A peptide synthesis system and method based on a digital droplet microfluidic chip

Abstract

This invention provides a peptide synthesis system and method based on a digital droplet microfluidic chip. The peptide synthesis system includes a fluid transport mechanism, a digital droplet microfluidic chip, and a control mechanism. The digital droplet microfluidic chip includes a first substrate and a second substrate disposed opposite each other, with a droplet-containing space formed between the first and second substrates. The first and second substrates form a peptide synthesis electrode array composed of a plurality of peptide synthesis electrode units. The control mechanism controls the fluid transport mechanism to drive the raw material droplets to each peptide synthesis region according to a planned droplet movement path to react with magnetic bead droplets for peptide synthesis. The peptide synthesis region is composed of one or more peptide synthesis electrode units. This invention utilizes microliter-level droplet synthesis for peptide synthesis. Due to the small volume of the microdroplets, the mass transfer rate within the droplets is very fast, shortening the time required for each step in the synthesis process.

Description

Technical Field

[0001] This invention relates to the field of solid-phase peptide synthesis technology, and in particular to a peptide synthesis system and method based on a digital droplet microfluidic chip. Background Technology

[0002] In 1963, Professor Bruce Merrifield of Rockefeller University proposed a solid-phase synthesis technique, a major breakthrough in peptide synthesis that has profoundly impacted the development of chemistry, medicine, immunology, and genetic science. The principle of solid-phase synthesis is to use resin or silica gel as a stationary phase, where amino acids are sequentially linked to form peptide chains through amino group deprotection and amide bond condensation reactions, ultimately releasing the peptide chains from the stationary phase. In late 1969, the world's first peptide synthesizer based on solid-phase peptide synthesis was officially launched, marking the beginning of automated production in chemical peptide synthesis. The advent of peptide synthesizers has significantly improved the efficiency and purity of chemical reactions, playing a vital role in peptide drug development, protein structure research, and antigen preparation.

[0003] Peptide drugs combine the advantages of both small molecule and protein drugs, and their research has received increasing attention in recent years. Peptide drugs are a novel type of drug with a molecular weight between that of small molecules and protein drugs (approximately 500-10,000). They combine the advantages of both, possessing high specificity, low immunogenicity, good stability, and high safety. Since the introduction of the first peptide drug, insulin, in 1922, nearly 100 peptide drugs have been successfully developed globally, covering 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 been increasing in recent years, including blockbuster products such as smegglutide and dulaglutide. According to Insight database statistics, the number of peptide drugs developed and launched globally and in China was 2 / 3 in 2018, increasing to 7 / 10 by 2022, showing a steady increase in the number of drugs developed and launched. As of June 2023, 167 peptide drugs were available globally for disease treatment, with 615 in clinical trials. The vast majority of the remaining drugs were still in early clinical or preclinical synthetic development stages, indicating significant room for growth in the peptide drug field. Compared to other drugs, the number of peptide drugs under development is still relatively small, accounting for only about 2% of all new drugs. Many factors limit this development, with the most challenging stage being the large-scale construction and high-throughput screening of preclinical peptide libraries. Peptide library construction requires the synthesis of 5,000-10,000 peptide compounds, from which 250 compounds are screened for preclinical research, involving a large amount of peptide compound preparation work. Therefore, the scientific and industrial communities hope that next-generation peptide synthesizers can meet the demands of high synthesis speed, a large number of channels, high automation, low production cost, and safety and reliability.

[0004] Commercially available peptide synthesizers such as CSBio, GYROS PROTEIN Technologies (formerly PTI), AppliedBiosystems Inc. (ABI), Advanced Automated Peptide Protein Technology (AAPPTec), Biotage, and CEM typically have only single-digit to double-digit channel counts, which is insufficient to meet the demands of high-throughput peptide synthesis for constructing peptide libraries. While the microwave peptide synthesizer CEMLiberty has seen strong growth in recent years, with a synthesis cycle of only 2.5 minutes per amino acid, its maximum channel count of 24 is still insufficient for constructing peptide libraries containing hundreds of millions of peptides. Therefore, there is an urgent need to develop a technology for large-scale, high-throughput, parallel peptide synthesis.

[0005] Current peptide synthesizers typically employ a cyclical process of amino group deprotection – (cleaning) – amino acid coupling – (cleaning) within a sealed, explosion-proof glass or plastic reactor. Deprotection reagents and activated amino acid monomers are continuously added, and nitrogen, agitation, or microwave technology is used to carry out the deprotection and synthesis reactions, ultimately synthesizing the peptide chain. However, peptide synthesizers based on explosion-proof glass, plastic reactors, and microchannel systems are limited by their space and system complexity, resulting in a limited number of channels and hindering the large-scale parallel synthesis of thousands of peptides. This limitation slows down drug development and increases costs. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a peptide synthesis system and method based on a digital droplet microfluidic chip, which solves the problem that the number of channels in existing peptide synthesizers is generally in the range of single digits to double digits, making it difficult to meet the needs of high-throughput peptide synthesis for constructing peptide libraries.

[0007] To achieve the above and other related objectives, the present invention provides a peptide synthesis system based on a digital droplet microfluidic chip, comprising a fluid transport mechanism, a digital droplet microfluidic chip, and a control mechanism. The digital droplet microfluidic chip includes a first substrate and a second substrate disposed opposite to each other, with a droplet-containing space formed between the first substrate and the second substrate. The first substrate and the second substrate form a peptide synthesis electrode array composed of a plurality of peptide synthesis electrode units. The control mechanism is used to control the fluid transport mechanism to drive the raw material droplets to each peptide synthesis region according to the droplet planning movement path to react with magnetic bead droplets for peptide synthesis. The peptide synthesis region is composed of one or more peptide synthesis electrode units.

[0008] This invention relates to a peptide synthesis system based on a digital droplet microfluidic chip. It enables peptide synthesis on this chip, using microliter-level droplets. Due to the small size of the droplets, mass transfer within them is rapid, significantly reducing the time required for each step of the synthesis process. Furthermore, by placing the droplets on the digital droplet microfluidic chip and controlling them via an external electronic system, automated droplet manipulation is achieved, resulting in fully automated peptide synthesis. More importantly, by manipulating a large number of droplets in parallel and arbitrarily arranging and combining them, high-throughput synthesis of peptides (or other biomolecules such as nucleic acids, proteins, and carbohydrates) can be realized. The peptide synthesis electrode array in the digital droplet microfluidic chip described in this application is scalable, overcoming limitations on the number of channels and easily enabling high-throughput parallel synthesis of thousands of peptides. Moreover, the rapid and flexible mass transfer of the droplets in the digital droplet microfluidic chip greatly improves peptide synthesis efficiency. This peptide synthesizer based on digital microfluidic technology is currently unreported and possesses significant innovation and practical value.

[0009] Preferably, each polypeptide synthesis zone is equipped 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 polypeptide synthesis.

[0010] More preferably, the heating mechanism is capable of precise temperature control within a range of 10~90°C to optimize reaction conditions and improve the reaction rate. The heating mechanism is located above or below the peptide synthesis region.

[0011] Preferably, each polypeptide synthesis zone has a liquid-solid separation zone and a mixing zone. The liquid-solid separation zone is provided with a magnetic component and a magnetic control mechanism for controlling the magnetic component to generate a magnetic field. The magnetic component 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.

[0012] In the above-described technical solution of this application, the magnetic control mechanism is a highly efficient separation system for magnetic beads and liquid phase. This system can employ a fixed-position magnetic component, or a three-axis motion system with X and Y-axis planar displacement and Z-axis lifting functions. The magnetic component can be a permanent magnet such as ferrite, neodymium iron boron, AlNiCo, or Samarium Cobalt, or it can be an electromagnet.

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

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

[0015] Preferably, 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, with the first hydrophobic layer and the second hydrophobic layer disposed opposite to each other.

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

[0017] More preferably, the dielectric material used in the dielectric layer is selected from one or more composite films selected from pyrene, alumina, silicon nitride, polyamide, polyethylene and polytetrafluoroethylene.

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

[0019] Preferably, the control mechanism includes a central processing unit and a control circuit disposed within the control electrode. The control circuit is communicatively connected to 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 peptide synthesis requirements, and through the control circuit, controls the fluid transport mechanism based on the electrowetting principle to drive the raw material droplets to each peptide synthesis zone to react with the magnetic bead droplets for peptide synthesis according to the droplet planning movement path.

[0020] In the above technical solutions of this 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 or a printed circuit board.

[0021] More preferably, the central processing unit can be an intelligent software control system. This intelligent software control system can 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 ease of operation and equipment integration of the peptide synthesis system.

[0022] This application also provides a peptide synthesis method based on a digital droplet microfluidic chip. The peptide synthesis system based on the digital droplet microfluidic chip includes the following steps: 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 region according to the droplet planning movement path to react with the magnetic bead droplets to synthesize peptides.

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

[0024] Furthermore, in large-scale peptide synthesis electrode arrays, different peptide synthesis regions can prepare peptides with different amino acid sequences and types in parallel, including but not limited to branched peptides, cyclic peptides, glycopeptides, N-methyl peptides, peptide nucleic acids (PNAs), peptide-like substances, peptide thioesters, and phosphopeptides, thereby achieving high-throughput synthesis of peptides. Different synthesis regions can also prepare the same peptide simultaneously to increase the yield of peptides. For the synthesis of long-chain peptides (length > 50 amino acids), this invention can also achieve rapid synthesis of long peptides by breaking down the long peptide chain into multiple short peptide fragments for parallel synthesis, and then splicing these short peptide fragments into a complete long peptide, thereby effectively solving the problem of long peptide synthesis.

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

[0026] Preferably, each polypeptide synthesis region has a liquid-solid separation region and a mixing region, wherein the liquid-solid separation region is provided with a magnetic component and a magnetic control mechanism;

[0027] After the reaction is complete, the droplets are driven to the liquid-solid separation zone. A magnetic control mechanism is used to generate a magnetic field by controlling the magnetic components, which separates 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.

[0028] Preferably, the above steps are repeated, and different amino acid types and sequences are sequentially connected in each polypeptide synthesis region, so that several identical or different peptide chains are synthesized in parallel within the polypeptide synthesis electrode array.

[0029] More preferably, the raw material droplets include, but are not limited to, 20 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.

[0030] More preferably, if the size of the polypeptide synthesis electrode array is n×n, then the number of polypeptide types that can be synthesized is n. 2 / 25.

[0031] As described above, the present invention has the following beneficial effects:

[0032] (1) The peptide synthesis electrode array in the digital droplet microfluidic chip of this application is scalable, which can overcome the limitation of the number of channels and easily realize the high-throughput parallel synthesis of thousands of peptides. Moreover, due to the fast droplet mass transfer speed and flexible control of the digital droplet microfluidic chip, the peptide synthesis efficiency is greatly improved.

[0033] (2) The synthesis of peptides using microliter-level droplets significantly reduces the amount of organic reagents used, achieving environmental friendliness. Simultaneously, due to the small volume of the microdroplets, the mass transfer rate within the droplets is very fast, shortening the time required for each step in the synthesis process;

[0034] (3) In a large-scale polypeptide synthesis electrode array, different synthesis regions can prepare polypeptides with different amino acid sequences and types in parallel, including but not limited to branched peptides, cyclic peptides, glycopeptides, N-methyl peptides, peptide nucleic acids (PNA), peptide-like substances, peptide thioesters and phosphopeptides, thereby achieving high-throughput synthesis of polypeptide substances. Attached Figure Description

[0035] Figure 1 This is one of the schematic diagrams of a peptide synthesis system based on a digital droplet microfluidic chip.

[0036] Figure 2 The diagram shows the structure of a digital droplet microfluidic chip with a 30×30 polypeptide synthesis electrode array.

[0037] Figure 3 The diagram shows a digital droplet microfluidic chip with a 30×30 polypeptide synthesis electrode array used in Example 1 for synthesizing different peptide chains.

[0038] Figure 4 The image shown is a mass spectrum of the pentapeptide product synthesized in a digital droplet microfluidic chip in Example 1.

[0039] Figure 5 The diagram shows the synthesis strategy of the 64 peptides in Example 2.

[0040] Figure 6 The diagram shown is a schematic of the 64-peptide synthesis process on the digital droplet microfluidic chip in Example 2.

[0041] Figure 7 This diagram illustrates a synthesis strategy for synthesizing the same polypeptide on a digital droplet microfluidic chip. Detailed Implementation

[0042] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0043] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0044] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0045] like Figure 1As shown in the figure, this application provides a peptide synthesis system based on a digital droplet microfluidic chip, including a fluid transport mechanism, a digital droplet microfluidic chip, and a control mechanism. The digital droplet microfluidic chip includes a first substrate and a second substrate disposed opposite to each other, with a droplet-containing space formed between the first substrate and the second substrate. The first substrate and the second substrate form a peptide synthesis electrode array composed of a plurality of peptide synthesis electrode units. The control mechanism is used to control the fluid transport mechanism to drive raw material droplets to each peptide synthesis zone according to the droplet planning path to react with magnetic bead droplets for peptide synthesis. The peptide synthesis zone is composed of one or more peptide synthesis electrode units. Each peptide synthesis zone is equipped with a heating mechanism. Each peptide synthesis zone forms a liquid-solid separation zone and a mixing zone. The liquid-solid separation zone is equipped with a magnetic element and a magnetic control mechanism for controlling the magnetic element to generate a magnetic field. The magnetic element is used to separate the peptide synthesis product formed on the surface of the magnetic beads from the waste liquid under the action of the magnetic field.

[0046] The fluid transport mechanism includes a transport pipeline, several raw material storage tanks and waste liquid tanks, and a transport electrode. The raw material storage tanks and waste liquid tanks are located at the edge of the digital droplet microfluidic chip and are connected to each peptide synthesis region 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, with the first and second hydrophobic layers arranged opposite to each other. The control mechanism includes a central processing unit and a control circuit located within the control electrode. The control circuit is communicatively connected to 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 peptide synthesis requirements, and the control circuit controls the fluid transport mechanism based on the electrowetting principle to drive the raw material droplets to each peptide synthesis region to react with the magnetic bead droplets for peptide synthesis.

[0047] Taking a digital droplet microfluidic chip with a 30×30 peptide synthesis electrode array as an example, each 5×5 peptide synthesis electrode unit constitutes an independent peptide synthesis region, forming a total of 6×6=36 peptide synthesis regions. Figure 2 As shown, under the action of electrowetting, raw material droplets are distributed from the raw material reservoir at the edge of the digital droplet microfluidic chip to each peptide synthesis zone. Following the programmed steps, a solid-phase peptide synthesis process of deprotection-washing-amino acid coupling-washing is performed, automating the synthesis of peptide chains in each reaction zone. A heating mechanism is located below each synthesis zone to accelerate the synthesis reaction and improve the purity of the synthesized peptides. The central black area of ​​the synthesis zone is the solid-liquid separation region. When a solid-liquid mixture droplet containing magnetic beads enters this region, the magnetic beads are separated from the waste liquid under the action of a magnetic field. The magnetic beads are retained for the next amino acid coupling reaction, forming the target peptide chain after multiple cycles, while the waste liquid is moved to a waste liquid pool.

[0048] Example 1

[0049] This application provides a peptide synthesis method based on a digital droplet microfluidic chip. The peptide synthesis system based on the digital droplet microfluidic chip described above, and the fabrication of the digital droplet microfluidic chip, include the following steps:

[0050] On an ITO glass or copper-clad resin substrate, exposure, development, and etching are performed according to a pre-designed mask pattern to form driving electrodes and control circuits. A dielectric layer is deposited on the surface of the driving electrodes, and then a hydrophobic material is coated onto its surface by spin coating or vapor deposition to form a second substrate. The electrode plates on the first substrate undergo the same hydrophobic treatment. The first and second substrates are combined to form a digital droplet microfluidic chip.

[0051] Figure 3 The image 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 region. There are 6 peptide synthesis regions arranged horizontally and 6 vertically on the chip, for a total of 36 synthesis regions. Each synthesis region can independently synthesize one peptide chain, thus the entire 30×30 chip can simultaneously synthesize 36 different peptide chains.

[0052] The steps for parallel synthesis of 36 peptide chains on the aforementioned digital droplet microfluidic chip include:

[0053] Step A: Magnetic bead droplets, piperidine, DMF and amino acid monomer solutions are loaded into each reservoir of the digital droplet microfluidic chip, a small amount of silicone oil is used to coat each droplet, and the driving electrode of the reservoir is pre-energized through the peripheral electronic control circuit and control software.

[0054] Step B: Perform a series of on / off operations on the electrodes adjacent to the raw material storage tank electrode unit to distribute raw material droplets of appropriate volume, and transport the droplets to each synthesis area through the connected channel electrodes;

[0055] Step C: Combine the magnetic bead droplets with the piperidine droplets and mix them at room temperature or with 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 complete, move the droplets to the central liquid-solid separation zone, where the magnetic beads and waste liquid are separated under magnetic force. The waste liquid is then moved to the waste liquid collection zone, while the magnetic beads remain in the synthesis zone for later use.

[0056] Step D: Clean the magnetic beads with DMF droplets. Dispense cleaning droplets from the DMF reservoir and mix them with the magnetic beads to thoroughly remove excess reagent. Repeat the cleaning process 1 to 3 times.

[0057] Step E: According to the predetermined peptide chain sequence, corresponding amino acid droplets are dispensed from the 20 amino acid monomer reservoirs and transported to 36 synthesis zones. Magnetic beads are mixed with the amino acid droplets, and after mixing at room temperature or with heating for a certain time, the amino groups on the surface of the magnetic beads and the carboxyl groups of the amino acids undergo a coupling reaction to form amide bonds. After the reaction is complete, the magnetic bead droplets are transferred to the liquid-solid separation zone, where the magnetic beads and waste liquid are separated under the action of a magnet. The waste liquid is transferred to the waste liquid collection zone, while the magnetic beads are retained in the peptide synthesis zone for later use.

[0058] Step F: Repeat steps B to E, sequentially linking different amino acid types and sequences within each polypeptide synthesis region, ultimately synthesizing 36 different peptide chains in parallel within 36 synthesis regions.

[0059] Figure 4 These are MALDI-TOF mass spectrometry results of the pentapeptide synthesized on a digital microfluidic chip. The experimental results confirm the feasibility of automated polypeptide chain synthesis 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 significantly reduced compared to the 1 hour of the traditional method.

[0060] The above embodiments are based on a digital microfluidic chip with a 30×30 electrode array, and are demonstrated with each 5×5 unit as an independent synthesis region. If the size of the peptide synthesis electrode array is n×n, then the number of peptide types that can be synthesized is n. 2 / 25. Currently, the digital microfluidic chip developed by the applicant has 1000×1000 electrodes, enabling the parallel synthesis of tens of thousands of polypeptides. With the continued increase of the n value, it is expected to achieve high-throughput parallel synthesis of millions of polypeptide chains.

[0061] 30×30 36

[0062] 100×100 400

[0063] 1000×1000 40000

[0064] ...

[0065] n×nn 2 / 25

[0066] Example 2

[0067] 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 through a stepwise decomposition: first, it is decomposed into two 32-peptides, then each 32-peptide is further decomposed into four 16-peptides, then the 16-peptides are decomposed into eight octapeptides, and finally decomposed into sixteen tetrapeptides. The synthesis process is as follows: Figure 6As shown, starting with the smallest unit, a tetrapeptide, 16 short tetrapeptides are synthesized simultaneously in 16 independent synthetic regions. Next, the synthesized tetrapeptides are paired to form octapeptides, which are then paired again to form 16-peptides. These 16-peptides are further paired to form 32-peptides, and finally, two 32-peptides are joined together to form a 64-peptide. This segmented synthesis method not only reduces the difficulty of synthesizing long peptides but also shortens the synthesis time.

[0068] Example 3

[0069] The difference between Example 3 and Example 1 is that Example 1 involved the parallel synthesis of different types of peptides within various synthesis regions of a digital microfluidic chip, while in this example, the same peptide is synthesized simultaneously within each synthesis region to increase the yield of the peptide. Following this method, by performing the same synthesis steps in multiple peptide synthesis regions, the large-scale production of a specific peptide can be achieved.

[0070] like Figure 7 As shown in the diagram. This method is suitable for situations requiring the large-scale preparation of specific peptides, such as the mass production of specific peptide drugs, proteins, or nucleic acids. By performing multiple synthesis reactions on a digital microfluidic chip, the yield can be significantly increased while maintaining high synthetic purity and reaction efficiency.

[0071] Furthermore, this embodiment allows for flexible modification of the number and size of the synthesis regions by adjusting the number and arrangement of the peptide synthesis electrode array on the digital droplet microfluidic chip, thus adapting to different synthesis tasks. Whether for the parallel synthesis of multiple peptides or the high-yield synthesis of a single peptide, this invention provides an efficient and flexible synthesis solution, effectively expanding the application scope of microfluidic technology in biosynthesis and chemical synthesis.

[0072] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should 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 device includes a fluid transport mechanism, a digital droplet microfluidic chip, and a control mechanism. The digital droplet microfluidic chip includes a first substrate and a second substrate arranged opposite to each other, with a droplet-containing space formed between the first and second substrates. The first and second substrates form a peptide synthesis electrode array composed of a plurality of peptide synthesis electrode units. The control mechanism is used to control the fluid transport mechanism to drive the raw material droplets to each peptide synthesis zone according to the droplet planning movement path to react with the magnetic bead droplets for peptide synthesis. The peptide synthesis zone is composed of one or more peptide synthesis electrode units. Each peptide synthesis zone is equipped with a heating mechanism. Each peptide synthesis zone forms a liquid-solid separation zone and a mixing zone. The liquid-solid separation zone is equipped with a magnetic element and a magnetic control mechanism for controlling the magnetic element to generate a magnetic field. The magnetic element is used to separate the peptide synthesis product formed on the surface of the magnetic beads from the waste liquid under the action of the magnetic field.

2. The peptide synthesis system based on a digital droplet microfluidic chip according to claim 1, characterized in that: The fluid transport mechanism includes a transport pipeline, several raw material storage tanks and waste liquid tanks, and a transport electrode. The raw material storage tanks and waste liquid tanks are located at the edge of the digital droplet microfluidic chip and are connected to each peptide synthesis region through the transport electrode.

3. The peptide synthesis system based on a 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, and the second base plate is composed of a control electrode, a dielectric layer and a second hydrophobic layer in sequence, with the first hydrophobic layer and the second hydrophobic layer disposed opposite to each other.

4. The peptide synthesis system based on a digital droplet microfluidic chip according to claim 2, characterized in that: The control mechanism includes a central processing unit and a control circuit located within the control electrode. The control circuit is communicatively connected to 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 peptide synthesis requirements, and through the control circuit, controls the fluid transport mechanism based on the electrowetting principle to drive the raw material droplets to each peptide synthesis zone to react with the magnetic bead droplets for peptide synthesis.

5. A method for peptide synthesis based on a digital droplet microfluidic chip, characterized in that: The peptide synthesis system based on a digital droplet microfluidic chip as described in any one of claims 1 to 4 includes the following steps: 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 region according to the droplet planning movement path to react with the magnetic bead droplets to synthesize peptides.

6. The polypeptide synthesis method based on a digital droplet microfluidic chip according to claim 5, characterized in that: Each peptide synthesis zone is equipped with a heating mechanism; during the reaction, the magnetic bead droplets are combined with the raw material droplets, and the heating mechanism is used to heat the peptide 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.

7. The polypeptide synthesis method based on a digital droplet microfluidic chip according to claim 6, characterized in that: Each polypeptide synthesis region is formed with a liquid-solid separation region and a mixing region, and the liquid-solid separation region is equipped with magnetic components and a magnetic control mechanism. After the reaction is complete, the droplets are driven to the liquid-solid separation zone. A magnetic control mechanism is used to generate a magnetic field by controlling the magnetic components, which separates 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.

8. The polypeptide synthesis method based on a digital droplet microfluidic chip according to claim 7, characterized in that: Repeat the above steps, sequentially linking different amino acid types and sequences within each polypeptide synthesis region, ultimately synthesizing several identical or different peptide chains in parallel within the polypeptide synthesis electrode array.

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