CN103266050B - For micro-fluidic chip of sorting and application thereof - Google Patents
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Abstract
The present invention relates to micro-fluidic chip for sorting and application thereof, specifically, disclose a kind of micro-fluidic chip for sorting, this chip includes: for flowing into the chip entrance treating sorting liquid；For for the main channel treating sorting liquid flowing；And for discharging the chip outlet of sorted rear liquid；Wherein, main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between access road and exit passageway, and trapping region passage be provided with multiple downward opening, collect the collection cell of target substance to be sorted.It addition, the method that present invention also offers the micro flow control chip device for sorting, micro-fluidic chip test kit and sorting target cell or other materials.The micro-fluidic chip of the present invention not only makes simply, operability and favorable repeatability, and greatly improves the area in cell sorting district, supports the cell sorting of high flow capacity.
Description
Technical field
The present invention relates to micro fluidic chip technical field, be specifically related to the method for separating of a kind of micro-fluidic chip for sorting, micro flow control chip device, micro-fluidic chip test kit and sorting object.
Background technology
Microfluidic chip technology is also known as chip lab (lab-on-a-chip), it is that the one proposed by Switzerland's research worker the nineties in 20th century utilizes micromachining technology, is integrated into basic steps such as separation, sampling, reaction and detections involved in biochemical reaction on the area about chip of several square centimeters and realizes omnidistance accurately to control.The microchannel of chip internal forms controlled microfluidic networks, it is possible to for replacing the biochemistry detection of routine, and microminiaturized with it, integrated, high flux and high-precision feature demonstrate huge development potentiality and using value in medical science and life science.
Be broadly divided into according to principle for CTCs (circulating tumor cell) micro-fluidic chip sorted: (1) based on the micro-fluidic chip (2) of cell biological physical features based on the micro-fluidic chip (3) of cellular affinity-microfluidic chromatography technology based on the micro-fluidic chip of immune magnetic sorting technology.
CTCs in conjunction with immunomagnetic isolation technology sorts micro-fluidic chip, it is utilize the immunomagnetic beads of surface coupling specificities Anti-tumor labelling molecular antibody to catch the cell expressing corresponding antigens, then adding outside is trapped in the magnetic field in micro-fluidic chip passage by magnetic bead-cell conjugate under the effect of electric magnet or permanent magnet, reaches the purpose of target cell and the sorting of magnetic bead unlabeled cells.Compared with traditional enrichment and detection method, such micro-fluidic chip can realize the single stage of CTCs in a small amount of blood (1 milliliter) and be continuously separated, having high sensitivity, high specificity and high flux feature, the CTCs caught can keep complete morphological feature and higher cell viability.
Saliba in 2010 etc. devise a kind of immunomagnetic beads micro-fluidic chip, this chip utilizes the method for the ink marking to inoculate the iron oxide magnetic substance spots of distribution in equilateral hexagon in the chip of polydimethylsiloxane material, when externally-applied magnetic field, when the suspension containing magnetic beads of binding antibody first passes through chip microchannel, the magnetic field on additional vertical chip surface can make magnetic bead spontaneously start to pile up and formed the magnetic microtrabeculae of the perpendicular shape of paramagnetic field direction at speckle locating place, again when blood sample flows through microchannel, it is possible to achieve CTCs catches.But such chip fabrication technique relative complex, experiment condition controls to require height, and the repeatability therefore tested need checking.Kang in 2012 etc. report a kind of magnetic separation micro-fluidic chip based on dual channel mode, same sidewall at two parallel microchannels is provided with for the CTCs cell collected, the outside permanent magnet of placing of cell end for producing sufficiently strong magnetic field in microchannel, when the tumor cell of marked by magnetic bead flows through microchannel, the deflection in flow velocity direction will be perpendicular under the action of a magnetic force, enter into the collection cell of sidewall, thus realizing the sorting of CTCs, but, the main channel limited width of this chip, thus chip flow and process sample ability are on the low side.Hoshino in 2011 etc. devise the another kind of CTCs based on immune magnetic sorting technology and sort micro-fluidic chip, this chip only includes the main channel that height is 500 microns, the length and width in cell sorting district is 20 millimeters, the bottom of main channel uses the coverslip of 150 micron thickness to close, three blocks of neodymium iron boron N42 Magnet are placed as externally-applied magnetic field in the lower section of slide, for catching of CTCs cell.But major downside is that of this chip, magnetic field force induced and adhere to immunomagnetic beads marked tumor cell on the cover slip and be still limited by stronger flow disturbance, be exposed under fluid shear stress, cause coming off and losing of part attached cell.
Therefore exploitation one makes simple, operability and favorable repeatability, and support high flow capacity cell sorting, the micro-fluidic chip that attached cell can be prevented effectively from come off and lose be current problem demanding prompt solution.
Summary of the invention
It is an object of the invention to provide a kind of micro-fluidic chip for sorting, micro-fluidic chip test kit, micro flow control chip device and application thereof.
First aspect present invention provides a kind of micro-fluidic chip for sorting, and described chip includes:
For flowing into the chip entrance treating sorting liquid；
For for the main channel treating sorting liquid flowing；And
Chip for discharging sorted rear liquid exports；
Wherein, described main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between described access road and exit passageway,
And described trapping region passage be provided with multiple downward opening, collect the collection cell of target substance to be sorted.
In another preference, it is circle, subcircular, ellipse or just polygon pit that described collection cell includes cross section.
In another preference, from chip entrance to trapping region channel direction, the width of described access road becomes larger.
In another preference, from trapping region passage to chip Way out, the width of described exit passageway tapers into.
In another preference, in described trapping region passage, the distance between opening and the lower surface of trapping region passage of described collection cell is 100-500 μm, it is preferred that be 300 μm.
In another preference, the material of described micro-fluidic chip includes polymer, glass.
In another preference, the material of described micro-fluidic chip is selected from lower group: polydimethylsiloxane, polystyrene, Polyethylene Glycol or its combination, it is preferred to polydimethylsiloxane.
In another preference, described collection cell is array distribution.
In another preference, it is the equilateral triangle of 50-200 μm that three of arbitrary neighborhood collect the kernel of section composition length of side of cell, it is preferred that the length of side is the equilateral triangle of 60-150 μm.
In another preference, the sectional area of described each collection cell is 1000-5000 square micron, it is preferred that 1500-2500 square micron.
In another preference, the cross section of described collection cell is radius is the circle of 15-50 μm.
In another preference, described collection cell height is 50-800 μm, it is preferred that for 75-500 μm.
In another preference, adjacent two collection cell distances are 25-100 μm, it is preferred that be 50 ± 10 μm.
In another preference, described trapping region passage is the rectangle of long 15～30mm, wide 15～30mm.
In another preference, described main channel is made up of the exit passageway of the access road of isosceles right triangle, square trapping region passage and isosceles right triangle, and the hypotenuse of two isosceles right triangles overlaps with foursquare the right and left respectively.
In another preference, described collection cell is positioned at the top of trapping region passage.
In another preference, the height of described main channel is 100-1000 μm, it is preferred that for 200-800 μm.
In another preference, the outlet of described chip and chip entrance are positioned at the bottom surface of described chip and lay respectively at the access road of isosceles right triangle and the right angle of exit passageway.
In another preference, the outlet of described chip and chip entrance are rectangle or circle.
In another preference, the cross section of the outlet of described chip and chip entrance is diameter is the circle of 200-2000 μm.
Second aspect present invention provides the another kind of micro-fluidic chip for sorting, described chip includes: the first substrate that can be mutually closed and the second substrate, and described first substrate closes with described second substrate, is formed for for the main channel treating that sorting liquid flows, wherein
Described first substrate is provided with the collection cell district for collecting target substance to be sorted, and described collection cell district is provided with multiple downward opening collection cell；With
Described second substrate is provided with the lower surface of chip entrance, chip outlet and main channel；
In another preference, described first substrate and the second substrate are one-body molded or split-type structural.
In another preference, described main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between described access road and exit passageway, and described collection cell district is positioned at trapping region channel upper so that described trapping region passage have multiple downward opening, collect the collection cell of target substance to be sorted.
In another preference, described micro-fluidic chip is for sorting cells (such as circulating tumor cell) or other materials from fluid.
In another preference, other materials described include DNA, mRNA, microRNA or protein.
In another preference, described cell includes in circulating tumor cell, colon cancer CD133 tumor stem cell, bone marrow tumor cell in tumor cell, ascites.
In another preference, the material of described first substrate and/or the second substrate includes polymer, glass.
In another preference, the material of described first substrate and/or the second substrate is selected from lower group: polydimethylsiloxane, polystyrene, Polyethylene Glycol or its combination, it is preferred to polydimethylsiloxane.
A third aspect of the present invention provides a kind of micro flow control chip device for sorting, and described chip apparatus includes:
The micro-fluidic chip for sorting described in first aspect present invention or second aspect；With
Being located at the Magnet being positioned at above described chip, described Magnet is used for producing magnetic field, so that target substance to be sorted is collected in the collection cell of described chip.
In another preference, described Magnet is permanent magnet or electric magnet.
In another preference, described chip apparatus is used for sorting in colon cancer CD133 tumor stem cell, bone marrow tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites.
A fourth aspect of the present invention provides a kind of micro-fluidic chip test kit (kit) for sorting, and described chip agent box includes:
The micro-fluidic chip for sorting described in first aspect present invention or second aspect；And operation instruction.
In another preference, described test kit also includes one or more assemblies being selected from lower group:
The immunomagnetic beads of (i) coupled antibody；
(ii) magnetic microsphere of non-coupled antibody；
(iii) Magnet.
In another preference, described immunomagnetic beads includes: the immunomagnetic beads of coupling Anti-Human's epithelial cell adhesion molecule antibody, Anti-Human's CD133 immunomagnetic beads.
In another preference, described chip agent box is used for sorting in colon cancer CD133 tumor stem cell, bone marrow tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites.
A fifth aspect of the present invention provides a kind of method for separating sorting target substance from fluid, and wherein said target substance includes cell or other materials, comprises the following steps:
(1) fluid is provided, in described fluid containing need sorting cell or other materials, cell wherein said to be sorted or other materials combine with magnetic microsphere, thus forming " cell-magnetic microsphere " or " other material-magnetic microspheres " complex；
(2) described fluid is flowed into described micro-fluidic chip from chip entrance, and flow through the main channel of described chip, so that " cell-magnetic microsphere " or " other material-magnetic microspheres " complex is collected in described collection cell, and sorted fluid is made to flow out the chip outlet of described chip；Wherein, the magnetic field being arranged over for adsorbing described complex of described main channel；Described chip includes: for flowing into the chip entrance treating sorting liquid；For for the main channel treating sorting liquid flowing；And for discharging the chip outlet of sorted rear liquid；Wherein, described main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between described access road and exit passageway, and described trapping region passage be provided with multiple downward opening, collect the collection cell of target substance to be sorted；Described chip also includes: the first substrate that can be mutually closed and the second substrate, described first substrate and described second substrate Guan Bi, formed for for the main channel treating sorting liquid flowing, wherein, described first substrate is provided with the collection cell district for collecting target substance to be sorted, and described collection cell district is provided with multiple downward opening collection cell；Described second substrate is provided with the lower surface of chip entrance, chip outlet and main channel；
(3) when removing magnetic field and/or setting is positioned at magnetic field below main channel, " cell-magnetic microsphere " or " other material-magnetic microspheres " complex that eluting is collected, thus obtaining the target substance of sorting.
In another preference, in step (2), described magnetic field is located at the top of the trapping region passage of described main channel.
In another preference, described target substance is circulating tumor cell.
A sixth aspect of the present invention provides the method for separating of a kind of circulating tumor cell, comprises the following steps:
(1) having the immunomagnetic beads of antibody to mix with coupling in the sample containing circulating tumor cell to be sorted, form the mixed liquor containing " circulating tumor cell-immunomagnetic beads " complex, wherein said antibody is the specificity antibody for described circulating tumor cell；
(2) gained mixed liquor is made to flow through the main channel of described chip, so that described " circulating tumor cell-magnetic microsphere " complex is collected in described collection cell；Wherein, described chip includes: for flowing into the chip entrance treating sorting liquid；For for the main channel treating sorting liquid flowing；And for discharging the chip outlet of sorted rear liquid；Wherein, described main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between described access road and exit passageway, and described trapping region passage be provided with multiple downward opening, collect the collection cell of target substance to be sorted；Described chip also includes: the first substrate that can be mutually closed and the second substrate, described first substrate and described second substrate Guan Bi, formed for for the main channel treating sorting liquid flowing, wherein, described first substrate is provided with the collection cell district for collecting target substance to be sorted, and described collection cell district is provided with multiple downward opening collection cell；Described second substrate is provided with the lower surface of chip entrance, chip outlet and main channel；
(3) chip described in eluting, thus obtaining described circulating tumor cell.
In another preference, between step (2) and (3), it is additionally provided with washing step.
In another preference, in step (2), it is arranged over magnetic field in described main channel, and removes the magnetic field above main channel in step (3).
In another preference, before step (2), also include the step that main channel is rinsed.
In another preference, use blocked polyethers F-108 that main channel is rinsed.
In another preference, in step (2), continue to enter main channel through inlet pump by mixed liquor according to the flow velocity of 1-20ml/h (being preferably 6ml/h).
A seventh aspect of the present invention provides the purposes of micro-fluidic chip described in first aspect present invention or second aspect, and described chip is used for sorting in circulating tumor cell, colon cancer CD133 tumor stem cell, bone marrow tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus constituting new or preferred technical scheme.As space is limited, tired no longer one by one state at this.
Accompanying drawing explanation
Fig. 1 is the structural representation of micro-fluidic chip of the present invention.
Fig. 2 is the top view of micro-fluidic chip of the present invention.
Fig. 3 is the floor map that micro-fluidic chip of the present invention collects cell array distribution.
Fig. 4 is the sectional view collecting cell and trapping region passage of micro-fluidic chip of the present invention.
Detailed description of the invention
Inventor through research deep for a long time, develops a kind of micro-fluidic chip for sorting, micro flow control chip device and micro-fluidic chip test kit and application thereof.By being arranged to the collection cell of sorting cells on the top of the trapping region passage of chip, and collect external magnetic field above cell district at chip, not only further increase the area in cell sorting district, improve chip processing capabilities, and make to catch CTCs and avoid being subject to the damage of flow disturbance and shear stress as far as possible, further increase the sensitivity catching cell.Complete the present invention on this basis.
Micro-fluidic chip
The first micro-fluidic chip for sorting provided by the invention, including:
For flowing into the chip entrance treating sorting liquid；
For for the main channel treating sorting liquid flowing；And
Chip for discharging sorted rear liquid exports；
Wherein, main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between access road and exit passageway,
And trapping region passage be provided with multiple downward opening, collect the collection cell of cell to be sorted or other materials.
It is circle, subcircular, ellipse or just polygon pit that collection cell includes cross section.From chip entrance to trapping region channel direction, the width of access road becomes larger.From trapping region passage to chip Way out, the width of exit passageway tapers into.
Other materials described include DNA, mRNA, microRNA or protein etc..
In the passage of trapping region, the distance collected between opening and the lower surface of trapping region passage of cell is 100-500 μm, it is preferred that be 300 μm.
The material of micro-fluidic chip includes polymer, glass.Polymer includes but not limited to polydimethylsiloxane, polystyrene, Polyethylene Glycol or its combination, it is preferred to polydimethylsiloxane.
Collection cell is array distribution.It is the equilateral triangle of 50-200 μm that three of arbitrary neighborhood collect the kernel of section composition length of side of cell, it is preferred that the length of side is the equilateral triangle of 60-150 μm.Each sectional area collecting cell is 1000-5000 square micron (note: the area of a circle that radius is 25 microns is about 1960 square micron), it is preferred that 1500-2500 square micron.Collect cell and be preferably the circle that cross section is radius 15-50 μm.Collect cell height and be 50-800 μm, it is preferred that for 75-500 μm.Adjacent two collection cell distances of same row or column are 25-100 μm, it is preferred that be 50 ± 10 μm.Such collection compact design can avoid the CTCs the caught damage being subject to flow disturbance and shear stress.
Collect the rectangle that little chamber region is long 20～30mm, wide 20～30mm.
Main channel is made up of the exit passageway of the access road of isosceles right triangle, square trapping region passage and isosceles right triangle, and the hypotenuse of two isosceles right triangles overlaps with foursquare the right and left respectively.Collect cell and be preferably placed at the top of trapping region passage.The height of main channel is 100-1000 μm, it is preferred that for 200-800 μm.The design of such passage can support the cell sorting of high flow capacity.
Chip outlet and chip entrance are positioned at the bottom surface of described chip and lay respectively at the access road of isosceles right triangle and the right angle of exit passageway.Size or the width of outlet and entrance are not particularly limited, and can be generally rectangle or circle, if diameter is the circle of 200-2000 μm.
The another kind provided by the invention micro-fluidic chip for sorting, described chip includes: the first substrate that can be mutually closed and the second substrate, when described first substrate above and the described second substrate Guan Bi being positioned below, is formed for for the main channel treating sorting liquid flowing, wherein
First substrate is provided with the collection cell district for collecting cell to be sorted or target substance, and described collection cell district is provided with multiple downward opening collection cell；With
Second substrate is provided with the lower surface of chip entrance, chip outlet and main channel.
Main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between access road and exit passageway, and collect cell district and be positioned at trapping region channel upper so that trapping region passage have multiple downward opening, collect the collection cell of cell to be sorted or other materials.
First substrate and the second substrate can be integrated formed structures, it is also possible to be for split-type structural.
When the first substrate and the second substrate are Split type structure, the first substrate and the second substrate are mutually closed the micro-fluidic chip namely forming the present invention.
As long as being provided with the channel part collecting cell, an all part for trapping region passage.Generally, trapping region passage is set to rectangle, it is also possible to be that other are variously-shaped, for instance the width of trapping region passage can be that two ends are less and centre is bigger.
The material of the first substrate and/or the second substrate includes but not limited to polymer, glass, and wherein polymer is selected from lower group: polydimethylsiloxane, polystyrene, Polyethylene Glycol or its combination, it is preferred to polydimethylsiloxane.
Micro flow control chip device and chip agent box
Being used for of the present invention sorts micro flow control chip device, including: the micro-fluidic chip for sorting that the present invention is above-mentioned；Be located at the Magnet being positioned at above described chip.Magnet includes permanent magnet or electric magnet, and described Magnet is used for producing magnetic field, so that target substance to be sorted is collected in the collection cell of described chip.
In the present invention, described target substance includes cell or other materials, wherein cell includes in circulating tumor cell, colon cancer CD133 tumor stem cell, bone marrow tumor cell etc. in tumor cell, ascites, and other materials include DNA, mRNA, microRNA, protein etc..
The chip apparatus of the present invention can be used for sorting in colon cancer CD133 tumor stem cell, bone marrow tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites.
Micro-fluidic chip test kit (kit) for sorting provided by the present invention, including: the micro-fluidic chip for sorting that the present invention is above-mentioned；And operation instruction.
Additionally, test kit also includes one or more assemblies being selected from lower group:
The immunomagnetic beads of (i) coupled antibody；
(ii) magnetic microsphere of non-coupled antibody；
(iii) Magnet.
Immunomagnetic beads can select according to the object being sorted or material, and in one preference of the present invention, the immunomagnetic beads of coupled antibody includes but not limited to: the immunomagnetic beads of coupling Anti-Human's epithelial cell adhesion molecule antibody, Anti-Human's CD133 immunomagnetic beads.
The chip agent box of the present invention can be used for sorting in colon cancer CD133 tumor stem cell, bone marrow tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites.
The method for separating of object is sorted from fluid
The method for separating sorting target substance from fluid of the present invention, comprises the following steps:
(1) by containing needing the cell of sorting or the fluid of other materials has the immunomagnetic beads of antibody mix with coupling, thus formation contains the fluid of " cell-magnetic microsphere " or " other material-magnetic microspheres " complex；
(2) fluid is flowed in the micro-fluidic chip of the present invention, and flow through the trapping region passage of chip, " cell-magnetic microsphere " or " other material-magnetic microspheres " complex is made to be collected in described collection cell, and make sorted fluid flow out chip, wherein, it is arranged over the magnetic field for adsorption complex in chip main channel；
(3) when removing magnetic field and/or setting is positioned at magnetic field below main channel, " cell-magnetic microsphere " or " other material-magnetic microspheres " complex that eluting is collected, thus obtaining the target substance of sorting.
Described target substance includes but not limited to: tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites in colon cancer CD133 tumor stem cell, bone marrow.
Between step (1) and step (2), also include the step that main channel is rinsed, as used blocked polyethers F-108 that main channel is rinsed.
Between step (2) and (3), it is additionally provided with washing step, for washing not untargeted cells by immunomagnetic beads labelling.
In another preference, the method sorting object from fluid specifically includes step:
(1) blocked polyethers F-108 is used to rinse micro-fluidic chip passage 30 minutes, to prevent non-specific cell or other materials from adhering to.
(2) immunomagnetic beads of coupled antibody and testing sample liquid fully react in test tube, make magnetic bead and target cell be formed magnetic bead-cell conjugate by the specific binding affinity of Ag-Ab.
(3) above the trapping region of micro-fluidic chip, permanent magnet is placed, in order to produce uneven magnetic field gradient in trapping region.
(4) sample of above-mentioned abundant reaction is all moved in micro-injection pump, continue to enter in micro-fluidic main channel through chip inlet pump by sample according to certain flow rate.
(5) magnetic bead-cell conjugate is subject to magnetic field force effect in chip trapping region and is perpendicular to the speed in flow velocity direction, and enters into collection cell；Not by the untargeted cells of immunomagnetic beads labelling, not magnetic field force induced effect, therefore flow out from chip outlet with liquid flowing.
(6) after cell sorting terminates, use wash buffer main channel three times, will be located in the Magnet above the passage of trapping region and move to original position offside, namely the bottom of trapping region passage it is placed on, magnetic bead-cell conjugate is subject to magnetic field force effect, and attracted out from collection cell, it is trapped in main channel.
(7) remove permanent magnet, the target cell of sorting can be obtained.
(8) using the main channel of wash buffer micro-fluidic chip, collect the target cell of sorting further from chip outlet, the cell that sorting obtains may be used for follow-up various experiment.
The features described above that the present invention mentions, or the feature that embodiment is mentioned can be in any combination.The disclosed all features of this case description can with any composition forms use, each feature disclosed in description, it is possible to any alternative characteristics being provided identical, impartial or similar purpose replaces.Therefore except having special instruction, disclosed feature to be only impartial or similar features general example.
Compared with prior art, the major advantage of the present invention includes:
(1) facture of microchip of the present invention is simple, operability and favorable repeatability.
(2) micro-fluidic chip of the present invention greatly improves the area in cell sorting district, supports the cell sorting of high flow capacity.
(3) external Magnet is placed on the top of passage, further increase the area in cell sorting district, pass through magnetic field optimization, further increase externally-applied magnetic field gradient, pass through flow field simulation, it is provided with the cell for cell harvesting, so that caught CTCs to avoid being subject to the damage of flow disturbance and shear stress as far as possible on the top in cell sorting district.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition or according to manufacturer it is proposed that condition.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words.Additionally, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that preferably implementation described in literary composition and material only present a demonstration.
Embodiment 1 micro-fluidic chip
As Figure 1-4, the chip of the present invention is formed in one, including:
(1) chip entrance 4: be positioned at the bottom surface of micro-fluidic chip 1, is shaped as the circle that diameter is 500 microns, and chip entrance 4 is connected with the main channel 3 of chip.
(2) chip main channel 3: main channel 3 is made up of the exit passageway 7 of the square trapping region passage 2 that the access road 6 of the isosceles right triangle that hypotenuse length is 25.4 millimeters, the length of side are 25.4 millimeters and isosceles right triangle that hypotenuse length is 25.4 millimeters, and the hypotenuse of two isosceles right triangles overlaps with foursquare the right and left respectively.Chip outlet 5 and chip entrance 4 lay respectively at the access road 6 of isosceles right triangle and the right angle of exit passageway 7, and the right angle of isosceles right triangle is substituted by the semicircle of diameter 500 microns.The height of main channel 3 is 300 microns.
Trapping region passage 2 be provided with multiple downward opening, collect the collection cell 8 of cell to be sorted or other materials.Collect the circle that cross section is diameter 50 microns of cell 8, each collection cell 8 is highly 100 microns, collect cell 8 in array distribution, form the foursquare collection cell district of 25.4 millimeters × 25.4 millimeters, the adjacent spacing collecting cell 8 of same row or column is 50 microns, and three of arbitrary neighborhood collect the kernel of section of cell 8 and constitute the equilateral triangle that the length of side is 100 microns.Collecting the opening down of cell 8, upper end is airtight, collects cell district and is positioned at the top of trapping region passage 2 so that trapping region passage 2 have multiple downward opening, collect the collection cell 8 of cell to be sorted or other materials.
(3) chip outlet 5: be positioned at the bottom surface of micro-fluidic chip 1, be shaped as the circle that diameter is 500 microns, chip outlet 5 is connected with the main channel 3 of chip.
Embodiment 2 micro-fluidic chip
As shown in Figure 1 and Figure 4, the chip of the present embodiment is essentially identical with the chip structure of embodiment 1, exports including chip entrance, chip main channel and chip.Being different in that chip is split-type structural, including the first substrate above and the second substrate being positioned below, both are mutually closed.First substrate is provided with the collection cell district for collecting cell to be sorted or other materials, collects cell district and is provided with multiple downward opening collection cell, and the second substrate is provided with the lower surface 9 of chip entrance, chip outlet and main channel.When the first substrate and the second substrate close, first substrate is provided with the main channel lower surface of the one side and the second substrate of collecting cell district and is collectively forming for for the chip main channel treating sorting liquid flowing, chip main channel includes access road, trapping region passage and exit passageway, wherein collect cell district and be positioned at the top of trapping region passage, constitute a part for trapping region passage.First substrate and the second substrate are made by polydimethylsiloxane.
Embodiment 3 utilizes the chip of the present invention to carry out the sorting of peripheral blood of patients with colonic cancer circulating tumor cell
(1) extracting colorectal cancer patients peripheric venous blood 5ml, remove beginning 2ml peripheral blood, it is to avoid skin epithelial cell pollutes, residue blood sample uses EDTA anticoagulant tube to collect, and 4 degrees Celsius are stored and transport, test in 4 hours.
(2) using blocked polyethers F-108 to rinse micro-fluidic chip passage 30 minutes, to prevent non-specific cell from adhering to, then placing magnetic field intensity above the trapping region of micro-fluidic chip is the permanent magnet of 500mT.
(3) according to 107The ratio of magnetic bead/ml blood, adds in the 3ml blood in step (1) by the immunomagnetic beads (purchased from Invitrogen company) adding 150 ul of conjugate Anti-Human's epithelial cell adhesion molecule antibody, hatches altogether 30 minutes at 4 DEG C.
(4) sample of fully reaction in step (3) is all moved in micro-injection pump, continue to enter in micro-fluidic main channel through chip inlet pump by sample according to the flow velocity of 6ml/h.
(5) magnetic bead-cell conjugate is subject to magnetic field force effect in chip trapping region and is perpendicular to the speed in flow velocity direction, and enters into collection cell；Not by the untargeted cells of immunomagnetic beads labelling, not magnetic field force induced effect, flow out from chip outlet with liquid flowing.
(6) after cell sorting terminates, use wash buffer main channel three times, then will be located in the Magnet above the passage of trapping region and move to original position offside, namely the bottom of trapping region passage it is placed on, magnetic bead-cell conjugate is subject to magnetic field force effect, and attracted out from collection cell, it is trapped in main channel.
(7) remove permanent magnet, the target cell of sorting can be obtained.
(8) use the main channel of wash buffer micro-fluidic chip, collect the cell of sorting further from chip outlet, use immunofluorescence dyeing labeled cell keratin-8/18/19, human leucocyte antigen CD45 further, then use DAPI to redye nucleus；The colon cancer circulating tumor cell that sorting obtains is identified by fluorescence microscope；The cell that sorting obtains can be also used for other subsequent experimental.
Embodiment 4 utilizes the chip of the present invention to carry out the sorting of CD133 colon cancer tumours stem cell
(1) using blocked polyethers F-108 to rinse micro-fluidic chip passage 30 minutes in advance, to prevent non-specific cell from adhering to, then placing magnetic field intensity above the trapping region of micro-fluidic chip is the permanent magnet of 500mT.
(2) single cell suspension sample is adjusted to 1X108Cell/300 microlitre, by 100 microlitre/108The ratio of cell adds blocking antibody and closes FcR receptor.
(3) according to every 1X108Cell adds the ratio of 100 microlitres Anti-Human's CD133 immunomagnetic beads (purchased from MiltenyiBiotec company), adds immunomagnetic beads in the reactant liquor in step (2), is sufficiently mixed, hatches 30 minutes for 4 degrees Celsius.
(4) sample of fully reaction in step (3) is all moved in micro-injection pump, continue to enter in micro-fluidic main channel through chip inlet pump by sample according to the flow velocity of 6ml/h.
(5) magnetic bead-cell conjugate is subject to magnetic field force effect in chip trapping region and is perpendicular to the speed in flow velocity direction, and enters into collection cell；Not by the untargeted cells of immunomagnetic beads labelling, not magnetic field force induced effect, flow out from chip outlet with liquid flowing.
(6) after cell sorting terminates, use wash buffer main channel three times, then will be located in the Magnet above the passage of trapping region and move to original position offside, namely the bottom of trapping region passage it is placed on, magnetic bead-cell conjugate is subject to magnetic field force effect, and attracted out from collection cell, it is trapped in main channel.
(7) remove permanent magnet, the target cell of sorting can be obtained.
(8) use the main channel of wash buffer micro-fluidic chip, collect the cell of sorting further from chip outlet, be human colon carcinoma CD133 tumor stem cell；The cell of sorting can use the research such as cell, molecular biology.
The preparation method of single cell suspension sample in this programme: aseptic cut human colon carcinoma tumor tissues, puts in sterilized petri dishes, uses the phosphate buffer without magnesium ion and calcium ion to rinse 3 times；Tumor tissues is cut into the fritter of 3 cubic millimeters of sizes；Putting in conical flask by the piece of tissue sheared, the tryptic digestive juice of the 0.25% of 30 times of volumes of addition, by flask as, on magnetic stirrer, digesting 45 minutes；Digest complete, pour centrifuge tube 800 revs/min into centrifugal 6 minutes, remove supernatant；Adopt Hanks liquid to rinse 3 times, be centrifuged and remove supernatant, twice totally；500 revs/min are centrifuged 5 minutes, remove supernatant, add certain culture fluid, filtered by the nylon wire of the meshes of 100 microns, remove indigested bigger piece of tissue, collect filtrate and are single cell suspension.
Result: through the cell of micro-fluidic chip sorting, uses flow cytometer to analyze CD133 positive cell and CD133 negative cells proportion further, and display CD133 positive cell ratio is more than 90%.
Comparative example utilizes existing micro-fluidic chip to carry out the sorting of colon cancer circulating tumor cell
Use existing micro-fluidic chip, test according to the method for embodiment 3.Chip structure reference literature: Labonachip2011；11 (20): 3449-57.
Experimental result is as shown in table 1
Table 1
As it can be seen from table 1 the chip of the present invention is when carrying out the sorting of colon cancer circulating tumor cell, the effective processing capacity of chip reaches as high as 20ml/h；With the treating capacity of 6ml/h when, tumor cell capture rate reaches as high as 88%, catches the sensitivity of cell up to 1-2 CTC/108-109Hemocyte；And when being circulated tumor cell sorting with the micro-fluidic chip that the know-why reported at present is similar, its maximum capture rate is about 86%, but average effective processes range of flow and is typically only 1ml/h-10ml/h, and sensitivity is about 1 CTC/107-109Hemocyte.
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
1. the micro-fluidic chip being used for sorting circulating tumor cell, it is characterised in that described chip includes:
For flowing into the chip entrance treating sorting liquid；
For for the main channel treating sorting liquid flowing；And
Chip for discharging sorted rear liquid exports；
Wherein, described main channel includes being positioned at the access road of chip porch, being positioned at the exit passageway in chip exit, and the trapping region passage between described access road and exit passageway,
And described trapping region passage be provided with multiple downward opening, collect the collection cell of target substance to be sorted；And described collection cell is positioned at the top of trapping region passage；
The sectional area of described each collection cell is 1000-5000 square micron；And it is circle, subcircular, ellipse or just polygon pit that described collection cell includes cross section；
Distance between opening and the lower surface of trapping region passage of described collection cell is 100-500 μm, and described collection cell height is 50-800 μm；
Adjacent two collection cell distances are 25-100 μm；
From chip entrance to trapping region channel direction, the width of described access road becomes larger；
From trapping region passage to chip Way out, the width of described exit passageway tapers into；
And the rectangle that described trapping region passage is long 15～30mm, wide 15～30mm.
2. chip as claimed in claim 1, it is characterised in that described collection cell is array distribution.
3. chip as claimed in claim 1, it is characterised in that adjacent two collection cell distances are 50 ± 10 μm.
4. chip as claimed in claim 1, it is characterised in that described collection cell height is 75-500 μm.
5. chip as claimed in claim 1, it is characterised in that described chip includes: the first substrate that can be mutually closed and the second substrate, described first substrate and described second substrate Guan Bi, form the main channel being used for for treating sorting liquid flowing, wherein,
Described first substrate is provided with the collection cell district for collecting target substance to be sorted, and described collection cell district is provided with multiple downward opening collection cell；The sectional area of wherein said each collection cell is 1000-5000 square micron, and described collection cell to include cross section be circle, subcircular, ellipse or just polygon pit；
Described second substrate is provided with the lower surface of chip entrance, chip outlet and main channel；
Distance between opening and the lower surface of trapping region passage of described collection cell is 100-500 μm, and described collection cell height is 50-800 μm.
6. the micro flow control chip device being used for sorting circulating tumor cell, it is characterised in that described chip apparatus includes:
The micro-fluidic chip for sorting described in claim 1；With
Being located at the Magnet being positioned at above described chip, described Magnet is used for producing magnetic field, so that target substance to be sorted is collected in the collection cell of described chip.
7. the micro-fluidic chip test kit being used for sorting circulating tumor cell, it is characterised in that described chip agent box includes:
The micro-fluidic chip for sorting described in claim 1；And operation instruction.
8. sorting a method for separating for target substance from fluid, wherein said target substance includes cell or other materials, it is characterised in that comprise the following steps:
(1) fluid is provided, in described fluid containing need sorting cell or other materials, cell wherein said to be sorted or other materials combine with magnetic microsphere, thus forming " cell-magnetic microsphere " or " other material-magnetic microspheres " complex；
(2) described fluid is flowed into micro-fluidic chip described in claim 1 from chip entrance, and flow through the main channel of described chip, so that " cell-magnetic microsphere " or " other material-magnetic microspheres " complex is collected in described collection cell, and sorted fluid is made to flow out the chip outlet of described chip；Wherein, the magnetic field being arranged over for adsorbing described complex of described main channel；
(3) when removing magnetic field and/or setting is positioned at magnetic field below main channel, " cell-magnetic microsphere " or " other material-magnetic microspheres " complex that eluting is collected, thus obtaining the target substance of sorting；
Wherein said " other materials " is DNA, mRNA, microRNA or protein.
9. the method for separating of a circulating tumor cell, it is characterised in that comprise the following steps:
(1) having the immunomagnetic beads of antibody to mix with coupling in the sample containing circulating tumor cell to be sorted, form the mixed liquor containing " circulating tumor cell-immunomagnetic beads " complex, wherein said antibody is the specificity antibody for described circulating tumor cell；
(2) gained mixed liquor is made to flow through the main channel of chip described in claim 1, so that described " circulating tumor cell-magnetic microsphere " complex is collected in described collection cell；
(3) chip described in eluting, thus obtaining described circulating tumor cell.
10. the purposes of micro-fluidic chip described in claim 1, it is characterised in that described chip is used for sorting in circulating tumor cell, colon cancer CD133 tumor stem cell, bone marrow tumor cell, DNA, mRNA, microRNA or protein in tumor cell, ascites.
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