CN100392384C - Entity molecule separating process on chip and required device and reagent - Google Patents

Abstract

The present invention provides a sample solution. After the sample solution is mixed with a sample, the sample solution has the characteristics that (a) at least one kind of dielectric property of at least one component of the sample is retouched selectively; (b) the sample solution has appropriate electric conductivity; one kind or a plurality of kinds of solid molecules of the sample can be separated by dielectrophoresis force. The present invention also provides a method for separating solid molecules of a sample by the solution.

Description

The method of entity molecule separating and sample solution on the chip

Technical field

The present invention relates to the bio-separation field, particularly on chip, entity molecule is carried out dielectrophoresis and magnetic separation.

Background technology

To biological sample or environmental sample carries out in genetic analysis, biochemical analysis or the bioanalysis, specimen preparation is wherein requisite link.Specimen preparation usually needs to isolate interested sample component from mixed system.This work is wasted time and energy, and is difficult to robotization.In many application, sample preparation steps need be carried out suitable purifying so that next step analysis to a kind of or several component in the sample.Dielectrophoresis in order to distinguish with " row ripple dielectrophoresis ", is also referred to as " conventional dielectrophoresis " sometimes, can be in non-homogeneous amplitude electric field to electric neutrality or charged corpuscle transports and locatees.Dielectric properties per sample, dielectrophoresis and row ripple dielectrophoresis can provide a kind of efficient, reliable, automated method, in order to the particulate in the sample separation, and can not destroy the structure of particulate.

Dielectrophoresis and row ripple dielectrophoresis have been used to separating mammary cancer cell from blood (Becker et al., Proc.Natl.Acad.Sci.USA 92:860-864 (1995)); Separation of bacterial from haemocyte (Hawkes et al., Microbios.73:81-86 (1993) and Cheng etal., Nat.Biotech.16:546-547 (1998)); Enrichment has the stem cell (Stephens et al., Bone Marrow Transplantation 18:777-782 (1996)) of CD34 activity from blood; Collect virion, submicron order pearl body and biomolecule (Washizu, et al, IEEETrans.Ind.App.30:835-843 (1994), Green and Morgan, J.Phys.D:Appl.Phys.30:L41-L44 (1997), Hughes et al., Biochim.Biophys.Acta1425:119-126 (1998), and Morgan et al., Biophys.J.77:516-525 (1999).Following patent has also been used method separating particles (comprising cell) (U.S.Patent No.4,390,403 issued Jun.28,1983 of dielectrophoresis; U.S.Patent No.4,326,934 issued April, 27,1982 to Pohl; U.S.Patent No.5,344,535issued Sept.6,1994 to Betts and Hawkes; U.S.Patent No.5,454,472issued Oct.3,1995 to Benecke et al.; U.S.Patent No.5,569,367 issuedOct.29,1996 to Betts et al.; U.S Patent No.5,653,859 issued Aug.5,1997; U.S.Patent No.5,795,457 issued Aug.18,1998 to Pethig, et al.; U.S Patent No.5,814,200 issued Sep.29,1998 to Pethig et al.; U.S.Patent No.5,858,192 issued Jan.12,1999 to Becker et al.; U.S.Patent No.5,888,370 issued Mar.30,1999; U.S.Patent No.5,993,630 issued Nov.30,1999 to Becker et al.; U.S.Patent No.5,993,631 issued Nov.30,1999to Parton et al.; And U.S.Patent No.5,993,632 issued Nov.30,1999 toBecker et al).

Dielectrophoresis is meant the translation motion of particulate in AC field (having uneven amplitude) of polarization.When particulate is inserted electric field, if there is certain difference in the dielectric properties of particulate and its surrounding medium, particulate will be polarized.Like this, will induce electric charge at particulate-medium interface.If extra electric field is an AC field heterogeneous, so, electric charge that particulate is induced and extra electric field interact, and the net effort that particulate is subjected to is non-vanishing, thereby particulate is moved to high field or feeble field zone.The suffered net effort of particulate is called dielectrophoresis force, and the motion of particulate is called as dielectrophoresis.The suffered dielectrophoresis force of particulate is by dielectric properties, the dielectric properties of particulate surrounding medium, the frequency of extra electric field, the amplitude distribution decision of particulate itself.In the literature, dielectrophoresis is also sometimes referred to as conventional dielectrophoresis (for example, Wang et al.Biochim.Biophys.Acta 1243:185-194 (1995)).For simplicity, in this patent, we use " dielectrophoresis " to refer to the motion of particulate in inhomogeneous field that polarizes.The power that acts on accordingly on the polarisable particle is called " dielectrophoresis force ".

Utilize the isolation technics of dielectrophoresis principle to have dielectric to be detained (or claiming dielectric to attract), dielectric migration and dielectric electric current/gravity field flow point from (DEP/G-FFF) technology.Dielectric delay technology is meant, one or more sample particulates are owing to be subjected to the forward dielectrophoresis force, thereby attracted to and remain on chip or the strong field of reaction tank (chamber), normally electrode edge zone.Using dielectric to be detained in the detachment process of technology, be subjected to the negative sense dielectrophoresis force or the component of faint forward dielectrophoresis force in the sample along with field flow is taken out of reaction tank.In dielectric migration, particulate is based on self suffered different dielectric electrophoretic force and separated.For example, one or more particulates are moved to a certain zone of reaction tank owing to being subjected to the negative sense dielectrophoresis force, and another or multiple particulate are moved to another zone of reaction tank owing to being subjected to the forward dielectrophoresis force.In (DEP/G-FFF), particulate be in differing heights in reaction tank, and the medium of differing heights has different mobilities in the reaction tank owing to be subjected to the negative sense dielectrophoresis force at dielectrophoresis/gravity field flow point, thereby the field flow of realizing particulate separates.

Row ripple dielectrophoresis (TW-DEP), relevant with above-mentioned dielectrophoresis (or being sometimes referred to as conventional dielectrophoresis), be called " traveling-wave field migration " or " TWFM " again.The electric field of row ripple dielectrophoresis has PHASE DISTRIBUTION heterogeneous.The polarization charge that causes by electric field that row ripple electric field action has on particulate, thus make particulate be subjected to corresponding acting force, make particulate along or move against the direction of transfer of row ripple electric field.The suffered capable ripple dielectrophoresis force of particulate is determined by the dielectric properties of particulate itself, the dielectric properties of particulate surrounding medium, frequency, amplitude and the PHASE DISTRIBUTION of row ripple electric field.In " two-dimentional dielectrophoresis " (2-D DEP), separation of particles is by applying dielectrophoresis force and the realization of row ripple dielectrophoresis force simultaneously on particulate.(De Gasperis et al.，Biomedical Microdevices 2：41-49(1999))。In addition, in " two-dimentional dielectrophoresis ", particulate also is to be in the field of flow.

The theory of dielectrophoresis and row ripple dielectrophoresis and use the method that dielectrophoresis handles particulate can be referring to below with reference to document: Wang et al.Biochim.Biophys.Acta1243:185-194 (1995); Wang et al.IEEE Transaction on IndustryApplications 33:660-669 (1997); Huang et al.J.Phys.D:Appl.Phys.26:1528-1535; Fuhr et al.Sensors and Materials 7:131-146; Wang et al.Biophys.J.72:1887-1899 (1997); And Becker et al.Proc.Natl.Acad.Sci.92:860-864 (1995).

Particulate to be handled comprises artificially produced particles, crystal, colloid, molecule, compound, molecular complex, cell and organelle.Use dielectrophoresis and row ripple dielectrophoresis to the maneuverability pattern of particulate comprise concentrated, assemble, catch, repulsion, suspension, separate, or make particulate do linearity or other directed motion.Particulate can be hunted down, be enriched in the specific region in the reaction tank.Particulate can be subdivided into each subclass on fine level, also can be transported on certain distance.Being used for the required Electric Field Distribution of special particulate manipulation can design by relevant dielectrophoresis theory and electric field simulation method, and the distribution of electric field is determined by the size and the geometric configuration of electrode in concrete the application.

In many cases, because the dielectric properties of entity molecule (as cell and molecule) to be separated is close in the sample, only be difficult to separately each component in the sample by dielectrophoresis force and/or row ripple dielectrophoresis force.In other situations,, also be difficult to from sample, separate because the size of entity molecule to be separated is less.Because the size of the dielectrophoresis force that entity molecule is suffered is proportional to the size of waiting the entity molecule that transports or locate, so entity molecule of reduced size, (for example: nucleic acid molecules and protein) manipulation need apply highfield, promptly needs to apply high pressure as molecule.And high pressure can destroy biological substance, as cell.High pressure also can bring thermal effect, and medium is heated up, and may certain destruction all be arranged to biology or abitotic substance.So need a kind of method that can carry out high efficiency separation to the entity molecule of various sizes, various dielectric propertiess.

The separation of blood sample and analysis are that sample separation and analysis field have challenging problem.Blood sample is easy to obtain, and a large amount of relevant with metabolism status, genetic, information with diagnosis and disease forecasting value can be provided.

But, the red blood cell of the abundant acellular nuclear that comprises in the blood, and a large amount of haemoglobins has but hindered normally carrying out of gene, metabolism, diagnostic test.The red blood cell of removing in the blood can be by method centrifugal and that filter, and is difficult with robotization as these step 1 of adding.In some other step, from blood sample, obtain red blood cell by the method that makes the red blood cell born of the same parents separate centrifugal then other cell of collection earlier.These red blood cells born of the same parents separate damping fluid has certain spinoff to leucocyte.This just requires the separation of dialogue cell to be limited in the regular hour to finish, and is destroyed to avoid leucocyte, and this brings very big inconvenience with regard to the experimenter who gives analytic sample.In addition, the additional step of centrifugal process needs is difficult to realize robotization.

No. 99120320.8 Chinese patent application that the present invention and the applicant submitted on September 16th, 1999 and No. 00122631.2 Chinese patent application of submitting on August 8th, 2000 are associated, and work of the present invention is the continuity to above-mentioned application.The content of these two parts of applications is hereby incorporated by reference.

The present invention has fully taken into account that the separation of each component can offer convenience for sample analysis in the sample, and this sometimes separation is necessary for sample analysis.Based on the dielectric properties of entity molecule in the sample, the dielectrophoresis isolation technics can provide a kind of efficient, reliable, automated method, in order to the entity molecule in the sample separation, and can not destroy the structure of entity molecule.The present invention has provided the entity molecule in the sample has been carried out the improved method of dielectrophoresis separation and required device and reagent.

At first, the present invention has provided a kind of like this solution, and solution should satisfy following demand: when with sample mix after, can change the dielectric properties of one or more components in the sample; The conductivity of solution is also suitable, so that one or more entity molecules in the sample can be separated by the dielectrophoresis method; Solution can be used for the sample analysis on the chip; Working as in addition to analyze needs to use bond, and comprising can be by dielectrophoresis force, when going the ripple dielectrophoresis force or the particulate of electromagnetic force manipulation, and this solution also can be suitable for.

Secondly, the present invention has provided a kind of method that can carry out the dielectrophoresis separation to one or more entity molecules in the sample, wherein used the solution that can change one or more component dielectric propertiess in the sample, and this solution has suitable conductivity, and the entity molecule in the sample can utilize dielectrophoresis force or row ripple dielectrophoresis force to be separated therein.This method can also be used energy and entity molecule coupling connection, and the particulate that can be handled, locate by dielectrophoresis.

Once more, the present invention has provided the method that a kind of use can separate one or more entity molecules in the sample with the magnetic bead of separated entity molecule combination, and this method has also been used and a kind ofly can modify erythrocytic solution and use electromagnetic force simultaneously.Electromagnetic force can be produced by electromagnetic component on the chip that is used to separate or structure.

Fig. 1 has shown a kind of design proposal of the present invention, sample solution wherein of the present invention is added into blood sample by branch road, mixed liquor is passed on the chip that contains interlaced parallel pole array subsequently, and leucocyte is separated by dielectric delay technology.

Fig. 2 has shown another kind of design proposal of the present invention, and sample solution and one group of particulate are added into blood sample earlier, and then mixed liquor is added the polynomial expression electrod-array, utilizes dielectric delay technology that entity molecule is separated.

Fig. 3 has shown another kind of design proposal of the present invention, and sample solution is added into blood sample earlier, and then mixed liquor is added the screw electrode array, utilizes dielectric delay technology that interested entity molecule is separated.

Fig. 4 has shown another kind of design proposal of the present invention, and sample solution is added into blood sample earlier, and then mixed liquor utilization row ripple dielectrophoresis technology is separated entity molecule.

Fig. 5 has shown another kind of design proposal of the present invention, and sample solution and one group of magnetic bead are added into blood sample earlier, and then utilize electromagnetic force that the entity molecule in the sample is separated.

Definition

Unless specified otherwise, herein all technology and scientific and technical terminology all with the present invention under the field in meaning consistent. Usually, herein and the employed term of production/process of the test in this area, all be known. Use some traditional methods in the process of the present invention, seen technology or the comprehensive list of references quoted. Orientation term "up" and "down", or " top " and " below " suchlike usage all refers to the orientation of the parts of employed device. As described below in this article with process of the test in the term that uses in association area, all be in daily use. When the term in the list of references and definition herein were variant, employed term was as the criterion with the definition of this paper in this article. Following term, unless other specified otherwise in addition, otherwise all be as the criterion with the following description:

" Hyposmolality " solution refers to that with respect to haemocyte be hypotonic solution, and osmotic pressure is lower than 300mOsm usually.

" alternative born of the same parents separate erythrocytic solution " refers to a kind of like this solution, when this solution with after comprising erythrocytic sample mix, born of the same parents occur red blood cell separates, in the cell membrane punching its permeability is strengthened, thereby or other effect so that the erythrocytic destroy integrity in the sample. Like this, the red blood cell in the sample mix liquid just can not be to responding in order to the dielectrophoresis force that separates other cell or entity molecule.

" low conductivity " solution is meant a kind of under certain dielectrophoresis separation condition, and interested entity molecule can carry out the forward dielectrophoresis therein.Best, the conductivity of the solution of low conductivity is between 1 μ S/cm to 1S/m; The conductivity of better solution is between 5 μ S/cm to 0.5S/m; Optimal cases is that the conductivity of solution is between 10 μ S/cm to 0.1S/m.

" dielectrophoresis " is the motion of polarisable particle in non-homogeneous amplitude electric field.The dielectrophoresis that two kinds of forms are arranged usually, forward dielectrophoresis and negative sense dielectrophoresis.In the forward dielectrophoresis, particulate is moved to strong field by the effect of dielectrophoresis force.In the negative sense dielectrophoresis, particulate is subjected to the effect of dielectrophoresis force to the feeble field regional movement.It still is the negative sense dielectrophoresis that particulate is made forward, is by the relative degree of polarization decision of particulate and medium.

" dielectrophoresis force " is the power that polarisable particle is subjected in non-homogeneous amplitude AC field.In the inhomogeneous field, radius is the dielectrophoresis force that the particulate of r is subjected to

Can be expressed as:

E wherein _RmsBe the root-mean-square value of field intensity, symbol

Be the symbol of expression gradient operation, ε _mIt is the dielectric coefficient of medium.χ _DEPBe the polarization factor of particulate, can be expressed as:

${\mathrm{\χ}}_{\mathrm{DEP}}=\mathrm{Re}\left(\frac{{\mathrm{\ϵ}}_p^{*}-{\mathrm{\ϵ}}_m^{*}}{{\mathrm{\ϵ}}_p^{*}+2{\mathrm{\ϵ}}_m^{*}}\right)$

Here Re refers to real, symbol ${\mathrm{\ϵ}}_x^{*}={\mathrm{\ϵ}}_x-j{\mathrm{\σ}}_x/\left(2\mathrm{\πf}\right)$ Be compound dielectric coefficient (wherein x=p is corresponding to particulate, and x=m is corresponding to medium).Parameter ε _p, σ _pBe respectively effective dielectric coefficient and conductivity (may be relevant) of particulate with impressed frequency.For example, typical biological cell will have conductivity and the dielectric coefficient (is because the cytoplasma membrane polarization causes) with frequency dependence to small part.

The formula of more than describing dielectrophoresis force also can be expressed as

Wherein p (z) is the distribution of the electric field value square of unit voltage excitation on the electrode (voltage V=1V), and V is an impressed voltage.

" row ripple dielectrophoresis force " is meant the power that is subjected on molecule in the ripple electric field of being expert at or the particulate.The feature of desirable traveling-wave field is that the phase value of AC field component is the linear function of particulate present position.To a radius is r, is subjected to row ripple electric field

The particulate of (the x component that is the E field is advanced along the z direction, the phase value of x component along z to the linear function that is the position) effect, effect capable ripple dielectrophoresis force thereon

For:

Wherein E is the field intensity size, ε _mBe the dielectric coefficient of medium, ζ _TwDEPBe the capable ripple dielectrophoresis polarization factor of particulate:

${\mathrm{\ζ}}_{\mathrm{twDEP}}=\mathrm{Im}\left(\frac{{\mathrm{\ϵ}}_p^{*}-{\mathrm{\ϵ}}_m^{*}}{{\mathrm{\ϵ}}_p^{*}+2{\mathrm{\ϵ}}_m^{*}}\right)$

Wherein Im refers to the imaginary part of corresponding plural number, symbol ${\mathrm{\ϵ}}_x^{*}={\mathrm{\ϵ}}_x-j{\mathrm{\σ}}_x/\left(2\mathrm{\πf}\right)$ Be compound dielectric coefficient, parameter ε _p, σ _pBe respectively effective dielectric coefficient and conductivity (may be relevant) of particulate with impressed frequency.

On the microelectrode that is arranged on the chip, apply suitable AC signal and just can produce capable ripple electric field.In order to produce capable ripple electric field, on electrode, should apply the electric signal of three groups of out of phase values at least.In the example of a capable ripple electric field, used the electric signal (being respectively 0,90,180,270 degree) of four kinds of out of phase values, be applied on the parallel pole that is arranged in chip surface of four wire, four such electrodes can be used as a basic repetitive in order to chip design.Needs according to using can get up unit cell arrangement such more than two, thus above the electrode or near generation row ripple electric field.As long as electrode unit is spatially lined up according to certain order, the signal of the out of phase that adds just can produce row ripple electric field at the electrode near zone.

" electric field intensity map " is meant Electric Field Distribution, and it is geometric configuration and the frequency of electric field modulation and/or the function of amplitude of electric field frequency, electric field magnitude, electrode structure.

" dielectric properties " to the small part of entity molecule is to be determined by the response of entity molecule to electric field.The dielectric properties of entity molecule comprises effective conductivity and effective dielectric coefficient of entity molecule.For by identical or the compound that analog structure is formed, for example, the polystyrene bead body, its effective conductivity and effectively dielectric coefficient and at least within a large range (for example 1 hertz to 100 megahertzes) have nothing to do with electric field frequency.Particulate with homogeneous composition has surperficial net charge, when these charged corpuscles are suspended in the medium, will form electric double layer at the surface of contact of particulate/medium.Extra electric field and this electric double layer interact and have caused particulate effective conductivity and the effectively change of dielectric coefficient.Extra electric field is normally relevant with frequency with the interaction of electric double layer, and therefore, the particulate effective conductivity is with effectively dielectric coefficient is also relevant with frequency.For the compound that constitutes by different component, for example, cell, its effective conductivity and effective dielectric coefficient are made up of two parts, be the effective conductivity of cell membrane and the effectively effective conductivity and the effective dielectric coefficient of dielectric coefficient and cell interior, and and the frequency dependence of electric field.In addition, the size of suffered dielectrophoresis force of entity molecule and entity molecule is relevant in the electric field, and like this, the size of entity molecule also should be taken into account among the dielectric properties of entity molecule.To the character of the contributive entity molecule of dielectric properties of entity molecule comprise it with net charge, its composition (comprise with chemical group, other entity molecule or the like), its size, surface parameter, surface institute electrically charged.

" magnetic field force " is meant the acting force that particulate is subjected in magnetic field.Usually, handle particulate in order to use magnetic field force, particulate must have magnetic or be magnetized.For a magnetic bead of typically making, be when this particulate is in magnetic induction intensity by paramagnetic material

Magnetic field in, the magnetic dipole that induces Can be expressed as:

V wherein _pBe the volume of particulate, χ _pAnd χ _mBe respectively the volume coefficient of magnetization of particulate and its surrounding medium, μ _mBe the magnetic permeability of medium,

It is magnetic field intensity.The magnetic field force F that particulate is suffered _MagneticDetermine by instantaneous magnetic dipole and magnetic field gradient:

Wherein " ● " and "

" represent dot product and gradient operational character respectively.The effect whether particulate is subjected to magnetic field force is by the difference decision of the volume coefficient of magnetization of particulate and surrounding medium.Normal conditions are, particulate (the volume coefficient of magnetization substantially exceeds zero) is suspended in (the volume coefficient of magnetization of medium is approximately zero) in liquid state, the nonmagnetic medium, and particulate just is subjected to the effect of magnetic field force like this.When the suffered magnetic field force of particulate and liquid viscous force reach balance, the speed v of particulate _ParticleCan be expressed as:

Wherein r is the radius of particulate, η _mBe the viscosity of particulate surrounding medium.

" component " of sample or " sample component " are meant any ingredient of sample, can be the particulate of ion, molecule, compound, molecular complex, organ, virus, cell, condensate or any kind, comprise colloid, condensate, microsome, crystal, mineral body etc.The composition of sample can be soluble also insoluble in sample media or sample buffering that provides or sample solution.The composition of sample can be gas, liquid or solid form.

" entity molecule " or " interested entity molecule " is meant any predetermined substance molecule that can handle with dielectrophoresis force, row ripple dielectrophoresis force or electromagnetic force.Entity molecule can be a solid, comprises the solid of suspension, also can be to exist with dissolved state.Entity molecule can be a molecule, the molecule that can be handled comprises, but be not limited only to, inorganic molecule (comprising ion and mineral compound), can be organic molecule also, comprise amino acid, peptide, protein, glycoprotein, lipoprotein, GLP, fat, fat, sterol, sugar, carbohydrates and other micromolecule or complicated organic molecule.Entity molecule also can be a molecular complex, such as organelle, one or more cells (comprising protokaryon and eukaryotic), pathogen (part that comprises virus, parasite, PrPC or these materials); Entity molecule also can be crystal, mineral matter, colloid, fragment or the like, can be made up of one or more dead matter such as polymkeric substance, metal, mineral, glass, pottery or the like; Entity molecule also can be molecule aggregate, compound, cell, organelle, virus, pathogen, crystal, colloid or other fragment.Cell can be the cell of any kind of, comprises protokaryon and eukaryotic.Wherein eukaryotic also can be an any kind.Common interested cell comprises, but is not limited only to, leucocyte, malignant cell, stem cell, the early stage cell of differentiation, fetal cell, by the cell of pathogenic infection, bacterial cell or the like.

" entity molecule in the cell " is meant and is positioned at intracellular entity molecule, promptly be arranged in tenuigenin or organelle substrate, on any cell inner membrance, be positioned on the plasma membrane (if existence), or be positioned at cell surface, promptly on the outside surface attached to cytoplasma membrane or cell membrane (if existence).

" manipulation " is meant and moves and handle these entity molecules, thereby cause entity molecule on the chip (be included on the single-chip or on many integrated chips or between, between a plurality of substrates in the substrate or in device) do the motion on one dimension, two dimension or the three-dimensional." manipulation " comprise, but be not limited only to, transport, focusing, enrichment, concentrate, assemble, catch, the moving of the entity molecule on repulsion, suspension, separation, fractionation, isolation, linearity or other direction.In order to realize handling efficiently, entity molecule to be handled and the physical force that is applied thereto should be coordinated.For example, the entity molecule with magnetic can apply with magnetic force.Similar, the entity molecule with electric charge can apply with electrostatic force (being electrophoretic force).In the situation of handling connector-entity molecule compound, the character of these entity molecules or corresponding connector and the physical force that is used to handle must be coordinated.For example, entity molecule or its connector have certain dielectric properties, can dielectric polarization, can use dielectrophoresis force.

" surface of entity molecule to be handled and connector fully combine " is meant that the entity molecule to be handled of certain percentage is combined in the surface of connector and can passes through manipulation connector and then the realization manipulation to entity molecule with suitable physical force.Usually, at least 0.1% entity molecule to be handled is the surface that is combined in connector.Preferably is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% entity molecule is the surface that is combined in connector.

" entity molecule to be handled is combined on the surface of connector fully " is meant that at least 90% entity molecule to be handled is combined on the surface of connector.Preferably is, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or 100% entity molecule to be handled is combined on the surface of connector.

" optionally modify erythrocytic solution " and be meant that this solution can change the character of erythroplastid, make it not disturb other cell in the blood sample or the dielectric separation of component, the simultaneously as far as possible little leukocytic integrality of influence, or do not influence the process of other component in leucocyte and the blood sample being separated with the method for dielectrophoresis.

" sample " is meant and anyly can therefrom separates or analyze the fluid of component.Sample can have various sources, can be from a biosome, and from a group biology of identical or different kind, also can be from environment, as take from a water body or soil, perhaps from food or industrial materials.Sample can be finished, also can be unprocessed.Sample can be a gas, liquid, and semisolid also can be solution or suspension.Sample can be a kind of extract, as takes from the liquid extract of soil or foodstuff samples, as throat or phallic extract, or as taking from the extract of faecal samples.

" blood sample " is meant at this and processed or unprocessed blood sample, such as, but be not limited only to, it can be the blood sample through centrifugal, filtration, extracting or other processing, can also be to have added anticoagulant, the blood sample of one or more reagent such as stabilizing agent.Blood sample can be an arbitrary volume, and the source is any, as animal or human's class.First-selected material is from the mankind.

" leucocyte " is meant white blood cell, or a class can in animal blood, find neither the non-again hematoblastic hemopoietic cell of granulophilocyte.White blood cell can comprise lymphocyte, as bone-marrow-derived lymphocyte or T lymphocyte.White blood cell also can comprise phagocyte, as monocyte, and macrophage and include basocyte, eosinocyte, the granular white blood cells of neutrophil cell.White blood cell also comprises mast cell.

" red blood cell " is meant red blood cell.

" tumour cell " is meant the faster abnormal cell of a class cell proliferation growth fraction normal cell, it in addition can still continued growth after the stimulation that is subjected to causing new growth retardation.Compare with normal structure, oncocyte tends to partly or entirely lack structure organization and functional harmony, it both may be optimum also may be pernicious.

" malignant cell " is meant that a class has local invasion property and the destructive cell of growing and shifting.

" stem cell " is meant the cell that a class is broken up, and it can be taken turns or many wheel cellses differentiation causes growth by one, generates at least a noble cells.

" break up early stage cell " and be meant that a class is broken up but must will cause the cell of growth, it is taken turns by one or the differentiation of many wheel cellses causes growth, generates at least a noble cells.More representationally be, a stem cell responds to a special stimulation or a series of stimulation, take turns or early stage cell of differentiation of many wheel cellses differentiation generation by one, break up early stage cell by this then a special stimulation or a series of stimulation are responded, generate one or more noble cellss.

" pathogen " is meant any pathogen, as bacterium, virus, parasite or PrPC that can infection biological.Pathogen can cause being subjected to the biology of its infection disease million to occur or be in morbid state.Human pathogen is meant and can infects human pathogen.Human pathogen both can be that specificity is at the mankind, as the human pathogen of specificity; Can infect multiple biology again, as mixing property human pathogen.

" object " is meant any biosome, as animal or human's class.Animal can comprise any animal body, as wild animal, and domestic animal such as cat or dog, agricultural animal such as pig or ox, or animal such as horse are used in amusement.

" reaction tank " is a kind of structure of compositing chip, and it can the receiving fluids sample.Reaction tank can have various sizes, and volume is from 0.001ml to 50ml.

" port " is meant the opening of reaction tank body, and fluid sample can be by its turnover reaction tank.Port can be any size, but suitable be to allow sample by pipette, syringe, conduit or other add the shape and size that sample loading mode adds reaction tank.

" conduit " is in order to liquid is transferred to a kind of mode of reaction tank by container in this patent.The suitable port that is applied to reaction tank of conduit.Conduit can be made up of the material that any permission liquid passes through.Relatively Shi Yi conduit is a tubular structure, such as rubber tube, polyfluortetraethylene pipe or polyethylene pipe.Conduit can be an arbitrary dimension, but suitable be that internal diameter is between 10 microns to 5 millimeters.

" chip " is meant that can carry out one or many on it handles as physics chemistry, biochemistry, the solid matter of biological treatment.These processing can be to analyze, and comprise biochemical, cell and chemical analysis; Also separate, comprise making electricity consumption, magnetic, physics, (comprising biochemical) acting force or interaction of chemistry; Can be chemical reaction, enzyme reaction and association reaction also, comprise and catching.Be shaped on the chip or be integrated with such as the little processing structure of groove, passage, electrode unit or the like, can carry out reaction or processing physics, biophysics, biological, chemistry thereon.Chip is a thin slice.Requirement to the chip surface area size is not harsh, such as from 1mm ²To 0.25m ²Can.Preferably used chip surface area is from 4mm ²To 25cm ²About.Chip can have difformity, shape such as rectangle, circle, ellipse or other irregular shape of rule.Chip surface can be flat, also can be uneven.The chip that has a non-planar surface should comprise from the teeth outwards by processing at chip surface or structures such as the groove that etching gets, reaction tank.

" separation " is meant one or more components in the sample and the other spatially separated process of one or more components.Detachment process can be carried out like this, one or more interested entity molecules are located in certain or some position in the discrete device, at least other component in the sample segment is located in other position, or removes from the position at interested entity molecule place.Perhaps, one or more components in the sample can be positioned at some position, and one or more interested entity molecules are removed from this position, collect again.Also one or more components in the sample can be positioned at some position, again one or more other interested entity molecules be navigated to other position.Separation can realize by using physical force, chemical force, electric field force or magnetic field force or the like, can also use gravity field, fluid, dielectrophoresis force, row ripple dielectrophoresis force and electromagnetic force or the like to realize.

" catch " and be meant such class separate mode, this mode is trapped in one or more zones of chip one or more entity molecules, applies physical force again and catches.

" analysis " is a test that component is carried out to sample or sample.The existence that analysis can detect certain component whether, the quantity of certain component or concentration, the composition of certain component, and the activity of certain component or the like.Analysis can be used with method of the present invention and device, reagent, comprises biochemical analysis, binding analysis, cell analysis and genetic analysis.

" reaction " is meant the chemistry or the biochemical component that can change one or more molecules or compound, perhaps changes the chemistry or the biochemical process of mutual relationship between one or more molecules and another or multiple molecule or compound.Reaction among the present invention can comprise by enzymatic, but is not limited only to, and degradation reaction arranged, synthetic reaction, modification reaction or association reaction.

" binding analysis " is a kind of analysis mode, it is by combining the predetermined substance molecule existence or the concentration that detects this predetermined substance molecule with specificity junction mixture, perhaps detect the possibility that combines of a predetermined substance molecule and another predetermined substance molecule, perhaps detect the binding affinity of a predetermined substance molecule and another predetermined substance molecule.The predetermined substance molecule can refer to an organic or inorganic molecule, and one by an organic and inorganic or organic-inorganic composite body, organelle, the molecule mixture that virus or cell are formed.Binding analysis can use detectable mark or can produce the signal generating system of detectable label in the presence of in conjunction with the predetermined substance molecule.The binding analysis of standard includes the dependence nucleic acid hybridization and detects special nucleotide sequence, relies on the combination of antibody to the predetermined substance molecule, and relies on the combination of part to acceptor.

" biochemical analysis " is meant a kind of existence of one or more components of sample, concentration or the active mode of analyzing.

" cell analysis " is meant a kind of analysis mode that detects cell processes, for example, but be not limited only to metabolic activities, catabolic activity, ion channel activity, intercellular signal conduction activity, receptor-mediated signaling activity, transcriptional activity, translation activity or secretion activity or the like.

" genetic analysis " is meant a kind of existence of certain hereditary unit or analysis mode of sequence of detecting.This hereditary unit can refer to any segment of DNA or RNA, comprises, but is not limited only to, and gene, repetitive sequence, transposable element, regulation and control unit, telomere, centriole or other have DNA or the RNA segment of not knowing function now as yet.The example of genetic analysis is as (but being not limited only to these examples), and nucleic acid hybridization technique comprises the nucleic acid sequencing reaction, can use one or more polymerases, for example the genetic analysis of PCR-based.Genetic analysis can be used one or more detectable marks, such as, but be not limited only to fluorescein, radioactive isotope or signal generating system.

" gene expression analysis " (or being called gene expression spectrum analysis) is the analysis that different genes expression product such as mRNA are carried out.The multiple mRNA of interested cell is detected simultaneously in the sample.In the different application, the quantity of detected mRNA can be different with type.

" protein expression analysis " (or being called the protein expression analysis of spectrum) is the analysis that multiple protein is carried out.The multiple protein of interested cell is detected simultaneously in the sample.In the different application, the quantity of detected protein can be different with type.

" electrode " is a kind of structural unit of being made by high conductive material.Here so-called high conductive material is meant that the conductance of material is much larger than surrounding medium or material.High conductive material comprises metal, as gold, chromium, platinum aluminium or the like, also can be nonmetal, as graphite, conducting polymer.Electrode can be made different shape, as rectangle, circle, castellated or the like.Also can comprise doped semiconductor in the electrode material, as mixing phosphorus on the silicon chip.

" groove " is the structural unit on the chip, has a lower surface and one or more side that extends out at a certain angle from the bottom surface.The parameter of side is very random, for example can have S shape or crooked or the side with a plurality of angles.The bottom surface of groove can be lower than, be higher than or and chip upper surface level.The material of side should be different with the material of the lower surface of chip.For example, the chip lower surface can be made of by the material that electric field force (comprising dielectrophoresis force, row ripple dielectrophoresis force and electromagnetic force) penetrates one deck, and the side of groove is made up of other material, as insulating material, can hinder penetrating of electric field force.The side of groove or the passage on the chip can be made by any suitable material, as silicon, glass, rubber, polymer, plastics, pottery, metal or the like.

" passage " is the structural unit on a kind of chip, has a lower bottom surface and at least two sides that extend up from the bottom surface, and the length of side is than big many of the distance between the side, and the inside of the cavity that liquid can form from bottom surface and side is flow through.It also can be open that passage can seal.

" continuous flow " is meant in detachment process of the present invention, and fluid is pumped into or injects reaction tank by continuous.Like this, the component that is not trapped in the chip by selectivity in the sample will be taken out of reaction tank along with fluid.

" connector " is meant any any material that can link to each other with specificity with certain compatibility with entity molecule, and can handle by corresponding physical force.Connector can be, but be not limited only to cell, organelle, virus, particulate, condensate or complex, the perhaps condensate of molecule or complex.

" particulate " is meant Any shape, and any composition has the particulate of any composite structure, can handle by corresponding physical force.Particle size can be from about 0.01 micron to about 10 centimetres.More suitably be, the size of the particulate in this method from about 0.1 micron to about several thousand microns.Particulate can be made by various suitable materials, and example comprises, but is not limited only to glass, pottery, polymkeric substance such as nylon, teflon (TEFLON ^TM), polystyrene, polyacrylamide, agarose, cellulose, glucosan or the like.The example of particulate comprises, but be not limited only to, plastic particles, ceramic particle, carbon particulate, polystyrene bead body, beaded glass, magnetic bead, Hollow Glass Sphere, metal particle and particle complex, little contactless microstructure that is processed to form (" Design of asynchronousdielectric micromotors ", Hagedorn et al., in Journal ofElectrostatics, 1994, Volume:33, Page 159-185) etc.The particulate of composite parts is meant the particulate that those are made up of multiple heterogeneity, and for example bag is by the Metal Ball of the non-conductive polymer film of one deck.

" coupling connection " is combination.For example, entity molecule can carry out special or non-specific combination with other particulate.In conjunction with being covalent bond, can be non-covalent bond also, be reversible combination also can the reversible combination of right and wrong.

" specific junction mixture " is meant in two such different moleculars, like this surface of molecule have specific morphosis can be specific and the space or the complementary combination of polar structure of another molecule.Specific junction mixture can be a kind of in two kinds of materials of specific bond in the immune family, as Ag-Ab, biotin-avidin, biotin-Streptavidin, ligand-receptor, double-strandednucleic acid, IgG-albumin A, DNA-DNA, DNA-RNA, RNA-RNA or the like.

" nucleic acid molecules " is meant polynucleotide.Nucleic acid molecules can be DNA, RNA or both combinations.Nucleic acid molecules also can comprise except ribose and ribodesose, form skeletal chain carbohydrate, can be the nucleic acid molecule outside DNA or the RNA.Nucleic acid molecules can be made up of the nucleotide base that exists naturally that exist naturally or non-, as xanthine, and the extension 2-aminoacyl purine of nucleoside base or analog etc.Nucleic acid molecules can be the peptide nucleic acid molecule.Nucleic acid molecules can be a random length, can be strand or two strands, or part strand and partially double stranded.

" mark that can be detected " is a kind of compound or molecule that can be detected, and they can produce output signal, the existing or method that can produce output signal that Future Development goes out as fluorescence, radioactivity, color, chemiluminescence or technical field.Output can be based on fluorescence, as by fluorescent marker, and Cy-3 for example, Cy-5, phycoerythrin, phycocyanin, allophycocyanin, fluorescein isothiocynate (FITC), rhodamine, or lanthanide series or the like, but be not only to be limited to above material; Also can be by fluorescin such as green fluorescent protein (GFP) and its variant, be based on the activity of enzyme reaction, such as, but be not limited only to β-nougat, beta-lactamase, horseradish peroxidase, alkaline phosphatase or luciferase or the like; Perhaps can based on radioactive isotope (as ³³P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, ³²P or ¹³¹I etc.).Label also can be by other base group modification base, such as, modify pyrimidine in the C5 position or modify purine in the N7 position.Modification group can be diversified, for example halogen, ether or polyethers, alkyl, lipid or polyester, or common XR, and here, X is a linking group, R is a modification group.In the modification technique field, exist many kinds effectively may be applicable to the method for modified nucleic acid molecule, oligonucleotide molecules and analog thereof.(A Practical Approach，Eckstein，ed.(1991)and inPCT/US94/00193.)

" signal generating system " can comprise one or more components, and one of them plants component is detectable bond.Signal generating system comprises that all are in order to produce or to strengthen the reagent that can survey signal (or act on mark signal to produce by certain method).Signal generating system provides a kind of mark that can use the external detection method to detect, and this method normally realizes by measurement absorbing light and radiative difference of wavelength.Signal generating system also can comprise chromogenic substrate and enzyme, and wherein chromogenic substrate absorbs the light of visible region or ultraviolet region or phosphorescence, fluorescence, thereby shows color under the effect of enzyme.Certainly, signal generating system also can use detectable label such as radioactive isotope so that detectable signal to be provided.

Signal generating system comprises at least a catalyzer (normally at least a enzyme) and at least a substrate, also can comprise two or more catalyzer and one group of substrate (such as comprising one group of enzyme, wherein a kind of substrate of enzyme is the product of another kind of enzyme).The operation of signal generating system is to produce detectable signal in predetermined site, promptly means in this predetermined site to have corresponding mark.

In order to obtain detectable signal, this just requires the signal that the mark at predetermined site produces is amplified.Like this, this mark generally is catalyzer, luminophor or radioactive isotope, preferably catalyzer.Catalyzer preferably can produce the enzyme or the coenzyme of multiple signal generation molecule from single marking.Enzyme or coenzyme can light absorbing products by producing, as dyestuff or be created in the product that irradiation can excite bright dipping down, as fluorescer to reach the purpose that desired signal is amplified.Perhaps, catalytic reaction also can directly produce exciting light, for example chemiluminescence.Many enzymes and coenzyme can be seen U.S. Patent No. 4,275 in order to this purpose, 149 and U.S.Pat.No.4, and 318,980 (in lists of references).In addition, many visible U.S. Patent No.s 4,160,645 of non-enzyme catalyzer (July 10,1979) that also can realize above-mentioned effect.

The product of above-mentioned enzyme reaction is dyestuff or fluorescer normally.In U.S. Patent No. 4,275, provided the explanation of a large amount of fluorescers in 149.

Other is used for herein term and has meaning consistent when using usually with this area, and explanation is all arranged in technical dictionary.

Patent is introduced

The present invention recognizes that dielectrophoresis provides a kind of method that entity molecule in the sample is carried out fast, efficiently separates with non-destructive.The present invention also recognized when the dielectric properties when each different component in the sample is close simultaneously, is difficult to the various entity molecules in the sample are separated with the method for dielectrophoresis.

The present invention has considered above aspect and some other demands in this area.One of purpose of the present invention just provides test required device and reagent, as dielectric separation efficient that can improve entity molecule in the sample and the sample solution that can be directly used in the dielectrophoresis separation.Another object of the present invention is to provide a kind of method of using sample solution of the present invention that entity molecule in the sample is separated.Also purpose of the present invention is a kind ofly used electromagnetic force and is used simultaneously and can optionally modify the method that erythrocytic solution separates the entity molecule in the blood sample with regard to providing.

As A brief introduction of the present invention, provide common and practical aspects more of the present invention (but being not limited only to these aspects) below, comprising:

1) a kind of solution, when with sample mix after, can modify the dielectric properties of at least a entity molecule in the sample, simultaneously the conductivity of solution also allows directly interested entity molecule to be separated in solution;

2) a kind of method that can carry out the dielectrophoresis separation to one or more entity molecules in the sample, wherein used the solution that to adjust one or more component dielectric propertiess in the sample, and this solution has suitable conductivity, and the entity molecule in the sample can utilize dielectrophoresis force to be separated therein;

3) a kind of method of using electromagnetic force that the entity molecule in the sample is separated has wherein been used and a kind ofly can optionally have been modified erythrocytic solution and used magnetic bead and be integrated in the structural unit that can produce electromagnetic force on the chip.

Can be by method described herein, the device that provides and reagent, and, just can realize these aspects of inventing with reference to corresponding document.More fully understand for the present invention being had one, must be appreciated that different aspect of the present invention can mutually combine, and realizes various needs.

The solution that can modify the dielectric properties of at least a component in the sample

The present invention includes provides a kind of like this sample solution, after this sample solution and sample mix, can modify the dielectric properties of at least a entity molecule in the sample, the conductivity of sample-sample solution potpourri also allows directly interested entity molecule to be separated in solution simultaneously.Preferably the conductivity of sample solution can satisfy such condition, when with sample mix after the degree of polarization of the interested entity molecule that separates than dielectrophoresis method to be used of the degree of polarization of sample-sample solution potpourri of obtaining low.Like this, just can separate with the interested entity molecule of particulate combination by the forward dielectrophoresis force.But this point requires for the present invention not necessarily.For example, when the degree of polarization of the interested entity molecule that separates than dielectrophoresis method to be used when the degree of polarization of the sample-sample solution potpourri that obtains after sample solution and the sample mix wants high, be also contained in the scope of the present invention.The degree of polarization of considering sample-sample solution potpourri may be than entity molecule height to be separated, and historical facts or anecdotes body molecule also should use the negative sense dielectrophoresis or be separated based on the method for negative sense dielectrophoresis.These methods comprise all dielectrophoresis isolation technics, for example, but are not limited only to, dielectric migration, dielectric are detained, dielectrophoresis/gravity field flow point from, based on the isolation technics and the two-dimentional dielectrophoresis of row ripple dielectrophoresis.

The conductivity of the sample among the present invention-sample solution potpourri is generally between 0.1 μ S/cm to 1S/m, better between 5 μ S/cm to 0.5S/m, preferably between 10 μ S/cm to 0.1S/m.The conductivity of the conductivity of sample solution and sample-sample solution potpourri can use commercially available conductivity meter to measure.The conductivity of solution can be regulated, as adding salt to increase the conductivity of solution in solution.

Sample solution among the present invention can dissolve each other with sample with any ratio, for example, sample can mix with 10: 1 to 1: 10000 ratio with sample solution, generally mix with 5: 1 to 1: 1000 ratio, be more preferably with 1: 1 to 1: 250 ratio and mix, the optimum mixture ratio example is between 1: 2 to 1: 50.

When with sample with after sample solution mixes, one or more components in the sample can be subjected to modification to a certain degree, utilize efficient that dielectrophoresis force separates one or more entity molecules in the sample to want high when not using sample solution like this.If the dielectric difference of other component can be strengthened in interested entity molecule and the sample, so just can improve separation efficiency to this entity molecule.Different response to same electric field can show as, the difference different or movement degree in electric field of direction of motion in electric field.After adding sample solution, one or more entity molecules that dielectric separation efficient is improved can be the components of being modified by solution in the sample, and the component of being modified by solution also can be other component in the sample except these entity molecules just certainly.For example, originally there was not corresponding sample component can in electric field, show the forward dielectrophoresis by modifying to electric field.Perhaps, the method for the application of the invention, the component in certain sample no longer shows the forward dielectrophoresis to electric field, so just can separate other component in the sample by the dielectric technology of being detained.

Component in the sample of being modified by sample solution can be those entity molecules that are trapped in a certain or some position in the reaction tank, or those are by a certain from reaction tank or entity molecule that some position repulsion is opened, also can be that those are neither attracted by a certain in the reaction tank or some positions, again not by a certain from reaction tank or entity molecule that some position repulsion is opened.The sample solution that plays modification can make the dielectric properties of one or more components in the sample change, and makes these original components that are not trapped in a certain or some position in the reaction tank by the forward dielectrophoresis to be trapped in a certain or some position in the reaction tank by the forward dielectrophoresis; Or make the component of originally not making the negative sense dielectrophoresis do the motion of negative sense dielectrophoresis; Can be so that the component of originally doing the motion of forward or negative sense dielectrophoresis no longer to electric field is responded, does not remake the motion of forward or negative sense dielectrophoresis yet.

Sample solution among the present invention can be modified the various ingredients in the sample, and wherein the mode that various ingredients is modified can be similar, also can be different.For example, a kind of sample solution of the present invention can be modified so that these two kinds of components are all done the motion of negative sense dielectrophoresis in given electric field two kinds of components.Perhaps, another kind of sample solution among the present invention can be modified two kinds of components, make a kind of component of originally in electric field, not making the forward dielectrophoresis do the motion of forward dielectrophoresis, and the another kind of component of originally not making the negative sense dielectrophoresis is done the motion of negative sense dielectrophoresis.The modification that the dielectric properties of one or more components in the sample is carried out does not need to change fully component corresponding to electric field, as long as certain change is arranged, for example, changes the responsiveness of component in given electric field.

Can be comprised by the sample component that sample solution is modified among the present invention molecule (as biomolecule, for example nucleic acid, protein, carbohydrates and lipid; Compound, for example various organism and inorganics), compound (as transcription complex or ribosomes), organelle (as mitochondria and nucleus), virus, parasite (as trypanosoma and plasmodium) and cell (comprising eucaryon and prokaryotic).The dielectric properties of these components is relevant with the dielectric coefficient of component, conductivity and size.These character have partly determined the amplitude and the direction of the dielectrophoresis force that particulate is suffered.

Radius is the suffered dielectrophoresis force of the particulate of r For

E wherein _RmsBe the root-mean-square value of field intensity,

Be the gradient functional symbol, ε _mIt is the dielectric coefficient of medium.Factor χ _CDEPBe the polarization factor of particulate, can be expressed as:

${\mathrm{\χ}}_{\mathrm{DEP}}=\mathrm{Re}\left(\frac{{\mathrm{\ϵ}}_p^{*}-{\mathrm{\ϵ}}_m^{*}}{{\mathrm{\ϵ}}_p^{*}+{2\mathrm{\ϵ}}_m^{*}}\right),---\left(2\right)$

Here Re refers to real, symbol ${\mathrm{\ϵ}}_x^{*}={\mathrm{\ϵ}}_x-j{\mathrm{\σ}}_x/\left(2\mathrm{\πf}\right)$ It is compound dielectric coefficient. $j=\sqrt{-1},$ Parameter ε _p, σ _pBe respectively effective dielectric coefficient and conductivity (may be relevant) of particulate with impressed frequency.When the dielectrophoresis polarization factor of a particulate is (χ just _CDEP＞0) time, particulate is moved to strong field by the dielectrophoresis force effect.When the dielectrophoresis polarization factor of a particulate is negative (χ _CDEP＜0) time, particulate be subjected to the dielectrophoresis force effect away from strong field to the feeble field regional movement.

To a radius is r, is subjected to row ripple electric field

The particulate of (the x component that is the E field is advanced along the z direction, the phase value of x component along z to the linear function that is the position) effect, the capable ripple dielectrophoresis force F of effect ideal row ripple electric field thereon _TwDEPFor

Wherein E is the field intensity size, ε _mBe the dielectric coefficient of medium, ζ _TwDEPBe the capable ripple dielectrophoresis polarization factor of particulate:

${\mathrm{\ζ}}_{\mathrm{TW}-\mathrm{DEP}}=\mathrm{Im}\left(\frac{{\mathrm{\ϵ}}_p^{*}-{\mathrm{\ϵ}}_m^{*}}{{\mathrm{\ϵ}}_p^{*}+2{\mathrm{\ϵ}}_m^{*}}\right)$

Wherein Im refers to the imaginary part of corresponding plural number, symbol ${\mathrm{\ϵ}}_x^{*}={\mathrm{\ϵ}}_x-j{\mathrm{\σ}}_x/\left(2\mathrm{\πf}\right)$ Be compound dielectric coefficient, parameter ε _p, σ _pBe respectively effective dielectric coefficient and conductivity (may be relevant) of particulate with impressed frequency.

Positive and negative according to row ripple dielectrophoresis polarization factor like this, the traveling-wave component of row ripple dielectrophoresis force will along or act on the particulate against the direction of propagation of traveling-wave field.If the capable ripple dielectrophoresis polarization factor of particulate is positive (ζ under frequency of operation _TWD＞0), the capable ripple dielectrophoresis force that then acts on the particulate will be opposite with the electric field direct of travel.If the capable ripple dielectrophoresis polarization factor of particulate is the (ζ that bears under frequency of operation _TWD＜0), the capable ripple dielectrophoresis force that then acts on the particulate will be identical with the electric field direct of travel.

Like this, the motion parts of the particulate in inhomogeneous field is by size (r), the dielectric coefficient (ε of particulate _p) and conductivity (σ _p) decision.The size of particulate is partly determining the size of the suffered dielectrophoresis force of particulate, and the dielectric coefficient of particulate and conductivity then can influence the direction of the dielectrophoresis of particulate in inhomogeneous field.Therefore, when the particulate with different dielectric character is in the same electric field, the effect of different conventional dielectrophoresis forces and different capable ripple dielectrophoresis force will be subjected to.

Below to the discussion of particulate dielectric properties as selective reagent among the present invention and illustrate that the background knowledge of reagent source provides, only be to be used as example, and do not mean that the present invention only only limits to following said part.

The dielectric coefficient of particulate (for example component in the sample) and conductivity are by the structurally associated of forming of component.For example, thus for example the polystyrene bead body has definite dielectric coefficient to the particulate of being made up of same structural unit and conductivity has definite effective dielectric coefficient and effective conductivity.These character (for example 1 hertz to 100 megahertzes) within a large range are irrelevant with electric field frequency.Particulate with homogeneous composition has surperficial net charge, when these charged corpuscles are suspended in the medium, will form electric double layer at the surface of contact of particulate/medium.Extra electric field and this electric double layer interact and have caused particulate effective conductivity and the effectively change of dielectric coefficient.Extra electric field is normally relevant with frequency with the interaction of electric double layer, and therefore, the particulate effective conductivity is with effectively dielectric coefficient is also relevant with frequency.

Correspondingly be, have particulate such as cell that heterogeneity is formed, have surperficial dielectric coefficient (claiming the film dielectric coefficient again) and inner dielectric coefficient, surface conductivity (membrane conductivity) and internal electrical conductance.Effective dielectric coefficient with particulate of heterogeneity composition is determined jointly that by surperficial dielectric coefficient and inner dielectric coefficient effective conductivity is also determined jointly by surface conductivity and internal electrical conductance.Effective dielectric coefficient with particulate that heterogeneity forms is relevant with electric field frequency with effective conductivity.Set up corresponding different dielectric model for different types of cell.Particularly, monoshell model correspondence mammalian cell, and wherein to be conducting sphere (corresponding intracellular matter) surrounded by the more weak shell of one deck electric conductivity (corresponding cell membrane) model of cell.The effective dielectric properties of cell is determined jointly by the dielectric parameter of intracellular matter and cell membrane.

Owing to have the particulate that heterogeneity forms such as the dielectric properties and the frequency dependence of cell, so the conventional dielectrophoresis polarization factor of particulate and row ripple dielectrophoresis polarization factor with regard to and frequency dependence.For mammalian cell, the dielectric properties of cell is subjected to the influence of the factors such as dielectric properties of the dielectric properties of cell size, film thickness, cell membrane and cell interior.In general, a living cells has a relatively poor cell membrane of electric conductivity (membrane conductivity is very little, less than 10 ^-4S/m), it has surrounded the medium intracellular matter (the internal electrical conductance is higher, greater than 0.1S/m) of electric conductivity.Under the low frequency situation, the electric field that applies mainly is added on the cell membrane like this, and the dielectric properties of cell membrane has just determined the dielectric properties of whole cell like this.At this moment the conventional dielectrophoresis factor of cell is negative value (χ usually _CDEP＜0), shows the conventional dielectrophoresis behavior of negative sense.Along with the increase of frequency, electric field permeates cell membranes gradually acts on the cell interior material.Like this, the conventional dielectrophoresis factor χ of cell _CDEPGradually from negative value become on the occasion of.General in such frequency range, the acting force that extra electric field is subjected to cell tends to make the capable ripple dielectrophoresis factor ζ of cell _TwDEPShow as on the occasion of (ζ _TwDEP＞0).When frequency further increases, the character of cell inclusion (promptly beginning is effective conductivity, is effective dielectric coefficient afterwards) has determined the reaction of cell.Beginning, the conventional dielectrophoresis polarization factor χ of cell _CDEPGreater than zero, then along with the rising of frequency, χ _CDEPReduce gradually.In this frequency range, row ripple dielectrophoresis polarization factor ζ _TwDEP＜0.The accurate frequency range of the conventional dielectrophoresis polarization factor of cell and row ripple dielectrophoresis polarization factor is decided by the dielectric properties of cell and the conductivity of the residing aqueous solution of cell.

The size of component has determined its response to electric field in particulate or the sample, also belongs to the part of dielectric properties.The dielectric coefficient of component, conductivity, size and other relevant therewith character can be changed by the solution among the present invention in the sample.

Some cells mainly are bacterium, fungi and vegetable cell, also have cell membrane outside cell membrane.The dielectric properties more complicated of these composite particles, the dielectrophoresis behavior under the specific electric field frequency is determined jointly by the dielectric coefficient and the conductivity of cell membrane, cell membrane and cell interior material.Cell membrane to the influence of microorganism dielectric properties and have the dielectrophoresis behavior of the microorganism of cell membrane can be referring to this piece article: Markx et al.Microbiology140:585-591 (1994).

Can be comprised the composition (inside that comprises compound is formed) of component, the net charge of sample component, the CHARGE DISTRIBUTION of sample component, the surface charge of sample component, the surface charge density of sample component, the configuration of surface of sample component and size of sample component or the like by the character of the sample component of sample solution change dielectric properties of the present invention.

Sample solution among the present invention can change the dielectric properties of one or more components in the sample by following mode: add some binding entity in sample component, or some combining unit of removal sample component, promptly pass through to change the size of component to realize this purpose.For example, the bond that certain is special such as antibody, can be discerned a kind of albumen that is connected on a certain particulate, adds antibody in the matter sample by separating certain cell born of the same parents, can use dielectrophoresis force to catch this albumen.In another example, can in the sample of forming by cell, add the solution that contains the lysis agent, such as washing agent, thereby make all or part cell generating unit divide or all degradeds, so just can not also have cell that the electric field of separating thing in order to the isolated cell born of the same parents is responded.Can be so that the hypotonic solution that some cell generation born of the same parents separates be can be in order to change the sample solution of one or more component dielectric propertiess in the sample among the present invention.This hypotonic solution can allow that one or more interested entity molecules in the sample are carried out dielectrophoresis to be separated.Can be by increasing or reducing the sample component size comprises specific bond with the sample solution of the dielectric properties that changes sample component, can with the bond of other predetermined substance molecular specificity combination, as particulate, enzyme, washing agent, surfactant, chaotropic agent, denaturant, salt, sequestrant or the like.

Sample solution among the present invention can also change the dielectric properties of one or more components in the sample by following mode: add some binding entity in sample component, or remove some combining unit of sample component, promptly the net charge by changing sample component, CHARGE DISTRIBUTION, surface charge, surface charge density are to realize this purpose.Here, the predetermined substance molecule that is added into sample component can be ion, molecule, chemical group, polymer, particulate or the like.Wherein, molecule can be the molecule of any kind, organic, inorganic or both assemblys, for example protein (comprising antibody), carbohydrates, lipid, steroids, washing agent or the like.The ingredient of sample component comprises the assembly and the chemical group of subunit (comprising accessory factor), protein, lipid, sterol, carbohydrates, nucleic acid, organic compound, mineral compound, organic compound and mineral compound.Ingredient in the sample component can be by using salt, sequestrant, denaturant, chaotropic agent, washing agent, surfactant, enzyme, chemical reagent (as hydrazine, piperidines and periodic acid) or the like.For example, have specific junction mixture such as antibody (antibody and a entity molecule among the present invention with a large amount of electric charges, link to each other as poly-D-lysine) sample solution add sample after, thereby antibody can combine the net charge or the surface charge density of change component with corresponding sample component.Thereby sample solution of the present invention also can be to include clean surface charge or the surface charge density that the surface that can be coated on component such as washing agent, lipid or polymer changes sample component.

In some example of the present invention, component to be separated is cell or organelle, and wherein the cell of one or more types is separated by a kind of or several composition born of the same parents in the solution or punched on cell membrane its permeability is strengthened.When being perforated on cell membrane, cell or organelle make its permeability strengthen such effect, this typically refers under this effect, the following compound of certain size can enter or leave cell by cell membrane, perhaps also can refer to have necessarily optionally mass exchange, as only specific material, can enter or leave cell such as potassium ion.Can born of the same parents separating or make the osmotic pressure of the solution that its permeability strengthens to separate or treat that punching is forced down the infiltration of its permeability enhanced cell on cell membrane by hypotonic born of the same parents than treating in punching on the cell membrane among the present invention to one or more cells in the sample.Perhaps, can cause among the present invention that born of the same parents separate or punching on the cell membrane make solution that its permeability strengthens can be from cell membrane the (albumen on the cell membrane for example, as transport protein and example channel protein) act on cell, can comprise salt, washing agent, surfactant, lipid, sterol, polymer, ethanol, enzyme, ionophore, metabolic poison, ion channel Inhibitors and ion channel modulators or the like reagent in the solution.The effect that the cell born of the same parents separate and punching strengthens its permeability on cell membrane can change electric density, net charge and the CHARGE DISTRIBUTION of cell.

Sample solution also can change sample component surface net charge or thereby surface charge density is modified the dielectric properties of component by enzyme or chemical reagent.Because the glycolipid of cell surface and the carbochain of glycoprotein have negative charge usually, so cell surface all is electronegative usually.Thereby the sample solution that includes neuraminidase can change cell surface electric density or cell surface net charge by the carbochain on incising cell surface.Proteinase, washing agent, reductive agent or sequestrant such as EDTA can remove albumen or other molecule of cell surface, thereby the dielectric properties of pair cell is modified.

Thereby sample solution also can be by carrying out surface charge, surface charge density or the net charge of chemical modification change sample component to the sample component surface.Oxygenant is periodic acid for example, and reductive agent for example dithiothreitol (DTT) can add sample solution so that they carry out chemical modification to the surface of sample component.By the surface of impelling sample component chemical reaction takes place, enzyme also can change the dielectric properties of sample component.Various enzymes such as kinases, phosphatase, reductase, oxidase can join in the sample solution and go.

Sample solution also can comprise by form that changes sample component or the compound that the mode of cell differentiation is modified the surface composition of sample component (for example cell).For example, the sample component that includes phytohemagglutinin or interleukin 2 (in order to stimulate the expression of MHC in the T lymphocyte) can increase the complexity of surface of cell membrane configuration, thereby change the dielectric properties (Huang et al., Biochimica Biophys.Acta 1417:51-62 (1999)) of cell membrane.

By the change that the inside with sample (as cell) component that heterogeneity forms is formed, sample solution of the present invention also can change the net charge and the CHARGE DISTRIBUTION of sample component.Solution among the present invention can change the inside of the sample component with non-homogeneous composition by following mode and form, in sample component, introduce new predetermined substance molecule, from sample component, remove certain predetermined substance molecule or make the inside of sample component form generation chemistry or modal variation.For example, charged molecule can be introduced in the cell, also can be used other commercially available carrier by liposome, as

(Life Technologies, Rockville, MD), gene porter ^TM(Gene Therapy Systems, San Diego, CA) or Influx ^TM(Eugene OR) or the like introduces molecule or compound (for example charged and uncharged polymer) in the cell Pinocytic Cell-Loading Reagent for Molecular Probes, Inc..Sample solution also can discharge the inside composition of molecule or the method for compound change cell by making cell.For example, can comprise one or more ionophores in the sample solution and make cell discharge some ion, thus the net charge and the electric density of change cell interior.Perhaps, also can directly change the memebrane protein such as the ionophorous protein of surface of cell membrane, thereby make the ion composition of cell interior change.

Sample solution also can comprise by the form that changes sample component or the mode of cell differentiation and sample interior be formed the compound of modifying.For example, the B cell can secrete immunoglobulin (Ig) under the effect of antigen and growth factor.When secretory volume increased, the inner structure of cell can change, and changed such as membrane structure in the cell.Such variation can change the dielectric coefficient and the conductivity of cell interior.Ionophore, ion channel Inhibitors, metabolic poison and other the medicine that can change the cell interior composition can be included in the sample solution of the present invention.

Those skilled in the art can select suitable sample solution according to the knowledge of sample component to be separated or to be removed.For example, when the cell (having bigger density and negative surface charge) with high glycosylation is arranged in the sample, can contain neuraminidase in the sample solution, promptly a kind of enzyme that can excise the cell surface carbochain.In another example, sample solution can comprise a kind of specific junction mixture (for example a kind of bivalent antibody), this antibody can with mitochondrial surface combination, thereby can be so that mitochondria can be separated by the method for dielectrophoresis.In going back an example, the univalent antibody that can comprise certain cell of specific recognition that is connected with group (as poly-D-lysine) in the sample solution with High Density Charge, when antibody with after corresponding cell combine, thereby can change the dielectric properties of the surface charge change cell of cell.Also can in sample solution, add the material which kind of effect those also do not know to produce specific sample component, for example, metabolic poison, ion channel Inhibitors or other medicine that can exert an influence to the inside composition of biological sample component.These changes can be carried out to a certain degree modification to their dielectric properties.

Can whether be changed sample solution with the dielectric properties of determining sample component by experimental test changes.For example, the technician can be by the dielectric properties of electric method judgement sample component of rotating.Can derive the dielectric properties of this entity molecule by measuring the rotating speed of entity molecule in rotating electric field, this method can be referring to Huang et al., Phys.Med.Biol.37:1499-1517 (1992); Huang et al.Phys.Med.Biol.40:1789-1806 (1995); Huang et al.Biochim.Biophys.Acta 1282:76-84 (1996); AndHuang et al.Biochim Biophys.Acta 1417:51-62 (1999); Also can determine the effective conductivity (as the conductivity of entity molecule before or after adding sample solution) of entity molecule by the absorptance of measuring the entity molecule suspension, referring to Price et al.Biochim.Biophys.Acts 964:221-230 (1988); Burt et al., and J.Phys.E Sci.Instrum. 22:952-957 (1989); In addition, by judging that entity molecule is under specified criteria, making the forward dielectrophoresis on the polynomial expression electrod-array still is the negative sense dielectrophoresis, just can test out entity molecule with sample solution dielectric properties before and after treatment, the visible Huang and of this method Pethig Meas Sci Technol 2:1142-1146 (1991) andMarkx et al., Microbiology 140:585-591.

Except the function with the dielectric properties of modifying sample component, the conductivity of sample solution also will be able to satisfy the needs of the dielectrophoresis separation of one or more entity molecules in the sample.At great majority but be not that the technician can have general estimation to the conductivity range of required sample-sample solution in the whole example.Sample solution and sample can be mixed with various ratio, with conductivity meter the electricity of solution be led again and measure.At great majority but be not in the whole example, need to use low conductivity solutions, low conductivity is meant that the conductivity of solution is lower than 0.5S/m.But, in some example, when needs use the negative sense dielectrophoresis to separate, be not less than entity molecule to be separated with regard to the conductivity that requires sample-sample solution mixed liquor.The conductivity of sample solution can be adjusted by adding salt (as NaCl) or sequestrant (as EDTA).

The sample that contains more than a kind of cell can carry out born of the same parents to one or more cells wherein to be separated, and for example can separate situation by the born of the same parents of microscopic examination cell after adding hypotonic solution.

Sample solution in the entity example of the present invention has lower conductivity, and after mixing with blood sample, sample solution can optionally be modified red blood cell, thereby makes and be not trapped in the reaction tank by dielectrophoresis force.In these entity examples, sample solution of the present invention is mainly modified red blood cell rather than leucocyte, and hereinafter such solution refers to that optionally born of the same parents separate erythrocytic solution.General requirement, after sample solution and blood sample mixed, the ratio of natural red blood cell and natural leucocyte was less than 20: 1 in the solution, was more preferably less than 10: 1, was preferably less than 5: 1.This can be by microscopic examination.Sample solution of the present invention can mix with 1: 10 to 10000: 1 ratio with blood sample, generally is to mix with 1: 5 to 1000: 1 ratio, is more preferably with 1: 1 to 250: 1 ratio and mixes, and preferably mixes with 2: 1 to 50: 1 ratio.The conductivity of these sample solutions is lower, and the degree of polarization of ie in solution in extra electric field compared low with entity molecule to be separated.

In the entity example, the sample solution among the present invention has lower osmotic pressure, and when sample solution added blood sample, haemocyte was arranged in hypotonic environment like this.In the entity example, the final osmotic pressure of general solution is between between the 20mOsm to 150mOsm, preferably between between the 30mOsm to 80mOsm.The suitable solute that can be used for hypotonic solution of the present invention comprises glycerine, sugar (as sucrose, glucose and mannose) and sugar alcohol (as sweet mellow wine and sorbierite).Uncharged zwitter-ion when other solute that can be used for hypotonic solution of the present invention also is included near neutral pH, (comprise D-as glycocoll, alanine, gamma-amino-butyric acid, halfcystine, histidine, L-, 3 (τ) methyl-and 1 (π) methyl-histidine), carnosine, pyrimidine, imidazoles and 4-ethyl-2-picoline (seeing Edman, et al.Nucl.AcidsRes.25:4907-4914 (1997)).

Electrod-array can be used for testing the present invention's sample solution.For example, can observe in electric field, originally did forward or the sample component of negative sense dielectrophoresis with the mixed moving situation of sample solution.For example, small amount of sample-sample solution mixed liquor can be added on the electrod-array, apply appropriate frequency (between the 10Hz to 500MHz) and amplitude (peak-to-peak value is less than 20 volts) sinusoidal electric signals on the electrode.The sample component of making the forward dielectrophoresis is collected at the end of array, and the sample component of making the negative sense dielectrophoresis accumulates in middle section (Huang and Pethig, the Meas.Sci.Technol.2:1142-1146 (1991) of electrod-array.

For the entity molecule that separates by dielectrophoresis is detected, should have detectable mark at least a entity molecule in the sample.For example, carry out dielectrophoresis when separating in that biological sample and sample solution of the present invention are mixed, can use a kind of cell or the antibody of component in the specific identification sample that certain cell or component are carried out mark.Can have detectable mark on the antibody, for example fluorescence molecule is (as rhodamine, fluorescein, red, the phycoerythrin of Texas, phycocyanin, green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescence protein or the like.Cell also can use and have the not different antibodies mark of isolabeling.Like this, having the residing position of fluorescently-labeled cell can be detected, and uses sample solution that sample component is carried out the effect that dielectrophoresis separates thereby can judge.Except extracellular sample component, can utilize antibody to carry out mark as organelle, virus, protein, compound and nucleic acid or the like, thereby the dielectrophoresis that detects them separate.

In addition, also having a kind of detection mode is at after separating, detects the existence of protein, nucleic acid and other compound by binding analysis.For example, after sample solution of the present invention and sample mix, carry out cell separation again, the effect of separation can or detect with the antibody of the expressed protein-specific combination of given cell and can obtain with the probe nucleic acid sequence of distinguished sequence combination in definite cell (as the bacterium of special kind) or the like method by detecting.Can or analyze (analyzing) by enzymatic testing process (as PCR) to representing the special cell type or the detection of the nucleotide sequence of specific cell component and protein as P450.

Can the detachment process of pair cell dielectrophoresis monitor by introduce detectable mark (as dyestuff) to cell interior.For example, can introduce BCECF-AM (Molecular Probes in the cell, Eugene, OR), this fluorescein probe can be absorbed by living cells, after dielectrophoresis separated, (Gascoyneet al.IEEE Transcactions 33:670-678 (1997)) can be determined in the position of this fluorescein molecule.Chip in order to isolated cell is proved to be and can observes with microscope, separated like this entity molecule can be washed out reaction tank, analyzes by micro measurement, Flow Cytometry, cell growth analysis or the like method (but be not limited only to these several) again.

Also can comprise the compound the material of dielectric properties of one or more entity molecules in can the selective modification sample in the sample solution of the present invention.These compounds can be, but be not limited only to salt, damping fluid, enzyme, stabilizing agent, antiseptic, sequestrant (as EDTA and EGTA), chaotropic agent, denaturant, washing agent, anticoagulant, specific bond and detectable mark or the like.

Reagent required for the present invention can provide with hybrid packed or the form of independent packing.Reagent can provide with the form of kit, wherein this kit comprises sample solution and one or more additive reagents that one or more can be modified the dielectric properties of one or more sample component at least, as particulate, specific bond, damping fluid, reagent corresponding and solution or the like and portion in order to the instructions of realizing function of the present invention (instructions can be separately provides with the form of a handbook, and the packing that also can be dispersed in each reagent is interior).

II uses sample solution of can selectivity the dielectric properties of one or more entity molecules in the sample being modified and the method for on chip the entity molecule in the sample being separated

The present invention includes a kind of method of on chip, the interested entity molecule in the sample being separated, wherein to use the sample solution among the present invention, after this solution and the sample mix, can modify the dielectric properties of at least a component in the sample, simultaneously the conductivity of sample-sample solution potpourri satisfies the needs that one or more entity molecules in the sample carry out dielectric separation.The process of this method is: add sample solution of the present invention in sample, the entity molecule in the sample is carried out dielectrophoresis separate.This method also can comprise use can with entity molecule coupling connection and the bond that can be handled by dielectrophoresis force.

Sample solution

Sample solution of the present invention can be the solution of any kind of that meets the following conditions: when with sample mix after, can modify the dielectric properties of one or more components in the sample, and the conductivity of solution can satisfy the needs of entity molecule dielectric separation in the sample.The sample solution that the present invention recommends is the selectivity born of the same parents to separate erythrocytic solution, such as hypotonic tygon glycerite, sugar juice or sugar alcohol solution, these solution be limited to blood sample and mix after, the osmotic pressure of sample-sample solution potpourri is between between the 20mOsm to 150mOsm.

Sample

Sample can be the fluid sample of any kind of, and for example environmental sample comprises air sample, water sample, food samples; Can be biological sample also, comprise the extract of biological sample.Biological sample can be blood, serum, saliva, urine, seminal fluid, tears, nose, throat, genitals or excremental extract.Biological sample also can be organ, tissue, cell culture medium sample (comprising nutrient culture media and cell line).First-selected sample is a blood sample.

Blood sample can be the blood sample of any kind of, from the fresh extracting of individuality, that from depots, obtain, from individual external acquisition, as clothing, furniture, apparatus or the like.Blood sample can obtain by extracting, for example, the article that have blood is immersed specific damping fluid or solution obtain.Blood sample can be to handle, also can be untreated, or section processes crosses, and the mode of processing can be centrifugal to remove serum, dialyse, carry out flow cytometry, to add reagent or the like.The treatment step of blood sample roughly comprises, but is not limited only to, and buffy coat is by separating such as flow cytometry technology, density gradient centrifugation and magnetic cell sorting technology or the like pair cell sample.Blood sample can be an arbitrary volume, for example less than 5 microlitres, or surpasses 5 liters, and this is needed to decide by test fully.

Bond

Anyly can combine with entity molecule and can be applied to method of the present invention with certain specificity and affinity by the bond that dielectrophoresis force is handled.The bond that uses in order to handle entity molecule in microfluid system can be referring to United States Patent (USP) " Methods forManipulating Moieties in Microfluidic Systems " (patent sequence number: 09/636,104, submit August 10 2000 date).Bond can be cell such as animal, plant, fungi and bacterial cell; Organelle such as nucleus, mitochondria, chloroplast, ribosomes, endoplasmic reticulum, golgiosome, lysosome, proteasome, secretory vacuole, vacuole and microbody; Virus; Particulate or their aggregation.First-selected bond is a particulate.

Be used for particulate of the present invention usually between 0.01 micron between the hundreds of micron.The particulate that uses in this method comprises, but be not limited only to, the particulate of plastic particles, ps particle, beaded glass, magnetic bead, hollow glass ball, composite parts, little contactless microstructure that is processed to form (" Design of asynchronous dielectric micromotors ", Hagedorn et al., in Journal of Electrostatics, 1994, Volume:33, Page 159-185) etc.The particulate of composite parts is meant the particulate that those are made up of multiple heterogeneity, and for example bag is by the Metal Ball of the non-conductive polymer film of one deck.

Type, material, composition, structure and the size of the bond of selecting must and application-specific in maneuverability pattern be complementary.For example, particulate must have suitable dielectric properties so that particulate is handled by dielectrophoresis force.

The selection of particulate is relevant with concrete manipulation process.For example, for the method for operating by dielectrophoresis separates the interested component in the sample mixture, necessarily require the dielectric properties of other component except component to be separated in the dielectric properties of particulate and the sample to differ greatly, like this, after particulate and the combination of interested entity molecule, interested entity molecule-bond compound can carry out Selective Separation with the method for dielectrophoresis.

Entity molecule to be handled can be by any feasible method and the surface combination of bond.For example, entity molecule can directly link to each other or link to each other by a linking arm with the surface of bond, preferably the linking arm that can cut.Entity molecule also can pass through covalency or non-covalent combination and bond surface combination.The connected mode on entity molecule and bond surface can be special, and is also can right and wrong special.Best, the connection between entity molecule and the bond can be cut, as the linking arm that can be cut off by chemistry, physics or enzyme reaction.

Linking arm (connecting key) is any predetermined substance molecule that entity molecule and bond can be coupled together.Such linking arm (connecting key) can comprise, but be not limited only to covalent bond between the amino acid or the connection of peptide bond, disulfide bond, thioether bond, interrupted thioether bond, the free radical (as amino and mercapto) or the like.The linking arm of other form comprises the linking arm that can be cut by acid, for example bismaleimideothoxy propane, acid labile-transferrin conjugates, these materials that can be cut under the slant acidity condition of adipic acid dihydrazide in cell; Also can be the glue crosslinking agent that can under ultraviolet light or visible light, be cut, for example IgG ₁In constant zone C _H1, C _H2 and C _H3 (Batra et al., Molecular Immunol., 30: 379-386 ((1993)).In some entity examples, can use multiple linking arm simultaneously, facilitate the use the advantage of various linking arms.The linking arm of other kind such as trityl linking arm come from trityl group, can discharge entity molecule (U.S.Patent No.5,612,474) under different Acidity of Aikalinity conditions.Also have some connect with entity molecule can vide infra (Huston et al., Proc.Natl.Acad.Sci.U.S.A., 85: 5879-5883 (1988), Whitlow, et al., Protein Engineering, 6: 989-995 (1993), Newton et al., Biochemistry, 35: 545-553 (1996), Cumber et al., Bioconj.Chem., 3: 397-401 (1992), Ladurner et al., J.Mol.Biol., 273: 330-337 (1997) and in U.S.Patent.No.4,894,443).Usually the connected mode of selecting for use among the present invention is biotin-Streptavidin interaction, antigen-antibody interaction, aglucon-acceptor interaction or nucleic acid array complementation effect.The linking arm and the method that are used to connect entity molecule and particulate can be referring to U.S. Patent applications, application serial 09/636,104 (submitting August 10 2000 date).

In some cases, when entity molecule-bond such as molecule-particle complex handled/be separated to preposition after, not next step reaction of disturbing molecule of the existence of particulate.Like this, just will not separate again with bond by molecule.But in some cases, such separation is again necessary.The feature of detachment process is by the character of entity molecule and bond, the surface appearance and the manipulation process decision of bond.Usually, the condition of the condition of separation and polymerization is just the opposite.For example, if the combination under high salt ionic concentration condition of entity molecule and bond, entity molecule can elute from bond under low salt ionic concentration condition so.Similar, if entity molecule was finished with being connected by specific linking arm of bond, the mode of Fen Liing is exactly to impose on the condition that this linking arm can specificity makes its fracture so.

Reaction tank on the chip

Reaction tank of the present invention is a kind of structure that can hold fluid.Reaction tank can be an arbitrary dimension, should hold the fluid of 0.001 μ l to 50ml usually, better should hold the fluid of 1 μ l to 20ml, and optimal cases is to hold the fluid of 10 μ l to 10ml.Generally speaking, reaction tank is one of structural unit on the chip.Reaction tank can be made by any suitable material, as silicon, glass, metal, pottery, polymkeric substance, plastics or the like, can be the quality stiff materials, also can be the material of quality softness.First-selected material is those not have the material of interference to dielectrophoresises motion of the entity molecule in the sample, for example those not with charged or the material that polar molecule combines, as the plastics or the polymer (as third rare acid amides, glass or the like) of silicon, some kind.

Reaction tank is one of ingredient of chip, and chip is a solid substrate, can carry out once thereon or separation repeatedly, analyzes, catches or the like process.The material of chip can be a kind of or several in metal, pottery, polymkeric substance, multipolymer, plastics, rubber, silicon, glass or the like.Chip can comprise several certain flexible materials that have.Chip can be made of porous materials, and also can be made by the material of densification.Be shaped on the chip or be integrated with such as the little processing structure of groove, passage, electrode unit or the like, can carry out reaction or processing physics, biophysics, biological, chemistry thereon.Chip is a thin slice.The chip surface area size is not done requirement, such as from 1mm ²To 0.25m ²Can.Preferably used chip surface area is from 4mm ²To 25cm ²About.Chip can have difformity, shape such as rectangle, circle, ellipse or other irregular shape of rule.Chip surface can be flat, also can be uneven.The chip that has a non-planar surface can comprise from the teeth outwards by processing at chip surface or structures such as the groove that etching gets, reaction tank.

It must be noted that,, must use chip with geometry in particular and parameter for the reaction tank with larger capacity (as above to 50ml).Chip can be produced on the flexible material, so that chip is folded to form the reaction tank that is similar to pipeline.Can use the polylith chip in one reaction pool, electrode unit also will satisfy and dielectrophoresis force can be applied in the reaction tank in the interesting areas.

In the entity example, wrap electrode (electrode be produced on the chip or chip in) in the reaction tank, but this is not requirement of the present invention.Electrode on the chip can have arbitrary shape, for example rectangle, castellated, triangle, circle or the like.The arrangement mode of electrode also has many kinds, such as spiral fashion, parallel shape, interlaced shape, polynomial expression shape or the like.The making of the chip power utmost point can be used various little processing and methods of micro-mechanics common in this area, for example plating, metal sputtering, photochemical etching.The chip that includes electrode has, but be not limited only to, be produced on dielectrophoresis electrod-array (the Dielectrophoretic Manipulationof Particles by Wang et al. on the glass substrate, in IEEE Transaction on IndustryApplications, Vol.33, No.3, May/June, 1997, pages 660-669), have can single gate array little processing bioelectronics chip (Preparation andHybridization Analysis of DNA/RNA from E.coli on MicrofabricatedBioelectronic Chips by Cheng et al., Nature Biotechnology, Vol.16,1998, pages 541-546) and capillary electrophoresis chip (Combination of Sample-Preconcentration and Capillary Electrophoresis On-Chip by Lichtenberg, et al., in Micro Total Analysis Systems 2000 edited by A.van den Berg etal., pages 307-310).

Among the present invention or the reaction tank that uses in this method has one or more ports, or on the wall of reaction tank, has opening.Port can be any size, but suitable be to allow sample to add the shape and size of reaction tank by conduit.Conduit can be the tubular structure that any permission liquid passes through, such as rubber tube, polyfluortetraethylene pipe or polyethylene pipe.Port can provide an opening so that introduce sample by pipette or syringe for the reaction tank wall.

Conduit can be introduced sample from one or several port by pump (for example peristaltic pump or inculcate pump), syringe or gravity to reaction tank.One or more reagent, damping fluid, solution or the like.The sample solution that can modify the dielectric properties of one or more entity molecules in the sample among the present invention can add reaction tank simultaneously with sample, or adds reaction tank prior to, back in sample.Also can be before entering reaction tank, earlier sample and reagent, damping fluid or solution are mixed earlier, such mixed process can be carried out in the conduit of fluid being introduced reaction tank, also can carry out in one or a plurality of and sample cell that conduit links to each other.

Sample solution adds sample

Sample, sample solution and additional solution, damping fluid, reagent can add reaction tank in any mode easily, for example shift by transfer pipet, injector to inject, utilize gravity to introduce by conduit (for example polyethylene catheter or the like).Generally, sample, sample solution and other solution, damping fluid, reagent add reaction tank in the mode of continuous flow, such continuous flow can be injected into or pump into reaction tank from least one inlet, then, do not contain sample component and other liquid and flow out reaction tank from least one outlet.

(see figure 1) in an entity example of the present invention, sample solution is added into blood sample by a side pipe.Transport the sample among the present invention and the peristaltic pump of sample solution by adjusting, can be so that sample mixes with different ratios with sample solution, to meet the requirement that in reaction tank, the entity molecule in the sample is separated.

Before sample adds reaction tank, can in sample, add sample solution earlier.Before sample-sample solution mixed liquor is added reaction tank, sample and sample solution jointly incubation from less than 1 second to several hrs time arbitrarily among several days.In the conduit that the mixing of sample and sample solution can occur in reaction tank is communicated with, as shown in Figure 1.Also can earlier sample be added reaction tank, then sample solution be added reaction tank again; Or earlier sample solution is added reaction tank, then sample is added reaction tank again.

Wherein bond such as the particulate that uses in the present invention can provide together or separately with sample solution.If bond provides separately, so bond can be before sample solution adds sample, add simultaneously or afterwards.

Use dielectrophoresis force to separate

Under the inhomogeneous field effect that in reaction tank, adds, can carry out dielectrophoresis to the entity molecule in the sample and separate.Best, the separation of entity molecule can be carried out being arranged on the chip of reaction tank, and the electric field that adds can be controlled outside reaction tank or chip.On electrode, apply electric signal by one or several voltage, frequency, electric signal generator that phase place is all adjustable, to produce inhomogenous AC field on the space.Be applied to voltage on the electrode generally between between the 0V to 100V, preferably between between the 0V to 15V; And the frequency of electric signal is generally between between the 0.01KHz to 500MHz, preferably between between the 1KHz to 20MHz.More than given frequency range only be the example effect, in concrete the application, the frequency of the electric signal that is used for the sample separation entity molecule that is applied is determined by the dielectric properties of entity molecule to be separated itself and the conductivity of the residing solution of entity molecule.

Can use any dielectrophoresis separation mechanism that entity molecule is separated, as dielectric delay, dielectric migration, dielectrophoresis/gravity field flow point from, based on the separation of row ripple dielectrophoresis, 2-D dielectrophoresis or the like.The example of the separation that below provides only plays the explanation explanation, and does not mean that the present invention only comprises the part that example is related.When using dielectric to be detained, interested entity molecule is by one or several zone in the reaction tank of being trapped in of selectivity, and other component of sample is gone out reaction tank by liquid stream wash-out.When using dielectric migration, interested entity molecule is transferred to a place or zone, many places on the chip by dielectrophoresis force, and other component in the sample is transferred out of these zones.When use dielectrophoresis/gravity field flow point from the time, under the acting in conjunction of dielectrophoresis force and gravity, different entity molecules is suspended in the differing heights place of reaction tank, or one or more entity molecules suspend, and other component in the sample accumulates in some position on the chip, like this, the fluid that has a different entities molecule is with the different flow velocitys reaction tank/chip of flowing through, thereby realizes separating.Also can interested entity molecule directly be migrated out reaction tank by using capable ripple dielectrophoresis force.The method that also has two-dimentional dielectrophoresis wherein, is used for dielectrophoresis force and row ripple dielectrophoresis force separate (De Gasperis et al., the Biomedical Microdevices 2:41-49 (1999)) of the interested entity molecule of sample simultaneously.

Owing to comprise some in the sample in the component of same electric field intermediary electricity electrophoresis behavior the unknown not, can by regulate electrode geometry, these parameters of electric field magnitude and electric field frequency be separated to optimize with the dielectrophoresis of realizing entity molecule.

Can be by catching and collect the entity molecule of making the forward dielectrophoresis at electrode edge, and the method for utilizing other acting force such as fluid force to remove other cell realizes lock out operation.Similar methods also can be used for separating the particulate of making the negative sense dielectrophoresis from cell mixture, if most cell all shows the motion of forward dielectrophoresis in electric field.When using dielectrophoresis/gravity field flow point from, row ripple dielectrophoresis or two-dimentional dielectrophoresis method, separation can be collected eluent from reaction tank to realize by substep.

The description of following entity example only plays a part to explain explanation, and does not mean that the present invention only comprises described part.

Entity example among Fig. 1, the sample solution of separating red blood cell 100 in order to born of the same parents optionally joins in the blood sample by a side pipe.Blood sample comprises red blood cell 100, leucocyte 120 and other blood constitutent (not providing among Fig. 1).Blood sample-sample solution flows to the dielectrophoresis chip with interlaced parallel pole array.Be detained technology can be trapped in leucocyte 120 electrode in inhomogeneous field surface by dielectric, the red blood cell born of the same parents separate thing 140 and other blood sample component (not providing in Fig. 1) is washed out from reaction tank by liquid.

Entity example among Fig. 2 includes specificity junction mixture 160 in the sample solution, this bond 160 is graphite particulates that surperficial covalent bond has antibody.In this example, antibody can with interested entity molecule 180 in sample protein bound for example.Sample-sample mixture can leave standstill a period of time earlier in advance, for example 10 to 120 minutes, sample-sample solution mixed liquor is fed on the chip of being made up of the polynomial expression electrod-array again.The process that leaves standstill can be so that entity molecule in the sample 180 and bond 160 abundant combinations form entity molecule-bond compound 200.Like this, in AC field, protein of interest matter-particle complex can be trapped in the surface of electrode.Other sample component can be washed away from reaction tank, for example passes through microsyringe.

In the entity example of Fig. 3, interested entity molecule 210, isolated stem cell from marrow for example, the sample solution that can be contained antibody is modified, and the antibody in the solution can combine with the antigen of cell surface, thereby changes the surface charge of stem cell.Except interested entity molecule 210, also comprise many other entity molecules in the sample, for example undifferentiated and cell that broken up is represented with 220,240,260 and 280 respectively among the figure.Sample-sample mixture can leave standstill a period of time earlier in advance, for example 5 to 60 minutes, sample-sample solution mixed liquor is fed on the chip that (as passing through syringe) be made up of the screw electrode array again.Like this, reformed stem cell 300 and other entity molecule enter together that chip etc. is to be separated, and sample solution is pumped to reaction tank, and stem cell 300 can be detained technology and differentiation and the cell that broken up be separated by dielectric.

In the entity example of Fig. 4, optionally born of the same parents separate erythrocytic sample solution and are added into blood sample.Blood sample comprises the cell (not providing) of red blood cell 320, malignant lymphatic oncocyte 340, non-pernicious leucocyte 360 and other type in Fig. 4.Sample solution is added blood sample, can be so that red blood cell 320 be separated by born of the same parents, and the cell of malignant lymphatic oncocyte 340, non-pernicious leucocyte 360 and other type remains unchanged.Again sample-sample solution mixed liquor is added the reaction tank of the chip of forming by parallel wire electrod-array, on electrode, pass to four phase electric signal, then utilize row ripple dielectrophoresis malignant lymphatic oncocyte 340 and non-pernicious leucocyte 360 can be separated.

Above-mentioned example all is to use can the dielectric properties of one or more components in the sample be modified among the present invention, and conductivity itself can satisfy one or more components in the sample and carry out using the dielectrophoresis method to separate after dielectrophoresis or row ripple dielectrophoresis separate the sample solution of needs.There is the method for many kinds of dielectrophoresises can be now in order to the entity molecule in separation and manipulated cell, biological particle and the sample mix liquid.These methods comprise, but are not limited only to, and dielectric migration, dielectric are detained, dielectrophoresis/gravity field flow point is from, row ripple dielectrophoresis and two-dimentional dielectrophoresis.Dielectrophoresis is handled and the technician of dielectrophoresis separation field can be according to specific circumstances, selects for use among the present invention accordingly method to separate and handle.Below listed some articles of reference: Wang about dielectrophoresis manipulation and dielectrophoresis isolation technics aspect, et al., Biochim.Biophys.Acta.1243:185-194 (1995), Wang, et al., IEEE Trans.Ind.Appl.33:660-669 (1997) (about the design of different electrode structures, the method for handling with dielectrophoresis and row ripple dielectrophoresis); Wang, et al., Biophys.J.72:1887-1899 (1997) (concentrate and separate) at the enterprising every trade ripple of screw type electrode dielectrophoresis; Wang, et al., Biophys.J.74:2689-2701 (1998), Huang, et al., Biophys.J.73:1118-1129 (1997) and Yang, et al., Anal.Chem.71 (5): 911-918 (1999) (particulate repulsion from the electrode is opened, and it is suspended in the medium, utilize dielectrophoresis/gravity field flow point to separate again) from technology; Gascoyne, et al., IEEE Trans.Ind.Apps.33 (3): 670-678 (1997), Becker, et al., Proc.Natl.Acad.Sci.USA 92:860-864 (1995) andBecker, et al., J.Phys.D:Appl.Phys.27:2659-2662 (1994) (to capture particles, repulsion, location, separation, utilize dielectric delay technology to separate, utilize the dielectric migration technology to separate); Huang, et al., J.Phys.D:Appl. Phys.26:1528-1535 (1993) (utilizing row ripple dielectrophoresis that particulate is transported, separates and catches); And Wang, et al., J.Phys.D:Appl.Phys.26:1278-1285 (1993) (utilizing the dielectric migration technology that particulate is separated) to capture particles, separation and repulsion.Other can use among the present invention method that particulate is handled with the paper of having delivered that separates has: about (Hawkes, et al., the Microbios.73:81-86 (1993) of separation of bacterial from blood cell or other cell type; And Cheng, et al., Nat.Biotech.16:547-546 (1998)); Enrichment has the stem cell (Stephens, et al., Bone MarrowTransplantation 18:777-782 (1996)) of CD34 activity from blood; Virion, sub-micron magnetic bead, biomolecule are carried out dielectrophoresis collect (Washizu, et al., IEEE Trans.Ind.Appl.30:835-843 (1994); Green and Morgan, J.Phys.D:Appl.Phys.30:L41-L44 (1997); Hughes, et al., Biochim.Biophys.Acta.1425:119-126 (1998); And Morgan, et al., Biophys be (1999) J.77:516-525); Make special cell suspension (Fuhr, et al., Biochim.Biophys.Acta.1108:215-233 (1992)); Homogeneous list particulate is handled (Washizu, et al., IEEE Trans.Ind.Appl.26:352-358 (1990); Fiedler, et al., Anal.Chem.70:1909-1915 (1998); AndM ü ller, et al., Biosensors and Bioelectronics 14:247-256 (1999)); Dielectrophoresis cage technology (Schnelle, et al., Biochim.Biophys.Acta.1157:127-140 (1993); Fiedler, et al. (1995); Fuhr, et al. (1995a); Fiedler, et al. (1998); M ü l ler, et al. (1999)); The capable ripple dielectrophoresis of pair cell is handled (Hagedorn, et al., Electrophoresis 13:49-54 (1992) on linear array; Fuhr, et al., Sensors and Actuators A:41:230-239 (1994); And Morgan, et al., J.Micromech.Microeng.7:65-70 (1997)).

III uses the method for can the selectivity born of the same parents separating erythrocytic sample solution and on electromagnetic chip the entity molecule in the blood sample being separated

The present invention includes a kind of method of on electromagnetic chip, the entity molecule in the blood sample being separated, wherein to use the sample solution among the present invention, this solution optionally born of the same parents is separated red blood cell, and this method is used the particulate that can be transported and locate by electromagnetic force.The process of this method is: adding is of the present invention in sample can the selectivity born of the same parents separate erythrocytic sample solution, adds one or more magnetic beads in blood sample, on electromagnetic chip the entity molecule in the blood sample is carried out dielectrophoresis and separates.

Sample solution

Optionally born of the same parents of the present invention separate the solution that erythrocytic sample solution can be any kind of that meets the following conditions: after mixing with blood sample, the overwhelming majority who is separated by born of the same parents is red blood cell rather than leucocyte.General requirement, after sample solution and blood sample mixed, the ratio of intact red cells and complete leucocyte was less than 20: 1 in the solution, was more preferably less than 10: 1, was preferably less than 5: 1.This can be by microscopic examination.Sample solution of the present invention can mix with 1: 10 to 10000: 1 ratio with blood sample, generally is to mix with 1: 5 to 1000: 1 ratio, is more preferably with 1: 1 to 250: 1 ratio and mixes, and preferably mixes with 2: 1 to 50: 1 ratio.

In the entity example, the sample solution among the present invention has lower osmotic pressure, and when sample solution added blood sample, haemocyte was arranged in hypotonic environment like this.In the entity example, the final osmotic pressure of general solution is between between the 20mOsm to 150mOsm, preferably between between the 30mOsm to 100mOsm.The suitable solute that can be used for hypotonic solution of the present invention comprises glycerine, sugar (as sucrose, glucose and mannose) and sugar alcohol (as sweet mellow wine and sorbierite).

Sample

Blood sample can be the blood sample of any kind of, from the fresh extracting of individuality, that from depots, obtain, from individual external acquisition, as clothing, furniture, apparatus or the like.Blood sample can obtain by extracting, for example, the article that have blood is immersed specific damping fluid or solution obtain.Blood sample can be to handle, also can be untreated, or section processes crosses, and the mode of processing can be centrifugal to remove serum, dialyse, carry out flow cytometry, to add reagent or the like.The treatment step of blood sample roughly comprises, but is not limited only to, and buffy coat is by separating such as flow cytometry technology, density gradient centrifugation and magnetic cell sorting technology or the like pair cell sample.Blood sample can be an arbitrary volume, for example less than 5 microlitres, or surpasses 5 liters, and this is needed to decide by test fully.

Magnetic bead

The magnetic bead that can be transported and locate by electromagnetic force in magnetic field can have the material of magnetic (as γ Fe by any ₂O ₃, promptly be in the Fe of γ phase ₂O ₃).The magnetization particulate can induce magnetic dipole when applying magnetic field, and when externally-applied magnetic field disappeared, the magnetic dipole that induces is vanishing thereupon also.Suitable magnetic material comprises, for example, and iron compound.Magnetic material can combine with other material, for example polymer.Magnetic bead surfaces can be wrapped by one or more compounds so that with sample in direct or indirect being connected of interested entity molecule.Magnetic bead can be an arbitrary shape, normally spherical and ovum shape, but this necessary requirement that is not the present invention.With during biological chemistry is separated, the application of magnetic bead is very extensive biological.Magnetic bead comprises magnetic bead that coupling has joined various specific junction mixtures all having obtained commercialization (DynalBiotech can using multiple magnetic bead among Lake the present invention.When using multiple magnetic bead, multiple magnetic bead has kinds of surface character, and they just can be connected with the different entities molecule in the sample like this.Like this, can use method of the present invention that multiple entity molecule is separated.The different surfaces character of magnetic bead can exchange mutually, for example, can be achieved by different compound in the reversible or irreversible combination of magnetic bead surfaces.

Magnetizable material is meant when applying magnetic field can induce magnetic dipole, and when externally-applied magnetic field disappeared, the magnetic dipole that induces is vanishing thereupon also.In application, can use commercially available magnetizable material or magnetic bead.The size of majority of particles below micron (for example 50 nanometers to 0.5 micron) to tens microns.Particulate can have different structures and composition.A kind of structure of magnetic particle is at small bits of iron magnetic material outer wrapping one deck colloid, as polystyrene layer.Can also nano level ferrimagnet and colloid as, polystyrene mixes and forms.The surface of these two kinds of particulates all is a polystyrene, can be further with various types of molecular modifications in the above.

Specificity junction mixture

In the present invention, the solution that comprises magnetic particle also can comprise one or more bonds.Bond can be one or more protein, as antibody, antibody fragment, antigen, aglucon (for example, but being not limited only to receptor-ligand), phytohemagglutin phytolectin or the like.Bond also can be organic or inorganic molecule, for example the receptor-ligand of nickel, glutathione, biotin, Avidin, Streptavidin, non-albumen or aglucon analog or the like.Bond also can be a nucleic acid, as RNA, DNA or the nucleic acid of any non-natural existence.Bond can or irreversible combine reversible with magnetic particle.At solid phase surface coupling connection molecule, all be method common in this area as the method for nucleic acid and albumen.

Electromagnetic chip

On electromagnetic chip, can the particulate with electromagnetic property be separated.Can provide the signal source of electric current to be positioned at the outside of chip, electromagnetic unit is integrated on the chip, and the electromagnetic force of handling usefulness can the person's of being operated control.Electromagnetic unit can produce magnetic field, thereby magnetic bead is applied magnetic field force.Electromagnetic unit can have different geometries.For example, electromagnetic unit can be one for example metal by conductor material round a ferromagnetic coil, this unit can be fabricated on the chip by the method for sputter, plating, deposition.Can comprise the electromagnetic unit (U.S. Patent application, the number of applying for a patent is 09/399,299, submits September 16 1999 date) in or a plurality of following patent on the electromagnetic chip.The example of other electromagnetic unit can referring to, but be not limited only to Ahn, C., et al., J.Microelectromechanical Systems.Volume 5:151-158 (1996); Ahn, C., et al., IEEE Trans.Magnetics.Volume 30:73-79 (1994); Liakopouloset al., in Transducers 97, pages 485-488, presented in 1997 InternationalConference on Silid-State Sensors and Actuators, Chicago, June 16-19,1997; US patent No.5,883,760 by Naoshi et al.These articles and just in the U.S. Patent application of application process (number of applying for a patent: 09/399,299, submit September 16 1999 date), also comprising required material, method and the producton order in electromagnetic structure unit that is used to make on the chip.

Electromagnetic chip can be made by following material, pottery, polymer, multipolymer, plastics, rubber, silicon, glass or the like.The surface area of electromagnetic chip can be from 1mm ²To 0.25m ²Preferably used chip surface area is from 4mm ²To 25cm ²About.Chip can have difformity, shape such as rectangle, circle, ellipse or other irregular shape of rule.The surface of chip should have by processing at chip surface or structures such as the groove that etching gets, reaction tank.

Electromagnetic chip can be an ingredient of reaction tank, and wherein reaction tank is in order to hold the structural unit of fluid sample.Reaction tank can be made by the impermeable material of any liquid, as silicon, glass, metal, pottery, polymkeric substance, plastics, acrylic acid or the like.

Reaction tank in the electromagnetic chip among the present invention or in this method has one or more ports, or has opening on the wall of reaction tank.Port can be any size, but suitable be to allow sample to add the shape and size of reaction tank by conduit.Conduit can be the tubular structure that any permission liquid passes through, such as rubber tube, polyfluortetraethylene pipe or polyethylene pipe.Port can provide an opening so that introduce sample by pipette or syringe for the reaction tank wall.

Conduit can be introduced sample from one or several port by pump (for example peristaltic pump or inculcate pump), syringe or gravity to reaction tank.One or more reagent, damping fluid, solution, for example, but be not limited only to the sample solution that to modify the dielectric properties of one or more entity molecules in the sample among the present invention, can add reaction tank simultaneously with sample, or add reaction tank in sample prior to, back.Also can be before entering reaction tank, earlier sample and reagent, damping fluid or solution are mixed earlier, such mixed process can be carried out in the conduit of fluid being introduced reaction tank, also can carry out in one or a plurality of and sample cell that conduit links to each other.

The reaction tank of electromagnetic chip of the present invention can be an arbitrary dimension, should hold the fluid of 1 μ l to 50ml usually, better should hold the fluid of 1 μ l to 20ml, and optimal cases is to hold the fluid of 10 μ l to 10ml.Reaction tank can be made by any suitable material, as silicon, glass, metal, pottery, polymkeric substance, plastics or the like, can be the quality stiff materials, also can be the material of quality softness.

It must be noted that,, must use chip with geometry in particular and parameter for the reaction tank with larger capacity (as above to 50ml).Chip can be produced on the flexible material, so that chip is folded to form the passage that is similar to reaction tank.Can use the polylith chip in one reaction pool, electromagnetic unit also will satisfy and can effectively electromagnetic force be applied in the reaction tank in the interesting areas.

Sample solution adds sample

Sample, sample solution and additional solution, damping fluid, reagent can add reaction tank in any mode easily, for example shift by transfer pipet, injector to inject, utilize gravity to introduce by conduit (for example polyethylene catheter or the like).Generally, sample, sample solution and other solution, damping fluid, reagent add reaction tank in the mode of continuous flow, such continuous flow can be injected into or pump into reaction tank from least one inlet, then, do not contain sample component and other liquid and flow out reaction tank from least one outlet.

Before sample adds reaction tank, can in sample, add sample solution earlier.Before sample-sample solution mixed liquor is added reaction tank, sample and sample solution jointly incubation from less than time arbitrarily among 1 second to several hours to several days.In the conduit that the mixing of sample and sample solution can occur in reaction tank is communicated with, as shown in Figure 1.Also can earlier sample be added reaction tank, then sample solution be added reaction tank again; Or earlier sample solution is added reaction tank, then sample is added reaction tank again.

The magnetic bead that uses in the present invention wherein, can with can the selectivity born of the same parents separate erythrocytic sample solution and provide together or separately.If magnetic bead provides separately, so magnetic bead can be before sample solution adds sample, add simultaneously or afterwards.

Sample is added on the electromagnetic chip

Sample solution can add sample or comprise in the reaction tank of electromagnetic chip and mix before sample deposition is to the electromagnetic chip.Sample-sample solution mixed liquor is being fed before reaction tank separates, sample and sample solution can leave standstill a period of time jointly, from can less than 1 second to several hours even several days.The mixed process of sample and sample solution can occur in the conduit that feeds reaction tank.Perhaps, can earlier sample be joined in the reaction tank, again sample solution be added reaction tank; Also can earlier sample solution be joined in the reaction tank, again sample be added reaction tank.

Usually, magnetic bead should be added sample and carry out leaving standstill a period of time before the magnetic bead separation, during this period of time can be from extremely several hours even several days a few minutes.Can the selectivity born of the same parents separate erythrocytic sample solution add sample before, simultaneously or in sample, add magnetic bead afterwards.

Sample, sample solution and additional solution, damping fluid, reagent can add reaction tank in any mode easily, for example shift by transfer pipet, injector to inject, utilize gravity to introduce by conduit (for example polyethylene catheter or the like).Generally, sample, sample solution and other solution, damping fluid, reagent add reaction tank in the mode of continuous flow, such continuous flow can be injected into or pump into reaction tank from least one inlet, then, do not contain sample component and other liquid and flow out reaction tank from least one outlet.

Can be by one or a plurality of conduit feeds one or more additive reagents in sample, as particulate, but this is not of the present invention necessarily requiring.For example, can add sample as particulate, leave standstill jointly after a period of time, again sample be joined in the reaction tank earlier with one or more additive reagents.Perhaps, particulate can contact with sample by the conduit that links to each other with reaction tank, and like this, particulate can mix with sample in sample flows into the process of reaction tank mutually.Also can be when sample be fed reaction tank or afterwards, with additive reagent such as particulate by one or a plurality of conduit feeds reaction tank.Use if desired more than a kind of additive reagent, then they can or add sample respectively according to the above-mentioned method while.

Use electromagnetic force to separate

Need on chip, produce corresponding Distribution of Magnetic Field on the electromagnetic chip magnetic bead being handled.A kind of mode that produces such magnetic field is to use electromagnetic unit.When on electromagnetic unit, applying electric current, can produce corresponding magnetic field.Can determine DISTRIBUTION OF MAGNETIC FIELD by ON/OFF signal and adjusting strength of current.According to required Distribution of Magnetic Field requirement, can design little electromagnetic unit with different structure and size.The example of electromagnetic unit can be referring to following list of references, but is not limited only to following content: Ahn, C., et al., J.Microelectromechanical Systems.Volume 5:151-158 (1996); Ahn, C., et al., IEEE Trans.Magnetics.Volume 30:73-79 (1994); Liakopouloset al., in Transducers 97, pages 485-488, presented in 1997 InternationalConference on Solid-State Sensors and Actuators, Chicago, June 16-19,1997; US patent No.5,883,760 by Naoshi et al.The example of other electromagnetic unit can be referring to the United States Patent (USP) in application process just, application serial 09/399,299, and submitting the date is on September 16th, 1999.

To the manipulation of magnetic particle comprise magnetic particle direct motion, focus on and catch.In magnetic field, the motion of magnetic particle can be called " magnetophoresis (magnetophoresis) ".Can be referring to below with reference to document about the theory of the particulate magnetophoresis that is used for cell separation and other application and method: Magnetic Microspheres in Cell Separation, by Kronick, P.L.in Methods of Cell Separation, Volume 3, edited by N.Catsimpoolas, 1980, pages 115-139; Use of magnetic techniques for the isolation ofcells, by Safarik I.And Safarikova M., in J.of Chromatography, 1999, Volume 722 (B), pages 33-53; A fully integrated micromachined magneticparticle separator, by Ahn C.H.et al., in J.of Microelectromechanicalsystems, 1996, Volume 5, pages 151-157.Relevant use electromagnetic chip carries out contents separated to the entity molecule that is combined with magnetic particle can be referring to U.S. Patent application (application number 09/399,299 be submitted September 16 1999 date).

In the entity example of Fig. 5, contain magnetic particle 400 in the sample solution of the present invention, have on the magnetic particle can with the antibody of leucocyte specific bond.Blood sample comprises leucocyte 420, red blood cell 450 and other entity molecule and cell (not providing) in Fig. 5.Add sample solution in the blood sample so that the red blood cell born of the same parents separate,, comprise that magnetic particle 400 feeds reaction tank again with sample-sample solution potpourri.Sample-sample solution should leave standstill a period of time in reaction tank jointly, so that the abundant combination of magnetic particle and leucocyte.Contain the electromagnetic chip of forming by little electromagnetic unit in the reaction tank.On little electromagnetic unit, apply electric current, can on chip, will catch with the magnetic particle of leucocyte combination.Other sample component can by pump as the liquid flush away.

Above-mentioned example can use other antibody catching the cell of other kind, and those can be coated on the surface of magnetic particle with the antibody of the special surface antigen combination of surface epithelial cell.Like this, above-mentioned method can detect and catch metastatic breast cancer cell and these epithelial cells of metastatic lung carcinoma cell on one's body from the peripheral blood that the patient extracts.The another one example is to catch fetal cell from maternal blood liquid.In this example, the magnetic particle pan coating can with the antibody of fetal cell specific bond.

Example

Following example has shown among the present invention characteristic and the using method in order to the sample solution of the entity molecule in the sample separation.

Example 1: to the analysis of can the selectivity born of the same parents separating erythrocytic sample solution

Following solution is separated erythrocytic effect in order to test selectivity born of the same parents.

(1.1) form 1: the sucrose damping fluid of human blood sample and 2% (mass percent) (approximately 60mOsm) mixes with 1: 19 ratio.Blood sample and the mixed different time of sucrose damping fluid, complete leucocyte (WBC) and red blood cell (RBC) in the blood sample can be examined under a microscope and count.According to the data of form 1,2% sucrose solution can be used as the sample solution among the present invention.

Detection time (minute)	Leucocyte (cell number/ml)	Red blood cell (cell number/ml)
			2	1.6×10 5	3.0×10 5
5	1.2×10 5	2.4×10 5
			10	1.1×10 5	2.0×10 5
15	1.0×10 5	2.0×10 5
			20	0.8×10 5	1.9×10 5
30	0.8×10 5	1.8×10 5

(1.2) form 2: the sucrose damping fluid of human blood sample and 2.2% (mass percent) (approximately 66mOsm) mixes with 1: 19 ratio.Blood sample and the mixed different time of sucrose damping fluid, natural leucocyte (WBC) and red blood cell (RBC) in the blood sample can be examined under a microscope and count.According to the data of form 2,2.2% sucrose solution can be used as the sample solution among the present invention.

Detection time (minute)	Leucocyte (cell number/ml)	Red blood cell (cell number/ml)
			2	2.0×10 5	8.1×10 5
5	1.9×10 5	8.0×10 5
			10	1.6×10 5	7.8×10 5
15	1.5×10 5	7.7×10 5
			20	1.2×10 5	6.6×10 5
30	0.8×10 5	4.9×10 5

(1.3) form 3: human blood sample and various sucrose damping fluid mix with 1: 9 ratio.Blood sample and the mixing of sucrose damping fluid are after 5-10 minute, and natural leucocyte (WBC) and red blood cell (RBC) in the blood sample can be examined under a microscope and count.According to the data of form 1, sucrose solution can be used as the sample solution among the present invention, and mixes with the ratio except 9: 1 with blood sample.

2.0％(-60mOsm)	＜＜1∶20	Remain a large amount of red blood cells	Most leucocytes are unaffected
				2.2％(-66mOsm)	＜＜1∶20	Remain a large amount of red blood cells	Most leucocytes are unaffected
2.4％(-72mOsm)	＜＜1∶20	The red blood cell that residue is very many	Most leucocytes are unaffected

Form 4: human blood sample and various sucrose damping fluid mix with 1: 24 ratio.Blood sample and the mixing of sucrose damping fluid be after 5-10 minute, the natural leucocyte in the blood sample

(WBC) and red blood cell (RBC) can examine under a microscope and count.

Sucrose concentration (mass percent)	Leucocyte: red blood cell	Red blood cell	The leucocyte state
				1.3％(-39mOsm)	-------	Remain few red blood cell	Remain few leucocyte
1.4％(-42mOsm)	-------	Remain a spot of red blood cell	Remain a spot of leucocyte
				1.5％(-45mOsm)	-------	The red blood cell of remainder	Remain a spot of leucocyte
1.6％(-48mOsm)	5∶1-2∶1	-------	Remain a spot of leucocyte
				1.8％(-54mOsm)	～1∶1	-------	Most leucocytes are unaffected
2.0％(-60mOsm)	-------	Remain a large amount of red blood cells	Most leucocytes are unaffected
				2.2％(-66mOsm)	＜＜1∶20	Remain a large amount of red blood cells	Most leucocytes are unaffected
2.4％(-72mOsm)	＜＜1∶20	The red blood cell that residue is very many	Most leucocytes are unaffected

(1.5) the hypotonic red blood cell born of the same parents of other type separate liquid (concentration are 0.5%, 0.65%, 0.7%, 0.75%, 0.8% and 0.85% glycerite, all be mass percent) test findings be: consider remaining leukocytic quantity and leucocyte and erythrocytic ratio, optimal cases is that 0.8% glycerite and blood sample are mixed with 9: 1 ratio.

(1.6) we also separate liquid to multiple red blood cell born of the same parents commonly used in the present biology laboratory and test, a kind of solution is to contain 0.826% ammonium chloride (mass/volume), 0.1% saleratus, with the solution of 0.0037% EDTA, also has the ammonium oxalate solution (210mOsm) of a kind of 1.1-1.2% of being (mass/volume).These solution can make the red blood cell born of the same parents separate, and have only a fraction of leucocyte to be affected in a period of time.But these solution have very high conductivity, and leukocytic dielectrophoresis is caught and collected and can not carry out therein.And these solution are " being harmful to " to leucocyte, because after a period of time (as 30 minutes), the most of leucocytes in the solution are also separated by born of the same parents.So these solution are unsuitable for as sample solution of the present invention.

Example 2: in hypotonic solution, utilize dielectric migration on chip, leucocyte to be carried out dielectrophoresis and separate with dielectric delay technology

The sucrose solution of blood sample and 2% is mixed with 1: 19 ratio.With microsyringe 40 μ l mixed liquors being injected volume again is the reaction tank of 40 μ l.Quantity of leucocyte in the 2 μ l blood samples is 10-20 * 10 ³Individual cell.Comprise a glass-chip in the reaction tank, be shaped on interlaced gold/platinum electrode array on it.The characteristic dimension of electrode (for example distance of electrode width, tip width, adjacent electrode) is 50 microns.What electric signal source (Hewlett Packard) produced is that frequency is 5MHz, and peak-to-peak value is the electric signal of 5V.Leucocyte is collected at electrode edge, and the bib that born of the same parents separate is suspended in the electrode plane top by the negative sense dielectrophoresis force, and the damping fluid that is introduced into is gone out reaction tank.The leucocyte that electrode surface is collected washes out and counts again.The leukocytic quantity that is collected is 4.65 * 10 ³

Example 3: can the selectivity born of the same parents separate in the erythrocytic solution, utilizing dielectric delay technology that leucocyte is carried out dielectrophoresis and separate

The sucrose solution of blood sample and 2% is mixed with 1: 19 ratio, at room temperature left standstill 5 minutes so that the red blood cell born of the same parents separate.Again sample-sample solution potpourri is added on the dielectrophoresis chip of being made up of the interlaced gold/platinum electrode that is produced on the glass substrate.The characteristic dimension of electrode (for example distance of electrode width, tip width, adjacent electrode) is 50 microns.What electric signal source (Hewlett Packard) produced is that frequency is 5MHz, and peak-to-peak value is the electric signal of 5V.Leucocyte is collected at electrode edge, and remaining sample-sample solution can be sucked out reaction tank not influencing under the leukocytic prerequisite that is collected.The leucocyte that electrode surface is collected washes out with the PBS damping fluid again.

The leucocyte sample of collecting is analyzed the DQB gene with round pcr again.PCR reaction system (25 μ l): the collection sample of 1 μ l, 19 μ l H2O, 2 μ l primers, 2.5 μ l PCR damping fluids, 0.5 μ l dNTP (10 mM), 1 μ l Taq enzyme.Wherein, special primer at the DQB gene is ordered to giving birth to worker (Sangon) company, and sequence is:

DQB1 primer 1:5 '-CATGTGCTACTTCACCAACGG-3 '

The DQB1 primer 2: 5 '-CTGGTAGTTGTGTCTGCACAC-3 '

At the Taq enzyme (Cetus) that adds a unit before, reaction mixture heating 3 minutes under 95 degree earlier, ice bath 3 minutes at once again.Such step repeats 3 times, and the PCR instrument that uses is GeneAmp PCR System 9700, and program is: 94 degree unwind 30 seconds, 55 degree annealing 30 seconds, and 72 degree extended 40 circulations 40 seconds.

PCR product electrophoresis on agarose gel then, special band show that red blood cell is seedless, does not contain the DQB gene to the separating of leucocyte success.

Example 4: use can the selectivity born of the same parents be separated erythrocytic sample solution and magnetic particle and from blood sample leucocyte is carried out magnetic force catch on electromagnetic chip

In order to prepare in conjunction with leukocytic magnetic particle is that the immunity of the anti-CD15 of 12.5 μ l separates magnetic bead (Dynal) separates magnetic bead (Dynal) with the immunity of the anti-CD45 of 12.5 μ l potpourri, is contained in the microcentrifugal tube.With a magnet magnetic bead is adsorbed onto on the tube wall earlier, remove other impurity, remove magnet, in microcentrifugal tube, add the PBS/BSA damping fluid (phosphate in the 25 μ l Dynal company leucocyte magnetic resolution kits, Ph 7.4, contain 0.1%BSA and 0.6% sodium citrate), magnetic bead is suspended.Hold magnetic bead with magnet, remove damping fluid, add damping fluid again, magnetic bead is redissolved therein.

Blood sample that 100 μ l are cold and 2% sucrose solution mix with 1: 19 ratio.At room temperature left standstill 5 minutes so that the red blood cell degraded.The immunity that the immunity of anti-CD15 is separated magnetic bead (Dynal) and anti-CD45 separates the potpourri adding sample-sample solution mixed liquor of magnetic bead (Dynal).With injector sample and magnetic bead are fully mixed, blood and magnetic bead were left standstill under the 2-8 degree 15 minutes.Again sample-sample solution-magnetic bead potpourri is added electromagnetic chip, wherein electromagnetic chip comprises the substrate of a silicon, is shaped on conductor coils on it, and hub of a spool has ferromagnetic core.

The DC source made from Keithley applies electric current to coil, can the leucocyte that be combined with magnetic bead be separated.Be combined with leukocytic magnetic bead like this and be attracted to the surface of chip, and other blood constitutent is washed out chip by fluid.

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Claims (52)

1. sample solution, when this sample solution with have after blood sample mixes:

(a) optionally modify at least one dielectric property of at least a composition in the sample;

(b) sample solution has suitable conductivity, can adopt dielectrophoresis force to come one or more entity molecules in the sample separation;

(c) the mixed final osmotic pressure of sample solution and blood sample is 20mOsm to 150mOsm.

2. the sample solution described in the claim 1, wherein the final osmotic pressure after sample solution and the sample mix is 30mOsm to 80mOsm.

3. the sample solution described in the claim 1, wherein said solution comprises one or more zwitter-ions.

4. the sample solution described in the claim 1, wherein said solution comprises one or more enzymes.

5. the sample solution described in the claim 1, wherein said solution comprises one or more washing agent.

6. the sample solution described in the claim 1, wherein said solution comprises one or more combinations that can combine with entity molecule in the sample.

7. the sample solution described in the claim 1, wherein said sample solution optionally break born of the same parents to red blood cell and handle.

8. the sample solution in the claim 7, wherein said sample solution comprises glycerine.

9. the solution described in the claim 8, the glycerol concentration that wherein said solution comprises guarantee that the concentration that mixes glycerine in the liquid is between 0.075% to 0.085% after solution and whole blood sample mix.

10. the sample solution described in the claim 7, wherein said sample solution comprises sucrose, mannose, sweet mellow wine or sorbierite.

11. the sample solution described in the claim 10, wherein said sample solution comprises sucrose.

12. the sample solution described in the claim 10, the sucrose concentration that comprises in the wherein said sample solution guarantee after solution and whole blood sample mix, and mixes in the liquid concentration of sucrose between 1.62% to 2.16%.

13. being used for the method for one or more entity molecules of sample separation comprises:

(a) add the sample solution described in the claim 1;

(b) utilize dielectrophoresis force to separate one or more entity molecules in the described blood sample.

14. the method described in the claim 13, wherein said entity molecule is a cell.

15. the method described in the claim 14, wherein said cell be leucocyte, tumour cell, stem cell, neoblast, fetal cell, by the cell of pathogenic infection or bacterial cell.

16. the method described in the claim 13, wherein said entity molecule are the parts of pathogen or pathogen.

17. the method described in the claim 13, wherein said blood sample are the blood samples from the people.

18. the method described in the claim 13, wherein said entity molecule are separated in the fluid pool on chip.

19. the method described in the claim 18, wherein said fluid pool are to be made of glass, at least a pottery, at least a plastics or at least a polymer.

20. the method described in the claim 18, wherein said fluid pool have a port at least.

21. the method described in the claim 20, wherein said fluid pool contain two ports.

22. the method described in the claim 20, at least one port connects one or more conduits on the wherein said fluid pool.

23. the method described in the claim 13, wherein said entity molecule are separated in the fluid pool on chip, described blood sample is to add in the fluid pool continuously.

24. the method described in the claim 18, wherein said sample solution is to add in the fluid pool continuously.

25. the method described in the claim 18, wherein said sample solution is to join in the fluid pool before sample.

26. the method described in the claim 18, wherein said sample are to join in the fluid pool before described sample solution adds.

27. the method described in the claim 18, the wherein said sample solution that adds in the sample occurs in sample and joins before the fluid pool.

28. the method described in the claim 18, wherein said sample and described sample solution join in the fluid pool simultaneously.

29. the method described in the claim 18, wherein said chip comprises at least two electrodes.

30. the method described in the claim 18, wherein said chip material are glass, silicon, rubber, plastics, pottery or at least a polymer.

31. the method described in the claim 13, wherein said solution comprise one or more combinations that can combine with entity molecule in the sample, this method further comprises combining between at least a entity molecule and at least a combination in the sample.

32. the method described in the claim 31, wherein said at least a combination is at least a particulate.

33. the method described in the claim 31, wherein said combination comprises at least one antibody or antibody fragment.

34. the method described in the claim 31, wherein said combination comprise biotin, avidin, antibiotin streptococcal protein.

35. the method described in the claim 32, wherein said at least a particulate comprises one or more nucleic acid molecules.

36. the method described in the claim 32, wherein said at least a microparticle material is metal, pottery, plastics, carbon, glass or polymer.

37. the method described in the claim 32, the size of wherein said mean particle dia is between 2 microns to 50 microns.

38. the method described in the claim 13, wherein said separation are to realize from, row ripple dielectrophoresis or two-dimentional dielectrophoresis by dielectric delay, dielectric migration, dielectric/gravity field flow point.

39. be used for the method for the one or more entity molecules of separating blood sample, comprise:

(a) add the sample solution described in the claim 1;

(b) in blood sample, add at least a solution for preparing that comprises one or more magnetic particles that can combine with entity molecule;

(c) described blood sample is joined on the electromagnetic chip;

(d) described blood sample is subjected to the effect of electromagnetic force, and interested entity molecule optionally is trapped in one or more zones on the described chip.

40. the method described in the claim 39, wherein said interested entity molecule is a cell.

41. the method described in the claim 40, wherein said cell comprise leucocyte, tumour cell, stem cell, neoblast, fetal cell, by the cell of pathogenic infection or bacterial cell.

42. the method described in the claim 39, wherein said interested entity molecule are virus.

43. the method described in the claim 39, wherein said interested entity molecule comprises one or more albumen.

44. the method described in the claim 39, wherein said interested entity molecule comprises one or more nucleic acid molecules.

45. the method described in the claim 39, wherein said blood sample are the blood samples from the people.

46. the method described in the claim 39, wherein said chip comprise that at least one can produce the parts of electromagnetic force.

47. the method described in the claim 39, wherein said magnetic particle comprise one or more combinations that can combine with entity molecule in the sample.

48. the method described in the claim 47, wherein said one or more combinations comprise at least one antibody or antibody fragment.

49. the method described in the claim 47, wherein said one or more combinations comprise biotin, avidin, antibiotin streptococcal protein.

50. the method described in the claim 47, wherein said at least one combination comprises one or more nucleic acid.

51. the method described in the claim 39 wherein saidly adds at least a solution that comprises one or more magnetic particles in the blood sample and is adding before the adding blood sample toward electromagnetic chip in.

52. the method described in the claim 39 wherein saidly adds at least a solution adding add blood sample toward electromagnetic chip in after that comprises one or more magnetic particles in the blood sample.

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