Micro-fluidic chip and micro-fluidic device

Abstract

The utility model relates to a micro-fluidic chip and a micro-fluidic device. The micro-fluidic chip comprises a cover plate and a substrate, and the substrate is provided with a front-end mixing unit, a rear-end mixing unit and a liquid outlet channel which are communicated in sequence. The micro-fluidic device comprises a tangential flow device, a liquid storage tank and one or at least two parallel micro-fluidic chips. The micro-fluidic chip disclosed by the utility model is provided with the annular asymmetric splitting-recombination unit, and fluid is subjected to continuous asymmetric splitting, extrusion and recombination, so that the mixing efficiency is improved; meanwhile, a plurality of gradients are arranged, feeding and mixing are performed in batches, turbulent flow of the fluid can be further increased, internal pressure is reduced, backflow of the liquid is avoided, and stable mixing of the fluid is better facilitated.

Description

Technical Field

[0001] The utility model relates to a microfluidic chip and a microfluidic device. Background Art

[0002] Microfluidics refers to the technology of precisely manipulating fluid flow in microchannels of different geometries and sizes (micrometers or millimeters). It has shown great application potential in medicine, physics, materials, mechanics, chemistry, energy and other disciplines.

[0003] Microfluidics has become a mainstream technology for lipid nanoparticle (LNP) manufacturing. It is an advanced method that utilizes microchannels to achieve controllable and efficient LNP preparation. By utilizing microchannels or microfluidic chips of varying structures, many technical bottlenecks in drug delivery have been overcome, such as the stable encapsulation and delivery of nucleic acid drugs. ONPATTRO (2018, Alnylam Pharmaceuticals, Inc., siRNA-LNP); COMIRNATY (2020, Pfizer-BioNTech, mRNA-LNP); and SPIKEVAX (2020, Moderna, mRNA-LNP) all benefited from microfluidics.

[0004] Particle size significantly impacts the stability and in vivo performance of LNPs. Therefore, microfluidics' ability to control LNP size is an essential technical challenge in LNP applications. The World Health Organization (WHO) and the Food and Drug Administration (FDA) have both designated LNP size and distribution as critical quality attributes (CQAs) for mRNA vaccine products.

[0005] Currently, several microchannel structures, such as staggered herringbone mixers (SHMs) and toroidal mixers (TrMs), have been used to produce LNPs loaded with nucleic acid drugs. However, the limited accessibility of microchannels for clinical translation of LNPs still faces many challenges, such as throughput, applicability, or consistency. Utility Model Content

[0006] The technical problem to be solved by the present invention is to overcome the stability defect of LNPs intermediates after preparation in the prior art and provide a microfluidic chip and a microfluidic device. The present invention can achieve efficient, controllable and continuous preparation of LNPs.

[0007] The utility model solves the above technical problems through the following technical solutions:

[0008] In a first aspect, the present invention provides a microfluidic chip comprising a cover plate and a base plate, wherein the base plate is provided with a front mixing unit, a rear mixing unit and a liquid outlet channel that are connected in sequence;

[0009] The front mixing unit includes a front main channel and at least two front annular microchannels connected in series, which are connected in sequence; the rear mixing unit includes a rear main channel and one or at least two rear annular microchannels connected in series, which are connected in sequence; the end of the front annular microchannel is connected to the front end of the rear main channel; at least one rear branch channel is provided on the rear main channel; the end of the rear annular microchannel is connected to the liquid outlet channel;

[0010] The front annular microchannel includes a front main subchannel and a front secondary subchannel connected end to end to form a ring, the front end of the front main subchannel and the front end of the front secondary subchannel are connected to the front splitting point, the end of the front main subchannel and the end of the front secondary subchannel are connected to the front combination point, the first front splitting point is connected to the end of the front main channel, and the last front combination point is connected to the front end of the rear main channel; the cross-sectional area S of the front main subchannel is 121 Greater than the cross-sectional area S of the previous sub-channel 122 ;

[0011] The rear annular microchannel includes a rear main sub-channel and a rear secondary sub-channel connected end to end to form a ring, the front end of the rear main sub-channel and the front end of the rear secondary sub-channel are connected to the rear splitting point, the end of the rear main sub-channel and the end of the rear secondary sub-channel are connected to the rear combination point, the first rear splitting point is connected to the end of the rear main channel, and the last rear combination point is connected to the front end of the liquid outlet channel; the cross-sectional area S of the rear main sub-channel is 321 Greater than the cross-sectional area S of the rear sub-channel 322 .

[0012] In the present invention, the flow channel depth H1 of the front mixing unit may be smaller than the flow channel depth H3 of the rear mixing unit. Preferably, H1:H3=1:(1.5-6), more preferably, H1:H3=1:(2-4).

[0013] In the present invention, the number N1 of the front annular microchannels may be greater than the number N3 of the rear annular microchannels.

[0014] In the present invention, the cross-sectional area S of the front main channel is 11 It can be smaller than the cross-sectional area S of the rear main channel 31 .

[0015] In the present invention, the width W of the front main channel is 11 Can be smaller than the width W of the rear main channel 31 .

[0016] In the present invention, a liquid outlet is provided at one end of the liquid outlet channel away from the rear end annular microchannel.

[0017]

Front-end mixing unit

[0018] In the present invention, the m+1th front splitting point is the mth front combining point, and n is a positive integer.

[0019] In some embodiments, N1=2-6, preferably 3-5.

[0020] In the present invention, the depth H of the front main channel is 11 The depth H of the front annular microchannel can be 12 Equal, preferably equal to the flow channel depth H1 of the front mixing unit.

[0021] In some embodiments, H1=10-500 μm, preferably 50-200 μm.

[0022] In the present invention, the width W of the front main channel is 11 The width W of the front main channel can be 121 equal.

[0023] In certain embodiments, W 11 =10-500 μm, preferably 50-300 μm.

[0024] In some embodiments, the length L of the front main channel 11 =100-800 μm, preferably 200-600 μm.

[0025] In the present invention, the width W of the front main sub-channel is 121 and the width W of the previous subchannel 122 The ratio can be W 121 : W 122 =(1-4): 1, preferably, W 121 : W 122 =(1.5-3): 1.

[0026] In the present invention, the depth H of the front main sub-channel is 121 and the depth H of the previous subchannel 122 It can be equal to, and preferably equal to, the flow channel depth H1 of the front mixing unit.

[0027] In the present invention, the curvature radius R of the front main sub-channel is 121 and the curvature radius R of the previous sub-channel 122 The ratio can be R 121 : R 122 =(0.8-2): 1, preferably (0.9-1.5): 1, more preferably (1-1.3): 1.

[0028] In some embodiments, the curvature radius R of the front main sub-channel is 121 =200-800 μm, preferably 500-700 μm.

[0029] In some embodiments, the curvature radius R of the previous sub-channel is 122 =300-700 μm, preferably 400-600 μm.

[0030]

Backend mixing unit

[0031] In the present invention, the o+1th post-splitting point is the oth post-combining point, where o is a positive integer.

[0032] In some embodiments, N3=1-4, preferably 2-3.

[0033] In the present invention, the depth H of the rear main channel is 31 The depth H of the rear end annular microchannel can be 32 Equal, preferably equal to the flow channel depth H3 of the rear mixing unit.

[0034] In certain embodiments, H 31 =10-500 μm, preferably 50-400 μm.

[0035] In the present invention, the width W of the rear main channel is 31 It can be equal to the width of the rear main sub-channel W 321 .

[0036] In certain embodiments, W 31 =100-2000 μm, preferably 200-1000 μm.

[0037] In the present invention, the width W of the rear main sub-channel is 321 and the width W of the rear sub-channel 322 The ratio can be W 321 : W 322 =(1-4): 1, preferably, W 321 : W 322 =(1.5-3): 1.

[0038] In the present invention, the depth H of the rear main sub-channel is 321 and the depth H of the rear sub-channel 322 It can be equal to, and preferably equal to, the flow channel depth H3 of the rear mixing unit.

[0039] In some embodiments, the length L of the rear main channel 31 =2000-5000 μm, preferably 2500-3500 μm.

[0040] In the present invention, the curvature radius R of the rear main sub-channel is 321 and the curvature radius R of the rear sub-channel 322 The ratio can be R 321 : R 322 =(0.8-2): 1, preferably (0.9-1.5): 1, more preferably (1-1.3): 1.

[0041] In some embodiments, the curvature radius R of the rear main sub-channel is 321 =500-3000 μm, preferably 1000-2000 μm.

[0042] In some embodiments, the curvature radius R of the rear sub-channel is 322 =500-2000 μm, preferably 800-1500 μm.

[0043] In the present invention, the angle θ3 formed by the rear end main channel and the rear end branch channel at the intersection is 15°-90°, preferably 30°-90°.

[0044] In the present invention, the width W of the rear main channel is 31 Can be less than or equal to the width W of the rear end branch channel 33 Preferably, W 31 : W 33 =1: (1-5), more preferably, W 31 : W 33 =1: (1-3).

[0045] In the present invention, a fourth liquid inlet may be provided at one end of the rear end branch channel away from the rear end main channel.

[0046]

Liquid inlet channel

[0047] In the present invention, a liquid inlet channel may also be provided on the substrate, and the liquid inlet channel includes one or at least two parallel liquid inlet channels, and the ends of the liquid inlet channels are connected to the front end of the front main channel after merging at a confluence point.

[0048] Among them, the width W of the front main channel 11 It can be less than or equal to the minimum value of the width W0 of each liquid inlet channel. 11 : W0=1: (1-5), more preferably, W 11 : W0=1: (1-3).

[0049] In some embodiments, the liquid inlet channel includes a second liquid inlet channel and a first liquid inlet channel connected in parallel. The width W of the second liquid inlet channel is 01 and the width W of the first liquid inlet channel 02 The second liquid inlet channel and the first liquid inlet channel form an angle θ1 = 0-180°, preferably 45°-180°. The second liquid inlet channel is provided with a second liquid inlet at one end away from the front main channel. The first liquid inlet channel is provided with a first liquid inlet at one end away from the front main channel.

[0050] The depth H0 of the liquid inlet channel may be equal to the flow channel depth H1 of the front mixing unit.

[0051]

Mid-range hybrid unit

[0052] In the present invention, the substrate may further be provided with one or at least two middle-end mixing units connected in series, and the middle-end mixing unit is connected between the front-end mixing unit and the rear-end mixing unit.

[0053] The mid-end mixing unit may include a mid-end main channel and one or at least two mid-end annular microchannels connected in series, wherein the front end of the mid-end main channel is connected to the end of the front annular microchannel, and the end of the mid-end annular microchannel is connected to the front end of the rear main channel. At least one mid-end branch channel is provided on the mid-end main channel.

[0054] In some embodiments, the cross-sectional area S of the middle main channel is 21 It can be smaller than the cross-sectional area S of the rear main channel 31 , which may be larger than the cross-sectional area S of the front main channel 11 .

[0055] In some embodiments, the width W of the middle main channel is 21 Can be smaller than the width W of the rear main channel 31 , which may be greater than the width W of the front main channel 11 Preferably, W 21 : W 31 =1: (1.5-6), preferably 1: (2-4).

[0056] In some embodiments, the width W of the middle main channel is 21 =50-1000 μm, preferably 100-500 μm.

[0057] The flow channel depth H2 of the middle-end mixing unit may be equal to the flow channel depth H3 of the rear-end mixing unit.

[0058] In some embodiments, the depth H of the middle main channel is 21 The depth H of the mid-end annular microchannel can be 22 Equal, preferably equal to the flow channel depth H3 of the rear mixing unit.

[0059] The number N2 of the middle annular microchannels may be greater than or equal to the number N3 of the rear end annular microchannels, and less than the number N1 of the front end annular microchannels.

[0060] In some embodiments, N2=1-4, preferably 2-3.

[0061] In certain specific embodiments, the middle annular microchannel comprises a middle main subchannel and a middle secondary subchannel connected end to end to form a ring, the front end of the middle main subchannel and the front end of the middle secondary subchannel are connected at a middle splitting point, the end of the middle main subchannel and the end of the middle secondary subchannel are connected at a middle combination point, the first middle splitting point is connected to the end of the middle main channel, and the last middle combination point is connected to the front end of the rear main channel. The cross-sectional area S of the middle main subchannel is 221 Greater than the cross-sectional area S of the secondary sub-channel 222 .

[0062] In some embodiments, the width W of the middle main channel is 21 The width W of the main sub-channel can be 221 equal.

[0063] In some embodiments, the width W of the middle main sub-channel is 221 and the width W of the middle sub-channel 222 The ratio can be W 221 : W 222 =(1-4): 1, preferably, W 221 : W 222 =(1.5-3): 1.

[0064] In some embodiments, the depth H of the middle main sub-channel is 221 and the depth H of the middle sub-channel 222 It can be equal to, and preferably equal to, the flow channel depth H3 of the rear mixing unit.

[0065] In some embodiments, the length L of the middle main channel is 21 =1000-4000 μm, preferably 1500-3000 μm.

[0066] In some embodiments, the curvature radius R of the middle main sub-channel is 221 and the curvature radius R of the middle sub-channel 222 The ratio can be R 221 : R 222 =(0.8-2): 1, preferably (0.9-1.5): 1, more preferably (1-1.3): 1.

[0067] In some embodiments, the curvature radius R of the middle main sub-channel is 221 =800-1800 μm, preferably 1000-1500 μm.

[0068] In some embodiments, the curvature radius R of the middle sub-channel is 222 =300-1000 μm, preferably 500-800 μm.

[0069] In some specific embodiments, the angle θ2 formed by the middle main channel and the middle branch channel at the intersection is 15°-90°, preferably 30°-90°.

[0070] In some embodiments, the width W of the middle main channel is 21 Can be less than or equal to the width W of the middle branch channel 23 Preferably, W 31 : W 33 =1: (1-4), more preferably, W 31 : W 33 =1: (1-2).

[0071] In some specific embodiments, a third liquid inlet may be provided at one end of the middle branch channel away from the middle main channel.

[0072] In the present invention, the materials of the cover plate and the substrate are conventionally selected in the art, such as metal, silicon, quartz, ceramic, glass, Hastelloy or polymer. The metal can be stainless steel. The polymer can be polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyamide (PI), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), photoresist, polyester (TPE), polyethylene glycol diacrylate (PEGDA), perfluorinated compound, polyurethane (PU), cycloolefin copolymer (COC) or cycloolefin polymer (COP). The photoresist can be SU-8 photoresist. The perfluorinated compound can be polyperfluoroethylene propylene (PFEP), perfluoroalkoxy (PFA) or perfluoropolyether (PFPE).

[0073] In a second aspect, the present invention provides a microfluidic device comprising a tangential flow device, a liquid storage tank, and one or at least two microfluidic chips as described above connected in parallel;

[0074] The tangential flow device is provided with a second feed inlet, a discharge port and a waste liquid discharge port; the liquid storage tank is provided with a first feed inlet, a product discharge port, a circulation feed inlet and a circulation discharge port;

[0075] Each liquid outlet is connected to the first feed port, the circulation outlet is connected to the second feed port via an intermediate pipeline, and the outlet is connected to the circulation feed port.

[0076] In the present invention, conventionally, the tangential flow device comprises a hollow fiber column and a filter membrane disposed in the hollow fiber column. The pore size of the filter membrane may be 300K.

[0077] In the present invention, the first liquid inlet can be connected to the ethanol storage tank via a first liquid inlet pipeline. The first liquid inlet pipeline can be provided with a first feed pump.

[0078] In the present invention, the second liquid inlet can be connected to the buffer phase material storage tank via a second liquid inlet pipeline. The second liquid inlet pipeline can be provided with a second feed pump.

[0079] In the present invention, the third liquid inlet can be connected to the dilute phase material storage tank via a third liquid inlet pipeline. The third liquid inlet pipeline can be provided with a third feed pump.

[0080] In the present invention, the fourth liquid inlet can be connected to the dilute phase material storage tank via a fourth liquid inlet pipeline. The fourth liquid inlet pipeline can be provided with a fourth feed pump.

[0081] In the present invention, the product discharge port can be connected to the product storage tank via a product discharge pipeline. A filter can be provided on the product discharge pipeline.

[0082] In the present invention, a peristaltic pump may be provided on the intermediate pipeline.

[0083] In the utility model, the waste liquid discharge port can be connected to the waste liquid storage tank through a product waste liquid pipeline.

[0084] The positive progress effect of this utility model is:

[0085] (1) The microfluidic chip of the utility model is provided with a ring-shaped asymmetric splitting-recombining unit. The fluid undergoes continuous asymmetric splitting, squeezing and recombination, thereby improving the mixing efficiency of the fluid;

[0086] (2) The microfluidic chip of the present invention can induce asymmetric splitting with higher intensity by adjusting the width ratio of each main sub-channel and each secondary sub-channel, which can further improve the mixing efficiency of the fluid;

[0087] (3) The microfluidic chip of the present invention is equipped with multiple gradients and batch mixing, which can further increase the turbulence of the fluid, reduce the internal pressure, avoid the backflow of the liquid, and is more conducive to the stable mixing of the fluid. It can realize the functions of self-assembly and rapid dilution respectively. After the lipid and buffer phase are mixed, the intermediate can be diluted within 3 seconds;

[0088] (4) The microfluidic device of the utility model can realize the continuous and efficient preparation of LNPs, greatly improving the quality of the product. The prepared LNPs have small particle size, uniform distribution and good encapsulation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Schematic diagram of the microfluidic chip in Example 1;

[0090] Figure 2 Schematic diagram of the microfluidic device in the present invention;

[0091] As shown in the figure, the numbers are as follows: first liquid inlet 1, second liquid inlet 2, third liquid inlet 3, fourth liquid inlet 4, liquid outlet 5, second liquid inlet flow channel 6, first liquid inlet flow channel 7, front main channel 8, front main sub-channel 9, front sub-channel 10, front annular microchannel 11, middle main channel 12, middle branch channel 13, middle main sub-channel 14, middle sub-channel 15, middle annular microchannel 16, rear main channel 17, rear branch channel 18, rear main sub-channel 19, rear sub-channel 20, rear annular microchannel 21, liquid outlet channel 22, ethanol material storage tank 23, buffer phase material storage tank 24, dilution phase material storage tank 25, first feed pump 26, second feed pump 27, fourth feed pump 28, third feed pump 29, tangential flow device 31, product storage tank 32, waste liquid storage tank 33, peristaltic pump 34, liquid storage tank 35, filter 36. DETAILED DESCRIPTION

[0092] A preferred embodiment is given below, and the present invention is described more clearly and completely in conjunction with the accompanying drawings.

[0093] In the following examples and application examples, DLin-MC3-DMA was produced by Lipoid GmbH; DSPC was produced by Lipoid GmbH; cholesterol was produced by Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.; DMG-PEG2000 was produced by Lipoid GmbH; and eGFP-mRNA was produced by CanSino Biologics Co., Ltd. All DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 were injection-grade.

[0094] Example 1

[0095] A microfluidic chip, such as Figure 1 , including a cover plate and a base plate, the base plate is provided with a liquid inlet channel, a front mixing unit, a middle mixing unit, a rear mixing unit and a liquid outlet channel 22 which are sequentially connected. The base plate is made of PDMS and the cover plate is made of glass.

[0096] In this embodiment, the liquid inlet channel includes a second liquid inlet channel 6 and a first liquid inlet channel 7 connected in parallel. The ends of the two liquid inlet channels merge at a junction and then communicate with the front end of the front main channel 8. The second liquid inlet channel 6 has a second liquid inlet port 2 at the end away from the front main channel 8. The first liquid inlet channel 7 has a first liquid inlet port 1 at the end away from the front main channel 8.

[0097] In this embodiment, the width W of the first liquid inlet channel 7 is 01 =300 μm, length L 01 =14 mm, depth H 01 =100 μm. In this embodiment, the width W of the second liquid inlet channel 6 is 02 =300 μm, length L02 =14 mm, depth H 02 =100 μm. The angle θ1 formed by the second liquid inlet channel 6 and the first liquid inlet channel 7 is 60°.

[0098] In this embodiment, the front-end mixing unit includes a front-end main channel 8 and four front-end annular microchannels 11 connected in series. In this embodiment, the front-end annular microchannel 11 includes a front main subchannel 9 and a front secondary subchannel 10 connected end to end in a ring. The front ends of the front main subchannel 9 and the front secondary subchannel 10 are connected at a front splitting point, and the ends of the front main subchannel 9 and the ends of the front secondary subchannel 10 are connected at a front combining point. The first front splitting point is connected to the end of the front main channel 8, and the last front combining point is connected to the front end of the rear main channel 17.

[0099] In this embodiment, the width W of the front main channel 8 is 11 =200 μm, depth H 11 =100 μm, length L 11 =500 μm. In this embodiment, the width W of the front main sub-channel 9 is 121 =200 μm, depth H 121 =100 μm, radius of curvature R 121 =550 μm. In this embodiment, the width W of the front sub-channel 10 122 =80 μm, depth H 122 =100 μm, radius of curvature R 122 =485 μm.

[0100] In this embodiment, the mid-end mixing unit includes a mid-end main channel 12 and two mid-end annular microchannels 16 connected in series. The front end of the mid-end main channel 12 is connected to the end of the front annular microchannel 11, and the end of the mid-end annular microchannel 16 is connected to the front end of the rear main channel 17. A mid-end branch channel 13 is opened on the mid-end main channel 12. In this embodiment, the mid-end annular microchannel 16 includes a middle main sub-channel 14 and a middle secondary sub-channel 15 connected end to end to form a ring. The front end of the middle main sub-channel 14 and the front end of the middle secondary sub-channel 15 are connected to the middle splitting point, and the end of the middle main sub-channel 14 and the end of the middle secondary sub-channel 15 are connected to the middle combination point. The first middle splitting point is connected to the end of the mid-end main channel 12, and the last middle combination point is connected to the front end of the rear main channel 17. In this embodiment, a third liquid inlet 3 is opened at the end of the mid-end branch channel 13 away from the mid-end main channel 12.

[0101] In this embodiment, the width W of the middle main channel 12 is 21 =300 μm, depth H 21 =300 μm, length L 21 =2400 μm. In this embodiment, the width W of the middle branch channel 13 is23 =300 μm, depth H 21 =300 μm. In this embodiment, the width W of the middle main sub-channel 14 is 221 =300 μm, depth H 221 =300 μm, curvature radius R 221 =750 μm. In this embodiment, the width W of the middle sub-channel 15 is 222 =100 μm, depth H 222 =300 μm, curvature radius R 222 =650 μm. In this embodiment, the included angle θ2 formed at the intersection of the middle main channel 12 and the middle branch channel 13 is 60°.

[0102] In this embodiment, the rear-end mixing unit includes a rear-end main channel 17 and two rear-end annular microchannels 21 connected in series. The end of the front-end annular microchannel 11 is connected to the front end of the rear-end main channel 17. A rear-end branch channel 18 is provided on the rear-end main channel 17. The rear-end annular microchannel 21 includes a rear-end main sub-channel 19 and a rear-end secondary sub-channel 20 connected end to end to form a ring. The front ends of the rear-end main sub-channel 19 and the front ends of the rear-end secondary sub-channel 20 are connected to a rear splitting point, and the ends of the rear-end main sub-channel 19 and the ends of the rear-end secondary sub-channel 20 are connected to a rear combination point. The first rear splitting point is connected to the end of the rear-end main channel 17, and the last rear combination point is connected to the front end of the liquid outlet channel 22. In this embodiment, a fourth liquid inlet 4 is provided at the end of the rear-end branch channel 18 away from the rear-end main channel 17.

[0103] In this embodiment, the width W of the rear main channel 17 is 31 =800 μm, depth H 31 =300 μm, length L 31 =3200 μm. In this embodiment, the width W of the rear branch channel 18 is 33 =800 μm, depth H 31 =300 μm. In this embodiment, the width W of the rear main sub-channel 19 is 321 =800 μm, depth H 321 =300 μm, curvature radius R 321 =1400 μm. In this embodiment, the width W of the rear sub-channel 20 is 322 =270 μm, depth H 322 =300 μm, curvature radius R 322 =1135 μm. In this embodiment, the angle θ3 formed at the intersection of the rear main channel 17 and the rear branch channel 18 is 60°.

[0104] In this embodiment, a liquid outlet 5 is formed at one end of the liquid outlet channel 22 away from the rear annular microchannel 21. In this embodiment, the liquid outlet channel 22 has a width W4 = 800 μm, a depth H4 = 300 μm, and a length L4 = 10 mm.

[0105] Example 2

[0106] A microfluidic device, reference Figure 2 , comprising a tangential flow device 31, a liquid storage tank 35, and one or at least two parallel microfluidic chips as described above; the tangential flow device 31 is provided with a second feed port, a discharge port, and a waste liquid discharge port; the liquid storage tank 35 is provided with a first feed port, a product discharge port, a circulating feed port, and a circulating discharge port; each liquid discharge port 5 is connected to the first feed port, and the circulating discharge port is connected to the second feed port via an intermediate pipeline, the intermediate pipeline being provided with a peristaltic pump 34, and the discharge port is connected to the circulating feed port. In this embodiment, the tangential flow device 31 comprises a hollow fiber column and a filter membrane disposed in the hollow fiber column. The pore size of the filter membrane can be 300K.

[0107] In this embodiment, the first liquid inlet 1 is connected to the ethanol material storage tank 23 via a first liquid inlet pipeline. A first feed pump 26 is provided on the first liquid inlet pipeline. In this embodiment, the second liquid inlet 2 is connected to the buffer phase material storage tank 24 via a second liquid inlet pipeline. A second feed pump 27 is provided on the second liquid inlet pipeline. In this embodiment, the third liquid inlet 3 is connected to the dilute phase material storage tank 25 via a third liquid inlet pipeline. A third feed pump 29 is provided on the third liquid inlet pipeline. In this embodiment, the fourth liquid inlet 4 is connected to the dilute phase material storage tank 25 via a fourth liquid inlet pipeline. A fourth feed pump 28 is provided on the fourth liquid inlet pipeline. In this embodiment, the waste liquid discharge port is connected to the waste liquid storage tank 33 via a product waste liquid pipeline.

[0108] Application Example 1

[0109] The microfluidic device in Example 2 was used to prepare lipid nanoparticles (LNPs). The specific steps were as follows:

[0110] S1. Set and confirm relevant parameters: the total flow rate of the four feed pumps is 24 mL / min, and the volume flow rate ratio of the corresponding fluids of the first feed pump 26, the second feed pump 27, the third feed pump 29, and the fourth feed pump 28 is 1.5: 4.5: 5: 12;

[0111] S2. DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 were mixed and dissolved in anhydrous ethanol in a molar ratio of 46.3:9.4:42.7:1.6 in an ethanol material storage tank 23 as a first material; eGFP-mRNA was dissolved in a 50 mM citric acid buffer solution at pH 4.0 in a buffer phase material storage tank 24 as a second material; and DPBS (pH 7.4) buffer or Tris buffer solution was placed in a dilution phase material storage tank 25 as a third material. The mass ratio of DLin-MC3-DMA to eGFP-mRNA was 20:1.

[0112] S3, first start the third feed pump 29 and the fourth feed pump 28, pre-introduce the third material as a diluent to keep the middle mixing unit, the rear mixing unit and the liquid outlet channel 22 fully filled;

[0113] S4, then simultaneously start the first feed pump 26 and the second feed pump 27 to mix the first material and the second material, and then enter the next stage for mixing;

[0114] S5. When the amount of LNPs prepared reaches 2 / 3 of the total planned batch, the peristaltic pump 34 is turned on to fully fill the liquid into the main pipeline of the tangential flow device 31 at zero pressure to prepare for ethanol removal;

[0115] S6. After the injection of the first material and the second material is completed, the first feed pump 26 and the second feed pump 27 are turned off to increase the pressure of the tangential flow device 31, and the flow rates of the third feed pump 29 and the fourth feed pump 28 are adjusted proportionally to maintain the total flow rate of the feed liquid at 4 mL / min. Until the pH of the feed liquid in the liquid storage tank 35 is greater than 7, the third feed pump 29 and the fourth feed pump 28 are turned off at this time, and the feed liquid in the liquid storage tank 35 is continued to be refluxed and concentrated by the peristaltic pump 34 until the theoretical concentration of mRNA reaches the desired concentration;

[0116] S7. Filter the lipid nanoparticle solution obtained in the liquid storage tank 35 using a 0.22 μm microporous filter membrane to obtain lipid nanoparticles.

[0117] Effect Example 1

[0118] The size and PDI of the lipid nanoparticles prepared in Application Example 1 were determined by dynamic light scattering; the encapsulation efficiency was determined using the Quant it Ribogreen RNA quantification kit; and the measured results are listed in Table 1 below.

[0119] Table 1

[0120]

[0121] The LNPs prepared in Example 1 had a particle size of approximately 80-100 nm, a uniform particle size distribution, and a high encapsulation efficiency. The particle size and encapsulation efficiency remained essentially stable when the sample was stored at 4°C ± 2°C for 6 months.

Claims

1. A microfluidic chip, characterized in that: It comprises a cover plate and a base plate, wherein the base plate is provided with a front mixing unit, a rear mixing unit and a liquid outlet channel (22) which are connected in sequence; The front mixing unit comprises a front main channel (8) and at least two front annular microchannels (11) connected in series; the rear mixing unit comprises a rear main channel (17) and one or at least two rear annular microchannels (21) connected in series; the end of the front annular microchannel (11) is connected to the front end of the rear main channel (17); at least one rear branch channel (18) is provided on the rear main channel (17); the end of the rear annular microchannel (21) is connected to the liquid outlet channel (22); The front annular microchannel (11) comprises a front main subchannel (9) and a front secondary subchannel (10) connected to form a ring at the ends, the front end of the front main subchannel (9) and the front end of the front secondary subchannel (10) are connected to a front splitting point, the end of the front main subchannel (9) and the end of the front secondary subchannel (10) are connected to a front combination point, the first front splitting point is connected to the end of the front main channel (8), and the last front combination point is connected to the front end of the rear main channel (17); the cross-sectional area S of the front main subchannel (9) is 121 Greater than the cross-sectional area S of the previous sub-channel (10) 122 ; The rear annular microchannel (21) comprises a rear main subchannel (19) and a rear secondary subchannel (20) connected end to end to form a ring, the front end of the rear main subchannel (19) and the front end of the rear secondary subchannel (20) are connected to a rear splitting point, the end of the rear main subchannel (19) and the end of the rear secondary subchannel (20) are connected to a rear combination point, the first rear splitting point is connected to the end of the rear main channel (17), and the last rear combination point is connected to the front end of the liquid outlet channel (22); the cross-sectional area S of the rear main subchannel (19) is 321 Greater than the cross-sectional area S of the rear sub-channel (20) 322 .

2. The microfluidic chip according to claim 1, wherein The flow channel depth H1 of the front mixing unit is smaller than the flow channel depth H3 of the rear mixing unit; The number N1 of the front annular microchannels (11) is greater than the number N3 of the rear annular microchannels (21); The cross-sectional area S of the front main channel (8) 11 smaller than the cross-sectional area S of the rear main channel (17) 31 ; The width W of the front main channel (8) 11 Smaller than the width W of the rear main channel (17) 31 ; A liquid outlet (5) is provided at one end of the liquid outlet channel (22) away from the rear end annular microchannel (21).

3. The microfluidic chip according to claim 1, wherein The depth H of the front main channel (8) 11 The depth H of the front annular microchannel (11) can be 12 Equal, preferably equal to the flow channel depth H1 of the front mixing unit; The width W of the front main channel (8) 11 The width W of the front main sub-channel (9) 121 equal; The width W of the front main sub-channel (9) 121 and the width W of the first sub-channel (10) 122 The ratio is W 121 : W 122 =(1-4): 1; The depth H of the front main sub-channel (9) 121 and the depth H of the previous sub-channel (10) 122 equal; The curvature radius R of the front main sub-channel (9) 121 and the curvature radius R of the front sub-channel (10) 122 The ratio is R 121 :R 122 =(0.8-2):

1.

4. The microfluidic chip according to claim 1, wherein The depth H of the rear main channel (17) 31 The depth H of the rear end annular microchannel (21) 32 equal; The width W of the rear main channel (17) 31 Equal to the width W of the rear main sub-channel (19) 321 ; The width W of the rear main sub-channel (19) 321 and the width W of the rear sub-channel (20) 322 The ratio is W 321 : W 322 =(1-4): 1; The depth H of the rear main sub-channel (19) 321 and the depth H of the rear sub-channel (20) 322 equal; The curvature radius R of the rear main sub-channel (19) 321 and the curvature radius R of the rear sub-channel (20) 322 The ratio is R 321 : R 322 =(0.8-2): 1; The angle θ3 formed at the intersection between the rear main channel (17) and the rear branch channel (18) is 15°-90°; The width W of the rear main channel (17) 31 Less than or equal to the width W of the rear end branch channel (18) 33 ; A fourth liquid inlet (4) is provided at one end of the rear end branch channel (18) away from the rear end main channel (17).

5. The microfluidic chip according to claim 1, wherein The substrate is also provided with a liquid inlet channel, which includes one or at least two parallel liquid inlet channels, and the ends of the liquid inlet channels are connected to the front end of the front main channel (8) after merging at a merging point; The substrate is further provided with one or at least two middle-end mixing units connected in series, and the middle-end mixing unit is connected between the front-end mixing unit and the rear-end mixing unit.

6. The microfluidic chip according to claim 5, characterized in that The width W of the front main channel (8) 11 Less than or equal to the minimum value of the width W0 of each liquid inlet channel; The depth H0 of the liquid inlet channel is equal to the flow channel depth H1 of the front mixing unit; The mid-end mixing unit comprises a mid-end main channel (12) and one or at least two mid-end annular microchannels (16) connected in series, the front end of the mid-end main channel (12) being connected to the end of the front annular microchannel (11), and the end of the mid-end annular microchannel (16) being connected to the front end of the rear main channel (17); at least one mid-end branch channel (13) is provided on the mid-end main channel (12); The flow channel depth H2 of the middle mixing unit is equal to the flow channel depth H3 of the rear mixing unit; The number N2 of the middle-end annular microchannels (16) is greater than or equal to the number N3 of the rear-end annular microchannels (21), and less than the number N1 of the front-end annular microchannels (11).

7. The microfluidic chip according to claim 6, wherein: The cross-sectional area S of the middle main channel (12) 21 smaller than the cross-sectional area S of the rear main channel (17) 31 , which is larger than the cross-sectional area S of the front main channel (8) 11 ; The width W of the middle main channel (12) 21 Smaller than the width W of the rear main channel (17) 31 , which may be greater than the width W of the front main channel (8) 11 ; The depth H of the middle main channel (12) 21 The depth H of the middle annular microchannel (16) 22 equal; The middle end annular microchannel (16) comprises a middle main subchannel (14) and a middle secondary subchannel (15) connected to form a ring at the ends, the front end of the middle main subchannel (14) and the front end of the middle secondary subchannel (15) are connected to a middle splitting point, the end of the middle main subchannel (14) and the end of the middle secondary subchannel (15) are connected to a middle combination point, the first middle splitting point is connected to the end of the middle end main channel (12), and the last middle combination point is connected to the front end of the rear end main channel (17); the cross-sectional area S of the middle main subchannel (14) is 221 Greater than the cross-sectional area S of the secondary sub-channel (15) 222 ; The width W of the middle main channel (12) 21 The width W of the main sub-channel (14) 221 equal; The width W of the middle main sub-channel (14) 221 and the width W of the middle sub-channel (15) 222 The ratio is W 221 : W 222 =(1-4): 1; The depth H of the middle main sub-channel (14) 221 and the depth H of the middle sub-channel (15) 222 equal; The curvature radius R of the middle main sub-channel (14) 221 and the curvature radius R of the middle sub-channel (15) 222 The ratio is R 221 : R 222 =(0.8-2): 1; The included angle θ2 formed by the middle end main channel (12) and the middle end branch channel (13) at the intersection is 15°-90°; The width W of the middle main channel (12) 21 Less than or equal to the width W of the middle branch channel (13) 23 ; A third liquid inlet (3) is provided at one end of the middle branch channel (13) away from the middle main channel (12).

8. The microfluidic chip according to claim 1, wherein The cover plate and the substrate are made of metal, silicon, quartz, ceramic, glass, Hastelloy or high molecular polymer.

9. A microfluidic device, characterized in that It comprises a tangential flow device (31), a liquid storage tank (35) and one or at least two microfluidic chips as described in any one of claims 1 to 8 connected in parallel; The tangential flow device (31) is provided with a second feed inlet, a discharge port, and a waste liquid discharge port; the liquid storage tank (35) is provided with a first feed inlet, a product discharge port, a circulation feed inlet, and a circulation discharge port; Each liquid outlet (5) is connected to the first feed port, the circulating outlet is connected to the second feed port via an intermediate pipeline, and the outlet is connected to the circulating feed port.

10. The microfluidic device according to claim 9, wherein The product discharge port is connected to the product storage tank (32) via a product discharge pipeline; a filter (36) is provided on the product discharge pipeline; A peristaltic pump (34) is provided on the intermediate pipeline; The waste liquid discharge port is connected to the waste liquid storage tank (33) via a product waste liquid pipeline.

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