A microfluidic chip device for continuous integration of inertial sorting of large-sized cells

Abstract

The application discloses a micro-fluidic chip device for continuously integrating inertial 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, a target ion solution outlet and an impurity particle outlet communicated with inlets and outlets of the continuous multi-stage sorting device. Particle sorting units of two adjacent stages of the sorting device are exponentially increased. The micro-fluidic chip device is provided with a side channel at a discharging end of a micro-groove, which can greatly improve the sorting efficiency of particles / cells, and the target particle / cell solution after capture can be directly collected without secondary treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, and particularly relates to a continuous integrated inertial sorting large-size cell microfluidic chip device. BACKGROUND

[0002] In recent years, the realization of particle / cell sorting based on microfluidic technology 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. Microfluidic technology, due to its ability to manipulate micro-volume fluids in microchannels, has become a new platform for cell sorting. The particle / cell sorting method based on the vortex cell in the microchannel groove completely utilizes vortex flow and particle / cell inertial migration, and can sort extremely low content of circulating tumor cells (CTC, particle size 15-30 μm) from complex whole blood cells (5-10 μm). 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] The application of micro-groove-based particle / cell sorting technology in 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, including the current micro-groove technology's limitation on the number of target particles / cells it can accommodate (usually 30-40), and the high shear force that may cause damage to target particles / cells during the sorting process. In addition, the current microfluidic device can usually only sort target particles / cells of a specific size threshold, which limits its flexibility and diversity in clinical applications. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a continuous integrated inertial sorting large-size cell microfluidic chip device, which creatively sets a side channel at the discharge end of the micro-groove, greatly improves the sorting efficiency of particles / cells, and the target particle / cell solution after capture can be directly collected without the need for secondary processing.

[0005] A continuous integrated inertial sorting large-size cell microfluidic chip device, comprising a housing and a continuous multi-stage sorting device arranged in the housing, the housing is provided with a mixed particle solution inlet, a target ion solution outlet, and an impurity particle outlet communicated with each stage of the continuous multi-stage sorting device, and the particle sorting units of adjacent two stages of the sorting device are exponentially increased.

[0006] As the preferred technical scheme of the above, the particle sorting unit comprises a micro-groove, a feeding main channel and a discharging main channel in communication with the micro-groove, and side channels symmetrically arranged on both sides of the discharging main channel and in communication with the micro-groove, the feeding main channel of the particle sorting unit of the first-stage sorting device is in communication with the mixed particle solution inlet, the side channels of all the particle sorting units of the last-stage sorting device are gathered and then in communication with the target ion solution outlet, the feeding main channel of the particle sorting unit of the last-stage sorting device is in communication with the side channel of the particle sorting unit of the previous-stage sorting device, and the discharging main channel of each group of the particle sorting units is in communication with one impurity particle outlet.

[0007] As the preferred technical scheme of the above, the micro-groove is symmetrically spliced by two identical circular-arc grooves, and the feeding main channel and the discharging main channel are arranged outside the splicing position of the two circular-arc grooves.

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

[0009] As the preferred technical scheme of the above, the diameter threshold of the sorting target particles / cells can be adjusted by changing the length of the outlet-external pipe of the side channel.

[0010] As the preferred technical scheme of the above, the diameter threshold of the sorting target particles / cells can be adjusted by changing the flow rate of the feeding main channel and the discharging main channel.

[0011] As the preferred technical scheme of the above, the shell comprises an upper cover plate and a lower bottom plate which are combined together, and the continuous multi-stage sorting device is fixed by oxygen ion up-down bonding between the lower cover plate.

[0012] Compared with the prior art, the microfluidic chip device has the following advantages:

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

[0014] 2. The microfluidic chip device is convenient to operate, and the target particle / cell solution after capture can be directly collected without secondary treatment, because the microfluidic chip device only needs the inertial force of the particles / cells in the flow field to drive the sorting, which greatly liberates manpower and does not need other large equipment assistance, while ensuring the breakthrough capture efficiency and maintaining the simplicity of the chip.

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

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

[0017] Figure 2 It is a structural schematic diagram of the particle sorting unit.

[0018] Figure 3 It is a flow field diagram of the micro-groove.

[0019] Figure 4 It is an experimental diagram of a single particle sorting unit, wherein a is a chip photograph, b is a schematic diagram of the flow field size in the chip, and c is a particle trajectory diagram taken by experiment.

[0020] Figure 5 It is a particle sorting mechanism diagram, wherein a is a particle sorting force diagram, and b is a streamline diagram when the particle is sorted.

[0021] Figure 6 It is a three-stage sorting diagram.

[0022] The reference signs are as follows: 1 - shell, 101 - upper cover plate, 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 - feed main channel, 403 - discharge main channel, 404 - side channel, 5 - first-stage sorting device, 6 - second-stage sorting device, 7 - third-stage sorting device, 8 - first-stage impurity particle outlet, 9 - second-stage impurity particle outlet, 10 - third-stage impurity particle outlet. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0024] The present application will be described in further detail below in combination with the drawings:

[0025] As Figure 1The microfluidic chip device for continuous integrated inertial sorting of large-sized cells includes a housing 1 and a continuous multi-stage sorting device disposed within the housing 1. The housing 1 is provided with a mixed particle solution inlet 2, a target ion solution outlet 3, and an impurity particle outlet, which are connected to the inlet and outlet of the continuous multi-stage sorting device. The particle sorting units 4 of adjacent sorting devices increase exponentially.

[0026] In this embodiment, the particle sorting unit 4 includes a microgroove 401, a main feed channel 402 and a main discharge channel 403 that are relatively connected to the microgroove 401, and side channels 404 symmetrically arranged on both sides of the main discharge channel 403 and connected to the microgroove 401, such as... 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 inlet 2 of the mixed particle solution. The side channels 404 of all the particle sorting units 4 of the last-stage sorting device converge and are connected to the outlet 3 of the target ion solution. The main feed channel 401 of the particle sorting unit 4 of the next-stage sorting device is connected to the side channel 404 of the particle sorting unit 4 of the previous-stage sorting device. The main discharge channel 403 of each group of 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-shaped grooves, and the main feed channel 402 and the main discharge channel 403 are located on the outer side of the splicing point of the two arc-shaped grooves. Specifically, the curvature of each arc-shaped groove is 4 / 5 of a circle of the same diameter.

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

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

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

[0031] In this embodiment, the housing 1 includes an upper cover plate 101 and a lower bottom plate 102 that are fitted together, and the continuous multi-stage sorting device is fixed to the lower cover plate 102 by oxygen ion bonding.

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

[0033] The present application takes three-stage sorting as an example, as shown in Figure 1 The continuous multi-stage sorting device includes a first-stage sorting device 5, a second-stage sorting device 6 and a third-stage sorting device 7. The first-stage sorting device 5 is provided with a group of particle sorting units 4. Correspondingly, the second-stage sorting device 6 and the third-stage sorting device 7 are respectively provided with two groups of particle sorting units 4 and four groups of particle sorting units 4. Correspondingly, the impurity particle outlet communicating with the discharge main channel 403 of the particle sorting unit 4 of the first-stage sorting device 5 is a first-stage impurity particle outlet 8, the impurity particle outlet communicating with the discharge main channel 403 of the particle sorting unit 4 of the second-stage sorting device 6 is a second-stage impurity particle outlet 9, and the impurity particle outlet communicating with the discharge main channel 403 of the particle sorting unit 4 of the third-stage sorting device 7 is a third-stage impurity particle outlet 10.

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

[0035] To verify the feasibility of the present application, experiments were conducted, as shown in Figure 4 The microchannel chip material used in the experiment is a high-molecular polymer polydimethylsiloxane (PDMS), which is made by standard photolithography etching technology. The micro-groove diameter D is 500 μm, and the opening length L c is 400 μm. The width W of the feed main channel 402 and the discharge main channel 403 is 80 μm, the height H is 100 μm, and the length L1 is 15 mm. The width W1 and W2 of the side channel 404 are 50 μm, and the inner diameter of the connecting pipe is 0.6 mm.

[0036] We define the ratio of the flow through the side outlet 404 to the flow of the main inlet channel as e, when e = 1 / 15, the length of the outer connecting pipe of the side channel 404 L2 = L3 = 156 mm; e = 1 / 10, the length of the outer connecting pipe of the side channel 404 L2 = L3 = 123 mm; e = 1 / 15, the length of the outer connecting pipe of the side channel 404 L2 = L3 = 61 mm. For the convenience of microchannel production, the channel height is the same, H = 100 μm. In order to make the streamline more fit the shape of the micro groove 401, a circular micro groove 401 is designed, the diameter D = 500 μm, the opening L c = 100 μm.

[0037] In this embodiment, first, according to the flow resistance of the side channel, the outer connecting pipe of different length is connected to the two side channels 401, and then the mixed particle / cell solution in the syringe (Hamilton 1000, Hamilton Company) is injected from the inlet 402 of the main inlet channel by using a syringe pump (Elite 1012, Harvard Apparatus), and the Reynolds number Re of the main inlet channel 402 is 9-36. The target large particles / cells are sorted into the side channel 404 when passing through the micro groove 401, and the non-target small particles / cells flow out through the main outlet channel 403 after passing through the particle sorting unit.

[0038] The particle / cell motion behavior is observed and recorded by an inverted microscope system (IX73, Olympus) and a high-speed camera (VEO 340L, Phantom), and the video recording resolution is 832x600, and the frame rate is 6000fps, and the experimental results are shown in (c) of Figure 4 The statistical data show that when the inlet Reynolds number Re is 18.5, the length of the outer connecting pipe of the side channel 404 L2 = L3 = 123 mm, i.e. e = 1 / 10, the sorting effect of the single particle / cell sorting unit 111 on the 20 μm particle / cell reaches the best, and the sorting efficiency can be as high as 98%, and the purity of the sorted 20 μm particles can reach 67.3%, and the removal efficiency of the impurity particles / cells is 97.1%. It can be seen that only the single particle / cell sorting unit has realized high-efficiency sorting of target particles / cells, which lays a foundation for subsequent parallelization of multi-polarization 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, as shown in Figure 5 (a).

[0040] The first step is the inertial focusing of the particles in the main inlet channel 402: when the solution containing mixed particles / cells is introduced into the microchannel, the particles are uniformly distributed on the cross section of the channel, and when the particles move downstream, they will be subjected to shear gradient induced lift force (F LS) and wall-induced lift force (F LW ), which compete with each other in the lateral direction. The net lift force F L is proportional to the fourth power of the particle diameter a. Therefore, larger particles experience a larger net lift force, and the equilibrium position of larger particles is closer to the wall of the main channel 402 than that of smaller 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 force (F LW ) generated by the hydrodynamic interaction between the particle and the wall disappears. The shear-gradient-induced lift force (F LS ) will act on the particle / cell alone, causing the particle to move across the streamlines into the sheath flow. The shear gradient responsible for the shear-gradient-induced lift force (F LS ) slowly decays in the expansion region. The lateral migration speed of the particle is proportional to the square of the particle diameter. Therefore, it can be predicted that larger particles will migrate much faster than smaller particles, and larger particles with higher lateral migration speed have the potential to cross the streamlines into 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 channel expansion-constriction region is three parts: the main flow showing an outwardly expanding circular arc, i.e. the formed main flow field 401a; the circular arc-shaped sheath flow flowing into the side channel 401, i.e. the formed sheath flow field 401b; and the vortex flow near the lower wall in the deep part of the micro-groove 401, i.e. the formed vortex flow field 401c. Due to the expansion of the channel, the fluid velocity in the channel decreases rapidly, and the fluid velocity in the y direction in the micro-groove 401 decreases monotonically exponentially. At this time, the particle / cell is only affected by the shear-gradient-induced lift force (F LS ) in the lateral direction. The force experienced by the particle is proportional to the square of the maximum fluid velocity, so the shear-gradient-induced lift force (F LS ) experienced by the particle in the lateral direction can be ignored at this time. The inertial effect on the particle / cell is proportional to the particle diameter, and the larger the particle diameter, the easier it is to cross the streamlines into the sheath flow field 401b under the influence of inertia.

[0043] In the above embodiment, the mixed particle / cell solution undergoes three-stage sorting, and the schematic diagram is as shown in Figure 6The target particle / cell solution sorted out by the first sorting device 5 flows into two particle sorting units 4 through two side channels 404 for secondary sorting, and the target particle / cell solution sorted out by the second sorting device 6 flows into four particle sorting units 4 through four side channels 404 for tertiary sorting. The multi-stage sorting can significantly improve the sorting purity of the chip of the present application. The preliminary experiment shows that the purity of the target particle / cell solution after the tertiary sorting is increased by 8.8 times, and the concentration of the target particle / cell is 137.5 times of the initial concentration, as shown in Table 1. Figure 6

[0044] The above merely illustrates the preferred embodiments of the present application, but should not be used to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.​

Claims

1. A microfluidic chip device for continuous integration inertial sorting of large size cells, characterized in that: The application relates to a continuous multi-stage sorting device, which 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, a target ion solution outlet and impurity particle outlets which are communicated with inlets and outlets of the continuous multi-stage sorting device, and particle sorting units of two adjacent stages of the sorting device are exponentially increased. The particle sorting unit comprises a micro-groove, a feeding main channel and a discharging main channel which are communicated with the micro-groove, and side channels which are symmetrically arranged on both sides of the discharging main channel and communicated with the micro-groove; the feeding main channel of the particle sorting unit of the first stage of the sorting device is communicated with the mixed particle solution inlet; the side channels of all the particle sorting units of the last stage of the sorting device are communicated with the target ion solution outlet after being converged; the feeding main channel of the particle sorting unit of the last stage of the sorting device is communicated with the side channel of the particle sorting unit of the first stage of the sorting device; and the discharging main channel of each group of the particle sorting unit is communicated with one of the impurity particle outlets. The micro-groove is symmetrically spliced by two identical circular arc grooves, and the feeding main channel and the discharging main channel are arranged outside the splicing position of the two circular arc grooves.

2. The microfluidic chip device of claim 1, wherein: The micro-groove is divided into a main flow field, a sheath flow field and a vortex cell flow field according to the flow direction of particles / cells.

3. The microfluidic chip device of claim 1, wherein: The diameter threshold of the sorting target particle / cell can be adjusted by changing the length of the outlet external pipe of the side channel.

4. The microfluidic chip device of claim 1, wherein: The diameter threshold of the sorting target particle / cell can be adjusted by changing the flow rate of the feeding main channel and the discharging main channel.

5. The microfluidic chip device of claim 1, wherein: The shell comprises an upper cover plate and a lower bottom plate which are combined together, and the continuous multi-stage sorting device is fixed by oxygen ion up-down bonding with the lower bottom plate.

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