CN107699485B

CN107699485B - Microelectrode flow control chip and parameter-adjustable single-cell electroporation device

Info

Publication number: CN107699485B
Application number: CN201711082795.5A
Authority: CN
Inventors: 朱真; 耿杨烨; 潘任豪; 王颖瀛
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2017-11-06
Filing date: 2017-11-06
Publication date: 2020-08-11
Anticipated expiration: 2037-11-06
Also published as: CN107699485A

Abstract

The invention discloses a microelectrode flow control chip and a parameter-adjustable single-cell electroporation device, wherein the microelectrode flow control chip comprises a transparent substrate and a microfluid channel layer, the microfluid channel layer is positioned above the transparent substrate, a microelectrode is integrated on the transparent substrate, the microelectrode comprises an interdigital electrode and a parallel electrode, the interdigital electrode and the parallel electrode are distributed below a microfluid control electroporation channel, and the interdigital electrode and the parallel electrode are sequentially distributed from a cell suspension liquid inlet to a cell outlet. The parameter-adjustable single-cell electroporation device comprises a voltage-stabilized power supply module, a function signal generation module, a PCB control circuit, a digital display module, a chip carrier and a microelectrode flow control chip. The invention can realize the sequential electroporation treatment of single cells; reducing cell damage and even mortality; the cell electrotransfection efficiency is improved; the kind and the number of the access electrodes are selected by the control circuit, the frequency, the amplitude and the duty ratio of the electric signals are changed, and the optimization of electroporation parameters is realized aiming at different cell lines.

Description

Microelectrode flow control chip and parameter-adjustable single-cell electroporation device

Technical Field

The invention belongs to the fields of micro-fluidic technology and single cell electroporation and electrotransfection, and particularly relates to a parameter-adjustable single cell electroporation device based on a microelectrode-fluidic chip.

Background

Microfluidics (Microfluidics) is a science and technology related to the processing or manipulation of nanoliter to microliter fluid systems using micro-channels with dimensions from a few micrometers to hundreds of micrometers, and is an emerging interdisciplinary subject related to the fields of micro-nano processing, physics, microelectronics, biology, chemistry, new materials and the like. The microfluidic Chip is characterized by miniaturization and integration, and is also called a Lab-on-a-Chip (Lab on a Chip) and a micro total analysis system (microTAS). Microfluidic technology is considered to have great development potential and wide application prospect in biomedical research.

When the micro-fluidic chip is used for detecting and analyzing biological and chemical samples, external electrical and optical signal excitation needs to be applied or parameters of the samples to be detected are reflected by outputting electrical and optical signals. Particularly, for the electrical detection and analysis technology, micro-processing technology is required to integrate electrodes on the microfluidic chip to realize interaction with external signals, so as to integrate the electrical detection method on the microfluidic chip. The micro-fluidic chip integrated with the micro-electrode has many excellent performances: when the biological sample is controlled and detected, the electric field generated by the microelectrode can promote cells to generate certain physiological reactions such as perforation, cracking and the like, or relevant electrical information (such as electrical impedance signals and the like) is fed back to an experimental system to realize cell detection, and the microelectrode has the advantages of high sensitivity, quick response and the like and has the potential of miniaturization of the device.

Electroporation (Electroporation), also known as electrotransfection, is a common approach used in transfection of cells. Because the cell membrane has selective permeability to external substances, the experiment for controlling the gene of the eukaryotic cell needs to input specific biological DNA and RNA fragments into the eukaryotic cell. Applying a certain intensity of potential difference on two sides of cell membrane and lasting for a period of time, the cell membrane can produce micropores, and the permeability of cell membrane can be enhanced. When the cell membrane is electroporated, its permeability and membrane conductance are transiently increased, so that hydrophilic molecules, DNA, proteins, virus particles, drug particles, etc. which normally cannot pass through the cell membrane, are introduced into the cell. After the potential difference is removed in a short time, the cell membrane can recover itself and become a selective permeability barrier again. Compared with traditional chemical transfection and virus transfection, electroporation has wide applicability and superiority: the method is suitable for plasmids and genome segments of dozens of KB, and has the advantages of no chemical virus pollution, no permanent damage to cells, transient transfection and the like. Therefore, the electroporation technology has wide application prospect in the fields of biophysics, molecular biology, clinical medicine and the like.

In the conventional cell electroporation apparatus, the size of the electrodes is large, the electrode distance is still in a macroscopic size, namely a ten-millimeter scale, and the size of the cells is in a micrometer scale, so that the applied voltage is large, about several hundred volts, the electric field is not uniform, the electric field environment of each cell is different, cells close to the electrodes are easy to die, cells at a weaker electric field cannot be transfected by the perforation, and the survival rate and the transfection efficiency are low. The electrode spacing and other parameters of the existing micro-fluidic electroporation device are fixed, the operability of the experiment and the adjustability of the electroporation parameters are limited by the design of a chip, and the universality is lacked. In addition, the key parameters of the commercial electrotransfection instrument, such as the frequency and the amplitude of the electric signal, are set by manufacturers, so that customers cannot perform optimal adjustment, and the transfection efficiency of special cells, such as primary cells, immune cells and the like, is not high.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a microelectrode flow control chip and a parameter-adjustable single-cell electroporation device, which aim to solve the problems in the prior art and enable the electroporation device to be universally used for various cells.

The technical scheme is as follows: a microelectrode flow control chip comprises a transparent substrate and a microfluidic channel layer, wherein the microfluidic channel layer is positioned above the transparent substrate and comprises a sheath inflow port, a cell suspension liquid inlet, a sheath flow channel, a cell suspension liquid channel, a microfluidic electroporation channel and a cell outlet; the sheath inflow port is communicated with the sheath flow channel, the cell suspension inlet is communicated with the cell suspension channel, the tail end of the sheath flow channel is converged with the tail end of the cell suspension channel and is communicated with one end of the microfluidic electroporation channel, and the other end of the microfluidic electroporation channel is communicated with the cell outlet; the microelectrode is integrated on the transparent substrate and comprises an interdigital electrode and a parallel electrode, the interdigital electrode and the parallel electrode are distributed below the micro-fluidic electroporation channel, and the interdigital electrode and the parallel electrode are sequentially distributed from a cell suspension liquid inlet to a cell outlet; the microelectrode comprises microelectrode leading-out ends which are distributed on two sides of the transparent substrate.

Preferably, the interdigital electrodes and/or the parallel electrodes are distributed in a plurality and uniformly spaced manner. The electric field distribution can be more uniform, the electric field environment of each cell is similar, and the survival rate and the transfection efficiency are improved.

Preferably, the sheath flow channel comprises two channel branches, the two channel branches bypass the cell suspension inlet from two sides of the cell suspension inlet and are communicated with the cell suspension channel, and the sheath flows of the two channel branches can effectively converge cells in the center of the microfluidic channel to flow, so that sequential electroporation treatment on single cells is realized.

Preferably, the device also comprises a cell suspension input pipe, a sheath flow input pipe and a cell output pipe, wherein the cell suspension input pipe is communicated with the cell suspension inlet, the sheath flow input pipe is communicated with the sheath flow inlet, and the cell output pipe is communicated with the cell output port.

Preferably, the transparent substrate is provided with a cross alignment mark for bonding, the microfluidic channel layer is provided with four square alignment marks for bonding matched with the cross alignment mark for bonding, and bonding enables the transparent substrate and the microfluidic channel layer to be combined more accurately so as to ensure that the electrode can be just positioned in the microfluidic electroporation channel.

Preferably, the finger pitch of the interdigital electrodes is 50-80 μm; the electrode pitch of the parallel electrodes is 150 to 200 μm.

A parameter-adjustable single-cell electroporation device using a microelectrode flow control chip comprises a voltage-stabilized power supply module, a function signal generation module, a PCB control circuit, a digital display module, a chip carrier and a microelectrode flow control chip, wherein a chip groove is formed in the chip carrier, the microelectrode flow control chip is fixed in the chip groove, the output ends of the PCB control circuit and the digital display module are spring probes, and the spring probes are in electrical contact with a microelectrode leading-out end; the stabilized voltage supply module is used for supplying power to the function signal generation module, the PCB control circuit, the digital display module and the microelectrode flow control chip; the function signal generation module is used for outputting signals required by power supply perforation.

Preferably, the PCB control circuit and the digital display module comprise a switch circuit and an amplifier, the switch circuit divides a single input into multiple outputs, and the amplifier is used for adjusting the amplitude of the output signal; the input end of the switching circuit is connected with the output end of the function signal generation module, and the output end of the switching circuit is connected with the input end of the amplifier; the output end of the amplifier is connected with the microelectrode leading-out end.

Preferably, the amplifier is a gain adjustable amplifier.

Preferably, the inter-finger electric field of the interdigital electrode is larger than the inter-electrode electric field of the parallel electrode.

Has the advantages that: compared with the prior art, the microelectrode in the microelectrode flow control chip comprises the interdigital electrode and the parallel electrode, wherein the interdigital electrode is positioned at the upstream of a micro-flow control electroporation channel to generate a strong electric field, and a cell suspension is subjected to the action of an electric field when passing through the interdigitated electrode, so that electroporation is carried out on a cell membrane; the parallel electrode is positioned at the downstream of the microfluidic electroporation channel, and applies and maintains a low electric field, so that the electroporation state of the cells can be maintained, and the transfection efficiency of the cells is enhanced and improved. The electric field environment of each cell is similar, so that the cell damage and even death caused by the strong electric field and the electric field intensity difference of the traditional electroporator are reduced.

Compared with the prior art, the device is 1) a complete single cell electroporation experiment system, can conveniently carry out single cell electroporation experiments, and is arranged on an inverted microscope to observe the dynamic process of cell electroporation; 2) the device uses a microelectrode flow control chip, the distance between electrodes is short, the voltage applied for achieving the effect of cell electroporation is 10V magnitude, the cell death rate is low, and the operation is safe; 3) the device carries out electroporation on single cells in sequence, and the electric field environment of each cell is similar, so that cell damage caused by uncontrollable factors in the experimental process is reduced; 4) the geometric parameters of the electrodes of the core microelectrode fluidic chip of the device can be designed according to experimental requirements, the type and the number of the electrodes used for electroporation are selected through a PCB control circuit, and the waveform, the frequency, the duty ratio and the amplitude of an electric signal are selected, so that the optimal adjustment of the parameters of the electroporation experiment is realized; 5) parallel electrodes are designed and processed on the downstream of a micro-fluid channel of the micro-electrode flow control chip of the device, and a low electric field is generated when cells flow through the region, so that the cell electrotransfection efficiency is effectively enhanced.

Drawings

FIG. 1 is a schematic diagram of the three-dimensional structure of a microelectrode fluidic chip in accordance with the present invention;

FIG. 2 is a schematic diagram showing the three-dimensional structure of a microfluidic channel layer in the microelectrode fluidic chip of the present invention;

FIG. 3 is a schematic diagram showing a three-dimensional structure of a transparent substrate on which microelectrodes are integrated in a microelectrode fluidic chip according to the present invention;

FIG. 4 is a schematic cross-sectional view of the microelectrode fluidic chip taken along line A-A;

FIG. 5 is a block diagram of the construction of the adjustable parameter single-cell electroporation apparatus of the present invention.

Detailed Description

The invention is further described with reference to the following figures and specific examples.

As shown in fig. 1, the microelectrode fluidic chip includes a transparent substrate 10 and a microfluidic channel layer 20, wherein the microfluidic channel layer 20 is disposed on the transparent substrate 10. As shown in fig. 2, the microfluidic channel layer 20 includes a sheath flow inlet 201, a cell suspension inlet 202, a sheath flow channel 203, a cell suspension channel 204, a microfluidic electroporation channel 205, and a cell outlet 206; the sheath flow inlet 201 is communicated with a sheath flow channel 203, the cell suspension inlet 202 is communicated with a cell suspension channel 204, the tail end of the sheath flow channel 203 is converged with the tail end of the cell suspension channel 204 and is communicated with one end of a microfluidic electroporation channel 205, and the other end of the microfluidic electroporation channel 205 is communicated with a cell outlet 206. As shown in fig. 3, the transparent substrate 10 is integrated with a microelectrode, the microelectrode comprises an interdigitated electrode 101 and a parallel electrode 102, the interdigitated electrode 101 and the parallel electrode 102 are distributed below a micro-fluidic electroporation channel 205, the micro-fluidic electroporation channel 205 passes through the middle of the interdigitated electrode 101 and the parallel electrode 102 from a top view, two stages of the electrodes are respectively located at two ends of the micro-fluidic electroporation channel 205, and the interdigitated electrode 101 and the parallel electrode 102 are sequentially distributed from a cell suspension inlet 202 to a cell outlet 206, that is, the interdigitated electrode 101 is close to the cell suspension inlet 202 and is located upstream; parallel electrodes 102 are near cell outlet 206, downstream; the micro-electrode includes micro-electrode terminals 103, and the micro-electrode terminals 103 are disposed on both sides of the transparent substrate 10.

In this embodiment, there are three interdigital electrodes 101, there is one parallel electrode 102, and actually, a corresponding number may be set as needed, and there may be a plurality of parallel electrodes 102. The plurality of microelectrodes are uniformly distributed at intervals, and the distance between the adjacent microelectrodes is designed according to requirements, so that the electric field distribution is more uniform, the electric field environment of each cell is similar, and the survival rate and the transfection efficiency are improved. The microelectrode spacing is embodied in the electrode geometry and layout design when the transparent substrate 10 is fabricated.

The sheath flow channel 203 comprises two channel branch flows, the two channel branch flows bypass the cell suspension inlet 202 from two sides of the cell suspension inlet 202 and are communicated with the cell suspension channel 204, and the sheath flows of the two channel branch flows can effectively converge cells to flow in the center of the microfluidic channel, so that the sequential electroporation treatment of single cells is realized.

As shown in fig. 4, the apparatus further comprises a sheath flow input tube 301, a cell suspension input tube 302, and a cell output tube 303, wherein the cell suspension input tube 302 is communicated with the cell suspension inlet 202, the sheath flow input tube 301 is communicated with the sheath flow inlet 201, and the cell output tube 303 is communicated with the cell output port 206, so as to facilitate the injection of the cell suspension and the sheath flow, and the outflow of the mixed liquid. The sheath flow inlet pipe 301, the cell suspension inlet pipe 302 and the cell outlet pipe 303 are typically teflon pipes, but stainless steel pipes may be used, and the specific diameters may be determined according to the pore diameters of the cell suspension inlet 202, the sheath flow inlet 201 and the cell outlet 206.

The transparent substrate 10 is provided with a cross alignment mark 104 for bonding, and the microfluidic channel layer 20 is provided with a four-square alignment mark 207 for bonding, which is matched with the cross alignment mark 104 for bonding, i.e. a field-shaped alignment mark or an alignment mark with other shapes can be adopted, and the effect is the same. The transparent substrate 10 is further provided with processing alignment marks 105 including a sheath flow inlet 201, a cell suspension inlet 202, and a cell outlet 206. The cross-shaped alignment mark 104 is just embedded into the square alignment mark 207, so that the transparent substrate 10 and the microfluidic channel layer 20 can be bonded more accurately, and the microfluidic electroporation channel 205 can be located in the microelectrode exactly. In the case of not requiring precise alignment, the bonding alignment mark may not be designed.

The distance between the electrodes at the two ends of the microelectrode can be designed according to the needs, and the finger distance of the interdigital electrode 101 in the embodiment is 50-80 μm; the electrode pitch of the parallel electrodes 102 is 150 to 200 μm. The variability of both is represented by the electrode parameter design at the time of designing the transparent substrate 10.

As shown in fig. 5, the parameter-adjustable single cell electroporation device using the microelectrode flow control chip comprises a voltage-stabilized power supply module, a function signal generation module, a PCB control circuit, a digital display module, a chip carrier and a microelectrode flow control chip, wherein the chip carrier is provided with a chip slot, the microelectrode flow control chip is fixed in the chip slot, the output ends of the PCB control circuit and the digital display module are spring probes, and the spring probes are in electrical contact with a microelectrode leading-out end.

The stabilized voltage supply module is used for supplying power to the function signal generation module, the PCB control circuit, the digital display module and the microelectrode flow control chip.

The function signal generation module can control the waveform, frequency, duty ratio and other parameters of the electric signal applied to the microelectrode and is used for outputting signals required by power supply perforation.

The PCB control circuit and the digital display module can adjust the amplitude of the electric signal applied to the micro electrode through the gain adjustable amplifier, so that the optimal adjustment of electroporation parameters aiming at different cell lines is realized. The PCB control circuit and the digital display module comprise a switch circuit and an amplifier, and the switch circuit divides a single-path input into multiple paths of outputs; the amplifier is used for adjusting the amplitude of the output signal; the input end of the switching circuit is connected with the output end of the function signal generation module, and the output end of the switching circuit is connected with the input end of the amplifier; the output end of the amplifier is connected with the microelectrode leading-out end. Because there are a plurality of microelectrodes, so there are a plurality of amplifiers, and each signal output from the switching circuit is amplified by the amplifier and then connected to the corresponding microelectrode. The amplifiers are gain adjustable amplifiers, and the gain of each amplifier can be adjusted according to the requirement, so that different amplitudes of signals applied to each microelectrode can be realized.

The voltage stabilizing circuit module can use switching power supply voltage stabilizing chips such as LM2575, MAX1715 and the like, the efficiency is high, the power consumption is low, and the specific chip can be determined according to the power supply voltage of the chip used by the PCB control circuit and the digital display module; the function signal generation module can be completed by using a monolithic integrated function generation chip MAX038, can generate high-frequency high-precision output waveforms, has small distortion, small drift and wide frequency range of the output waveforms, can generate sine waves, square waves and other waveforms, and has adjustable frequency and duty ratio; the PCB control circuit uses a switch circuit to realize single-path input and multi-path output of electric signals, and uses a gain adjustable amplifier to realize the gain adjustable function. In this embodiment, there are three sets of interdigital electrodes and one set of parallel electrodes, so the PCB control circuit has a single input and four outputs, and the gains are set to 0 times, 1 times, 2 times and 3 times, respectively.

The on-off of the electric signals on the micro-electrodes is controlled by a chip and a switch on a PCB control circuit module, the chip realizes the input of single-path electric signals and the output of multi-path electric signals, the type and the number of the accessed electrodes are adjusted by the PCB control circuit switch, the gating of the multi-path electrodes is realized, the time of exposing cells to an electric field is changed, and thus the working condition of cell electroporation is controlled.

The inter-finger electric field of the interdigital electrode 101 is larger than that of the parallel electrode 102, and when the cell passes through the upstream, the cell can be subjected to electroporation; the electroporated cells continue to move downstream, and the electric field between the electrodes of the parallel electrodes 102 is low, so that the electroporation state of the cells can be maintained, and the transfection efficiency of the cells is enhanced and improved.

The process of manufacturing and installing the microelectrode flow control chip into the parameter-adjustable single-cell electroporation device is as follows:

(1) manufacturing a gold microelectrode on a 4-inch transparent substrate by using a lift-off process; manufacturing a PDMS microfluidic channel layer on a 4-inch silicon wafer by using a soft lithography process based on SU-8 photoresist; the transparent substrate can be made of transparent insulating materials such as glass, polymethyl methacrylate (PMMA) and the like; the microelectrode can be manufactured by using noble metal materials such as gold, platinum and the like through processes such as electroplating or deposition. The transparent substrate material ensures that optical microscopic observation can be carried out in the experimental process; the gold, platinum and other materials have strong chemical inertness, good conductivity and no biotoxicity.

(2) As shown in fig. 1 to 4, holes were punched in the PDMS microfluidic channel layer under observation by a stereomicroscope to fabricate a cell suspension inlet, a sheath inlet, and a cell outlet.

(3) And respectively cleaning and drying the transparent substrate and the PDMS microfluidic channel layer, and then placing the transparent substrate and the PDMS microfluidic channel layer in an oxygen plasma cleaning machine for surface modification treatment to realize permanent bonding. During bonding, the four square alignment marks for bonding on the PDMS microfluidic channel layer are overlapped with the cross alignment mark for bonding on the transparent substrate under observation of a body mirror, so as to ensure that the electrode is just positioned in the microfluidic electroporation channel. Then, as shown in FIG. 4, a cell suspension input tube, a sheath flow input tube and a cell output tube are inserted into the microelectrode fluidic chip.

(4) As shown in fig. 5, the microelectrode flow control chip is fixed in a chip groove in a chip carrier, a PCB control circuit module is fastened with the chip carrier by screws, so that a spring probe of the control circuit is electrically contacted with the microelectrode under pressure, and then a stabilized voltage supply module and a function signal generation module are connected to form the parameter-adjustable single cell electroporation device based on the microelectrode flow control chip.

Claims

1. A microelectrode fluidic chip is characterized by comprising a transparent substrate (10) and a microfluidic channel layer (20), wherein the microfluidic channel layer (20) is positioned above the transparent substrate (10), and the microfluidic channel layer (20) comprises a sheath inflow port (201), a cell suspension inlet (202), a sheath flow channel (203), a cell suspension channel (204), a microfluidic electroporation channel (205) and a cell outlet (206); the sheath inflow port (201) is communicated with the sheath flow channel (203), the cell suspension inlet (202) is communicated with the cell suspension channel (204), the tail end of the sheath flow channel (203) is converged with the tail end of the cell suspension channel (204) and is communicated with one end of the microfluidic electroporation channel (205), and the other end of the microfluidic electroporation channel (205) is communicated with the cell outlet (206); the transparent substrate (10) is integrated with a microelectrode which comprises an interdigital electrode (101) and a parallel electrode (102), the interdigital electrode (101) and the parallel electrode (102) are distributed below the micro-fluidic electroporation channel (205), and the interdigital electrode (101) and the parallel electrode (102) are sequentially distributed from a cell suspension inlet (202) to a cell outlet (206); the microelectrode comprises microelectrode leading-out ends (103), and the microelectrode leading-out ends (103) are distributed on two sides of the transparent substrate (10).

2. The microelectrode fluidic chip of claim 1, wherein the interdigitated electrodes (101) and/or the parallel electrodes (102) are distributed in a plurality and uniformly spaced apart.

3. The microelectrode fluidic chip of claim 1, wherein the sheath flow channel (203) comprises two channel branches, and the two channel branches bypass the cell suspension inlet (202) from two sides of the cell suspension inlet (202) and are communicated with the cell suspension channel (204).

4. The microelectrode fluidic chip of claim 1, further comprising a sheath flow input tube (301), a cell suspension input tube (302), and a cell output tube (303), wherein the cell suspension input tube (302) is connected to the cell suspension inlet (202), the sheath flow input tube (301) is connected to the sheath flow inlet (201), and the cell output tube (303) is connected to the cell output port (206).

5. The microelectrode fluidic chip of claim 1, wherein the transparent substrate (10) has a cross-shaped alignment mark (104) for bonding, and the microfluidic channel layer (20) has a four-square-shaped alignment mark (207) for bonding matching the cross-shaped alignment mark (104) for bonding.

6. The microelectrode flow control chip of claim 1, wherein the inter-digital distance between the interdigitated electrodes (101) is 50 to 80 μm; the electrode pitch of the parallel electrodes (102) is 150 to 200 μm.

7. A parameter-adjustable single cell electroporation device using the microelectrode flow control chip of any one of claims 1 to 5, which comprises a voltage-stabilized power supply module, a function signal generation module, a PCB control circuit, a digital display module, a chip carrier and the microelectrode flow control chip, wherein the chip carrier is provided with a chip groove, the microelectrode flow control chip is fixed in the chip groove, the output ends of the PCB control circuit and the digital display module are spring probes, and the spring probes are electrically contacted with a microelectrode leading-out end (103); the stabilized voltage supply module is used for supplying power to the function signal generation module, the PCB control circuit, the digital display module and the microelectrode flow control chip; the function signal generation module is used for outputting signals required by power supply perforation.

8. The adjustable parameter single cell electroporation device of claim 6, wherein the PCB control circuit and digital display module comprises a switch circuit and an amplifier, the switch circuit divides a single input into multiple outputs, and the amplifier is used for adjusting the amplitude of the output signal; the input end of the switching circuit is connected with the output end of the function signal generation module, and the output end of the switching circuit is connected with the input end of the amplifier; the output end of the amplifier is connected with the microelectrode leading-out end.

9. The adjustable parameter single cell electroporation device of claim 7, wherein the amplifier is a gain adjustable amplifier.

10. The tunable parameter single-cell electroporation device according to claim 7, wherein the inter-finger electric field of the inter-finger electrodes (101) is larger than the inter-electrode electric field of the parallel electrodes (102).