CN109414694A

CN109414694A - For collecting the equipment, system and method for target substance

Info

Publication number: CN109414694A
Application number: CN201680086993.6A
Authority: CN
Inventors: R·休伯特; L·乌仁
Original assignee: Rarecyte Inc
Current assignee: Rarecyte Inc
Priority date: 2016-06-03
Filing date: 2016-09-13
Publication date: 2019-03-01
Also published as: WO2017209780A1; EP3463659A1; EP3463659A4

Abstract

This disclosure relates to the equipment, system and method for recycling target substance from suspension.System includes process container, displacement fluid and pipe.The pipe includes funnel, set and cavity.The set allows the fluid communication between the funnel and the cavity, so that the process container is inserted into the cavity, and the set extends into the process container.

Description

For collecting the equipment, system and method for target substance

Cross-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 2015 The part continuation application for the application number 14/610,522 submitted January 30, the latter require on 2 4th, the 2014 interim Shens submitted Please 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 24th On May 26th, 9,039,999,2016 authorization) part continuation application, the latter is the application number submitted on November 26th, 2013 14/090,337 part continuation application, it is required that the following equity applied: on November 30th, 2012 Provisional Application No. submitted 61/732,029；The Provisional Application No. 61/745,094 that on December 21st, 2012 submits；The interim Shen that on March 15th, 2013 submits It 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, 2013 When 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 26th The 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 suspension Matter.

Background technique

Suspension often includes being difficult to detect, extract and separate the target substance for analysis.For example, whole blood is that substance exists Suspension in fluid.The substance include billions of red blood cells in the protein fluid of referred to as blood plasma and leucocyte with And 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 ovum Son, fetal cell, endothelial cell, helminth, bacterium and inflammatory cell and virus, including HIV, cytomegalovirus, the third type liver Scorching virus and epstein-Barr virus.Currently, practitioner, researcher and with blood sample work those of people try Separately, certain components of separation and extraction peripheral blood sample are for checking.Typical technology for analyzing blood sample include with Lower step: by blood film smear on glass slide, and so that certain components can pass through bright-field or fluorescence microscopy inspection Mode 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 suspension If energy) use many prior art detection and analysises.So practitioner, researcher and with suspension work those of People 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 processing Container, displacement fluid (displacement fluid) and pipe.The pipe includes funnel, set (cannula) and cavity.The set is permitted Perhaps the fluid communication between the described funnel and the cavity, so that the process container is inserted into the cavity, and the set prolongs It 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 made Viewgraph of cross-section.Pipe 100 can be any size appropriate or cross-sectional shape (such as cylinder, ellipse, triangle, just Rectangular, rectangle etc.).Pipe 100 include main body 102, the main body 102 have connection first end 104, the second end 118 and The side wall of collection section (segment) 106 between the first end 104 and the second end 118.Pipe 100 can be with Including cap 108 first end 104 is sealed or closed, the first end 104 can be unlimited.Collection section 106 includes leakage Bucket 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 110 Mouth 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 funnel 110 mouth smaller diameter.Funnel 110 is formed by tapered wall, and the tapered wall can be straight, is curve, arc etc..Leakage Bucket 110 can be any proper shape, including but not limited to, tubulose, hemispheric, paraboloidal, conical, square Shape, 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 recess The vertex in space 114 extends and enters in cavity 114.Cavity 114 is aperture 120 from the second end 118 to first end 104 dented spaces extended.Cavity 114 can receive and support process container (not shown).Cavity 114 can be any appropriate Depth 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 point Portion, 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 groups At, including but not limited to, ceramics；Metal；Organic or inorganic material；And plastic material, such as polypropylene, acrylic acid, poly- carbonic acid Ester 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 resealable The material of glue or other suitable resealables composition, the material of the resealable can use syringe needle or other sharp devices Tool 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 can Alternatively, cap 108 can be screw-cap, the complementary threads with screw thread to be bonded on inside or around first end 104.It can Alternatively, 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 end The 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, cap 108 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 welding Sonic soldering connects), adherency (such as adhesive), clamp (clamping) (such as sealing ring, crimping machine, clip etc.) or for temporarily Or 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 114 In to inhibit fluid to escape from from set 112.Plug 116 can be by the rubber or other suitable resealables of resealable The material of material composition, the resealable can be pierced through and when the set 112 is removed Shi Zaimi repeatedly with set 112 Envelope.Alternatively, plug 116 can be screw-cap, with screw thread to be bonded in cavity 114 or in the second end 118 Complementary 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 100 To increase the pressure applied on first end 104 from plug 116 to provide with stronger.The fixture can be double Component ring, component ring around pipe 100 entire periphery or a component ring around less than pipe 100 entire periphery, such as Half (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, aromatics Polyamide-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 acyl Imines, polyetheretherketone, polyethylene terephthalate, polyimide, polymethacrylates, are gathered polyethers ketone Alkene (for example, polyethylene, polypropylene), gathers polyallmerDiazole, polyphenyl ethers (PPO), changes Parylene The PPO class of property, polystyrene, polysulfones, fluoropolymer (such as polytetrafluoroethylene (PTFE)), polyurethane, polyvinyl acetate, polyethylene Alcohol, 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 pipe 200.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 container 202 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 open End 206 has the size for receiving cap 208.Cap 208 can be by the rubber or other suitable resealables of resealable Material composition, the material of the resealable can be pierced through repeatedly with syringe needle or other sharp instruments with approach processing hold The content of 202 internal storage of device, and sealed again when the syringe needle or utensil are removed.Alternatively, process container 202 Also two open ends be can have, the open end has the size for receiving cap.Process container 202 can have direction and open Put the taper geometry that end 206 broadens or narrows；Process container 202 can have geometry generally cylindrical in shape；Or Person, process container 202 can have geometry generally cylindrical in shape in the first paragraph and the cone shape in second segment Geometry, wherein first segment and second segment are connected to each other and are continuous.Although at least one section of process container 202 With circular cross section, but in other embodiments, at least one described section can have ellipse, square, three Angular, 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 center Axis 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 substance 212 exchange target substances.Closing end 204 threaded can be connected with providing with the screw thread of the threaded cavity (not shown) of pipe 100 It 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 can The material resealed can be pierced through with syringe needle or other sharp instruments repeatedly to approach in 202 internal storage of process container It 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 action To maintain the sealing between plug 210 and process container 202 in 210 insertion process container 202 of plug, such as matched by interference It closes (interference fit).It alternatively, can be in the closing end of process container 202 using heated liquid rubber Plug 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 for The adhesive that plug 210 is attached to the inner wall of process container can be to adhesive, epoxy resin, contact-type based on polymer Adhesive or any other suitable material for being bonded or establishing heat bonding.Alternatively, it is possible to by 210 injection of plug It 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 202 Inner 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 held The inner chamber body of device 202 accommodates target substance.Set 112 can be covered by sleeve (sleeve) (not shown) of resealable to prevent Target substance outflow, unless process container 202 is to reach to allow to cover 112 depths for just breaking through process container 202 in cavity 114 Degree.The sleeve (not shown) covering jacket 112 of the resealable, has spring, can be penetrated with quilt cover 112, and By 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 substance It 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 suspension The 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 unlimited In: with the solution of polyvinylpyrrolidone (such as Percoll) coated colloidal silica particle, polysaccharide solution (such as Ficoll), Iodixanol (such as OptiPrep), organic solvent, liquid wax, oil, gas, and combinations thereof；Olive oil, mineral It is oil, silicone oil (silicone oil), impregnation oils, mineral oil, paraffin oil, silicone oil (silicon oil), fluorosilicone, complete Fluoronaphthalene alkane, perfluor perhydro luxuriant and rich with fragrance (perfluoroperhydrophenanthrene), perfluoro bromide octane, and combinations thereof；It is organic molten Agent, 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 complete Fluorine cyclohexanone), fluorination ketone (fluorinated ketones), hydrofluoroether class, hydrofluorocarbon, perfluoro alkane class, perfluoropolyether Class, 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 202 Conversion to target substance.Processing solution (not shown) can be preservative, cell adherence solution, dyestuff etc..Different from displacement fluid 212, the processing solution (not shown) of most of (if not all) is stayed in after centrifugation in process container 202, thus with one Kind or another way (that is, anti-corrosion, increase adhesion property etc.) realize conversion to target substance.Processing solution (can not shown Show) it is introduced as liquid or as the liquid being accommodated in shell (casing).The shell, which can be, is dissolvable in water water Solution, 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 will Shell 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 volume And on substrate (such as glass slide or orifice plate).Cap 208 can be flexible, or cap 208 can be removed and will be flexible Cap is inserted into open end 206.Alternatively, process container 202 can be attached to (that is, after accumulating target substance) or can be with Including distributor, the target substance of predetermined volume can be assigned to another substrate (such as from process container 202 by the distributor Microscopic slide) on.The distributor can repeatedly pierce through the cap 208 of resealable or be compressed in process container Substance 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 208 It removes and distributor (not shown) can be inserted directly into process container 202 to distribute buffy coat (buffy Coat)-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 made View.Pipe 300 is similar to pipe 100, but pipe 300 includes that valve 302 substitutes set 112, with provide funnel 110 and cavity 114 it Between access.When undergoing centrifugation, valve 302 is opened to allow the fluid between funnel 110 and cavity 114 to flow.When without When going through centrifugation, valve 302 is closed to inhibit the fluid between funnel 110 and cavity 114 to flow.Valve 302 may include but unlimited In 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.Institute State predetermined amount may include but be not limited to 2g, 5g, 10g, 100g, 1000g, 2000g, 2500g, 3000g, 5000g or 10000g, wherein g is gravity.

Collecting end 106 may include breaking point 304, allows cavity 114 and valve 302 to separate from funnel 110 here, makes Obtaining target substance can be removed and be retained in single container for subsequent processing.Breaking point 304 may include screw thread, tongue and groove joint Conjunction, 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 114 Displacement fluid 212.Displacement fluid 212 replaces target substance, so that displacement fluid 212 flows out process chambers 114 when pipe 300 undergoes and is centrifuged And 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 all Such 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 made Figure.Pipe 400 is similar to pipe 100, but pipe 400 includes that hole 402 substitutes set 112, with provide funnel 110 and cavity 114 it Between access.Hole 402, which a diameter that, to be sized to inhibit funnel 110 and cavity before and after centrifugation Fluid communication between 114, and allowing the fluid communication between funnel 110 and cavity 114 in centrifugal process.For example, described Diameter 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 114 Displacement fluid 212.Displacement fluid 212 replaces target substance, so that displacement fluid 212 flows out process chambers 114 when pipe 400 undergoes and is centrifuged And enter funnel 110 via hole 402, and by replacement, (being lifted up substance) is such as replaced by buoyancy, push target Substance passes through hole 402 and enters cavity 114.In centrifugal process, cavity 114 can be sealed by lid (not shown), the lid Son 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 line The center of table sealing ring 300 or highest symmetry axis.Sealing ring 300 includes inner wall 304, outer wall 306 and cavity 308.Scheming In 3B, R_IWIt represents from the center of sealing ring 300 to the radial distance of inner wall 304, R_OWIt represents from the center of sealing ring 300 to outer The 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 308 Shape 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 through Uniform 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 308 And 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 300 Uniform force is applied to primary tank, thereby results in primary tank contraction.When removing the radial force from sealing ring 300, sealing ring 300 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 shown The 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 polyhedron Seal 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 310 Including inner wall 314, outer wall 316 and cavity 318.The sealing ring can by metal (such as brass), polymer, or combinations thereof group At.

Alternatively, as shown in fig. 3e, sealing ring 320 can be made of piezoelectric material.Fig. 3 F shows sealing ring 320 top view.Chain-dotted line 322 represents center or the highest symmetry axis of sealing ring 320.Sealing ring 320 can be via One lead 324 and the second lead 326 are connected to 328, such as battery potential source (electric potential source).Electricity Position 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 includes Inner wall 330, outer wall 332 and cavity 334.In Fig. 3 F, R_IWIt represents from the center of sealing ring 320 to the radial distance of inner wall 330, R_OWIt represents from the center of sealing ring 320 to the radial distance of outer wall 332.Alternatively, sealing ring 320 may be at nature tension State.When applying current potential, sealing ring 320 is expanded.Alternatively, a part of of sealing ring can be made of piezoelectric material, make The piezoelectric is obtained as actuator to work that the other parts of sealing ring is caused to become tight and apply base on primary tank Thus 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 R_IDRegulating mechanism 348.The contractile ring includes first end 342 and the second end 346, and first end 342 and the second end 346 pass through knot Part 344 is closed to connect.First end 342 and the second end 346 include the complementary portion of regulating mechanism 348.Regulating mechanism 348 wraps Include 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 to It shrinks.Alternatively, the thermal element can be such that primary tank melts to provide the sealing more adhered to.Alternatively, the heat member Part 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 to It is so limited in their application range.The method can make together with any kind of suspension in practice With.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 half Solid.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, tire Youngster 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 membrane It is naturally occurring or manually prepare microscopic units (microscopic unit), (such as spirillum such as causes Lay to helminth The 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 made For 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 them Application range on be so limited.For example, can be without using centrifugation, and gravity (i.e. 1g) can be used to allow fluid Exchange and/or the separation of fluid.Alternatively, the density of fluid described below can be huge or fluid density Between difference can be it is huge so that fluid separation and/or exchange in the presence of there is no centrifugation.Even if not from The 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 small When, 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 114 Display) insertion tube 100 cavity 114 in.Filling device (not shown) (such as pump or syringe) includes stratified fluid, is had The 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 institute State 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 target The 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 Jirong Agent, 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, second Nitrile, 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, perfluor Polyethers, 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.Figure 5A 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 and The support part for being radially spaced from and being axially directed on the body.Alternatively, float 504 can not include any Hold 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 respective It is independently varied.The support part is also possible to disconnecting or segmentation.The main body has the internal diameter less than pipe 100 Thus 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 it Between the surface of main body can be flat, curved, or there is another suitable geometry.Support part and main body can To 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, circle Cone shape or any other suitable shape.In other embodiments, the main body of float 504 may include a variety of differences Support structure, for separating sample in centrifugal process, supporting tube wall or guiding around float suspension.Implement Scheme 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 replacing For in embodiment, thus it is possible to vary the number and pattern of bump.Main body may include single continuous helicoidal structure or Spiral shape is formed around main body to form the shoulder (shoulder) of helical channel.In other embodiments, described Helical form shoulder can form round or disconnect or be segmented to allow fluid between the adjacent turn (turns) of helical form shoulder Flowing.In different implementation scenarios, the spacing and rib thickness of the helical form shoulder can be changed independently.At another In 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 content Plastics；Sintering metal；The metal of machining；Plastic material and combinations thereof.Pipe 100 can have inner wall and first diameter.It can To 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 float The axial movement of son 504.When being centrifuged stopping, inner wall, which reduces, to be restored to first diameter to induce interference fit.Alternatively, interior Wall, which can not be expanded and is interference fitted, can not occur between float 504 and pipe 100, so that the float is in centrifugation Before, 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 shell Nail, any other proper method at least two components to keep together or a combination thereof, end cap can be connected to Main body.

Return to Fig. 4 A, in box 408, primary tank, float and whole blood undergo the separation based on density, such as by from Thus 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 shown Go 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 centrifuged Whole blood include three fractions.For convenience, three fractions include blood plasma, buffy coat and red blood cell.But when When 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 close Degree.Suspension undergoes axial separation to be separated into three fractions based on density and along the length of pipe, wherein red blood cell 514 In bottom, blood plasma 510 is located at top, and buffy coat 512 is positioned there between, as shown in FIG. 8.Float 504 can have In one for thering is any density appropriate to be deposited in the fraction.It can choose the density of float 504, so that float 504 Extend buffy coat 512 between the main body of float and the inner wall of primary tank.Buffy coat 512 can be trapped in floating In 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 managed In.The description fluid can provide target substance and between any non-target substance of the target substance above and or below Further separation.At least one description fluid 520 can have the density more than or less than target substance.For example, when wishing into one Step separation buffy coat 512 and when red blood cell 514, the description fluid can have greater than buffy coat 512 and small In 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 close The region of envelope, because there is bigger description and separation between buffy coat 512 and red blood cell 514.Regardless of whether making With float, at least one description fluid 520 can be used.The example of suitable description fluid includes but is not limited to: with poly- second Solution, the polysaccharide solution (such as Ficoll), iodine gram of alkene pyrrolidone (such as Percoll) coated colloidal silica particle It is husky alcohol (such as OptiPrep), cesium chloride, sucrose, the solution based on sugar, the solution based on polymer, surfactant, organic Solvent, liquid wax, oil, gas, and combinations thereof；Olive oil, mineral oil, silicone oil, impregnation oils, mineral oil, paraffin oil, silicon Oil, 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, nitro Benzene, toluene, octanol, octane, propene carbonate, tetramethylene sulfone and ion fluid；Solution based on polymer；Surface-active Agent；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 sealing Ring.

Fig. 5 D shows two part seals comprising the soft inner component that be inserted into primary tank and external compression portion Part, 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 tank Point.The external compression component can also make soft inner component shrink and deform.Two part seal prevents fluid from existing Mobile in primary tank is more than two part seal and the movement for preventing the soft inner component.For convenience, with reference to floating Son 504 describes the soft inner component, and describes the external compression component with reference to sealing ring 300, but describe System 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 float On 504.Enlarged drawing 522 shows the sealing ring 300 being fastened to around float 504 and pipe 100.Have been placed in description fluid 520 and the sealing ring 300 of interface of red blood cell 514 cause pipe 100 inwardly to collapse until between pipe 100 and float 504 Form 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 pipe 100 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 protect Sealing is held, this can prevent fluid upper in any direction mobile more than the sealing.Sealing ring 300 can also keep tensioning state.It can It 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 slightly Expansion, 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 100 Circumferentially it is applied to sealing ring 300.After pipe 100 undergoes the separation (such as passing through centrifugation) based on density, by sealing ring 300 It 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 ring 300 support to lean against on pipe 100.It is that the operation of the fixture can be automation or can manually execute.Alternatively, institute State 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 to Sealing is formed between float 504 and pipe 100, such as by ultrasonic welding, or by applying heat or temperature gradient so that pipe 100 deformations/are melted to float 504.For convenience, method is described with reference to sealing ring, but method as described below is not intended to Application 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) or The 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 motor When driving, pressure member is remain stationary.When pressure member is by motor drive, collet is remain stationary.The fixture can wrap Buckle 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, band Ring folder or setting fastening (such as zipper setting fastening).Can in the case where no sealing ring using fixture to provide between float and pipe Sealing.

Return to Fig. 4 A, in box 418, and as seen in Fig. 5 E, pipe 100 can be overturn, can by plug 116 from Pipe 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 should It 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 pipe Process container 202.With displacement fluid 212 (it has greater than buffy coat 512 but is less than the density of description fluid 520) from Process container 202 flows into pipe 100, and buffy coat 512 passes through set 214 in pipe 100 and moves outwardly and into process container 202。

Then process container 202 (including buffy coat 512) can be taken out from pipe 100 to be further processed, divide with experience Analysis, storage etc..After taking out process container 202, processing solution can be added, although at the recycling foregoing description of target substance Reason 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 fibre Yellow 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 side It, can be by coloring agent 606, negative enrichment reagents (negative enrichment in frame 436, and as seen in fig. 6b Reagent) 604 and description fluid 602 be added pipe 100.The primary tank is added to mark target substance in coloring agent 606.To also The 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 604 Density.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 than The 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 but It 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 with The mixing of target substance.For example, coloring agent can be the solution containing fluorescence probe, the fluorescence probe can be used for target substance It dyes and thus marks, thus the fluorescence signal for identifying and characterizing is provided.Before container is added in suspension, it will hang Supernatant liquid is added after container but has been subjected to after centrifugation before centrifugation or in suspension, can will contain fluorescence probe Solution be added suspension.Fluorescence probe includes the fluorescent molecule with ligand binding.Target substance can have many different types Surface 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 glimmering The concrete type of optical molecule conjugation and the determining target substance being present in suspension.The example of suitable fluorescent molecule include but It 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- phenyl Indoles ") and TRITC (" isothiocyanic acid tetramethylrhodamine ")；Dyestuff, the combination of such as CY5PE, CY7APC and CY7PE；And conjunction At molecule, such as self assembly nucleic acid structure.Many solution can be used, so that every kind of solution includes in conjunction with different ligands Different 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 size It 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 from Pipe 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 should It 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 pipe Process container 202.With displacement fluid 212 (it has greater than buffy coat 512 but is less than the density of description fluid 520) from Process container 202 flows into pipe 100, and buffy coat 512 passes through set 214 in pipe 100 and moves outwardly and into process container 202.In addition, the fluid 604 (it is mixed to form high density blood plasma 608 with blood plasma 510) for changing density causes high density Blood plasma 608 has the density at least more than buffy coat 502 and description fluid 602.

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, four Dimer 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.Example Such 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 changed Matter 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 hour When, 4 hours, 8 hours, 24 hours etc.).The example of suitable positive enrichment reagents includes but is not limited to: using polyvinylpyrrolidone The solution of (such as Percoll) coated colloidal silica particle, polysaccharide solution (such as Ficoll), Iodixanol (such as OptiPrep), 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, silicon At 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 target Matter.Continuous density classification separation is by gradually or sample is divided into fraction or a fraction of sample is divided into Asia by continuous process Fraction, so that each step or sequence are caused from the above-mentioned fraction or substate different with consecutive steps or sequence collection or separation Point.In other words, continuous density classification separates each subpopulation that a group can be provided by series of steps or a group Each sub- subpopulation of the subpopulation of body.For example, buffy coat is the fraction of whole blood sample.The buffy coat fraction can To 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 separation It gets by oneself.

According to the number for separating and collecting desired fraction or subfraction, can be used two or more process containers and Various 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 substance Degree is greater than the density of desired target substance；For example, displacement fluid can have bigger than the density of desired target substance about 0.001 To about 0.005g/cm³Density.Similarly, every kind of continuous fraction or subfraction are bigger than preceding fraction or subfraction.It collects Afterwards, can analyze continuous subfraction, such as diagnosing, prognosis, research purpose, to determine component characteristics (i.e. complete haemocyte Count), 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 Daosuo There is subfraction to be collected or until desired subfraction is collected.Although being with float and close by continuous density classification separation expression Seal ring executes, but continuous density classification separation can execute in the case where no float, sealing ring or both.Here is to use In in a kind of embodiment for executing the execution continuous density classification separation after all steps of the insertion of process container Method:

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 as N=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 depositing Shelves 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 picking Analysis.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 any Substrate or container appropriate.

It can analyze target substance using any analysis method appropriate or technology, although more specifically using extracellular and thin Analysis intracellular, including intracellular protein label；Color staining；Foranalysis of nucleic acids, including but not limited to DNA array, expression battle array Column, protein array and DNA hybridization array；(" ISH "-is a kind of for analyzing the tool of DNA and/or RNA, all in situ hybridization Such as copy number changes)；Polymerase chain reaction (" PCR ")；Reverse transcription PCR；Or (" bDNA "-one kind is used for branched DNA Analyze 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 one Step processing is to test such fetal abnormality, including but not limited to, chromosome abnormality (such as fetus aneuploidy, Tang Shi are comprehensive Sign, trisomy 13, trisomy 18 or sex chromosomal abnormality, such as Turner syndrome), lesser subchromoso is abnormal, gender inspection It 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 marked In 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, day Aspartic acid specific cysteine protease, survivin, p27^kip, 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 egg White, Slug, Ki-67, M30, MAGEA3, the receptor kinase of phosphorylation, modified histone, the relevant albumen of chromatin and MAGE.For fixation, infiltration or label, fixative (such as formaldehyde, formalin, methanol, acetone, paraformaldehyde can be used Or glutaraldehyde), detergent (such as saponin(e, polyoxyethylene, digitonin, octyl beta-glucosidase, octyl β-sulfur sugar Glycosides, 1-S- octyl-β-D- thioglucopyranoside, Tween-20, CHAPS, CHAPSO, (1,1,3,3- tetramethyl fourth Base) 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 target Matter absorbs or intake weight), or can reduce (such as by the way that buoy (buoy) is attached to target substance, or by making target substance Absorb or take in buoy).The weight or the buoy can be bound to ligand.The target substance can have many inhomogeneities The 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 weight The 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 can Alternatively, weight or buoy can be attached to non-target substance to change the density of non-target substance to obtain purer target substance Sample.

For purposes of explanation, the description of front provides the thorough understanding of present disclosure using specific term.But Those skilled in the art are not it is clear that require the detail for implementing system and method described herein.In order to illustrate With 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 this Disclosure is confined to described precise forms.In view of the introduction of front, many modifications and variations are possible.Display is simultaneously The embodiment is described most preferably to explain the principle and practical application of present disclosure, thus makes those skilled in the art Present disclosure and various embodiments can be most preferably utilized, wherein various modifications are suitable for covered particular use.This Scope of the disclosure is intended to the equivalents by following following claims and they.

Claims

1. system comprising:

Pipe comprising

First end,

The second end including aperture,

Biological suspensions comprising target substance, and

Collection section between the first end and the second end, the collection section include

Funnel comprising in the mouth of the first end nearside and on the vertex in the first end distal side,

Cavity extends from the aperture in the second end to the vertex of the funnel, and

Set, extends into the cavity from the vertex of the funnel；With

Process container comprising the displacement fluid of closing end and the density with the density for being greater than the target substance,

Wherein at least part of the process container is located in the cavity of the pipe, and

Wherein the closing end for wearing the process container extends.

2. system according to claim 1, wherein the displacement fluid is selected from: using the coated colloidal state two of polyvinylpyrrolidone Solution, polysaccharide solution, Iodixanol, organic solvent, liquid wax, oil, gas, olive oil, mineral oil, the silicon oxygen of silicon oxide particle Alkane oil, impregnation oils, mineral oil, paraffin oil, silicone oil, fluorosilicone, perfluorodecalin, perfluor perhydro phenanthrene, perfluoro bromide octane, You Jirong Agent, 1,4- bis-Alkane, acetonitrile, ethyl acetate, the tert-butyl alcohol, cyclohexanone, methylene chloride, tert-pentyl alcohol, t-butyl methyl ether, acetic acid Butyl ester, hexanol, nitrobenzene, toluene, octanol, octane, propene carbonate, tetramethylene sulfone, ion fluid, based on the molten of polymer Liquid, surfactant, perfluor ketone, perfluorocyclopentanone, perfluorocyclohexanone, fluorinated ketone, hydrofluoroether, hydrofluorocarbon, perfluoro alkane, perfluor Polyethers, silicon, the liquid based on silicon, phenyl methyl siloxane, and combinations thereof.

3. system according to claim 1, the process container be further contained in the closing end can be again The plug of sealing, wherein the plug for wearing the resealable extends.

4. system according to claim 1, wherein the set is pipe, syringe needle or non-coring syringe needle.

5. system according to claim 1, the process container further includes processing solution to realize to the target The conversion of matter.

6. system according to claim 1, the biological suspensions further include non-target substance, and the pipe is further Change the density of the non-target substance comprising negative enrichment reagents.

7. system according to claim 6, wherein the suitable negative enrichment reagents are coated with polyvinylpyrrolidone The solution of colloidal silica particle, polysaccharide solution, Iodixanol, branched chain glucans, cesium chloride, sucrose, based on the molten of sugar Liquid, polymer solution, multiphase polymer solution, tetrameric antibody compound, particle, pearl, nano particle, the change based on metal Close object, metal complex, lipid or sugar.

8. system according to claim 1, the system further comprises the cap being inserted in the first end of the pipe.

9. system according to claim 8, the system further comprises the first end for being placed on the cap and the pipe Fixture above.

10. system according to claim 1, the system further comprises floating at the lengthwise position in the pipe Son.

11. system according to claim 10, the system further comprises sealing ring, the sealing ring with the pipe It is circumferentially located at around the pipe at the identical lengthwise position of at least part of interior float.

12. system according to claim 1, wherein the first end of the pipe is bottom end, the second end of the pipe It is top end part, and the process container is upwardly extended from the second end of the pipe.

13. system according to claim 1, wherein the target substance be buffy coat, cell, ovum, fetal material, Trophoderm, the red blood cell for having core, fetus red blood cell, fetus white blood corpuscle, foetal DNA, fetal rna, the tumour of circulation are thin Born of the same parents, the endothelial cell of circulation, immunocyte, vesica, liposome, albumen, nucleic acid, biomolecule, naturally depositing with close membrane Microscopic units, the microscopic units manually prepared, helminth, microorganism, virus, inflammatory cell or decomposition with close membrane Biosolids.

14. system according to claim 1, the pipe further includes positive enrichment reagents to change the close of the target substance Degree.

15. system according to claim 14, wherein the suitable positive enrichment reagents are with polyvinylpyrrolidone packet The solution of the colloidal silica particle of quilt, polysaccharide solution, Iodixanol, branched chain glucans, cesium chloride, sucrose, based on sugar Solution, polymer solution, multiphase polymer solution, tetrameric antibody compound, particle, pearl, nano particle, based on metal Compound, metal complex, lipid or sugar.

16. method comprising following step:

Pipe is provided comprising

First end,

The second end including aperture,

Biological suspensions comprising target substance, and

Set, extends into the cavity from the vertex of the funnel；

It will include that the process container of closing end is inserted into the cavity of the pipe, wherein the closing for wearing the process container End extends；

Displacement fluid is added to the process container, there is the density of the density greater than the target substance；With

The pipe, the displacement fluid and the process container are centrifuged, wherein the displacement fluid is via described in centrifugal process Set flows into the pipe to replace the target substance from the pipe, passes through the set and flows into the process container.

17. the method according to claim 11, the method further includes:

The pipe is added to change the density of the target substance in positive enrichment reagents.

18. according to the method for claim 16, wherein the target substance is buffy coat, cell, ovum, fetus object Matter, trophoderm, the red blood cell for having core, fetus red blood cell, fetus white blood corpuscle, foetal DNA, fetal rna, circulation tumour Cell, the endothelial cell of circulation, immunocyte, vesica, liposome, albumen, nucleic acid, biomolecule, with the natural of close membrane Existing microscopic units, the microscopic units manually prepared with close membrane, helminth, microorganism, virus, inflammatory cell divide The biosolids of solution.

19. the method according to claim 11, the method further includes:

The pipe is added in negative enrichment reagents to change the density of non-target substance, the biological suspensions further include non-target Matter,

The biological suspensions further include non-target substance.

20. according to the method for claim 16, the method further includes following step:

Filling device including closing end and stratified fluid is inserted into the cavity of the pipe, wherein described wore described fill out The closing end set is filled to extend；

At least part of the stratified fluid is transferred to the pipe from the filling device and enters institute to replace from the pipe State the air in filling device；With

The filling device is taken out from the cavity of the pipe,

The insertion of the filling device is wherein executed before the process container is inserted into the cavity of the pipe, is shifted and is taken Step out.