Polypeptide synthesis system and method based on digital liquid drop microfluidic chipTechnical Field
The invention relates to the technical field of solid-phase polypeptide synthesis, in particular to a polypeptide synthesis system and method based on a digital liquid drop microfluidic chip.
Background
In 1963, professor Bruce Merrifield, university of rockfield, presented a solid phase synthesis technique which is a major breakthrough in the field of polypeptide synthesis, and has profound significance for the development of chemistry, medicine, immunity and genetic science. The solid phase synthesis principle is a technology that resin or silica gel is used as a stationary phase, amino acid is sequentially connected to the stationary phase through amino deprotection and amide bond condensation reaction to form peptide chains, and finally the peptide chains are released from the stationary phase. At the end of 1969, the first polypeptide synthesizer in the world based on solid-phase polypeptide synthesis was formally introduced, marking the move of chemical peptide synthesis to the automated production era. The appearance of the polypeptide synthesizer greatly improves the synthesis efficiency and purity of chemical reaction, and has important roles in the fields of polypeptide medicine development, protein structure research, antigen preparation and the like.
Polypeptide drugs have the advantages of both small molecules and protein drugs, and in recent years, research on polypeptide drugs has been receiving more and more attention. The polypeptide drug is a novel drug with molecular weight between small molecules and protein drugs (molecular weight is about 500-10000), combines the advantages of the small molecules and the protein drugs, and has the advantages of strong specificity, low immunogenicity, good stability, high safety and the like. Since the first polypeptide drug insulin in 1922, more than hundred polypeptide drugs have been developed successfully worldwide, and the polypeptide drugs relate to various fields such as diabetes, cancer, osteoporosis and rare diseases. With the continuous development of new preparation technology, the varieties of polypeptide drugs on the market in recent years are increasing, and the polypeptide drugs comprise heavy-weight research and development products such as semaglutinin, dolapride and the like. According to the statistics of the weight database, the number of polypeptide drugs developed and marketed in 2018 worldwide and China is 3/2, and the number of developed and marketed is steadily increased by 2022 to 10/7. By 2023 and 6 months, 167 polypeptide drugs are available worldwide, 615 are in clinical experimental stage, the rest most are still in clinical early stage and preclinical synthesis research and development stage, and the field of polypeptide drugs still has a large development space. Compared with other medicines, the development quantity of polypeptide medicines is still less, and only accounts for about 2% of the total new medicines, and the restriction factors are many, wherein the most challenging stage is large-scale construction and high-throughput screening of preclinical polypeptide libraries. The construction of polypeptide library requires synthesis of 5000-10000 polypeptide compounds, and 250 compounds are screened out from the polypeptide library to enter preclinical research, which involves preparation of a large number of polypeptide compounds. Therefore, the scientific research and industry hope that the new generation of polypeptide synthesizer can meet the requirements of high synthesis speed, large number of channels, high degree of automation, low production cost, safety, reliability and the like.
In recent years, the development of a microwave polypeptide synthesizer CEMLiberty has been powerful, and the synthesis period of each amino acid is only 2.5 min, but the number of channels is only 24 at most, and the requirement of constructing hundreds of millions of polypeptide libraries cannot be met, so that the development of a technology capable of synthesizing polypeptides in a large-scale high-throughput parallel manner is needed.
The conventional polypeptide synthesizer is prepared through the steps of amino deprotection, cleaning, amino acid coupling and cleaning in sealed explosion-proof glass or plastic reactor, adding deprotection reagent and activated amino acid monomer, and final deprotection and synthesis reaction with nitrogen, vibration stirring or microwave technology. The polypeptide synthesizer based on explosion-proof glass, plastic reactor and micro-pipe system technology is limited by the space and system complexity, the number of channels is small, and the large-scale parallel synthesis of thousands of polypeptides is difficult to realize, so that the limitation affects the speed of drug research and development and increases the cost.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention is directed to a polypeptide synthesis system and method based on a digital droplet microfluidic chip, which are used for solving the problem that the number of channels of the existing polypeptide synthesizer is generally in the range from one digit to two digits, and it is difficult to meet the requirement of high-throughput synthesis of polypeptides to construct a polypeptide library.
In order to achieve the above and other related objects, the invention provides a polypeptide synthesis system based on a digital droplet microfluidic chip, which comprises a fluid transportation mechanism, the digital droplet microfluidic chip and a control mechanism, wherein the digital droplet microfluidic chip comprises a first substrate and a second substrate which are oppositely arranged, a droplet accommodating space is formed between the first substrate and the second substrate, a polypeptide synthesis electrode array formed by a plurality of polypeptide synthesis electrode units is formed on the first substrate and the second substrate, the control mechanism is used for controlling the fluid transportation mechanism to drive raw material droplets to each polypeptide synthesis region to react with magnetic bead droplets according to a droplet planning moving path so as to synthesize polypeptides, and the polypeptide synthesis region is formed by combining one or more of the polypeptide synthesis electrode units.
The polypeptide synthesis system based on the digital liquid drop microfluidic chip can realize the synthesis operation of polypeptide on the digital liquid drop microfluidic chip, and adopts the liquid drop with the micro-liter level to synthesize polypeptide substances. Meanwhile, the liquid drops are arranged on the digital liquid drop micro-fluidic chip, and the automatic control of the liquid drops can be realized through a peripheral electronic control system, so that the full-automatic synthesis of polypeptide substances is realized. More importantly, through the parallel operation of a large number of liquid drops, the high-throughput synthesis of polypeptides (also can be biological macromolecules such as nucleic acid, protein, saccharides and the like) can be realized through arbitrary arrangement and combination. The scale of the polypeptide synthesis electrode array in the digital liquid drop microfluidic chip can be expanded, the limit of the number of channels can be broken through, and high-flux parallel synthesis of thousands of polypeptides can be easily realized. In addition, the mass transfer speed of the liquid drops of the digital liquid drop micro-fluidic chip is high and the liquid drops can be flexibly controlled, so that the polypeptide synthesis efficiency is greatly improved. The polypeptide synthesizer based on the digital microfluidic technology provided by the application has no report at present, and has certain innovation and practical value.
Preferably, each polypeptide synthesis zone is provided with a heating mechanism, and the reaction temperature can be flexibly controlled according to the synthesis of the target polypeptide so as to accelerate the synthesis reaction and improve the purity of the polypeptide synthesis.
More preferably, the heating mechanism can be precisely controlled within a temperature range of 10-90 ℃ so as to optimize reaction conditions and improve reaction rate. The heating mechanism is arranged above or below the polypeptide synthesis region.
Preferably, each polypeptide synthesis region is formed with a liquid-solid separation region and a liquid mixing region, a magnetic part and a magnetic control mechanism for controlling the magnetic part to generate a magnetic field are arranged in the liquid-solid separation region, and the magnetic part is used for carrying out solid-liquid separation on polypeptide synthesis products formed on the surfaces of the magnetic beads and waste liquid under the action of the magnetic field.
In the technical scheme of the application, the magnetic control mechanism is a high-efficiency separation system for magnetic beads and liquid phase. The system can adopt a magnetic part at a fixed position or a triaxial motion system with X and Y axis plane displacement and Z axis lifting functions, and the magnetic part can be a permanent magnet such as ferrite, rubidium-iron-boron, alnico, samarium-cobalt or the like, or an electromagnet.
Preferably, the fluid conveying mechanism comprises a conveying pipeline, a plurality of raw material liquid storage tanks, a plurality of waste liquid tanks and conveying electrodes, wherein the raw material liquid storage tanks and the waste liquid tanks are arranged at the edges of the digital liquid drop microfluidic chip and are communicated with each polypeptide synthesis region through the conveying electrodes.
More preferably, the fluid delivery mechanism is a multiple parameter adjustment module for delivering a feedstock solution onto a digital droplet microfluidic chip, wherein the control parameters include flow rate, feedstock type, delivery sequence, fluid pressure, and fluid flow rate.
Preferably, 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 oppositely arranged.
More preferably, the conductive electrode and the control electrode are made of one of gold, silver, chromium, copper, aluminum and ITO conductive materials, and the deposition method of the conductive electrode and the control electrode can be magnetron sputtering, electron beam thermal evaporation, and the like, and can also be a printed circuit board.
More preferably, the dielectric material used for the dielectric layer is selected from a composite film of one or more of parylene, aluminum oxide, silicon nitride, polyamide, polyethylene and polytetrafluoroethylene.
More preferably, the hydrophobic material used for the first and second hydrophobic layers is 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.
The control mechanism comprises a central processor and a control circuit arranged in the control electrode, wherein the control circuit is in communication connection with the central processor and the fluid transport mechanism, the central processor is used for presetting a liquid drop planning moving path according to the polypeptide synthesis requirement of a customer, and the control circuit is used for controlling the fluid transport mechanism to drive raw material liquid drops to each polypeptide synthesis zone according to the liquid drop planning moving path to react with magnetic bead liquid drops to synthesize the polypeptide.
In the above-described embodiments of the present application, the control circuit may be a passive circuit, such as a chromium plate or an ITO plate, or an active matrix circuit, such as a thin film transistor, a printed circuit board, or the like.
More preferably, the central processing unit may be an intelligent software control system, where the intelligent software control system may implement droplet path planning, a heating mechanism, a droplet transport mechanism, a magnetic control mechanism, and other modules of the digital droplet microfluidic chip, where multiple control programs are integrated into the same operation software, so as to improve operation convenience and equipment integration level of the polypeptide synthesis system.
The application also provides a polypeptide synthesis method based on the digital droplet microfluidic chip, which adopts the polypeptide synthesis system based on the digital droplet microfluidic chip and comprises the following steps of respectively loading magnetic bead droplets and raw material liquid into a fluid conveying mechanism, controlling the fluid conveying mechanism to distribute the raw material liquid by adopting a control mechanism, and driving the raw material liquid to react with the magnetic bead droplets in each polypeptide synthesis zone according to a planned movement path of the liquid droplets to synthesize the polypeptide.
The digital droplet microfluidic chip of the 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, the number of the electrodes of the microfluidic chip based on the active matrix thin film transistor control circuit can be extended to 100×100, 1000×1000, or even 10000×10000. In the polypeptide synthesis electrode array, hundreds of independent polypeptide synthesis regions can be configured, and each polypeptide synthesis region can rapidly perform solid-phase polypeptide synthesis of liquid drops, so that synthesis of a large number of polypeptide compounds is realized in a large-scale array.
In addition, in large scale polypeptide synthesis electrode arrays, different polypeptide synthesis regions can produce polypeptides of different amino acid sequences and species in parallel, including but not limited to branched peptides, cyclic peptides, glycopeptides, N-methyl peptides, peptide Nucleic Acids (PNAs), peptoids, peptide thioesters, and phosphopeptides, thereby enabling high throughput synthesis of polypeptide materials. Different synthesis regions can also simultaneously produce the same polypeptide to increase the yield of polypeptide material. For the synthesis of long-chain polypeptide (with the length of more than 50 amino acids), the invention can also realize the rapid synthesis of long peptide by decomposing a long peptide chain into a plurality of short peptide fragments for parallel synthesis and then splicing the short peptide fragments into complete long peptide, thereby effectively solving the difficult problem of long peptide synthesis.
Preferably, each polypeptide synthesis zone is provided with a heating mechanism, and during the reaction, the magnetic bead droplets are combined with the raw material droplets, and the heating mechanism is used for heating the polypeptide synthesis zone to completely remove the amino protecting groups on the surface of the magnetic beads, and the carboxyl groups on the next amino acid are quickly connected so as to form stable peptide bonds.
Preferably, each polypeptide synthesis region is formed with a liquid-solid separation region and a liquid mixing region, and a magnetic part and a magnetic control mechanism are arranged in the liquid-solid separation region;
after the reaction is finished, driving the liquid drops to a liquid-solid separation area, adopting a magnetic control mechanism to control a magnetic piece to generate a magnetic field, carrying out solid-liquid separation on a polypeptide synthesis product formed on the surface of the magnetic beads and the waste liquid, and reserving the magnetic beads for the next polypeptide synthesis reaction.
Preferably, the above steps are repeated to sequentially ligate different amino acid species and sequences in each polypeptide synthesis region, and finally a plurality of identical or different peptide chains are synthesized in parallel in the polypeptide synthesis electrode array.
More preferably, the feedstock droplets include, but are not limited to, 20 amino acid monomers with Fmoc or Boc protecting groups, piperidines, DMF, DIPEA, HATU, HBTU, DCC, HOBt, DIC, EDC, TFA, NMP, TIS, ligands, magnetic beads, and the like.
More preferably, the size of the polypeptide synthesis electrode array is n×n, and the number of polypeptide species that can be synthesized is n2/25.
As described above, the present invention has the following advantageous effects:
(1) The scale of the polypeptide synthesis electrode array in the digital liquid drop microfluidic chip can be expanded, the limit of the number of channels can be broken through, and high-flux parallel synthesis of thousands of polypeptides can be easily realized. In addition, as the digital liquid drop micro-fluidic chip has high liquid drop mass transfer speed and can be flexibly controlled, the polypeptide synthesis efficiency is greatly improved
(2) The polypeptide substance is synthesized in the micro-liter liquid drops, so that the use amount of the organic reagent is obviously reduced, and the environmental friendliness is realized. Meanwhile, as the volume of the trace liquid drops is small, the mass transfer speed in the liquid drops is high, and the time required by each step in the synthesis process is shortened;
(3) In large scale polypeptide synthesis electrode arrays, different synthesis regions can be used to produce polypeptides of different amino acid sequences and species in parallel, including but not limited to branched peptides, cyclic peptides, glycopeptides, N-methyl peptides, peptide Nucleic Acids (PNAs), peptoids, peptide thioesters, and phosphopeptides, thereby achieving high throughput synthesis of polypeptide materials.
Drawings
Fig. 1 shows one of the schematic structural diagrams of a polypeptide synthesis system based on a digital droplet microfluidic chip.
Fig. 2 shows a schematic structure of a digital droplet microfluidic chip of a 30×30 polypeptide synthesis electrode array.
FIG. 3 is a schematic diagram of a digital droplet microfluidic chip of example 1 using a 30×30 array of polypeptide synthesis electrodes for synthesizing different peptide chains.
Fig. 4 shows a mass spectrum of a pentapeptide product synthesized in a digital droplet microfluidic chip of example 1.
FIG. 5 shows a schematic of the synthetic strategy of the 64 peptide of example 2.
FIG. 6 is a schematic diagram showing the synthesis of 64 peptides on a digital droplet microfluidic chip according to example 2.
Fig. 7 shows a schematic representation of a synthetic strategy for synthesizing the same polypeptide substance on a digital droplet microfluidic chip.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
It should be understood that the process equipment or devices not specifically identified in the examples below are all conventional in the art.
It is to be further understood that the use of one or more method steps in the present invention does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between the explicitly mentioned steps, unless otherwise indicated, and that the use of a combined connection between one or more devices/means in the present invention does not exclude the presence of other devices/means before or after the combination device/means or the insertion of other devices/means between the explicitly mentioned two devices/means, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
The embodiment of the application provides a polypeptide synthesis system based on a digital liquid drop micro-fluidic chip, which comprises a fluid transportation mechanism, the digital liquid drop micro-fluidic chip and a control mechanism, wherein the digital liquid drop micro-fluidic chip comprises a first substrate and a second substrate which are oppositely arranged, a liquid drop accommodating space is formed between the first substrate and the second substrate, a polypeptide synthesis electrode array formed by a plurality of polypeptide synthesis electrode units is formed on the first substrate and the second substrate, the control mechanism is used for controlling the fluid transportation mechanism to drive raw material liquid drops to each polypeptide synthesis area to react with magnetic bead liquid drops according to a liquid drop planning moving path so as to synthesize polypeptides, and the polypeptide synthesis area is formed by combining one or more of the polypeptide synthesis electrode units. Each polypeptide synthesis zone is provided with a heating mechanism. Each polypeptide synthesis region is provided with a liquid-solid separation region and a liquid mixing region, a magnetic part and a magnetic control mechanism for controlling the magnetic part to generate a magnetic field are arranged in the liquid-solid separation region, and the magnetic part is used for carrying out solid-liquid separation on polypeptide synthesis products formed on the surfaces of the magnetic beads and waste liquid under the action of the magnetic field.
The fluid conveying mechanism comprises a conveying pipeline, a plurality of raw material liquid storage tanks, a plurality of waste liquid tanks and conveying electrodes, wherein the raw material liquid storage tanks and the waste liquid tanks are arranged at the edges of the digital liquid drop microfluidic chip and are communicated with each polypeptide synthesis area through the conveying electrodes. The first base plate 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 oppositely arranged. The control mechanism comprises a central processor and a control circuit arranged in the control electrode, wherein the control circuit is in communication connection with the central processor and the fluid transport mechanism, the central processor is used for presetting a liquid drop planning moving path according to the polypeptide synthesis requirement of a customer, and controlling the fluid transport mechanism to drive raw liquid drops to each polypeptide synthesis zone according to the liquid drop planning moving path by the control circuit based on the electrowetting principle so as to react with magnetic bead liquid drops to synthesize polypeptides.
Taking a digital droplet microfluidic chip of a 30×30 polypeptide synthesis electrode array as an example, each 5×5 polypeptide synthesis electrode units constitutes an independent polypeptide synthesis region, and 6×6=36 polypeptide synthesis regions are formed in total, as shown in fig. 2. Under the action of electrowetting drive, the raw material liquid drops are distributed to each polypeptide synthesis region from a raw material liquid storage pool at the edge of the digital liquid drop microfluidic chip. The solid-phase polypeptide synthesis process of deprotection-cleaning-amino acid coupling-cleaning is performed according to the programmed steps, so that the peptide chain is automatically synthesized in each reaction zone. A heating mechanism is arranged below each synthesis zone to accelerate the synthesis reaction and improve the purity of the polypeptide synthesis. The black position in the center of the synthesis zone is a solid-liquid separation zone, and after the solid-liquid mixed liquid drop containing the magnetic beads enters the solid-liquid separation zone, the magnetic beads are separated from the waste liquid under the action of a magnetic field. The magnetic beads are reserved for the amino acid coupling reaction of the next step, the target polypeptide chain is formed after a plurality of cycles, and the waste liquid is moved into a waste liquid pool.
Example 1
The embodiment of the application provides a polypeptide synthesis method based on a digital liquid drop micro-fluidic chip, which adopts the polypeptide synthesis system based on the digital liquid drop micro-fluidic chip, and the preparation of the digital liquid drop micro-fluidic chip comprises the following steps:
And exposing, developing and etching the ITO glass or the copper-coated resin substrate according to a pre-designed mask pattern to form a driving electrode and a control circuit. And depositing a dielectric layer on the surface of the driving electrode, coating a layer of hydrophobic material on the surface of the dielectric layer by spin coating or vapor deposition to prepare a second substrate, and carrying out the same hydrophobic treatment on the polar plate on the first substrate. The first substrate and the second substrate are combined to form the digital droplet microfluidic chip.
FIG. 3 shows an example of the synthesis of different peptide chains on a digital droplet microfluidic chip of a 30X 30 array of polypeptide synthesis electrodes. Every 5×5 polypeptide synthesis electrode units form an independent polypeptide synthesis region, 6 polypeptide synthesis regions are transversely arranged on the chip, and the longitudinal direction is also 6 polypeptide synthesis regions, and total 36 synthesis regions are formed. Each synthesis region can synthesize one peptide chain independently, so that the whole 30×30 chip can synthesize 36 different peptide chains simultaneously.
The step of synthesizing 36 peptide chains in parallel on the digital droplet microfluidic chip comprises the following steps:
Step A, magnetic bead liquid drops, piperidine, DMF and amino acid monomer solution are respectively loaded in each liquid storage tank of a digital liquid drop microfluidic chip, trace silicone oil is taken to wrap each liquid drop, and a driving electrode of the liquid storage tank is pre-electrified through a peripheral electronic control circuit and control software;
step B, a series of on-off operation is carried out on the adjacent electrodes of the electrode units of the raw material liquid storage tank, so that raw material liquid drops with proper volumes are distributed, and the liquid drops are transported to each synthesis area through the connected channel electrodes;
And C, combining the magnetic bead liquid drop and the piperidine liquid drop, and mixing at normal temperature or under heating for a certain time to completely remove amino protecting groups on the surface of the magnetic bead. After the reaction is finished, the liquid drops are moved to a central liquid-solid separation area, solid-liquid separation of the magnetic beads and the waste liquid is realized under the action of magnetic force, then the waste liquid is moved to a waste liquid collection area, and the magnetic beads are left in a synthesis area for standby;
Step D, using DMF liquid drops to clean the magnetic beads, distributing the cleaning liquid drops from a DMF liquid storage tank, and mixing with the magnetic beads to thoroughly remove excessive reagent, and repeating the cleaning process for 1 to 3 times;
and E, distributing corresponding amino acid droplets from a 20 amino acid monomer liquid storage tank according to a preset peptide chain sequence, conveying the amino acid droplets to 36 synthesis areas, mixing magnetic beads with the amino acid droplets, and performing coupling reaction on amino groups on the surfaces of the magnetic beads and carboxyl groups of the amino acids to form amide bonds after mixing at normal temperature or under heating for a certain time. After the reaction is finished, the magnetic bead liquid drops are moved to a liquid-solid separation area, the magnetic beads and the waste liquid are separated under the action of a magnet, the waste liquid is moved to a waste liquid collection area, and the magnetic beads are reserved in a polypeptide synthesis area for standby;
And F, repeating the steps B to E, gradually connecting different amino acid types and sequences in sequence in each polypeptide synthesis region, and finally synthesizing 36 different peptide chains in parallel in 36 synthesis regions.
FIG. 4 shows MALDI-TOF mass spectrometry results of pentapeptides synthesized on a digital microfluidic chip. Experimental results demonstrate the feasibility of automated synthesis of polypeptide chains on digital droplet microfluidic chips. Because of the small volume of the liquid drops, the reaction time of each step is shortened to 1 min, and compared with 1h of the traditional method, the synthesis time is greatly shortened.
The above embodiment is a digital microfluidic chip based on a 30×30 electrode array and is exemplified with one independent synthesis region per 5×5 unit. If the size of the polypeptide synthesis electrode array is n×n, the number of polypeptide species that can be synthesized is n2/25. The number of the electrodes of the digital microfluidic chip developed by the applicant of the present application is 1000 multiplied by 1000, and the number of the parallel synthesized polypeptide types can reach tens of thousands. With the continued increase in the value of n, it is expected to achieve high-throughput parallel synthesis of millions of polypeptide chains.
30×30 36
100×100 400
1000×1000 40000
......
n×n n2/25
Example 2
The rapid synthesis of long peptides can also be realized on a digital droplet microfluidic chip. FIG. 5 shows a long peptide synthesis strategy, exemplified by 64 peptide. In this strategy, 64 peptide synthesis can be performed by stepwise decomposition, first, by decomposing it into 232 peptides, then further decomposing each 32 peptide into 4 16 peptides, then decomposing 16 peptides into 8 peptides, and finally decomposing it into 16 4 peptides. The synthesis procedure is shown in FIG. 6, starting from the smallest unit 4 peptide, 16 short peptide 4 peptides were synthesized simultaneously in 16 independent synthesis regions. Next, the synthesized 4 peptides were combined two by two to form 8 peptides, the 8 peptides were spliced two by two to form 16 peptides, the 16 peptides were further combined two by two to form 32 peptides, and finally the two 32 peptides were spliced to 64 peptides. The sectional synthesis method not only reduces the synthesis difficulty of the long peptide, but also shortens the synthesis time of the long peptide.
Example 3
Embodiment 3 differs from embodiment 1 in that in embodiment 1, different kinds of polypeptide substances are synthesized in parallel in each synthesis region of the digital microfluidic chip, and in this embodiment, the same kind of polypeptide is synthesized simultaneously in each synthesis region, so as to achieve the purpose of improving the yield of the polypeptide substances. According to this approach, mass production of a particular polypeptide substance can be achieved by performing the same synthesis steps in multiple polypeptide synthesis regions.
As shown in fig. 7. The method is suitable for cases where a large amount of a specific polypeptide substance is required, such as large-scale production of a specific polypeptide drug, protein or nucleic acid. The yield can be remarkably improved by carrying out multiple synthesis reactions on the digital microfluidic chip, and meanwhile, the synthesis purity and the reaction efficiency are still higher.
Furthermore, the number and the size of the synthesis regions can be flexibly changed by adjusting the number and the arrangement mode of the polypeptide synthesis electrode arrays on the digital liquid drop microfluidic chip so as to adapt to different synthesis tasks. The invention provides a solution which can be synthesized efficiently and flexibly, and effectively expands the application range of the microfluidic technology in the fields of biosynthesis and chemical synthesis, whether the parallel synthesis of a plurality of polypeptide substances or the high-yield synthesis of a single polypeptide substance is aimed at.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.