Method for automated high-throughput preparation of mRNA vaccine and drug

Abstract

Disclosed in the present invention is a method for automated high-throughput preparation of an mRNA vaccine and drug. The method uses a microfluidic chip and a preparation system. The microfluidic chip has a curved sample injection channel and a mixing channel composed of alternately communicated transverse and longitudinal flow channels and is capable of automated and efficient mixing of an aqueous phase and an organic phase for high-throughput preparation of LNPs. The preparation system comprises the microfluidic chip and is further integrated with a variety of peripheral devices and pipetting workstations, and can achieve end-to-end, high-throughput, rapid, integrated automated preparation involving mRNA preparation and purification, LNP encapsulation, in vitro transfection, and cell level characterization in the research of mRNA vaccines and drugs.

Description

A method for automated high-throughput preparation of mRNA vaccines and drugs

[0001] This application claims priority from the following two earlier applications:

[0002] The prior application filed with the China National Intellectual Property Administration on January 15, 2025, with patent application number 2025200941437 and invention title "A microfluidic chip for preparing LNP, an LNP synthesis module and an automated preparation system for mRNA vaccines and drugs";

[0003] The prior application filed with the China National Intellectual Property Administration on January 15, 2025, with patent application number 2025100643786 and invention title "A method for automated high-throughput preparation of mRNA vaccines and drugs";

[0004] The full text of the prior application is incorporated herein by reference. Technical Field

[0005] This invention belongs to the field of biomedical technology, specifically relating to an automated, high-throughput method for preparing mRNA vaccines and drugs. Background Technology

[0006] mRNA vaccines, which stimulate an immune response by delivering mRNA containing genes encoding key parts of a pathogen into human cells, have the potential to be a game-changer in the fight against many diseases. Compared to traditional vaccines, mRNA vaccines offer several advantages: they have shorter development times and can be deployed rapidly; mRNA does not integrate into the genome, thus avoiding concerns about insertional mutations; they can be manufactured in a cell-free manner, enabling rapid, economical, and efficient production; and a single mRNA vaccine can encode multiple antigens, targeting multiple microorganisms or viral variants at once.

[0007] Similar to mRNA vaccines, mRNA therapeutics utilize synthetic mRNA that encodes therapeutic proteins needed to be produced in the body. Compared to traditional drugs, mRNA drugs have broader application potential, including cancer treatment, genetic diseases, cardiovascular diseases, and autoimmune diseases. Furthermore, they can be personalized based on individual genetic information, potentially enabling personalized treatment in areas such as oncology.

[0008] With the widespread use of mRNA vaccines and drugs in the prevention and treatment of major diseases such as infectious diseases and cancer, the demand for their production is constantly increasing. Against this backdrop, high-throughput preparation has become crucial for improving the efficiency of mRNA vaccine and drug production. However, the production process of mRNA vaccines and drugs involves complex steps, including mRNA synthesis, encapsulation, transfection, and subsequent validation. Traditional mRNA vaccine preparation methods have limitations in terms of efficiency, accuracy, and reproducibility. Especially with the increasing demand for large-scale production and diversified vaccines and drugs, developing more efficient and automated production processes and systems is of paramount importance. Summary of the Invention

[0009] The inventors discovered the following technical challenges in establishing automated processes and devices for high-throughput preparation of mRNA vaccines and drugs:

[0010] 1. Rapid, Integrated, and Automated Process: mRNA development requires rapid, high-volume, and stable synthesis. However, mRNA preparation involves numerous and complex steps, necessitating high-throughput automated synthesis and preparation to improve experimental efficiency and accuracy. Currently, however, no single-machine system, domestically or internationally, can complete the entire experimental process from mRNA synthesis, purification, lipid nanoparticle encapsulation, and cellular characterization in mRNA vaccine research.

[0011] 2. High-throughput preparation of vaccine and drug carriers: Lipid nanoparticles (LNPs) are currently the only clinically approved effective delivery carrier for mRNA vaccines. However, due to patent restrictions on lipids, overcoming international lipid patent limitations and establishing their own lipid formulation libraries has become a core competitive advantage for mRNA vaccine companies. There is an urgent need for a technological method to achieve high-throughput, automated, and unattended LNP formulation preparation to accelerate the LNP formulation process.

[0012] 3. Standardization of the preparation process: The complex purification steps and GMP supervision in mRNA preparation are the main constraints on rapid production throughout the entire process. Traditional methods have certain limitations in terms of accuracy and reproducibility of standardized operations, while high-throughput automated platforms can achieve standardization of the entire preparation process.

[0013] Therefore, designing an automated process and encapsulation device for high-throughput preparation of mRNA vaccines and drugs can not only significantly improve production speed and reduce operational complexity, but also ensure precise control of each step throughout the process, reducing human error and operational costs. Furthermore, automation technology enables researchers to conduct large-scale experimental screenings, more quickly optimize the design and production of mRNA drugs, and thus accelerate the process of bringing new drugs to market.

[0014] To address the aforementioned technical problems, this invention provides an automated, high-throughput method for preparing mRNA vaccines and drugs. This method utilizes a microfluidic chip for preparing LNPs, an LNP synthesis module, and an mRNA vaccine preparation system, achieving high-throughput, automated preparation of LNPs and mRNA.

[0015] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0016] This invention provides a microfluidic chip, comprising one or more chip units, wherein the chip unit includes a first liquid reservoir, a second liquid reservoir, a first inlet channel, a second inlet channel, and a mixing channel;

[0017] The bottom liquid outlet of the first liquid storage chamber is connected to the mixing channel through a first inlet channel, and the bottom liquid outlet of the second liquid storage chamber is connected to the mixing channel through a second inlet channel.

[0018] The liquid outlets of the first inlet channel and the second inlet channel converge at the single liquid inlet of the mixing channel.

[0019] According to an embodiment of the present invention, the number of chip units is one or more, such as two, three or four.

[0020] According to embodiments of the present invention, the first and second liquid storage chambers have a funnel-shaped structure to facilitate the inflow of liquid into the inlet channel. In some embodiments, the first and second liquid storage chambers have a two-part structure: a cylindrical (e.g., a cylindrical) structure at the upper end and a funnel-shaped structure at the lower end.

[0021] In some embodiments, the first reservoir is used to store the aqueous phase for synthesizing LNP, and the second reservoir is used to store the organic phase for synthesizing LNP; in other embodiments, the liquids stored in the first and second reservoirs may be reversed.

[0022] According to an embodiment of the present invention, the first inlet channel and the second inlet channel are curved injection channels, with the number of bends N≥2 (and preferably an even number), for example, N=2 or 4. In some embodiments, the first inlet channel and the second inlet channel each have two bends and three straight sections (relative to the bends), connected in the order of the first straight section, the first bend, the second straight section, the second bend, and the third straight section. The first straight section is also connected to the bottom liquid outlet of the first storage chamber, and the third straight section is also connected to the only liquid inlet of the mixing channel.

[0023] According to an embodiment of the present invention, the cross-section of the first inlet channel and the second inlet channel is circular or square.

[0024] According to an embodiment of the present invention, the cross-sectional areas of the first inlet channel and the second inlet channel are different, for example, the ratio of their cross-sectional areas is (1.5 to 9):1, for example (2 to 5):1, and in some embodiments the ratio of their cross-sectional areas is 2.5:1.

[0025] According to an embodiment of the present invention, the mixing channel has a mixed flow channel composed of alternating transverse flow channels and vertical flow channels; further, the number of transverse flow channels is 10 to 30, for example 12, 15, 17, 20, 22, 25, 27, or 30; further, the transverse length of the transverse flow channels is 5.2 mm to 10.4 mm, for example 6.0 mm, 6.5 mm, 7.0 mm, 7.5 mm, 7.8 mm, 8.0 mm, 8.5 mm, 9.0 mm, 9.5 mm, or 10.0 mm.

[0026] According to an embodiment of the present invention, one or more fishbone-shaped structures are provided inside the transverse flow channel to increase the mixing effect of the organic phase and the aqueous phase.

[0027] According to an embodiment of the present invention, the mixing channel further has a non-mixing channel communicating with the mixing channel, disposed below the mixing channel, serving as the bottom sample outlet of the microfluidic chip.

[0028] According to an embodiment of the present invention, the height ratio of the mixed flow channel to the non-mixed flow channel is 1:2 to 2:1, for example, the ratio is 1:1.1.

[0029] According to an embodiment of the present invention, the height ratio of the mixing channel to the microfluidic chip is 1:4 to 1:2.

[0030] According to an embodiment of the present invention, the height ratio of the inlet channel (first inlet channel or second inlet channel) to the mixing channel is 6:20 to 9:20.

[0031] According to an embodiment of the present invention, the height of the microfluidic chip is determined according to the actual designed module, with a typical height range of 50mm to 70mm.

[0032] According to an embodiment of the present invention, the inlet channel (first inlet channel or second inlet channel), the transverse flow channel, the vertical flow channel and the non-mixed flow channel have the same or different dimensions, for example, 260 micrometers × 260 micrometers and 130 micrometers × 130 micrometers.

[0033] According to an embodiment of the present invention, the microfluidic chip is made of a polymer material, such as PMMA (polymethyl methacrylate).

[0034] The present invention also provides an LNP synthesis module, including the microfluidic chip described above.

[0035] According to an embodiment of the present invention, the LNP synthesis module further includes an upper-layer module and a lower-layer module;

[0036] The upper module is provided with a chip slot for assembling the microfluidic chip;

[0037] The lower module is provided with a sample receiving slot, which corresponds to the bottom sample outlet of the microfluidic chip.

[0038] According to an embodiment of the present invention, the upper module and the lower module are detachable.

[0039] According to some embodiments of the present invention, the number of sample slots is the same as the number of chip cells.

[0040] According to an embodiment of the present invention, the number of chip slots is one or more, such as two, three, four or five; when the number of chip slots is two or more, the chip slots are arranged in parallel (preferably parallel and equally spaced).

[0041] According to an embodiment of the present invention, the LNP synthesis module further includes a robotic arm gripper, disposed at the lower edge of the upper module and / or the lower module.

[0042] According to an embodiment of the present invention, the lower module is further provided with a base connecting groove and a base connecting post connected to the base connecting groove.

[0043] The present invention also provides a system for preparing mRNA products (e.g., mRNA vaccines and / or mRNA drugs), comprising the microfluidic chip or the LNP synthesis module described above.

[0044] According to an embodiment of the present invention, the preparation system includes a pipetting workstation and peripheral devices, which are connected by a connector;

[0045] The peripheral equipment includes, but is not limited to, PCR instruments, centrifuges, CO2 incubators, microplate readers, consumable stacks, and plate washers;

[0046] The connector includes three guide rails and a robotic arm.

[0047] According to an embodiment of the present invention, the pipetting workstation includes a pipetting workstation main unit and a mechanical gripper;

[0048] Furthermore, the main unit of the pipetting workstation includes one or more pipettes and plate positions;

[0049] The mechanical gripper is mounted on the pipette.

[0050] According to an embodiment of the present invention, the pipetting workstation host is equipped with a pipette tip cleaning station, and / or the microplate reader, PCR instrument and plate washer are located near the pipetting workstation host.

[0051] According to an embodiment of the present invention, a shaking incubator is provided in the middle of the main unit of the pipetting workstation to facilitate operation by two mechanical grippers.

[0052] According to an embodiment of the present invention, the consumable stack is connected to the main unit of the pipetting workstation via a first guide rail, the CO2 incubator is connected to the main unit of the pipetting workstation via a second guide rail, and the centrifuge is connected to the main unit of the pipetting workstation via a third guide rail and a robotic arm.

[0053] In some implementations, the three guide rails are all linear shuttle guide rails, and / or the robotic arm is a Cartesian coordinate cooperative robotic arm.

[0054] According to an embodiment of the present invention, a barcode scanner is also provided on any one or more guide rails, which can scan the barcode on the consumable and correspond to its data result.

[0055] In some implementations, the pipette includes a 96-channel pipette and a flexible 8-channel pipette.

[0056] According to embodiments of the present invention, the plate positions include, but are not limited to, pipette tip loading positions, temperature-controlled incubation positions, shaking incubation positions, plate washer plate loading positions, microplate reader plate loading positions, consumables stack plate loading positions, PCR instrument plate loading positions, centrifuge plate loading positions, CO2 incubator loading and unloading positions, and pipetting workstation main unit and guide rail exchange positions.

[0057] The present invention also provides the application of the above-mentioned microfluidic chip in the synthesis of LNP.

[0058] The present invention also provides a method for simultaneously preparing two or more LNPs, including using the microfluidic chip described above.

[0059] The present invention also provides the application of the above-described LNP synthesis module or system in the preparation of mRNA products (e.g., mRNA vaccines and / or mRNA drugs).

[0060] The present invention also provides a method for preparing mRNA products (e.g., mRNA vaccines and / or mRNA drugs), including using the microfluidic chip, LNP synthesis module or system described above.

[0061] The beneficial effects of this invention are:

[0062] Firstly, the microfluidic chip provided by this invention can automatically and fully mix the aqueous and organic phases, and can simultaneously prepare LNP vectors for multiple different mRNA vaccines and drugs, thus accelerating the development of mRNA vaccines and drugs. The prepared LNPs have uniform particle size, high reproducibility, and encapsulation efficiency and concentration that are superior to existing methods.

[0063] Secondly, this invention introduces a microfluidic chip into the preparation system of mRNA vaccines and drugs through an LNP synthesis module. This system also integrates a variety of peripheral devices and a pipetting workstation, and for the first time completes the entire process of mRNA preparation and purification, LNP encapsulation, in vitro transfection, and cell-level characterization in mRNA vaccine and drug research in a high-throughput, rapid, and automated manner (see Figure 22), laying the foundation for the standardization of the entire mRNA preparation process.

[0064] Terminology Definitions and Explanations

[0065] Unless otherwise stated, the numerical ranges described in this specification and claims correspond to each specific consecutive or integer value. For example, the cross-sectional area of ​​the first inlet channel and the second inlet channel is (1.5 to 9):1, which describes a ratio of decimal to integer within that range. For example, the number of transverse flow channels is 10 to 30, which describes each specific integer value within that range.

[0066] "mRNA vaccine and drug preparation system", "(high-throughput) automated (work) platform" and "automated workstation" have the same meaning.

[0067] "Microfluidic chip" and "centrifugal microfluidic chip" have the same meaning. Attached Figure Description

[0068] Figure 1 is a schematic diagram of the high-throughput automation platform that has been built;

[0069] Figure labels: 1-PCR instrument, 2-Online plate centrifuge, 3-CO2 incubator, 4-ELISA reader, 5-Consumables stack, 6-Plate washer, 7-Pipeline workstation host, 8-Cartesian coordinate cooperative robotic arm, 9-Linear shuttle rail, 10-96-channel gantry crane, 11-8-channel gantry crane, 12-Bar scanner, 13-Mechanical gripper, 14-96-channel pipette, 15-8-channel pipette, 16-Infrared sensor.

[0070] Figure 2 is a schematic diagram of the LNP synthesis module;

[0071] Figure labels: 2-1 Upper-layer module, 2-2 Microfluidic chip, 2-3 Lower-layer module;

[0072] Figure 3 is a schematic diagram of the upper-level module of the LNP synthesis module;

[0073] Attached reference numerals: 2-1 upper module, 3-1 chip slot, 3-2 robotic arm gripper, 3-3 base connecting column;

[0074] Figure 4 is a schematic diagram of the microfluidic chip structure;

[0075] Reference numerals: 4-1 First liquid storage chamber, 4-2 Second liquid storage chamber, 4-3 First inlet channel, 4-4 Second inlet channel, 4-5 First straight channel, 4-6 First bend, 4-7 Second straight channel, 4-8 Second bend, 4-9 Third straight channel, 4-10 Mixing channel, 4-11 Horizontal flow channel, 4-12 Vertical flow channel;

[0076] Figure 5 is a schematic diagram of the structure of the lower-level module of the LNP synthesis module;

[0077] Attached reference numerals: 5-1 base mating groove, 5-2 sample receiving groove, 5-3 lower module body, 3-2 robotic arm gripping the outer edge;

[0078] Figure 6 shows the plasmid map of the automated platform verification model;

[0079] Figure 7 shows the capillary electrophoresis results of the automatically prepared mRNA sample;

[0080] Figure 8 shows the comparison results of capillary electrophoresis of mRNA prepared automatically and manually;

[0081] Figure 9 shows the comparison results of poly(A) capillary electrophoresis obtained from automatically / manually prepared mRNA;

[0082] Figure 10 shows the in vitro expression results of fluorescent insect luciferase mRNA prepared automatically / manually;

[0083] Figure 11 shows the in vivo expression results of fluorescent insect luciferase mRNA prepared automatically / manually;

[0084] Figure 12 shows a comparison of the particle size, encapsulation efficiency, and encapsulation concentration of LNPs prepared by the two methods;

[0085] Figure 13 shows the animal expression verification of LNP prepared by the two methods;

[0086] Figure 14 shows the results of transfection of luciferase mRNA using the automated platform;

[0087] Figure 15 shows the results of automated / manual transfection of luciferase mRNA;

[0088] Figure 16 shows the preparation of mRNAs containing different 5'UTRs from 901 using an automated platform;

[0089] Figure 17 shows a comparison of the automated preparation results with the ten-fold cross-correlation of the public dataset;

[0090] Figure 18 shows the in vitro expression results of fluorescent insect luciferase mRNA containing 5'UTR-L and 5'UTR-H;

[0091] Figure 19 shows the in vivo expression results of fluorescent insect luciferase mRNA containing 5'UTR-L and 5'UTR-H;

[0092] Figure 20 shows the in vitro expression results of green fluorescent protein mRNA containing 5'UTR-L and 5'UTR-H;

[0093] Figure 21 shows the in vitro expression results of the omeprion Spike protein mRNA containing 5'UTR-L and 5'UTR-H;

[0094] Figure 22 shows an automated preparation system for mRNA vaccines and drugs that integrates mRNA preparation, LNP encapsulation, in vitro transfection, and evaluation / characterization. Detailed Implementation

[0095] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0096] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0097] Building an automation platform

[0098] Example 1: Construction of an automated platform for the preparation of mRNA vaccines and drugs

[0099] The mRNA vaccine and drug preparation system shown in Figure 1 includes an LNP synthesis module, a pipetting workstation, and peripheral devices, which are connected by connectors.

[0100] The LNP synthesis module is described in Example 3;

[0101] Peripheral equipment includes, but is not limited to, PCR instrument 1, online plate centrifuge 2, CO2 incubator 3, microplate reader 4, consumables stack 5, and plate washer 6;

[0102] The connector includes three guide rails and one robotic arm. Specifically, the three guide rails are all linear shuttle guide rails 9, and the robotic arm is a rectangular coordinate cooperative robotic arm 8.

[0103] The pipetting workstation includes a main unit 7 and a mechanical gripper 13. The main unit 7 includes one or more pipettes and plate positions; the mechanical gripper 13 is mounted on the pipette. Specifically, the pipette includes a 96-channel pipette 14 and a flexible 8-channel pipette 15, and each pipette set is equipped with one mechanical gripper.

[0104] The main unit 7 of the pipetting workstation is equipped with a tip cleaning station (such as an 8-channel tip cleaning station or a 96-channel tip cleaning station), which is used to clean the tips and reduce cross-contamination. The microplate reader, PCR instrument, and plate washer are located near the main unit of the pipetting workstation, and the microplates are handled by mechanical grippers mounted on the 96-channel / flexible 8-channel pipette.

[0105] A shaking incubator is installed in the middle of the main unit 7 of the pipetting workstation to facilitate operation by the two mechanical grippers.

[0106] The consumable stack 5 is connected to the pipetting workstation host 7 via the first guide rail, the CO2 incubator 3 is connected to the pipetting workstation host 7 via the second guide rail, and the online plate centrifuge 2 is connected to the pipetting workstation host 7 via the third guide rail and the robotic arm.

[0107] A barcode scanner 12 is also installed on one or more guide rails, which can scan the barcode on the consumables and match the data results.

[0108] Those skilled in the art can configure the plate positions as needed; for example, in some implementations, the plate positions include, but are not limited to, 96-channel pipette tip loading positions, temperature-controlled incubation positions, shaking incubation positions, plate washer plate loading positions, microplate reader plate loading positions, consumables stack plate loading positions, PCR instrument plate loading positions, centrifuge plate loading positions, incubator loading and unloading positions, pipette tip recycling baskets, main unit and track exchange positions, barcode scanning positions, consumables stack and main unit exchange positions, etc.

[0109] The preparation system also includes a 96-channel gantry 10, an 8-channel gantry 11, and an infrared sensor 16.

[0110] This mRNA vaccine and drug preparation system is a one-stop mRNA drug preparation / research and development workstation system with an automated pipetting workstation as its core. It integrates peripheral equipment such as consumable stacks, microplate readers, online plate centrifuges, plate washers, PCR instruments, and online CO2 incubators, as well as multiple plate positions, providing ample operating space.

[0111] Example 2: High-throughput preparation of mRNA using an mRNA vaccine and drug preparation system

[0112] The four steps required for plasmid to mRNA conversion—plasmid linearization and purification, in vitro transcription and purification of mRNA—were programmed using automated control software and integrated into the mRNA vaccine and drug preparation system of Example 1. The specific steps are as follows:

[0113] (1) Plasmid linearization: The 120nt poly(A) tail required for in vitro transcription was designed on the reverse primer sequence and integrated into the plasmid template by polymerase chain reaction (PCR). The workstation control software was programmed according to the instructions of the standard PCR enzyme. In particular, considering the insufficient accuracy of low pipetting volume and to reduce robotic arm operation, the PCR enzyme, dNTP and reaction buffer were premixed together, and the two primers were premixed together. The reaction was carried out by the online PCR instrument integrated with the platform.

[0114] (2) Plasmid linearization purification: The PCR reaction solution was purified using a magnetic bead PCR product purification kit. The magnetic beads are modified with carboxyl functional groups, enabling the formation of nucleic acid-salt ion-carboxyl ion bridges under high-salt conditions to adsorb nucleic acids. Under low-salt conditions, these ion bridges are disentangled, thus purifying the linearized DNA. Liquid replacement was performed using a magnetic separation plate adapted to the workstation. The workflow was written according to the instructions of the magnetic bead purification kit. The concentration of the purified linearized DNA template was determined using the integrated microplate reader on the platform.

[0115] (3) In vitro mRNA transcription: Following the instructions of the T7 co-transcription kit, premix the appropriate concentrations of T7 transcriptase mixture, ATP, GTP, CTP, UTP, Cap1 analog, and transcription buffer in a 1.5 ml centrifuge tube in a chilled metal module and place it in a temperature-controlled incubation plate position. After diluting the template DNA to the same concentration using an automated platform, use 96-channel and 8-channel pipette tips to aspirate the DNA template and transcription premix into new 96-well plates, respectively. After mixing by pipetting, transfer to a PCR instrument and incubate at 37°C for 2 hours. Then, add DNase I and incubate at 37°C for an appropriate time to remove the linearized DNA template.

[0116] (4) mRNA purification: The in vitro transcripts were purified using a magnetic bead mRNA purification kit. The purification procedure was basically the same as point (2) in Example 2. The difference was that the principle of the RNA purification magnetic beads was that the oligo-dT coupled on the magnetic beads and the poly(A) on the mRNA were complementary and adsorbed.

[0117] Example 3: Microfluidic Chip and LNP Synthesis Module

[0118] After automated mRNA synthesis, to deliver the mRNA into cells or animals to perform its function, it needs to be mixed with lipids to form lipid nanoparticles (LNPs). Therefore, the synthesis of LNPs also needs to be automated. The traditional LNP synthesis process involves dissolving mRNA in citrate at a specific concentration (the aqueous phase), and then preparing liposomes (the organic phase) containing four lipids: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol at specific concentrations. A syringe pump then pushes the organic and aqueous phases through a microfluidic chip containing this mixed structure at a specific volume and flow rate ratio to form LNPs. However, the syringe pump and microfluidic chip required for this process are not available in automated workstations. Even if the syringe pump, catheter, and microfluidic chip were introduced into the workstation, it would be difficult to control them through automated software, making the process quite challenging.

[0119] Therefore, in order to integrate the LNP synthesis process into the automated workstation, this embodiment provides an LNP synthesis module that can be matched with the automated system of Embodiment 1. This module includes an upper module 2-1, a microfluidic chip 2-2, and a lower module 2-3. The upper module 2-1 and the lower module 2-3 are detachable (as shown in Figure 2).

[0120] (1) The upper module 2-1 (as shown in Figure 3) is equipped with chip slots 3-1 for assembling and supporting microfluidic chips; it is also equipped with robotic arm grippers 3-2 so that the LNP synthesis module can be manipulated by the robotic arm and added to the platform. The number of chip slots can be adjusted as needed, for example, five can be set, with each chip slot parallel and equally spaced;

[0121] (2) Microfluidic chip 2-2 (as shown in Figure 4) is a centrifugal microfluidic chip, which is the core of the entire LNP synthesis module and the entire process.

[0122] The microfluidic chip includes one or more chip units. In this embodiment, it has three chip units. Each chip unit includes a first liquid storage chamber 4-1, a second liquid storage chamber 4-2, a first inlet channel 4-3, a second inlet channel 4-4, and a mixing channel 4-10.

[0123] The bottom liquid outlet of the first liquid storage chamber 4-1 is connected to the mixing channel 4-10 through the first inlet channel 4-3, and the bottom liquid outlet of the second liquid storage chamber 4-2 is connected to the mixing channel 4-10 through the second inlet channel 4-4.

[0124] The liquid outlets of the first inlet channel 4-3 and the second inlet channel 4-4 converge at the single liquid inlet of the mixing channel 4-10.

[0125] Two storage chambers are used to pre-store the organic and aqueous phases. When the liquid flows through the channel, under the action of centrifugal force, the organic and aqueous phases are mixed and self-assembled into LNP in the mixing channel. Specifically:

[0126] The first liquid storage chamber 4-1 and the second liquid storage chamber 4-2 have two parts: a columnar (e.g., cylindrical) structure at the upper end and a funnel-shaped structure at the lower end.

[0127] The first storage chamber 4-1 is used to store the aqueous phase for synthesizing LNP, and the second storage chamber 4-2 is used to store the organic phase for synthesizing LNP; in other embodiments, the liquids stored in the first storage chamber 4-1 and the second storage chamber 4-2 may be reversed.

[0128] The first inlet channel 4-3 and the second inlet channel 4-4 are curved injection channels with a number of bends N=2. That is, the first inlet channel 4-3 and the second inlet channel 4-4 each have two bends and three straight channels (relative to the bends). They are connected in the order of the first straight channel 4-5, the first bend 4-6, the second straight channel 4-7, the second bend 4-8 and the third straight channel 4-9. The first straight channel 4-5 is connected to the bottom liquid outlet of the first liquid storage chamber 4-1, and the third straight channel 4-9 is connected to the only liquid inlet of the mixing channel 4-10.

[0129] The first inlet channel 4-3 and the second inlet channel 4-4 have circular cross-sections. The cross-sectional areas of the first inlet channel 4-3 and the second inlet channel 4-4 are different, depending on the flow-volume ratio during LNP synthesis. For example, the ratio of their cross-sectional areas is (1.5 to 9):1. In this embodiment, the larger inlet channel cross-sectional size is 260μm × 260μm, and the smaller inlet channel cross-section is 100μm × 100μm.

[0130] The mixing channel 4-10 comprises alternating transverse channels 4-11 and vertical channels 4-12. The number of transverse channels 4-11 is 10 to 30, for example, 12, 15, 17, 20, 22, 25, 27, or 30. The transverse length of the transverse channels 4-11 is 5.2 mm to 10.4 mm, for example, 6.0 mm, 6.5 mm, 7.0 mm, 7.5 mm, 7.8 mm, 8.0 mm, 8.5 mm, 9.0 mm, 9.5 mm, or 10.0 mm. In this embodiment, the transverse channel structure length is 7.8 mm. Furthermore, one or more herringbone-shaped structures are provided inside the transverse channels to increase the mixing effect of the organic phase and the aqueous phase.

[0131] The mixing channel 4-10 also has a non-mixing channel that communicates with the mixing channel and is located below the mixing channel as the bottom sample outlet of the microfluidic chip.

[0132] The height ratio of the mixed flow channel to the non-mixed flow channel is 1:2 to 2:1, and in this embodiment, the ratio is 1:1.1.

[0133] The height ratio of the mixing channel to the microfluidic chip is 1:4 to 1:2, and the height ratio of the inlet channel (first inlet channel or second inlet channel) to the mixing channel is 6:20 to 9:20.

[0134] Microfluidic chips are made of polymer materials, such as PMMA (polymethyl methacrylate) acrylic sheets. The overall size depends on the application requirements, with a typical height range of 50mm to 70mm.

[0135] (3) Lower module 2-3 (as shown in Figure 5) is used to transfer LNPs prepared from the microfluidic chip.

[0136] The lower module body 5-3 is equipped with a sample receiving slot 5-2, which corresponds to the bottom sample outlet of the microfluidic chip 2-2. It is used to receive the LNP centrifuged from the microfluidic chip for subsequent operation.

[0137] The lower module body 5-3 is also provided with a base connecting groove 5-1, a base connecting column 3-3 connected to the base connecting groove 5-1, and a robotic arm gripping eave 3-2 set on the edge of the lower module.

[0138] The dimensions of the lower-level module vary depending on the specific automated workstation. The guiding principle is that the dimensions ensure stable placement on the workstation's panel and allow for smooth gripping, unloading, separation, and assembly by the workstation's robotic arm. In this example, the lower-level module of the LNP synthesis module measures 120mm × 81mm and features a 3-row, 5-column sample collection slot to collect the LNP liquid flowing from the chip. The size of the sample collection slots can be adjusted based on the volume of the liquid to be collected. In this example, the sample collection slots are cylindrical with a radius of 3.1mm and a height of 6mm.

[0139] Example 4: High-throughput preparation of mRNA-loaded LNPs using an automated workstation

[0140] The mRNA from the 96-well plates containing different mRNAs prepared in Example 2 was transferred to a new 96-well plate, and the mRNA was diluted to a certain concentration using citrate buffer. A certain amount of liposomes was placed in another sample well. In this example, the liposome formulation used was mRNA-1273, with a lipid composition ratio of SM102:DSPC:Cholesterol:DMG-PEG2000 = 50:10:38.5:1.5. The specific LNP synthesis steps are as follows:

[0141] (1) Place the pipette tip to be used, the 96-well plate containing mRNA, the sample loading groove containing liposomes, and the LNP synthesis module containing microfluidic chip on the workstation plate position in advance (see Example 3).

[0142] (2) Use workstation control software to write a workflow, which includes:

[0143] ① Use the pipette at the workstation to add 3 volumes of mRNA and 1 volume of liposomes to the two wells of a mixing channel. Depending on the number of LNPs to be prepared, each LNP synthesis module can prepare up to 15 LNPs.

[0144] ② Send each LNP synthesis module into a centrifuge for centrifugation. In this example, the centrifuge is operated at 1000 rpm for 10 seconds.

[0145] ③ Remove the LNP synthesis module from the centrifuge and separate the upper module from the lower module to expose the sample collection slot containing the LNPs sample collected from the microfluidic chip.

[0146] ④ Remove the LNP liquid from the sample collection tank and place it into a new 96-well plate.

[0147] ⑤ Reassemble the LNP synthesis module, add enzyme-free water to each sample well for washing, and repeat step one until all mRNAs in the 96-well plate have been prepared into LNPs.

[0148] Example 5: High-throughput in vitro transfection and detection of mRNA using an automated workstation

[0149] Traditionally, when evaluating mRNA expression in vitro using 96-well plates, considering the varying evaporation losses due to prolonged cell culture, the outermost ring of the 96-well plate is often discarded and replaced with the same amount of phosphate buffer or water, resulting in 60 wells actually being used. Control samples are also required; when each sample requires 3, 4, or 5 parallel wells (n=3, 4, 5), the actual number of test samples that can be detected in a 96-well plate is 19, 14, or 11, respectively. This method has the following problems: ① Low experimental throughput, and when the number of sample plates is large, the amount of data from control samples is excessive, leading to data waste; ② The well positions of the mRNA transfection plate and the cell transfection plate are not in a one-to-one correspondence, which can easily cause confusion.

[0150] This embodiment uses a parallel plate method for cell transfection: 59 of the 60 usable wells in each 96-well plate are used as test sample wells, and the remaining well is used as a control sample well. 3-5 parallel plates are used to reduce experimental error. The difference in sample volume between this method and the traditional method is shown in Table 1.

[0151] Table 1 shows the difference in sample quantity that can be tested by the method in this embodiment and the traditional method.

[0152] The experimental steps are as follows:

[0153] (1) Through preliminary experiments, we explored the appropriate cell seeding concentration and the time required for cells to grow to the appropriate degree of confluence.

[0154] (2) A 96-well cell culture plate, pre-seeded with cells at an appropriate concentration, was used as the input plate and placed in a 37°C, 5% CO2 saturated humidity incubator integrated into the workstation for overnight incubation. After the time determined in the preliminary experiment, the cell culture plate was placed in the designated position in the automated workstation using a conveyor belt and mechanical gripper. The automated workstation replaced the culture plate with new culture medium, and the LNPs containing mRNA prepared in Example 5 were added to the corresponding wells of the parallel plates. The transfected parallel plates were then sequentially transferred back to the saturated humidity incubator for subsequent operations.

[0155] (3) Detection of firefly luciferase: After the transfected 96-well cell culture plates were cultured in an incubator for 24 hours, 90 μL of luciferase detection substrate was added to each well. The PCR plate cover (hereinafter referred to as the cover plate) was then placed on the plate to keep it in the dark. After mixing in the dark by transferring the plate to the shaking module, the supernatant of each well was transferred to a white flat-bottomed 96-well plate. Bioluminescence was measured using a multi-functional microplate reader integrated into the workstation. Different sample plates were distinguished by the barcode on the side of the plate and the barcode reader integrated into the platform.

[0156] (4) Detection of the target protein: The target protein was detected using an indirect enzyme-linked immunosorbent assay (ELISA). The specific procedure involved using a plate washer integrated into the workstation, incubating and washing the antibody according to the ELISA kit instructions, adding the substrate working solution and stop solution, and immediately measuring the OD value at 450 nm using a microplate reader.

[0157] Automated platform verification

[0158] To facilitate rapid validation of the entire Qualcomm automated work platform, the gene encoding firefly luciferase was selected as the open reading frame. Firefly luciferase is a protein with a molecular weight of approximately 61 kDa. In the presence of ATP, magnesium ions, and oxygen, it catalyzes the oxidation of luciferin substrate (D-luciferin potassium salt), emitting bioluminescence at a wavelength of approximately 560 nm during the oxidation process. This bioluminescence can be measured using a chemiluminescence analyzer or a liquid scintillation analyzer. By using this bioluminescent system of luciferin substrate and luciferase, after transfecting the luciferase-encoding mRNA into cells, only cell lysis buffer and luciferin substrate are needed for highly sensitive and efficient detection of mRNA expression.

[0159] Example 6: Detection of mRNA prepared by the automated platform (compared to manual synthesis)

[0160] (1) Construction of plasmid template for firefly luciferase mRNA for verification: The T7 promoter, 5'Cap binding site, 5'UTR, open reading frame encoding firefly luciferase, and 3'UTR, which are essential elements for in vitro transcription of mRNA, were sequentially cloned into the pUC57-Kan plasmid vector. After transformation into competent E. coli cells, the plasmid was extracted and pre-loaded into a 96-well PCR plate. The specific plasmid map is shown in Figure 6. The above plasmid was simultaneously transcribed in vitro according to the method described in Example 2 and the conventional manual method, denoted as automated / manual preparation.

[0161] (2) After detecting the in vitro transcribed mRNA using an ultra-micro UV spectrophotometer, it was found that the A260 / 280 ratio of the mRNA prepared by this high-throughput automated platform was between 1.8 and 2.2, proving that the prepared mRNA was free of protein contamination. An A260 / 230 ratio greater than 2.0 proved that the prepared mRNA was free of residual salt ions or alcohol / phenol contamination. Both of these indicate that the prepared mRNA was relatively pure; specific results are shown in Table 2.

[0162] Table 2. Ultraviolet spectrophotometer of mRNA prepared by the automated platform.

[0163] (3) Capillary electrophoresis was used to analyze the quality of the in vitro transcribed mRNA. The measured peak diagram showed that, apart from the marker peak used as a control, only one pure single peak existed in the sample region. Furthermore, smear analysis of the sample peaks revealed that the peak area accounted for more than 90%, proving that the prepared mRNA was free from degradation and mutation. It was also relatively pure; some sample detection results are shown in Figure 7. The results showed that although the peak times of different samples varied due to differences in the length of the non-coding region sequence, all sample peaks were pure single peaks. A comparison of the detection peak of one sample with that of a manually prepared mRNA sample (Figure 8) revealed overlap, demonstrating that there was no significant difference between automated and manually prepared mRNA.

[0164] (4) The tailing length of the automated / manually prepared mRNA was determined using capillary electrophoresis. The specific processing method is as follows: After denatured mRNA samples were randomly fragmented using Nase T1, oligo-dT affinity beads were used to collect samples containing poly(A) using the principle of AT pairing. The samples were then eluted with methanol and detected. The results are shown in Figure 9. The results show that the poly(A) samples obtained from the automated / manually prepared mRNA all exhibited a normal distribution with multiple peaks. The highest peaks were relatively overlapping, and the overall normal distribution trend was the same, indicating that the tailing length of the automated / manually prepared mRNA was basically consistent.

[0165] (5) In vitro expression evaluation of automated / manually prepared mRNA: Automated / manually prepared mRNA was transfected into human cervical cancer cells (HeLa) and human embryonic kidney cells (293T), respectively. After incubation at 37°C for 24 hours, luciferase detection substrate was added, and the supernatant was transferred to white flat-bottomed 96-well plates. Bioluminescence was measured using a multi-functional microplate reader. The detection results are shown in Figure 10. After performing a t-test on the detection results, there was no significant difference in fluorescence intensity between the automated / manually prepared fluorescent insect luciferase mRNA in the two cell types (left: human cervical cancer cells HeLa, right: human embryonic kidney cells 293T), indicating that there was no difference in in vitro expression evaluation between the two.

[0166] (6) Evaluation of in vivo expression of automated / manually prepared mRNA: LNP containing firefly luciferase mRNA was injected intramuscularly into the left thigh of each female Balb / C mouse. Six hours later, D-luciferin potassium salt was injected intraperitoneally, and the mice were imaged using an in vivo imaging system. The imaging results (left) and fluorescence intensity analysis (right) are shown in Figure 11. The results showed that the automated / manually prepared firefly luciferase mRNA expressed fluorescence intensity in vivo, and there was no significant difference after t-test analysis.

[0167] Example 7: Detection of LNPs containing mRNA prepared by an automated platform

[0168] In the LNPs prepared in the following examples, the molar ratio of nitrogen in the composite ionizable lipid to phosphorus in the encapsulated nucleic acid drug is 5.67:1, and the volume ratio of the aqueous phase to the organic phase is 3:1. The aqueous phase refers to the solution of the mRNA obtained in Example 6 in a 50 mM sodium citrate buffer solution at pH 4, wherein the concentration of the nucleic acid drug is 0.17 mg / mL. The organic phase refers to the solution of SM102 lipid:DSPC:cholesterol:DMG-PEG2000 in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5. The structure of the centrifugal microfluidic chip and the LNP synthesis module is described in Example 3, and the structure of the automated platform is described in Example 1.

[0169] (1) Preparation of LNP using centrifugal microfluidic chip: 45 μl of aqueous phase containing 170 ng / μl luciferase mRNA was added to the first reservoir with an inlet channel size of 300 μm × 300 μm. 15 μl of organic phase was then added to the second reservoir with an inlet channel size of 170 μm × 170 μm in the same mixing channel. The automated platform was used to send the LNP synthesis module with the centrifugal microfluidic chip into a centrifuge. The module was centrifuged at 1500 rpm for 30 seconds. The LNP synthesis module was then removed from the centrifuge. The robotic arm was used to remove the lower module, and the prepared LNP was obtained in the sample collection tank.

[0170] (2) Preparation of LNP using a pipetting platform: Use a 96-channel pipette to aspirate the aqueous phase containing luciferase nucleic acid and quickly add it to the organic phase. Then, rapidly pipette the mixture more than 30 times and let it stand at room temperature for 10 minutes after mixing.

[0171] (3) Comparison of particle size and encapsulation effect of LNP prepared by centrifugal microfluidic chip / pipette platform: The particle size of LNP prepared by the two methods was detected by a particle size analyzer, and the encapsulation efficiency and encapsulation concentration were detected by RiboGreen kit. The differences in particle size, encapsulation efficiency and encapsulation concentration of LNP prepared by the two methods were compared. The results, as shown in Figure 12, show that LNP prepared by centrifugal microfluidic chip is significantly better than LNP prepared by pipetting platform in terms of particle size, encapsulation efficiency and encapsulation concentration.

[0172] (4) Differences in LNP expression levels in animals prepared by centrifugal microfluidic chip / pipette platform: LNPs containing firefly luciferase mRNA prepared by both methods were intramuscularly injected into the left thigh of each female Balb / C mouse. Six hours later, D-luciferin potassium salt was injected intraperitoneally, and the mice were imaged using an in vivo imaging system. The imaging results (left) and fluorescence intensity analysis (right) are shown in Figure 13. The results showed that the LNPs prepared by centrifugal microfluidic chip had significantly stronger expression intensity in animals than the LNPs prepared by mixing using a pipetting platform, indicating that the developed centrifugal microfluidic chip module can not only achieve automated LNP preparation, but also has a superior preparation effect.

[0173] Example 8: Detection of mRNA transfection using an automated platform

[0174] (1) Following the method described in point (2) of Example 6, the LNP containing 60 luciferase mRNAs prepared in Example 6 was transfected into cervical cancer cells (HeLa). Three parallel plates were set up. After waiting for 24 hours, the results were detected as described in point (3) of Example 6. The detection results are shown in Figure 14. According to the detection results, the errors shown by the three parallel results for each sample were all within an acceptable range, proving that the automated platform for mRNA transfection is feasible.

[0175] (2) To ensure consistent reaction time, six 96-well culture plates seeded with human cervical cancer cells (HeLa) at the same cell density were divided into two groups of three: an automated transfection group and a manual transfection group. The luciferase mRNA prepared in Example 6 was transfected using both the automated platform and the manual transfection method. The mRNA and encapsulation method were kept consistent across all wells. The transfection results are shown in Figure 15. A t-test showed no significant difference in bioluminescence data between automated and manual transfection, and the data obtained from automated transfection had a lower error.

[0176] Practical applications of automation platforms

[0177] Example 9: Preparation of 5' UTR using an automated platform

[0178] To facilitate rapid validation of the entire high-throughput automated work platform, the gene encoding firefly luciferase was selected as the open reading frame, and the 5' untranslated region (5'UTR) was selected as the differential element. 901 5'UTRs from the GSE database were constructed into plasmid templates using high-throughput automation.

[0179] The 5'UTR, located before the start codon (AUG) in the mRNA structure, is part of the mRNA but does not encode any protein. The 5'UTR plays diverse and important roles in mRNA, including regulating translation initiation efficiency, influencing mRNA stability, and responding to changes in the cellular environment. Optimization targeting the 5'UTR is a highly targeted and stable approach that does not alter the RNA coding sequence, does not affect vaccine immunogenicity, and can significantly improve RNA translation efficiency and stability. By using the luciferase-encoding gene as the open reading frame, the translation efficiency of the 5'UTR and its corresponding mRNA can be rapidly obtained.

[0180] Following the high-throughput automated platform constructed in Example 1 above, 901 plasmids containing different luciferases with different 5'UTRs were transcribed into mRNA and transcribed into human cervical cancer cells (HeLa). The bioluminescence of the expressed luciferases was detected, and the luminescence intensity of the mRNA containing the 5'UTR of human α-hemoglobin (HBA1) was used as a standard. The luminescence intensities of all measured mRNAs were homogenized to quantify the expression intensity of the mRNAs. The detection results are shown in Figure 16, where Muscle, PJI, PC3, and HEK293 are the tissue names from which the 5'UTRs originated.

[0181] Example 10: Quality Analysis of Automated Preparation of 5' UTR Data

[0182] (1) Quality analysis of 5'UTR data: The model was trained using the dataset labeled with fluorescence intensity prepared in this embodiment and the dataset labeled with TE value (transfection efficiency) in the existing GSE database (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE176581). After performing a 10-fold cross-correlation analysis on the Pearson correlation coefficients of the two datasets, the results are shown in Figure 17.

[0183] As can be seen, the experimental dataset prepared by this embodiment using fluorescence intensity as a label is of higher quality than the datasets currently available in the public datasets that use TE values. The dataset prepared by automation can have higher prediction accuracy when applied to training models such as machine learning.

[0184] Example 11: Accuracy Verification of Automated Preparation of 5' UTR Data

[0185] Two types of 5'UTRs were selected from the prepared 5'UTRs and designated as 5'UTR-L and 5'UTR-H. The results of the two 5'UTRs, with luciferase as the coding region, were verified in vitro in different cells, in vivo in animals, and with different genes as the coding regions, to ensure consistency with the results of automated preparation.

[0186] (1) In vitro validation of 5'UTR-L and 5'UTR-H using luciferase as the coding region: mRNAs containing the two 5'UTRs were transfected into human cervical cancer cells (HeLa) and human embryonic kidney cells (293T), respectively. After incubation at 37°C for 24 hours, luciferase detection substrate was added, and the supernatant was transferred to a white flat-bottomed 96-well plate. Bioluminescence was measured using a multi-functional microplate reader. The detection results are shown in Figure 18. The fluorescence intensity of the fluorescent insect luciferase mRNAs containing 5'UTR-L and 5'UTR-H expressed in both cell types (left: human cervical cancer cells HeLa, right: human embryonic kidney cells 293T) was consistent with the trend of the preparation results.

[0187] (2) In vivo validation of 5'UTR-L and 5'UTR-H as coding regions for luciferase: LNPs containing firefly luciferase mRNA of 5'UTR-L and 5'UTR-H were intramuscularly injected into the left thigh of each female Balb / C mouse. Six hours later, D-luciferin potassium was injected intraperitoneally, and the mice were imaged using an in vivo imaging system. The imaging results (left) and fluorescence intensity analysis (right) are shown in Figure 19. The results showed that the fluorescence intensity of firefly luciferase mRNA containing 5'UTR-L and 5'UTR-H was consistent with the trend of the preparation results.

[0188] (3) In vitro validation of 5'UTR-L and 5'UTR-H as coding regions of green fluorescent protein: 5'UTR-L and 5'UTR-H were constructed into the coding region of green fluorescent protein via Gibson linker and transcribed into mRNA in vitro. Human cervical cancer cells (HeLa) were seeded in 10 mm confocal cell culture dishes and transfected with LNPs of 5'UTR-L and 5'UTR-H green fluorescent protein mRNA. After 24 hours of culture, Hoechst 33342 dye was added and the images were observed under a confocal microscope. The imaging results are shown in Figure 20A. At the same time, cells transfected in two other groups of confocal culture dishes were digested with trypsin, and the cells were collected and detected by flow cytometry. The detection results are shown in Figure 20B (from left to right: PBS, 5'UTR-L and 5'UTR-H groups) and C. The results showed that the results obtained using green fluorescent protein as the coding region were consistent with the trends of the results obtained by automated preparation using luciferase as the coding region.

[0189] (4) In vitro validation of 5'UTR-L and 5'UTR-H using the novel coronavirus variant - Omeprone Spike protein (hereinafter referred to as Omeprone Spike protein) as the coding region: 5'UTR-L and 5'UTR-H were constructed into the coding region of Omeprone Spike protein via Gibson linking and transcribed into mRNA in vitro. Human cervical cancer cells (HeLa) were seeded in 6-well plates and transfected with LNPs containing Omeprone Spike protein mRNA containing 5'UTR-L and 5'UTR-H. After culturing for 48 hours, the target protein was detected by incubation with a commercially available anti-Omeprone Spike protein antibody using the Weston Blot method. β-actin was selected as an internal control for protein quantification. The detection results are shown in Figure 21, indicating that the results obtained using Omeprone Spike protein as the coding region are consistent with the results obtained by automated preparation using luciferase as the coding region.

[0190] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic chip, characterized in that, The microfluidic chip includes one or more chip units, and each chip unit includes a first liquid storage chamber, a second liquid storage chamber, a first inlet channel, a second inlet channel, and a mixing channel; The bottom liquid outlet of the first liquid storage chamber is connected to the mixing channel through a first inlet channel, and the bottom liquid outlet of the second liquid storage chamber is connected to the mixing channel through a second inlet channel. The liquid outlets of the first inlet channel and the second inlet channel converge at the single liquid inlet of the mixing channel.

2. The microfluidic chip according to claim 1, characterized in that, The first and second storage chambers have funnel-shaped structures and are used to store the aqueous phase and organic phase for the synthesis of LNP, respectively. Preferably, the first liquid storage chamber and the second liquid storage chamber have two parts: a columnar structure at the upper end and a funnel-shaped structure at the lower end.

3. The microfluidic chip according to claim 1, characterized in that, The first and second inlet channels are curved injection channels with the number of bends N≥2 (and preferably an even number); For example, the first inlet channel and the second inlet channel each have two bends and three straight sections, which are connected in the order of the first straight section, the first bend, the second straight section, the second bend, and the third straight section. The first straight section is also connected to the bottom liquid outlet of the first liquid storage chamber, and the third straight section is also connected to the only liquid inlet of the mixing channel. And / or, the cross-section of the first inlet channel and the second inlet channel is circular or square; And / or, the ratio of the cross-sectional area of ​​the first inlet channel to the cross-sectional area of ​​the second inlet channel is (1.5 to 9):

1.

4. The microfluidic chip according to claim 1, characterized in that, The mixing channel has a mixed flow channel consisting of alternating horizontal and vertical flow channels; And / or, the number of the transverse flow channels is 10 to 30; And / or, the lateral length of the transverse flow channel is 5.2 mm to 10.4 mm; And / or, the interior of the transverse flow channel is provided with one or more fishbone-shaped structures; And / or, the mixing channel also has a non-mixing channel communicating with the mixing channel, disposed below the mixing channel, serving as the bottom sample outlet of the microfluidic chip; Preferably, the height ratio of the mixed flow channel to the non-mixed flow channel is 1:2 to 2:

1.

5. The microfluidic chip according to claim 1, characterized in that, The height ratio of the mixing channel to the microfluidic chip is 1:4 to 1:2; And / or, the height ratio of the inlet channel (first inlet channel or second inlet channel) to the mixing channel is 6:20 to 9:20; And / or, the height of the microfluidic chip is 50mm to 70mm; And / or, the microfluidic chip is made of polymer material.

6. An LNP synthesis module, characterized in that, The LNP synthesis module includes the microfluidic chip according to any one of claims 1-5; Preferably, the LNP synthesis module further includes an upper-layer module and a lower-layer module; The upper module is provided with a chip slot for assembling the microfluidic chip; The lower module is provided with a sample receiving slot, which corresponds to the bottom sample outlet of the microfluidic chip; The upper and lower modules are detachable; And / or, the LNP synthesis module further includes a robotic arm gripper edge, disposed at the edge of the upper module and / or the lower module; And / or, the lower module is further provided with a base coupling groove and a base coupling column connected to the base coupling groove.

7. A system for preparing an mRNA product (e.g., an mRNA vaccine and / or a drug), characterized in that, The preparation system includes the microfluidic chip according to any one of claims 1-5 or the LNP synthesis module according to claim 6; Preferably, the preparation system includes a pipetting workstation and peripheral devices, which are connected by connectors. The peripheral equipment includes, but is not limited to, PCR instruments, centrifuges, CO2 incubators, microplate readers, consumable stacks, and plate washers; The connector includes three guide rails and a robotic arm.

8. The mRNA preparation system according to claim 7, characterized in that, The pipetting workstation includes a main unit and a mechanical gripper; The main unit of the pipetting workstation includes one or more pipettes and plate positions; The mechanical gripper is mounted on the pipette; And / or, the main unit of the pipetting workstation is equipped with a pipette tip cleaning station, and / or, the microplate reader, PCR instrument and plate washer are located near the main unit of the pipetting workstation; And / or, an oscillating incubator is provided in the middle of the main unit of the pipetting workstation; And / or, the consumable stack is connected to the main unit of the pipetting workstation via the first guide rail, the CO2 incubator is connected to the main unit of the pipetting workstation via the second guide rail, and the centrifuge is connected to the main unit of the pipetting workstation via the third guide rail and the robotic arm; And / or, all three guide rails are linear shuttle guide rails, and / or, the robotic arm is a Cartesian coordinate cooperative robotic arm. And / or, barcode scanners are also installed on one or more guide rails, which can scan the barcodes on the consumables and match them with the data results.

9. A high-throughput method for synthesizing LNPs, characterized in that, The method includes using the microfluidic chip according to any one of claims 1-5.

10. The use of the LNP synthesis module of claim 6 or the preparation system of any one of claims 7-8 in the preparation of mRNA products (e.g., mRNA vaccines and / or drugs).

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