CN106190770A - A kind of double layer micro fluidic chip for tumor cell sorting - Google Patents
A kind of double layer micro fluidic chip for tumor cell sorting Download PDFInfo
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Abstract
The invention discloses a kind of double layer micro fluidic chip for tumor cell sorting, it includes the first chip and is positioned at the second chip of the first beneath chips, the surface of described first chip has sample channel, the surface of second chip relative with described sample channel has buffer passage, and described sample channel intersects with buffer passage and communicates.Relative to CellSearch detection method, the technology utilizing this double layer micro fluidic chip to analyze tumor cell need not many more manipulations, can a step be continuously finished, simple to operate, faster, the CTCs purity of enrichment is higher for detection speed, and owing to not using antibody, the CTCs that the method obtains keeps higher cytoactive, can preferably be used for late detection and tomour specific Journal of Sex Research.
Description
Technical field
The present invention relates to a kind of micro-fluidic chip, particularly relate to a kind of double layer micro fluidic chip, further to
A kind of double layer micro fluidic chip for tumor cell sorting.
Background technology
Circulating tumor cell (circulating tumor cells, CTCs) is an off primary lesion, and invasion and attack are gone forward side by side
Enter the cancerous cell of blood circulation, can as diagnosis patient's prognosis or tumor recurrence, announcement neoplasm metastasis behavior,
The index rationally instructing clinical individualization to treat, is the study hotspot of current oncology.But oncosis human blood
In liquid, the number of CTCs is the most rare, contains only 1～100 CTCs in every milliliter of blood, and every milliliter of blood
Liquid there are up to a million leukocyte and the more erythrocyte of number, so wanting high efficiency sorting from blood
The CTCs difficulty going out purity higher is the biggest.
Microfluidic chip technology prepares the sample involved by the fields such as biological and chemical, chemical reaction, separation
The operating units such as enrichment, detection method are integrated on the slide of a piece several square centimeters.Compared to conventional richness
Collection technology, it have controllability, high flux, low consumption, can be integrated etc. feature, decrease in assorting room
Damage and pollution to purpose cell.Chip channel diameter is typically in the range of between 5～100 μm, with internal micro-blood
Pipe and cell size are close, can realize the cultivation to cell, observe, the purpose such as detection, and can be at small chi
High throughput analysis is realized under very little, the most time-consuming but also save reagent, have in clinical diagnosis and disorder in screening field
There is good prospect.
At present CTCs detection includes enrichment and detection two steps: conventional beneficiation technologies mainly have immune magnetic separation,
Density gradient centrifugation and cell filtration etc..Detection technique includes RT-polymerase chain reaction (RT-PCR), exempts from
Epidemic disease tissue chemical technology (ICC), fluorescence in situ hybridization technique (FISH) and flow cytometry (FCM) etc..
In recent years, micro-fluidic chip is combined by many scholars with existing CTCs sorting detection technique, devises one
Series of CT Cs sorting chip, mainly includes following a few class:
1. the method for separating of physical size is utilized
Under normal circumstances, the diameter of CTCs between 12～25 μm, the diameter of leukocyte between 8～14 μm,
Red blood cell diameter is between 6～8 μm, and compared to leukocyte, the volume of CTCs is relatively big, but deformability is not so good as
Leukocyte.According to tumor cell and hemocyte difference in terms of particle diameter and deformability, can be with different
CTCs is sorted by physical method.
2. the enrichment method of biological property is utilized
CTCs can borrow other cells that its surface marker is different from blood, such as swelling of most epithelial origins
Oncocyte high expressed signal transduction factor (epithelial cellular adhesion molecule,
EpCAM), low expression CD45, and the EpCAM expression of leukocyte is relatively low, the expression water of CD45
Flat the highest, therefore, it can utilize the difference of different cellular elements expression that CTCs is sorted.
At present, the CellSearch of only U.S. Veridex company research and developmentTMSystem becomes a standard test side
Case, in 2004 by FDA (Food and Drug Adminstration) (FDA) access clinical practice.This is one and summarizes
The sorting detection technique of immune magnetic sorting technology and immunocytochemical method, by fixing on magnetic bead
EpCAM antibody and specifically identify, combine target cell, under the effect of externally-applied magnetic field, complete tumor cell
Screening.
The method of commercialization at present is the CellSearch of Veridex company of U.S. research and developmentTMSystem, this is
System can use immune nano magnetic granule technology that cell carries out capture and analyze, but the shortcoming of the method is price
Costliness, operation complexity, need multistep to complete, false positive and false-negative result often occur.
The most frequently used physical size screening technique is that the method using similar membrane filtration is by less hemocyte mistake
Filter, retain the CTCs that particle diameter is bigger, thus reach the purpose of CTCs sorting enrichment.Lacking of the method
Point is that separation velocity is too slow, and efficiency is on the low side, and chip is easily blocked by hemocyte, causes sorting operation cannot be carried out.
Summary of the invention
The technical problem to be solved provides a kind of for the state of the art and sorts for tumor cell
Double layer micro fluidic chip, it can quickly sort enrichment CTCs.
The present invention solves the technical scheme that above-mentioned technical problem used: a kind of for tumor cell sorting
Double layer micro fluidic chip, including the first chip and the second chip being positioned at the first beneath chips, described first core
The surface of sheet has sample channel, and the surface of second chip relative with described sample channel has buffer and leads to
Road, described sample channel intersects with buffer passage and communicates.
Wherein, described first chip has a sample inlet, and described sample channel includes that some groups of samples lead to
Road group, often organizes described sample channel group and includes some blood flow passage, and described sample inlet passes through sample water conservancy diversion
Passage group is connected with the arrival end of every blood flow passage；Described first chip correspondence is often organized sample channel group and is provided with
One waste liquid outlet, each described waste liquid outlet passes through the sample that waste liquid flow-guiding channel group is corresponding with this waste liquid outlet
The port of export of every blood flow passage of product passage group connects.
Wherein, described sample channel includes even number set sample channel group, often organizes described sample channel group and includes 2n
Bar blood flow passage, wherein n is positive integer, and described sample flow-guiding channel group includes Multilayer Samples flow-guiding channel,
Every layer of sample flow-guiding channel includes horizontal sample flow-guiding channel respectively and is positioned at horizontal sample flow-guiding channel two ends
Vertical sample flow-guiding channel, the vertical sample water conservancy diversion closest to the innermost layer sample flow-guiding channel of sample channel leads to
Road connects with two adjacent blood flow passage respectively, is positioned at two vertical samples of the sample flow-guiding channel of outer layer
Flow-guiding channel connects with the horizontal sample flow-guiding channel of the sample flow-guiding channel of two adjacent internal layers respectively, with
This type of advances the stacking of row sample flow-guiding channel to connect, and described sample inlet is arranged on outermost layer sample water conservancy diversion and leads to
On the horizontal sample flow-guiding channel in road.
Wherein, often organize described waste liquid flow-guiding channel group and include that multilamellar waste liquid flow-guiding channel, every layer of waste liquid water conservancy diversion lead to
Road includes horizontal waste liquid flow-guiding channel respectively and the vertical waste liquid water conservancy diversion being positioned at horizontal waste liquid flow-guiding channel two ends leads to
Road, closest to the vertical waste liquid flow-guiding channel of internal layer waste liquid flow-guiding channel of waste fluid channel respectively with adjacent two
Bar blood flow passage connect, two vertical waste liquid flow-guiding channels of the waste liquid flow-guiding channel being positioned at outer layer respectively with phase
The horizontal waste liquid flow-guiding channel of the waste liquid flow-guiding channel of two adjacent internal layers connects, and carries out waste liquid by that analogy and leads
The stacking of circulation road connects, and the corresponding waste liquid outlet often organizing described sample channel group is arranged in this group sample
On the horizontal waste liquid flow-guiding channel of passage group outermost waste liquid flow-guiding channel.
Wherein, described vertical sample flow-guiding channel, vertical waste liquid flow-guiding channel are respectively a diameter of 5～50 μm
Column structure.
Wherein, the adjacent distance between two described vertical sample flow-guiding channels is 10～100 μm, adjacent
Two described vertical waste liquid flow-guiding channels between distance be 10～100 μm.
Wherein, the width of described blood flow passage is 10～500 μm, and thickness is 3～200 μm.
Wherein, the angle between described blood flow passage and buffer passage is less than 180 ° more than 0 °.
Wherein, the angle between described sample channel and buffer passage is 15 °, 30 °, 45 ° or 60 °.
Wherein, when carrying out tumor cell sorting, buffer flow velocity in described buffer passage and blood
Sample velocity ratio in sample channel is 1:0.5～1:5.
Compared with prior art, it is an advantage of the current invention that: it is different that this double layer micro fluidic chip devises height
Double-layer structure, it is on the middle and senior level for sample channel, low layer for buffer passage.When with this bilayer miniflow
When control chip carries out tumor cell sorting, the CTCs that yardstick is bigger can be along higher passage i.e. sample
Channel flow, leukocyte and erythrocyte that yardstick is less then can be by buffer (i.e. buffer) from relatively low runners
I.e. buffer passage is taken away, thus reaches quickly to sort the effect of enrichment CTCs.Relative to
CellSearch detection method, the technology utilizing this double layer micro fluidic chip to analyze tumor cell need not multistep
Operation, can a step be continuously finished, simple to operate, and detection speed is more, and the CTCs purity of enrichment is high, and
And the CTCs owing to not using antibody, the method to obtain keeps higher cytoactive, can preferably use
In late detection and tomour specific Journal of Sex Research.
Accompanying drawing explanation
Fig. 1 is the structural representation of embodiment of the present invention double layer micro fluidic chip.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
As it is shown in figure 1, the double layer micro fluidic chip for tumor cell sorting of the present embodiment, including first
Chip 1 and be positioned at the second chip 2 below the first chip 1, the surface of the first chip 1 has sample channel,
The surface of second chip 2 relative with sample channel has buffer passage 11, and buffer passage 11 has slow
Rush liquid entrance 19 and buffer outlet 20.
First chip 1 has a sample inlet 4, and sample channel includes some groups of sample channel groups 21, often
Group sample channel group 21 includes some blood flow passage 23, and sample inlet 4 is by sample flow-guiding channel group 22
It is connected with the arrival end of every blood flow passage 23；First chip 1 correspondence is often organized sample channel group 21 and is provided with one
Individual waste liquid outlet 3, each waste liquid outlet 3 is by waste liquid flow-guiding channel group 8 sample corresponding with this waste liquid outlet 3
The port of export of the blood flow passage 23 of product passage group 21 connects.
Blood flow passage 23 intersects with buffer passage 11 and communicates, between sample channel and buffer passage 11
Angle is less than 180 ° more than 0 °, such as 15 °, 30 °, 45 ° or 60 °.
In the present embodiment, sample channel includes 4 groups of sample channel groups 21, and often group sample channel group 21 includes
Article 4, blood flow passage 23, sample flow-guiding channel group 22 includes 5 layers of sample flow-guiding channel, waste liquid flow-guiding channel group
8 include 3 layers of waste liquid flow-guiding channel.
Sample flow-guiding channel group 22 has four layers, is to include ground floor sample flow-guiding channel 13, second layer sample respectively
Flow-guiding channel 14, third layer sample flow-guiding channel 15 and the 4th layer of sample flow-guiding channel 16, every layer of sample is led
Circulation road includes horizontal sample flow-guiding channel 17 respectively and is positioned at the vertical of horizontal sample flow-guiding channel 17 two ends
Sample flow-guiding channel 18.Two vertical sample flow-guiding channels 18 of ground floor sample flow-guiding channel 13 respectively with
The outfan of two adjacent blood flow passage 23 connects, two vertical samples of second layer sample flow-guiding channel 14
Product flow-guiding channel 18 connects with the horizontal sample flow-guiding channel 17 of adjacent ground floor sample flow-guiding channel 13 respectively
Connect, two vertical sample flow-guiding channels 18 of third layer sample flow-guiding channel 15 respectively with the adjacent second layer
The horizontal sample flow-guiding channel 17 of sample flow-guiding channel 14 connects, two of the 4th layer of sample flow-guiding channel 16
Vertical sample flow-guiding channel 18 leads to the horizontal sample water conservancy diversion of adjacent third layer sample flow-guiding channel 15 respectively
Road 17 connects, and finally, sample inlet 4 is arranged on the horizontal sample water conservancy diversion of the 4th layer of sample flow-guiding channel 16
On passage 17.
First chip 1 of the present embodiment is provided with 4 waste liquid outlets 3, each waste liquid outlet 3 and sample channel
Annexation as follows: each waste liquid outlet 3 is by one group of waste liquid flow-guiding channel group 8 and this waste liquid outlet 3
The blood flow passage 23 of corresponding sample channel group 21 connects, and often group waste liquid flow-guiding channel group 8 has 2 layers, point
Not Wei ground floor waste liquid flow-guiding channel 6 and second layer waste liquid flow-guiding channel 5, every layer of waste liquid flow-guiding channel wraps respectively
Include horizontal waste liquid flow-guiding channel 10 and be positioned at the vertical waste liquid flow-guiding channel at horizontal waste liquid flow-guiding channel 10 two ends
9.Two vertical waste liquid flow-guiding channels 9 of ground floor waste liquid flow-guiding channel 6 respectively with two adjacent blood streams
The outfan in road 23 connects, two vertical waste liquid flow-guiding channels 9 of second layer waste liquid flow-guiding channel 5 respectively with
The horizontal waste liquid flow-guiding channel 10 of adjacent ground floor waste liquid flow-guiding channel 6 connects, and correspondence often organize sample lead to
The waste liquid outlet 3 of road group 21 is arranged in the horizontal stroke of the second layer waste liquid flow-guiding channel 5 of this group sample channel group
On waste liquid flow-guiding channel 10.
The vertical sample flow-guiding channel 18 of the present embodiment, vertical waste liquid flow-guiding channel 9 are the most a diameter of
5～50 μm, such as 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, the column structure of 45 μm.
The adjacent distance between two vertical sample flow-guiding channels 18 is 10～100 μm, such as 20 μm,
30 μm, 50 μm, 60 μm, 70 μm, 80 μm, between two adjacent vertical waste liquid flow-guiding channels 9 away from
From being also 10～100 μm, such as 20 μm, 40 μm, 50 μm, 60 μm, 80 μm.
The width of the blood flow passage 23 of the present embodiment is 10～500 μm, such as 20 μm, 50 μm, 100 μm,
200 μm, 300 μm, 400 μm, thickness is 3～200 μm, such as 10 μm, 50 μm, 100 μm, 150 μm,
160μm、180μm。
When carrying out tumor cell sorting by the double layer micro fluidic chip of the present embodiment, if controlling buffer flow rate
Time suitable with the ratio of sample flow rate, the CTCs that yardstick is bigger can flow along higher sample channel, and chi
Spend less leukocyte and erythrocyte then can be carried from relatively low buffer passage 11 by PBS (phosphate buffer)
Walk, thus reach quickly to sort the effect of enrichment CTCs.Thus can be fast as a kind of novel blood cell
Speed sorting unit, provides effective technological means for peripheral blood detection circulating tumor cell.
In this enforcement, buffer flow velocity in buffer passage 11 and blood sample stream in sample channel
Speed ratio is 1:0.5～1:5, such as 1:1,1:2,1:3,1:4.
The preparation method of the double layer micro fluidic chip of the present embodiment is as follows:
(1) Double-layer photoetching prepares chip template: coat on the substrate silicon chip for preparation the second chip
Thickness is the photoresist SU 8 of 5 μm～10 μm；Use film mask plate to make photoengraving pattern, carry out first
Secondary exposure, time of exposure is between 5s～20s；Using the structure after exposure for the first time as the base of the first chip
Plate, applied atop thickness is the photoresist SU 8 of 25 μm～40 μm, uses film mask plate to make light needle drawing
Case, then by the template of the microscope alignment lower level structure on litho machine with higher level structure, carries out the
Re-expose, time of exposure is between 5s～20s, and layers of chips height altogether, more than 40 μm, prepares
Template；
(2) by the template for preparing under the conditions of 150 DEG C, toast 30min, make template firm；
(3) template is put in a clean glass dish, TMCS (trim,ethylchlorosilane) suffocating treatment
5min, thus prevent template from adhering to each other with PDMS；
(4) SYLGRAD 184 and PDMS 1:5～1:20 in mass ratio is cast in template surface, very
Empty pumping removes the air between PDMS and silicon chip (i.e. substrate), toasts in the constant temperature blast drying oven of 80 DEG C
45min；
(5) chip cuts out the size and shape of needs with guarded blade utility knife, and card punch punches；Clean with one afterwards
Microscope slide put into O together2Plasma processes 10s～30s；
(6) PDMS chip is sealed with microscope slide, dry in the constant temperature blast drying oven of 80 DEG C
Roasting 2h, it is thus achieved that the double layer micro fluidic chip of the present embodiment.
Above content is only presently preferred embodiments of the present invention, for those of ordinary skill in the art, according to this
The thought of invention, the most all will change, and this specification content is not
It is interpreted as limitation of the present invention.
Claims (10)
1. the double layer micro fluidic chip for tumor cell sorting, it is characterised in that: include the first core
Sheet (1) and be positioned at the first chip (1) lower section the second chip (2), the surface of described first chip (1) has sample
Passage, the surface of second chip (2) relative with described sample channel has buffer passage (11), described sample
Product passage intersects with buffer passage (11) and communicates.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 1, its feature exists
In: described first chip (1) has a sample inlet (4), and described sample channel includes some groups of sample channels
Group (21), often organizes described sample channel group (21) and includes some blood flow passage (23), and described sample inlet (4) leads to
Cross sample flow-guiding channel group (22) to be connected with the arrival end of every blood flow passage (23)；Described first chip (1) is corresponding
Often group sample channel group (21) is provided with a waste liquid outlet (3), and each described waste liquid outlet (3) passes through waste liquid water conservancy diversion
The port of export of every blood flow passage (23) of the sample channel group (21) that passage group (8) is corresponding with this waste liquid outlet (3)
Connect.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 2, its feature exists
In: described sample channel includes even number set sample channel group (21), often organizes described sample channel group (21) and includes 2n
Bar blood flow passage (23), wherein n is positive integer, and described sample flow-guiding channel group (22) includes Multilayer Samples water conservancy diversion
Passage, every layer of sample flow-guiding channel includes horizontal sample flow-guiding channel (17) respectively and is positioned at horizontal sample water conservancy diversion and leads to
The vertical sample flow-guiding channel (18) at road (17) two ends, closest to the innermost layer sample flow-guiding channel of sample channel
Vertical sample flow-guiding channel (18) connects with two adjacent blood flow passage (23) respectively, and the sample being positioned at outer layer is led
Two vertical sample flow-guiding channels (18) of circulation road respectively with the sample flow-guiding channel of two adjacent internal layers
Laterally sample flow-guiding channel (17) connects, and the stacking carrying out sample flow-guiding channel by that analogy connects, described sample
Entrance (4) is arranged on the horizontal sample flow-guiding channel (17) of outermost layer sample flow-guiding channel.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 3, its feature exists
In: often organize described waste liquid flow-guiding channel group (8) and include that multilamellar waste liquid flow-guiding channel, every layer of waste liquid flow-guiding channel divide
Do not include horizontal waste liquid flow-guiding channel (10) and be positioned at the vertical waste liquid at horizontal waste liquid flow-guiding channel (10) two ends and lead
Circulation road (9), closest to waste fluid channel internal layer waste liquid flow-guiding channel vertical waste liquid flow-guiding channel (9) respectively with
Two adjacent blood flow passage (23) connect, and two vertical waste liquid water conservancy diversion of the waste liquid flow-guiding channel being positioned at outer layer lead to
Road (9) connects with the horizontal waste liquid flow-guiding channel (10) of the waste liquid flow-guiding channel of two adjacent internal layers respectively, with
This type of advances the stacking of row waste liquid flow-guiding channel to connect, and correspondence often organizes the waste liquid outlet of described sample channel group (21)
(3) the horizontal waste liquid flow-guiding channel of this group sample channel group (21) outermost waste liquid flow-guiding channel it is arranged in
(10) on.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 4, its feature exists
In: described vertical sample flow-guiding channel (18), vertical waste liquid flow-guiding channel (9) are respectively a diameter of 5～50 μm
Column structure.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 4, its feature exists
In: the adjacent distance between two described vertical sample flow-guiding channels (18) is 10～100 μm, adjacent institute
Stating the distance between two vertical waste liquid flow-guiding channels (9) is 10～100 μm.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 2, its feature exists
In: the width of described blood flow passage (23) is 10～500 μm, and thickness is 3～200 μm.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 2, its feature exists
In: the angle between described blood flow passage (23) and buffer passage (11) is less than 180 ° more than 0 °.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 8, its feature exists
In: the angle between described sample channel and buffer passage (11) is 15 °, 30 °, 45 ° or 60 °.
Double layer micro fluidic chip for tumor cell sorting the most according to claim 1, its feature
It is: when carrying out tumor cell sorting, buffer flow velocity in described buffer passage (11) and blood sample
Product velocity ratio in sample channel is 1:0.5～1:5.
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