Circulating tumor cell sorting and enriching chip based on inertial microfluidic technology

Abstract

The invention provides a circulating tumor cell sorting and enriching chip based on an inertial micro-fluidic technology. The inertial micro-fluidic chip designed by the invention is formed by nesting a sorting chip with a trapezoidal cross section and a concentrating chip for enriching. And one side of the concentrated chip inlet is used for realizing vertical stacking of the concentrated chip inlet and the separated chip and communication of the upper chip and the lower chip through bonding an inner outlet of the separated chip. During working, sample liquid carrying cells passes through the preset flow channel at a set flow speed through the injection pump, most of the cells are gathered to the outer side of the spiral flow channel and are discharged from the outer outlet under the combined action of a microfluid inertia effect and Dean vortex, and circulating tumor cells are concentrated on the inner side of the spiral flow channel and enter a next enrichment link through the inner outlet. The sorted circulating tumor cells are focused to the center of the flow channel under the same physical effect through the three-stage sinusoidal asymmetric snake-shaped flow channel, then cell-free liquid is removed through the branch flow channels on the two sides, and finally the circulating tumor cells are discharged from a middle outlet, so that the enrichment process is completed.

Description

Technical Field

[0001] The invention belongs to the fields of molecular and cell biology, biotechnology and medical technology, and in particular relates to a circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology. Background Art

[0002] The key to early diagnosis and precision medicine of cancer is to improve the treatment effect and improve the prognosis of patients. However, early diagnosis is difficult because early tumors are small in size and have no obvious symptoms, and early screening may require invasive examinations, which may lead to the risk of over-medicalization. Liquid biopsy technology provides new ideas for early diagnosis and precision medicine by detecting tumor markers. This technology relies on indicators such as circulating tumor cells (CTCs) and cell-free nucleic acids. In particular, CTCs, due to their cell membrane integrity and proliferation ability, can provide rich tumor genetic, transcriptional and protein information, and are effective biomarkers for tumor diagnosis and treatment evaluation. Although CTCs liquid biopsy is valuable in clinical research, it has technical difficulties. CTCs are scarce in biopsy samples and may undergo epithelial to mesenchymal transformation in the circulation, resulting in a decrease in markers (EpCAM) and the appearance of a mesenchymal phenotype, resulting in phenotypic heterogeneity. In addition, although CTCs vary in cell morphology, the size difference with WBCs is less than 5μm, which reduces the efficiency of size-based sorting and detection.

[0003] Driven by new cell sorting theories and chip manufacturing processes, CTCs sorting and enrichment instrument prototypes based on microfluidic technology have been successfully developed in recent years and have been initially applied in biological science research and clinical diagnosis. They mainly include the Cell Search system, Parsortix cell sorting system, ClearCe ll FX1 system and VTX-1 liquid biopsy system.

[0004] The Cell Search system is a circulating tumor cell (CTCs) detection platform based on immunomagnetic bead sorting technology. Its working mechanism is to identify and capture CTCs expressing epithelial cell adhesion molecule (EpCAM) through specific antibodies. However, the Cell Search system also has some obvious limitations. For example, its ability to capture cells is limited by the expression of EpCAM, which will lead to insufficient capture of non-epithelial or epithelial-mesenchymal transition (EMT) CTCs, which may produce false negative results. In addition, the high cost of the system and its dependence on professional equipment have limited its popularity in medical institutions. The antibody-based capture method of the Cell Search system may also affect subsequent biosignature analysis, such as gene expression.

[0005] The Parsort ix cell sorting system is a liquid biopsy platform developed based on microfluidic chip technology, which captures CTCs in the Parsort ix chip through cell size and deformability. The operating procedures of the Parsort ix system cover the collection of blood samples, the direct connection of samples to the device without pretreatment, and the automatic blood filtration process of the system. In this process, due to the relatively large size and low deformability of CTCs, they are effectively captured in the Parsort ix chip. However, the limitation of the Parsort ix system is that the CTCs captured by filtration need to be backwashed for secondary collection, which may result in the loss of CTC quantity and the expansion of the volume of the collection fluid, which is not conducive to subsequent analysis. In addition, the Parsort ix system is currently for research use only and cannot be used for diagnostic procedures.

[0006] Another successfully commercialized microfluidic sorting technology is the VTX-1 liquid biopsy system, whose core principle is to use the vortices generated in the contraction-expansion structure to sort and capture large-sized cells. The system contains a microfluidic channel with a bidirectional contraction-expansion array structure, which is designed to efficiently sort and capture CTCs from liquid biopsy samples, and can process samples at a throughput of 500μL / min, significantly improving the purity of cells. Although the VTX-1 system has certain advantages in capture technology, it still faces some challenges in the sensitivity and specificity of CTCs sorting, especially in distinguishing tumor cells from blood cells of similar size.

[0007] From the overall situation, there are significant defects in the commercial circulating tumor cell (CTCs) sorting instruments on the market. On the one hand, instruments such as Cell Search and LiqudBiopsy have excessive cell loss during the sorting process, resulting in impaired cell activity and difficulty in subsequent research and analysis. On the other hand, such as Parsort ix and VTX-1, their chip structure design is complex, difficult to operate, and expensive, and the stability is poor. In view of this, there is an urgent need for a new type of chip that can achieve rapid, convenient, and accurate sorting and enrichment of circulating tumor cells, while having the advantages of low cost, high throughput, and high concentration. Summary of the invention

[0008] To solve the above problems, the present invention discloses a circulating tumor cell (CTCs) sorting and enrichment chip based on inertial microfluidics technology, which has a simple and reliable structure and low cost, and has high throughput and high precision characteristics and a preparation method thereof.

[0009] A circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology, comprising a sorting chip, wherein the sorting chip is composed of a spiral flow channel; and also comprising a concentration chip used for enrichment; wherein a "Y-shaped" flow channel is provided at the outer outlet of the sorting chip; one side of the concentration chip is bonded to the inner outlet of the sorting chip to achieve vertical stacking and connection between the two; the concentration chip used for enrichment is composed of three levels of wavy concentration flow channels with different widths and the same height, namely, the first, second and third level concentration flow channels.

[0010] Furthermore, the "Y-shaped" flow channel includes an outer outlet and an inner outlet; wherein the concentration chip is connected to the inner outlet, in order to receive the circulating tumor cell liquid with a relatively low concentration and further concentrate it to meet the requirements.

[0011] Furthermore, the cross section of the sorting chip is a right-angled trapezoid.

[0012] Since the physical quantity values ​​of CTCs and normal blood cells are quite close, the focusing equilibrium positions of the two are very close or partially overlapped, resulting in low sorting accuracy of CTCs in the existing rectangular spiral flow channel.

[0013] This chip induces asymmetric distribution of Dean flow through a right-angled trapezoidal cross-section and utilizes the position boundary between the two modes of cell inertial focusing and Dean vortex focusing to achieve separation of CTCs and blood cells at the focusing position on the flow channel cross-section.

[0014] Furthermore, the spiral flow channel is a six-turn spiral flow channel; the initial spiral inner diameter of the spiral flow channel is set to 12 mm, the spiral pitch is 4 mm, and the final spiral outer diameter of six turns is 60 mm; a right-angled trapezoidal cross-section design with a long side of 165 μm and a short side of 65 μm is adopted, and the width of the flow channel is uniformly set to 500 μm.

[0015] Furthermore, the first, second and third level concentrating flow channels are composed of three asymmetric sinusoidal units, and the flow channel height is 100 μm; the width of the first level concentrating flow channel is 270 μm, the width of the second level concentrating flow channel is 150 μm, and the width of the third level concentrating flow channel is 60 μm; wherein each of the asymmetric sinusoidal units is composed of a group of 3 parallel arranged concentrating flow channels, wherein the discharge ends of the first level concentrating flow channel, the second level concentrating flow channel and the third level concentrating flow channel are all connected to the short straight flow channel.

[0016] Furthermore, each of the short straight flow channels is provided with a graded discharge pipe, and each of the graded discharge pipe is provided with a symmetrical discharge outlet. When the sample solution containing circulating tumor cells passes through the spiral sorting chip and enters the enrichment chip, as the tissue fluid continuously flows out of the graded discharge pipe, the expected concentration of circulating tumor cells can be received at the outlet of the tertiary concentration flow channel of the tertiary concentration flow channel.

[0017] Furthermore, a method for operating a circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology comprises the following steps:

[0018] S21. Remove the large background blood cells from the blood sample by chemical lysis or other methods;

[0019] S22. To ensure the chip effect, dilute the sample to a solution with a total cell concentration of <7.5×105 cells / mL;

[0020] S23. Pre-staining with calcein stain makes it easier to distinguish circulating tumor cells from leukocytes;

[0021] S24. Use a syringe pump to deliver the sample into the chip at a flow rate of 3000 μL / min. After waiting for an appropriate period of time, receive the sample solution from the outlet of the tertiary concentration flow channel of the tertiary concentration flow channel. In microscopic observation, tumor cells appear red under fluorescence excitation.

[0022] Through the operation method of the present invention, in microscopic observation, tumor cells appear red under fluorescence excitation, and the sorting and enrichment of circulating tumor cells after pre-staining can facilitate the subsequent separate culture and specific electrical impedance detection of circulating tumor cells.

[0023] Beneficial effects of the present invention:

[0024] 1. In the process of completing cell sorting, the present invention has designed a six-turn spiral flow channel, which completes the separation and enrichment of target cells and other cells through the composite action mechanism between fluid inertial lift and Dean drag, thereby maximizing the high-throughput and non-labeling advantages of microfluidic technology, and realizing high-throughput and high-precision sorting and enrichment of target cells. In addition, due to the design of an enrichment chip with good sealing performance, the present invention can better maintain the activity of CTCs to meet subsequent research needs.

[0025] 2. The present invention achieves cell separation through different manifestations of cell characteristics in fluids and cell enrichment through physical phenomena such as particle focusing, showing significant automation and stability, thereby reducing the threshold for the use of professional knowledge and operating techniques in practical applications of the chip. At the same time, the highly enclosed and integrated equipment improves the invention's resistance to external environmental interference factors, and is more convenient for further development and integration with other equipment to develop more diverse functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the complete structure of the circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology in the present invention;

[0027] Figure 2It is a schematic diagram of the flow channel cross section in the circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology in the present invention. DETAILED DESCRIPTION

[0028] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. It should be noted that the words "front", "rear", "left", "right", "upper" and "lower" used in the following description refer to directions in the accompanying drawings, and the words "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.

[0029] like Figure 1-2 As shown, this embodiment discloses a circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology, including a sorting chip 1 and a concentration chip 2 used for enrichment, wherein the cross-section of the sorting chip is a right-angle trapezoid.

[0030] A "Y-shaped" flow channel 3 is provided at the outer outlet of the sorting chip 1; the "Y-shaped" flow channel 3 includes an outer outlet 3-1 and an inner outlet 3-2; wherein the concentration chip 2 is connected to the inner outlet 3-2. The concentration chip 2 used for enrichment is composed of three levels of wavy concentration flow channels 5 with different widths and the same height, which are the first, second and third level concentration flow channels.

[0031] The spiral flow channel 4 of this embodiment is a six-turn spiral flow channel; the initial spiral inner diameter of the spiral flow channel 4 is set to 12mm, the spiral pitch is 4mm, and the final spiral outer diameter of six turns is 60mm; a right-angled trapezoidal cross-section design with a long side of 165μm and a short side of 65μm is adopted, and the width of the flow channel is uniformly set to 500μm.

[0032] The first, second and third level concentrating channels are composed of three asymmetric sinusoidal units 6, and the channel height is 100 μm; the width of the first level concentrating channel 6-1 is 270 μm, the width of the second level concentrating channel 6-2 is 150 μm, and the width of the third level concentrating channel 6-3 is 60 μm; each of the asymmetric sinusoidal units 6 is composed of three parallel arranged concentrating channels, wherein the discharge ends of the first level concentrating channel 6-1, the second level concentrating channel 6-2 and the third level concentrating channel 6-3 are all connected to the short straight channel 2-1; each of the short straight channel 2-1 is provided with a graded discharge pipe 7, and each of the graded discharge pipes is provided with a symmetrical discharge outlet 7-1.

[0033] The present invention includes an operating method of the circulating tumor cell sorting and enrichment chip, comprising:

[0034] Cells are sorted by flowing through a spiral sorting chip and enriched by flowing through a multi-stage concentration chip.

[0035] Specifically, the following steps are performed during cell sorting:

[0036] S11. Inject the pretreated cell sample into the inlet of the sorting chip via a syringe pump at 3000 μL / min.

[0037] S12. The velocity difference caused by the fluid dynamic viscosity will cause the cells near the center line of the flow channel to move toward the wall. Then the velocity difference between the cells and the fluid at the center of mass will generate a wall-induced lift pointing toward the center of the flow channel, causing the cells to move toward the center of the flow channel. After passing through a sufficiently long flow channel, the cells will be focused from the dispersed state at the time of entry to a specific position in the flow channel.

[0038] S13. The asymmetric trapezoidal cross-section of the flow channel induces asymmetric distribution of Dean flow. The suspended cells migrate further toward the outer wall of the flow channel under the guidance of Dean drag force, thereby expanding the focus position boundary between different cells. Finally, the target cells are focused on the inner side of the flow channel and enter the next link through the inner outlet of the Y-shaped outlet, and enter the concentration chip through the inner outlet of the Y-shaped outlet to obtain a higher concentration of circulating tumor cells.

[0039] Other cells and tissue fluid are focused on the outside of the flow channel and flow out through the outside of the Y-shaped outlet, thus completing cell sorting.

[0040] Cell enrichment includes the following steps:

[0041] S21. The sorted samples enter the concentration chip through the interface between the two chips.

[0042] S22. In a bend of a certain size, the fluid will be thrown toward the wall due to the greater centrifugal force. Under the influence of Dean vortex and other effects, the target cells will be further focused in the center of the flow channel.

[0043] S23. There are three centrally symmetrical outlets at the end of each flow channel. The cell-free liquid will be discharged from the two side outlets to complete the concentration and achieve cell enrichment. The target cells will flow from the middle outlet into the next level of concentration flow channel, ultimately achieving a good cell enrichment effect.

[0044] This embodiment also includes a method for preparing a circulating tumor cell sorting and enrichment chip, comprising the following steps:

[0045] S31. Using ultraviolet laser engraving technology to process chip structure and positioning holes on the surface of polymer film;

[0046] S32. After the bonding surface is subjected to plasma treatment, the base plate, the inner wall film, the outer wall film and the cover plate are assembled layer by layer from bottom to top in the order;

[0047] S33 is repeated and uniformly rolled, so that the lower surface of the cover plate is simultaneously attached to the upper surfaces of the inner wall film and the outer wall film, and finally a closed trapezoidal cross-section spiral microchannel is formed.

[0048] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above technical features.

Claims

1. A circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology, comprising a sorting chip (1), wherein the sorting chip (1) is composed of a spiral flow channel (4); characterized in that: It also includes a concentration chip (2) used for enrichment; wherein a "Y-shaped" flow channel (3) is provided at the outer outlet of the sorting chip (1); one side of the concentration chip (2) is bonded to the inner outlet of the sorting chip (1) to achieve vertical stacking and connection between the two; The concentration chip (2) used for enrichment is composed of three levels of wavy concentration channels (5) with different widths and the same height, namely the first, second and third level concentration channels.

2. The circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology according to claim 1, characterized in that; The Y-shaped flow channel (3) comprises an outer outlet (3-1) and an inner outlet (3-2); wherein the concentration chip (2) is connected to the inner outlet (3-2).

3. The circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology according to claim 1, characterized in that: The cross section of the sorting chip (1) is a right-angle trapezoid.

4. The circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology according to claim 1, characterized in that: The spiral flow channel (4) is a six-turn spiral flow channel; the initial spiral inner diameter of the spiral flow channel (4) is set to 12 mm, the spiral pitch is 4 mm, and the final spiral outer diameter of the six turns is 60 mm; a right-angled trapezoidal cross-section design with a long side of 165 μm and a short side of 65 μm is adopted, and the width of the flow channel is uniformly set to 500 μm.

5. The circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology according to claim 1, characterized in that: The first, second and third level concentrating flow channels are composed of three asymmetric sinusoidal units (6), and the flow channel height is 100 μm; the width of the first level concentrating flow channel (6-1) is 270 μm, the width of the second level concentrating flow channel (6-2) is 150 μm, and the width of the third level concentrating flow channel (6-3) is 60 μm; each of the asymmetric sinusoidal units (6) is composed of three parallel-arranged concentrating flow channels, and the discharge ends of the first level concentrating flow channel (6-1), the second level concentrating flow channel (6-2) and the third level concentrating flow channel (6-3) are all connected to the short straight flow channel (2-1).

6. The circulating tumor cell sorting and enrichment chip based on inertial microfluidics technology according to claim 5, characterized in that: Each of the short straight channels (2-1) is provided with a graded discharge pipeline (7), and each of the graded discharge pipelines is provided with a symmetrical discharge outlet (7-1).

7. An operating method based on the circulating tumor cell sorting and enrichment chip according to any one of claims 1 to 6, characterized in that: The following steps are involved: S21. Remove the large background blood cells from the blood sample by chemical lysis or other methods; S22. To ensure the chip effect, dilute the sample to a total cell concentration of <7.5×105 cells / mL solution; S23. Pre-staining with calcein stain makes it easier to distinguish circulating tumor cells from leukocytes; S24. Use a syringe pump to deliver the sample into the chip at a flow rate of 3000 μL / min. After waiting for an appropriate period of time, receive the sample solution from the tertiary concentration channel outlet (7-2) of the tertiary concentration channel (6-3). In microscopic observation, tumor cells should appear red under fluorescence excitation.

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