CN114717085A - Micro-fluidic chip based on dielectrophoresis single-cell capture and electroporation - Google Patents
Micro-fluidic chip based on dielectrophoresis single-cell capture and electroporation Download PDFInfo
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- 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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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- 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
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- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
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Abstract
The application discloses micro-fluidic chip based on dielectrophoresis unicell is caught and electroporation, its characterized in that includes: the device comprises a PDMS cover plate, ITO conductive glass, an outlet, a capillary injection tube, a positioning micro-tube, an inlet and a micro-channel; the PDMS cover plate is arranged on the upper surface of the ITO conductive glass, and a micro-channel is formed between the PDMS cover plate and the ITO conductive glass; the upper surface of PDMS apron is provided with: the micro-channel comprises an inlet, an outlet and a positioning micro-tube, wherein the inlet and the outlet are communicated with the micro-channel; the capillary injection tube is inserted into the positioning microtube and is communicated with the microchannel; the upper surface of the ITO conductive glass is provided with a conductive coating; and the outer surfaces of the positioning microtube and the capillary injection tube are both provided with metal coatings. The micro-fluidic chip can be used in the fields of in vitro fertilization, transgenosis, micro biopsy, cell drug therapy and the like.
Description
Technical Field
The invention belongs to the technical field of medical instruments, and particularly relates to a cell microinjection microfluidic chip based on dielectrophoresis single cell capture and electroporation.
Background
Cell microinjection is a very important micromanipulation technique in cell engineering, and generally, a precise micro-positioning device is used to fix cells at a specified position, and then a cell membrane is punctured by an injection needle, so that exogenous substances are injected into cytoplasm or nucleus. At present, the most common method is a micro-pipette method, wherein the position of a cell is found through microscope observation, the cell is adsorbed at the opening of a micro-tube by using negative pressure, then the cell is approached, extruded and punctured by using a micro-injector filled with a target substance, and an exogenous substance is further injected into the cell to complete micro-injection of the cell. It can be seen that the micropipette method is a static cell injection technique, and can only perform microinjection on a single cell at a time, resulting in very low manipulation flux; in addition, the complicated micro-manipulation steps also increase certain labor intensity. With the increasing demand for automation, it is necessary to provide a method capable of realizing integration of high-throughput single-cell autonomous capture and microinjection so as to improve the microinjection efficiency of cells.
The Dielectrophoresis (DEP) effect is one of the most common particle manipulation techniques in the field of microfluidics, and describes the phenomenon of translational motion of micro-nano particles (such as cells, viruses, bacteria, biological macromolecules and the like) suspended in a solution in a non-uniform electric field due to the difference of the polarization degree with the surrounding solution, so that dielectrophoresis is a non-contact and non-destructive particle manipulation technique, and has been widely applied to the researches on capturing and sorting of cells. When the polarization degree of the particles is stronger than that of the solution, the particles move to a high electric field intensity area, which is called positive dielectrophoresis effect (pDOP); conversely, if the particle polarization is weaker than the solution polarization, the particles will move to a region of lower electric field strength, referred to as the negative dielectrophoresis effect (nDEP). For example, when cell capture is performed in a specific electrolyte, the cell can be adsorbed at the edge of the electrode (the position where the electric field intensity is maximum) by positive dielectrophoresis force in a certain electric signal frequency range; by changing the electric field frequency, the cells can also be subjected to negative dielectrophoretic forces and repelled away from the electrode edges. Therefore, dielectrophoretic forces are closely related to the electrical properties of the sample (including particles and suspensions), with the potential to manipulate the electric field parameters and the electrical properties of the sample for a variety of particle manipulations.
The electroporation technology is to instantaneously increase the permeability of cell membranes by the action of a high-intensity electric field, thereby forming pores with a certain size on the surface of the cell membranes, and absorbing exogenous substances in the surrounding medium by using the pores. The electroporation technology can introduce nucleotide, DNA, RNA, protein, saccharide, dye, virus particle, etc. into prokaryotic and eukaryotic cells, is one key technology for delivering medicine or gene into cell, and has the features of wide application, high efficiency, no residual toxicity, easy control of parameters, etc. Conventional electroporation techniques are performed in specialized vessels, requiring the application of ultra-high voltages, resulting in high cell mortality.
With the rapid development of micro-nano processing technology, electroporation technology under the micro scale begins to be very diverse, and scholars begin to perform cell electrotransfection in a micro channel of tens of microns, so that transfection voltage can be greatly reduced, and the survival rate of cells can be effectively improved. The key premise for performing cell electroporation on a microscale is to perform precise manipulation on cells, and usually, a single cell is captured to an electroporation position or a cell group is arranged into a cell bundle to flow through an electroporation region, and the cells are subjected to electroporation by using a pulse electrical signal.
Therefore, the micro-scale electroporation technology is an effective combination of the cell electroporation technology and the fluidic cell manipulation technology, and can provide a reference for micro-injection of cells.
By combining the analysis, dielectrophoresis is a powerful microfluidic cell capture technology and can be perfectly applied to cell capture and positioning operation in micro-scale electroporation; electroporation is an effective method for delivering foreign substances into cells. Therefore, the dielectrophoresis cell capture technology and the electroporation exogenous substance introduction technology are combined, and an ideal and feasible scheme can be provided for developing a micro-fluidic chip integrating cell capture and injection kinetic energy. Is beneficial to promoting the development of high-flux, high-efficiency and intelligent cell microinjection.
Disclosure of Invention
The present invention is directed to a miniaturized nucleic acid sample processing apparatus and a pipetting method to solve the problems of the prior art.
In order to achieve the above purpose, the technical solution proposed by the present application is as follows:
a cell microinjection microfluidic chip based on dielectrophoretic single-cell capture and electroporation, comprising: the device comprises a PDMS cover plate, ITO conductive glass, an outlet, a capillary injection tube, a positioning micro-tube, an inlet and a micro-channel;
the PDMS cover plate is arranged on the upper surface of the ITO conductive glass, and a micro-channel is formed between the PDMS cover plate and the ITO conductive glass;
the upper surface of PDMS apron is provided with: the micro-channel comprises an inlet, an outlet and a positioning micro-tube, wherein the inlet and the outlet are communicated with the micro-channel;
the capillary injection tube is inserted into the positioning microtube and is communicated with the microchannel.
Furthermore, the number of the positioning micro-tubes is multiple, and the positioning micro-tubes are arranged on the PDMS cover plate in an array manner.
Further, a conductive coating is arranged on the upper surface of the ITO conductive glass; and the outer surfaces of the positioning microtube and the capillary injection tube are both provided with metal coatings.
Furthermore, the upper surface of the ITO conductive glass is provided with a conductive coating, and the outer surfaces of the positioning microtube and the capillary injection tube are both provided with metal coatings which are AU.
Further, the glass substrate of the ITO conductive glass is 60mm long, 50mm wide and 1.1mm high, and the thickness of the ITO coating is 0.5 mu m.
Further, the PDMS microchannel has a width of 5mm, a length of 16mm and a height of 200 μm.
Furthermore, the inner diameter of the positioning micro-tube is 0.58mm, the outer diameter is 1.03mm, and the length is 10 mm.
Further, the capillary injection tube has an inner diameter of 100 μm, an outer diameter of 170 μm, and a length of 16 mm.
A method for using a cell microinjection microfluidic chip based on dielectrophoresis single cell capture and electroporation comprises the following steps:
first, a cell suspension is injected into the microchannel from the inlet: the voltage amplitude and the electric field frequency applied to the positioning microtube are regulated and controlled to enable the cells to be acted by positive dielectrophoresis force, so that the cells are captured at the position of the tube opening of the positioning microtube;
then, applying a proper pulse signal to the capillary injection tube in the interior to ensure that the cells are subjected to electroporation, and forming a series of microporous structures on the surfaces of the cells;
then, delivering the substance in the capillary injection tube into the cell; and then adjusting the current signal of the frequency of the positioning microtube to enable the cells to be subjected to the action of positive dielectrophoresis force, repelling the cells away from the positioning microtube, and introducing the cell suspension to perform the next cell microinjection operation.
The beneficial effect of this application:
the core design of the application is that the PDMS cover plate is arranged on the upper surface of the ITO conductive glass, and a micro-channel is formed between the PDMS cover plate and the ITO conductive glass; the upper surface of PDMS apron is provided with: the micro-channel comprises an inlet, an outlet and a positioning micro-tube, wherein the inlet and the outlet are communicated with the micro-channel; the capillary injection tube is inserted into the positioning micro-tube and is communicated with the micro-channel, and a conductive coating is arranged on the upper surface of the ITO conductive glass; the outer surfaces of the positioning microtube and the capillary injection tube are both provided with metal coatings, thereby being practical for cell microfluidics. Compared with the prior art, the invention is suitable for the fields of cell gene modification, in vitro fertilization, micro biopsy, single cell electrophysiological property research, cell drug therapy and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a three-dimensional overall structure diagram of a micro-injection chip based on dielectrophoretic single-cell capture and electroporation.
Fig. 2 is a schematic plan view of a micro-injection chip.
Fig. 3 is a schematic sectional view a-a of fig. 2.
FIG. 4 is a PDMS cover with channels and hole-like structures.
Figure 5 is a nested configuration of positioning microtubes and capillary injection tubes.
FIG. 6 is a design drawing of the dimensions of the micro-injection chip of the present application.
The reference numerals are explained below:
1-PDMS cover plate; 2-ITO conductive glass; 3-an outlet; 4-capillary injection tube 3; 5-positioning the microtube; 6-inlet; 7-micro-channel.
Detailed Description
The present invention will be described in detail below with reference to embodiments shown in the drawings. The embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to the embodiments are included in the scope of the present invention.
(1) Chip design processing
As can be seen from fig. 1-3 and fig. 6: the key structural parameters of the chip are as follows: the glass substrate with the indium tin film (ITO) conductive coating is 60mm long, 50mm wide and 1.1mm high, and the thickness of the ITO coating is 0.5 mu m. PDMS micro channel width 5mm, length 16mm, height 200 m. The inner diameter of the positioning micro-tube is 0.58mm, the outer diameter is 1.03mm, the length is 10mm, the inner conical part of the channel is 60 micrometers, the inner diameter of the conical tube opening is 60 micrometers, the inner diameter of the capillary injection tube is 100 micrometers, the outer diameter is 170 micrometers, the length is 16mm, the inner conical part of the channel is 60 micrometers, and the inner diameter of the conical opening is 10 micrometers.
1. Processing and bonding of the chip: (1) processing a channel mold by using a DuPont dry film based on a standard soft lithography technology, further pouring PDMS on the channel mold, and processing a PDMS cover plate with a channel structure; (2) preparing a gold (Au) film with the thickness of 200nm on the outer surface of the positioning microtube and the capillary injection tube respectively by using a magnetron sputtering instrument; (3) punching holes of 2mm at the inlet and the outlet of a channel by using a puncher, punching 6 multiplied by 3 array micro-tube holes of 1mm at the middle position of the channel, inserting a capillary injection tube into a positioning micro-tube, and packaging together by AB glue; (4) carrying out hydrophilic treatment on PDMS and a glass substrate by using a plasma machine and then bonding; (5) and respectively inserting rubber tubes with the diameter of 2mm into the inlet and the outlet, and bonding the rubber tubes by using AB glue.
2. Sample preparation: (1) before the experiment, the micro-fluidic chip is processed by a plasma machine to change the inner surface of the channel into a hydrophilic surface, so that the cell suspension liquid flows smoothly and the adhesion of bubbles on the surface of the channel when the solution is injected is prevented; (2) suspending the egg cells and the sperm cells in DMEM/F12 cell culture solution respectively, calibrating the conductivity of the suspension by using a conductivity meter so as to select the proper cell capture frequency, injecting the egg cell suspension from the inlet of the channel, and transferring the sperm cell suspension into a capillary injection tube.
3. The micro-fluidic chip is electrically connected with the configuration: the conductive glass substrate is used as a common electrode and is grounded, the Au thin film on the outer surface of the positioning micro-tube is connected with the anode of a high-frequency alternating current signal, and the Au thin film on the outer surface of the capillary injection tube is connected with a pulse electrical signal.
4. And (3) experimental operation: (1) opening an alternating current signal, and adjusting a proper frequency to enable the egg cells to be acted by positive dielectrophoresis force; (2) slowly injecting an egg cell culture solution into the channel until the whole channel is filled, and observing that the egg cells are captured at the position of the pipe opening of the positioning pipe due to the positive dielectrophoresis force; (3) opening a pulse electric signal, adjusting a proper pulse amplitude and a proper pulse width to enable reversible electroporation to be carried out on the surface of the egg cell, closing the pulse electric signal, and slowly injecting a sperm cell culture solution into the egg cell through the perforation by using a high-precision micro-injection pump; (4) adjusting the alternating current signal to a proper frequency to enable the egg cells to be acted by negative dielectrophoresis force, so that the positioning microtubes repel the egg cells from leaving the microtubes; (5) and (4) introducing the egg cell culture solution again, repeating the steps, and entering a new round of in vitro fertilization experiment.
5. And (3) processing experimental data: fertilized egg cells were collected from the outlet and cultured in vitro, and their survival and growth status were observed and recorded.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. A microfluidic chip based on dielectrophoretic single-cell capture and electroporation, comprising: the device comprises a PDMS cover plate, ITO conductive glass, an outlet, a capillary injection tube, a positioning micro-tube, an inlet and a micro-channel;
the PDMS cover plate is arranged on the upper surface of the ITO conductive glass, and a micro-channel is formed between the PDMS cover plate and the ITO conductive glass;
the upper surface of PDMS apron is provided with: the micro-channel comprises an inlet, an outlet and a positioning micro-tube, wherein the inlet and the outlet are communicated with the micro-channel;
the capillary injection tube is inserted into the positioning microtube and is communicated with the microchannel;
the upper surface of the ITO conductive glass is provided with a conductive coating; and the outer surfaces of the positioning microtube and the capillary injection tube are both provided with metal coatings.
2. The microfluidic chip according to claim 1, wherein the number of the positioning microtubes is multiple and the positioning microtubes are arranged in an array on the PDMS cover plate.
3. A microfluidic chip based on dielectrophoretic single-cell capture and electroporation according to claim 1, wherein the metal coating provided on the outer surface of the positioning microtubes and the capillary injection tubes is 200nm gold thin film.
4. A dielectrophoresis-based single-cell capture and electroporation-based microfluidic chip according to claim 2, wherein the array of positioning microtubes is distributed between the inlet and the outlet.
5. A microfluidic chip according to any one of claims 1 to 4, wherein the glass substrate of ITO conductive glass has a length of 60mm, a width of 50mm, a height of 1.1mm, and a thickness of 0.5 μm.
6. A microfluidic chip according to any of claims 1 to 4, wherein said PDMS microchannel has a width of 5mm, a length of 16mm and a height of 200 μm.
7. The microfluidic chip for dielectrophoresis-based single-cell capture and electroporation according to any one of claims 1 to 4, wherein the positioning microtube has an inner diameter of 0.58mm, an outer diameter of 1.03mm and a length of 10 mm.
8. A microfluidic chip according to any of claims 1 to 4, wherein the capillary injection tube has an inner diameter of 100 μm, an outer diameter of 170 μm and a length of 16 mm.
9. A microfluidic chip according to claim 1, wherein the following steps are adopted when in use:
first, a cell suspension is injected into the microchannel from the inlet: the voltage amplitude and the electric field frequency applied to the positioning microtube are regulated and controlled to enable the cells to be acted by positive dielectrophoresis force, so that the cells are captured at the position of the tube opening of the positioning microtube;
then, applying a proper pulse signal to the capillary injection tube in the interior to electroporate the cells, and forming a series of micropore structures on the surfaces of the cells;
then, delivering the substance in the capillary injection tube into the cell; and then adjusting the current signal of the frequency of the positioning microtube to enable the cells to be subjected to the action of positive dielectrophoresis force, repelling the cells away from the positioning microtube, and introducing the cell suspension to perform the next cell microinjection operation.
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| CN202210468435.3A CN114717085A (en) | 2022-04-29 | 2022-04-29 | Micro-fluidic chip based on dielectrophoresis single-cell capture and electroporation |
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| CN202210468435.3A CN114717085A (en) | 2022-04-29 | 2022-04-29 | Micro-fluidic chip based on dielectrophoresis single-cell capture and electroporation |
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| CN (1) | CN114717085A (en) |
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2022
- 2022-04-29 CN CN202210468435.3A patent/CN114717085A/en not_active Withdrawn
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Application publication date: 20220708 |
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