CN107557285B - Microfluidic device for realizing low-voltage electro-cell lysis and cell lysis method thereof - Google Patents
Microfluidic device for realizing low-voltage electro-cell lysis and cell lysis method thereof Download PDFInfo
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Abstract
A micro-fluidic device for realizing low-voltage electro cell lysis and a cell lysis method thereof belong to the technical field of micro-fluidic devices. The device consists of conductive coated glass, a channel layer and an organic glass cover plate which are bonded into a whole; a linear cell microchannel is arranged in the channel layer, an electrode pair formed by two conductive coatings with the distance of 10-100 micrometers is manufactured on the coating layer of the conductive coated glass contacted with the channel layer, and a signal generator inputs a phase difference of 180 DEG and an amplitude of 10-30V to two electrodes of the electrode pair respectivelyp‑pAnd an alternating current signal with a frequency of 50 Hz-200 kHz; the strip-shaped structure is vertical to the cell microchannel; a sample inlet and a sample outlet are arranged on the surface of the organic glass cover plate; the width of the cell microchannel is 0.05 mm-1 mm, the depth of the cell microchannel is the same as the thickness of the channel layer, and the distance between the electrode pairs is the same as the width of the strip-shaped structure and is 10-100 micrometers. The invention has simple preparation and low cost, and can realize the continuous cell lysis under the condition of low voltage.
Description
Technical Field
The invention belongs to the technical field of microfluidic devices, and particularly relates to a microfluidic device for realizing low-voltage electro cell lysis and a cell lysis method thereof.
Background
Cell lysis is a key step in biological experiments to extract cell contents such as DNA, RNA, and proteins. According to different principles of cell lysis methods, cell lysis methods can be divided into: physical, mechanical, chemical, electrical, and the like. The physical method is to destroy cells by using osmotic pressure or pressure. Mechanical methods use shear and stress to destroy cells. Chemical methods utilize chemical solvents or antibiotics to disrupt cell membranes. Conventional electrical lysis methods employ the application of a high electric field to the cell to allow penetration of the cell membrane by extracellular substances, a sufficiently high electric field causing irreversible mechanical destruction of the cell membrane, followed by destruction of the cell by osmotic shock. Compared with a chemical method requiring a reagent and a mechanical method relying on high pressure, an electrical method using a high electric field is more convenient and is more suitable for application of micro total analysis systems (micro total analysis systems) and microfluidic chip laboratories (lab-on-a-chips) technologies.
Current high voltage cell lysis devices generally use direct current and all require high operating voltages (greater than 1000V) to achieve high electric field strengths to lyse cells. However, the requirement of high operating voltage limits the application of this method in micro total analysis systems and microfluidic chip laboratories. If the operating voltage is to be reduced, two general approaches are used: the electric field strength is increased by reducing the distance between the electrodes, or the pH value of the buffer solution near the electrodes is changed to achieve the purpose of cell lysis. However, the process of reducing the inter-electrode distance makes the microfluidic chip more complicated and difficult to process, and changing the pH may result in disruption of the cell contents after lysis. Therefore, in order to realize simple, efficient and rapid cell lysis on a chip, it is urgent to develop a cell lysis method that can be integrated into a lab-on-a-chip system using a low voltage.
Disclosure of Invention
The invention aims to provide a microfluidic device for realizing low-voltage electro cell lysis, and solves the problems that the existing electric cell lysis method needs high voltage, cell contents are easy to damage, the structure is complex and the like.
It is another object of the present invention to provide a method for lysing cells under low voltage conditions using the above microfluidic device.
The invention is realized by the following technical scheme:
a microfluidic device for realizing low-voltage electrogenerated cell lysis is composed of a conductive coated glass (the conductive coating is gold, silver, aluminum or Indium Tin Oxide (ITO) with a thickness of 2-10 microns), a channel layer (a double-sided acrylate pressure-sensitive adhesive tape or other transparent double-sided adhesive tapes with a thickness of 10-200 microns) and an organic glass cover plate (an acrylic organic glass cover plate or other organic glass materials with a thickness of 0.5-2 mm) which are bonded into a wholeComposition is carried out; a linear cell microchannel is arranged in the channel layer; etching a strip-shaped structure with the width of 10-100 microns on a film coating layer of the conductive coated glass contacted with the channel layer by using laser, forming an electrode pair by the remaining two parts of conductive coatings, and respectively inputting a phase difference of 180 DEG and an amplitude of 10-30V to two electrodes of the electrode pair by a signal generatorp-pAnd an alternating current signal with a frequency of 50 Hz-200 kHz; the strip-shaped structure is vertical to the cell microchannel and can be crossed with the cell microchannel at any position according to the requirement; the surface of the organic glass cover plate is provided with channel openings corresponding to the positions of the cell microchannels, namely a sample inlet and a sample outlet; the width of the cell microchannel is 0.05 mm-1 mm, and the depth of the cell microchannel is 10-200 microns, which is the same as the thickness of the channel layer; the thickness of the electrode pairs is 2-10 micrometers, and the distance between the electrode pairs is 10-100 micrometers, which is the same as the width of the strip-shaped structure.
A method for cracking cells under the condition of low voltage by using the microfluidic device is characterized in that the cells enter a cell microchannel from a sample inlet, a signal generator is used for respectively inputting a phase difference of 180 DEG and an amplitude of 10-30V to two electrodes of an electrode pairp-pWhen the cell flows to the middle of the electrode pair, the cell membrane is perforated under the action of an electric field, so that the osmotic pressure between cytoplasm and a medium outside the cell membrane is unbalanced, and the cell is finally ruptured; the cell lysate, including DNA, RNA and proteins, continues to flow along the cell microchannel and is eventually collected at the sample outlet.
Adopt above-mentioned technical scheme's positive effect: the invention has simple manufacture and low cost, can crack cells by applying a voltage alternating current electric field, and then collects cell lysate from a sample outlet of the microfluidic device. The device can continuously crack cells and can not be blocked. The separation parameters of the invention can be adjusted at any time, and the sample injection speed of the cells can be adjusted according to different actual requirements so as to adapt to the requirements of different biological experiments.
Drawings
Fig. 1 is a schematic view of a split structure of a microfluidic device according to the present invention.
The names of the parts are: the device comprises conductive coated glass 3, a channel layer 2 and an organic glass cover plate 1, wherein the conductive coated glass 3, the channel layer 2 and the organic glass cover plate 1 are bonded into a whole; a linear cell microchannel 4 is arranged on the channel layer; and etching a strip-shaped structure on a coating layer of the conductive coated glass by laser to form an electrode pair 5, wherein the axis of the strip-shaped structure is vertical to the axis 4 of the cell microchannel, and the cell is cracked according to the requirement and the cell microchannel 4 are crossed at any position of the cell microchannel 4 so as to be convenient for upstream and downstream integration with other microfluidic functional units.
The surface of the organic glass cover plate 1 is provided with channel openings corresponding to the corresponding channel positions, namely a sample inlet 6 and a sample outlet 7. The signal generator 8 inputs alternating signals having a phase difference of 180 ° to the two electrodes of the electrode pair 5, respectively.
Fig. 2 is a schematic diagram of the microfluidic device for low voltage electro-lysis in example 1.
FIG. 3 shows the microfluidic device of the invention at 16V in example 1p-pAnd (3) a microscope image of the breast cancer MCF-7 cell lysis under the electric field condition of 10 kHz. Wherein Panel A is a case where MCF-7 cells flow in a cell microchannel before power-up; panel B is a case where a part of MCF-7 cells were lysed 7 seconds after the electric field was applied; panel C shows the lysis of all MCF-7 cells after 15 seconds of application of the electric field.
Detailed Description
Example 1
Referring to fig. 1, a microfluidic device is composed of an acrylic organic glass cover plate 1, a double-sided acrylate pressure sensitive adhesive tape 2 and indium tin oxide ITO coated glass 3, wherein the acrylic organic glass cover plate 1 and the indium tin oxide ITO coated glass 3 are bonded by the double-sided acrylate pressure sensitive adhesive tape 2 and then sealed to form a microchannel structure, a cell microchannel 4 is etched on the double-sided acrylate pressure sensitive adhesive tape 2, a strip-shaped structure with a width of about 50 micrometers is etched on a coating layer of the ITO coated glass 3 by laser to form a pair of transparent electrode pairs 5, and a sample inlet 6 and a sample outlet 7 are formed on the acrylic organic glass cover plate 1.
Fig. 2 is a schematic structural diagram of a microfluidic device according to the present invention, and as shown in the figure, the microfluidic device has a long-strip microchannel, and the microchannel has an opening on the surface of the acrylic organic glass cover plate 1, which is a sample inlet 6 and a sample outlet 7. The sample inlet 6 and the sample outlet 7 are used for injecting cell samples and collecting cell lysates, respectively. A bar-shaped electrode pair 5 is arranged on the bottom surface of the cell microchannel 4, the axis of the bar-shaped structure is vertical to the cell microchannel, the bar-shaped structure can be crossed with the cell microchannel at any position of the cell microchannel according to the required cell lysis position, and alternating current signals are respectively input into the two electrodes.
The principle on which the present invention is based includes: the cell enters the cell microchannel from the sample inlet, and the signal generator is used for respectively inputting a phase difference of 180 DEG and an amplitude of 10-30V to two electrodes of the electrode pairp-pWhen the cell flows to the middle of the electrode pair, the cell membrane is perforated under the action of an electric field, so that the osmotic pressure between cytoplasm and a medium outside the cell membrane is unbalanced, and the cell is finally ruptured; the cell lysate, which contains DNA, RNA and proteins, continues to flow along the cell microchannel and can eventually be collected at the sample outlet.
The specific manufacturing method of the microfluidic device comprises the following steps:
(1) firstly, designing and manufacturing a micro-channel of a microfluidic device on a pressure-sensitive double-sided adhesive tape: and cutting and processing the pressure-sensitive double-sided adhesive tape with the length of 20mm, the width of 10mm and the thickness of 76 microns by adopting a cutting mode to obtain the micro-channel, wherein the width of the cell micro-channel is 120 microns, and the depth of the cell micro-channel is 76 microns.
(2) The ITO conductive coated glass is 20mm long, 20mm wide and 1mm thick, the thickness of the ITO coated layer is 5 microns, the ITO coated layer is burned in a high-power laser burning mode, the coated layer is divided into two electrodes which are 50 microns apart, and the electrode pair 5 used for generating the non-uniform electric field in the figure 2 is obtained.
(3) The organic glass cover sheet is an acrylic sheet with a transmittance of 95%. The organic glass cover plate is 20mm long, 20mm wide and 1mm thick. Two holes with the diameter of 0.8mm are punched on the cover plate corresponding to the head end of the cell microchannel 4 and the tail end of the cell microchannel 5, and are respectively a sample inlet 6 and a sample outlet 7.
(4) After the conductive coated glass and the organic glass cover plate are respectively cleaned, the ITO conductive coated glass layer 3, the pressure-sensitive double-sided adhesive layer 2 and the organic glass cover plate 1 are aligned in sequence from bottom to top in the figure 1 and are adhered by the pressure-sensitive double-sided adhesive layer 2.
Example 2
A method for lysing cells under AC low voltage condition by using microfluidic device comprises suspending human breast cancer MCF-7 cells in PBS buffer solution with syringe pump (Longpurmp)TMLSP02-2, run length resolution: 0.03125 μm) is injected into the cell microchannel 4 of the microfluidic chip through the injection port 6, and the injection speed is adjusted to be 0.2-5 μ l/min, 2 μ l/min in this embodiment, as shown in fig. 3A. The cell microchannel 4 has a bottom surface on which electrode pairs 5 are disposed in a bar-shaped configuration having an axis perpendicular to the flow direction of the fluid in the cell microchannel. The two electrodes are respectively input with a phase difference of 180 DEG and a voltage of 20V by a signal generatorp-pAnd an alternating current signal having a frequency of 10 kHz. When the cell is pushed between the two electrodes of the pair 5, the cell membrane is perforated by the electric field, which causes an imbalance in the osmotic pressure between the cytoplasm and the extracellular medium, resulting in the final rupture of the cell, as shown in fig. 3B and 3C. The cell lysate continues to flow along the cell microchannel and may eventually be collected at the outlet 7.
Claims (6)
1. A micro-fluidic device for realizing low-voltage electro cell lysis is characterized in that: the device consists of conductive coated glass, a channel layer and an organic glass cover plate which are bonded into a whole; a linear cell microchannel is arranged in the channel layer, a strip-shaped structure with the width of 10-100 micrometers is etched on a coating layer of the conductive coated glass contacted with the channel layer by laser, the rest two parts of conductive coatings form an electrode pair, a signal generator respectively inputs a phase difference of 180 DEG and an amplitude of 10-30V to two electrodes of the electrode pairp-pAnd an alternating current signal with a frequency of 50 Hz-200 kHz; the strip-shaped structure is vertical to the cell microchannel; the surface of the organic glass cover plate is provided with channel openings corresponding to the positions of the cell microchannels, namely a sample inlet and a sample outlet; the width and depth of the cell micro-channel are 0.05-1 mmThe thickness of the electrode pair is the same as that of the channel layer, and the distance between the electrode pairs is the same as the width of the strip-shaped structure and is 10-100 micrometers;
the principle comprises the following steps: the cell enters the cell microchannel from the sample inlet, and the signal generator is used for respectively inputting a phase difference of 180 DEG and an amplitude of 10-30V to two electrodes of the electrode pairp-pWhen the cell flows to the middle of the electrode pair, the cell membrane is perforated under the action of an electric field, so that the osmotic pressure between cytoplasm and a medium outside the cell membrane is unbalanced, and the cell is finally broken; the cell lysate, which contains DNA, RNA and proteins, continues to flow along the cell microchannel and is eventually collected at the sample outlet.
2. The microfluidic device for performing low voltage electro-lysis of cells according to claim 1, wherein: the coating layer of the conductive coated glass is gold, silver, aluminum or ITO, and the thickness of the coating layer is 2-10 microns.
3. The microfluidic device for performing low voltage electro-lysis of cells according to claim 1, wherein: the channel layer is a double-sided acrylate pressure sensitive adhesive tape, and the thickness of the channel layer is 10-200 microns.
4. The microfluidic device for performing low voltage electro-lysis of cells according to claim 1, wherein: the organic glass cover plate is an acrylic organic glass cover plate, and the thickness is 0.5-2 mm.
5. A method of effecting low voltage electro-lysis, comprising: the microfluidic device for realizing low-voltage electro-lysis of cells according to any one of claims 1 to 4, wherein the cells enter the cell microchannel from the sample inlet, and the signal generator is used for respectively inputting the phase difference of 180 degrees and the amplitude of 10 to 30V to the two electrodes of the electrode pairp-pWhen the cell flows to the middle of the electrode pair, the cell membrane is perforated under the action of the electric field, and the osmotic pressure between cytoplasm and the medium outside the cell membrane is unevenEquilibration, leading to eventual rupture of the cells; the cell lysate, including DNA, RNA and proteins, continues to flow along the cell microchannel and is eventually collected at the sample outlet.
6. A method of performing low voltage electro-lysis as claimed in claim 5, wherein: the sample injection speed is 0.2 microliter per minute to 5 microliter per minute.
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| CN113000078A (en) * | 2019-12-19 | 2021-06-22 | 深圳华大生命科学研究院 | Chip and preparation method thereof |
| CN111205966B (en) | 2020-04-18 | 2020-07-21 | 博奥生物集团有限公司 | Sample extraction chip and biological reaction device |
| CN112011446A (en) * | 2020-08-31 | 2020-12-01 | 吉林大学 | Micro-fluidic chip for bacterial lysis, bacterial lysis device and application thereof |
| CN112730560B (en) * | 2020-12-10 | 2023-09-22 | 东南大学 | Microfluidic impedance cytometer and preparation method thereof |
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