A microfluidic chip for pre-focused cell sorting and a sorting method

Abstract

This invention discloses a microfluidic chip for pre-focused cell sorting. Newtonian and viscoelastic fluids are introduced into different inlets of the chip. Through the polygonal flow channel shape within the microfluidic chip, a stable viscoelastic-Newtonian interface is generated by the co-flow of the two solutions. The inertial and viscoelastic effects generated within the microfluidic channels induce inertial lift at the microchannel walls and viscoelastic lift in the viscoelastic solution, both acting on the cell particles within the channels. Furthermore, the driving forces for the passive separation of cell particles, such as inertial and viscoelastic lift, strongly depend on the particle diameter. Particles of different diameters are affected by different resultant forces. Therefore, cell particles of different diameters accumulate different distances during migration within the microchannels, pre-arranging the lateral migration patterns of the particles and ultimately achieving sorting at different channel outlets.

Description

Technical Field

[0001] This application relates to the field of microfluidics, and in particular to a microfluidic chip for pre-focused cell sorting and a sorting method. Background Technology

[0002] Viscoelastic focusing is a typical passive microfluidic manipulation technology that does not require the application of external active fields, such as sound fields, light fields, electric fields, magnetic fields, etc. It can achieve high-precision and high-efficiency manipulation of particles solely based on the fluid's own properties and the simple flow channel structure of the microchannel.

[0003] In addition, most polymer solutions in nature (such as PEO, PVP, HA, etc.) and most biological fluids (such as blood, saliva, lymph, etc.) have a certain degree of viscoelasticity. Therefore, using the viscoelasticity of solutions to manipulate particles or cells is an ideal sample pretreatment method, and it still has untapped potential in the biomedical field.

[0004] Currently, microfluidics technology has been successfully applied to particle mixing, enrichment, focusing, sorting, filtering, and detection.

[0005] Microfluidic technology can be mainly divided into two categories according to its implementation principle: active manipulation and passive manipulation. Passive manipulation often uses microfluidic effects induced by specially structured microchannels or the interaction between cells and microstructures to achieve cell sorting based on the shape and size of particles. The key parameters are generally the deformability and size of the particles.

[0006] Passive focusing sorting chips achieve particle manipulation through a special flow channel structure, and their advantages are low cost and high throughput.

[0007] Therefore, the development of passive sorting chips using viscoelastic focusing has important applications in fields such as pathological analysis, cancer diagnosis, and precise culture of rare cells.

[0008] For example, patent CN111592965A, "A Microfluidic Chip Detection System and Method for Cell Sorting and Focusing," uses only the viscoelastic effect in its sorting principle, lacking the inertial lift generated by the inertial effect described in this patent. Therefore, the maneuverability of cell particles is reduced. Due to its weak maneuverability, pre-focusing of cell particles before sorting cannot be achieved, resulting in a decrease in the final cell particle sorting efficiency and preventing the implementation of high-throughput cell particle sorting in experimental settings. Furthermore, the expansion / contraction structure design in this application is theoretically suitable for specific cell particle sorting at low Reynolds numbers (Re) and low flow rates, thus limiting its applicability.

[0009] For example, patent CN114073997A, "A Microfluidic Chip and Method for Rapid and Precise Cell Sorting at Low Flow Rates," describes a shrink-expansion array channel comprising one or more expansion channels and one or more contraction channels. These expansion and contraction channels are arranged alternately, and adjacent expansion and contraction channels are interconnected. The width of the expansion channel is greater than the width of the contraction channel; the width of the sudden expansion sorting channel is greater than the width of the direct sorting channel. In this application, the shrink-expansion array channel only describes the sequential connection of the contraction and expansion structures, without specifying concrete design parameters. In actual operation, it fails to achieve the original shrink-expansion structure effect and may even have the opposite effect of clogging particles and reducing sorting efficiency.

[0010] In summary, existing microfluidic chips suffer from problems such as insufficient lateral migration distance of cell particles, inability to efficiently pre-focus and sort cell particles, and inability to act on cells of different sizes. Summary of the Invention

[0011] The purpose of this invention is to address the shortcomings of the prior art and provide a microfluidic chip and method for pre-focused cell sorting that is applicable to cells of different sizes and enables convenient, label-free, and highly efficient focused sorting of tiny cell particles.

[0012] The beneficial effects of this invention are as follows:

[0013] 1. The microfluidic chip for pre-focusing and sorting cells provided in this application generates viscoelastic and inertial effects respectively within the microchannel through the injection of viscoelastic and Newtonian fluids. This induces viscoelastic lift in the viscoelastic solution and inertial lift on the microchannel wall, and the resultant force can act on cells of different sizes. With the polygonal focusing unit set in the chip, when cell particles enter the channel, the velocity of the particles will increase sharply in a short time due to the sudden reduction of the channel width. With the help of multiple subsequent focusing units, different cells and particles can be flexibly and quickly focused at different positions in the microchannel space, providing an easy-to-control, efficient and low-cost experimental environment for subsequent cell sorting. This application makes full use of the hydrodynamic effect of carrier media such as Newtonian fluid and viscoelastic fluid, solving the problem of the small lateral migration distance of cell particles and the inability to efficiently pre-focus and sort cell particles, and realizing the function of convenient, label-free and highly efficient focusing and sorting of tiny cell particles.

[0014] 2. Based on experimental testing and cell applications, and considering the sorting performance of cell particles in different viscoelastic solutions and at different flow rates, a polygonal focusing region was designed. Specifically, it is a polygonal structure symmetrical about its axis of symmetry. Viewed from left to right in the flow direction of the solution, the upstream edge of this structure forms a 120° angle with the flow channel. 。 The included angle is 135 degrees between the downstream side and the flow channel. 。Angle; This polygonal focusing element is suitable for sorting trajectories of most cells with a size of 8-12μm in viscoelastic solutions under different flow rates;

[0015] Due to the sudden increase in space, the high-speed fluid forms vortices within it. Smaller particles are more easily captured by the polygonal structure due to the elastic lift of the guiding interface. At the same time, the capture effect of the multi-structure further improves the particle sorting efficiency. Therefore, this design can better enhance the lateral migration effect of the expansion and contraction structure in the sorting process, avoid mutual interference of particles under high throughput, and has a good effect on improving sorting efficiency and increasing cell particle throughput.

[0016] 3. From left to right, the system consists of a fluid confluence zone, a fluid co-flow zone, a polygonal focusing zone, and a cell particle sorting zone. The fluid confluence zone has equidistantly connected viscoelastic and Newtonian fluid inlets at its front ends. The viscoelastic fluid inlet contains a mixture of large cell particles, viscoelastic solution, and small cell particles. The cell particle sorting zone has outlets for small and large cell particles at its ends. Because the driving forces for passive separation of cell particles, such as inertial lift and viscoelastic lift, strongly depend on particle diameter, particles of different diameters are affected by different resultant forces. Therefore, in the microchannel, cell particles of different diameters migrate and accumulate different distances, pre-arranging their lateral migration patterns, ultimately flowing into different channel outlets to achieve sorting. This overall process realizes the experimental process of "focusing first, then sorting" cell particles. This method not only improves the sorting efficiency in the cell sorting process but also further increases the throughput of cell particle sorting. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of the microfluidic chip in this invention;

[0018] Figure 2 This is a schematic diagram of the specific structure of the polygonal focusing unit in this invention;

[0019] Figure 3 This is a schematic diagram of the focusing and sorting principle within the microchannel structure in an example of the present invention;

[0020] Figure 4 This is a schematic diagram of cell particle migration inside the microfluidic chip for focusing and sorting cells in an example of the present invention;

[0021] Figure 5 This is a graph showing the results of a focusing and sorting experiment of cell particles in Newtonian fluid / viscoelastic sheath fluid = PBS / 500ppmPEO = 80 / 80μL / min in an example of this invention;

[0022] Figure 6This is a graph showing the focusing and sorting experiment results of cell particles using Newtonian fluid / viscoelastic sheath fluid = PBS / 500ppmPEO = 80 / 240μL / min in an example of this invention;

[0023] In the diagram: 1-Viscoelastic fluid inlet; 2-Newtonian fluid inlet; 21-Newtonian fluid solution; 3-Fluid confluence zone; 4-Fluid co-flow zone; 5-Polygonal focusing zone; 50-Polygonal focusing unit; 51-Contraction / expansion channel; 6-Cell particle sorting zone; 7-Small cell particle outlet; 8-Large cell particle outlet; 11-Large cell particle; 12-Viscoelastic solution; 13-Small cell particle. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0025] like Figure 1 As shown, it includes a fluid confluence zone 3, a fluid co-flow zone 4, a polygonal focusing zone 5, and a cell particle sorting zone 6 connected from left to right; the fluid co-flow zone 4 has the same channel width as the fluid confluence zone 3.

[0026] The front end of the fluid junction zone 3 is connected to the viscoelastic fluid inlet 1 and the Newtonian fluid inlet 2 at equal distances; the width of the flow channel connecting the viscoelastic fluid inlet 1 to the fluid junction zone 3 is the same as the width of the flow channel connecting the Newtonian fluid inlet 2 to the fluid junction zone 3, which is half the width of the flow channel of the fluid junction zone 3.

[0027] The viscoelastic fluid inlet 1 is a mixed fluid inlet containing large cell particles 11, viscoelastic solution 12 and small cell particles 13, wherein the viscoelastic solution is one or more of polyethylene oxide (PEO), polyvinylpyrrolidone (PVP) and hyaluronic acid (HA).

[0028] Newtonian fluid inlet 2 is the inlet of Newtonian fluid solution 21; Newtonian fluid solution 21 is one or more of deionized water (DIWATER), phosphate-balanced physiological saline (PBS), and distilled water.

[0029] The cell particle sorting zone 6 has a small cell particle outlet 7, a large cell particle outlet 8 and a waste liquid outlet 9 at its end; the flow channel width of the cell particle sorting zone 6 is the same as the maximum vertical width of the polygonal focusing zone 5, and is three times the width of the one-to-three claw microchannel.

[0030] The polygonal focusing area 5 includes several polygonal focusing units 50, and two adjacent polygonal focusing units 50 are connected by a narrowing and expanding flow channel 51; the width of the narrowing and expanding flow channel 51 is 1 / 4 of the maximum flow channel width of the polygonal focusing unit 50.

[0031] The width of the expansion and contraction channel 51 is less than the maximum width of the polygonal focusing unit 50 and less than the width of the fluid co-flow region 4.

[0032] Specifically, at the inlet end of the polygonal focusing unit 50, the angle A between the contraction-expansion channel 51 and the inlet of the polygonal focusing unit 50 is 120°; at the outlet end of the polygonal focusing unit 50, the angle A between the contraction-expansion channel 51 and the outlet of the polygonal focusing unit 50 is 135°.

[0033] The polygonal focusing unit 50 is symmetrically arranged about the narrowing and expanding flow channel 51, including an inlet section 510, a smoothing section 511 and an outlet section 512; the width of the smoothing section 511 is the maximum flow channel width of the polygonal focusing unit 50.

[0034] This microfluidic chip was fabricated using photolithography and PDMS material. In the experiment, a mold was used to create... Figure 1 The designed microchannel shape ultimately bonds PDMS onto a glass slide for biological particle focusing and sorting applications.

[0035] In microfluidic experiments, the passive control of microparticles largely depends on the hydrodynamic effects of carrier media such as Newtonian fluids and viscoelastic fluids, such as inertia and viscoelasticity. This solution environment enables effective manipulation of cellular particles in a label-free and force-free manner.

[0036] Among them, the driving force for passive separation is inertial lift. F L or viscoelastic lift F e It strongly depends on the particle diameter a in the microfluidic. F L ~ a 4 ，F e ~ a 3 Therefore, cell particles with different diameters 'a' will be subject to different migration effects.

[0037] This invention utilizes the co-flow of Newtonian fluid PBS and viscoelastic fluid PEO within a microfluidic chip to perform pre-focusing and sorting functions based on the size-dependent differences in microparticles. For example... Figure 2 As shown, in order to improve the separation performance of inertial or viscoelastic microfluidics, this chip utilizes the combined action of sheath fluid and inertial force or elastic lift to pre-arrange the lateral migration of particles.

[0038] Since Newtonian fluids lack elastic stress, the elastic lift acting on the particles changes significantly. Compared to experimental methods using pure Newtonian fluids or pure viscoelastic flows, co-flow conditions generate stable viscoelastic / Newtonian interfaces with greater separation distances between particles of different sizes.

[0039] During the experiment, cell particles initially align along the sidewall through the protective flow of the viscoelastic fluid inlet 1. As the two fluids co-flow, larger cell particles are pushed away from the sidewall by the inertial lift guided by the centerline. Through the co-flow interface, they gradually move towards the center and focus. Smaller particles, due to the elastic lift of the wall-guided interface, cannot pass through the interface between the Newtonian and viscoelastic media and remain focused in the viscoelastic flow, thus achieving particle sorting in the microchannel.

[0040] like Figure 4 As shown, in practical applications, a viscoelastic enhancer is first added to the sample solution of the cell particles to be sorted to prepare a viscoelastic sample solution PEO with a certain mass percentage concentration, wherein the added viscoelastic enhancer accounts for ≤20% of the mass percentage concentration of the sample solution.

[0041] A viscoelastic fluid inlet 1 is injected at a stable flow rate using a syringe pump, while Newtonian fluid PBS is introduced through Newtonian fluid inlet 2. The two sample solutions flow through fluid junction zone 3; due to the difference in fluid viscosity, only a slight mixing effect occurs at the interface. A stable viscoelastic / Newtonian interface is formed after passing through fluid co-flow zone 4. At this point, according to... Figure 2 The migration principle in the flow channel involves large-diameter cellular particles gradually moving towards the centerline of the flow channel under the combined effects of inertia and viscoelasticity. The fluid then enters the polygonal focusing zone 5. Due to the sudden increase in space, the high-speed fluid forms vortices within these vortices. Smaller-diameter particles are more easily captured by the polygonal structure due to the elastic lift of the guiding interface. Furthermore, the capture effect of the multi-structure further enhances the particle sorting efficiency.

[0042] Furthermore, the polygonal focusing areas are connected by narrow converging and expanding channels 51. When cell particles enter the channels, the sudden decrease in width causes a rapid increase in particle velocity within a short period. Combined with subsequent focusing structures, this structure further accelerates the separation of different particles, improving subsequent sorting efficiency. Ultimately, through the focusing and sorting of cell particles, particles of different sizes are separated into cell particle samples of different diameters at the small cell particle outlet 7 and the large cell particle outlet 8 in the cell particle sorting area.

[0043] like Figure 5 The figure shows the results of a focusing and sorting experiment of cell particles under the conditions of Newtonian fluid / viscoelastic sheath fluid = PBS / 500ppmPEO = 80 / 80μL / min.

[0044] Figure 6 The figure shows the results of a focusing sorting experiment under the conditions of Newtonian fluid / viscoelastic sheath fluid = PBS / 500ppmPEO = 80 / 80μL / min.

[0045] The experimental results show that particles of different sizes effectively produced focusing and sorting effects under different experimental conditions, and the experimental results are consistent with the theoretical analysis.

[0046] This application fully utilizes the co-flow of Newtonian fluid PBS and viscoelastic fluid PEO to perform pre-focusing and sorting functions based on the size-dependent differences of particles. Compared with existing hydrodynamic separation techniques using single-phase Newtonian or viscoelastic flow conditions, the stable co-flow conditions produce significant interfacial viscoelastic and inertial effects. The interaction of these two forces results in a large separation distance between particles of different sizes, leading to high sorting efficiency and high separation purity. Furthermore, the polymer concentration used under co-flow conditions is much lower than that under pure viscous conditions. However, because the interfacial viscoelastic lift is greater than the bulk viscoelastic lift, particles in the microchannel exhibit more efficient focusing and sorting, while the low-concentration PEO environment provides more suitable conditions for handling biological samples.

[0047] Therefore, this microfluidic chip provides a universal, label-free, and highly efficient focusing and sorting method for tiny cellular particles.

[0048] Those skilled in the art will understand that, unless otherwise defined, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the meaning consistent with their meaning in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless defined as herein.

[0049] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A microfluidic chip for pre-focused cell sorting, characterized in that: It includes a fluid confluence zone (3), a fluid co-flow zone (4), a polygonal focusing zone (5), and a cell particle sorting zone (6) connected from left to right. The front end of the fluid confluence zone (3) is connected at equal distances to the viscoelastic fluid inlet (1) and the Newtonian fluid inlet (2); The viscoelastic fluid inlet (1) is a mixed fluid inlet containing large cell particles (11), viscoelastic solution (12) and small cell particles (13); The cell particle sorting area (6) is provided with a small cell particle outlet (7), a large cell particle outlet (8) and a waste liquid outlet (9) at its end. The Newtonian fluid inlet (2) is the inlet of the Newtonian fluid solution (21); The polygonal focusing area (5) includes a plurality of polygonal focusing units (50), and two adjacent polygonal focusing units (50) are connected by a shrinking and expanding flow channel (51). The width of the expansion and contraction channel (51) is smaller than the maximum width of the polygonal focusing unit (50) and smaller than the width of the fluid co-flow region (4); The channel width of the cell particle sorting area (6) is the same as the maximum width in the vertical direction of the polygonal focusing area (5); The flow channel width of the small cell particle outlet (7) is the same as that of the large cell particle outlet (8), which is 1 / 3 of the flow channel width of the cell particle sorting area (6). At the inlet end of the polygon focusing unit (50), the angle A between the constriction-expansion channel (51) and the inlet of the polygon focusing unit (50) is 120°; at the outlet end of the polygon focusing unit (50), the angle A between the constriction-expansion channel (51) and the outlet of the polygon focusing unit (50) is 135°. The width of the expansion channel (51) is 1 / 4 of the maximum channel width of the polygonal focusing unit (50).

2. The microfluidic chip according to claim 1, characterized in that: The width of the flow channel connecting the viscoelastic fluid inlet (1) to the fluid junction (3) is the same as the width of the flow channel connecting the Newtonian fluid inlet (2) to the fluid junction (3), which is half the width of the flow channel in the fluid junction (3). The flow channel width of the fluid co-flow zone (4) is the same as that of the fluid confluence zone (3).

3. The microfluidic chip according to claim 1, characterized in that: The polygonal focusing unit (50) is symmetrically arranged about the contraction and expansion channel (51), including an inlet section (510), a smooth section (511) and an outlet section (512); the width of the smooth section (511) is the maximum width of the polygonal focusing unit (50).

4. The microfluidic chip according to claim 1, characterized in that: The viscoelastic solution is one or more of polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), and hyaluronic acid (HA). The Newtonian fluid solution (21) is one or more of deionized water (DIWATER), phosphate-balanced saline (PBS), and distilled water.

5. A sorting method for a microfluidic chip with pre-focused cell sorting, characterized in that: Includes the following steps: S1. Add a viscoelastic enhancer to the sample solution of cell particles to be sorted to prepare a viscoelastic sample solution with a certain mass percentage concentration, and prepare a Newtonian fluid solution. S2. Based on the microfluidic chip for pre-focused sorted cells as described in any of claims 1-4, a viscoelastic sample solution is introduced into the viscoelastic fluid inlet (1) at a stable flow rate, while a Newtonian fluid inlet (2) is introduced into the Newtonian fluid solution. S3. After the viscoelastic sample solution and the Newtonian fluid solution converge in the fluid confluence zone (3), they flow sequentially through the fluid co-flow zone (4) and the polygonal focusing zone (5). In the cell particle sorting zone (6), cell particle samples of different diameters are separated from the small cell particle outlet (7) and the large cell particle outlet (8), respectively. The remaining impurities and waste liquid are discharged from the waste liquid outlet (9).

6. The sorting method for a microfluidic chip with pre-focused cell sorting according to claim 5, characterized in that: The viscoelasticity enhancer is one or more of polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), and hyaluronic acid (HA). The Newtonian fluid solution is one or more of deionized water (DIWATER), phosphate-balanced physiological saline (PBS), and distilled water.

Images