Methods of detecting amplified product

• US 8,067,159 B2
• Filed: 08/13/2007
• Issued: 11/29/2011
• Est. Priority Date: 04/17/1997
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Inventors: Silver, Jonathan E., Brown, James F.
• Primary Examiner(s): Chin; Christopher L
• Application Number:
  US11/837,600
• Publication Number:
  US 20080169184A1
• Time in Patent Office: 1,569 Days
• Field of Search: None
• 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 microfluidic device comprising a first surface portion and a first sample retaining element, which have differing affinities to a fluid, and a method comprising supplying a sample to such a device. In some embodiments, the differing affinity is a result of plasma, ion embedding, surface charging, chemical, optical, electronic and/or electromagnetic treatment. Also, a microfluidic device comprising at least one microcapillary device having a sample retaining element, at least one surface of which exhibits hydrophobicity, hydrophilicity, electromagnetic force exertion and electrostatic force exertion. Also, a microfluidic device comprising a first element having a hydrophilic pattern comprising at least a first sample retaining element. Also, a method comprising supplying a sample to a channel between a first element and a second element, and inducing in the first element at least one hydrophilic pattern by electrets or by internal or external electrodes to provide a charged surface.

Claims

1. A method comprising:

providing a sample containing (1) at least one target nucleic acid molecule to be amplified and (2) constituents for enabling amplification of the at least one target nucleic acid molecule;

loading the sample into a device;

in the device, subdividing the loaded sample into a plurality of sample volumes, wherein at least one of said sample volumes contains said at least one target nucleic acid molecule;

subjecting the sample volumes to a homogeneous assay for detecting amplified product, the homogeneous assay comprising;

a single round of amplification of said at least one target nucleic acid molecule, wherein the single round of amplification yields a detectable amplified product of the at least one target molecule contained in the at least one of said sample volumes, and

detection of amplified product in at least one of said sample volumes; and

determining the number of said sample volumes that contain amplified product, wherein said sample volumes each have a volume of about 10 nl or less.

2. A method as claimed in claim 1, wherein said constituents include at least one probe that hybridizes to amplified target molecules and has a fluorescence property that changes upon hybridization or as a consequence of hybridization, and wherein determining the number of said sample volumes comprises detecting the fluorescence change consequence to hybridization of said at least one probe.

3. The method of claim 1, wherein said sample volumes are contained in sample chambers of the device.

4. The method of claim 1, wherein said device comprises an assembly comprising:

a plurality of sample chambers each confined in at least one dimension by opposing barriers separated by about 500 microns or less;

means for sealing said plurality of sample chambers to prevent evaporation and contamination of sample constituents confined within said plurality of sample chambers; and

means for restraining amplification product.

5. The method of claim 4, wherein said opposing barriers are separated by 100 microns or less.

6. The method of claim 1, wherein said device comprises an assembly, said assembly comprising:

a plurality of sample chambers, each sample chamber being confined in at least one dimension by opposing barriers separated by about 500 microns or less;

means for sealing said plurality of sample chambers to prevent evaporation and contamination of sample constituents confined within said plurality of sample chambers; and

means for restraining amplification product comprising a patterned layer disposed on at least one of said opposing barriers and which at least partially defines said plurality of sample chambers.

7. The method of claim 6, wherein said opposing barriers are separated by 100 microns or less.

8. The method of claim 1, wherein said device comprises an assembly, said assembly comprising:

a substrate and a cover having facing surfaces spaced from one another, the facing surface of at least one of said substrate and said cover comprising a first material;

a flow-through channel between said substrate and said cover; and

a plurality of sample chambers bounded on at least one side thereof by said first material and being in communication with said flow-through channel, said sample chamber having a first affinity to said sample and a second affinity to a displacing fluid, said flow-through channel having a third affinity to the sample and a fourth affinity to said displacing fluid, wherein said first, second, third and fourth affinities are such that the sample chambers retain the sample volumes while the displacing fluid displaces sample from the flow-through channel without substantially displacing said sample volumes from the sample chambers.

9. The method of claim 8, wherein each of said sample chambers is confined in at least one dimension by opposing barriers separated by a dimension of about 100 microns or less.

10. The method of claim 8, wherein said flow-through channel contains a curable displacing fluid which isolates and seals off each of said sample chambers and prevents evaporation of said sample volumes from said sample chambers.

11. The method of claim 1, wherein said sample volumes are contained within respective sample chambers each including a cavity having an open upper end, and said device includes:

means for loading said sample volumes into said sample chambers;

means for displacing sample away from the open upper ends of said sample cavities to isolate said sample volumes within said cavities; and

means for sealing the open upper ends of said cavities immediately after displacing sample fluid away from said open upper ends.

12. The method of claim 11, wherein said means for displacing comprises a curable adhesive.

13. The method of claim 11, wherein said means for displacing and said means for sealing both comprise a common single surface.

14. The method of claim 1, wherein a single round of amplification comprises 30 cycles of polymerase chain reaction.

15. The method of claim 1, wherein at least one of the sample volumes contains at least one target nucleic acid molecule and wherein at least one of the sample volumes contains no nucleic acid molecules.