reserved air port of the liquid distribution injection pump is used to suck air and inject the residual reaction liquid into the buffer bottle 10.

[0091] In a second aspect, the present application provides a method for synchronous synthesis of multiple polypeptides. Each synthesis channel includes the following steps:

[0092] (1) Preliminary preparation:

[0093] The prepared N,N-dimethylformamide (DMF), deprotection solution, and each amino acid activation solution are filled into the DMF storage bottle 4, deprotection solution storage bottle 5, and each amino acid storage bottle (6, 7, 8, 9); and the resin with Fmoc protecting group is swollen with dichloromethane (DCM), and then configured into a suspension with DMF and sucked into the microfluidic chip 15 by the reciprocating injection pump 16.

[0094] (2) Washing step:

[0095] Control the multi-channel injection pump 1, multi-channel switching valve 2, first two-way three-way solenoid valve 11, second two-way three-way solenoid valve 12, and reciprocating injection pump 16, and cooperate with the DMF storage bottle 4, buffer bottle 10, and waste liquid bottle 3 to realize the washing of the connected pipeline and the microfluidic chip 15.

[0096] (3) Deprotection step:

[0097] 1) Deprotection preparation:

[0098] Control the multi-channel injection pump 1 and multi-channel switching valve 2, and inject the deprotection solution storage bottle 5 into the buffer bottle 10.

[0099] 2) Deprotection:

[0100] Control the first two-position three-way solenoid valve 11, the reciprocating injection pump 16, and the second two-position three-way solenoid valve 12 to inject the deprotection solution in the buffer bottle 10 into the microfluidic chip 15, and set the system reciprocating time and speed parameters. The reciprocating injection pump 16 performs reciprocating displacement so that the liquid passes back and forth through the microfluidic chip 15;

[0101] When the set reciprocating time ends, control the second two-position three-way solenoid valve 12 and the reciprocating injection pump 16 to discharge the reagent in the microfluidic chip 15 into the waste liquid bottle 3;

[0102] (4) Coupling step:

[0103] 1) Coupling preparation:

[0104] Complete the washing according to the washing step in (2); then control the multi-channel injection pump 1 and the multi-channel switching valve 2 to inject the corresponding amino acid activation solution into the buffer bottle 10;

[0105] 2) Coupling:

[0106] Control the first two-position three-way solenoid valve 11, the reciprocating injection pump 16, and the second two-position three-way solenoid valve 12 to inject the amino acid activation solution in the buffer bottle 10 into the microfluidic chip 15, and set the system reciprocating time and speed parameters. The reciprocating injection pump 16 performs reciprocating displacement so that the liquid passes back and forth through the microfluidic chip 15;

[0107] When the set reciprocating time ends, control the second two-position three-way solenoid valve 12 and the reciprocating injection pump 16 to discharge the reagent in the microfluidic chip 15 into the waste liquid bottle 3;

[0108] Thus, the coupling of one amino acid is completed. Subsequently, repeat steps (1)-(3) to sequentially connect the other amino acids of the polypeptide to be synthesized.

[0109] A method for synchronous synthesis of multiple polypeptides by reciprocation, and each synthesis channel specifically includes the following steps:

[0110] (1) Preliminary preparation:

[0111] Load the configured N,N-dimethylformamide (DMF), deprotection solution, and each amino acid activation solution into the DMF storage bottle 4, deprotection solution storage bottle 5, and each amino acid storage bottle (6, 7, 8, 9); and swell the resin with Fmoc protection group with dichloromethane (DCM), and then configure it with DMF into a suspension and suck it into the retention sand core by the reciprocating injection pump 16. The second two-way three-way solenoid valve 12 switches the passage to the air end, the reciprocating injection pump 16 sucks a certain amount of air, the second two-way three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, the first two-way three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end, the piston of the reciprocating injection pump 16 advances, and the resin is retained by the first retention sand core 13, and the reagent is discharged to the waste liquid bottle 3.

[0112] (2) Washing step:

[0113] 1) The multi-channel injection pump 1 switches the channel to suck DMF from the DMF storage bottle 4, then the multi-channel injection pump 1 switches the channel to be connected to the multi-channel switching valve 2, and the multi-channel switching valve 2 switches the channel to the buffer bottle 10, and the multi-channel injection pump 1 injects DMF into the buffer bottle 10; the multi-channel injection pump 1 switches the channel to the air end to suck air, and then switches to be connected to the multi-channel switching valve 2 to discharge the remaining solvent in the air pipeline to the buffer bottle 10;

[0114] 2) The first two-way three-way solenoid valve 11 switches to the buffer bottle 10 end, and the reciprocating injection pump 16 completely sucks the liquid in the buffer bottle 10; the second two-way three-way solenoid valve 12 switches the passage to the air end, the piston of the reciprocating injection pump 16 advances 1 / 2 stroke, the second two-way three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, the first two-way three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end, the system sets the reciprocating time and speed parameters, and the reciprocating injection pump 16 makes a reciprocating displacement to carry the resin solid-liquid mixture back and forth through the microfluidic chip 15;

[0115] 3) When the set reciprocating time ends, the second two-way three-way solenoid valve 12 switches the passage to the air end, the reciprocating injection pump 16 sucks air, the second two-way three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the piston of the reciprocating injection pump 16 advances to discharge the reagent to the waste liquid bottle 3;

[0116] (3) Deprotection step:

[0117] 1) Deprotection preparation:

[0118] The multi-channel injection pump 1 switches channels to suck the deprotection solution from the deprotection solution storage bottle 5, and then the multi-channel injection pump 1 switches channels to be connected to the multi-channel switching valve 2. The multi-channel switching valve 2 switches channels to the buffer bottle 10, and the multi-channel injection pump 1 injects the deprotection solution into the buffer bottle 10; the multi-channel injection pump 1 switches channels to the air end to suck air, and then switches to be connected to the multi-channel switching valve 2 to discharge the remaining solvent in the pipeline through the air to the buffer bottle 10;

[0119] 2) Deprotection:

[0120] The first two-position three-way solenoid valve 11 switches to the buffer bottle 10 end, and the reciprocating injection pump 16 completely sucks the liquid in the buffer bottle 10; the second two-position three-way solenoid valve 12 switches the passage to the air end, the piston of the reciprocating injection pump 16 advances 1 / 2 stroke, the second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, the first two-position three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end, the system sets the reciprocating time and speed parameters, and the reciprocating injection pump 16 makes a reciprocating displacement, carrying the resin solid-liquid mixture back and forth through the microfluidic chip 15.

[0121] When the set reciprocating time ends, the second two-position three-way solenoid valve 12 switches the passage to the air end, the reciprocating injection pump 16 sucks a certain amount of air, the second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the piston of the reciprocating injection pump 16 advances to discharge the reagent to the waste liquid bottle 3.

[0122] (4) Coupling step:

[0123] 1) Coupling preparation:

[0124] After the washing is completed according to the (2) washing step, the multi-channel injection pump 1 switches channels to suck the corresponding amino acid activation solution from the amino acid solution storage bottle, and then the multi-channel injection pump 1 switches channels to be connected to the multi-channel switching valve 2. The multi-channel switching valve 2 switches channels to the buffer bottle 10, and the multi-channel injection pump 1 injects the amino acid activation solution into the buffer bottle 10; the multi-channel injection pump 1 switches channels to the air end to suck air, and then switches to be connected to the multi-channel switching valve 2 to discharge the remaining amino acid activation solution in the pipeline through the air to the buffer bottle 10;

[0125] 2) Coupling:

[0126] The first two-position three-way solenoid valve 11 switches to the buffer bottle 10 end, and the reciprocating injection pump 16 completely sucks the amino acid activation solution in the buffer bottle 10; the second two-position three-way solenoid valve 12 switches the passage to the air end, the reciprocating injection pump 16 advances 1 / 2 stroke, the second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, the first two-position three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end, the system sets the reciprocating time and speed parameters, and the reciprocating injection pump 16 makes a reciprocating displacement, carrying the resin solid-liquid mixture back and forth through the microfluidic chip 15;

[0127] When the reciprocating time to be set ends, the second two-position three-way solenoid valve 12 switches the passage to the air end, the reciprocating syringe pump 16 sucks air, the second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the reciprocating syringe pump 16 advances to discharge the reagent into the waste liquid bottle 3;

[0128] Thus, the coupling of one amino acid is completed, and the subsequent steps (1)-(3) are repeated to sequentially connect the other amino acids of the polypeptide to be synthesized.

[0129] Further, before the deprotection preparation, it also includes: the multi-channel syringe pump 1 switches the channel to suck a certain amount of deprotection solution from the deprotection solution storage bottle 5, then the multi-channel syringe pump 1 switches the channel to be connected to the multi-channel switching valve 2, the multi-channel switching valve 2 switches the channel to the waste liquid bottle 3, the multi-channel syringe pump 1 discharges the deprotection solution, then the multi-channel syringe pump 1 switches the channel to the air port, sucks a certain amount of air and then switches to be connected to the multi-channel switching valve 2, and uses the air to discharge the remaining deprotection solution in the pipeline to complete the rinsing of the pump chamber of the multi-channel syringe pump 1 with the deprotection solution.

[0130] Further, the washing step is repeated 2-3 times.

[0131] Further, the multi-channel switching valve 2 is connected to a plurality of reciprocating reaction control modules composed of a buffer bottle 10, a two-position three-way solenoid valve, a retention sand core, a microfluidic chip 15 and a reciprocating syringe pump 16 to complete the coupling steps of the same or different polypeptides.

[0132] Further, different reciprocating reaction control modules perform the synthesis of polypeptides synchronously.

[0133] Further, the speed of the reciprocating syringe pump 16 is 200 ul / s.

[0134] Further, during the deprotection and coupling, the reciprocating stroke of the reciprocating syringe pump 16 is 5 ml.

[0135] Further, the length of the pipeline connecting the first two-position three-way solenoid valve 11 and the waste liquid bottle 3 is 2-3 times the reciprocating stroke of the reciprocating syringe pump 16.

[0136] By lengthening the pipeline and specifying the reciprocating displacement distance, the reaction solution is prevented from flowing into the waste liquid bottle 3. At the same time, to prevent the waste liquid in the waste liquid bottle 3 from entering the microfluidic chip 15 and affecting the reaction, the pipeline is only connected to the port of the waste liquid bottle 3 and does not extend into the bottom of the waste liquid bottle 3.

[0137] Example

[0138] Hereinafter, embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application. For those technologies or conditions not specified in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0139] Example 1:

[0140] Synthesis of leucine enkephalin, with the amino acid sequence: H-Tyr-Gly-Gly-Phe-Leu-OH

[0141] The prepared N,N-dimethylformamide (DMF), deprotection solution, tyrosine activation solution (Phe), glycine activation solution (Gly), and phenylalanine activation solution (Tyr) were respectively filled into the DMF storage bottle 4, deprotection solution storage bottle 5, and the corresponding amino acid storage bottles 6, 7, 8, and 9.

[0142] The resin raw material is leucine Wang resin with Fmoc protecting group (Fmoc-Leu-Wang).

[0143] First, the Fmoc-Leu-Wang resin was swollen with dichloromethane (DCM), and then configured into a suspension with DMF and sucked into the second retention sand core by the reciprocating injection pump 16. The second two-position three-way solenoid valve 12 switched the passage to the air end, the reciprocating injection pump 16 sucked 25 ml of air, set the speed to 400 μl / s, the second two-position three-way solenoid valve 12 switched the passage to the microfluidic chip 15 end, the first two-position three-way solenoid valve 11 switched the passage to the waste liquid bottle end, and the piston of the reciprocating injection pump 16 was advanced from 25 ml to 0 ml. The resin was retained by the first retention sand core 13, and the reagent was discharged to the waste liquid bottle 3.

[0144] (1) Washing step:

[0145] The high-precision multi-channel injection pump 1 switched the channel to suck 3 ml of DMF from the DMF storage bottle 4, then the high-precision multi-channel injection pump 1 switched the channel to be connected to the multi-channel switching valve 2, and the multi-channel switching valve 2 switched the channel to the buffer bottle 10. The high-precision multi-channel injection pump 1 injected the DMF into the buffer bottle 10; the high-precision multi-channel injection pump 1 switched the channel to the air end to suck 1 ml of air, and then switched to be connected to the multi-channel switching valve 2 to discharge the remaining solvent in the pipeline through the air to the buffer bottle 10.

[0146] The first two-position three-way solenoid valve 11 is switched to the buffer bottle 10 end, and the piston position of the reciprocating syringe pump 16 is displaced from 0 ml to 10 ml, completely sucking the liquid in the buffer bottle 10. The second two-position three-way solenoid valve 12 switches the passage to the air end, and the piston position of the reciprocating syringe pump 16 is displaced from 10 ml to 5 ml (depending on the set parameters, the set parameter is reciprocating 5 ml). The second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the first two-position three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end. The system sets the reciprocating time to 1 min, and the reciprocating syringe pump 16 sets the speed parameter to 200 ul / s. The reciprocating syringe pump 16 repeats the displacement from 5 ml - 0 ml and 0 ml - 5 ml.

[0147] When the set reciprocating time ends, the second two-position three-way solenoid valve 12 switches the passage to the air end, and the reciprocating syringe pump 16 sucks air and is displaced to 25 ml. The second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the piston of the reciprocating syringe pump 16 is displaced from 25 ml to 0 ml, discharging the reagent to the waste liquid bottle 3.

[0148] Due to the need for multiple washings, the above (1) washing steps are repeated 2 - 3 times.

[0149] (2) Preparation for deprotection:

[0150] When the last washing is in progress, the high-precision multi-channel syringe pump 1 cavity is pre-rinsed with the deprotection solution in advance. The high-precision multi-channel syringe pump 1 switches the channel to suck 3 ml of the deprotection solution from the deprotection solution storage bottle 5, and then the high-precision multi-channel syringe pump 1 switches the channel to be connected to the multi-channel switching valve 2. The multi-channel switching valve 2 switches the channel to the waste liquid bottle 3. The high-precision multi-channel syringe pump 1 is displaced from 3 ml to 0 ml to discharge the deprotection solution. Then the high-precision multi-channel syringe pump 1 switches the channel to the air port, sucks a certain amount of air and then switches to be connected to the multi-channel switching valve 2, and uses the air to discharge the remaining deprotection solution in the pipeline.

[0151] The high-precision multi-channel syringe pump 1 switches the channel to suck 3 ml of the deprotection solution from the deprotection solution storage bottle 5, and then the high-precision multi-channel syringe pump 1 switches the channel to be connected to the multi-channel switching valve 2. The multi-channel switching valve 2 switches the channel to the buffer bottle 10. The high-precision multi-channel syringe pump 1 injects the deprotection solution into the buffer bottle 10; the high-precision multi-channel syringe pump 1 switches the channel to the air end to suck 1 ml of air, and then switches to be connected to the multi-channel switching valve 2, and discharges the remaining solvent in the pipeline to the buffer bottle 10 through the air.

[0152] (3) Deprotection step:

[0153] The first two-position three-way solenoid valve 11 is switched to the buffer bottle 10 end, and the piston position of the reciprocating syringe pump 16 is displaced from 0 ml to 10 ml, completely sucking the liquid in the buffer bottle 10. The second two-position three-way solenoid valve 12 switches the passage to the air end, and the piston position of the reciprocating syringe pump 16 is displaced from 10 ml to 5 ml (depending on the set parameters, the set parameter is reciprocating 5 ml). The second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the first two-position three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end. The system sets the reciprocating time to 5 min, the reciprocating syringe pump 16 sets the speed parameter to 200 ul / s, and the reciprocating syringe pump 16 repeats the displacement from 5 ml - 0 ml and 0 ml - 5 ml.

[0154] When the set reciprocating time of 5 min ends, the second two-position three-way solenoid valve 12 switches the passage to the air end, and the reciprocating syringe pump 16 sucks air and is displaced to 25 ml. The second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the piston of the reciprocating syringe pump 16 is displaced from 25 ml to 0 ml, discharging the reagent to the waste liquid bottle 3.

[0155] Repeat the washing step (1) 2 - 3 times.

[0156] (4) Coupling preparation:

[0157] After the last washing is completed, enter the coupling step. The high-precision multi-channel syringe pump 1 switches the channel to suck 3 ml of Phe activation solution from the Phe (tyrosine) activation solution storage bottle 6, and then the high-precision multi-channel syringe pump 1 switches the channel to be connected to the multi-channel switching valve 2. The multi-channel switching valve 2 switches the channel to the buffer bottle 10, and the high-precision multi-channel syringe pump 1 injects the Phe activation solution into the buffer bottle 10; the high-precision multi-channel syringe pump 1 switches the channel to the air end to suck 1 ml of air, and then switches to be connected to the multi-channel switching valve 2, and discharges the remaining Phe activation solution in the pipeline to the buffer bottle 10 through the air.

[0158] (5) Coupling step:

[0159] The first two-position three-way solenoid valve 11 is switched to the buffer bottle 10 end, and the piston position of the reciprocating syringe pump 16 is displaced from 0 ml to 10 ml, completely sucking the Phe amino acid activation solution in the buffer bottle 10. The second two-position three-way solenoid valve 12 switches the passage to the air end, and the piston position of the reciprocating syringe pump 16 is displaced from 10 ml to 5 ml (depending on the set parameters, the set parameter is reciprocating 5 ml). The second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end, and the first two-position three-way solenoid valve 11 switches the passage to the waste liquid bottle 3 end. The system sets the reciprocating time to 5 - 10 min, the reciprocating syringe pump 16 sets the speed parameter to 200 ul / s, and the reciprocating syringe pump 16 repeats the displacement from 5 ml - 0 ml and 0 ml - 5 ml.

[0160] The reciprocating time to be set, 5 minutes, ends. The second two-position three-way solenoid valve 12 switches the passage to the air end. The reciprocating syringe pump 16 sucks air and displaces to 25 ml. The second two-position three-way solenoid valve 12 switches the passage to the microfluidic chip 15 end. The piston of the reciprocating syringe pump 16 displaces from 25 ml to 0 ml, and the reagent is discharged into the waste liquid bottle 3.

[0161] Thus, the coupling of one amino acid is completed. Subsequently, steps 1-5 are repeated to sequentially connect Gly, Gly, and Tyr to complete the synthesis of leucine enkephalin.

[0162] Example 2:

[0163] Synchronous synthesis method of different polypeptides

[0164] Channel 1: Amino acid sequence: H-Tyr-Gly-Gly-Phe-Leu-OH

[0165] Channel 2: Amino acid sequence: H-Ala-Leu-Phe-Ala-Leu-OH

[0166] The prepared N,N-dimethylformamide (DMF), deprotection solution, tyrosine activation solution (Phe), glycine activation solution (Gly), phenylalanine activation solution (Tyr), and alanine (Ala) are respectively filled into the DMF storage bottle 4, deprotection solution storage bottle 5, and the corresponding amino acid storage bottles 6, 7, 8, and 9.

[0167] First, two portions of Fmoc-Leu-Wang resin are respectively swollen with dichloromethane (DCM) and then configured into a suspension with DMF and respectively sucked into the second retention sand core by the reciprocating syringe pumps 16 and 20. The second two-position three-way solenoid valves 12 and 19 switch the passage to the air end. The reciprocating syringe pumps 16 and 20 suck 25 ml of air, and the set speed is 400 ul / s. The second two-position three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 ends. The first two-position three-way solenoid valves 11 and 18 switch the passage to the waste liquid bottle end. The pistons of the reciprocating syringe pumps 16 and 20 are advanced from 25 ml to 0 ml. The resin is retained by the first retention sand cores 13 and 21, and the reagent is discharged into the waste liquid bottle 3.

[0168] (1) Washing step:

[0169] The high-precision multi-channel syringe pump 1 switches channels to aspirate 6 ml of DMF from the DMF reservoir bottle 4. Then, the high-precision multi-channel syringe pump 1 switches channels to be connected to the multi-channel switching valve 2, and the multi-channel switching valve 2 switches channels to the buffer bottle 10. The high-precision multi-channel syringe pump 1 injects 3 ml of DMF into the buffer bottle 10. The high-precision multi-channel syringe pump 1 switches channels to the buffer bottle 17, injects the remaining 3 ml of DMF into the buffer bottle 17, and then switches to be connected to the multi-channel switching valve 2. By injecting air into the pipeline, the remaining solvent is discharged to the buffer bottle 17.

[0170] The first and second two-way three-way solenoid valves 11 and 18 switch to the buffer bottle 10 and 17 ends. The piston positions of the reciprocating syringe pumps 16 and 20 are displaced from 0 ml to 10 ml to completely aspirate the liquid in the buffer bottles 10 and 17. The second two-way three-way solenoid valves 12 and 19 switch the passage to the air end. The piston positions of the reciprocating syringe pumps 16 and 20 are displaced from 10 ml to 5 ml (depending on the set parameters, the set parameter is reciprocating 5 ml). The second two-way three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 ends. The first two-way three-way solenoid valves 11 and 18 switch the passage to the waste liquid bottle 3 end. The system sets the reciprocating time to 1 min, and the set speed parameter of the reciprocating syringe pumps 16 and 20 is 200 ul / s. The reciprocating syringe pumps 16 and 20 displace from 5 ml to 0 ml and 0 ml to 5 ml repeatedly.

[0171] When the set reciprocating time ends, the second two-way three-way solenoid valves 12 and 19 switch the passage to the air end. The reciprocating syringe pumps 16 and 20 aspirate air and displace to 25 ml. The second two-way three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 ends. The pistons of the reciprocating syringe pumps 16 and 20 are displaced from 25 ml to 0 ml to discharge the reagent to the waste liquid bottle 3.

[0172] Due to the need for multiple washings, the above washing steps are repeated 2 - 3 times.

[0173] (2) Deprotection preparation:

[0174] After the last washing, it should be the deprotection step. Therefore, the pump cavity of the high-precision multi-channel syringe pump 1 is pre-rinsed with the deprotection solution in advance. The high-precision multi-channel syringe pump 1 switches channels to aspirate 3 ml of the deprotection solution from the deprotection solution reservoir bottle 5. Then, the high-precision multi-channel syringe pump 1 switches channels to be connected to the multi-channel switching valve 2, and the multi-channel switching valve 2 switches channels to the waste liquid bottle 3. The high-precision multi-channel syringe pump 1 displaces from 3 ml to 0 ml to discharge the deprotection solution. Then, the high-precision multi-channel syringe pump 1 switches channels to the air port, aspirates several ml of air, and then switches to be connected to the multi-channel switching valve 2 to use air to discharge the remaining deprotection solution in the pipeline.

[0175] The high-precision multi-channel injection pump 1 switches channels to draw 6 ml of deprotection solution from the deprotection solution storage bottle 5. Then, the high-precision multi-channel injection pump 1 switches channels to connect to the multi-channel switching valve 2. The multi-channel switching valve 2 switches channels to the buffer bottles 10 and 17. The high-precision multi-channel injection pump 1 injects 3 ml of deprotection solution into the buffer bottles 10 and 17. The multi-channel switching valve 2 switches channels to the buffer bottles 17 and 17. The high-precision multi-channel injection pump 1 injects 3 ml of deprotection solution into the buffer bottles 17 and 17.

[0176] (3) Deprotection step:

[0177] The first and second two-way three-way solenoid valves 11 and 18 are switched to the buffer bottle end. The piston positions of the reciprocating injection pumps 16 and 20 are displaced from 0 ml to 10 ml to completely draw the liquid in the buffer bottle 10. The second two-way three-way solenoid valves 12 and 19 switch the passage to the air end. The piston positions of the reciprocating injection pumps 16 and 20 are displaced from 10 ml to 5 ml (depending on the set parameters, the set parameter is reciprocating 5 ml). The second two-way three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 end. The first two-way three-way solenoid valves 11 and 18 switch the passage to the waste liquid bottle end. The system sets the reciprocating time to 5 min. The set speed parameter of the reciprocating injection pumps 16 and 20 is 200 μl / s. The reciprocating injection pumps 16 and 20 are displaced from 5 ml - 0 ml and 0 ml - 5 ml repeatedly.

[0178] When the set reciprocating time of 5 min ends, the second two-way three-way solenoid valves 12 and 19 switch the passage to the air end. The reciprocating injection pumps 16 and 20 draw air and are displaced to 25 ml. The second two-way three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 end. The pistons of the reciprocating injection pumps 16 and 20 are displaced from 25 ml to 0 ml to discharge the reagent to the waste liquid bottle 3.

[0179] Repeat the (1) washing step 2 - 3 times.

[0180] (4) Coupling preparation:

[0181] After the last washing is completed, it should be the coupling step. The high-precision multi-channel injection pump 1 switches channels to draw 3 ml of Phe activation solution from the tyrosine (Phe) activation solution storage bottle 6. Then, the high-precision multi-channel injection pump 1 switches channels to connect to the multi-channel switching valve 2. The multi-channel switching valve 2 switches channels to the buffer bottle 10. The high-precision multi-channel injection pump 1 injects the Phe activation solution into the buffer bottle 10; the high-precision multi-channel injection pump 1 switches channels to the air end to draw 1 ml of air, and then switches to connect to the multi-channel switching valve 2 to discharge the remaining Phe activation solution in the pipeline to the buffer bottle through the air.

[0182] The high-precision multi-channel syringe pump 1 switches channels to aspirate 5 ml of DMF from the DMF reservoir bottle 4. Then, the high-precision multi-channel syringe pump 1 switches channels to be connected to the multi-channel switching valve 2, and the multi-channel switching valve 2 switches channels to the waste liquid bottle 3. The high-precision multi-channel syringe pump 1 discharges 5 ml of DMF into the waste liquid bottle to clean the residual Phe amino acid activation solution in the pipeline.

[0183] The high-precision multi-channel syringe pump 1 switches channels to aspirate 3 ml of Ala activation solution from the alanine (Ala) activation solution reservoir bottle 8. Then, the high-precision multi-channel syringe pump 1 switches channels to be connected to the multi-channel switching valve 2, and the multi-channel switching valve 2 switches channels to the buffer bottle 17. The high-precision multi-channel syringe pump 1 injects the Ala activation solution into the buffer bottle 17; the high-precision multi-channel syringe pump 1 switches channels to the air end to aspirate 1 ml of air, then switches to be connected to the multi-channel switching valve 2, and discharges the remaining Ala activation solution in the pipeline to the buffer bottle through the air.

[0184] (5) Coupling step:

[0185] The first and second two-way three-way solenoid valves 11 and 18 switch to the buffer bottle end. The piston positions of the reciprocating syringe pumps 16 and 20 are displaced from 0 ml to 10 ml to completely aspirate the amino acid liquids in the buffer bottles 10 and 17 respectively. The second two-way three-way solenoid valves 12 and 19 switch the passage to the air end. The piston positions of the reciprocating syringe pumps 16 and 20 are displaced from 10 ml to 5 ml (depending on the set parameters, the set parameter is reciprocating 5 ml). The second two-way three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 ends. The first two-way three-way solenoid valves 11 and 18 switch the passage to the waste liquid bottle end. The system sets the reciprocating time to 5 - 10 min, and the set speed parameter of the reciprocating syringe pumps 16 and 20 is 200 μl / s. The reciprocating syringe pumps 16 and 20 repeat the displacement from 5 ml - 0 ml and 0 ml - 5 ml.

[0186] When the set reciprocating time of 5 min ends, the second two-way three-way solenoid valves 12 and 19 switch the passage to the air end. The reciprocating syringe pumps 16 and 20 aspirate air and are displaced to 25 ml. The second two-way three-way solenoid valves 12 and 19 switch the passage to the microfluidic chips 15 and 23 ends. The pistons of the reciprocating syringe pumps 16 and 20 are displaced from 25 ml to 0 ml to discharge the reagent into the waste liquid bottle 3.

[0187] At this point, each of the two polypeptides has completed the coupling of one amino acid. Subsequently, repeat the washing, deprotection, washing, and coupling steps of 1 - 5 to sequentially connect (Gly, Gly, Tyr) and (Phe, Leu, Ala) respectively to complete the synthesis of their respective peptide chains.

[0188] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A multi-channel synthesis device for synchronous synthesis of multiple polypeptides, characterized in that, It includes a multi-channel injection pump, a multi-channel switching valve, a liquid storage bottle, a waste liquid bottle and a reciprocating reaction control module. The liquid storage bottle is provided with a DMF liquid storage bottle, a deprotection liquid storage bottle and several amino acid liquid storage bottles which are respectively communicated with the feed inlet of the multi-channel injection pump. The discharge outlet of the multi-channel injection pump is communicated with the liquid inlet of the multi-channel switching valve. There are at least 2 reciprocating reaction control modules. The waste liquid bottle and the reciprocating reaction control module are respectively communicated with the liquid outlet of the multi-channel switching valve, and the reciprocating reaction control module is communicated with the waste liquid bottle.

2. The multi-channel synthesis device according to claim 1, wherein The reciprocating reaction control module is provided with a buffer bottle, a first two-position three-way solenoid valve, a retention sand core, a microfluidic chip and a reciprocating injection pump. The retention sand core is provided with a first retention sand core and a second retention sand core. The buffer bottle, the first two-position three-way solenoid valve, the first retention sand core, the microfluidic chip, the second retention sand core and the reciprocating injection pump are sequentially communicated. The upper end of the buffer bottle is communicated with the waste liquid bottle. The lower end of the buffer bottle is a sharp mouth, and the bottom of the sharp mouth is communicated with the first two-position three-way solenoid valve through a pipeline.

3. The multi-channel synthesis device according to claim 3, characterized in that, The end of the second retention sand core is provided with a second two-position three-way solenoid valve which is communicated with the reciprocating injection pump.

4. The multi-channel synthesis device according to claim 1, characterized in that, The first retention sand core is a filter rod, and the second retention sand core is a filter ball.

5. The multi-channel synthesis device according to claim 1, characterized in that, The multi-channel injection pump is reserved with an air inlet.

6. A method for synchronous solid-phase synthesis of multiple polypeptides. According to the multi-channel synthesis device described in any one of claims 1-5, each synthesis channel includes the following steps: (1) Preliminary preparation: Load the configured N,N-dimethylformamide (DMF), deprotection liquid, and each amino acid activation liquid into the DMF liquid storage bottle, the deprotection liquid storage bottle and each amino acid liquid storage bottle; and swell the resin with Fmoc protection group with dichloromethane (DCM), and then configure it into a suspension with DMF and suck it into the microfluidic chip by a reciprocating injection pump. (2) Washing step: Control the multi-channel injection pump, the multi-channel switching valve, the first two-position three-way solenoid valve, the second two-position three-way solenoid valve and the reciprocating injection pump, and cooperate with the DMF liquid storage bottle, the buffer bottle and the waste liquid bottle to realize the washing of the connected pipeline and the microfluidic chip. (3) Deprotection step: 1) Deprotection preparation: Control the multi-channel injection pump and the multi-channel switching valve to inject the deprotection liquid storage bottle into the buffer bottle. 2) Deprotection: Control the first two-position three-way solenoid valve, the reciprocating injection pump, and the second two-position three-way solenoid valve to inject the deprotection liquid in the buffer bottle into the microfluidic chip, and set the system reciprocating time and speed parameters. The reciprocating injection pump performs reciprocating displacement, so that the liquid passes through the microfluidic chip back and forth. When the set reciprocating time ends, control the second two-position three-way solenoid valve and the reciprocating injection pump to discharge the reagent in the microfluidic chip to the waste liquid bottle. (4) Coupling step: 1) Coupling preparation: Complete the washing according to the (2) washing step. Then control the multi-channel injection pump and the multi-channel switching valve to inject the corresponding amino acid activation liquid into the buffer bottle. 2) Coupling: Control the first two-position three-way solenoid valve, reciprocating injection pump, and second two-position three-way solenoid valve to inject the amino acid activation solution in the buffer bottle into the microfluidic chip, and set the reciprocating time and speed parameters of the system. The reciprocating injection pump performs reciprocating displacement, causing the liquid to pass back and forth through the microfluidic chip; When the set reciprocating time ends, control the second two-position three-way solenoid valve and the reciprocating injection pump to discharge the reagent in the microfluidic chip into the waste liquid bottle; Thus, the coupling of one amino acid is completed. Subsequently, repeat steps (1)-(3) to sequentially connect the other amino acids of the polypeptide to be synthesized.

7. The synthesis method according to claim 6, characterized in that, Specifically, it includes the following steps: (1) Preliminary preparation: Load the prepared N,N-dimethylformamide (DMF), deprotection solution, and each amino acid activation solution into the DMF storage bottle, deprotection solution storage bottle, and each amino acid storage bottle; swell the resin with Fmoc protection group with dichloromethane (DCM), and then configure it into a suspension with DMF and suck it into the retention sand core by the reciprocating injection pump; the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating injection pump sucks a certain amount of air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, and the reciprocating injection pump piston advances, and the resin is intercepted by the sand core, and the reagent is discharged into the waste liquid bottle; (2) Washing steps: 1) The multi-channel injection pump switches the channel to suck DMF from the DMF storage bottle, and then the multi-channel injection pump switches the channel to be connected to the multi-channel switching valve. The multi-channel switching valve switches the channel to the buffer bottle, and the multi-channel injection pump injects DMF into the buffer bottle; the multi-channel injection pump switches the channel to the air end to suck air, and then switches to be connected to the multi-channel switching valve, and discharges the remaining solvent in the pipeline to the buffer bottle through the air; 2) The first two-position three-way solenoid valve switches to the buffer bottle end, and the reciprocating injection pump completely sucks the liquid in the buffer bottle; the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating injection pump piston advances 1 / 2 stroke, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, the system sets the reciprocating time and speed parameters, and the reciprocating injection pump performs reciprocating displacement, causing the liquid to pass back and forth through the microfluidic chip; 3) When the set reciprocating time ends, the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating injection pump sucks air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, and the reciprocating injection pump piston advances to discharge the reagent into the waste liquid bottle; (3) Deprotection steps: 1) Deprotection preparation: The multi-channel injection pump switches the channel to suck the deprotection solution from the deprotection solution storage bottle, and then the multi-channel injection pump switches the channel to be connected to the multi-channel switching valve. The multi-channel switching valve switches the channel to the buffer bottle, and the multi-channel injection pump injects the deprotection solution into the buffer bottle; the multi-channel injection pump switches the channel to the air end to suck air, and then switches to be connected to the multi-channel switching valve, and discharges the remaining solvent in the pipeline to the buffer bottle through the air; 2) Deprotection: The first two-position three-way solenoid valve is switched to the buffer bottle end, and the reciprocating syringe pump completely aspirates the liquid in the buffer bottle; the second two-position three-way solenoid valve switches the passage to the air end, the piston of the reciprocating syringe pump advances by 1 / 2 stroke, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, and the system sets the reciprocating time and speed parameters, and the reciprocating syringe pump performs reciprocating displacement, so that the liquid passes through the microfluidic chip back and forth; When the set reciprocating time ends, the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating syringe pump aspirates a certain amount of air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, and the piston of the reciprocating syringe pump advances to discharge the reagent to the waste liquid bottle. (4) Coupling step: 1) Coupling preparation: After completing the washing according to the washing step in (2), the multi-channel syringe pump switches the channel to aspirate the corresponding amino acid activation solution from the amino acid solution storage bottle, and then the multi-channel syringe pump switches the channel to be connected to the multi-channel switching valve, the multi-channel switching valve switches the channel to the buffer bottle, and the multi-channel syringe pump injects the amino acid activation solution into the buffer bottle; the multi-channel syringe pump switches the channel to the air end to aspirate air, and then switches to be connected to the multi-channel switching valve, and discharges the remaining amino acid activation solution in the pipeline to the buffer bottle through the air; 2) Coupling: The first two-position three-way solenoid valve is switched to the buffer bottle end, and the reciprocating syringe pump completely aspirates the amino acid activation solution in the buffer bottle; the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating syringe pump advances by 1 / 2 stroke, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, the first two-position three-way solenoid valve switches the passage to the waste liquid bottle end, and the system sets the reciprocating time and speed parameters, and the reciprocating syringe pump performs reciprocating displacement, so that the liquid passes through the microfluidic chip back and forth; When the set reciprocating time ends, the second two-position three-way solenoid valve switches the passage to the air end, the reciprocating syringe pump aspirates air, the second two-position three-way solenoid valve switches the passage to the microfluidic chip end, and the reciprocating syringe pump advances to discharge the reagent to the waste liquid bottle; Thus, the coupling of one amino acid is completed, and the subsequent steps (1)-(3) are repeated to sequentially connect the other amino acids of the polypeptide to be synthesized.

8. The synthesis method according to claim 7, characterized in that Before the deprotection preparation, it also includes: the multi-channel syringe pump switches the channel to aspirate a certain amount of deprotection solution from the deprotection solution storage bottle, and then the multi-channel syringe pump switches the channel to be connected to the multi-channel switching valve, the multi-channel switching valve switches the channel to the waste liquid bottle, the multi-channel syringe pump discharges the deprotection solution, and then the multi-channel syringe pump switches the channel to the air port, aspirates a certain amount of air and then switches to be connected to the multi-channel switching valve, and uses the air to discharge the remaining deprotection solution in the pipeline to complete the flushing of the pump cavity of the multi-channel syringe pump by the deprotection solution.

9. The synthesis method according to claim 6 or 7, characterized in that, The speed of the reciprocating syringe pump is 200-400 ul / s; during deprotection and coupling, the reciprocating stroke of the reciprocating syringe pump is 5 ml.

10. The synthesis method according to claim 6 or 7, characterized in that, The length of the pipeline connecting the first two-position three-way solenoid valve to the waste liquid bottle is 2-3 times the reciprocating stroke of the reciprocating syringe pump.

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