Optically-based nanopore analysis with reduced background

Abstract

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.

What is claimed is: 
     
         1 . A method of determining characteristics of polymers, the method comprising:
 providing a nanopore array comprising a solid phase membrane and an opaque layer co-extensive therewith, the nanopore array comprising a plurality of apertures and separating a first chamber and a second chamber, 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 polymers from the first chamber to the second chamber through the apertures, each polymer having one or more optical labels attached thereto capable of generating an optical signal having at least a first wavelength indicative of a characteristic of the polymer;   exciting with an excitation beam having a second wavelength the optical labels of the polymers as they translocate through the signal generation regions of the apertures, wherein the optical labels in the detection regions generate optical signals whose first wavelength is different than the second wavelength;   detecting optical signals from the optical labels in the signal generation regions to determine the characteristics of the polymers.   
     
     
         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 a metal selected from the group consisting of Al, Au, Ag and Cu. 
     
     
         4 . The method of  claim 1  wherein said polymers are polynucleotides and wherein said characteristic is a nucleotide sequence thereof. 
     
     
         5 . The method of  claim 4  wherein said optical labels are fluorescent labels and wherein said optical signals are fluorescent signals. 
     
     
         6 . The method of  claim 5  wherein said signal generation region of each of said apertures extends from a surface of said opaque layer closest to said second chamber into said second chamber. 
     
     
         7 . The method of  claim 6  wherein different kinds of nucleotides of said polynucleotide are labeled with different fluorescent labels that generate distinguishable fluorescent signals and wherein each of said apertures constrains nucleotides of a polynucleotide to move single file through said signal generation region. 
     
     
         8 . The method of  claim 7  further including a step of quenching said fluorescent signals from excited fluorescent labels outside of said signal generation region using a non-fluorescent quenching agent. 
     
     
         9 . The method of  claim 8  wherein said quenching agent binds to said polynucleotides. 
     
     
         10 . The method of  claim 9  wherein said quenching agent is disposed in said second chamber. 
     
     
         11 . The method of  claim 5  further including a step of quenching said fluorescent signals from excited fluorescent labels outside of said signal generation region by selecting said fluorescent labels to be mutually self-quenching. 
     
     
         12 . The method of  claim 1  wherein said excitation beam is directed to said nanopore array through said second chamber so that said opaque layer substantially prevents excitation of optical labels in said first chamber. 
     
     
         13 . The method of  claim 1  wherein each of said apertures of said nanopore array comprises a protein nanopore immobilized therein. 
     
     
         14 . The method of  claim 1  wherein said optical labels are acceptor labels and wherein said excitation beam excites a donor label at each of said apertures which excites acceptor labels as they translocate through said signal generation region. 
     
     
         15 . A method of determining sequences of polynucleotides, the method comprising:
 providing a nanopore array comprising a solid phase membrane and an opaque layer co-extensive therewith, the nanopore array comprising a plurality of apertures and separating a first chamber and a second chamber, 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 different kinds of nucleotides of the polynucleotides are labeled with different fluorescent labels that generate distinguishable fluorescent signals and wherein each of said apertures constrains nucleotides of a polynucleotide to move single file through the signal generation region;   exciting with an excitation beam the fluorescent labels of the polynucleotides as they translocate through the signal generation regions of the apertures;   detecting fluorescent signals from the fluorescent labels in the signal generation regions to determine the characteristics of the polymers; and   determining a sequence of nucleotides from the fluorescent signals detected at the signal generation region of each aperture.   
     
     
         16 . The method of  claim 15  wherein said opaque layer is a metal layer. 
     
     
         17 . The method of  claim 16  wherein said metal layer comprises an aluminum layer or a gold layer. 
     
     
         18 . The method of  claim 16  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. 
     
     
         19 . The method of  claim 16  wherein said 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. 
     
     
         20 . The method of  claim 16  further including a step of quenching said fluorescent signals from excited fluorescent labels outside of said signal generation region using a non-fluorescent quenching agent. 
     
     
         21 . The method of  claim 20  wherein said quenching agent binds to said polynucleotides. 
     
     
         22 . The method of  claim 21  wherein said quenching agent is disposed in said second chamber. 
     
     
         23 . The method of  claim 16  further including a step of quenching said fluorescent signals from excited fluorescent labels outside of said signal generation region by selecting said fluorescent labels to be mutually self-quenching. 
     
     
         24 . The method of  claim 16  wherein said steps of exciting and detecting are implemented with an epi-illumination system. 
     
     
         25 . The method of  claim 16  wherein each of said apertures of said nanopore array comprises a protein nanopore immobilized therein. 
     
     
         26 . The method of  claim 25  wherein each of said protein nanopores are immobilized in a lipid bilayer disposed across said apertures. 
     
     
         27 . The method of  claim 16  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.