Polypeptide synthesis system and method based on digital droplet micro-fluidic chip

Abstract

The invention provides a polypeptide synthesis system and method based on a digital droplet micro-fluidic chip, the polypeptide synthesis system comprises a fluid transport mechanism, the digital droplet micro-fluidic chip and a control mechanism, the digital droplet micro-fluidic chip comprises a first substrate and a second substrate which are oppositely arranged, a liquid drop containing space is formed between the first substrate and the second substrate, and the first substrate and the second substrate form a polypeptide synthesis electrode array composed of a plurality of polypeptide synthesis electrode units. The control mechanism is used for controlling the fluid conveying mechanism to drive the raw material liquid drops to each polypeptide synthesis area according to a planned liquid drop moving path to react with the magnetic bead liquid drops for polypeptide synthesis; and the polypeptide synthesis region is formed by combining one or more of the polypeptide synthesis electrode units. According to the method, polypeptide substances are synthesized in micro-liter-magnitude liquid drops, and due to the fact that the volume of the micro liquid drops is small, the mass transfer speed in the liquid drops is high, and the time needed by each step in the synthesis process is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of solid phase peptide synthesis, and in particular to a peptide synthesis system and method based on a digital droplet microfluidic chip. Background Art

[0002] In 1963, Professor Bruce Merrifield of Rockefeller University in the United States proposed a solid phase synthesis technology, which was a major breakthrough in the field of peptide synthesis and had far-reaching significance for the development of chemistry, medicine, immunology and genetic science. The principle of solid phase synthesis is to use resin or silica gel as the stationary phase, connect amino acids in sequence on the stationary phase to form a peptide chain through amino deprotection and amide bond condensation reaction, and finally release the peptide chain from the stationary phase. At the end of 1969, the world's first peptide synthesizer based on solid phase peptide synthesis was officially launched, marking the entry of chemical peptide synthesis into the era of automated production. The emergence of peptide synthesizers has greatly improved the synthesis efficiency and purity of chemical reactions, and plays a very important role in the development of peptide drugs, protein structure research, antigen preparation and other fields.

[0003] Peptide drugs have the advantages of both small molecules and protein drugs. In recent years, the research on peptide drugs has received more and more attention. Peptide drugs are a new type of drug with a molecular weight between small molecules and protein drugs (molecular weight of about 500~10000). It combines the advantages of both, and has many advantages such as strong specificity, low immunogenicity, good stability, and high safety. Since the advent of the first peptide drug insulin in 1922, nearly 100 peptide drugs have been successfully developed in the world, involving various fields such as diabetes, cancer, osteoporosis, and rare diseases. With the continuous development of new preparation technologies, the number of peptide drugs approved for marketing has continued to increase in recent years, including blockbuster R&D products such as semaglutide and dulaglutide. According to the Insight database, the number of peptide drugs developed and marketed in the world and China in 2018 was 3 / 2, and it will increase to 10 / 7 by 2022, and the number of R&D and marketing has steadily increased. As of June 2023, there are 167 peptide drugs available for disease treatment worldwide, 615 of which are in the clinical trial stage, and the vast majority of the rest are still in the early clinical and preclinical synthesis and research and development stages. There is still a lot of room for development in the field of peptide drugs. Compared with other drugs, the number of peptide drug research and development is still small, accounting for only about 2% of all new drugs. There are many constraints, and the most challenging stage is the large-scale construction and high-throughput screening of preclinical peptide libraries. The construction of a peptide library requires the synthesis of 5,000-10,000 peptide compounds, and then 250 compounds are screened out for preclinical research, which involves a large amount of peptide compound preparation work. Therefore, the scientific research community and the industry hope that the new generation of peptide synthesizers can meet the requirements of fast synthesis speed, large number of channels, high degree of automation, low production cost, safety and reliability.

[0004] The number of channels of peptide synthesizers on the market, such as CSBio, GYROS PROTEIN Technologies (formerly PTI), Applied Biosystems Inc. (ABI), Advanced Automated Peptide Protein Technology (AAPPTec), Biotage, CEM, etc., is generally in the single-digit to double-digit range, which is difficult to meet the needs of high-throughput peptide synthesis to build peptide libraries. Although the microwave peptide synthesizer CEM Liberty has developed strongly in recent years, and the synthesis cycle of each amino acid is only 2.5 minutes, its maximum number of channels is only 24, which still cannot meet the needs of building hundreds of millions of peptide libraries. Therefore, it is urgent to develop a technology that can synthesize peptides in large-scale high-throughput parallel.

[0005] The common technology of existing peptide synthesizers is to continuously add deprotection reagents and activated amino acid monomers in a closed explosion-proof glass or plastic reactor according to the cycle steps of amino deprotection-(cleaning)-amino acid coupling-(cleaning), and use nitrogen, oscillation stirring or microwave technology to carry out deprotection and synthesis reactions to finally synthesize polypeptide chains. Peptide synthesizers based on explosion-proof glass, plastic reactors and micro-pipeline system technology are limited by their space and system complexity, and the number of channels is small, making it difficult to achieve large-scale parallel synthesis of thousands of peptides. This limitation affects the speed of drug development and increases costs. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a peptide synthesis system and method based on a digital droplet microfluidic chip, which is used to solve the problem that the number of channels of existing peptide synthesizers is generally in the single digit to double digit range, which is difficult to meet the needs of high-throughput synthesis of peptides to construct a peptide library.

[0007] To achieve the above-mentioned purpose and other related purposes, the present invention provides a polypeptide synthesis system based on a digital droplet microfluidic chip, comprising a fluid transport mechanism, a digital droplet microfluidic chip and a control mechanism, wherein the digital droplet microfluidic chip comprises a first substrate and a second substrate arranged opposite to each other, a droplet accommodating space is formed between the first substrate and the second substrate, and a polypeptide synthesis electrode array composed of a plurality of polypeptide synthesis electrode units is formed on the first substrate and the second substrate, and the control mechanism is used to control the fluid transport mechanism to drive the raw material droplets to each polypeptide synthesis area according to the planned droplet movement path to react with the magnetic bead droplets for polypeptide synthesis; the polypeptide synthesis area is composed of one or more combinations of the polypeptide synthesis electrode units.

[0008] The polypeptide synthesis system based on the digital droplet microfluidic chip of the present invention can realize the polypeptide synthesis operation on the digital droplet microfluidic chip, and synthesize the polypeptide material in the droplet of the microliter level. Due to the small volume of the micro droplet, the mass transfer speed in the droplet is very fast, and the time required for each step in the synthesis process is shortened. At the same time, the droplet is placed on the digital droplet microfluidic chip, and the droplet can be automatically controlled through the peripheral electronic control system, thereby realizing the full-automatic synthesis of the polypeptide material. More importantly, through the parallel control of a large number of droplets, through any arrangement and combination, high-throughput synthesis of polypeptides (which can also be nucleic acids, proteins, carbohydrates and other biological macromolecules) can be achieved. The scale of the polypeptide synthesis electrode array in the digital droplet microfluidic chip described in the present application can be expanded, and it can break through the limitation of the number of channels, and it is easy to realize the high-throughput parallel synthesis of thousands of polypeptides. Moreover, since the droplet mass transfer speed of the digital droplet microfluidic chip is fast and can be flexibly controlled, the efficiency of polypeptide synthesis is greatly improved. The polypeptide synthesizer based on digital microfluidic technology proposed by the present invention has not been reported yet, and has certain innovation and practical value.

[0009] Preferably, each polypeptide synthesis zone is provided with a heating mechanism, which can flexibly control the reaction temperature according to the synthesis of the target polypeptide, so as to accelerate the synthesis reaction and improve the purity of the polypeptide synthesis.

[0010] More preferably, the heating mechanism can be precisely controlled within a temperature range of 10-90° C. to optimize reaction conditions and increase reaction rate. The heating mechanism is disposed above or below the polypeptide synthesis zone.

[0011] Preferably, each polypeptide synthesis area is formed with a liquid-solid separation area and a mixed liquid area, and a magnetic component and a magnetic control mechanism for controlling the magnetic component to generate a magnetic field are provided in the liquid-solid separation area, and the magnetic component is used to perform solid-liquid separation of the polypeptide synthesis product formed on the surface of the magnetic beads from the waste liquid under the action of the magnetic field.

[0012] In the above technical solution of the present application, the magnetron mechanism is an efficient separation system for magnetic beads and liquid phase. The system can use a fixed-position magnetic part, or a three-axis motion system with X, Y axis plane displacement and Z axis lifting function. The type of magnetic part can be a permanent magnet such as ferrite, nepheline iron boron, aluminum nickel cobalt, samarium cobalt, etc., or an electromagnet.

[0013] Preferably, the fluid transport mechanism includes a transport pipeline, a plurality of raw material storage tanks and waste liquid tanks and a transport electrode. The raw material storage tanks and waste liquid tanks are arranged at the edge of the digital droplet microfluidic chip and are connected to each polypeptide synthesis area through the transport electrode.

[0014] More preferably, the fluid transport mechanism is a multiple parameter adjustment module for transporting raw material solution to a digital droplet microfluidic chip, and its control parameters include flow rate, raw material type, transport sequence, fluid pressure and fluid flow rate.

[0015] Preferably, the first substrate is sequentially composed of a conductive electrode and a first hydrophobic layer, the second base plate is sequentially composed of a control electrode, a dielectric layer and a second hydrophobic layer, and the first hydrophobic layer and the second hydrophobic layer are arranged opposite to each other.

[0016] More preferably, the conductive electrode and the control electrode are made of one of gold, silver, chromium, copper, aluminum and ITO conductive materials; the deposition method of the conductive electrode and the control electrode can be magnetron sputtering, electron beam thermal evaporation, etc., or a printed circuit board.

[0017] More preferably, the dielectric material used for the dielectric layer is a composite film of one or more selected from parylene, aluminum oxide, silicon nitride, polyamide, polyethylene and polytetrafluoroethylene.

[0018] More preferably, the hydrophobic material used for the first hydrophobic layer and the second hydrophobic layer is selected from one or more of CYTOP, PTFE, FEP, ECTE, ETFE, PFA, Teflon, polyolefin, polyamide, polyacrylonitrile, molten paraffin, PDMS, alkoxysilane hybrid material and fluorinated polyethylene.

[0019] Preferably, the control mechanism includes a central processing unit and a control circuit disposed in a control electrode, and the control circuit is communicatively connected with the central processing unit and the fluid transport mechanism; the central processing unit is used to preset a planned droplet movement path according to the customer's polypeptide synthesis requirements, and control the fluid transport mechanism through the control circuit based on the electrowetting principle to drive the raw material droplets to each polypeptide synthesis area according to the planned droplet movement path to react with the magnetic bead droplets for polypeptide synthesis.

[0020] In the above technical solution of the present application, the control circuit can be a passive circuit, such as a chrome plate or an ITO plate, or an active matrix circuit, such as a thin film transistor, a printed circuit board, etc.

[0021] More preferably, the central processing unit may be specifically an intelligent software control system, which may integrate multiple control programs of modules such as droplet path planning, heating mechanism, droplet transport mechanism, and magnetic control mechanism of the digital droplet microfluidic chip into the same operating software, thereby improving the operational convenience and equipment integration of the polypeptide synthesis system.

[0022] The present application also provides a method for peptide synthesis based on a digital droplet microfluidic chip, which uses the above-mentioned peptide synthesis system based on a digital droplet microfluidic chip, including the following steps: respectively loading magnetic bead droplets and raw material liquid into a fluid transport mechanism, using a control mechanism to control the fluid transport mechanism to distribute raw material droplets, and driving the raw material droplets to each peptide synthesis area according to the planned droplet movement path to react with the magnetic bead droplets to perform peptide synthesis.

[0023] The digital droplet microfluidic chip described in the present application can be a passive chromium / copper / silver / ITO electrode plate, an active matrix printed circuit board, and an active matrix thin film transistor. For example, a microfluidic chip based on an active matrix thin film transistor control circuit is used, and the number of electrodes can be expanded to 100×100, 1000×1000, or even 10000×10000. In the peptide synthesis electrode array, hundreds or thousands of independent peptide synthesis areas can be configured, and each peptide synthesis area can quickly perform solid-phase peptide synthesis of droplets, thereby realizing the synthesis of a large number of peptide compounds in a large-scale array.

[0024] In addition, in large-scale peptide synthesis electrode arrays, different peptide synthesis areas can prepare peptides of different amino acid sequences and types in parallel, including but not limited to branched peptides, cyclized peptides, glycopeptides, N-methyl peptides, peptide nucleic acids (PNA), peptoids, peptide thioesters and phosphopeptides, thereby achieving high-throughput synthesis of peptide substances. Different synthesis areas can also prepare the same polypeptide at the same time to increase the yield of peptide substances. For the synthesis of long-chain polypeptides (length > 50 amino acids), the present invention can also achieve rapid synthesis of long peptides by decomposing the long peptide chain into multiple short peptide fragments for parallel synthesis, and then splicing these short peptide fragments into complete long peptides, thereby effectively solving the problem of long peptide synthesis.

[0025] Preferably, each polypeptide synthesis zone is provided with a heating mechanism; during the reaction, the magnetic bead droplets are merged with the raw material droplets, and the heating mechanism is used to heat the polypeptide synthesis zone to completely remove the amino protecting groups on the surface of the magnetic beads, and quickly connect the carboxyl group on the next amino acid to form a stable peptide bond.

[0026] Preferably, each polypeptide synthesis area is formed with a liquid-solid separation area and a liquid mixing area, and a magnetic element and a magnetic control mechanism are provided in the liquid-solid separation area; After the reaction is completed, the droplets are driven to the liquid-solid separation area, and the magnetic control mechanism is used to control the magnetic parts to generate a magnetic field to separate the polypeptide synthesis products formed on the surface of the magnetic beads from the waste liquid. The magnetic beads are retained for the next polypeptide synthesis reaction.

[0027] Preferably, the above steps are repeated to sequentially and gradually connect different amino acid types and sequences in each polypeptide synthesis area, and finally several identical or different peptide chains are synthesized in parallel in the polypeptide synthesis electrode array.

[0028] More preferably, the raw material droplets include but are not limited to 20 kinds of amino acid monomers with Fmoc or Boc protecting groups, piperidine, DMF, DIPEA, HATU, HBTU, DCC, HOBt, DIC, EDC, TFA, NMP, TIS, ligands, magnetic beads, etc.

[0029] More preferably, the size of the polypeptide synthesis electrode array is n×n, and the number of polypeptide types that can be synthesized is n. 2 / 25.

[0030] As described above, the present invention has the following beneficial effects: (1) The peptide synthesis electrode array in the digital droplet microfluidic chip of the present application is scalable, which can break through the limitation of the number of channels and easily realize the high-throughput parallel synthesis of thousands of peptides. Moreover, since the droplet mass transfer speed of the digital droplet microfluidic chip is fast and can be flexibly controlled, the efficiency of peptide synthesis is greatly improved. (2) The synthesis of peptide substances is carried out in microliter droplets, which significantly reduces the amount of organic reagents used and achieves environmental friendliness. At the same time, due to the small volume of the micro droplets, the mass transfer rate in the droplets is very fast, which shortens the time required for each step in the synthesis process; (3) In the large-scale peptide synthesis electrode array, different synthesis areas can prepare peptides of different amino acid sequences and types in parallel, including but not limited to branched peptides, cyclized peptides, glycopeptides, N-methyl peptides, peptide nucleic acids (PNA), peptoids, peptide thioesters and phosphopeptides, thereby achieving high-throughput synthesis of peptide substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown is one of the structural schematic diagrams of the peptide synthesis system based on the digital droplet microfluidic chip.

[0032] Figure 2 Schematic diagram of the structure of the digital droplet microfluidic chip showing a 30×30 peptide synthesis electrode array.

[0033] Figure 3 Shown is a schematic diagram of the digital droplet microfluidic chip with 30×30 polypeptide synthesis electrode arrays in Example 1 used to synthesize different peptide chains.

[0034] Figure 4 Shown is the mass spectrum of the pentapeptide product synthesized in the digital droplet microfluidic chip in Example 1.

[0035] Figure 5 Shown is a schematic diagram of the synthesis strategy of the 64 peptides in Example 2.

[0036] Figure 6Shown is a schematic diagram of the synthesis process of 64 peptides on the digital droplet microfluidic chip in Example 2.

[0037] Figure 7 Shown is a schematic diagram of the synthesis strategy for the same polypeptide substance on a digital droplet microfluidic chip. DETAILED DESCRIPTION

[0038] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0039] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0040] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0041] like Figure 1As shown, the embodiment of the present application provides a polypeptide synthesis system based on a digital droplet microfluidic chip, including a fluid transport mechanism, a digital droplet microfluidic chip and a control mechanism, wherein the digital droplet microfluidic chip includes a first substrate and a second substrate arranged opposite to each other, a droplet accommodating space is formed between the first substrate and the second substrate, and a polypeptide synthesis electrode array composed of a plurality of polypeptide synthesis electrode units is formed on the first substrate and the second substrate, and the control mechanism is used to control the fluid transport mechanism to drive the raw material droplets to each polypeptide synthesis area according to the droplet planning moving path to react with the magnetic bead droplets for polypeptide synthesis; the polypeptide synthesis area is composed of one or more combinations of the polypeptide synthesis electrode units. A heating mechanism is provided in each polypeptide synthesis area. A liquid-solid separation area and a mixed liquid area are formed in each polypeptide synthesis area, and a magnetic part and a magnetic control mechanism for controlling the magnetic part to generate a magnetic field are provided in the liquid-solid separation area, and the magnetic part is used to separate the polypeptide synthesis product formed on the surface of the magnetic bead from the waste liquid under the action of the magnetic field.

[0042] The fluid transport mechanism includes a transport pipeline, a plurality of raw material storage tanks and waste liquid tanks, and a transport electrode. The raw material storage tanks and waste liquid tanks are arranged at the edge of the digital droplet microfluidic chip and are connected to each polypeptide synthesis area through the transport electrode. The first substrate is composed of a conductive electrode and a first hydrophobic layer in sequence, and the second base plate is composed of a control electrode, a dielectric layer and a second hydrophobic layer in sequence, and the first hydrophobic layer and the second hydrophobic layer are arranged opposite to each other. The control mechanism includes a central processing unit and a control circuit arranged in the control electrode, and the control circuit is communicatively connected with the central processing unit and the fluid transport mechanism; the central processing unit is used to preset the droplet planning movement path according to the customer's polypeptide synthesis needs, and control the fluid transport mechanism based on the electrowetting principle through the control circuit to drive the raw material droplets to each polypeptide synthesis area according to the droplet planning movement path to react with the magnetic bead droplets for polypeptide synthesis.

[0043] Taking the digital droplet microfluidic chip with 30×30 peptide synthesis electrode array as an example, each 5×5 peptide synthesis electrode unit constitutes an independent peptide synthesis area, forming a total of 6×6=36 peptide synthesis areas, such as Figure 2 As shown. Under the action of electrowetting drive, the raw material droplets are distributed from the raw material reservoir at the edge of the digital droplet microfluidic chip to each peptide synthesis area. According to the steps set by the program, the solid-phase peptide synthesis process of deprotection-cleaning-amino acid coupling-cleaning is carried out, so that the peptide chain is automatically synthesized in each reaction area. A heating mechanism is provided under each synthesis area to accelerate the synthesis reaction and improve the purity of peptide synthesis. The black position in the center of the synthesis area is the solid-liquid separation area. When the solid-liquid mixed droplets containing magnetic beads enter this area, the magnetic beads are separated from the waste liquid under the action of the magnetic field. The magnetic beads are retained for the next amino acid coupling reaction. After multiple cycles, the target polypeptide chain is formed, and the waste liquid is moved to the waste liquid pool.

[0044] Example 1 The embodiment of the present application provides a method for synthesizing a peptide based on a digital droplet microfluidic chip. The preparation of the digital droplet microfluidic chip using the above-mentioned peptide synthesis system based on the digital droplet microfluidic chip includes the following steps: On an ITO glass or copper-clad resin substrate, exposure, development, and etching are performed according to a pre-designed mask pattern to form a driving electrode and a control circuit. A dielectric layer is deposited on the surface of the driving electrode, and then a layer of hydrophobic material is coated on its surface by spin coating or evaporation to form a second substrate. The upper electrode of the first substrate is subjected to the same hydrophobic treatment. The first substrate and the second substrate are combined to form a digital droplet microfluidic chip.

[0045] Figure 3 The figure shows an example of synthesizing different peptide chains on a digital droplet microfluidic chip with a 30×30 peptide synthesis electrode array. Each 5×5 peptide synthesis electrode unit constitutes an independent peptide synthesis area. There are 6 peptide synthesis areas horizontally and 6 peptide synthesis areas vertically on the chip, totaling 36 synthesis areas. Each synthesis area can independently synthesize a peptide chain, so the entire 30×30 chip can synthesize 36 different peptide chains at the same time.

[0046] The steps of synthesizing 36 peptide chains in parallel on the digital droplet microfluidic chip include: Step A: The magnetic bead droplets, piperidine, DMF and amino acid monomer solution are loaded into each liquid reservoir of the digital droplet microfluidic chip, a trace amount of silicone oil is taken to wrap each droplet, and the driving electrode of the liquid reservoir is pre-energized through the peripheral electronic control circuit and control software; Step B: performing a series of on-off operations on the electrodes adjacent to the raw material reservoir electrode unit, thereby distributing raw material droplets of appropriate volume, and transporting the droplets to various synthesis areas through the connected channel electrodes; Step C: Combine the magnetic bead droplets with the piperidine droplets, and mix them at room temperature or under heating for a certain period of time to completely remove the amino protecting groups on the surface of the magnetic beads. After the reaction is completed, move the droplets to the central liquid-solid separation area, and achieve solid-liquid separation of the magnetic beads and the waste liquid under the action of magnetic force. Then, move the waste liquid to the waste liquid collection area, and leave the magnetic beads in the synthesis area for standby use; Step D: Wash the magnetic beads with DMF droplets. Dispense wash droplets from the DMF reservoir and mix with the magnetic beads to completely remove excess reagent. Repeat the washing process 1 to 3 times. Step E: According to the predetermined peptide chain sequence, the corresponding amino acid droplets are distributed from the 20 amino acid monomer reservoirs and transported to 36 synthesis areas, and the magnetic beads are mixed with the amino acid droplets. After a certain period of mixing at room temperature or heating, the amino groups on the surface of the magnetic beads react with the carboxyl groups of the amino acids to form amide bonds. After the reaction is completed, the magnetic bead droplets are moved to the liquid-solid separation area, and the magnetic beads and waste liquid are separated under the action of a magnet, and the waste liquid is moved to the waste liquid collection area, and the magnetic beads are retained in the peptide synthesis area for standby use; Step F: Repeat steps B to E, and gradually connect different amino acid types and sequences in order in each polypeptide synthesis area, and finally synthesize 36 different peptide chains in parallel in 36 synthesis areas.

[0047] Figure 4 The MALDI-TOF mass spectrometry results of the synthesis of pentapeptides on a digital microfluidic chip. The experimental results confirm the feasibility of automated synthesis of polypeptide chains on a digital droplet microfluidic chip. Due to the small droplet volume, the reaction time for each step is shortened to 1 minute, which is much shorter than the 1 hour of the traditional method.

[0048] The above embodiment is based on a digital microfluidic chip with a 30×30 electrode array, and is demonstrated with each 5×5 unit as an independent synthesis area. If the scale of the peptide synthesis electrode array is n×n, the number of peptide types that can be synthesized is n. 2 / 25. At present, the number of electrodes of the digital microfluidic chip developed by the applicant of this application is 1000×1000, and tens of thousands of peptides can be synthesized in parallel. As the value of n continues to increase, it is expected to achieve high-throughput parallel synthesis of millions of peptide chains.

[0049] 30×30 36 100×100 400 1000×1000 40000 … n×nn 2 / 25 Example 2 Rapid synthesis of long peptides can also be achieved on digital droplet microfluidic chips. Figure 5 A long peptide synthesis strategy using a 64-peptide as an example is demonstrated. In this strategy, the synthesis of the 64-peptide can be carried out by step-by-step decomposition: first, it is decomposed into two 32-peptides, and then each 32-peptide is further decomposed into four 16-peptides, and then the 16-peptide is decomposed into eight 8-peptides, and finally decomposed into 16 4-peptides. The synthesis process is as follows Figure 6As shown in the figure, starting from the smallest unit 4 peptides, 16 short peptides 4 peptides are synthesized simultaneously in 16 independent synthesis regions. Next, the synthesized 4 peptides are combined in pairs to form 8 peptides, and the 8 peptides are spliced ​​in pairs to form 16 peptides. The 16 peptides are further combined in pairs to form 32 peptides, and finally two 32 peptides are spliced ​​into 64 peptides. This segmented synthesis method not only reduces the difficulty of long peptide synthesis, but also shortens the synthesis time of long peptides.

[0050] Example 3 The difference between Example 3 and Example 1 is that: in Example 1, different types of polypeptide substances are synthesized in parallel in each synthesis area of ​​the digital microfluidic chip, while in this example, the same polypeptide is synthesized simultaneously in each synthesis area to achieve the purpose of increasing the yield of the polypeptide substance. According to this method, by performing the same synthesis steps in multiple polypeptide synthesis areas, large-scale production of a specific polypeptide substance can be achieved.

[0051] like Figure 7 This method is suitable for situations where a large number of specific polypeptide substances need to be prepared, such as large-scale production of specific polypeptide drugs, proteins or nucleic acids. By performing multiple synthesis reactions on a digital microfluidic chip, the yield can be significantly increased while still maintaining high synthesis purity and reaction efficiency.

[0052] Furthermore, this embodiment can flexibly change the number and size of the synthesis areas by adjusting the number and arrangement of the peptide synthesis electrode arrays on the digital droplet microfluidic chip to adapt to different synthesis tasks. Whether it is for the parallel synthesis of multiple peptide substances or the high-yield synthesis of a single peptide substance, the present invention provides a solution that can achieve efficient and flexible synthesis, effectively expanding the application scope of microfluidic technology in the fields of biosynthesis and chemical synthesis.

[0053] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A peptide synthesis system based on a digital droplet microfluidic chip, characterized in that: The invention comprises a fluid transport mechanism, a digital droplet microfluidic chip and a control mechanism. The digital droplet microfluidic chip comprises a first substrate and a second substrate arranged opposite to each other, a droplet accommodating space is formed between the first substrate and the second substrate, a polypeptide synthesis electrode array composed of a plurality of polypeptide synthesis electrode units is formed on the first substrate and the second substrate, and the control mechanism is used to control the fluid transport mechanism to drive the raw material droplets to each polypeptide synthesis area according to the planned droplet movement path to react with the magnetic bead droplets for polypeptide synthesis; the polypeptide synthesis area is composed of one or more combinations of the polypeptide synthesis electrode units.

2. The peptide synthesis system based on digital droplet microfluidic chip according to claim 1, characterized in that: Each peptide synthesis area is provided with a heating mechanism.

3. The peptide synthesis system based on digital droplet microfluidic chip according to claim 1, characterized in that: Each polypeptide synthesis area is formed with a liquid-solid separation area and a mixed liquid area. The liquid-solid separation area is provided with a magnetic part and a magnetic control mechanism for controlling the magnetic part to generate a magnetic field. The magnetic part is used to perform solid-liquid separation of the polypeptide synthesis product formed on the surface of the magnetic beads from the waste liquid under the action of the magnetic field.

4. The peptide synthesis system based on digital droplet microfluidic chip according to claim 1, characterized in that: The fluid transport mechanism comprises a transport pipeline, a plurality of raw material storage tanks and waste liquid tanks and transport electrodes. The raw material storage tanks and waste liquid tanks are arranged at the edge of the digital droplet microfluidic chip and are connected with each polypeptide synthesis area through the transport electrodes.

5. The peptide synthesis system based on digital droplet microfluidic chip according to claim 1, characterized in that: The first substrate is composed of a conductive electrode and a first hydrophobic layer in sequence, the second base plate is composed of a control electrode, a dielectric layer and a second hydrophobic layer in sequence, and the first hydrophobic layer and the second hydrophobic layer are arranged opposite to each other.

6. The peptide synthesis system based on digital droplet microfluidic chip according to claim 5, characterized in that: The control mechanism includes a central processing unit and a control circuit arranged in a control electrode, and the control circuit is in communication with the central processing unit and the fluid transport mechanism; the central processing unit is used to preset a planned droplet movement path according to the customer's polypeptide synthesis needs, and control the fluid transport mechanism based on the electrowetting principle through the control circuit to drive the raw material droplets to each polypeptide synthesis area according to the planned droplet movement path to react with the magnetic bead droplets to perform polypeptide synthesis.

7. A method for peptide synthesis based on a digital droplet microfluidic chip, characterized in that: The peptide synthesis system based on the digital droplet microfluidic chip as described in any one of claims 1 to 6 comprises the following steps: respectively loading magnetic bead droplets and raw material liquid into a fluid transport mechanism, using a control mechanism to control the fluid transport mechanism to distribute the raw material droplets, and driving the raw material droplets to each peptide synthesis area according to the planned droplet movement path to react with the magnetic bead droplets for peptide synthesis.

8. The method for synthesizing polypeptides based on a digital droplet microfluidic chip according to claim 7, characterized in that: Each peptide synthesis area is provided with a heating mechanism; during the reaction, the magnetic bead droplets are merged with the raw material droplets, and the heating mechanism is used to heat the peptide synthesis area to completely remove the amino protecting groups on the surface of the magnetic beads and quickly connect the carboxyl group on the next amino acid to form a stable peptide bond.

9. The method for synthesizing polypeptides based on a digital droplet microfluidic chip according to claim 8, characterized in that: Each polypeptide synthesis area is formed with a liquid-solid separation area and a liquid mixing area, and a magnetic part and a magnetic control mechanism are arranged in the liquid-solid separation area; After the reaction is completed, the droplets are driven to the liquid-solid separation area, and the magnetic control mechanism is used to control the magnetic parts to generate a magnetic field to separate the polypeptide synthesis products formed on the surface of the magnetic beads from the waste liquid. The magnetic beads are retained for the next polypeptide synthesis reaction.

10. The method for synthesizing polypeptides based on a digital droplet microfluidic chip according to claim 9, characterized in that: Repeat the above steps to connect different amino acid types and sequences in order in each peptide synthesis area, and finally synthesize several identical or different peptide chains in parallel in the peptide synthesis electrode array.

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