CN1566951A - Cell sorting method based on micro flow control chips and special-purpose chips therefor - Google Patents
Cell sorting method based on micro flow control chips and special-purpose chips therefor Download PDFInfo
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- CN1566951A CN1566951A CN 03133463 CN03133463A CN1566951A CN 1566951 A CN1566951 A CN 1566951A CN 03133463 CN03133463 CN 03133463 CN 03133463 A CN03133463 A CN 03133463A CN 1566951 A CN1566951 A CN 1566951A
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Abstract
A distinguishing method for cell based on micro=flow control chip is characterized by the following: first to exert a local electricity field on both sides of tunnel wall of the micro-flow control chip to rearrange the cells there according to their different surface charge properties; then to exert on the cross of tunnel a vertical electromotion and pressure which drives the rearranged cells to flow to different space to realize the distinguishing of cells. This method is based on the force difference in the static field caused by cell surface charge density of cell and is free of signal feedback and molecule pre-marks on the cells.
Description
Technical field:
The present invention is chiefly directed to the method that realizes cell sorting on the micro-fluidic chip.
Background technology:
Cell sorting is a necessary means of obtaining the target cell of character homogeneous from complex environment.Present stage realizes that the main tool of cell sorting is a flow cytometer.Existing commercial flow cytometer is used widely in each hospital, scientific research institutions.But this equipment significant disadvantages is to cost an arm and a leg, and complex structure is bulky, is difficult for popularizing.The appearance of micro-fluidic chip is expected to the cell sorter microminiaturization.
The approach that realizes cell sorting on micro-fluidic chip comes branch to mainly contain two big classes by mechanism.A kind of be with feedback signal judgment mode decision stream of cells to method for separating.Such as document [1 Fu AY, Spence C, Scherer A et al..Nat.Biotech.1999,17,1109-1111.2?Fu?AY，Chou?HP，Spence?C?et?al..Anal.Chem.2002，74，2451-2457。3 Wolff A, Perch-Nielsen IR, Larsen UD et al..LabChip, 2003,3,22-27] report that the fluorescence that goes out with laser excitation is as fluorescence excitation cell sorting (Fluorescence Activated Cell Sorter, the FACS) method of stream of cells to criterion.This method principle and flow cytometer are similar.Except that fluorescence signal, the resistance variations that document [4 Gawad S, Schild L, Renaud Ph, Lab on a Chip, 2001,1,76-82] causes when passing electrode in the passage with cell is as the criterion of distinguishing target cell.The information of collecting in the said method relevant with cell is (as fluorescence, scattered light, electronic signal etc.) through judging (the size of power that applies that influences driving force as feedback signal, direction etc.), thus will satisfy the cell that sets in advance criterion flows to assigned direction under certain driving force (electric power, pressure etc.).
Another kind of sorting mode need not signal feedback, mainly relies on the amplification of certain species diversity in certain field of force of cell self to finish cell sorting.Such as, cell surface can cause the sorting [5 Sun Ls, Zborowski W, Moore LR et al..Cytometry, 1998,33,469-475] of cell in magnetic field in conjunction with the capacity variance of magnetic material.
No matter be above-mentioned which kind of method, all do not have really on micro-fluidic chip, to realize efficient cell sorting, still lack a kind of universally recognized sorting mode at present.
Summary of the invention:
The object of the present invention is to provide the different method of finishing cell sorting on micro-fluidic chip of a kind of and existing sorting schemes, the method need not signal feedback, does not also need cell various molecules (as the magnetic material molecule) on the mark in advance.
The invention provides a kind of cell sorting method, it is characterized in that: at first impose internal field, cell is rearranged according to the surface charge property difference at channel cross-section in the conduit wall both sides of micro-fluidic chip based on micro-fluidic chip; Right-angled intersection place at passage imposes orthogonal electric power and pressure again, and orthogonal pair of power effect will make the stream of cells of reorientating to different spaces, thereby realizes the sorting of cell.
The present invention is based in the cell sorting method of micro-fluidic chip, local electric field strength at 100V/cm to 1000V/cm, be added in pressure on the cell from 1 handkerchief to 1000 handkerchiefs; Electric field intensity is from 10V/cm to 1000V/cm.
The principle that the present invention is based on is, the cell under the pressure-driven enters sample intake passage, flows to a minute constituency (decussation district), and its position at the sample intake passage xsect is a stochastic distribution.When flowing through the internal field district, cell surface electric charge and internal field interact, and cell is arranged according to the size of cell surface electric density again in the position of sample intake passage xsect.Continue to flow to the branch constituency under pressure through the cell of reorientating.The branch constituency is made of orthogonal connection microchannel, and wherein the mobile driving force of fluid is respectively electric power and pressure, and two power are vertical.The cell that flow to the branch constituency under electric power and pressure synergy, according in the position of sample intake passage xsect various flows to different spaces.
A kind of micro-fluidic chip that is specifically designed to above-mentioned cell sorting method of the present invention is characterized in that this micro-fluidic chip has double-decker; One layer surface is carved with cross passage, and the liquid pool at vertical channel two ends is respectively cell pool and empty pond, and the liquid pool at two ends, horizontal channel is respectively Buffer Pool and waste liquid pool; One layer surface is coated with paired keeper electrode in addition; After the above-mentioned two-layer relative sealing-in, described electrode is positioned at described vertical microchannel just.
Among the present invention sample intake passage positioning electrode number can be a pair of also can be many to as Fig. 2, probe in the passage electrode width from several microns to several centimetres.
In the micro-fluidic chip of the present invention, the intersection region of horizontal channel and vertical channel can be one or more (as Fig. 9).The electrode certain position (as Fig. 8) that can directly relatively also can stagger.Chip material can be a multiple polymers, glass, pottery or their complex.Channel width is between 100 microns to 2000 microns.Cell can be the cell without any processing, also can be the cell that indicates various antibody or other molecules.
The present invention is suitable equally for acellular charged particle (diameter is at tens microns).
Description of drawings:
Fig. 1 is chip structure figure, and light-colored part is sort channel and liquid storage tank, and dark part is a keeper electrode;
Fig. 2 is that stream of cells is illustrated to right-angled intersection place track under pressure and local electric field force effect;
Fig. 3 illustrates along the pressure direction flow process for uniting under the driving cell at electric power, pressure;
Fig. 4 is for uniting cell two-way flow process signal under the driving at electric power, pressure;
Fig. 5 illustrates along electric power direction flow process for uniting under the driving cell at electric power, pressure;
Fig. 6 is for uniting cell signal along pressure against the direction flow process under the driving at electric power, pressure;
Fig. 7 is the carp erythrocyte speed transition graph through different disposal;
The another kind of position location of Fig. 8;
The connect chip in two right-angled intersection districts of Fig. 9.
Embodiment:
Micro-fluidic chip with cross passage is an operating platform, and the liquid pool at vertical channel two ends is respectively cell pool and empty pond.The liquid pool at two ends, horizontal channel is respectively Buffer Pool and waste liquid pool, and connects high voltage and ground connection respectively.Chip is by two-layer composition up and down.Lower floor plates the platinum electrode of required form and position, and after the sealing-in of upper strata, electrode is positioned at the microchannel.This electrode is called keeper electrode, sees Fig. 1;
Damping fluid is joined in Buffer Pool and the waste liquid pool, make damping fluid be full of all passages, then cell liquid is added in the cell pool, under the effect of pressure, cell flows to empty pond from cell pool;
The cell of the keeper electrode of flowing through moves to channel wall under internal field's effect, the cell of different charged character and density is radially arranged again at passage, the cell flow trace is seen synoptic diagram 2, the light cell among Fig. 2 and dark cell through the location electric field after in passage position change radially;
Under pressure and electrodynamic effect, the cell flow direction that flow to right-angled intersection place can be divided into pressure direction stream according to the size of two kinds of power, bidirectional flow, and electric power direction stream, synoptic diagram 3-6 is seen in four intervals such as pressure direction adverse current.At bidirectional flow interval (Fig. 4), the flow direction of cell depends on the residing position of cell when entering the intersection region, and the cell that is in different radial positions flows to different directions under two power effects.
Buffer pool and waste liquid pool successively add 13 microlitre 10mMPBS damping fluids, after microscopically is observed and all is full of damping fluid continuously in each passage, insert platinum electrode at buffer pool and waste liquid pool, connect 100V and ground respectively.Three pairs of keeper electrodes on the sample intake passage all insert equidirectional electric potential difference 5V, are 50V/cm thereby radially produce electric field intensity at passage.Add 13 microlitre carp red blood cell liquid (cell concentrations about 10
8Individual mL) in cell pool.Cell flows to empty pond under the static pressure effect.Cell departs from former pressure-driven track when flowing through keeper electrode, and more close channel side wall is seen Fig. 2.Difference through carp red blood cell migration velocity under electric field of different disposal is seen Fig. 7 and table 1.
Intersection region electric field intensity is 50V/cm, keeps being in the intersection region two-way flow interval.Through passage potential electrical field decided at the higher level but not officially announced when the stream of cells of ranking is crossed the zone of intersection again, according to the cell present position to different channel flow (see figure 4)s.
Table 1 cell is handled item and corresponding cell mobility
| Group number | The mobility cm of cell 2/v.s | Carp is handled item |
| NO.1 | ?1.1572E-04 | Contain the water quality cleanser in the breeding environment, not at carp tail muscles injection cyhalothrin |
| NO.2 | ?1.0472E-05 | Contain the water quality cleanser in the breeding environment, and injected the cyhalothrin of 6ug/kg concentration in the carp tail muscles |
| NO.3 | ?-8.328E-05 | Do not contain the water quality cleanser in the breeding environment, and injected the cyhalothrin of 6ug/kg concentration in the carp tail muscles |
Claims (7)
1, a kind of cell sorting method based on micro-fluidic chip is characterized in that: at first impose internal field in the conduit wall both sides of micro-fluidic chip, cell is rearranged according to the surface charge property difference at channel cross-section; Right-angled intersection place at passage imposes orthogonal electric power and pressure again, and orthogonal pair of power effect will make the stream of cells of reorientating to different spaces, thereby realizes the sorting of cell.
2, according to the described cell sorting method of claim 1 based on micro-fluidic chip, it is characterized in that local electric field strength at 100V/cm to 1000V/cm, be added in pressure on the cell from 1 handkerchief to 1000 handkerchiefs; Electric field is from 10V/cm to 1000V/cm.
3,, it is characterized in that described cell is the cell that indicates various antibody or other molecules according to the described cell sorting method of claim 1 based on micro-fluidic chip.
4, a kind of micro-fluidic chip that is specifically designed to claim 1,2 or 3 described cell sorting methods is characterized in that this micro-fluidic chip has double-decker; One layer surface is carved with cross passage, and the liquid pool at vertical channel two ends is respectively cell pool and empty pond, and the liquid pool at two ends, horizontal channel is respectively Buffer Pool and waste liquid pool; One layer surface is coated with paired keeper electrode in addition; After the above-mentioned two-layer relative sealing-in, described electrode is positioned at described vertical microchannel just.
5,, it is characterized in that described channel width is between 100 microns to 2000 microns according to the described micro-fluidic chip that is used for cell sorting method of claim 4.
6,, it is characterized in that the described width that is insinuated into the keeper electrode in the passage is from 1 micron to 10 centimetre according to the described micro-fluidic chip that is used for cell sorting method of claim 5.
7, according to claim 4, the 5 or 6 described micro-fluidic chips that are used for cell sorting method, it is characterized in that described chip material is a multiple polymers, glass, pottery or their complex.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 03133463 CN1566951A (en) | 2003-06-16 | 2003-06-16 | Cell sorting method based on micro flow control chips and special-purpose chips therefor |
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| CN 03133463 CN1566951A (en) | 2003-06-16 | 2003-06-16 | Cell sorting method based on micro flow control chips and special-purpose chips therefor |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053160A (en) * | 2009-11-06 | 2011-05-11 | 上海交通大学附属第一人民医院 | Micro-flow control cell chip capable of capturing various cells simultaneously |
| CN102262162A (en) * | 2010-05-26 | 2011-11-30 | 中国科学院大连化学物理研究所 | Microfluidic chip system for studying mechanical behaviors of cells |
| CN102530829A (en) * | 2010-12-16 | 2012-07-04 | 上海交通大学附属第一人民医院 | Method for preparing micro-fluidic chip by freeze drying process |
| CN104923322A (en) * | 2015-06-26 | 2015-09-23 | 河北工业大学 | Dielectrophoresis particle separation micro-fluidic chip |
| CN105378481A (en) * | 2013-07-09 | 2016-03-02 | 雀巢产品技术援助有限公司 | Microfluidic collaborative enzyme enhanced reactive ceer immunoassay |
| CN112834573A (en) * | 2020-12-30 | 2021-05-25 | 武汉大学 | Intermediate frequency micro-electric field biological effect rapid test method |
-
2003
- 2003-06-16 CN CN 03133463 patent/CN1566951A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053160A (en) * | 2009-11-06 | 2011-05-11 | 上海交通大学附属第一人民医院 | Micro-flow control cell chip capable of capturing various cells simultaneously |
| CN102053160B (en) * | 2009-11-06 | 2013-09-18 | 上海交通大学附属第一人民医院 | Micro-flow control cell chip capable of capturing various cells simultaneously |
| CN102262162A (en) * | 2010-05-26 | 2011-11-30 | 中国科学院大连化学物理研究所 | Microfluidic chip system for studying mechanical behaviors of cells |
| CN102530829A (en) * | 2010-12-16 | 2012-07-04 | 上海交通大学附属第一人民医院 | Method for preparing micro-fluidic chip by freeze drying process |
| CN102530829B (en) * | 2010-12-16 | 2014-11-05 | 上海交通大学附属第一人民医院 | Method for preparing micro-fluidic chip by freeze drying process |
| CN105378481A (en) * | 2013-07-09 | 2016-03-02 | 雀巢产品技术援助有限公司 | Microfluidic collaborative enzyme enhanced reactive ceer immunoassay |
| CN104923322A (en) * | 2015-06-26 | 2015-09-23 | 河北工业大学 | Dielectrophoresis particle separation micro-fluidic chip |
| CN112834573A (en) * | 2020-12-30 | 2021-05-25 | 武汉大学 | Intermediate frequency micro-electric field biological effect rapid test method |
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