Devices, systems, and methods for detecting nucleic acids using sedimentation

Patent metadata

Field	Value
Publication number	US-10384202-B2
Application number	US-201715704860-A
Country	US
Kind code	B2
Filing date	Sep 14, 2017
Priority date	Jul 8, 2010
Publication date	Aug 20, 2019
Grant date	Aug 20, 2019

Abstract

Embodiments of the present invention are directed toward devices, systems, and method for conducting nucleic acid purification and quantification using sedimentation. In one example, a method includes generating complexes which bind to a plurality of beads in a fluid sample, individual ones of the complexes comprising a nucleic acid molecule such as DNA or RNA and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

First claim

What is claimed is:

1. A method of conducting an assay, the method comprising:
generating complexes which bind to a plurality of beads in a fluid sample, individual ones of the complexes comprising a negatively-charged target analyte bound, using a charge, directly to a positively charged surface of the bead, and a labeling agent bound to the target analyte, wherein the generating occurs in the presence of a binding buffer configured to provide an acidic pH to maintain the negative charge of the target analyte;
transporting the plurality of beads including the complexes through a density media, wherein the density media has a density less than a density of the beads and greater than a density of the fluid sample, and wherein the transporting occurs, at least in part, by a sedimentation force; and
detecting a signal from the labeling agents of the complexes.

2. The method of claim 1 , wherein the target analyte includes a nucleic acid.

3. The method of claim 2 , wherein the labeling agent comprises a nucleic acid dye.

4. The method of claim 3 wherein the nucleic acid dye has a binding constant <20 nM.

5. The method of claim 3 , wherein the beads have a positively-charged surface in the fluid sample and the nucleic acid has a negative charge.

6. The method of claim 5 , wherein the fluid sample includes kosmotropes or order-forming salts.

7. The method of claim 5 , wherein the fluid sample has an acidic pH<4 in order to cause contaminating proteins to exhibit positive charge.

8. The method of claim 3 , wherein the fluid sample includes chaotropic salts.

9. The method of claim 1 , wherein said fluid sample comprises whole blood, wherein the density media has a density greater than a density of red blood cells, and wherein the method further comprises:
separating the red blood cells from the plurality of beads at least in part by applying a sedimentation force to the fluid sample such that the red blood cells remain at an interface between the density media and the fluid sample.

10. The method of claim 1 , wherein a surface of the beads comprises amines.

11. The method of claim 1 , wherein the density media comprises a detergent.

12. The method of claim 2 , wherein the nucleic acid is a cell-free DNA.

13. The method of claim 1 , wherein the sedimentation force comprises a centrifugal force.

14. The method of claim 1 , wherein the transporting forms a pellet.

15. The method of claim 1 , wherein the density media comprises sucrose, dextran, or a buffered density media.

16. The method of claim 1 , wherein the binding buffer comprises a high salt concentration and a low pH.

17. The method of claim 1 , wherein the acidic pH is less than 4.

18. A method of conducting an assay, the method comprising:
generating complexes which bind to a plurality of beads in a fluid sample, individual ones of the complexes comprising a negatively-charged target analyte bound, using a charge, directly to a positively charged surface of the bead, and a labeling agent bound to the target analyte, wherein the generating occurs in the presence of a binding buffer configured to provide an acidic pH to maintain the negative charge of the target analyte and a high salt concentration;
transporting the plurality of beads including the complexes through a density media, wherein the density media has a density less than a density of the beads and greater than a density of the fluid sample, and wherein the transporting occurs, at least in part, by a sedimentation force; and
detecting a signal from the labeling agents of the complexes.

Assignees

• Nat Tech & Eng Solutions Sandia Llc

Inventors

• Koh Chung-Yan
• Schaff Ulrich Y
• Sommer Gregory J

Classifications

• G01N15/05

  in blood · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title

• G01N2015/045

  by optical analysis · CPC title

• G01N15/042

  by centrifuging and investigating centrifugates (centrifuges per se B04B) · CPC title

• B01L2300/0803

  Disc shape · CPC title