Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

• US 9,944,922 B2
• Filed: 12/08/2015
• Issued: 04/17/2018
• Est. Priority Date: 05/05/2008
• Status: Active Grant

• Current Assignee: TRIAD National Security, LLC
• Original Assignee: Los Alamos National Security LLC (Government of the United States of America)
• Inventors: Cary, Robert B.
• Primary Examiner(s): Chin, Chris L
• Application Number:
  US14/962,967
• Publication Number:
  US 20160083716A1
• Time in Patent Office: 861 Days
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387

Abstract

Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

Claims

1. A method for processing a sample comprising target nucleic acids, the method comprising:

adding a sample to a sample receiving zone and a first solution to a first solution zone;

transporting at least some of the sample to a capture zone via capillary flow along a central capillary flow path;

binding the target nucleic acids to the capture zone; and

delivering via capillary flow, at a desired time or sequence after the adding step, a first portion of the first solution via a first solution capillary flow path to the central capillary flow path or the capture zone, and a second portion of the first solution via a second solution capillary flow path to the central capillary flow path or the capture zone;

wherein the first solution is useful for processing the target nucleic acids prior to detecting the target nucleic acids.

2. The method of claim 1 further comprising the first portion of the first solution and the second portion of the first solution flowing in a direction along a length of the central capillary flow path after the delivering step.

3. The method of claim 1 wherein the binding step increases a concentration of the target nucleic acids relative to inhibitory matrix constituents present in the capture zone.

4. The method of claim 3 further comprising removing the inhibitory matrix constituents from the capture zone.

5. The method of claim 4 wherein the first solution comprises a wash buffer for removing residual sample from the central capillary flow path and removing the inhibitory matrix constituents from the capture zone.

6. The method of claim 1 wherein the adding step further comprises adding a second solution to a second solution zone, the method further comprising delivering via capillary flow, at a desired time or sequence after the transporting step, a first portion of the second solution via a third solution capillary flow path to the central capillary flow path or the capture zone, and a second portion of the second solution via a fourth solution capillary flow path to the central capillary flow path or the capture zone;

wherein the second solution is useful for processing the target nucleic acids prior to detecting the target nucleic acids.

7. The method of claim 6 wherein the second solution comprises a wash buffer, an amplification buffer, an amplification reagent, a dye, a high ionic strength buffer, a low ionic strength buffer, a staining buffer, an exchange buffer, guanidinium buffer, guanidinium isothiocyanate buffer, and/or an elution buffer.

8. The method of claim 1 wherein the binding step comprises binding DNA with silica, binding RNA with silica, binding the target nucleic acids to ligands immobilized in the capture zone, binding the target nucleic acids to a glass fiber nucleic acid affinity matrix, binding the target nucleic acids to a polvethyleneimine (PEI) or diethyl aminoethyl (DEAE) membrane, or providing a functionalized substrate for anion or cation exchange mediated enrichment of nucleic acids, proteins, or small molecules.

9. The method of claim 1 further comprising releasing the target nucleic acids from the capture zone.

10. The method of claim 1 further comprising amplifying the target nucleic acids.

11. The method of claim 10 further comprising detecting a presence of the amplified target nucleic acids.

12. The method of claim 1 wherein the first solution comprises a wash buffer, an amplification buffer, an amplification reagent, a dye, a high ionic strength buffer, a low ionic strength buffer, a staining buffer, an exchange buffer, guanidinium buffer, guanidinium isothiocyanate buffer, and/or an elution buffer.

13. A method for processing a sample comprising biological particles comprising target nucleic acids, the method comprising:

adding a sample to a sample receiving zone, a lysis buffer to a buffer zone, and a first solution to a first solution zone;

delivering the lysis buffer to the sample at a desired time or sequence after the adding step;

lysing the biological particles in the sample, thereby liberating the target nucleic acids contained therein,

transporting at least some constituents of the sample to a capture zone via capillary flow along a central capillary flow path;

binding the target nucleic acids to the capture zone; and

delivering via capillary flow, at a desired time or sequence after the adding step, a first portion of the first solution via a first solution capillary flow path to the central capillary flow path or the capture zone, and a second portion of the first solution via a second solution capillary flow path to the central capillary flow path or the capture zone;

wherein the first solution is useful for processing the target nucleic acids prior to detecting the target nucleic acids.

14. The method of claim 13 wherein the biological particles are selected from the group consisting of cells, viruses, and bacteria.

15. The method of claim 13 further comprising capturing the biological particles by reacting ligands with surfaces of the biological particles prior to the lysing step.

16. The method of claim 15 wherein the ligands comprise antibodies or carbohydrates.

17. The method of claim 15 wherein the method further comprises delivering via capillary flow, at a desired time or sequence after the adding step and prior to the transporting step, a first portion of the lysis buffer via a third solution capillary flow path to the captured biological particles, and a second portion of the lysis buffer via a fourth solution capillary flow path to the captured biological particles.

18. The method of claim 13 further comprising the first portion of the first solution and the second portion of the first solution flowing in a direction along a length of the central capillary flow path after the delivering step.

19. The method of claim 13 wherein the binding step increases a concentration of the target nucleic acids relative to inhibitory matrix constituents present in the capture zone.

20. The method of claim 19 further comprising removing the inhibitory matrix constituents from the capture zone.

21. The method of claim 20 wherein the first solution comprises a wash buffer, the wash buffer removing residual sample and lysis buffer from the central capillary flow path and removing the inhibitory matrix constituents and lysis buffer from the capture zone.

22. The method of claim 13 wherein the adding step further comprises adding a second solution to a second solution zone, the method further comprising delivering via capillary flow, at a desired time or sequence after the transporting step, a first portion of the second solution via a fifth solution capillary flow path to the central capillary flow path or the capture zone, and a second portion of the second solution via a sixth solution capillary flow path to the central capillary flow path or the capture zone;

wherein the second solution is useful for processing the target nucleic acids prior to detecting the target nucleic acids.

23. The method of claim 22 wherein the second solution comprises a wash buffer, the wash buffer removing some of the first solution from the capture zone.

24. The method of claim 22 wherein the second solution comprises a wash buffer, an amplification buffer, an amplification reagent, a dye, a high ionic strength buffer, a low ionic strength buffer, a staining buffer, an exchange buffer, guanidinium buffer, guanidinium isothiocyanate buffer, and/or an elution buffer.

25. The method of claim 13 wherein the binding step comprises binding DNA with silica, binding RNA with silica, binding the target nucleic acids to ligands immobilized in the capture zone, binding the target nucleic acids to a glass fiber nucleic acid affinity matrix, binding the target nucleic acids to a polvethyleneimine (PEI) or diethyl aminoethyl (DEAE) membrane, or providing a functionalized substrate for anion or cation exchange mediated enrichment of nucleic acids, proteins, or small molecules.

26. The method of claim 13 further comprising releasing the target nucleic acids from the capture zone.

27. The method of claim 13 further comprising amplifying the target nucleic acids.

28. The method of claim 27 further comprising detecting a presence of the amplified target nucleic acids.

29. The method of claim 13 wherein the first solution comprises a wash buffer, an amplification buffer, an amplification reagent, a dye, a high ionic strength buffer, a low ionic strength buffer, a staining buffer, an exchange buffer, guanidinium buffer, guanidinium isothiocyanate buffer, and/or an elution buffer.