CN107267382B - Microfluidic chip based on dielectrophoresis and preparation method and application thereof - Google Patents
Microfluidic chip based on dielectrophoresis and preparation method and application thereof Download PDFInfo
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims 3
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- 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
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- 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
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- 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
- B01L3/50273—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 means or forces applied to move the fluids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- 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
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- 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
- B01L2200/12—Specific details about manufacturing devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
Abstract
The invention relates to a microfluidic chip based on dielectrophoresis, and a preparation method and application thereof, wherein the microfluidic chip comprises a planar chip (16) at the bottom and a micro-channel (13) at the top, and the planar chip has a four-layer structure which is sequentially overlapped; the first layer structure (1) of the planar chip comprises electrodes on a glass substrate, wherein the electrodes are interdigital electrodes (2); the second layer structure (3) of the planar chip comprises a microcavity array (8); the third layer structure (4) of the planar chip comprises a conductive layer (10); the fourth layer structure (5) of the planar chip comprises a microcavity array (12) and a micro baffle (11) positioned in the microcavity array; the second layer structure (3) and the fourth layer structure (5) comprise microfluidic channels (9) thereon. The microfluidic chip can realize high-flux single cell capturing and pairing.
Description
Technical Field
The invention belongs to the technical field of single-cell capturing and pairing, and relates to a micro-fluidic chip based on dielectrophoresis and a preparation method and application thereof.
Background
Cell fusion, also known as cell hybridization, is a process in which two or more homologous or heterologous cells are allowed to form heterozygous cells under ex vivo conditions by means of induction and culture. Has become an important means in modern bioengineering technology research. The cell fusion technology has wide application in research fields of genetics, immunology, developmental biology, drug or gene delivery, hybridization breeding and the like. Accurate single cell capture and pairing are the preconditions for developing cell fusion studies, so an effective single cell capture and pairing experimental platform needs to be designed and constructed.
Dielectrophoresis (DEP) is a technique for manipulating particles in a non-uniform electric field according to their dielectric properties. By changing the conditions such as the frequency of the applied voltage, the particles can be controlled to move to a high electric field under the action of positive dielectrophoresis force or to move to a low electric field under the action of negative dielectrophoresis force, so that the particles can be manipulated. Thus, DEP-based methods can achieve efficient high-throughput cell capture and pairing processes.
The Yasukawa subject group of the university of Website in Japan reports a vertical paired cell microfluidic device based on a dielectrophoresis method, which mainly utilizes positive dielectrophoresis force generated by a top ITO electrode and a bottom patterned microelectrode to capture two cells in sequence in a microcavity array, so as to realize a vertical high-flux cell pairing process. The Matsue subject group of university of northeast China reports a micro-fluidic chip based on a dielectrophoresis method for cell pairing, wherein the chip comprises a miniature interdigital electrode and a calabash-shaped microcavity array, and the cell pairing process is a process of capturing two cells in sequence by utilizing positive dielectrophoresis force generated by an ITO electrode at the top and an interdigital electrode at the bottom so as to form a cell pair.
The existing high-flux cell pairing microfluidic chip based on the DEP method adopts a mode that an ITO electrode is arranged at the top and a microelectrode is arranged at the bottom to generate DEP, and the defect of the structure is that: (1) Polydimethylsiloxane (PDMS) is the most common material for preparing a micro-channel in a microfluidic chip, is easy to package with the chip, and can be used for preparing an inlet and an outlet of the chip on the PDMS through a simple puncher, and the packaging process and the preparation of the inlet and the outlet are more troublesome when an ITO electrode is arranged at the top; (2) The DEP captures cells by applying an alternating signal between electrodes to generate a non-uniform electric field so as to lead the cells to move directionally, in the pairing process, ITO is used as a common electrode for capturing two kinds of cells at the top, and when capturing a first kind of cells, the position for capturing the other kind of cells is occupied by the first kind of cells due to the action of an induced electric field, so that the pairing efficiency of the chip is affected.
Therefore, how to simplify the chip packaging process and improve the pairing efficiency of chips is a current urgent problem to be solved.
Disclosure of Invention
Aiming at the problems, the main purpose of the invention is to develop a micro-fluidic chip based on dielectrophoresis, a preparation method and application thereof, wherein the micro-fluidic chip not only simplifies the chip packaging process, but also can effectively improve the pairing efficiency of the chip by changing the structure of the micro-fluidic chip into a planar structure.
To achieve the purpose, the invention adopts the following technical scheme:
the invention provides a micro-fluidic chip based on dielectrophoresis, which is characterized by comprising a planar chip 16 at the bottom and a micro-channel 13 at the top, wherein the planar chip has a four-layer structure which is sequentially overlapped;
The first layer structure 1 of the planar chip comprises electrodes on a glass substrate, wherein the electrodes are interdigital electrodes 2;
The second layer structure 3 of the planar chip comprises a microcavity array 8 for cell capture;
the third layer structure 4 of the planar chip comprises a conductive layer 10 for forming conductive areas at two ends of the interdigital electrode;
The fourth layer structure 5 of the planar chip comprises a microcavity array 12 and a micro baffle 11 positioned in the microcavity array, wherein the microcavity array 12 is used for cell pairing, and the micro baffle 11 is used for cell contact;
The second layer structure (3) and the fourth layer structure (5) also comprise microfluidic channels (9) for flushing cells in each microcavity array to the position of the micro baffle after cell pairing.
In the invention, the interdigital electrode generates a nonuniform electric field under the condition of applying an electric signal, and cells are captured to the surface of the electrode under the action of forward dielectrophoresis force.
According to the present invention, it is a transparent conductive material, preferably any one or a combination of at least two of ITO (indium tin oxide), AZO (aluminum-doped zinc oxide transparent conductive glass) or graphene, and preferably ITO.
Preferably, the number of the interdigital electrodes 2 is 2-10, for example, 2,3, 4,5, 6, 7, 8, 9 or 10, preferably 2.
In the invention, the number of the interdigital electrodes is more than 2, and as more than two kinds of fluorescent cells are captured and paired subsequently, the interdigital electrodes are required to correspond to the types of the fluorescent cells so as to realize the matching of different fluorescent cells and different interdigital electrodes, thereby separately realizing capturing and pairing.
Preferably, the width of the interdigital electrode (2) is 10 to 30. Mu.m, for example, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm, preferably 15 to 25 μm, further preferably 20 μm.
Preferably, the inter-digital electrode (6) has a spacing between two electrodes of 3-10 μm, for example 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm,9 μm or 10 μm, preferably 6 μm.
Preferably, the inter-digital electrodes (6) and (7) are spaced apart by a distance of 5-50 μm, for example 5μm、6μm、7μm、8μm、9μm、10μm、11μm、12μm、13μm、14μm、15μm、16μm、18μm、20μm、22μm、23μm、25μm、28μm、30μm、32μm、33μm、35μm、38μm、40μm、42μm、45μm、48μm or 50 μm, preferably 10 μm.
In the invention, the length of the interdigital electrode can be adjusted according to the size of the planar chip.
In the application, the interdigital electrode with the size can generate a strong enough electric field so as to form enough dielectrophoresis force for attracting single cells, the depth and the distance are related, and the capturing and pairing of the single cells can be realized only within the scope of the application.
According to the invention, the material of the second layer structure 3 is a negative glue, preferably SU-8 negative glue.
Preferably, the diameter of the microcavity array 8 is designed according to the size of the individual cells, and the thickness of the microcavity is related to the size of the dielectrophoretic force, and the smaller the dielectrophoretic force is, the larger the dielectrophoretic force is, the diameter of the microcavity array 8 in the present invention is 10 to 30 μm, for example, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm,20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm, preferably 15 to 25 μm, and more preferably 20 μm.
Preferably, the microcavity array 8 has a depth of 1-20. Mu.m, for example, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 17 μm, 18 μm, 19 μm or 20 μm, preferably 5-15 μm, and more preferably 10 μm.
According to the present invention, the material of the conductive layer 10 is any one or a combination of at least two of copper, silver, chromium or ITO, preferably copper.
Preferably, the conductive layer 10 has a thickness of 0.5-5 μm, for example, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm,1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm or 5 μm, preferably 1 μm.
In the invention, since the conductive layer is located in the third layer structure and the second layer structure is made of negative adhesive material, the second layer structure is provided with the cavity for communicating the conductive layer with the first layer structure for conducting electricity, and the size of the cavity is only required to meet the condition that the conductive layer is communicated with the first layer structure for conducting electricity by a person skilled in the art, and the invention is not limited in particular.
Preferably, the positions of the micro-flow channels 9 on the second layer structure 3 and the fourth layer structure 5 correspond to each other, and the sizes are the same.
In the present invention, the microfluidic channel 9 on the fourth layer structure 5 passes through the micro-baffle 11, and can flow out from the position of the micro-baffle after the liquid has washed out the cells.
Preferably, the microfluidic channel 9 has a width of 3-15 μm, for example 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm or 15 μm, preferably 8 μm.
According to the invention, the material of the fourth layer structure 5 is a negative glue, preferably SU-8 negative glue.
Preferably, the microcavity array 12 has a diameter of 130-200. Mu.m, for example 130 μm, 132 μm, 135 μm, 138 μm, 140 μm, 145 μm, 150 μm, 155 μm, 170 μm, 175 μm, 180 μm, 185 μm, 190 μm, 195 μm or 200 μm, preferably 150-170 μm, further preferably 170 μm.
Preferably, the microcavity array 12 has a depth of 10-30. Mu.m, for example, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm, preferably 15-25 μm, and more preferably 20 μm.
Preferably, the shape of the micro-baffle 11 is a micro-baffle capable of realizing shielding function for the paired cells to be flushed into the microcavity array, and the shape can be adjusted according to the need by a person skilled in the art, and any one or a combination of at least two of semi-circular shape, square shape and rectangle shape is adopted.
Preferably, the size of the micro-barrier 11 is adjusted according to the size of the micro flow channel, the size of the micro-barrier is such that it can shield cells that are flushed from the micro flow channel into the micro cavity array, the width of the micro-barrier 11 of the present application may be 30-50 μm, for example 30 μm, 31 μm, 32 μm, 33 μm, 35 μm, 36 μm, 38 μm, 40 μm, 41 μm, 43 μm, 45 μm, 48 μm or 50 μm, preferably 40 μm, and the length of the micro-barrier 11 may be 50-70 μm, for example 50 μm, 52 μm, 53 μm, 55 μm, 56 μm, 58 μm, 60 μm, 62 μm, 65 μm, 68 μm or 70 μm, preferably 60 μm.
According to the present invention, the material of the micro flow channel 13 is a transparent silica gel material, preferably PDMS.
Preferably, the micro flow channel 13 includes a sample injection flow channel 14, a sample outlet flow channel 15 and a working flow channel 17.
Preferably, the sample inlet channel 14 and the sample outlet channel 15 are branched step by step.
Preferably, the depth of the injection flow channel 14 is 100-500 μm, for example 100μm、102μm、103μm、105μm、110μm、115μm、120μm、125μm、130μm、132μm、135μm、138μm、140μm、145μm、150μm、160μm、170μm、180μm、200μm、220μm、250μm、260μm、280μm、300μm、320μm、350μm、380μm、400μm、420μm、450μm、480μm or 500 μm, preferably 120-200 μm, and more preferably 125 μm.
Preferably, the depth of the sample outlet channel 15 is 100-500 μm, for example 100μm、102μm、103μm、105μm、110μm、115μm、120μm、125μm、130μm、132μm、135μm、138μm、140μm、145μm、150μm、160μm、170μm、180μm、200μm、220μm、250μm、260μm、280μm、300μm、320μm、350μm、380μm、400μm、420μm、450μm、480μm or 500 μm, preferably 120-200 μm, preferably 120-130 μm, and more preferably 125 μm.
Preferably, the depth of the working channel 17 is 50-150 μm, for example 50μm、51μm、53μm、55μm、58μm、60μm、61μm、63μm、65μm、68μm、70μm、75μm、80μm、85μm、90μm、95μm、100μm、105μm、110μm、115μm、120μm、125μm、130μm、135μm、140μm、145μm or 150 μm, preferably 70-100 μm, more preferably 80 μm.
In a second aspect, the present invention provides a method for preparing a microfluidic chip according to the first aspect, comprising the steps of:
(1) Preparing a four-layer structure of the planar chip by adopting a photoetching method, a wet etching method and a magnetron sputtering method;
(2) Preparing a mould of the micro-channel by adopting a 3D printing mode, pouring a PDMS precursor in the mould, and performing heat drying at 50-80 ℃ preferably at 55 ℃ for 1-5 hours, preferably 3 hours to obtain the micro-channel;
(3) And assembling the planar chip and the micro-flow channel to obtain the micro-fluidic chip.
In the invention, a specific first layer structure is prepared by combining a photolithography method and wet etching, a second layer structure is prepared by using the photolithography method, a third layer structure is prepared by using a magnetron sputtering method, and a fourth layer structure is prepared by using the photolithography method.
In a third aspect, the present invention provides a microfluidic chip as described in the first aspect for single cell capture and pairing.
According to the invention, the cell capture and pairing comprises the following steps:
1) Preparing two cell dispersions of fluorescent markers with different colors;
2) In a sample injection flow channel 14 of a micro flow channel 13 on a green fluorescent cell dispersion liquid micro flow control chip, a nonuniform electric field is generated between interdigital electrodes A6 applying sinusoidal alternating current signals, so that cells are captured into a microcavity array 8 on one side under the action of positive dielectrophoresis force, and redundant cells are washed away by using buffer liquid, so that the capture of the green fluorescent cell array is realized;
3) Red fluorescent cell dispersion liquid is added into a sample injection flow channel 14 of a micro flow channel 13 on a micro flow control chip, a nonuniform electric field is generated between interdigital electrodes B7 applying sinusoidal alternating current signals, so that cells are captured into a microcavity array 8 at the other side under the action of positive dielectrophoresis force, redundant cells are washed away by using buffer liquid, capturing of the red fluorescent cell array is realized, and meanwhile, two fluorescent cells form cell pairing;
4) The micro flow channel 13 is tightly attached to the planar chip 16 by a clamp, buffer solution is introduced from the sample injection flow channel 14 and flows in through the micro flow channel 9, and two paired cells in each microcavity array 8 are flushed onto the micro baffle 11 of the microcavity array 12, so that the contact of the cells is realized.
Preferably, the preparation of the cell dispersion in step 1) specifically comprises: the cells were stained with two fluorescent labeling dyes, respectively, and the red-and green-emitting cells were dispersed in a low conductivity buffer to give a dispersion of cell concentration of 2X 10 5-2×107/mL, preferably 2X 10 6/mL.
Preferably, the flow rate of the green fluorescent cell dispersion liquid in the step 2) in the sample injection flow channel is 5-20. Mu.L/min, for example 5μL/min、6μL/min、7μL/min、8μL/min、9μL/min、10μL/min、11μL/min、12μL/min、13μL/min、14μL/min、15μL/min、17μL/min、17μL/min、18μL/min、19μL/min or 20. Mu.L/min, preferably 10. Mu.L/min.
Preferably, the capturing time in step 2) is 1-10min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min, preferably 3-5min.
Preferably, the flow rate of the red fluorescent cell dispersion liquid in the step 3) in the sample injection flow channel is 5-20 mu L/min, for example 5μL/min、6μL/min、7μL/min、8μL/min、9μL/min、10μL/min、11μL/min、12μL/min、13μL/min、14μL/min、15μL/min、17μL/min、17μL/min、18μL/min、19μL/min or 20 mu L/min, preferably 10 mu L/min.
Preferably, the capturing time in the step 3) is 1-10min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min, preferably 3-5min.
Preferably, the sinusoidal ac signal in step 2) and step 3) is a sinusoidal ac, and the peak voltage of the ac is 5-20Vpp, which may be, for example, 5Vpp, 6Vpp, 7Vpp, 8Vpp, 9Vpp, 10Vpp, 11Vpp, 12Vpp, 13Vpp, 14Vpp, 15Vpp, 16Vpp, 17Vpp, 18Vpp, 19Vpp or 20Vpp, preferably 12Vpp.
Preferably, the frequency of the alternating current is 2-10MHz, which may be, for example, 2MHz, 3MHz, 4MHz, 5MHz, 6MHz, 7MHz, 8MHz, 9MHz or 10MHz, preferably 4MHz.
The working principle of the microfluidic chip comprises the following steps:
1) Two cell dispersions of fluorescent markers of different colors were prepared: respectively staining cells with two fluorescent labeling dyes, and dispersing the two cells emitting red light and green light in a low-conductivity buffer solution to obtain a dispersion liquid with the cell concentration of 2X 10 5-2×107/mL;
2) In a sample injection flow channel 14 of a micro flow channel 13 on a green fluorescent cell dispersion microfluidic chip, the flow speed is 5-20 mu L/min, a nonuniform electric field is generated between interdigital electrodes 6 applying sinusoidal alternating current signals, so that cells are captured into a microcavity array 8 at one side under the action of positive dielectrophoresis force for 1-10min, and redundant cells are washed away by using buffer solution, so that the capture of the green fluorescent cell array is realized;
3) Adding red fluorescent cell dispersion liquid into a sample injection flow channel 14 of a micro flow channel 13 on a micro flow control chip, wherein the flow speed is 5-20 mu L/min, generating a non-uniform electric field between interdigital electrodes 7 applying sinusoidal alternating current signals, further capturing cells into a microcavity array 8 at the other side under the action of positive dielectrophoresis force for 1-10min, flushing redundant cells by using buffer solution, capturing the red fluorescent cell array, and simultaneously forming cell pairing by two fluorescent cells;
Wherein, the voltage peak value of the alternating current applied with the sinusoidal alternating current signal is 5-20Vpp, and the frequency of the alternating current is 2-10MHz;
4) The micro flow channel 13 is tightly attached to the planar chip 17 by a clamp, buffer solution is introduced from the sample injection flow channel 14 and flows in through the micro flow channel 9, and two paired cells in each microcavity array 8 are flushed onto the micro baffle 11 of the microcavity array 12, so that the contact of the cells is realized.
Compared with the prior art, the invention has the following beneficial effects:
(1) The planar structure of the microfluidic chip is easy to package and has high flux;
(2) The microfluidic chip provided by the invention is used for capturing and pairing single cells with high flux, the pairing efficiency is high, the pairing efficiency can reach 60%, and the local pairing efficiency can reach 80%.
Drawings
Fig. 1 is a schematic structural diagram of a planar chip in a dielectrophoresis-based microfluidic chip according to the present invention; wherein, in the figure, the structure comprises a 1-first layer structure, a 2-interdigital electrode, a 3-second layer structure, a 4-third layer structure and a 5-fourth layer structure;
FIG. 2 is a schematic view of a first layer structure of a planar chip according to the present invention; wherein, 6-interdigital electrode A and 7-interdigital electrode B in the figure;
FIG. 3 is a schematic diagram of a second layer structure of a planar chip according to the present invention; wherein, 8-microcavity array, 9-microfluidic channel in the figure;
FIG. 4 is a schematic structural view of a third layer structure of the planar chip of the present invention; wherein 10-is a conductive layer;
FIG. 5 is a schematic diagram of a fourth layer structure of the planar chip of the present invention; wherein, in the figure, the array of 11-micro baffle plates and 12-micro cavities;
Fig. 6 is a schematic structural diagram of a dielectrophoresis-based microfluidic chip according to the present invention; in the figure, a 13-micro flow channel, a 14-sample injection flow channel, a 15-sample discharge flow channel, a 16-plane chip and a 17-working flow channel are shown;
FIG. 7 is a graph showing the results of cell pairing according to the invention.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below by the specific embodiments in combination with the accompanying drawings, but the invention is not limited to the examples.
Example 1: microfluidic chip based on dielectrophoresis
Fig. 6 is a schematic diagram of a micro-fluidic chip based on dielectrophoresis according to an embodiment of the present invention, where a specific structure of the micro-fluidic chip is shown in fig. 1 to 5, and the micro-fluidic chip includes a planar chip 16 at the bottom and a micro-channel 13 at the top, and the planar chip has a four-layer structure stacked in sequence;
The first layer structure 1 of the planar chip comprises ITO electrodes on a glass substrate, wherein the ITO electrodes are interdigital electrodes 2, the number of the interdigital electrodes 2 is 2-10, the width of each interdigital electrode 2 is 10-30 mu m, the interval between two electrodes in each interdigital electrode 6 is 3-10 mu m, and the interval between each interdigital electrode 6 and each interdigital electrode 7 is 5-50 mu m;
The second layer structure 3 of the planar chip is SU-8 negative adhesive, the second layer structure 3 comprises a microcavity array 8 for capturing cells, the diameter of the microcavity array 8 is 10-30 mu m, and the depth of the microcavity array 8 is 1-20 mu m;
the third layer structure 4 of the planar chip comprises a copper conductive layer 10 for forming conductive areas at two ends of the interdigital electrode, wherein the thickness of the copper conductive layer 10 is 0.5-5 mu m;
The fourth layer structure 5 of the planar chip comprises a microcavity array 12 and a micro baffle 11 positioned in the microcavity array, wherein the microcavity array 12 is used for cell pairing, the micro baffle 11 is used for cell contact, the diameter of the microcavity array 12 is 130-200 mu m, the depth of the microcavity array 12 is 10-30 mu m, the micro baffle 11 is semicircular, the width of the micro baffle 11 is 30-50 mu m, and the length of the micro baffle 11 is 50-70 mu m.
The second layer structure 3 and the fourth layer structure 5 further comprise micro-flow channels 9, which are used for flushing cells in each micro-cavity array to the positions of the micro-baffles after the cells are paired, and the width of each micro-flow channel 9 is 3-15 mu m;
The micro flow channel 13 comprises a sample injection flow channel 14, a sample outlet flow channel 15 and a working flow channel 17;
The sample inlet flow channel 14 and the sample outlet flow channel 15 are branched step by step;
the depth of the sample injection flow channel 14 is 100-500 mu m, and the depth of the sample outlet flow channel 15 is 100-500 mu m;
The depth of the working channel 17 is 50-150 μm.
The working principle of the microfluidic chip comprises the following steps:
1) Two cell dispersions of fluorescent markers of different colors were prepared: respectively staining cells with two fluorescent labeling dyes, and dispersing the two cells emitting red light and green light in a low-conductivity buffer solution to obtain a dispersion liquid with the cell concentration of 2X 10 5-2×107/mL;
2) In a sample injection flow channel 14 of a micro flow channel 13 on a green fluorescent cell dispersion microfluidic chip, the flow speed is 5-20 mu L/min, a nonuniform electric field is generated between interdigital electrodes 6 applying sinusoidal alternating current signals, so that cells are captured into a microcavity array 8 at one side under the action of positive dielectrophoresis force for 1-10min, and redundant cells are washed away by using buffer solution, so that the capture of the green fluorescent cell array is realized;
3) Adding red fluorescent cell dispersion liquid into a sample injection flow channel 14 of a micro flow channel 13 on a micro flow control chip, wherein the flow speed is 5-20 mu L/min, generating a non-uniform electric field between interdigital electrodes 7 applying sinusoidal alternating current signals, further capturing cells into a microcavity array 8 at the other side under the action of positive dielectrophoresis force for 1-10min, flushing redundant cells by using buffer solution, capturing the red fluorescent cell array, and simultaneously forming cell pairing by two fluorescent cells;
Wherein, the voltage peak value of the alternating current applied with the sinusoidal alternating current signal is 5-20Vpp, and the frequency of the alternating current is 2-10MHz;
4) The micro flow channel 13 is tightly attached to the planar chip 17 by a clamp, buffer solution is introduced from the sample injection flow channel 14 and flows in through the micro flow channel 9, and two paired cells in each microcavity array 8 are flushed onto the micro baffle 11 of the microcavity array 12, so that the contact of the cells is realized.
Example 2: microfluidic chip based on dielectrophoresis
The microfluidic chip comprises a planar chip 16 at the bottom and a micro-channel 13 at the top, and the planar chip has a four-layer structure which is sequentially overlapped;
The first layer structure 1 of the planar chip comprises ITO electrodes on a glass substrate, wherein the ITO electrodes are interdigital electrodes 2, the number of the interdigital electrodes 2 is 2, the width of each interdigital electrode 2 is 20 mu m, the interval between two electrodes in each interdigital electrode 6 is 6 mu m, and the interval between each interdigital electrode 6 and each interdigital electrode 7 is 10 mu m;
the second layer structure 3 of the planar chip is SU-8 negative adhesive, the second layer structure 3 comprises a microcavity array 8 for capturing cells, the diameter of the microcavity array 8 is 20 mu m, and the depth of the microcavity array 8 is 10 mu m;
The third layer structure 4 of the planar chip comprises a copper conductive layer 10 for forming conductive areas at two ends of the interdigital electrode, wherein the thickness of the copper conductive layer 10 is 1 μm;
The fourth layer structure 5 of the planar chip comprises a microcavity array 12 and a micro baffle 11 positioned in the microcavity array, wherein the microcavity array 12 is used for cell pairing, the micro baffle 11 is used for cell contact, the diameter of the microcavity array 12 is 170 mu m, the depth of the microcavity array 12 is 20 mu m, the shape of the micro baffle 11 is semicircular, the width of the micro baffle 11 is 40 mu m, and the length of the micro baffle 11 is 60 mu m;
A micro-flow channel 9 is further arranged between the second layer structure 3 and the third layer structure 4 and is used for flushing the cells in each microcavity array to the position of the micro-baffle after the cells are paired, and the width of the micro-flow channel 9 is 8 mu m;
The micro flow channel 13 comprises a sample injection flow channel 14, a sample outlet flow channel 15 and a working flow channel 17;
The sample inlet flow channel 14 and the sample outlet flow channel 15 are branched step by step;
The depth of the sample injection flow channel 14 is 125 μm, and the depth of the sample outlet flow channel 15 is 125 μm;
the depth of the working channel 17 is 80 μm.
Example 3: cell capture and pairing
The method for capturing and pairing cells by using the dielectrophoresis-based microfluidic chip described in example 2 comprises the following specific steps:
1) Two cell dispersions of fluorescent markers of different colors were prepared: respectively staining cells with two fluorescent labeling dyes, and dispersing the two cells emitting red light and green light in a low-conductivity buffer solution to obtain a dispersion liquid with the cell concentration of 2X 10 6/mL;
2) In a sample injection flow channel 14 of a micro flow channel 13 on a green fluorescent cell dispersion microfluidic chip, the flow speed is 10 mu L/min, a nonuniform electric field is generated between interdigital electrodes 6 applying sinusoidal alternating current signals, so that cells are captured into a microcavity array 8 on one side under the action of positive dielectrophoresis force for 3-5min, and redundant cells are washed away by using buffer solution, so that the capture of the green fluorescent cell array is realized;
3) Adding red fluorescent cell dispersion liquid into a sample injection flow channel 14 of a micro flow channel 13 on a micro flow control chip, wherein the flow speed is 10 mu L/min, and a nonuniform electric field is generated between interdigital electrodes 7 applying sinusoidal alternating current signals, so that cells are captured into a microcavity array 8 at the other side under the action of positive dielectrophoresis force for 3-5min, and extra cells are washed away by using buffer solution to capture the red fluorescent cell array, and meanwhile, two fluorescent cells form cell pairing;
Wherein, the voltage peak value of the alternating current applied with the sinusoidal alternating current signal is 12Vpp, and the frequency of the alternating current is 4MHz;
4) The micro flow channel 13 is tightly attached to the planar chip 17 by a clamp, buffer solution is introduced from the sample injection flow channel 14 and flows in through the micro flow channel 9, and two paired cells in each microcavity array 8 are flushed onto the micro baffle 11 of the microcavity array 12, so that the contact of the cells is realized.
Detecting the capturing efficiency of the cells by using a fluorescence microscope, capturing green fluorescent cells, exciting the green fluorescent cells by using blue laser to emit green light, recording images of the green fluorescent cells, capturing red fluorescent cells, exciting the red fluorescent cells by using the green laser to emit red light, recording images of the red fluorescent cells, combining the two fluorescent cell images together by using software, and calculating the pairing efficiency of the single cells.
The results are shown in FIG. 7, and the calculated cell pairing efficiency after pairing by single cell capture is 80%.
Example 4
The other components were the same as in example 2 except that the width of the interdigital electrode 2 was 10 μm, the pitch between two electrodes in the interdigital electrode 6 was 3 μm, and the pitch between the interdigital electrode 6 and the interdigital electrode 7 was 5 μm, as compared with example 2.
Example 5
The other components were the same as in example 2 except that the width of the interdigital electrode 2 was 30 μm, the pitch between two electrodes in the interdigital electrode 6 was 10 μm, and the pitch between the interdigital electrode 6 and the interdigital electrode 7 was 50 μm, as compared with example 2.
Comparative example 1
The procedure was the same as in example 2 except that the gap between the two electrodes in the interdigital electrode 6 was 15. Mu.m, as compared with example 2.
Comparative example 2
The procedure was the same as in example 2 except that the pitch between the interdigital electrode 6 and the interdigital electrode 7 was 55. Mu.m.
Comparative example 3
In comparison with example 2, the other components were the same as in example 2 except that the pitch between the interdigital electrode 6 and the interdigital electrode 7 was 1. Mu.m, and the interdigital electrodes were too closely spaced to form a sufficient dielectrophoresis force.
Comparative example 4
The procedure was the same as in example 2 except that the depth of the microcavity array 8 was 25. Mu.m, as compared with example 2.
Comparative example 5
The structure was the same as that of example 2, and the dielectrophoresis force was too small to capture cells as in example 3, except that the voltage peak value 2Vpp of the alternating current to which the sinusoidal alternating current signal was applied was the same as that of example 3.
The results of the tests of example 2, examples 4-5 and comparative examples 1,2, 4 are shown in Table 1 below:
TABLE 1
| Example 2 | Example 4 | Example 5 | Comparative example 1 | Comparative example 2 | Comparative example 4 | |
| Pairing efficiency | 80％ | 68％ | 65％ | 48％ | 43％ | 40％ |
As can be seen from table 1, the size of the interdigital electrode has an effect on the pairing efficiency, and the pairing efficiency in example 2 can reach 80%, but when the size of the interdigital electrode is not within the scope of the present application, it cannot generate a sufficient electric field, a sufficient dielectrophoresis force for attracting single cells cannot be formed, and the respective sizes of the interdigital electrodes are interrelated, and only within the scope of the interdigital electrode of the present application, effective pairing of single cells can be achieved.
According to the method, the pairing efficiency is high, the pairing efficiency can reach more than 60%, and the local pairing efficiency can reach 80%.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (17)
1. The micro-fluidic chip based on dielectrophoresis is characterized by comprising a planar chip (16) at the bottom and a micro-channel (13) at the top, wherein the planar chip has a four-layer structure which is sequentially overlapped;
the first layer structure (1) of the planar chip comprises electrodes on a glass substrate, wherein the electrodes are interdigital electrodes (2);
The second layer structure (3) of the planar chip comprises a microcavity array (8) for cell capture;
the third layer structure (4) of the planar chip comprises a conductive layer (10) for forming conductive areas at both ends of the interdigital electrode;
The fourth layer structure (5) of the planar chip comprises a microcavity array (12) and a micro baffle (11) positioned in the microcavity array (12), wherein the microcavity array (12) is used for cell pairing, and the micro baffle (11) is used for cell contact;
The second layer structure (3) and the fourth layer structure (5) also comprise micro-flow channels (9) which are used for flushing cells in each micro-cavity array (8) to the positions of micro-baffles after the cells are paired;
The electrode is made of any one or a combination of at least two of ITO, AZO or graphene;
The number of the interdigital electrodes (2) is 2-10;
the width of the interdigital electrode (2) is 10-30 mu m;
The interval between two electrodes in the interdigital electrode (6) is 3-10 mu m;
the interval between the interdigital electrode (6) and the interdigital electrode (7) is 5-50 mu m;
the material of the second layer structure (3) is negative adhesive;
the diameter of the microcavity array (8) is 10-30 μm;
The depth of the microcavity array (8) is 1-20 mu m;
The microcavity array (12) has a diameter of 130-200 μm;
the microcavity array (12) has a depth of 10-30 μm;
The width of the micro baffle plate (11) is 30-50 mu m;
the length of the micro baffle plate (11) is 50-70 mu m.
2. Microfluidic chip according to claim 1, characterized in that the number of interdigital electrodes (2) is 2;
the width of the interdigital electrode (2) is 15-25 mu m;
The interval between two electrodes in the interdigital electrode (6) is 6 mu m;
the interval between the interdigital electrode (6) and the interdigital electrode (7) is 10 mu m;
The material of the second layer structure (3) is SU-8 negative adhesive;
the diameter of the microcavity array (8) is 15-25 μm;
the microcavity array (8) has a depth of 5-15 μm.
3. The microfluidic chip according to claim 2, wherein the material of the electrode is ITO;
the width of the interdigital electrode (2) is 20 mu m;
the microcavity array (8) has a diameter of 20 μm;
the microcavity array (8) has a depth of 10 μm.
4. A microfluidic chip according to any one of claims 1-3, characterized in that the material of the conductive layer (10) is any one or a combination of at least two of copper, silver, chromium or ITO;
The thickness of the conductive layer (10) is 0.5-5 mu m;
The positions of the micro-flow channels (9) on the second layer structure (3) and the fourth layer structure (5) correspond to each other, and the sizes are the same;
the width of the microfluidic channel (9) is 3-15 μm.
5. The microfluidic chip according to claim 4, wherein the material of the conductive layer (10) is copper;
The thickness of the conductive layer (10) is 1 [ mu ] m;
the width of the microfluidic channel (9) is 8 μm.
6. A microfluidic chip according to any one of claims 1-3, wherein the material of the fourth layer structure (5) is a negative glue;
the shape of the micro baffle plate (11) is any one or a combination of at least two of semicircle, square or rectangle.
7. The microfluidic chip according to claim 6, wherein the material of the fourth layer structure (5) is SU-8 negative glue;
the microcavity array (12) has a diameter of 150-170 μm;
The microcavity array (12) has a depth of 15-25 μm;
the width of the micro baffle plate (11) is 40 mu m;
the length of the micro baffle plate (11) is 60 mu m.
8. The microfluidic chip according to claim 7, wherein the microcavity array (12) has a diameter of 170 μm;
the microcavity array (12) has a depth of 20 μm.
9. A microfluidic chip according to any one of claims 1-3, wherein the material of the micro flow channels (13) is a transparent silica gel type material;
The micro flow channel (13) comprises a sample injection flow channel (14), a sample outlet flow channel (15) and a working flow channel (17);
The sample inlet flow channel (14) and the sample outlet flow channel (15) are branched step by step;
the depth of the sample injection flow channel (14) is 100-500 mu m;
the depth of the sample outlet flow channel (15) is 100-500 mu m;
the depth of the working flow channel (17) is 50-150 mu m.
10. The microfluidic chip according to claim 9, wherein the material of the micro flow channels (13) is PDMS;
the depth of the sample injection flow channel (14) is 120-200 mu m;
the depth of the sample outlet flow channel (15) is 120-200 mu m;
The depth of the working flow channel (17) is 70-100 mu m.
11. The microfluidic chip according to claim 10, wherein,
The depth of the sample injection flow channel (14) is 125 mu m;
The depth of the sample outlet flow channel (15) is 125 mu m;
the depth of the working flow channel (17) is 80 mu m.
12. A method of manufacturing a microfluidic chip according to any one of claims 1 to 11, comprising the steps of:
(1) Preparing a four-layer structure of the planar chip by adopting a photoetching method, a wet etching method and a magnetron sputtering method;
(2) Preparing a mould of the micro-channel by adopting a 3D printing mode, pouring a PDMS precursor into the mould, and thermally drying at 50-80 ℃ for 1-5 hours to obtain the micro-channel;
(3) And assembling the planar chip and the micro-flow channel to obtain the micro-fluidic chip.
13. The method for manufacturing a microfluidic chip according to claim 12, wherein,
And pouring the PDMS precursor into a die, and heating at 55 ℃ for 3 hours to obtain the micro-channel.
14. Use of a microfluidic chip according to any one of claims 1-11 for single cell capture and pairing.
15. The use according to claim 14, characterized by the steps of:
1) Preparing two cell dispersions of fluorescent markers with different colors;
2) In a sample injection flow channel (14) of a micro flow channel (13) on a green fluorescent cell dispersion liquid micro flow control chip, a nonuniform electric field is generated between interdigital electrodes A (6) applying sinusoidal alternating current signals, so that cells are captured into a microcavity array (8) on one side under the action of positive dielectrophoresis force, and redundant cells are washed away by using buffer liquid, so that the capture of the green fluorescent cell array is realized;
3) Adding red fluorescent cell dispersion liquid into a sample injection flow channel (14) of a micro flow channel (13) on a micro flow control chip, generating a non-uniform electric field between interdigital electrodes B (7) applying sinusoidal alternating current signals, further capturing cells into a microcavity array (8) at the other side under the action of positive dielectrophoresis force, flushing away redundant cells by using buffer liquid, capturing the red fluorescent cell array, and simultaneously forming cell pairing by two fluorescent cells;
4) The micro flow channels (13) are tightly attached to the planar chip (16) by a clamp, buffer solution is introduced from the sample injection flow channels (14), the buffer solution flows in through the micro flow channels (9), and two paired cells in each micro cavity array (8) are flushed onto the micro baffle plates (11) of the micro cavity array (12), so that the contact of the cells is realized.
16. The use according to claim 15, wherein the preparation of the cell dispersion of step 1) comprises in particular: respectively staining cells with two fluorescent labeling dyes, and dispersing the two cells emitting red light and green light in a low-conductivity buffer solution to obtain a dispersion liquid with the cell concentration of 2X 10 5-2×107/mL;
The flow rate of the green fluorescent cell dispersion liquid in the step 2) in a sample injection flow channel is 5-20 mu L/min;
the capturing time in the step 2) is 1-10min;
the flow rate of the red fluorescent cell dispersion liquid in the step 3) in a sample injection flow channel is 5-20 mu L/min;
the capturing time in the step 3) is 1-10min;
the sinusoidal alternating current signals in the step 2) and the step 3) are sinusoidal pulse alternating current, and the voltage peak-to-peak value of the alternating current is 5-20Vpp;
The frequency of the alternating current is 2-10MHz.
17. The use according to claim 16, wherein the preparation of the cell dispersion of step 1) comprises in particular: respectively staining cells with two fluorescent labeling dyes, and dispersing the two cells emitting red light and green light in a low-conductivity buffer solution to obtain a dispersion liquid with the cell concentration of 2X 10 6/mL;
The flow rate of the green fluorescent cell dispersion liquid in the step 2) in a sample injection flow channel is 10 mu L/min;
the capturing time in the step 2) is 3-5min;
the flow rate of the red fluorescent cell dispersion liquid in the step 3) in a sample injection flow channel is 10 mu L/min;
The capturing time in the step 3) is 3-5min;
The sinusoidal alternating current signals in the step 2) and the step 3) are sinusoidal pulse alternating current, and the voltage peak-to-peak value 12Vpp of the alternating current;
The frequency of the alternating current is 4MHz.
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