CN101717718A - Micro mesh sieve structure used for cell sorting and application method thereof - Google Patents
Micro mesh sieve structure used for cell sorting and application method thereof Download PDFInfo
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Abstract
The invention discloses a micro mesh structure used for cell sorting and an application method thereof, belonging to the field of microfluidic chips. The structure comprises at least two layers of sorting thin films which are provided with a plurality of sorting pores, wherein at least one layer of the sorting thin film is connected with a driving device. When in use, an overlapping area of the corresponding sorting pores of the two layers of adjacent sorting thin films is adjusted through the driving device to realize a successive sorting on all cells to be sorted or a sorting on cells with specific size dimensions. By using the structure, the cell sorting has the advantages of high sorting efficiency, high precision, continuously adjustable pore diameter, large sorting range, uneasy channel blocking, necessity free of learning dimension distribution conditions of the cells to be sorted in advance, and the like; moreover, beside the cell sorting, the invention is also suitable for sorting micro-particles with diameters ranging from below ten micrometer to hundreds of micrometers.
Description
One, affiliated field:
The present invention relates to a kind of micro mesh sieve structure and using method thereof that is used for cell sorting, belong to the micro-fluidic chip field.
Two, background technology:
Cell sorting is a necessary means of obtaining the target cell of character homogeneous from complex environment.Cell sorting is the key link of cell physiological and pathological study, and it is for the diagnosis of major disease and treat significant.Always be that for cell sorting method and The Research of Relevant Technology one of medical circle and association area is studied focus.Along with the appearance of MEMS (micro electro mechanical system) (MEMS) technology and in the application of biomedical sector, many new cell sorting methods have been produced.Present stage with the flow cytometer is that the conventional cell sorter of representative has that volume is big, complex structure, shortcoming such as with high costs, can be with the cell sorter microminiaturization based on the micro-fluidic chip of MEMS technology.With respect to conventional cell sorting method such as Flow Cytometry, immunity magnetic micropearls sortings, the micro-fluidic chip cell sorting method need not the complicated sample set-up procedure, and has separation purity height, plurality of advantages such as easy and simple to handle, with low cost.
Because the biological cell size is minimum and of a great variety, the sorting difficulty is very big.Cell sorting method based on micro-fluidic chip mainly comprises: utilize cell size difference, utilize dielectrophoresis, utilize strong magnetic field gradient variation etc.Wherein, utilize cell size difference to carry out the method for cell sorting because need not pair cell or protein carries out specific marker, pair cell is not damage almost, and has compact construction, efficiency of separation height, actively preserves plurality of advantages such as good.
2004, a kind of cell sorting method based on different apertures micro channel array (Hisham Mohamed has invented at Wadsworth health research center in the U.S., Leslie D.McCurdy, et al.Development of a rare cell fractionation device:application for cancer detection.IEEE Transactions on Nanobioscience.2004, and be used for the sorting and the detection of cancer cells 3:251～256.).Its core texture is four groups of micro channel arrays with different apertures, has realized the discrete sorting of cancer cells in the 5-20 mu m range by the micro channel array in this different apertures.After microchannel in this method formed, designed aperture, microchannel and distribution just can't change again, only can carry out sorting at the cell of specific discontinuous size section, and need know the distribution of sizes situation for the treatment of sorting cells in advance.In addition, the passage obstruction also appears in this method easily because of passage is long and narrow.
Three, summary of the invention:
The objective of the invention is to overcome the existing deficiency of utilizing cell size difference to carry out cell sorting method: after 1) microchannel formed, designed aperture, microchannel and distribution just can't change again, only can carry out sorting at the cell of specific discontinuous size section; 2) need know the distribution of sizes situation for the treatment of sorting cells in advance; 3) the long and narrow passage that occurs of passage stops up.Thus, the present invention proposes the cell sorting method based on micro mesh sieve structure, can realize continuous variable and the dynamic real-time adjustment of cell sorting size on a large scale, and passage is difficult for stopping up, and need not to know in advance the distribution of sizes situation for the treatment of sorting cells.
For achieving the above object, technical scheme of the present invention is: a kind of micro mesh sieve structure that is used for cell sorting is provided, this structure comprises that double-layer separate at least is furnished with the sorting film in some sortings hole, and each layer sorting film spacing g is not less than 1 μ m, and wherein one deck sorting film is connected with drive unit at least; Adjacent two layers sorting film motion stepping accuracy mutually is t, and promptly sharpness of separation is t, and t satisfies t≤0.1 * g; Described sorting hole shape is any, and the little circumscribed circle diameter of sorting Kongzui is D
0, the big inscribed circle diameter of sorting Kongzui is D
1, sorting film relative movement displacement is l, then cell sorting scope D satisfies g＜D≤min (D
1, l) for reducing and eliminating relative movement displacement of each layer sorting film up and down and the nonlinear relationship between the pore size, so the aperture shape in sorting hole is preferably regular polygons such as equilateral triangle, square, regular pentagon; The material of micro mesh sieve structure is polymkeric substance, glass, pottery, silicon or their complex body.
As a kind of preferred embodiment: the sorting hole becomes regular array arrangement on the sorting film, and the position is arranged in correspondence with each other in the sorting hole on each layer sorting film; The direction of motion of described drive unit is consistent with adjacent sorting centerline hole.
As a kind of preferred embodiment: adjacent sorting pitch of holes is not less than the little circumscribed circle diameter of sorting Kongzui.
The present invention also provides the using method of above-mentioned micro mesh sieve structure, comprises using micro mesh sieve structure to sub-elect the using method of all treating sorting cells one by one, and the using method of using the cell of micro mesh sieve structure sorting specific dimensions size.
Because the micro mesh sieve structure that is used for cell sorting provided by the invention, form by two-layer at least sorting film, wherein one deck sorting film is connected with drive unit at least, in following using method, by regulating drive unit, accurately the relative movement between each adjacent two layers sorting film of control realizes that sorting hole lap size changes, thereby realizes cell sorting.Therefore, following method at be adjacent two-layer movable sorting film arbitrarily.
One, uses micro mesh sieve structure to sub-elect the using method of all treating sorting cells one by one, comprise the steps:
1. hypothesis will sub-elect diameter one by one and be respectively d
1, d
2... d
h... d
kCell, d wherein
1＜d
2＜... d
h...＜d
k, be t for sorting film spacing g and sharpness of separation, always there is a natural number i, satisfy the t of it＜g≤(i+1);
2. initialize drive unit makes the maximum inscribed circle diameter M of the initial lap in sorting hole
0Satisfy (i+1) t≤M
0＜(i+2) t;
3. will treat that the sorting cells nutrient solution flows to micro mesh sieve structure;
4. the adjusting drive unit is accurately controlled the relative movement between each adjacent two layers sorting film, makes the maximum inscribed circle diameter of sorting hole lap from M
0Increase t gradually, up to satisfying jt＜d
1≤ (j+1) t, this moment, the maximum inscribed circle diameter of sorting hole lap was M
1, i.e. M
1=M
0+ (j-i) t, at this moment, it is d that the cell by microgrid screening constituency can be considered diameter
1Cell;
5. continue to regulate drive unit, make the maximum inscribed circle diameter of sorting hole lap from M
1Increase t gradually, up to satisfying nt＜d
2≤ (n+1) t, this moment, the maximum inscribed circle diameter of sorting hole lap was M
2, i.e. M
2=M
1+ (n-j) t, then to can be considered diameter be d to the cell by microgrid screening constituency
2Cell;
6. circulation step 5 is successively all treated sorting cells up to sub-electing.
Two, use the using method of the cell of micro mesh sieve structure sorting specific dimensions size, comprise the steps:
1. hypothesis treats that the cell dia of sorting specific dimensions size is d
h, then always there is a natural number m, satisfy mt＜d
h≤ (m+1) t;
2. initialize drive unit makes the maximum inscribed circle diameter M of the initial lap in sorting hole
0Satisfy (i+1) t＜M
0≤ (i+2) t;
3. will treat that the sorting cells nutrient solution flows to micro mesh sieve structure;
4. the adjusting drive unit is accurately controlled the relative movement between each layer sorting film, makes the maximum inscribed circle increasing diameter of sorting hole lap be added to M
3, satisfy M
3=mt then sieves the cell dia in constituency smaller or equal to M by microgrid
3
5. continue to regulate drive unit, make the maximum inscribed circle diameter M of sorting hole lap
3Increase t, promptly the maximum inscribed circle diameter of sorting hole lap is from M
3Be increased to M
4, M
4=(m+1) t, then the cell dia by microgrid screening constituency satisfies M
3＜d
h≤ M
4, being about to the specific dimensions size is d
hCell sorting come out.
More than two kinds of using method in order to reach better separating effect, using micro mesh sieve structure to carry out applying vibration in the cell sorting.
The invention has the beneficial effects as follows:
(1) efficiency of separation height: the sorting divided thin film of micro mesh sieve structure is furnished with some sortings hole, can sub-elect the sorting cells that remains fast one by one; When using the cell of micro mesh sieve structure sorting specific dimensions size, only need can sub-elect the cell of specific dimensions size for twice;
(2) sharpness of separation height: the sharpness of separation of micro mesh sieve structure is an adjacent two layers sorting film motion stepping accuracy mutually, and prior art can reach nano-precision;
(3) aperture is adjustable continuously: but the adjusting of the maximum inscribed circle diameter real-time continuous of sorting hole lap, to reach good separating effect;
(4) sorting scope is big: the sorting scope D of micro mesh sieve structure satisfies g＜D≤min (D
1, l), therefore can realize large-scale cell sorting;
(5) passage is difficult for stopping up: the maximum inscribed circle diameter of sorting hole lap can be regulated in real time, and passage is difficult for stopping up;
(6) need not to know in advance the distribution of sizes situation for the treatment of sorting cells: the maximum inscribed circle diameter of sorting hole lap can be regulated in real time, need not to know in advance that the distribution of sizes situation for the treatment of sorting cells can carry out cell sorting.
For the change in the actual design processing, as: the sorting scope, stepping accuracy, microgrid sieve type of drive, the present invention such as microgrid mesh size shape are suitable equally.
Sorting unit and method that the present invention proposes not only are confined to cell sorting, are equally applicable to diameter several microns microparticle sortings of arriving in the hundreds of micrometer range.
The present invention is further described below in conjunction with drawings and Examples.
Four, description of drawings (accompanying drawing in literal quote explanation)
The micro mesh sieve structure synoptic diagram of Fig. 1 specific embodiment 1
The micro mesh sieve structure assorting room synoptic diagram of Fig. 2 specific embodiment 1
The micro mesh sieve structure assorting room vertical view of Fig. 3 specific embodiment 1
Wherein, 1-upper strata sorting film, 2-lower floor sorting film, 3-drive unit, 4-sorting hole
Five, embodiment:
Specific embodiment 1:
Consult Fig. 1, present embodiment provides a kind of micro mesh sieve structure that is used for cell sorting, this structure comprises upper strata sorting film 1 and the lower floor's sorting film 2 that is furnished with 3 * 3 array sorting holes 4 respectively, the position is arranged in correspondence with each other in sorting hole on the two-layer up and down sorting film, two-layer sorting film spacing g is 2 μ m, wherein lower floor's sorting film 2 is connected with drive unit 3, and the direction of motion of drive unit 3 is consistent with adjacent sorting centerline hole; Two-layer sorting film motion stepping accuracy t mutually is 0.15 μ m; Described sorting hole 4 aperture shape are that the length of side is the square of 50 μ m, sorting hole 4 minimum circumscribed circle diameter D
0For
Sorting hole 4 maximum inscribed circle diameter D
1Be 50 μ m, sorting hole 4 minimum spacings are that S is 50 μ m; Sorting film relative movement displacement l is 100 μ m, and cell sorting scope D satisfies g＜D≤min (D
1, l), i.e. 2 μ m＜D≤50 μ m; The micro mesh sieve structure material is a polysilicon.
Consult Fig. 2, Fig. 3, the micro mesh sieve structure of use present embodiment sub-elects diameter one by one and is respectively 7, the cell of 8...10...12 μ m, using method comprises the steps:
1. be t for sorting film spacing g and sharpness of separation in the present embodiment, have a natural number i=13, satisfy the t of it＜g≤(i+1), promptly 13 * 0.15＜2≤(13+1) * 0.1, promptly 1.95＜2＜2.1;
2. the initialize drive unit 3, make the maximum inscribed circle diameter M of the initial lap in sorting hole
0Satisfy (i+1) t≤M
0＜(i+2) t, i.e. (13+1) * 0.15≤2.1＜(13+2) * 0.15, promptly 2.1≤2.1＜2.25;
3. will treat that the sorting cells nutrient solution flows to microgrid screening constituency;
4. regulate drive unit 3, accurately control the relative movement between each layer sorting film, make the maximum inscribed circle diameter of sorting hole lap from M
0Increase t gradually, up to satisfying jt＜d
1≤ (j+1) t, promptly 46 * 0.15＜7＜(46+1) * 0.15, promptly 6.9＜7＜7.05, this moment, the maximum inscribed circle diameter of sorting hole lap was M
1, i.e. M
1=M
0+ (j-i) t, i.e. 7.05=2.1+ (46-13) * 0.15, at this moment, it is d that the cell by microgrid screening constituency can be considered diameter
1The cell of=7 μ m;
5. continue to regulate drive unit 3, make the maximum inscribed circle diameter of sorting hole lap from M
1Increase t gradually, up to satisfying nt＜d
2≤ (n+1) t, promptly 53 * 0.15＜8＜(53+1) * 0.15, promptly 7.95＜8＜8.1, this moment, the maximum inscribed circle diameter of sorting hole lap was M
2, i.e. M
2=M
1+ (n-j) t, i.e. 8.1=7.05+ (53-46) * 0.15, at this moment, it is d that the cell by microgrid screening constituency can be considered diameter
2The cell of=8 μ m;
6. circulation step 5 is successively all treated sorting cells up to sub-electing residue.
Specific embodiment 2:
The invention provides a kind of micro mesh sieve structure that is used for cell sorting, this structure is made up of three layers of sorting film that is distributed with 20 * 20 array sorting holes, the position is arranged in correspondence with each other in sorting hole on each layer sorting film, each layer sorting film spacing g is 3 μ m, wherein sorting film in middle layer is connected with drive unit, and the direction of motion of drive unit is consistent with adjacent sorting centerline hole; Two-layer sorting film motion stepping accuracy t mutually is 0.2 μ m; Described sorting hole aperture shape is that the length of side is the regular hexagon of 20 μ m, the little circumscribed circle diameter D of sorting Kongzui
0Be 40 μ m, the big inscribed circle diameter D of sorting Kongzui
1For
The little spacing of sorting Kongzui is that S is 40 μ m; Sorting film relative movement displacement l is 50 μ m, and cell sorting scope D satisfies g＜D≤min (D
1, l), promptly
The micro mesh sieve structure material is polydimethylsiloxane (PDMS).
The micro mesh sieve structure that uses present embodiment one by one sorting specific dimensions size cell dia d
hBe 7.9 μ m, its method comprises the steps:
1. for the cell dia d that wants sorting specific dimensions size
hBe 7.9 μ m, have a natural number m=39, satisfy mt＜d
h≤ (m+1) t, promptly 39 * 0.2＜7.9＜(39+1) * 0.2, promptly 7.8＜7.9＜8;
2. initialize drive unit makes the maximum inscribed circle diameter M of the initial lap in sorting hole
0Satisfy (i+1) t≤M
0＜(i+2) t, i.e. (14+1) * 0.2≤3＜(14+2) * 0.2, promptly 3≤3＜3.2;
3. will treat that the sorting cells nutrient solution flows to micro mesh sieve structure;
4. the adjusting drive unit is accurately controlled the relative movement between each layer sorting film, makes the maximum inscribed circle increasing diameter of sorting hole lap be added to M
3, satisfy M
3=mt, promptly 7.8=39 * 0.2 applies vibration simultaneously, then sieves the cell dia in constituency less than M by microgrid
3, promptly sieve the cell dia in constituency smaller or equal to 7.8 μ m by microgrid;
5. continue to regulate drive unit, make the maximum inscribed circle diameter M of sorting hole lap
3Increase t, promptly the maximum inscribed circle diameter of sorting hole lap is from M
3Be increased to M
4, M
4=(m+1) t, promptly 8=(39+1) * 0.2 applies vibration simultaneously, and then the cell dia by microgrid screening constituency satisfies M
3＜d
h≤ M
4, promptly 7.8＜7.9＜8, the cell sorting that is about to the specific dimensions size and is 7.9 μ m comes out.
Claims (8)
1. a micro mesh sieve structure that is used for cell sorting is characterized in that, comprises that double-layer separate at least is furnished with the sorting film in some sortings hole, and each layer sorting film spacing g is not less than 1 μ m, and wherein one deck sorting film is connected with drive unit at least; Adjacent two layers sorting film motion stepping accuracy mutually is t, and t satisfies t≤0.1 * g.
2. a micro mesh sieve structure that is used for cell sorting as claimed in claim 1 is characterized in that, the material of micro mesh sieve structure is polymkeric substance, glass, pottery, silicon or their complex body.
3. a micro mesh sieve structure that is used for cell sorting as claimed in claim 1 is characterized in that, the sorting hole becomes regular array arrangement on the sorting film, and the position is arranged in correspondence with each other in the sorting hole on each layer sorting film; The direction of motion of described drive unit is consistent with adjacent sorting centerline hole.
4. a micro mesh sieve structure that is used for cell sorting as claimed in claim 1 is characterized in that, adjacent sorting pitch of holes is not less than the little circumscribed circle diameter of sorting Kongzui.
5. a micro mesh sieve structure that is used for cell sorting as claimed in claim 1 is characterized in that, the aperture shape in described sorting hole is a regular polygon.
6. one kind is used according to claim 1 that micro mesh sieve structure sub-elects the using method of all treating sorting cells one by one, it is characterized in that, comprises the steps:
Step 1: suppose that sub-elect diameter one by one is respectively d
1, d
2... d
h... d
kCell, d wherein
1＜d
2＜... d
h...＜d
k, be t for sorting film spacing g and sharpness of separation, always there is a natural number i, satisfy the t of it＜g≤(i+1);
Step 2: the initialize drive unit makes the maximum inscribed circle diameter M of the initial lap in sorting hole
0Satisfy (i+1) t≤M
0＜(i+2) t;
Step 3: will treat that the sorting cells nutrient solution flows to micro mesh sieve structure;
Step 4: regulate drive unit, accurately control the relative movement between each adjacent two layers sorting film, make the maximum inscribed circle diameter of sorting hole lap from M
0Increase t gradually, up to satisfying jt＜d
1≤ (j+1) t, this moment, the maximum inscribed circle diameter of sorting hole lap was M
1, i.e. M
1=M
0+ (j-i) t, at this moment, it is d that the cell by microgrid screening constituency can be considered diameter
1Cell;
Step 5: continue to regulate drive unit, make the maximum inscribed circle diameter of sorting hole lap from M
1Increase t gradually, up to satisfying nt＜d
2≤ (n+1) t, this moment, the maximum inscribed circle diameter of sorting hole lap was M
2, i.e. M
2=M
1+ (n-j) t, then to can be considered diameter be d to the cell by microgrid screening constituency
2Cell;
Step 6: circulation step 5 successively, all treat sorting cells up to sub-electing.
7. one kind is used the using method of the cell of micro mesh sieve structure sorting specific dimensions size according to claim 1, it is characterized in that, comprises the steps:
Step 1: suppose to treat that the cell dia of sorting specific dimensions size is d
h, then always there is a natural number m, satisfy mt＜d
h≤ (m+1) t;
Step 2: the initialize drive unit makes the maximum inscribed circle diameter M of the initial lap in sorting hole
0Satisfy (i+1) t＜M
0≤ (i+2) t;
Step 3: will treat that the sorting cells nutrient solution flows to micro mesh sieve structure;
Step 4: regulate drive unit, accurately control the relative movement between each layer sorting film, make the maximum inscribed circle increasing diameter of sorting hole lap be added to M
3, satisfy M
3=mt then sieves the cell dia in constituency smaller or equal to M by microgrid
3
Step 5: continue to regulate drive unit, make the maximum inscribed circle diameter M of sorting hole lap
3Increase t, promptly the maximum inscribed circle diameter of sorting hole lap is from M
3Be increased to M
4, M
4=(m+1) t, then the cell dia by microgrid screening constituency satisfies M
3＜d
h≤ M
4, being about to the specific dimensions size is d
hCell sorting come out.
8. one kind as claim 6 or 7 described micro mesh sieve structure using method, it is characterized in that, apply vibration when being expert at cell sorting.
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| CN101717718B CN101717718B (en) | 2012-10-10 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948739A (en) * | 2010-09-09 | 2011-01-19 | 西北工业大学 | Rotary micro screen structure for cell sorting |
| CN103387935A (en) * | 2012-05-09 | 2013-11-13 | 中国人民解放军军械工程学院 | Microfluidic array chip for cell capture |
| CN104004650A (en) * | 2014-05-28 | 2014-08-27 | 上海丽恒光微电子科技有限公司 | Biofluid macromolecular detection device and detection method |
| CN105420086A (en) * | 2015-12-28 | 2016-03-23 | 苏州浚惠生物科技有限公司 | Single-cell positioning microporous membrane, application and single-cell automatic acquisition device |
| CN106047706A (en) * | 2016-06-15 | 2016-10-26 | 西北工业大学 | Chip for implementing cellular localization culture based on single-cell capture and using and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6881315B2 (en) * | 2001-08-03 | 2005-04-19 | Nec Corporation | Fractionating apparatus having colonies of pillars arranged in migration passage at interval and process for fabricating pillars |
| US7160730B2 (en) * | 2002-10-21 | 2007-01-09 | Bach David T | Method and apparatus for cell sorting |
| US20080213821A1 (en) * | 2004-05-06 | 2008-09-04 | Nanyang Technological University | Microfluidic Cell Sorter System |
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2009
- 2009-12-03 CN CN 200910219282 patent/CN101717718B/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948739A (en) * | 2010-09-09 | 2011-01-19 | 西北工业大学 | Rotary micro screen structure for cell sorting |
| CN103387935A (en) * | 2012-05-09 | 2013-11-13 | 中国人民解放军军械工程学院 | Microfluidic array chip for cell capture |
| CN104004650A (en) * | 2014-05-28 | 2014-08-27 | 上海丽恒光微电子科技有限公司 | Biofluid macromolecular detection device and detection method |
| CN104004650B (en) * | 2014-05-28 | 2016-03-02 | 上海珏芯光电科技有限公司 | Biofluid polymer means of detection and detection method |
| CN105420086A (en) * | 2015-12-28 | 2016-03-23 | 苏州浚惠生物科技有限公司 | Single-cell positioning microporous membrane, application and single-cell automatic acquisition device |
| CN106047706A (en) * | 2016-06-15 | 2016-10-26 | 西北工业大学 | Chip for implementing cellular localization culture based on single-cell capture and using and preparation method thereof |
| CN106047706B (en) * | 2016-06-15 | 2018-11-16 | 西北工业大学 | One kind realizing cellular localization culture chip and its use and preparation method based on unicellular capture |
| CN111073797A (en) * | 2020-03-04 | 2020-04-28 | 山东第一医科大学(山东省医学科学院) | Circulating tumor cell step-by-step separation device |
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