CN109082364B - A kind of micro-structural device of physical method clasmatosis and its clasmatosis and processing method - Google Patents
A kind of micro-structural device of physical method clasmatosis and its clasmatosis and processing method Download PDFInfo
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- CN109082364B CN109082364B CN201810888197.5A CN201810888197A CN109082364B CN 109082364 B CN109082364 B CN 109082364B CN 201810888197 A CN201810888197 A CN 201810888197A CN 109082364 B CN109082364 B CN 109082364B
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Abstract
The structure of the micro-structural device of physical method clasmatosis of the present invention are as follows: nitrogen input channel, cell suspending liquid input channel, broken chamber and crushing shell.Broken chamber and its connected runner are processed using organic polymer method of molding, the sealing-in of broken chamber is then realized with thermal bonding, other parts can realize connection with the method for welding.Clasmatosis system ruptures cell using the method for physical impacts, extracts target component in cell and carries out next step experiment.For cell suspending liquid with spray form by pipe outlet, the partial size of spraying granule is about 5 microns.High velocity carrier gas nitrogen flow rate is 200-300m/s, spray form particulate is sent into broken chamber, high-speed impact crushing shell is crushed.Then cell residue object is collected in broken chamber and is exported from outlet carries out further microfluidic control experiment.
Description
Technical field
The present invention relates to a kind of micro-structural device of physical method clasmatosis and its clasmatosis and processing method, belong to thin
Born of the same parents are crushed field.
Background technique
Micro-fluidic chip (microfluidic chip) is current micro-total analysis system (Miniaturized Total
Analysis Systems) development hot fields.Its target is the function entire laboratory, including sampling, dilution plus
Multiple links such as reagent, reaction, separation, detection are integrated on microchip, and can be used for multiple times, but are with cell (bacterium)
The micro-fluidic chip of operation object all the time without the feasible clasmatosis scheme of system.Common cell breaking technology can
It is divided into Mechanical Method and on-mechanical method, Mechanical Method includes:
High-pressure homogenization crush method (homogenization), oscillation pearl hit crush method (Skaking Bead), high-speed stirred
Pearl grinds crush method (fine grinding), ultrasonic fragmentation (ultrasonication)；On-mechanical method includes: osmotic pressure
Impact crushing method (osmotic shock), freeze thawing crush method (freezing and thawing), the molten crush method (enzyme of enzyme
Lysis), chemically fragmenting method (chemical treatment), detergent crush method (detergents).
Method of cell disruption is applied to the generally existing Railway Project of micro-fluidic chip at present:
1, crushing system volume itself is excessive, cannot be combined with microfluidic system.
2, chemical impurity is introduced, contamination of cells fragment influences the subsequent operation of experiment.
3, the degree of crushing of cell can not be adjusted, and be likely to result in the overcrushing or broken inadequate of cell.
4, cataclasitlc structure is complicated, structure processing difficulties.
Summary of the invention
Technical problem: in order to overcome the shortcomings of above-mentioned existing cell breaking technology, the present invention provides one kind to be based on miniflow
The novel cell cataclasitlc structure of chip is controlled, it can effectively be crushed complete cell or bacterium, guarantee intracellular organelle
Integrality lay a good foundation for subsequent experimental on micro-fluidic chip.
Technical solution: in order to achieve the above technical purposes, the present invention adopts the following technical scheme:
A kind of micro-structural device of physical method clasmatosis, comprising:
Broken chamber, for collecting cell residue object, the top of broken chamber is equipped with the lid that can cover conjunction or opening；
Cell suspending liquid input channel, one end are connected to connection, the other end and cell with the side cavity wall of the broken chamber
The connection of suspension conveying equipment；
Crushing shell is arranged in crusher chamber room and is located at cell suspending liquid input channel exit, towards cell on crushing shell
The one side of suspension input channel outlet end is equipped with puncture structure；
High velocity carrier gas input channel, one end are connected to connection with the cell suspending liquid input channel, and the other end is defeated with high pressure
Gas equipment connection, for by the cell suspending liquid in cell suspending liquid input channel in spray form and flow velocity be not less than
The initial velocity of 200m/s hits the crushing shell；Gas transmission equipmen is pressed to be equipped with the regulating valve for adjusting high velocity carrier gas flow velocity；
Flushing liquor input port is opened in the side for being located at puncture structure on crushing shell on crusher chamber chamber wall, passes through pipeline
Connect with flushing liquor conveying equipment, for broken chamber inner wall and crushing shell be rinsed；
Gas delivery port is set on lid, for discharging the indoor high pressure gas of crusher chamber；
Target liquid delivery outlet, connect with extraction module, for collecting Objective extraction liquid.
The cell suspending liquid input channel includes that the different cell suspending liquid of two diameters inputs pipe unit, is respectively
Diameter thinner first cell suspending liquid input pipe unit and thicker the second cell suspending liquid of diameter input pipe unit, wherein
One end of first cell suspending liquid input pipe unit is directly connected to setting, also, the first cell with the side cavity wall of broken chamber
The side side wall that suspension inputs the axis of pipe unit and the crushing shell is equipped with puncture structure is perpendicular；Second cell suspends
Liquid inputs the intersection setting between pipe unit and first cell suspending liquid input pipe unit at an angle, and first
The intersection of cell suspending liquid input pipe unit and the second cell suspending liquid input pipe unit has one to cross slot；
The high velocity carrier gas input channel includes the different high velocity carrier gas input channel of two diameters, be diameter respectively compared with
Thin first high velocity carrier gas input pipe unit and thicker the second high velocity carrier gas of diameter input pipe unit, wherein the first high speed carries
One end of gas input pipe unit is connected with the slot that crosses, also, the first high velocity carrier gas input pipe unit and the first cell suspend
Liquid inputs the coaxial setting of pipe unit, and the first high velocity carrier gas inputs the other end of pipe unit and the second high velocity carrier gas input pipe unit connects
It connects.
The caliber of the first cell suspending liquid input pipe unit is 5 microns, and the second cell suspending liquid inputs pipe unit
Caliber is 15 microns.
The caliber of the first high velocity carrier gas input pipe unit is 3 microns, and the caliber that the second carrier gas inputs pipe unit is 10
Micron.
Puncture structure is pointed cone array structure on the crushing shell, and the distance between each pointed cone is 300nm.
A kind of method of cell disruption of the micro-structural device based on the physical method clasmatosis, including following step
It is rapid:
S1, high pressure gas transmission equipmen is opened, high pressure gas transmission equipmen is inputted by high velocity carrier gas input channel to cell suspending liquid
Conveying high-pressure carrier gas in pipeline, by adjusting the gas flow rate of valve regulation high pressure gas transmission equipmen to reaching requirement；
S2, cell suspending liquid conveying equipment is opened, cell suspending liquid is in the feeding of spray form under the carrying of high pressure carrier gas
Broken chamber, high-speed impact crushing shell, cell or bacterium are crushed；
After S3, clasmatosis, extraction module collects Objective extraction liquid by target liquid delivery outlet；
After S4, Objective extraction liquid are collected, cleaning solution is conveyed into crusher chamber room by washing lotion conveying equipment, will be broken
The inner wall and crushing shell of broken chamber are rinsed.
The high velocity carrier gas is nitrogen.
The flow velocity of the high velocity carrier gas is 200-300m/s.
The micro-structural device of the physical method clasmatosis is applied in the Integrated manufacture of micro-fluidic chip.
A kind of processing method of such as micro-structural device of the physical method clasmatosis, is added using organic polymer method of molding
Work goes out broken chamber and coupled runner, the sealing-in of broken chamber is then realized using thermal bonding, on elastomeric stamp
Groove in fill up macromolecule prepolymer, be buckled on substrate, after solidification, remove mould, macromolecule material is printed on substrate
Expect the figure constituted, the substrate processed and the cover plate of same material cleaned after drying alignment is close to and lain in high temperature furnace,
One piece of polished graphite plate is respectively put in substrate and cover plate upper and lower, presses one piece of weight 0.5Kg again on graphite plate above not
Become rusty bloom, and bonding is heated in high temperature furnace, and bonding temperature is 1000 DEG C or more.
A kind of micro-structural device of physical method clasmatosis of the present invention and its beneficial effect of method of cell disruption are:
The present invention is crushed cell (bacterium) using physical method, and the broken cell of this method has the following characteristics that
First, clasmatosis occurs over just a flash hit with crushing shell, and clasmatosis is more uniform, can avoid cell repeatedly
Overcrushing phenomenon occurs for stress；
2nd, clasmatosis degree can be controlled by electrodeless adjustment nebulizer gas pressure (flow velocity), avoid structural damage intracellular,
Recycling suitable for organelle.
The micro-structural device of 3rd, physical method clasmatosis of the present invention is simple for structure, and design is easy to process, can quickly have
Effect ground, which is realized, is crushed cell, ensure that the progress of subsequent experimental on micro-fluidic chip, to the miniflow for promoting Highgrade integration
Control system has very profound significance.
Detailed description of the invention
Fig. 1 is the stereoscopic schematic diagram of the non-cap body of micro-structural device of physical method clasmatosis of the present invention；
Fig. 2 is that the micro-structural device of physical method clasmatosis of the present invention is stamped the stereoscopic schematic diagram of lid；
Fig. 3 is the structural schematic diagram of clasmatosis plate；
Fig. 4 is the cross-sectional view of the micro-structural device of physical method clasmatosis of the present invention；
Fig. 5 is the micro-structural device procedure of processing flow chart of physical method clasmatosis of the present invention；
Have in figure: 1, crushing shell；2, cell suspending liquid input channel；3, high velocity carrier gas input channel；4, it is crushed chamber；
5, nitrogen and cell liquid river conjunction；6, cell suspending liquid delivery outlet；7, flushing liquor input port；8, target liquid delivery outlet；9, exhaust gas
Outlet；10, gas delivery port.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment to the micro-structural device of physical method clasmatosis of the present invention and
The technical solution of its breaking method is described in further detail.
As shown in Figures 1 to 3, a kind of micro-structural device of physical method clasmatosis, comprising:
Broken chamber 4 occurs room for clasmatosis, collects cell residue object；
Cell suspending liquid input channel 2, one end are connected to connection, the other end and cell with the side cavity wall of the broken chamber
The connection of suspension conveying equipment；
Crushing shell 1 is arranged in broken chamber 4 and is located at cell suspending liquid input channel exit, towards thin on crushing shell 1
The one side of born of the same parents' suspension input channel outlet end is equipped with puncture structure；
High velocity carrier gas input channel 3, one end are connected to connection, the other end and high pressure with the cell suspending liquid input channel 2
Gas transmission equipmen connection, for by the cell suspending liquid in cell suspending liquid input channel in spray form and flow velocity be not less than
The initial velocity of 200m/s hits the crushing shell.To realize spray form effect, cell suspending liquid delivery outlet 6 uses array of orifices knot
Structure, the diameter in hole are about 5 microns；Gas transmission equipmen is pressed to be equipped with the regulating valve for adjusting high velocity carrier gas flow velocity；
Flushing liquor input port 7 is opened in the side for being located at puncture structure on crushing shell on crusher chamber chamber wall, passes through pipeline
Connect with flushing liquor conveying equipment, for broken chamber inner wall and crushing shell be rinsed；
Gas delivery port, for discharging the indoor high pressure gas of crusher chamber；
Target liquid delivery outlet 8, connect with extraction module, for collecting Objective extraction liquid.
Further, the cell suspending liquid input channel includes the different cell suspending liquid input pipe list of two diameters
Member is diameter thinner first cell suspending liquid input pipe unit and thicker the second cell suspending liquid input pipe list of diameter respectively
Member, wherein one end of the first cell suspending liquid input pipe unit is directly connected to setting with the side cavity wall of broken chamber, also,
The side side wall that first cell suspending liquid inputs the axis of pipe unit and the crushing shell is equipped with puncture structure is perpendicular；Second
Cell suspending liquid inputs the intersection setting between pipe unit and first cell suspending liquid input pipe unit at an angle, and
And have one to cross slot in the intersection that the first cell suspending liquid inputs pipe unit and the second cell suspending liquid input pipe unit；
The high velocity carrier gas input channel includes the different high velocity carrier gas input channel of two diameters, be diameter respectively compared with
Thin first high velocity carrier gas input pipe unit and thicker the second high velocity carrier gas of diameter input pipe unit, wherein the first high speed carries
One end of gas input pipe unit is connected with the slot that crosses, also, the first high velocity carrier gas input pipe unit and the first cell suspend
Liquid inputs the coaxial setting of pipe unit, and the first high velocity carrier gas inputs the other end of pipe unit and the second high velocity carrier gas input pipe unit connects
It connects.
Embodiment 1
The caliber of first cell suspending liquid input pipe unit of the present invention is 5 microns, the second cell suspending liquid input pipe
The caliber of unit is 100 microns.Purpose be in order to by cell suspending liquid be dispersed as partial size be 5 microns particle and hit broken
Plate.The caliber of the first high velocity carrier gas input pipe unit is 50 microns, and the caliber that the second carrier gas inputs pipe unit is 100 micro-
Rice, it is therefore an objective to improve the output flow velocity of nitrogen.It, can be by this structure process to micro-fluidic chip because the structural volume is small
In.
Embodiment 2
The caliber of first cell suspending liquid input pipe unit of the present invention is 5 microns, the second cell suspending liquid input pipe
The caliber of unit is 100 microns.Purpose be in order to by cell suspending liquid be dispersed as partial size be 5 microns particle and hit broken
Plate.The caliber of the first high velocity carrier gas input pipe unit is 50 microns, and the caliber that the second carrier gas inputs pipe unit is 100 micro-
Rice, it is therefore an objective to improve the output flow velocity of nitrogen.Filter device is added in the output end of broken chamber, filters out in cell residue object
DNA or RNA, then output liquid stream is entered in PCR instrument to replicate, which can simplify experimental procedure, save real
Test the operating time.
As the further preferred scheme of technical solution of the present invention, puncture structure is pointed cone array junctions on the crushing shell
Structure, the distance between each pointed cone are 300nm-10 μm.Guarantee that each cell (bacterium) for theoretically hitting crushing shell is equal
It can be broken.
The present invention further discloses a kind of clasmatosis of micro-structural device based on the physical method clasmatosis
Method, including the following steps:
S1, the gas delivery port for opening broken chamber, then open high pressure gas transmission equipmen, and high pressure gas transmission equipmen passes through high speed
Carrier gas input channel conveying high-pressure carrier gas into cell suspending liquid input channel, by the gas for adjusting valve regulation high pressure gas transmission equipmen
Body flow velocity is to reaching requirement；
S2, cell suspending liquid conveying equipment is opened, cell suspending liquid is in the feeding of spray form under the carrying of high pressure carrier gas
Broken chamber, high-speed impact crushing shell, cell or bacterium are crushed；
After S3, clasmatosis, extraction module collects Objective extraction liquid by target liquid delivery outlet；
After S4, Objective extraction liquid are collected, cleaning solution is conveyed into crusher chamber room by washing lotion conveying equipment, will be broken
The inner wall and crushing shell of broken chamber are rinsed.
Further, the high velocity carrier gas is nitrogen.
Further, the flow velocity of the high velocity carrier gas is 200-300m/s.
Further, the micro-structural device of the physical method clasmatosis is applied in the Integrated manufacture of micro-fluidic chip.
As shown in figure 4, the present invention processes broken chamber and coupled runner using organic polymer method of molding,
Then the sealing-in of broken chamber is realized using thermal bonding.The material that crushing system uses is monocrystalline silicon.Specific processing technology is as follows:
Macromolecule prepolymer is filled up in groove on elastomeric stamp, is buckled on substrate, after solidification, mould is removed, on substrate
The figure for just having stamped high molecular material composition will be after the substrate that processed and the clean drying alignment abutting of the cover plate of same material
It lies in high temperature furnace, respectively puts one piece of polished graphite plate in substrate and cover plate upper and lower, pressed again on graphite plate above
The piece of stainless steel of one piece of weight 0.5Kg, heats bonding in high temperature furnace.Bonding temperature is 1000 DEG C or more.
Bacteria breaking system ruptures cell (bacterium) using the method for physical impacts, extracts mesh in cell (bacterium)
It marks ingredient and carries out next step experiment.For cell suspending liquid with spray form by pipe outlet, the partial size of spraying granule is about 5 micro-
Rice.Spray form particulate is sent into broken chamber by high velocity carrier gas --- nitrogen flow rate 200-300m/s, and high-speed impact is broken
Plate is crushed.Then cell residue object is collected in broken chamber and is exported from outlet carries out further microfluidic control experiment.
Fluid course diameter prepared by the present invention is micron level, and required precision is not high therefore method of molding can be used and added
Work, distance is 300 nanometers between the pointed cone of crushing shell, and design can be adjusted according to cell size.With existing clasmatosis side
Method is compared, and this method is simple for structure, is crushed uniform and complete, is facilitated processing and is carried out subsequent experimental to Related Component in cell,
It will be applied in the Integrated manufacture of micro-fluidic chip, realize automation, integration.
Claims (8)
1. a kind of micro-structural device of physical method clasmatosis characterized by comprising
Broken chamber, for collecting cell residue object, the top of broken chamber is equipped with the lid that can cover conjunction or opening；
Cell suspending liquid input channel, one end are connected to connection with the side cavity wall of the broken chamber, and the other end and cell suspend
The connection of liquid conveying equipment；
Crushing shell is arranged in crusher chamber room and is located at cell suspending liquid input channel exit, suspends on crushing shell towards cell
The one side of liquid input channel outlet end is equipped with puncture structure；
High velocity carrier gas input channel, one end are connected to connection with the cell suspending liquid input channel, and the other end is set with high pressure gas transmission
Standby connection, for by the cell suspending liquid in cell suspending liquid input channel in spray form and flow velocity not less than 200m/s's
Initial velocity hits the crushing shell；Gas transmission equipmen is pressed to be equipped with the regulating valve for adjusting high velocity carrier gas flow velocity；
Flushing liquor input port is opened in the side for being located at puncture structure on crushing shell on crusher chamber chamber wall, passes through pipeline and punching
Washing lotion conveying equipment connection, for broken chamber inner wall and crushing shell be rinsed；
Gas delivery port is set on lid, for discharging the indoor high pressure gas of crusher chamber；
Target liquid delivery outlet, connect with extraction module, for collecting Objective extraction liquid；
The cell suspending liquid input channel includes that the different cell suspending liquid of two diameters inputs pipe unit, is diameter respectively
Thinner first cell suspending liquid input pipe unit and thicker the second cell suspending liquid of diameter input pipe unit, wherein first
One end of cell suspending liquid input pipe unit is directly connected to setting with the side cavity wall of broken chamber, also, the first cell suspends
The side side wall that liquid inputs the axis of pipe unit and the crushing shell is equipped with puncture structure is perpendicular；Second cell suspending liquid is defeated
Enter intersection setting at an angle between pipe unit and first cell suspending liquid input pipe unit, and in the first cell
The intersection of suspension input pipe unit and the second cell suspending liquid input pipe unit has one to cross slot；
The high velocity carrier gas input channel includes the different high velocity carrier gas input channel of two diameters, is that diameter is thinner respectively
First high velocity carrier gas inputs pipe unit and thicker the second high velocity carrier gas of diameter inputs pipe unit, wherein the first high velocity carrier gas is defeated
The one end for entering pipe unit is connected with the slot that crosses, also, the first high velocity carrier gas input pipe unit and the first cell suspending liquid are defeated
Enter the coaxial setting of pipe unit, the first high velocity carrier gas inputs the other end and the input pipe unit connection of the second high velocity carrier gas of pipe unit.
2. the micro-structural device of physical method clasmatosis according to claim 1, which is characterized in that first cell is outstanding
The caliber that supernatant liquid inputs pipe unit is 5 microns, and the caliber that the second cell suspending liquid inputs pipe unit is 15 microns.
3. the micro-structural device of physical method clasmatosis according to claim 1, which is characterized in that first high speed carries
The caliber that gas inputs pipe unit is 3 microns, and the caliber that the second carrier gas inputs pipe unit is 10 microns.
4. the micro-structural device of physical method clasmatosis according to claim 1, which is characterized in that pierced on the crushing shell
Broken structure is pointed cone array structure, and the distance between each pointed cone is 300nm.
5. method of cell disruption of the one kind based on the micro-structural device of any physical method clasmatosis in claim 1 ~ 4,
It is characterised in that it includes following steps:
S1, high pressure gas transmission equipmen is opened, high pressure gas transmission equipmen passes through high velocity carrier gas input channel to cell suspending liquid input channel
Interior conveying high-pressure carrier gas, by adjusting the gas flow rate of valve regulation high pressure gas transmission equipmen to reaching requirement；
S2, cell suspending liquid conveying equipment is opened, cell suspending liquid is broken in the feeding of spray form under the carrying of high pressure carrier gas
Chamber, high-speed impact crushing shell, cell or bacterium are crushed；
After S3, clasmatosis, extraction module collects Objective extraction liquid by target liquid delivery outlet；
After S4, Objective extraction liquid are collected, cleaning solution is conveyed into crusher chamber room by washing lotion conveying equipment, by crusher chamber
The inner wall and crushing shell of room are rinsed.
6. the method for cell disruption of the micro-structural device of physical method clasmatosis according to claim 5, which is characterized in that
The high velocity carrier gas is nitrogen.
7. the method for cell disruption of the micro-structural device of physical method clasmatosis according to claim 5, which is characterized in that
The flow velocity of the high velocity carrier gas is 200-300m/s.
8. a kind of processing method such as the micro-structural device of any physical method clasmatosis in claim 1 ~ 4, using having
Machine polymer molding method processes broken chamber and coupled pipeline, and the envelope of broken chamber is then realized using thermal bonding
It connects, which is characterized in that fill up macromolecule prepolymer in the groove on elastomeric stamp, be buckled on substrate, after solidification, removed
Elastomeric stamp is printed on the figure of high molecular material composition on substrate, the cover plate of the substrate processed and same material is cleaned
Drying alignment is lain in high temperature furnace after being close to, and one piece of polished graphite plate is respectively put in substrate and cover plate upper and lower, above
Graphite plate on press the piece of stainless steel of one piece of 0.5 Kg of weight again, bonding is heated in high temperature furnace, bonding temperature is 1000 DEG C or more.
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Cited By (1)
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| EP4296352A1 (en) * | 2022-06-20 | 2023-12-27 | LenioBio GmbH | Tunable disruption of eukaryotic protoplast to release intact cellular organelles |
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