CN108117969A - Single celled micro-fluidic chip of a kind of high throughput automatic capture and preparation method thereof - Google Patents
Single celled micro-fluidic chip of a kind of high throughput automatic capture and preparation method thereof Download PDFInfo
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- CN108117969A CN108117969A CN201611063004.XA CN201611063004A CN108117969A CN 108117969 A CN108117969 A CN 108117969A CN 201611063004 A CN201611063004 A CN 201611063004A CN 108117969 A CN108117969 A CN 108117969A
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Abstract
The present invention provides single celled micro-fluidic chips of a kind of high-throughput automatic capture and preparation method thereof, and the chip is formed by two layers, and upper strata is flow path entrance layer；Lower floor is flow path key-course；The flow path entrance layer has liquid flow path feeder connection (1) and liquid flow path channel outlet (2)；The flow path key-course is made of unicellular capture flow path channel (3), gas channels (4), drop formation unit (5)；The chip prepares the 8 chip templates of SU of channel part protrusion using photoetching and caustic solution, and PDMS chips are obtained by dimethyl silicone polymer (PDMS) reverse.The chip possesses high-throughput drop formation unit, with the controllable gas flow path passage of air pressure, it can be achieved that high-throughput unicellular capture is, it can be achieved that the automation of unicellular acquisition procedure, reduces cell and reagent dosage, have wide range of applications.
Description
Technical field
A kind of fields such as processing, making the invention belongs to microflow control technique and polymer chip, and in particular to high throughput
Single celled micro-fluidic chip of automatic capture and preparation method thereof.
Background technology
Microfluid based Lab on a chip is sample preparation involved in the fields such as chemistry and biology, reaction, separation, detection
And the basic operation units such as cell culture, separation, division are integrated on the chip of one piece several square centimeters (or even smaller), by micro-
Passage forms network, with controlled fluid through whole system, to substitute the various functions of conventional chemical and biology laboratory
A kind of technology platform.The essential characteristic and sharpest edges of micro-fluidic chip are a variety of monotechnicses in whole controllable small platform
Upper flexible combination, scale integrate.High throughput is a kind of form that scale integrates, and the operating unit or one-element group being simply integrated are
Identical.
Biology and medical research in recent years finds there is important meaning for the RESEARCH ON CELL-BIOLOGY of individual cells
Justice.Same cell may also have very big difference between different cell individuals, and the most significant cell that makes a variation tends to take off
Show important biomolecule phenomenon or prompt the generation of major disease, disclose pathogenesis of cancer mechanism, understand that cell differentiation is sent out with tissue
Principle is educated, identifies gene expression characteristics and cell characteristic.Therefore, accurately capture the single cell population of significant difference and carry out accurate
Identification is the key technology being badly in need of in biomedical research.
In recent years, with the rapid development of microflow control technique, a kind of side for manipulating micro-meter scale fluid is provided for people
Method.The method that the development of micro-processing technology also provides the manufacture micro-structure similar to cell dimensions simultaneously, at unicellular point
Application in analysis causes more and more concerns.Cell factory on chip can be controlled effectively as unicellular microreactor
System diffusion, improves detection sensitivity, has been successfully applied in a variety of single cell analysis.In addition, from substantial amounts of sample (such as blood
Liquid) in accurately capture specific cells and carry out identification identification to it and traditional technology is difficult to realize (such as flow cytometer).
The present invention is prepared for a kind of high-throughput automatic capture list using micro-fluidic and polymer chip processing, manufacturing technology
The micro-fluidic chip of cell, whole device is simple in structure, without any complicated and expensive equipment, it can be achieved that high throughput, operation
Facilitate automation, unicellular suspension culture, reduction cellular damage can be captured for a long time and be convenient for adding in medicine irritation.
The content of the invention
The object of the present invention is to provide a kind of high-throughput single celled micro-fluidic chip of automatic capture and preparation method thereof, with
Solve present in previous unicellular acquisition procedure it is complex for operation step it is complicated, to be easy to cause cellular damage, capture rate low etc.
Limitation, preparation process of the present invention is stablized, easy to operate, and integrated level is high, automation can be achieved.
A kind of single celled micro-fluidic chip of high throughput automatic capture, the chip are formed by two layers, and upper strata goes out for flow path
Entry level；Lower floor is flow path key-course；
The flow path entrance layer has liquid flow path feeder connection and liquid flow path channel outlet；
The flow path key-course is by unicellular capture flow path channel, gas channels, (the unicellular capture of drop formation unit
Trap) composition；
The unicellular capture flow path channel is a long-channel being made of the U-shaped passage of multiple interactive connection, long logical
Road both ends connect liquid flow path feeder connection and liquid flow path channel outlet respectively, and the straight channel of each U-shaped passage has therewith
Parallel gas flow path passage is each dispersed with the drop life of several cylindrical depression pitting structures in the straight channel of U-shaped passage
Into unit.
After the chip vacuumizes, gas channels are in negative pressure state, and due to the gas permeability of PDMS, gas circuit drives surrounding
Fluid path passage forms negative pressure, can automatically suck single cell suspension from fluid path feeder connection, for unicellular feed liquor and capture.
The unicellular capture flow path channel is 100~300 μm wide, a length of 40~50mm of straight channel of U-shaped passage, straight channel
Spacing is 0.5~1mm.
The gas channels width is 200~500 μm, a length of 40~50mm, and spacing is 0.5~1mm.
The unicellular capture flow path channel and gas channels spacing are 0.5~1.5mm.
The drop formation unit is cylindrical depression pitting structure, shares 6000~10000, pitting a diameter of 15~
It is 45 μm, 80~200 μm high, 80~150 μm of pitting spacing.
A kind of preparation method of the single celled micro-fluidic chip of high throughput automatic capture, the chip preparation process are as follows：
(1) the SU-8 templates of channel part protrusion are prepared using photoetching and caustic solution：The SU-8 templates are by bilayer
Structure composition, first layer are made of unicellular capture flow path channel and gas channels, and the second layer is made of drop formation unit；
The preparation of SU-8 templates by whirl coating twice, front baking, exposure, after dry after, once development, post bake obtains；
(2) the PDMS modules being made based on SU-8 templates：By PDMS and initiator with volume ratio 10：1 is uniformly mixed, and pours
It notes in SU-8 templates, 75~85 DEG C of 30~60min of curing oven, PDMS and SU-8 templates is removed, obtain on structured
Layer chip；By upper strata chip and lower layer chip sealing-in after 1~4min of corona treatment with flow path entrance.
A kind of application of the single celled micro-fluidic chip of high throughput automatic capture, the micro-fluidic chip can be used as high throughput
Automatic unicellular capture can carry out the researchs such as unicellular Culture in situ, administration, multiplication, induction differentiation.
The micro-fluidic chip can be used as high-throughput automatic unicellular capture, can carry out unicellular Culture in situ, administration, increasing
It grows, induce the researchs such as differentiation.
The advantage of the invention is that：
1st, it is easy to operate, quick；
2nd, cell and reagent dosage are few, and experimental cost is cheap；
3rd, Highgrade integration, automation；
4th, have wide range of applications；
Description of the drawings
Fig. 1：Chip structure schematic diagram；(a) overall structure figure, (b) are partial enlarged view；
Fig. 2：Micro-fluidic chip side partial enlarged view.
Wherein：1 be liquid flow path feeder connection, 2 be liquid flow path channel outlet, 3 be unicellular capture flow path channel, 4
It is drop formation unit (unicellular capture trap) for gas flow path passage, 5,6 be unicellular；
Fig. 3：Micro-fluidic chip pictorial diagram.
Specific embodiment
A kind of single celled micro-fluidic chip of high throughput automatic capture, as shown in Figure 1, Figure 2, Figure 3 shows；The chip is by two layers
Composition, upper strata are flow path entrance layer；Lower floor is flow path key-course；
The flow path entrance layer has liquid flow path feeder connection 1 and liquid flow path channel outlet 2；
The flow path key-course is by unicellular capture flow path channel 3, gas channels 4, (the unicellular capture of drop formation unit
Trap) 5 compositions；
The unicellular capture flow path channel 3 is a long-channel being made of the U-shaped passage of multiple interactive connection, long logical
Road both ends connect liquid flow path feeder connection 1 and liquid flow path channel outlet 2 respectively, the straight channel of each U-shaped passage have with
Parallel gas flow path passage 4, the drops of several cylindrical depression pitting structures is dispersed in the straight channel of each U-shaped passage
Generation unit 5.
The unicellular capture flow path channel is 100~300 μm wide, a length of 40~50mm of straight channel of U-shaped passage, straight channel
Spacing is 0.5~1mm.
The gas channels width is 200~500 μm, a length of 40~50mm, and spacing is 0.5~1mm.
The unicellular capture flow path channel and gas channels spacing are 0.5~1.5mm.
The drop formation unit is cylindrical depression pitting structure, shares 6000~10000, pitting a diameter of 15~
It is 45 μm, 80~200 μm high, 80~150 μm of pitting spacing.
After chip vacuumizes, gas flow path passage 4 is in negative pressure state, and due to the gas permeability of PDMS, gas circuit drives week
The liquid flow path passage 3 enclosed forms negative pressure, and MCF-7 single cell suspensions 6 are caught under suction function through feeder connection 1 into unicellular
Flow path channel 3 is obtained, after drop formation unit 5, unicellular 6 are retained in the drop of generation, and remaining single cell suspension is through logical
Road entrance 2 flows out, and realizes unicellular automatic capture.Embodiment 1
The preparation of the high-throughput single celled micro-fluidic chip SU-8 templates of automatic capture
Micro-fluidic chip prepares the SU-8 templates of channel part protrusion using photoetching and caustic solution；First, first layer
SU-8 glue thickness is got rid of as 100 μm, 95 DEG C of front baking 20min, mask is placed in SU-8 glue washers, uv-exposure by Temperature fall
30s dries 20min, Temperature fall after 95 DEG C；Secondly, based on the basis of first layer SU-8 glue, the second layer gets rid of SU-8 glue thickness and is
150 μm, 95 DEG C of front baking 30min, mask is placed in SU-8 glue washers by Temperature fall, uv-exposure 40s, is dried after 95 DEG C
40min, Temperature fall；Finally, above-mentioned SU-8 glue is developed 10min, 180 DEG C of post bake 2h using ethyl lactate, Temperature fall is standby
With.Embodiment 2
The preparation of the high-throughput single celled PDMS chips of automatic capture
By PDMS and initiator with volume ratio 10：1 is uniformly mixed, and is cast in the SU-8 templates of preparation early period, 80 DEG C of baking ovens
Cure 30min, PDMS and SU-8 templates are removed, obtained with structured micro-fluidic chip (1)；By PDMS and initiator with body
Product ratio 10：1 is uniformly mixed, and is cast in blank glass plate, and PDMS and SU-8 templates are removed, obtained by 80 DEG C of curing oven 30min
Blank PDMS chips (2)；Two holes are made a call on chip (2) with card punch, correspond to flow path entrance respectively, by chip 1 and chip 2
Sealing-in is spare after corona treatment 120s.
Claims (7)
1. a kind of single celled micro-fluidic chip of high throughput automatic capture, it is characterised in that：The chip is formed by two layers, upper strata
For flow path entrance layer；Lower floor is flow path key-course；
The flow path entrance layer has liquid flow path feeder connection (1) and liquid flow path channel outlet (2)；
The flow path key-course is made of unicellular capture flow path channel (3), gas channels (4), drop formation unit (5)；
The unicellular capture flow path channel (3) is a long-channel being made of the U-shaped passage of multiple interactive connection, long-channel
Both ends connect liquid flow path feeder connection (1) and liquid flow path channel outlet (2) respectively, and the straight channel of each U-shaped passage has
Gas flow path passage (4) parallel with it is each dispersed with several cylindrical depression pitting structures in the straight channel of U-shaped passage
Drop formation unit (5).
2. according to the single celled micro-fluidic chip of a kind of high throughput automatic capture described in claim 1, it is characterised in that：The list
Cell capture flow path channel is 100~300 μm wide, a length of 40~50mm of straight channel of U-shaped passage, straight channel spacing for 0.5~
1mm。
3. according to the single celled micro-fluidic chip of a kind of high throughput automatic capture described in claim 1, it is characterised in that：The gas
Paths width is 200~500 μm, a length of 40~50mm, and spacing is 0.5~1mm.
4. according to the single celled micro-fluidic chip of a kind of high throughput automatic capture described in claim 1, it is characterised in that：The list
Cell capture flow path channel is 0.5~1.5mm with gas channels spacing.
5. according to the single celled micro-fluidic chip of a kind of high throughput automatic capture described in claim 1, it is characterised in that：The liquid
Drop generation unit is cylindrical depression pitting structure, shares 6000~10000, a diameter of 15~45 μm of pitting is high by 80~200
μm, 80~150 μm of pitting spacing.
6. according to a kind of preparation method of the single celled micro-fluidic chip of high throughput automatic capture described in claim 1, feature
It is：The PDMS chips preparation process is as follows：
(1) the SU-8 templates of channel part protrusion are prepared using photoetching and caustic solution：The SU-8 templates are by double-layer structure
Composition, first layer are made of unicellular capture flow path channel and gas channels, and the second layer is made of drop formation unit；SU-8 moulds
The preparation of plate by whirl coating twice, front baking, exposure, after dry after, once development, post bake obtains；
(2) the PDMS modules being made based on SU-8 templates：By PDMS and initiator with volume ratio 10：1 is uniformly mixed, and is cast in
PDMS and SU-8 templates are removed, obtained with structured upper strata core by SU-8 templates, 75~85 DEG C of 30~60min of curing oven
Piece；By upper strata chip and lower layer chip sealing-in after 1~4min of corona treatment with flow path entrance.
7. according to a kind of application of the single celled micro-fluidic chip of high throughput automatic capture described in claim 1, it is characterised in that：
The micro-fluidic chip can be used as high-throughput automatic unicellular capture, can carry out unicellular Culture in situ, administration, multiplication, induction
The researchs such as differentiation.
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| CN201611063004.XA CN108117969A (en) | 2016-11-28 | 2016-11-28 | Single celled micro-fluidic chip of a kind of high throughput automatic capture and preparation method thereof |
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| CN201611063004.XA CN108117969A (en) | 2016-11-28 | 2016-11-28 | Single celled micro-fluidic chip of a kind of high throughput automatic capture and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113234583A (en) * | 2021-05-17 | 2021-08-10 | 苏州新格元生物科技有限公司 | Cell reaction module integrated with gas circuit control and method for carrying out cell reaction by adopting cell reaction module |
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| CN113234583A (en) * | 2021-05-17 | 2021-08-10 | 苏州新格元生物科技有限公司 | Cell reaction module integrated with gas circuit control and method for carrying out cell reaction by adopting cell reaction module |
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Application publication date: 20180605 |