CN206986150U - A kind of urine Exfoliated tumor cells micro-fluidic chip for bladder transitional cell carcinoma - Google Patents
A kind of urine Exfoliated tumor cells micro-fluidic chip for bladder transitional cell carcinoma Download PDFInfo
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Abstract
The utility model belongs to biological fluid vitro detection field, and in particular to a kind of urine Exfoliated tumor cells micro-fluidic chip for bladder transitional cell carcinoma.The micro-fluidic chip includes sample entrance port, cell capture region and the sample exit port being sequentially connected, the cell capture region is provided with multiple cell sorters, the cell sorter is made up of the columnar projections of three curved arrangements of entirety, gap between columnar projections be present, arc opening is as liquid flow inlet, the gap of middle column projection both sides is symmetric as fluid outlet, two fluid outlets.The micro-fluidic chip realizes in urine the separating trap of various types of cells and compatible a variety of downstream cellular colouring methods are to realize cell recognition, improve the accuracy in detection of the urine Exfoliated tumor cells in human urine, dependence of the conventional method for pathologist subjective experience has been broken away from, and it is whole noninvasive.In addition, its lossless cell capture, way of recycling are laid a good foundation for the molecular biological analysis in downstream.
Description
Technical field
The utility model belongs to biological fluid vitro detection field, and in particular to a kind of urine for bladder transitional cell carcinoma comes off
Tumour cell micro-fluidic chip.
Background technology
1st, clinical active service technology-urine sediment inspection：Urine sediment inspection (Cytology) is current clinic
On the bladder transitional cell carcinoma detection method that generally uses.To find tumour cell, this method relies on pathologist and smear is led
Observation (factor such as nucleus, chromatin, endochylema for cell is judged cell), mainly has the disadvantage that：
(1) detection sensitivity is low, typically only 30-50%.
(2) diagosis places one's entire reliance upon the subjective experience of pathologist, judged result between different pathological doctor or differs
Cause.
(3) dyeing course is cumbersome, and artificial participation is big, working strength is big.Cell dyeing effect is by personnel's experience, proficiency
Influence.
2nd, clinical active service technology-cystoscope, Flexible ureteroscope inspection
Cystoscope, Flexible ureteroscope inspection are the goldstandards of current clinical diagnosis bladder transitional cell carcinoma.It will be peeped by conduit
Mirror per urethra is sent to target area, and suspicious occupy-place is imaged, observed.But still have the disadvantage that：
(1) intrusive mood inspection is belonged to, the pain that patient is born is big；
(2) tumor development to naked eyes it is visible when can just be observed, be unfavorable for early diagnose
3rd, other are in the technology of grinding-urine cytoscopy based on microfiltration membranes
To overcome traditional urine sediment to check the shortcomings that susceptibility is low, urine detects using microfiltration membranes in some seminar
Exfoliated tumor cells.Compared to traditional urine sediment inspection, this method is come off carefully using the principle capture urine of membrane filtration
Born of the same parents, susceptibility is slightly lifted, but still has following defect：
(1) after capturing cell, still only with traditional cytological stains method (pap staining), this method is such as clinic
The urine sediment inspection of active service, height rely on Pathologis, restricted by subjective factor；
(2) the filter opening bore of the filter membrane is single, is 7.5um；But the cell component in bladder transitional cell carcinoma Urine in Patients is answered
Miscellaneous, except tumour cell, the Urothelial cell also normally to come off, leucocyte, red blood cell etc., these cell sizes differ
Cause, if using single aperture, cell component confusion in the visual field will be caused, disturb follow-up discriminatory analysis；
(3) real time imagery, monitoring, regulation and control can not be carried out to the acquisition procedure of cell, such as：(a) led in cell concentration surplus
In the case of causing filter membrane blocking, process can not be terminated in time；(b) flow velocity is too high cause cell damage serious situation under, nothing
The real-time coutroi velocity of method；
(4) limited by maximum load amount, when cell quantity is too high, all filter openings are occupied, and the filter membrane fails immediately；
The cell then come will all be jammed in filter membrane, cause the visual field chaotic, it is difficult to carry out the interpretation of next step cell aspect.
Utility model content
In order to overcome the problems of in the prior art, the purpose of this utility model is to provide a kind of on urinary tract
The urine Exfoliated tumor cells micro-fluidic chip of skin cancer.
To achieve these goals and other related purposes, the utility model adopt the following technical scheme that：
First aspect of the present utility model, there is provided a kind of micro-fluidic chip, including sample entrance port, the cell being sequentially connected
Capture region and sample exit port, the cell capture region are provided with multiple cell sorters, and the cell sorter is by three
The columnar projections of overall curved arrangement are formed, and gap be present between columnar projections, arc opening is as liquid flow inlet, intermediolateral column
The gap of shape projection both sides is symmetric as fluid outlet, two fluid outlets.
Preferably, the cell capture region is provided with the cell sorter of three kinds of different dimensions, respectively the first chi
Very little cell sorter, the second size cell sorter and the 3rd size cell sorter, the first size cell sorter,
The size of two size cell sorters and the 3rd size cell sorter is sequentially reduced.
Preferably, adjacent cell sorter size is different.
Preferably, the micro-fluidic chip includes multiple cell sorting units, and each cell sorting unit is by one
First size cell sorter, a second size cell sorter and a 3rd size cell sorter composition.
The arrangement mode for being preferably located at each size cell sorter in the cell sorting unit with a line is identical.
Preferably, laterally adjacent cell sorter, back gauge are identical.
Preferably, laterally a first size cell sorter of back gauge arrangement, a second size cell point such as successively
Device and a 3rd size cell sorter is selected to form a first cell sorting unit；Laterally one of back gauge arrangement such as successively
First size cell sorter, a 3rd size cell sorter and a second size cell sorter form one second
Cell sorting unit；Multiple first cell sorting unit transverse circulation arrangements form the first cell sorting array, and multiple second is thin
The arrangement of born of the same parents' separation unit traverse cycle forms the second cell sorting array, a first cell sorting array being parallel to each other and one
Individual second capture array forms a cell sorting two level array, multiple cell sortings two being parallel to each other in cell capture region
The back gauge circulation arrangement such as level array longitudinal direction.
Preferably, in each cell sorting two level array, the first size cell sorter of the second cell sorting array
Liquid flow inlet midpoint and corresponding first cell sorting array the 3rd size cell sorter and the second size cell sorting
Device widthwise edge away from midpoint alignment.
Preferably, first cell sorter lengthwise position of the first trip of each odd-numbered line cell sorting two level array is alignd；Each idol
The more adjacent previous odd-numbered line cell sorting two level array of several rows of cell sorting two level arrays pair-wise offset to the right.
Preferably, the width of the liquid flow inlet of first size cell sorter is 60 ± 5 μm, and the width of fluid outlet is
22±5μm；The width of the liquid flow inlet of second size cell sorter is 30 ± 3 μm, and the width of fluid outlet is 10 ± 3 μm；
The width of the liquid flow inlet of 3rd size cell sorter is 16 μ ± 1m, and the width of fluid outlet is 4 ± 1 μm.
Second aspect of the present utility model, there is provided the preparation method of foregoing micro-fluidic chip, including：According to poly dimethyl silicon
Oxygen alkane standard technology, using conventional slide as substrate, it is bonded by plasma treatment, prepares micro-fluidic chip.
The third aspect of the present utility model, there is provided foregoing micro-fluidic chip is used for the use for capturing or reclaiming Urine exfoliative cell
On the way.
Preferably, urine Exfoliated tumor cells are contained in the Urine exfoliative cell.
Preferably, urothelial cancer cells are contained in the Urine exfoliative cell.The epithelioma includes carcinoma of urinary bladder, ureter
Cancer, carcinoma of renal pelvis.
Fourth aspect of the present utility model, there is provided a kind of method for capturing or reclaiming Urine exfoliative cell, including step：Will
Pending urine specimen or the suspension of arena, it is passed through micro-fluidic chip of the present utility model.
Preferably, urine Exfoliated tumor cells are contained in the Urine exfoliative cell.
Preferably, urothelial cancer cells are contained in the Urine exfoliative cell.The epithelioma includes carcinoma of urinary bladder, ureter
Cancer, carcinoma of renal pelvis.
5th aspect of the present utility model, there is provided a kind of Papanicolau staining process, including step：First with of the present utility model micro-
Fluidic chip captures and sorting Urine exfoliative cell, and the Urine exfoliative cell of capture then is carried out into pap staining.
Compared with prior art, the utility model has the advantages that：
For susceptibility present in current bladder transitional cell carcinoma clinical diagnosis it is low, rely on pathologist subjective judgement, have
The problems such as creating, be poorly efficient, micro-fluidic chip provided by the utility model can be realized all kinds of in human urine according to physical size difference
The separating trap of cell and compatible a variety of downstream cellular colouring methods are to realize cell recognition.This technique improves human urine
The accuracy in detection of urine Exfoliated tumor cells in liquid, has broken away from dependence of the conventional method for pathologist subjective experience, and
It is whole noninvasive.In addition, its lossless cell capture, way of recycling are laid a good foundation for the molecular biological analysis in downstream.
Brief description of the drawings
Fig. 1：Microfluidic chip structure and basic principle schematic.
Fig. 2：The structural representation of cell sorter.
Fig. 3：Various sizes of cell sorter is according to cell size difference classification capture cell principle schematic diagram.
Fig. 4：Cell sorting cell schematics in micro-fluidic chip cell capture area.
Fig. 5：Cell capture array schematic diagram in micro-fluidic chip cell capture area.
Fig. 6：ROC curve obtained by bladder transitional cell carcinoma Urine exfoliative cell miniflow detection technique.
Fig. 7：Unicellular sequencing is carried out using 6 Urine exfoliative cells of the capture of the utility model micro-fluidic chip, recovery, and
Analyze its CNV situation.
Component label instructions
1-1 sample entrance ports
1-2 cell captures region
1-3 sample exit ports
2 cell sorters
201 columnar projections
202 liquid flow inlets
203 fluid outlets
2-1 first size cell sorters
2-2 the second size cell sorters
The size cell sorters of 2-3 the 3rd
3 cell sorting units
3-1 the first cell sorting units
3-2 the second cell sorting units
4 cell sorting two level arrays
4-1 the first cell sorting arrays
4-2 the second cell sorting arrays
Embodiment
First, micro-fluidic chip
The structure and principle schematic of micro-fluidic chip of the present utility model can be found in Fig. 1.Specifically, the micro-fluidic core
Piece, including sample entrance port 1-1, cell capture region 1-2 and the sample exit port 1-3 being sequentially connected along liquid flow path direction A.It is pending
Liquid sample such as urine, enter micro-fluidic chip from sample entrance port 1-1, cell capture region 1-2 is the capture of target cell
With the nucleus of sorting, target cell such as Urine exfoliative cell B are caught during sorting during approach cell capture region 1-2
Obtain, remaining liq flows out micro-fluidic chip from sample exit port 1-3.
For the sorting and capture suitable for target cell such as Urine exfoliative cell, cell capture region 1-2 is provided with multiple thin
Born of the same parents' sorter 2, as shown in Fig. 2 the cell sorter 2 is made up of the columnar projections 201 of three curved arrangements of entirety, column
Gap between projection be present, arc opening 202 is used as liquid flow inlet, and the gap 203 of middle column projection both sides goes out as liquid stream
Mouthful, two fluid outlets are symmetric.
Further, the bottom of each cell sorter is in arcuation, helps at utmost to keep cellular morphology complete, this sets
Meter is a kind of optimization design for being adapted to the captured cell of recovery.When reclaiming captured cell, buffer solution can be from reverse flow
Enter chip (flowing to sample entrance port 1-1 directions from sample exit port 1-3 directions), when cell is rushed out archaeocyte sorter, will meet
Other cell sorters numerous to front.Now, because the bottom of cell sorter is arc, cell is just easier to bypass cell
Sorter and smoothly reclaimed, so as to greatly reduce the physical damnification that cell is subject to.
Further, as shown in figure 3, in order to be set according to cell size difference classification capture cell, capture region 1-2
There are the cell sorter 2 of three kinds of different dimensions, respectively first size cell sorter 2-1, the second size cell sorting
Device 2-2 and the 3rd size cell sorter 2-3, first size cell sorter 2-1, the second size cell sorter 2-2 and
Three size cell sorter 2-3 size is sequentially reduced.Adjacent cell sorter size is different, in whole cell capture region
Disorderly flow conditions are built in 1-2.Therefore, the target cell in liquid sample, as Urine exfoliative cell flow through at random it is each not
, can be from large scale cell point after the cell of small size enters large scale cell sorter with the cell sorter of dimensions
Two fluid outlets of device bottom are selected, and as liquid stream continues toward moving ahead, until running into the cell sorter that size matches
It is captured.So various sizes of cell is only captured by the cell sorter of correspondingly-sized, different size can be pointedly captured
Target cell such as Urine exfoliative cell.Small size cell will not occupy the space of large scale cell sorter, greatly improved each
The utilization rate of cell sorter.Listed in some embodiments of the present utility model：First size cell sorter 2-1 liquid
The width of inflow entrance is 60 ± 5 μm, and the width of two fluid outlets is 22 ± 5 μm, can capture diameter in 22~60 μ ms
Interior cell (or cell mass).The width of second size cell sorter 2-2 liquid flow inlet is 30 ± 3 μm, and two liquid streams go out
The width of mouth is 10 ± 3 μm, can capture cell of the diameter in 10~22 μ ms.3rd size cell sorter 2-3's
The width of liquid flow inlet is 16 μ ± 1m, and the width of two fluid outlets is 4 ± 1 μm, can capture diameter in 4~10 μ ms
Interior cell.Cell in urine is generally bigger than in blood, and is usually present the situation of cell mass, is set for urine cell is specific
The cell sorter of three kinds of different dimensions of meter, the defects of cell mass can be avoided the occurrence of.
As shown in figure 4, the capture region 1-2 includes multiple cell sorting units 3, each cell sorting unit 3
By a first size cell sorter 2-1, a second size cell sorter 2-2 and a 3rd size cell sorter
2-3 is formed.Further, the arrangement mode of each size cell sorter is identical in the cell sorting unit with a line.It is horizontal
To adjacent cell sorter, back gauge d1 is identical.In some embodiments of the utility model, laterally adjacent cell point is listed
It is 60 ± 3 μm to select the back gauge d1 between device, and the other impurities that the size allows to be likely to occur in urine pass through, and avoid core
The blocking of piece.Even when having substantial amounts of cell in Urine in Patients and all cell sorters in micro-fluidic chip is occupied
According to when, 60 ± 3 μm of spacing can also allow that the cell of surplus by the way that the visual field in traditional filter membrane method is chaotic to ask so as to avoid
Topic.
As shown in figure 5, laterally a first size cell sorter 2-1 of back gauge arrangement, second size such as successively
Cell sorter 2-2 and a 3rd size cell sorter 2-3 form a first cell sorting unit 3-1；Laterally successively
Etc. a first size cell sorter 2-1, a 3rd size cell sorter 2-3 and second size of back gauge arrangement
Cell sorter 2-2 forms a second cell sorting unit 3-2；Multiple first cell sorting unit 3-1 traverse cycles arrangements
The first cell sorting array 4-1 is formed, multiple second cell sorting unit 3-2 traverse cycles arrangements form the second cell sorting battle array
4-2 is arranged, a first cell sorting array being parallel to each other and second capture array form a cell sorting two level battle array
Row 4, the back gauge circulation arrangement such as multiple cell sorting two level arrays longitudinal directions being parallel to each other in cell capture region.
Further, in each cell sorting two level array 4, the second cell sorting array 4-2 first size cell
The midpoint of sorter 2-1 liquid flow inlet and corresponding first cell sorting array 4-1 the 3rd size cell sorter 2-2 and
2nd 2-3 size cell sorter widthwise edges away from midpoint alignment.
Further, first cell sorter lengthwise position of the first trip of each odd-numbered line cell sorting two level array is alignd；Respectively
The more adjacent previous odd-numbered line cell sorting two level array of even number line cell sorting two level array pair-wise offset to the right.Contribute to
Disorderly, random flow conditions are built, avoid cell from being orientated along single-pathway, so as to lift the capture rate of micro-fluidic chip.
All target cells such as Urine exfoliative cell for flowing through foregoing micro-fluidic chip of the present utility model will be with same equiprobability
Cell sorters at different levels are met with, so as to reach the purpose of " according to cell size difference classification capture cell ".When cell can be according to
It is captured separately and comes according to size difference, then field of view is cleaner and tidier, cellular morphology is apparent, is advantageous to be directed to cell in next step
The analysis interpretation of form (such as general urine sediment inspection clinical now).
2nd, the preparation method of micro-fluidic chip
, using conventional slide as substrate, plasma can be passed through according to dimethyl silicone polymer (PDMS) standard technology
Processing bonding, to prepare micro-fluidic chip of the present utility model.
PDMS is the english abbreviation of dimethyl silicone polymer.Dimethyl silicone polymer belongs to curing type polymer, curing type
After polymer mixes with curing agent, it is hardened to obtain the micro-fluidic chip of certain structure through curable after a while.
3rd, the purposes of micro-fluidic chip
Micro-fluidic chip of the present utility model can be used for capturing or reclaiming Urine exfoliative cell.
4th, the method for capturing or reclaiming Urine exfoliative cell
Capture or recovery Urine exfoliative cell of the present utility model, including step：(1) capture：Pending urine specimen or
The suspension of person's arena, micro-fluidic chip of the present utility model, cell quilt are entered by sample entrance port under the promotion of syringe pump
It is trapped in micro-fluidic chip, waste liquid flows out from sample exit port；(2) reclaim：Phosphate buffer under the promotion of syringe pump by
Sample exit port enters micro-fluidic chip of the present utility model, flows, is reclaimed along the direction opposite with cell capture process
Cell flows out from sample entrance port.
5th, Papanicolau staining process in situ in micro-fluidic chip
Papanicolau staining process of the present utility model, including step：First captured and divided with micro-fluidic chip of the present utility model
Urine exfoliative cell is selected, the Urine exfoliative cell of capture is then subjected to pap staining in situ in micro-fluidic chip.The micro-fluidic core
Pap staining in situ can use method of the prior art in piece.
Illustrated in some embodiments of the present utility model：Pap staining detailed step in situ is in micro-fluidic chip：
(1) under the control of syringe pump, the alcohol that concentration is 95% has urine to come off with 0.5-4ml/h flow velocity by capture
The micro-fluidic chip of cell, maintain 15min；
(2) under the control of syringe pump, clean clear water, by micro-fluidic chip, maintains 1min with 0.5-4ml/h flow velocity；
(3) under the control of syringe pump, haematoxylin dye liquor, by micro-fluidic chip, is maintained with 0.5-4ml/h flow velocity
3min；
(4) under the control of syringe pump, clean clear water, by micro-fluidic chip, maintains 1min with 0.5-4ml/h flow velocity；
(5) under the control of syringe pump, Lithium carbonate solution, by micro-fluidic chip, is maintained with 0.5-4ml/h flow velocity
30s；
(6) under the control of syringe pump, clean clear water, by micro-fluidic chip, maintains 1min with 0.5-4ml/h flow velocity；
(7) under the control of syringe pump, EA-OG solution, by micro-fluidic chip, is maintained with 0.5-4ml/h flow velocity
3min；
(8) under the control of syringe pump, the alcohol that concentration is 95% with 0.5-4ml/h flow velocity by micro-fluidic chip,
Maintain 1min；
(9) under the control of syringe pump, absolute alcohol, by micro-fluidic chip, maintains 1min with 0.5-4ml/h flow velocity；
(10) Microscopic observation.
6th, immunofluorescence dyeing method in situ in micro-fluidic chip
Immunofluorescence dyeing method of the present utility model, including step：First captured with micro-fluidic chip of the present utility model
With sorting Urine exfoliative cell, the Urine exfoliative cell of capture is then subjected to immunofluorescence dyeing in situ in micro-fluidic chip.It is described
Immunofluorescence dyeing in situ can use method of the prior art in micro-fluidic chip.
Illustrated in some embodiments of the present utility model：Immunofluorescence dyeing detailed step in situ in micro-fluidic chip
For：
(1) under the control of syringe pump, paraformaldehyde has Urine exfoliative cell with 0.5~4ml/h flow velocity by capture
Micro-fluidic chip, maintain 30min；
(2) under the control of syringe pump, PBS, by the micro-fluidic chip, maintains 3- with 0.5~4ml/h flow velocity
5min；
(3) under the control of syringe pump, the Triton X-100 solution that concentration is 0.1% is led to 0.5~4ml/h flow velocity
The micro-fluidic chip is crossed, maintains 10min；
(4) under the control of syringe pump, PBS, by the micro-fluidic chip, maintains 3- with 0.5~4ml/h flow velocity
5min；
(5) under the control of syringe pump, BSA solution, by the micro-fluidic chip, is maintained with 0.5~4ml/h flow velocity
30min；
(6) under the control of syringe pump, anti-CK20 primary antibodies (abcam, ab76126), anti-CD44v6 primary antibodies (abcam,
Ab78960) mixed solution maintains 60min with 0.5ml/h flow velocity by the micro-fluidic chip；
(7) under the control of syringe pump, PBS, by micro-fluidic chip, maintains 3-5min with 0.5~4ml/h flow velocity；
(8) under the control of syringe pump, CK20 corresponds to secondary antibody (Invitrogen, A11070), CD44v6 corresponds to secondary antibody
(abcam, ab150116), DAPI (Invitrogen, MP01306) mixed solutions pass through the miniflow with 0.5ml/h flow velocity
Chip is controlled, maintains 30min；
(9) under the control of syringe pump, PBS, by the micro-fluidic chip, maintains 3- with 0.5~4ml/h flow velocity
5min；
(10) under the control of syringe pump, finally, under the exciting light of fluorescence microscope corresponding wavelength, searching CK20,
The simultaneously positive cell of CD44v6, DAPI three, to urinate Exfoliated tumor cells.
Advantage of the present utility model is that the cell that (1) captures through micro-fluidic chip is distributed according to size difference,
So that field of view is clear, Pathology Doctors ' diagosis (2) agents useful for same is facilitated to be controlled by miniflow syringe pump, dyeing time is accurate, dye
Color crosses process automation.
Before the utility model embodiment is further described, it should be appreciated that the scope of protection of the utility model is not
It is confined to following specific specific embodiments；It is also understood that the term used in the utility model embodiment is to retouch
Specific specific embodiment is stated, rather than in order to limit the scope of protection of the utility model.Unreceipted tool in the following example
The test method of concrete conditions in the establishment of a specific crime, generally according to normal condition, or the condition proposed by according to each manufacturer.
When embodiment provides number range, it should be appreciated that unless the utility model is otherwise noted, the two of each number range
Any one numerical value can be selected between individual end points and two end points.Unless otherwise defined, the institute used in the utility model
There are technology and scientific terminology identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific side used in embodiment
Method, equipment, outside material, according to grasp of the those skilled in the art to prior art and record of the present utility model, also
Any side of the prior art similar or equivalent with the method described in the utility model embodiment, equipment, material can be used
Method, equipment and material realize the utility model.
Unless otherwise indicated, the experimental method disclosed in the utility model, detection method, preparation method use this skill
Art field conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA skill
The routine techniques of art and association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The micro-fluidic chip of embodiment 1 captures Urine exfoliative cell
As shown in Fig. 1~5, micro-fluidic chip of the present utility model, including the sample entrance port being sequentially connected along liquid flow path direction A
1-1, cell capture region 1-2 and sample exit port 1-3.Cell capture region 1-2 is provided with multiple cell sorters 2, such as Fig. 2 institutes
To show, the cell sorter 2 is made up of the columnar projections 201 of three curved arrangements of entirety, gap between columnar projections be present,
Arc opening 202 is used as liquid flow inlet, and the gap 203 of middle column projection both sides is used as fluid outlet, and two fluid outlets are in
It is symmetrical.Cell capture region 1-2 is provided with the cell sorter 2 of three kinds of different dimensions, respectively first size cell
Sorter 2-1, the second size cell sorter 2-2 and the 3rd size cell sorter 2-3, first size cell sorter 2-1,
Second size cell sorter 2-2 and the 3rd size cell sorter 2-3 size are sequentially reduced.Wherein, first size cell
The width of sorter 2-1 liquid flow inlet is 60 μm, and the width of two fluid outlets is 22 μm, can capture diameter 22~60
Cell in μ m.The width of second size cell sorter 2-1 liquid flow inlet is 30 μm, the width of two fluid outlets
Degree is 10 μm, can capture cell of the diameter in 10~22 μ ms.3rd size cell sorter 2-3 liquid flow inlet
Width is 16 μm, and the width of two fluid outlets is 4 μm, can capture cell of the diameter in 4~10 μ ms.Adjacent
Cell sorter size is different, and the flow conditions of disorder are built in whole cell capture region 1-2.
The capture region 1-2 includes multiple cell sorting units 3, and each cell sorting unit 3 is by one first
Size cell sorter 2-1, a second size cell sorter 2-2 and a 3rd size cell sorter 2-3 composition.Enter
One step, the arrangement mode of each size cell sorter is identical in the cell sorting unit with a line.Laterally adjacent is thin
Born of the same parents' sorter, back gauge d1 is identical, is 60 μm.Laterally first size cell sorter 2-1, one of back gauge arrangement such as successively
Individual second size cell sorter 2-2 and a 3rd size cell sorter 2-3 form a first cell sorting unit 3-
1；A laterally first size cell sorter 2-1, a 3rd size cell sorter 2-3 and one for back gauge arrangement such as successively
Individual second size cell sorter 2-2 forms a second cell sorting unit 3-2；Multiple first cell sorting unit 3-1 are horizontal
The first cell sorting array 4-1 is formed to circulation arrangement, multiple second cell sorting unit 3-2 traverse cycles arrangements form second
Cell sorting array 4-2, a first cell sorting array being parallel to each other and second capture array form a cell
Sort two level array 4, back gauge (the d3=120 μ such as multiple cell sorting two level arrays longitudinal directions being parallel to each other in cell capture region
M) circulation arrangement.
The first trip of each odd-numbered line cell sorting two level array first cell sorter lengthwise position alignment；Each even number line cell
Sort equidistant (d2=50 μm) skew to the right of the more adjacent previous odd-numbered line cell sorting two level array of two level array.Advise in due order
Rule, until filling up total length and overall width is 6000 μm of micro-fluidic chip capture region 1-2.
According to dimethyl silicone polymer (PDMS) standard technology, using conventional 2.5*7.5cm slides as substrate, pass through
Plasma treatment is bonded, to prepare micro-fluidic chip of the present utility model.
Under the control of syringe pump, PBS (phosphate buffer) suspension of pending urine specimen or arena,
Enter micro-fluidic chip from sample entrance port 1-1, Urine exfoliative cell is captured during approach cell capture region 1-2, remaining liq from
Sample exit port 1-3 flows out micro-fluidic chip.Urine exfoliative cell B in liquid sample flows through the thin of each different dimensions at random
Born of the same parents' sorter, various sizes of Urine exfoliative cell B are captured by the cell sorter 2 of correspondingly-sized.Therefore, various sizes of cell
Come, get a clear view in chip internal regular distribution, cellular morphology is intact, the analysis especially suitable for next step.
The immunofluorescence technique of embodiment 2 judges urine Exfoliated tumor cells
After Urine exfoliative cell is carried out the micro-fluidic chip capture in example 1, various sizes of cell is in chip internal rule
It is distributed and, gets a clear view, cellular morphology is intact, the analysis especially suitable for next step.
The present embodiment distinguishes urine Exfoliated tumor cells and normal Urothelial cell by immunofluorescence technique.
Immunofluorescence dyeing step：Paraformaldehyde has the miniflow of Urine exfoliative cell with 0.5~4ml/h flow velocity by capture
Chip is controlled, maintains 30min；PBS, by the micro-fluidic chip, maintains 3-5min with 0.5~4ml/h flow velocity；Concentration is
0.1% Triton X-100 solution, by the micro-fluidic chip, maintains 10min with 0.5~4ml/h flow velocity；PBS with
0.5~4ml/h flow velocity maintains 3-5min by the micro-fluidic chip；BSA solution is passed through with 0.5~4ml/h flow velocity
The micro-fluidic chip, maintain 30min.Anti- CK20 primary antibodies (abcam, ab76126), anti-CD44v6 primary antibodies (abcam,
Ab78960) mixed solution maintains 60min with 0.5ml/h flow velocity by the micro-fluidic chip；PBS is with 0.5~4ml/h
Flow velocity pass through micro-fluidic chip, maintain 3-5min；CK20 corresponds to secondary antibody (Invitrogen, A11070), CD44v6 corresponding two
Anti- (abcam, ab150116), DAPI (Invitrogen, MP01306) mixed solutions are passed through described micro- with 0.5ml/h flow velocity
Fluidic chip, maintain 30min；PBS, by the micro-fluidic chip, maintains 3-5min with 0.5~4ml/h flow velocity.
Finally, under the exciting light of fluorescence microscope corresponding wavelength, it is simultaneously positive that CK20, CD44v6, DAPI three are found
Cell, for urinate Exfoliated tumor cells.
Specifically, CK20：Excitation wavelength 450nm-480nm, launch wavelength 515nm；CD44v6:Excitation wavelength 515nm-
585nm, launch wavelength 610nm；DAPI:Excitation wavelength 330nm-385nm, launch wavelength 420nm.
3 Detection accuracy of the present utility model of embodiment
In order to verify accuracy rate of the present utility model, we have collected 50 bladder transitional cell carcinoma made a definite diagnosis patients, 13 it is non-
The urine of cancer volunteer uses the method in above-described embodiment 2 to be detected.Find CK20, CD44v6, DAPI three simultaneously
Positive cell, to urinate Exfoliated tumor cells.ROC curve is drawn based on detected tumor cell number in all case urines
(as shown in Figure 6), obtaining susceptibility of the present utility model is：88.9%；Specificity is：76.9%, area is under ROC curve
0.808。
In addition, we also implement the one-to-one comparison with clinical active service urine sediment inspection.17 urine are gathered altogether
The urine of road epithelioma patient diagnosed, wherein half urine are sent to hospital pathology department and carry out urine sediment inspection, and in addition one
Half urine is sent to laboratory and detected using the method in the utility model above-described embodiment 2.Searching CK20, CD44v6,
The simultaneously positive cell of DAPI three, to urinate Exfoliated tumor cells.As a result it is：In 17 patients diagnosed, traditional Urine exfoliative cell
Only 4 that inspection is positive and (includes suspected case) are learned, accuracy rate 23.5%；And the utility model detects in 17 patients
15 positive findingses, accuracy rate 88.2%.Refer to shown in table 1 below：
Table 1：The utility model and the comparison of traditional urine sediment inspection
N:It is negative；S:It is doubtful；P:It is positive
The Papanicolaou's vaginal smear technique of embodiment 4 judges urine Exfoliated tumor cells
In terms of the judgement of urine Exfoliated tumor cells, the utility model is also compatible with except above-mentioned immunofluorescence technique can be used
Traditional cytological stains method.Pap staining in chip, agents useful for same and flow and clinical use bar are carried out after cell is captured
Albert'stain Albert method is consistent.Cellular morphology can clearly be presented after dyeing, for pathologist diagosis.
Embodiment 5 reclaims cell and carries out unicellular sequencing
The analysis for carrying out molecular level to tumour cell has huge clinical value, has also fully demonstrated accurate medical
Theory.A big advantage of the present utility model is：It lossless can capture, reclaim single urine Exfoliated tumor cells, so as to the list for downstream
Cell sequencing lays the first stone.We are captured using the micro-fluidic chip in the utility model above-described embodiment 1 and have reclaimed one
6 Urine exfoliative cells (1 normal Urothelial cell, 5 tumour cells) of bladder transitional cell carcinoma patient are then thin to this 6
Born of the same parents carry out unicellular sequencing, its copy number variation (CNV) situation of ultimate analysis respectively.
As a result showing, there is the disorderly situation of copy number as expected in tumour cell, and normal Urothelial cell
Copy number then meets normal diploid feature.
This example fully demonstrates the utility model and can efficiently capture and reclaim single Urine exfoliative cell, overall process
Damage to target cell is minimum, can be connected a series of analysis of molecules means in downstream, therefore have substantial worth and potentiality.
Described above, preferred embodiment only of the present utility model is not any to the utility model in form and substantive
On limitation, it is noted that for those skilled in the art, do not departing from the premise of the utility model method
Under, some improvement and supplement can be also made, these are improved and supplement also should be regarded as the scope of protection of the utility model.It is all ripe
Professional and technical personnel is known, in the case where not departing from spirit and scope of the present utility model, when using disclosed above
Technology contents and the equivalent variations of a little variation, modification and evolution made, be equivalent embodiment of the present utility model；Together
When, the variation, modification and evolution of all any equivalent variations made according to substantial technological of the present utility model to above-described embodiment,
In the range of still falling within the technical solution of the utility model.
Claims (10)
1. a kind of micro-fluidic chip, including sample entrance port, cell capture region and the sample exit port being sequentially connected, the cell
Capture region is provided with multiple cell sorters, and the cell sorter is made up of the columnar projections of three curved arrangements of entirety,
Gap between columnar projections be present, arc opening as liquid flow inlet, the gaps of middle column projection both sides as fluid outlet,
Two fluid outlets are symmetric.
2. micro-fluidic chip according to claim 1, it is characterised in that the cell capture region is provided with three kinds of different chis
The cell sorter of very little specification, respectively first size cell sorter, the second size cell sorter and the 3rd size cell
Sorter, the size of the first size cell sorter, the second size cell sorter and the 3rd size cell sorter according to
Secondary reduction.
3. micro-fluidic chip according to claim 2, it is characterised in that adjacent cell sorter size is different.
4. micro-fluidic chip according to claim 3, it is characterised in that the micro-fluidic chip includes multiple cell sortings
Unit, each cell sorting unit is by a first size cell sorter, a second size cell sorter and one
Individual 3rd size cell sorter composition.
5. micro-fluidic chip according to claim 4, it is characterised in that each chi in the cell sorting unit of same a line
The arrangement mode of very little cell sorter is identical.
6. micro-fluidic chip according to claim 5, it is characterised in that laterally adjacent cell sorter, back gauge are identical.
7. micro-fluidic chip according to claim 6, it is characterised in that laterally first chi of back gauge arrangement such as successively
Very little cell sorter, a second size cell sorter and a 3rd size cell sorter form first cell point
Menu member；A laterally first size cell sorter, a 3rd size cell sorter and one for back gauge arrangement such as successively
Individual second size cell sorter forms a second cell sorting unit；Multiple first cell sorting unit transverse circulation arrangements
The first cell sorting array is formed, multiple second cell sorting unit transverse circulation arrangements form the second cell sorting array, phase
Mutually a parallel first cell sorting array and second capture array form a cell sorting two level array, and cell is caught
Obtain multiple cell sorting two level array longitudinal direction equidistantly circulation arrangements being parallel to each other in region.
8. micro-fluidic chip according to claim 7, it is characterised in that in each cell sorting two level array, second
The midpoint of the liquid flow inlet of the first size cell sorter of cell sorting array and the 3rd of corresponding first cell sorting array
Size cell sorter and the second size cell sorter widthwise edge away from midpoint alignment.
9. micro-fluidic chip according to claim 8, it is characterised in that the first trip of each odd-numbered line cell sorting two level array
First cell sorter lengthwise position is alignd；The more adjacent previous odd-numbered line cell sorting of each even number line cell sorting two level array
Two level array pair-wise offset to the right.
10. according to the micro-fluidic chip described in any one of claim 2~8, it is characterised in that first size cell sorter
The width of liquid flow inlet is 60 ± 5 μm, and the width of fluid outlet is 22 ± 5 μm；The liquid flow inlet of second size cell sorter
Width be 30 ± 3 μm, the width of fluid outlet is 10 ± 3 μm；The width of the liquid flow inlet of 3rd size cell sorter is
16 ± 1 μm, the width of fluid outlet is 4 ± 1 μm.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867867A (en) * | 2017-01-24 | 2017-06-20 | 浙江大学 | A kind of urine Exfoliated tumor cells micro-fluidic chip detection technique for bladder transitional cell carcinoma |
| CN109097245A (en) * | 2018-07-25 | 2018-12-28 | 大连理工大学 | A kind of new microfluidic array arrested for cell high-efficient |
| CN110564588A (en) * | 2019-09-30 | 2019-12-13 | 深圳市儿童医院 | Structure and chip for capturing peripheral blood circulation tumor cells and using method thereof |
| CN110835596A (en) * | 2019-10-09 | 2020-02-25 | 山东大学 | Microfluidic device and method for cell sorting and detection |
| CN114085731A (en) * | 2021-10-21 | 2022-02-25 | 华南理工大学 | Cell capturing device and processing method thereof |
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2017
- 2017-01-24 CN CN201720093752.6U patent/CN206986150U/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867867A (en) * | 2017-01-24 | 2017-06-20 | 浙江大学 | A kind of urine Exfoliated tumor cells micro-fluidic chip detection technique for bladder transitional cell carcinoma |
| CN109097245A (en) * | 2018-07-25 | 2018-12-28 | 大连理工大学 | A kind of new microfluidic array arrested for cell high-efficient |
| CN110564588A (en) * | 2019-09-30 | 2019-12-13 | 深圳市儿童医院 | Structure and chip for capturing peripheral blood circulation tumor cells and using method thereof |
| CN110835596A (en) * | 2019-10-09 | 2020-02-25 | 山东大学 | Microfluidic device and method for cell sorting and detection |
| CN110835596B (en) * | 2019-10-09 | 2022-08-02 | 山东大学 | Microfluidic device and method for cell sorting and detection |
| CN114085731A (en) * | 2021-10-21 | 2022-02-25 | 华南理工大学 | Cell capturing device and processing method thereof |
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