CN116099581A - A microfluidic chip and sorting method for pre-focused sorting cells - Google Patents

Abstract

The invention discloses a micro-fluidic chip for prefocusing and sorting cells, which is characterized in that Newtonian fluid and viscoelastic fluid are introduced into different inlets of the chip, a stable viscoelastic-Newtonian interface is generated by co-flow of two solutions through a polygonal runner shape arranged in the micro-fluidic chip, and an inertial effect and a viscoelastic effect generated in the runner micro-fluid are utilized, so that an inertial lift force is induced on the wall surface of the micro-runner and a viscoelastic lift force is induced in the viscoelastic solution to jointly act on cell particles in the runner. In addition, the driving force of passive separation of the cell particles, such as inertial lifting force and viscoelastic lifting force, is strongly dependent on particle diameters, and particles with different diameters are affected by different resultant forces, so that the migration and accumulation of the cell particles with different diameters in the micro-channel generate different distance differences in the micro-channel, the particles are prearranged in a transverse migration mode, and finally the sorting effect is realized at different channel outlets.

Description

A microfluidic chip and sorting method for pre-focused sorting cells

technical field

The present application relates to the field of microfluidic technology, in particular to a microfluidic chip and a sorting method for pre-focusing and sorting cells.

Background technique

Viscoelastic focusing is a typical passive microfluidic manipulation technology. It does not require external active fields, such as sound fields, light fields, electric fields, magnetic fields, etc., and can be completed only by the characteristics of the fluid itself and the simple channel structure of the microchannel. High-precision and high-efficiency manipulation of particles.

In addition, most polymer solutions in nature (such as PEO, PVP, HA, etc.) and most biological fluids (such as blood, saliva, lymph, etc.) have certain viscoelasticity, so the viscoelasticity of the solution is used to manipulate particles Or cells are an ideal means of sample pretreatment, and there is still untapped potential in the field of biomedicine.

Nowadays, microfluidic technology has been maturely applied to the mixing, enrichment, focusing, sorting, filtering and detection of particles.

According to its realization principle, microfluidic technology can be divided into two categories, namely active control and passive control. Among them, passive manipulation often uses the microfluidic effect induced by special structured microchannels or the interaction between cells and microstructures to achieve cell sorting according to the shape and size of particles. The key parameters are generally the deformability of particles and their size size.

The passive focusing sorting chip realizes the manipulation of particles through a special flow channel structure, and its advantages are low cost and high throughput.

Therefore, the use of viscoelastic focusing to develop passive sorting chips plays an important role in pathological analysis, cancer diagnosis, and precise cultivation of rare cells.

Such as patent CN111592965A - a microfluidic chip detection system and method for cell sorting and focusing. The sorting principle of the microfluidic chip only uses the viscoelastic effect, and there is no inertial lift generated by the inertial effect in this patent. , so the manipulation of cell particles will be reduced. Due to its weak controllability, the pre-focusing phenomenon of cell particles before sorting cannot be realized, which will lead to a decrease in the final cell particle sorting efficiency, and the large-throughput cell particle sorting function cannot be realized in the experimental setup. Secondly, in this application, the design of the contraction-expansion structure is theoretically suitable for the sorting of specific cell particles at low Reynolds number Re and small flow rate, and the trial range has certain limitations.

Another example is patent CN114073997A - a microfluidic chip and method for realizing rapid and accurate cell sorting at low flow rates. The expansion channels and contraction channels are arranged alternately, and the adjacent expansion channels and contraction channels are connected to each other. The width of the expansion channels is greater than the width of the contraction channels; The width of the road. In this application, the contraction and expansion array channel only shows that its contraction and expansion structures are connected in sequence, and there is no specific design parameter standard. The reaction to election efficiency.

To sum up, the existing microfluidic chips have the problems that the lateral migration distance of cell particles is too small, cannot efficiently pre-focus and sort cell particles, and cannot act on cells of different sizes.

Contents of the invention

The purpose of the present invention is to solve the deficiencies in the prior art, to provide a microcell for pre-focused sorting that is applicable to cells of different sizes and realizes convenient, label-free, and high-efficiency focused sorting of tiny cell particles. Fluidic chip and method.

The technical solution adopted in the present invention: the present invention discloses a microfluidic chip for pre-focusing and sorting cells, including a fluid confluence area, a fluid co-flow area, a polygonal focus area, and a cell particle sorting area that are sequentially connected from left to right ;

The front end of the fluid confluence area is respectively equidistantly connected to the viscoelastic fluid inlet and the Newtonian fluid inlet;

The viscoelastic fluid inlet is a mixed fluid inlet comprising large cell particles, viscoelastic solution and small cell particles;

The end of the cell particle sorting area is provided with a small cell particle outlet, a large cell particle outlet and a waste liquid outlet;

The Newtonian fluid inlet is a Newtonian fluid solution inlet;

The polygonal focusing area includes several polygonal focusing units, and two adjacent polygonal focusing units are communicated through shrinking and expanding channels;

The channel width of the narrowing and expanding channel is smaller than the maximum channel width of the polygonal focusing unit, and smaller than the channel width of the fluid co-flow area.

Further, the flow channel width of the viscoelastic fluid inlet connected to the fluid confluence area is the same as the flow channel width of the Newtonian fluid inlet connected to the fluid confluence area, which is half of the flow channel width of the fluid confluence area;

The flow path width of the fluid co-flow area and the fluid confluence area is the same.

Further, the flow channel width of the cell particle sorting area is the same as the maximum vertical width of the polygonal focus area;

The flow channel width of the small cell particle outlet is the same as that of the large cell particle outlet, which is 1/3 of the flow channel width of the cell particle sorting area.

Further, at the inlet end of the polygonal focusing unit, the included angle A between the shrinking and expanding channel and the inlet of the polygonal focusing unit is 120°; at the outlet end of the polygonal focusing unit, the contracting and expanding channel and the The included angle A at the outlet of the polygonal focusing unit is 135°.

Further, the polygonal focusing unit is arranged symmetrically with respect to the shrinking and expanding channel, including an inlet section, a smooth section and an outlet section; the width of the smooth section channel is the maximum channel width of the polygonal focusing unit.

Further, the viscoelastic solution 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.

Further, the width of the shrinking and expanding channel is 1/4 of the maximum channel width of the polygonal focusing unit.

The invention also discloses a sorting method of a microfluidic chip for pre-focusing and sorting cells, which includes the following steps:

S1. Add a viscoelastic enhancer to the sample solution of the cell particles to be sorted to prepare a viscoelastic sample solution with a concentration of a certain mass percentage, and configure a Newtonian fluid solution;

S2. Based on the above-mentioned microfluidic chip for pre-focusing and sorting cells, the viscoelastic sample solution is introduced into the viscoelastic fluid inlet at a stable flow rate, and the Newtonian fluid inlet is passed into the Newtonian fluid solution at the same time;

S3. After the viscoelastic sample solution and the Newtonian fluid solution are converged in the fluid confluence area, they flow through the fluid co-flow area and the polygonal focus area in turn, and then separate the small cell particle outlet and the large cell particle outlet with different diameters in the cell particle sorting area. The cell particle sample of the same size, and the rest of the impurity waste liquid is discharged from the waste liquid outlet.

Further, the viscoelastic 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.

The beneficial effects of the present invention are:

1. The microfluidic chip for pre-focused and sorted cells provided by this application generates viscoelastic and inertial effects in the microchannel through the injected viscoelastic fluid and Newtonian fluid, thereby inducing viscoelasticity in the viscoelastic solution. lift and induce inertial lift on the wall of the microchannel, and its resultant force can act on cells of different sizes; with the polygonal focusing unit set in the chip, when the cell particles enter the flow channel, due to the sudden decrease in the width of the flow channel, the particles are short The speed will increase sharply within a short period of time. With subsequent multiple focusing units, it will flexibly and quickly focus on different cells and particles at different positions in the microchannel space, providing an easy-to-control, high-efficiency and low-cost method for the subsequent sorting of cells. In the experimental environment, this application makes full use of the hydrodynamic effect of carrier media such as Newtonian fluid and viscoelastic fluid to solve the problem that the lateral migration distance of cell particles is too small and cannot efficiently pre-focus and sort cell particles, and realizes the convenience of tiny cell particles, Marker-free, high-efficiency focused sorting capabilities.

2. On the basis of experimental tests and cell applications, according to the sorting performance of cell particles in different viscoelastic solutions and different flow rates, the polygonal focus area is designed, specifically, it is a polygonal structure that is symmetrical about the symmetry axis, From the perspective of the flow direction of the solution from left to right, the angle between the upstream side of the structure and the flow channel is 120°, and the angle between the downstream side and the flow channel is 135°; this polygonal focusing unit is suitable for different Sorting trajectories of most cells with a size of 8-12 μm in a viscoelastic solution at a flow rate;

Due to the sudden increase in space, the fluid under high-speed flow forms a vortex in it, and the particles with smaller diameters are more likely to be captured by the polygonal structure due to the elastic lift force of the wall-guiding interface. At the same time, through the capture effect of the multi-structure, Further improve the sorting efficiency of particles; therefore, this design can better improve the lateral migration effect of the shrinking and expanding structure in the sorting process, avoid the mutual interference of particles under high flux, improve the sorting efficiency and The improvement of cell particle flux has a better effect.

3. The fluid confluence area, the fluid co-flow area, the polygonal focus area and the cell particle sorting area are sequentially connected from left to right; the front end of the fluid confluence area is respectively equidistantly connected to the viscoelastic fluid inlet and the Newtonian fluid inlet; the viscoelastic fluid inlet is included The mixed fluid inlet of large cell particles, viscoelastic solution and small cell particles; the end of the cell particle sorting area is equipped with small cell particle outlets and large cell particle outlets; because of the driving force of passive separation of cell particles, such as inertial lift and viscoelastic lift, Strongly dependent on particle diameter, particles of different diameters are affected by different resultant forces. Therefore, cell particles with different diameters migrate in the micro-channel to produce different distance differences, and the way of lateral migration of the particles is pre-arranged, and finally flow into different channel outlets to achieve the effect of sorting, and the overall realization of cell The experimental process of "focusing first, then sorting" particles. This method can not only improve the sorting efficiency in the cell sorting process, but also further improve the throughput of cell particle sorting.

Description of drawings

1 is a schematic diagram of the overall structure of the microfluidic chip in the present invention;

Fig. 2 is the concrete structure schematic diagram of polygon focusing unit among the present invention;

Fig. 3 is a schematic diagram of the principle of focusing and sorting in a microchannel structure in an example of the present invention;

Fig. 4 is a schematic diagram of the migration of cell particles inside the microfluidic chip for focusing and sorting cells in the example of the present invention;

Fig. 5 is a graph showing the results of a focus sorting experiment of cell particles in Newtonian fluid/viscoelastic sheath fluid=PBS/500ppmPEO=80/80μL/min in an example of the present invention;

Fig. 6 is a result diagram of the focus sorting experiment of cell particle Newtonian fluid/viscoelastic sheath fluid=PBS/500ppmPEO=80/240μL/min in the example of the present invention;

In the figure: 1-viscoelastic fluid inlet; 2-Newtonian fluid inlet; 21-Newtonian fluid solution; 3-fluid confluence area; 4-fluid co-flow area; Expanding channel; 6-cell particle sorting area; 7-small cell particle outlet; 8-large cell particle outlet; 11-large cell particle; 12-viscoelastic polymer; 13-small cell particle.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

As shown in Figure 1, it includes the fluid confluence area 3, the fluid co-flow area 4, the polygonal focus area 5, and the cell particle sorting area 6, which are sequentially connected from left to right; the flow channel width of the fluid co-flow area 4 and the fluid confluence area 3 same.

The front end of the fluid confluence area 3 is equidistantly connected to the viscoelastic fluid inlet 1 and the Newtonian fluid inlet 2 respectively; half of the width of the flow channel in the fluid confluence area 3;

Among them, the viscoelastic fluid inlet 1 is a mixed fluid inlet including large cell particles 11, viscoelastic solution 12 and small cell particles 13, wherein the viscoelastic solution is polyethylene oxide PEO, polyvinylpyrrolidone PVP and hyaluronic acid HA. one or more of .

The Newtonian fluid inlet 2 is the Newtonian fluid solution 21 inlet; the Newtonian fluid solution 21 is one or more of deionized water DIWATER, phosphate balanced physiological saline PBS and distilled water.

The end of 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; the flow path width of the cell particle sorting area 6 is the same as the maximum vertical width of the polygonal focus area 5, and is one minute. Three times the width of the three-jaw microchannel.

The polygonal focusing area 5 includes several polygonal focusing units 50 , and two adjacent polygonal focusing units 50 are communicated through shrinking and expanding flow channels 51 ;

The channel width of the narrowing and expanding channel 51 is smaller than the maximum channel width of the polygonal focusing unit 50 and smaller than the channel width of the fluid co-flow area 4 .

Wherein, at the inlet end of the polygonal focusing unit 50, the included angle A between the shrinking and expanding channel 51 and the inlet of the polygonal focusing unit 50 is 120°; Angle A is 135°.

The polygonal focusing unit 50 is arranged symmetrically with respect to the shrinking and expanding channel 51 , including an inlet section 510 , a smooth section 511 and an outlet section 512 ; the channel width of the smooth section 511 is the maximum channel width of the polygonal focusing unit 50 .

The microfluidic chip is made of photolithography technology and PDMS material. In the experiment, the shape of the microfluidic channel designed in Figure 1 is molded out, and finally the PDMS is bonded to a glass slide for focusing and sorting applications of biological particles.

The passive control of particles in microfluidic experiments largely relies on the hydrodynamic effects of carrier media such as Newtonian fluids and viscoelastic fluids, such as inertia and viscoelasticity, which can be achieved in a label-free and force-field-free manner in this solution environment. To achieve efficient cell particle manipulation.

Among them, the driving force of passive separation, such as inertial lift _FL or viscoelastic lift F _e , strongly depends on the particle diameters aF _L ~ a ⁴ , F _e ~ a ³ in microfluidics, so cell particles with different diameters a will be subject to different migration effects.

This invention is based on the co-flow of Newtonian fluid PBS and viscoelastic fluid PEO in the microfluidic chip, and performs pre-focusing and sorting functions according to the size-dependent difference of particles. As shown in Figure 2, in order to improve the separation performance of inertial or viscoelastic microfluidics, this chip uses the combined action of sheath fluid and inertial force or elastic lift force to pre-arrange the way particles migrate laterally.

Since Newtonian fluid has no elastic stress, the elastic lift force acting on the particle changes significantly. Co-flow conditions yield a stable viscoelastic/Newtonian interface with larger separation distances between particles of different sizes than experimental approaches for pure Newtonian fluids or pure viscoelastic flows.

During the experiment, the cell particles are initially arranged along the sidewall through the protective flow of the viscoelastic fluid inlet 1, and with the co-flow of the two fluids, the cell particles with larger particle diameters are pushed away from the sidewall by the inertial lift guided by the centerline , through the co-flow interface, gradually move closer to the center and focus, while small particles cannot pass through the interface between the Newtonian medium and the viscoelastic medium due to the elastic lift force of the wall-guided interface, and still focus in the viscoelastic flow, thus in the micro Particle sorting is realized in the flow channel.

As shown in Figure 4, in practical application, firstly, a viscoelasticity enhancer is added to the sample solution of the cell particles to be sorted to prepare a viscoelastic sample solution PEO with a concentration of a certain mass percentage, wherein the added viscoelasticity enhancer accounts for 1% of the sample solution. Mass percent concentration≤20%.

Use a syringe pump to push the viscoelastic fluid into the inlet 1 at a steady flow rate, and at the same time pass the Newtonian fluid PBS into the Newtonian fluid inlet 2; the two sample solutions flow through the fluid intersection area 3, because of the difference in fluid viscosity, there is only a small amount at the interface. The mixing effect is produced, and a stable viscoelastic/Newtonian interface can be produced after passing through the fluid co-flow zone 4. At this time, according to the migration principle in Figure 2, the cell particles with large diameters are gradually under the joint action of the inertial effect and the viscoelastic effect. down toward the centerline of the runner. Afterwards, the fluid enters the polygonal focus area 5. Due to the sudden increase in space, the fluid under high-speed flow forms a vortex in it, and the particles with smaller diameters are more likely to be captured by the polygonal structure due to the elastic lift force of the wall-guiding interface. At the same time, the sorting efficiency of particles is further improved through the capture effect of multiple structures.

In addition, the polygonal focus areas are connected by a narrow narrow expansion flow channel 51. When the cell particles enter the flow channel, due to the sudden decrease in width, the particle speed suddenly increases in a short period of time. With the subsequent multiple focus structures , this structure will further speed up the separation of different particles and improve the subsequent sorting efficiency. Finally, with the focused sorting of the cell particles, the particles of different sizes in the cell particle sorting area are separated from the small cell particle outlet 7 and the large cell particle outlet 8 to separate cell particle samples with different diameters.

Figure 5 is the results of the focusing sorting experiment of cell particles under the conditions of Newtonian fluid/viscoelastic sheath fluid=PBS/500ppmPEO=80/80μL/min;

Fig. 6 is a graph showing the experimental results of focus sorting under the condition of Newtonian fluid/viscoelastic sheath fluid=PBS/500ppmPEO=80/80μL/min.

It can be seen from the figure of the experimental results that particles of different sizes can effectively produce the effects of focusing and sorting under different experimental conditions, and the experimental results are consistent with the theoretical analysis.

This application makes full use of the co-flow of Newtonian fluid PBS and viscoelastic fluid PEO, and performs pre-focusing and sorting functions according to the size-dependent differences of particles, which is comparable to the existing hydrodynamic separation technology using single-phase Newtonian or viscoelastic flow conditions. In contrast, stable co-flow conditions will produce obvious interfacial viscoelastic effects and inertial effects. The interaction of these two forces leads to a large separation distance between particles of different sizes, resulting in high separation efficiency and high separation purity. In addition, the concentration of polymer used under co-flow conditions is much lower than that under purely viscous conditions, but since the viscoelastic lift force at the interface is greater than the viscoelastic lift force at the bulk, the particles in the microfluidic channel will have more efficient focusing and sorting efficiency, The low concentration of PEO environment will also provide a more suitable condition for the operation of biological samples.

Therefore, this microfluidic chip provides a general, label-free, high-efficiency focused sorting method for tiny cell particles.

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

Inspired by the above-mentioned ideal embodiment according to the present invention, through the above-mentioned description content, relevant workers can make various changes and modifications within the scope of not departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, but must be determined according to the scope of the claims.

Claims (9)

1. A microfluidic chip for pre-focusing and sorting cells, characterized in that: it includes a fluid confluence area (3), a fluid co-flow area (4), a polygonal focus area (5) and a cell that are sequentially connected from left to right Particle sorting area (6); The front end of the fluid confluence area (3) is equidistantly connected to the viscoelastic fluid inlet (1) and the Newtonian fluid inlet (2); The viscoelastic fluid inlet (1) is a mixed fluid inlet comprising large cell particles (11), viscoelastic solution (12) and small cell particles (13); The end of 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); The Newtonian fluid inlet (2) is a Newtonian fluid solution (21) inlet; The polygonal focusing area (5) includes several polygonal focusing units (50), and two adjacent polygonal focusing units (50) are communicated through shrinking and expanding channels (51); The channel width of the narrowing and expanding channel (51) is smaller than the maximum channel width of the polygonal focusing unit (50), and smaller than the channel width of the fluid co-flow area (4). 2. The microfluidic chip according to claim 1, characterized in that: the viscoelastic fluid inlet (1) communicates with the flow channel width of the fluid confluence region (3) and communicates with the Newtonian fluid inlet (2) The flow channel width of the fluid confluence area (3) is the same, which is half of the flow channel width of the fluid confluence area (3); The flow channel width of the fluid co-flow area (4) and the fluid confluence area (3) is the same. 3. The microfluidic chip according to claim 1, characterized in that: the flow channel width of the cell particle sorting area (6) is the same as the maximum vertical width of the polygonal focus 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). 4. The microfluidic chip according to claim 1, characterized in that: at the inlet end of the polygonal focusing unit (50), the shrinking and expanding channel (51) is connected to the inlet of the polygonal focusing unit (50). The included angle A is 120°; at the outlet end of the polygonal focusing unit (50), the included angle A between the shrinking and expanding channel (51) and the outlet of the polygonal focusing unit (50) is 135°. 5. The microfluidic chip according to claim 4, characterized in that: the polygonal focusing unit (50) is arranged symmetrically with respect to the shrinking and expanding channel (51), including an inlet section (510), a smooth section (511 ) and the outlet section (512); the flow channel width of the smooth section (511) is the maximum flow channel width of the polygonal focusing unit (50). 6. 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 physiological saline PBS and distilled water. 7. The microfluidic chip according to claim 4, characterized in that: the width of the narrowing and expanding channel (51) is 1/4 of the maximum channel width of the polygonal focusing unit (50). 8. A sorting method of a microfluidic chip for pre-focused sorting cells, characterized in that: comprising the steps of: S1. Add a viscoelastic enhancer to the sample solution of the cell particles to be sorted to prepare a viscoelastic sample solution with a concentration of a certain mass percentage, and configure a Newtonian fluid solution; S2. Based on the microfluidic chip of any one of claims 1-5, the viscoelastic sample solution is introduced into the viscoelastic fluid inlet (1) with a steady flow rate, while the Newtonian fluid inlet (2 ) into the Newtonian fluid solution; S3. The viscoelastic sample solution and the Newtonian fluid solution flow through the fluid co-flow area (4) and the polygonal focus area (5) sequentially after converging in the fluid confluence area (3), and then flow from small to small in the cell particle sorting area (6) respectively. The cell particle outlet (7) and the large cell particle outlet (8) separate cell particle samples with different diameters, and the waste liquid of other impurities is discharged from the waste liquid outlet (9). 9. The sorting method of the microfluidic chip of pre-focus sorting cells according to claim 8, characterized in that: the viscoelastic enhancer is polyethylene oxide PEO, polyvinylpyrrolidone PVP and hyaluronic acid One or more of HA; The Newtonian fluid solution is one or more of deionized water DIWATER, phosphate balanced physiological saline PBS and distilled water.

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