CN116121031A - Multistage microfluidic chip for single cell screening and preparation method thereof - Google Patents
Multistage microfluidic chip for single cell screening and preparation method thereof Download PDFInfo
- Publication number
- CN116121031A CN116121031A CN202211666131.4A CN202211666131A CN116121031A CN 116121031 A CN116121031 A CN 116121031A CN 202211666131 A CN202211666131 A CN 202211666131A CN 116121031 A CN116121031 A CN 116121031A
- Authority
- CN
- China
- Prior art keywords
- sample
- micro
- channel
- multistage
- single cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012216 screening Methods 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 claims description 25
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical group C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 18
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 13
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 13
- 229920002120 photoresistant polymer Polymers 0.000 claims description 13
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 13
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 13
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 12
- 239000010931 gold Substances 0.000 claims description 12
- 229910052737 gold Inorganic materials 0.000 claims description 12
- 238000009832 plasma treatment Methods 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 238000009849 vacuum degassing Methods 0.000 claims description 7
- 238000005530 etching Methods 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 238000002174 soft lithography Methods 0.000 claims description 6
- 238000004528 spin coating Methods 0.000 claims description 6
- 238000004544 sputter deposition Methods 0.000 claims description 6
- 238000011161 development Methods 0.000 claims description 5
- 238000001259 photo etching Methods 0.000 claims description 5
- 238000001755 magnetron sputter deposition Methods 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 abstract description 35
- 238000013461 design Methods 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 238000004720 dielectrophoresis Methods 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 89
- 238000000926 separation method Methods 0.000 description 31
- 239000000243 solution Substances 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 7
- 230000006872 improvement Effects 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 238000007599 discharging Methods 0.000 description 4
- 230000013011 mating Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000003320 cell separation method Methods 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- -1 electronics Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the field of microfluidic chips and discloses a multistage microfluidic chip for single-cell screening and a preparation method thereof. The invention designs the micro-trap array for capturing cells/particles, the baffle structure of the capturing area can greatly improve the utilization rate of the sample, the matched electrode array is used for releasing negative dielectrophoresis force, and the multi-outlet and multi-flow-path chip channel structure can realize the gradual enrichment, purification and target sample collection and recovery of the sample.
Description
Technical Field
The invention relates to the field of microfluidic chips, in particular to a multistage microfluidic chip for single cell screening and a preparation method thereof.
Background
Cell sorting is a process of separating a cell from a multicellular sample, and common cell sorting methods include antibody labeling separation, immunodensity centrifugation, immunomagnetic bead separation, and flow cell separation techniques. Among them, the flow cell separation technology is widely used in the field because of its characteristics of high flux, mature equipment, etc. However, the flow cell separation technique still has the following disadvantages when in use: which requires expensive instrumentation and complex operational procedures.
The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip, and can automatically complete the whole analysis process. The microfluidic chip has the characteristics of controllable liquid flow, extremely small consumption of samples and reagents, ten times or hundred times improvement of analysis speed and the like, has great potential in the fields of biology, chemistry, medicine and the like, and has been developed into a brand-new research field of intersection of subjects of biology, chemistry, medicine, fluid, electronics, materials, machinery and the like. At present, the micro-fluidic technology has been applied to cell separation, the cell separation method in the micro-fluidic field is concentrated on group cell separation, and a single cell level monitoring, controlling and analyzing platform is crucial to the development of the biomedical field, and comprises cell identification, classification, separation, culture, pairing, cell interaction and the like; single cell capture and release are the premise and important components of single cell level research, but no mature single cell capture and addressable release technology exists in the prior art. Conventional techniques for manipulating cells/particles using fluid and dielectrophoresis generally only capable of achieving a preliminary low-precision separation of cells from a population, but cannot accurately select a small number of target cells or particles from a large number of samples.
Disclosure of Invention
The invention aims to provide a multistage micro-fluidic chip for single-cell screening and a preparation method thereof, so as to solve the problem that the prior art cannot capture cells/particles and controllably release and collect a small amount of targets on a low-cost micro-fluidic chip.
In order to achieve the above purpose, the invention adopts the following technical scheme: a multistage micro-fluidic chip for single cell screening, including PDMS structural layer and the supporting electrode that bonds with it, be provided with the microchannel structure that is used for carrying out cell capture and sorting in the PDMS structural layer, the microchannel structure is including the multistage separation unit that communicates in proper order, and separation unit all includes the separation chamber, and the separation chamber includes dispersion district, capture district and play appearance district.
On the other hand, the technical scheme also provides a preparation method of the multistage micro-fluidic chip for single cell screening, which comprises the following steps:
step one, drawing a micro-channel structure;
step two, designing an electrode array;
preparing a micro-channel structure given to PDMS by utilizing a soft lithography process;
preparing a gold electrode by adopting a sputtering-photoetching method;
and fifthly, carrying out surface plasma treatment on the electrode array and the micro-channel structure, and bonding and packaging.
The principle and the advantages of the scheme are as follows: in practical application, in the technical scheme, the structure of the microfluidic chip is improved and upgraded aiming at the technical problems that cells/particles are difficult to capture and a small amount of target objects are controlled and released accurately in the prior art, and the aim of single-cell sorting is achieved. Firstly, through setting up multistage capture and separation unit, realize the capture of cell/particle through micro trap array, then realize the accurate screening of cell/particle that catches in the different separation units through the power up of supporting electrode, cell/particle can get into next stage separation unit and carry out again and catch and select separately after last stage separation unit selects separately, through multistage, multichannel structural design, can realize the process of enrichment step by step, purification and target sample screening collection of sample. Experiments prove that the chip adopting the scheme can realize visual and addressable single-cell/particle accurate release, and can greatly improve the capture rate (namely the sample utilization rate), and the capture rate of 15-micrometer particles can reach more than 90%. The multi-stage sorting can sort and screen target cells/particles from a large number of samples on the same microfluidic chip, and has the advantages of low cost, convenient operation, high sample capturing rate and high utilization rate.
Preferably, as an improvement, a plurality of split microcolumns are arranged in the dispersing area, and the split microcolumns are arranged in a staggered manner.
In the technical scheme, the dispersing area is used for dispersing samples, and through the diversion microcolumns which are arranged in a staggered manner, the dispersing intervention can be carried out on the flow direction of the samples after entering, so that the samples are uniformly dispersed into each sample channel, and the capturing and sorting efficiency is improved.
Preferably, as an improvement, a plurality of sample channels are arranged in the capturing area, a plurality of capturing chambers are arranged in the sample channels in an array mode, and a baffle is arranged at one end, close to the sample outlet area, of each sample channel.
In the technical scheme, the sample channel is a flow channel for capturing/sorting samples, and high-flux sample capturing and purifying can be realized through the capturing chambers arranged in the array and the strip. In the early stage of research and development, the problem of low capture rate of capturing single particles/cells by utilizing fluid power and a micro-column structure still exists, and the flow resistance of a channel can be changed by arranging a baffle in the sample channel, so that the particles/cells can be captured efficiently.
Preferably, as an improvement, the sample outlet area is communicated with the sample channel, the sample outlet area is communicated with the sample outlet channel, and the sample outlet channels are internally and fixedly provided with obliquely arranged interception plates.
In the technical scheme, the interception plate plays a role in interception and slows down sample discharge.
Preferably, as an improvement, the mating electrode has a metal electrode array structure, and the mating electrode includes multiple electrode array units corresponding to the multiple sorting units respectively.
In the technical scheme, the matched electrodes are arranged to be of the array electrode structures respectively corresponding to the multi-stage sorting units, so that the precise power-on control sorting of different sorting areas (sorting areas of different levels) can be realized, and the visual precise release of single cells/particles is further realized.
Preferably, as an improvement, in the third step, the specific fabrication of the micro-channel structure by using the soft lithography process is as follows: spin-coating 20-200 μm photoresist on a silicon wafer, soft-baking with a hot plate, exposing, then developing in a developing solution, hard-baking with a hot plate to manufacture a channel template, and demolding with PDMS to obtain the micro-channel structure layer.
In the technical scheme, the micro-channel structure is constructed by adopting a soft lithography technology, the technology is mature, and the prepared micro-channel structure can meet the cell sorting and enriching requirements of the technical scheme.
Preferably, as an improvement, in the fourth step, the specific method for preparing the gold electrode is as follows: sputtering a metal layer of 5-10nm on quartz glass by using a magnetron sputtering instrument, sputtering gold of 100-300 nm thick, spin-coating a photoresist layer, performing soft baking by using a hot plate, performing exposure treatment, then placing into a developing solution for development, placing into an etching solution for etching, and finally removing the photoresist by using a photoresist removing solution to obtain the gold electrode.
In the technical scheme, the gold electrode is prepared by adopting a magnetron sputtering mode, and the operation is simple and convenient.
Preferably, as a modification, in the fifth step, the plasma treatment time is 10-15s, and the bonding temperature is 100-150 ℃.
In the technical scheme, the plasma treatment time mainly influences the bonding effect in the later period, and too long plasma treatment time can lead to too tight bonding, so that sample liquid is difficult to enter the structure; too short plasma treatment time can lead to loose bonding and easy leakage.
Preferably, as a modification, the bonded chip is subjected to vacuum degassing treatment, and the time of the vacuum degassing treatment is more than 30 minutes.
In the technical scheme, each inlet and outlet of the chip is immersed in the buffer solution, and is degassed for more than 30min in a vacuum box of-1.5 to-1.2 psi, so that the channel is completely filled with liquid, and the influence of bubbles on the flow of microfluid is avoided
Drawings
Fig. 1 is a schematic structural diagram of a PDMS structural layer according to an embodiment of the present invention.
Fig. 2 is a partial view of the primary sorting unit of fig. 1.
Fig. 3 is a schematic structural diagram of a mating electrode according to an embodiment of the present invention.
Fig. 4 is a schematic structural diagram of a bonding state of a multistage microfluidic chip according to an embodiment of the present invention.
Detailed Description
The following is a detailed description of embodiments, but embodiments of the invention are not limited thereto. The technical means used in the following embodiments are conventional means well known to those skilled in the art unless otherwise specified; the experimental methods used are all conventional methods; the materials, reagents, and the like used are all commercially available.
Reference numerals in the drawings of the specification include: the device comprises a first-stage separation unit 1, a second-stage separation unit 2, a third-stage separation unit 3, a sample inlet 4, a split micro-column 5, a first-stage sample channel 6, a first-stage capturing chamber 7, a conveying channel 8, a first-stage sample outlet 9, a second-stage sample outlet 10, a third-stage sample outlet 11, a baffle 12, a interception plate 13 and a matched electrode 14.
Example 1
An example is substantially as shown in figures 1 to 4 of the accompanying drawings: a multistage microfluidic chip for single cell screening comprising a PDMS structural layer and a mating electrode 14 bonded thereto (fig. 3).
Referring to fig. 1 and 2, a micro-channel structure is arranged in the PDMS structure layer and used for capturing and sorting cells, and comprises a primary sorting unit 1, a secondary sorting unit 2 and a tertiary sorting unit 3 which are sequentially communicated; the primary sorting unit 1 comprises a primary sorting chamber which comprises a dispersing area, a capturing area and a sample outlet area. The dispersing area is provided with a sample inlet 4, a plurality of split micro-columns 5 are fixed in the dispersing area, and the split micro-columns 5 are staggered; seven groups of transversely arranged primary sample channels 6 are arranged in the capturing area, a plurality of primary capturing chambers 7 arranged in an array are arranged in the primary sample channels 6, the primary capturing chambers 7 are surrounded by PDMS insulating microcolumns and channel walls to form a micro-trap structure array, and a return baffle 12 is arranged at one end of the primary sample channels 6 close to the sample outlet area; the sample outlet area is communicated with the primary channel, the sample outlet area is communicated with the primary sample outlet channel and the primary conveying channel 8, the primary sample outlet channel is provided with the primary sample outlet 9, the primary sample outlet channel is fixedly provided with the inclined retaining plate 13, the interception of sample solution is mainly realized, and the sample utilization rate is improved. One end of the primary transfer channel 8, which is far away from the sample outlet area, is communicated with the secondary separation unit 2.
The secondary separation unit 2 is basically similar to the primary separation unit 1 in structure and comprises a secondary separation cavity, wherein the primary separation cavity comprises a dispersion area, a capturing area and a sample outlet area, the dispersion area is communicated with a primary conveying channel 8, and a plurality of diversion microcolumns 5 are also fixed in the dispersion area; five groups of transversely arranged secondary sample channels are arranged in the capture area, a plurality of secondary capture chambers arranged in an array are arranged in the secondary sample channels, the secondary capture chambers are surrounded by PDMS insulating microcolumns and channel walls to form a micro-trap structure array, and a return baffle 12 is arranged at one end, close to the sample outlet area, of each secondary sample channel; the sample outlet area is communicated with a secondary sample channel, the sample outlet area is communicated with a secondary sample outlet channel and a secondary conveying channel 8, a secondary sample outlet 10 is arranged on the secondary sample outlet channel, and a blocking and retaining plate 13 which is obliquely arranged is fixed on the secondary sample outlet channel; the secondary transfer passage 8 communicates with the tertiary sorting unit 3.
The tertiary separation unit 3 includes tertiary separation chamber, and tertiary separation intracavity is provided with tertiary sample channel, is provided with the tertiary cavity of catching that a plurality of horizontal arrays arranged in the tertiary sample channel, and tertiary cavity of catching is enclosed by PDMS insulating microcolumn and passageway wall, and tertiary sample channel is close to the one end of going out the sample district and also is provided with the baffle 12 of returning shape. One end of the tertiary separation cavity, which is far away from the secondary conveying channel 8, is provided with an inclined blocking plate 13, and one end of the tertiary separation cavity, which is far away from the secondary conveying channel 8, is communicated with a tertiary sample outlet channel and a collecting channel, and a tertiary sample outlet 11 is arranged on the tertiary sample outlet channel.
The matched electrode 14 is of a metal electrode array structure and comprises a primary electrode array unit, a secondary electrode array unit and a tertiary electrode array unit, which are respectively used for controlling the primary sorting unit 1, the secondary sorting unit 2 and the tertiary sorting unit 3, so that the step-by-step enrichment, purification and target sample collection and recovery of samples are realized.
The preparation method of the multistage microfluidic chip for single cell screening comprises the following steps:
drawing a micro-channel structure of a PDMS structure layer by using software, introducing simulation software for calculation and analysis, and optimizing the structure;
step two, designing an electrode array corresponding to the structure, performing electric field calculation and optimizing electrode arrangement;
step three, manufacturing a corresponding mask/chromium plate, and manufacturing a micro-channel structure based on PDMS by using a soft lithography method, wherein the specific preparation method comprises the following steps: spin-coating a layer of photoresist with the thickness of 20-200 mu m on a silicon wafer with the thickness of 3-5 inches by using a spin coater, performing soft baking by using a hot plate, performing exposure by using a photoetching machine, then placing into a developing solution for development, performing hard baking by using the hot plate, manufacturing a channel template, and performing demoulding by using PDMS to obtain a micro-channel structure layer;
preparing an array electrode, namely preparing a gold electrode by adopting a sputtering-photoetching method, wherein the specific preparation method comprises the following steps of: sputtering a layer of titanium metal with the thickness of 5-10nm on quartz glass with the thickness of 3-5 inches by using a magnetron sputtering instrument, sputtering a layer of gold with the thickness of 100-300 nm, spin-coating a layer of photoresist with the thickness of 5-10 mu m by using a photoresist homogenizing machine, performing soft baking by using a hot plate, exposing by using the photoetching machine, then placing into developing solution for development, placing into etching solution for etching, and finally removing photoresist by using photoresist removing solution to obtain a gold electrode with a required shape;
step five, carrying out surface plasma treatment and bonding encapsulation on the array electrode and the micro-channel structure layer to obtain a multistage micro-fluidic chip for single cell screening; the plasma treatment time is 10-15s, and the bonding temperature is 100-150 ℃;
and step six, carrying out vacuum degassing treatment on the multistage microfluidic chip for single cell screening, wherein the vacuum degassing treatment time is more than 30min.
When the multistage microfluidic chip for single cell screening is used for cell sorting, PBS/PM buffer solution is firstly configured, and buffer solution infiltration and surface treatment are carried out on a channel structure inside the chip by using a negative pressure pump.
And then opening a sample inlet, and connecting the chip with the signal generator and the negative pressure pump, wherein an outlet of the chip is connected with the negative pressure pump, and an electrode part of the chip is connected with the signal generator. And adding a sample at the sample inlet, dispersing the sample into each primary sample channel under the dispersion action of the split microcolumn, capturing the sample in the primary capturing cavity, discharging the residual sample along the primary sample discharging channel, and collecting the residual sample in a container at the primary sample outlet.
And then closing the primary sample outlet, opening the secondary sample outlet, selecting a target sample in the primary sorting area, electrifying a primary electrode array corresponding to the primary sorting area, releasing target sample cells/particle groups into the secondary sorting area, and discharging and collecting the residual sample along the secondary sample outlet. And then closing the secondary sample outlet, opening the tertiary sample outlet, selecting the sample in the secondary sorting area, powering on the secondary electrode array, releasing the target sample cell/particle group into the tertiary sorting area, and collecting the rest sample in a container of the tertiary sample outlet. And then closing the three-stage sample outlet, opening the collection port of the collection channel, selecting a target sample in the three-stage sorting area, powering up the three-stage electrode array, and releasing the target sample cell/particle group into the final sample collection port.
The multistage sorting microfluidic chip can realize the gradual enrichment and accurate screening of a large number of samples in the same chip, can realize the visualization, addressable single cell/particle capture and release, and can be used for cell sorting, cell counting and subsequent single cell analysis.
Comparative example 1
The difference between this comparative example and example 1 is that: in this comparative example, no baffle structure was provided, resulting in a very low capture rate, and the analysis was due to: most of the incoming sample follows the fluid directly to the outlet rather than being immobilized within the trap structure.
Experiment one dip test
The influence of different shape baffles to the capture rate is mainly investigated in this experiment, and the inclination in the inclination test refers to the baffle structure of every capture channel exit, has investigated outer inclined baffle, vertical baffle, interior inclined baffle and the influence of returning shape baffle structure to the capture rate respectively, and the concrete operation of experiment is:
step one, manufacturing a chip, wherein the specific method is shown in the embodiment 1;
step two, preparing a buffer solution and a solution containing particles/cells;
immersing the chip into a buffer solution for vacuum degassing, and discharging bubbles;
step four, connecting the chip with a pump and a signal generator;
step five, adding a solution containing particles/cells from an inlet and observing under a microscope, wherein the particles/cells enter a channel along with the fluid and are captured in each row of micro traps, counting the number of single particles, the number of multiple particles and empty traps captured in each row through the microscope observation, and recording and calculating the trap occupation number (namely, the trapped particles are in the trap, whether the single particles or the multiple particles exist), wherein the occupation rate=the occupation number/the total trap number. The results are shown in Table 1, wherein single particles refer to: only one particle is in one trap; multiparticulates refer to two or more particles in a trap.
TABLE 1
The results show that: counting and capturing immediately after sample injection, wherein the outlet of the 5 th row structure is blocked (the channel is deformed, <15 um); preliminary statistics, the 4 th row has higher hole occupancy rate and single particle rate, and the subsequent chip design can adopt the structure, namely the square baffle plate, the width is 25 microns, and the bottom edge extends to 50 microns.
Experiment two
Escape and capture experiments were performed using the chip prepared in example 1, capturing conditions at 5, 10, and 15 seconds were observed under a microscope, and the time point when escape was first observed was recorded, and then occupancy and single-particle and multi-particle capturing rates at each time point were counted by video playback, and the results are shown in table 2.
TABLE 2
From the data in table 2, the capture rate reaches 100% in the first 20s, 2 particles escape after 22s without particle escape under visual observation, and a larger amount of particles escape after 25s with the chip capture capacity approaching the optimal capture limit, which indicates that the chip capture capacity reaches the limit and continues to keep sample injection, and the site occupancy rate can reach 100%, but the loss is more. The structure has ideal capture rate and is not easy to cause blockage.
The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
1. A multistage micro-fluidic chip for single cell screening, its characterized in that: the cell sorting device comprises a PDMS structure layer and a matched electrode bonded with the PDMS structure layer, wherein a micro-channel structure used for capturing and sorting cells is arranged in the PDMS structure layer, the micro-channel structure comprises a multi-stage sorting unit which is sequentially communicated, each sorting unit comprises a sorting cavity, and each sorting cavity comprises a dispersing area, a capturing area and a sample outlet area.
2. The multistage microfluidic chip for single cell screening according to claim 1, wherein: a plurality of split micro-columns are arranged in the dispersing area, and the split micro-columns are arranged in a staggered mode.
3. The multistage microfluidic chip for single cell screening and the preparation method thereof according to claim 2, wherein: a plurality of sample channels are arranged in the capturing area, a plurality of capturing chambers are arranged in the sample channels in an array mode, and a baffle is arranged at one end, close to the sample outlet area, of each sample channel.
4. A multistage microfluidic chip for single cell screening according to claim 3, wherein: the sample outlet area is communicated with the sample channel, the sample outlet area is communicated with the sample outlet channel, and the sample outlet channel is internally and fixedly provided with a interception plate which is obliquely arranged.
5. The multistage microfluidic chip for single cell screening according to claim 4, wherein: the matched electrode is of a metal electrode array structure, and comprises multistage electrode array units respectively corresponding to the multistage sorting units.
6. The method for preparing a multistage microfluidic chip for single cell screening according to any one of claims 1 to 5, comprising the steps of:
step one, drawing a micro-channel structure;
step two, designing an electrode array;
preparing a micro-channel structure given to PDMS by utilizing a soft lithography process;
preparing a gold electrode by adopting a sputtering-photoetching method;
and fifthly, carrying out surface plasma treatment on the electrode array and the micro-channel structure, and bonding and packaging.
7. The method for preparing the multistage microfluidic chip for single cell screening according to claim 6, wherein the method comprises the following steps: in the third step, the specific fabrication of the micro-channel structure prepared by using the soft lithography process is as follows: spin-coating 20-200 μm photoresist on a silicon wafer, soft-baking with a hot plate, exposing, then developing in a developing solution, hard-baking with a hot plate to manufacture a channel template, and demolding with PDMS to obtain the micro-channel structure layer.
8. The method for preparing the multistage microfluidic chip for single cell screening according to claim 7, wherein: in the fourth step, the specific method for preparing the gold electrode comprises the following steps: sputtering a metal layer of 5-10nm on quartz glass by using a magnetron sputtering instrument, sputtering gold of 100-300 nm thick, spin-coating a photoresist layer, performing soft baking by using a hot plate, performing exposure treatment, then placing into a developing solution for development, placing into an etching solution for etching, and finally removing the photoresist by using a photoresist removing solution to obtain the gold electrode.
9. The method for preparing the multistage microfluidic chip for single cell screening according to claim 8, wherein the method comprises the following steps: in the fifth step, the plasma treatment time is 10-15s, and the bonding temperature is 100-150 ℃.
10. The method for preparing the multistage microfluidic chip for single cell screening according to claim 9, wherein the method comprises the following steps: and carrying out vacuum degassing treatment on the bonded chips, wherein the time of the vacuum degassing treatment is more than 30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211666131.4A CN116121031B (en) | 2022-12-23 | 2022-12-23 | Multistage microfluidic chip for single cell screening and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211666131.4A CN116121031B (en) | 2022-12-23 | 2022-12-23 | Multistage microfluidic chip for single cell screening and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116121031A true CN116121031A (en) | 2023-05-16 |
| CN116121031B CN116121031B (en) | 2024-03-12 |
Family
ID=86307248
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211666131.4A Active CN116121031B (en) | 2022-12-23 | 2022-12-23 | Multistage microfluidic chip for single cell screening and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116121031B (en) |
Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040191789A1 (en) * | 2001-05-02 | 2004-09-30 | Nicolo Manaresi | Method and apparatus for high-throughput biological-activity screening of cells and/or compounds |
| US20130296196A1 (en) * | 2012-02-29 | 2013-11-07 | Fluidigm Corporation | Methods, systems and devices for multiple single-cell capturing and processing using microfluidics |
| CN103392124A (en) * | 2010-12-03 | 2013-11-13 | 麦德西斯科学实验室 | Microanalysis of cellular function |
| CN108728328A (en) * | 2018-05-30 | 2018-11-02 | 中北大学 | The micro-current controlled cell for integrating unicellular capture sorts chip |
| CN109251841A (en) * | 2018-08-21 | 2019-01-22 | 中国科学院上海微系统与信息技术研究所 | The unicellular sorting chip of one kind and its manufacturing method and unicellular method for separating |
| CN109456874A (en) * | 2018-10-16 | 2019-03-12 | 上海交通大学 | A kind of unicellular manipulation micro-fluidic chip of the two-way dielectrophoresis of cell |
| CN109722385A (en) * | 2019-01-29 | 2019-05-07 | 厦门大学 | A kind of micro-fluidic chip and its application of accurate manipulation and the single particle of pairing |
| CN110004043A (en) * | 2019-04-09 | 2019-07-12 | 东北大学 | A kind of unicellular capture micro-fluidic chip |
| CN110302849A (en) * | 2019-05-31 | 2019-10-08 | 昆明理工大学 | A kind of micro fluidic device and method based on dielectrophoresis principle sorting drop |
| CN110305788A (en) * | 2019-05-31 | 2019-10-08 | 西安医学院 | The array chip and its preparation and operating method of cell capture and the culture of tumour ball |
| CN110496655A (en) * | 2019-07-26 | 2019-11-26 | 北京工业大学 | A kind of tumour cell detection chip based on microflow control technique |
| CN111295245A (en) * | 2017-08-29 | 2020-06-16 | 赛尔希诊断公司 | Systems and methods for separating and analyzing cells |
| US20200238286A1 (en) * | 2017-11-01 | 2020-07-30 | Zigzag Biotechnology Co., Ltd. | Chip for separating and capturing cell and application of chip in tumor cell sorting thereof |
| CN113322167A (en) * | 2021-04-26 | 2021-08-31 | 上海交通大学 | Micro-fluidic chip |
| CN114149915A (en) * | 2021-12-28 | 2022-03-08 | 西安交通大学 | Exosome sorting and detecting chip, system and method based on surface acoustic waves |
| CN216073750U (en) * | 2021-07-13 | 2022-03-18 | 广东省科学院健康医学研究所 | Chip and device for separating, capturing and recovering rare cells in whole blood |
| CN114574357A (en) * | 2022-02-14 | 2022-06-03 | 大连交通大学 | Microalgae and bacteria sorting device based on combination of deterministic lateral displacement and dielectrophoresis technologies |
| CN114940937A (en) * | 2021-08-17 | 2022-08-26 | 南京微欣利康科技有限公司 | Microfluidic chip and application thereof |
-
2022
- 2022-12-23 CN CN202211666131.4A patent/CN116121031B/en active Active
Patent Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040191789A1 (en) * | 2001-05-02 | 2004-09-30 | Nicolo Manaresi | Method and apparatus for high-throughput biological-activity screening of cells and/or compounds |
| CN103392124A (en) * | 2010-12-03 | 2013-11-13 | 麦德西斯科学实验室 | Microanalysis of cellular function |
| US20130296196A1 (en) * | 2012-02-29 | 2013-11-07 | Fluidigm Corporation | Methods, systems and devices for multiple single-cell capturing and processing using microfluidics |
| CN111295245A (en) * | 2017-08-29 | 2020-06-16 | 赛尔希诊断公司 | Systems and methods for separating and analyzing cells |
| US20200238286A1 (en) * | 2017-11-01 | 2020-07-30 | Zigzag Biotechnology Co., Ltd. | Chip for separating and capturing cell and application of chip in tumor cell sorting thereof |
| CN108728328A (en) * | 2018-05-30 | 2018-11-02 | 中北大学 | The micro-current controlled cell for integrating unicellular capture sorts chip |
| CN109251841A (en) * | 2018-08-21 | 2019-01-22 | 中国科学院上海微系统与信息技术研究所 | The unicellular sorting chip of one kind and its manufacturing method and unicellular method for separating |
| CN109456874A (en) * | 2018-10-16 | 2019-03-12 | 上海交通大学 | A kind of unicellular manipulation micro-fluidic chip of the two-way dielectrophoresis of cell |
| CN109722385A (en) * | 2019-01-29 | 2019-05-07 | 厦门大学 | A kind of micro-fluidic chip and its application of accurate manipulation and the single particle of pairing |
| CN110004043A (en) * | 2019-04-09 | 2019-07-12 | 东北大学 | A kind of unicellular capture micro-fluidic chip |
| CN110302849A (en) * | 2019-05-31 | 2019-10-08 | 昆明理工大学 | A kind of micro fluidic device and method based on dielectrophoresis principle sorting drop |
| CN110305788A (en) * | 2019-05-31 | 2019-10-08 | 西安医学院 | The array chip and its preparation and operating method of cell capture and the culture of tumour ball |
| CN110496655A (en) * | 2019-07-26 | 2019-11-26 | 北京工业大学 | A kind of tumour cell detection chip based on microflow control technique |
| CN113322167A (en) * | 2021-04-26 | 2021-08-31 | 上海交通大学 | Micro-fluidic chip |
| CN216073750U (en) * | 2021-07-13 | 2022-03-18 | 广东省科学院健康医学研究所 | Chip and device for separating, capturing and recovering rare cells in whole blood |
| CN114940937A (en) * | 2021-08-17 | 2022-08-26 | 南京微欣利康科技有限公司 | Microfluidic chip and application thereof |
| CN114149915A (en) * | 2021-12-28 | 2022-03-08 | 西安交通大学 | Exosome sorting and detecting chip, system and method based on surface acoustic waves |
| CN114574357A (en) * | 2022-02-14 | 2022-06-03 | 大连交通大学 | Microalgae and bacteria sorting device based on combination of deterministic lateral displacement and dielectrophoresis technologies |
Non-Patent Citations (1)
| Title |
|---|
| 赵坚勇: "《等离子体显示（PDP）技术》", 河南科学技术出版社, pages: 124 - 125 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116121031B (en) | 2024-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109852530B (en) | Micro-fluidic chip integrating capture, lysis and nucleic acid detection of circulating tumor cells, device and method thereof | |
| US10351894B2 (en) | Method and device for isolating cells from heterogeneous solution using microfluidic trapping vortices | |
| CN106434302B (en) | A kind of micro-current controlled cell separating chips in Portable no-power source | |
| CN106824313A (en) | A kind of digital pcr chip and preparation method thereof | |
| CN109913352B (en) | Microfluidic device and method for controlling and capturing microparticles and cells based on non-contact dielectrophoresis force | |
| CN210916029U (en) | Simple micro-fluidic chip for separating and detecting circulating tumor cells | |
| EP3527985B1 (en) | Method for capturing specific cells | |
| Chai et al. | A microfluidic device enabling deterministic single cell trapping and release | |
| CN108117968A (en) | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip | |
| CN109722385A (en) | A kind of micro-fluidic chip and its application of accurate manipulation and the single particle of pairing | |
| CN116121031B (en) | Multistage microfluidic chip for single cell screening and preparation method thereof | |
| WO2023236760A1 (en) | Printing apparatus matched with microfluidic chip and printing method | |
| CN206512222U (en) | A kind of single celled micro-fluidic chip of high flux automatic capture | |
| KR101416634B1 (en) | Microfluidic Chip, Fabricating Method Thereof And Microfluidic Separation System | |
| CN113101989A (en) | Cell capturing and stretching integrated arrayed microfluidic chip | |
| LU100914B1 (en) | Microfluidic systems for yeast aging analysis | |
| CN110551617A (en) | Chip for separating body fluid bacteria and cells, manufacturing method and using method thereof | |
| TWI805068B (en) | High-efficiency single-cell collection method | |
| CN108117969A (en) | Single celled micro-fluidic chip of a kind of high throughput automatic capture and preparation method thereof | |
| CN114632564B (en) | Integrated micro-fluidic chip and primary circulating tumor cell in-vitro treatment method | |
| JP2010051291A (en) | Apparatus for capturing cell and method for capturing cell | |
| CN218573685U (en) | Micro-fluidic chip | |
| CN116179351A (en) | Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method | |
| Laki et al. | Microvesicle fractionation using deterministic lateral displacement effect | |
| WO2023178222A1 (en) | Devices, methods, and systems for enriching cellular samples by physical properties |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |