Solid gel amplification method and apparatus for platform molecular diagnostics

Patent metadata

Field	Value
Publication number	US-9957559-B2
Application number	US-201314399637-A
Country	US
Kind code	B2
Filing date	Feb 25, 2013
Priority date	May 9, 2012
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

The present invention provides for a novel system and method for amplification and detection of nucleic acids within a miniaturized device wherein sample administration occurs via capillary forces through a channel created by drying a hydrogel containing all components needed for a cell-free, enzymatic, nucleic-acid amplification system other than the template nucleic acid or precursor thereto, and wherein an aqueous sample is provided to the desiccated hydrogel, and the hydrogel is rehydrated, through capillary forces.

First claim

What is claimed is:

1. A cassette for performing interrogations for the presence of a nucleic acid within an aqueous sample comprising
a pan containing within it solid wax with at least one trench on its surface;
a multiplicity of capillaries with two opposing apertures;

wherein
said capillaries contain a desiccated hydrogel containing all components needed for a cell-free nucleic acid amplification other than the template nucleic acid,
within the capillaries there exists a path described by the inner diameter of said capillary and said desiccated hydrogel which allows atmospheric communication between opposing apertures and through said capillaries,
said capillaries are within said trench with the longitudinal axis of said capillary parallel to the longitudinal axis of said trench,
said trench resists the motion of said capillaries through friction between the inner longitudinal surface of said trench and the longitudinal surface of said capillaries with at least one end of said capillary is capable of receiving an aqueous sample,
said wax is optically transparent to at least two wavelengths of electromagnetic radiation wherein said at least two wavelengths are capable of differentiation by an optical detector, and
said pan is capable of containing the wax when in a molten state.

2. The cassette of claim 1 wherein the wax is Paraffin wax.

3. The cassette of claim 1 wherein said capillaries are less than 2 mm in diameter.

4. The cassette of claim 3 wherein said capillaries have an inner diameter of 1.1 mm.

5. The cassette of claim 1 wherein said pan is thermally conductive.

6. The cassette of claim 5 wherein said pan is aluminum.

7. The cassette of claim 1 wherein said pan absorbs electromagnetic radiation of at least one of said at least two wavelengths.

8. The cassette of claim 1 wherein said desiccated hydrogel is polymerized acrylamide and bis-acrylamide.

9. The cassette of claim 8 wherein said desiccated hydrogel is 4% acrylamide and 0.4% bis-acrylamide.

10. The cassette of claim 1 wherein said components needed for a cell free nucleic acid amplification system comprises the enzymes, substrates and primers needed for a polymerase chain reaction.

11. The cassette of claim 10 wherein said components needed for a cell free nucleic acid amplifications system comprises tris-sulfate, (NH 4 ) 2 SO 4 , MgCl 2 , all four deoxyribonucleotides, Bovine Serum Albumin, at least two primers designed to hybridize with a target nucleotide sequence, a heat stable DNA polymerase, azobis, TEMED and water.

12. The cassette of claim 11 wherein said components needed for a cell free nucleic acid amplifications system includes LC Green.

13. A system for detecting the presence of a nucleic acid within an aqueous sample comprising
a pan containing within it solid wax with at least one trench on its surface;
a multiplicity of capillaries with two opposing apertures;
means for controlling temperature in thermal communication with said pan;
illumination source; and
optical detector,

wherein
The amplification of a nucleic acid is detected through an increase in an optical signal received by said optical detector resulting from the interaction of an illumination wavelength provided by said illumination source,
said capillaries contain a desiccated hydrogel containing all components needed for a cell-free nucleic acid amplification other than the template nucleic acid,
within the capillaries there exists a path described by the inner diameter of said capillary and said desiccated hydrogel which allows atmospheric communication between opposing apertures and through said capillaries;
said capillaries are within said trench with the longitudinal axis of said capillary parallel to the longitudinal axis of said trench,
said trench resists the motion of said capillaries through friction between the inner longitudinal surface of said trench and the outer longitudinal surface of said capillaries with at least one end of said capillary is capable of receiving an aqueous sample,
said wax is optically transparent to at least two wavelengths of electromagnetic radiation provided by said illumination source wherein said at least two wavelengths are capable of differentiation by an optical detector, and
said pan is capable of containing the wax when in a molten state.

14. The system of claim 13 wherein the wax is Paraffin wax.

15. The system of claim 13 wherein said capillaries are less than 2 min in diameter.

16. The system of claim 15 wherein said capillaries have an inner diameter of 1.1 mm.

17. The system of claim 13 wherein said pan is thermally conductive.

18. The system of claim 17 wherein said pan is aluminum.

19. The system of claim 13 wherein said pan absorbs electromagnetic radiation of at least one of said at least two wavelengths.

20. The system of claim 13 wherein said desiccated hydrogel is polymerized acrylamide and bis-acrylamide.

21. The system of claim 20 wherein said desiccated hydrogel is 4% acrylamide and 0.4% bis-acrylamide.

22. The system of claim 13 wherein said components needed for a cell free nucleic acid amplification system comprises the enzymes, substrates and primers needed for a polymerase chain reaction.

23. The system of claim 22 wherein said components needed for a cell free nucleic acid amplifications system comprises tris-sulfate, (NH 4 ) 2 SO 4 , MgCl 2 , all four deoxyribonucleotides, Bovine Serum Albumin, at least two primers designed to hybridize with a target nucleotide sequence, a heat stable DNA polymerase, azobis, TEMED and water.

24. The system of claim 23 wherein said components needed for a cell free nucleic acid amplifications system includes LC Green.

25. The system of claim 24 wherein said illumination means is a laser emitting light at a wavelength of 445 nm.

26. The system of claim 13 where said optical detection means is a CCD camera with a band-pass interference filter centered at 530 nm.

27. A cassette for performing interrogations for the presence of a nucleic acid within an aqueous sample comprising
a pan containing within it solid wax with at least one trench on its surface;
a multiplicity of capillaries with two opposing apertures;

wherein
said capillaries contain a desiccated hydrogel containing all components needed for a cell-free nucleic acid amplification other than the template nucleic acid,
within the capillaries there exists a path described by the inner diameter of said capillary and said desiccated hydrogel which allows atmospheric communication between opposing apertures and through said capillaries;
said capillaries are within said trench with the longitudinal axis of said capillary parallel to the longitudinal axis of said trench, and
said trench resists the motion of said capillaries through friction between the inner longitudinal surface of said trench and the longitudinal surface of said capillaries with at least one end of said capillary is capable of receiving an aqueous sample.

Assignees

• Manage Damikka
• Univ Alberta

Inventors

• Manage Damikka

Classifications

• B01L2200/0689

  Sealing · CPC title

• B01L2300/069

  Absorbents; Gels to retain a fluid · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• C12M21/18

  Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes · CPC title

• C12M43/00

  Combinations of bioreactors or fermenters with other apparatus · CPC title