CN109251841B - Single cell sorting chip, manufacturing method thereof and single cell sorting method - Google Patents
Single cell sorting chip, manufacturing method thereof and single cell sorting method Download PDFInfo
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- CN109251841B CN109251841B CN201810955552.6A CN201810955552A CN109251841B CN 109251841 B CN109251841 B CN 109251841B CN 201810955552 A CN201810955552 A CN 201810955552A CN 109251841 B CN109251841 B CN 109251841B
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Abstract
The invention provides a single-cell sorting chip, which comprises an upper chip and a glass sheet which are sequentially attached from top to bottom; the lower surface of the upper chip is a structural surface of the upper chip, a main channel is etched on the lower surface of the upper chip, the main channel comprises a plurality of U-shaped parts, a capturing channel is etched between two straight sections of each U-shaped part, the center of each capturing channel is connected with a release micro valve etched on the structural surface through a release channel, and the size of the capturing channel is set to enable the flow resistance of the capturing channel to be smaller than that of the main channel. The invention also provides a manufacturing method thereof and a single cell sorting method. The single cell sorting chip of the invention realizes the capture of single cells and the selective release of target cells by pressing the release micro valve based on the flow resistance difference distribution and the size constraint of the capture channel and the arrangement of the release micro valve, thereby improving the capture rate and the selectivity of cell sorting; the functional units can be designed into various sizes to meet the requirements of different cell sorting.
Description
Technical Field
The invention belongs to the field of biosensors, and particularly relates to a single cell sorting chip, a manufacturing method thereof and a single cell sorting method.
Background
The incidence and mortality of lung cancer in China are the first of malignant tumors, and have become the first major lung cancer world. Statistics shows that the survival rate of 5 years of patients with low early diagnosis rate of lung cancer in China is only 10%, and 70-90% of cancer patients die of tumor metastasis and recurrence within 5 years. Through long-term research, it is found that Circulating Tumor Cells (CTC) play an important role in the process of tumor metastasis. The detection and analysis of CTC can realize early diagnosis of cancer, improve survival rate within 5 years, can be used for curative effect evaluation and monitoring of recurrence and metastasis, and has important significance in the aspects of prognosis evaluation and individualized treatment.
Various methods are currently used for the enrichment and recovery of CTCs. Among these, one approach is based on immunocapture, which requires the combination of multiple tumor markers to increase capture rate and reduce the loss of heterogeneous CTCs; the other method is based on physical separation, does not need additional markers, is simple to operate, has poor specificity, cannot screen out leukocytes with the same physical characteristics as CTC, and has higher false positive. Most enrichment recovery methods are based on bulk analysis and are not selective, resulting in poor purity of the CTCs obtained.
The enrichment method based on the single cell level can improve the purity of CTC to the maximum extent, improve the accuracy of subsequent research and provide a technical basis for single cell analysis. The existing single cell level enrichment methods, such as micromanipulation and laser cutting, have the disadvantages of complicated operation and expensive instrument. There is a need for a method for single cell enrichment that is easy to operate, low cost and highly selective.
Disclosure of Invention
The invention aims to provide a single cell sorting chip which can realize single cell sorting with high capture rate and high selectivity.
In order to achieve the purpose, the invention provides a single-cell sorting chip, which comprises an upper chip and a glass sheet which are sequentially attached from top to bottom; the lower surface of the upper chip is a structural surface of the upper chip, a main channel is etched on the lower surface of the upper chip, the main channel comprises a plurality of U-shaped portions which are communicated with each other, a capturing channel is etched between two straight sections of each U-shaped portion, each capturing channel is connected with a release micro valve etched on the structural surface of the upper chip through a release channel, and the size of each capturing channel is set to enable the flow resistance of the capturing channel to be smaller than that of the main channel.
The intersection of the capture channel with the main channel is sized to accommodate only a single cell.
Still be equipped with the first introduction port, the second introduction port, first appearance mouth and the second appearance mouth that link up on the upper chip, just still the sculpture has the connection on the structural plane of upper chip the inlet channel of two introduction ports to and connect two appearance passageways of two appearance mouths, just inlet channel and appearance passageway link to each other with the both ends of main entrance respectively.
And a lower chip is arranged between the upper chip and the glass sheet, the lower surface of the lower chip is a structural surface of the lower chip, 4 extrusion channels are etched on the lower chip, and the extrusion channels are respectively corresponding to the lower parts of the sample feeding channel or the sample discharging channel and are vertical to the sample feeding channel or the sample discharging channel.
One end of each extrusion channel is provided with a flow control micro valve which penetrates through the upper chip and the lower chip and is communicated with the extrusion channel.
The upper chip and the lower chip are made of PDMS.
In another aspect, the present invention also provides a method for manufacturing the single-cell sorting chip according to claim 5, comprising the steps of: s1: making an upper chip, comprising: s11: manufacturing a required mask according to the structure of the upper chip; s12: carrying out photoetching and deep reactive ion etching by using the mask of S11 and a silicon wafer as a substrate, and removing residual glue after the etching is finished to obtain an upper silicon wafer mold with a microstructure; s13: silanization is carried out on the manufactured upper layer silicon wafer mold, then the first material is poured on the upper layer silicon wafer mold and heated together until solidification, the solidified first material is stripped, and holes are punched at the sample inlet, the sample outlet and the release micro valve, so as to obtain an upper layer middle mold; s14: pouring a second material into the upper-layer middle mould, filling the punching position of the upper-layer middle mould with the second material, curing to form a column corresponding to the sample inlet, the sample outlet and the release micro valve, and stripping the cured second material to obtain an upper-layer final mould; s15: pouring a first material into the upper layer final die to enable the liquid level of the first material to be higher than the pillars on the upper layer final die, heating to be cured, stripping the cured first material, perforating at the sample inlet and the sample outlet, and reserving the film at the position of the release micro valve to obtain the upper layer chip; s2: making a lower chip, comprising: s21: adopting a photoetching process on a silicon wafer to obtain a lower-layer silicon wafer die; s22: spin-coating a first material on a lower-layer silicon wafer mold and heating the first material and the lower-layer silicon wafer mold together until the first material and the lower-layer silicon wafer mold are solidified to obtain a lower-layer silicon wafer mold and a lower-layer chip which are attached together; s3: making a single-cell sorting chip comprising: s31: bonding the structural surface of the upper chip prepared in S1 and the lower chip prepared in S2 together, and heating; s32: stripping the upper chip and the lower chip from the silicon wafer together, and punching a hole at the lower micro valve; s33: and bonding the structural surface of the lower chip (2) and the glass sheet together to obtain the single cell sorting chip.
The first material is PDMS, and the first material is obtained by preparing a prepolymer and curing agent mixture, uniformly stirring, and vacuumizing until bubbles disappear.
The second material is epoxy glue.
The bonding of step S31 and step S33 is achieved by plasma treatment.
In another aspect, the present invention also provides a single-cell sorting method based on the single-cell sorting chip of claim 5, which comprises the following steps: a1: vacuumizing the single-cell sorting chip, and then introducing a buffer solution into the upper chip to completely fill liquid in the upper chip; a2: when capturing cells, opening a first sample inlet and a first sample outlet, closing a second sample inlet and a second sample outlet, and injecting a cell suspension subjected to fluorescent staining into the first sample inlet; a3: when the cells are recovered, the second sample inlet and the second sample outlet are opened, the first sample inlet and the first sample outlet are closed, buffer solution is injected into the second sample inlet, and one or more release micro valves are manually pressed.
And the first sample inlet, the first sample outlet, the second sample inlet and the second sample outlet are respectively opened and closed by extracting and injecting liquid into the corresponding flow control micro valve by using an injector.
The buffer solution is 0.05% of tween-20.
A single cell sorting chip comprises an upper chip and a glass sheet which are sequentially attached from top to bottom; the lower surface of the upper chip is a structural surface of the upper chip, a main channel is etched on the upper chip, the main channel comprises a plurality of U-shaped parts, a capturing channel is etched between two straight sections of each U-shaped part, and the center of each capturing channel is connected with a release micro valve etched on the structural surface of the upper chip through a release channel.
The capture channel has a flow resistance less than that of the main channel and the intersection of the capture channel with the main channel is sized to accommodate only a single cell.
Still be equipped with the first introduction port, the second introduction port, first appearance mouth and the second appearance mouth that link up on the upper chip, just it connects respectively still to etch on the structural plane of upper chip the one end of main entrance and two inlet channels of two introduction ports to and connect respectively the other end of main entrance and two outlet channels of two appearance mouths.
And a lower chip is arranged between the upper chip and the glass sheet, the lower surface of the lower chip is a structural surface of the lower chip, 4 extrusion channels are etched on the lower chip and are respectively positioned under the sample introduction channel or the sample discharge channel and are vertical to the sample introduction channel or the sample discharge channel.
One end of each extrusion channel is provided with a flow control micro valve which penetrates through the upper chip and the lower chip and is communicated with the extrusion channel.
The upper chip and the lower chip are made of PDMS.
In another aspect, the present invention also provides a method for manufacturing a single-cell sorting chip as described above, comprising the steps of: s1: making an upper chip, comprising: s11: manufacturing a required mask according to the structure of the upper chip; s12: carrying out photoetching and deep reactive ion etching by using the mask of S11 and a silicon wafer as a substrate, and removing residual glue after the etching is finished to obtain an upper silicon wafer mold with a microstructure; s13: silanization is carried out on the manufactured upper layer silicon wafer mold, then the first material is poured on the upper layer silicon wafer mold and heated together until solidification, the solidified first material is stripped, and holes are punched at the sample inlet, the sample outlet and the release micro valve, so as to obtain an upper layer middle mold; s14: pouring a second material into the upper-layer middle mould, filling the punching position of the upper-layer middle mould with the second material, curing to form a column corresponding to the sample inlet, the sample outlet and the release micro valve, and stripping the cured second material to obtain an upper-layer final mould; s15: pouring a first material into the upper layer final die to enable the liquid level of the first material to be higher than the pillars on the upper layer final die, heating to be cured, stripping the cured first material, perforating at the sample inlet and the sample outlet, and reserving the film at the position of the release micro valve to obtain the upper layer chip; s2: making a single-cell sorting chip comprising: s21: adopting a photoetching process on the corresponding position of the silicon wafer to obtain a lower-layer silicon wafer die; s22: spin-coating a first material on a lower-layer silicon wafer mold and heating the first material and the lower-layer silicon wafer mold together until the first material and the lower-layer silicon wafer mold are solidified to obtain a lower-layer silicon wafer mold and a lower-layer chip which are attached together; s23: bonding the structural surface of the upper chip in S1 and the lower chip in S22 together, and heating; s24: stripping the upper chip and the lower chip from the silicon wafer together, and punching a hole at the lower micro valve; s25: and bonding the structural surface of the lower chip and the glass sheet together to obtain the single cell sorting chip.
The first material is PDMS, and the first material is obtained by preparing a prepolymer and curing agent mixture, uniformly stirring, and vacuumizing until bubbles disappear.
The second material is epoxy glue.
The bonding of step S23 and step S23 is achieved by plasma treatment.
In another aspect, the present invention also provides a single-cell sorting method based on the single-cell sorting chip of claim 5, which comprises the following steps: a1: vacuumizing the single-cell sorting chip, and then introducing a buffer solution into the upper chip to completely fill liquid in the upper chip; a2: when capturing cells, opening a first sample inlet and a first sample outlet, closing a second sample inlet and a second sample outlet, and injecting cell suspension into the first sample inlet; a3: when the cells are recovered, the second sample inlet and the second sample outlet are opened, the first sample inlet and the first sample outlet are closed, buffer solution is injected into the second sample inlet, and one or more release micro valves are manually pressed.
And the first sample inlet, the first sample outlet, the second sample inlet and the second sample outlet are opened and closed by extracting and injecting liquid into the corresponding flow control micro valve by using the injector.
The buffer solution is 0.05% of tween-20.
The single cell sorting chip can realize the capture of single cells and the selective release of target cells by pressing the release micro valve based on the flow resistance difference distribution and the size constraint of a capture channel, and the single cell sorting chip can judge whether the captured CTC or the leukocyte is the CTC or the leukocyte according to the fluorescence color of the CTC and the leukocyte in a dyed cell suspension, and then selectively release the required cells and remove the blocked leukocyte by using the release micro valve of the single cell sorting chip when the cells are recovered, so that the purity is improved, the capture rate and the selectivity of the cell sorting are improved, the cost of the chip required by the single cell sorting is low, the operation is simple, and the cells can be recovered only by pressing the release micro valve; the functional units can be designed into various sizes to meet the requirements of different cell sorting. The introduction of the flow control micro valve in the lower chip enables the micro valve to be driven by injected liquid, and the deformation of the micro valve film is caused by water pressure, so that the channel of the upper chip is sealed and opened, the flow direction of the liquid is controlled, the sample inlet and the sample outlet can be switched on and off at any time, the conversion between sample liquid and buffer liquid entering from different sample inlets and the seamless connection between target cells (CTC) and other cells (leucocytes) recovered from different sample outlets are realized, the introduction of bubbles in the chip is avoided, the experimental operation is facilitated, single cells can be sorted and recovered in real time as required, and the subsequent cell research is facilitated. In addition, the single cell sorting method of the invention adopts 0.05 percent of Tween-20 as a buffer solution to effectively solve the problem of cell nonspecific adsorption.
Drawings
FIG. 1 is an exploded view of a single-cell sorting chip according to one embodiment of the present invention;
FIG. 2 is a schematic top view of an upper chip of the single-cell sorting chip shown in FIG. 1;
FIG. 3 is an enlarged view of a portion of FIG. 2;
FIG. 4 is a schematic top view of the lower chip of the single-cell sorting chip shown in FIG. 1;
FIG. 5 is an image of a microscope image of single cell capture in the single cell sorting chip of the present invention, showing the result of single cell capture in different U-shaped sections;
FIG. 6 is an image taken by a microscope of the single cell release in the single cell sorting chip of the present invention, which shows the release of cells in the same U-shaped section with time upon the release of a single cell.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
FIG. 1 shows a single-cell sorting chip according to one embodiment of the present invention, which comprises an upper chip 1, a lower chip 2 and a glass plate 3, which are sequentially attached from top to bottom.
The lower surface of the upper chip 1 is a structural surface, and the upper chip 1 of the single-cell sorting chip shown in FIG. 1 is viewed from above, whereby a schematic structural view of the structural surface of the upper chip 1 shown in FIG. 2 can be obtained. The structural surface of the upper chip 1 is etched with a main channel 11. The main channel 11 comprises a plurality of U-shaped parts 111 which are communicated with each other, each U-shaped part 111 is composed of a bent section and two straight sections which are respectively positioned at two sides of the bent section and are parallel to each other, a capturing channel 12 which is vertical to the straight sections is etched between the two straight sections, and the center of each capturing channel 12 is connected with a release micro valve 13 through a release channel, so that a three-way structure is formed between the capturing channel 12 and the release channel 13. The release micro valve 13 is etched on the structural surface of the upper chip 1, and then each U-shaped portion 111 and the connected capture channel 12 and release micro valve 13 form a functional unit, and the functional units are communicated with each other through the main channel 11. The upper chip 1 is preferably made of PDMS (polydimethylsiloxane), the structure surface of the upper chip is attached to the upper surface of the lower chip 2, and the upper surface of the lower chip 2 plays a role in sealing the etching structure on the structure surface of the upper chip 1, so that the release micro valve 13 can make the liquid in the release micro valve flow out through the capture channel 12 by pressing and drive the cells to be released from the capture channel 12.
As shown in fig. 3, the capture channel 12 and the main channel 11 of the chip are designed based on the differential distribution of the flow resistance. The capture channel 12 is dimensioned such that the flow resistance of the capture channel 12 is smaller than the flow resistance of the main channel 11, thereby allowing the cells to preferentially flow through the capture channel 12. Wherein the flow resistance of the main channel 11 and the capturing channel 12 is calculated by the formula,
l, W, H represents the length, width and height of the main channel 11 or the capture channel 12, respectively, and μ represents the viscosity coefficient of the fluid.
When the cell is intercepted by the capture channel 12, the flow resistance of the capture channel 12 is increased instantaneously, and the cell will select the main channel 11 to flow to the next functional unit. This design avoids clogging of cells in individual functional units. At the same time, as shown in fig. 3, the intersection of the capture channel 12 with the main channel 11 is dimensioned to accommodate only a single cell, providing for sorting at the single cell level.
Referring to fig. 2 again, the upper chip 1 is further provided with a first sample inlet 141, a second sample inlet 142 and a sample channel 16 connecting the two sample inlets; and a first sample outlet 151 and a second sample outlet 152 and a sample outlet channel 17 connecting the two sample outlets, and the sample inlet channel 16 and the sample outlet channel 17 are connected to two ends of the main channel 11, respectively. The sample inlets 141 and 142 and the sample outlets 151 and 152 penetrate through the upper chip 1, and the sample inlet channel 16 and the sample outlet channel 17 are etched on the structural surface of the upper chip 1.
The lower surface of the lower chip 2 is a structural surface of the lower chip 2, and the other surface is a flat surface bonded to the upper chip 1. The structure of the lower chip 2 shown in FIG. 4 can be obtained by looking down the lower chip 2 of the single-cell sorting chip shown in FIG. 1. The structural surface of the lower chip 2 is etched with 4 extrusion channels 21, which are respectively corresponding to and perpendicular to the sample inlet channel 16 or the sample outlet channel 17 of the upper chip 1. In addition, one end of each extrusion channel 21 is provided with a flow control micro valve 22 which penetrates the upper chip 1 and the lower chip 2 and is communicated with the extrusion channel 21. When the device is used, air or liquid is injected into the extrusion channel 21 and the flow control micro valve 22, the air pressure or the hydraulic pressure at the position of the extrusion channel 21 causes the deformation of the upper chip 1 through extruding the flow control micro valve 22, and the deformation is used for sealing the sample inlet channel 16 or the sample outlet channel 17. The lower-layer chip 2 is made of PDMS, the structural surface of the lower-layer chip is attached to the lower glass sheet 3, and the glass sheet 3 plays a role in sealing the etching structure on the structural surface of the lower-layer chip 2.
The invention also provides a manufacturing method of the single-cell sorting chip, which comprises the following steps:
s1: manufacturing an upper chip 1, including:
s11: manufacturing a required mask by utilizing CAD software according to the structure of the upper chip 1;
s12: carrying out photoetching and deep reactive ion etching by using the mask described in S11 and a silicon wafer as a substrate, and removing residual glue by using a plasma dry method after etching is finished to obtain an upper silicon wafer mold with a microstructure;
the lithography includes: spin-coating photoresist on the cleaned silicon wafer, and patterning the photoresist on the silicon wafer according to the pattern of the mask plate through a series of photoetching processes such as exposure, development and the like, wherein the thickness of the photoresist is 3 mu m; and etching the silicon wafer by using the photoresist pattern after photoetching as a mask by the deep reactive ion etching, wherein the etching depth is 20 mu m.
S13: silanization (including fumigating silane and vacuum overnight) is carried out on the manufactured upper silicon wafer mould, the first material is obtained by preparing a mixture of a prepolymer of the first material and a curing agent, the mixture is uniformly stirred and then vacuumized until bubbles disappear, then the first material is poured on the upper silicon wafer mould and is placed on a hot plate together to be heated until curing is carried out, the heating temperature is 65-95 ℃, the cured first material is peeled off, and holes are formed at the sample inlets 141 and 142, the sample outlets 151 and 152 and the release micro valve 13, so that an upper middle mould is obtained. In this embodiment, the first material is PDMS, and a mass ratio of the prepolymer of the PDMS to the curing agent is 10: 1.
S14: and pouring a second material into the upper middle mould, filling the punched positions of the upper middle mould with the second material, curing to form pillars corresponding to the sample inlets 141 and 142, the sample outlets 151 and 152 and the release micro valves 13, and stripping the cured second material to obtain an upper final mould. In this embodiment, the second material is an epoxy glue.
S15: and pouring the first material into the upper final die to enable the liquid level of the first material to be higher than the pillars on the upper final die, then placing the upper final die on a hot plate, heating to be solidified at the temperature of 50-70 ℃, stripping the solidified first material, perforating at the sample inlets 141 and 142 and the sample outlets 151 and 152, and reserving the film at the position of the release micro valve to obtain the upper chip 1 made of the first material.
S2: manufacturing a lower chip 2, comprising:
s21: adopting a photoetching process on a silicon wafer to obtain a lower silicon wafer mold of a male mold with an extrusion channel (21) and a flow control micro valve (22);
s22: preparing a mixture of a prepolymer and a curing agent of a first material, uniformly stirring, and vacuumizing until bubbles disappear to obtain the first material; and spin-coating the first material on a lower silicon wafer mold at 1300-1500 rpm, placing the first material on a hot plate, and heating the first material and the lower silicon wafer mold to be solidified to obtain the lower silicon wafer mold and the lower chip 2 which are attached together. The lower chip 2 is made of a first material (i.e., PDMS), but the mass ratio of the PDMS prepolymer to the curing agent is 20: 1, improving the flexibility of PDMS, wherein the heating temperature is 90-105 ℃.
S3: making a single-cell sorting chip comprising:
s31: and bonding the structural surface of the upper chip 1 prepared in the step S1 and the flat surface of the lower chip 2 prepared in the step S2 together by using plasma treatment, and heating the bonded upper chip and the flat surface on a hot plate at 90-105 ℃ for 5-10 min.
S32: the upper chip 1 and the lower chip 2 are peeled off together from the silicon wafer and perforated at the lower microvalve 22.
S33: and (3) bonding the structural surface of the lower chip 2 and the glass sheet 3 together by utilizing plasma treatment again to obtain the single cell sorting chip.
According to the single-cell sorting chip, the invention also provides a single-cell sorting method based on the single-cell sorting chip, which comprises the following steps:
a1: the single cell sorting chip is vacuumized, and then the buffer solution is introduced into the upper chip 1 from the second sample inlet 142, so that the upper chip 1 is completely filled with the liquid. The vacuum pumping realizes a negative pressure sample introduction mode, the injection of the sample liquid and the buffer liquid is simpler and more convenient by the negative pressure sample introduction, and the possibility of introducing bubbles into the chip is reduced. In this example, the buffer is 0.05% tween-20, which is used to eliminate non-specific adsorption of cells in the microfluidic channel. Generally, cells are non-specifically adsorbed to the walls of the microfluidic channel through hydrophobic interactions, and the adsorbed cells interfere with the flow of subsequent cells, thereby affecting the capture and release of the cells. And the Tween-20 is a nonionic surfactant, can change the hydrophilicity and hydrophobicity of an interface, and effectively solves the problem of non-specific adsorption by adopting 0.05 percent Tween-20 as a sample injection buffer solution.
A2: when capturing the cells C, the first sample inlet 141 and the first sample outlet 151 are opened, the second sample inlet 142 and the second sample outlet 152 are closed, and the cell suspension that is subjected to fluorescent staining is injected into the first sample inlet 141. Due to the difference in flow resistance between the main channel 11 and the capture channel 12, as shown in fig. 5, the cell C preferentially flows through the capture channel and is captured therein, and the subsequent cell C bypasses the cell C, flows through the main channel 11 to the next capture channel 12, and finally flows to the first outlet 151 after the number of cells greater than the number of the capture channels 12. The step a2 may further include: the release microvalve 13 corresponding to the trapping channel 12 where the leukocytes are trapped is pressed to release the leukocytes, thereby removing them from the single-cell sorting chip.
A3: when cell recovery is performed, the second sample inlet 142 and the second sample outlet 151 are opened, the first sample inlet 141 and the first sample outlet 151 are closed, and a buffer solution is injected into the second sample inlet 142, and simultaneously, the release microvalves 13 corresponding to one or more capture channels 12 in which desired CTCs are captured are manually pressed to release the CTC cells and recover the CTC cells. As shown in fig. 6, the deformation of the release microvalve 13 caused by the pressing causes the disturbance of the fluid, which gives an impact on the previously captured cells C, so that the cells C re-enter the main channel 11 from the capture channel 12 and move first upward and then downward to the right in the flow direction of the liquid in the main channel 11, leaving the U-shaped portion 111 in the main channel, thereby achieving the release of the cells C, the number of the released cells C being equal to the number of the pressed release microvalve 13, and the released cells flow to the second sample outlet, achieving the recovery of the cells.
Wherein the first sample inlet 141, the first sample outlet 151, the second sample inlet 142, and the second sample outlet 152 are opened and closed by respectively drawing and injecting a liquid (e.g., water) into the fluidic micro-valve 22 corresponding thereto by using a syringe. The positive pressure applied by the injected liquid in the fluidic micro valve 22 presses the sample inlet channel 16 or the sample outlet channel 17 to deform and close the sample inlet 141, 142 or the sample outlet 151, 152. When the liquid in the micro flow control valve 22 is extracted and a negative pressure is applied to the sample inlet channel 16 or the sample outlet channel 17, the deformation of the sample inlet channel 16 or the sample outlet channel 17 is recovered, so as to open the sample inlets 141 and 142 or the sample outlets 151 and 152. The invention uses the injector to extract and inject the liquid to apply negative pressure and positive pressure to the sample inlet channel 16 or the sample outlet channel 17 so as to realize the opening and closing of the sample inlet channel 16 or the sample outlet channel 17, compared with the air, the liquid has the incompressible performance, so the sample inlet channel 16 or the sample outlet channel 17 has faster response on deformation.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. For example, the single-cell sorting of the present invention may include only the upper chip and the glass sheet, omitting the lower chip, the buffer may be replaced with a phosphate buffer, the materials of the upper chip and the lower chip, i.e., the first material, may be replaced with plastic, and the second material may be replaced with other epoxy materials having similar properties. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (12)
1. A single cell sorting chip comprises an upper chip (1) and a glass sheet (3) which are sequentially attached from top to bottom; the chip is characterized in that the lower surface of the upper chip (1) is a structural surface of the upper chip (1), a main channel (11) is etched on the lower surface, the main channel (11) comprises a plurality of mutually communicated U-shaped parts (111), a capture channel (12) is etched between two straight sections of each U-shaped part (111), each capture channel (12) is connected with a release micro valve (13) etched on the structural surface of the upper chip (1) through a release channel, and the size of each capture channel (12) is set to enable the flow resistance of the capture channel (12) to be smaller than that of the main channel (11);
the intersection of the capture channel (12) with the main channel (11) is dimensioned to accommodate only a single cell.
2. The single-cell sorting chip according to claim 1, wherein the upper chip (1) is further provided with a first sample inlet (141), a second sample inlet (142), a first sample outlet (151) and a second sample outlet (152) which are connected through, and a sample channel (16) for connecting the two sample inlets (141, 142) and two sample channels (17) for connecting the two sample outlets (151, 152) are further etched on the structural surface of the upper chip (1), and the sample channel (16) and the sample channels (17) are respectively connected with two ends of the main channel (11).
3. The single-cell sorting chip according to claim 2, wherein a lower chip (2) is disposed between the upper chip (1) and the glass sheet (3), and the lower surface of the lower chip (2) is a structural surface of the lower chip (2) on which 4 extrusion channels (21) are etched, which correspond to and are perpendicular to the lower side of the sample inlet channel (16) or the sample outlet channel (17), respectively.
4. The single-cell sorting chip according to claim 3, wherein one end of each extrusion channel (21) is provided with a fluidic micro-valve (22) which penetrates the upper chip (1) and the lower chip (2) and is communicated with the extrusion channel (21).
5. The single-cell sorting chip of claim 3, wherein the upper chip (1) and the lower chip (2) are made of PDMS or plastic.
6. A method for manufacturing the single-cell sorting chip according to claim 4, comprising the steps of:
s1: manufacturing an upper chip (1) comprising:
s11: manufacturing a required mask according to the structure of the upper chip (1);
s12: carrying out photoetching and deep reactive ion etching by using the mask of S11 and a silicon wafer as a substrate, and removing residual glue after the etching is finished to obtain an upper silicon wafer mold with a microstructure;
s13: silanization is carried out on the manufactured upper layer silicon wafer mold, then the first material is poured on the upper layer silicon wafer mold and heated together to be solidified, the solidified first material is stripped, and holes are punched at the injection ports (141, 142), the sample outlets (151, 152) and the release micro valve (13), so as to obtain an upper layer middle mold;
s14: pouring a second material into the upper middle mould, filling the second material into the punching position of the upper middle mould, curing to form columns corresponding to the sample inlets (141, 142), the sample outlets (151, 152) and the release micro valves (13), and stripping the cured second material to obtain an upper final mould;
s15: pouring the first material into the upper layer final mould, leading the liquid level of the first material to be over the pillars on the upper layer final mould, then heating to be solidified, stripping the solidified first material, perforating at the sample inlets (141, 142) and the sample outlets (151, 152), and reserving the film at the position of the release micro valve (13) to obtain the upper layer chip (1);
s2: -manufacturing a lower chip (2) comprising:
s21: adopting a photoetching process on a silicon wafer to obtain a lower-layer silicon wafer die;
s22: spin-coating a first material on a lower silicon wafer mold and heating the first material and the lower silicon wafer mold together until the first material is solidified to obtain a lower silicon wafer mold and a lower chip (2) which are attached together;
s3: making a single-cell sorting chip comprising:
s31: bonding the structural surface of the upper chip (1) prepared in S1 and the lower chip (2) prepared in S2 together, and heating;
s32: stripping the upper chip (1) and the lower chip (2) from the silicon chip together, and punching a hole at the position of the flow control micro valve (22);
s33: and bonding the structural surface of the lower chip (2) and the glass sheet (3) together to obtain the single cell sorting chip.
7. The method of manufacturing the single-cell sorting chip of claim 6, wherein the first material is PDMS or plastic, and the first material is obtained by mixing a prepolymer and a curing agent, stirring them uniformly, and then evacuating until the bubbles disappear.
8. The method of manufacturing the single-cell sorting chip of claim 6, wherein the second material is epoxy glue.
9. The method for manufacturing a single-cell sorting chip according to claim 6, wherein the bonding of step S31 and step S33 are both achieved by plasma treatment.
10. A single-cell sorting method based on the single-cell sorting chip of claim 4, comprising the steps of:
a1: vacuumizing the single-cell sorting chip, and then introducing a buffer solution into the upper-layer chip (1) to ensure that the upper-layer chip (1) is completely filled with liquid;
a2: when the cells are captured, opening a first sample inlet (141) and a first sample outlet (151), closing a second sample inlet (142) and a second sample outlet (152), and injecting a cell suspension subjected to fluorescent staining into the first sample inlet (141);
a3: when cell recovery is carried out, the second sample inlet (142) and the second sample outlet (151) are opened, the first sample inlet (141) and the first sample outlet (151) are closed, buffer solution is injected into the second sample inlet (142), and one or more release micro valves (13) are manually pressed.
11. The single-cell sorting method according to claim 10, wherein the opening and closing of the first sample inlet (141), the first sample outlet (151), the second sample inlet (142), and the second sample outlet (152) are performed by respectively drawing and injecting a liquid into a fluidic micro-valve (22) corresponding thereto by using a syringe.
12. The single-cell sorting method of claim 10, wherein the buffer is 0.05% tween-20 or phosphate buffer.
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| CN110711608B (en) * | 2019-09-23 | 2022-03-15 | 中国科学院上海微系统与信息技术研究所 | Microfluidic chip for cell detection and preparation method thereof |
| CN111763612B (en) * | 2020-06-10 | 2022-10-18 | 宁波大学 | Single cell gene detection chip and manufacturing method and detection method thereof |
| CN112023991A (en) * | 2020-07-24 | 2020-12-04 | 中国科学院上海微系统与信息技术研究所 | Single cell dielectric detection chip |
| CN114426922B (en) * | 2022-01-24 | 2023-12-22 | 国科温州研究院(温州生物材料与工程研究所) | High-flux micro-fluidic chip for measuring cell mechanical parameters in wide pressure range and application |
| WO2023201942A1 (en) * | 2022-04-22 | 2023-10-26 | 上海傲睿科技有限公司 | Single-cell sorting microfluidic chip and single-cell sorting method |
| CN116121031B (en) * | 2022-12-23 | 2024-03-12 | 重庆大学 | Multistage microfluidic chip for single cell screening and preparation method thereof |
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