A multi-stage flow channel coupling-based circulating tumor cell sorting and enrichment chip

Abstract

This invention discloses a circulating tumor cell (CTC) sorting and enrichment chip based on multi-stage flow channel coupling. It is composed of a cover layer, a flow channel layer, and a base layer bonded together, and integrates a trapezoidal spiral sorting unit, a sinusoidal flow channel enrichment unit, and a contraction-expansion spiral purification unit. Samples flow sequentially through each unit, achieving initial cell separation, waste liquid discharge for enrichment, and vortex-based three-stage deep purification and precise focusing, respectively. This invention effectively improves the purity and efficiency of CTC sorting through the synergistic effect of multi-stage flow channels while maintaining cell viability, thus possessing clinical application value.

Description

Technical Field

[0001] This invention relates to the fields of molecular and cell biology, biotechnology and medical technology, and in particular to a circulating tumor cell sorting and enrichment chip based on multi-level flow channel coupling. Background Technology

[0002] Early cancer diagnosis and precision medicine are key to improving treatment outcomes and patient prognosis. However, early diagnosis is challenging because early-stage tumors are small, asymptomatic, and early screening may require invasive procedures, posing a risk of overtreatment. Liquid biopsy technology can provide new insights for early diagnosis and precision treatment by detecting circulating tumor markers in peripheral blood, such as circulating tumor cells (CTCs), circulating DNA, and exosomes. CTCs, due to their cell membrane integrity and proliferative capacity, provide rich information on tumor genetics, transcription, and proteins, making them effective biomarkers for tumor diagnosis and treatment assessment. However, CTCs are very rare in each milliliter of blood and may undergo epithelial-to-mesenchymal transition during circulation, leading to a reduction in epithelial phenotype and the emergence of mesenchymal phenotype, resulting in phenotypic heterogeneity. Furthermore, although CTCs exhibit morphological variations, their size difference from leukocytes is less than 5 μm, and they overlap, reducing the efficiency of size-based sorting and detection.

[0003] Inertial microfluidic cell sorting utilizes the inertial force generated by high-speed fluid within micrometer-scale channels to automatically focus, sort, and classify cells within specific microstructures based on differences in cell size and deformation, without the need for external field excitation such as electric fields, magnetic fields, ultrasound, or light fields. This technology can achieve the sorting and enrichment of CTCs.

[0004] In inertial microfluidics, the channel structure is a key factor determining chip function and performance. Reported channel structures include straight channels, sinusoidal channels, helical channels, and other corresponding improvements. The core working principle of inertial microfluidics is based on inertial migration and secondary flow effects: inertial migration refers to the gradual migration of cells to a stable equilibrium position under the combined action of shear-induced lift and wall-induced lift in laminar flow; while secondary flow is due to the momentum difference of fluid in curved channels or disturbed straight channels, forming Dean vortices. Cell extraction using inertial microfluidics utilizes the aforementioned inertial lift and Dean vortex drag to adjust the position of cells within the channel.

[0005] Trapezoidal cross sections (such as inverted trapezoidal channels where the outer wall depth is greater than the inner wall) disrupt the symmetry of Dean vortices in rectangular cross sections, causing the vortex core to shift towards the outer wall. This effectively increases the lateral spacing between particles of different sizes, significantly improving sorting resolution and purity, while maintaining the focusing position of large particles on the inner wall. The sinusoidal flow channel is composed of alternating, oppositely oriented curvature units connected in series. The direction and intensity of the Dean flow change periodically along the channel. Particles undergo repeated acceleration and reversal in this non-uniform secondary flow field, eventually converging to a single equilibrium position under specific conditions. Simultaneously, an opening at the upper end of the channel allows for the discharge and enrichment of impurity liquid. The contraction-expansion channel consists of alternating narrow contraction and wide expansion sections. When fluid abruptly enters the expansion chamber from the contraction section, the sudden velocity change triggers boundary layer separation, inducing planar microvortices within the expansion region. Simultaneously, the streamline curvature and centrifugal force differences generated by the alternating structure also induce Dean-like secondary flows within the cross-section. Large particles, due to their greater inertia, are captured by micro-vortices in the expansion zone and remain along specific streamlines; small particles, due to insufficient inertial lift to resist the drag force of the vortex, escape with the mainstream and migrate downstream, thus achieving sorting and enrichment based on size differences.

[0006] Current methods for detecting circulating tumor cells, such as immunoaffinity enrichment, molecular nucleic acid detection, and flow cytometry, while technically mature and highly standardized in clinical practice, still face limitations such as cell viability damage, false positives, and high costs. Many passively controlled microfluidic chips have emerged to sort cells based on size characteristics; however, existing microfluidic chips suffer cell loss during the sorting process, leading to impaired cell viability and hindering their use in subsequent research and analysis. Summary of the Invention

[0007] Purpose of the invention: The purpose of this invention is to provide a circulating tumor cell sorting and enrichment chip based on multi-level flow channel coupling. Through the synergistic effect of the multi-level flow channel structure, it can achieve continuous, efficient, and high-purity sorting and enrichment of cells, and solve the technical defects of the existing technology, such as insufficient sorting resolution of single flow channel structure, easy disruption of cell focusing by subsequent flow channel disturbance, and difficulty in achieving both high throughput and high purity.

[0008] Technical solution: The circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling described in this invention includes: a cover layer, a flow channel layer, and a base layer, wherein, within the flow channel layer, a trapezoidal spiral sorting unit, a sinusoidal flow channel enrichment unit, and a contraction-expansion spiral purification unit are sequentially connected in series along the fluid flow direction; wherein, The trapezoidal spiral sorting unit has a spiral flow channel with an asymmetric cross section, which is used to focus target cells and blood cells to different lateral positions. Downstream, there is a first diversion structure to introduce the fluid enriched with target cells into the next unit and discharge waste liquid. The sinusoidal flow channel enrichment unit is composed of multiple sinusoidal waveform flow channel segments connected in series. At the peak position of at least one sinusoidal waveform segment, a waste discharge channel extending to the outside of the chip is opened on the top wall of the flow channel. This channel is used to discharge near-wall waste liquid while the cells are focused in the middle of the flow channel, thereby achieving volume concentration. The shrinking-expanding spiral purification unit is spiral-shaped and has alternating shrinking and expanding sections along its length. The vortex generated by the expanding section retains non-target cells and keeps the target cells in the main flow. The end is equipped with a second diversion structure to separately discharge the purified target cells and residual waste liquid. The units are connected by interlayer transition holes to form a continuous sorting and enrichment flow path.

[0009] Furthermore, the asymmetric cross-section of the trapezoidal spiral sorting unit is a right trapezoid, which is used to disrupt the Dean vortex symmetry, causing target cells to focus inward and blood cells to focus outward.

[0010] Furthermore, the first diversion structure is a Y-shaped branch structure, with the inner branch supporting the target cell flow and the outer branch discharging waste liquid.

[0011] Furthermore, the front section of the sinusoidal flow channel enrichment unit is used for stable focusing, and the peak of the rear section of the sinusoidal wave is provided with a waste discharge channel, which is connected to the waste liquid outlet of the cover plate through interlayer holes.

[0012] Furthermore, in the shrinking-expanding spiral purification unit, the expanding section faces outwards from the spiral, and the shrinking and expanding sections are arranged alternately to generate planar microvortices in the expansion cavity.

[0013] Furthermore, the second diversion structure is a Y-shaped branch structure, with its inner branch being a cell collection channel and its outer branch being a waste liquid discharge channel.

[0014] Furthermore, the flow channel layer includes three layers. The first layer is provided with a shrinking and expanding spiral purification unit and its second diversion structure. The third layer is provided with a trapezoidal spiral sorting unit, a first diversion structure and a sinusoidal flow channel enrichment unit. The second layer is a separator layer and has transition holes connecting the layers.

[0015] The present invention discloses a microfluidic chip-based method for sorting and enriching circulating tumor cells, which is implemented using any of the aforementioned chips. The method includes: injecting a pre-treated sample into the chip, sequentially passing it through a trapezoidal spiral sorting unit for inertial focusing and preliminary diversion, then through a sinusoidal flow channel enrichment unit for volume concentration via top drainage, followed by deep purification using a shrinking and expanding spiral purification unit with vortex, and finally obtaining high-purity target cells from the cell collection outlet.

[0016] Furthermore, a method for fabricating a microfluidic chip, using any one of the chips described above, includes: processing a cover layer, a multilayer channel layer, and a substrate layer respectively and opening positioning holes; aligning and stacking the layers in sequence, and sealing them by plasma-assisted or thermo-press bonding to form a closed microfluidic channel network.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: Due to inertial effects and Dean flow, the fluid in the spiral channel aggregates circulating tumor cells and other cells at different equilibrium positions. Then, a Y-shaped separation structure guides the circulating tumor cells into the cell-focusing channel, while other cells are extracted. The cell-focusing channel is a sinusoidal enrichment channel combined with a contraction-expansion array structure, which can generate precise cross-sectional vortices. Under the coupling effect of local vortex-induced lift and inertial lift, circulating tumor cells focus on one side of the channel; it can effectively distinguish cells with minute size differences, significantly improving the concentration and purity of target cells, maximizing the preservation of the original cell activity and biological characteristics, and achieving an integrated operation of "sorting-enrichment-purification". Attached Figure Description

[0018] Figure 1 This is a top view of the structure of the present invention; Figure 2 This is a schematic diagram of the overall structure of the present invention; Figure 3 This is a schematic diagram of the cover plate layer and the flow channel holes thereon of the present invention; Figure 4 This is a schematic diagram of the first flow channel layer of the present invention (including a contraction-expansion spiral purification flow channel and an end branch outlet structure); Figure 5 This is a schematic diagram of the positioning of the second flow channel layer and the flow channel holes thereon in this invention; Figure 6 This is a schematic diagram of the third flow channel layer of the present invention (including a trapezoidal spiral sorting flow channel, a Y-shaped separation structure, and a sinusoidal enrichment flow channel); Figure 7 This is a schematic diagram of the base layer of the present invention.

[0019] The structure comprises: 1. A trapezoidal spiral sorting channel; 2. An outer branch of the Y-shaped separation structure for discharging preliminary sorting waste liquid; 3. An inner branch of the Y-shaped separation structure for guiding target cells to the sinusoidal enrichment channel; 4. The sinusoidal enrichment channel, i.e., the sinusoidal enrichment unit; 5. An impurity channel, i.e., the discharge pipe at the top of the latter half of the sinusoidal channel, for discharging waste liquid from the enrichment stage; 6. A contraction-expansion spiral purification channel, i.e., the contraction-expansion spiral purification unit; 7. An outer branch of the terminal branch outlet structure for discharging residual impurity waste liquid; 8. An inner branch of the terminal branch outlet structure, i.e., the cell collection outlet, for discharging the final high-purity target cells; 9. A cover plate layer; 10. A first channel layer, which includes a contraction-expansion spiral purification channel and a terminal branch outlet structure; 11. A second channel layer, i.e., a separator layer, with transition holes for interlayer communication; 12. A third channel layer, which includes a trapezoidal spiral sorting channel, a Y-shaped separation structure, and a sinusoidal enrichment channel; and 13. A base layer. 14 is the sample inlet, located in the cover layer, for injecting the sample to be processed; 15 is the first waste liquid outlet, located in the cover layer, for discharging the primary sorting waste liquid; 16 is the third waste liquid outlet, located in the cover layer, for discharging the final residual waste liquid; 17 is the cell outlet, located in the cover layer, for collecting the final target cells; 18 is the second waste liquid outlet, located in the cover layer, for discharging the enrichment stage waste liquid; 19 is a connecting hole, located in the first flow channel layer, for connecting the sample inlet and the flow channel inlet, guiding the sample into the trapezoidal spiral sorting flow channel; 20 is a connecting hole, located in the first flow channel layer, for connecting the outer outlet of the Y-shaped separation structure and the first waste liquid outlet, discharging the primary waste liquid. ; 21 is the outer waste liquid outlet, located on the outer side of the end of the flow channel layer, connected to the third waste liquid outlet of the cover plate layer; 22 is the cell collection outlet, located on the inner side of the end of the flow channel layer, connected to the cell outlet of the cover plate layer; 23 is the flow channel inlet, located in the first flow channel layer, and is the starting inlet of the contraction-expansion spiral purification flow channel; 24 is the connecting hole, located in the first flow channel layer, used to connect the sinusoidal flow channel outlet and the second waste liquid outlet to discharge the waste liquid from the enrichment stage; 25 is the connecting hole, located in the second flow channel layer, used to connect the sample inlet and the flow channel inlet; 26 is a connecting hole located in the second flow channel layer; 27 is a connecting hole located in the second flow channel layer; 28 is a connecting hole located in the second flow channel layer, used to connect the sinusoidal enrichment flow channel and the contraction-expansion spiral purification flow channel; 29 is a flow channel inlet located in the second flow channel layer, which is the starting inlet of the trapezoidal spiral sorting flow channel; 30 is an outer outlet located in the third flow channel layer, which is the outlet of the outer branch of the Y-shaped separation structure; 31 is a flow channel outlet located in the third flow channel layer, which is the final outlet of the sinusoidal enrichment flow channel; 32 is the cell outlet of the sinusoidal enrichment flow channel, located in the third flow channel layer. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0021] like Figures 1-2As shown, this embodiment of the invention provides a circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling, including a cover layer 9, a first flow channel layer 10, a second flow channel layer 11, a third flow channel layer 12, and a base layer 13. Within the flow channel layers, a trapezoidal spiral sorting unit 1, a sinusoidal flow channel enrichment unit 4, and a contraction-expansion spiral purification unit 6 are sequentially connected in series along the fluid flow direction.

[0022] The trapezoidal spiral sorting unit 1 is used for preliminary inertial focusing and sorting of cells in the sample. Downstream, the Y-shaped separation structure 2 guides the fluid enriched with target cells into the next unit, while simultaneously discharging waste liquid. The flow channel of the trapezoidal spiral sorting unit 1 has a right-angled trapezoidal cross-section, with a long side length of 125~205μm, a short side length of approximately 40%~60% of the long side length, a flow channel width of 400~600μm, 6 spiral turns, an initial spiral inner diameter of 12mm, a spiral spacing of 4mm, a short side inside the flow channel, a long side outside the flow channel, and an average cross-sectional depth of 82.5~164μm.

[0023] The upper end of the sinusoidal flow channel enrichment unit 4 is provided with an opening structure for discharging waste liquid during the enrichment process. This unit is composed of multiple sinusoidal wave units with a rectangular flow channel shape. The front section of the sinusoidal wave is used to stabilize fluid focusing, and the peak of the rear section of the sinusoidal wave is provided with a waste discharge channel, which is connected to the waste liquid outlet of the cover plate through interlayer holes to achieve volume concentration.

[0024] The contraction-expansion spiral purification unit 6 has alternating contraction and expansion sections along its length, with the expansion section facing outwards. The alternating arrangement of the contraction and expansion sections creates planar microvortices within the expansion chamber, which retain non-target cells and maintain the mainstream migration of target cells. The end of the contraction-expansion spiral purification unit 6 is connected to a Y-shaped branch structure, with the inner side serving as the target cell outlet and the outer side as the waste liquid outlet.

[0025] A sample inlet 14 is provided on the cover plate layer 9 for injecting the cell mixture to be treated, which connects to the trapezoidal spiral sorting channel 1. The outer branch 2 of the Y-shaped separation structure is used to discharge the initial sorting waste liquid, and the inner branch 3 connects to the sinusoidal enrichment channel 4. The latter half of the sinusoidal enrichment channel has an opening, and impurities after deep sorting are discharged through the impurity channel 5. The end of the sinusoidal enrichment channel 4 connects to the contraction-expansion spiral purification channel 6, and the outer side 7 of the end branch outlet structure discharges residual impurities, while the inner cell collection outlet 8 is used to collect the final high-purity target cells.

[0026] The units are connected by interlayer transition holes to form a continuous sorting and enrichment flow path. The second flow channel layer 11 is a separator layer with transition holes for connecting the layers. The first flow channel layer 10 is equipped with a contraction-expansion spiral purification flow channel and an end-branch outlet structure. The third flow channel layer 12 is equipped with a trapezoidal spiral sorting flow channel, a Y-shaped separation structure, and a sinusoidal enrichment flow channel.

[0027] This invention also provides a method for sorting and enriching circulating tumor cells using a microfluidic chip, the specific operation method of which is as follows: (1) At the sorting chip inlet 14, the pre-treated cell sample is injected by a syringe pump at a flow rate of 3000 μL / min.

[0028] (2) The sample solution enters the trapezoidal spiral sorting channel 1 from the sorting chip inlet 14 through the connecting holes 19 and 25 on the second channel layer 11 and the channel inlet 29. Larger target cells (such as CTCs) are focused towards the inside of the channel under the combined action of inertial lift and Dean drag, while smaller blood cells and tissue fluid are focused on the outside of the channel. Subsequently, the fluid enters the Y-shaped separation structure, and the outer outlet 30, carrying most of the blood cells, is discharged as waste liquid through the connecting holes 26 and 20 and the first waste liquid outlet 15 of the cover layer. The fluid rich in target cells is introduced into the next stage sinusoidal enrichment channel 4 through the inner branch 3.

[0029] (3) Fluid rich in target cells enters the sinusoidal enrichment channel 4. Under the periodic disturbance of the sinusoidal channel, the target cells are precisely focused in the center of the channel. At the same time, a discharge pipe 5 is provided on the short straight channel at the upper end of the latter half of the sinusoidal channel. Due to the pressure difference, some of the outer liquid without cells is discharged from the discharge pipe 5 and discharged through the channel outlet 31, connecting holes 27 and 24 and the second waste liquid outlet 18 of the cover plate layer, thereby achieving volume concentration and cell enrichment.

[0030] (4) The cell flow through the sinusoidal channel enters the contraction-expansion spiral purification channel 6 through the channel outlet 32, the connecting hole 28 and the channel inlet 23. Cells that do not meet the size requirements or are not the target cells are pushed to the outside of the channel or captured in the vortex under the action of the micro vortex generated in the expansion section, while the target cells that meet the size requirements remain focused and migrate along the main channel to complete the deep purification and sorting.

[0031] (5) Finally, the fluid reaches the end branch outlet structure. After the aforementioned multi-stage inertial separation and vortex focusing, the high-purity target cells flow out stably from the inner cell collection outlet 22 and are collected through the cover plate cell outlet 17, which facilitates subsequent microscopic observation and specific detection; the remaining impurity cells and liquid are discharged from the outer waste liquid outlet 21 and collected through the cover plate third waste liquid outlet 16.

[0032] This embodiment also includes a method for preparing a circulating tumor cell sorting and enrichment chip, the specific steps of which are as follows: (1) Using multilayer polymethyl methacrylate (PMMA) or polydimethylsiloxane (PDMS) materials, the base layer 13, the first flow channel layer 10, the second flow channel layer 11, the third flow channel layer 12 and the cover plate layer 9 are processed by ultraviolet laser cutting, and the flow channel structure and positioning holes are processed on each layer.

[0033] (2) Plasma treatment is applied to the bonding surfaces of each layer.

[0034] (3) The layers are aligned and stacked from bottom to top in the order of base layer 13, third flow channel layer 12, second flow channel layer 11, first flow channel layer 10 and cover plate layer 9. The positioning holes are used to ensure that each layer is accurately positioned and then bonded and packaged.

[0035] (4) Through repeated and uniform rolling, the layers are tightly bonded together, eventually forming a closed microfluidic channel network.

Claims

1. A circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling, comprising: The system comprises a cover plate layer, a flow channel layer, and a base layer, characterized in that the flow channel layer contains, sequentially connected in series along the fluid flow direction, a trapezoidal spiral sorting unit, a sinusoidal flow channel enrichment unit, and a contraction-expansion spiral purification unit; wherein... The trapezoidal spiral sorting unit has a spiral flow channel with an asymmetric cross section, which is used to focus target cells and blood cells to different lateral positions. Downstream, there is a first diversion structure to introduce the fluid enriched with target cells into the next unit and discharge waste liquid. The sinusoidal flow channel enrichment unit is composed of multiple sinusoidal waveform flow channel segments connected in series. At the peak position of at least one sinusoidal waveform segment, a waste discharge channel extending to the outside of the chip is opened on the top wall of the flow channel. This channel is used to discharge near-wall waste liquid while the cells are focused in the middle of the flow channel, thereby achieving volume concentration. The shrinking-expanding spiral purification unit is spiral-shaped and has alternating shrinking and expanding sections along its length. The vortex generated by the expanding section retains non-target cells and keeps the target cells in the main flow. The end is equipped with a second diversion structure to separately discharge the purified target cells and residual waste liquid. The units are connected by interlayer transition holes to form a continuous sorting and enrichment flow path.

2. The circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling according to claim 1, characterized in that, The asymmetric cross-section of the trapezoidal spiral sorting unit is a right trapezoid, which is used to disrupt the Dean vortex symmetry, causing target cells to focus inward and blood cells to focus outward.

3. The circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling according to claim 1, characterized in that, The first diversion structure is a Y-shaped branch structure, with the inner branch supporting the target cell flow and the outer branch discharging waste liquid.

4. The circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling according to claim 1, characterized in that, The front section of the sinusoidal flow channel enrichment unit is used for stable focusing, and the peak of the rear section of the sinusoidal wave is provided with a waste discharge channel, which is connected to the waste liquid outlet of the cover plate through the interlayer hole.

5. A circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling according to claim 1, characterized in that, In the shrinking-expanding spiral purification unit, the expanding section faces the outside of the spiral, and the shrinking and expanding sections are arranged alternately to generate planar microvortices in the expansion chamber.

6. The circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling according to claim 1, characterized in that, The second diversion structure is a Y-shaped branch structure, with the inner branch being the cell collection channel and the outer branch being the waste liquid discharge channel.

7. A circulating tumor cell sorting and enrichment chip based on multi-stage flow channel coupling according to claim 1, characterized in that, The flow channel layer consists of three layers. The first layer is equipped with a shrinking and expanding spiral purification unit and its second diversion structure. The third layer is equipped with a trapezoidal spiral sorting unit, a first diversion structure and a sinusoidal flow channel enrichment unit. The second layer is a separator layer and has transition holes that connect the layers.

8. A method for sorting and enriching circulating tumor cells using a microfluidic chip, implemented using the chip described in any one of claims 1-7, characterized in that, Includes the following steps: The pre-treated sample is injected into the chip and sequentially passes through the trapezoidal spiral sorting unit for inertial focusing and initial diversion. It then passes through the sinusoidal flow channel enrichment unit for volume concentration through top drainage. Finally, it passes through the shrinking and expanding spiral purification unit for deep purification using vortex. High-purity target cells are obtained from the cell collection outlet.

9. A method for fabricating a microfluidic chip, implemented using the chip described in any one of claims 1-7, characterized in that, The process includes the following steps: processing the cover plate layer, multi-layer flow channel layer and base layer separately and opening positioning holes; aligning and stacking each layer in sequence, and sealing them by plasma-assisted or thermo-press bonding to form a closed microfluidic flow channel network.

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