Micro-fluidic chip

Abstract

The utility model provides a microfluidic chip, and belongs to the field of microfluidics. A micro-channel is arranged in the micro-fluidic chip, the micro-channel comprises an inlet and a mixed micro-channel, the inlet is an L-shaped micro-channel, one end of the L-shaped micro-channel is communicated with the mixed micro-channel, the mixed micro-channel comprises a mixing unit, the mixing unit comprises a semi-circular micro-channel, the semi-circular micro-channel is spatially overlapped to form the mixing unit, and the semi-circular micro-channel is communicated with the mixed micro-channel. And one end of the semicircular micro-channel is communicated with the contact end of the other semicircular micro-channel. According to the utility model, the micro-fluidic chip design is adopted, mixed material fluid is repeatedly subjected to the vortex mixing-convergence-divergence process, and the mixing effect is good; the fluid is large in flux, small in resistance and high in mixing efficiency; the mixing effect on fluid with low Reynolds number is good; the LNP prepared by the device is good in particle size uniformity and good in encapsulation effect.

Description

TECHNICAL FIELD

[0001] The utility model relates to micro -fluidic field especially relates to a micro -fluidic chip. BACKGROUND

[0002] Lipid nanoparticles (LNP) as non-viral vectors play a key role in the delivery of mRNA vaccines and drugs. LNP can efficiently encapsulate mRNA molecules, protect them from degradation, and promote their release and expression in cells. However, traditional LNP preparation methods have problems such as complicated steps, poor reproducibility, and uneven particle size distribution, which limit their widespread application in drug research and development.

[0003] Microfluidic chip technology, with its precise liquid control ability and efficient micro-mixing characteristics, provides a new approach to solving the LNP preparation problem. Through microfluidic chips, precise control can be achieved during the LNP preparation process, including the ratio of lipids to mRNA, mixing speed, and environmental conditions, thereby significantly improving the efficiency and quality of LNP preparation, achieving rapid, efficient, and reproducible LNP preparation.

[0004] Prior art CN214020875U discloses a LNP nucleic acid vaccine preparation instrument that uses a wave-shaped microfluidic chip to enhance mixing effect, suitable for industrialized mass production requirements; prior art CN116492888A discloses a mixing unit, mixer, microfluidic chip, mixing device, application and process, which uses a designed mixing unit to enhance mixing effect and improve the flux of mixed liquid to adapt to industrialization requirements, but still has problems of poor fluid mixing effect at low Reynolds number, uneven LNP particle size, and poor encapsulation effect. UTILITY MODEL CONTENT

[0005] To solve the above technical problems, the purpose of the utility model is to meet the rapid, efficient, and reproducible preparation of LNP, large flux, good uniformity of LNP particle size, and good encapsulation effect of a microfluidic chip.

[0006] The utility model provides a micro -fluidic chip, the micro -fluidic chip is opened in and has micro -fluidic channel, the micro -fluidic channel includes entrance and mixed micro -fluidic channel, the entrance is L shaped micro -fluidic channel, L shaped micro -fluidic channel one end with the mixed micro -fluidic channel is linked together, the mixed micro -fluidic channel includes mixing unit, the mixing unit includes semicircle ring micro -fluidic channel, the semicircle ring micro -fluidic channel is C shaped micro -fluidic channel, the semicircle ring micro -fluidic channel is stacked along Z axle direction and constitutes the mixing unit, the semicircle ring micro -fluidic channel one end with another semicircle ring micro -fluidic channel contact one end is linked together, the width range of semicircle ring micro -fluidic channel is 0.2mm 1mm, the depth range is 0.2mm 1mm.

[0007] Preferably, the number of the mixing units is at least one.

[0008] Preferably, the semi-circular ring micro flow channel is centrally symmetric; the mixing unit comprises a feeding port, a first shunt pipeline, a second shunt pipeline and a discharging port; when the mixing unit is multiple, the multiple mixing units are sequentially connected in a head-to-tail manner between the feeding port and the discharging port.

[0009] Preferably, the first shunt pipeline of the mixing unit is arranged at one end of the same plane as the feeding port, and the second shunt pipeline is arranged at the bottom end of the same plane as the feeding port.

[0010] Preferably, the second shunt pipeline of the mixing unit is arranged at one end of the same plane as the feeding port, and the first shunt pipeline is arranged at the top end of the same plane as the feeding port.

[0011] Preferably, one end of the orthogonal part of the L-shaped micro flow channel is connected with the mixing micro flow channel.

[0012] Working principle:

[0013] Two materials enter the feeding port through two ends of the L-shaped micro flow channel entrance, and are preliminarily mixed at the feeding port and then enter the mixing unit for shunting. Because the extension direction of the inner wall of the two semi-circular ring micro flow channels in the shunt pipeline is different from the direction of the fluid formed by the mixed materials, the flow rates of the fluid formed by the mixed materials are different after shunting, and then the two fluids with different flow rates are mixed again at the next semi-circular ring micro flow channel convergence. The fluid mixed by the previous stage of shunt pipeline enters the next shunt pipeline again, and is mixed again. The materials repeatedly undergo vortex mixing-converging-diverging, thereby enhancing the mixing effect.

[0014] Compared with the prior art, the utility model has the advantages that:

[0015] The utility model discloses a micro fluidic chip design, and the mixed material fluid repeatedly undergoes vortex mixing-converging-diverging, and the mixing effect is good. The fluid has large flux and small resistance, and the mixing efficiency is high. The utility model discloses a space superposition design, and the fluid disturbance is enhanced when the fluid passes through, and the mixing effect of the fluid with low Reynolds number is better. The LNP prepared by using the utility model has good particle size uniformity and good encapsulation effect. DRAWINGS

[0016] Figure 1 It is a top view schematic diagram of the micro fluidic chip of the space superposition mode of the utility model;

[0017] Figure 2 It is a bird's eye view schematic diagram of the micro fluidic chip of the space superposition mode of the utility model;

[0018] Figure 3 A bird's eye view schematic diagram of the microfluidic chip in a plane superposition mode of the utility model;

[0019] Figure 4 Simulation operation results of the mixing effect of different types of chips of the utility model;

[0020] Figure 5 A microfluidic chip usage mode schematic diagram of the utility model;

[0021] Figure 6 For Figure 2 The mRNA-LNP particle size detection graph prepared by the total flow rate of 40ml / min in the chip structure embodiment.

[0022] In the figure:

[0023] 1, inlet; 2, mixing micro flow channel; 3, mixing unit; 31, feed port; 32, first shunt pipeline; 33, second shunt pipeline; 34, discharge port; 10, water phase filling; 11, pump; 12, lipid solution filling; 13, pipeline; 14, microfluidic chip; 15, chip packaging; 16, sample receiving bag. DETAILED DESCRIPTION

[0024] The specific embodiments of the utility model will be described in detail below with reference to the accompanying drawings. In addition, unless specifically stated, the first shunt pipeline indicated in each drawing is the shunt pipeline on the left side of the fluid advancing direction, and the second shunt pipeline indicated in the drawing is the shunt pipeline on the right side of the fluid advancing direction.

[0025] It should be noted that the following description uses the words "first", "second", etc. to limit the components, which are only used to

[0026] For the convenience of distinguishing the corresponding components, unless otherwise stated, the above words have no special meaning and do not represent primary and secondary, because

[0027] This cannot be understood as a limitation on the scope of protection of the present application.

[0028] The utility model provides a kind of microfluidic chip, such as Figures 1-2As shown, the microfluidic chip is provided with a microfluid channel, which comprises an inlet (1) and a mixing microfluid channel (2); the inlet (1) is an L-shaped microfluid channel, one end of the L-shaped microfluid channel is communicated with the mixing microfluid channel (2); the mixing microfluid channel (2) comprises a mixing unit (3), the mixing unit (3) comprises a semi-circular ring microfluid channel, the semi-circular ring microfluid channel is a C-shaped microfluid channel, the semi-circular ring microfluid channels are stacked along the Z-axis direction to form the mixing unit (3), one end of the semi-circular ring microfluid channel is communicated with one end of another semi-circular ring microfluid channel; the width of the semi-circular ring microfluid channel ranges from 0.2 mm to 1 mm, and the depth ranges from 0.2 mm to 1 mm.

[0029] According to one specific embodiment of the present application, the number of the mixing unit (3) is at least one.

[0030] According to one specific embodiment of the present application, the semi-circular ring microfluid channel is centrally symmetric; the mixing unit (3) comprises a feeding port (31), a first shunt pipeline (32), a second shunt pipeline (33) and a discharging port (34); when the mixing unit (3) is multiple, the multiple mixing units are sequentially connected end to end between the feeding port (31) and the discharging port (34).

[0031] According to one specific embodiment of the present application, the first shunt pipeline (32) of the mixing unit (3) is arranged at one end of the same plane where the feeding port (31) is located, and the second shunt pipeline (33) is arranged at the bottom end of the same plane where the feeding port (31) is located.

[0032] According to one specific embodiment of the present application, the second shunt pipeline (33) of the mixing unit (3) is arranged at one end of the same plane where the feeding port (31) is located, and the first shunt pipeline (32) is arranged at the top end of the same plane where the feeding port (31) is located.

[0033] According to one specific embodiment of the present application, one end of the L-shaped microfluid channel at the orthogonal position is communicated with the mixing microfluid channel.

[0034] According to one specific embodiment of the present application, as shown in Figure 3 When the semi-circular ring microfluid channels are stacked along the Z-axis, there is no height difference in the Z-axis direction.

[0035] According to one specific embodiment of the present application, as shown in Figure 4 The mixing performance of two different structure chips is simulated by using COMSOL Multiphysics simulation software, and in the case of low Re Figure 3 The mixing performance of the single layer is better than Figure 2 The mixing performance of the stack is slightly worse, and there is no obvious difference in the mixing performance of the two with the increase of Re.

[0036] According to one specific embodiment of the present application, as shown in Figure 5 Figure 1, a schematic diagram of an apparatus using a microfluidic chip is disclosed, the apparatus comprising a water phase reservoir (10), a lipid solution reservoir (12), a pump (11), a pipeline (13), a microfluidic chip (14), a chip package (15) and a sample receiving bag (16).

[0037] According to one specific embodiment of the present application, as shown in Figure 6 Figure 2, a schematic diagram of an apparatus using a microfluidic chip is disclosed, the apparatus comprising a water phase reservoir (10), a lipid solution reservoir (12), a pump (11), a pipeline (13), a microfluidic chip (14), a chip package (15) and a sample receiving bag (16). Figure 2 Figure 3 shows a mRNA-LNP particle size detection chart prepared using the microfluidic chip shown in Figure 2, the total flow rate of mixing is 40 ml / min, and the mixing ratio of mRNA / sodium citrate solution to lipid ethanol solution is 3:1.

[0038] Example 1

[0039] COMSOL Multiphysics 6.2 version was used to simulate the convective mixing effect of incompressible fluid in the three-dimensional structure model by using the multi-physics turbulent flow k~ω or laminar flow and dilute substance transfer model, and the mixing effect (Mixing index, MI) calculation formula is as follows:

[0040] MI=

[0041] Wherein, N is the number of sampling points on the cross section, C is the initial concentration, Ci is the optimal concentration of sufficient mixing, and C is the average concentration of the fluid in the analysis area. MI is the mixing index. The value of MI is from 0 to 1, which means no mixing at 0, and complete mixing at 1, so the value of MI can be regarded as the mixing effect in the experiment.

[0042] Water and ethanol were selected as working fluids in the simulation. In the simulation, the inlet temperature was set to be constant at 293.12 K (20℃), and the densities of water (w) and ethanol (o) were 9.98×10 2 kg / m 3 and 7.98×10 2 kg / m 3 , respectively. The dynamic viscosities of water and ethanol were 1.004×10 -3 Pa·s and 1.2×10 -3 Pa·s, respectively. The diffusion coefficients of water and ethanol were both 1×10 -9 m 2 / s. The inlet was fully developed fluid, and the outlet was set to be zero static pressure. The convection mechanism was added in the simulation by twice discretization of the fluid concentration. In fluid mechanics, the Reynolds number is the most important dimensionless number, which represents the ratio of the importance of inertial effect to the importance of viscous effect in flow. The Reynolds number Re is obtained by the following formula:

[0043]

[0044] where ρ is the fluid density (kg / m 3 ), u is the characteristic flow velocity of the fluid (m / s), μ is the dynamic viscosity (Pa·s), D is the characteristic length of the geometry (m), D = 2W x H / (W + H), W is the channel width (m), and H is the channel height (m);

[0045] The Reynolds number is used to describe the fluid flow, and at a low Reynolds number (Re≤2000), the fluid is laminar flow.

[0046] In the model, the cross-sectional height W and the width H are both 1 mm.

[0047] The simulation investigated the mixing effect of the two fluids at different mixing ratios under different Re, and the simulation results are as follows:

[0048]

[0049] The simulation results show that at a low Re, the superposition effect is more obvious for the mixing effect of the two fluids, and at a high Re, there is no obvious difference between them.

[0050] Example 2

[0051] As shown in Figure 5 , the microfluidic chip is printed by using a 3D printing technology, the manufactured microfluidic chip is packaged by using a specific clamp, a conduit is used to connect the pump and the microfluidic chip, the aqueous phase (such as water) and the organic phase (such as ethanol) are introduced into the microfluidic chip by using the pump, and the pump provides power for the fluid. After the fluid enters the microfluidic chip, it is mixed, and the mixed fluid is discharged from the outlet and enters the sample collection bag.

[0052] Example 3

[0053] ALC-0315 lipid, ALC-0159, DSPC and cholesterol lipid formula are used to encapsulate mRNA. Each lipid is weighed according to the following prescription, and each lipid is dissolved in anhydrous ethanol at about 40°C to prepare a mixed lipid solution, and the total lipid concentration is 12 mM.

[0054]

[0055] The mRNA stock solution is diluted with 100 mM sodium citrate (pH 4.0) to prepare an mRNA / sodium citrate solution, and the concentration of mRNA in the solution is 100 μg / ml.

[0056] Using a plunger pump, the lipid solution and mRNA / sodium citrate solution were mixed at a 1:3 ratio, with total flow rates of 12 ml / min, 20 ml / min, 28 ml / min, and 40 ml / min, respectively. Figure 1 and Figure 2 The structured chips were mixed, with channel dimensions of 0.3 mm in both width and depth. The prepared mRNA-LNP was diluted 5-fold with 100 mM sodium citrate solution (pH 4.0) and the particle size was detected using a Malvern laser particle size analyzer (Zetasizer Ultra). The material RI was set to 1.35, material absorption to 0.01, dispersant RI to 1.33, dispersant viscosity to 0.887, and dispersant dielectric constant to 78.5. The detection temperature was 25 °C.

[0057]

[0058] The total flow rate was 40 ml / min. Figure 2 The mRNA-LNP sample prepared by the microfluidic chip was added to the Slide-A-Lyzer dialysis box 20 K MWCO (Thermo), and then dialyzed in at least 50 volumes of 1×TBS solution (18 mM Tirs, 137 mM sodium chloride, pH 7.4) at room temperature for 4 h. After 4 h, the solution was replaced with an equal volume of 1×TBS solution and dialyzed overnight. The sample was collected and placed in an ultrafiltration tube (Pall, 100 kD), centrifuged at 1000 g at room temperature for 30 min, and ultrafiltered to 1 / 4 of the original volume.

[0059] The mRNA content and encapsulation efficiency of the ultrafiltered mRNA-LNP were determined using Ribogreen fluorescence staining. The mRNA content was 90.7 μg / ml, and the encapsulation efficiency was 97.51%.

[0060] Performance verification of microfluidic chips using the publicly available ALC-0315 formulation lipids, including mRNA-LNP particle size, PDI, mRNA content, and encapsulation efficiency data, shows that the microfluidic chip of this invention can be well used to manufacture mRNA-LNPs, and the manufactured mRNA-LNPs have uniform particle size and high encapsulation efficiency.

[0061] The above description is merely a preferred embodiment of this utility model and is not intended to limit the utility model. Various modifications and variations can be made to this utility model by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this utility model are included within the protection scope of this utility model.

Claims

1. A microfluidic chip, characterized by, The microfluidic chip is provided with a micro flow channel, which comprises an inlet (1) and a mixing micro flow channel (2); the inlet (1) is an L-shaped micro flow channel, one end of the L-shaped micro flow channel is communicated with the mixing micro flow channel (2); the mixing micro flow channel (2) comprises a mixing unit (3), the mixing unit (3) comprises a semi-circular ring micro flow channel, the semi-circular ring micro flow channel is a C-shaped micro flow channel, the semi-circular ring micro flow channels are stacked along the Z-axis direction to form the mixing unit (3), one end of the semi-circular ring micro flow channel is communicated with the other end of the semi-circular ring micro flow channel in contact; the width of the semi-circular ring micro flow channel ranges from 0.2 mm to 1 mm, and the depth ranges from 0.2 mm to 1 mm.

2. The microfluidic chip according to claim 1, wherein, The number of the mixing unit (3) is at least one.

3. The microfluidic chip of claim 2, wherein, The semi-circular ring micro flow channel is centrally symmetric; the mixing unit (3) comprises a feed inlet (31), a first shunt pipeline (32), a second shunt pipeline (33) and a discharge outlet (34); when the mixing unit (3) is multiple, the multiple mixing units are sequentially connected end to end between the feed inlet (31) and the discharge outlet (34).

4. The microfluidic chip according to claim 3, wherein, The first shunt pipeline (32) of the mixing unit (3) is arranged at one end of the same plane where the feed inlet (31) is located, and the second shunt pipeline (33) is arranged at the bottom end of the same plane where the feed inlet (31) is located.

5. The microfluidic chip of claim 3, wherein, The second shunt pipeline (33) of the mixing unit (3) is arranged at one end of the same plane where the feed inlet (31) is located, and the first shunt pipeline (32) is arranged at the top end of the same plane where the feed inlet (31) is located.

6. The microfluidic chip of claim 1, wherein, One end of the L-shaped micro flow channel at the orthogonal position is communicated with the mixing micro flow channel.

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