Miniaturized lateral flow device for rapid and sensitive detection of proteins or nucleic acids

• US 10,458,978 B2
• Filed: 02/13/2017
• Issued: 10/29/2019
• Est. Priority Date: 08/22/2006
• Status: Active Grant

• Current Assignee: TRIAD National Security, LLC
• Original Assignee: TRIAD National Security, LLC
• Inventors: Cary, Robert B.
• Primary Examiner(s): Crow, Robert T.
• Application Number:
  US15/431,705
• Publication Number:
  US 20170160271A1
• Time in Patent Office: 988 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: G01N 33/523 the element being adapted for a specific analyte

Abstract

The invention provides miniaturized lateral flow chromatographic and lateral flow chromatographic microarray devices (LFM). The miniaturization of lateral flow nucleic acid detection achieved by the present invention offers reduced reagent use, femtomole sensitivity, excellent linear dynamic range, and rapid detection. Moreover, the small feature sizes of capture oligonucleotides renders the potential information capacity of the platform comparable to more traditional spotted fluorescence microarrays as well as improving sensitivity. The LFM devices exemplified herein enable analytes to be detected within 10 seconds from the time of sample introduction to the LFM device. Sample volumes may be as low as about 10 microliters, significantly reducing assay costs and ameliorating reagent storage logistics. Additionally, the miniaturization of lateral flow opens the door to highly multiplexed assays, allowing many proteins or nucleic acids to be detected in a single assay.

Claims

1. A device for detecting the presence of a target nucleic acid in a fluid sample, the device comprising:

a porous lateral flow strip having a width of 3 mm or less; and

a microarray of spots on said porous lateral flow strip, each spot comprising a plurality of capture oligonucleotides;

wherein each capture oligonucleotide is complementary to a capture sequence of the target nucleic acid;

wherein each spot was printed with a solution having a concentration of capture oligonucleotides between 200 μ

M and 800 μ

M; and

wherein said microarray of spots provides a quantitative visual colorimetric signal indicating an amount of from 250 amol to 1 fmol of the target nucleic acid in the fluid sample comprising at least 150 mM salt in less than two minutes by capillary flow.

2. The device of claim 1 wherein said lateral flow strip comprises an area of approximately 60 mm².

3. The device of claim 1 wherein each spot has a diameter of between 50 and 300 μ

m.

4. The device of claim 1 wherein each spot has a diameter of between 50 and 250 μ

m.

5. The device of claim 1 wherein each spot has a diameter of between 50 and 200 μ

m.

6. The device of claim 1 wherein the porous lateral flow strip has a pore size of between 0.2 and 20 μ

m.

7. The device of claim 1 comprising a zone for receiving a labeled colorimetric detection oligonucleotide, which detection oligonucleotide is complementary to a detection sequence of the target nucleic acid.

8. The device of claim 7 wherein the detection oligonucleotide is a branched nucleic acid molecule or a dendrimeric nucleic acid molecule.

9. The device of claim 7 wherein the detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μ

m in diameter.

10. The device of claim 9 wherein the detectable particle is selected from the group consisting of polystyrene microspheres, latex particles, nano-gold particles, colloidal gold particles, metal particles, magnetic particles, and semiconductor nanocrystals.

11. The device of claim 7 wherein a detection oligonucleotide comprises a first portion having a sequence complementary to a part of the detection sequence and a second portion having a non-target specific sequence of at least 9 nucleotides, which second portion is adjacent to a label.

12. The device of claim 11 wherein the second portion has a poly (A) or poly (T) sequence of at least 9 nucleotides.

13. The device of claim 7 wherein the capture sequence and detection sequence of the target nucleic acid are adjacent within 2 bases.

14. The device of claim 1 wherein the porous strip comprises lateral flow compatible nitrocellulose.

15. The device of claim 14 wherein said nitrocellulose has pore sizes range between 0.2 and 20 μ

m.

16. The device of claim 1 wherein said visual colorimetric signal is viewable by the unaided human eye.

17. The device of claim 1 comprising a sample pad and/or a conjugate release pad.

18. The device of claim 17 wherein said sample pad and/or said conjugate release pad has been impregnated with a substance selected from the group consisting of 4-1(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glucol, SDS, BSA, a hydrophilic polysaccharide, polyvinyl pyrolidone, and combinations thereof.