Patent ID: 12257580

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A number of preferred examples of this application will be introduced below with reference to the accompanying drawings of the specification, such that the technical content can be clearly and easily understood. This application can be embodied through examples of many different forms, and the protection scope of this application is not limited to the examples mentioned herein.

In the drawings, components with the same structure are denoted by the same numeral, and components with similar structures or functions are denoted by similar numerals. The size and thickness of each component are randomly shown in the drawings, and this application does not limit the size and thickness of each component. In order to make the illustration clearer, a thickness of a component is appropriately exaggerated in some places of the drawings.

The PDMS mentioned in this application refers to polydimethylsiloxane.

Example 1

As shown inFIG.1andFIG.2, the microfluidic chip100provided by this application includes: a circulating tumor cell separation unit10, which has a channel11for a cell sample to pass through and can rapidly separate circulating tumor cells from a complex cell sample; a single-cell capture unit20, a front end of which communicates with a tail end of the circulating tumor cell separation unit10, and the single-cell capture unit can capture single cells from separated and enriched target cells and further subject the captured single cells to closed lysis; and a gel layer30which is arranged at the single-cell capture unit20, where the single-cell western blotting analysis can be completed on the gel layer30.

The microfluidic chip100proposed in this application is a fast, sensitive, and stable chip to realize label-free high-throughput separation and single-cell western blotting analysis of circulating tumor cells. This application combines the microfluidic chip100and the western blotting technology, that is, through the structural design of the microfluidic chip100, the cell separation and single-cell western blotting technologies are integrated on the microfluidic chip100, which fundamentally solves the problems of cell number limitation, slow analysis speed, low detection sensitivity, and the like in the circulating tumor cell separation on a microchip and single-cell western blotting.

The circulating tumor cell separation unit10is provided to separate circulating tumor cells from a cell sample. In the channel11of the circulating tumor cell separation unit, circulating tumor cells and white blood cells are under different stresses in the channel11due to different sizes and thus separated. Specifically, the properties of a fluid at a microscale and the different sizes of circulating tumor cells and white blood cells enable the separation of circulating tumor cells and white blood cells. At a microscale, when the Reynolds number of the microfluidic chip100is less than 2,300, a fluid in the microfluidic chip100forms a laminar flow, and particles with different sizes occupy different equilibrium positions in the laminar flow due to the equilibrium of inertial force and Dean's drag force. In some implementations, the channel11has at least one arc portion111, and when a cell sample passes through the arc portion111, particles with different sizes are separated due to different stresses. In some implementations, the channel11includes a serpentine channel110, which is formed by connecting a plurality of linear portions112and arc portions111in sequence. In some implementations, the channel11can also be arranged as a spiral channel or a channel with another irregular shape and an arc portion. In some implementations, the channel11also includes a semicircular channel113in communication with the serpentine channel110, and a diameter of the semicircular channel113gradually increases in a direction from the serpentine channel110to the single-cell capture unit20, such that separated cells can leave the channel11; and a radius R of the semicircular channel113can be set according to actual requirements, and as shown inFIG.3, the radius R is 6 mm. In some implementations, the separation unit10further includes a drainage channel114, which is arranged in front of the arc portion111of the channel11to drain a cell sample from the outside into the channel11. As shown inFIG.3, according to different circulating tumor cell sizes, various portions of the channel11and the drainage channel114can have different diameters. For example, the drainage channel114and the serpentine channel110can have the same diameter r of 0.2 mm.

With the separation of breast cancer cell line MCF-7 (with a diameter of about 22 μm to 28 μm) from white blood cells (with a diameter of about 8 μm to 18 μm) as an example, as shown in the figure, when a flow rate is set to 1.4 mL/min, the maximum separation distance can be achieved for the two. The flow rate can be adjusted and optimized according to circulating tumor cells with different sizes. In this application, a sample only needs to be subjected to conventional red blood cell lysis, and then a resulting supernatant is removed. Compared with other existing technologies, this application reduces the time for incubating cells with immunomagnetic beads and reduces the cost. The process has high throughput, a short separation time at a sample flow rate of 1.4 mL/min, and a separation rate of higher than 68%. Moreover, in the entire separation process, no work field (such as electric field and magnetic field) needs to be applied additionally, there is no need to modify cells (such as incubation with immunomagnetic beads), and cells flowing through the chip100have unaffected activity and are intact.

The single-cell capture unit20can capture single cells from separated and enriched cells and subject captured cells to closed lysis. After passing through the separation unit10, a cell sample enters the single-cell capture unit20. The single-cell capture unit20can capture single circulating tumor cells. Specifically, a gel layer30is provided at the single-cell capture unit20, and a cell well21is formed on the gel layer30. The cell well21is a hole-shaped structure formed on the gel layer30, and a diameter of the cell well is adapted to a size of circulating tumor cells. After entering the gel layer30, circulating tumor cells flow with a liquid and sink into the cell well21, such that a single circulating tumor cell enters a hole, thereby achieving the capture of single cells. In some implementations, there can be a plurality of cell wells21, and preferably, the plurality of cell wells21form a microhole array and are uniformly distributed, such that a plurality of circulating tumor cells can be captured. A diameter of the cell well21can be adjusted according to a size of circulating tumor cells to be separated. For example, for the analysis of breast cancer circulating tumor cells (with a diameter of about 22 μm to 28 μm), a cell well21with a diameter of 30 μm is adopted to ensure that a single cell can enter the hole. After a cell enters the hole, a single-cell lysis buffer preheated to 55° C. can be added to achieve the lysis of the captured single cell. It should be understood that different lysis buffers can be used for different circulating tumor cells, and the preheating temperature can also be set according to actual needs.

In some implementations, the microfluidic chip100further includes a filter membrane70, which is arranged at a tail end of the separation unit10to further purify and concentrate a separated cell cluster. After passing through the separation unit10, a cell sample is filtered through the filter membrane70to remove white blood cells with a particle size smaller than a pore size of the filter membrane70and excess buffer. The pore size of the filter membrane70can be adjusted according to a size of circulating tumor cells to be separated. For example, for the separation of breast cancer circulating tumor cells (with a diameter of about 22 μm to 28 μm), a filter membrane pore size of 20 μm can be adopted. When a liquid just enters the chip100, it takes a specified time to stabilize the fluid, and thus some white blood cells will appear at an outlet of circulating tumor cells. Therefore, a filter membrane70is pasted on the outlet of circulating tumor cells to further purify circulating tumor cells, thereby eliminating the influence of background cells.

Circulating tumor cells, after being captured by the single-cell capture unit20, can be subjected to western blotting analysis. In some implementations, after the circulating tumor cells are captured and lysed by the single-cell capture unit20, a resulting cell lysate can be directly recognized by and bound with a specific primary antibody and a secondary antibody labeled with a fluorescent or luminescent group, and then an observation device such as a laser confocal fluorescence microscope is used to determine a fluorescence signal intensity of the target protein molecule. In some implementations, after the circulating tumor cells are captured and lysed by the single-cell capture unit20, an electric field is applied to the microfluidic chip100. For example, the microfluidic chip100can be placed in a device capable of generating an electric field, such that a protein enters the gel layer30of the chip100under the action of the electric field to start electrophoretic separation. After the electrophoresis is over, the surface of the gel is irradiated with excitation light of a specified wavelength and intensity to achieve the in situ polymerization of the protein molecule and the gel monomer molecule in a protein band of the gel coating. Then the western blotting analysis is conducted, where an immobilized protein molecule is recognized by and bound with a specific primary antibody and a secondary antibody labeled with a fluorescent or luminescent group, and a laser confocal fluorescence microscope is used to determine a fluorescence signal intensity of the target protein molecule. For the light-sensitive immobilization and western blotting analysis of a protein, the Chinese Patent No. CN112390763A is incorporated herein by reference in its entirety.

In some implementations, the microfluidic chip100has a multilayer structure, and the circulating tumor cell separation unit10and the single-cell capture unit20may be arranged in different layers. Specifically, the microfluidic chip100includes a basal layer40, a second interlayer50arranged on the basal layer40, and a first interlayer60arranged on the second interlayer50, where the circulating tumor cell separation unit10is arranged on the first interlayer60and the single-cell capture unit20is arranged on the second interlayer50. During a manufacture process, the circulating tumor cell separation unit10is formed on the first interlayer60according to a preset pattern of the channel11; a tail end of the separation unit10is provided with a first hole61that penetrates through the first interlayer60in a thickness direction of the first interlayer60, and the first hole61communicates with the channel11; and a second hole51is provided on the second interlayer50at a position corresponding to the first hole61, and a gel layer30with a structure of the cell well21is provided in the second hole51. Preferably, the filter membrane70covers a side of the first hole61facing towards the second interlayer50. The first hole61and the second hole51can be square, and a side length can be set according to actual requirements, for example, the side length is set to 10 mm in the figure.

In some implementations, an inlet and an outlet of the channel11may each be connected with a conduit, where the conduit at the inlet is provided to introduce a cell sample and the conduit at the outlet is provided to draw out excess liquid.

Example 2

This application provides a method for analyzing circulating tumor cells using the microfluidic chip100, where the separation and western blotting analysis of circulating tumor cells are integrated on the microfluidic chip100, which improves the analysis efficiency and accuracy, and provides a new method for single-cell protein quantitative analysis, single-cell omics, and cell heterogeneity research.

A basic process of the method of this application is as follows:

Step S1: Pretreatment of a blood sample. This step can be conducted by any method known in the art, which does not limit this application. In some implementations, a blood sample is subjected to red blood cell lysis, then centrifuged, and diluted and suspended with a large volume of PBS or normal saline.

Step S2: A pretreated cell sample is introduced into the circulating tumor cell separation unit10of the microfluidic chip100to complete the separation and enrichment of the target cell. Due to the channel11of the separation unit10, the circulating tumor cells and white blood cells can be separated by using the principle that they are different in size and are under different stresses in the microfluidic channel11.

Step S3: Single cells are captured from the separated cells and then lysed. A target cell exiting the channel11enters the single-cell capture unit20of the microfluidic chip100and is further introduced into the cell well21, such that a circulating tumor cell enters a cell well21. When a plurality of cell wells21are provided, a plurality of circulating tumor cells can be captured at the same time. A captured circulating tumor cell is lysed through the lysis buffer in the cell well21.

Step S4: A circulating tumor cell lysate is subjected to western blotting analysis.

In some implementations, in order to further purify and concentrate circulating tumor cells, after step S2, step S21can be added to filter a separated cell solution: a filter membrane70is added to remove white blood cells smaller than pores of the filter membrane70and excess buffer, thereby eliminating the influence of background cells.

The step S2, step S3, and step S4are all completed on the microfluidic chip100, and the filtration step S21is also completed on the microfluidic chip100.

Using different western blotting analysis methods, step S4can also include different operations. In some implementations, a target protein of circulating tumor cell obtained after lysis directly bind to a specific antibody and then is subjected to staining and destaining, and different fluorescence intensities of different proteins are determined by an observation device, where the observation device can be a laser confocal fluorescence microscope, a fluorescence scanning array system, or the like. In some implementations, step S4further includes:

S41: An electric field (for example, 40 V/min) is applied on the microfluidic chip100to achieve electrophoretic separation. The electric field can be applied by any means known in the art. After the electric field is applied, the protein in the circulating tumor cells enter the gel layer30under the action of the electric field to start electrophoretic separation.

S42: Light-sensitive immobilization of protein. After the gel electrophoresis is over, the surface of the gel is irradiated with excitation light of a specified wavelength and intensity to achieve the in situ polymerization of the protein molecule and the gel monomer molecule in a protein band of the gel layer30.

S43: Western blotting analysis. Then an immobilized protein molecule is recognized by and bound with a specific primary antibody and a secondary antibody labeled with a fluorescent or luminescent group, and an observation device is used to determine a fluorescence signal intensity of the target protein molecule.

Example 3

The breast cancer cell line MCF-7 cells are used to simulate circulating tumor cells in a breast cancer patient, and MCF-7 cells are mixed into normal human blood to simulate a blood sample collected from a breast cancer patient to verify the function of the microfluidic chip100.

A screenshot of cell distribution in the separation channel11when MCF-7 cells are passing through the separation unit10of the microfluidic chip100is shown inFIG.8.FIG.9shows the separation effect, where a separation efficiency of MCF-7 is 68% and a purity is 96%. As shown inFIG.10, MCF-7 cells show a higher purity after being filtered, concentrated, and purified by the filter membrane70. As shown inFIG.11, circulating tumor cells are captured by the cell well21, then subjected to in situ lysis and electrophoresis, then irradiated with ultraviolet light to immobilize a protein, and recognized by a primary antibody and a secondary antibody, and a fluorescence image and data processing results show that the protein analysis at the single-cell level is achieved on the separated circulating tumor cells.

Example 4

A manufacture method of the microfluidic chip100is provided in this example, specifically as follows:

Step S100: Preparation of a polydimethylsiloxane (PDMS) mixed colloid. An appropriate amount of polydimethylsiloxane and an appropriate amount of polydimethylsiloxane curing agent are weighed, added to a container successively, and then thoroughly stirred to remove bubbles in the mixed colloid. A ratio of the polydimethylsiloxane to the polydimethylsiloxane curing agent is 12:1 to 8:1. The stirring can be conducted with a glass rod. In some implementations, vacuum-pumping can be conducted in a vacuum environment to ensure that there are no bubbles in the mixed colloid. Other methods can also be used to remove bubbles. Preferably, the preparation process is conducted in a clean room environment.

Step S200: Manufacture of a first interlayer60with a separation unit10. The mixed colloid undergoing vacuum-pumping is introduced into a petri dish in which a silicon wafer with a target pattern (the target pattern corresponds to a shape of a channel11of the separation unit10), such that the mixed colloid covers a surface of the silicon wafer; and the mixed colloid is dried, and a formed first interlayer60is removed from the silicon wafer. In some implementations, after the mixed colloid covers the silicon wafer, bubbles between the silicon wafer and the petri dish can be removed before the drying operation. During a drying process, the flatness of the first interlayer60should be ensured to prevent an experimental result from being affected. Specifically, vacuum-pumping is conducted such that there are no bubbles between the silicon wafer and a bottom of the petri dish; the petri dish is dried in an electrothermal constant-temperature drying box; a shelf in the drying box must be horizontal, otherwise, the first interlayer60obtained after the drying is uneven, which will affect an experimental result to some extent; and then the petri dish is taken out, the first interlayer60is slowly peeled off from a surface of the silicon wafer with a utility knife, and the patterned part is cut into a square, and then perforated to form an inlet and an outlet. The following three points should be noted in this step: 1. In a process of peeling off the first interlayer60, a utility knife should not touch the silicon wafer, otherwise, the silicon wafer will be broken. 2. During perforation, a corresponding inlet or outlet position on the channel11must be aligned, and it should be ensured that a perforator is vertically inserted into the first interlayer60. 3. After the perforation is completed, an formed inlet or outlet needs to be poked with a fine wire to remove excess PDMS in the inlet or outlet. Four sides of a rectangular groove are cut through with a utility knife to form a rectangular frame with two transparent sides, which is the first hole61.

Step S300: Paste of a filter membrane70. The filter membrane is pasted on the rectangular frame at a side without the channel11, and then a glue is dried. Before the paste, the filter membrane can be soaked in BSA and then dried. If the filter membrane70is not required, this step can be omitted.

Step S400: Manufacture of a second interlayer50. A PDMS mixed colloid is prepared (the same as in step100), and then coated on the basal layer40. The basal layer40can be a glass sheet. Specifically, the PDMS mixed colloid can be coated by a spin-coater. That is, a PDMS mixed colloid layer with a thickness of 200 μm is spin-coated on a 75 mm×75 mm glass sheet by the spin-coater, and then dried in an oven. After the PDMS is peeled off, a rectangular frame (namely, the second hole51) is cut at a corresponding position according to a size of the chip100.

Step S500: Manufacture of a single-cell capture unit20. The surfaces of the second interlayer50and the basal layer are cleaned with a transparent glue to ensure a clean surface, and then the second interlayer and the basal layer are cleaned in a plasma cleaning machine. The plasma cleaning machine is turned on, and a cleaning time is set to 50 s; vacuum-pumping is started, and a pressure in a chamber of the plasma cleaning machine is observed; when the pressure is reduced to a preset value, the vacuum-pumping is stopped, and a switch corresponding to the highest glow intensity is turned on; and when a purple glow appears in the chamber, timing is started. The second interlayer50and the glass slide are taken out and then immediately glued together. A rectangular or square groove on the basal layer with the second interlayer50is subjected to silanization for 0.5 h. An acrylamide gelatinization solution is added to the rectangular groove on the basal layer with the second interlayer50after the silanization, and a manufactured silicon wafer mold with a cylinder array is placed inversely on the rectangular groove. After the gelatinization, the mold is removed, and a gel with a microhole array is formed in the groove.

Step S600: Bonding of the first interlayer60, the second interlayer50with the single-cell capture unit20, and the basal layer. The first interlayer60and the basal layer treated in step S500are bonded together through Plasma to obtain the microfluidic chip100. Before the two are subjected to surface-plasma treatment, DI water can be added to the groove with hydrogel to prevent the hydrogel from being dried out during the large-area surface-plasma process. In order to further strengthen the bonding, the microfluidic chip100can be dried in an oven, during which a glue needs to be prevented from being dried out and turned up.

Step S700: Insertion of conduits. After the drying, a tetrafluoroethylene tube with an outer diameter of 0.8 mm is inserted into each of the inlet and outlet to avoid liquid leakage from the inlet and outlet during an experiment, and the inlet and outlet are sealed with a PDMS mixture.

Preferred specific examples of this application are described in detail above. It should be understood that, a person of ordinary skill in the art can make various modifications and variations according to the concept of this application without creative efforts. Therefore, all technical solutions that can be obtained by a person skilled in the art based on the prior art through logical analysis, deduction, or limited experiments according to the concept of this application should fall within the protection scope defined by the claims.