Abstract: The invention comprises a method for determining a nucleotide sequence of a polynucleotide. In some embodiments, the method comprises providing a solid state substrate comprising a cis side and a trans side. The fluorescently labeled polynucleotide strand comprises (i) a proximal end that is attached to the carrier particle, (ii) a distal end that is cleavable by an exonuclease, and (iii) at least one fluorescently labeled nucleotide comprising a fluorescent label. The trans side of the substrate is illuminated with excitation light to create a fluorescence excitation zone. While the substrate is illuminated, the fluorescently labeled polynucleotide strand is reacted with an exonuclease so that mononucleotides are released serially from the distal end of the strand and diffuse through the fluorescence excitation zone, so that fluorescently labeled mononucleotides in the excitation zone emit fluorescent signals.

Abstract: The invention is directed to a method for determining a monomer sequence of a polymer that is translocated through a nanopore. Monomers of the polymer are labeled with fluorescent labels such that in free solution fluorescent labels of adjacent monomers substantially quench each other and wherein the nanopore constrains fluorescent labels within its bore into a constrained state wherein no detectable fluorescent signal can be generated. By exciting the fluorescent label of each monomer as it exits the nanopore and transitions from a constrained state to a quenched state with an adjacent fluorescent label, a fluorescent signal can be generated by the exiting fluorescent label that allows its monomer to be identified, thereby permitting a monomer sequence to be determined from a sequence of fluorescent signals as the polymer translocates through the nanopore.

Abstract: The invention is directed to optically based methods for nanopore sequencing of polynucleotides which comprise steps of (i) translocating a polynucleotide through a bore of a nanopore at a translocation speed, wherein nucleotides of the polynucleotide are labeled with fluorescent labels such that in free solution fluorescent labels of nucleotides are substantially quenched and wherein fluorescent labels within the bore are constrained such that substantially no detectable fluorescent signal is generated therein; (ii) exciting the fluorescent label of each nucleotide upon exiting the nanopore and prior to quenching with a preceding mutually quenching fluorescent label or a quenching agent; (iii) measuring a fluorescent signal generated by fluorescent labels exiting the nanopore, wherein the translocation speed is selected so that the measured fluorescent signal comprises fluorescence from substantially a single fluorescent label; and (iv) determining a nucleotide sequence of the polynucleotide from a sequence

Abstract: The invention is directed to nanopore-based methods for analyzing polymers, such as polynucleotides or proteins, containing optical labels specific for different kinds of monomers. In some embodiments, methods of the invention include steps of (a) translocating a polymer through a nanopore, wherein different kinds of monomers of the polymer are labeled with different optical labels that generate distinguishable optical signals and wherein the nanopore constrains the monomers to move single file through an excitation zone that encompasses a plurality of monomers; (b) detecting a time-ordered set of optical signals from the monomers as the polymer passes they pass through the excitation zone; (c) separating optical signals from different kinds of monomers to form monomer-specific time-ordered sets of optical signals; and (d) determining a sequence of monomers from the monomer-specific time-ordered sets of optical signals from the polymer.

Abstract: The invention is directed to methods for analyzing polymers comprising linear chains of at least two types of monomers, such as polynucleotides, including DNA, RNA, and the like, using nanopores and optical detection. In some embodiments, as few as two different kinds of nucleotide are labeled with different optical labels that generate distinguishable optical signals for the selected kinds of nucleotide in both sense strands and antisense strands of target polynucleotides. Labeled strands are translocated through nanopores where nucleotides of the strands are constrained to pass sequentially through an optical detection region where their labels generate a sequence of optical signals making up an optical signature. In some embodiments, information from optical signatures from both sense and antisense strands are combined to determine complete nucleotide sequences of target polynucleotides.

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.

Abstract: The invention is directed to methods of making stable lipid bilayers supported by a solid state nanopore array. Exemplary methods include the steps of masking a first layer on a planar support to form dry etch zones; dry etching the dry etch zones to form an array of apertures extending into but not through the first layer; masking a second side of the planar support body to form an etch region aligned with the array of apertures; wet etching the etch region to expose a surface of the first layer; dry etching the exposed surface of the first layer to a depth overlapping the apertures so that apertures of the array provide fluid communication across the first layer; and disposing a lipid bilayer on a surface of the first layer on a side opposite the planar support which encompasses the array of apertures.

Abstract: The invention provides methods for analyzing polynucleotides using nanopores that allow passage of single stranded polynucleotides but not double stranded polynucleotides. In accordance with some embodiments, a double-stranded product is produced that comprises a labeled strand with a single stranded tail or overhang. The double stranded product is exposed to one or more nanopores in the presence of an electric field across the one or more nanopores such that the single stranded tail may be captured and the labeled strand translocated by unzipping from the double stranded product. The ionic composition of the reaction mixture and electric field strength are selected so that nucleotides translocate a nanopore at a rate of less than 1000 nucleotides per second.

Abstract: The invention is directed to optically based methods for nanopore sequencing of polynucleotides which comprise steps of (i) translocating a polynucleotide through a bore of a nanopore at a translocation speed, wherein nucleotides of the polynucleotide are labeled with fluorescent labels such that in free solution fluorescent labels of nucleotides are substantially quenched and wherein fluorescent labels within the bore are constrained such that substantially no detectable fluorescent signal is generated therein; (ii) exciting the fluorescent label of each nucleotide upon exiting the nanopore and prior to quenching with a preceding mutually quenching fluorescent label or a quenching agent; (iii) measuring a fluorescent signal generated by fluorescent labels exiting the nanopore, wherein the translocation speed is selected so that the measured fluorescent signal comprises fluorescence from substantially a single fluorescent label; and (iv) determining a nucleotide sequence of the polynucleotide from a sequence

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.

Abstract: The invention is directed to methods and apparatus for detecting sequences of optical signals from parallel reactions on an array of nanostructures, such as nanopores, nanowells, or nanoparticles. In accordance with the invention, an array of nanostructures is provided, each nanostructure comprising a reaction site and each capable of confining a reaction that generates a sequence of optical signals, and the nanostructures of the array being arranged in clusters each comprising a number of nanostructures. Each different cluster is disposed within a different resolution limited area and the number of nanostructures in each cluster is either greater than one or a random variable with an average value greater than zero. Optical signals from reactions in the nanostructures are detected by an optical system operatively associated with the array.

Abstract: The invention is directed to methods of modifying metal oxide or hydroxylated polymer surfaces using compositions of water soluble polymers that adsorb onto such surfaces and that contain functional groups which directly modify such surfaces without further processing. In some embodiments, compositions used in such methods include water-soluble oxide-adsorbing polymers having water solubility in an indicated temperature range, an indicated concentration in the aqueous solution, and a molecular weight range, wherein each of the water-soluble oxide-adsorbing polymers comprises a linear copolymer comprising a first monomer having at least one hydrophilic moiety and a second monomer having at least one lipophilic moiety and wherein a first monomer: second monomer molecular ratio is at least 3:1.

Abstract: Various methods, systems and devices for optical detection and analysis of polymers, such as polynucleotides, using nanopores, e.g., for determining sequences of nucleic acids, are provided herein. In certain variations, methods and systems for determining a nucleotide sequence of a polynucleotide, which include measuring mixed FRET signals as a polynucleotide translocates through a nanopore and determining a nucleotide sequence of the polynucleotide from the mixed FRET signals, are provided.

Abstract: The invention is directed to a method for determining a monomer sequence of a polymer that is translocated through a nanopore. Monomers of the polymer are labeled with fluorescent labels such that in free solution fluorescent labels of adjacent monomers substantially quench each other and wherein the nanopore constrains fluorescent labels within its bore into a constrained state wherein no detectable fluorescent signal can be generated. By exciting the fluorescent label of each monomer as it exits the nanopore and transitions from a constrained state to a quenched state with an adjacent fluorescent label, a fluorescent signal can be generated by the exiting fluorescent label that allows its monomer to be identified, thereby permitting a monomer sequence to be determined from a sequence of fluorescent signals as the polymer translocates through the nanopore.

Abstract: In some aspects the invention is directed to methods of analyzing a polynucleotide which include steps of directing to a nanopore an excitation beam having a predetermined polarization state; translocating a polynucleotide through the nanopore, wherein nucleotides of the polynucleotide are labeled with fluorescent labels having absorption dipoles and wherein the nanopore spatially orients the fluorescent labels so that during translocation the adsorption dipoles are substantially unresponsive to the excitation beam; detecting changes in fluorescent signals generated by the fluorescent labels as nucleotides with fluorescent labels exit the nanopore and absorption dipoles thereof become responsive to excitation by the excitation beam with the predetermined polarization state; and identifying nucleotides exiting the nanopore from the changes in fluorescent signals.

Abstract: The invention is directed to methods of making stable lipid bilayers supported by a solid state nanopore array. Exemplary methods include the steps of masking a first layer on a planar support to form dry etch zones; dry etching the dry etch zones to form an array of apertures extending into but not through the first layer; masking a second side of the planar support body to form an etch region aligned with the array of apertures; wet etching the etch region to expose a surface of the first layer; dry etching the exposed surface of the first layer to a depth overlapping the apertures so that apertures of the array provide fluid communication across the first layer; and disposing a lipid bilayer on a surface of the first layer on a side opposite the planar support which encompasses the array of apertures.

Abstract: The invention is directed to methods of modifying metal oxide or hydroxylated polymer surfaces using compositions of water soluble polymers that adsorb onto such surfaces and that contain functional groups which directly modify such surfaces without further processing. In some embodiments, compositions used in such methods include water-soluble oxide-adsorbing polymers having water solubility in an indicated temperature range, an indicated concentration in the aqueous solution, and a molecular weight range, wherein each of the water-soluble oxide-adsorbing polymers comprises a linear copolymer comprising a first monomer having at least one hydrophilic moiety and a second monomer having at least one lipophilic moiety and wherein a first monomer: second monomer molecular ratio is at least 3:1.

Abstract: The invention is directed to a method for determining a monomer sequence of a polymer that is translocated through a nanopore. Monomers of the polymer are labeled with fluorescent labels such that in free solution fluorescent labels of adjacent monomers substantially quench each other and wherein the nanopore constrains fluorescent labels within its bore into a constrained state wherein no detectable fluorescent signal can be generated. By exciting the fluorescent label of each monomer as it exits the nanopore and transitions from a constrained state to a quenched state with an adjacent fluorescent label, a fluorescent signal can be generated by the exiting fluorescent label that allows its monomer to be identified, thereby permitting a monomer sequence to be determined from a sequence of fluorescent signals as the polymer translocates through the nanopore.

Abstract: The invention is directed to methods and apparatus for detecting sequences of optical signals from parallel reactions on an array of nanostructures, such as nanopores, nanowells, or nanoparticles. In accordance with the invention, an array of nanostructures is provided, each nanostructure comprising a reaction site and each capable of confining a reaction that generates a sequence of optical signals, and the nanostructures of the array being arranged in clusters each comprising a number of nanostructures. Each different cluster is disposed within a different resolution limited area and the number of nanostructures in each cluster is either greater than one or a random variable with an average value greater than zero. Optical signals from reactions in the nanostructures are detected by an optical system operatively associated with the array.

Abstract: Various methods, systems and devices for optical detection and analysis of polymers, such as polynucleotides, using nanopores, e.g., for determining sequences of nucleic acids, are provided herein. In certain variations, methods and systems for determining a nucleotide sequence of a polynucleotide, which include measuring mixed FRET signals as a polynucleotide translocates through a nanopore and determining a nucleotide sequence of the polynucleotide from the mixed FRET signals, are provided.