CN109609344A - A kind of micro- magnet cell capture device and preparation method thereof - Google Patents
A kind of micro- magnet cell capture device and preparation method thereof Download PDFInfo
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- CN109609344A CN109609344A CN201910031923.6A CN201910031923A CN109609344A CN 109609344 A CN109609344 A CN 109609344A CN 201910031923 A CN201910031923 A CN 201910031923A CN 109609344 A CN109609344 A CN 109609344A
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Abstract
The invention discloses a kind of micro- magnet cell capture devices and preparation method thereof, device includes microfluidic channel layer, cell capture chip and the permanent magnet for being from top to bottom successively close to setting, the bottom setting of microfluidic channel layer is fluted, microfluidic channel is formed between groove and the upper surface of cell capture chip, feature is: cell capture chip includes substrate, the upper surface of substrate and microfluidic channel corresponding position form cell capture area, are provided with multiple equally distributed micro- magnets in cell capture area；Advantage is: the present invention can be improved cell capture rate, reduce loss cell, and can reduce cell and assemble in microfluidic channel leading portion, be conducive to the research of cell subsequent analysis.
Description
Technical field
The present invention relates to biological technology application more particularly to a kind of micro- magnet cell capture device and its production sides
Method.
Background technique
Circulating tumor cell (CTC, circulating tumor cell) is that all kinds of tumours that are present in peripheral blood are thin
The general designation of born of the same parents is discharged by tumor focus, is split away off to enter in blood from solid tumor lesion (primary tumor, transfer stove) and be followed
The rare cell of ring, quantity are the important indicators of tumor patient disease progression and transfer.The detection of circulating tumor cell helps
In the diagnosis of early stage metastatic tumour patient, the recurrence of monitoring postoperative patient tumour and transfer, the sensibility for assessing anti-tumor drug
With patient more after and selection individuation therapeutic strategy.But the quantity of circulating tumor cell is very rare in blood, it is average
106~107Just have 1 in a leucocyte, therefore, how from blood separating trap and precise Identification go out circulating tumor cell
Face huge challenge.
Currently, circulating tumor cell detection technique is mainly by immune magnetic particle capture cell (i.e. for most of sheet
Body does not have magnetic cell, is manipulated after being adsorbed onto the nanometer magnetic bead surface of functionalization), and it is glimmering in conjunction with being immunized
Light staining technique identifies tumour cell.Traditional immune magnetic particle catching method uses common glass slide as cell capture base
Bottom, uses centimetre or the permanent magnet of mm size is as flux source, by the blood for flowing through microfluidic channel with nanometer magnetic bead
In conjunction with target cell be adsorbed to slide surface.
However, there are still following defects in use for the above method: on the one hand, blood flows through microfluidic channel process
In, it is limited by downfield gradient, the capture effect of cell is not strong, may result in the loss of cell；On the other hand, effectively
Magnetic well density is low, may cause cell aggregation in microfluidic channel leading portion, and cell aggregation will affect the knot of circulating tumor cell
Structure integrality and fluorescence signal interfere cell imaging and identification.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art, the present invention provide a kind of micro- magnet cell capture device and its
Production method can be improved cell capture rate, reduce loss cell, and can reduce cell and assemble in microfluidic channel leading portion,
Conducive to cell subsequent analysis research.
The technical scheme of the invention to solve the technical problem is: a kind of micro- magnet cell capture device, including
From top to bottom successively it is close to microfluidic channel layer, cell capture chip and the permanent magnet of setting, the microfluidic channel layer
Bottom setting is fluted, forms microfluidic channel between the upper surface of the groove and the cell capture chip, described
Permanent magnet it is corresponding with the position of the microfluidic channel, the cell capture chip includes substrate, the substrate
Upper surface and microfluidic channel corresponding position form cell capture area, be provided in the cell capture area it is multiple uniformly point
Micro- magnet of cloth.
In some embodiments, multiple micro- magnets are in direction array distribution in length and breadth in the cell capture area
It is interior, micro- magnet array is formed, the spacing in micro- magnet array between adjacent micro- magnet is 100~200 μm.By
This has preferably cell capture effect.
In some embodiments, micro- magnet array by square type main part and respectively with the main body
The connection of portion both ends is constituted in the front end of square type and tail end, is offered on the microfluidic channel layer micro- described in connection
The inlet and liquid outlet of fluid channel, the front end of the front end to the distance between the caudal end of tail end
No more than the inlet to the distance between the liquid outlet.
In some embodiments, each micro- magnet is made of multiple nanometers of magnetic particles, multiple nanometers
Magnetic particle is annular in shape in cross-sectional direction aggregation, and the internal diameter of micro- magnet is 22~30 μm, the outer diameter of micro- magnet
It is 44~53 μm；Micro- magnet with a thickness of 200~350nm.The nanometer magnetic particle of such high concentration assembles shape at edge
At local maxima magnetic gradient, to improve magnetic field gradient, the capture rate of label cancer cell is improved.
In some embodiments, it is 7~15nm's that the upper surface of the cell capture chip, which is provided with a layer thickness,
SiO2Film, the SiO2Film covers micro- magnet described in whole.As a result, to protect micro- magnet structure to prevent by microfluid
The influence of blood flow in channel.
In some embodiments, the permanent magnet is made of the identical permanent magnet unit of multiple sizes, multiple described
Permanent magnet unit plate is spliced on two-dimensional surface；Upper surface and the cell capture in the permanent magnet unit
Liquid flow direction front end is provided with upper spacer between the lower surface of chip, the upper spacer is along liquid flow direction
It is gradually reduced from front to back in thickness step-like.Thereby, it is possible to reduce the magnetic field of permanent magnet front end, target cell is avoided excessively to collect
In be captured in front end, the magnetic field of uniform balance makes target cell more towards being distributed in entire capture region.
In some embodiments, the lower surface of the permanent magnet unit is provided with down along the rear end of liquid flow direction
Spacer, the lower spacer along liquid flow direction be in from front to back thickness be gradually increased it is step-like.
A kind of production method of micro- magnet cell capture device, comprising:
Numerous magnetic nano-particles are made to be formed according to the Structural assignments of setting in the cell capture area of upper surface of substrate
Multiple equally distributed micro- magnets, obtain cell capture chip；
The uniform SiO of a layer thickness is covered in the upper surface of cell capture chip2Film；
Microfluidic channel layer is prepared, its bottom is made to form groove；
Microfluidic channel layer, cell capture chip and permanent magnet are successively fitted closely, microfluidic channel layer bottom is made
Groove is corresponding with the position in cell capture area of cell capture chip upper surface, makes to form microfluidic channel between them, will
Permanent magnet is corresponding with the position of microfluidic channel, obtains micro- magnet cell capture device.
Specifically includes the following steps:
(1) ink-jet printing is used to prepare micro- magnet: by concentration for the magnetic nano-particle suspension of 8~15mg/ml with
Ink medium is uniformly mixed according to 2~5:100 volume ratio, and mixed ink is made, and preheats mixed ink, preheating temperature using printer
Degree is 30~50 DEG C, then prints mixed ink to the cell of upper surface of substrate with being evenly distributed according to the structure plan of setting
In trapping region, the heating temperature to substrate is 50~70 DEG C, and the substrate with nanometer magnetic particle is obtained after the completion of printing, will be had
The substrate of nanometer magnetic particle, which is placed on hot plate, to be evaporated, and nanometer magnetic particle is made to stay in the upper surface of substrate, and is formed uniform
Micro- magnet of distribution, obtains cell capture chip；
(2) using plasma enhances chemical vapour deposition technique, coats one layer in the upper surface of entire cell capture chip
Thickness is uniformly and with a thickness of the SiO of 7~15nm2Film；
(3) prepare PDMS microfluidic channel layer: coating photoresist on silicon wafer, and prepared using photoetching technique have into
PDMS is injected in mold, is cut into and is sized after demoulding, obtain microfluid by the mold of liquid mouth, liquid outlet and bottom groove
Channel layer；
(4) microfluidic channel layer, cell capture chip and permanent magnet are successively fitted closely, makes microfluidic channel layer bottom
Groove it is corresponding with the position in cell capture area of cell capture chip upper surface, make to form microfluidic channel between them,
Permanent magnet is corresponding with the position of microfluidic channel, micro- magnet cell capture device is obtained, is led to so that blood flows through microfluid
When road, micro- magnet is flowed through by the target cell that nanometer magnetic bead marks, and be captured under permanent magnet effect.
Ink medium is the mixture of glycerol and deionized water in the step (1), the glycerol and described is gone
Ionized water is uniformly mixed according to the mixed proportion of volume ratio 1:0.6~1:0.8.
Compared with the prior art, the advantages of the present invention are as follows:
(1) micro- magnet is set in microfluidic channel, and micro- magnet generates strong by external magnetic field (permanent magnet) after local high-intensity magnetic field
Change, thus increase the magnetic force to target cell, the process for promoting target cell to separate from blood, so as to reduce loss cell,
Circulating tumor cell capture rate is greatly improved, there are higher sensitivity and specificity；
(2) micro- magnet is set in microfluidic channel, and is combined with the improved permanent magnet of the present invention, the two composition
Cell capture device can be avoided target cell concentrations and be captured in front end, the magnetic field of uniform balance make target cell more towards
Be distributed in entire capture region, so as to reduce cell aggregation, be conducive to the subsequent analysis and research of cell, such as dyeing at
Picture；
(3) cell capture apparatus structure of the invention and manufacture craft are simple and fast, and operability and repeatability are good,
Detection application low in cost, particularly suitable for circulating tumor cell；
(4) present invention is directed to different target cells, such as the target cell of various cancers type or various cancers by stages,
Redjustment and modification are convenient for, to promote CTC clinic Transformation Application to provide a good technology platform.
Detailed description of the invention
Fig. 1 is the schematic diagram of cell capture in the present invention；
Fig. 2 is a kind of structural decomposition diagram of micro- one embodiment of magnet cell capture device of the present invention；
Fig. 3 is the process schematic representation for making micro- magnet cell capture device in the present invention based on inkjet printing；
Fig. 4 is the structural design drawing of micro- magnet array in an embodiment of the present invention；
Fig. 5 is scanning electron microscope (SEM) figure of micro- magnet array in an embodiment of the present invention；
Fig. 6 is the experimental result picture that 205 cell of COLO is captured in the embodiment of the present invention five；
Fig. 7 is the distribution results figure for capturing 205 cell of COLO in embodiment five using cell capture chip of the invention；
Fig. 8 is the distribution results figure for capturing 205 cell of COLO in embodiment five using common chip.
Wherein, microfluidic channel layer 1, cell capture chip 2, permanent magnet 3, microfluidic channel 4, substrate 5, cell capture area
6, micro- magnet 7, micro- magnet array 8, main part 9, front end 10, tail end 11, inlet 12, liquid outlet 13, nanometer magnetic particle
14, SiO2Film 15, permanent magnet unit 16, upper spacer 17, distance piece 18, nozzle 19, ink droplet 20, lower spacer 21.
Specific embodiment
A kind of micro- magnet cell capture device of the present invention and preparation method thereof is made further specifically below in conjunction with attached drawing
It is bright but not as a limitation of the invention.
Embodiment one
As shown, a kind of micro- magnet cell capture device of the invention, including being from top to bottom successively close to the micro- of setting
The bottom setting of fluid channel layer 1, cell capture chip 2 and permanent magnet 3, microfluidic channel layer 1 is fluted, and groove is caught with cell
It is opposite with the position of microfluidic channel 4 to obtain formation microfluidic channel 4, the setting position of permanent magnet 3 between the upper surface of chip 2
It answers, cell capture chip 2 includes substrate 5, and 5 upper surface of substrate and 4 corresponding position of microfluidic channel form cell capture area 6, carefully
Born of the same parents' trapping region 6 is preferably hexagon, is provided with multiple equally distributed micro- magnets 7 in cell capture area 6.
In the present embodiment, microfluidic channel 4 is a flat cavity, and the height of cavity is 500 μm, and thus, it is possible to increase
The contact area of sample and magnet, and reduce the distance between microfluidic channel and magnet.
In the present embodiment, multiple micro- magnets 7 form micro- magnet battle array in cell capture area 6 in direction array distribution in length and breadth
Column 8, the spacing in micro- magnet array 8 between adjacent micro- magnet 7 are 100~200 μm, in the present embodiment between adjacent micro- magnet 7
Spacing d be 150 μm, in other embodiments, the spacing between adjacent micro- magnet 7 can be 100 μm, 130 μm, 170 μm, 200 μ
M etc..
The front end in square type that micro- magnet array 8 is connect by the main part 9 in square type and respectively with the both ends of main part 9
Portion 10 and tail end 11 are constituted, and the inlet 12 and liquid outlet 13 of connection microfluidic channel 4 are offered on microfluidic channel layer 1,
The front end of front end 10 to the distance between the caudal end of tail end 11 no more than inlet 12 between liquid outlet 13 away from
From, and the foremost position of micro- magnet array 8 is without departing from 12 position of inlet, last end position without departing from 13 position of liquid outlet,
I.e. 12 channel of inlet that does not cover microfluidic channel 4 of front end 10, tail end 11 do not cover 13 channel of liquid outlet.
Each micro- magnet 7 is made of multiple nanometers of magnetic particles 14, and multiple nanometers of magnetic particles 14 are in cross-sectional direction aggregation
Ring-type, the internal diameter of micro- magnet 7 are 22~30 μm, and the outer diameter of micro- magnet 7 is 44~53 μm, micro- magnet 7 on substrate 5 with a thickness of
200~350nm.As shown in figure 5, numerous nanometers of magnetic particles 14 form the micro- magnet 7 of multiple cyclic structures on substrate 5, absolutely mostly
Several nanometers of magnetic particles 14 are gathered in the edge of ring, and a small amount of nanometer magnetic particle 14 is dispersed in the centre of ring.Due to being steamed in manufacturing process
The Thermodynamic effect in hair stage, the ink evaporation rate in edge are higher than center, outside convection current are produced, by nano particle
It is brought in edge, magnetic nanoparticle is caused to form cyclic structure.The nanometer magnetic particle of such high concentration assembles shape at edge
At local maxima magnetic gradient, to improve magnetic field gradient, the capture rate of label cancer cell is improved.Make in the present embodiment
Printed patterns are imaged with light field microscope, and are measured using ImageJ, the internal diameter of micro- 7 cyclic structure of magnet is obtained
Between 22~30 μm, thus the outer diameter of micro- magnet 7 has preferably effect between 44~53 μm, and integral array is arranged
Relatively rationally.The thickness of micro- magnet 7 is about 300nm in the present embodiment, and in other embodiments, the thickness of micro- magnet 7 can be
Thus 200nm, 250nm, 350nm etc. have preferably capture effect.
The upper surface of cell capture chip 2 is provided with the uniform SiO of a layer thickness2Film 15, SiO in the present embodiment2Film 15 is thick
Spending is 10nm, in other embodiments, SiO215 thickness of film can be 7nm, 9nm, 11nm, 13nm, 15nm etc., SiO2Film 15 covers
Firmly whole micro- magnets 7.As a result, to protect micro- magnet structure to prevent the influence by blood flow in microfluidic channel.
Embodiment two
As shown, a kind of micro- magnet cell capture device, remaining structure are the same as example 1, difference exists
In: in the present embodiment, permanent magnet 3 is made of the identical permanent magnet unit 16 of multiple geomeries, and multiple permanent magnet units 16 exist
Plate is spliced on two-dimensional surface.In the present embodiment, be made of 36 permanent magnet units 16 one 6 × 6 gusts of permanent magnet 3
Column, wherein permanent magnet unit 16 selects model NB001-N48, the cube magnet block that the side length of Plano, TX are 1/8 inch.
Important component of the permanent magnet as immunomagnetic beads method capture cell, compared to traditional monolith magnet structure, the present invention
Magnet structure magnetic field alternant polarization, be shown as on microchannel dispersion potential well, make Distribution of Magnetic Field more evenly.
In the present embodiment, flowed between the upper surface of permanent magnet unit 16 and the lower surface of cell capture chip 2 along liquid
Direction front end is provided with spacer 17, and upper spacer 17 is in the step that thickness is gradually reduced along liquid flow direction from front to back
Shape.Upper spacer 17 is arranged between 3 leading portion of permanent magnet and microfluidic channel 4 as a result, can reduce the magnetic of permanent magnet front end
, avoid target cell concentrations from being captured in front end, the magnetic field of uniform balance catches target cell entirely more towards being distributed to
It obtains in region.
In the present embodiment, by the identical distance piece 18 of multiple geomeries, closely arrangement is constituted upper spacer 17.This implementation
In example, distance piece 18 selects the flat thin magnet (thickness 1mm, AFG-13346 flat thin magnet, ProMAG) of weakly magnetization, through measuring: by being spaced
Magnetic field caused by piece, they may diminish to ignore, and upper spacer is only covered on the front end of permanent magnet, and thus microfluid is logical
The high-intensity magnetic field of road back segment it is prevented that CTC potential loss.In other embodiments, distance piece 18 can select surface to have gold
As long as belonging to the tablet of coating or the object that can play similar interval magnetic fields of other structures shape.In the present embodiment,
The thickness ratio of partition 18 and permanent magnet unit 2 is 1:3.175, and thus Distribution of Magnetic Field is more reasonable, has preferably structure and effect,
Cell can be made to be preferably minimized in the enrichment of permanent magnet leading portion, in other embodiments, the thickness ratio of distance piece and magnet block can be with
For 1:3~1:4.
In the present embodiment, the rear end of liquid flow direction is provided with lower spacer in the lower surface of permanent magnet unit 16
21, lower spacer 21 is in that thickness is gradually increased step-like along liquid flow direction from front to back, and lower spacer 21 is equally by more
The close arrangement of a identical distance piece 18 of geomery is constituted.It is consequently for polishing permanent magnet whole height, makes all permanent magnetism
The cuboid of body unit and spacer overall structure rule, thus optimize 3 overall structure of permanent magnet more, manufacture craft letter
It is single.
Compared to traditional monolith magnet structure, the magnetic field of permanent magnet alternant polarization of this structure, in microfluidic channel
It is shown as the potential well of dispersion, makes Distribution of Magnetic Field more evenly, reduces loss cell and aggregation；In addition, spacer is arranged in permanent magnet
Between leading portion and microfluidic channel, the magnetic field of permanent magnet front end can be reduced, avoid target cell concentrations captured in front end
Arrive, and the high-intensity magnetic field of channel rear end can then prevent the potential loss of cell, the magnetic field of uniform balance make target cell more towards point
Cloth is further reduced cell aggregation into posterior segment and entire capture region, is conducive to subsequent identification and analysis.
Embodiment three
A kind of production method of micro- magnet cell capture device of the present invention, comprising:
By numerous magnetic nano-particles according to the Structural assignments of setting on substrate 5 in the cell capture area 6 on surface, and shape
At multiple equally distributed micro- magnets 7, cell capture chip 2 is obtained；
The uniform SiO of a layer thickness is covered in the upper surface of cell capture chip 22Film 15 makes micro- magnet 7 not by microfluid
The influence that liquid flows in channel 4；
Microfluidic channel layer 1 is prepared, its bottom is made to form groove；
Microfluidic channel layer 1, cell capture chip 2 and permanent magnet 3 are successively fitted closely, 1 bottom of microfluidic channel layer is made
The groove in portion is corresponding with the position in cell capture area 6 of 2 upper surface of cell capture chip, keeps formation microfluid between them logical
Road 4, permanent magnet 3 is corresponding with the position of microfluidic channel 4, obtain micro- magnet cell capture device.
Example IV
A kind of production method of micro- magnet cell capture device of the present invention, specifically includes the following steps:
(1) ink-jet printing is used to prepare micro- magnet: by concentration for the magnetic nano-particle suspension (partial size of 10mg/ml
It is uniformly mixed with ink medium according to 3:100 volume ratio for 100nm or so), mixed ink is made, use the pre- hot mixing of printer
Ink, preheating temperature are 30~50 DEG C, then print mixed ink to substrate with being evenly distributed according to the structure plan of setting
In the cell capture area of upper surface, the heating temperature to substrate is 50~70 DEG C, is obtained after the completion of printing with nanometer magnetic particle
Substrate, the substrate with nanometer magnetic particle is placed on hot plate and is evaporated, nanometer magnetic particle is made to stay in the upper table of substrate
Face, and equally distributed micro- magnet is formed, obtain cell capture chip；
(2) using plasma enhances chemical vapour deposition technique, coats one layer in the upper surface of entire cell capture chip
Thickness is uniformly and with a thickness of the SiO of 10nm2Film；
(3) prepare PDMS microfluidic channel layer: coating photoresist on silicon wafer, and prepared using photoetching technique have into
PDMS is injected in mold, is cut into and is sized after demoulding, obtain microfluid by the mold of liquid mouth, liquid outlet and bottom groove
Channel layer；
(4) microfluidic channel layer, cell capture chip and permanent magnet are successively fitted closely, makes microfluidic channel layer bottom
Groove it is corresponding with the position in cell capture area of cell capture chip upper surface, make to form microfluidic channel between them,
Permanent magnet is corresponding with the position of microfluidic channel, micro- magnet cell capture device is obtained, is led to so that blood flows through microfluid
When road, micro- magnet is flowed through by the target cell that nanometer magnetic bead marks, and be captured under permanent magnet effect.
In the present embodiment, ink medium is the mixture of glycerol and deionized water in step (1), and glycerol and deionized water are pressed
It is uniformly mixed according to the mixed proportion of volume ratio 1:0.75, there is preferably dielectric viscosity as a result, printing effect is best, at other
Glycerol and deionized water can be uniformly mixed according to mixed proportions such as volume ratio 1:0.6,1:0.7,1:0.8 in embodiment, when sweet
When oil concentration is higher, the viscosity of medium becomes larger.In the present embodiment, the concentration of magnetic nano-particle suspension uses 10mg/ml,
Concentration can be 8mg/ml, 12mg/ml, 15mg/ml etc. in other embodiments.In the present embodiment, in entire cell capture chip
Upper surface coat a layer thickness uniformly and with a thickness of the SiO of 10nm2Film, in other embodiments thickness can for 7nm,
12nm, 15nm etc..
In the present embodiment, micro- magnet is made using inkjet printing, inkjet printing technology is that contactless, the layer-by-layer nothing of one kind is covered
The method of mould can deposit specified magnetic pattern in any substrate such as sheet glass, silicon wafer, form Micro Magnet.Miniature magnetic
Body is magnetized after generating local high-intensity magnetic field by external magnetic field, promotes cell separation process to the magnetic force that target cell applies to increase.This
Outside, inkjet printing technology is a kind of method of rapid shaping, easily can be integrated in one with substrate as a kind of mold making technology
It rises, has many advantages, such as that at low cost, precision is high, flexibility and high reproducibility.It is printed in the present embodiment using DMP-2800 piezoelectric type
Machine, if Fig. 3 schematically shows ink jet printing process, 19 be nozzle in figure, and 20 be the mixed ink containing nanometer magnetic particle 14
Ink droplet 20.The suitable preheating temperature of mixed ink is 40 DEG C, in other embodiments, and heating temperature is different with the viscosity of mixed ink
It is adaptively adjusted, substrate is preheating to 60 DEG C according to setting pattern printing.
Embodiment five
By taking 205 analog sample of tumour cell COLO as an example, cell capture chip and common chip of the invention is respectively adopted
The experiment of 205 cell capture of COLO is carried out, but not as limitation of the present invention, the specific method is as follows:
(1) cell culture:
205 cell suspension of COLO and trypsase (0.05%Trypsin-EDTA (1X), phenol red, Life
Technology) mixing is incubated for 5 minutes, breaks cell cluster, guarantees cell separately through microchannel.Observation is thin under the microscope
Otherwise born of the same parents increase brooding time, and inspection in every 30 seconds separates situation until 90% or more cell individually disperses.Then it will wait
The cell culture medium (RPMI 1640,5% cow's serum) of amount is added in suspension, to neutralize trypsase.
(2) sample process:
Cell suspending liquid comprising about 150 cells is added in 2.5ml healthy human peripheral blood sample, density level bands is passed through
Spend centrifugal process centrifugation removal blood plasma and red blood cell (800g, 10min), and with dilution buffer (Veridex, Janssen
Diagnostic LLC, NJ) supernatant containing blood plasma is replaced, make total volume 3.5mL.Then being added has epithelial cell function
The Fe of energy adhesion molecule (anti-EpCAM)3O4Magnetic nano-particle suspension (partial size average diameter is 100nm or so), is incubated for
10min。
(3) cell separates:
Microfluidic channel is filled with PBS buffer solution, the gas in passing away, flow velocity 5ml/h；It will be through with syringe pump
It crosses above-mentioned pretreated blood sample and microfluidic channel is passed through with the flow velocity of 2.5ml/h；With PBS buffer solution irrigation channel, flow velocity
For 5ml/h；After flushing, with the flow velocity of 2.5ml/h, 1ml acetone is introduced in microfluidic channel, tumour cell is fixed on chip
On；Then chip is taken out, 37 DEG C of dryings are dried, and are deposited in 4 DEG C of refrigerators.
(4) cell dyeing:
After screening, the chip of taking-up is dyed with three kinds of fluorescent dyes (anti-CK/CD45/DAPI), is used for cell
Identification and counting.Wherein, specific immunofluorescence dyeing process are as follows: the chip after drying is immersed in PBST (0.1%tween20
PBS) in cleaning solution, stand 5min；After taking out chip drying, pre-configured anti-CK (mouse anti-cell angle egg is added dropwise
It is white, pan-FITC, Sigma-Aldrich) and anti-CD45 (AlexaFluor 594, Invitrogen) dyeing liquor (buffer
Fluorescent marker is carried out for 1XPBS, 0.1%tween20,1%BSA) to be floated after being incubated for 45mim under the conditions of 37 DEG C with PBST buffer
It washes；Then it is dyed, and is stored in 4 DEG C of refrigerators with DAPI (dye nucleus, Vector Laboratories).
(5) cellular identification:
Chip after dyeing is placed under Olympus inverted fluorescence microscope and is observed, and is carried out carefully according to coloration result
Born of the same parents' identification and counting.As a result as shown in fig. 6, the immunofluorescence chromogenic reaction of cell meet CK be positive, CD45 be it is negative and
DAPI it is positive be judged as tumour cell, and CK be negative, CD45 be positive and DAPI it is positive be judged as leucocyte.
Interpretation of result: under identical external magnetic field and experiment condition, being computed, and uses traditional common chip, tumour
The average capture efficiency of cell is 69.1 ± 12.6% (N=4)；Use the micro- magnet array cell capture chip of the present invention, tumour
The average capture efficiency of cell is 95.6 ± 6% (N=4), and capture rate improves 26%, wherein the calculating side of capture rate
Method is to send out the cancer cell number captured from addition sample divided by control slide (without the process of cell capture) is middle
Existing cancer cell number.As shown in fig. 7, wherein dark point indicates that the cell for being connected to micro- magnet, light color point indicate to be not attached to
The cell of micro- magnet.The result shows that in the tumour cell being captured to using the device of the invention and method, 78.5% be by
Micro- magnet array capture, only 21.5% cell is not attached to any micro- magnet array.Therefore, cell of the invention is caught
Obtaining device and method can be improved the capture rate of tumour cell.
On the other hand, the improved magnet structure of the present invention combines the capture of composition with micro- magnet cell capture chip
Device, in microfluidic channel leading portion, the accumulation rate of tumour cell is reduced to 61%, and in traditional structure 95% or more tumour
Cell can be captured in the leading portion of permanent magnet, and therefore, cell capture device and method of the invention can make target cell more become
To being distributed in entire capture region, to reduce cell aggregation, it is conducive to the subsequent analysis and research of cell.
Embodiment six
By taking the peripheral blood sample of 13 tumor patients as an example, CTC is carried out using micro- magnet cell capture chip of the invention
Cell capture, to verify the feasibility and validity of the method for the present invention, but not as limitation of the present invention, specific method is such as
Under:
(1) sample collection: 13 different blood of cancer patients 5-10ml are extracted, CellSave is placed inTMIn anticoagulant tube,
Immediately every sample is examined；
(2) sample process: being centrifuged removal blood plasma and red blood cell (800g, 10min) by density-gradient centrifugation method, and with dilute
It releases buffer (Veridex, Janssen Diagnostic LLC, NJ) and replaces supernatant containing blood plasma, make total volume
Then the Fe with epithelial cell adhesion molecule (anti-EpCAM) is added in 3.5mL3O4Magnetic nano-particle suspension (grain
Diameter average diameter is 100nm or so), it is incubated for 10min；
(3) cell separates: microfluidic channel is filled with PBS buffer solution, the gas in passing away, and flow velocity 5ml/h；With
Syringe pump will pass through microfluidic channel by above-mentioned pretreated blood sample with the flow velocity of 2.5ml/h；It is rushed with PBS buffer solution
Wash channel, flow velocity 5ml/h；After flushing, with the flow velocity of 2.5ml/h, 1ml acetone is introduced in microfluidic channel, tumour is thin
Born of the same parents are fixed on chip；Then chip is taken out, 37 DEG C of dryings are dried, and are deposited in 4 DEG C of refrigerators；
(4) cell dyeing: after screening, the chip of taking-up is contaminated with three kinds of fluorescent dye anti-CK/CD45/DAPI
Color for cellular identification and counts；
(5) cellular identification: the chip after dyeing is placed under Olympus inverted fluorescence microscope and observes, and according to dye
Color result carries out cellular identification and counts.
(6) result counts: 13 tumor patients have capture to obtain CTC, as shown in table 1:
The CTC quantity statistics table captured in 1:13 tumor patient blood of table
A kind of micro- magnet cell capture device of the invention and preparation method thereof, is arranged micro- magnet in microfluidic channel,
Micro- magnet is strengthened after generating local high-intensity magnetic field by external magnetic field (permanent magnet), to increase the magnetic force to target cell, promotes target cell
The process separated from blood greatly improves circulating tumor cell capture rate so as to reduce loss cell, has higher
Sensitivity and specificity；Micro- magnet is set in microfluidic channel, and is combined with the improved permanent magnet of the present invention, the two
The cell capture device of composition can be avoided target cell concentrations and be captured in front end, and the magnetic field of uniform balance makes target cell
More towards being distributed in entire capture region, so as to reduce cell aggregation, it is conducive to the subsequent analysis and research of cell, such as contaminate
Color and imaging；Cell capture apparatus structure of the invention and manufacture craft are simple and fast, and operability and repeatability are good, at
This cheap, detection application particularly suitable for circulating tumor cell；The present invention is directed to different target cells, such as various cancers
The target cell of type or various cancers by stages, is convenient for redjustment and modification, to promote CTC clinic Transformation Application to provide one
Good technology platform.
It is worth noting that, the foregoing is merely presently preferred embodiments of the present invention, patent of the invention is not thereby limited
Protection scope, the present invention can also carry out the improvement of material and structure to the construction of above-mentioned various components, or use skill
Art equivalent is replaced.Therefore it is all with equivalent structure made by specification and diagramatic content of the invention change, or directly or
Apply to other correlative technology fields indirectly to be similarly all contained in the range of of the invention cover.
Claims (10)
1. a kind of micro- magnet cell capture device, microfluidic channel layer, cell capture including being from top to bottom successively close to setting
The bottom setting of chip and permanent magnet, the microfluidic channel layer is fluted, the groove and the cell capture core
Microfluidic channel is formed between the upper surface of piece, the permanent magnet is corresponding with the position of the microfluidic channel, special
Sign is that the cell capture chip includes substrate, and upper surface and the microfluidic channel corresponding position of the substrate are formed
Cell capture area is provided with multiple equally distributed micro- magnets in the cell capture area.
2. a kind of micro- magnet cell capture device according to claim 1, which is characterized in that multiple micro- magnets are in
Direction array distribution forms micro- magnet array, adjacent institute in micro- magnet array in the cell capture area in length and breadth
The spacing between micro- magnet stated is 100~200 μm.
3. a kind of micro- magnet cell capture device according to claim 2, which is characterized in that micro- magnet array by
In square type main part and respectively connect with the main part both ends constituted in the front end of square type and tail end, it is described
Microfluidic channel layer on offer the inlet and liquid outlet of the connection microfluidic channel, the front end most before
End is to the distance between the caudal end of tail end no more than the inlet to the distance between the liquid outlet.
4. a kind of micro- magnet cell capture device according to claim 1-3, which is characterized in that described in each
Micro- magnet is made of multiple nanometers of magnetic particles, and multiple nanometer magnetic particles are annular in shape in cross-sectional direction aggregation, described
The internal diameter of micro- magnet is 22~30 μm, and the outer diameter of micro- magnet is 44~53 μm；Micro- magnet with a thickness of 200~
350nm。
5. a kind of micro- magnet cell capture device according to claim 1, which is characterized in that the cell capture chip
Upper surface be provided with a layer thickness be 7~15nm SiO2Film, the SiO2Film covers micro- magnet described in whole.
6. a kind of micro- magnet cell capture device according to claim 1, which is characterized in that the permanent magnet is by multiple
The identical permanent magnet unit of size is constituted, and multiple permanent magnet units are spliced into plate on two-dimensional surface；Described
It is provided between the upper surface of permanent magnet unit and the lower surface of the cell capture chip along liquid flow direction front end
Spacer, the upper spacer along liquid flow direction be in from front to back thickness be gradually reduced it is step-like.
7. a kind of micro- magnet cell capture device according to claim 6, which is characterized in that the permanent magnet unit
Lower surface is provided with lower spacer along the rear end of liquid flow direction, the lower spacer along liquid flow direction from front to back
It is gradually increased in thickness step-like.
8. a kind of production method of micro- magnet cell capture device characterized by comprising
Numerous magnetic nano-particles are made to be formed multiple according to the Structural assignments of setting in the cell capture area of upper surface of substrate
Equally distributed micro- magnet, obtains cell capture chip；
The uniform SiO of a layer thickness is covered in the upper surface of cell capture chip2Film；
Microfluidic channel layer is prepared, its bottom is made to form groove；
Microfluidic channel layer, cell capture chip and permanent magnet are successively fitted closely, the groove of microfluidic channel layer bottom is made
It is corresponding with the position in cell capture area of cell capture chip upper surface, make to form microfluidic channel between them, by permanent magnetism
Body is corresponding with the position of microfluidic channel, obtains micro- magnet cell capture device.
9. a kind of production method of micro- magnet cell capture device according to claim 8, which is characterized in that specifically include
Following steps:
(1) ink-jet printing is used to prepare micro- magnet: by concentration for the magnetic nano-particle suspension and ink of 8~15mg/ml
Medium is uniformly mixed according to 2~5:100 volume ratio, and mixed ink is made, and preheats mixed ink using printer, preheating temperature is
30~50 DEG C, then mixed ink is printed to the cell capture of upper surface of substrate with being evenly distributed according to the structure plan of setting
In area, the heating temperature to substrate is 50~70 DEG C, and the substrate with nanometer magnetic particle is obtained after the completion of printing, will have nanometer
The substrate of magnetic particle is placed on hot plate and is evaporated, and so that nanometer magnetic particle is stayed in the upper surface of substrate, and formed and be uniformly distributed
Micro- magnet, obtain cell capture chip；
(2) using plasma enhances chemical vapour deposition technique, coats a layer thickness in the upper surface of entire cell capture chip
Uniformly and with a thickness of the SiO of 7~15nm2Film；
(3) it prepares PDMS microfluidic channel layer: coating photoresist on silicon wafer, and prepared using photoetching technique with feed liquor
PDMS is injected in mold, is cut into and is sized after demoulding by the mold of mouth, liquid outlet and bottom groove, and it is logical to obtain microfluid
Channel layer；
(4) microfluidic channel layer, cell capture chip and permanent magnet are successively fitted closely, makes the recessed of microfluidic channel layer bottom
Slot is corresponding with the position in cell capture area of cell capture chip upper surface, makes to form microfluidic channel between them, will forever
Magnet is corresponding with the position of microfluidic channel, obtains micro- magnet cell capture device, thus when blood flows through microfluidic channel,
Micro- magnet is flowed through by the target cell that nanometer magnetic bead marks, and is captured under permanent magnet effect.
10. a kind of production method of micro- magnet cell capture device according to claim 9, which is characterized in that described
Ink medium is the mixture of glycerol and deionized water in step (1), and the glycerol and the deionized water are according to volume
Mixed proportion than 1:0.6~1:0.8 is uniformly mixed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI796893B (en) * | 2021-12-23 | 2023-03-21 | 財團法人工業技術研究院 | Cell and tissue sheet forming package and cell injection equipment |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005233931A (en) * | 2004-01-19 | 2005-09-02 | Hitachi Metals Ltd | Magnetic separator |
| CN101376121A (en) * | 2007-08-31 | 2009-03-04 | 泰肯贸易股份公司 | Microplate support with magnets |
| CN101458251A (en) * | 2009-01-04 | 2009-06-17 | 东南大学 | Magnetic particle micro array apparatus for high throughput detecting biomacromolecule and its use method |
-
2019
- 2019-01-14 CN CN201910031923.6A patent/CN109609344A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005233931A (en) * | 2004-01-19 | 2005-09-02 | Hitachi Metals Ltd | Magnetic separator |
| CN101376121A (en) * | 2007-08-31 | 2009-03-04 | 泰肯贸易股份公司 | Microplate support with magnets |
| US20090064800A1 (en) * | 2007-08-31 | 2009-03-12 | Gerhard Fasching | Microplate carrier having magnets |
| CN101458251A (en) * | 2009-01-04 | 2009-06-17 | 东南大学 | Magnetic particle micro array apparatus for high throughput detecting biomacromolecule and its use method |
Non-Patent Citations (1)
| Title |
|---|
| PENG CHEN等: "Inkjet-Print Micromagnet Array on Glass Slides for Immunomagnetic Enrichment of Circulating Tumor Cells", 《ANN BIOMED ENG》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI796893B (en) * | 2021-12-23 | 2023-03-21 | 財團法人工業技術研究院 | Cell and tissue sheet forming package and cell injection equipment |
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