CN108977343B - Micro-fluidic chip for cell separation and capture based on dielectrophoresis principle - Google Patents
Micro-fluidic chip for cell separation and capture based on dielectrophoresis principle Download PDFInfo
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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
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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
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Abstract
A micro-fluidic chip for cell separation and capture based on a dielectrophoresis principle belongs to the technical field of micro-fluidic and solves the problems that the existing cell analysis work is low in efficiency due to long time consumption of a cell sorting link and non-continuity of the cell sorting link and a single cell capturing link. The micro-fluidic chip for cell separation and capture based on the dielectrophoresis principle applies an electric field to a cell mixed solution in a separation area through an inclined driving electrode array, utilizes the difference of dielectric properties among different cells, and sorts the different cells based on positive dielectrophoresis force and negative dielectrophoresis force. After the cell sorting is completed, the micro-fluidic chip for cell separation and capture based on the dielectrophoresis principle realizes the single cell capture by arranging the bipolar electrode array in the capture area.
Description
Technical Field
The invention relates to a micro-fluidic chip, and belongs to the technical field of micro-fluidic.
Background
Cell sorting and single cell capture are necessary pretreatment links in cell analysis work. The existing cell sorting mode mainly comprises fluorescence activated cell sorting and magnetic field activated cell sorting, however, both the fluorescence activated cell sorting and the magnetic field activated cell sorting require complicated sample processing steps. For example, cells need to be fluorescently labeled prior to fluorescence activated cell sorting and magnetically labeled prior to magnetic field activated cell sorting. Therefore, the existing cell sorting method takes a long time, and the efficiency of cell analysis work is seriously reduced.
On the other hand, the existing cell sorting and single cell capturing are realized by corresponding experimental instruments respectively, so that the cell transfer link after cell sorting is increased, and the efficiency of cell analysis work is reduced to a certain extent.
A Micro fluidic Chip (also called Lab-on-a-Chip) refers to a Chip that integrates basic operation units related to biological and chemical fields, such as sample preparation, reaction, separation, detection, etc., or basically on a Chip of several square centimeters (even smaller), and a network is formed by microchannels to automatically complete an analysis process, and has been developed into a research field with a cross application prospect in multiple disciplines, such as machinery, chemistry, biology, medicine, hydromechanics, etc.
Disclosure of Invention
The invention provides a micro-fluidic chip for separating and capturing cells based on a dielectrophoresis principle, which aims to solve the problems that the existing cell analysis work is low in efficiency due to longer time consumption in a cell sorting link and non-continuity between the cell sorting link and a single cell capturing link.
The invention relates to a micro-fluidic chip for cell separation and capture based on a dielectrophoresis principle, which comprises a glass substrate 1, a PDMS cover plate 2, a driving electrode array 3 and first to third bipolar electrode arrays 4 to 6;
the lower surface of the PDMS cover plate 2 is provided with a first flow channel 7 to a ninth flow channel 15, a separation zone 16, a first capture zone 17 to a third capture zone 19, a first inflow groove 20 to a third inflow groove 22 and a first outflow groove 23 to a third outflow groove 25 in an invagination manner;
the first inflow groove 20 to the third inflow groove 22 are respectively communicated with an inflow port of the first flow channel 7 to an inflow port of the third flow channel 9, an outflow port of the first flow channel 7 to an outflow port of the third flow channel 9 are respectively communicated with an inflow port of the separation region 16, an outflow port of the separation region 16 is simultaneously communicated with an inflow port of the fourth flow channel 10 to an inflow port of the sixth flow channel 12, an outflow port of the fourth flow channel 10 to an outflow port of the sixth flow channel 12 are respectively communicated with an inflow port of the first trapping region 17 to an inflow port of the third trapping region 19, an outflow port of the first trapping region 17 to an outflow port of the third trapping region 19 are respectively communicated with an inflow port of the seventh flow channel 13 to an inflow port of the ninth flow channel 15, and an outflow port of the seventh flow channel 13 to an outflow port of the ninth flow channel 15 are respectively communicated with the first outflow groove 23 to the third outflow groove 25;
first inflow through holes to third inflow through holes are respectively formed in the groove bottom of the first inflow groove 20 to the groove bottom of the third inflow groove 22, first outflow through holes to third outflow through holes are respectively formed in the groove bottom of the first outflow groove 23 to the groove bottom of the third outflow groove 25, and the first inflow through holes to the third inflow through holes and the first outflow through holes to the third outflow through holes all penetrate through the PDMS cover plate 2;
first to sixth metal connectors are respectively arranged on the inflow ports of the first to third inflow through holes and the outflow ports of the first to third outflow through holes;
the driving electrode array 3 and the first to third bipolar electrode arrays 4 to 6 are all arranged on the upper surface of the glass substrate 1, and the upper surface of the glass substrate 1 is opposite to and closely attached to the lower surface of the PDMS cover plate 2;
the driving electrode array comprises a first driving electrode 26 to an eighth driving electrode 33, the first driving electrode 26, the eighth driving electrode 33, a second driving electrode 27, a seventh driving electrode 32, a third driving electrode 28, a sixth driving electrode 31, a fourth driving electrode 29 and a fifth driving electrode 30 respectively form a first splayed structure to a fourth splayed structure, the first splayed structure to the fourth splayed structure are sequentially distributed between an outflow port of the separation area 16 and an inflow port of the separation area 16 in a row, small opening sides of the first splayed structure to the fourth splayed structure face the outflow port of the separation area 16 and are opposite to the inflow port of the fifth flow channel 11, and two ends of the first splayed structure from the large opening side to two ends of the fourth splayed structure from the large opening side respectively exceed two sides of the separation area 16;
both ends of the large opening side of the first splayed structure and both ends of the large opening side of the third splayed structure are connected with alternating voltage, and both ends of the large opening side of the second splayed structure and both ends of the large opening side of the fourth splayed structure are grounded;
the first bipolar electrode array 4 to the third bipolar electrode array 6 are respectively positioned in the coverage range of the first capture area 17 to the third capture area 19;
the first metal connector to the third metal connector are respectively a channel for allowing cell mixed liquor, buffer solution and cell mixed liquor to enter the microfluidic chip;
the two cells contained in the cell mixture have different dielectric constants.
Preferably, the first to eighth driving electrodes 26 to 33 have the same structure, and the opening distances on the small opening side of the first to fourth splay structures are equal to each other.
Preferably, each of the splayed structures is connected to a voltage source or power ground via lead electrodes 34.
Preferably, the first bipolar electrode array 4 to the third bipolar electrode array 6 have the same structure and are all wireless bipolar electrode arrays;
the first bipolar electrode array 4 comprises ninth to twelfth driving electrodes 35 to 38 and a bipolar electrode array body 39;
alternating voltages are applied to two ends of the ninth driving electrode 35 and two ends of the twelfth driving electrode 38;
for the ninth drive electrode 35 to the twelfth drive electrode 38, the phase angle of the voltage across the former and the phase angle of the voltage across the latter differ by 90 °;
after power is supplied, the ninth to twelfth driving electrodes 35 to 38 are commonly used for driving the bipolar electrode array body 39.
Preferably, the lower surface of the PDMS cover plate 2 is further provided with tenth to fifteenth flow channels and first to sixth auxiliary grooves in an inward recessed manner;
the first auxiliary groove and the second auxiliary groove are respectively communicated with the first capturing area 17 through a tenth flow passage and an eleventh flow passage, the third auxiliary groove and the fourth auxiliary groove are respectively communicated with the second capturing area 18 through a twelfth flow passage and a thirteenth flow passage, and the fifth auxiliary groove and the sixth auxiliary groove are respectively communicated with the third capturing area 19 through a fourteenth flow passage and a fifteenth flow passage.
Preferably, separation region 16 is rectangular, and the width of the inflow and outflow ports of separation region 16 are equal to the width of separation region 16;
the separation zone 16 has a length L and a width W of 4000 μm and 1400 μm, respectively;
width W of outflow port of first flow channel 7i1Outflow port W of third flow channel 9i3Are 500 μm, 400 μm and 500 μm, respectively;
width W of inflow port of fourth flow channel 10o1Width W of inflow port of sixth flow path 12o3550 μm, 300 μm and 550 μm, respectively;
minimum spacing L between the small open side of the first splayed configuration and the outflow port of the separation zone 16dAnd 300 μm.
Preferably, the bipolar electrode array body 39 is square, and the ninth to twelfth driving electrodes 35 to 38 are equal in length and are all 1400 μm;
the ninth to twelfth driving electrodes 35 to 38 are distributed around the bipolar electrode array body 39 in the clockwise direction, and are parallel to four sides of the bipolar electrode array body 39, respectively;
the ninth to twelfth driving electrodes 35 to 38 are all equal to the bipolar electrode array body 39 in minimum distance;
the minimum separation G between the ninth drive electrode 35 and the eleventh drive electrode 37 is 2000 μm.
Preferably, the first to third inflow grooves 20 to 22, the first to third trapping regions 17 to 19, the first to third outflow grooves 23 to 25, and the first to sixth auxiliary grooves are all circular and have diameters of 5000 μm.
Preferably, the height of the invaginated region of the PDMS cover plate 2 is 20 μm.
Preferably, the electrodes related to the microfluidic chip are all ITO thin film electrodes or metal thin film electrodes.
The micro-fluidic chip for separating and capturing cells based on the dielectrophoresis principle realizes the cell sorting of cell mixed liquid by arranging the driving electrode array 3 in the separation area 16, and realizes the single-cell capturing link by respectively arranging the first bipolar electrode array 4 to the third bipolar electrode array 6 in the first capturing area 17 to the third capturing area 19.
Among them, the basic principle of cell sorting is:
a part of cell mixed liquor enters the separation region 16 through the first metal connector, the first inflow through hole, the first inflow groove 20 and the first flow channel 7 in sequence, buffer solution enters the separation region 16 through the second metal connector, the second inflow through hole, the second inflow groove 21 and the second flow channel 8 in sequence, and the other part of cell mixed liquor enters the separation region 16 through the third metal connector, the third inflow through hole, the third inflow groove 22 and the third flow channel 9 in sequence.
Both ends of the large opening side of the first splayed structure and both ends of the large opening side of the third splayed structure are both connected with alternating voltage, and both ends of the large opening side of the second splayed structure and both ends of the large opening side of the fourth splayed structure are both grounded.
And adjusting the amplitude and frequency of alternating voltage at two ends of the large opening side of the first splayed structure and two ends of the large opening side of the third splayed structure to enable the two cells to be respectively subjected to positive dielectrophoresis attraction force and negative dielectrophoresis repulsion force. The flow rate of the buffer solution is set between the moving speed of the cells under the positive dielectrophoretic attraction force and the moving speed of the cells under the negative dielectrophoretic repulsion force. Cells moving at a velocity greater than the flow velocity of the buffer solution will move along the tilted driving electrode array 3 and finally leave the separation region 16 through the fifth flow channel 11. Cells moving at a velocity less than the flow velocity of the buffer solution will enter the fourth flow channel 10 and the sixth flow channel 12 with the buffer solution.
The micro-fluidic chip for separating and capturing cells based on the dielectrophoresis principle realizes the sorting of the cells based on the dielectrophoresis principle. Compared with the existing fluorescence activated cell sorting and magnetic field activated cell sorting, the cell sorting method provided by the invention does not need to label the cells in advance, so that the time consumption is relatively short, and the efficiency of cell analysis work is improved. On the other hand, the micro-fluidic chip for cell separation and capture based on the dielectrophoresis principle integrates the sorting link and the single cell capture link, so that the cell transfer link after cell sorting is omitted, and the efficiency of cell analysis work is improved to a certain extent.
Drawings
The microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to the present invention will be described in more detail hereinafter on the basis of an embodiment and with reference to the accompanying drawings, in which:
FIG. 1 is a perspective view of the structure of a microfluidic chip for cell separation and capture based on the principle of dielectrophoresis according to an embodiment;
FIG. 2 is a schematic structural view of a separation zone according to the example;
FIG. 3 is a schematic structural diagram of a first bipolar electrode array according to an embodiment, wherein LeThe length of the ninth drive electrode 35;
FIG. 4 is a graph showing the relationship between Claus-Moxoti factors of yeast cells and PS microspheres with respect to voltage and frequency, wherein the dotted-PS microsphere curve, the solid-PS microsphere curve, the dotted-yeast curve and the solid-yeast curve are the imaginary Claus-Moxoti factor of PS microspheres, the real Claus-Moxoti factor of PS microspheres, the imaginary Claus-Moxoti factor of yeast cells and the real Claus-Moxoti factor of yeast cells with respect to voltage and frequency;
FIG. 5 is a diagram of the experimental separation process of yeast cells and PS microspheres under the conditions that the amplitude of the voltage is 20Vpp, the frequency of the voltage is 1MHz, and the flow rate of the buffer solution is 0.1mm/s, wherein the voltage is applied to the first splayed structure and the third splayed structure;
FIG. 6 is the embodiment mentioned with the overlay for 10 seconds of FIG. 5;
FIG. 7 is an experimental diagram of the capturing of single yeast cell by the bipolar electrode array body 39 when the diameter of a single bipolar electrode is 25 μm, the gap between two adjacent bipolar electrodes is 50 μm, the frequency of the applied voltage is 500KHz, and the amplitude is 10 Vpp;
FIG. 8 is a flowchart of PDMS channel processing according to the embodiment, wherein a is a silicon substrate, b is a photoresist, c is a flow channel template, d is a mixture of PDMS and a curing agent, and UV is ultraviolet light;
FIG. 9 is a flow chart of the ITO thin film electrode processing mentioned in the examples, wherein e is an ITO thin film and f is an electrode template;
FIG. 10 is the bonding diagram of the PDMS cover and the ITO substrate mentioned in the example.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, wherein the microfluidic chip for cell separation and capture is based on the dielectrophoresis principle.
Example (b): the present embodiment will be described in detail with reference to fig. 1 to 10.
The micro-fluidic chip for cell separation and capture based on the dielectrophoresis principle comprises a glass substrate 1, a PDMS cover plate 2, a driving electrode array 3 and first to third bipolar electrode arrays 4 to 6;
the lower surface of the PDMS cover plate 2 is provided with a first flow channel 7 to a ninth flow channel 15, a separation zone 16, a first capture zone 17 to a third capture zone 19, a first inflow groove 20 to a third inflow groove 22 and a first outflow groove 23 to a third outflow groove 25 in an invagination manner;
the first inflow groove 20 to the third inflow groove 22 are respectively communicated with an inflow port of the first flow channel 7 to an inflow port of the third flow channel 9, an outflow port of the first flow channel 7 to an outflow port of the third flow channel 9 are respectively communicated with an inflow port of the separation region 16, an outflow port of the separation region 16 is simultaneously communicated with an inflow port of the fourth flow channel 10 to an inflow port of the sixth flow channel 12, an outflow port of the fourth flow channel 10 to an outflow port of the sixth flow channel 12 are respectively communicated with an inflow port of the first trapping region 17 to an inflow port of the third trapping region 19, an outflow port of the first trapping region 17 to an outflow port of the third trapping region 19 are respectively communicated with an inflow port of the seventh flow channel 13 to an inflow port of the ninth flow channel 15, and an outflow port of the seventh flow channel 13 to an outflow port of the ninth flow channel 15 are respectively communicated with the first outflow groove 23 to the third outflow groove 25;
first inflow through holes to third inflow through holes are respectively formed in the groove bottom of the first inflow groove 20 to the groove bottom of the third inflow groove 22, first outflow through holes to third outflow through holes are respectively formed in the groove bottom of the first outflow groove 23 to the groove bottom of the third outflow groove 25, and the first inflow through holes to the third inflow through holes and the first outflow through holes to the third outflow through holes all penetrate through the PDMS cover plate 2;
first to sixth metal connectors are respectively arranged on the inflow ports of the first to third inflow through holes and the outflow ports of the first to third outflow through holes;
the driving electrode array 3 and the first to third bipolar electrode arrays 4 to 6 are all arranged on the upper surface of the glass substrate 1, and the upper surface of the glass substrate 1 is opposite to and closely attached to the lower surface of the PDMS cover plate 2;
the driving electrode array comprises a first driving electrode 26 to an eighth driving electrode 33, the first driving electrode 26, the eighth driving electrode 33, a second driving electrode 27, a seventh driving electrode 32, a third driving electrode 28, a sixth driving electrode 31, a fourth driving electrode 29 and a fifth driving electrode 30 respectively form a first splayed structure to a fourth splayed structure, the first splayed structure to the fourth splayed structure are sequentially distributed between an outflow port of the separation area 16 and an inflow port of the separation area 16 in a row, small opening sides of the first splayed structure to the fourth splayed structure face the outflow port of the separation area 16 and are opposite to the inflow port of the fifth flow channel 11, and two ends of the first splayed structure from the large opening side to two ends of the fourth splayed structure from the large opening side respectively exceed two sides of the separation area 16;
both ends of the large opening side of the first splayed structure and both ends of the large opening side of the third splayed structure are connected with alternating voltage, and both ends of the large opening side of the second splayed structure and both ends of the large opening side of the fourth splayed structure are grounded;
the first bipolar electrode array 4 to the third bipolar electrode array 6 are respectively positioned in the coverage range of the first capture area 17 to the third capture area 19;
the first metal connector to the third metal connector are respectively a channel for allowing cell mixed liquor, buffer solution and cell mixed liquor to enter the microfluidic chip;
the two cells contained in the cell mixture have different dielectric constants.
The first to eighth driving electrodes 26 to 33 of the present embodiment have the same structure, and the opening distances on the small opening side of the first to fourth splay structures are equal to each other.
Each of the figure-of-eight structures of this embodiment is connected to a voltage source or power ground via lead electrodes 34.
The first bipolar electrode array 4 to the third bipolar electrode array 6 of the present embodiment have the same structure, and are all wireless bipolar electrode arrays;
the first bipolar electrode array 4 comprises ninth to twelfth driving electrodes 35 to 38 and a bipolar electrode array body 39;
alternating voltages are applied to two ends of the ninth driving electrode 35 and two ends of the twelfth driving electrode 38;
for the ninth drive electrode 35 to the twelfth drive electrode 38, the phase angle of the voltage across the former and the phase angle of the voltage across the latter differ by 90 °;
after power is supplied, the ninth to twelfth driving electrodes 35 to 38 are commonly used for driving the bipolar electrode array body 39.
The lower surface of the PDMS cover plate 2 of the embodiment is also provided with tenth to fifteenth flow channels and first to sixth auxiliary grooves in an inwards-recessed manner;
the first auxiliary groove and the second auxiliary groove are respectively communicated with the first capturing area 17 through a tenth flow passage and an eleventh flow passage, the third auxiliary groove and the fourth auxiliary groove are respectively communicated with the second capturing area 18 through a twelfth flow passage and a thirteenth flow passage, and the fifth auxiliary groove and the sixth auxiliary groove are respectively communicated with the third capturing area 19 through a fourteenth flow passage and a fifteenth flow passage.
The separation area 16 of the present embodiment is rectangular, and the width of the inflow port and the outflow port of the separation area 16 is equal to the width of the separation area 16;
the separation zone 16 has a length L and a width W of 4000 μm and 1400 μm, respectively;
width W of outflow port of first flow channel 7i1Outflow port W of third flow channel 9i3Are 500 μm, 400 μm and 500 μm, respectively;
width W of inflow port of fourth flow channel 10o1Width W of inflow port of sixth flow path 12o3550 μm, 300 μm and 550 μm, respectively;
minimum spacing L between the small open side of the first splayed configuration and the outflow port of the separation zone 16dAnd 300 μm.
The bipolar electrode array body 39 of the present embodiment is square, and the ninth driving electrode 35 to the twelfth driving electrode 38 are equal in length and are all 1400 μm;
the ninth to twelfth driving electrodes 35 to 38 are distributed around the bipolar electrode array body 39 in the clockwise direction, and are parallel to four sides of the bipolar electrode array body 39, respectively;
the ninth to twelfth driving electrodes 35 to 38 are all equal to the bipolar electrode array body 39 in minimum distance;
the minimum separation G between the ninth drive electrode 35 and the eleventh drive electrode 37 is 2000 μm.
In the embodiment, the first to third inflow grooves 20 to 22, the first to third trapping regions 17 to 19, the first to third outflow grooves 23 to 25, and the first to sixth auxiliary grooves are all circular and have diameters of 5000 μm.
The height of the recessed area of the PDMS cover 2 of this example was 20 μm.
The electrodes of the microfluidic chip for cell separation and capture based on the dielectrophoresis principle are all ITO thin film electrodes or metal thin film electrodes.
The working principle of the microfluidic chip for cell separation and capture based on the dielectrophoresis principle described in this embodiment is described in detail below with a mixed solution composed of yeast cells and PS microspheres as an implementation object:
a cell sorting part:
the expression of the time-averaged dielectrophoretic force acting on the particles when the electric field is applied is:
ε*＝ε-j(σ/ω) (3)
in the formula (I), the compound is shown in the specification,<FD>for the time-averaged dielectrophoretic force acting on the particles, r is the particle radius, K (w) is the Clausius-Moxolitin factor, Re [ K (w)]And Im [ K (w)]Respectively the real part and the imaginary part of the Clausius-Moxoy factor, E is the electric field intensity, the wave line is the complex amplitude, and the complex conjugate is the complex number;
ε represents a dielectric constant, and σ represents an electric conductivity.
From equation 1, the dielectrophoretic force is mainly dependent on the non-uniformity of the electric field and k (w). The first term of equation 1 is the conventional dielectrophoretic force, when Re [ k (w) ], is positive or negative, the particles will be subjected to either positive or negative dielectrophoretic forces, causing the particles to be attracted to or away from the strong electric field area. The second term of equation 1 is traveling wave dielectrophoresis (twDEP), when Im [ K (w) ] is positive or negative, the particles will move in the direction of the electric field phase increase or decrease.
FIG. 4 is a graph of Clausius-Moxoti factor as a function of voltage frequency for yeast cells and PS microspheres. As can be seen from FIG. 4, when the voltage frequency is in the range of 100kHz-40MHz, the yeast cells are subjected to positive dielectrophoretic force, and the PS microspheres are subjected to negative dielectrophoretic force.
Experiments show that when the amplitude of the voltage is 10V and the frequency is more than 1MHz, the negative dielectrophoresis repulsion velocity of the PS microspheres in the buffer solution with the conductivity of 0.1S/m is about 0.17mm/S, and the positive dielectrophoresis attraction velocity of the yeast cells in the buffer solution with the conductivity of 0.1S/m is about 0.03 mm/S. Therefore, when the flow rate of the buffer solution is 0.04mm/s to 0.16mm/s, the PS microspheres will move along the tilted driving electrode array 3 due to the moving speed of the PS microspheres being greater than the flow rate of the buffer solution, and finally leave the separation region 16 through the fifth flow channel 11. Since the moving speed of the yeast cells is less than the flow speed of the buffer solution, the yeast cells will enter the fourth flow channel 10 and the sixth flow channel 12 along with the buffer solution.
FIG. 5 is a diagram showing the separation process of yeast cells and PS microspheres under the conditions that the first splayed structure and the third splayed structure are applied with voltage with the amplitude of 20Vpp and the frequency of 1MHz and the flow rate of the buffer solution is 0.1 mm/s. Fig. 6 is the overlay of fig. 5 after 10 seconds. As can be seen from fig. 5 and 6, the microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to this embodiment can effectively separate yeast cells from PS microspheres.
The single cell capture section is illustrated by the first bipolar electrode array 4: bipolar electrode means a conductor immersed in the electrolyte between the anode and the cathode that is not connected to an external power source. When a bipolar electrode is placed in a microfluidic channel, a potential drop is formed in the solution when a certain driving DC potential is applied. The bipolar electrode is an equipotential body and the potentials are the same, thus creating an overpotential at the interface of the bipolar electrode and the solution. An electric double layer is formed at both ends of the bipolar electrode, and when an overpotential on the bipolar electrode is sufficiently large, an electrochemical reaction will occur. Based on the characteristics of the bipolar electrodes, an electrochemical array of a plurality of wireless bipolar electrodes is designed between two driving electrodes, and the two driving electrodes can drive each bipolar electrode to generate electrochemical reaction. When the applied driving voltage is a high-frequency alternating current signal rather than a direct current signal (i.e. the frequency of the electric field is higher than the rate of electron transfer in the redox reaction), the electrochemical reaction is suppressed and the electric double layer at both ends of the bipolar electrode will generate capacitive charge-discharge effect. The electric field strength at the edges of the bipolar electrode will be greatest and the electric field strength in the middle region of the bipolar electrode will be smallest. Therefore, the bipolar electrode is not only suitable for large-scale wireless array design, but also the distribution of the electric field can be adjusted by changing the size of the bipolar electrode.
In the first bipolar electrode array 4 of the present embodiment, the ninth to twelfth driving electrodes 35 to 38 are used to apply a rotating electric field around the bipolar electrode array body 39, and further capture cells to a region with low field intensity energy by using negative dielectrophoretic force. Compared with the existing single cell capturing mode of capturing the cells to the region with high field intensity energy by using positive dielectrophoresis force, the single cell capturing mode of the embodiment has less adverse effect on the cells because the cells are captured to the region with low field intensity energy, and is beneficial to subsequent cell analysis work.
FIG. 7 is a diagram of an experiment showing the capture of single yeast cell by the bipolar electrode array body 39 when the diameter of a single bipolar electrode is 25 μm, the gap between two adjacent bipolar electrodes is 50 μm, the frequency of the applied voltage is 500KHz, and the amplitude is 10 Vpp. According to experimental data, the capture efficiency of the single saccharomycete cell is up to 75%. Therefore, the microfluidic chip for cell separation and capture based on the dielectrophoresis principle described in the embodiment can perform efficient capture on single cells.
The preparation method of the microfluidic chip for cell separation and capture based on the dielectrophoresis principle described in this embodiment is performed according to the following steps:
firstly, processing a PDMS channel:
(1) and cleaning the silicon substrate: first, the silicon substrate is hand-washed with a cleaning agent. And secondly, sequentially placing the silicon substrate in acetone and isopropanol to respectively ultrasonically clean for 10 minutes. And thirdly, flushing the silicon substrate by using plasma water and drying by using nitrogen. Finally, the dried silicon substrate is placed in a baking oven and heated for 15 minutes at the temperature of 80 ℃.
(2) And flatly paving the photoresist: firstly, a layer of photoresist is coated on the upper surface of a silicon substrate. Secondly, the silicon substrate is placed on a spin coater and rotated at the speed of 1500r/s until the thickness of the photoresist is 100 microns. Finally, the silicon substrate was subjected to pre-baking, placed on a hot plate at 60 ℃, heated to 95 ℃, and heated at that temperature for 1 hour. The photoresist is SU-82050 type negative photoresist.
(3) And exposure: first, a runner template is placed on the photoresist surface. And secondly, pressing the flow channel template and the photoresist surface by using a light-transmitting plate. Finally, the ultraviolet lamp tube is used for exposure.
(4) And developing: first, the exposed silicon substrate was subjected to post-baking, placed on a hot plate at 60 ℃, heated to 95 ℃ and heated at that temperature for 35 minutes. Next, the cooled silicon substrate was placed in an SU-8 developer for development for 10 minutes. And thirdly, carrying out plasma water cleaning and nitrogen blow-drying on the silicon substrate. Finally, the silicon substrate was placed in a baking oven and heated at a temperature of 80 ℃ for 10 to 20 minutes to obtain a PDMS runner mold.
(5) And pouring PDMS: first, PDMS and a curing agent were mixed in a mass ratio of 10:1, and stirred using a clean glass rod for 15 to 20 minutes to be uniformly mixed. Next, the mixture of PDMS and the curing agent was evacuated for 30 minutes using a vacuum pump to eliminate air bubbles in the mixture. And performing silanization treatment on the PDMS flow channel mold to deposit a layer of silane on the surface of the PDMS flow channel mold. Finally, a mixture of PDMS and a curing agent was poured onto the silane side of the PDMS runner mold, and the mixture was evacuated for 20 minutes using a vacuum pump to eliminate air bubbles in the mixture and heated at 80 ℃ for 2 hours to cure the mixture.
The silane layer on the surface of the PDMS flow channel mold serves to prevent the PDMS flow channel mold from sticking to the mixture.
(6) And PDMS channel treatment: first, the cured PDMS was slowly removed from the PDMS runner mold. Next, it was cut into a shape matching the glass substrate with a blade. And finally, grooving and punching the cured PDMS by adopting a grooving machine and a punching machine to obtain the PDMS cover plate 2.
Fig. 8 is a flow chart of PDMS channel processing.
Secondly, processing an ITO thin film electrode:
(1) and cleaning the ITO substrate: the ITO substrate comprises a glass substrate 1 and an ITO thin film, and the cleaning method of the ITO substrate is the same as that of the silicon substrate.
(2) And flatly paving the photoresist: first, a layer of photoresist is coated on the ITO film. Next, the ITO substrate was placed on a spin coater and rotated at 3100r/s for 40 seconds. Finally, the ITO substrate was soft-baked, and placed on a hot plate at 100 ℃ and heated for 6 minutes.
The photoresist is AZ4620 type photoresist.
(3) And exposure: the ITO substrate was placed under an ultraviolet lamp tube for exposure.
(4) And developing: and (3) placing the exposed ITO substrate in an AZ developing solution, and developing for 4 to 5 minutes.
(5) And etching the ITO film: and (3) placing the developed ITO substrate in a hydrochloric acid solution with the mass ratio of 60%, adding ferric chloride serving as a catalyst, soaking for 40 minutes, and corroding the ITO film. In the process, the exposed and cured photoresist layer plays a role in protecting the ITO film, and the ITO film which is not covered by the photoresist is corroded.
(6) And removing the photoresist: and after the ITO film is corroded, soaking the ITO substrate in 5% NaOH solution by mass ratio, and removing the cured photoresist to obtain the ITO film electrode.
FIG. 9 is a flow chart of ITO thin film electrode processing.
Bonding of PDMS cover plate 2 and ITO substrate
Firstly, the PDMS cover plate 2 is arranged on an ITO substrate and is placed in a cavity of a plasma machine, and plasma treatment is carried out according to the using steps of the plasma machine, so that the PDMS cover plate 2 and the ITO substrate are arranged in a sealing mode, and the microfluidic chip is formed.
And secondly, taking out the microfluidic chip, and calibrating the relative position of the internal structure of the microfluidic chip under a microscope.
Finally, after the calibration is completed, the micro-fluidic chip is pressed for a few minutes by force, then placed in a baking oven and heated for 30 minutes at 80 ℃ to obtain the micro-fluidic chip.
FIG. 10 is a bonding diagram of the PDMS cover 2 to the ITO substrate.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims. It should be understood that features described in different dependent claims and herein may be combined in ways different from those described in the original claims. It is also to be understood that features described in connection with individual embodiments may be used in other described embodiments.
Claims (10)
1. The microfluidic chip for cell separation and capture based on the dielectrophoresis principle is characterized by comprising a glass substrate (1), a PDMS cover plate (2), a driving electrode array (3) and first to third bipolar electrode arrays (4 to 6);
a first flow channel (7) to a ninth flow channel (15), a separation zone (16), a first capture zone (17) to a third capture zone (19), a first inflow groove (20) to a third inflow groove (22) and a first outflow groove (23) to a third outflow groove (25) are arranged on the lower surface of the PDMS cover plate (2) in a sunken way;
the first inflow groove (20) to the third inflow groove (22) are respectively communicated with inflow ports of the first flow passage (7) to the third flow passage (9), outflow ports of the first flow passage (7) to the third flow passage (9) are respectively communicated with inflow ports of the separation region (16), outflow ports of the separation region (16) are simultaneously communicated with inflow ports of the fourth flow passage (10) to the sixth flow passage (12), outflow ports of the fourth flow passage (10) to the sixth flow passage (12) are respectively communicated with inflow ports of the first capture region (17) to the third capture region (19), outflow ports of the first capture region (17) to the third capture region (19) are respectively communicated with inflow ports of the seventh flow passage (13) to the ninth flow passage (15), and outflow ports of the seventh flow passage (13) to the ninth flow passage (15) are respectively communicated with outflow ports of the first outflow groove (7) The grooves (23) are communicated with the third outflow groove (25);
a first inflow through hole, a second inflow through hole, a third inflow through hole, a first outflow through hole, a third outflow through hole, a PDMS cover plate (2) and a third inflow through hole are respectively arranged on the groove bottom of the first inflow groove (20) to the groove bottom of the third inflow groove (22), a first outflow through hole, a second outflow through hole and a third outflow through hole are respectively arranged on the groove bottom of the first outflow groove (23) to the groove bottom of the third outflow groove (25), and the first inflow through hole, the second inflow through hole, the third inflow through hole and the first outflow through hole, the second outflow through hole and the third outflow through hole penetrate through the PDMS cover plate (2);
first to sixth metal connectors are respectively arranged on the inflow ports of the first to third inflow through holes and the outflow ports of the first to third outflow through holes;
the driving electrode array (3) and the first bipolar electrode array (4) to the third bipolar electrode array (6) are all arranged on the upper surface of the glass substrate (1), and the upper surface of the glass substrate (1) is opposite to and closely attached to the lower surface of the PDMS cover plate (2);
the driving electrode array comprises a first driving electrode (26) to an eighth driving electrode (33), the first driving electrode (26) and the eighth driving electrode (33), a second driving electrode (27) and a seventh driving electrode (32), the third driving electrode (28) and the sixth driving electrode (31) as well as the fourth driving electrode (29) and the fifth driving electrode (30) respectively form a first splayed structure to a fourth splayed structure, the first splayed structure to the fourth splayed structure are sequentially distributed between the outflow port of the separation area (16) and the inflow port of the separation area (16) in a row, the small opening side of the first splayed structure to the small opening side of the fourth splayed structure face the outflow port of the separation area (16) and are opposite to the inflow port of the fifth flow channel (11), and the two ends of the large opening side of the first splayed structure to the two ends of the large opening side of the fourth splayed structure respectively exceed the two sides of the separation area (16);
both ends of the large opening side of the first splayed structure and both ends of the large opening side of the third splayed structure are connected with alternating voltage, and both ends of the large opening side of the second splayed structure and both ends of the large opening side of the fourth splayed structure are grounded;
the first bipolar electrode array (4) to the third bipolar electrode array (6) are respectively positioned in the coverage range of the first capture area (17) to the third capture area (19);
the first metal connector to the third metal connector are respectively a channel for allowing cell mixed liquor, buffer solution and cell mixed liquor to enter the microfluidic chip;
the two cells contained in the cell mixture have different dielectric constants.
2. The microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to claim 1, wherein the first to eighth driving electrodes (26) to (33) have the same structure, and the opening distances from the small-opening side of the first to fourth splay structures are equal.
3. The microfluidic chip for cell separation and capture based on dielectrophoresis principle according to claim 2, wherein each of the splayed structures is connected to a voltage source or a power ground via a lead electrode (34).
4. The microfluidic chip for cell separation and capture based on dielectrophoresis principle according to claim 3, wherein the first bipolar electrode array (4) to the third bipolar electrode array (6) have the same structure and are wireless bipolar electrode arrays;
the first bipolar electrode array (4) comprises ninth driving electrodes (35) to twelfth driving electrodes (38) and a bipolar electrode array body (39);
alternating voltages are applied to two ends of the ninth driving electrode (35) to two ends of the twelfth driving electrode (38);
for the ninth drive electrode (35) to the twelfth drive electrode (38), the phase angle of the voltage at the two ends of the former and the phase angle of the voltage at the two ends of the latter are different by 90 degrees;
after the power is connected, the ninth driving electrode (35) to the twelfth driving electrode (38) are used for driving the bipolar electrode array body (39) together.
5. The microfluidic chip for cell separation and capture based on dielectrophoresis principle according to claim 4, wherein the lower surface of the PDMS cover plate (2) is further provided with tenth to fifteenth flow channels and first to sixth auxiliary grooves in a sunken manner;
the first auxiliary groove and the second auxiliary groove are respectively communicated with the first capture area (17) through a tenth flow passage and an eleventh flow passage, the third auxiliary groove and the fourth auxiliary groove are respectively communicated with the second capture area (18) through a twelfth flow passage and a thirteenth flow passage, and the fifth auxiliary groove and the sixth auxiliary groove are respectively communicated with the third capture area (19) through a fourteenth flow passage and a fifteenth flow passage.
6. The microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to claim 5, wherein the separation region (16) has a rectangular shape, and the width of the inflow port and the outflow port of the separation region (16) are equal to the width of the separation region (16);
the separation zone (16) has a length L and a width W of 4000 μm and 1400 μm, respectively;
of the outflow port of the first flow passage (7)Width Wi1-outflow port W of third flow channel (9)i3Are 500 μm, 400 μm and 500 μm, respectively;
width W of inflow port of fourth flow channel 10o1Width W of inflow port of sixth flow path 12o3550 μm, 300 μm and 550 μm, respectively;
the minimum distance L between the small opening side of the first splayed structure and the outflow port of the separation zone (16)dAnd 300 μm.
7. The microfluidic chip for cell separation and capture based on dielectrophoresis principle according to claim 6, wherein the bipolar electrode array body (39) is square, and the ninth driving electrode (35) to the twelfth driving electrode (38) are all 1400 μm in length;
the ninth driving electrode (35) to the twelfth driving electrode (38) are distributed around the bipolar electrode array body (39) along the clockwise direction and are respectively parallel to four sides of the bipolar electrode array body (39);
the minimum distances between the ninth driving electrode (35) to the twelfth driving electrode (38) and the bipolar electrode array body (39) are all equal;
the minimum distance G between the ninth driving electrode (35) and the eleventh driving electrode (37) is 2000 μm.
8. The microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to claim 7, wherein the first inflow groove (20) to the third inflow groove (22), the first capture region (17) to the third capture region (19), the first outflow groove (23) to the third outflow groove (25), and the first auxiliary groove to the sixth auxiliary groove are all circular and have a diameter of 5000 μm.
9. The microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to claim 8, wherein the height of the invaginated region of the PDMS cover plate (2) is 20 μm each.
10. The microfluidic chip for cell separation and capture based on the dielectrophoresis principle according to claim 9, wherein the electrodes involved in the microfluidic chip are all ITO thin film electrodes or metal thin film electrodes.
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