WO2024124033A1 - Dispositifs nanoélectriques et leur utilisation - Google Patents

Dispositifs nanoélectriques et leur utilisation Download PDF

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Publication number
WO2024124033A1
WO2024124033A1 PCT/US2023/082940 US2023082940W WO2024124033A1 WO 2024124033 A1 WO2024124033 A1 WO 2024124033A1 US 2023082940 W US2023082940 W US 2023082940W WO 2024124033 A1 WO2024124033 A1 WO 2024124033A1
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electrode
item
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devices
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PCT/US2023/082940
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Jeremy Lackey
Andres Fernandez
David Dodd
Alexander GORYAYNOV
Ahmed SHARIQ
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Twist Bioscience Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Definitions

  • Biomolecule based information storage systems e.g., DNA-based
  • the single molecule comprises a biomolecule.
  • the biomolecule comprises a nucleic acid.
  • the nucleic acid comprises DNA, RNA, or a mixture thereof.
  • the charge sensor comprises a graphene-enabled field effect transistor (GeFET) or CMOS device.
  • the molecular sensor is in electrical communication with a charge sensor. Further provided herein are methods wherein the molecular sensor comprises a polymerase. Further provided herein are methods wherein the molecular sensor comprises an isothermal polymerase. Further provided herein are methods wherein the molecular sensor comprises Phi29 polymerase or variant thereof. Further provided herein are methods wherein the charge sensor comprises a nanowire. Further provided herein are methods wherein the charge sensor bridges a gap between a first electrode and a second electrode. Further provided herein are methods wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • a surface of the first electrode or the second electrode is functionalized with a thiolbiotin, a terminal cysteine, or a cysteamine.
  • the charge sensor comprises a linker between a surface of the first electrode or the second electrode and the polymerase.
  • the linker comprises one or more components.
  • the one or more components comprises a biotin-streptavidin construct.
  • the one or more components comprises a SpyCatcher or a SpyTag.
  • the one or more components comprises a peptide linker.
  • the one or more components comprises a protein.
  • the protein comprises a Clq/TNF- related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.
  • the at least one charge modulator comprises a negative or positive charge.
  • the at least one charge modulator comprises a charge modulating nucleotide (CMN).
  • contacting comprising incorporating a CMN into a polynucleotide primer.
  • the CMN comprises at least one modification relative to a canonical nucleotide.
  • the modification comprises a modification to the base of the CMN. Further provided herein are methods wherein the modification comprises a 7-deaza or 8-bromo modified base. Further provided herein are methods wherein the CMN comprises a modification to the 5’ position. Further provided herein are methods wherein the modification comprises a modification to a 5’ polyphosphate or chemical variant thereof. Further provided herein are methods wherein the modification comprises a thiolated or bromated phosphate. Further provided herein are methods wherein the polyphosphate comprises at least 3, 4, 5, 6, 8, or 10 phosphates or variants thereof. Further provided herein are methods wherein the modification comprises a modification to a terminal 5 ’ polyphosphate or chemical variant thereof.
  • the modification comprises a polymer.
  • the polymer comprises one or more of a nucleic acid chain, a peptide chain, a polysaccharide, a lipid, a synthetic polymer, and a dendrimer.
  • the nucleic acid chain comprises 25-5000 bases.
  • the nucleic acid chain is branched (dendrimeric).
  • the nucleic acid chain comprises a secondary structure.
  • the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadraplex, and an I-motif.
  • nucleic acid chain comprises a single strand, double strand, or triple strand. Further provided herein are methods wherein the nucleic acid chain comprises at least one charge modulating chemical modification. Further provided herein are methods wherein the at least one charge modulating chemical modification increases the charge of the CMN relative to an unmodified nucleotide. Further provided herein are methods wherein the at least one charge modulating chemical modification comprises one or more of an amine, an alkylamine, a guanidinium, a quaternary amine, an imidazolium, a pyridinium, and a pyrrolidinium.
  • the at least one charge modulating chemical modification decreases the charge of the CMN relative to an unmodified nucleotide.
  • the at least one charge modulating chemical modification comprises one or more of a phosphate, a phosphite, a sulfonate, a sulfite, a carboxylate, a xanthate, a thiocarboxylic acid, a boranophosphonate, and a boric acid.
  • the nucleic acid chain comprises at least one sugar-modified nucleotide.
  • the sugar-modified nucleotide comprises a deoxy or dideoxy nucleotide.
  • the nucleic acid chain comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid.
  • the nucleic acid chain comprises a phosphate modification.
  • the phosphate modification comprises a hydrophobic group.
  • the hydrophobic group comprises a straight or branched alkyl chain.
  • the phosphate modification comprises a hydrophilic group.
  • the hydrophilic group comprises polyethylene glycol.
  • the polyethylene glycol comprises a molecular weight of 1000-100,000 daltons. Further provided herein are methods wherein the peptide chain is 1-100 amino acids in length. Further provided herein are methods wherein the CMN comprises a charged small molecule. Further provided herein are methods wherein the charged small molecule comprises one or more of a chelator, a dye, and a metal complex. Further provided herein are methods wherein the metal complex comprises a ferrocene, Ru-dipy, and bis- cyclopentadienyl diiron. Further provided herein are methods wherein the change in current is 1 nanoamp to 100 picoamps. Further provided herein are methods wherein the change in current is 100 picoamps to 1 microamp.
  • chemically-sensitive field effect transistor devices for sensing single molecules comprising: a solid support, wherein the solid support comprises a plurality of loci, wherein each loci comprises: a graphene layer; a gate electrode and a drain electrode, where the gate electrode and the drain electrode are in electrical communication via the graphene layer; and at least one insulating layer, where the insulating layer is located between the gate electrode and the drain electrode; wherein the loci have a pitch of 50-1000 nanometers.
  • the device further comprises at least one ground shield.
  • the at least one ground shield is at ground potential.
  • the device further comprises at least one buried gate.
  • the at least one ground shield comprises an opening which permits electrical communication between the graphene layer and the least one buried gate. Further provided herein are devices wherein the graphene layer is approximately one atom thick. Further provided herein are devices wherein the device comprises 100 to 1 billion loci. Further provided herein are devices wherein each loci is 50-200 nm in size. Further provided herein are devices wherein each loci is a well, channel, or is substantially planer. Further provided herein are devices wherein the device is 4 to 2000 mm 2 . Further provided herein are devices wherein the device is 4 to 16 mm 2 . Further provided herein are devices wherein the device is 200 to 900 mm 2 . Further provided herein are devices wherein the device is 4 to 900 mm 2 .
  • the graphene binder comprises an aromatic group.
  • the graphene binder comprises an aryl or heteroaryl group. Further provided herein are methods wherein the graphene binder comprises a C6-C30 aryl or heteroaryl group. Further provided herein are methods wherein the graphene binder comprises an aromatic hydrocarbon.
  • the graphene binder comprises naphthalene, biphenyl, fluorene, anthracene, phenanthrene, tetracene, chrysene, triphenylene, pyrene, pentacene, perylene, benzo [a]pyrene, corannulene, benzo[ghi]perylene, coronene, ovalene, or benzo [c] fluorene.
  • step c) comprises washing the surface.
  • the ternary complex is attached to the graphene binder via the primer, the polymerase, or the polynucleotide library.
  • ternary complex is attached via a linker.
  • methods wherein the ternary complex is attached via a linker using a conjugation.
  • the conjugation comprises nucleophile/carbonyl; an azide/phosphine; 1,4 Michael addition, 1,3-dipolar cycloaddition, inverse electron demand cycloaddition; olefin metathesis; or cross-coupling reaction.
  • removing comprises contacting the surface with a solvent.
  • solvent comprises an organic solvent.
  • the organic solvent comprises MeCN, methanol, ethanol, 2-propanol, acetone, DMF, formamide, THF, or DMSO. Further provided herein are methods wherein the organic solvent is heated. Further provided herein are methods wherein the polymerase comprises a Phi29 polymerase or variant thereof. Further provided herein are methods wherein the polymerase is configured for incorporation charge modified nucleotides described herein. Further provided herein are methods wherein the polymerase is bound to the surface in step a). Further provided herein are methods wherein the polymerase is not bound to the surface in step a). Further provided herein are methods wherein the plurality of polynucleotides comprises at least 100,000 unique polynucleotides.
  • detecting comprises contacting the ternary complexes with at least one nucleotide. Further provided herein are methods wherein detecting comprises measuring a change in current when a CMN is incorporated. Further provided herein are methods wherein the buried gate has a positive potential during step (a). Further provided herein are methods wherein the buried gate has a positive or negative potential during step (b).
  • devices for molecular sensing comprising: a first electrode, wherein the first electrode comprises a neck region; a passive layer; a second electrode, and wherein the first electrode and the second electrode are located above the first base layer; wherein a first portion of the neck region overlaps a first portion of the second electrode, such that the first electrode and the second electrode are separated by a nanogap; wherein a second portion of the first electrode and a second portion of the second electrode are separated by the passive layer; and a first base layer and a second base layer, wherein the first base layer is located above the second base layer, and the first electrode and the second electrode are located above the base layers.
  • the nanogap is 1 - 50 nm. Further provided herein are devices wherein the nanogap is 10-30 nm. Further provided herein are devices wherein the nanogap is no more than 50 nm. Further provided herein are devices wherein the passive layer comprises an oxide. Further provided herein are devices wherein the oxide comprises silicon, nitride, or carbide. Further provided herein are devices wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium. Further provided herein are devices wherein the first electrode and the second electrode each comprise a superficial layer of gold. Further provided herein are devices wherein the layer of gold is no more than 75 angstroms thick.
  • the superficial layer of gold is deposited above an adhesion layer.
  • the adhesion layer comprises titanium or chromium.
  • the first electrode and the second electrode each comprise gold nano-islands.
  • the first base layer comprises silicon oxide or silicon nitride.
  • the second base layer comprises silicon.
  • the device further comprises a charge sensor, wherein the charge sensor is in electrical communication with the first electrode and the second electrode.
  • the charge sensor is attached to the first electrode and the second electrode.
  • the charge sensor comprises a polymer.
  • the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.
  • the charge sensor comprises carbon.
  • the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine.
  • the charge sensor is further attached to a molecular sensor via a tether.
  • the tether comprises one or more components.
  • the one or more components comprises a biotin-streptavidin construct. Further provided herein are devices wherein the one or more components comprises a Spy Catcher or a SpyTag. Further provided herein are devices wherein the one or more components comprises a peptide linker. Further provided herein are devices wherein the one or more components comprises a protein. Further provided herein are devices wherein the protein comprises a Clq/TNF- related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein are devices wherein the molecular sensor comprises an enzyme. Further provided herein are devices wherein the molecular sensor comprises an antibody.
  • devices wherein the enzyme comprises a polymerase. Further provided herein are devices wherein the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein are devices wherein the longest linear dimension of the second electrode is parallel to the neck region.
  • the first electrode is undercut relative to the passive layer. Further provided herein are devices wherein the surface area of the first electrode is less than the surface area of the second electrode. Further provided herein are devices wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region. Further provided herein are devices wherein the first electrode and the second electrode each comprise a superficial layer of gold. Further provided herein are devices wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein are devices wherein the second base layer comprises silicon. Further provided herein are devices wherein the neck region has a width of no more than 200 nm.
  • devices for molecular sensing comprising: a first electrode, wherein the first electrode is located above the first base layer; a second electrode, wherein the second electrode comprises a neck region; wherein a first portion of the neck region overlaps a first portion of the first electrode, such that the first electrode and the second electrode are separated by a nanogap; wherein a second portion of the first electrode and a second portion of the second electrode are separated by a passive layer; a first base layer and a second base layer, wherein the first base layer is located above the second base layer, and the first electrode and the second electrode are located above the base layers.
  • the passive layer is configured to overlap with a portion of the second electrode.
  • the passive layer is configured to passivate electrode traces. Further provided herein are devices wherein the nanogap is 1-50 nm. Further provided herein are devices wherein the nanogap is 10-30 nm. Further provided herein are devices wherein the nanogap is no more than 50 nm. Further provided herein are devices wherein the passive layer comprises an oxide. Further provided herein are devices wherein the oxide comprises silicon, nitride, or carbide. Further provided herein are devices wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium. Further provided herein are devices wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • the layer of gold is no more than 75 angstroms thick. Further provided herein are devices wherein the superficial layer of gold is deposited above an adhesion layer. Further provided herein are devices wherein the adhesion layer comprises titanium or chromium. Further provided herein are devices wherein the first electrode and the second electrode each comprise gold nano-islands. Further provided herein are devices wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein are devices wherein the second base layer comprises silicon. Further provided herein are devices wherein the device further comprises a charge sensor, wherein the charge sensor is in electrical communication with the first electrode and the second electrode. Further provided herein are devices wherein the charge sensor is attached to the first electrode and the second electrode.
  • the charge sensor comprises a polymer. Further provided herein are devices wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid. Further provided herein are devices wherein the charge sensor comprises carbon. Further provided herein are devices wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction. Further provided herein are devices wherein a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine. Further provided herein are devices wherein the charge sensor is further attached to a molecular sensor via a tether. Further provided herein are devices wherein the tether comprises one or more components.
  • the one or more components comprises a biotin-streptavidin construct. Further provided herein are devices wherein the one or more components comprises a Spy Catcher or a SpyTag. Further provided herein are devices wherein the one or more components comprises a peptide linker. Further provided herein are devices wherein the one or more components comprises a protein. Further provided herein are devices wherein the protein comprises a Clq/TNF- related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein are devices wherein the molecular sensor comprises an enzyme. Further provided herein are devices wherein the molecular sensor comprises an antibody.
  • the enzyme comprises a polymerase. Further provided herein are devices wherein the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein are devices wherein the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein are devices wherein at least one edge of the first electrode is undercut relative to the passive layer. Further provided herein are devices wherein the surface area of the first electrode is less than the surface area of the second electrode. Further provided herein are devices wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region. Further provided herein are devices wherein the first electrode and the second electrode each comprise a superficial layer of gold. Further provided herein are devices wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein are devices wherein the second base layer comprises silicon. Further provided herein are devices wherein the neck region has a width of no more than 200 nm.
  • devices for molecular sensing comprising: a first electrode, wherein the first electrode comprises a neck region; a passive layer, wherein the passive layer comprises a channel or well, where the bottom of the well or channel comprises the first base layer; a second electrode, and wherein the first electrode and the second electrode are located above the first base layer, and the second electrode is at least partially embedded in the passive layer; wherein a first portion of the neck region overlaps a first portion of the second electrode, such that the first electrode and the second electrode are separated by a nanogap; wherein a second portion of the first electrode and a second portion of the second electrode are separated by the passive layer; and a first base layer and a second base layer, wherein the first base layer is located above the second base layer, and the first electrode and the second electrode are located above the base layers.
  • the nanogap is 1 - 50 nm. Further provided herein are devices wherein the nanogap is 10-30 nm. Further provided herein are devices wherein the nanogap is no more than 50 nm. Further provided herein are devices wherein the passive layer comprises an oxide. Further provided herein are devices wherein the oxide comprises silicon, nitride, or carbide. Further provided herein are devices wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium. Further provided herein are devices wherein the first electrode and the second electrode each comprise a superficial layer of gold. Further provided herein are devices wherein the layer of gold is no more than 75 angstroms thick.
  • the superficial layer of gold is deposited above an adhesion layer.
  • the adhesion layer comprises titanium or chromium.
  • the first electrode and the second electrode each comprise gold nano-islands.
  • the first base layer comprises silicon oxide or silicon nitride.
  • the second base layer comprises silicon.
  • the device further comprises a charge sensor, wherein the charge sensor is in electrical communication with the first electrode and the second electrode.
  • the charge sensor is attached to the first electrode and the second electrode.
  • the charge sensor comprises a polymer.
  • the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.
  • the charge sensor comprises carbon.
  • the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine.
  • the charge sensor is further attached to a molecular sensor via a tether.
  • the tether comprises one or more components.
  • the one or more components comprises a biotin-streptavidin construct. Further provided herein are devices wherein the one or more components comprises a Spy Catcher or a SpyTag. Further provided herein are devices wherein the one or more components comprises a peptide linker. Further provided herein are devices wherein the one or more components comprises a protein. Further provided herein are devices wherein the protein comprises a Clq/TNF- related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein are devices wherein the molecular sensor comprises an enzyme. Further provided herein are devices wherein the molecular sensor comprises an antibody.
  • the enzyme comprises a polymerase. Further provided herein are devices wherein the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein are devices wherein the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein are devices wherein at least one edge of the first electrode is undercut relative to the passive layer. Further provided herein are devices wherein the surface area of the first electrode is less than the surface area of the second electrode. Further provided herein are devices wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region. Further provided herein are devices wherein the first electrode and the second electrode each comprise a superficial layer of gold. Further provided herein are devices wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein are devices wherein the second base layer comprises silicon. Further provided herein are devices wherein the neck region has a width of no more than 200 nm.
  • arrays wherein the array comprises at least 5000 devices of any one of the devices described herein. Further provided herein are arrays wherein the array comprises at least 100,000 devices of any one of the devices described herein. Further provided herein are arrays wherein the pitch distance of the nanogaps of at least some of the devices is no more than 2 micron. Further provided herein are arrays wherein the array further comprises a plurality of vias, wherein the plurality of vias are configured to connect at least two vertical layers of the device. Further provided herein are arrays wherein the array further comprises a plurality of routing connections, wherein the plurality of routing connections are configured for addressable control of each device in the array.
  • any one of the devices described herein comprises: a) providing one or more base layers; b) depositing material to generate a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to generate a passive layer; f) depositing material to generate a first electrode; g) patterning the first electrode; and h) isotropically etching the passive layer, such that the edges of the first electrode are undercut. Further provided herein are methods wherein the method further comprises depositing gold on the first top layer of the device. Further provided herein are methods wherein the one or more base layers comprise thermal oxide on silicon.
  • the method comprises etching or lithography. Further provided herein are methods wherein the method comprises RIE (reactive ion etching). Further provided herein are methods wherein patterning comprises lithography and/or RIE. Further provided herein are methods wherein the method does not comprise e-beam or DUV (deep ultraviolet light) lithography. Further provided herein are methods wherein the method comprises deposition of gold on the first electrode and the second electrode. Further provided herein are methods wherein the first electrode and the second electrode are separated by a nanogap. Further provided herein are methods wherein the nanogap is 1-50 nm. Further provided herein are methods wherein the nanogap is 10-30 nm.
  • the nanogap is no more than 50 nm.
  • the passive layer comprises an oxide.
  • the oxide comprises silicon, nitride, or carbide.
  • the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.
  • the method further comprises analyzing the electrical signal to establish the identity of the at least one nucleotide triphosphate.
  • the at least one nucleotide triphosphate comprises a non-canonical base.
  • the at least one nucleotide triphosphate comprises a terminator which is configured to prevent chain extension.
  • methods wherein the method is repeated to establish the identity of at least 20 bases. Further provided herein are methods wherein the method is repeated to establish the identity of at least 100 bases. Further provided herein are methods wherein the method is repeated to establish the identity of at least 1000 bases.
  • FIG. 1 illustrates a non-limiting example of a scheme for polynucleotides synthesis and sequencing according to some embodiments.
  • FIG. 2 illustrates a non-limiting example of a graphene device described herein according to some embodiments.
  • FIG. 3 depicts an example of a charge-modulating nucleotide for use with the devices and methods described herein according to some embodiments.
  • FIG. 4A depicts a top view of a graphene device for polynucleotide sequencing having a buried gate and shield electrode according to some embodiments.
  • FIG. 4B depicts a side view of a graphene device for polynucleotide sequencing having a buried gate and shield electrodes according to some embodiments.
  • FIGs. 5A-5C depict a zoom in of a flexible structure, having spots, channels, or wells, respectively, according to some embodiments.
  • FIG. 6A is a schema of solid support comprising an active area and fluidics interface according to some embodiments.
  • FIG. 6B is a front side of an example of a solid support array according to some embodiments.
  • Such arrays in some instances may comprise thousands or millions of polynucleotide synthesis devices as described herein;
  • FIG. 6C is a back side of an example of a solid support array according to some embodiments.
  • FIG. 6D is an example of rack-style instrument according to some embodiments. Such instruments may comprise hundreds or thousands of solid support arrays.
  • FIG. 7 illustrates an example of a computer system according to some embodiments.
  • FIG. 8 is a block diagram illustrating architecture of a computer system according to some embodiments.
  • FIG. 9 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS) according to some embodiments.
  • NAS Network Attached Storage
  • FIG. 10 is a block diagram of a multiprocessor computer system using a shared virtual address memory space according to some embodiments.
  • FIG. 11 depicts a nanoelectric device according to some embodiments.
  • An analyte (nucleic acid shown as an example only) is in communication with a molecular sensor connected to the charge sensor which spans a loci.
  • FIG. 12A depicts a top view of an edge finger device according to some embodiments.
  • FIG. 12B depicts a side view of an edge finger device according to some embodiments.
  • FIG. 12C depicts a side view of an edge finger device according to some embodiments.
  • the dotted line indicates a coating, and a nanowire is depicted as bridging two electrodes.
  • FIG. 13A depicts a top view of a crossed fingers device according to some embodiments.
  • FIG. 13B depicts a side view of a crossed fingers device according to some embodiments.
  • FIG. 14A depicts a top view of a crossed fingers with mask device according to some embodiments.
  • FIG. 14B depicts a side view of a crossed fingers with mask device according to some embodiments.
  • FIG. 15 depicts an array of multiple crossed fingers device according to some embodiments.
  • FIG. 16A depicts a top view of a self-aligned finger etch device according to some embodiments.
  • FIG. 16B depicts a side view of a self-aligned finger etch device according to some embodiments.
  • FIG. 17A depicts a sideview of a nanoelectric device comprising metal crisscrosses without an undercut, according to some embodiments.
  • FIG. 17B depicts a sideview of a nanoelectric device comprising metal crisscrosses with an undercut, according to some embodiments.
  • FIG. 18A depicts a sideview of a nanoelectric device with gold deposition without an adhesion layer, resulting in growth of gold islands, according to some embodiments.
  • FIG. 18B depicts a picture of the sideview of a nanoelectric device with gold deposition without an adhesion layer, according to some embodiments.
  • FIG. 18C depicts a sideview of a nanoelectric device with gold deposition with an adhesion layer, resulting in a continuous gold film, according to some embodiments.
  • FIG. 19A depicts a sideview of a nanoelectric device with gold islands, with a biomolecule conjugated between two gold islands, according to some embodiments.
  • FIG. 19B depicts a sideview of a nanoelectric device with a biomolecule conjugated directly onto electrodes, according to some embodiments.
  • FIG. 20 depicts a sideview of a nanoelectric device employing a bi-layer passivation, such as oxide and carbide/nitride, according to some embodiments.
  • FIG. 21 depicts an exemplary molecular wire construct, according to some embodiments.
  • FIG. 22A depicts an exemplary schematic for a functionalization of a surface, according to some embodiments.
  • FIG. 22B depicts an exemplary functionalization scheme on a gold surface, according to some embodiments.
  • FIG. 23A depicts an exemplary schematic for further functionalization of a surface, according to some embodiments.
  • FIG. 23B depicts an exemplary schematic for an enzyme linker structure, according to some embodiments.
  • FIG. 23C depicts an exemplary schematic of a map for a dual-biotin tagged P29 Variant RPN, according to some embodiments.
  • FIG. 24 depicts an exemplary schematic for assembling a molecular nanowire construct, according to some embodiments.
  • FIG. 25 depicts an exemplary schematic for characterization of the tagged surfaces using a gold nanoparticle, according to some embodiments.
  • FIG. 26 depicts an exemplary schematic illustrating a molecular wire construct comprising gold nanoparticles, according to some embodiments.
  • FIG. 27 depicts an exemplary schematic illustrating a molecular wire construct comprising biotin and monomeric streptavidin, according to some embodiments.
  • FIG. 28 depicts an exemplary schematic illustrating a molecular wire construct comprising cysteamine, according to some embodiments.
  • FIG. 29 depicts an exemplary schematic illustrating a molecular wire construct comprising biotin and streptavidin, according to some embodiments.
  • FIG. 30 depicts an exemplary schematic illustrating a molecular wire construct comprising SpyCatcher and SpyTag, where the SpyTag is conjugated to a polymerase, according to some embodiments.
  • FIG. 31 depicts an exemplary schematic illustrating a molecular wire construct comprising SpyCatcher and SpyTag, where the SpyCatcher is conjugated to a polymerase, according to some embodiments.
  • FIG. 32 depicts an exemplary schematic illustrating a molecular wire construct comprising SpyCatcher and SpyTag, as well as a CTRP peptide, according to some embodiments.
  • FIG. 33 depicts an exemplary schematic illustrating a molecular wire construct comprising linkers comprising peptides, according to come embodiments.
  • a biomolecule such as a DNA molecule provides a suitable host for information storage in-part due to its stability over time and capacity for four bit (or other) information coding, as opposed to traditional binary information coding.
  • Provided herein are methods devices, and systems for real-time sensing of single molecules (e.g., biomolecules).
  • preselected sequence As used herein, the terms “preselected sequence”, “predefined sequence” or “predetermined sequence” are used interchangeably. The terms mean that the sequence of the polymer is known and chosen before synthesis or assembly of the polymer. In particular, various aspects are described herein primarily with regard to the preparation of nucleic acids molecules, the sequence of the polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules.
  • symbol generally refers to a representation of a unit of digital information. Digital information may be divided or translated into one or more symbols. In an example, a symbol may be a bit and the bit may have a numerical value. In some examples, a symbol may have a value of ‘0’ or ‘ 1’. In some examples, digital information may be represented as a sequence of symbols or a string of symbols. In some examples, the sequence of symbols or the string of symbols may comprise binary data.
  • nucleic acids may be, unless stated otherwise, comprise DNA or RNA or an analog or derivative thereof.
  • nucleic acids polynucleotides, oligonucleotides, oligos, oligonucleic acids are used synonymously throughout to represent a polymer of nucleoside monomers.
  • nucleic acids are connected via phosphate or sulfur-containing linkages.
  • Nucleic acids in some instances comprise DNA, RNA, non-canonical nucleic acids, unnatural nucleic acids, or other nucleoside.
  • nucleotides comprise non-canonical bases, sugars, or other moiety.
  • nucleotides comprise terminators which are configured to prevent extension reactions. In some instances, such terminators are removed before addition of subsequent nucleotides to the growing chain.
  • Sensing in some instances comprises detecting the presence or absence of, concentration, and/or identity of a biomolecule or portion of a biomolecule thereof.
  • a biomolecule comprises a polymer.
  • devices, methods, compositions, and systems are used for sequencing (e.g., nucleic acids).
  • solid supports comprise surfaces.
  • pluralities of devices which are combined to form larger arrays or chips.
  • electrical voltages or currents are measured in order to sense molecular changes associated with a charge sensor, molecular sensor, or complex thereof.
  • Arrays of such devices in some instances provide for high-throughput reading of digital information encoded in nucleic acids.
  • devices are arrayed on solid supports with each device or groups of devices at an addressable loci. Such larger groups of devices in some instances are present in serve rack units.
  • Electrodes may be configured to detect changes in voltage, current or resistance to sense biomolecules.
  • a device for molecular sensing comprises one or more electrodes.
  • a device for molecular sensing comprises one, two, three, four, five or more than five electrodes.
  • a device for molecular sensing comprises a source electrode and a drain electrode.
  • an electrode of the one or more electrodes is configured as a source.
  • an electrode of the one or more electrodes is configured as a drain.
  • a device for molecular sensing comprises a passive layer.
  • the passive layer comprise an oxide layer.
  • the passive layer comprise a nitride layer. In some examples, the passive layer reduces reactivity of the surface to which it is applied.
  • a device for molecular sensing comprises a charge sensor binding layer. In some examples, the charge sensor binding layer is a metal, such as gold.
  • the one or more electrodes are in electrical communication. In some instances, the electrical communication between the one or more electrodes forms a gap. In some examples, the gap is a nanogap. In some examples, the gap (e.g., nanogap) is spanned by a charge sensor. In some examples, the charge sensor comprises graphene.
  • the charge sensor comprises a graphene-enabled field effect transistor (GeFET) or CMOS device.
  • a charge sensor is attached to a tether, such as those provided herein.
  • the tether is attached to a charge sensor, a molecular sensor, or both.
  • the molecular sensor and the charge sensor is in electrical communication.
  • the molecular sensor comprises a polymerase.
  • the polymerase is an isothermal polymerase, a Phi29 polymerase, or a variant thereof.
  • the one or more electrodes are located above one or more base layers.
  • the one or more base layers is a passive layer or gate layer.
  • a device in some instances comprises a first electrode 1105a, and a second electrode 1105b. In some instances, a portion of the first electrode 1105a and a portion of the second electrode 1105b are partially covered with a passive layer (1106a and 1106b, respectively). In some instances a portion of the first electrode 1105a and a portion of the second electrode 1105b are partially covered with a charge sensor binding layer (e.g., gold, 1107a and 1107b, respectively).
  • a charge sensor binding layer e.g., gold, 1107a and 1107b, respectively.
  • the distance between the first charge sensor binding layer 1107a in electrical communication with the first electrode 1105a and the second charge sensor binding layer 1107b in electrical communication with the second electrode 1105b forms a nanogap 1112. In some instances, the nanogap is spanned by a charge sensor 1108.
  • the first electrode 1105a is configured as a source. In some instances, the first electrode 1105b is configured as a drain.
  • the charge sensor 1108 is attached to a tether 1109. In some instances the tether 1109 is attached to the charge sensor 1108 and a molecular sensor 1110. In some instances, the molecular sensor 1110 is configured to detection an analyte 1111.
  • first electrode 1105a and the second electrode 1105b are located above one or more base layers.
  • one or more base layers comprise passive layers or gate layers.
  • first electrode 1105a and/or the second electrode 1105b are separated by at least one passive base layer (1104 or 1103).
  • one or more base layers e.g., 1103 and 1101 are separated by a gate layer 1102.
  • a gate layer 1102 is located above a base layer 1101. Such devices are in some instances combined into larger arrays. In some instances, devices are integrated into electronics such as a CMOS and connected to a computer. [0076] Provided herein is a first device 1200 for molecular sensing (FIGs. 12A-12B).
  • the device comprises a first electrode 1202a. In some instances, the first electrode comprises a neck region 1202b. In some instances, the device comprises a second electrode 1203. In some instances, the first electrode 1202a and the second electrode 1202b are located above one or more base layers. In some instances, the first electrode 1202a and the second electrode 1202b are located above a first base layer 1201. In some instances, a first portion of the neck region 1202b overlaps a first portion of the second electrode 1203, such that the first electrode 1202a and the second electrode 1203 are separated by a gap 1212 (e.g., nanogap). In some instances, the nanogap is the smallest defined dimension of a device.
  • a gap 1212 e.g., nanogap
  • a second portion of the first electrode 1202a and a second portion of the second electrode 1203 are separated by the passive layer 1205.
  • the first base layer 1201 is located above the second base layer 1204, and the first electrode 1202a and the second electrode 1203 are located above the base layers 1201 and 1204.
  • a device described herein comprises a coating 1206 (FIG. 12C).
  • a coating 1206 is configured to bind a charge sensor 1207.
  • a charge sensor 1207 comprises a nanowire 1207.
  • a nanowire 1207 comprises a nucleic acid.
  • the device comprises a first electrode 1302.
  • the first electrode is located above the first base layer 1301.
  • the device comprises a second electrode 1303a.
  • the second electrode 1303a comprises a neck region 1303b.
  • a first portion of the neck region 1303b overlaps a first portion of the first electrode 1302, such that the first electrode 1302 and the second electrode 1303a are separated by a nanogap 1312.
  • a second portion of the first electrode 1302 and a second portion of the second electrode 1303a are separated by a passive layer 1305.
  • a device comprises a first base layer 1301 and a second base layer 1304.
  • the first base layer 1301 is located above the second base layer 1302, and the first electrode 1302 and the second electrode 1303a are located above the first base layer 1301.
  • the device further comprises a second passive layer 1406.
  • the second passive layer 1406 is further configured to overlap with a portion of the second electrode 1403 (FIGs. 14A-14B).
  • the second passive layer 1406 passivates the electrode traces.
  • the device comprises a first electrode 1402.
  • the first electrode 1402 is located above the first base layer 1401.
  • the device comprises a second electrode 1403a, wherein the second electrode 1403a comprises a neck region 1403b.
  • a first portion of the neck region 1403b overlaps a first portion of the first electrode 1402, such that the first electrode 1402 and the second electrode 1403a are separated by a nanogap 1412.
  • a second portion of the first electrode 1402 and a second portion of the second electrode 1403a are separated by a passive layer 1405.
  • a device comprises a first base layer 1401 and a second base layer 1404.
  • the first base layer 1401 is located above the second base layer 1402, and the first electrode 1402 and the second electrode 1403a are located above the first base layer 1401.
  • the device further comprises a second passive layer 1406.
  • the first electrode, the second electrode, or both on the devices described herein may be deposited with gold.
  • gold is deposited without an undercut as shown in the schematic of FIG. 17A.
  • a shorting path between top and bottom electrodes may be formed over the oxide as illustrated in the FIG. 17A.
  • a precise, ultra-thin gold film may be deposited in order to avoid shorting.
  • gold is deposited with an undercut between the first electrode (top electrode) and the second electrode (bottom electrode).
  • An exemplary schematic is provided in FIG. 17B.
  • wet chemistry is used to obtain the oxide undercut.
  • metal electrodes with an oxide undercut can help prevent a short between the top and bottom electrodes post gold deposition. This can allow tuning to requisite gold thickness.
  • an adhesion layer is deposited onto the first electrode (top electrode), the second electrode (bottom electrode), or both. In some instances, an adhesion layer is not deposited onto the first electrode (top electrode), the second electrode (bottom electrode), or both. In some instances, the adhesion layer comprises a titanium (Ti), chromium (Cr), or both.
  • gold is deposited directly onto the electrodes (e.g., no adhesion layer), as shown, for example in FIGs. 18A-18B. As illustrated, gold nano-islands are formed in the absence of the adhesion layer. In some examples, gold deposition with an adhesion layer leads to a continuous gold film, as shown, for example, in FIG. 18C.
  • Biomolecules may be conjugated on the electrodes.
  • a biomolecule is conjugated to the gold deposited on the surface of the electrodes.
  • biomolecules are conjugated to one or more surfaces deposited with gold on the electrodes (e.g., extending between a nanogap).
  • a biomolecule is conjugated to a surface on the first electrode (top electrode) and the second electrode (bottom electrode), both deposited with gold.
  • a biomolecule is conjugated to one or more gold islands (e.g., extending between the nanogap).
  • An exemplary schematic is provided in FIG. 19A.
  • a biomolecule is conjugated to a gold island on the first electrode (top electrode) and a gold island on the second electrode (bottom electrode).
  • a biomolecule is conjugated directly to the one or more electrodes, as illustrated, for example in FIG. 19B.
  • the gold nano islands, as illustrated in FIG. 19A provide lower contact resistance compared to direct binding of biomolecule on the continuous metal electrode, as illustrated in FIG. 19B, resulting in a higher current.
  • the electrode surface e.g., TiN
  • the oxide layer inhibits the direct conjugation of biomolecules on the electrodes that are not made of noble metals.
  • the device comprises a passivation around the metal crisscrosses of the device.
  • An exemplary diagram is provided in FIG. 20.
  • a passivation layer can be utilized to safeguard the field and metal routings.
  • an adhesion layer to form a continuous gold film (e.g., FIG. 18C)
  • a bilayer passivation such as for example, an oxide and carbide/nitride, can be employed.
  • the device comprises a first electrode 1602a.
  • the first electrode 1602a comprises a neck region 1602b.
  • the device comprises a passive layer such as 1605 and 1606.
  • the passive layer 1605/1606 comprises a channel or well.
  • the bottom of the well or channel (or trench) 1607 comprises (or is exposed to) a first base layer 1601.
  • the device comprises a second electrode 1603.
  • the first electrode 1602a and the second electrode 1603 are located above the first base layer 1601, and the second electrode 1603 is at least partially embedded in the passive layer 1605.
  • a first portion of the neck region 1602b overlaps a first portion of the second electrode 1603, such that the first electrode 1602a and the second electrode 1603 are separated by a nanogap 1612.
  • a second portion of the first electrode 1602a and a second portion of the second electrode 1603 are separated by the passive layer 1605.
  • the device comprises a first base layer 1601 and a second base layer 1604.
  • the first base layer 1601 is located above the second base layer 1604, and the first electrode 1602a and the second electrode 1603 are located above the base layers 1601/1604.
  • a device stack is etched using the same hard mask so that electrode edges are aligned and a deeper trench (channel or well) is etched below (FIGs. 16A-16B).
  • devices comprise a graphene layer which is configured to detect changes in local currents.
  • such layers are coupled to polymerases which provide unique signals (sensing) corresponding to nucleotide incorporation events.
  • devices provided herein are used for sequencing nucleic acids.
  • devices comprise one or more of a charge sensor and a molecular sensor.
  • molecular sensors interact with a biomolecule and convey information about the biomolecule to the charge sensor via changes in current.
  • a charge sensor comprises a graphene layer.
  • information about the biomolecule comprises information about a monomer of the biomolecule.
  • the information about the biomolecule comprises information of the nucleotide.
  • information about the nucleotide comprises information about the bases (e.g., A, T, C, and G).
  • the information about the peptide comprise information about the amino acids.
  • the charge sensor comprises a graphene layer.
  • the one or more electrodes comprises a gate electrode.
  • the one or more electrodes comprises a drain electrode.
  • the gate electrode and the drain electrode are in electrical communication via the charge sensor.
  • the gate electrode and the drain electrode are in electrical communication via a graphene layer.
  • an insulating layer is located between the gate electrode and the drain electrode.
  • the solid support comprises a plurality of loci.
  • the device further comprises at least one ground shield.
  • the device further comprises at least one buried gate (electrode).
  • the device comprises a solid support 204, a graphene layer 201, a gate electrode 203a, a drain electrode 203b, and at least one insulating layer 202a/202b.
  • the insulating layer is located between the gate electrode 203a and the drain electrode 203b.
  • the gate electrode 203a and the drain electrode 203b are in electrical communication via the graphene layer 201.
  • the gate electrode 203a and the drain electrode 203b comprise platinum.
  • the solid support 204 or insulating layer 202a/202b comprises silicon or silicon nitride.
  • the device comprises a solid support 408, a graphene layer 401, a gate electrode 403a, a drain electrode 403b, at least one insulating layer 409, at least one ground shield 407a/407b, and a buried gate 405.
  • the at least one ground shield 407a/407b comprises an opening which allows electrical contact between the graphene layer 401 and the buried gate 405.
  • the insulating layer 409 is located between two or more of the gate electrode 403a, the drain electrode 403b, at least one ground shield 407a/407b, a buried gate 405, and the graphene layer 401.
  • the gate electrode 403a and the drain electrode 403b are in electrical communication via the graphene layer 401.
  • one or more of the gate electrode 403a, the drain electrode 403b, the buried gate 405, and at least one ground shield 407a/407b comprise platinum.
  • the solid support or insulating layer 409 comprises silicon or silicon nitride.
  • the buried gate 405 is charged with a voltage to attract or repel molecules from the charge sensor (e.g., graphene layer 401). In some instances, the buried gate 405 is charged with a voltage to modulate currents produced by biomolecules interacting with a molecular sensor described herein.
  • any devices described above may be arrayed on a solid support.
  • devices are arrayed on a solid support such that at least some or a portion of the devices are addressable.
  • devices 1500 are arrayed on a solid support 1501 in a configuration.
  • the first electrodes are placed on the x-axis, and second electrodes 1502 are placed on the y-axis (FIG. 15). Any number of devices are in some instances present in an array.
  • arrays comprise 10- 1,000,000, 10-100,000, 10-10,000, 10-5,000, 50-1,000,000, 50-100,000, 50-10,000, 50-5,000, 100- 1,000,000, 100-100,000, 100-10,000, 100-5,000 or 500-1,00,000 devices.
  • arrays comprise at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, or at least 500,000 devices.
  • devices are arrayed into larger devices such.
  • the density of an array can be from 2 to as many as a billion or more different reaction sites (devices, or loci) per square cm.
  • the density of an array is at least 10,000,000 reaction sites/cm 2 , at least 100,000,000; reaction sites/cm 2 , at least 1,000,000,000 reaction sites/cm 2 , at least 2,000,000,000 reaction sites/cm 2 ; at least 100,000 reaction sites/cm 2 ; at least 10,000,000 reaction sites/cm 2 ; at least 100,000 reaction sites/cm 2 ; or at least 10,000 reaction sites/cm 2 .
  • the density of the array is 10,000-100,000 reaction sites/cm 2 ; 100,000-500,000 reaction sites/cm 2 ; 100,000- 1,000,000 reaction sites/cm 2 ; 10,000-1,000,000 reaction sites/cm 2 ; or 100,000-1,000,000,000 reaction sites/cm 2 .
  • Devices in some instances further comprise vias and/or other connections for electrical communication. In some instances, devices comprise electrodes placed in the z-axis.
  • Devices described herein may comprise a solid support.
  • An exemplary solid support can be seen in FIGS. 6B-6C.
  • FIG. 6B shows a front side of the solid support made of glass and comprising a clear window for array and fluidic ports.
  • FIG. 6C shows a back side of the solid support that is a circuit comprising electrical contacts (for example, an LGA 1 mm pitch) and a thermal interface under the solid support area.
  • Solid supports as described herein comprise an active area.
  • the active area comprises addressable solid supports, regions, or loci for molecular sensing.
  • the active area comprises addressable regions or loci for nucleic acid storage.
  • an active area is in fluid communication with solvents or other reagents.
  • the active area comprises varying dimensions.
  • the dimension of the active area is between about 1 mm to about 50 mm by about 1 mm to about 50 mm.
  • the active area comprises a width of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm.
  • the active area comprises a height of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm.
  • the dimension of the active area is between about 1 pm to about 50 pm by about 1 pm to about 50 pm.
  • the active area comprises a width of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 pm.
  • the active area comprises a height of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 pm.
  • the active area is 4 to 900, 4 to 500, 4 to 250, 2 to 900, 10 to 900, 25 to 900, 50 to 900, 100 to 900, or 400 to 900 mm 2 .
  • the active and any passive area is 4 to 900, 4 to 500, 4 to 250, 2 to 900, 10 to 900, 25 to 900, 50 to 900, 100 to 900, or 400 to 900 mm 2 .
  • the active area is 4 to 900, 4 to 500, 4 to 250, 2 to 900, 10 to 900, 25 to 900, 50 to 900, 100 to 900, or 400 to 900 mm 2 .
  • the active and any passive area is 4 to 900, 4 to 500, 4 to 250, 2 to 900, 10 to 900, 25 to 900, 50 to 900, 100 to 900, or 400 to 900 mm 2 for each side of a device described herein.
  • An exemplary active area within a solid support is seen in FIG. 6A.
  • a package 607 comprises an active area 605 within a solid support 603.
  • the package 607 also comprises a fluidics interface 601.
  • the solid support has a number of sites or positions for molecular sensors. In some instances, the solid support comprises up to or about 10,000 by 10,000 positions in an area. In some instances, the solid support comprises between about 1000 and 20,000 by between about 1000 and 20,000 positions in an area.
  • the solid support comprises at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions by least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions in an area.
  • the area is up to 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 inches squared.
  • the solid support comprises addressable loci having a pitch of at least or about 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, or more than 10 um. In some instances, the solid support comprises addressable loci having a pitch of no more than about 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, or no more than 10 um. In some instances, the solid support comprises addressable loci having a pitch of about 5 um.
  • the solid support comprises addressable loci having a pitch of about 2 um. In some instances, the solid support comprises addressable loci having a pitch of about 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.01 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.02 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.05 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.08 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 um.
  • the solid support comprises addressable loci having a pitch of about 0.05 um to about 10 um, about 0. 05 to about 1 um, about 0.05 to about 1 um, about 0. 1 um to about 1 um, about 0.2 um to about 0.8 um, about 0.3 um to about 0.5 um, about 1 um to about 3 um or about 0.05 um to about 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0. 1 um to about 3 um.
  • the solid support comprises addressable loci having a pitch of at least or about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.1, 0.15, .02, 0.25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more than 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.5 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 um. In some instances, the solid support comprises addressable loci having a pitch of about 0. 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.02 um.
  • the solid support comprises addressable loci having a pitch of about 0.02 um to about 1 um, about 0.02 to about 0.8 um, about 0.05 to about 0. 1 um, about 0.1 um to about 1 um, about 0.2 um to about 0.8 um, about 0.3 um to about 0.5 um, about 0. 1 um to about 0.3 um or about 0.05 um to about 0.3 um.
  • the solid support comprises addressable loci having a pitch of about 0.01 um to about 0.3 um.
  • the solid support comprises addressable loci having a pitch of about 0.05 um to about 1 um.
  • Devices may comprise gaps or distances between one or more electrodes of various lengths.
  • electrodes on each side of the gap are attached to a charge sensor.
  • electrodes on each side of the gap are attached to at least one charge sensor.
  • electrodes on each side of the gap are attached to at least one shared charge sensor.
  • the length of a nanogap is about 5, 10, 15, 20, 25, 30, 35, 40, or about 50 nm. In some instances, the length of a nanogap is about 5-50, 5-25, 5-20, 10-50, 10-30, 15-25, 15-30, 20-40, or 25-50 nm.
  • the length of a nanogap is no more than 5, 10, 15, 20, 25, 30, 35, 40, or no more than 50 nm. In some instances, the length of a nanogap is at least 5, 10, 15, 20, 25, 30, 35, 40, or at least 50 nm.
  • Devices described herein may comprise a neck region.
  • an electrode comprises a neck region.
  • the neck region is proximal to a nanogap.
  • the neck region has width which is at least 5%, 10%, 20%, 30%, 50%, 75% 90%, or more than 95% shorter than the largest dimension of the electrode.
  • the neck region has width which is at least 0.5, 1, 1.5, 2, 5, 10, 20, 50, or 100 fold shorter than the largest dimension of the electrode.
  • a first electrode comprises a neck region.
  • a second electrode comprises a neck region.
  • a first electrode comprises a neck region, and a second electrode does not comprise a neck region.
  • the neck region has a width of no more than 250, 200, 150, 125, 110, 100, 90, 75, or no more than 50 nm. In some instances, the neck region has a width of about 250, 200, 150, 125, 110, 100, 90, 75, or about 50 nm.
  • each locus of the structure has a width of about 1 um and a distance between the center of each structure of about 2.1 um. In some instances, each locus of the structure has a width of about 0.5 um and a distance between the center of each structure of about 2 um. In some instances, each locus of the structure has a width of about 0.1 um and a distance between the center of each structure of about 0.2 um. Loci may comprise, without limitation, circular, rectangular, tapered, or rounded shapes. Alternatively or in combination, the structures are rigid. In some instances, the rigid structures comprise loci for molecular sensing. In some instances, the rigid structures comprise substantially planar regions, channels, or wells for molecular sensing.
  • FIGS. 5A-5C show a zoom in of the locus in the flexible structure.
  • Each locus in a portion of the flexible structure 501 may be a substantially planar spot 503 (e.g., flat), a channel 505, or a well 507.
  • Loci may comprise, without limitation, circular, rectangular, tapered, or rounded shapes.
  • the structures are rigid. In some instances, the rigid structures comprise loci, channels, or wells for polynucleotide synthesis.
  • a well described herein has a width to depth (or height) ratio of 1 to 0.01. In some instances, the width is a measurement of the width at the narrowest segment of the well. In some instances, a well described herein has a width to depth (or height) ratio of 0.5 to 0.01. In some instances, the width is a measurement of the width at the narrowest segment of the well. In some instances, a well described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1. Provided herein are structures for molecular sensing comprising a plurality of discrete loci for molecular sensing.
  • Exemplary structures for the loci include, without limitation, substantially planar regions, channels, wells or protrusions. Structures described herein may comprise a plurality of clusters, each cluster comprising a plurality of wells, loci or channels. Alternatively, described herein may comprise a homogeneous arrangement of wells, loci or channels. Structures provided herein may comprise wells having a height or depth from about 0.1 um to about 5 um, from about 0.1 um to about 400 um, from about 0. 1 um to about 300 um, from about 0.1 um to about 200 um, from about 0. 1 um to about 100 um, from about 0.1 um to about 0.5 um, or from about 0.01 um to about 0.5 um.
  • the height of a well is less than 0.10 um, less than .08 um, less than 0.6 um, less than 0.40 um or less than 0.2 um. In some instances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nm or more. In some instances, the height or depth of the well is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm.
  • the height or depth of the well is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some instances, the height or depth of the well is in a range of about 50 nm to about 1 um. In some instances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900 or about 1000 nm.
  • Structures for molecular sensing provided herein may comprise channels.
  • the channels may have a width to depth (or height) ratio of 1 to 0.01.
  • the width is a measurement of the width at the narrowest segment of the microchannel.
  • a channel described herein has a width to depth (or height) ratio of 0.5 to 0.01.
  • the width is a measurement of the width at the narrowest segment of the microchannel.
  • a channel described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1.
  • structures for molecular sensing comprising a plurality of discrete loci.
  • Structures comprise, without limitation, substantially planar regions, channels, protrusions, or wells for molecular sensing.
  • structures described herein are provided comprising a plurality of channels, wherein the height or depth of the channel is from about 5 um to about 500 um, from about 5 um to about 400 um, from about 5 um to about 300 um, from about 5 um to about 200 um, from about 5 um to about 100 um, from about 5 um to about 50 um, or from about 10 um to about 50 um.
  • the height of a channel is less than 100 um, less than 80 um, less than 60 um, less than 40 um or less than 20 um.
  • channel height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 um or more. In some instances, the height or depth of the channel is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the height or depth of the channel is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. Channels described herein may be arranged on a surface in clusters or as a homogeneous field.
  • the width of a locus on the surface of a structure for molecular sensing described herein may be from about 0. 1 um to about 500 um, from about 0.5 um to about 500 um, from about 1 um to about 200 um, from about 1 um to about 100 um, from about 5 um to about 100 um, or from about 0.1 um to about 100 um, for example, about 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um, 10 um, 5 um, 1 um or 0.5 um. In some instances, the width of a locus is less than about 100 um, 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.
  • the width of a locus is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the width of a locus is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some instances, the width of a locus is in a range of about 50 nm to about 1000 nm. In some instances, the distance between the center of two adjacent loci is from about 0.
  • the total width of a locus is about 5 um, 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, or 100 um. In some instances, the total width of a locus is about 1 um to 100 um, 30 um to 100 um, or 50 um to 70 um.
  • the distance between the center of two adjacent loci is from about 0.5 um to about 2 um, 0.5 um to about 2 um, from about 0.75 um to about 2 um, from about 1 um to about 2 um, from about 0.2 um to about 1 um, from about 0.5 um to about 1.5 um, from about 0.5 um to about 0.8 um, or from about 0.5 um to about 1 um, for example, about 1 um.
  • the total width of a locus is about 50 nm, 0.1 um, 0.2 um, 0.3 um, 0.4 um, 0.5 um, 0.6 um, 0.7 um, 0.8 um, 0.9 um, 1 um, 1.1 um, 1.2 um, 1.3 um, 1.4 um, or 1.5 um. In some instances, the total width of a locus is about 0.5 um to 2 um, 0.75 um to 1 um, or 0.9 um to 2 um.
  • each locus supports the sensing of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus.
  • surfaces which comprise at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.
  • surfaces which comprise more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;
  • each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more loci. In some cases, each cluster includes 50 to 500, 50 to 200, 50 to 150, or 100 to 150 loci. In some cases, each cluster includes 100 to 150 loci. In some instances, each cluster includes 109, 121, 130 or 137 loci.
  • loci having a width at the longest segment of 5 to 100 um. In some cases, the loci have a width at the longest segment of about 30, 35, 40, 45, 50, 55 or 60 um. In some cases, the loci are channels having multiple segments. In some instances, each segment has a center to center distance apart of 5 to 50 um. In some cases, the center to center distance apart for each segment is about 5, 10, 15, 20 or 25 um.
  • loci having a width at the longest segment of 5 to 500 nm.
  • the loci have a width at the longest segment of about 30, 35, 40, 45, 50, 55, 60, 80, or 100 nm.
  • the loci are channels having multiple segments.
  • each segment has a center to center distance apart of 5 to 50 nm.
  • the center to center distance apart for each segment is about 5, 10, 15, 20, 25, 50, 100, or 200 nm.
  • the number of distinct polynucleotides synthesized on the surface of a structure described herein is dependent on the number of distinct loci available in the substrate.
  • the density of loci within a cluster of a substrate is at least or about 1 locus per mm 2 , 10 loci per mm 2 , 25 loci per mm 2 , 50 loci per mm 2 , 65 loci per mm 2 , 75 loci per mm 2 , 100 loci per mm 2 , 130 loci per mm 2 , 150 loci per mm 2 , 175 loci per mm 2 , 200 loci per mm 2 , 300 loci per mm 2 , 400 loci per mm 2 , 500 loci per mm 2 , 1,000 loci per mm 2 10 4 loci per mm 2 , 10 5 loci per mm 2 , 10 6 loci per mm 2 , or more.
  • a substrate comprises from about 10 loci per mm 2 to about 500 mm 2 , from about 25 loci per mm 2 to about 400 mm 2 , from about 50 loci per mm 2 to about 500 mm 2 , from about 100 loci per mm 2 to about 500 mm 2 , from about 150 loci per mm 2 to about 500 mm 2 , from about 10 loci per mm 2 to about 250 mm 2 , from about 50 loci per mm 2 to about 250 mm 2 , from about 10 loci per mm 2 to about 200 mm 2 , or from about 50 loci per mm 2 to about 200 mm 2 .
  • a substrate comprises from about 10 4 loci per mm 2 to about 10 5 mm 2 .
  • a substrate comprises from about 10 5 loci per mm 2 to about 10 7 mm 2 . In some cases, a substrate comprises at least IO 5 loci per mm 2 . In some cases, a substrate comprises at least IO 6 loci per mm 2 . In some cases, a substrate comprises at least IO 7 loci per mm 2 . In some cases, a substrate comprises from about 10 4 loci per mm 2 to about IO 5 mm 2 .
  • the density of loci within a cluster of a substrate is at least or about 1 locus per um 2 , 10 loci per um 2 , 25 loci per um 2 , 50 loci per um 2 , 65 loci per um 2 , 75 loci per um 2 , 100 loci per um 2 , 130 loci per um 2 , 150 loci per um 2 , 175 loci per um 2 , 200 loci per um 2 , 300 loci per um 2 , 400 loci per um 2 , 500 loci per um 2 , 1,000 loci per um 2 or more.
  • a substrate comprises from about 10 loci per um 2 to about 500 um 2 , from about 25 loci per um 2 to about 400 um 2 , from about 50 loci per um 2 to about 500 um 2 , from about 100 loci per um 2 to about 500 um 2 , from about 150 loci per um 2 to about 500 um 2 , from about 10 loci per um 2 to about 250 um 2 , from about 50 loci per um 2 to about 250 um 2 , from about 10 loci per um 2 to about 200 um 2 , or from about 50 loci per um 2 to about 200 um 2 .
  • the distance between the centers of two adjacent loci within a cluster is from about 10 um to about 500 um, from about 10 um to about 200 um, or from about 10 um to about 100 um. In some cases, the distance between two centers of adjacent loci is greater than about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um or 100 um. In some cases, the distance between the centers of two adjacent loci is less than about 200 um, 150 um, 100 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.
  • the distance between the centers of two adjacent loci is less than about 10000 nm, 8000 nm, 6000 nm, 4000 nm, 2000 nm 1000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm, 100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or 10 nm.
  • each square meter of a structure described herein allows for at least I0 7 , 10 8 , 10 9 , 10 10 , 10 11 loci.
  • each locus supports one polynucleotide.
  • 10 9 polynucleotides are supported on less than about 6, 5, 4, 3, 2 or 1 m 2 of a structure described herein.
  • a structure described herein provides support for the sensing of more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;
  • the structure provides support for the sensing of more than 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences.
  • at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence.
  • the structure provides a surface environment for the growth of polynucleotides having at least 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.
  • structures for molecular sensing described herein comprise sites for molecular sensing in a uniform arrangement.
  • polynucleotides are synthesized on distinct loci of a structure. In some instances, each locus supports the sensing of a population of polynucleotides.
  • each locus supports the sensing of a population of polynucleotides having a different sequence than a population of polynucleotides sensed on another locus.
  • the loci of a structure are located within a plurality of clusters.
  • a structure comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.
  • a structure comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci.
  • each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150 or more loci. In some instances, each cluster includes 50 to 500, 100 to 150, or 100 to 200 loci. In some instances, each cluster includes 109, 121, 130 or 137 loci. In some instances, each cluster includes 5, 6, 7, 8, 9, 10, 11 or 12 loci.
  • polynucleotides from distinct loci within one cluster have sequences that, when assembled, encode for a contiguous longer polynucleotide of a predetermined sequence. In some instances, each of the polynucleotides comprise a plurality of different nucleotide bases (e.g., A, T, C, G, etc.).
  • a structure described herein is about the size of a plate (e.g., chip), for example between about 40 and 120 mm by between about 25 and 100 mm. In some instances, a structure described herein has a diameter less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm.
  • the diameter of a substrate is between about 25 mm and 1000 mm, between about 25 mm and about 800 mm, between about 25 mm and about 600 mm, between about 25 mm and about 500 mm, between about 25 mm and about 400 mm, between about 25 mm and about 300 mm, or between about 25 mm and about 200.
  • substrate size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 84 mm, 76 mm, 54 mm, 51 mm and 25 mm.
  • a substrate has a planar surface area of at least 100 mm 2 ; 200 mm 2 ; 500 mm 2 ; 1,000 mm 2 ; 2,000 mm 2 ; 4,500 mm 2 ; 5,000 mm 2 ; 10,000 mm 2 ; 12,000 mm 2 ; 15,000 mm 2 ; 20,000 mm 2 ; 30,000 mm 2 ; 40,000 mm 2 ; 50,000 mm 2 or more.
  • the thickness is between about 50 mm and about 2000 mm, between about 50 mm and about 1000 mm, between about 100 mm and about 1000 mm, between about 200 mm and about 1000 mm, or between about 250 mm and about 1000 mm.
  • Non-limiting examples thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In some instances, the thickness is at least or about 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than 4.0 mm. In some cases, the thickness of varies with diameter and depends on the composition of the substrate. For example, a structure comprising materials other than silicon may have a different thickness than a silicon structure of the same diameter. Structure thickness may be determined by the mechanical strength of the material used and the structure must be thick enough to support its own weight without cracking during handling.
  • Described herein are devices where two or more solid supports are assembled.
  • solid supports are interfaced together on a larger unit.
  • Interfacing may comprise exchange of fluids, electrical signals, or other medium of exchange between solid supports.
  • This unit may be capable of interfacing with any number of servers, computers, or networked devices.
  • a plurality of solid support is integrated onto a rack unit or mounted onto a rack unit, which can be conveniently inserted or removed from a server rack.
  • the rack unit may comprise any number of solid supports. In some instances the rack unit comprises about 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000 or 100,000 solid supports.
  • the rack unit comprises at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000 or more than 100,000 solid supports. In some instances, the rack unit comprises at most 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000 or 100,000 solid supports. In some instances, all or a portion of the solid supports of a rack unit are in fluidic communication, electronic communication, or both. In some instances, the server rack comprises about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some instances, the server rack comprises at least about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units.
  • the server rack comprises at most about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some instances, all or a portion of the rack units of a rack server are in fluidic communication, electronic communication, or both. In some instances, two or more solid supports are not interfaced with each other. In some instances, two or more rack units comprising solid supports, such as those described herein, are stacked vertically. Fluidic communication, electronic communication, or both may be formed using, by way of non-limiting example, one or more tubes (e.g., microfluidic tubes), valves, actuators, robotics, etc.
  • tubes e.g., microfluidic tubes
  • Nucleic acids (and the information stored in them) present on solid supports can be accessed from the rack unit. See e.g., FIG. 6D.
  • Access includes removal of polynucleotides from solid supports, direct analysis of polynucleotides on the solid support, or any other method which allows the information stored in the nucleic acids to be manipulated or identified.
  • Information in some instances is accessed from a plurality of racks, a single rack, a single solid support in a rack, a portion of the solid support, or a single locus on a solid support.
  • access comprises interfacing nucleic acids with additional devices such as mass spectrometers, HPLC, sequencing instruments, PCR thermocyclers, or other device for manipulating nucleic acids.
  • Access to nucleic acid information in some instances is achieved by cleavage of polynucleotides from all or a portion of a solid support.
  • the rack unit or rack server is located in a data center.
  • the data center employs mechanical structures used for mounting conventional computing and data storage resources in rack units, for example, openings adapted to support disk drives, processing blades, or other computer equipment.
  • computer systems such as those provided herein, are used to retrieve polynucleotides from one or more rack units on one or more rack servers.
  • a user e.g., technician, researcher, customer, etc.
  • computer system or both directs retrieval of one or more rack units on one or more rack servers.
  • a rack unit can be retrieved from a rack server using a robotic system, such as a robotic arm.
  • the robotic system is in communication with the computer system.
  • the robotic system may be used to interface any component of a data storage system with another component of the data storage system.
  • interfacing comprises transferring, storing, moving, processing, or retrieving.
  • the robotic system moves a solid support between components (e.g., units or chambers) of the data storage system.
  • a component may comprise, by way of non-limiting example, synthesis unit, storage unit, amplification unit, etc.
  • Rack units or servers in some instances comprise units for inflow of reagents or outflow of waste or synthesis products.
  • Cleavage in some instances comprises exposure to chemical reagents (ammonia or other reagent), electrical potential, radiation, heat, light, acoustics, or other form of energy capable of manipulating chemical bonds.
  • cleavage occurs by charging one or more electrodes in the vicinity of the polynucleotides.
  • electromagnetic radiation in the form of UV light is used for cleavage of polynucleotides.
  • a lamp is used for cleavage of polynucleotides, and a mask mediates exposure locations of the UV light to the surface.
  • a laser is used for cleavage of polynucleotides, and a shutter opened/closed state controls exposure of the UV light to the surface.
  • a computer system such as those provided herein, directs the opened/closed state of the shutter.
  • access to nucleic acid information is completely automated (e.g., using computer systems provided herein).
  • chips have one or more contacts. In some instances, chips comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, or more than 200 contacts.
  • Devices herein may comprise biomolecules attached to one or more electrodes.
  • the biomolecule is a charge sensor.
  • the charge sensor bridges or spans at least two electrodes.
  • the charge sensor comprises a nanowire.
  • the charge sensor comprises a polymer.
  • the polymer comprises nucleic acids.
  • the charge sensor is configured to bind to one or more electrodes of the devices described herein.
  • a charge sensor comprises a detection device that translates perturbations at its surface or in its surrounding electrical field into an electrical signal. For example, a charge sensor can translate the arrival or departure of a reaction component into an electrical signal.
  • a charge sensor can also translate interactions between two reaction components, or conformational changes in a single reaction component, into an electrical signal.
  • a charge sensor comprises a nanowire.
  • a charge sensor comprises a GeFET.
  • An exemplary charge sensor is a field effect transistor (FET) such as a single-walled carbon nanotube (SWNT) based FET, silicon nanowire (SiNW) FET, graphene nanoribbon FET (and related nanoribbon FETs fabricated from 2D materials such as M0S2, silicene, or other material), tunnel FET (TFET), and steep subthreshold slope devices.
  • FET field effect transistor
  • SWNT single-walled carbon nanotube
  • SiNW silicon nanowire
  • graphene nanoribbon FET and related nanoribbon FETs fabricated from 2D materials such as M0S2, silicene, or other material
  • TFET tunnel FET
  • steep subthreshold slope devices such as M0S2, silicene, or other material
  • the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.
  • the charge sensor comprises carbon.
  • the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • the charge sensor is further attached to a molecular sensor via a tether, such as those described herein.
  • the charge sensor comprises nucleic acids.
  • the charge sensor comprises DNA or RNA.
  • the charge sensor comprises 20-500, 50-500, 100-500, 150-1000, 150-500, 250-1000, 500-1000, or 600-1000 nucleic acids.
  • the charge sensor comprises no more than 1000, 750, 500, 250, 200, 100, 50, or no more than 20 nucleic acids. In some instances, the charge sensor comprises about 1000, 750, 500, 250, 200, 100, 50, or about 20 nucleic acids. In some instances, the charge sensor comprises at least 1000, 750, 500, 250, 200, 100, 50, or at least 20 nucleic acids. In some instances, at least one nucleic acid is attached to a tether. In some instances, the polymer comprises a moiety for attachment to one or more electrodes. In some instances, the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • length of the charge sensor is about 10, 15, 20, 25, 30, 35, or about 40 nm. In some instances, then length of the charge sensor is no more than 10, 15, 20, 25, 30, 35, or no more than 40 nm. In some instances, at least 1, 2, 5, 10, 15, 20, 25, 50, 75, or 80% of the electrodes are attached to a molecular sensor.
  • Devices herein may comprise molecular sensors.
  • a molecular sensor is configured to detect the presence or absence of an analyte.
  • a molecular sensor is in electrical communication with a charge sensor, optionally through a tether.
  • the molecular sensor comprises a polymerase. Any of a variety of polymerases are in some instances used in a method or composition including protein-based enzymes isolated from biological systems and functional variants thereof.
  • the polymerase is configured to catalyze the polymerization of a nucleic acid strand using an existing nucleic acid as a template.
  • Polymerases include but are not limited to DNA polymerases and RNA polymerases.
  • Exemplary DNA polymerases include those that have been classified by structural homology into families identified as A, B, C, D, X, Y, and RT.
  • DNA Polymerases in Family A include T7 DNA polymerase, eukaryotic mitochondrial DNA Polymerase gamma, E. coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus stearothermophilis Pol I.
  • DNA Polymerases in Family B include, eukaryotic DNA polymerases alpha, delta, and epsilon; DNA polymerase ; T4 DNA polymerase, Phi29 DNA polymerase, and RB69 bacteriophage DNA polymerase.
  • Family C includes the E. coli DNA Polymerase III alpha subunit.
  • Family D includes polymerases derived from the Euryarchaeota subdomain of Archaea.
  • DNA Polymerases in Family X include eukaryotic polymerases Pol beta, pol sigma, Pol X, and Pol p. and .S', cerevisiae Pol4.
  • DNA polymerases in Family Y include Pol eta, Pol iota, Pol kappa, E. coli Pol IV (DINB) and E. coli Pol V (UmuD'2C).
  • the RT (reverse transcriptase) family of DNA polymerases includes retrovirus reverse transcriptases and eukaryotic telomerases.
  • RNA polymerases include but are not limited to viral RNA polymerases such as T7 RNA polymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and Archaea RNA polymerase.
  • a molecular sensor may be attached to a charge sensor via a tether (or linkage).
  • a molecular sensor is attached to a charge sensor by a non-covalent linkage such as one formed between a receptor and a ligand.
  • Linkages include but are not limited to those between streptavidin (or variants or analogs thereof) and biotin (or its analogs), those between complementary nucleic acids, those between antibodies and epitopes and the like.
  • a conducting tether is used to attach a molecular sensor to a charge sensor.
  • Exemplary conducting tethers include those having a structure that includes doped polythiophene, poly(3,4-ethylenedioxythiophene), polyacetylenes, polypyrroles, polyanilines, polyfluorenes, polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes, polycarbazoles, polyindoles, or polyazepines. Charge doping of tether structures I some instances is achieved by oxidation of the polymer. Exemplary conducting tethers and methods for their creation are set forth in Vernitskaya et al. Russ. Chem. Rev. 66:44311 (1997); MacDiarmid, Angew. Chem., mt. Ed.
  • the molecular sensor is a polymerase.
  • the charge sensor is a nanowire.
  • a molecular sensor is attached to a charge sensor via a noncovalent interaction, such as pi bonding, electrostatics, F-S interaction, or other noncovalent binding modality.
  • molecular sensor is attached to a charge sensor via a linker using a conjugation.
  • the conjugation comprises nucleophile/carbonyl; an azide/phosphine; 1,4 Michael addition, 1,3-dipolar cycloaddition, inverse electron demand cycloaddition; olefin metathesis; or cross-coupling reaction.
  • a ternary complex is formed among a molecular sensor, a biomolecule, and a primer.
  • a ternary complex is attached to the charge sensor via a primer.
  • a ternary complex is attached to the charge sensor via a molecular sensor.
  • a ternary complex is attached to the charge sensor via a biomolecule.
  • a molecular sensor may comprise a graphene-binding moiety configured to attach the molecular sensor to a charge sensor comprising a graphene layer. Such moieties may bind to other pi- based charge sensors such as a graphene layer.
  • graphene layers are 1-5 atoms thick. In some instances, graphene layers are about 1 atom thick.
  • the graphene binder comprises an aromatic group. In some instances, the graphene binder comprises an aryl or heteroaryl group. In some instances, the graphene binder comprises a Ce-Cso aryl or heteroaryl group. In some instances, the graphene binder comprises a C6-C20 aryl or heteroaryl group.
  • the graphene binder comprises a Ce-Cis aryl or heteroaryl group. In some instances, the graphene binder comprises a Ce-Cio aryl or heteroaryl group. In some instances, the graphene binder comprises a C10-C30 aryl or heteroaryl group. In some instances, the graphene binder comprises a C15-C30 aryl or heteroaryl group. In some instances, the graphene binder comprises an aromatic hydrocarbon.
  • the graphene binder comprises naphthalene, biphenyl, fluorene, anthracene, phenanthrene, tetracene, chrysene, triphenylene, pyrene, pentacene, perylene, benzo[a]pyrene, corannulene, benzo [ghi]perylene, coronene, ovalene, or benzo [c] fluorene.
  • a ternary complex (comprising a molecular sensor, biomolecule, and a primer) is attached to the graphene binder via the primer, the polymerase, or the biomolecule.
  • FIG. 21 An exemplary schematic for a molecular wire construct is provided in FIG. 21.
  • the wire extends between two surface, such as the surface of first electrode and the surface of the second electrode.
  • the wire may extend between the nanogaps of devices described herein.
  • the molecular wire construct may comprise a molecular sensor (e.g., a polymerase) and a charge sensor (e.g., nanowire).
  • the molecular sensor and the charge sensor may be in electrical communication as described herein.
  • the wire construct schematically illustrated in FIG. 21 may extend between gaps shown in the devices of any one of FIGs. 11-20.
  • the wire extends from the surface of a first electrode 2105a and the surface of a second electrode 2105b.
  • the surfaces of the first or second electrode are deposited with gold, as described herein.
  • the wire extends from a gold surface of a first electrode 2105a and a gold surface of a second electrode 2105b.
  • one or both of the surfaces may comprise a tether 2125, a linking moiety 2110, or both.
  • the tether 2125 or the linking moiety 2110 is connect to the molecular wire construct.
  • the tether 2125 is biotin and the linking moiety 2110 is streptavidin.
  • the molecular wire construct may comprise one or more linkers that can associate with the tether or linking moiety of the surfaces.
  • the molecular wire construct comprises a molecular sensor 2115, such as for example, a polymerase.
  • the molecular sensor comprises one or more linkers 2120 that can associate with the tether 2125 or the linking moiety 2110, or both.
  • the one or more linkers 2120 comprises a protein.
  • the one or more linkers 2120 comprises a peptide linker.
  • the one or more linkers 2120 comprises a biotin.
  • the one or more linkers 2120 comprises a streptavidin.
  • the one or more linkers 2120 comprises SpyCatcher.
  • the one or more linkers 2120 comprises SpyTag.
  • a molecular wire construct as described herein can have one or more components, as illustrated by way of non-limiting example in FIG. 21.
  • a molecular wire construct can have, by way of non-limiting example, a charge sensor (e.g., polymerase), one or more linker constructs, such as biotin- SA construct or a SpyCatcher and/or SpyTag construct, a gold nanoparticle, or surface tethers or linkers (e.g., cysteamine, cysteine, thiol-biotin, proteins, peptides, etc.).
  • a linker construct comprises Clq/TNF -related protein (CTRP peptide).
  • a linker construct comprises a tryptophan-zipper pentamer.
  • the peptide linker comprises a five-stranded phenylalanine zipper.
  • the biotin-streptavidin (SA) construct is used as the molecular wire construct.
  • a DNA wire construct is more resistive compared to a biotin-SA construct.
  • currents in low nA range are achieved with biotin-SA wires.
  • a high concentration of thiol-DNA, long reaction times in buffer, or both can be required for Au conjugation.
  • Au conjugation comprises the used of greater than about 100 uM of thiol-DNA.
  • Au conjugation comprises a reaction time of about 48 hours, or more.
  • biotin-SA wires may be readily assembled.
  • a large concentration of building blocks are readily available, with shorter reaction conditions.
  • a polymerase can be inserted in the biotin-SA wire, which leads to a higher current in the field (e.g., higher signal).
  • a molecular wire construct is about 5 nm to 20 nm in length. In some examples, the length of the molecular wire construct is tuned based by adjusting the length of the one or more linkers 2120 or the tether 2125. The one or more linkers may be a peptide linker. In some instances, the molecular wire construct is about 5, 8, 10, 12, 15, 18, or 20 nm in length. In some instances, the molecular wire construct is at least about 5, 8, 10, 12, 15, or 18 nm in length. In some instances, the molecular wire construct is at most about 8, 10, 12, 15, 18, or 20 nm in length.
  • the molecular wire construct is about 5-8, 5-10, 5-12, 5-15, 5-18, 5-20, 8-10, 8-12, 8-15, 8-18, 8-20, 10-12, 10-15, 10-17, 10-20, 12-15, 12-18, 12-20, 15-18, 15-20, or 18-20 nm in length.
  • FIGs. 22A-22B provide a schematic illustration functionalization of the surface.
  • the surface 2205 comprises an electrode surface, which may comprise gold, as described herein.
  • the surface can be functionalized with a tether.
  • the tether comprises a first moiety extending from the surface 2210 and a second moiety 2215 available for association or conjugation with another molecule.
  • the first moiety 2210 comprises cysteamine and the second moiety 2215 comprises biotin.
  • FIG. 22B A non-limiting example of a scheme for the functionalization of a gold surface with cysteamine and biotin is provided in FIG. 22B.
  • a gold surface may be functionalized with cysteamine by exposing the surface to 18 mM cysteamine in water for 4 hours. The surface may then be treated with biotin-NHS in DIPEA and BMF at 60 °C for 4 hours.
  • a surface is further functionalized with a linking moiety 2305 as described herein.
  • An exemplary schematic is provided in FIG. 23A.
  • a biotinylated surface is functionalized with SA.
  • the surface is functionalized with SA by exposing the biotinylated surface to 100 uM of SA in 0. IX PBS for 10 minutes.
  • the biotinylated surface can be characterized using fluorescently labeled SA.
  • a molecular wire construct for association with surfaces described herein is schematically illustrated in FIG. 23B.
  • the molecular wire can comprise a molecular sensor 2315.
  • the molecular sensor can be in communication with a charge sensor as described herein.
  • the molecular sensor can comprise, in some cases, a polymerase, such as those described herein.
  • the molecular sensor can comprise one or more linkers 2320, such as a peptide linker.
  • a peptide linker comprises a terminal moiety 2310 for association with a functionalized surface.
  • the terminal moiety comprises biotin.
  • the molecular wire comprises a bis biotinylated polymerase at the C- and N-terminus.
  • the biotin on the molecular sensor can be used to link the molecular wire to a surface, for example, SA on a surface as described herein.
  • the length of a linker e.g., peptide linker
  • the length of the linker can be adjusted depending on empirical testing based on a nanoelectric device, such as, but not limited to, those described herein.
  • FIG. 23C provides a map illustrating a dual-biotin tagged P29 variant RPN that may be used for generation of a bis-biotinylated polymerase.
  • FIG. 24 An exemplary schematic for the assembly of a molecular wire is provided FIG. 24.
  • a surface e.g., gold
  • the molecular wire may comprise a DNA for sequencing.
  • the molecular wire may be assembled for sequencing.
  • the molecular wire may be assembled using 10 mM MOPS at pH 7.5, with 10 mM KOAc, and 5 mM Mg 2+ .
  • the surface is functionalized with SA and the molecular wire comprises biotin, forming a molecular wire construct.
  • the molecular wire construct can be used to sequence DNA using the devices and methods described herein.
  • a molecular wire construct may comprise a gold nanoparticle.
  • gold nanoparticles may be used for characterizing streptavidin or biotin binding.
  • An exemplary schematic is provided in FIG. 25.
  • the surface is functionalized with biotin and the gold nanoparticles are functionalized with SA, or vice versa.
  • the nanoparticle is about 1, 1.5, 1.8, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, 8, 10, 20, 30, 40, or about 50 run.
  • the nanoparticle is at least about 1, 1.5, 1.8, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, 8, 10, 20, 30, 40, or about 45 nm.
  • the nanoparticle is at most about 1.5, 1.8, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, 8, 15, 20, 30, 40, or about 50 nm. In some instances, the nanoparticle is about 1-2, 1-3, 1-5, 1-8, 1-10, 1-15, 1-20, 2-3, 2-5, 2-8, 2- 10, 2-15, 2-20, 3-5, 3-8, 3-10, 3-15, 3-20, 4-5, 4-8, 4-10, 4-15, 4-20, 5-8, 5-10, 5-15, 5-20, 8-10, 8-15, 8- 20, 10-20, 10-30, 10-40, 10-50, 20-25, 20-30, 20-35, 20-40, 20-50, 25-30, 25-35, 25-40, 25-50, 30-35, 30-40, 30-50, 35-40, 35-50, 40-45, 40-50, or about 45-50 nm.
  • FIG. 26 An example of a gold nanoparticle in a molecular wire construct is illustrated in FIG. 26.
  • a molecular wire construct may be linked between gold coated surfaces.
  • the surfaces can be electrodes on a device described herein.
  • the gold surface is functionalized with a thiolbiotin.
  • the thiol-biotin may be functionalized with streptavidin.
  • the streptavidin may bind to biotin labeled gold nanoparticles.
  • the biotin labeled gold nanoparticles may further be bound to monomeric streptavidin.
  • the monomeric streptavidin may be linked to via a peptide linker to a polymerase.
  • the molecular wire construct may not comprise a gold nanoparticle.
  • the gold coated surface may be functionalized with thiol biotin which may linked to a polymerase via a monomeric streptavidin and a peptide linker.
  • the gold coated surface is functionalized with thiol biotin which may be linked to a polymerase via a streptavidin and a peptide linker (e.g., AVI tag), as shown in FIG. 29.
  • the gold coated surface may be functionalized with cysteamine, as shown for example in FIG. 28.
  • the cysteamine can functionalized with a biotin PEG aldehyde that can be used to link the polymerase via a monomeric streptavidin and a peptide linker, as described herein.
  • the gold coated surface may be functionalized with a terminal cysteine.
  • a SpyCatcher moiety or a SpyTag may be used to link the polymerase between the surfaces.
  • cysteine on the gold coated surface is functionalized with a SpyCatcher, as shown in FIG. 30.
  • a SpyTag may further be used to link the polymerase.
  • cysteine on the gold coated surface is functionalized with a SpyTag, as shown in FIG. 31.
  • a SpyCatcher moiety may further be used to link the polymerase.
  • the gold coated surface is partially functionalized, for example, as shown in FIG. 30.
  • the gold coated surface is fully functionalized, for example, as shown in FIG. 31.
  • the linkage in the molecular wire construct further comprises a peptide.
  • the peptide comprises a Clq/TNF -related protein (CTRP peptide).
  • CTRP peptide As terminal cysteine residue on a gold coated surface may be functionalized with a CTRP peptide.
  • the CTRP peptide may further link to a SpyTag, which in term may be connected to the polymerase via a SpyCatcher, as shown in FIG. 32.
  • a peptide linker comprises a peptide zipper.
  • a peptide zipper may comprise a tryptophan-zipper pentamer or a five-stranded phenylalanine zipper.
  • a gold coated surface may be functionalized with a terminal cysteine.
  • a peptide zipper such as a tryptophan-zipper pentamer or a five-stranded phenylalanine zipper, or both, may be used to connect the polymerase, as shown in FIG. 33.
  • methods herein comprise deposition materials onto one or more base layers. In some instances, methods comprise depositing materials to generate electrodes or passive layers. In some instances, methods comprise patterning electrodes or passive layers. In some instances, methods described herein comprise etching. In some instances, methods described herein comprise isotropic or substantially isotropic etching. In some instances, etching generates edges of an electrode that are undercut.
  • a method comprises at least some of the steps a) providing one or more base layers; b) depositing material to generate a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to generate a passive layer; f) depositing material to generate the first electrode; g) patterning the first electrode; and h) isotropically etching the passive layer, such that the edges of the first electrode are undercut.
  • methods further comprise depositing a material configured to bind to a nanowire.
  • methods further comprise depositing gold on the top layer of the device.
  • methods further comprise depositing gold on one or more electrodes.
  • the method comprises etching or lithography.
  • the method comprises RIE (reactive ion etching). In some instances, patterning comprises lithography and/or RIE. In some instances, the method does not comprise e-beam or DUV (deep ultraviolet light) lithography. In some instances, the method comprises deposition of gold on the first electrode and the second electrode.
  • RIE reactive ion etching
  • patterning comprises lithography and/or RIE. In some instances, the method does not comprise e-beam or DUV (deep ultraviolet light) lithography. In some instances, the method comprises deposition of gold on the first electrode and the second electrode.
  • a surface of a structure described herein comprises a material and/or is coated with a material that facilitates a coupling reaction with the biomolecule for attachment.
  • surface modifications may be employed that chemically and/or physically alter the substrate surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of the surface.
  • surface modification involves (1) changing the wetting properties of a surface, (2) functionalizing a surface, e.g., providing, modifying or substituting surface functional groups, (3) defiinctionalizing a surface, e.g., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.
  • the surface of a structure is selectively functionalized to produce two or more distinct areas on a structure, wherein at least one area has a different surface or chemical property that another area of the same structure.
  • Such properties include, without limitation, surface energy, chemical termination, surface concentration of a chemical moiety, and the like.
  • the surfaces provided herein can have an active area, a passive area, or both.
  • the active area may refer to as an actively functionalized surface.
  • the active area is functionalized with an active material.
  • the passive area is functionalized with a passive material.
  • a surface of a structure disclosed herein is modified to comprise one or more actively functionalized surfaces configured to bind to both the surface of the substrate and a biomolecule, thereby supporting a coupling reaction to the surface.
  • the surface is also functionalized with a passive material that does not efficiently bind the biomolecule.
  • the surface functionalized with a passive material prevents biomolecule attachment at sites where the passive functionalization agent is bound.
  • the surface comprises an active layer only defining distinct loci for biomolecule support.
  • functionalization comprises deposition of a functionalization agent to a structure by any deposition technique, including, but not limiting to, chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma enhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD), physical vapor deposition (e.g., sputter deposition, evaporative deposition), and molecular layer deposition (MLD).
  • CVD chemical vapor deposition
  • ALD atomic layer deposition
  • PECVD plasma enhanced CVD
  • PEALD plasma enhanced ALD
  • MOCVD metal organic CVD
  • HWCVD hot wire CVD
  • iCVD initiated CVD
  • MCVD vapor axial deposition
  • OTD vapor axial deposition
  • MLD molecular layer deposition
  • a substrate is first cleaned, for example, using a piranha solution.
  • An example of a cleaning process includes soaking a substrate in a piranha solution (e.g., 90% H2SO4, 10% H2O2) at an elevated temperature (e.g., 120 °C) and washing (e.g., water) and drying the substrate (e.g., nitrogen gas).
  • the process optionally includes a post piranha treatment comprising soaking the piranha treated substrate in a basic solution (e.g., NH4OH) followed by an aqueous wash (e.g., water).
  • a surface of a structure is plasma cleaned, optionally following the piranha soak and optional post piranha treatment.
  • An example of a plasma cleaning process comprises an oxygen plasma etch.
  • the surface is deposited with an active functionalization agent following by vaporization.
  • the substrate is actively functionalized prior to cleaning, for example, by piranha treatment and/or plasma cleaning.
  • the process for surface functionalization optionally comprises a resist coat and a resist strip.
  • the substrate is spin coated with a resist, for example, SPRTM 3612 positive photoresist.
  • the process for surface functionalization in various instances, comprises lithography with patterned functionalization. In some instances, photolithography is performed following resist coating. In some instances, after lithography, the surface is visually inspected for lithography defects.
  • the process for surface functionalization in some instances, comprises a cleaning step, whereby residues of the substrate are removed, for example, by plasma cleaning or etching. In some instances, the plasma cleaning step is performed at some step after the lithography step.
  • a surface coated with a resist is treated to remove the resist, for example, after functionalization and/or after lithography.
  • the resist is removed with a solvent, for example, with a stripping solution comprising N-methyl-2 -pyrrolidone.
  • resist stripping comprises sonication or ultrasonication.
  • a resist is coated and stripped, followed by active functionalization of the exposed areas to create a desired differential functionalization pattern.
  • the methods and compositions described herein relate to the application of photoresist for the generation of modified surface properties in selective areas.
  • the application of the photoresist relies on the fluidic properties of the surface defining the spatial distribution of the photoresist.
  • surface tension effects related to the applied fluid may define the flow of the photoresist.
  • surface tension and/or capillary action effects may facilitate drawing of the photoresist into small structures in a controlled fashion before the resist solvents evaporate.
  • resist contact points are pinned by sharp edges, thereby controlling the advance of the fluid.
  • the underlying structures may be designed based on the desired flow patterns that are used to apply photoresist during the manufacturing and functionalization processes.
  • a solid organic layer left behind after solvents evaporate may be used to pursue the subsequent steps of the manufacturing process.
  • Structures may be designed to control the flow of fluids by facilitating or inhibiting wicking effects into neighboring fluidic paths.
  • a structure is designed to avoid overlap between top and bottom edges, which facilitates the keeping of the fluid in top structures allowing for a particular disposition of the resist.
  • the top and bottom edges overlap, leading to the wicking of the applied fluid into bottom structures. Appropriate designs may be selected accordingly, depending on the desired application of the resist.
  • a structure described herein has a surface that comprises a material having thickness of at least or at least 0. 1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm or 25 nm that comprises a reactive group capable of molecular sensors.
  • exemplary materials include, without limitation, gold, glass and silicon, such as silicon dioxide and silicon nitride.
  • exemplary surfaces include nylon and PMMA.
  • electromagnetic radiation in the form of UV light is used for surface patterning.
  • a lamp is used for surface patterning, and a mask mediates exposure locations of the UV light to the surface.
  • a laser is used for surface patterning, and a shutter opened/closed state controls exposure of the UV light to the surface.
  • the laser arrangement may be used in combination with a flexible structure that is capable of moving. In such an arrangement, the coordination of laser exposure and flexible structure movement is used to create patterns of one or more agents having differing nucleoside coupling capabilities.
  • a surface After loading of charge sensors, a surface may be bathed, washed, cleaned, baked, etched, or otherwise functionally restored to a condition suitable for subsequent molecular sensing.
  • the number of times a surface is reused and the methods for recycling/preparing the surface for reuse vary depending on subsequent applications. Surfaces prepared for reuse are in some instances reused about 1, 2, 3, 5, 10, 20, 50, 100, 1,000 or more times. Surfaces prepared for reuse are in some instances reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1,000 or more times. In some instances, the remaining “life” or number of times a surface is suitable for reuse is measured or predicted.
  • layers of a device are integrated into a solid support.
  • layers comprise electrodes or are configured for use as electrodes.
  • Devices in some instances comprise at least 2, 3, 4, 5, 6, 10, 20, or more electrodes per device.
  • electrodes are configured as sources, drains, or gates.
  • layers comprise a metal oxide layer.
  • layers comprise a metal oxide layer comprising a continuous metal layer beneath it.
  • Electrodes in some instances comprise at least one conductor, and are fabricated of materials well known in the art.
  • electrodes comprise at least one conductor and one or more insulators or semi-conductors.
  • electrodes comprise platinum, titanium, or titanium nitride.
  • electrodes comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of one or more of platinum, titanium, or titanium nitride.
  • Materials in some instances comprise metals, non-metals, mixed- metal oxides, nitrides, carbides, silicon-based materials, or other material.
  • metal oxides include TiCE, Ta2Os, IrCE, RuCE, RhCE, bft ⁇ CE, AI2O3, BaO, Y2O3, HfC SrO or other metal oxide known in the art.
  • metal carbides include TiC, WC, TI1C2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbide known in the art.
  • metal nitrides include GaN, InN, BN, Be 3 N 2 , Cr 2 N, MoN, Si 3 N 4 , TaN, Th 2 N 2 , VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitride known in the art.
  • a device disclosed herein is manufactured with a combination of materials listed herein or any other suitable material known in the art.
  • layers described herein are coated with an additional metal.
  • layers described herein are coated with an additional material which is configured for attachment of a nanowire.
  • one or more layers are coated with gold.
  • one or more electrodes are coated in gold.
  • gold is depositied using direct, thermal evaporation.
  • gold is depositied using direct, thermal evaporation at a fixed angle.
  • gold is depositied using electrodeposition.
  • the thickness of the gold layer is about 10, 25, 30, 40, 50, 60, or about 75 angstroms. In some instances, the thickness of the gold layer is at least 10, 25, 30, 40, 50, 60, or at least 75 angstroms.
  • the thickness of the gold layer is no more than 10, 25, 30, 40, 50, 60, or no more than 75 angstroms. In some instances, the gold layer is added to a layer comprising another metal. In some instances, the gold layer is added to a layer comprising titanium.
  • devices are contacted with one or more charge sensors.
  • charge sensors attach to one or more electrodes.
  • charge sensors span at least two electrodes.
  • Such charge sensors in some instances are attached to tether.
  • the tether is further attached to a molecular sensor.
  • charge sensors comprise nanowires.
  • the nanowire comprises nucleic acids.
  • charge sensors (such as nucleic acids) are loaded onto the device surface.
  • Loading in some instances comprises one or more of steps: (a) applying a voltage to the electrodes to attract the nucleic strands to the electrodes; (b) monitoring a current path between electrodes to determine if a nanowire has bridged two electrodes; and (c) when the current spikes (e.g., contact is made) the voltage is turned off so that no more DNA is attracted.
  • loading voltages are aboutlO-lOOV, 10-50V, 10-25V, 10-75V, or 25-100V. In some instances, the loading voltage is about 10 V, 15 V, 20 V, 25 V, 30 V, 40 V, 50 V, 60 V, 70 V, 75 V, 80 V, 90 V, or 100 V. In some instances, loading voltages are applied for no more than about 0.01, 0.05, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 90, or no more than 120 seconds.
  • devices comprising a surface.
  • the surface is modified to support molecular sensing at predetermined locations.
  • surfaces of devices for molecular sensing provided herein are fabricated from a variety of materials capable of modification to support molecular sensing.
  • the devices are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of the devices.
  • Devices described herein may comprise a flexible material. Exemplary flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. Devices described herein may comprise a rigid material.
  • Exemplary rigid materials include, without limitation, glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics (for example, polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and metals (for example, gold, platinum).
  • Devices disclosed herein may be fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, devices disclosed herein are manufactured with a combination of materials listed herein or any other suitable material known in the art.
  • Devices described herein may comprise material having a range of tensile strength.
  • Exemplary materials having a range of tensile strengths include, but are not limited to, nylon (70 MPa), nitrocellulose (1.5 MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa), agarose (1- 10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane (PDMS) (3.9-10.8 MPa).
  • Solid supports described herein can have a tensile strength from 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 MPa.
  • Solid supports described herein can have a tensile strength of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 270, or more MPa.
  • a device described herein comprises a solid support for molecular sensing that is in the form of a flexible material capable of being stored in a continuous loop or reel, such as a tape or flexible sheet.
  • Young’s modulus measures the resistance of a material to elastic (recoverable) deformation under load.
  • Exemplary materials having a range of Young’s modulus stiffness include, but are not limited to, nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10 GPa), polydimethylsiloxane (PDMS) (1-10 GPa).
  • Solid supports described herein can have a Young’s moduli from 1 to 500, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 GPa.
  • Solid supports described herein can have a Young’s moduli of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa, or more. As the relationship between flexibility and stiffness are inverse to each other, a flexible material has a low Young’s modulus and changes its shape considerably under load. In some instances, a solid support described herein has a surface with a flexibility of at least nylon.
  • devices disclosed herein comprise a silicon dioxide base and a surface layer of silicon oxide.
  • the devices may have a base of silicon oxide.
  • Surface of the devices provided here may be textured, resulting in an increase overall surface area for molecular sensing.
  • Devices disclosed herein in some instances comprise at least 5 %, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon.
  • Devices disclosed herein in some instances are fabricated from silicon on insulator (SOI) wafer.
  • SOI silicon on insulator
  • the materials from which the substrates/ solid supports of the comprising the invention are fabricated exhibit a low level of polynucleotide binding.
  • material that are transparent to visible and/or UV light can be employed.
  • Materials that are sufficiently conductive e.g. those that can form uniform electric fields across all or a portion of the substrates/solids support described herein, can be utilized. In some instances, such materials may be connected to an electric ground.
  • the substrate or solid support can be heat conductive or insulated.
  • the materials can be chemical resistant and heat resistant to support chemical or biochemical reactions.
  • materials of interest can include: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like.
  • specific materials of interest include: glass; fuse silica; silicon, plastics (for example polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like).
  • the structure can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass.
  • the substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.
  • a substrate disclosed herein comprises a computer readable material.
  • Computer readable materials include, without limitation, magnetic media, reel-to-reel tape, cartridge tape, cassette tape, flexible disk, paper media, film, microfiche, continuous tape (e.g., a belt) and any media suitable for storing electronic instructions.
  • the substrate comprises magnetic reel-to-reel tape or a magnetic belt.
  • the substrate comprises a flexible printed circuit board.
  • Structures described herein may be transparent to visible and/or UV light. In some instances, structures described herein are sufficiently conductive to form uniform electric fields across all or a portion of a structure. In some instances, structures described herein are heat conductive or insulated. In some instances, the structures are chemical resistant and heat resistant to support a chemical reaction. In some instances, the substrate is magnetic. In some instances, the structures comprise a metal or a metal alloy. Structures described herein may be integrated into a rack, such as a rack unit in a rack server described herein.
  • an analyte interacts with a molecular sensor in electrical communication with one or more electrodes.
  • interaction with the analyte results in a change in voltage, current, or resistance which is detectable at the device (signal or signal pattern).
  • the analyte comprises nucleotide triphosphates.
  • one or more nucleotide triphosphates generate a unique signal (are distinguishable).
  • each type of nucleotide triphosphate correlates to the identity of a unique base.
  • the sequence or identity of nucleoside in a nucleic is in some instances determined.
  • the molecular sensor comprises a polymerase.
  • binding of a nucleotide triphosphate to the polymerase generates a measurable signal within a device described herein.
  • the one or more nucleotide triphosphates comprise non-canonical or unnatural amino acids.
  • nucleotide triphosphates comprise an unnatural or non-canonical bases.
  • unnatural or non-canonical bases are configured to generate unique signals or signal patterns. Such signals in some instances are measured between one or more electrodes.
  • a method described herein is provided in FIG. 1.
  • a method comprises any one of the steps in FIG. 1.
  • Polynucleotides 103 attached (and/or synthesized) from a surface 101 are cleaved 102.
  • Primers 104 are added 105, followed by a molecular sensor (e.g., polymerases) conjugated to a sensor-binding moiety 115 (graphene binder shown in FIG. 1, via optionally linker) to form a ternary complex 108.
  • the ternary complex 108 is then contacted 109 with a graphene device 110 to bind the ternary complex 108 to a graphene layer.
  • Charge modulating nucleotides (CMNs, indicated with base letter and an asterisk) 111 are added, which extend the primer of the ternary complex. Changes in current resulting from incorporation of CMNs 111 produce a signal which can be measured 114, thus identifying the incorporated base.
  • a charge sensor comprises a graphene layer, and a molecular sensor comprises a polymerase.
  • the method can include one or more steps of (a) providing a polymerase attached to a solid support charge sensor; (b) contacting the polymerase with a mixture of nucleotides; (c) detecting the incorporation of the nucleotides via the charge sensor; (d) repeating steps (b) and (c) using the polymerase, the template nucleic acid, and a second mixture of nucleotides; and (e) comparing the first and second signal patterns to determine the sequence of the template nucleic acid.
  • the mixture in (b) includes different types of nucleotides.
  • a first type of the nucleotide is in a distinguishable state compared to the other types of nucleotides in the mixture of (b).
  • a second type of the nucleotides is not in the distinguishable state compared to the other types of nucleotides in the mixture of (b).
  • the polymerase incorporates nucleotides from the mixture in (b) into a nascent strand against a template nucleic acid strand.
  • detecting the incorporation of the nucleotides via the charge comprises the first type of the nucleotides producing a signal that is unique compared to signals produced by other nucleotides in the mixture, thereby acquiring a first signal pattern.
  • repeating steps (b) and (c) using the polymerase comprises the second type of the nucleotides being in a distinguishable state compared to the other types of nucleotides in the second mixture. In some instances, repeating steps (b) and (c) using the polymerase comprises the first type of the nucleotides not being in the distinguishable state compared to the other types of nucleotides in the second mixture, thereby acquiring a second signal pattern.
  • a method of nucleic acid sequencing that includes steps of (a) providing a polymerase attached to a solid support charge sensor; (b) contacting the polymerase with a mixture of nucleotides; (c) detecting the incorporation of the nucleotides via the charge sensor; (d) repeating steps (b) and (c) using the polymerase, the template nucleic acid, and a second mixture of nucleotides, wherein; and (e) comparing the first and second signal patterns to determine the sequence of the template nucleic acid.
  • the mixture in (b) includes different types of nucleotides.
  • a first two types of the nucleotides are in a first distinguishable state compared to a second two types of the nucleotides in the mixture of (b).
  • the polymerase incorporates nucleotides from the mixture in (b) into a nascent strand against a template nucleic acid strand.
  • detecting the incorporation of the nucleotides via the charge sensor comprises the first two types of the nucleotides producing a signal that distinguished from signals produced by second two types of the nucleotides in the mixture, thereby acquiring a first signal pattern.
  • one or more non-natural nucleotide that are present in a mixture will produce a signal change having an inverted polarity compared to other nucleotides in the mixture.
  • one or more non-natural nucleotide that is used in the mixture will produce a delay in nucleotide incorporation or reduced rate of incorporation.
  • one or more non-natural nucleotide that is used in the method will produce a significantly altered signal height.
  • method of using a device described herein comprises one or more steps of providing an analyte; reacting, binding, or other allowing the analyte to interact with the sensor; and measuring an electrical signal generated from the sensor.
  • method of using a device described herein comprises one or more steps of providing at least one nucleotide, at least one template (nucleic acid), and at least one primer; extending the primer by the at least one nucleotide; and measuring an electrical signal generated from the polymerase.
  • electrical signals are analyzed to establish the identity of the at least one nucleotide incorporated by a polymerase.
  • at least one nucleotide comprises a terminator which is configured to prevent chain extension.
  • methods described herein are used to sequence at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, or more than 1000 bases. In some instances, methods described herein are used to sequence about 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1500, or about 2000 bases. In some instances, methods described herein are used to sequence 10-1000, 20-1000, 50-1000, 100-1000, 50-2000, 25-500, 25-200, 50-500, or 50-750 bases. In some instances, nucleotides comprise charge modulating nucleotides.
  • Interactions between a molecular sensor and an analyte may generate detectable signals.
  • the analyte comprises a charge modulating nucleotide.
  • a non-natural moiety or modification that is present in the nonnatural nucleotide(s) produces a change in polymerase conformation (compared to the conformation produced by a nucleotide that lacks the moiety or modification) thereby producing a unique signature in one or more signal parameter detected by a charge sensor to which the polymerase is attached.
  • Exemplary signal parameters include, but are not limited to, signal duration, signal height, signal rise time, signal fall time, signal polarity, signal noise, signal shape, and the like.
  • methods described herein utilize a mixture of four different types of nucleotide triphosphates in which one of the nucleotide triphosphate types is present in a substantially lower amount or concentration (e.g., the ‘low’ abundance nucleotide) compared to the other three types (e.g., the ‘high’ abundance nucleotides).
  • the ‘low’ abundance nucleotide compared to the other three types
  • incorporation of the low nucleotide will be detectable as a relative delay or decreased incorporation rate.
  • This signature in some instances is exploited to identify the location in the template of the nucleotide type that complements the low nucleotide.
  • Several sequencing runs in some instances are completed for the same template, wherein each run is carried out with a different nucleotide in the low state.
  • the signal patterns from the different runs in some instances are compared to determine the sequence of the template.
  • the method comprises use of 3 high-1 low mixture of nucleotide triphosphates. Other ratios of high to low are also described herein, mixtures as well including, for example, a one high-three low mixture, or a two high-two low mixture. Further useful configurations of mixtures with regard to using nucleotides having different concentrations are set forth in U.S. Pat. No.
  • the template nucleic acid is circular.
  • the use of a circular template in some instances provides a convenient format for repeated sequencing runs since the polymerase need not be replaced and can instead make multiple laps around the template, each lap being effectively a repeated sequencing of the template.
  • the polymerase includes a 5' exonuclease activity to digest a nucleic acid strand that is to be displaced from the circular template when the polymerase proceeds multiple times around the template.
  • a different primer in some instances is used for different sequencing runs carried out on the same template. The different primers in some instances are designed to hybridize at different locations on the template.
  • each of the runs start at a different location in the template, but there is in some instances substantial overlap between the portions of the template that are sequenced in each run.
  • the signal patterns resulting from each run in some instances is aligned based on the expected start sites for each run in order to facilitate sequence calling and error checking.
  • a charge sensor that is used in a method set forth herein in some instances detects nucleotide incorporation by polymerase via a field effect using a SWNT FET, nanowire FET, FinFET, trigate FET, tunneling FET, or another field sensitive device.
  • the sensor is magnetic, electrochemical, or nanoelectromechanical.
  • each nucleotide triphosphate generates a distinguishable state.
  • distinguishable states applies to a particular type of nucleotide triphosphate having a characteristic or property that manifests uniquely under a detection condition compared to other nucleotide triphosphates.
  • Exemplary distinguishable states include, but are not limited to, being present in a quantity or concentration that is substantially less than the quantity or concentration of the other types of nucleotide triphosphates in the mixture, being present in a quantity or concentration that is substantially greater than the quantity or concentration of the other types of nucleotide triphosphates in the mixture, having a chemical moiety or modification that is not present on other types of nucleotide triphosphates in the mixture, or lacking a chemical moiety or modification that is present on other types of nucleotide triphosphates in the mixture.
  • the distinguishable state can manifest when the nucleotide type interacts with a polymerase.
  • signals are detected from conformational changes, such as the appearance, disappearance, or alteration of a detectable signal from a molecule in response to a change in the structure, shape or arrangement of parts of the molecule.
  • the signal change can be due to a change in the interaction of a label with a first portion of the molecule to interact with a second portion of the molecule.
  • Detectable signals may comprise changes to current, resistance, or voltage.
  • the detectable signal comprises a change in current.
  • the change in current is 1 nanoamp to 100 picoamps, 1 nanoamp to 50 picoamps, 1 nanoamp to 25 picoamps, 1 nanoamp to 10 picoamps, 1 nanoamp to 1 picoamp, 1 nanoamp to 500 nanoamps, 1 nanoamp to 250 nanoamps, 1 nanoamp to 100 nanoamps, 100 nanoamps to 100 picoamps, 100 nanoamps to 10 picoamps, 100 nanoamps to 1 picoamp, 100 nanoamps to 500 nanoamps, 500 nanoamps to 100 picoamps, 500 nanoamps to 50 picoamps, 500 nanoamps to 10 picoamps, 1 picoamp to 100 picoamps, 10 picoamps to 100 picoamps, or 250 nanoamps to 750 nanoamps.
  • a detectable signal is measured as a change in signal relative to a background signal (e.g., absence of analyte).
  • the change in current is 1.01-3, 1.01-2.75, 1.01-2.50, 1.01-2.25, 1.01-2, 1.01-1.95, 1.01-1.75, 1.01-1.5, 1.01-1.25, 1.25-3. 1.35-3, 1.5-3, 2-3, or 2.5-3 relative to a background signal.
  • Methods described herein may allow for rapid analysis of analytes.
  • 1-200, 1-500, 1-300, 1-150, 1-100, 10-500, 10-300, 50-300, 50-200, 100-200, or 150-400 biomolecules are analyzed per second.
  • at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, or at least 600 biomolecules are analyzed per second.
  • the biomolecule comprises a nucleotide or variant thereof.
  • the biomolecule comprises a charge modulating nucleotide (CMN).
  • CPN charge modulating nucleotide
  • Analyte may comprise nucleotides.
  • an analyte comprises a charge modulating nucleotide.
  • the modification comprises a modification to the base (nucleobase) of the CMN, such as a modification to a C, T, G, or C base.
  • An exemplary CMN is depicted in FIG. 3.
  • the modification comprises a deaza or halogen modified base.
  • the modification comprises a 7-deaza or 8-bromo modified base.
  • the CMN comprises a modification to the 5’ position.
  • the modification comprises a modification to a 5’ polyphosphate or chemical variant thereof.
  • the modification comprises a thiolated or bromated phosphate.
  • the polyphosphate comprises at least 3, 4, 5, 6, 8, or 10 phosphates or variants thereof.
  • the modification comprises a modification to a terminal 5 ’ polyphosphate or chemical variant thereof.
  • the modification comprises a polymer.
  • the polymer comprises one or more of a nucleic acid chain, a peptide chain, a polysaccharide, a lipid, a synthetic polymer, and a dendrimer.
  • the nucleic acid chain comprises at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 2500, 3000, or at least 5000 bases. In some instances, the nucleic acid chain comprises no more than 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 2500, 3000, or no more than 5000 bases.
  • the nucleic acid chain comprises about 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 2500, 3000, or about 5000 bases.
  • the nucleic acid chain comprises 25-300, 25-500, 25-400, 25-300, 25-250, 50-500, 50-300, 75-300, 75-250, 75-200, 100-500, 125-500, 200-500, 300-500, 10-250, 25-5000, 50-5000, 100-5000, 200-5000, 500-5000, 1000-5000, 2000-5000 or 10-5000 bases.
  • the nucleic acid chain is branched (dendrimeric).
  • the nucleic acid chain comprises a secondary structure.
  • the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadraplex, and an I-motif.
  • the nucleic acid chain comprises a single strand, double strand, or triple strand.
  • the nucleic acid chain comprises at least one charge modulating chemical modification. In some instances, the at least one charge modulating chemical modification increases the charge of the CMN relative to an unmodified nucleotide.
  • the at least one charge modulating chemical modification comprises one or more of an amine, an alkylamine, a guanidinium, a quaternary amine, an imidazolium, a pyridinium, and a pyrrolidinium. In some instances, the at least one charge modulating chemical modification decreases the charge of the CMN relative to an unmodified nucleotide.
  • the at least one charge modulating chemical modification comprises one or more of a phosphate, a phosphite, a sulfonate, a sulfite, a carboxylate, a xanthate, a thiocarboxylic acid, a boranophosphonate, and a boric acid.
  • the nucleic acid chain comprises at least one sugar-modified nucleotide.
  • the sugar-modified nucleotide comprises a deoxy or dideoxy nucleotide.
  • the nucleic acid chain comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid.
  • a nucleic acid chain may comprise a phosphate modification.
  • the phosphate modification comprises a hydrophobic group.
  • the hydrophobic group comprises a straight or branched alkyl chain.
  • the hydrophobic group comprises a C5-C50 aliphatic chain.
  • the phosphate modification comprises a hydrophilic group.
  • the hydrophilic group comprises polyethylene glycol (PEG).
  • the polyethylene glycol comprises a molecular weight of 1000-100,000, 100-500,000, 100-250,000, 100-75,000, 100-50,000, 100-25,000, 100-10,000, 100-7500, 100-5000, 100-3000, or 100-2000 daltons.
  • the polyethylene glycol comprises a molecular weight of about 100, 200, 300, 500, 1000, 2000, 2500, 3000, 5000, 7500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, or 50,000 daltons. In some instances, the polyethylene glycol comprises a molecular weight of at least about 100, 200, 300, 500, 1000, 2000, 2500, 3000, 5000, 7500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, or 50,000 daltons.
  • the polyethylene glycol comprises a molecular weight of at most about 100, 200, 300, 500, 1000, 2000, 2500, 3000, 5000, 7500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, or 50,000 daltons. In some instances, the polyethylene glycol comprises 10-600 monomers. In some instances, the polyethylene glycol comprises about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 monomers. In some instances, the polyethylene glycol comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 monomers.
  • the polyethylene glycol comprises at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 monomers.
  • the peptide chain is 1-100 amino acids in length. In some instances, the peptide chain is about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In some instances, the peptide chain is at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In some instances, the peptide chain is at most about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length.
  • the CMN comprises a charged small molecule.
  • the charged small molecule comprises one or more of a chelator, a dye, and a metal complex.
  • the metal complex comprises a ferrocene, Ru-dipy, and bis-cyclopentadienyl diiron.
  • conformational labeling comprises at least one label that is responsive to a change in the structure of the molecule, a change in the shape of the molecule, or a change in the arrangement of parts of the molecule.
  • the molecule is in some instances a polymerase, reverse transcriptase, exonuclease or other nucleic acid enzyme.
  • the parts of the molecule can be, for example, atoms that change relative location due to rotation about one or more chemical bonds occurring in the molecular structure between the atoms.
  • the parts of the molecule can be domains of a macromolecule such as those commonly known in the relevant art.
  • polymerases include domains referred to as the finger, palm and thumb domains.
  • the parts can be regions of secondary, tertiary or quaternary structure.
  • the label(s) can be attached to the molecule, for example, via a covalent linkage. However, the label(s) need not be attached to the molecule, being, for example, located in proximity to the molecule. In particular embodiments, the label is not attached to a reactant or product of the molecule such as a nucleotide or nucleic acid.
  • a molecular sensor comprises a polymerase.
  • a method comprises one or more of contacting a plurality of polynucleotides with at least one primer and at least one polymerase to form a plurality of ternary complexes, wherein the ternary complex comprising a graphene binder; detecting one or more bases of the polynucleotides, wherein detection occurs when the plurality of ternary complexes are bound to a graphene layer; removing the ternary complexes from the surface; and repeating previous steps to sequence the polynucleotides.
  • ternary complexes are attracted to the graphene layer by applying a positive charge to one or more electrodes buried underneath the graphene layer.
  • the plurality of polynucleotides comprises at least 10,000, 50,000, 100,000 250,000, 300,000, 400,000, 500,000, 600,000, 700,000, 750,000, 800,000, 900,000, 1 million, 10 million, 100 million, 200 million, 500 million, or at least 750 million unique polynucleotides.
  • the plurality of polynucleotides comprises about 10,000, 50,000, 100,000 250,000, 300,000, 400,000, 500,000, 600,000, 700,000, 750,000, 800,000, 900,000, 1 million, 10 million, 100 million, 200 million, 500 million, or at least 750 million unique polynucleotides.
  • the plurality of polynucleotides are 50-30,000, 50-10,000, 50-1000, 50-750, 50-500, 50-400, 50-300, 50-200, 1000- 30,000, 1000-20,000, 1000-10,000, 2000-5000, 2000-10,000, 5000-30,000, or 10,000-30,000 bases in length.
  • the plurality of polynucleotides are about 10, 50, 100, 200, 300, 400, 500, 750, 1000, 2000, 2500, 5000, 10,000, 20,000, or 30,000 bases in length.
  • a digital sequence encoding an item of information (e.g., digital information in a binary code for processing by a computer) is received.
  • An encryption scheme is applied to convert the digital sequence from one or more symbols (e.g., a binary code) to a nucleic acid sequence.
  • a surface material for nucleic acid extension, a design for loci for nucleic acid extension (e.g., arrangement spots), and reagents for nucleic acid synthesis are selected.
  • the surface of a structure is prepared for nucleic acid synthesis. De novo polynucleotide synthesis is in some instances performed.
  • the synthesized polynucleotides are stored and available for subsequent release, in whole or in part.
  • pools of pre-determined polynucleotides are assembled into larger polynucleotides which represent digital information.
  • the polynucleotides, in whole or in part are sequenced using the devices, systems, and methods described herein, subject to decryption to convert nucleic sequence back to digital sequence.
  • the digital sequence is then assembled to obtain an alignment encoding for the original item of information.
  • polynucleotides are sequenced using the methods and devices described herein.
  • Nucleic acids encoding digital information may comprise error correction component.
  • the error correction component comprises an error correction code, such as a Reed-Solomon (RS) code, a LDPC code, a polar code, a turbo code.
  • the error correction code spreads the digital data to be stored over many polynucleotides. In some instances, spreading the data over a plurality of polynucleotides builds redundancy to correct for erasures (e.g., lost oligos).
  • the digital information can be recovered in the presence of errors.
  • the error correction component comprises a parity base. In some instances, the error correction component comprises an index sequence.
  • the index sequences define the location or address of the digital information encoded in the nucleic acid. In some instances, the index sequences define the source of the digital information. Nucleic acids encoding digital information in some instances comprise overlap with one or more nucleic acids in the same library or set. In some instances, the error correction component comprises an overlap or redundancy region. In some instances, algorithms are applied to sequenced nucleic acids to reduce errors. In some instances, error corrective algorithms comprise consensus sequencing, HEDGES (Hash Encoded, Decoded by Greedy Exhaustive Search), or other method.
  • Nucleic acids encoding for digital information may be stored in different media.
  • nucleic acids are stored as essentially dry or lyophilized powders.
  • nucleic acids are stored in buffers.
  • nucleic acids are stored on chips, wafers, or other silicon solid support.
  • nucleic acids are stored inside an organism (or population of organisms), such as a plasmid or genome.
  • an early step of data storage process disclosed herein includes obtaining or receiving one or more items of information in the form of an initial code.
  • Items of information include, without limitation, text, audio and visual information.
  • Exemplary sources for items of information include, without limitation, books, periodicals, electronic databases, medical records, letters, forms, voice recordings, animal recordings, biological profdes, broadcasts, fdms, short videos, emails, bookkeeping phone logs, internet activity logs, drawings, paintings, prints, photographs, pixelated graphics, and software code.
  • Exemplary biological profile sources for items of information include, without limitation, gene libraries, genomes, gene expression data, and protein activity data.
  • Exemplary formats for items of information include, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls,
  • the amount of individual file sizes encoding for an item of information, or a plurality of files encoding for items of information, in digital format include, without limitation, up to 1024 bytes (equal to 1 KB), 1024 KB (equal to 1MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1TB), 1024 TB (equal to 1PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyte or more.
  • an amount of digital information is at least 1 gigabyte (GB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 gigabytes. In some instances, the amount of digital information is at least 1 terabyte (TB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 terabytes. In some instances, the amount of digital information is at least 1 petabyte (PB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 petabytes.
  • PB petabyte
  • the solid support for molecular sensing as described herein comprises a high capacity for reading of data.
  • the capacity of the solid support is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 petabytes.
  • the capacity of the solid support is between about 1 to about 10 petabytes or between about 1 to about 100 petabytes.
  • the capacity of the solid support is about 100 petabytes.
  • the data is stored as addressable arrays of packets as droplets. In some instances, the data is stored as addressable arrays of packets as droplets on a spot.
  • the data is stored as addressable arrays of packets as dry wells.
  • the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 gigabytes of data.
  • the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 terabytes of data.
  • an item of information is stored in a background of data. For example, an item of information encodes for about 10 to about 100 megabytes of data and is stored in 1 petabyte of background data.
  • an item of information encodes for at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 megabytes of data and is stored in 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 petabytes of background data.
  • any of the systems described herein are operably linked to a computer and are optionally automated through a computer either locally or remotely.
  • the methods and systems of the invention further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the invention.
  • the computer systems are programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.
  • a computer system such as the system shown in FIG. 7 or FIG.
  • a program may be used for encoding data represented as a set of symbols to another set of symbols.
  • the data may be represented as numerical symbols, such as binary values of “0”s and “l”s and the computer system may execute a program comprising an error correction code (e.g., Reed-Solomon (RS) code, low-density parity-check (LDPC) code, Turbo code, etc.).
  • RS Reed-Solomon
  • LDPC low-density parity-check
  • Turbo code etc.
  • the computer system executes a program to convert the data to a plurality of nucleic acid sequences, convert a plurality of nucleic acid sequences to data, or both.
  • a program may be a machine learning algorithm.
  • the machine learning algorithm may determine a nucleotide base based on a signal (e.g., electrical signal, such as current or voltage).
  • a program may be executed on a computer system provided herein.
  • a program comprises a statistical algorithm or a machine learning algorithm.
  • an algorithm comprising machine learning (ML) is used to associate the signal (e.g., electrical currents/voltages) to the nucleoside monomer added to the polynucleotide.
  • the algorithm comprising ML may be trained with training data in order to associate the signal (e.g., electrical currents/voltages) to the nucleoside monomer added to the polynucleotide.
  • the algorithm comprises classical ML algorithms for classification and/or clustering (e.g., K-means clustering, meanshift clustering, density-based spatial clustering of applications with noise (DBSCAN), expectationmaximization (EM) clustering, agglomerative hierarchical clustering, logistic regression, naive Bayes, K- nearest neighbors, random forests or decision trees, gradient boosting, support vector machines (SVMs), or a combination thereof).
  • K-means clustering meanshift clustering
  • DBSCAN density-based spatial clustering of applications with noise
  • EM expectationmaximization
  • agglomerative hierarchical clustering logistic regression
  • naive Bayes K- nearest neighbors
  • random forests or decision trees boosting
  • SVMs support vector machines
  • the algorithm comprises a learning algorithm comprising layers, such as one or more neural networks.
  • Neural networks may comprise connected nodes in a network, which may perform functions, such as transforming or translating input data.
  • the output from a given node may be passed on as input to another node.
  • the nodes in the network may comprise input units, hidden units, output units, or a combination thereof.
  • an input node may be connected to one or more hidden units.
  • one or more hidden units may be connected to an output unit.
  • the nodes may take in input and may generate an output based on an activation function.
  • the input or output may be a tensor, a matrix, a vector, an array, or a scalar.
  • the activation function may be a Rectified Linear Unit (ReLU) activation function, a sigmoid activation function, or a hyperbolic tangent activation function.
  • the activation function may be a Softmax activation function.
  • the connections between nodes may further comprise weights for adjusting input data to a given node (e.g., to activate input data or deactivate input data).
  • the weights may be learned by the neural network.
  • the neural network may be trained using gradient-based optimizations.
  • the gradient-based optimization may comprise of one or more loss functions.
  • the gradient-based optimization may be conjugate gradient descent, stochastic gradient descent, or a variation thereof (e.g., adaptive moment estimation (Adam)).
  • the gradient in the gradient-based optimization may be computed using backpropagation.
  • the nodes may be organized into graphs to generate a network (e.g., graph neural networks).
  • the nodes may be organized into one or more layers to generate a network (e.g., feed forward neural networks, convolutional neural networks (CNNs), recurrent neural networks (RNNs), etc.).
  • the neural network may be a deep neural network comprising of more than one layer.
  • the neural network may comprise one or more recurrent layer.
  • the one or more recurrent layer may be one or more long short-term memory (LSTM) layers or gated recurrent unit (GRU), which may perform sequential data classification and clustering.
  • the neural network may comprise one or more convolutional layers.
  • the input and output may be a tensor representing of variables or attributes in a data set (e.g., features), which may be referred to as a feature map (or activation map).
  • the convolutions may be one dimensional (ID) convolutions, two dimensional (2D) convolutions, three dimensional (3D) convolutions, or any combination thereof.
  • the convolutions may be ID transpose convolutions, 2D transpose convolutions, 3D transpose convolutions, or any combination thereof.
  • onedimensional convolutional layers may be suited for time series data since it may classify time series through parallel convolutions.
  • convolutional layers may be used for analyzing a signal or patterns in a signal (e.g., electrical currents/voltages) to the nucleoside monomer added to the polynucleotide.
  • the layers in a neural network may further comprise one or more pooling layers before or after a convolutional layer.
  • the one or more pooling layers may reduce the dimensionality of the feature map using fdters that summarize regions of a matrix. This may down sample the number of outputs, and thus reduce the parameters and computational resources needed for the neural network.
  • the one or more pooling layers may be max pooling, min pooling, average pooling, global pooling, norm pooling, or a combination thereof. Max pooling may reduce the dimensionality of the data by taking only the maximums values in the region of the matrix, which helps capture the significant feature.
  • the one or more pooling layers may be one dimensional (ID), two dimensional (2D), three dimensional (3D), or any combination thereof.
  • the neural network may further comprise of one or more flattening layers, which may flatten the input to be passed on to the next layer.
  • the input may be flattened by reducing it to a one-dimensional array.
  • the flattened inputs may be used to output a classification of an object (e.g., classification of signals (e.g., electrical currents/voltages) to a nucleoside monomer added to the polynucleotide, etc.).
  • the neural networks may further comprise one or more dropout layers.
  • Dropout layers may be used during training of the neural network (e.g., to perform binary or multi -class classifications).
  • the one or more dropout layers may randomly set certain weights as 0, which may set corresponding elements in the feature map as 0, so the neural network may avoid overfitting.
  • the neural network may further comprise one or more dense layers, which comprise a fully connected network. In the dense layer, information may be passed through the fully connected network to generate a predicted classification of an object, and the error may be calculated. In some embodiments, the error may be backpropagated to improve the prediction.
  • the one or more dense layers may comprise a Softmax activation function, which may convert a vector of numbers to a vector of probabilities. These probabilities may be subsequently used in classifications, such as classifications of signals (e.g., electrical currents and/or voltages) to the nucleoside monomer added to the polynucleotide.
  • the computer system 700 illustrated in FIG. 7 may be understood as a logical apparatus that can read instructions from media 711 and/or a network port 705, which can optionally be connected to server 709 having fixed media 712.
  • the system can include a CPU 701, disk drives 703, optional input devices such as keyboard 715 and/or mouse 716 and optional monitor 707.
  • Data communication can be achieved through the indicated communication medium to a server at a local or a remote location.
  • the communication medium can include any means of transmitting and/or receiving data.
  • the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 722.
  • FIG. 8 is a block diagram illustrating a first example architecture of a computer system that can be used in connection with example instances of the present invention.
  • the example computer system can include a processor 802 for processing instructions.
  • processors include: Intel XeonTM processor, AMD OpteronTM processor, Samsung 32-bit RISC ARM 1176JZ(F)-S vl.OTM processor, ARM Cortex-A8 Samsung S5PC100TM processor, ARM Cortex- A8 Apple A4TM processor, Marvell PXA 930TM processor, or a fimctionally-equivalent processor. Multiple threads of execution can be used for parallel processing.
  • multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.
  • a high speed cache 804 can be connected to, or incorporated in, the processor 802 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor 802.
  • the processor 802 is connected to a north bridge 806 by a processor bus 808.
  • the north bridge 806 is connected to random access memory (RAM) 810 by a memory bus 812 and manages access to the RAM 810 by the processor 802.
  • RAM random access memory
  • the north bridge 806 is also connected to a south bridge 814 by a chipset bus 816.
  • the south bridge 814 is, in turn, connected to a peripheral bus 818.
  • the peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus.
  • the north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 818.
  • the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip.
  • a system 800 can include an accelerator card 822 attached to the peripheral bus 818.
  • the accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing.
  • FPGAs field programmable gate arrays
  • an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.
  • Software and data are stored in external storage 824 and can be loaded into RAM 810 and/or cache 804 for use by the processor.
  • the system 800 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, WindowsTM, MACOSTM, BlackBerry OSTM, iOSTM, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example embodiments of the present invention.
  • system 800 also includes network interface cards (NICs) 820 and 821 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.
  • NICs network interface cards
  • NAS Network Attached Storage
  • FIG. 9 is a diagram showing a network 900 with a plurality of computer systems 902a, and 902b, a plurality of cell phones and personal data assistants 902c, and Network Attached Storage (NAS) 904a, and 904b.
  • systems 902a, 902b, and 902c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 904a and 904b.
  • a mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 902a, and 902b, and cell phone and personal data assistant systems 902c.
  • Computer systems 902a, and 902b, and cell phone and personal data assistant systems 902c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 904a and 904b.
  • FIG. 9 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various embodiments of the present invention.
  • a blade server can be used to provide parallel processing.
  • Processor blades can be connected through a back plane to provide parallel processing.
  • Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface.
  • NAS Network Attached Storage
  • processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors.
  • some or all of the processors can use a shared virtual address memory space.
  • FIG. 10 is a block diagram of a multiprocessor computer system 1000 using a shared virtual address memory space in accordance with an example embodiment.
  • the system includes a plurality of processors 1002a-f that can access a shared memory subsystem 1004.
  • the system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 1006a-f in the memory subsystem 1004.
  • MAPs programmable hardware memory algorithm processors
  • Each MAP 1006a-f can comprise a memory 1008a-f and one or more field programmable gate arrays (FPGAs) lOlOa-f.
  • the MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs lOlOa-f for processing in close coordination with a respective processor.
  • the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example embodiments.
  • each MAP is globally accessible by all of the processors for these purposes.
  • each MAP can use Direct Memory Access (DMA) to access an associated memory 1008a-f, allowing it to execute tasks independently of, and asynchronously from, the respective microprocessor 1002a-f.
  • DMA Direct Memory Access
  • a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.
  • the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems.
  • the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements.
  • FPGAs field programmable gate arrays
  • SOCs system on chips
  • ASICs application specific integrated circuits
  • the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card.
  • EXAMPLE 1 Fabrication of a graphene device
  • Any array of addressable devices shown in FIG. 2 is constructed into a chip having a size of 4 to 16 mm 2 , and a pitch distance of 50-1000 nm using the general methods of graphene FET fabrication described in US 9,859,394, incorporated by reference in its entirety.
  • EXAMPLE 2 Sequencing with a graphene device
  • a device of Example 1 is used to sequence nucleic acids.
  • a device is contacted with a nucleic acid template, at least one primer which is configured to bind to the nucleic acid template, and a polymerase.
  • the polymerase comprises a pyrene moiety linked to a Phi29 polymerase.
  • the primer, sensor, and nucleic acid template form a ternary complex which is bound to the graphene surface of the device,
  • a mixture of four nucleotides is contacted with the device and the template, wherein at least one nucleic acid generates a uniquely detectable signal from the device upon interaction with the polymerase.
  • terminators are removed from the incorporated nucleotides. The process is repeated to establish the identity of each base added, thus determining the sequence of the nucleic acid template.
  • the ternary complexes are washed off the device and the device is reused.
  • EXAMPLE 3 Fabrication of a graphene device with buried gate and shield
  • Any array of addressable devices shown in FIG. 4A-4B is constructed into a chip having a size of 4 to 16 mm 2 , and a pitch distance of 50-1000 nm using the general methods of graphene FET fabrication described in Example 1.
  • EXAMPLE 4 Sequencing with a graphene device having a buried gate and shield
  • a device of Example 3 is used to sequence nucleic acids using the general methods of Example 2 with modification.
  • a positive voltage is applied to the buried gate. This will attract negatively charged DNA (or other similar moieties) to towards the surface and the graphene.
  • the shield layer which is at ground potential, is configured with small openings that allow the positive potential from the gate to leak out to specific locations on the chip surface. The localization of the potential will concentrate the DNA (or similar moieties) to the locations of interest on the graphene. This promotes higher loading of the devices.
  • the gate can be used to modulate the graphene potential to maximize the signal change associated with molecular events occurring on or near the graphene layer.
  • An array of devices having the general structure of FIGs. 12A-12C is fabricated by a) providing one or more base layers; b) depositing material to generate a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to generate a passive layer; f) depositing material to generate a first electrode; g) patterning the first electrode; and h) isotropically etching the passive layer, such that the edges of the first electrode are undercut and the device comprises a nanogap of about 20 nm.
  • Each electrode is fabricated from one or more of titanium, platinum, and titanium nitride.
  • the passive layer comprises an oxide.
  • a layer of gold is deposited over the electrodes.
  • the device is contacted with one or more nanowires comprising nucleic acids, wherein at least some of the nanowires bridge one or more first electrodes and second electrodes.
  • Each nanowire comprises at least one biotin functional handle (optionally connected via a tether).
  • Loading of the nanowires comprises (a) applying a voltage to the electrodes to attract the nucleic strands to the electrodes; (b) monitoring a current path between electrodes to determine if a nanowire has bridged two electrodes; and (c) when the current spikes (i.e., contact is made) turning off the voltage so that no more DNA is attracted.
  • nanowires are contacted with polymerases tethered to streptavidin to facilitate attachment of polymerases to the nanowires (e.g., similar to FIG. 11).
  • FIG. 13A-13B Following the general procedure or Example 5, an array of devices of FIGs. 13A-13B is fabricated. Devices are arrayed according to the general arrangement of FIG. 15.
  • EXAMPLE 7 Crossed fingers device with oxide layer
  • EXAMPLE 8 Self-aligned finger etch
  • a device of any of Examples 5-8 is used to sequence nucleic acids.
  • a device is contacted with a nucleic acid template, at least one primer which is configured to bind to the nucleic acid template, and a mixture of four nucleotide triphosphates (and/or CMNs) is contacted with the device and the template, wherein at least one nucleic acid generates a uniquely detectable signal from the device upon interaction with the polymerase.
  • terminators are removed from the incorporated nucleotides. The process is repeated to establish the identity of each base added (e.g., A, T, C, or G), thus determining the sequence of the nucleic acid template.
  • Gold is deposited onto the electrodes of the devices fabricated according to the general procedure in Example 6. Gold islands growth is observed without an adhesion layer, as schematically illustrated in FIG. 18A and depicted in FIG. 18B. Gold deposition after formation of an adhesion layer on the electrodes results in a continuous gold layer, as schematically illustrated in FIG. 18C.
  • Biomolecules are conjugated at the nanogaps of the device, between gold islands on the electrode surface, as illustrated in FIG. 19A, for sequencing following the general procedure of Example 9.
  • the conjugation to the gold nano islands provides lower contact resistance compared to direct binding (e.g., FIG. 19B).
  • a passivation is further utilized as shown in FIG. 20 to safeguard the field and metal routings, as described herein.
  • EXAMPLE 11 Molecular wire construct with biotin-streptavidin
  • a molecular wire construct is assembled across a nanogap in a device with gold coated electrodes as generally described in Example 10.
  • the molecular wire construct includes a biotinstreptavidin (SA) construct (e.g., FIG. 21) that can be used to link the polymerase to between electrode surfaces.
  • SA biotinstreptavidin
  • a gold coated surface is exposed to 18 mM cysteamine in water for 4 hours to functionalize the surface. Once functionalized with cysteamine, the surface is exposed to biotin-NHS in DIPEA and DMF at 60 °C for 4 hours, resulting in a gold coated surface functionalized with biotin (e.g., FIG. 22A-22B). The surface is further exposed to streptavidin to create a surface with an available streptavidin (e.g., FIG. 23A).
  • a bis-biotinylated polymerase e.g., FIG. 23B is construct using a dual -biotin tagged P29 variant RPN (FIG. 23C).
  • a bis-biotinylated polymerase is assembled to form the molecular wire construct across a nanogap of a device under the following conditions: 10 mM MOPS, pH 7.5, 10 mM KO Ac, and 5 mM Mg 2+ (FIG. 24).
  • the molecular wire construct is used for sequencing as generally described in Example 9.
  • Item 1 A method for single molecule sensing comprising: a. contacting a molecular sensor with at least one charge modulator, wherein contacting generates a change in current; b. measuring the change in current; and c. correlating the change in current with the presence or absence of the at least one charge modulator, thereby sensing the single molecule.
  • Item 2 The method of item 1, wherein the single molecule comprises a biomolecule.
  • Item 3 The method of item 2, wherein the biomolecule comprises a nucleic acid.
  • Item 4 The method of any one of items 1-3, wherein the nucleic acid comprises DNA, RNA, or a mixture thereof.
  • Item 5 The method of any one of items 1-4, wherein the charge sensor comprises a graphene- enabled field effect transistor (GeFET) or CMOS device.
  • GaFET graphene- enabled field effect transistor
  • Item 6 The method of any one of items 1-5, wherein the molecular sensor is in electrical communication with a charge sensor.
  • Item 7 The method of any one of items 1-6, wherein the molecular sensor comprises a polymerase.
  • Item 8 The method of item 7, wherein the molecular sensor comprises an isothermal polymerase.
  • Item 9 The method of item 7, wherein the molecular sensor comprises Phi29 polymerase or variant thereof.
  • Item 10 The method of any one of items 6-9, wherein the charge sensor comprises a nanowire.
  • Item 11 The method of any one of items 6-10, wherein the charge sensor bridges a gap between a first electrode and a second electrode.
  • Item 12 The method of item 11, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • Item 13 The method of item 11 or 12, wherein a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine.
  • Item 14 The method of any one of items 10-13, wherein the charge sensor comprises a linker between a surface of the first electrode or the second electrode and the polymerase.
  • Item 15 The method of item 14, wherein the linker comprises one or more components.
  • Item 16 The method of item 15, wherein the one or more components comprises a biotinstreptavidin construct.
  • Item 17 The method of item 15, wherein the one or more components comprises a SpyCatcher or a SpyTag.
  • Item 18 The method of item 15, wherein the one or more components comprises a peptide linker.
  • Item 19 The method of item 15, wherein the one or more components comprises a protein.
  • Item 20 The method of item 19, wherein the protein comprises a Clq/TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.
  • Item 21 The method of any one of items 1-20, wherein the at least one charge modulator comprises a negative or positive charge.
  • Item 22 The method of any one of items 1-21, wherein the at least one charge modulator comprises a charge modulating nucleotide (CMN).
  • CPN charge modulating nucleotide
  • Item 23 The method of any one of items 1-22, wherein contacting comprising incorporating a CMN into a polynucleotide primer.
  • Item 24 The method of item 22 or 23, wherein the CMN comprises at least one modification relative to a canonical nucleotide.
  • Item 25 The method of item 24, wherein the at least one modification comprises a modification to the base of the CMN.
  • Item 26 The method of item 25, wherein the at least one modification comprises a 7-deaza or 8-bromo modified base.
  • Item 27 The method of any one of items 24-26, wherein the CMN comprises a modification to the 5’ position.
  • Item 28 The method of item 27, wherein the at least one modification comprises a modification to a 5 ’ polyphosphate or chemical variant thereof.
  • Item 29 The method of any one of items 24-27, wherein the at least one modification comprises a thiolated or bromated phosphate.
  • Item 30 The method of item 28 or 29, wherein the polyphosphate comprises at least 3, 4, 5, 6, 8, or 10 phosphates or variants thereof.
  • Item 31 The method of any one of items 24-27, wherein the at least one modification comprises a modification to a terminal 5 ’ polyphosphate or chemical variant thereof.
  • Item 32 The method of any one of items 24-31, wherein the at least one modification comprises a polymer.
  • Item 33 The method of item 32, wherein the polymer comprises one or more of a nucleic acid chain, a peptide chain, a polysaccharide, a lipid, a synthetic polymer, and a dendrimer.
  • Item 34 The method of item 33, wherein the nucleic acid chain comprises 25-5000 bases.
  • Item 35 The method of item 33 or 34, wherein the nucleic acid chain is branched (dendrimeric).
  • Item 36 The method of any one of items 33-35, wherein the nucleic acid chain comprises a secondary structure.
  • Item 37 The method of item 36, wherein the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadraplex, and an I-motif.
  • Item 38 The method of any one of items 27-37, wherein the nucleic acid chain comprises a single strand, double strand, or triple strand.
  • Item 39 The method of any one of items 27-38, wherein the nucleic acid chain comprises at least one charge modulating chemical modification.
  • Item 40 The method of item 39, wherein the at least one charge modulating chemical modification increases the charge of the CMN relative to an unmodified nucleotide.
  • Item 41 The method of item 39, wherein the at least one charge modulating chemical modification comprises one or more of an amine, an alkylamine, a guanidinium, a quaternary amine, an imidazolium, a pyridinium, and a pyrrolidinium.
  • Item 42 The method of item 39, wherein the at least one charge modulating chemical modification decreases the charge of the CMN relative to an unmodified nucleotide.
  • Item 43 The method of item 39, wherein the at least one charge modulating chemical modification comprises one or more of a phosphate, a phosphite, a sulfonate, a sulfite, a carboxylate, a xanthate, a thiocarboxylic acid, a boranophosphonate, and a boric acid.
  • Item 44 The method of any one of items 27-43, wherein the nucleic acid chain comprises at least one sugar-modified nucleotide.
  • Item 45 The method of item 44, wherein the sugar-modified nucleotide comprises a deoxy or dideoxy nucleotide.
  • Item 46 The method of any one of items 27-45, wherein the nucleic acid chain comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid.
  • Item 47 The method of any one of items 27-46, wherein the nucleic acid chain comprises a phosphate modification.
  • Item 48 The method of item 47, wherein the phosphate modification comprises a hydrophobic group.
  • Item 49 The method of item 48, wherein the hydrophobic group comprises a straight or branched alkyl chain.
  • the phosphate modification comprises a hydrophilic group.
  • Item 51 The method of item 50, wherein the hydrophilic group comprises polyethylene glycol.
  • Item 52 The method of item 51, wherein the polyethylene glycol comprises a molecular weight of 1000-100,000 daltons.
  • Item 53 The method of item 33, wherein the peptide chain is 1-100 amino acids in length.
  • Item 54 The method of item 11, wherein the CMN comprises a charged small molecule.
  • Item 55 The method of item 54, wherein the charged small molecule comprises one or more of a chelator, a dye, and a metal complex.
  • Item 56 The method of item 55, wherein the metal complex comprises a ferrocene, Ru-dipy, and bis-cyclopentadienyl diiron.
  • Item 57 The method of any one of items 1-56, wherein the change in current is 1 nanoamp to 100 picoamps.
  • Item 58 The method of any one of items 1-56, wherein the change in current is 100 picoamps to 1 microamp.
  • Item 59 The method of any one of items 1-56, wherein the change in current is at least 1.01-3 times the background current.
  • Item 60 The method of any one of items 1-56, wherein steps a-c are repeated at least 50 times.
  • Item 61 The method of any one of items 1-56, wherein method is configured to detect 1-200 single molecules per second.
  • a chemically-sensitive field effect transistor device for sensing single molecules comprising: a solid support, wherein the solid support comprises a plurality of loci, wherein each loci comprises: a graphene layer; a gate electrode and a drain electrode, where the gate electrode and the drain electrode are in electrical communication via the graphene layer; and at least one insulating layer, where the insulating layer is located between the gate electrode and the drain electrode; wherein the loci have a pitch of 50-1000 nanometers.
  • Item 63 The device of 62, wherein the device further comprises at least one ground shield.
  • Item 64 The device of item 63, wherein the at least one ground shield is at ground potential.
  • Item 65 The device of any one of items 62-64, wherein the device further comprises at least one buried gate.
  • Item 66 The device of any one of items 63-65, wherein the at least one ground shield comprises an opening which permits electrical communication between the graphene layer and the least one buried gate.
  • Item 67 The device of any one of items 62-66, wherein the graphene layer is approximately one atom thick.
  • Item 68 The device of any one of items 62-67, wherein the device comprises 100 to 1 billion loci.
  • Item 69 The device of any one of items 62-68, wherein each loci is 50-200 nm in size.
  • Item 70 The device of any one of items 62-69, wherein each loci is a well, channel, or is substantially planer.
  • Item 71 The device of any one of items 62-70, wherein the device is 4 to 900 mm 2 .
  • Item 72 The device of any one of items 62-70, wherein the device is 4 to 16 mm 2 .
  • Item 73 The device of any one of items 62-70, wherein the device is 200 to 2000 mm 2 .
  • Item 74 A method for single molecule polynucleotide sequencing comprising: a) contacting a plurality of polynucleotides with at least one primer and at least one polymerase to form a plurality of ternary complexes, wherein the ternary complex comprising a graphene binder; b) detecting one or more bases of the polynucleotides in real time, wherein detection occurs when the plurality of ternary complexes are bound to the graphene layer of any one of items 62-73; c) removing the ternary complexes from the surface; and d) repeating steps a-c to sequence the polynucleotides.
  • Item 75 The method of item 74, wherein the graphene binder comprises an aromatic group.
  • Item 76 The method of item 74 or 75, wherein the graphene binder comprises an aryl or heteroaryl group.
  • Item 77 The method of any one of items 74-76, wherein the graphene binder comprises a Ce- C30 aryl or heteroaryl group.
  • Item 78 The method of any one of items 74-77, wherein the graphene binder comprises an aromatic hydrocarbon.
  • Item 79 The method of any one of items 74-78, wherein the graphene binder comprises naphthalene, biphenyl, fluorene, anthracene, phenanthrene, tetracene, chrysene, triphenylene, pyrene, pentacene, perylene, benzo [a]pyrene, corannulene, benzo [ghi]perylene, coronene, ovalene, or benzofc] fluorene.
  • the graphene binder comprises naphthalene, biphenyl, fluorene, anthracene, phenanthrene, tetracene, chrysene, triphenylene, pyrene, pentacene, perylene, benzo [a]pyrene, corannulene, benzo [ghi]perylene, coronene, ovalene, or benzofc] fluorene.
  • Item 80 The method of any one of items 74-79, wherein step c) comprises washing the surface.
  • Item 81 The method of items 74-80, wherein the ternary complex is attached to the graphene binder via the primer, the polymerase, or the polynucleotide library.
  • Item 82 The method of items 74-81, wherein the ternary complex is attached via a linker.
  • Item 83 The method of items 74-82, wherein the ternary complex is attached via a linker using a conjugation.
  • Item 84 The method of items 83, wherein the conjugation comprises nucleophile/carbonyl; an azide/phosphine; 1,4 Michael addition, 1,3-dipolar cycloaddition, inverse electron demand cycloaddition; olefin metathesis; or cross-coupling reaction.
  • Item 85 The method of any one of items 74-84, wherein removing comprises contacting the surface with a solvent.
  • Item 86 The method of item 85, wherein the solvent comprises an organic solvent.
  • Item 87 The method of item 86, wherein the organic solvent comprises MeCN, methanol, ethanol, 2-propanol, acetone, DMF, formamide, THF, or DMSO.
  • Item 88 The method of item 86 or 87, wherein the organic solvent is heated.
  • Item 89 The method of any one of items 74-88, wherein the polymerase comprises a Phi29 polymerase or variant thereof.
  • Item 90 The method of any one of items 74-89, wherein the polymerase is configured for incorporation charge modified nucleotides of any one of items 22-56.
  • Item 91 The method of any one of items 74-90, wherein the polymerase is bound to the surface in step a).
  • Item 92 The method of any one of items 74-90, wherein the polymerase is not bound to the surface in step a).
  • Item 93 The method of any one of items 74-92, wherein the plurality of polynucleotides comprises at least 100,000 unique polynucleotides.
  • Item 94 The method of any one of items 74-93, wherein the plurality of polynucleotides are 50-30,000 bases in length.
  • Item 95 The method of any one of items 74-94, wherein detecting comprises contacting the ternary complexes with at least one nucleotide.
  • Item 96 The method of any one of items 74-95, wherein detecting comprises measuring a change in current when a CMN is incorporated.
  • Item 97 The method of any one of items 74-96, wherein the buried gate has a positive potential during step (a).
  • Item 98 The method of any one of items 74-97, wherein the buried gate has a positive or negative potential during step (b).
  • a device for molecular sensing comprising: a first electrode, wherein the first electrode comprises a neck region; a passive layer; a second electrode, and wherein the first electrode and the second electrode are located above the first base layer; wherein a first portion of the neck region overlaps a first portion of the second electrode, such that the first electrode and the second electrode are separated by a nanogap; wherein a second portion of the first electrode and a second portion of the second electrode are separated by the passive layer; and a first base layer and a second base layer, wherein the first base layer is located above the second base layer, and the first electrode and the second electrode are located above the base layers.
  • Item 100 The device of item 99, wherein the nanogap is 1-50 nm.
  • Item 101 The device of item 99, wherein the nanogap is 10-30 nm.
  • Item 102 The device of item 99, wherein the nanogap is no more than 50 nm.
  • Item 103 The device of any one of items 99-102, wherein the passive layer comprises an oxide.
  • Item 104 The device of item 103, wherein the oxide comprises silicon, nitride, or carbide.
  • Item 105 The device of any one of items 99-104, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.
  • Item 106 The device of any one of items 99-105, wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • Item 107 The device of item 106, wherein the layer of gold is no more than 75 angstroms thick.
  • Item 108 The device of item 106, wherein the superficial layer of gold is deposited above an adhesion layer.
  • Item 109 The device of item 108, wherein the adhesion layer comprises titanium or chromium.
  • Item 110 The device of any one of items 99-109, wherein the first electrode and the second electrode each comprise gold nano-islands.
  • Item 111 The device of any one of items 99-107, wherein the first base layer comprises silicon oxide or silicon nitride.
  • Item 112. The device of any one of items 99-111, wherein the second base layer comprises silicon.
  • Item 113 The device of any one of items 99-112, wherein the device further comprises a charge sensor, wherein the charge sensor is in electrical communication with the first electrode and the second electrode.
  • Item 114 The device of item 113, wherein the charge sensor is attached to the first electrode and the second electrode.
  • Item 115 The device of any one of items 113-114, wherein the charge sensor comprises a polymer.
  • Item 116 The device of item 115, wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.
  • Item 117 The device of any one of items 113-116, wherein the charge sensor comprises carbon.
  • Item 118 The device of any one of items 113-117, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • Item 119 The device of item 118, wherein a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine.
  • Item 120 The device of any one of items 113-118, wherein the charge sensor is further attached to a molecular sensor via a tether.
  • Item 121 The device of item 120, wherein the tether comprises one or more components.
  • Item 122 The device of item 121, wherein the one or more components comprises a biotinstreptavidin construct.
  • Item 123 The device of item 122, wherein the one or more components comprises a SpyCatcher or a SpyTag.
  • Item 124 The device of item 122, wherein the one or more components comprises a peptide linker.
  • Item 125 The device of item 122, wherein the one or more components comprises a protein.
  • Item 126 The device of item 125, wherein the protein comprises a Clq/TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.
  • Item 127 The device of item 120, wherein the molecular sensor comprises an enzyme.
  • Item 128 The device of item 120, wherein the molecular sensor comprises an antibody.
  • Item 129 The device of item 127, wherein the enzyme comprises a polymerase.
  • Item 130 The device of any one of items 99-129, wherein the longest linear dimension of the second electrode is perpendicular to the neck region.
  • Item 131 The device of any one of items 99-129, wherein the longest linear dimension of the second electrode is parallel to the neck region.
  • Item 132 The device of any one of items 99-131, wherein at least one edge of the first electrode is undercut relative to the passive layer.
  • Item 133 The device of any one of items 99-132, wherein the surface area of the first electrode is less than the surface area of the second electrode.
  • Item 134 The device of any one of items 99-133, wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region.
  • Item 135. The device of any one of items 99-134, wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • Item 136 The device of item 135, wherein the superficial layer of gold is deposited with an adhesion layer.
  • Item 137 The device of item 136, wherein the adhesion layer comprises titanium or chromium.
  • Item 138 The device of any one of items 99-137, wherein the first electrode and the second electrode each comprise gold nano-islands.
  • Item 139 The device of any one of items 99-138, wherein the first base layer comprises silicon oxide or silicon nitride.
  • Item 140 The device of any one of items 99-139, wherein the second base layer comprises silicon.
  • Item 141 The device of any one of items 99-140, wherein the neck region has a width of no more than 200 nm.
  • a device for molecular sensing comprising: a first electrode, wherein the first electrode is located above the first base layer; a second electrode, wherein the second electrode comprises a neck region; wherein a first portion of the neck region overlaps a first portion of the first electrode, such that the first electrode and the second electrode are separated by a nanogap; wherein a second portion of the first electrode and a second portion of the second electrode are separated by a passive layer; a first base layer and a second base layer, wherein the first base layer is located above the second base layer, and the first electrode and the second electrode are located above the base layers.
  • Item 143 The device of item 142, wherein the passive layer is configured to overlap with a portion of the second electrode.
  • Item 144 The device of item 142, wherein the passive layer is configured to passivate electrode traces.
  • Item 145 The device of any one of items 142-144, wherein the nanogap is 1-50 nm.
  • Item 146 The device of any one of items 142-144, wherein the nanogap is 10-30 nm.
  • Item 147 The device of any one of items 142-144, wherein the nanogap is no more than 50 nm.
  • Item 148 The device of any one of items 142-147, wherein the passive layer comprises an oxide.
  • Item 149 The device of item 148, wherein the oxide comprises silicon, nitride, or carbide.
  • Item 150 The device of any one of items 142-149, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.
  • Item 151 The device of any one of items 142-150, wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • Item 152 The device of item 151, wherein the superficial layer of gold is no more than 75 angstroms thick.
  • Item 153 The device of item 151 wherein the superficial layer of gold is deposited above an adhesion layer.
  • Item 154 The device of item 152, wherein the adhesion layer comprises titanium or chromium.
  • Item 155 The device of any one of items 142-150, wherein the first electrode and the second electrode each comprise gold nano-islands.
  • Item 156 The device of any one of items 142-152, wherein the first base layer comprises silicon oxide or silicon nitride.
  • Item 157 The device of any one of items 142-156, wherein the second base layer comprises silicon.
  • Item 158 The device of any one of items 142-157, wherein the device further comprises a charge sensor, wherein the charge sensor is in electrical communication with the first electrode and the second electrode.
  • Item 159 The device of item 158, wherein the charge sensor is attached to the first electrode and the second electrode.
  • Item 160 The device of any one of items 158-159, wherein the charge sensor comprises a polymer.
  • Item 161 The device of item 160, wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.
  • Item 162 The device of any one of items 158-161, wherein the charge sensor comprises carbon.
  • Item 163. The device of any one of items 158-162, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • Item 164 The device of item 163, wherein a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine.
  • Item 165 The device of any one of items 158-163, wherein the charge sensor is further attached to a molecular sensor via a tether.
  • Item 166 The device of item 165, wherein the tether comprises one or more components.
  • Item 167 The device of item 166, wherein the one or more components comprises a biotinstreptavidin construct.
  • Item 168 The device of item 167, wherein the one or more components comprises a SpyCatcher or a SpyTag.
  • Item 169 The device of item 167, wherein the one or more components comprises a peptide linker.
  • Item 170 The device of item 167, wherein the one or more components comprises a protein.
  • Item 171 The device of item 170, wherein the protein comprises a Clq/TNF -related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.
  • Item 172 The device of item 165, wherein the molecular sensor comprises an enzyme.
  • Item 173. The device of item 165, wherein the molecular sensor comprises an antibody.
  • Item 174 The device of item 172, wherein the enzyme comprises a polymerase.
  • Item 175. The device of any one of items 142-174, wherein the longest linear dimension of the second electrode is perpendicular to the neck region.
  • Item 176 The device of any one of items 142-174, wherein the longest linear dimension of the second electrode is parallel to the neck region.
  • Item 177 The device of any one of items 142-176, wherein at least one edge of the first electrode is undercut relative to the passive layer.
  • Item 178 The device of any one of items 142-177, wherein the surface area of the first electrode is less than the surface area of the second electrode.
  • Item 179 The device of any one of items 142-178, wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region.
  • Item 180 The device of any one of items 142-179, wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • Item 181. The device of any one of items 142-180, wherein the first base layer comprises silicon oxide or silicon nitride.
  • Item 182. The device of any one of items 142-181, wherein the second base layer comprises silicon.
  • Item 183 The device of any one of items 142-182, wherein the neck region has a width of no more than 200 nm.
  • a device for molecular sensing comprising: a first electrode, wherein the first electrode comprises a neck region; a passive layer, wherein the passive layer comprises a channel or well, where the bottom of the well or channel comprises the first base layer; a second electrode, and wherein the first electrode and the second electrode are located above the first base layer, and the second electrode is at least partially embedded in the passive layer; wherein a first portion of the neck region overlaps a first portion of the second electrode, such that the first electrode and the second electrode are separated by a nanogap; wherein a second portion of the first electrode and a second portion of the second electrode are separated by the passive layer; and a first base layer and a second base layer, wherein the first base layer is located above the second base layer, and the first electrode and the second electrode are located above the base layers.
  • Item 185 The device of item 184, wherein the nanogap is 1-50 nm.
  • Item 186 The device of item 184, wherein the nanogap is 10-30 nm.
  • Item 187 The device of item 184, wherein the nanogap is no more than 50 nm.
  • Item 188 The device of any one of items 184-187, wherein the passive layer comprises an oxide.
  • Item 189 The device of items 188, wherein the oxide comprises silicon, nitride, or carbide.
  • Item 190 The device of any one of items 184-189, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.
  • Item 191. The device of any one of items 184-190, wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • Item 192 The device of item 191, wherein the layer of gold is no more than 75 angstroms thick.
  • Item 193 The device of item 191, wherein the superficial layer of gold is deposited above an adhesion layer.
  • Item 194. The device of item 192, wherein the adhesion layer comprises titanium or chromium.
  • Item 195 The device of any one of items 184-190, wherein the first electrode and the second electrode each comprise gold nano-islands.
  • Item 196 The device of any one of items 184-192, wherein the first base layer comprises silicon oxide or silicon nitride.
  • Item 197 The device of any one of items 184-196, wherein the second base layer comprises silicon.
  • Item 198 The device of any one of items 184-197, wherein the device further comprises a charge sensor, wherein the charge sensor is in electrical communication with the first electrode and the second electrode.
  • Item 199 The device of item 198, wherein the charge sensor is attached to the first electrode and the second electrode.
  • Item 200 The device of any one of items 198-199, wherein the charge sensor comprises a polymer.
  • Item 201 The device of item 200, wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.
  • Item 202 The device of any one of items 198-201, wherein the charge sensor comprises carbon.
  • Item 203 The device of any one of items 198-202, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interaction.
  • Item 204 The device of item 203, wherein a surface of the first electrode or the second electrode is functionalized with a thiol-biotin, a terminal cysteine, or a cysteamine.
  • Item 205 The device of any one of items 198-203, wherein the charge sensor is further attached to a molecular sensor via a tether.
  • Item 206 The device of item 205, wherein the tether comprises one or more components.
  • Item 207 The device of item 206, wherein the one or more components comprises a biotinstreptavidin construct.
  • Item 208 The device of item 206, wherein the one or more components comprises a SpyCatcher or a SpyTag.
  • Item 209 The device of item 206, wherein the one or more components comprises a peptide linker.
  • Item 210 The device of item 206, wherein the one or more components comprises a protein.
  • Item 211 The device of item 210, wherein the protein comprises a Clq/TNF -related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.
  • Item 212 The device of item 205, wherein the molecular sensor comprises an enzyme.
  • Item 213. The device of item 205, wherein the molecular sensor comprises an antibody.
  • Item 214 The device of item 212, wherein the enzyme comprises a polymerase.
  • Item 215. The device of any one of items 184-214, wherein the longest linear dimension of the second electrode is perpendicular to the neck region.
  • Item 216 The device of any one of items 184-214, wherein the longest linear dimension of the second electrode is parallel to the neck region.
  • Item 217 The device of any one of items 184-216, wherein at least one edge of the first electrode is undercut relative to the passive layer.
  • Item 218 The device of any one of items 184-217, wherein the surface area of the first electrode is less than the surface area of the second electrode.
  • Item 219. The device of any one of items 184-218, wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region.
  • Item 220 The device of any one of items 184-219, wherein the first electrode and the second electrode each comprise a superficial layer of gold.
  • Item 221. The device of any one of items 184-220, wherein the first base layer comprises silicon oxide or silicon nitride.
  • Item 222 The device of any one of items 184-221, wherein the second base layer comprises silicon.
  • Item 223. The device of any one of items 184-222, wherein the neck region has a width of no more than 200 nm.
  • Item 224 An array of any one of devices 99-223, wherein at least some of the first electrodes and second electrodes are independently addressable.
  • Item 225 The array of item 224, wherein the pitch distance of the nanogaps of at least some of the devices is no more than 200 nanometers.
  • Item 226 The array of any one of items 224-225, wherein at least some of the first electrode and second electrode are independently addressable.
  • Item 227 The array of any one of items 224-226, wherein at least some of the first electrode and second electrode are independently addressable on an x-y axis.
  • Item 228 The array of any one of items 224-226, wherein at least some of the first electrode and second electrode are independently addressable on a z axis.
  • Item 229. The array of any one of items 224-228, wherein the array comprises at least 50 devices of any one of items 99-223.
  • Item 230 The array of any one of items 224-229, wherein the array comprises at least 5000 devices of any one of items 99-223.
  • Item 231. The array of any one of items 224-230, wherein the array comprises at least 100,000 devices of any one of items 99-223.
  • Item 232 The array of any one of items 224-231, wherein the pitch distance of the nanogaps of at least some of the devices is no more than 2 micron.
  • Item 233 The array of any one of items 224-232, wherein the array further comprises a plurality of vias, wherein the plurality of vias are configured to connect at least two vertical layers of the device.
  • Item 234. The array of any one of items 224-233, wherein the array further comprises a plurality of routing connections, wherein the plurality of routing connections are configured for addressable control of each device in the array.
  • Item 235 A method of fabricating the device of any one of items 99-223, wherein the method comprises: a) providing one or more base layers; b) depositing material to generate a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to generate a passive layer; f) depositing material to generate a first electrode; g) patterning the first electrode; and h) isotropically etching the passive layer, such that the edges of the first electrode are undercut. [00447] Item 236. The method of items 235, wherein the method further comprises depositing gold on the first top layer of the device.
  • Item 237 The method of any one of items 235-236, wherein the one or more base layers comprise thermal oxide on silicon.
  • Item 238 The method of any one of items 235-237, wherein the method comprises etching or lithography.
  • Item 239. The method of any one of items 235-238, wherein the method comprises RIE (reactive ion etching).
  • Item 240 The method of any one of items 235-239, wherein patterning comprises lithography and/or RIE.
  • Item 241. The method of any one of items 235-240, wherein the method does not comprise e- beam or DUV (deep ultraviolet light) lithography.
  • Item 242. The method of any one of items 235-241, wherein the method comprises deposition of gold on the first electrode and the second electrode.
  • Item 243 The method of any one of items 235-242, wherein the first electrode and the second electrode are separated by a nanogap.
  • Item 244. The method of item 243, wherein the nanogap is 1-50 nm.
  • Item 245. The method of item 243, wherein the nanogap is 10-30 nm.
  • Item 246 The method of item 243, wherein the nanogap is no more than 50 nm.
  • Item 247 The method of any one of items 235-246, wherein the passive layer comprises an oxide.
  • Item 248 The method of items 247, wherein the oxide comprises silicon, nitride, or carbide.
  • Item 249. The method of any one of items 235-248, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.
  • Item 250 A method of using a device of any one of items 99-223 for molecular sensing, comprising: a. providing an analyte; b. reacting, binding, or allowing the analyte to interact with the sensor; and c. measuring an electrical signal generated from the sensor.
  • Item 251 The method of item 250, wherein the method comprises: a. providing at least one nucleotide triphosphate, at least one template, and at least one primer; b. extending the primer by the at least one nucleotide triphosphate; and c. measuring an electrical signal generated from the polymerase.
  • Item 252 The method of item 251, wherein the method further comprises analyzing the electrical signal to establish the identity of the at least one nucleotide triphosphate.
  • Item 253 The method of any one of items 251-252, wherein the at least one nucleotide triphosphate comprises a non-canonical base.
  • Item 254 The method of any one of items 251-253, wherein the at least one nucleotide triphosphate comprises a terminator which is configured to prevent chain extension.
  • Item 255 The method of any one of items 251-254, wherein the method is repeated to establish the identity of at least 20 bases.
  • Item 256 The method of any one of items 251-255, wherein the method is repeated to establish the identity of at least 100 bases.
  • Item 257 The method of any one of items 251-256, wherein the method is repeated to establish the identity of at least 1000 bases.

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  • Biophysics (AREA)
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Abstract

L'invention concerne des compositions, des dispositifs, des systèmes et des méthodes de détection de molécule unique. L'invention concerne en outre des dispositifs de séquençage d'acides nucléiques. Les compositions, les dispositifs, les systèmes et les méthodes décrits ici permettent une récupération améliorée d'informations basées sur des biomolécules.
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