CN107557285A - A kind of method for the micro-fluidic device and its cell lysis for realizing the electroluminescent cell cracking of low-voltage - Google Patents
A kind of method for the micro-fluidic device and its cell lysis for realizing the electroluminescent cell cracking of low-voltage Download PDFInfo
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Abstract
A kind of method for the micro-fluidic device and its cell lysis for realizing the electroluminescent cell cracking of low-voltage, belongs to micro-fluidic device technical field.Conductive coated glass, channel layer and the lucite cover plate being integrated by bonding form；Linear pattern cell microchannel is provided with channel layer, on the film plating layer for the conductive coated glass being in contact with channel layer, make the electrode pair having by being formed at a distance of 10~100 microns of two parts conduction plated film, from signal generator to the two of electrode pair electrodes respectively input phase difference be 180 °, amplitude be 10~30Vp‑p, frequency be 50Hz~200kHz AC signal；Strip structure is perpendicular to cell microchannel；Injection port and outlet are provided with the surface of lucite cover plate；The width of cell microchannel is 0.05mm~1mm, and depth is identical with the thickness of channel layer, and distance is identical with the width of strip structure between electrode pair, is 10~100 microns.Present invention making is simple, cost is low, it is possible to achieve the lasting cracking of cell under low voltage condition.
Description
Technical field
The invention belongs to micro-fluidic device technical field, and in particular to a kind of miniflow for realizing the electroluminescent cell cracking of low-voltage
The method for controlling device and its cell lysis.
Background technology
Cell cracking is to extract the committed step in cellular content such as DNA, RNA and protein Bioexperiment.According to
The different principle of cell lysing methods, cell lysing methods can be divided into：Physical method, mechanical means, chemical method and electricity side
Method etc..Physical method is to destroy cell using osmotic pressure or pressure.Mechanical means utilizes shearing force and stress rupture cell.Chemistry
Method then destroys cell membrane using chemical solvent or antibiotic.Conventional electrical cleavage method then by cell apply high electric field with
Make the permeable cell membrane of ECM, sufficiently high electric field causes the irreversible mechanical damage of cell membrane, then passed through
Osmotic shock destroys cell.Compared with needing the chemical method of reagent and the mechanical means by high pressure, high electric field is used
Electrical method is more convenient, is more suitable for micro-total analysis system (micro total analysis system) and micro-fluidic core
Piece laboratory (lab-on-a-chip) technology application.
Current high voltage cell lysis device is generally adopted by direct current, and is required for higher operating voltage (big
In 1000V), carry out cell lysis to obtain high electric-field intensity.But the requirement of high working voltage limits this method at micro- complete point
The application of analysis system and Microfluid based Lab on a chip.To reduce operating voltage, then typically pass through two ways：By reducing electricity
Distance reaches the mesh of cell lysis to increase electric-field intensity, or by changing the pH value of the cushioning liquid near electrode between pole
's.Make the processing of micro-fluidic chip more complicated and difficult however, reducing the method for interelectrode distance, changing pH value may then lead
Cellular content is destroyed after fracturing solution.Therefore, in order to realize, easy, efficient, quick cell cracking, exploitation are sharp on chip
Turn into the task of top priority with low-voltage and cell lysing methods that array experiment chamber system can be integrated in.
The content of the invention
It is an object of the invention to provide a kind of micro-fluidic device for realizing the electroluminescent cell cracking of low-voltage, solves existing electricity
Learn cell lysing methods faced need high-tension electricity, cellular content to be more easily damaged, complex structure and other problems.
It is a further object of the present invention to provide the method using above-mentioned micro-fluidic device cell lysis under low voltage condition.
The present invention is achieved through the following technical solutions：
A kind of micro-fluidic device for realizing the electroluminescent cell cracking of low-voltage, the conductive coated glass being integrated by bonding (are led
Electroplating film is gold, silver, aluminium or tin indium oxide ITO, and film plating layer thickness is 2 microns~10 microns), channel layer (sided acrylic ester
Pressure sensitive adhesive tape or other transparent double-sided tapes, its thickness are 10~200 microns) and lucite cover plate (acrylic lucite
Cover plate or other pmma materials, thickness are 0.5~2mm) composition；Linear pattern cell microchannel is provided with channel layer；
It it is 10~100 microns with one width of laser incising eating away on the film plating layer for the conductive coated glass being in contact with channel layer
Strip structure, remaining two parts conduction plated film form electrode pair, defeated to the two of electrode pair electrode difference from signal generator
Applying aspect difference is 180 °, amplitude is 10~30Vp-p, frequency be 50Hz~200kHz AC signal；Strip structure is perpendicular to thin
Born of the same parents microchannel, and optional position can be intersected at cell microchannel as needed；It is provided with the surface of lucite cover plate
The access portal corresponding with cell microchannel position, respectively injection port and outlet；The width of cell microchannel is
0.05mm~1mm, depth is identical with the thickness of channel layer, is 10~200 microns；The thickness of electrode pair is 2 microns~10 microns,
Distance is identical with the width of strip structure between electrode pair, is 10~100 microns.
A kind of method using above-mentioned micro-fluidic device cell lysis under low voltage condition, cell are entered thin by injection port
Born of the same parents microchannel, with two electrodes from signal generator to electrode pair respectively input phase difference for 180 °, amplitude be 10~30Vp-p、
Frequency is 50Hz~200kHz AC signal, and when cell is flow among electrode pair, cell membrane goes out in the presence of electric field
Now perforate, in turn result in the imbalance of osmotic pressure between cytoplasm and the outer medium of cell membrane, cause the final rupture of cell；Comprising
Cell pyrolysis liquid including DNA, RNA and protein then continues to flow along cell microchannel, is finally collected in outlet.
Using the good effect of above-mentioned technical proposal：Present invention making is simple, cost is low, applied voltage AC field, i.e.,
Cell can be cracked, then collect cell pyrolysis liquid from the outlet of micro-fluidic device.The sustainable cell lysis of the device, and not
It can block.The separation parameter of the present invention can be adjusted at any time, can be according to the different sample introduction speed for being actually needed regulation cell
Degree, to adapt to the needs of different Bioexperiment.
Brief description of the drawings
Fig. 1 is the micro-fluidic device fractionation structural representation of the present invention.
Each several part is entitled：Conductive coated glass 3, channel layer 2 and lucite cover plate 1, conductive coated glass 3, passage
Layer 2 and the bonding of lucite cover plate 1 are integrated；Linear pattern cell microchannel 4 is provided with channel layer；In conductive coated glass
Film plating layer on, pass through the strip structure of laser incising eating away one, form electrode pair 5, the axis of strip structure and cell microchannel
Axis 4 is vertical, and the position of cell lysis intersects at the optional position of cell microchannel 4 with cell microchannel 4 as needed, with
It is easy to carry out upstream and downstream integration with other micro-fluidic functional units.
The access portal corresponding with respective channel position is provided with the surface of lucite cover plate 1, be respectively into
Sample mouth 6, outlet 7.Signal generator 8 distinguishes the AC signal that input phase difference is 180 ° to two electrodes of electrode pair 5.
Fig. 2 is the micro-fluidic device schematic diagram of the electroluminescent cell cracking of low-voltage in embodiment 1.
Fig. 3 is micro-fluidic device of the invention in embodiment 1 in 16Vp-p, 10kHz current field condition under crack breast cancer
The microscope imaging figure of MCF-7 cells.Wherein scheme the situation that MCF-7 cells flow in cell microchannel before A is power-up；Figure
B is the situation that part MCF-7 cells are cleaved after electric field 7 seconds is added；Figure C is then whole MCF-7 after electric field 15 seconds is added
The situation that cell is cleaved.
Embodiment
Embodiment 1
With reference to Fig. 1, a kind of micro-fluidic device, by acrylic lucite cover plate 1, the and of sided acrylic ester pressure-sensitive band 2
Tin indium oxide ito coated glass 3 forms, and acrylic lucite cover plate 1 and tin indium oxide ito coated glass 3 pass through two-sided third
Sealing forms MCA after olefin(e) acid ester pressure-sensitive band 2 bonds, wherein, it is carved with cell on sided acrylic ester pressure-sensitive band 2
Microchannel 4, the strip structure that the film plating layer of ito coated glass 3 is about 50 microns by one wide laser incising eating away are formed a pair
Transparency electrode has injection port 6 and outlet 7 to 5 on acrylic lucite cover plate 1.
Fig. 2 is the micro-fluidic device structural representation of the present invention, as illustrated, have microchannel in the micro-fluidic device, it is micro-
Passage is strip, and microchannel openings are on the surface of acrylic lucite cover plate 1, respectively injection port 6, outlet 7.Sample introduction
Mouth 6 and outlet 7 are respectively used to inject cell sample and collect cell pyrolysis liquid.One is laid on the bottom surface of cell microchannel 4
For strip electrode to 5, the axis of strip structure is vertical with cell microchannel, strip structure can according to the position that required cell cracks with
Cell microchannel intersects at the optional position of cell microchannel, and two electrodes distinguish input AC electric signal.
The present invention based on principle include：Cell enters cell microchannel by injection port, with signal generator to electrode pair
Two electrodes respectively input phase difference for 180 °, amplitude be 10~30Vp-p, frequency be 50Hz~200kHz AC signal,
When cell is flow among electrode pair, cell membrane is perforated in the presence of electric field, in turn results in cytoplasm and cell membrane
The imbalance of osmotic pressure between outer medium, cause the final rupture of cell；Cell pyrolysis liquid comprising DNA, RNA and protein then after
It is continuous to be flowed along cell microchannel, it can be finally collected in outlet.
The specific preparation method of micro-fluidic device is as follows：
(1) first, the microchannel of micro-fluidic device is designed, made on double coated pressure-sensitive adhesive tape：By the way of cutting
Long 20mm, wide 10mm, thickness are that cutting processing obtains microchannel on 76 microns of double coated pressure-sensitive adhesive tape, cell microchannel it is a width of
120 microns, depth is 76 microns.
(2) the long 20mm of ITO conductive coated glass, wide 20mm, thickness 1mm, ITO film plating layer thickness are 5 microns, utilize Gao Gong
The mode of rate laser burn burns ITO film plating layers, film plating layer is divided into two electrodes at a distance of 50 microns, obtains and is used in Fig. 2
In the electrode pair 5 for producing inhomogeneous field.
(3) lucite cover plate is the acrylic board that light transmittance is 95%.Plexiglass cover length of a film 20mm, wide 20mm, thickness
Spend for 1mm.The head end of cell microchannel 4 is corresponded on the cover plate, the position of the end of cell microchannel 5 make a call to two it is a diameter of
0.8mm hole, respectively injection port 6 and outlet 7.
(4) after conductive coated glass and lucite cover plate are respectively washed totally, according to the conductive platings of ITO from bottom to top in Fig. 1
The order alignment of film glass layer 3, double-coated pressure-sensitive glue-line 2, lucite cover plate 1, is bonded by double-coated pressure-sensitive glue-line 2.
Embodiment 2
It is a kind of using micro-fluidic device under the conditions of low AC voltage cell lysis method, PBS will be suspended in
In MCF-7 Human Breast Cancer Cells syringe pump (LongerPumpTMLSP02-2, stroke resolution ratio：0.03125 μm) by sample introduction
Mouthfuls 6 cell microchannels 4 injected in micro-fluidic chips, adjust sample introduction speed, sample introduction speed 0.2 microlitre per minute~it is per minute
5 microlitres, the present embodiment is 2 microlitres per minute, as shown in Figure 3A.A strip structure is laid on the bottom surface of cell microchannel 4
Electrode pair 5, the axis of strip structure are vertical with fluid flow direction in cell microchannel.It is defeated to two electrode difference with signal generator
Applying aspect difference is 180 °, voltage 20Vp-p, frequency be 10kHz AC signal.When cell is pushed to two electricity of electrode pair 5
When between pole, cell membrane is perforated in the presence of electric field, in turn results in osmotic pressure between cytoplasm and the outer medium of cell membrane
Imbalance, cause the final rupture of cell, as shown in figs. 3 b and 3 c.Cell pyrolysis liquid then continues to flow along cell microchannel,
It can be finally collected in outlet 7.
Claims (6)
- A kind of 1. micro-fluidic device for realizing the electroluminescent cell cracking of low-voltage, it is characterised in that：The conductive plating being integrated by bonding Film glass, channel layer and lucite cover plate composition；Linear pattern cell microchannel is provided with channel layer, with channel layer phase It is remaining with the strip structure that one width of laser incising eating away is 10~100 microns on the film plating layer of the conductive coated glass of contact Two parts conduction plated film formed electrode pair, distinguishing input phase difference to the two of electrode pair electrodes from signal generator is 180 °, amplitude be 10~30Vp-p, frequency be 50Hz~200kHz AC signal；Strip structure is perpendicular to cell microchannel； It is provided with the access portal corresponding with cell microchannel position on the surface of lucite cover plate, respectively injection port and goes out sample Mouthful；The width of cell microchannel is 0.05mm~1mm, and depth is identical with the thickness of channel layer, distance and bar shaped between electrode pair The width of structure is identical, is 10~100 microns.
- A kind of 2. micro-fluidic device for realizing the electroluminescent cell cracking of low-voltage as claimed in claim 1, it is characterised in that：It is conductive The film plating layer of coated glass is gold, silver, aluminium or ITO, and film plating layer thickness is 2 microns~10 microns.
- A kind of 3. micro-fluidic device for realizing the electroluminescent cell cracking of low-voltage as claimed in claim 1, it is characterised in that：Passage Layer is sided acrylic ester pressure-sensitive band or other transparent double-sided tapes, and its thickness is 10~200 microns.
- A kind of 4. micro-fluidic device for realizing the electroluminescent cell cracking of low-voltage as claimed in claim 1, it is characterised in that：It is organic Cover glass is acrylic lucite cover plate or other pmma materials, and thickness is 0.5~2mm.
- A kind of 5. method for realizing the electroluminescent cell cracking of low-voltage, it is characterised in that：Utilize any one institute of Claims 1 to 44 That states realizes the micro-fluidic device of the electroluminescent cell cracking of low-voltage, and cell is entered into cell microchannel by injection port, sent out with signal Two electrodes from raw device to electrode pair respectively input phase difference be 180 °, amplitude be 10~30Vp-p, frequency be 50Hz~200kHz AC signal, when cell is flow among electrode pair, cell membrane is perforated in the presence of electric field, in turn results in cell The imbalance of osmotic pressure, causes the final rupture of cell between matter and the outer medium of cell membrane；Including DNA, RNA and protein Cell pyrolysis liquid then continues to flow along cell microchannel, is finally collected in outlet.
- A kind of 6. method for realizing the electroluminescent cell cracking of low-voltage as claimed in claim 5, it is characterised in that：Sample introduction speed is 0.2 microlitre per minute~per minute 5 microlitres.
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