CN115369012A - Single cell phenotype determination micro-fluidic chip, preparation method, determination method and application thereof - Google Patents

Single cell phenotype determination micro-fluidic chip, preparation method, determination method and application thereof Download PDF

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CN115369012A
CN115369012A CN202211199868.XA CN202211199868A CN115369012A CN 115369012 A CN115369012 A CN 115369012A CN 202211199868 A CN202211199868 A CN 202211199868A CN 115369012 A CN115369012 A CN 115369012A
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cell
upper chamber
microfluidic chip
micro
channel
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刘小诗
唐源
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Suzhou Hopson Medical Technology Co ltd
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Suzhou Hopson Medical Technology Co ltd
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    • 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
    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention relates to a single cell phenotype determination microfluidic chip and a preparation method, a determination method and application thereof, and the single cell phenotype determination microfluidic chip comprises a first upper chamber, a second upper chamber, a first lower chamber and a second lower chamber, wherein a single cell filter is arranged in a connecting branch channel of the first upper chamber connected with a main connecting channel, or a single cell filter is arranged in a connecting branch channel of the second upper chamber connected with the main connecting channel, a parallel micro-channel array is arranged in the main connecting channel, the parallel micro-channel array comprises a plurality of micro-channels which are uniformly arranged in parallel from left to right, and an L-shaped structure trap structure is arranged in each micro-channel. The capture trap structure of the microfluidic chip is an L-shaped structure with an etched structure, and forms a single-cell capture trap structure with unidirectional flow capture capacity together with the microchannel wall, so that single-wall backflow of liquid can be reduced, and single-cell capture efficiency is improved.

Description

Single cell phenotype determination micro-fluidic chip, preparation method, determination method and application thereof
Technical Field
The invention relates to the technical field of microstructure processing and cell biomedicine intersection, in particular to a single-cell phenotype determination micro-fluidic chip, and a preparation method, a determination method and application thereof, and specifically relates to a lung cancer cell phenotype determination micro-fluidic chip, and a preparation method, a determination method and an application thereof.
Background
In recent years, with the rapid development of new technologies in the biomedical field, various single-cell omics technologies including single-cell genomics, single-cell transcriptomics, single-cell proteomics, single-cell metabonomics and the like are applied to the biomedical research aspect to play an increasingly important role, and the single-cell metabonomics are applied to a plurality of advanced fields such as tumors, immunity, cell physiology and the like, so that the complex cell map of an organism can be analyzed at the single-cell level, the types and the compositions of the cells of the organism can be explored, and the disease occurrence and development process or the pharmaceutical effect can be explored. However, the technology for single cell phenotyping is still slow to progress.
Different types of tissue cells have different physiological functions and thus exhibit different phenotypic characteristics in vitro and in vivo. Traditional phenotypic assays such as cell migration, chemotaxis, adhesion, proliferation, killing, etc. are often based on cell populations, and single cell based phenotypic assay techniques currently lack effective tools.
Tumors are important diseases threatening human life, metastasis and recurrence of tumors are serious problems in tumor therapy, and most of tumor-related deaths are caused by metastasis and recurrence. The tumor metastasis and recurrence is closely related to the migration ability, invasion ability and chemotaxis ability of tumor cells.
At present, the conventional determination of the in vitro migration ability and chemotactic ability of tumor cells adopts a scratch test and a Transwell migration test, however, the methods are all used for determining the collective migration ability of the cells, and the determination of the migration ability, chemotaxis ability and invasion ability can not be carried out at a single cell level.
The single cell capture is a key link of a single cell determination technology, and due to the inherent advantages of the microfluidic technology, the application of the microfluidic technology in the separation and immobilization of single cells is more and more extensive, but the microfluidic technology for determining the migration and chemotaxis capability of single cells is still immature at present.
In view of the above-mentioned drawbacks, the present designer has made active research and innovation to create a single cell phenotype assay microfluidic chip, and a preparation method, an assay method and an application thereof, so that the single cell phenotype assay microfluidic chip has industrial utility value.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a single-cell phenotype determination microfluidic chip, and a preparation method, a determination method and an application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
one of the objects of the present invention is:
the single cell phenotype determination microfluidic chip comprises a first upper chamber, a second upper chamber, a first lower chamber and a second lower chamber, wherein the first upper chamber and the second upper chamber are sequentially arranged from left to right; the single cell filter is arranged in a connecting branch channel connected with the connecting main channel of the first upper chamber, or the single cell filter is arranged in a connecting branch channel connected with the connecting main channel of the second upper chamber, a parallel micro-channel array is arranged in the connecting main channel, the parallel micro-channel array comprises a plurality of micro-channels which are sequentially and evenly arranged in parallel from left to right, and a capture trap structure with an L-shaped structure is arranged in the micro-channels.
As a further improvement of the invention, the trap structure comprises a parallel trap section parallel to the microchannel wall and a vertical trap section vertical to the microchannel wall, and an incised structure is arranged between the parallel trap section and the vertical trap section.
As a further improvement of the invention, the length L1 of the parallel section of the trap is 15-80 μm, the length L2 of the vertical section of the trap is 8-20 μm, the length L3 of the incised structure is 2-10 μm, and the distance L4 between the vertical section of the trap and the wall of the microchannel is 2-10 μm.
As a further improvement of the invention, the area of the first upper chamber, the second upper chamber, the first lower chamber and the second lower chamber is 9-100mm 2 The pore diameter of the micro-channel is 20-90 μm, and the single cell filter is composed of columnar arrays with the diameter of 3-30 μm and the distance of 10-100 μm.
The second object of the present invention is:
the preparation method of the single cell phenotype determination microfluidic chip sequentially comprises the following steps:
step 1, sequentially coating a binder and photoresist on the clean surface of a silicon wafer, placing a mask plate containing a trap trapping structure/parallel microchannel array on a photoresist layer, exposing in photoetching equipment, removing the mask plate, and washing uncured photoresist in the photoresist layer by using an anode photoresist developing solution to obtain a photoetched material;
step 2, depositing silicon dioxide on the surface of the material after photoetching, and then removing the residual photoresist on the surface of the silicon wafer to obtain a silicon dioxide mask non-photoresist silicon wafer;
step 3, placing the silicon wafer without the silicon dioxide mask in an inductively coupled plasma etching machine for dry etching to obtain a silicon wafer template with a capture trap structure/parallel micro-channel array;
and 4, placing the silicon wafer template prepared by pouring the uniformly stirred bubble-free PDMS into an oven for curing, completely separating the PDMS layer from the silicon wafer template, and bonding the upper bottom plate and the lower bottom plate after two demolding operations to form the final microfluidic chip.
As a further improvement of the present invention, wherein,
in the step 1, the silicon wafer is a polished silicon wafer, and the thickness of the silicon wafer is 100-1000 microns; the binder is hexamethyldisilazane, and the coating mode of the binder is spin coating, spray coating or vacuum volatilization; the photoresist is positive photoresist, and the coating mode of the photoresist is spin coating or spray coating;
in the step 2, depositing a silicon dioxide layer by adopting an evaporation or magnetron sputtering method, wherein the thickness of the silicon dioxide layer is 10-300 nanometers; the method for removing the residual photoresist on the surface of the silicon wafer is acetone soaking;
in the step 3, the dry etching gas comprises one or a combination of a plurality of gases of carbon tetrafluoride gas, sulfur hexafluoride gas, octafluorocyclobutane gas, oxygen gas, argon gas and helium gas, the working pressure in an etching cavity in an etching machine is less than 50mTorr, and the etching time is 1-120 minutes;
in the step 4, the proportion of PDMS to the curing agent is 10:1 or 8:2 or 7:3, the temperature of the oven is 50-90 ℃, and the curing time is 30-240 min.
The third object of the present invention is:
the method for measuring the single cell phenotype micro-fluidic chip sequentially comprises a step A, a single cell capturing step, a step B and a measuring step, wherein,
step A, a single cell capturing step, which sequentially comprises:
a1, preparing a cell suspension by enzymatic digestion of cells, and dripping the cell suspension into a first upper chamber 1;
step A2, opening a negative pressure aspirator, forming gas negative pressure in the first lower chamber 3 or the second lower chamber 4, and driving the negative pressure to suck the cell suspension into the microchannel 8 and capture the cell suspension by the capture trap structure 9;
step A3, adding a physiological/culture solution into the first upper chamber 1, cleaning redundant cells by using negative pressure, and sucking away waste liquid;
step A4, adding a cell culture medium into the first upper chamber 1, and then placing the mixture into a cell culture box for culture;
step B, a determination step, which sequentially comprises:
step B1, after single cell capture, adding a culture medium containing a drug to be detected into the second upper chamber 2, and culturing for 12-120 hours in a cell culture box;
b2, after culturing, carrying out microscopic imaging on each micro-channel, measuring the distance of each single cell from the trap structure, and taking an average value;
wherein, mobility = sample single cell migration distance/control single cell migration distance 100%;
chemotactic intensity = migration of single cells of sample/migration of single cells of control 100%.
The fourth purpose of the invention is that:
the application of the micro-fluidic chip for single cell phenotype determination is that the micro-fluidic chip is applied to the determination of the migration capability of single cells, the single cells are normal tissue cells or tumor cells or microorganism cells, and the microorganisms are microorganisms with the mobility capability.
The fifth purpose of the invention is:
the application of the single cell phenotype determination microfluidic chip is to determine the single cell migration capacity of the microfluidic chip under a 3D culture condition, wherein the 3D culture comprises pouring a 3D culture medium into the chip.
The sixth purpose of the invention is:
the application of the single cell phenotype determination microfluidic chip, the application of the microfluidic chip in screening and evaluating the anti-tumor migration/metastasis drugs, and the tumor cells are solid tumor cells and mononuclear/macrophage tumor cells.
By the scheme, the invention at least has the following advantages:
1. the invention is based on the novel micro-fluidic chip of the fluid mechanics principle of the minimum flow resistance path, adopts a negative pressure active single cell trapping technology, and can nondestructively and efficiently trap single cells in a micro-channel and adsorb and fix the single cells in a trap;
2. the invention can be used for measuring the migration rate and chemotactic capacity of single cells under the conventional cell culture condition;
3. after the 3D culture medium is added into the micro-fluidic chip, the micro-fluidic chip can also be used for measuring the single cell migration rate and chemotaxis capacity in a 3D culture environment;
4. the micro-fluidic chip can be used for screening drugs for inhibiting tumor migration and metastasis under the condition of adding the drug-containing culture medium.
5. The capture trap structure of the microfluidic chip is an L-shaped structure with an etched structure, and forms a single-cell capture trap structure with unidirectional flow capture capacity together with the microchannel wall, so that single-wall backflow of liquid can be reduced, and single-cell capture efficiency is improved;
6. the design of the notch structure can generate certain adsorption capacity on the captured cells under the condition of low pressure difference, avoid dislocation of single cells, reduce low shearing force of liquid flow, stably capture and fix the single cells more stably and keep the activity of the cells;
7. the micro-fluidic chip provided by the invention accords with the fluid dynamics characteristics, and the layout of the upper chamber, the lower chamber and the parallel micro-channel array can conveniently realize various application scenes such as single cell migration capability determination, chemotactic factor chemotaxis capability determination, migration inhibition drug screening, migration capability determination under 3D culture conditions and the like;
8. the micro-fluidic chip of the invention realizes single cell capture by adopting a negative pressure method, can be well jointed with the traditional cell culture facility conditions, and does not need additional instrument and equipment;
9. the method for preparing the microfluidic chip accurately transfers the patterns of the mask to the surface of the silicon wafer by utilizing the photoetching technology, and is suitable for flexible processing of complex patterns;
10. the mask plate can be repeatedly used, and the production cost is reduced.
11. According to the preparation method of the microfluidic chip, regular and uniform trap structures are obtained on the surface of monocrystalline silicon, and then the microfluidic chip system is obtained through the PDMS demolding step, so that the obtained trap structures are arranged in order and the size is controllable;
12. the invention has convenient processing and low cost, and the silicon-based template can be repeatedly utilized.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to make the technical solutions of the present invention practical in accordance with the contents of the specification, the following detailed description is given of preferred embodiments of the present invention with reference to the accompanying drawings.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application, generally described and illustrated in the figures herein, can be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present application, presented in the accompanying drawings, is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
FIG. 1 is a schematic diagram of the structure of a single-cell phenotyping microfluidic chip according to the present invention;
FIG. 2 is a schematic view of a partial enlarged structure of the parallel microchannel array of FIG. 1;
FIG. 3 is an enlarged partial schematic view of FIG. 2 at A;
FIG. 4 is a schematic diagram of the structure of the single-cell filter of FIG. 1;
FIG. 5 is a photograph of a silicon-based chip template and portions thereof prepared in a first embodiment of the present invention;
FIG. 6 is a photograph of a microfluidic chip prepared in a first embodiment of the present invention;
FIG. 7 is a photomicrograph of a cell capture of a parallel microchannel array and capture trap structures according to a second embodiment of the invention;
FIG. 8 is a determination of the migratory capacity of lung cancer cells A549 and H1299 in a second embodiment of the present invention;
FIG. 9 is a measurement of the chemotactic ability of lung cancer cells A549 by TGF-. Beta.according to the third embodiment of the present invention;
FIG. 10 is a measurement of the migration inhibitory ability of the drug against lung cancer cell A549 in the fourth example of the present invention;
FIG. 11 is a graph showing the measurement of the migration ability of lung cancer cells H1299 in 3D culture conditions in the fifth embodiment of the present invention.
Wherein the meanings of each reference numeral in the figures are as follows.
1. First upper chamber 2 second upper chamber
3. First lower chamber 4 second lower chamber
5. Connecting branch channel 6 single cell filter
7. Connect main road channel 8 microchannel
9. Capture trap structure 10 microchannel walls
11. Trap parallel segment 12 trap vertical segment
L1 parallel segment length L2 vertical segment length
L3 length of the notch
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
Examples
As shown in the figures 1-11 of the drawings,
one of the objects of the present invention is:
the single cell phenotype determination micro-fluidic chip comprises a first upper chamber 1, a second upper chamber 2, a first lower chamber 3 and a second lower chamber 4, wherein the first upper chamber 1 and the second upper chamber 2 are sequentially arranged from left to right, the first lower chamber 3 is positioned under the first upper chamber 1, the second lower chamber 4 is positioned under the second upper chamber 2, and the first upper chamber 1, the second upper chamber 2, the first lower chamber 3 and the second lower chamber 4 are respectively connected with a main connecting path channel 7 positioned in the middle through a branch connecting channel 5; a single cell filter 6 is arranged in a connecting branch channel 5 connected with a connecting main channel 7 of the first upper chamber 1, or a single cell filter 6 is arranged in a connecting branch channel 5 connected with a connecting main channel 7 of the second upper chamber 2, a parallel micro-channel array is arranged in the connecting main channel 7, the parallel micro-channel array comprises a plurality of micro-channels 8 which are sequentially and evenly arranged in parallel from left to right, and a capture trap structure 9 with an L-shaped structure is arranged in each micro-channel 8.
Preferably, the trapping trap structure 9 comprises a parallel trap section 11 parallel to the microchannel wall 10 and a perpendicular trap section 12 perpendicular to the microchannel wall 10, and an incised structure is arranged between the parallel trap section 11 and the perpendicular trap section 12.
Preferably, the length L1 of the parallel trap segments 11 is 15-80 μm, the length L2 of the vertical trap segments 12 is 8-20 μm, the length L3 of the etched structures is 2-10 μm, and the distance L4 between the vertical trap segments 12 and the microchannel walls 10 is 2-10 μm.
Preferably, the first upper chamber 1, the second upper chamber 2, the first lower chamber 3 and the second lower chamber 4 have an area of 9-100mm2, the micro-channel 8 has a pore size of 20-90 μm, and the single cell filter 6 is composed of columnar arrays having a diameter of 3-30 μm and a pitch of 10-100 μm.
The second object of the present invention is:
the preparation method of the single cell phenotype determination microfluidic chip sequentially comprises the following steps:
step 1, sequentially coating a binder and photoresist on the surface of a clean silicon wafer, placing a mask plate containing a trap structure/parallel microchannel array on a photoresist layer, exposing in photoetching equipment, removing the mask plate, and washing uncured photoresist in the photoresist layer by using positive photoresist developing solution to obtain a material after photoetching;
step 2, depositing silicon dioxide on the surface of the material after photoetching, and then removing the residual photoresist on the surface of the silicon wafer to obtain a silicon dioxide mask-free silicon wafer;
step 3, placing the silicon wafer without the silicon dioxide mask in an inductively coupled plasma etching machine for dry etching to obtain a silicon wafer template with a capture trap structure/parallel micro-channel array;
and 4, placing the silicon wafer template prepared by pouring the uniformly stirred bubble-free PDMS into an oven for curing, then completely separating the PDMS layer from the silicon wafer template, and bonding the upper base plate and the lower base plate after two demolding operations to form the final microfluidic chip.
In a preferred embodiment of the method of the invention,
wherein the content of the first and second substances,
in the step 1, the silicon wafer is a polished silicon wafer, and the thickness of the silicon wafer is 100-1000 microns; the binder is hexamethyldisilazane, and the coating mode of the binder is spin coating, spray coating or vacuum volatilization; the photoresist is positive photoresist, and the coating mode of the photoresist is spin coating or spray coating;
in the step 2, depositing a silicon dioxide layer by adopting an evaporation or magnetron sputtering method, wherein the thickness of the silicon dioxide layer is 10-300 nanometers; the method for removing the residual photoresist on the surface of the silicon wafer is acetone soaking;
in the step 3, the dry etching gas comprises one or a combination of a plurality of gases of carbon tetrafluoride gas, sulfur hexafluoride gas, octafluorocyclobutane gas, oxygen gas, argon gas and helium gas, the working pressure in an etching cavity in an etching machine is less than 50mTorr, and the etching time is 1-120 minutes;
in the step 4, the proportion of PDMS to the curing agent is 10:1 or 8:2 or 7: and 3, keeping the temperature of the oven at 50-90 ℃ for curing for 30-240 min.
The third object of the present invention is:
the measuring method of the single cell phenotype measuring microfluidic chip sequentially comprises a step A, a single cell capturing step, a step B and a measuring step, wherein,
step A, a single cell capturing step, which sequentially comprises:
a1, preparing a cell suspension by enzymatic digestion of cells, and dripping the cell suspension into a first upper chamber 1;
step A2, starting a negative pressure aspirator, forming gas negative pressure in the first lower chamber 3 or the second lower chamber 4, and enabling the cell suspension to be sucked into the microchannel 8 by negative pressure driving and captured by the capture trap structure 9;
step A3, adding a physiological/culture solution into the first upper chamber 1, cleaning redundant cells by using negative pressure, and sucking away waste liquid;
step A4, adding a cell culture medium into the first upper chamber 1, and then placing the mixture into a cell culture box for culture;
step B, a determination step, which sequentially comprises:
step B1, after single cell capture, adding a culture medium containing a drug to be detected into the second upper chamber 2, and culturing for 12-120 hours in a cell culture box;
b2, after culturing, carrying out microscopic imaging on each micro-channel, measuring the distance of each single cell from the trap structure, and taking an average value;
wherein, mobility = sample single cell migration distance/control single cell migration distance 100%;
chemotactic intensity = migration distance of single cells of sample/migration distance of single cells of control x 100%.
The fourth purpose of the invention is that:
the micro-fluidic chip is applied to the determination of the migration capacity of single cells, the single cells are normal tissue cells or tumor cells or microbial cells, and the microbes are microbes with the mobility.
The fifth purpose of the invention is:
the microfluidic chip is applied to the determination of the migration capacity of the single cell under the 3D culture condition, and the 3D culture comprises the step of pouring a 3D culture medium into the chip.
The sixth purpose of the invention is: the micro-fluidic chip is applied to screening and evaluating of anti-tumor migration/metastasis drugs, and tumor cells are solid tumor cells and mononuclear/macrophage tumor cells.
The micro-fluidic chip provided by the invention has a specific single cell filter structure and a specific single cell capture trap structure, can realize the determination of the migration, chemotaxis and invasion capacities of single cells (such as lung cancer cells, breast cancer cells, cervical cancer cells and the like), and is used for screening single cell phenomics and anti-migration/transfer drugs.
The invention designs and manufactures a novel micro-fluidic chip based on the fluid mechanics principle of the minimum flow resistance path, adopts a negative pressure active single cell trapping technology, and can nondestructively and efficiently trap single cells in a micro-channel and be adsorbed and fixed in a trapping trap. The micro-fluidic chip can be used for measuring the migration rate and chemotactic capacity of single cells under the conventional cell culture condition.
Meanwhile, after the 3D culture medium is added, the microfluidic chip can also be used for measuring the single cell mobility and chemotactic capacity in a 3D culture environment. The micro-fluidic chip can be used for screening drugs for inhibiting tumor migration and metastasis under the condition of adding a drug-containing culture medium.
The invention aims to provide a multi-channel micro-fluidic chip which is used for measuring the migration, chemotaxis and invasion capacities of single cells, has a single cell filter structure and a specific single cell capture trap structure, and realizes the measurement of the migration, chemotaxis and invasion capacities of single cancer cells (such as lung cancer cells, breast cancer cells, cervical cancer cells and the like).
According to the invention, a chip mold based on a silicon wafer is manufactured by combining various micro-nano processing technologies and accurately controlling a trap structure on the surface of monocrystalline silicon, and then a PDMS (polydimethylsiloxane) pouring mold is adopted and assembled at a later stage to prepare a micro-fluidic chip, so that the measurement of the migration, chemotaxis and invasion capacities of single cells is realized.
The design of the microfluidic chip of the project mainly adopts the principle of 'minimum flow resistance path' hydrodynamics, combines with cell biomechanics and cell culture technology, and prepares the novel microfluidic chip for measuring the migration, chemotaxis and invasion capacity of single cells through simulation optimization and improvement.
The chip is composed of a first upper chamber 1, a second upper chamber 2, a first lower chamber 3 and a second lower chamber 4, and parallel micro-channels and connecting channels connecting the upper chambers and the lower chambers, wherein the connecting channel between one of the upper chambers and the micro-channel has a single-cell filter structure, and the micro-channel has a specific single-cell trap structure therein.
The layout of the microfluidic chip related to the invention is shown in the attached figures 1-4, the chip is provided with 2 upper chambers, namely a first upper chamber 1 and a second upper chamber 2; the lower chambers 2 are a first lower chamber 3 and a second lower chamber 4, respectively. The first upper chamber 1 is used for adding cell suspension and cleaning solution, the second upper chamber 2 is used for adding a drug-containing culture medium, and the first lower chamber 3 and the second lower chamber 4 are used for sucking out culture waste liquid or cleaning solution under negative pressure.
Wherein, the first upper chamber 1 or the second upper chamber 2 and the connecting branch channel 5 of the micro channel are provided with single cell filters 6, which ensure that all entering the micro channel are single cells, the single cell filters 6 are composed of columnar arrays with the diameter of 3-30 μm and the distance of 10-100 μm, and the specific structure is shown in figure 4.
A parallel microchannel array is arranged between an upper chamber and a lower chamber, a single-cell trapping trap structure 9 is arranged in each microchannel, the trapping trap structure 9 is designed into an L-shaped structure with an indentation, wherein the length L1 of a parallel section relative to a microchannel wall is 15-80 microns, the length L2 of a vertical section is 8-20 microns, the lengths L3 of the parallel section and the vertical section are 2-10 microns, and the L shape with the indentation is 2-10 microns away from the microchannel wall L4 on one side to form the single-cell trapping trap structure with unidirectional flow trapping capability together with the microchannel wall.
The trap structure 9 is innovatively designed after hydrodynamics simulation calculation, wherein the notched L-shaped trap structure design can reduce liquid single-wall backflow and improve single-cell trapping efficiency; the design of the notch structure can form a low-pressure area in the trap, and a certain adsorption capacity is generated on the captured cells, so that dislocation of the single cells is avoided, and the single cells are stably captured and fixed under low flow rate and low shearing force. The specific structure of the trap involved in the present invention is shown in fig. 3.
The area of each upper chamber and each lower chamber is 9-100mm 2 The aperture of the parallel micro-channel is 20-90 μm, and the single cell filter is composed of cylindrical arrays with the diameter of 3-30 μm and the distance of 10-100 μm.
The microfluidic chip and the substrate of the capture trap array structure are prepared by the following method:
step 1, sequentially coating a binder and photoresist on the clean surface of a silicon wafer, placing a mask plate with a trap array/micro-channel structure on a photoresist layer, exposing in photoetching equipment, removing the mask plate, and washing uncured photoresist in the photoresist layer by using an anode photoresist developing solution to obtain a photoetched material;
step 2, depositing silicon dioxide on the surface of the material after photoetching, and then removing the residual photoresist on the surface of the silicon wafer to obtain a silicon dioxide mask-free silicon wafer;
step 3, placing the silicon wafer without the silicon dioxide mask in an ICP-RIE (inductively coupled plasma etching) machine for dry etching to obtain a silicon wafer template with a trap array/micro-channel structure;
and 4, placing the silicon wafer template prepared by pouring the uniformly stirred bubble-free PDMS into an oven for curing for several hours, then carefully separating the PDMS layer from the silicon wafer template completely, and bonding the upper and lower bottom plates after two demoulding operations to form the final microfluidic chip.
Further, in step 1, the silicon wafer is a polished silicon wafer, and the thickness of the silicon wafer is 100-1000 microns; the binder is hexamethyldisilazane and the like, and the coating mode of the binder is spin coating, spray coating or vacuum volatilization and the like; the photoresist is a positive photoresist, and the coating mode of the photoresist is spin coating or spray coating.
Further, in the step 2, a silicon dioxide layer is deposited by adopting an evaporation or magnetron sputtering method, and the thickness of the silicon dioxide layer is 10-300 nanometers; the method for removing the residual photoresist on the surface of the silicon wafer is acetone soaking.
Further, in step 3, the dry etching gas includes one or more of carbon tetrafluoride gas, sulfur hexafluoride gas, octafluorocyclobutane gas, oxygen gas, argon gas, helium gas, etc., the working pressure in the etching chamber is generally less than 50mTorr, and the etching time is 1-120 minutes.
Further, in step 4, the ratio of PDMS to the curing agent may be 10: 1. 8: 2. 7:3, the temperature of the oven can be 50-90 ℃, and the curing time is kept for 30-240 min.
The micro-fluidic chip realizes single cell capture by a negative pressure method, and the principle is that cell suspension is driven to pass through a micro-channel by the pressure difference of gas negative pressure and is captured and fixed by capture traps, only one cell is captured by one trap, so that a single cell state at a fixed position is formed, and redundant cells are sucked away by negative pressure cleaning of physiological salt/culture solution. The single cell can migrate upwards in the incubator by taking the capture trap as a starting point, and the migration capability of the cell, the chemotactic capability of the medicine or the migration inhibition capability of the medicine can be evaluated by measuring the migration distance.
The method mainly comprises the following steps:
1. cells are digested by an enzyme method to prepare cell suspension, and the cell suspension is dripped into the first upper chamber 1;
2. starting a negative pressure aspirator, forming gas negative pressure in the first lower chamber 3 or the second lower chamber 4, and enabling the cell suspension to be sucked into the microchannel under the drive of the negative pressure and captured by the single cell capture trap structure;
3. adding physiological/culture solution into the first upper chamber 1, cleaning redundant cells by using negative pressure, and sucking waste liquid away;
4. after the cell culture medium is added into the first upper chamber 1, the cell culture medium is placed in a cell culture box for culture. If the migration or chemotaxis of the drug on single cells is to be determined, a corresponding volume of drug-containing medium is added to the second upper chamber 2.
5. After the required time of incubation, individual microchannels were microscopically imaged and the distance of migration of each single cell from the trap was measured.
The mobility calculation formula is: multichannel cell migration distance was averaged, mobility = sample single cell migration distance/control single cell migration distance 100%.
The calculation formula for chemotaxis is: chemotactic intensity = migration of single cells of sample/migration of single cells of control 100%.
Furthermore, the micro-fluidic chip is applied to the measurement of the migration capacity of single cells, wherein the single cells are normal tissue cells, tumor cells or microorganism cells, and the microorganisms are microorganisms with the mobility.
Furthermore, the microfluidic chip provided by the invention is applied to the determination of the migration capacity of single cells under 3D culture conditions, and the method is that a culture solution containing a 3D matrix is added into the second upper chamber 2 and is sucked into the microchannel through the negative pressure of the first lower chamber 3 or the second lower chamber 4, so that the migration or chemotactic capacity of the cells can be observed and measured under the 3D culture conditions.
Furthermore, the micro-fluidic chip related to the invention is applied to the determination of the chemotactic capacity of single cells, and the method is that a culture solution containing the chemotactic factors is added into the second upper chamber 2 and is sucked into the micro-channel through the negative pressure of the first lower chamber 3 or the second lower chamber 4, so that the influence of the chemotactic factors on the chemotactic capacity of the cells can be observed and measured.
Furthermore, the microfluidic chip provided by the invention is applied to screening and evaluation of anti-tumor migration/metastasis drugs, and the tumor cells are solid tumor cells and mononuclear/macrophage tumor cells. The method comprises the steps of adding solid tumor cells or mononuclear/macrophage tumor cells into the first upper chamber 1, adding culture solution containing a drug to be detected into the second upper chamber 2, and sucking the culture solution into the microchannel through the negative pressure of the first lower chamber 3 or the second lower chamber 4, so that the influence of the drug to be detected on the migration and chemotactic capacity of the cells can be observed and measured under the action of the drug, and a new anti-tumor metastasis drug can be screened and researched.
The first embodiment:
design, mold preparation and overprint pouring of microfluidic chip
The method is characterized in that a micro-fluidic chip containing a specific trap array structure is prepared, the migration, chemotaxis and invasion capacity determination of single cells (such as lung cancer cells, breast cancer cells, cervical cancer cells and the like) is systematically realized, and the trap array/micro-channel structure substrate is prepared through the following steps:
1) The mask pattern of the microfluidic chip was designed using AutoCAD.
2) Cutting a clean silicon wafer with the thickness of 600 micrometers into a square with the side length of 3cm, immersing the square in concentrated sulfuric acid, and heating for 3 hours at 90 ℃;
then, sequentially carrying out ultrasonic treatment on the silicon wafer in ethanol, acetone and ultrapure water for 15min, and drying by using nitrogen;
adding hexamethyldisilazane dropwise on the surface of the silicon wafer, and spin-coating at the rotating speed of 1000rpm for 2min to form a uniform adhesive layer;
pouring AZ5217 photoresist with bubbles removed onto the surface of a silicon wafer, and spin-coating at 3000rpm for 1min to form a uniform photoresist layer;
using a chromium plate containing a trap array pattern as a mask plate to mask the silicon wafer, and exposing the silicon wafer for 120s under an ultraviolet lamp;
and immersing the exposed silicon wafer into positive photoresist developing solution, and continuously washing the surface uncured photoresist by using the developing solution.
3) Depositing a 100-nanometer thick silicon dioxide layer on the surface of the material obtained in the step 2) in a magnetron sputtering mode, and then soaking the silicon dioxide layer in acetone for 15 minutes to remove the residual photoresist on the surface of the silicon wafer to obtain the silicon dioxide mask glue-free silicon wafer.
4) And (3) carrying out dry etching on the silicon dioxide mask non-glue silicon wafer substrate for 30 minutes to obtain the silicon wafer containing the substrate with the trap array/micro-channel structure, wherein the result photo is shown in figure 5.
5) Curing agent according to the ratio of 1: adding the 10 columns into PDMS, stirring uniformly for 2min, placing into a vacuum device for bubble removal, pouring foamless PDMS into the prepared silicon-based template, placing into an oven at 80 ℃ for curing for 1.5 h, finally, carefully separating the PDMS layer from the silicon-based template completely, bonding the upper and lower bottom plates after two demolding operations to form the final microfluidic chip, and the result is shown in FIG. 6.
The second embodiment:
a549 and H1299 single cell migration capacity assay
1) Lung cancer cell lines A549 and H1299 were cultured in DMEM medium containing 10% FCS,80U/mL penicillin and 100U/mL streptomycin at 37 ℃ in A5% CO2 incubator.
2) Digesting A549 and H1299 by pancreatin-EDTA, preparing cell suspension, sampling, dyeing by trypan blue, and measuring the number of cells by a cell counter;
after centrifugation at 1500rpm/min for 5min, the supernatant was decanted, the corresponding volume of medium was added, and the cell density was adjusted to 105 cells/mL.
3) Taking 2 microfluidic chips prepared in the project, firstly adding 200uL of DMEM medium into the first upper chamber 1, then adding 100uL of A549 cell suspension and H1299 cell suspension respectively, and gently sucking and uniformly beating.
4) Starting the negative pressure aspirator, adjusting the pressure to 0.005-0.1MPa, slowly inserting the aspirator tip into the first lower chamber 3, observing the liquid flow condition until all the cell suspension is sucked into the microchannel for single cell capture, and obtaining the result shown in figure 7.
5) 300uL of DMEM medium is added into the first upper chamber 1, the suction head is inserted into the bottom of the first lower chamber 3, all liquid is sucked up, and the cleaning process is repeated for 3 times.
6) 300uL of low serum (1% FCS) DMEM medium was added to the first upper chamber 1 and the medium was aspirated into the microchannel using the tip as above.
7) The first upper chamber 1 and the second upper chamber 2 were each charged with 300uL of low serum (1% FCS) DMEM medium, and the microfluidic chip with the captured cells was placed in a 37 ℃ and 5% CO2 incubator for 2 hours to allow the cells to adhere to the surface.
8) And taking out the microfluidic chip, observing and recording single cells in the capture trap of each microchannel by using a photomicrography system, calculating capture efficiency, and recording the initial position of the single cells.
9) The microfluidic chip was placed in a 37 ℃ C., 5% CO2 incubator and incubated for 24 hours to allow the cells to migrate along the microchannel.
10 The micro-fluidic chip is taken out, the position of single cells in the capture trap of each micro-channel is observed and recorded by a micro-photographic system, and the migration distance of each single cell is measured by a ranging scale of the micro-photographic system.
11 Calculate the 24 hour mean migration distance of a549 and H1299 cells.
The results are shown in FIG. 8, which shows that the migration capacity of A549 is stronger than that of H1299 cells under the single-cell microfluidic chip assay.
The third embodiment:
chemotactic ability assay of TGF-beta for A549 single cells
1) The lung cancer cell line A549 was cultured in DMEM medium containing 10% FCS,80U/mL penicillin and 100U/mL streptomycin at 37 ℃ in A5% CO2 incubator;
digesting A549 cells by pancreatin-EDTA (ethylene diamine tetraacetic acid), preparing a cell suspension, sampling, dyeing by trypan blue, and measuring the number of cells by using a cell counter;
after centrifugation at 1500rpm/min for 5min, the supernatant was decanted, the corresponding volume of medium was added and the cell density was adjusted to 105 cells/mL.
2) Taking 4 microfluidic chips prepared by the method, firstly adding 200uL DMEM culture medium into the first upper chamber 1, then adding 100uL A549 cell suspension into the first upper chamber respectively, and gently sucking and beating uniformly;
starting the negative pressure aspirator, adjusting the pressure to 0.005-0.1MPa, slowly inserting the aspirator into the first lower chamber 3 by using an aspirator tip, observing the liquid flow condition until all cell suspension is sucked into the microchannel for single cell capture.
3) Adding 300uL DMEM medium into the first upper chamber 1, inserting the suction head into the bottom of the first lower chamber 3, completely sucking all liquid, and repeating the cleaning process for 3 times;
300uL of low serum (1% FCS) DMEM medium was added to the first upper chamber 1, and the medium was aspirated into the microchannel using the aspirator tip as above.
4) For the 4 microfluidic chips in which the trapping was completed, 300uL, 0ug/mL, 1ug/mL, 5ug/mL, and 10ug/mL of low serum (1 FCS) DMEM medium for TGF-. Beta.was added to each of the first upper chamber 1 and the second upper chamber 2, and the mixture was cultured in a 37 ℃ C., 5-vol CO2 incubator for 2 hours to attach the cells to the wall.
5) Taking out the microfluidic chip, observing and recording single cells in the capture traps of all the microchannels by using a photomicrography system, calculating capture efficiency, and recording the starting positions of the single cells;
the microfluidic chip was placed in a 37 ℃ C., 5% CO2 incubator and incubated for 24 hours to allow the cells to migrate along the microchannel.
6) And taking out the microfluidic chip, observing and recording the positions of single cells in the capture traps of each microchannel by using a photomicrography system, and measuring the migration distance of each single cell by using a distance measuring scale of the photomicrography system.
7) And calculating the 24-hour average mobility of TGF-beta to A549 under different concentration gradients by taking 0ug/mL TGF-beta as a reference, and calculating the chemotactic capacity of the TGF-beta at different gradient concentrations.
The result is shown in figure 9, which shows that the TGF-beta has chemotactic ability to A549 under the determination of the single-cell microfluidic chip, and the migration rate and the TGF-beta concentration present dose dependence.
The fourth embodiment:
determination of migration inhibition capacity of PI3K inhibitor LY294002 on A549 lung cancer cells
1) The lung cancer cell strain A549 is cultured and cell suspension is prepared as above.
2) The microfluidic chip prepared by 4 items was used, and the A549 single cell capture process was the same as above.
3) 4 microfluidic chips in which A549 was captured, 300uL of low serum (1% FCS) DMEM medium containing 0uM, 0.5uM, 2uM, and 5uM LY294002 was added to each of the first upper chamber 1 and the second upper chamber 2, and the mixture was incubated in an incubator at 37 ℃ and 5% CO2 for 2 hours to allow the cells to adhere.
4) Taking out the microfluidic chip, observing and recording single cells in the capture traps of each microchannel by using a photomicrography system, calculating capture efficiency, and recording the initial positions of the single cells;
the microfluidic chip was incubated for 24 hours.
5) And taking out the microfluidic chip, observing and recording the positions of single cells in the capture traps of each microchannel by using a microscopic photography system, and measuring the migration distance of each single cell by using a ranging scale of the microscopic system.
6) The 24-hour average mobility of different concentrations of LY294002 to A549 is calculated by taking 0ug/mL LY294002 as a control, and the inhibition capability of the PI3K inhibitor LY294002 to the migration of A549 cells is examined.
The result is shown in fig. 10, which shows that the PI3K inhibitor LY294002 has inhibition on the migration of A549 under the determination of the single-cell microfluidic chip, and the migration rate of the PI3K inhibitor LY294002 is obviously reduced along with the concentration of LY 294002.
Fifth embodiment:
determination of migration rate of H1299 lung cancer cells under 3D culture condition
1) Culturing lung cancer cell strain H1299, and preparing cell suspension.
2) The microfluidic chip prepared by 2 items was taken, and the H1299 single cell capture process was the same as above.
3) Placing 2 microfluidic chips captured by H1299 single cells on ice for precooling for 30 minutes;
one upper chamber 1 and one lower chamber 2 were each supplemented with 300uL of low serum (1% FCS) DMEM medium;
another 300uL of pre-cooled matrigel and 2% FCS DMEM medium 1: the mixture was aspirated into the microchannel at a ratio of 1, and incubated in a 37 ℃ C., 5% CO2 incubator for 2 hours to coagulate matrigel, thereby forming a 3D culture environment.
4) Taking out the microfluidic chip, observing and recording single cells in the capture traps of all the microchannels by using a photomicrography system, calculating capture efficiency, and recording the starting positions of the single cells;
the microfluidic chip was incubated for 24 hours.
5) And taking out the microfluidic chip, observing and recording the positions of single cells in the capture traps of each microchannel by using a microscopic photography system, and measuring the migration distance of each single cell by using a ranging scale of the microscopic system.
6) The 24 hour average mobility of H1299 cells under 2D and 3D culture conditions was calculated and the mobility of H1299 cells in 3D medium was examined.
The results are shown in fig. 11, which shows that the migration capacity of H1299 in Matrigel 3D medium under the single cell microfluidic chip assay is significantly higher than that of conventional 2D culture.
The beneficial effects applied to the embodiment are as follows:
1. the invention is based on the novel micro-fluidic chip of the fluid mechanics principle of the minimum flow resistance path, adopts a negative pressure active single cell trapping technology, and can nondestructively and efficiently trap single cells in a micro-channel and adsorb and fix the single cells in a trap;
2. the invention can be used for measuring the mobility and chemotactic capacity of single cells under the conventional cell culture condition;
3. after the 3D culture medium is added into the micro-fluidic chip, the micro-fluidic chip can also be used for measuring the single cell mobility and chemotactic capacity in a 3D culture environment;
4. the micro-fluidic chip can be used for screening drugs for inhibiting tumor migration and metastasis under the condition of adding the drug-containing culture medium.
5. The capture trap structure of the microfluidic chip is an L-shaped structure with an etched structure, and forms a single-cell capture trap structure with unidirectional flow capture capacity together with the microchannel wall, so that single-wall backflow of liquid can be reduced, and single-cell capture efficiency is improved;
6. the design of the notch structure can generate certain adsorption capacity on the captured cells under the condition of low pressure difference, avoid dislocation of single cells, reduce low shearing force of liquid flow, stably capture and fix the single cells more stably and keep the activity of the cells;
7. the micro-fluidic chip provided by the invention accords with the hydrodynamic characteristics, and the layout of the upper chamber, the lower chamber and the parallel micro-channel array can conveniently realize various application scenes such as single cell migration capability determination, chemotactic factor chemotactic capability determination, migration inhibition drug screening, migration capability determination under 3D culture conditions and the like;
8. the micro-fluidic chip of the invention realizes single cell capture by adopting a negative pressure method, can be well jointed with the traditional cell culture facility conditions, and does not need additional instrument and equipment;
9. the method for preparing the microfluidic chip accurately transfers the patterns of the mask to the surface of the silicon wafer by utilizing the photoetching technology, and is suitable for flexible processing of complex patterns;
10. the mask plate can be repeatedly used, and the production cost is reduced.
11. According to the preparation method of the micro-fluidic chip, regular and uniform trap structures are obtained on the surface of monocrystalline silicon, a micro-fluidic chip system is obtained through a PDMS (polydimethylsiloxane) demolding step, and the obtained trap structures are arranged in order and controllable in size;
12. the invention has convenient processing and low cost, and the silicon-based template can be repeatedly utilized.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly referring to the number of technical features being grined. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, a fixed connection, a detachable connection, or an integral connection: either mechanically or electrically: the terms may be directly connected or indirectly connected through an intermediate agent, or may be a communication between two elements.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims (10)

1. The single cell phenotype determination microfluidic chip comprises a first upper chamber (1), a second upper chamber (2), a first lower chamber (3) and a second lower chamber (4), wherein the first upper chamber (1) and the second upper chamber (2) are sequentially arranged from left to right, the first lower chamber (3) is positioned under the first upper chamber (1), the second lower chamber (4) is positioned under the second upper chamber (2), and the first upper chamber (1), the second upper chamber (2), the first lower chamber (3) and the second lower chamber (4) are respectively connected with a main connecting path channel (7) positioned in the middle through a branch connecting path channel (5); the device is characterized in that a single cell filter (6) is arranged in a connecting branch channel (5) of a first upper chamber (1) connected with a connecting main channel (7), or a single cell filter (6) is arranged in a connecting branch channel (5) of a second upper chamber (2) connected with the connecting main channel (7), a parallel micro-channel array is arranged in the connecting main channel (7), the parallel micro-channel array comprises a plurality of micro-channels (8) which are sequentially and uniformly arranged in parallel from left to right, and a capture trap structure (9) with an L-shaped structure is arranged in each micro-channel (8).
2. The single-cell phenotyping microfluidic chip of claim 1, wherein said trapping trap structure (9) comprises a parallel trap section (11) parallel to the microchannel wall (10) and a vertical trap section (12) perpendicular to the microchannel wall (10), and an indentation structure is disposed between said parallel trap section (11) and said vertical trap section (12).
3. The single-cell phenotyping microfluidic chip of claim 2, wherein the length (L1) of the parallel trap segment (11) is 15-80 μm, the length (L2) of the vertical trap segment (12) is 8-20 μm, the length (L3) of the notch structure is 2-10 μm, and the distance (L4) between the vertical trap segment (12) and the microchannel wall (10) is 2-10 μm.
4. The single-cell phenotyping microfluidic chip according to claim 1, wherein said first upper chamber (1), said second upper chamber (2), said first lower chamber (3) and said second lower chamber (4) have an area of 9-100mm 2 The pore diameter of the micro-channel (8) is 20-90 μm, and the single cell filter (6) is composed of columnar arrays with the diameter of 3-30 μm and the distance of 10-100 μm.
5. The method for preparing a single-cell phenotyping microfluidic chip according to claim 1, comprising the following steps in sequence:
step 1, sequentially coating a binder and photoresist on the surface of a clean silicon wafer, placing a mask plate containing a trap structure/parallel microchannel array on a photoresist layer, exposing in photoetching equipment, removing the mask plate, and washing uncured photoresist in the photoresist layer by using positive photoresist developing solution to obtain a material after photoetching;
step 2, depositing silicon dioxide on the surface of the material after photoetching, and then removing the residual photoresist on the surface of the silicon wafer to obtain a silicon dioxide mask non-photoresist silicon wafer;
step 3, placing the silicon wafer without the silicon dioxide mask in an inductively coupled plasma etching machine for dry etching to obtain a silicon wafer template with a trap structure/parallel micro-channel array;
and 4, placing the silicon wafer template prepared by pouring the uniformly stirred bubble-free PDMS into an oven for curing, then completely separating the PDMS layer from the silicon wafer template, and bonding the upper base plate and the lower base plate after two demolding operations to form the final microfluidic chip.
6. The method for preparing a single-cell phenotyping microfluidic chip according to claim 5, wherein,
in the step 1, the silicon wafer is a polished silicon wafer, and the thickness of the silicon wafer is 100-1000 microns; the binder is hexamethyldisilazane, and the coating mode of the binder is spin coating, spray coating or vacuum volatilization; the photoresist is a positive photoresist, and the coating mode of the photoresist is spin coating or spray coating;
in the step 2, a silicon dioxide layer is deposited by adopting an evaporation or magnetron sputtering method, and the thickness of the silicon dioxide layer is 10-300 nanometers; the method for removing the residual photoresist on the surface of the silicon wafer is acetone soaking;
in the step 3, the dry etching gas comprises one or a combination of a plurality of gases of carbon tetrafluoride gas, sulfur hexafluoride gas, octafluorocyclobutane gas, oxygen gas, argon gas and helium gas, the working pressure in an etching cavity in an etching machine is less than 50mTorr, and the etching time is 1-120 minutes;
in the step 4, the proportion of PDMS to the curing agent is 10:1 or 8:2 or 7: and 3, keeping the temperature of the oven at 50-90 ℃ for curing for 30-240 min.
7. The method for determining the single-cell phenotype assay microfluidic chip of claim 1, comprising the steps of A, capturing single cell, B and determining in sequence, wherein,
step A, single cell capturing step, which comprises the following steps in sequence:
a1, preparing a cell suspension by digesting cells with an enzyme method, and dripping the cell suspension into a first upper chamber (1);
step A2, starting a negative pressure aspirator, forming gas negative pressure in the first lower chamber (3) or the second lower chamber (4), and enabling cell suspension to be sucked into the microchannel (8) under the drive of the negative pressure and captured by the capture trap structure (9);
step A3, adding a physiological/culture solution into the first upper chamber (1), cleaning redundant cells by utilizing negative pressure, and sucking away waste liquid;
step A4, adding a cell culture medium into the first upper chamber (1), and then placing the mixture into a cell culture box for culture;
step B, a determination step, which sequentially comprises:
step B1, after single cell capture, adding a culture medium containing a drug to be detected into the second upper chamber (2), and culturing for 12-120 hours in a cell culture box;
b2, after culturing, carrying out microscopic imaging on each micro-channel, measuring the distance of each single cell from the trap structure, and taking an average value;
wherein, mobility = sample single cell migration distance/control single cell migration distance 100%;
chemotactic intensity = migration distance of single cells of sample/migration distance of single cells of control x 100%.
8. The use of the microfluidic chip for single-cell phenotypic assay according to claim 1 wherein said microfluidic chip is used for single-cell mobility assay, wherein the single cell is a normal tissue cell or a tumor cell or a microorganism cell, and the microorganism is a microorganism with mobility.
9. The use of the single-cell phenotyping microfluidic chip according to claim 1, wherein said microfluidic chip is used for measuring the migration ability of single cells under 3D culture conditions, and wherein 3D culture comprises 3D medium perfusion in the chip.
10. The use of the single-cell phenotyping microfluidic chip of claim 1, wherein said microfluidic chip is used for screening and evaluating drugs against tumor migration/metastasis, and said tumor cells are solid tumor cells and mononuclear/macrophage tumor cells.
CN202211199868.XA 2022-09-29 2022-09-29 Single cell phenotype determination micro-fluidic chip, preparation method, determination method and application thereof Pending CN115369012A (en)

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