Optically-based nanopore analysis with reduced background
The invention is directed to nanopore arrays comprising opaque layers that reduce background fluorescence in optical signal collected in applications of such arrays for analyzing molecules. In some embodiments, such arrays are used to determine characteristics of polymers, such as polynucleotides, in methods comprising the steps of translocating polymers through nanopores of such arrays wherein polymers have one or more optical labels, exciting optical labels of the polymers in a signal generation region of each nanopore extending from the opaque layer toward the direction of the excitation beam, detecting optical signals from the signal generation regions of each nanopore to determine characteristics of the polymer translocating therethrough.
1. A method of determining sequences of polynucleotides, the method comprising:
providing a nanopore array comprising (a) a first side and a second side, (b) a solid phase membrane and an opaque layer co-extensive therewith, and (c) a plurality of apertures, the solid phase membrane separating a first chamber on the first side of the nanopore array and a second chamber on the second side of the nanopore array, wherein each aperture provides fluid communication between the first chamber and the second chamber and has a signal generation region and wherein the opaque layer substantially prevents light from passing through the nanopore array;
translocating polynucleotides from the first chamber to the second chamber through the apertures, wherein
(i) each polynucleotide comprises a polynucleotide strand in which different kinds of nucleotides of the polynucleotide strand are labeled with different fluorescent labels that generate distinguishable fluorescent signals,
(ii) each of said apertures constrains nucleotides of each said polynucleotide strand to move single file through the signal generation region of each aperture; and
(iii) the fluorescent labels of each polynucleotide strand are mutually self-quenching, such that fluorescent signals from excited mutually self-quenching labels are quenched when the labels are outside of the signal generation region;
exciting with an excitation beam the fluorescent labels of the polynucleotide strands as they translocate through the signal generation regions of the apertures, wherein the excitation beam is directed to said nanopore array through said second chamber so that said metal layer substantially prevents excitation of optical labels in said first chamber;
detecting fluorescent signals from the fluorescent labels in the signal generation regions to determine the characteristics of the polynucleotide strands; and
determining a sequence of nucleotides of the polynucleotide strands from the fluorescent signals detected at the signal generation region of each aperture.
2. The method of claim 1 wherein said opaque layer is a metal layer.
3. The method of claim 2 wherein said metal layer comprises an aluminum layer or a gold layer.
4. The method of claim 2 wherein said signal generation region of each of said apertures extends from a surface of said metal layer closest to said second chamber into said second chamber.
5. The method of claim 2 wherein said steps of exciting and detecting are implemented with an epi-illumination system.
6. The method of claim 2 wherein each of said apertures of said nanopore array comprises a protein nanopore immobilized therein.
7. The method of claim 6 wherein each of said protein nanopores are immobilized in a lipid bilayer disposed across said apertures.
8. The method of claim 2 wherein said fluorescent labels are acceptor labels and wherein said excitation beam excites a donor label at each of said apertures which donor labels excite the acceptor labels as they translocate through said signal generation region.