CN111744565B - Microfluidic device and system for multi-channel parallel detection of cell deformability - Google Patents
Microfluidic device and system for multi-channel parallel detection of cell deformability Download PDFInfo
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Abstract
The invention discloses a micro-fluidic device and a system for multi-channel parallel detection of cell deformability, and the micro-fluidic device comprises a sample inlet, a spiral flow channel, a Y-shaped double outlet and a deformability detection flow channel; the sample inlet below communicates spiral runner one end, the spiral runner other end intercommunication Y type exit channel, the first branch road of Y type exit channel communicates first export, and the second branch road communicates deformability and detects the runner, the deformability detects and is provided with a plurality of parallel passageways in the runner, deformability detects the runner intercommunication second export. The invention integrates inertial sorting, and effectively realizes high-flux sorting of cells in whole blood; the cell deformation is realized by utilizing the fluid pressure, and the multichannel parallel detection realizes the high-flux detection of the cell deformation, and the detected cells still have high activity, thereby identifying the residual white blood cells and cancer cells.
Description
Technical Field
The invention relates to a microfluidic device and a system, in particular to a microfluidic device and a system for multi-channel parallel detection of cell deformability.
Background
In recent years, a circulating tumor cell sorting and enriching method based on a microfluidic technology has attracted extensive attention and made breakthrough progress. However, conventional analytical methods such as immunocytology, flow cytometry, and nucleic acid detection techniques are still commonly used for the identification and characterization of circulating tumor cells after sorting. These methods all use a biomolecular marker as an analysis object, not only affect the cell activity, but also cannot realize the detection of circulating tumor cells that do not express a specific molecular marker, for example, some tumor cells may have epithelial mesenchymal transition in the process of metastasis and lose epithelial cell markers. In addition, the methods have the common defects of complex operation, low detection efficiency, difficult integration and the like.
Studies have shown that the mechanical properties of cells are closely related to the pathological state of the cells, e.g. cancerous cells are softer than healthy cells. Thanks to the vigorous development of microfabrication technology, microfluidic devices capable of analyzing mechanical properties of single cells have been developed successfully, such as measuring deformability of cells using dielectrophoresis-induced deformation technology, analyzing mechanical response of cells by compression, tension and fluid shear stress, and characterizing contractility of cells by patterning microcolumn substrates. The microfluidic platforms can effectively analyze the mechanical characteristics of single cells, but the cells need to be captured and fixed in the experimental process, so that the whole measurement process consumes a long time, and the detection flux of the devices is greatly limited.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a micro-fluidic device for multi-channel parallel detection of cell deformability, and solves the problems that cells need to be captured and fixed, the time consumption is long, and the detection flux is limited in the existing detection.
The technical scheme is as follows: the microfluidic device for multi-channel parallel detection of cell deformability comprises a sample inlet, wherein the lower part of the sample inlet is communicated with one end of a spiral flow channel, the other end of the spiral flow channel is communicated with a Y-shaped outlet channel, a first branch of the Y-shaped outlet channel is communicated with a first outlet, a second branch of the Y-shaped outlet channel is communicated with a deformability detection flow channel, a plurality of parallel channels are arranged in the deformability detection flow channel, and the deformability detection flow channel is communicated with a second outlet.
In order to capture impurities larger than the corresponding size when the impurities flow through and reduce the risk of blocking a flow channel, the sample inlet is provided with a filter sieve, and the filter sieve consists of micro-column columns which are arranged at equal intervals.
In order to focus the cell particles in the spiral flow channel, the cross-sectional height of the spiral flow channel satisfies 0.07<ap/h<0.3 wherein apThe cell diameter and h the cross-sectional height of the spiral flow channel.
In order to better utilize the acting force of microfluid to realize the sorting of cells, the cross section of the spiral flow channel is a rectangle with the ratio of height to width of 1/2-1/4 or a trapezoid with two sides with different heights.
And the purity of the tumor cells of the second branch is improved, and the width ratio of the first branch to the second branch of the Y-shaped outlet channel is 1.5-3.
The cells are gently dispersed and enter the plurality of parallel micro-channels to generate deformation, two sides of the deformability detection flow channel are of conical structures, a sudden expansion area of each conical structure forms an included angle of 10-60 degrees with the wall of the flow channel, and then the cells are expanded to the required width through straight extension.
The cell can be ensured to be obviously deformed under the action of fluid shearing force and pressure, the deformability detection flow channel is provided with a plurality of parallel equidistant microcolumn columns which are divided into a plurality of channels, the cross section of each channel is square, and the relationship between the side length of each channel and the diameter of the cell to be detected is as follows: the cell diameter is 50-90% of the side length.
The invention relates to a detection system comprising a microfluidic device for multi-channel parallel detection of cell deformability, and further comprising an injector, an illuminating device, an image shooting device and a PC, wherein the injector is connected with a sample inlet, the illuminating device is arranged above the deformability detection channel, the image shooting device is arranged below the deformability detection channel, the image shooting device is in signal connection with the PC, the injector injects a sample liquid into the sample inlet, the fluid is processed by the microfluidic device, and the image shooting device shoots deformed cells and then transmits images to the computer.
The technical principle is as follows: the sample liquid is injected into the spiral flow channel from the sample inlet, and enters the spiral flow channel after being screened by the filter sieve to separate out most blood cells, the circulating tumor cells and a small amount of white blood cells with similar sizes jointly enter the deformability detection flow channel, the cells are deformed by fluid shearing force and pressure when passing through a narrow channel in the deformability detection flow channel, an image shooting device shoots the deformation and transmits the image to a computer, and meanwhile, in order to reduce motion blur of the cells when the cells are shifted through the narrow flow channel, a high-power LED is adopted to carry out sample illumination by using pulse current, and a camera shutter triggers pulse to ensure synchronous exposure; the computer receives the image and processes the image by the programmed program to obtain the roundness value and the cross-sectional area of the deformed cell, so as to analyze the deformability of the cell and further identify whether the cell is a circulating tumor cell or a leukocyte.
Has the advantages that: the invention adopts integrated inertial sorting, thus effectively realizing high-flux sorting of target cells in whole blood; the multi-channel parallel detection greatly improves the detection flux, and realizes cell deformation by utilizing the fluid pressure, so that the cell deformation is more obvious; the cell is identified by the difference of the mechanical properties of the cell, and the detected cell has higher biological activity than the cell identified by using a biological molecular marker.
Drawings
FIG. 1 is a top view of the overall structure of the present invention;
FIG. 2 is a partially enlarged view of a microcolumn array according to the present invention;
FIG. 3 is a schematic diagram of the spiral flow channel inertial sorting principle of the present invention;
FIG. 4 is a schematic diagram of the force exerted by a cell in a narrow passage according to the present invention;
FIG. 5 is a schematic diagram of the deformation process of the cells in the narrow passage according to the present invention;
FIG. 6 is a flow chart of a computer processing cell image according to the present invention;
FIG. 7 is a schematic diagram of the experimental platform of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
As shown in figure 1, the invention discloses a microfluidic device for multi-channel parallel detection of cell deformability, which comprises a sample inlet 1, a filter sieve 2, a spiral flow channel 3, a Y-shaped outlet channel 4, a first outlet 5 of blood cells, a deformability detection flow channel 6 and a second outlet 7 of circulating tumor cells and white blood cells with similar sizes.
One end is equipped with sample entry 1 on the integrated device, and sample liquid passes through syringe pump promotion syringe 8 and pours sample liquid into the device into from sample entry 1 into, and sample entry 1 department sets up filter sieve 2, and when sample liquid stream passed through filter sieve 2, large granule impurity was intercepted to avoid the runner of device to block up. The lower part of the sample inlet 1 is communicated with the spiral flow channel 3, the circulating tumor cells and the white blood cells with similar sizes in the sample liquid are close to the inner wall surface 8 of the flow channel and most of the blood cells are close to the outer wall surface 9 under the combined action of inertia force and dean drag force after the sample liquid enters the spiral flow channel 3, when the circulating tumor cells and the white blood cells with similar sizes reach the Y-shaped outlet channel 4, the circulating tumor cells and the white blood cells with similar sizes flow into the deformability detection flow channel 6, and most of the blood cells flow out of the device through the first outlet 5; after entering the deformability detection flow channel 6, the cells pass through a plurality of parallel narrow channels, deform under the action of fluid shearing force and pressure, and finally flow out through the second outlet 7. The preparation material of each runner is Polydimethylsiloxane (PDMS), and can also be made of materials with good optical performance such as glass, epoxy resin, polymethyl methacrylate (PMMA), Polycarbonate (PC) and the like, and the prototype device is prepared by a soft lithography processing technology and specifically comprises the steps of photoetching an SU-8 male mold, PDMS pouring, PDMS-glass bonding and packaging and the like. In addition, the preparation of the male die can also be realized by means of wet/deep reactive ion etching of silicon, ultra-precision machining, metal plating and etching processing of a photosensitive circuit board.
When the microfluidic device is used for detection, an image shooting device 10 such as a high-speed camera is arranged below the deformability detection channel 6, and the high-speed camera shoots deformed cells and then transmits the images to a PC (personal computer) 11; meanwhile, in order to reduce the motion blur of the cells when the cells are displaced by contraction, an illuminating device 9 such as a high-power LED is adopted to illuminate the sample by using pulse current, and a camera shutter triggers pulses to ensure synchronous exposure; and the computer receives the image and processes the image by an image processing program to obtain the roundness value and the cross-sectional area of the deformed cell, so as to analyze the deformability of the cell and further identify whether the cell is a circulating tumor cell or a leukocyte.
As shown in fig. 2, a filter sieve 2 is provided at the sample inlet, and large particle impurities are captured when the sample liquid flows through the filter sieve 2, thereby preventing the flow channel of the device from being blocked.
As shown in fig. 3, the inner wall surface 8 and the outer wall surface 9 of the spiral flow channel 3 are shown in the figure, which is the sorting principle of the primary spiral flow channel 3. After the particle suspension is injected into the spiral flow channel 3 through the sample inlet 1 at a specific flow speed, because the fluid near the central line in the bent flow channel has a higher flow speed than the fluid near the wall surface, the fluid flows outwards under the action of unbalanced centrifugal force and radial pressure gradient; meanwhile, the outer wall surface 9 is close to based on the mass conservation in the closed flow channelThe fluid flows back along the upper and lower walls of the spiral flow channel 3, and two vortices with opposite rotation directions are generated in the vertical main flow direction, which are called dean flow or secondary flow. In addition, because the flow velocity of the fluid in the flow channel is distributed in a parabolic manner from the center of the fluid to the wall surface of the flow channel, the formed velocity gradient induces and generates a shear induced lift force pointing to the wall surface of the flow channel, so that the particles in the shear induced lift force move to the wall surface of the flow channel, and simultaneously, the wall surface and the fluid act together to generate a wall surface induced lift force driving the particles to leave the wall surface, and the resultant force of the two lift forces is called as an inertial lift force FL. At inertial lift force FLAnd dean drag force F induced by dean flowDWill reach stable equilibrium positions a, b, and particles of different sizes will have different equilibrium positions.
As shown in FIG. 4, the particle is subjected to the fluid shear force and pressure in the narrow channel as shown in the figure, and the flexible cell is deformed after being stressed, and the magnitude of the deformation is determined by the deformability of the cell, i.e. the flexibility of the cell.
As shown in fig. 5, after entering the flow channel 6 for detecting deformability, the cells pass through a plurality of parallel narrow channels and deform under the action of fluid shear force and pressure, which shows the shape of each stage of cell deformation when the cells move from left to right, the high-speed camera captures the deformation of the cells and transmits the images to the computer, and the cross-sectional area and roundness values of the deformed cells are obtained by processing the images by the programmed program, so that the deformability of the cells is analyzed, and whether the cells are circulating tumor cells or leukocytes is identified.
As shown in fig. 6, the computer first obtains a single frame image from the camera, assigns a unique handle to the image, and transmits it to the system responsible for image pre-processing for background subtraction and thresholding to create a binary image. Next, it is detected whether cells are present in the image, and if so, the contour of the cells is obtained using a boundary tracking algorithm. The algorithm derives the cross-sectional area, perimeter and location of the cell from its contour and calculates the roundness c of the cell. As shown in fig. 7, the main equipment of the experimental platform includes a syringe, a microfluidic device, a high-power LED, a high-speed camera, an inverted microscope and a computer.
The invention integrates inertial sorting, and effectively realizes high-throughput sorting of target cells in whole blood. The multi-channel parallel detection greatly improves the detection flux; meanwhile, the cell deformation is realized by utilizing the fluid pressure, the deformation is more obvious, and the detection flux and the cell deformation are more obvious compared with the denaturation modes such as dielectrophoresis induced deformation, atomic force microscopic deformation, microtubule sucking and the like. In addition, the circulating tumor cells and the white blood cells are identified through the difference of the mechanical properties of the cells, and the detected cells have higher biological activity than the cells identified by using the biological molecular markers.
Claims (6)
1. The microfluidic device for multi-channel parallel detection of cell deformability is characterized by comprising a sample inlet (1), wherein the lower part of the sample inlet (1) is communicated with one end of a spiral flow channel (3), the other end of the spiral flow channel (3) is communicated with a Y-shaped outlet channel (4), a first branch of the Y-shaped outlet channel (4) is communicated with a first outlet (5), a second branch of the Y-shaped outlet channel is communicated with a deformability detection flow channel (6), a plurality of parallel channels are arranged in the deformability detection flow channel (6), the deformability detection flow channel is communicated with a second outlet (7), two sides of the deformability detection flow channel (6) are of conical structures, a sudden expansion area of each conical structure and a flow channel wall form an included angle of 10-60 degrees, the sudden expansion area is expanded to a required width through flat extension, and the deformability detection flow channel (6) is divided into a plurality of channels by a plurality of parallel and equidistant microcolumns, the channel cross section is square, and the relationship between the side length and the measured cell diameter is as follows: the diameter of the cells is 50% -90% of the side length.
2. Microfluidic device for the multichannel parallel detection of cell deformability according to claim 1, characterized in that the sample inlet (1) is provided with a filter sieve (2), the filter sieve (2) consisting of an array of micro-columns arranged at equal distances.
3. Microfluidic device for multichannel parallel detection of cell deformability as claimed in claim 1Control device, characterized in that the cross-sectional height of the spiral flow channel (3) satisfies 0.07<ap/h<0.3 wherein apThe cell diameter and h the cross-sectional height of the spiral flow channel.
4. The microfluidic device for multi-channel parallel detection of cell deformability as claimed in claim 1, wherein the cross section of the spiral flow channel (3) is rectangular with a height-width ratio of 1/2-1/4 or trapezoidal with two sides having different heights.
5. The microfluidic device for multi-channel parallel detection of cell deformability according to claim 1, wherein the width ratio of the first branch and the second branch of the Y-shaped outlet channel (4) is 1.5-3.
6. A detection system comprising a microfluidic device for multi-channel parallel detection of cell deformability as claimed in any one of claims 1-5, further comprising an injector (8) connected to the sample inlet, an illumination device (9) disposed above the deformability detection channel, an image capturing device (10) connected to the signal of the PC for injecting the sample liquid into the sample inlet, and a PC (11) for transmitting the image to the computer after the deformed cells are captured by the image capturing device.
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| CN113533171A (en) * | 2021-07-01 | 2021-10-22 | 芯峰科技(广州)有限公司 | Cell deformation detection method and system based on deep learning and microfluidic chip |
| CN113533178B (en) * | 2021-07-30 | 2022-11-11 | 东南大学 | Multi-physical-characteristic fusion-sensing cell flow detection method |
| CN114870913B (en) * | 2022-04-18 | 2024-02-02 | 东南大学 | Microfluidic device and system integrating elasticity-inertial focusing and virtual flow channel |
| EP4296645A1 (en) * | 2022-06-24 | 2023-12-27 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Method and apparatus for determining rheological properties of deformable bodies |
| CN115463698A (en) * | 2022-09-23 | 2022-12-13 | 浙江大学 | Microfluidic chip for detecting stem cell deformation performance and preparation method thereof |
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