Microfluidic device for enriching circulating tumor cells in blood

Abstract

The invention discloses a microfluidic device for enriching circulating tumor cells in blood, and belongs to the field of medical instruments. The device is composed of a spaced double-helix disc structure, and double helixes are connected through an S-shaped knot. The spaced double-spiral disc structure comprises three stages, the first stage is a single channel, the second stage is double channels, and the third stage is three channels. Sample liquid enters from an inlet of the first stage and is closer to the inner wall due to the large size of flowing circulating tumor cells, and red blood cells are closer to the outer wall. When the circulating tumor cells reach the second stage, the circulating tumor cells flow out from the thin channel of the second stage, the white blood cells and the red blood cells enter the wide channel of the second stage, the white blood cells and the red blood cells reach the focusing positions again due to the change of the channel width, and the white blood cells are close to the inner wall and the red blood cells are close to the outer wall. The circulating tumor cells, the white blood cells and the red blood cells are separated from the three outlets respectively when the circulating tumor cells reach the third stage, so that the circulating tumor cells are separated from the blood and enriched.

Description

Technical Field

[0001] The invention relates to a microfluidic device for enriching circulating tumor cells in blood, and belongs to the field of medical devices. Background Art

[0002] Circulating tumor cells refer to malignant tumor cells that fall off from the primary tumor or metastatic lesion, undergo epithelial-mesenchymal transformation and enter the patient's peripheral blood circulation. It is well known that early detection of cancer is of great significance for the treatment of patients. Therefore, if circulating tumor cells can be accurately detected in advance from the patient's blood, it can be used as a basis for more accurate diagnosis of early cancer than conventional means. Moreover, according to the number of circulating tumor cells in the blood sample, it can provide a reference for post-healing detection of cancer patients, the therapeutic effect of anticancer drugs, and the adjustment of treatment plans. However, CTCs will grab the nutrients of the surrounding normal human cells during their growth, so the size is quite different from that of normal human blood cells CTCs. And its content is very small: there are about 109 blood cells per milliliter in human blood, and circulating tumor cells account for about 1 to 100. Therefore, the sorting of circulating tumor cells requires the sorting technology to have the characteristics of high throughput and efficient separation.

[0003] Microfluidics, also known as the full microanalysis system and lab-on-a-chip, is an integrated and miniaturized technology that integrates sample preparation, sample detection, sample screening and other operating units in chemistry, integrates these programs into a chip of a few square centimeters or even a few square millimeters, and realizes various functions of conventional biochemical laboratories by controlling the fluid entering the microchannel. Microfluidics can further affect the movement of microfluids by changing the microchannel structure design, especially affecting the movement of particles of different sizes carried by the fluid, so as to achieve precise control of microfluids. This technology has great application prospects in the separation and screening of microfluids. For example, the particle sorting technology for cells, bacteria and other particles with sizes ranging from micrometers to nanometers is widely used in clinical diagnosis, chemical analysis and other fields.

[0004] Since the channels of microfluidic chips match the size of cells, they also show great application potential in cell sorting. In order to enrich CTCs from a large number of blood cells in peripheral blood, people have developed many new methods in combination with microfluidic chip technology, such as immunoaffinity method, deterministic lateral displacement, microporous sieve, and spiral microfluidic chip. The above methods can be roughly divided into two categories according to their different working principles: physical method and biochemical method. The physical method uses the characteristics that CTCs are larger than ordinary blood cells (the cell size of CTCs is about 15-20μm, and the size of blood cells is 5-15μm), combined with the different effects of the chip on cells of different sizes to screen out CTCs.

[0005] In summary, the present invention aims at solving the problem of how to screen and measure the content of circulating tumor cells in human blood, and proposes a microfluidic device for enriching circulating tumor cells in blood, so as to achieve the separation of circulating tumor cells in blood. Summary of the invention

[0006] In order to solve the problem that circulating tumor cells are present in low levels in the blood and are difficult to capture, the purpose of the present invention is to provide a microfluidic device for enriching circulating tumor cells in the blood. By changing the radius of curvature of the microfluidic channel and thereby changing the focusing position of particles of different sizes, precise control of the particles can be achieved, thereby separating and enriching circulating tumor cells in the blood.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention discloses a microfluidic device for enriching circulating tumor cells in blood, which is composed of a double helix disc structure with intervals, and the double helixes are connected by an S-knot. The double helix disc structure with intervals includes three stages, the first stage is a single channel, the second stage is a double channel, and the third stage is a three-channel. The sample liquid enters from the entrance of the first stage, and the circulating tumor cells are closer to the inner wall due to their larger size, and the red blood cells are closer to the outer wall. When it reaches the second stage, the circulating tumor cells flow out from the thinner channel of the second stage, and the white blood cells and the red blood cells enter the wider channel of the second stage. At this time, due to the change in channel width, the white blood cells and the red blood cells are under the action of Dean drag, and the white blood cells and the red blood cells re-reach the focusing position. The white blood cells are closer to the inner wall due to their larger size, and the red blood cells are closer to the outer wall due to their smaller size. At this time, when it reaches the third stage, the white blood cells flow out from the thinner channel of the third stage with the fluid, and the red blood cells flow out from the wider channel of the third stage with the fluid. Then, the circulating tumor cells, white blood cells and red blood cells are separated from the three outlets respectively, and the circulating tumor cells are separated and enriched from the blood.

[0009] Preferably, the spaced double helix disc structure is a spaced ten-turn double helix disc structure.

[0010] The present invention discloses a microfluidic device for enriching circulating tumor cells in blood, which utilizes the inertial lift and Dean vortex in a curved channel to enhance the ability to sort tumor cells. By precisely manipulating the fluid in the microchannel, the curved channel microfluidic chip can quickly and efficiently capture tumor cells CTCs from the blood without using external force or chemically labeling the cells.

[0011] A microfluidic device for enriching circulating tumor cells in blood, which is a double-helix disc structure with intervals as a whole; it includes three stages, named as the first stage, the second stage and the third stage; the first stage includes an inlet and two outlets; the second stage is a dual-channel stage; the third stage is a three-channel stage, and circulating tumor cells, white blood cells and red blood cells flow out from three outlets;

[0012] The minimum lengths of the first and second stages are obtained by the following formulas:

[0013] C L =3.437Re-0.715

[0014] Re=ρU m D h / μ

[0015] D h =2wh / (w+h)

[0016] Among them, C L is the particle position, Re is the Reynolds number, U m is the flow rate, D h is the hydraulic diameter, ρ is the density, μ is the dynamic viscosity, w is the channel width, and h is the channel height.

[0017] In the second stage, before reaching the first stage, cells of different particle sizes reach the focusing equilibrium position, and the larger circulating tumor cells are first separated from the second stage separation port. At the same time, the cross-sectional size of the microchannel becomes smaller, and the smaller white blood cells and red blood cells can reach the focusing equilibrium position again and be separated from the third stage separation port.

[0018] Preferably, the radius of curvature of the S-shaped junction at the center of the double helix is ​​2.7 mm.

[0019] Preferably, the cross section of the first-stage channel is 100 μm high and 355 μm wide, and the spacing between adjacent channels is 455 μm.

[0020] Preferably, the width of the circulating tumor cell channel at the small first-order separation port in the first stage is 95 μm, and the width of the wide microfluidic channel is 260 μm.

[0021] In the middle of the second circle of the spiral channel at the outlet, a separation port is set, which is called the second stage. Before the separation port, through the optimization design of the channel size, the largest particle among the three types of large, medium and small particles gathers at the equilibrium position and flows out from the leftmost channel of the first-order separation port. The remaining small and medium particles do not reach the aggregation conditions and continue to move along the right channel. The small and medium particles continue to separate in the channel on the right side of the first-order separation port. Compared with the previous channel, the channel size for the movement of small and medium particles is narrowed at this time, and the various parameters of the channel change accordingly. The medium particles that did not reach the aggregation conditions before gradually gather and balance with the change of the pipeline, while the small particles are outside the channel due to their small size. When the small and medium particles gradually move to the third stage with the flow of the fluid, the medium particles have gathered at the equilibrium position close to the inner wall and flow out from the left channel of the third stage, and the small particles flow out from the wider right channel with the fluid. With the two separations of the double helix channel, it is possible to separate particles of three different sizes.

[0022] Beneficial effects:

[0023] 1. The present invention discloses a microfluidic device for enriching circulating tumor cells in the blood, which is composed of a spaced double helix disc structure, and the double helix is ​​connected by an S-knot. The spaced double helix disc structure includes three stages, the first stage is a single channel, the second stage is a double channel, and the third stage is a triple channel. The inertial lift and Dean vortex in the curved channel work together to improve the ability to sort tumor cells. By precisely manipulating the fluid in the microchannel control, the curved channel microfluidic chip can quickly and efficiently capture tumor cells CTCs from the blood without using external force and chemically labeling the cells.

[0024] 2. The present invention discloses a microfluidic device for enriching circulating tumor cells in blood, which constructs a formula for determining the minimum value of the length of the first stage and the second stage, thereby realizing quantitative selection of the length of the first stage and the second stage of the double helix disk structure.

[0025] 3. The present invention discloses a microfluidic device for enriching circulating tumor cells in blood, which changes the cross-sectional size, channel length, channel height, radius of curvature, etc. of the microfluidic, and analyzes the focusing range distance of cells in the curved channel and the collection efficiency of cells under the change of Reynolds number. According to the focusing range of the cells, the diameter of the S-type junction is preferably 2.7 mm. Compared with the traditional double-helix microchannel, the radius of curvature of the microfluidic channel can be changed to change the focusing position of particles of different sizes to achieve precise control of the particles.

[0026] 4. The present invention discloses a microfluidic device for enriching circulating tumor cells in blood, in which a separation port is arranged in the middle of the second circle of the spiral channel at the outlet. Compared with the traditional separation microfluidic channel, the second stage makes the largest particles among the three types of large, medium and small particles gather at the equilibrium position and flow out from the leftmost channel of the first-order separation port. The remaining small and medium particles do not meet the aggregation conditions and continue to move along the right channel, so that the circulating tumor cells can be separated in advance and do not participate in the subsequent sorting of white blood cells and red blood cells.

[0027] 5. The present invention discloses a microfluidic device for enriching circulating tumor cells in blood. The narrowing of the channel in the second stage can re-change the focusing position of white blood cells and red blood cells, allowing white blood cells to be closer to the inner wall and red blood cells to be closer to the outer wall, thereby better separating them. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic diagram of a microfluidic device for enriching circulating tumor cells in blood as exemplified in this application.

[0029] Figure 2 Schematic diagram of the microfluidic dimensions height, cross-sectional width, and channel spacing of the example in this application.

[0030] Figure 3 Schematic diagram of the dimensions of the second and third stages of the microfluidic system of this application example.

[0031] Figure 4 A schematic diagram of the dimensions of the S-junction at the connection of the microfluidic double helix structure exemplified in this application.

[0032] Figure 5 Schematic diagram of the positions of cells with different diameters in the second and third stages of microfluidics as exemplified in this application, wherein: Figure 5 a is a schematic diagram of the second stage separation port. Figure 5 b is a schematic diagram of the separation port of the third stage.

[0033] Among them: 1—liquid inlet, 2—circulating tumor cell outlet, 3—leukocyte outlet, 4—red blood cell and waste fluid outlet, 5—S-knot at the spiral junction, 6—second stage, 7—third stage. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings:

[0035] In microfluidics, due to the inertial effect, particles are subjected to inertial lift and Dean drag, which causes lateral movement to achieve particle enrichment. This example selects key parameters that affect CTCs enrichment by analyzing the force on particles in the double-helix microfluidic channel; uses UG software to build double-helix microfluidic chip models with different structures, and then performs meshing in Meshing software; refers to the main cell sizes in tumor liquid biopsy blood, uses 8μm particles to represent smaller cells such as red blood cells and platelets, 12μm particles to represent white blood cells, and 20μm particles to represent CTCs, and uses Fluent software to simulate the enrichment effect of CTCs in chips with different structures at different flow rates.

[0036] By changing the cross-sectional dimensions, channel length, channel height, curvature radius, etc. of the microfluidics, the focusing range distance of cells in the curved channel and the cell collection efficiency under the change of Reynolds number are analyzed. Based on the focusing range of cells, a microfluidic device for enriching circulating tumor cells in blood is provided.

[0037] Embodiment 1:

[0038] The present embodiment discloses a microfluidic device for enriching circulating tumor cells in blood. First, a double-helix microfluidic chip model with different structures is designed through UG modeling software, which has a liquid inlet 1, an S-junction 5, a three-outlet microfluidic chip model with a circulating tumor cell outlet 2 for the first stage, the second stage and the third stage, a white blood cell outlet 3 and a red blood cell and waste liquid outlet 4.

[0039] like Figure 2 As shown in the figure, the width of the microfluidic is 355μm, the interval between the two microchannels is 455μm, the radius of curvature of the S junction 5 is 2.7mm, and the double helix has a total of 10 turns. When the flow rate at the inlet is 80ml / h, the distance between various particle sizes is the largest, and the enrichment effect is the best.

[0040] Double helix connected S junction 5 Figure 4As shown in the figure, the focusing position of cells with different particle sizes can be changed by changing the curvature radius of the S-junction 5 at the double helix interface. In the microfluidic channel with an S-junction diameter of r = 2.25 mm, when the flow rate is 50 mL / h, the three particles can be effectively focused, but the interval between the 12 μm particles and the 8 μm particles is too small and difficult to separate; when the flow rate is 80 mL / h, although the three particles can be separated, the 12 μm particles appear to be scattered and gathered, and at other Reynolds numbers, the particles of the three particle sizes cannot be separated. When the diameter of the S-junction is r = 2.70 mm, when the flow rates are 50 mL / h, 80 mL / h, and 110 mL / h, the three particles can be effectively focused without scattering and gathering. When r = 3.15 mm, higher flow rates of 80 mL / h and 110 mL / h are required to effectively focus the three particles, and at other Reynolds numbers, the particles of the three particle sizes cannot be separated. The diameter of the S-junction 5 is selected to be 2.7 mm.

[0041] Phase II 6 Figure 3 As shown, before reaching the second stage 6, cells of different particle sizes reach the focusing equilibrium position, and the larger circulating tumor cells are separated from the separation port first. At the same time, the cross-sectional size of the microfluidic channel becomes smaller, and the smaller white blood cells and red blood cells can reach the focusing equilibrium position again and be separated from the third stage.

[0042] The second stage 6, before this stage, through the reasonable design of the channel size, the largest particle among the three types of large, medium and small particles can be gathered at the equilibrium position and flow out from the leftmost channel of the second stage 6 as shown in FIG. Figure 5 As shown in a, the remaining small and medium particles have not reached the aggregation condition and continue to move along the right channel. After passing the second stage 6, the largest particles no longer participate in the separation movement and finally flow out of the channel along the channel on the left side of the second stage 6. Small and medium particles continue to separate in the channel on the right side of the second stage 6. Compared with the previous channel, the channel size for the movement of small and medium particles has become narrower at this time, and various parameters of the channel have changed accordingly. The medium particles that did not reach the aggregation condition before gradually gathered and moved to the equilibrium position as the pipeline changed, while the small particles still did not reach the aggregation condition due to their small size. In this way, when the small and medium particles gradually move to the third stage 7 with the flow of the fluid, the medium particles have already gathered at the equilibrium position close to the inner wall. From the third stage 7 as shown in Figure 5 b) flows out from the left channel, while small particles are subject to the heavier effect of Dean drag, and although there is a tendency to aggregate, the particle movement trajectory is still relatively dispersed, and flows out from the wider right channel with the fluid. In this way, with the two separations of the double helix channel, three particles of different sizes can be separated.

[0043] Particles with diameters of 8μm, 12μm, and 20μm are released uniformly at the entrance and move forward along the pipe under the action of the fluid. In the first 5 circles, as the particles move toward the center, the two particles with larger diameters gradually gather at the equilibrium position, but the 20μm particles are closer to the inner wall and the particle trajectory is narrower. The 8μm particles with smaller diameters never gather at the equilibrium position, and the particle trajectory is also wide. This is because smaller particles are more susceptible to the Dean drag, and the inertial lift and Dean drag are not balanced. When the particles enter the S interface, the original outer wall of the channel becomes the inner wall, and the original outer wall becomes the inner wall, and the equilibrium position of the particles also changes. After passing through the S-shaped interface, the particles begin the spiral motion of the last 5 circles. As the radius of the last 5 circles gradually increases, the Dean drag is also gradually decreasing, and the inertial lift received by the particles is more likely to be balanced with the Dean drag, so that they gather at the equilibrium position. Compared with 12μm particles, the distribution range of the 20μm particle trajectory is narrower. When the particles move to the second stage 6, the 20μm particles closest to the inner wall flow into the left channel, while the 12μm particles and 8μm particles enter the right channel to continue separation. Due to the bifurcation of the channel, the width of the channel on the right becomes narrower, and the 12μm particles that were not well aggregated before are better aggregated in the right channel, while the 8μm particles continue to move with the Dean vortex because they do not meet the aggregation conditions. When the particles reach the third stage, the 12μm particles close to the inner wall flow out of the middle channel, and the remaining 8μm particles flow out of the right channel, achieving one-time multi-particle separation.

[0044] Embodiment 2:

[0045] A simulation verification example of a microfluidic device for enriching circulating tumor cells in blood disclosed in this embodiment is as follows:

[0046] Model design: The microfluidic pattern was designed using UG modeling software. After Fluent simulation verification, the channel height was determined to be 100 μm, the channel width was 355 μm, the interval between channels was 455 μm, and after passing through the first-order separation port, the circulating tumor cell channel was 95 μm and the second-order channel was 260 μm.

[0047] Simulation verification: First, the continuous fluid domain is iteratively calculated. After the calculation converges to obtain the steady-state flow field, its solution is used as the input of the discrete term model to solve the flow condition of the particles. Three diameter particles are set in the simulation process: circulating tumor cells are set to 20μm, white blood cells are set to 12μm, and red blood cells are set to 8μm. The density is set to 1.05kg / cm 3 .

[0048] Calculation result display: After the calculation process is completed, use Fluent's own post-processing module or CFD POST module to display the calculation results to obtain visual and intuitive results.

[0049] The specific description above further illustrates the purpose, technical solutions and beneficial effects of the invention in detail. It should be understood that the above is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A microfluidic device for enriching circulating tumor cells in blood, characterized in that: It is composed of a double helix disc structure with intervals, and the double helix is ​​connected by an S-knot; the double helix disc structure with intervals includes three stages, the first stage is a single channel, the second stage is a double channel, and the third stage is a triple channel; the sample enters from the entrance of the first stage, and the circulating tumor cells are closer to the inner wall due to their larger size, and the red blood cells are closer to the outer wall after flowing; when it reaches the second stage, the circulating tumor cells flow out from the thinner channel of the second stage, and the white blood cells and red blood cells enter the wider channel of the second stage. At this time, due to the change in channel width, the white blood cells and red blood cells return to the focusing position under the action of Dean drag, and the white blood cells are closer to the inner wall due to their larger size, and the red blood cells are closer to the outer wall due to their smaller size. At this time, it reaches the third stage, and the white blood cells flow out from the thinner channel of the third stage with the fluid, and the red blood cells flow out from the wider channel of the third stage with the fluid. Tumor cells, white blood cells and red blood cells are separated from the three outlets respectively, so as to separate and enrich circulating tumor cells from the blood.

2. A microfluidic device for enriching circulating tumor cells in blood according to claim 1, characterized in that: The spaced double spiral disc structure adopts a spaced ten-turn double spiral disc structure.

3. A microfluidic device for enriching circulating tumor cells in blood according to claim 1 or 2, characterized in that: The inertial lift and Dean vortex in the curved channel work together to improve the ability to sort tumor cells; by precisely manipulating the fluid in the microchannel, the curved channel microfluidic chip can quickly and efficiently capture tumor cells CTCs from the blood without using external force or chemically labeling the cells.

4. A microfluidic device for enriching circulating tumor cells in blood according to claim 1 or 2, characterized in that: The minimum lengths of the first and second stages are obtained by the following formulas: C L =3.437Re-0.715 Re=ρU m D h / m D h =2wh / (w+h) Among them, C L is the particle position, Re is the Reynolds number, U m is the flow rate, D h is the hydraulic diameter, ρ is the density, μ is the dynamic viscosity, w is the channel width, and h is the channel height.

5. A microfluidic device for enriching circulating tumor cells in blood according to claim 1 or 2, characterized in that: In the second stage, before reaching the first stage, cells of different particle sizes reach the focusing equilibrium position, and the larger circulating tumor cells are first separated from the second stage separation port. At the same time, the cross-sectional size of the microchannel becomes smaller, and the smaller white blood cells and red blood cells can reach the focusing equilibrium position again and be separated from the third stage separation port.

6. A microfluidic device for enriching circulating tumor cells in blood according to claim 1 or 2, characterized in that: The radius of curvature of the S-junction connection at the center of the double helix is ​​2.7 mm.

7. A microfluidic device for enriching circulating tumor cells in blood according to claim 1 or 2, characterized in that: The cross-section of the first-stage channel is 100 μm high and 355 μm wide; the spacing between adjacent channels is 455 μm.

8. A microfluidic device for enriching circulating tumor cells in blood according to claim 1 or 2, characterized in that: The width of the circulating tumor cell channel at the small first-order separation port in the second stage is 95 μm, and the width of the wide microfluidic channel is 260 μm.

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