Microfluidic chip and application thereof

Abstract

The application provides a micro-fluidic chip and application thereof. The micro-fluidic chip comprises a substrate and a cover plate which is attached to the substrate; the substrate is provided with a liquid inlet channel and a curved mixing channel which are connected in sequence, and an outlet is formed in the substrate and communicates with an output end of the curved mixing channel; a stopper is arranged at the bottom of an inner cavity of the liquid inlet channel; a first hole and a second hole are formed in the cover plate, the bottom end aperture of the first hole is larger than that of the second hole, and the first hole and the second hole both communicate with the liquid inlet channel; the first hole, the stopper and the second hole are arranged in sequence along the flow direction of the liquid. The micro-fluidic chip can ensure that the materials are uniformly mixed within several milliseconds to tens of milliseconds, and can ensure high mixing effect for operating conditions within a wide flow range.

Description

Technical Field

[0001] This invention belongs to the field of chip technology and relates to a microfluidic chip and its applications. Background Technology

[0002] Liposomes are bilayer vesicle structures composed of lipid molecules, similar in structure to biological membranes, and possess both hydrophilic and hydrophobic properties. Utilizing this property, hydrophobic drugs can be encapsulated in the hydrophobic regions within the lipid bilayer, while hydrophilic drugs can be encapsulated in the hydrophilic regions inside the liposome. Antibody proteins can bind to the liposome surface, exhibiting targeting capabilities. Drugs encapsulated in liposomes possess cell affinity and tissue compatibility; therefore, unstable and easily degraded mRNA molecules can be successfully delivered into the cytoplasm after encapsulation using liposomes, thus greatly advancing the development of mRNA vaccines and drugs.

[0003] Liposome preparation methods include thin-film hydration, extrusion, and homogenization. Among these, microfluidic technology is currently the most advanced method due to its ease of operation, high-throughput screening, low sample consumption, high controllability of liposome particle size, good reproducibility, and ease of scale-up. The contact and mixing structure of materials on a microfluidic chip is crucial to the chip's performance. For mRNA liposome preparation, the mixing time after the lipid and aqueous phases come into contact is extremely short; generally, mixing within milliseconds is necessary to obtain liposomes with high encapsulation efficiency and uniform particle size.

[0004] CN217341433U discloses a microfluidic chip for synthesizing mRNA lipid nanoparticles, comprising a shell, with injection ports fixed at both the front and rear ends of the left side of the top outer wall of the shell, and a drain port fixed at the center of the right side of the top outer wall of the shell. The inner wall of the shell is provided with a mixing component, which includes an injection chamber, a delivery tank, a mixing chamber, a storage tank, and a mixing tube. The injection chamber is located at the front and rear ends of the left side of the inner wall of the shell, and the bottom end of the injection port communicates with the top end of the injection chamber, using a Y-shaped channel for injection.

[0005] The article "Advanced Microfluidic Technologies for Lipid Nano-Microsystems from Synthesis to Biological Application" (DOI:10.3390 / pharmaceutics14010141) lists commonly used mixed structures for liposome preparation, including T-type, Y-type, and fishbone-type structures. These structures are relatively simple, and although the equipment is easy to manufacture, the quality of liposome particles prepared using these structures is greatly affected by flow rate fluctuations, especially the quality of liposome particles prepared at low flow rates.

[0006] Therefore, there is an urgent need to develop a microfluidic chip that can adapt to a wide flow range and complete material mixing in a short time. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a microfluidic chip and its applications. The mixing structure of the present invention enhances the interfacial contact between the lipid and aqueous phases, facilitating subsequent diffusion of interphase components. Simultaneously, the lipid and aqueous phases can form a secondary flow perpendicular to the flow direction within the tortuous mixing channel, further improving the mixing efficiency of the two phases. This ensures uniform mixing of materials within milliseconds to tens of milliseconds, and maintains high mixing performance under operating conditions over a wide flow range, producing high-quality liposome particles.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a microfluidic chip, the microfluidic chip comprising a substrate and a cover plate attached to the substrate;

[0010] The substrate is provided with an inlet channel and a curved mixing channel connected in sequence. The substrate has an outlet that is connected to the output end of the curved mixing channel. A baffle is provided at the bottom of the inner cavity of the inlet channel.

[0011] The cover plate has a first hole and a second hole. The bottom diameter of the first hole is larger than that of the second hole. Both the first hole and the second hole are connected to the liquid inlet channel. The first hole, the baffle and the second hole are arranged in sequence along the liquid flow direction.

[0012] This invention provides a microfluidic chip with a baffle in its inlet channel that divides the aqueous phase injected through the first orifice into two solutions. These two solutions sandwich the lipid phase injected through the second orifice, allowing the lipid and aqueous phases to contact at a large interface, which facilitates subsequent diffusion of interphase components. Simultaneously, the lipid and aqueous phases can form a secondary flow perpendicular to the flow direction within the tortuous mixing channel, further improving the mixing efficiency. This mixing structure ensures uniform mixing of materials within milliseconds to tens of milliseconds and maintains high mixing performance under operating conditions over a wide flow range, producing high-quality liposome particles. Furthermore, this structure is easily scalable, thus meeting high-volume processing requirements.

[0013] In this invention, the microfluidic chip can be modularly combined. For example, multiple microfluidic chips can be connected in series, or multiple microfluidic chips can be connected in series and in parallel, depending on the actual process.

[0014] Preferably, the liquid inlet channel is a liquid inlet groove recessed in the substrate.

[0015] Preferably, the curved mixing channel is a curved mixing groove recessed into the substrate.

[0016] Preferably, the holes and channels on the cover plate and the substrate are completed by high-precision machining or laser engraving.

[0017] Preferably, the curved mixing channel is an S-shaped mixing channel.

[0018] Preferably, the curved mixing channel includes at least one unit curved channel, such as 1, 2, 5, 8, 10, 20, 30, 40, 50, 60, 80, 100, 120 or 150, etc., preferably 1-100.

[0019] Preferably, the shape of the unit curved flow channel includes a semi-ellipse or a semi-circle.

[0020] Preferably, the ratio of the major axis of the semi-ellipse to the width of the curved mixing channel is 1-2, for example, it can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0021] Preferably, the eccentricity of the semi-ellipse is 0-0.95 and is not 0, for example, it can be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 0.95, etc.

[0022] It should be noted that the major axis of the semi-ellipse refers to the radius of the middle semi-ellipse, which is located between the two semi-ellipses of the semi-elliptical unit curved flow channel, and the distance between the middle semi-ellipse and the two semi-ellipses is equal. The eccentricity of the semi-ellipse is defined similarly.

[0023] Preferably, the ratio of the radius of the semicircle to the width of the curved mixing channel is 1-1.5, for example, it can be 1, 1.1, 1.2, 1.3, 1.4 or 1.5, etc.

[0024] It should be noted that the radius of the semicircle refers to the radius of the middle semicircle, which is located between the two semicircles of the semicircular unit curved flow channel, and the distance between the middle semicircle and the two semicircles is equal.

[0025] Preferably, the width of the curved mixing channel and the width of the liquid inlet channel are each independently 0.1-5 mm, for example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.7 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm or 5 mm, preferably 0.1-1 mm, and more preferably 0.1-0.4 mm.

[0026] Preferably, the width of the curved mixing channel is equal to the width of the liquid inlet channel.

[0027] Preferably, the ratio of the depth to the width of the curved mixing channel is 0.5-1.2, for example, it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1 or 1.2.

[0028] Preferably, the ratio of the depth to the width of the liquid inlet channel is 0.5-1.2, for example, it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1 or 1.2, etc.

[0029] Preferably, the first hole is a variable diameter hole, and the ratio of the top diameter to the bottom diameter of the first hole is 1.8-2.5, for example, it can be 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4 or 2.5, etc.

[0030] Preferably, the bottom diameter of the first hole is equal to the width of the liquid inlet channel.

[0031] Preferably, the top end of the first hole is connected to an injection line.

[0032] Preferably, the first hole includes a first equal diameter section and a second equal diameter section, and a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 45°-60°, for example, it can be 45°, 47°, 50°, 52°, 55°, 57° or 60°.

[0033] Preferably, the second hole is a variable diameter hole, and the ratio of the top diameter to the bottom diameter of the second hole is 1.8-2.5, for example, it can be 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4 or 2.5, etc.

[0034] Preferably, the bottom diameter of the second hole is equal to the width of the stop.

[0035] Preferably, the ratio of the distance between the bottom end of the second hole and the bottom surface of the liquid inlet channel to the height of the liquid inlet channel is 0-0.5, for example, it can be 0, 0.1, 0.2, 0.3, 0.4 or 0.5, etc.

[0036] Preferably, the portion of the second hole located within the liquid inlet channel is in close contact with the baffle.

[0037] Preferably, the top end of the second hole is connected to a liquid inlet pipe, and a one-way valve is provided on the liquid inlet pipe.

[0038] In this invention, a one-way valve is provided to prevent the grease phase from being unable to enter the chip when the flow ratio of the aqueous phase to the grease phase is too large.

[0039] Preferably, the second hole includes a first equal diameter section and a second equal diameter section, and a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 45°-60°, for example, it can be 45°, 47°, 50°, 52°, 55°, 57° or 60°.

[0040] Preferably, the line connecting the center of the first hole and the center of the second hole lies in the same plane as the axis of the stop.

[0041] Preferably, the line connecting the center of the first hole and the center of the second hole is parallel to the length direction of the liquid inlet channel.

[0042] Preferably, the ratio of the width of the baffle to the width of the liquid inlet channel is 0.1-0.9, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, and preferably 0.4-0.6.

[0043] Preferably, the height of the baffle is equal to the depth of the liquid inlet channel.

[0044] Preferably, bolt structures are provided at all four corners of the cover plate and the base plate. The cover plate and the base plate are pressed together by the bolt structures.

[0045] Preferably, the material of the cover plate and the material of the substrate each independently include a metallic material or a non-metallic material.

[0046] Preferably, the cover plate is made of the same material as the substrate.

[0047] Preferably, the metal material includes stainless steel, and more preferably 304 stainless steel or 316 stainless steel.

[0048] Preferably, the non-metallic material includes a polymer or glass, and more preferably polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA).

[0049] In addition to being fastened with bolts at the four corners, the cover plate and substrate can be further sealed using different methods depending on the substrate material. For example, when both the cover plate and substrate are made of metal, a groove is machined around the material mixing area (including the inlet channel and the curved mixing channel) and the bolt holes on the cover plate, and a corresponding boss is machined on the substrate, forming a sealing structure with a convex-concave surface fit. The flatness requirement for the contact surfaces of the cover plate and substrate is no higher than 0.8 μm.

[0050] In addition to the aforementioned concave-convex surface sealing, gasket sealing can also be used. This involves machining a groove around both the cover plate and the base plate, placing a gasket within the groove, and then tightening the upper and lower plates (cover plate and base plate) with bolts to achieve a seal. Non-metallic materials are preferred for the gaskets, such as polytetrafluoroethylene (PTFE) gaskets or polypropylene (PP) gaskets.

[0051] When both the cover plate and the substrate are made of non-metallic materials, the upper and lower plates (cover plate and substrate) can be sealed by bonding technology, preferably hot-press bonding or oxygen plasma surface treatment bonding.

[0052] In a second aspect, the present invention provides a method for preparing mRNA liposomes using the microfluidic chip described in the first aspect, the method comprising:

[0053] mRNA buffer (aqueous phase) and lipid organic solution (lipid phase) are injected into the inlet channel through the first and second wells, respectively. The mRNA buffer is divided into two solutions by the baffle. The two solutions, along with the lipid organic solution, enter the curved mixing channel and mix to obtain the mRNA liposomes.

[0054] The preparation method of this invention is a continuous operation process, which is highly efficient, produces products with good consistency, and is economical.

[0055] Preferably, the total flow rate of the mRNA buffer and the lipid organic solution is 0.5-2500 mL / min, for example, it can be 0.5 mL / min, 1 mL / min, 2 mL / min, 3 mL / min, 5 mL / min, 7 mL / min, 9 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 80 mL / min, 100 mL / min, 200 mL / min, 500 mL / min, 800 mL / min, 1000 mL / min, 1200 mL / min, 1500 mL / min, 1800 mL / min, 2000 mL / min, 2200 mL / min or 2500 mL / min, etc., preferably 0.5-100 mL / min, and more preferably 0.5-10 mL / min.

[0056] Preferably, the flow rate ratio of the lipid organic liquid to the mRNA buffer is 1:(1-10), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc.

[0057] In this invention, when preparing mRNA liposomes using the microfluidic chip, a higher encapsulation rate and a more uniform nanoparticle size can be obtained by adjusting process parameters such as the flow ratio of the lipid organic liquid and the mRNA buffer solution and the total flow rate.

[0058] Preferably, the time required to prepare the mRNA liposome is 1-100ms, for example, it can be 1ms, 2ms, 5ms, 10ms, 20ms, 30ms, 40ms, 50ms, 60ms, 70ms, 80ms, 90ms or 100ms.

[0059] Preferably, in the method for preparing the mRNA liposomes, the pressure drop of the microfluidic chip is less than or equal to 5 MPa, for example, it can be 4.5 MPa, 4 MPa, 3.5 MPa, 3 MPa or 2.5 MPa, etc.

[0060] Preferably, multiple microfluidic chips are connected in series and / or in parallel, which is applicable to the mixing of multiple materials. For example, when three materials are mixed, two microfluidic chips can be connected in series, referred to as chip one and chip two respectively; after material 1 and material 2 are mixed through chip one, they flow out from the outlet and then flow into the inlet (first hole or second hole) of chip two, while material 3 flows in from another inlet and completes a second mixing in chip two. Finally, the mixture of the three materials flows out from the outlet of chip two.

[0061] For example, when six materials are mixed, five microfluidic chips connected in series and parallel can be used, denoted as chip one, chip two, chip three, chip four, and chip five, respectively. Among them, chip two, chip three, and chip four are connected in parallel, and chip one, the three parallel chips, and chip five are connected in series. Material 1 and material 2 are injected into chip one respectively, and after mixing, a mixture is obtained. The mixture is then divided into three streams and injected into chip two, chip three, and chip four respectively. At the same time, material 3, material 4, and material 5 are also injected into chip two, chip three, and chip four respectively. The solution after mixing in chip two, chip three, and chip four is injected into the same inlet of chip five. At the same time, material 6 is also injected into chip five for mixing. Finally, the mixture of the six materials flows out from the outlet of chip five.

[0062] Thirdly, the present invention provides an mRNA liposome, which is prepared by the method described in the second aspect.

[0063] Preferably, the particle size of the mRNA liposomes is 50-200nm, for example, it can be 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 120nm, 150nm, 170nm or 200nm, etc.

[0064] Preferably, the encapsulation rate of the mRNA liposomes is 20-100%, for example, it can be 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, etc., preferably 70-80%.

[0065] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0066] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0067] This invention provides a microfluidic chip with a baffle in its inlet channel that divides the aqueous phase injected through the first orifice into two solutions. These two solutions sandwich the lipid phase injected through the second orifice, allowing the lipid and aqueous phases to contact at a large interface, which facilitates subsequent diffusion of interphase components. Simultaneously, the lipid and aqueous phases can form a secondary flow perpendicular to the flow direction within the tortuous mixing channel, further improving the mixing efficiency. This mixing structure ensures uniform mixing of materials within milliseconds to tens of milliseconds and maintains high mixing performance under operating conditions over a wide flow range, producing high-quality liposome particles. Furthermore, this structure is easily scalable, thus meeting high-volume processing requirements. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of the microfluidic chip provided in Embodiment 1 of the present invention;

[0069] Figure 2-4 These are, respectively, a front view, a top view, and a left view of the microfluidic chip provided in Embodiment 1 of the present invention;

[0070] Figure 5 This is a schematic diagram of a partially curved mixing channel in the microfluidic chip provided in Embodiment 1 of the present invention;

[0071] Figure 6 A schematic diagram of the sealing structure of the microfluidic chip provided in Embodiment 1 of the present invention;

[0072] Figure 7 This is a partially enlarged schematic diagram of the sealing structure of the microfluidic chip provided in Embodiment 1 of the present invention;

[0073] Figure 8A schematic diagram of the microfluidic chip provided in Embodiment 2 of the present invention;

[0074] Figure 9 This is a schematic diagram of a partially curved mixing channel in the microfluidic chip provided in Embodiment 2 of the present invention;

[0075] Figure 10 This is a partially enlarged schematic diagram of the sealing structure of the microfluidic chip provided in Embodiment 2 of the present invention;

[0076] Among them, 1-cover plate; 2-base plate; 3-bolt hole; 4-first hole; 5-second hole; 6-outlet; 7-curved mixing channel; 8-liquid inlet channel; 9-stop block; 10-concave-convex mating sealing structure. Detailed Implementation

[0077] It should be understood that in the description of this invention, the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0078] It should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "set," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0079] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0080] Example 1

[0081] This embodiment provides a microfluidic chip, such as Figure 1-4 As shown, it includes a substrate 2 and a cover plate 1 attached to the substrate 2. The material of the cover plate 1 and the material of the substrate 2 are both 316L stainless steel.

[0082] The substrate 2 is provided with a liquid inlet channel 8 and a curved mixing channel 7 connected in sequence. Both channels have a width of 2.5 mm and a depth of 2 mm. An outlet 6 is provided on the substrate 2, which is connected to the output end of the curved mixing channel 7. The curved mixing channel 7 is an S-shaped mixing channel, comprising four semi-circular curved unit channels with a radius of 2.5 mm, such as... Figure 5 As shown;

[0083] A baffle 9 is provided at the bottom of the inner cavity of the liquid inlet channel 8. The width of the baffle 9 is 1 mm, and the height of the baffle 9 is equal to the depth of the liquid inlet channel 8.

[0084] The cover plate 1 has a first hole 4 and a second hole 5, both of which are connected to the liquid inlet channel 8. The first hole 4 is a variable diameter hole, with a top diameter to bottom diameter ratio of 2. The bottom diameter of the first hole 4 is equal to the width of the liquid inlet channel 8. The first hole 4 includes a first equal diameter section and a second equal diameter section, as well as a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 60°.

[0085] The second hole 5 is a variable diameter hole, with a top diameter to bottom diameter ratio of 2. The bottom diameter of the second hole 5 is equal to the width of the stop block 9. The distance between the bottom of the second hole 5 and the bottom surface of the liquid inlet channel 8 is 1 mm. The portion of the second hole 5 located inside the liquid inlet channel 8 is in close contact with the stop block 9. The second hole 5 includes a first equal diameter section and a second equal diameter section, as well as a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 60°.

[0086] The first hole 4, the baffle 9, and the second hole 5 are arranged sequentially along the direction of liquid flow. The line connecting the center of the first hole 4 and the center of the second hole 5 is in the same plane as the axis of the baffle 9, and the line connecting the center of the first hole 4 and the center of the second hole 5 is parallel to the length direction of the liquid inlet channel 8.

[0087] Bolt holes 3 are provided at the four corners of both the cover plate 1 and the base plate 2, and are fastened with bolts. A sealing structure is provided between the liquid inlet channel 8 and the curved mixing channel 7 and the bolt holes 3. A groove is machined on the cover plate 1, and a boss is machined at the corresponding position on the base plate 2 to form a concave-convex fitting sealing structure 10. Figure 6 and Figure 7 As shown.

[0088] Example 2

[0089] This embodiment provides a microfluidic chip, such as Figure 8 As shown, it includes a substrate 2 and a cover plate 1 attached to the substrate 2. The material of the cover plate 1 and the material of the substrate 2 are both 316L stainless steel.

[0090] The substrate 2 is provided with a liquid inlet channel 8 and a curved mixing channel 7 connected in sequence. Both channels have a width of 1 mm and a depth of 0.8 mm. An outlet 6 is provided on the substrate 2, which is connected to the output end of the curved mixing channel 7. The curved mixing channel 7 is an S-shaped mixing channel, comprising four semi-elliptical unit curved channels. The major axis of each semi-elliptical unit curved channel is 1.5 mm, and the minor axis is 0.75 mm. Figure 9 As shown;

[0091] A baffle 9 is provided at the bottom of the inner cavity of the liquid inlet channel 8. The width of the baffle 9 is 0.4 mm, and the height of the baffle 9 is equal to the depth of the liquid inlet channel 8.

[0092] The cover plate 1 has a first hole 4 and a second hole 5, both of which are connected to the liquid inlet channel 8. The first hole 4 is a variable diameter hole, with a top diameter to bottom diameter ratio of 2. The bottom diameter of the first hole 4 is equal to the width of the liquid inlet channel 8. The first hole 4 includes a first equal diameter section and a second equal diameter section, as well as a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 60°.

[0093] The second hole 5 is a variable diameter hole, with a top diameter to bottom diameter ratio of 2. The bottom diameter of the second hole 5 is equal to the width of the stop block 9. The distance between the bottom of the second hole 5 and the bottom surface of the liquid inlet channel 8 is 1 mm. The portion of the second hole 5 located inside the liquid inlet channel 8 is in close contact with the stop block 9. The second hole 5 includes a first equal diameter section and a second equal diameter section, as well as a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 60°.

[0094] The first hole 4, the baffle 9, and the second hole 5 are arranged sequentially along the direction of liquid flow. The line connecting the center of the first hole 4 and the center of the second hole 5 is in the same plane as the axis of the baffle 9, and the line connecting the center of the first hole 4 and the center of the second hole 5 is parallel to the length direction of the liquid inlet channel 8.

[0095] Bolt holes 3 are provided at each of the four corners of the cover plate 1 and the base plate 2, and are fastened with bolts. A sealing structure is provided between the liquid inlet channel 8 and the curved mixing channel 7 and the bolt holes 3; that is, a groove is machined on both the cover plate 1 and the base plate 2, and a polytetrafluoroethylene (PTFE) gasket is placed in the groove. Figure 10 As shown, a sealing effect is achieved by tightening the bolts.

[0096] Example 3

[0097] This embodiment provides a microfluidic chip, including a substrate and a cover plate attached to the substrate. The material of the cover plate and the material of the substrate are both polydimethylsiloxane (PDMS).

[0098] The substrate is provided with a liquid inlet channel and a curved mixing channel connected in sequence. Both channels have a width of 0.6 mm and a depth of 0.4 mm. An outlet is provided on the substrate, and the outlet is connected to the output end of the curved mixing channel. The curved mixing channel is an S-shaped mixing channel, which includes four semi-circular unit curved channels with a radius of 0.6 mm.

[0099] A baffle is provided at the bottom of the inner cavity of the liquid inlet channel. The width of the baffle is 0.2 mm, and the height of the baffle is equal to the depth of the liquid inlet channel.

[0100] The cover plate has a first hole and a second hole, both of which are connected to the liquid inlet channel. The first hole is a variable diameter hole, with a top diameter to bottom diameter ratio of 2. The bottom diameter of the first hole is equal to the width of the liquid inlet channel. The first hole includes a first equal diameter section and a second equal diameter section, as well as a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 60°.

[0101] The second hole is a variable diameter hole, with a top diameter to bottom diameter ratio of 2. The bottom diameter of the second hole is equal to the width of the stop block. The distance between the bottom of the second hole and the bottom surface of the liquid inlet channel is 1 mm. The portion of the second hole located inside the liquid inlet channel is in close contact with the stop block. The second hole includes a first equal diameter section and a second equal diameter section, as well as a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section. The angle between the inclined surface of the variable diameter transition section and the radial section is 60°.

[0102] The first hole, the baffle, and the second hole are arranged sequentially along the direction of liquid flow. The line connecting the center of the first hole and the center of the second hole is in the same plane as the axis of the baffle, and the line connecting the center of the first hole and the center of the second hole is parallel to the length direction of the liquid inlet channel.

[0103] Bolt holes are provided at the four corners of the cover plate and the base plate, and they are fastened with bolts. A sealing structure is formed between the liquid inlet channel and the curved mixing channel and the bolt holes by thermo-press bonding.

[0104] Application Example 1

[0105] This application example provides a method for preparing mRNA liposomes using the microfluidic chip of Example 3, the method comprising:

[0106] mRNA buffer and lipid organic solution were injected into the inlet channel through the first and second wells, respectively. The total flow rate of mRNA buffer and lipid organic solution was 4 mL / min, and the flow rate ratio of lipid organic solution to mRNA buffer was 1:3. The mRNA buffer was divided into two solutions by the baffle. The two solutions, along with the lipid organic solution, entered the curved mixing channel and were mixed to obtain the mRNA liposomes. The preparation time of mRNA liposomes was 25 ms, and the pressure drop of the microfluidic chip was 8 kPa.

[0107] The mRNA liposomes prepared by the above method have a particle size of 50-200 nm and an encapsulation efficiency of 75%.

[0108] Application Example 2

[0109] This application example provides a method for preparing mRNA liposomes using the microfluidic chip of Example 2, the method comprising:

[0110] mRNA buffer and lipid organic solution were injected into the inlet channel through the first and second wells, respectively. The total flow rate of mRNA buffer and lipid organic solution was 50 mL / min, and the flow rate ratio of lipid organic solution to mRNA buffer was 1:10. The mRNA buffer was divided into two solutions by the baffle. The two solutions, along with the lipid organic solution, entered the curved mixing channel and were mixed to obtain the mRNA liposomes. The preparation time of mRNA liposomes was 12 ms, and the pressure drop of the microfluidic chip was 16 kPa.

[0111] The mRNA liposomes prepared by the above method have a particle size of 50-200 nm and an encapsulation efficiency of 70%.

[0112] Application Example 3

[0113] This application example provides a method for preparing mRNA liposomes using the microfluidic chip of Example 1, the method comprising:

[0114] mRNA buffer and lipid organic solution were injected into the inlet channel through the first and second wells, respectively. The total flow rate of mRNA buffer and lipid organic solution was 1000 mL / min, and the flow rate ratio of lipid organic solution to mRNA buffer was 1:5. The mRNA buffer was divided into two solutions by the baffle. The two solutions, along with the lipid organic solution, entered the curved mixing channel and were mixed to obtain the mRNA liposomes. The preparation time of mRNA liposomes was 2 ms, and the pressure drop of the microfluidic chip was 5 MPa.

[0115] The mRNA liposomes prepared by the above method have a particle size of 50-200 nm and an encapsulation efficiency of 70%.

[0116] In summary, the microfluidic chip provided by this invention features a baffle within its inlet channel that divides the aqueous phase injected through the first orifice into two solutions. These two solutions can sandwich the lipid phase injected through the second orifice, allowing the lipid and aqueous phases to contact at a large interface, which facilitates subsequent diffusion of interphase components. Simultaneously, the lipid and aqueous phases can form a secondary flow perpendicular to the flow direction within the tortuous mixing channel, further improving the two-phase mixing efficiency. This mixing structure ensures uniform mixing of materials within milliseconds to tens of milliseconds and maintains high mixing efficiency under operating conditions over a wide flow range, producing high-quality liposome particles. Furthermore, this structure is easily scalable to meet high throughput requirements and can be modularly combined to mix multiple material streams.

[0117] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A microfluidic chip for producing high-quality liposome particles, characterized in that, The microfluidic chip includes a substrate and a cover plate attached to the substrate; The substrate is provided with a liquid inlet channel and a curved mixing channel connected in sequence, the width of the curved mixing channel being equal to the width of the liquid inlet channel; An outlet is provided on the substrate, and the outlet is connected to the output end of the curved mixing channel; a baffle is provided at the bottom of the inner cavity of the liquid inlet channel; The cover plate has a first hole and a second hole. The first hole is a variable diameter hole, and the ratio of the top diameter to the bottom diameter of the first hole is 1.8-2.

5. The second hole is a variable diameter hole, and the ratio of the top diameter to the bottom diameter of the second hole is 1.8-2.

5. The bottom diameter of the first hole is larger than that of the second hole, and both the first hole and the second hole are connected to the liquid inlet channel; the first hole, the baffle and the second hole are arranged sequentially along the liquid flow direction; The portion of the second hole located within the liquid inlet channel is in close contact with the baffle. The line connecting the center of the first hole and the center of the second hole lies in the same plane as the axis of the stop block; The first hole is used for injecting the aqueous phase; The second hole is used for injecting the lipid phase; The lipid phase and the aqueous phase can form a secondary flow perpendicular to the flow direction within the curved mixing channel; the ratio of the width of the baffle to the width of the inlet channel is 0.4-0.6, and the height of the baffle is equal to the depth of the inlet channel; The total flow rate of the aqueous phase and lipid phase is 0.5-2500 mL / min.

2. The microfluidic chip according to claim 1, characterized in that, The curved mixing channel is an S-shaped mixing channel.

3. The microfluidic chip according to claim 1, characterized in that, The curved mixing channel includes at least one unit curved channel.

4. The microfluidic chip according to claim 3, characterized in that, The curved mixing channel consists of 1-100 unit curved channels.

5. The microfluidic chip according to claim 3, characterized in that, The shape of the unit curved flow channel includes a semi-ellipse or a semi-circle.

6. The microfluidic chip according to claim 5, characterized in that, The ratio of the major axis of the semi-ellipse to the width of the curved mixing channel is 1-2.

7. The microfluidic chip according to claim 5, characterized in that, The eccentricity of the semi-ellipse is 0-0.95 and is not 0.

8. The microfluidic chip according to claim 5, characterized in that, The ratio of the radius of the semicircle to the width of the curved mixing channel is 1-1.

5.

9. The microfluidic chip according to claim 1, characterized in that, The width of the curved mixing channel and the width of the liquid inlet channel are each independently 0.1-5 mm.

10. The microfluidic chip according to claim 9, characterized in that, The width of the curved mixing channel and the width of the liquid inlet channel are each independently 0.1-1 mm.

11. The microfluidic chip according to claim 10, characterized in that, The width of the curved mixing channel and the width of the liquid inlet channel are each independently 0.1-0.4 mm.

12. The microfluidic chip according to claim 1, characterized in that, The depth-to-width ratio of the curved mixing channel is 0.5-1.

2.

13. The microfluidic chip according to claim 1, characterized in that, The ratio of the depth to the width of the inlet channel is 0.5-1.

2.

14. The microfluidic chip according to claim 1, characterized in that, The bottom diameter of the first hole is equal to the width of the liquid inlet channel.

15. The microfluidic chip according to claim 1, characterized in that, The first hole includes a first equal diameter section and a second equal diameter section, and a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section, wherein the angle between the inclined surface of the variable diameter transition section and the radial section is 45°-60°.

16. The microfluidic chip according to claim 1, characterized in that, The bottom diameter of the second hole is equal to the width of the stop block.

17. The microfluidic chip according to claim 1, characterized in that, The ratio of the distance between the bottom end of the second hole and the bottom surface of the liquid inlet channel to the height of the liquid inlet channel is 0-0.

5.

18. The microfluidic chip according to claim 1, characterized in that, The top of the second hole is connected to an inlet pipe, and a one-way valve is installed on the inlet pipe.

19. The microfluidic chip according to claim 1, characterized in that, The second hole includes a first equal diameter section and a second equal diameter section, and a variable diameter transition section disposed between the first equal diameter section and the second equal diameter section, wherein the angle between the inclined surface of the variable diameter transition section and the radial section is 45°-60°.

20. The microfluidic chip according to claim 1, characterized in that, The line connecting the center of the first hole and the center of the second hole is parallel to the length direction of the liquid inlet channel.

21. A method for preparing mRNA liposomes using the microfluidic chip according to any one of claims 1-20, characterized in that, The method includes: mRNA buffer and lipid organic solution are injected into the inlet channel through the first and second wells, respectively. The mRNA buffer is divided into two solutions by the baffle. The two solutions, along with the lipid organic solution, are mixed in the curved mixing channel to obtain the mRNA liposomes. The total flow rate of the mRNA buffer and the lipid organic solution is 0.5-2500 mL / min.

22. The method according to claim 21, characterized in that, The total flow rate of the mRNA buffer and the lipid organic solution is 0.5-100 mL / min.

23. The method according to claim 21, characterized in that, The total flow rate of the mRNA buffer and the lipid organic solution is 0.5-10 mL / min.

24. The method according to claim 21, characterized in that, The flow rate ratio of the lipid organic liquid to the mRNA buffer is 1:(1-10).

25. The method according to claim 21, characterized in that, The preparation time for the mRNA liposomes is 1-100 ms.

26. The method according to claim 21, characterized in that, In the method for preparing the mRNA liposomes, the pressure drop of the microfluidic chip is less than or equal to 5 MPa.

27. An mRNA liposome, characterized in that, The mRNA liposomes are prepared by the method according to any one of claims 21-26.

28. The mRNA liposome according to claim 27, characterized in that, The particle size of the mRNA liposomes is 50-200 nm.

29. The mRNA liposome according to claim 27, characterized in that, The encapsulation rate of the mRNA liposomes is 20-100%.

30. The mRNA liposome according to claim 29, characterized in that, The encapsulation rate of the mRNA liposomes was 70-80%.

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