A dual-channel single-cell sorting biochip and method

Abstract

The present invention relates to microfluidic biochip technology, specifically a dual-channel single-cell sorting biochip and method, wherein the sorting biochip includes a chip body, which is provided with several sorting areas, each of which is provided with a sampling hole and several stepped holes with a large upper diameter and a small lower diameter. A lower channel and an upper channel are provided in the chip body of each sorting area. A cell sample injected into the lower part of the sampling hole is sorted into single cells through the lower channel and then flows into each small hole. A culture fluid injected into the upper part of the sampling hole flows into each large hole through the upper channel to culture the cells in the corresponding small hole, thereby realizing single cell sorting and culture on a single chip; and after the single cells are sorted, the culture fluid does not need to be added one by one, but is added to all single cells simultaneously through the upper channel, greatly improving efficiency.

Description

Technical Field

[0001] The present invention relates to microfluidic biochip technology, in particular to a dual-channel single cell sorting biochip and method. Background Art

[0002] In biology, medicine, and other fields, cell sorting and characterization technologies can rapidly isolate desired subpopulations for identification and monitoring for clinical diagnosis. For example, understanding the heterogeneity of a patient's solid tumor at the single-cell level can enable therapies tailored to multiple cell subtypes, thereby improving survival rates. Existing single-cell sorting methods primarily include microneedle aspiration, microdissection, limiting dilution, microwell arrays, and microfluidics-based sorting. Microfluidic biochip technology offers significant advantages for cell sorting, consolidating tasks previously performed in a comprehensive laboratory onto a single chip. This not only reduces testing and reagent consumption, significantly lowering costs, but also significantly increases analysis speed. Currently, after single cells are sorted using microfluidic biochips, they must be cultured for application. Existing methods, however, involve removing single cells and culturing them individually, or directly adding culture medium to existing cells one by one, which is inefficient. Summary of the Invention

[0003] In response to the above technical problems, the present invention provides a dual-channel single-cell sorting biochip and method with high sorting and culture efficiency.

[0004] The technical solution adopted by the present invention to solve the above technical problems is: a dual-channel single-cell sorting biochip, which includes a chip body, and several sorting areas are provided on the chip body. Each sorting area is provided with an injection hole and several step holes with a large upper diameter and a small lower diameter. A lower channel and an upper channel are provided in the chip body of each sorting area. The lower part of the injection hole is connected to one end of the lower channel, and the small hole of each step hole is connected to the other end of the lower channel; the upper part of the injection hole is connected to one end of the upper channel, and the large hole of each step hole is connected to the other end of the upper channel; the cell sample injected into the lower part of the injection hole is sorted into single cells by the lower channel and then flows into each small hole, and the culture fluid injected into the upper part of the injection hole flows into each large hole through the upper channel to culture the cells in the corresponding small hole.

[0005] Preferably, a hollow plunger is provided in the injection hole, and a pipette injects the cell sample into the lower channel through the inner cavity of the hollow plunger.

[0006] Preferably, a combined plunger is provided in the injection hole, the lower part of which blocks one end of the lower channel, and the upper part of the combined plunger is provided with an injection hole connected to one end of the upper channel, and a pipette injects culture fluid into the upper channel through the injection hole.

[0007] Preferably, a blocking plunger is provided in the injection hole, and the blocking plunger blocks one end of the upper channel and one end of the lower channel at the same time.

[0008] Preferably, the lower channel includes an array of main channels connected to the lower part of the injection hole and sorting microchannels respectively connected to each group of main channels. The cell sample injected from the injection hole first flows into each group of main channels, and then single cells are sorted out by the corresponding sorting microchannel before flowing into the corresponding small hole.

[0009] Preferably, the lower channel includes four groups of main channels, each group of main channels includes two main channels, and each main channel is connected to four sorting microchannels.

[0010] Preferably, the upper channel includes an array of primary flow channels, an array of secondary flow channels and an array of tertiary flow channels connected to the upper part of the sampling hole, and a number of primary holes and a number of secondary holes are correspondingly provided in the chip body. The culture fluid injected from the sampling hole first flows into the corresponding primary hole through each group of primary flow channels, then flows into the corresponding secondary hole through each group of secondary flow channels, and then flows into each corresponding large hole through each group of tertiary flow channels.

[0011] Preferably, three sorting zones are provided, and in each sorting zone, the length of the flow channel from the injection hole through the upper channel or the lower channel to each step hole is equal.

[0012] The present invention also provides a method for sorting single cells using the dual-channel single cell sorting biochip, which comprises the following steps:

[0013] (1) Place the hollow plunger on the nozzle of the pipette containing the cell sample;

[0014] (2) Pushing the hollow plunger into the injection hole so that the bottom end of the hollow plunger is higher than the lower channel, and then pushing the pipette gun, the cell sample flowing out of the gun nozzle flows through the inner cavity of the hollow plunger into the lower part of the injection hole, and then flows into the lower channel;

[0015] (3) The cell sample is sorted into single cells through the lower channel and flows into the small holes of each step hole;

[0016] (4) Remove the hollow plunger and pipette, and then inject the culture medium into the injection hole through the pipette filled with culture medium;

[0017] (5) The culture medium flows into the large hole of each step hole through the upper channel.

[0018] Preferably, the combined plunger is put on the nozzle of a pipette filled with culture fluid, and the combined plunger is pushed into the injection hole so that the lower part of the combined plunger blocks one end of the lower channel; then the pipette is pushed, and the culture fluid flowing out of the nozzle of the pipette flows into the upper channel through the injection hole on the upper part of the combined plunger, and then flows into the large hole of each step hole; after the injection of the culture fluid is completed, the combined plunger and the pipette are removed, and then the blocking plunger is pushed into the injection hole to simultaneously block one end of the upper channel and one end of the lower channel.

[0019] From the above technical solution, it can be seen that the present invention utilizes the structural characteristics of the sorting chip to provide a dual channel, wherein the lower channel is injected with cell samples to achieve single cell sorting, and the upper channel is injected with culture fluid to achieve single cell culture, thereby achieving single cell sorting and culture on one chip; and after the single cells are sorted, there is no need to add culture fluid one by one, but culture fluid is added to all single cells at the same time through the upper channel, which greatly improves efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the external structure of the biochip of the present invention.

[0021] Figure 2 It is a schematic diagram of the preferred structural mode of the lower channel in the present invention.

[0022] Figure 3 It is a schematic structural diagram of a preferred embodiment of the upper channel in the present invention.

[0023] Figure 4 It is a schematic diagram of the cross-sectional structure of the step hole in the present invention.

[0024] Figure 5 It is a schematic diagram of the matching structure of the hollow plunger and the injection hole of the present invention.

[0025] Figure 6 It is a schematic diagram of the matching structure of the combined plunger and the injection hole of the present invention.

[0026] Figure 7 It is a schematic diagram of the matching structure of the blocking plunger and the injection hole of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings. The exemplary embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0028] See also Figure 1 、 Figure 2 、 Figure 3 and Figure 4The present invention provides a dual-channel single-cell sorting biochip, which includes a chip body 11, which can be circular, square, or other shapes. The chip body is provided with several sorting areas 12, each of which is provided with an injection hole 13 and several stepped holes 14 with a large upper diameter and a small lower diameter. The injection holes can be cylindrical or conical. A lower channel 2 and an upper channel 3 are provided in the chip body of each sorting area. The lower portion of the injection hole is connected to one end of the lower channel, and the small hole 141 of each stepped hole is connected to the other end of the lower channel. The upper portion of the injection hole is connected to one end of the upper channel, and the large hole 142 of each stepped hole is connected to the other end of the upper channel. A cell sample injected into the lower portion of the injection hole is sorted into a single cell by the lower channel and then flows into each small hole, thereby achieving single cell sorting. A culture medium injected into the upper portion of the injection hole flows into each large hole through the upper channel to culture the cells in the corresponding small hole. Thus, the present invention achieves single cell sorting and culture simultaneously through the single-cell sorting biochip, greatly improving work efficiency.

[0029] like Figure 5 As a preferred embodiment, a hollow plunger 15 is provided in the injection hole. When the pipette injects the cell sample into the lower channel through the inner cavity 151 of the hollow plunger, the hollow plunger can block the upper part of the injection hole, which not only prevents the cell sample from overflowing when the pipette is working, but also ensures that the cell sample remains in the lower channel along the inner cavity of the hollow plunger. Specifically, the injection hole adopts a tapered hole with a large diameter at the upper end and a small diameter at the lower end, and the outer portion of the hollow plunger is correspondingly tapered, which not only facilitates the insertion and removal of the hollow plunger, but also improves the sealing between the two. A through hole is opened in the axial direction on the tapered hollow plunger, thereby forming a hollow inner cavity of the hollow plunger to facilitate the injection of cell samples by the pipette.

[0030] like Figure 6As another preferred embodiment, a combination plunger 16 is provided in the injection hole. The lower part of the combination plunger blocks one end of the lower channel, and the upper part of the combination plunger is provided with an injection hole 161 connected to one end of the upper channel. After the pipette is inserted into the combination plunger, the culture medium is injected into the upper channel through the injection hole. Specifically, the injection hole adopts a tapered hole with a large diameter at the upper end and a small diameter at the lower end. The outer part of the combination plunger is correspondingly tapered, which not only facilitates the insertion and removal of the combination plunger, but also improves the sealing between the two. A blind hole 162 is axially opened on the upper part of the tapered combination plunger to insert the pipette. The injection hole connected to the blind hole is radially provided at the bottom of the blind hole on the upper part of the combination plunger. The lower part of the combination plunger is a solid structure to achieve the purpose of blocking. The upper and lower parts of the combination plunger are an integrally molded structure, which is simple and convenient to process. During implementation, after the cell sample is injected, the hollow plunger and the pipette must be removed together. At this time, the cell sample may flow back into the injection hole. Therefore, the present invention sets a combined plunger in the injection hole, which can block the lower channel to prevent the cell sample from flowing back; on the other hand, the injection hole above the injection hole can be used to inject the culture fluid, thereby realizing the addition of cell samples and culture fluid. Figure 7 As another preferred embodiment, a blocking plunger 17 is provided within the injection hole. This blocking plunger simultaneously blocks one end of the upper channel and one end of the lower channel. This means that after the culture fluid is injected, the blocking plunger within the injection hole further prevents backflow of the cell sample and culture fluid. Similarly, the injection hole utilizes a tapered hole with a larger diameter at the upper end and a smaller diameter at the lower end. The blocking plunger is a solid structure with a correspondingly tapered exterior, which not only facilitates insertion and removal of the blocking plunger but also improves the seal between the two.

[0031] During implementation, the lower channel 2 includes an array of main channels 21 connected to the lower part of the injection hole and a sorting microchannel 22 connected to each group of main channels. The cell sample injected from the injection hole first flows into each group of main channels, and then flows into the corresponding small hole after sorting out single cells through the corresponding sorting microchannel. During implementation, the cross-sectional diameter of the sorting microchannel is about 30-50 microns, which is only for a single cell of about 30 microns to pass through, thereby realizing single cell sorting. The volume of the small hole is about 20 microliters, which can accommodate a single cell solution. Specifically, the lower channel includes four groups of main channels, each group of main channels includes two main channels, and each main channel is connected to four sorting microchannels. Thus, one injection hole can simultaneously add cell samples to 32 small holes, thereby sorting out 32 single cells at the same time. The sorting biochip of the present invention has three sorting zones, i.e., three sampling holes. Cell samples or culture fluid can be added simultaneously using three pipettes, thereby enabling the sorting and culture of 96 single cells. Approximately 20 milliliters of culture fluid can be added at a time, significantly improving efficiency. Preferably, the flow path length from the sampling hole through the upper or lower channel to each stepped hole in each sorting zone is equal, ensuring that the added culture fluid or cell sample reaches the stepped holes simultaneously, further improving efficiency.

[0032] The upper channel 3 of the present invention includes an array of primary flow channels 31, an array of secondary flow channels 32, and an array of tertiary flow channels 33 connected to the upper part of the injection hole. Several primary holes 34 and several secondary holes 35 are correspondingly provided in the chip body. The culture fluid injected from the injection hole first flows into the corresponding primary holes through each group of primary flow channels, then flows into the corresponding secondary holes through each group of secondary flow channels, and then flows into each corresponding large hole through each group of tertiary flow channels. During the implementation process, the cross-sectional diameters of the primary flow channels, secondary flow channels, and tertiary flow channels decrease in sequence; correspondingly, the diameters of the primary holes and secondary holes decrease in sequence. Therefore, by designing the levels of flow channels and holes, the number of flow channels can be reduced, making processing easier. Taking the example of the aforementioned injection hole filling 32 stepped holes, one injection hole connects to two groups of primary flow channels, each group of primary flow channels uses a large-diameter flow channel, each primary flow channel connects to a primary hole, each primary hole connects to two groups of secondary flow channels, each group of secondary flow channels uses two smaller-diameter flow channels, each secondary flow channel connects to a secondary hole, and each secondary hole delivers culture fluid to four large holes through a group of four tertiary flow channels. Thus, the present invention achieves simultaneous filling of 32 stepped holes with culture fluid in one sorting zone using two primary flow channels, two primary holes, eight secondary flow channels, eight secondary holes, and 32 tertiary flow channels. Therefore, by setting up three sorting zones and utilizing three injection holes, culture fluid can be simultaneously added to 96 stepped holes.

[0033] The present invention also provides a method for sorting single cells using the dual-channel single cell sorting biochip, which comprises the following steps:

[0034] First, the hollow plunger is put on the nozzle of the pipette containing the cell sample, and then the hollow plunger is pushed into the sampling hole so that the bottom end of the hollow plunger is higher than the lower channel to ensure that the lower channel is unobstructed; then the pipette is pushed, and the cell sample flowing out of the nozzle flows into the lower part of the sampling hole through the inner cavity of the hollow plunger, and then flows into the lower channel, that is, first flows into the main channel, and then flows into the sorting microchannel, thereby sorting out single cells and then flowing into the small holes of each step hole to achieve single cell sorting.

[0035] Next, the hollow plunger and pipette are taken out, and then the culture fluid is injected into the injection hole through the pipette filled with culture fluid. Specifically, the combined plunger is put on the nozzle of the pipette filled with culture fluid, and the combined plunger is pushed into the injection hole so that the lower part of the combined plunger blocks one end of the lower channel to prevent the cell sample from flowing back. Then, the pipette is pushed, and the culture fluid flowing out of the nozzle of the pipette flows into the upper channel through the injection hole on the upper part of the combined plunger, that is, first flows into the primary hole through the primary flow channel, then flows into the secondary hole through the secondary flow channel, and then flows into the large hole of each step hole through the tertiary flow channel, thereby realizing the filling of the culture fluid. After the injection of the culture fluid is completed, the combined plunger and pipette are taken out, and the blocking plunger is pushed into the injection hole to simultaneously block one end of the upper channel and one end of the lower channel to prevent the cell sample and culture fluid from flowing back. The present invention utilizes a single cell sorting biochip to achieve the addition of culture fluid, and realizes the sorting and culture of cells on a single cell sorting biochip without the need to add culture fluid one by one, thereby greatly improving efficiency.

Claims

1. A dual-channel single-cell sorting biochip, comprising a chip body with a plurality of sorting zones, characterized in that: Each sorting area is provided with an injection hole and several stepped holes with a large diameter at the upper end and a small diameter at the lower end. A lower channel and an upper channel are provided in the chip body of each sorting area. The lower part of the injection hole is connected to one end of the lower channel, and the small hole of each stepped hole is connected to the other end of the lower channel; the upper part of the injection hole is connected to one end of the upper channel, and the large hole of each stepped hole is connected to the other end of the upper channel; the cell sample injected into the lower part of the injection hole is sorted into single cells by the lower channel and then flows into each small hole, and the culture fluid injected into the upper part of the injection hole flows into each large hole through the upper channel to culture the cells in the corresponding small hole; a hollow plunger is provided in the injection hole, and a pipette injects the cell sample into the lower channel through the inner cavity of the hollow plunger; a combination plunger is provided in the injection hole, the lower part of the combination plunger blocks one end of the lower channel, and the upper part of the combination plunger is provided with an injection hole connected to one end of the upper channel, and the pipette injects the culture fluid into the upper channel through the injection hole.

2. The dual-channel single-cell sorting biochip according to claim 1, characterized in that: A blocking plunger is provided in the injection hole, and the blocking plunger blocks one end of the upper channel and one end of the lower channel at the same time.

3. The dual-channel single-cell sorting biochip according to claim 1, characterized in that: The lower channel includes an array of main channels connected to the lower part of the injection hole and a sorting microchannel connected to each group of main channels. The cell sample injected from the injection hole first flows into each group of main channels, and then single cells are sorted out by the corresponding sorting microchannel before flowing into the corresponding small hole.

4. The dual-channel single-cell sorting biochip according to claim 3, characterized in that: The lower channel includes four groups of main channels, each group of main channels includes two main channels, and each main channel is connected to four sorting microchannels.

5. The dual-channel single-cell sorting biochip according to any one of claims 1 to 4, characterized in that: The upper channel includes an array of primary flow channels, an array of secondary flow channels and an array of tertiary flow channels connected to the upper part of the sampling hole. Several primary holes and several secondary holes are correspondingly provided in the chip body. The culture fluid injected from the sampling hole first flows into the corresponding primary hole through each group of primary flow channels, then flows into the corresponding secondary hole through each group of secondary flow channels, and then flows into each corresponding large hole through each group of tertiary flow channels.

6. The dual-channel single-cell sorting biochip according to claim 1, characterized in that: Three sorting areas are provided, and in each sorting area, the length of the flow channel from the injection hole through the upper channel or the lower channel to each step hole is equal.

7. A method for sorting single cells using the dual-channel single cell sorting biochip according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Place the hollow plunger on the nozzle of the pipette containing the cell sample; (2) Pushing the hollow plunger into the injection hole so that the bottom end of the hollow plunger is higher than the lower channel, and then pushing the pipette gun, the cell sample flowing out of the gun nozzle flows through the inner cavity of the hollow plunger into the lower part of the injection hole, and then flows into the lower channel; (3) The cell sample is sorted into single cells through the lower channel and flows into the small holes of each step hole; (4) Remove the hollow plunger and pipette, and then inject the culture medium into the injection hole through the pipette filled with culture medium; (5) The culture medium flows into the large hole of each step hole through the upper channel.

8. The method for sorting single cells according to claim 7, wherein: The combined plunger is placed on the nozzle of a pipette filled with culture medium, and the combined plunger is pushed into the injection hole so that the lower portion of the combined plunger blocks one end of the lower channel; the pipette is then pushed, and the culture medium flowing out of the nozzle of the pipette flows through the injection hole on the upper portion of the combined plunger into the upper channel and then into the large hole of each stepped hole; After the culture solution is injected, the combined plunger and the pipette are taken out, and the blocking plunger is pushed into the injection hole to block one end of the upper channel and one end of the lower channel at the same time.

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