CN107828651A - A kind of micro-fluidic chip for unicellular microlayer model sample preparation - Google Patents
A kind of micro-fluidic chip for unicellular microlayer model sample preparation Download PDFInfo
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- CN107828651A CN107828651A CN201710891682.3A CN201710891682A CN107828651A CN 107828651 A CN107828651 A CN 107828651A CN 201710891682 A CN201710891682 A CN 201710891682A CN 107828651 A CN107828651 A CN 107828651A
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Abstract
The present invention relates to micro fluidic chip technical field, a kind of specifically micro-fluidic chip for unicellular microlayer model sample preparation, connected including chip body and the unicellular microlayer model sample preparation unit being distributed on chip body and blank drop removal unit, described unicellular microlayer model sample preparation unit with blank drop removal unit by the microchannel of micro-fluidic chip；The present invention realizes the unicellular microlayer model sample preparation of high flux, high accounting by unicellular microlayer model sample preparation unit and blank drop removal unit；By the size for controlling blank drop removal unit micro-structural, it is possible to achieve different cell samples are screened；So as to realize 95% of microlayer model in cell microlayer model sample preparation parcel cell, biological reagent utilization rate is improved for the subsequently cell culture based on microlayer model, the detection of unicellular fluorescence in situ, singe-cell PCR etc., improves experimental result screening efficiency, reduces workload and biological reagent cost.
Description
Technical field
It is specifically a kind of for unicellular microlayer model sample preparation the present invention relates to micro fluidic chip technical field
Micro-fluidic chip.
Background technology
Biological sample processing, including cell, bacterium, protein, DNA etc., are often played most important in Bioexperiment
Effect, especially relate to unicellular sequencing, unicellular screening, digital pcr of forefront etc. biology and medical experiment.Separately
Outside, the early diagnosis of cancer, clinical treatment, more afterwards detection, in drug screening process, be related to a kind of circulating tumor cell.
Circulating tumor cell is to be entered after being departed from by the cancer cell in primary tumor in Peripheral Circulation system, because it has former cancer
Cellular informatics, it is significant in cancer research using it as mark.However, circulating tumor cell is in peripheral blood
Quantity is very rare, is 1/100000000th of the normal plasma cells such as red blood cell or leucocyte.Therefore, how to separate and purify, even
Circulating tumor cell is extracted, there is very big challenge.At present, the capture of circulating tumor cell and separation needs come by antigen-antibody
Realize, but low separation efficiency, cell purity is low, for subsequent cell and Molecular level study, impacts.How to reduce normal
The interference of cell, it is very crucial in sample pre-treatments.Cell sample pre-treatment at present, realized by gradient centrifugation,
On the other hand realized by micro-fluidic chip microlayer model technology.It is therefore, micro-fluidic because the size of cell is typically in micron order
Chip microlayer model technology is used widely in cell sample processing.Cell can avoid and other cells in microlayer model
Interfere.However, preparing cell sample using micro-fluidic chip, have some limitations.It is empty in the sample prepared
White liquor drop accounting is larger, wraps up a cell, two cells, the drop proportion of three or more than three cells, presents just
It is distributed very much, but the microlayer model containing cell, population proportion is less than 30%.Because the volume of microlayer model is very small, picoliters or nanoliter
The order of magnitude, after general biological sample is prepared into microlayer model state, quantity is in terms of necessarily.Therefore, blank drop how is removed, for
Unicellular sample preparation is extremely important, reduces cell processing quantity, saves time cost.Therefore, the logical of micro-fluidic chip is passed through
Road designs, and to realize the removal of blank drop, improves the ratio of unicellular drop, has important applied field.
The content of the invention
In order to solve problem present in above-mentioned existing micro-fluidic chip, the present invention provides one kind and is used for unicellular microlayer model
The micro-fluidic chip of sample preparation, solving blank solution drop accounting in existing micro-fluidic chip cell microlayer model sample treatment causes greatly
The technical problems such as workload is big in subsequent bio-analysis, efficiency is low, time-consuming.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of micro-fluidic chip for unicellular microlayer model sample preparation, including chip body and be distributed on chip body
Unicellular microlayer model sample preparation unit and blank drop removal unit, the unicellular microlayer model sample preparation unit include connecting
Connect cell sample sample injection unit, the oil phase sample injection unit and microlayer model sample preparation list of the outside oil phase of connection of external cellular sample
Member, the microlayer model sample preparation unit are with T-shaped or decussate texture connection cell sample sample injection unit and oil phase sample introduction list
Member simultaneously realizes the preparation of microlayer model by hydrodynamic shear；The blank drop removal unit includes the second of the outside oil phase of connection
Oil phase sample injection unit, blank drop remove double-height micro-structural, the unicellular microlayer model for connecting external sample collector
Collector unit and the blank microlayer model collector unit for connecting outside blank microlayer model collector, the second oil phase sample introduction list
The microlayer model is set to flow through blank drop separation micro-structural after member the second oil phase of access, it is micro- that the blank drop removes double-height
Structure has the characteristic of some strength by difference in height and nucleus, realizes blank microlayer model and is enclosed with the microlayer model of cell
Separation, described unicellular microlayer model sample preparation unit and blank drop removal unit are connected by the microchannel of micro-fluidic chip
Connect.
Further, the microlayer model sample preparation unit includes：T-shaped raceway groove, cross raceway groove, Y types raceway groove, coaxial type lead to
Road.
Further, the blank drop removal unit includes blank microlayer model split tunnel and split tunnel support knot
Structure.
Preferably, the height of the blank microlayer model split tunnel is 1~15 micron, and width is at 1~200 micron, length
At 10~1000 microns.
Preferably, the split tunnel support structure height is 1~15 micron, width at 1~200 micron, length 1~
100 microns.
Further, the material of the micro-fluidic chip is dimethyl silicone polymer, glass, dimethyl silicone polymer, is gathered
Carbonic ester, polymethyl methacrylate, polyethylene terephthalate.
Preferably, the micro-fluidic chip is by by the polydimethylsiloxane containing microchannel, makrolon, poly- methyl
Methyl acrylate, polyethylene terephthalate are bonded by oxygen plasma with substrate of glass and are prepared.
Preferably, described micro-fluidic chip is by by the polydimethylsiloxane containing microchannel, makrolon, poly- first
Base methyl acrylate, polyethylene terephthalate and dimethyl silicone polymer substrate by oxygen plasma be bonded preparation and
Into.
Further, the cell sample sample injection unit, oil phase sample injection unit and the second oil phase sample injection unit are to pass through note
Infusion is penetrated to inject into micro-fluidic chip.
Preferably, cell sample sample injection unit, oil phase sample injection unit and the second oil phase sample injection unit be caused by air pressure just
Compressing makes sample enter micro-fluidic chip.
Further, the micro-fluidic chip microchannel, except blank microlayer model split tunnel, the height of remaining microchannel
For 10~200 microns, width is 10~500 microns.
Further, the mask layer of mask layer and other microchannels containing the blank microlayer model split tunnel passes through double
Layer photoetching is realized.
Relative to prior art, the beneficial effects of the invention are as follows：Unicellular microlayer model sample preparation provided by the invention
Micro-fluidic chip, high flux, high accounting list are realized by unicellular microlayer model sample preparation unit and blank drop removal unit
Cell microlayer model sample preparation；And further, by the size for controlling blank drop removal unit micro-structural, it is possible to achieve right
Different cell samples are screened；So as to realize 95% of microlayer model in cell microlayer model sample preparation parcel cell, for follow-up
Cell culture based on microlayer model, the detection of unicellular fluorescence in situ, singe-cell PCR etc. improve biological reagent utilization rate, improved in fact
Result screening efficiency is tested, reduces workload and biological reagent cost.
Brief description of the drawings
The present invention is further described with reference to the accompanying drawings and examples.
Fig. 1 is the microfluidic chip structure schematic diagram of the present invention；
Fig. 2 is the micro-fluidic chip schematic diagram of the present invention；
Fig. 3 is the blank drop removal unit micro-structural schematic diagram of the present invention；
Fig. 4 is the blank drop removal unit micro-structural side view of the present invention；
Fig. 5 is the micro-fluidic chip photolithographic mask layer one of the present invention；
Fig. 6 is the micro-fluidic chip photolithographic mask layer two of the present invention；
Fig. 7 is the micro-fluidic chip photolithographic mask layer one of the present invention and the micro-fluidic chip for unicellular microlayer model sample preparation
The alignment result figure of photolithographic mask layer two.
Fig. 8 is the electronic scanner microscope figure of the blank microlayer model split tunnel of the present invention.
In figure：0th, micro-fluidic chip, 1, unicellular microlayer model sample preparation unit, 11, oil phase sample injection unit, 12, cell
Sample feeding unit, 13, microlayer model sample preparation unit, 2, blank drop removal unit, the 21, second oil phase sample injection unit, 22,
Blank microlayer model collector unit, 23, unicellular microlayer model collector unit, 24, blank drop remove double-height micro-structural, 241,
Blank microlayer model split tunnel, 242, split tunnel supporting construction, 33, microlayer model prepare passage, 34, blank microlayer model collect it is logical
Road, 4, micro-fluidic chip photolithographic mask layer one, 41~43, lithography alignment figure one, 5, micro-fluidic chip photolithographic mask layer two, 51
~53, lithography alignment figure two.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
As Figure 1-Figure 8, a kind of micro-fluidic chip for unicellular microlayer model sample preparation of the present invention, bag
Include chip body and unicellular microlayer model sample preparation unit 1 and blank the drop removal unit 2 being distributed on chip body, institute
Stating unicellular microlayer model sample preparation unit 1 includes the cell sample sample injection unit 12 of connection external cellular sample, connection outside
The oil phase sample injection unit 11 and microlayer model sample preparation unit 13 of oil phase, the microlayer model sample preparation unit 13 be with T-shaped or
Decussate texture connection cell sample sample injection unit 12 and oil phase sample injection unit 11 simultaneously realize microlayer model by hydrodynamic shear
Prepare；The blank drop removal unit 2 includes the second oil phase sample injection unit 21 of the outside oil phase of connection, and blank drop removes double
Weight height micro-structural 24, the unicellular microlayer model collector unit 23 for connecting external sample collector and for connecting outside sky
The blank microlayer model collector unit 22 of white microlayer model collector, the second oil phase sample injection unit 21 access after the second oil phase make it is micro-
Drop flows through blank drop separation micro-structural, and the blank drop removes double-height micro-structural 24 and passes through difference in height and nucleus
The characteristic of had some strength, realize that blank microlayer model separates with the microlayer model for being enclosed with cell, described unicellular micro- liquid
Drop sample preparation unit 1 is connected with blank drop removal unit 2 by the microchannel of micro-fluidic chip.
Further, the microlayer model sample preparation unit 1 includes：T-shaped raceway groove, cross raceway groove, Y types raceway groove, coaxial type lead to
Road.
Further, the blank drop removal unit 2 includes blank microlayer model split tunnel 241 and split tunnel supports
Structure 242.
Preferably, the height of the blank microlayer model split tunnel 241 is 1~15 micron, and width is long at 1~200 micron
Degree is at 10~1000 microns.
Preferably, the height of split tunnel supporting construction 242 is 1~15 micron, and at 1~200 micron, length exists width
1~100 micron.
Further, the material of the micro-fluidic chip is dimethyl silicone polymer, glass, dimethyl silicone polymer, is gathered
Carbonic ester, polymethyl methacrylate, polyethylene terephthalate.
Preferably, the micro-fluidic chip is by by the polydimethylsiloxane containing microchannel, makrolon, poly- methyl
Methyl acrylate, polyethylene terephthalate are bonded by oxygen plasma with substrate of glass and are prepared.
Preferably, described micro-fluidic chip is by by the polydimethylsiloxane containing microchannel, makrolon, poly- first
Base methyl acrylate, polyethylene terephthalate and dimethyl silicone polymer substrate by oxygen plasma be bonded preparation and
Into.
Further, the cell sample sample injection unit 12, the oil phase sample injection unit 21 of oil phase sample injection unit 11 and second are
Micro-fluidic chip is entered by ejection of syringe pump.
Preferably, cell sample sample injection unit 12, the oil phase sample injection unit 21 of oil phase sample injection unit 11 and second are air pressure productions
Raw malleation forces sample to enter micro-fluidic chip.
Further, the micro-fluidic chip microchannel, except blank microlayer model split tunnel, the height of remaining microchannel
For 10~200 microns, width is 10~500 microns.
Further, mask layer and the mask layer of other microchannels containing the blank microlayer model split tunnel 241 lead to
Double-layer photoetching is crossed to realize.
The micro-fluidic chip of unicellular microlayer model sample preparation provided by the invention, passes through unicellular microlayer model sample preparation
Unit and blank drop removal unit realize the unicellular microlayer model sample preparation of high flux, high accounting；And further, pass through control
The size of blank drop removal unit micro-structural processed, it is possible to achieve different cell samples are screened；So as to realize that cell is micro-
95% parcel cell of microlayer model in prepared by droplet sample, for the subsequently cell culture based on microlayer model, unicellular fluorescence in situ
Detection, singe-cell PCR etc. improve biological reagent utilization rate, improve experimental result screening efficiency, reduce workload and biology examination
Agent cost.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technical staff of the industry
It should be appreciated that the present invention is not limited to the above embodiments, simply illustrate the present invention described in above-described embodiment and specification
Principle, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these change and
Improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent
Thing defines.
Claims (9)
1. a kind of micro-fluidic chip for unicellular microlayer model sample preparation, it is characterised in that including chip body and distribution
Unicellular microlayer model sample preparation unit and blank drop removal unit on chip body, the unicellular microlayer model sample
Preparation unit includes the cell sample sample injection unit of connection external cellular sample, the oil phase sample injection unit of the outside oil phase of connection and micro-
Droplet sample preparation unit, the microlayer model sample preparation unit are with T-shaped or decussate texture connection cell sample sample introduction list
Member and oil phase sample injection unit simultaneously realize the preparation of microlayer model by hydrodynamic shear；The blank drop removal unit includes connection
Second oil phase sample injection unit of outside oil phase, blank drop remove double-height micro-structural, for connecting external sample collector
Unicellular microlayer model collector unit and blank microlayer model collector unit for connecting outside blank microlayer model collector, it is described
Second oil phase sample injection unit makes the microlayer model flow through blank drop separation micro-structural, the blank drop after accessing the second oil phase
Removing double-height micro-structural has the characteristic of some strength by difference in height and nucleus, realizes blank microlayer model and parcel
There is the microlayer model separation of cell, described unicellular microlayer model sample preparation unit and blank drop removal unit pass through micro-fluidic
The microchannel connection of chip.
A kind of 2. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 1, it is characterised in that：
The microlayer model sample preparation unit includes：T-shaped raceway groove, cross raceway groove, Y types raceway groove, coaxial type passage.
A kind of 3. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 1, it is characterised in that：
The blank drop removal unit includes blank microlayer model split tunnel and split tunnel supporting construction, the blank microlayer model point
Height from passage is 1~15 micron, and width is at 1~200 micron, and length is at 10~1000 microns, the split tunnel support
Structure height is 1~15 micron, and width is at 1~200 micron, and length is at 1~100 micron.
A kind of 4. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 1, it is characterised in that：
The material of the micro-fluidic chip is dimethyl silicone polymer, glass, dimethyl silicone polymer, makrolon, polymethyl
Sour methyl esters, polyethylene terephthalate.
A kind of 5. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 4, it is characterised in that：
The micro-fluidic chip be by by the polydimethylsiloxane containing microchannel, makrolon, polymethyl methacrylate, poly- pair
PET is bonded by oxygen plasma with substrate of glass and is prepared.
A kind of 6. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 4, it is characterised in that：
Described micro-fluidic chip is by by the polydimethylsiloxane containing microchannel, makrolon, polymethyl methacrylate, poly-
Ethylene glycol terephthalate is bonded by oxygen plasma with dimethyl silicone polymer substrate and is prepared.
A kind of 7. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 1, it is characterised in that：
The cell sample sample injection unit, oil phase sample injection unit and the second oil phase sample injection unit be by ejection of syringe pump enter it is micro-fluidic
Chip.
A kind of 8. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 1, it is characterised in that：
The cell sample sample injection unit, oil phase sample injection unit and the second oil phase sample injection unit are that malleation forces sample to enter caused by air pressure
Enter micro-fluidic chip.
A kind of 9. micro-fluidic chip for unicellular microlayer model sample preparation according to claim 1, it is characterised in that：
The micro-fluidic chip microchannel, except blank microlayer model split tunnel, the height of remaining microchannel is 10~200 microns,
Width is 10~500 microns.
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