METHOD AND DEVICE FOR DETECTING THE PRESENCE OF A SINGLE TARGET NUCLEIC ACID IN A SAMPLE

• US 20080171326A1
• Filed: 08/13/2007
• Published: 07/17/2008
• Est. Priority Date: 04/17/1997
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America), Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Cytonix, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Silver, Jonathan E., Brown, James F.
• Application Number:
  US11/837,651
• Publication Number:
  US 20080171326A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method comprising depositing a sample portion in a sample retaining means, and forcing a curable fluid across an exposed surface of the sample retaining means, thereby displacing excess first sample from the exposed surface without displacing the sample from the sample retaining means. Also, a method comprising depositing a sample portion in a sample retaining means, removing excess sample from an exposed surface of the sample retaining means, and depositing a curable fluid on the exposed surface. Also, a microfluidic device comprising a sample chamber, a first sample portion in the sample chamber, and a curable fluid and/or a cured fluid positioned on an exposed surface of a sample retaining means.

Claims

1. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

depositing at least a first sample portion in a sample retaining means, said first sample portion comprising at least a portion of a first sample;

forcing a curable fluid across an exposed surface of said sample retaining means, thereby displacing any excess first sample from said exposed surface without displacing said sample from said sample retaining means.

2. A method as recited in claim 1, wherein as said curable fluid is forced across said exposed surface, a layer of said curable fluid is deposited on said exposed surface and coats a top of said first sample portion.

3. A method as recited in claim 2, wherein said method further comprises curing said curable fluid to form a seal for the first sample portion retained in said sample retaining means, whereby said first sample portion is isolated from other sample portions and from excess first sample.

4. A method as recited in claim 2, wherein said curable fluid is immiscible with said first sample

5. A method as recited in claim 1, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

6. A method as recited in claim 5, wherein said method further comprises:

subjecting said first sample portion in said first sample chamber to at least a first amplification step; and

then

determining whether said first sample portion contains at least one molecule of said target nucleic acid.

7. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

depositing at least a first sample portion in a sample retaining means, said first sample portion comprising at least a portion of a first sample;

removing excess first sample from said exposed surface; and

depositing at least a first curable fluid on said exposed surface of said sample retaining means.

8. A method as recited in claim 7, wherein said method further comprises curing said curable fluid to form a seal for the first sample portion retained in said sample retaining means, whereby said first sample portion is isolated from other sample portions and from excess first sample.

9. A method as recited in claim 8, wherein said curing said curable fluid is conducted immediately after said removing excess first sample from said exposed surface.

10. A method as recited in claim 7, wherein said curable fluid is immiscible with said first sample

11. A method as recited in claim 7, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

12. A method as recited in claim 11, wherein said method further comprises:

subjecting said first sample portion in said first sample chamber to at least a first amplification step; and

then

determining whether said first sample portion contains at least one molecule of said target nucleic acid.

13. A microfluidic device comprising:

a first sample chamber; and

a first sample portion, said first sample portion being positioned in said first sample chamber; and

a curable fluid positioned on an exposed surface of said sample retaining means, said curable fluid covering at least said first sample portion.

14. A microfluidic device as recited in claim 13, wherein said curable fluid is immiscible with said first sample

15. A microfluidic device as recited in claim 13, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

16. A microfluidic device as recited in claim 13, wherein said microfluidic device further comprises at least one amplification targeting reagent positioned in said first sample portion.

17. A microfluidic device comprising:

a first sample chamber; and

a first sample portion, said first sample portion being positioned in said first sample chamber; and

a cured fluid positioned on an exposed surface of said sample retaining means, said cured fluid covering at least said first sample portion.

18. A microfluidic device as recited in claim 17, wherein said cured fluid forms a seal for the first sample portion retained in said sample retaining means, whereby said first sample portion is isolated from other sample portions and from excess first sample.

19. A microfluidic device as recited in claim 17, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

20. A microfluidic device as recited in claim 17, wherein said microfluidic device further comprises at least one amplification targeting reagent positioned in said first sample portion.