Apparatus for in-situ synthesis of polypeptide chip, and liquid supply system
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-08-13
Smart Images
Figure CN2025148181_13082026_PF_FP_ABST
Abstract
Description
In-situ polypeptide chip synthesis device and liquid supply system TECHNICAL FIELD
[0001] The present application relates to the technical field of in-situ polypeptide chip synthesis, and particularly relates to an in-situ polypeptide chip synthesis device and a liquid supply system. BACKGROUND
[0002] A polypeptide chip is a powerful bio-information analysis platform and tool, and can provide assistance for disease diagnosis, new drug development and biomarker screening. This is because a large number of active functional polypeptide probes are fixed on the surface of the polypeptide chip in a patterned form.
[0003] The preparation methods of the polypeptide chip mainly include spotting and in-situ synthesis. Spotting is to transfer the polypeptides synthesized in advance to the substrate surface by a spotting needle or an inkjet method to form a microarray, and the array points are usually several hundred microns to millimeters in size, the density is low, and the number of probes is several tens to several hundreds. The application potential of the polypeptide chip also drives the continuous development of the preparation process method, and at present, the polypeptide chip with high throughput, high density and small feature size is prepared by in-situ synthesis technology.
[0004] Typical in-situ polypeptide synthesis methods mainly include two kinds. One method is to directly print monomers and other required reaction reagents on the substrate surface by inkjet printing technology. A representative product of this technology is the in-situ polypeptide chip synthesized by a 24-color inkjet printer of PEPperPRINT company. The dot size is 150 x 300 microns, the center distance is 254 x 508 microns, and only a few points can be printed on each square millimeter of area, and about 6000 polypeptide sequence arrays can be synthesized on a standard glass slide with a size of 25 mm x 75 mm. Another method is a light-guided patterning method, such as accurately deprotecting the protecting group at the N-terminal of the amino acid sequence on the substrate at a specific position by a semiconductor photolithography process, and then coupling to extend the peptide chain, so as to realize in-situ synthesis, such as preparation of a high-throughput polypeptide chip for health monitoring. In the inkjet printing method, the amino acid is wrapped by carbon powder and transferred by electrostatic force. The photolithography method needs to provide a reaction solution by a liquid supply system.
[0005] Photolithography is used to pattern and deprotect the surface of peptide chips, enabling the in-situ synthesis of amino acids with different sequences onto a substrate to create peptide chips containing thousands of different sequences. In this process, effectively transporting the amino acid monomer solution with protecting groups to the chip surface shares similarities with the liquid supply system of a peptide synthesizer, but also has its own unique characteristics. Peptides prepared by solid-phase peptide synthesizers often require purification to obtain high-purity target peptides. In peptide synthesizers, the mixed and activated amino acid monomers are typically transported to a synthesis column packed with resin, and amino acid monomers are added one by one for peptide synthesis. The liquid path is fixed, requiring repeated washing to reduce contamination, and post-synthesis purification to ensure purity.
[0006] However, in the process of in-situ synthesis of peptide chips, the large number of synthesized peptide sequences, which are directly synthesized and fixed on the substrate surface, make purification impossible, posing a challenge to providing high-quality peptide chips. Summary of the Invention
[0007] This application aims to address the problem of liquid path contamination in in situ peptide synthesis and proposes a new solution.
[0008] One aspect of this application proposes a liquid supply system for an in-situ peptide synthesis chip device, comprising: an amino acid coupling module including multiple synthesis units, each synthesis unit being used to couple amino acids to a matrix to complete peptide synthesis; an amino acid liquid supply module for activating a liquid mixture of amino acids and a coupling agent and then supplying it to the corresponding synthesis unit of the amino acid coupling module; wherein the amino acid liquid supply module includes: multiple amino acid liquid supply units, each amino acid liquid supply unit containing one amino acid monomer; each amino acid liquid supply unit is equipped with an output control switch; a coupling agent liquid supply unit including an independent coupling agent container and an output pipeline, as well as multiple output control branches; the number of output control branches is related to the number of amino acid liquid supply units. The number of units corresponds to the number of amino acid supply units; the cleaning agent supply unit includes an independent cleaning agent container and output pipeline, as well as multiple output control branches; the number of output control branches corresponds to the number of amino acid supply units; multiple mixing units are used to mix the corresponding amino acid and coupling agent in a container to form an activation solution; wherein, the number of mixing units is consistent with the number of synthesis units and corresponds one-to-one; wherein, each mixing unit includes an input port and an output port and a control switch, the input port is connected to the output control switch of the corresponding amino acid supply unit, the output control branch switch of the coupling agent supply unit, and the output control branch switch of the cleaning agent supply unit through different interfaces, and the output port is connected to the corresponding synthesis unit through the control switch.
[0009] Another aspect of this application proposes an in-situ synthesis peptide chip device, which includes the above-described liquid supply system and an exposure module for transferring deprotected sites onto the chip in a patterned manner using a mask.
[0010] The present invention has at least the following advantages: the liquid circuit system for independent supply of amino acid monomers is in a one-to-one correspondence with AAn-Hn, which eliminates the risk of cross-contamination, reduces reagent consumption and saves cleaning time, and meets the requirements of the in-situ synthesis of peptide chip automation system to achieve stability, efficiency and automation.
[0011] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description
[0012] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:
[0013] Figure 1A is a flowchart of a photolithography-based synthesis of polypeptide chips according to an embodiment of this application.
[0014] Figure 1B is a flowchart of the photolithography method for synthesizing polypeptide chips according to another embodiment of this application.
[0015] Figure 2A is a block diagram of the polypeptide chip in-situ synthesis system of the embodiment shown in Figure 1A.
[0016] Figure 2B is a block diagram of the polypeptide chip in-situ synthesis system of the embodiment shown in Figure 1B.
[0017] Figure 3 is a schematic diagram of the liquid supply system structure of the device shown in Figures 2A and 2B.
[0018] Figure 4 is a schematic diagram of the amino acid supply module structure of the supply system shown in Figure 3.
[0019] Figure 5 is a liquid circuit diagram of the overall liquid supply system shown in Figure 3.
[0020] Figure 6 is a schematic diagram of the amino acid supply module in another embodiment.
[0021] Figure 7 is a liquid circuit diagram of the overall liquid supply in the embodiment shown in Figure 6. Embodiments of the present invention
[0022] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0023] This application has many variations that can be expected by those skilled in the art, and can achieve the same effects as the application.
[0024] The use of terms to define space or location in this application, such as inside, outside, top, bottom, left, right, top, bottom, front, and back, is based on the placement of the object in its usage state.
[0025] In this application, the term "in-situ synthesis" refers to the process of directly synthesizing a polypeptide sequence at the desired application site on a matrix or substrate. In some exemplary embodiments, a linker with protecting groups is deposited on the substrate surface, which can be removed, followed by the addition of an amino acid layer, and polypeptide synthesis is performed by chemically coupling amino acid monomers. In some exemplary embodiments, a specially designed photoliographic mask is used to protect the sites that do not need to be synthesized, while exposing the sites that need to be synthesized. A photoresist, under light, generates an alkaline substance that removes the protecting groups on amino groups, allowing for the coupling of free hydroxyl groups to proceed with polypeptide synthesis.
[0026] In this application, the term "matrix or substrate" refers to a carrier in the process of biopeptide synthesis that has various chemical groups and can immobilize biomolecules such as proteins, antibodies, enzymes, peptides, DNA, etc.
[0027] In this application, the term "linker molecule" refers to a linker molecule laid on a treated glass slide in in situ synthesis, and amino acids can be synthesized into peptides based on linker molecules.
[0028] In this application, the term "protecting group" is used to protect the active side chain of amino acids. The protection of amino acids in peptide chemical synthesis is crucial and directly determines the success of the synthesis. These protecting groups are stable during the synthesis process, have no side reactions, and can be completely and quantitatively removed after the synthesis is completed.
[0029] In this application, the term "BOC protecting group" refers to t-Butyloxycarbonyl, an abbreviation for an amino protecting group used in organic synthesis, particularly in peptide synthesis.
[0030] In this application, the term "chemical modification" refers to the process of attaching active groups or catalytic substances to the electrode surface by means of adsorption, coating, polymerization, chemical reaction, etc., to protect the electrode or improve the electrode's characteristic functions.
[0031] In this application, the term "activation" refers to the activation reaction. In protein biosynthesis, various amino acids must be activated before they can be incorporated into the peptide chain. They are then carried by their specific tRNA to the ribosome, where they can be condensed into a peptide chain using mRNA as a template.
[0032] In this application, the term "valve assembly" or "multi-channel valve assembly" refers to a valve having multiple channels, including a one-inlet-multiple-outlet valve assembly and a multi-inlet-one-outlet valve assembly.
[0033] In this application, the term "rotary selection valve" includes multiple channels and is typically used to redirect fluid from a single source to one or more destinations.
[0034] In this application, the term "manifold" refers to a valve assembly that brings together fluid from multiple channels to a single outlet.
[0035] In this application, the term "comprising" means including, but not limited to, the following elements, that is, it does not exclude other elements.
[0036] In this application, the terms "about" and "approximately" represent a range of accuracy that a person skilled in the art would understand while still ensuring the accuracy of the technical effects of the features in question. This term typically indicates a deviation of 10% from the numerical values shown, preferably 5%.
[0037] One aspect of this application proposes a liquid supply system for an in-situ peptide synthesis chip device, comprising: an amino acid coupling module including multiple synthesis units, each synthesis unit being used to couple amino acids to a matrix to complete peptide synthesis; an amino acid liquid supply module for activating a liquid mixture of amino acids and a coupling agent and then supplying it to the corresponding synthesis unit of the amino acid coupling module; wherein the amino acid liquid supply module includes: multiple amino acid liquid supply units, each amino acid liquid supply unit containing one amino acid monomer; each amino acid liquid supply unit is equipped with an output control switch; a coupling agent liquid supply unit including an independent coupling agent container and an output pipeline, as well as multiple output control branches; the number of output control branches is related to the number of amino acid liquid supply units. The number of units corresponds to the number of amino acid supply units; the cleaning agent supply unit includes an independent cleaning agent container and output pipeline, as well as multiple output control branches; the number of output control branches corresponds to the number of amino acid supply units; multiple mixing units are used to mix the corresponding amino acid and coupling agent in a container to form an activation solution; wherein, the number of mixing units is consistent with the number of synthesis units and corresponds one-to-one; wherein, each mixing unit includes an input port and an output port and a control switch, the input port is connected to the output control switch of the corresponding amino acid supply unit, the output control branch switch of the coupling agent supply unit, and the output control branch switch of the cleaning agent supply unit through different interfaces, and the output port is connected to the corresponding synthesis unit through the control switch.
[0038] In at least one embodiment, the output control switch is an injection pump, which serves as a switch for the corresponding pipeline and controls the liquid output of the corresponding pipeline.
[0039] In at least one embodiment, the output control switch is a valve, and a volumetric meter is provided between the output control switches of each mixing unit and the amino acid supply unit, coupling agent supply unit, and cleaning agent supply unit to control the liquid output of amino acids, coupling agents, and cleaning agents to the mixing unit.
[0040] In at least one embodiment, the liquid supply system further includes a nitrogen supply unit for filling the connected container or pipe with nitrogen, causing the liquid in the container or pipe to bubble and flow to the output port of the container or pipe.
[0041] In at least one embodiment, the amino acid supply module further includes an organic base supply unit, which includes an organic base container and an output pipe, wherein the organic base is either DIEA or TEA.
[0042] In at least one embodiment, the input port of the mixing unit is connected to the nitrogen supply unit; the input port of the amino acid supply unit is connected to the nitrogen supply unit; and the input port of the synthesis unit is connected to the nitrogen supply unit.
[0043] In at least one embodiment, a waste liquid collection unit is also provided between the synthesis unit and the mixing unit.
[0044] In at least one embodiment, the amino acid supply unit is positioned vertically below the height of the mixing unit, and after supplying the amino acid to the mixing unit, the amino acid supply unit returns the solution by gravity.
[0045] In at least one embodiment, the output control branches of the coupling agent supply unit, the cleaning agent supply unit, and the organic alkali supply unit are generated by a one-in-multiple-out distribution valve.
[0046] In at least one embodiment, the coupling agent supply unit contains any one of the coupling agents HATU, BTU, HCTU, TBTU, and PyBop, or a mixture of any one coupling agent and any one of the excipients selected from HOAt, HOBt, and HOSU.
[0047] In at least one embodiment, the cleaning agent supply unit is provided with any one of acetonitrile, N-methylpyrrolidone, dichloromethane, and DMF to flush crystals generated inside the flushing fluid path.
[0048] Another aspect of this application proposes an in-situ synthesis peptide chip device, which includes the liquid supply system of any of the above embodiments, and an exposure module for transferring deprotected sites onto the chip in a patterned form using a mask.
[0049] In at least one embodiment, the device further includes a photoresist spin coating and pre-baking module for photoresist spin coating and pre-baking; and a heating amplification and deprotection module for amplifying the protection of the N-terminus of the exposure-excited sites on the chip surface to generate acid / alkali and remove the protection of the sites.
[0050] The specific implementation process of the embodiments of the present invention will be described in detail below with reference to the accompanying drawings:
[0051] In-situ synthesis of peptide chips using photolithography involves the parallel chemical synthesis of peptide probes via photoconductive mapping on a substrate surface. A mask is used to pattern the peptide probes. Based on the deprotection method of the N-terminal protecting group of amino acid monomers, two types are used: one where the N-terminal protecting group is removed with acid or alkali, requiring photoresist (as shown in Figure 1A); and another where the N-terminal protecting group is photosensitive, allowing direct deprotection by light of a specific wavelength (as shown in Figure 1B). These two methods also determine whether spin-coating with photoresist is necessary. Probe synthesis follows a cycle of "deprotection-washing-coupling-washing" in solid-phase peptide synthesis.
[0052] This application addresses the characteristics of in-situ synthesis of peptide chips using photolithography, combining semiconductor industry technology with solid-state synthesis technology. In at least one embodiment, an automated system for in-situ synthesis of peptide chips using photolithography is provided, as shown in Figure 2A. The system comprises five subsystems: a photoresist spin coating and pre-baking module 300, an exposure module 400, a heating amplification module 500, a heating spin coating amino acid coupling module 200, and an amino acid supply module 100. The photoresist spin coating and pre-baking module 300 is responsible for photoresist spin coating and pre-baking, and consists of several spin coaters and hot plates. The exposure module 400 uses a mask to transfer the deprotected sites onto the chip in a patterned form, mainly consisting of a photolithography machine. The heating amplification module 500 further amplifies the sites on the chip surface excited by exposure to generate acid / alkali and remove the protection at the N-terminus of the sites, and consists of hot plates. The heating spin coating amino acid coupling module 200 cleans the photoresist and removed protective groups from the chip surface, and performs amino acid coupling and post-coupling cleaning, and consists of several heating spin coaters. The amino acid supply module 100 is responsible for the delivery and mixing of amino acid coupling reagents. It is a supply system with independent supply channels for amino acid monomers. The liquid circuit system is AAn-Hn in a one-to-one correspondence, with no crossover and no contamination.
[0053] Compared to the system shown in Figure 2A, the system shown in Figure 2B only includes an amino acid supply module 100, an exposure module 400, and a heated spin-coating amino acid coupling module 200. It does not require the participation of a photoresist spin-coating and post-baking module or a heated amplification and deprotection module. The working process of the heated spin-coating amino acid coupling module 200 and the amino acid supply module 100 is consistent with the working process of the corresponding modules in the system shown in Figure 2A. The liquid circuit system for the independent supply of amino acid monomers is also in a one-to-one correspondence of AAn-Hn.
[0054] The system framework shown in Figure 3 is an amino acid liquid supply module provided by this application for the systems shown in Figures 2A and 2B. Specifically, the liquid supply system for this amino acid liquid supply module is also called an independent liquid supply system for amino acid monomers. It sets up a mixing unit for each amino acid monomer, eliminating cross-contamination. Therefore, compared with other liquid supply systems where multiple amino acid monomers share a single mixing unit, this system eliminates much cleaning during the liquid supply process to the synthesis unit, saving reagent consumption. The in-situ synthesis of peptide chips automated system is characterized by stability, high efficiency, and the ability to automate mass production. Peptide probes are synthesized in situ and fixed on the substrate surface, and cannot undergo purification. Therefore, the synthesis of peptide probes is crucial to the quality of peptide chips, making an efficient liquid supply system particularly important. The independent liquid supply system for amino acid monomers eliminates the risk of cross-contamination, reduces reagent consumption, and saves cleaning time, meeting the requirements of a stable, efficient, and automated in-situ synthesis of peptide chips automated system.
[0055] Figure 4 shows a schematic diagram of the module structure for supplying amino acid solutions to a device comprising n synthesis units based on the system framework shown in Figure 3. R1, R2, and R3 are three common reagents. R1 is a cleaning reagent used for system maintenance to flush out crystallization within the liquid path. It can be an organic solvent such as acetonitrile, N-methylpyrrolidone, dichloromethane, or DMF; DMF is preferred in this case. R2 and R3 are two coupling agents or excipients, compatible with solid-phase peptide synthesis coupling agent types. For example, R2 can be a onium salt coupling agent such as HATU / HBTU / HCTU / TBTU / PyBop, or a 1:1 mixture of such coupling agents with excipients such as HOAt / HOBt / HOSU. R3 can be an organic base such as DIEA / TEA. When activation is performed using only the coupling agent and no coupling auxiliary reagent is needed, such as CDI, only one of R2 or R3 is required. Preferably, R2 and R3 are HATU and DIEA. Where n represents the number of synthesis units, n = 01, 02, 03, ...; the number of mixing units is the same as the number of synthesis units. AVn is the volumetric liquid dispenser corresponding to the mixing unit and synthesis unit. MVn is the mixing unit corresponding to the volumetric liquid dispenser and synthesis unit. Amino acid monomer supply uses nitrogen extrusion, followed by gravity backflow after nitrogen is turned off. The liquid level in the reagent bottle must be lower than the mixing unit in the direction of gravity; alternatively, a valve can be installed on the amino acid monomer pipeline for control. The volumetric liquid dispenser AVn corresponds to the mixing unit MVn and is used to control the amount of liquid flowing into the dispenser. N2 is the nitrogen released by the nitrogen supply unit through the corresponding branch pipeline, used to extrude the liquid at the corresponding valve; V0001-V0003 are the corresponding control valves for R1-R3, and the branch control valves for R1-R3 of the nth synthesis unit (Vn01-Vn03); Vn04 is the control valve of the volumetric liquid dispenser at the nth synthesis unit; AAn is the amino acid monomer of the nth synthesis unit.
[0056] Figure 5 shows the overall liquid supply circuit diagram of the liquid supply system shown in Figure 3. In this example, 22 amino acid monomers are supplied, including 20 natural amino acid monomers and 2 reserved non-natural amino acid monomers. R1 is DMF, R2 is HATU, and R3 is DIEA. The liquid circuit system is constructed according to the liquid circuit diagram in Figure 4 when n=20. The liquid circuit system is connected to the heating spin-coating amino acid coupling module as shown in Figure 2a. The volumetric liquid dispensers AV1-AVn correspond one-to-one with the mixing units MV1-MVn, and are used to control the amount of liquid flowing into the volumetric liquid dispensers. N2 is the nitrogen gas released by the nitrogen supply unit through the corresponding branch pipe, used to extrude the liquid at the corresponding valve; V0001-V0003 are the control valves corresponding to R1-R3, and Vn01-Vn03 are the branch control valves of R1-R3 in the nth synthesis unit; Vn04 is the control valve of the volumetric liquid metering device in the nth synthesis unit; Vn05 is the nitrogen control valve at the mixing unit of the nth synthesis unit; Vn06 is the liquid control valve of the mixing unit of the nth synthesis unit; Vn07 is the waste liquid control valve of the nth synthesis unit; AA1-AAn are the amino acid monomers corresponding to the nth synthesis units. After all modules are built and debugged, an automated program is set to execute the in-situ synthesis production task of peptide chips, using 25 pre-treated substrates. 60 synthesis cycles. Only one amino acid monomer is mixed in each unit, i.e., AAn-Hn, so there is no cross-contamination. After mixing, the coupling reaction is carried out on the corresponding heated spin coater. Of course, one heated spin coater can also correspond to multiple amino acid mixing units, but the amino acid liquid paths do not cross; the spray lines are simply physically connected in parallel on the spray arm. A single cycle process is as follows:
[0057] 1. Spin-coating photoresist onto the substrate and pre-baking;
[0058] 1) The substrate enters the photoresist spin coating and pre-baking module from the boat box. After cleaning the surface on the spin coater, a certain thickness of photoresist is spin coated.
[0059] 2) Place the product on the pre-baking hot plate for pre-baking. Once the baking time is up, transfer it to the cold plate to cool to room temperature.
[0060] 3) The substrate is transferred to the exposure module, and the activation of the coupled amino acid monomers is carried out simultaneously.
[0061] 2. Exposure Patterning
[0062] 1) The substrate is placed into the exposure unit with the mask containing the current cycle pattern. After alignment, the shutter is opened for exposure.
[0063] 2) Multiple films are exposed sequentially, and the substrates are transferred from the exposure machine to the heating, amplification, and deprotection module.
[0064] 3. Heating amplification protection module
[0065] 1) The exposed substrate is transferred to the hot plate of the heating amplification and deprotection module to generate acid, which removes the protective substrate of the patterned sites, and then transferred to the cold plate to return to room temperature;
[0066] 2) The substrate was removed and transferred to a heated spin coater (Hn) for amino acid coupling.
[0067] 4. Activation of coupled amino acids
[0068] 1) When valve Vn08 is opened to the bottle passage, the AAn solution is sent to AVn. When the liquid level in the AVn tube reaches the set level, valve Vn08 is closed, valve Vn04 is opened, and the AAn solution is put into MVn. Then valve Vn04 is closed.
[0069] 2) When valve V0002 is opened to the bottle passage, valve Vn02 is opened, and HATU solution is sent to AVn. When the liquid level in the AVn tube reaches the set level, valve V0002 is closed, valve Vn04 is opened, and HATU solution is put into MVn. Then valve Vn04 is closed.
[0070] 3) When valve V0003 opens to the bottle passage and valve Vn03 opens, DIEA solution is sent to AVn. When the liquid level in AVn reaches the set level, valve V0003 closes and valve Vn04 opens to put HATU solution into MVn. Then, valve Vn04 closes.
[0071] 4) Open the nitrogen line on valves Vn06 and Vn07, bubble and stir, then close valves Vn06 and Vn07 and turn off the nitrogen.
[0072] 5) Open the spraying passage with Vn06, open valve Vn05 to push the mixture into the spraying pipe, close valve Vn05, and the activation solution is ready.
[0073] 5. Amino acid coupling reaction
[0074] 1) The substrate enters the heated spin coater Hn to clean the surface photoresist and the removed protective substrate;
[0075] 2) After rinsing, open the Vn05 valve to spray the liquid, and close the Vn05 valve to stop spraying the liquid. The activation solution is spin-coated onto the substrate to form a film reaction. The reaction is repeated once after a short while.
[0076] 3) After the amino acid coupling is completed, the Vn05 valve is opened to empty the pipeline, and all valves return to normal.
[0077] 4) Clean the surface of the substrate after the reaction, spin dry, and return it to the boat box. This cycle is now complete, and we are ready to start the next cycle.
[0078] After the synthesis process is complete, the substrate is post-processed, and the quality control sample is pre-processed for mass spectrometry before data acquisition. The spectral data shows that there is no cross-contamination in the liquid path during probe synthesis. No cleaning is required during use; reagent delivery and solution preparation can be performed directly, providing ample time for activation, while also requiring low reagent volumes.
[0079] In another embodiment, as shown in Figure 6, a metering pump such as a syringe pump (Sn1) is used instead of a volumetric tubing. This eliminates the need for nitrogen propulsion and volumetric tubing, simplifying the liquid path and making maintenance easier. However, the syringe pump method requires degassing the tubing, resulting in higher reagent usage compared to the nitrogen-propelled and volumetric tubing method. Alternatively, a syringe pump can be used to replace nitrogen propulsion in the spray tubing, providing more precise quantitative control. Of course, both methods can be replaced simultaneously.
[0080] In another embodiment, as shown in Figure 7, a rotary selector valve group with one inlet and multiple outlet channels distributes common reagents such as R1, R2, and R3, so that the number of liquid path branches corresponds to the number of synthesis units. The remaining configuration is the amino acid liquid supply module shown in Figure 4.
[0081] In this application, the liquid circuit system with independent supply of amino acid monomers is a design for high-throughput mass production of peptide chips. Because there are no shared liquid circuits, there is no cross-contamination, eliminating the need for cleaning processes, saving reagents and cleaning time, and improving the efficiency of liquid supply and mixing activation. The liquid circuit system with independent supply of amino acid monomers is one mixing unit to one synthesis unit, i.e., AAn~Hn, which is more suitable for the needs of large-scale production.
[0082] The methods provided in the exemplary embodiments in this specification are merely examples, and an example of one method does not limit an example of another method. Apparatus / methods discussed in one drawing may be added to or interchanged with apparatus / methods in other drawings. Furthermore, specific numerical data values (e.g., specific quantities, numbers, categories, etc.) or other specific information are used only for discussing exemplary embodiments and are not intended to limit the exemplary embodiments.
[0083] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A liquid supply system for an in-situ peptide chip device, comprising: The amino acid coupling module includes multiple synthetic units, each of which is used to couple amino acids to a matrix to complete peptide synthesis; An amino acid supply module is used to deliver an activated liquid containing amino acids and a coupling agent into the synthesis unit corresponding to the amino acid coupling module. The amino acid supply module includes: Multiple amino acid supply units, each containing one type of amino acid monomer; each amino acid supply unit is equipped with an output control switch; The coupling agent supply unit includes a separate coupling agent container and an output pipeline, as well as multiple output control branches; the number of the output control branches corresponds to the number of the amino acid supply units. The cleaning agent supply unit includes a separate cleaning agent container and an output pipeline, as well as multiple output control branches; the number of the output control branches corresponds to the number of the amino acid supply units. Multiple mixing units are used to mix corresponding amino acids and coupling agents in a single container to form an activation solution; The number of mixing units is the same as the number of synthesis units and they correspond one-to-one. Each of the mixing units includes an input port, an output port, and a control switch. The input port is connected to the output control switch of the corresponding amino acid supply unit, the output control branch switch of the coupling agent supply unit, and the output control branch switch of the cleaning agent supply unit through different interfaces. The output port is connected to the corresponding synthesis unit through the control switch.
2. The liquid supply system according to claim 1, characterized in that: The output control switch is an injection pump, which serves as a switch for the corresponding pipeline and controls the liquid output of the corresponding pipeline.
3. The liquid supply system according to claim 1, characterized in that: The output control switch is a valve. A volumetric meter is provided between the output control switches of each mixing unit and the amino acid supply unit, coupling agent supply unit, and cleaning agent supply unit to control the liquid output of amino acids, coupling agents, and cleaning agents to the mixing unit.
4. The liquid supply system according to claim 1, characterized in that: The liquid supply system also includes a nitrogen supply unit, which is used to fill the connected container or pipe with nitrogen, causing the liquid in the container or pipe to bubble and flow to the output port of the container or pipe.
5. The liquid supply system according to claim 1, characterized in that: The amino acid supply module also includes an organic base supply unit, which includes an organic base container and an output pipe, wherein the organic base is either DIEA or TEA.
6. The liquid supply system according to claim 4, characterized in that: The input port of the mixing unit is connected to the nitrogen supply unit; the input port of the amino acid supply unit is connected to the nitrogen supply unit; and the input port of the synthesis unit is connected to the nitrogen supply unit.
7. The liquid supply system according to claim 1, characterized in that: A waste liquid collection unit is also provided between the synthesis unit and the mixing unit.
8. The liquid supply system according to claim 1, characterized in that: The amino acid supply unit is positioned vertically below the mixing unit. After supplying the amino acid to the mixing unit, the amino acid supply unit returns the solution by gravity.
9. The liquid supply system according to claim 5, characterized in that: The output control branches of the coupling agent supply unit, the cleaning agent supply unit, and the organic alkali supply unit are generated by a one-in-multiple-out distribution valve.
10. The liquid supply system according to claim 1, characterized in that: The coupling agent supply unit contains any one of the coupling agents HATU, BTU, HCTU, TBTU, and PyBop, or a mixture of any one of the coupling agents and any one of the excipients selected from HOAt, HOBt, and HOSU.
11. The liquid supply system according to claim 1, characterized in that: The cleaning agent supply unit contains any one of acetonitrile, N-methylpyrrolidone, dichloromethane, or DMF to flush out crystals generated inside the flushing fluid path.
12. An in-situ polypeptide chip synthesis device, comprising the liquid supply system as described in claim 1, and The exposure module is used to transfer deprotected sites onto the chip in a patterned manner using a mask.
13. The device according to claim 12, characterized in that: The equipment also includes a photoresist spin coating and pre-baking module, which is used for photoresist spin coating and pre-baking; and The heating amplification deprotection module is used to amplify the protection of the N-terminus of the acid / alkali removal site at the site excited by exposure on the chip surface.