Methods of detecting amplified product
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.
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.