Continuous integrated micro-fluidic chip device for inertia sorting of large-size cells

Abstract

The invention discloses a continuous integrated micro-fluidic chip device for inertia sorting of large-size cells, which comprises a shell and a continuous multi-stage sorting device arranged in the shell, the shell is provided with a mixed particle solution inlet and a target ion solution outlet which are communicated with an inlet and an outlet of the continuous multi-stage sorting device, and an impurity particle outlet which is communicated with each stage of sorting device in the continuous multi-stage sorting device, and particle sorting units of two adjacent stages of sorting devices are in exponential increase. According to the micro-fluidic chip device, the side channel is creatively arranged at the discharging end of the micro-groove, so that the sorting efficiency of particles / cells can be greatly improved, and a captured target particle / cell solution can be directly collected without secondary treatment.

Description

Technical Field

[0001] The invention relates to the field of microfluidic technology, and in particular to a microfluidic chip device for continuously integrating inertial sorting of large-sized cells. Background Art

[0002] In recent years, particle / cell sorting based on microfluidics has become a key technology for tumor cell detection. In cell biology research and clinical diagnosis, sorting and purifying target cells from complex cell samples is a crucial sample processing step. Microfluidics has become a new platform for cell sorting because of its ability to manipulate tiny volumes of fluid in microchannels. The particle / cell sorting method based on vortex cells in microchannel grooves fully utilizes vortex cell flow and particle / cell inertial migration to sort extremely low circulating tumor cells (CTC, particle size 15-30μm) from complex whole blood cells (5-10μm). Due to its unique advantages, it stands out from a variety of microfluidic particle / cell sorting methods and is expected to become the core technology of cancer "liquid biopsy".

[0003] Applying microgroove-based particle / cell sorting technology to the clinical field is a challenge that has attracted much attention. Although many scholars have conducted in-depth research, there are still some technical limitations and bottlenecks. These limitations include the current microgroove technology’s limitation on the number of target particles / cells that can be accommodated (usually 30-40), and the high shear force that may be caused to the target particles / cells during the sorting process, resulting in particle / cell damage. In addition, current microfluidic devices can usually only sort target particles / cells of a specific size threshold, which limits their flexibility and diversity in clinical applications. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a microfluidic chip device for continuous integrated inertial sorting of large-sized cells. The microfluidic chip device creatively sets a side channel at the discharge end of the microgroove, which can greatly improve the particle / cell sorting efficiency, and the captured target particle / cell solution can be directly collected without secondary processing.

[0005] A microfluidic chip device for continuous integrated inertial sorting of large-sized cells comprises a shell and a continuous multi-stage sorting device arranged in the shell, wherein the shell is provided with a mixed particle solution inlet connected to the inlet and outlet of the continuous multi-stage sorting device, a target ion solution outlet and an impurity particle outlet connected to each stage of the sorting device in the continuous multi-stage sorting device, and the particle sorting units of the sorting devices in two adjacent stages increase exponentially.

[0006] As a preferred embodiment of the above technical solution, the particle sorting unit includes micro-grooves, a main feed channel and a main discharge channel relatively connected to the micro-grooves, and side channels symmetrically arranged on both sides of the main discharge channel and connected to the micro-grooves. The main feed channel of the particle sorting unit of the first-stage sorting device is connected to the inlet of the mixed particle solution, the side channels of all the particle sorting units of the last-stage sorting device are converged and connected to the outlet of the target ion solution, the main feed channel of the particle sorting unit of the latter-stage sorting device is connected to the side channel of the particle sorting unit of the former-stage sorting device, and the main discharge channel of each group of the particle sorting units is connected to one of the impurity particle outlets.

[0007] As a preferred embodiment of the above technical solution, the micro groove is formed by symmetrically splicing two identical arc-shaped grooves, and the main feed channel and the main discharge channel are arranged on the outside of the splicing of the two arc-shaped grooves.

[0008] As a preferred embodiment of the above technical solution, the micro-groove is divided into a mainstream flow field, a sheath flow field and a vortex cell flow field according to the flow direction of particles / cells.

[0009] As a preferred embodiment of the above technical solution, the diameter threshold of the target particles / cells to be sorted can be adjusted by changing the length of the outlet external pipe of the side channel.

[0010] As a preferred embodiment of the above technical solution, the diameter threshold of the target particles / cells to be sorted can be adjusted by changing the flow rates of the main feed channel and the main discharge channel.

[0011] As a preferred embodiment of the above technical solution, the shell includes an upper cover plate and a lower base plate which are covered together, and the continuous multi-stage sorting device is fixed to the lower cover plate by upper and lower bonding through oxygen ions.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The microfluidic chip device creatively adds a side channel 114 on the rear wall of the micro-groove, so that the sorting efficiency of target particles / cells reaches a breakthrough of 98%, which is much higher than that of traditional closed grooves (HeLa 10%, MCF7 23%).

[0014] 2. The microfluidic chip device is easy to operate, and the captured target particles / cell solutions can be directly collected without secondary processing, because the microfluidic chip device does not require other external forces and only requires the inertial force of the particles / cells flowing in the flow field to drive the sorting, which greatly liberates manpower and does not require the assistance of other large equipment. It maintains the simplicity of the chip while ensuring breakthrough capture efficiency.

[0015] 3. This microfluidic chip device is suitable for particle / cell sorting at low flow rates (Re~18), and can maximize the activity of target cells in clinical applications, which provides new ideas for the application of microfluidics in cell biology research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural schematic diagram of the present invention.

[0017] Figure 2 Schematic diagram of the structure of the particle sorting unit.

[0018] Figure 3 This is the flow field differentiation diagram of the micro groove.

[0019] Figure 4 These are experimental diagrams of a single particle sorting unit, where a is a real shot of the chip, b is a schematic diagram of the flow domain size within the chip, and c is a particle trajectory diagram taken during the experiment.

[0020] Figure 5 It is a diagram of particle sorting mechanism, where a is the force diagram of particle sorting, and b is the streamline diagram during particle sorting.

[0021] Figure 6 This is a conceptual diagram of three-level sorting.

[0022] The figure numbers are as follows: 1-shell, 101-upper cover, 102-lower bottom plate, 2-mixed particle solution inlet, 3-target ion solution outlet, 4-particle sorting unit, 401-micro groove, 401a-main flow field, 401b-sheath flow field, 401c-vortex cell flow field, 402-main feed channel, 403-main discharge channel, 404-side channel, 5-primary sorting device, 6-secondary sorting device, 7-tertiary sorting device, 8-primary impurity particle outlet, 9-secondary impurity particle outlet, 10-tertiary impurity particle outlet. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0025] like Figure 1The microfluidic chip device for continuous integrated inertial sorting of large-sized cells comprises a shell 1 and a continuous multi-stage sorting device arranged in the shell 1, the shell 1 is provided with a mixed particle solution inlet 2 connected to the inlet and outlet of the continuous multi-stage sorting device, a target ion solution outlet 3 and an impurity particle outlet connected to each stage of the sorting device in the continuous multi-stage sorting device, and the particle sorting units 4 of the sorting devices in two adjacent stages increase exponentially.

[0026] In this embodiment, the particle sorting unit 4 includes a micro-groove 401, a main feed channel 402 and a main discharge channel 403 relatively connected to the micro-groove 401, and a side channel 404 symmetrically arranged on both sides of the main discharge channel 403 and connected to the micro-groove 401. Figure 2 As shown, the main feed channel 401 of the particle sorting unit 4 of the first-stage sorting device is connected to the mixed particle solution inlet 2, the side channels 404 of all the particle sorting units 4 of the last-stage sorting device are converged and connected to the target ion solution outlet 3, the main feed channel 401 of the particle sorting unit 4 of the latter-stage sorting device is connected to the side channel 404 of the particle sorting unit 4 of the former-stage sorting device, and the main discharge channel 403 of each group of the particle sorting units 4 is connected to one of the impurity particle outlets.

[0027] In this embodiment, the micro groove 401 is formed by symmetrically splicing two identical arc grooves, and the main feeding channel 402 and the main discharging channel 403 are arranged outside the splicing of the two arc grooves. Specifically, the curvature of each arc groove is 4 / 5 of the same diameter circle.

[0028] In this embodiment, the micro groove 401 is divided into a mainstream flow field 401a, a sheath flow field 401b and a vortex cell flow field 401c according to the flow direction of particles / cells. Figure 3 shown.

[0029] In this embodiment, the diameter threshold for sorting target particles / cells can be adjusted by changing the length of the outlet external pipe of the side channel 404 .

[0030] In this embodiment, the diameter threshold of the target particles / cells to be sorted can be adjusted by changing the flow rates of the feed main channel 402 and the discharge main channel 403 .

[0031] In this embodiment, the housing 1 includes an upper cover plate 101 and a lower base plate 102 that are covered together, and the continuous multi-stage separation device is fixed to the lower cover plate 102 by upper and lower bonding via oxygen ions.

[0032] In view of the three major limitations of the existing particle / cell sorting technology based on micro-grooves, namely, the size of the groove limits the capacity of the captured target particles / cells (30-40); under high Reynolds numbers (Re), the high shear force generated by the flow field seriously damages the cells; most grooves can only perform dual-modal separation at a single cutoff diameter, the present invention first proposes a microfluidic chip device for continuous integrated inertial sorting of large-sized cells. The micro-groove 1 structure and chip structure of the present application are simple, the operation is simple, the sorting process does not require additional equipment assistance, and the sorting efficiency is extremely high.

[0033] This embodiment takes three-stage sorting as an example. Figure 1 As shown, the continuous multi-stage sorting device includes a primary sorting device 5, a secondary sorting device 6 and a tertiary sorting device 7, wherein the primary sorting device 5 is provided with a group of particle sorting units 4, and correspondingly, the secondary sorting device 6 and the tertiary sorting device 7 are provided with two groups of particle sorting units 4 and four groups of particle sorting units 4, respectively. Correspondingly, the impurity particle outlet connected to the main discharge channel 403 of the particle sorting unit 4 of the primary sorting device 5 is the primary impurity particle outlet 8, the impurity particle outlet connected to the main discharge channel 403 of the particle sorting unit 4 of the secondary sorting device 6 is the secondary impurity particle outlet 9, and the impurity particle outlet connected to the main discharge channel 403 of the particle sorting unit 4 of the tertiary sorting device 7 is the tertiary impurity particle outlet 10.

[0034] The specific method of use of the present application is very simple: use a syringe pump to inject the mixed particle solution at a constant flow rate into the mixed particle solution inlet 2 on the chip, and after three-stage sorting, the mixed particle solution outputs a high-purity target particle solution through the target particle solution outlet 3, wherein the target particle solution has only one outlet, and the target solution can be directly collected at this outlet without secondary operation, to prevent contamination and damage to the target solution. The continuous multi-stage sorting device in the present application can be formed by processing on the bottom surface of the upper cover plate 101 through standard photolithography technology, and the height of each stage of the sorting device is the same, and the sorting devices of the same height can greatly simplify the production of the chip.

[0035] To verify the feasibility of the present invention, an experimental verification was carried out. Figure 4 The microchannel chip material used in the experiment is a high molecular polymer polydimethylsiloxane (PDMS), which is made by standard photolithography technology. The micro-groove diameter D is set to 500 μm and the opening length is L. c =400 μm. The width of the main feed channel 402 and the main discharge channel 403 is W=80 μm, the height is H=100 μm, and the length is L1=15 mm. The width of the side channel 404 is W1=W2=50 μm, and the inner diameter of the external pipe is 0.6 mm.

[0036] We define the ratio of the flow rate flowing through the side outlet 404 to the flow rate of the main feed channel as e. When e=1 / 15, the length of the external pipe of the side channel 404 is L2=L3=156mm; when e=1 / 10, the length of the external pipe of the side channel 404 is L2=L3=123mm; when e=1 / 15, the length of the external pipe of the side channel 404 is L2=L3=61mm. To facilitate the production of microchannels, the channel height is the same, H=100μm. In order to make the streamlines more closely fit the shape of the micro-groove 401, a circular micro-groove 401 is designed, with a diameter of D=500μm and an opening of L c =100μm.

[0037] In this embodiment, firstly, external pipes of different lengths are selected according to the different flow resistance settings of the side channels to be connected to the two side channels 401, and then a syringe pump (Elite 1012, Harvard Apparatus) is used to inject the mixed particle / cell solution in a syringe (Hamilton 1000, Hamilton Company) from the inlet 402 of the main feed channel, and the inlet Reynolds number of the main feed channel 402 is Re=9 to 36. The target large particles / cells are sorted into the side channel 404 when passing through the micro grooves 401, and the non-target small particles / cells flow out through the discharge main channel 403 after passing through the particle sorting unit.

[0038] The particle / cell motion behavior was observed and recorded by an inverted microscope system (IX73, Olympus) and a high-speed camera (VEO 340L, Phantom). The video recording resolution was 832×600 and the frame rate was 6000fps. The experimental results are shown in Figure 2. Figure 4 (c) is shown. The experimental data are statistically analyzed to show that when the inlet Reynolds number Re = 18.5 and the length of the external tube of the side channel 404 L2 = L3 = 123 mm, that is, e = 1 / 10, the sorting effect of a single particle / cell sorting unit 111 on 20μm particles / cells is optimal, and the sorting efficiency can reach up to 98%, the purity of the sorted 20μm particles can reach 67.3%, and the removal efficiency of impurity particles / cells is 97.1%. It can be clearly seen that only a single particle / cell sorting unit has achieved high-efficiency target particle / cell sorting, laying the foundation for the subsequent parallel multipolarization application of this micro-groove structure.

[0039] In the above embodiment, the mixed particle / cell solution undergoes three steps of sorting when passing through the particle sorting unit, such as Figure 5 (a) shown.

[0040] The first step is the inertial focusing of the particles in the feed main channel 402: when the solution containing mixed particles / cells is introduced into the microchannel, the particles are initially evenly distributed on the channel cross section, and when the particles move downstream, they are subjected to the shear gradient induced lift force (F LS) and wall-induced lift (F LW ), these two forces compete with each other in the lateral direction. The net lift force F on the particle in the lateral direction is L The horizontal is proportional to the fourth power of the particle diameter a. Therefore, larger particles are subject to a greater net lift force, and the equilibrium position of larger particles is closer to the wall of the main feed channel 402 than that of small particles;

[0041] The second step is the inertial transition of the particle when it reaches the micro-groove 401: when the focused particle reaches the micro-groove 401, the adjacent channel wall suddenly disappears. Therefore, the wall-induced lift (F LW ) disappeared. The shear gradient induced lift (F LS ) will act on the particle / cell alone, causing the particle to move across the streamline toward the sheath flow. LS ) The shear gradient decays slowly in the expansion region. The lateral migration velocity of the particle is proportional to the square of the particle diameter. Therefore, it can be expected that larger particles will migrate much faster than smaller particles, and larger particles with higher lateral migration velocities are likely to pass through the streamline and enter the sheath flow field 401b;

[0042] The third step is that when the particle reaches the deep part of the micro-groove 401, due to the existence of the side channel 404 and the micro-groove 401, the fluid distribution in the expansion-contraction area of ​​the channel is divided into three parts: the main stream presenting an outwardly expanding arc shape, that is, the main stream flow field 401a; the arc-shaped long strip sheath flow flowing into the side channel 401, that is, the sheath flow field 401b; the vortex near the lower wall in the deep part of the micro-groove 401, that is, the vortex flow field 401c. Due to the expansion of the channel, the fluid velocity in the channel decreases rapidly, and the velocity of the fluid inside the micro-groove 401 along the y direction decreases monotonically exponentially. At this time, the particle / cell is only subjected to the shear gradient induced lift (F LS ) is affected by the shear gradient, and the force on the particle is proportional to the square of the maximum velocity of the fluid. Therefore, the shear gradient induced lift (F LS ) can be ignored, the inertial effect on particles / cells is proportional to the particle diameter. The larger the particle diameter, the easier it is for the particle to cross the streamline under the influence of inertia and enter the sheath flow field 401b.

[0043] In the above embodiment, when the mixed particle / cell solution undergoes three-stage sorting, the schematic diagram is as follows: Figure 6As shown. The target particle / cell solution sorted out by the primary sorting device 5 flows into two particle sorting units 4 through two side channels 404 for secondary sorting; the target particle / cell solution sorted out by the secondary sorting device 6 flows into four particle sorting units 4 through four side channels 404 for tertiary sorting. Multi-stage sorting can significantly improve the sorting purity of the chip of this invention. Preliminary experiments show that the purity of the target particles / cells in the target particle / cell solution after the three-stage sorting is increased by 8.8 times, and the concentration of the target particles / cells is 137.5 times the initial concentration. Figure 6 shown.

[0044] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A microfluidic chip device for continuous integrated inertial sorting of large-sized cells, characterized in that: It comprises a shell and a continuous multi-stage sorting device arranged in the shell, wherein the shell is provided with a mixed particle solution inlet connected to the inlet and outlet of the continuous multi-stage sorting device, a target ion solution outlet and an impurity particle outlet connected to each stage of the sorting device in the continuous multi-stage sorting device, and the particle sorting units of the sorting devices in two adjacent stages increase exponentially.

2. The microfluidic chip device according to claim 1, characterized in that: The particle sorting unit includes a micro-groove, a main feed channel and a main discharge channel relatively connected to the micro-groove, and side channels symmetrically arranged on both sides of the main discharge channel and connected to the micro-groove. The main feed channel of the particle sorting unit of the first-stage sorting device is connected to the inlet of the mixed particle solution, the side channels of all the particle sorting units of the last-stage sorting device converge and are connected to the outlet of the target ion solution, the main feed channel of the particle sorting unit of the latter-stage sorting device is connected to the side channel of the particle sorting unit of the former-stage sorting device, and the main discharge channel of each group of the particle sorting units is connected to one of the impurity particle outlets.

3. The microfluidic chip device according to claim 2, characterized in that: The micro groove is formed by symmetrically splicing two identical arc-shaped grooves, and the main feed channel and the main discharge channel are arranged on the outside of the splicing point of the two arc-shaped grooves.

4. The microfluidic chip device according to claim 2, characterized in that: The micro-groove is divided into a mainstream flow field, a sheath flow field and a vortex cell flow field according to the flow direction of particles / cells.

5. The microfluidic chip device according to claim 2, characterized in that: The diameter threshold for sorting target particles / cells can be adjusted by changing the length of the outlet outer tube of the side channel.

6. The microfluidic chip device according to claim 1, characterized in that: The diameter threshold for sorting target particles / cells can be adjusted by changing the flow rates of the feed main channel and the discharge main channel.

7. The microfluidic chip device according to claim 1, characterized in that: The shell comprises an upper cover plate and a lower bottom plate which are covered together, and the continuous multi-stage sorting device is fixed to the lower cover plate by upper and lower bonding via oxygen ions.

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