Apparatus, system, and method for collecting a target material

Patent metadata

Field	Value
Publication number	US-9625360-B2
Application number	US-201514883071-A
Country	US
Kind code	B2
Filing date	Oct 14, 2015
Priority date	Nov 30, 2012
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

This disclosure is directed to an apparatus, system and method for retrieving a target material from a sample. A fraction-density-altering solution may be added to a vessel that contains the sample to change the density of a first fraction of the sample without changing the density of the target material or the density of any other sample fraction. A collector may be inserted into the vessel to funnel the target material from the sample into the collector or into a processing receptacle included in the collector. In one implementation, the collector may include a cannula which extends from a funnel into a chamber to hold the processing receptacle. In another implementation, the processing receptacle may be inserted into a bore within the collector.

First claim

We claim:

1. A method comprising the steps of:
providing a primary vessel comprising
an open end, and
a sample comprising a target material and at least one other sample fraction;

adding at least one fluorescent probe to the primary vessel;
adding a density-altering agent to the vessel to change the density of the at least one other sample fraction such that the density of the target material is less than a changed density of the at least one other sample fraction;
inserting a collector into the open end of the primary vessel;
adding a layering fluid to the primary vessel, the layering fluid having a density greater than the target material;
inserting a first processing vessel into the collector, wherein the first processing vessel includes a first collection fluid having a density greater than at least a first sub-fraction of the target material; and
centrifuging the primary vessel, the collector, and the first processing vessel, such that during centrifugation the first collection fluid flows into the primary vessel via a cannula of the collector and at least the first sub-fraction of the target material flows into the first processing vessel via the cannula of the collector,
wherein centrifuging the primary vessel, the collector, and the first processing vessel is performed after inserting the collector into the primary vessel and after inserting the first processing vessel into the collector.

2. The method of claim 1 , wherein the density-altering agent is selected from the group consisting of a solution of colloidal silica particles coated with polyvinylpyrrolidone, a polysaccharide solution, iodixanol, a complex branched glucan, cesium chloride, sucrose, a sugar-based solution, a polymer solution, and a multi-phase polymer solution.

3. The method of claim 1 , further comprising the steps of:
removing the first processing vessel comprising at least the first sub-fraction from the collector;
inserting a second processing vessel comprising a second collection fluid into the collector; and
re-centrifuging the primary vessel with the collector and the second processing vessel,
wherein the second collection fluid flows into the primary vessel via the cannula of the collector and a second sub-fraction of the target material flows into the second processing receptacle via the cannula of the collector,
wherein the second collection fluid has a density greater than the second sub-fraction and the first collection fluid, and wherein the second sub-fraction has a density greater than the density of the first sub-fraction.

4. The method of claim 3 , wherein the removing, inserting, and re-centrifuging steps are repeated with an n th processing vessel including an n th collection fluid, wherein n th is equal to or greater than 3 rd , and wherein the removing, inserting, and re-centrifuging steps are repeated until all desired sub-fractions of the target material are obtained from the sample, wherein each successive collection fluid has a density greater than each preceding collection fluid.

5. The method of claim 1 , wherein the first processing vessel includes a plug in a closed end.

6. The method of claim 5 , wherein the first processing vessel includes cap to seal an open end.

7. The method of claim 5 , wherein the cannula of the collector extends at least partially through the plug.

8. The method of claim 1 , wherein adding the layering fluid is performed before inserting the collector into the primary vessel.

9. The method of claim 1 , wherein adding the layering fluid is performed after inserting the collector into the primary vessel by inserting a fluid laying device into the collector and forcing the layering fluid through the cannula of the collector and into the primary vessel.

10. The method of claim 1 , further comprising the step of adding a depletion reagent to the primary vessel.

11. The method of claim 10 , wherein the depletion reagent lyses at least one non-target material.

12. The method of claim 10 , wherein the depletion reagent changes the density of at least one non-target material.

13. The method of claim 1 , wherein the first collection fluid and the layering fluid are selected from the group consisting of: a solution of colloidal silica particles coated with polyvinylpyrrolidone, a polysaccharide solution, iodixanol, an organic solvent, a liquid wax, an oil, a gas, olive oil, silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil, fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide, 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate, tetramethylene sulfones, ionic liquids, a polymer-based solution, a surfactant, a perfluoroketone, perfluorocyclopentanone, perfluorocyclohexanone, a fluorinated ketone, a hydrofluoroether, a hydrofluorocarbon, a perfluorocarbon, a perfluoropolyether, silicon, a silicon-based liquid, phenylmethyl siloxane, and combinations thereof.

14. The method of claim 1 , the primary vessel further comprising a float comprising a main body.

15. The method of claim 14 , further comprising the step of centrifuging the primary vessel comprising the sample and the float thereby effecting a density-based separation of the sample into the target material and the at least one other sample fraction and trapping the target material between the main body of the float and an inner wall of the primary vessel,
wherein the centrifuging to effect the density-based separation is performed after providing the primary vessel comprising the sample and the float, and before adding the at least one fluorescent probe.

16. The method of claim 15 , further comprising the step of adding a clearing fluid having a density greater than the target material to the primary vessel, wherein the clearing fluid is added to the primary vessel after centrifuging to effect the density-based separation.

17. The method of claim 16 , further comprising the step of forming a first seal between a lower end of the main body of the float and the inner wall of the primary vessel, wherein forming the first seal is performed after centrifuging to effect the density-based separation and before adding clearing fluid.

18. The method of claim 17 , further comprising the step of moving the target material at least above the main body of the float by re-centrifuging the primary vessel, wherein the moving step is performed after the adding the clearing fluid, after centrifuging to effect the density-based separation, and before adding the at least one fluorescent probe.

19. The method of claim 18 , further comprising the step of forming a second seal between an upper end of the main body of the float and the inner wall of the primary vessel, wherein forming the second seal is performed after the moving step and before adding the at least one fluorescent probe to the primary vessel.

Assignees

• Rarecyte Inc

Inventors

• U'Ren Lance
• Chang Elizabeth
• Chow Jennifer

Classifications

• G01N1/4077 Primary

  by other techniques involving separation of suspended solids · CPC title

• B01L2300/042

  Caps; Plugs · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01L2200/0689

  Sealing · CPC title

• B01L3/5021

  Test tubes specially adapted for centrifugation purposes · CPC title