For collecting the equipment, system and method for target substanceCross-reference to related applications
This application claims the equity for the Provisional Application No. 62/345,172 that on June 3rd, 2016 submits, and are also 2015The part continuation application for the application number 14/610,522 submitted January 30, the latter require on 2 4th, the 2014 interim Shens submittedPlease numbers 61/935,457 equity, and be also 14/495,449 (the present patent No. of application number that September in 2014 is submitted on the 24thOn May 26th, 9,039,999,2016 authorization) part continuation application, the latter is the application number submitted on November 26th, 201314/090,337 part continuation application, it is required that the following equity applied: on November 30th, 2012 Provisional Application No. submitted61/732,029；The Provisional Application No. 61/745,094 that on December 21st, 2012 submits；The interim Shen that on March 15th, 2013 submitsIt please number 61/791,883；The Provisional Application No. 61/818,301 that on May 1st, 2013 submits；Face with what is submitted on August 26th, 2013When application number 61/869,866；And it is also the part continuation application for the application number 14/266,939 submitted on May 1st, 2014,The Provisional Application No. 61/ that its 61/818,301,2013 year August of Provisional Application No. for requiring on May 1st, 2013 to submit is submitted on the 26thThe equity of 869,866 and 2014 years 2 months Provisional Application No. 61/935,457 submitted for 4th.
Technical field
Present disclosure is generally related to the separation of the fluid based on density, and target is in particular to recycled from suspensionMatter.
Background technique
Suspension often includes being difficult to detect, extract and separate the target substance for analysis.For example, whole blood is that substance existsSuspension in fluid.The substance include billions of red blood cells in the protein fluid of referred to as blood plasma and leucocyte withAnd blood platelet.It is routinely directed to the presence of aberrant biological or cell and checks whole blood, the aberrant biological or cell are such as ovumSon, fetal cell, endothelial cell, helminth, bacterium and inflammatory cell and virus, including HIV, cytomegalovirus, the third type liverScorching virus and epstein-Barr virus.Currently, practitioner, researcher and with blood sample work those of people trySeparately, certain components of separation and extraction peripheral blood sample are for checking.Typical technology for analyzing blood sample include withLower step: by blood film smear on glass slide, and so that certain components can pass through bright-field or fluorescence microscopy inspectionMode the film is dyed.
On the other hand, it is particularly difficult to the target substance that low-down concentration occurs (if not can not in suspensionIf energy) use many prior art detection and analysises.So practitioner, researcher and with suspension work those ofPeople continues to look for the existence or non-existence for being directed to rare target substance and the system and method for accurately analyzing suspension.
Detailed description of the invention
Figure 1A -1B shows one embodiment pipe (tube).
Fig. 2A -2C shows one embodiment pipe-process container (processing vessel) system.
Fig. 3 A-3B shows one embodiment sealing ring.
Fig. 3 C-3D shows one embodiment sealing ring.
Fig. 3 E-3F shows one embodiment sealing ring.
Fig. 3 G shows one embodiment sealing ring.
Fig. 4 A shows the flow chart of the embodiment method for recycling target substance.
Fig. 4 B shows the flow chart of the embodiment method for recycling target substance.
Fig. 5 A-5F shows the embodiment system of recycling target substance.
Fig. 6 A-6E shows the embodiment system of recycling target substance.
Detailed description of the invention
This disclosure relates to the equipment, system and method for recycling target substance from suspension.System includes processingContainer, displacement fluid (displacement fluid) and pipe.The pipe includes funnel, set (cannula) and cavity.The set is permittedPerhaps the fluid communication between the described funnel and the cavity, so that the process container is inserted into the cavity, and the set prolongsIt puts in the process container.
Figure 1A shows the isometric views of pipe 100.Figure 1B is shown along the line I-I shown in figure 1A pipe 100 madeViewgraph of cross-section.Pipe 100 can be any size appropriate or cross-sectional shape (such as cylinder, ellipse, triangle, justRectangular, rectangle etc.).Pipe 100 include main body 102, the main body 102 have connection first end 104, the second end 118 andThe side wall of collection section (segment) 106 between the first end 104 and the second end 118.Pipe 100 can be withIncluding cap 108 first end 104 is sealed or closed, the first end 104 can be unlimited.Collection section 106 includes leakageBucket 110, set 112 and cavity 114.
Funnel 110 is gradually reduced from first end 104 to the second end 118.Funnel 110 is by target substance from funnel 110Mouth is conveyed into set 112, and the set 112 connect and is in fluid communication with the vertex of funnel 110.The vertex of funnel 110 has than funnel110 mouth smaller diameter.Funnel 110 is formed by tapered wall, and the tapered wall can be straight, is curve, arc etc..LeakageBucket 110 can be any proper shape, including but not limited to, tubulose, hemispheric, paraboloidal, conical, squareShape, cone-shaped etc..
112 (such as pipe or syringe needle, including but not limited to non-coring syringe needles (non-coring needle)) are covered from recessThe vertex in space 114 extends and enters in cavity 114.Cavity 114 is aperture 120 from the second end 118 to first end104 dented spaces extended.Cavity 114 can receive and support process container (not shown).Cavity 114 can be any appropriateDepth to receive and support process container (not shown).Cavity 114 can be threaded with joining process container (not shown)Threaded section.Set 112 can extend any appropriately distance into cavity 114 so as to extend into process container (not shown),Pierce through the base portion or insertion process container (not shown) of process container (not shown).Set 112 may include flat tip, beveling pointPortion, sharpened tip or tapered tip portion.In addition, cavity 114 can be any proper shape, including but not limited to, tubulose, it is hemispheric, paraboloidal, conical, rectangle, cone-shaped etc..Set 112 can be by a variety of different material groupsAt, including but not limited to, ceramics；Metal；Organic or inorganic material；And plastic material, such as polypropylene, acrylic acid, poly- carbonic acidEster etc.；And combinations thereof.Set 112 can have tip along the longitudinal axis of the set.
First end 104 has the size for receiving cap 108.Cap 108 can be by (re-sealable) rubber of resealableThe material of glue or other suitable resealables composition, the material of the resealable can use syringe needle or other sharp devicesTool is pierced through repeatedly to approach the content in 100 internal storage of pipe, and is sealed again when the syringe needle or utensil are removed.It canAlternatively, cap 108 can be screw-cap, the complementary threads with screw thread to be bonded on inside or around first end 104.It canAlternatively, fixture (clip) can be placed on to 104 outside of first end of cap 108 and pipe 100 to increase by cap 108 in first endThe pressure applied in portion 104 is to provide stronger cooperation.The fixture can be double component rings (two-piece ring),One component ring around pipe 100 entire periphery or a component ring around less than pipe 100 entire periphery, such as two/One (1/2), 5/8ths (5/8), 2/3rds (2/3), 3/4ths (3/4), 7/8ths (7/8) etc..Alternatively, cap108 can either temporarily or permanently be attached to the main body 102 of pipe 100 via first end 104, such as (such as super by weldingSonic soldering connects), adherency (such as adhesive), clamp (clamping) (such as sealing ring, crimping machine, clip etc.) or for temporarilyOr for good and all attach any other appropriate ways of two components.
Pipe 100 can also include plug (plug) 116, and when covering 112 under, the plug 116 is inserted into cavity 114In to inhibit fluid to escape from from set 112.Plug 116 can be by the rubber or other suitable resealables of resealableThe material of material composition, the resealable can be pierced through and when the set 112 is removed Shi Zaimi repeatedly with set 112Envelope.Alternatively, plug 116 can be screw-cap, with screw thread to be bonded in cavity 114 or in the second end 118Complementary threads on outside or surface.Alternatively, it is possible to which fixture to be placed on to 104 outside of first end of plug 116 and pipe 100To increase the pressure applied on first end 104 from plug 116 to provide with stronger.The fixture can be doubleComponent ring, component ring around pipe 100 entire periphery or a component ring around less than pipe 100 entire periphery, such asHalf (1/2), 5/8ths (5/8), 2/3rds (2/3), 3/4ths (3/4), 7/8ths (7/8) etc..
Pipe 100 can be made of a variety of different materials, including but not limited to: ceramics；Metal；Organic or inorganic material；And plastic material, such as polyformaldehydePolystyrene, acrylonitrile-butadiene-styrene (ABS) (" ABS ") copolymer,Aromatic polycarbonate class, aromatic polyester class, carboxymethyl cellulose, ethyl cellulose, ethylene-vinyl acetate copolymer, nylon,Polyacetals, poly-vinegar esters of gallic acid, polyacrylonitrile and other nitrile resins, acrylonitrile-vinyl chloride copolymer, polyamide-based, aromaticsPolyamide-based (" aramid fiber "), polyamide-imides, polyarylate class, polyarylene oxides, poly-arylene sulfide, polyarylsulfone (PAS)Class, polybenzimidazoles, polybutylene terephthalate (PBT), polycarbonate, polyester, polyesterimide class, polyether sulfone, polyethers acylImines, polyetheretherketone, polyethylene terephthalate, polyimide, polymethacrylates, are gathered polyethers ketoneAlkene (for example, polyethylene, polypropylene), gathers polyallmerDiazole, polyphenyl ethers (PPO), changes ParyleneThe PPO class of property, polystyrene, polysulfones, fluoropolymer (such as polytetrafluoroethylene (PTFE)), polyurethane, polyvinyl acetate, polyethyleneAlcohol, polyvinyl chloride acetate copolymer, polyvinylpyrrolidone, gathers inclined dichloro at polyethylene halogen class (such as polyvinyl chloride)Ethylene, specialty polymer, polystyrene, polycarbonate, polypropylene, acrylonitrile-butadiene-styrene copolymer, butyl rubber,Ethylene-propylendiene monomer；And combinations thereof.Pipe 100 can be rigid or flexible.
Fig. 2A shows the exploded view of embodiment pipe 100 and process container 202.Fig. 2 B show process container 202 etc.Axonometric drawing, the process container 202 are inserted into cavity 114 at the collection section of pipe 100 106 to form the containment system of processing pipe200.Fig. 2 C shows the viewgraph of cross-section along the process container 202 made of line II-II shown in Fig. 2 B, the process container202 are inserted into cavity 114 at the collection section of pipe 100 106.Pipe 100 and process container 202 form pipe-process container system 200.Process container 202 can be eppendorf pipe, syringe or test tube, and have closing end 204 and open end 206.It is openEnd 206 has the size for receiving cap 208.Cap 208 can be by the rubber or other suitable resealables of resealableMaterial composition, the material of the resealable can be pierced through repeatedly with syringe needle or other sharp instruments with approach processing holdThe content of 202 internal storage of device, and sealed again when the syringe needle or utensil are removed.Alternatively, process container 202Also two open ends be can have, the open end has the size for receiving cap.Process container 202 can have direction and openPut the taper geometry that end 206 broadens or narrows；Process container 202 can have geometry generally cylindrical in shape；OrPerson, process container 202 can have geometry generally cylindrical in shape in the first paragraph and the cone shape in second segmentGeometry, wherein first segment and second segment are connected to each other and are continuous.Although at least one section of process container 202With circular cross section, but in other embodiments, at least one described section can have ellipse, square, threeAngular, rectangle, octagonal or any other suitable cross-sectional shape.Process container 202 can by it is transparent, translucent,Opaque or sub- transparent material (such as plastics or another suitable material) composition.The process container includes centerAxis 214, the central axis 214 is coaxial with the central axis of pipe 100 when in process container insertion cavity 114.Process container 202 can also include the plug 210 at closing end 204 to allow introducing or and the displacement fluid of target substance212 exchange target substances.Closing end 204 threaded can be connected with providing with the screw thread of the threaded cavity (not shown) of pipe 100It connects.Process container 202 can be made of glass, plastics or other suitable materials.
Plug 210 can be made of the rubber of resealable or the material of other suitable resealables, it is described canThe material resealed can be pierced through with syringe needle or other sharp instruments repeatedly to approach in 202 internal storage of process containerIt is tolerant or allow to be introduced into content in process container 202, and sealed again when the syringe needle or utensil are removed.It can incite somebody to actionTo maintain the sealing between plug 210 and process container 202 in 210 insertion process container 202 of plug, such as matched by interferenceIt closes (interference fit).It alternatively, can be in the closing end of process container 202 using heated liquid rubberPlug 210 is formed in 204, the rubber can be shaped in warm or heat and be hardened as the rubber is cooling.It can be used forThe adhesive that plug 210 is attached to the inner wall of process container can be to adhesive, epoxy resin, contact-type based on polymerAdhesive or any other suitable material for being bonded or establishing heat bonding.Alternatively, it is possible to by 210 injection of plugIt manages in container 202.Alternatively, it is possible to which plug 210 is heat-bonded to process container 202.
For example, set 112 can have tapered tip portion, the tapered tip portion pierces through plug 210 and extends into process container 202Inner chamber body, the axis for covering 112 do not extend into the inner chamber body of process container 202.As being explained in greater detail below, processing is heldThe inner chamber body of device 202 accommodates target substance.Set 112 can be covered by sleeve (sleeve) (not shown) of resealable to preventTarget substance outflow, unless process container 202 is to reach to allow to cover 112 depths for just breaking through process container 202 in cavity 114Degree.The sleeve (not shown) covering jacket 112 of the resealable, has spring, can be penetrated with quilt cover 112, andBy being resistant to pierce through repeatedly while the elastomeric material of sealing still being maintained to be made.
Before being inserted into inlet pipe 100, displacement fluid 212 can be loaded to process container 202.Displacement fluid 212 replaces target substance,So that when the insertion of process container 202 is included the primary tank pipe 100 of target substance, pipe 100 and the experience centrifugation of process container 202,Displacement fluid 212 flows out into pipe 100 from process container 202, and by replacement, is such as replaced by buoyancy (i.e. by substanceIt is lifted up), it pushes target substance to pass through set 112 and enters process container 202.
(density can be greater than to suspend the density of density of the displacement fluid 212 with the expectation target substance greater than suspensionThe a subset (subset) of liquid fraction or the density of all suspension fractions), and be inert for suspended material.For example, the displacement fluid can have it is bigger than the density for it is expected target substance by about 0.001 to about 0.005g/cm3Density.Displacement fluid 212 can be miscible or immiscible in suspension.The example of suitable displacement fluid includes but unlimitedIn: with the solution of polyvinylpyrrolidone (such as Percoll) coated colloidal silica particle, polysaccharide solution (such asFicoll), Iodixanol (such as OptiPrep), organic solvent, liquid wax, oil, gas, and combinations thereof；Olive oil, mineralIt is oil, silicone oil (silicone oil), impregnation oils, mineral oil, paraffin oil, silicone oil (silicon oil), fluorosilicone, completeFluoronaphthalene alkane, perfluor perhydro luxuriant and rich with fragrance (perfluoroperhydrophenanthrene), perfluoro bromide octane, and combinations thereof；It is organic moltenAgent, such as Isosorbide-5-Nitrae-twoAlkane, acetonitrile, ethyl acetate, the tert-butyl alcohol, cyclohexanone, methylene chloride, tert-pentyl alcohol, t-butyl methyl ether,Butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propene carbonate, tetramethylene sulfone (tetramethylene) and ion fluid sulfones；Solution based on polymer；Surfactant；Perfluoroketone (such as perfluorocyclopentanone and completeFluorine cyclohexanone), fluorination ketone (fluorinated ketones), hydrofluoroether class, hydrofluorocarbon, perfluoro alkane class, perfluoropolyetherClass, silicon and the liquid (such as phenyl methyl siloxane) based on silicon；And combinations thereof.
Process container 202 can also include processing solution (not shown) with the realization when target substance enters process container 202Conversion to target substance.Processing solution (not shown) can be preservative, cell adherence solution, dyestuff etc..Different from displacement fluid212, the processing solution (not shown) of most of (if not all) is stayed in after centrifugation in process container 202, thus with oneKind or another way (that is, anti-corrosion, increase adhesion property etc.) realize conversion to target substance.Processing solution (can not shownShow) it is introduced as liquid or as the liquid being accommodated in shell (casing).The shell, which can be, is dissolvable in water waterSolution, but it is not dissolved in displacement fluid 212 (such as silicon rubber cup)；Alternatively, the shell can be it is breakable so that when willShell breakage when being shaken in turbine mixer of process container 202.Further, it is possible to use being more than a kind of processing solution.
Process container 202 may include flexible cap, and the flexible cap can be pushed so as to from wherein distributing scheduled volumeAnd on substrate (such as glass slide or orifice plate).Cap 208 can be flexible, or cap 208 can be removed and will be flexibleCap is inserted into open end 206.Alternatively, process container 202 can be attached to (that is, after accumulating target substance) or can be withIncluding distributor, the target substance of predetermined volume can be assigned to another substrate (such as from process container 202 by the distributorMicroscopic slide) on.The distributor can repeatedly pierce through the cap 208 of resealable or be compressed in process containerSubstance inside 202 by the target substance of predetermined volume to take out and be assigned on the substrate.Alternatively, it is possible to by cap 208It removes and distributor (not shown) can be inserted directly into process container 202 to distribute buffy coat (buffyCoat)-processing solution mixture.
Fig. 3 A shows the isometric views of pipe 300.Fig. 3 B shows the cross section along the line III-III pipe 300 madeView.Pipe 300 is similar to pipe 100, but pipe 300 includes that valve 302 substitutes set 112, with provide funnel 110 and cavity 114 itBetween access.When undergoing centrifugation, valve 302 is opened to allow the fluid between funnel 110 and cavity 114 to flow.When withoutWhen going through centrifugation, valve 302 is closed to inhibit the fluid between funnel 110 and cavity 114 to flow.Valve 302 may include but unlimitedIn ball check valve, diaphragm check valve, revolution check valve, turnover disc check valve, promote check valve and duck mouthful valve.Alternatively,When centrifugal force is less than or equal to predetermined amount, valve 302 is closed, and when centrifugal force is greater than or equal to predetermined amount, valve 320 is opened.InstituteState predetermined amount may include but be not limited to 2g, 5g, 10g, 100g, 1000g, 2000g, 2500g, 3000g, 5000g or10000g, wherein g is gravity.
Collecting end 106 may include breaking point 304, allows cavity 114 and valve 302 to separate from funnel 110 here, makesObtaining target substance can be removed and be retained in single container for subsequent processing.Breaking point 304 may include screw thread, tongue and groove jointConjunction, dovetail joint or any connection method appropriate.
Before centrifugation and after overturning pipe 300, plug 116 can be removed from cavity 114 and can be loaded to cavity 114Displacement fluid 212.Displacement fluid 212 replaces target substance, so that displacement fluid 212 flows out process chambers 114 when pipe 300 undergoes and is centrifugedAnd enter funnel 110 via open valve 302, and by replacement, (being lifted up substance) is such as replaced by buoyancy,It pushes target substance to pass through open valve 302 and enters cavity 114.In centrifugal process, cavity 114 can be by lid (not shown)Sealing, the lid may include screw thread, can be it is pierceable and resealable, or can be it is disposable, it is allSuch as foil.
Fig. 4 A shows the isometric views of pipe 400.Fig. 4 B shows the cross section view along the line IV-IV pipe 400 madeFigure.Pipe 400 is similar to pipe 100, but pipe 400 includes that hole 402 substitutes set 112, with provide funnel 110 and cavity 114 itBetween access.Hole 402, which a diameter that, to be sized to inhibit funnel 110 and cavity before and after centrifugationFluid communication between 114, and allowing the fluid communication between funnel 110 and cavity 114 in centrifugal process.For example, describedDiameter can be based on displacement fluid 212 and the respective surface tension of target substance.
Before centrifugation and after overturning pipe 400, plug 116 can be removed from cavity 114 and can be loaded to cavity 114Displacement fluid 212.Displacement fluid 212 replaces target substance, so that displacement fluid 212 flows out process chambers 114 when pipe 400 undergoes and is centrifugedAnd enter funnel 110 via hole 402, and by replacement, (being lifted up substance) is such as replaced by buoyancy, push targetSubstance passes through hole 402 and enters cavity 114.In centrifugal process, cavity 114 can be sealed by lid (not shown), the lidSon may include screw thread, can be pierceable and resealable, or can be disposable, such as foil.
Sealing ring
Fig. 3 A shows the isometric views of sealing ring 300.Fig. 3 B shows the top view of sealing ring 300.302 generation of chain-dotted lineThe center of table sealing ring 300 or highest symmetry axis.Sealing ring 300 includes inner wall 304, outer wall 306 and cavity 308.SchemingIn 3B, RIWIt represents from the center of sealing ring 300 to the radial distance of inner wall 304, ROWIt represents from the center of sealing ring 300 to outerThe radial distance of wall 306.Sealing ring 300 is configured to mate to around primary tank (such as managing).The size and shape of cavity 308Shape is suitble to receive primary tank.Sealing ring 300 can be made to become tightly, the big of the central axis 302 of sealing ring 300 will be directed toward will pass throughUniform radial force (radial force such as generated by fixture) is caused to be applied to outer wall 306 around and reduce the size of cavity 308And the radius of inner wall 304 and outer wall 306.When sealing ring 300 is fastened on the surrounding of primary tank, it is applied to sealing ring 300Uniform force is applied to primary tank, thereby results in primary tank contraction.When removing the radial force from sealing ring 300, sealing ring300 are still fastened and are tightened in around primary tank.
The sealing ring can be any shape, including but not limited to: round, triangle or polyhedron.Fig. 3 C is shownThe isometric views of sealing ring 310.Fig. 3 D shows the top view of sealing ring 310.It is close other than sealing ring 310 is polyhedronSeal ring 310 is similar to sealing ring 300.Chain-dotted line 312 represents center or the highest symmetry axis of sealing ring 310.Sealing ring 310Including inner wall 314, outer wall 316 and cavity 318.The sealing ring can by metal (such as brass), polymer, or combinations thereof groupAt.
Alternatively, as shown in fig. 3e, sealing ring 320 can be made of piezoelectric material.Fig. 3 F shows sealing ring320 top view.Chain-dotted line 322 represents center or the highest symmetry axis of sealing ring 320.Sealing ring 320 can be viaOne lead 324 and the second lead 326 are connected to 328, such as battery potential source (electric potential source).ElectricityPosition source 328 generates mechanical strain, and sealing ring 320 is caused to become tight (that is, the radius of sealing ring 320 reduces).Sealing ring 320 includesInner wall 330, outer wall 332 and cavity 334.In Fig. 3 F, RIWIt represents from the center of sealing ring 320 to the radial distance of inner wall 330,ROWIt represents from the center of sealing ring 320 to the radial distance of outer wall 332.Alternatively, sealing ring 320 may be at nature tensionState.When applying current potential, sealing ring 320 is expanded.Alternatively, a part of of sealing ring can be made of piezoelectric material, makeThe piezoelectric is obtained as actuator to work that the other parts of sealing ring is caused to become tight and apply base on primary tankThus uniform ambient pressure in sheet shrinks primary tank to form sealing.
Fig. 3 G shows the isometric views of sealing ring 340.The sealing ring includes for adjusting internal diameter RIDRegulating mechanism348.The contractile ring includes first end 342 and the second end 346, and first end 342 and the second end 346 pass through knotPart 344 is closed to connect.First end 342 and the second end 346 include the complementary portion of regulating mechanism 348.Regulating mechanism 348 wrapsInclude but be not limited to ratchet, tenon and slot, pawl etc..
The sealing ring can also include thermal element, such as heater strip.The thermal element primary tank can be made to soften so as toIt shrinks.Alternatively, the thermal element can be such that primary tank melts to provide the sealing more adhered to.Alternatively, the heat memberPart can cause sealing ring to compress, the sealing being thus formed between primary tank and float.
Method
For convenience, method is described with reference to the embodiment suspension of anticoagulated whole blood.But method described below is not intended toIt is so limited in their application range.The method can make together with any kind of suspension in practiceWith.For example, sample suspension can be urine, blood, marrow, cyst fluid, ascites fluid, excrement, sperm, cerebrospinal fluid, nipple aspirate fluid,Saliva, amniotic fluid, vaginal fluid, mucosal secretion, aqueous humor, vitreous humor, vomitus and any other physiological fluid or halfSolid.It is also understood that target substance can be a part of sample suspension, such as buffy coat, cell such as egg cell,Fetal material (fetal material) (such as trophoderm, erythroblast, fetus red blood cell, fetus white blood corpuscle, tireYoungster DNA, fetal rna etc.) or the tumour cell (" CTC ") of circulation, the endothelial cell of circulation, immunocyte (i.e. inmature (naive)Or memory B cell or inmature or memory T cell), vesica, liposome, protein, nucleic acid, biomolecule, with close membraneIt is naturally occurring or manually prepare microscopic units (microscopic unit), (such as spirillum such as causes Lay to helminthThe B. burgdorferi (Borrelia burgdorferi) of nurse disease；Malaria inducer), microorganism, virus or inflammatory cell.Alternatively, the sample can be biosolids, such as organize, and be decomposed before or after primary tank is added(such as passing through clostridiopetidase A).
For example, target substance enrichment is the process for purifying target substance relative to non-target substance.For example, target substance phase can be madeFor non-target material collection, thus have non-to 30,000,000 parts down to 1 part of target substance (unicellular, albumen, DNA etc.)The ratio of target substance.Other ratios may include but be not limited to, down to about 1:25,000,000,1:15, and 000,000,1:10,000,000、1:5,000,000、1:1,000,000、1:250,000、1:100,000、1:50,000、1:25,000、1:10,000,1:1,000,1:100,1:10 or 1:1.
In addition, for convenience, with reference to centrifugation (such as 2g, 5g, 10g, 100g, 1000g, 1250g, 1500g, 2000g,2500g, 3000g, 5000g or 10000g) method is described, wherein g is gravity.But method as described below is without being intended to themApplication range on be so limited.For example, can be without using centrifugation, and gravity (i.e. 1g) can be used to allow fluidExchange and/or the separation of fluid.Alternatively, the density of fluid described below can be huge or fluid densityBetween difference can be it is huge so that fluid separation and/or exchange in the presence of there is no centrifugation.Even if not fromThe heart can execute the method in the time of any appropriate amount, including but not limited to, less than 1 hour (i.e. 1min, 5min,10min, 15min, 20min, 30min, 45min etc.), 1 hour (i.e. 60min) or more than 1 hour (i.e. 90min, 2 hours, it is 4 smallWhen, 8 hours, 24 hours etc.).
In addition, in all methods, it, can be by filling device (not before process container 202 is inserted into cavity 114Display) insertion tube 100 cavity 114 in.Filling device (not shown) (such as pump or syringe) includes stratified fluid, is hadThe density of target substance can be less than or greater than.Set 112 extends into filling device (not shown) to approach at least partly by instituteState the internal volume of stratified fluid filling.It is replaced or is removed or replaced in pipe with stratified fluid using filling device (not shown)Air, such as by experience be centrifuged or by provide barometric gradient.The stratified fluid can have more than or less than targetThe density of matter.The example of suitable stratified fluid includes but is not limited to: being coated with polyvinylpyrrolidone (such as Percoll)Colloidal silica particle solution, polysaccharide solution (such as Ficoll), Iodixanol (such as OptiPrep), You JirongAgent, liquid wax, oil, gas, and combinations thereof；Olive oil, mineral oil, silicone oil, impregnation oils, mineral oil, paraffin oil, silicone oil,Fluorosilicone, perfluorodecalin, perfluor perhydro phenanthrene, perfluoro bromide octane, and combinations thereof；Organic solvent, such as Isosorbide-5-Nitrae-twoAlkane, secondNitrile, ethyl acetate, the tert-butyl alcohol, cyclohexanone, methylene chloride, tert-pentyl alcohol, t-butyl methyl ether, butyl acetate, hexanol, nitrobenzene,Toluene, octanol, octane, propene carbonate, tetramethylene sulfone and ion fluid；Solution based on polymer；Surfactant；Perfluoroketone (such as perfluorocyclopentanone and perfluorocyclohexanone), fluorination ketone, hydrofluoroether class, hydrofluorocarbon, perfluoro alkane class, perfluorPolyethers, silicon and the liquid (such as phenyl methyl siloxane) based on silicon；And combinations thereof.
Method I
Fig. 4 A shows the flow chart of the embodiment method for recycling target substance.In block 402, suspension is obtained,Such as anticoagulated whole blood.In box 404, primary tank is added in whole blood, is such as managed.In box 406, float is added and is managed.Figure5A shows whole blood sample 502 and the float 504 that pipe 100 is added.Float 504 include main body, the end cap of two tear-drop shapes andThe support part for being radially spaced from and being axially directed on the body.Alternatively, float 504 can not include anyHold component.Alternatively, float 504 may include the support part not engaged with the inner wall of pipe 100.
In an alternate embodiment, the number of support part, the spacing of support part and support part thickness can be respectiveIt is independently varied.The support part is also possible to disconnecting or segmentation.The main body has the internal diameter less than pipe 100Thus fluid reserve channel is limited between the outer surface of main body and the inner wall of pipe 100 by the size of outer diameter.Support part itBetween the surface of main body can be flat, curved, or there is another suitable geometry.Support part and main body canTo be single structure, or it can be independent structure.
Embodiment includes the other types of geometry for float end cap.Top end cap can have tear-drop shape,Domed shape, cone shape or any other proper shape.Bottom end cap can have tear-drop shape, domed shape, circleCone shape or any other suitable shape.In other embodiments, the main body of float 504 may include a variety of differencesSupport structure, for separating sample in centrifugal process, supporting tube wall or guiding around float suspension.ImplementScheme is not intended to be limited to these examples.Main body may include that many for pipe provides the bump (protrusion) of support.It is replacingFor in embodiment, thus it is possible to vary the number and pattern of bump.Main body may include single continuous helicoidal structure orSpiral shape is formed around main body to form the shoulder (shoulder) of helical channel.In other embodiments, describedHelical form shoulder can form round or disconnect or be segmented to allow fluid between the adjacent turn (turns) of helical form shoulderFlowing.In different implementation scenarios, the spacing and rib thickness of the helical form shoulder can be changed independently.At anotherIn embodiment, the main body may include the support part for extending radially from the body and circumferentially extending around main body.In another embodiment, the support part can be taper.
Float 504 can be composed of a variety of different materials, including but not limited to: metal；Organic or inorganic material；Iron contentPlastics；Sintering metal；The metal of machining；Plastic material and combinations thereof.Pipe 100 can have inner wall and first diameter.It canTo be captured float 504 in pipe 100 by interference fit, so that expansion occurs for the inner wall in centrifugation down tube 100 to allow to floatThe axial movement of son 504.When being centrifuged stopping, inner wall, which reduces, to be restored to first diameter to induce interference fit.Alternatively, interiorWall, which can not be expanded and is interference fitted, can not occur between float 504 and pipe 100, so that the float is in centrifugationBefore, moved freely through in the pipe in or after the process.It, can be by institute by machining, injection molding, adding technique etc.A part of main body is made in the end cap for stating float, thus becomes a single structure；Alternatively, passing through press-fit, adhesive, spiral shellNail, any other proper method at least two components to keep together or a combination thereof, end cap can be connected toMain body.
Return to Fig. 4 A, in box 408, primary tank, float and whole blood undergo the separation based on density, such as by fromThus the heart allows to be separated into whole blood based on density the fraction based on density of the axial position along pipe.Fig. 5 B is shownGo through the pipe 100 of the separation (such as passing through centrifugation) based on density, the isometric views of float 504 and blood.For example, it is assumed that through being centrifugedWhole blood include three fractions.For convenience, three fractions include blood plasma, buffy coat and red blood cell.But whenWhen the experience centrifugation of another suspension, it is understood that there may be more than, be less than or equal number of fraction, each fraction has different closeDegree.Suspension undergoes axial separation to be separated into three fractions based on density and along the length of pipe, wherein red blood cell 514In bottom, blood plasma 510 is located at top, and buffy coat 512 is positioned there between, as shown in FIG. 8.Float 504 can haveIn one for thering is any density appropriate to be deposited in the fraction.It can choose the density of float 504, so that float 504Extend buffy coat 512 between the main body of float and the inner wall of primary tank.Buffy coat 512 can be trapped in floatingIn region between son 504 and primary tank 702.
Fig. 4 A is returned, in box 410, blood plasma 510 can be taken out from pipe 100, by liquid relief, aspirate, topple over.In block 412, as seen in figure 5 c, at least one description fluid (delineation fluid) 520 can be added and is managedIn.The description fluid can provide target substance and between any non-target substance of the target substance above and or belowFurther separation.At least one description fluid 520 can have the density more than or less than target substance.For example, when wishing into oneStep separation buffy coat 512 and when red blood cell 514, the description fluid can have greater than buffy coat 512 and smallIn the density of red blood cell 514.At least one description fluid 520 can be it is miscible or immiscible with suspension,It and is inert for suspended material.At least one description fluid 520 also can be provided in wherein that primary tank 702 is closeThe region of envelope, because there is bigger description and separation between buffy coat 512 and red blood cell 514.Regardless of whether makingWith float, at least one description fluid 520 can be used.The example of suitable description fluid includes but is not limited to: with poly- secondSolution, the polysaccharide solution (such as Ficoll), iodine gram of alkene pyrrolidone (such as Percoll) coated colloidal silica particleIt is husky alcohol (such as OptiPrep), cesium chloride, sucrose, the solution based on sugar, the solution based on polymer, surfactant, organicSolvent, liquid wax, oil, gas, and combinations thereof；Olive oil, mineral oil, silicone oil, impregnation oils, mineral oil, paraffin oil, siliconOil, fluorosilicone, perfluorodecalin, perfluor perhydro phenanthrene, perfluoro bromide octane and combinations thereof；Organic solvent, such as Isosorbide-5-Nitrae-twoAlkane,Acetonitrile, ethyl acetate, the tert-butyl alcohol, cyclohexanone, methylene chloride, tert-pentyl alcohol, t-butyl methyl ether, butyl acetate, hexanol, nitroBenzene, toluene, octanol, octane, propene carbonate, tetramethylene sulfone and ion fluid；Solution based on polymer；Surface-activeAgent；Perfluoroketone (such as perfluorocyclopentanone and perfluorocyclohexanone), fluorination ketone, hydrofluoroether class, hydrofluorocarbon, perfluoro alkane class,Perfluoropolyether, silicon and the liquid (such as phenyl methyl siloxane) based on silicon；And combinations thereof.
In block 414, pipe, float, description fluid and residual fraction experience are centrifuged again.In block 416, using sealingRing.
Fig. 5 D shows two part seals comprising the soft inner component that be inserted into primary tank and external compression portionPart, the external compression component at least make primary tank shrink and deform to seal the top of primary tank from the lower part of primary tankPoint.The external compression component can also make soft inner component shrink and deform.Two part seal prevents fluid from existingMobile in primary tank is more than two part seal and the movement for preventing the soft inner component.For convenience, with reference to floatingSon 504 describes the soft inner component, and describes the external compression component with reference to sealing ring 300, but describeSystem is not intended to be so limited, and may include any float appropriate and any external compression device appropriate.
Sealing ring 300 applies circumferential or radial power on pipe 100, thereby results in pipe 100 and inwardly collapses and lean against floatOn 504.Enlarged drawing 522 shows the sealing ring 300 being fastened to around float 504 and pipe 100.Have been placed in description fluid520 and the sealing ring 300 of interface of red blood cell 514 cause pipe 100 inwardly to collapse until between pipe 100 and float 504Form sealing.The outer wall of sealing ring 300 can be flushed with the outer wall of pipe 100；The outer wall of sealing ring 300 can extend beyond pipe100 outer wall；Alternatively, the outer wall of pipe 100 can extend beyond the outer wall of sealing ring 300.Sealing ring 300 is still fastened to protectSealing is held, this can prevent fluid upper in any direction mobile more than the sealing.Sealing ring 300 can also keep tensioning state.It canIt alternatively, can be by the excessive power compressed and then removing is applied to sealing ring 300 of sealing ring 300.Sealing ring 300 can be slightlyExpansion, although still maintaining contraction.
In order to which sealing ring 300 and sealing is consequently formed in application, fixture can be used will be towards the power of the central axis of pipe 100Circumferentially it is applied to sealing ring 300.After pipe 100 undergoes the separation (such as passing through centrifugation) based on density, by sealing ring 300It is placed on around pipe 100.Then sealing ring 300 and pipe 100 are placed in fixture.The fixture may include frame with by sealing ring300 support to lean against on pipe 100.It is that the operation of the fixture can be automation or can manually execute.Alternatively, instituteState the sealing that fixture can be formed in the case where not including sealing ring 300 between float 504 and pipe 100.Alternatively, it is possible toSealing is formed between float 504 and pipe 100, such as by ultrasonic welding, or by applying heat or temperature gradient so that pipe100 deformations/are melted to float 504.For convenience, method is described with reference to sealing ring, but method as described below is not intended toApplication at them is above so limited, and can be implemented in the case where no sealing ring.
When making the operation automation of fixture, motor (motor) cause collet (collet) (including collet finger) orThe translation (translation) of pressure member is to cause the contraction of collet finger.Motor can pass through axis (such as camshaft)And one or more gears and be connected to collet or pressure member.Base portion engages and catches the object.When collet is by motorWhen driving, pressure member is remain stationary.When pressure member is by motor drive, collet is remain stationary.The fixture can wrapBuckle releaser (release) is included, is skidded off to cause stress component from collet finger 904, thus eliminates clamping force.
Alternatively, the fixture can be but be not limited to: collet fixture, O-ring, pipe clamp, hose clamp, spring clip, bandRing folder or setting fastening (such as zipper setting fastening).Can in the case where no sealing ring using fixture to provide between float and pipeSealing.
Return to Fig. 4 A, in box 418, and as seen in Fig. 5 E, pipe 100 can be overturn, can by plug 116 fromPipe 100 takes out, can be by 202 insertion tube 200 of process container, and process container 202 can be added in displacement fluid 212.It shouldIt points out, before or after by 202 insertion tube 200 of process container, process container 202 can be added in displacement fluid 212.
Fig. 4 A is returned in block 420 then to be centrifuged the system again.Fig. 5 F, which is shown, is centrifuged later 100 He of pipeProcess container 202.With displacement fluid 212 (it has greater than buffy coat 512 but is less than the density of description fluid 520) fromProcess container 202 flows into pipe 100, and buffy coat 512 passes through set 214 in pipe 100 and moves outwardly and into process container202。
Then process container 202 (including buffy coat 512) can be taken out from pipe 100 to be further processed, divide with experienceAnalysis, storage etc..After taking out process container 202, processing solution can be added, although at the recycling foregoing description of target substanceReason solution is in process container.Turbine mixer can such as be passed through with shake process container.It then can be by processing solution(not shown) (it is added in liquid form or in soluble shell or in breakable shell before shake)It is mixed with buffy coat to realize conversion and form buffy coat-processing solution mixture.It then can be by erythrocyte sedimentation rate palm fibreYellow layer-processing solution mixture is assigned on substrate (such as microscopic slide).
Method II
Fig. 4 B shows the flow chart of the embodiment method for recycling target substance.In box 432, suspension is obtained,Such as anticoagulated whole blood 502.In box 434, and as seen in fig. 6, pipe 100 is added in whole blood 502.Fig. 4 B is returned to, in sideIt, can be by coloring agent 606, negative enrichment reagents (negative enrichment in frame 436, and as seen in fig. 6bReagent) 604 and description fluid 602 be added pipe 100.The primary tank is added to mark target substance in coloring agent 606.To alsoThe primary tank is added to change the density of non-target substance (for example, blood plasma 510), without changing target substance in negative enrichment reagents 604Density.For example, negative enrichment reagents 604 can change the density of non-target substance, change so that the density of target substance is less than or greater thanThe non-target material density of change.It can permit coloring agent 606 and negative enrichment reagents 604 incubate the time of any appropriate amount, including butIt is not limited to, less than 1 hour (i.e. 1min, 5min, 10min, 15min, 20min, 30min, 45min etc.), 1 hour (i.e. 60min)Or more than 1 hour (i.e. 90min, 2 hours, 4 hours, 8 hours, 24 hours etc.).
In incubation period, can by pipe 100 be vortexed, shake, shake or it is any it is appropriate movement with enhance coloring agent withThe mixing of target substance.For example, coloring agent can be the solution containing fluorescence probe, the fluorescence probe can be used for target substanceIt dyes and thus marks, thus the fluorescence signal for identifying and characterizing is provided.Before container is added in suspension, it will hangSupernatant liquid is added after container but has been subjected to after centrifugation before centrifugation or in suspension, can will contain fluorescence probeSolution be added suspension.Fluorescence probe includes the fluorescent molecule with ligand binding.Target substance can have many different typesSurface marker.Each type of surface marker be can be attached particular ligand (such as antibody) molecule it is (such as anti-It is former).Therefore, can with ligand by target qualitative classification, and the ligand by making to be attached to particular surface marker with it is specific glimmeringThe concrete type of optical molecule conjugation and the determining target substance being present in suspension.The example of suitable fluorescent molecule include butIt is not limited to: quantum dot；The dyestuff being obtained commercially, such as fluorescein, FITC (" fluorescein isothiocynate "), R-PE(" PE "), texas Red, allophycocyanin, Cy5, Cy7, cascade blue, Hoechst, DAPI (" 4', 6- diamidino -2- phenylIndoles ") and TRITC (" isothiocyanic acid tetramethylrhodamine ")；Dyestuff, the combination of such as CY5PE, CY7APC and CY7PE；And conjunctionAt molecule, such as self assembly nucleic acid structure.Many solution can be used, so that every kind of solution includes in conjunction with different ligandsDifferent types of fluorescent molecule.In addition, the core of target substance can have the size different from the core of non-target substance.Determine core sizeIt can be with supplementary globe target substance and non-target substance.
Return to Fig. 4 B, in box 438, and as seen in figure 6d, pipe 100 can be overturn, can by plug 116 fromPipe 100 takes out, can be by 202 insertion tube 200 of process container, and process container 202 can be added in displacement fluid 212.It shouldIt points out, before or after by 202 insertion tube 200 of process container, process container 202 can be added in displacement fluid 212.
Fig. 4 B is returned then to be centrifuged the system again in box 440.Fig. 6 E, which is shown, is centrifuged later 100 He of pipeProcess container 202.With displacement fluid 212 (it has greater than buffy coat 512 but is less than the density of description fluid 520) fromProcess container 202 flows into pipe 100, and buffy coat 512 passes through set 214 in pipe 100 and moves outwardly and into process container202.In addition, the fluid 604 (it is mixed to form high density blood plasma 608 with blood plasma 510) for changing density causes high densityBlood plasma 608 has the density at least more than buffy coat 502 and description fluid 602.
Then process container 202 (including buffy coat 512) can be taken out from pipe 100 to be further processed, divide with experienceAnalysis, storage etc..After taking out process container 202, processing solution can be added, although at the recycling foregoing description of target substanceReason solution is in process container.Turbine mixer can such as be passed through with shake process container.It then can be by processing solution(not shown) (it is added in liquid form or in soluble shell or in breakable shell before shake)It is mixed with buffy coat to realize conversion and form buffy coat-processing solution mixture.It then can be by erythrocyte sedimentation rate palm fibreYellow layer-processing solution mixture is assigned on substrate (such as microscopic slide).
The example of suitable negative enrichment reagents includes but is not limited to: being coated with polyvinylpyrrolidone (such as Percoll)The solution of colloidal silica particle, polysaccharide solution (such as Ficoll), Iodixanol (such as OptiPrep), compound,Branched chain glucans (such as glucan), cesium chloride, sucrose, the solution based on sugar, polymer solution, multiphase polymer solution, fourDimer antibody compound (such as RosetteSep) etc.；Or particle, pearl is (by metal, silica gel, glass, polymer etc.At least one composition), nano particle, the compound based on metal, metal complex, lipid, sugar etc..
The density that primary tank also is added to change the density of target substance, without changing non-target substance in positive enrichment reagents.ExampleSuch as, the positive enrichment reagents can change the density of target substance, so that the density of non-target substance is less than or greater than the target changedMatter density.Can permit the time that positive enrichment reagents incubate any appropriate amount, including but not limited to, less than 1 hour (i.e. 1min,5min, 10min, 15min, 20min, 30min, 45min etc.), 1 hour (i.e. 60min) or (i.e. 90min, 2 small more than 1 hourWhen, 4 hours, 8 hours, 24 hours etc.).The example of suitable positive enrichment reagents includes but is not limited to: using polyvinylpyrrolidoneThe solution of (such as Percoll) coated colloidal silica particle, polysaccharide solution (such as Ficoll), Iodixanol (such asOptiPrep), compound, branched chain glucans (such as glucan), cesium chloride, sucrose, based on sugar solution, polymer solution,Multiphase polymer solution, tetrameric antibody compound (such as RosetteSep) etc.；Or particle, such as pearl is (by metal, siliconAt least one of glue, glass, polymer etc. composition), nano particle, the compound based on metal, metal complex, lipid,Sugar etc..
Fraction recycling
In one example, it may be desirable that, the entire fraction of blood sample is removed to obtain target substance.
In another example, it may be desirable that, a part of the fraction of blood sample is removed to obtain targetMatter.Continuous density classification separation is by gradually or sample is divided into fraction or a fraction of sample is divided into Asia by continuous processFraction, so that each step or sequence are caused from the above-mentioned fraction or substate different with consecutive steps or sequence collection or separationPoint.In other words, continuous density classification separates each subpopulation that a group can be provided by series of steps or a groupEach sub- subpopulation of the subpopulation of body.For example, buffy coat is the fraction of whole blood sample.The buffy coat fraction canTo be further separated into subfraction, including but not limited to, granulophilocyte, granulocyte, lymphocyte/monocyte and blood platelet.The buffy coat can also contain desired target substance.These subfractions can be single by executing continuous density classification separationIt gets by oneself.
According to the number for separating and collecting desired fraction or subfraction, can be used two or more process containers andVarious displacement fluids.Every kind of continuous displacement fluid is bigger than preceding displacement fluid density.In addition, being used to collect the close of the displacement fluid of target substanceDegree is greater than the density of desired target substance；For example, displacement fluid can have bigger than the density of desired target substance about 0.001To about 0.005g/cm3Density.Similarly, every kind of continuous fraction or subfraction are bigger than preceding fraction or subfraction.It collectsAfterwards, can analyze continuous subfraction, such as diagnosing, prognosis, research purpose, to determine component characteristics (i.e. complete haemocyteCount), how those features change at any time, etc..
Subsequent process container and displacement fluid can be used to collect the other subfraction of buffy coat 512, Zhi DaosuoThere is subfraction to be collected or until desired subfraction is collected.Although being with float and close by continuous density classification separation expressionSeal ring executes, but continuous density classification separation can execute in the case where no float, sealing ring or both.Here is to useIn in a kind of embodiment for executing the execution continuous density classification separation after all steps of the insertion of process containerMethod:
1. by the cavity in (n-y) a process container insertion tube,
2. (n-y) a process container is added in (n-y) kind displacement fluid, (n-y) plants displacement fluid；
3. primary tank and (n-y) a process container are centrifuged together, (n-y) plants displacement fluid and flows into primary tank via pipe(n-y) a subfraction from the suspension of primary tank is entered (n-y) a process container across pipe replacement via set；
4. taking out (n-y) a process container, including (n-y) a subfraction from pipe.
For example, n may be greater than or any number equal to 1, and y may be greater than or any number equal to 0, such asN=2 and y=1.In addition, n can be the sum of desired subfraction, and y=(number for the subfraction that n-1- has been collected).
After recycling target substance, target substance can be put and be used to be imaged and detect on substrate (and then storage for depositingShelves purpose).Then at least part of the target substance of detection can be taken out from substrate, it is further to undergo such as by pickingAnalysis.Isolated target substance can be stored into PCR pipe, the hole of orifice plate, glass slide or be used to execute further analyze it is anySubstrate or container appropriate.
It can analyze target substance using any analysis method appropriate or technology, although more specifically using extracellular and thinAnalysis intracellular, including intracellular protein label；Color staining；Foranalysis of nucleic acids, including but not limited to DNA array, expression battle arrayColumn, protein array and DNA hybridization array；(" ISH "-is a kind of for analyzing the tool of DNA and/or RNA, all in situ hybridizationSuch as copy number changes)；Polymerase chain reaction (" PCR ")；Reverse transcription PCR；Or (" bDNA "-one kind is used for branched DNAAnalyze the tool of DNA and/or RNA, such as mRNA expression) analysis.The red blood cell for having core of separation can be undergone into oneStep processing is to test such fetal abnormality, including but not limited to, chromosome abnormality (such as fetus aneuploidy, Tang Shi are comprehensiveSign, trisomy 13, trisomy 18 or sex chromosomal abnormality, such as Turner syndrome), lesser subchromoso is abnormal, gender inspectionIt tests, mutation analysis and rhesus blood group are examined.
These technologies may need before analysis to fix target substance, permeabilization and separation.The intracellular protein that can be markedIn matter it is some including but not limited to: cytokeratin (" CK "), actin, Arp2/3, coronin, dystrophin,FtsZ, myosin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc, dayAspartic acid specific cysteine protease, survivin, p27kip, FOXC2, BRAF, phosphoric acid-Akt1 and 2, phosphoric acid-Erk1/2,Erk1/2, P38MAPK, vimentin, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twist1, Snail1, ZEB1, fine even eggWhite, Slug, Ki-67, M30, MAGEA3, the receptor kinase of phosphorylation, modified histone, the relevant albumen of chromatin andMAGE.For fixation, infiltration or label, fixative (such as formaldehyde, formalin, methanol, acetone, paraformaldehyde can be usedOr glutaraldehyde), detergent (such as saponin(e, polyoxyethylene, digitonin, octyl beta-glucosidase, octyl β-sulfur sugarGlycosides, 1-S- octyl-β-D- thioglucopyranoside, Tween-20, CHAPS, CHAPSO, (1,1,3,3- tetramethyl fourthBase) phenyl-polyethylene glycol or octylphenol ethylene oxide) or marking agent (antibody of such as fluorescent marker, enzyme conjugation antibody,Pap coloring agent, Giemsa staining agent or h and E coloring agent).
The density of target substance can be increased (such as by the way that weight (weight) is attached to target substance, or by making targetMatter absorbs or intake weight), or can reduce (such as by the way that buoy (buoy) is attached to target substance, or by making target substanceAbsorb or take in buoy).The weight or the buoy can be bound to ligand.The target substance can have many inhomogeneitiesThe surface marker of type.Each type of surface marker be can be attached particular ligand (such as antibody) molecule it is (such as anti-It is former).It is thereby possible to select ligand changes the density of the target substance to be specifically attached to target substance.Suitable weightThe example of amount and/or buoy includes but is not limited to the pearl being made of metal, glass, ceramics, plastics or their combination.It canAlternatively, weight or buoy can be attached to non-target substance to change the density of non-target substance to obtain purer target substanceSample.
For purposes of explanation, the description of front provides the thorough understanding of present disclosure using specific term.ButThose skilled in the art are not it is clear that require the detail for implementing system and method described herein.In order to illustrateWith the purpose of description, before the description of specific embodiment is presented as example.They are not intended as exhaustive, or incite somebody to action thisDisclosure is confined to described precise forms.In view of the introduction of front, many modifications and variations are possible.Display is simultaneouslyThe embodiment is described most preferably to explain the principle and practical application of present disclosure, thus makes those skilled in the artPresent disclosure and various embodiments can be most preferably utilized, wherein various modifications are suitable for covered particular use.ThisScope of the disclosure is intended to the equivalents by following following claims and they.