Nanoelectrical devices and uses thereof

Abstract

Provided herein are compositions, devices, systems, and methods for single molecule sensing. Further provided are devices for nucleic acid sequencing. The compositions, devices, systems, and methods described herein provide improved retrieval of biomolecule-based information.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 386,466, filed December 7, 2022, and U.S. Provisional Patent Application No. 63 / 506,670, filed June 7, 2023, which are incorporated by reference herein in their entireties. All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Background technology]

[0002] Biomolecule-based information storage systems, such as those based on DNA, have large storage capacities and stability over time. However, there is a need for scalable, automated, highly accurate, and efficient systems for generating and reading biomolecules for information storage. Summary of the Invention

[0003] Provided herein are devices and methods for the detection of biomolecules.

[0004] Provided herein is a method for biomolecular sensing, comprising: (a) contacting a molecular sensor with at least one charge modulator, wherein the contact results in a change in current; (b) measuring the change in current; and (c) sensing a single molecule by correlating the change in current with the presence or absence of the at least one charge modulator. Further provided herein is a method wherein the single molecule comprises a biomolecule. Further provided herein is a method wherein the biomolecule comprises a nucleic acid. Further provided herein is a method wherein the nucleic acid comprises DNA, RNA, or a mixture thereof. Further provided herein is a method wherein the charge sensor comprises a graphene-enabled field-effect transistor (GeFET) or a CMOS device. Further provided herein is a method wherein the molecular sensor is in electrical communication with the charge sensor. Further provided herein is a method wherein the molecular sensor comprises a polymerase. Further provided herein is a method wherein the molecular sensor comprises an isothermal polymerase. Further provided herein is a method wherein the molecular sensor comprises Phi29 polymerase or a variant thereof. Further provided herein is a method wherein the charge sensor comprises a nanowire. Further provided herein is a method in which a charge sensor fills the gap between the first electrode and the second electrode. Further provided herein is a method in which the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interactions. Further provided herein is a method in which the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine. Further provided herein is a method in which the charge sensor comprises a linker between the surface of the first electrode or the second electrode and the polymerase. Further provided herein is a method in which the linker comprises one or more components. Further provided herein is a method in which one or more components comprises a biotin-streptavidin construct. Further provided herein is a method in which one or more components comprises SpyCatcher or SpyTag. Further provided herein is a method in which one or more components comprises a peptide linker. Further provided herein is a method in which one or more components comprises a protein.Further provided herein are methods in which the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein are methods in which at least one charge modulator comprises a negative or positive charge. Further provided herein are methods in which at least one charge modulator comprises a charge-modifying nucleotide (CMN). Further provided herein are methods in which the contacting step comprises incorporating a CMN into a polynucleotide primer. Further provided herein are methods in which the CMN comprises at least one modification relative to a reference nucleotide. Further provided herein are methods in which the modification comprises a modification to the base of the CMN. Further provided herein are methods in which the modification comprises a 7-deaza or 8-bromo modified base. Further provided herein are methods in which the CMN comprises a modification to the 5' position. Further provided herein are methods in which the modification comprises a modification to a 5' polyphosphate or chemical variant thereof. Further provided herein are methods in which the modification comprises a thiolated or brominated 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 a chemical variant thereof. Further provided herein are methods wherein the modification comprises a polymer. Further provided herein are methods 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. Further provided herein are methods wherein the nucleic acid chain comprises 25 to 5,000 bases. Further provided herein are methods wherein the nucleic acid chain is branched (dendrimer). Further provided herein are methods wherein the nucleic acid chain comprises a secondary structure. Further provided herein are methods wherein the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadruplex, and an I-motif. Further provided herein are methods wherein the nucleic acid chain comprises a single strand, a double strand, or a 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 in which at least one charge-modulating chemical modification increases the charge of CMN relative to an unmodified nucleotide. Further provided herein are methods in which at least one charge-modulating chemical modification comprises one or more of an amine, alkylamine, guanidinium, quaternary amine, imidazolium, pyridinium, and pyrrolidinium. Further provided herein are methods in which at least one charge-modulating chemical modification decreases the charge of CMN relative to an unmodified nucleotide. Further provided herein are methods in which at least one charge-modulating chemical modification comprises one or more of a phosphate, phosphite, sulfonate, sulfite, carboxylate, xanthate, thiocarboxylic acid, boranophosphonate, and boric acid. Further provided herein are methods in which the nucleic acid strand comprises at least one sugar-modified nucleotide. Further provided herein are methods in which the sugar-modified nucleotide comprises a deoxynucleotide or dideoxynucleotide. Further provided herein are methods in which the nucleic acid strand comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid. Further provided herein are methods wherein the nucleic acid chain comprises a phosphate modification. Further provided herein are methods wherein the phosphate modification comprises a hydrophobic group. Further provided herein are methods wherein the hydrophobic group comprises a straight-chain or branched-chain alkyl. Further provided herein are methods wherein the phosphate modification comprises a hydrophilic group. Further provided herein are methods wherein the hydrophilic group comprises polyethylene glycol. Further provided herein are methods wherein the polyethylene glycol comprises a molecular weight of 1,000 to 100,000 daltons. Further provided herein are methods wherein the peptide chain is 1 to 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 ferrocene, Ru-dipy, and bis-cyclopentadienyldiirone. Further provided herein are methods wherein the change in current is 1 nanoampere to 100 picoamperes.Further provided herein is a method wherein the change in current is between 100 picoamperes and 1 microampere. Further provided herein is a method wherein the change in current is at least 1.01 to 3 times the background current. Further provided herein is a method wherein steps a to c are repeated at least 50 times. Further provided herein is a method configured to detect between 1 and 200 biomolecules per second.

[0005] Provided herein is a chemically sensitive field effect transistor device comprising: a solid support including a plurality of loci each including a graphene layer; gate and drain electrodes in electrical communication through the graphene layer; and at least one insulating layer positioned between the gate and drain electrodes, wherein the loci have a pitch of 50 to 1000 nanometers. Further provided herein is a device further comprising at least one ground shield. Further provided herein is a device wherein the at least one ground shield is at ground potential. Further provided herein is a device further comprising at least one buried gate. Further provided herein is a device wherein the at least one ground shield comprises an opening that allows electrical communication between the graphene layer and the at least one buried gate. Further provided herein is a device wherein the graphene layer is approximately one atom thick. Further provided herein is a device comprising 10 to 1 billion loci. Further provided herein is a device wherein each loci is 50 to 200 nm in size. Further provided herein is a device wherein each loci is a well, a channel, or is substantially planar. Further provided herein is a device wherein the size is 4 to 2000 mm. 2 Further provided herein is a device having a diameter of 4 to 16 mm. 2 Further provided herein is a device having a thickness of 200 to 900 mm 2 Further provided herein is a device having a thickness of 4 to 900 mm 2 Further provided herein is a device,

[0006] Provided herein is a method for sequencing single-molecule polynucleotides, the method 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 complexes comprise a graphene binder; b) detecting one or more bases of the polynucleotides in real time, wherein the detection occurs upon binding of the plurality of ternary complexes to a graphene layer described herein; c) removing the ternary complexes from the surface; and d) repeating steps a-c to sequence the polynucleotides. Further provided herein is a method wherein the graphene binder comprises an aromatic group. Further provided herein is a method wherein the graphene binder comprises an aryl or heteroaryl group. Further provided herein is a method wherein the graphene binder comprises a C6-C30 aryl or heteroaryl group. Further provided herein is a method wherein the graphene binder comprises an aromatic hydrocarbon. Further provided herein is a method in which 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. Further provided herein is a method in which step c) comprises washing the surface. Further provided herein is a method in which the ternary complex is bound to the graphene binder via a primer, a polymerase, or a polynucleotide library. Further provided herein is a method in which the ternary complex is bound via a linker. Further provided herein is a method in which the ternary complex is bound via a linker using conjugation. Further provided herein is a method in which the conjugation comprises a nucleophile / carbonyl, azide / phosphine, 1,4 Michael addition, 1,3-dipolar cycloaddition, inverse electron demand cycloaddition, olefin metathesis, or cross-coupling reaction. Further provided herein is a method, wherein the removing step comprises contacting the surface with a solvent. Further provided herein is a method, wherein the solvent comprises an organic solvent.Further provided herein are methods wherein 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 Phi29 polymerase or a variant thereof. Further provided herein are methods wherein the polymerase is configured for incorporation of charge-modified nucleotides as described herein. Further provided herein are methods wherein the polymerase is bound to a surface in step a). Further provided herein are methods wherein the polymerase is not bound to a surface in step a). Further provided herein are methods wherein the plurality of polynucleotides comprises at least 100,000 unique polynucleotides. Further provided herein are methods wherein the plurality of polynucleotides is 50-30,000 bases in length. Further provided herein are methods wherein the detecting step comprises contacting the ternary complex with at least one nucleotide. Further provided herein are methods wherein the detecting step comprises measuring a change in current when CMN is incorporated. Further provided herein is a method wherein during step (a) the buried gate has a positive potential. Further provided herein is a method wherein during step (b) the buried gate has a positive or negative potential.

[0007] Provided herein is a device for molecular sensing, comprising: a first electrode including a neck region; a passivation layer; a second electrode, wherein the first electrode and the second electrode are disposed on a first base layer, a first portion of the neck region overlapping a first portion of the second electrode such that the first electrode and the second electrode are separated by a nanogap, and a second portion of the first electrode and a second portion of the second electrode are separated by a passivation layer; and first and second base layers, wherein the first base layer is disposed on the second base layer, and the first electrode and the second electrode are disposed on the base layer. Further provided herein is a device having a nanogap of 1 to 50 nm. Further provided herein is a device having a nanogap of 10 to 30 nm. Further provided herein is a device having a nanogap of 50 nm or less. Further provided herein is a device in which the passivation layer comprises an oxide. Further provided herein is a device in which the oxide comprises silicon, nitride, or carbide. Further provided herein is a device in which the first electrode and the second electrode comprise platinum, titanium nitride, or titanium. Further provided herein is a device in which the first electrode and the second electrode each comprise a gold surface layer. Further provided herein is a device in which the gold layer is 75 angstroms or less in thickness. Further provided herein is a device in which the gold surface layer is deposited on an adhesion layer. Further provided herein is a device in which the adhesion layer comprises titanium or chromium. Further provided herein is a device in which the first electrode and the second electrode each comprise gold nanoislands. Further provided herein is a device in which the first base layer comprises silicon oxide or silicon nitride. Further provided herein is a device in which the second base layer comprises silicon. Further provided herein is a device in which the device further comprises a charge sensor, the charge sensor being in electrical communication with the first electrode and the second electrode. Further provided herein is a device in which the charge sensor is attached to the first electrode and the second electrode. Further provided herein is a device, wherein the charge sensor comprises a polymer.Further provided herein is a device wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid. Further provided herein is a device wherein the charge sensor comprises carbon. Further provided herein is a device wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interactions. Further provided herein is a device wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine. Further provided herein is a device wherein the charge sensor is further attached to the molecular sensor via a tether. Further provided herein is a device wherein the tether comprises one or more components. Further provided herein is a device wherein one or more components comprises a biotin-streptavidin construct. Further provided herein is a device wherein one or more components comprises SpyCatcher or SpyTag. Further provided herein is a device wherein one or more components comprises a peptide linker. Further provided herein is a device wherein one or more components comprises a protein. Further provided herein is a device wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein is a device wherein the molecular sensor comprises an enzyme. Further provided herein is a device wherein the molecular sensor comprises an antibody. Further provided herein is a device wherein the enzyme comprises a polymerase. Further provided herein is a device wherein the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein is a device wherein the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein is a device wherein at least one edge of the first electrode is undercut relative to the passivation layer. Further provided herein is a device wherein the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode. Further provided herein is a device wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region. Further provided herein is a device wherein each of the first electrode and the second electrode comprises a gold surface layer. Further provided herein is a device, wherein the first base layer comprises silicon oxide or silicon nitride.Further provided herein is a device, wherein the second base layer comprises silicon.Further provided herein is a device, wherein the neck region is 200 nm or less in width.

[0008] Provided herein is a device for molecular sensing, comprising: a first electrode disposed on a first base layer; a second electrode including 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, and a second portion of the first electrode and a second portion of the second electrode are separated by a passivation layer; and first and second base layers, wherein the first base layer is disposed on the second base layer and the first electrode and the second electrode are disposed on the base layer. Further provided herein is a device in which the passivation layer is configured to overlap a portion of the second electrode. Further provided herein is a device in which the passivation layer is configured to passivate electrode traces. Further provided herein is a device in which the nanogap is 1 to 50 nm. Further provided herein is a device in which the nanogap is 10 to 30 nm. Further provided herein is a device in which the nanogap is 50 nm or less. Further provided herein is a device in which the passivation layer comprises an oxide. Further provided herein is a device in which the oxide comprises silicon, nitride, or carbide. Further provided herein is a device in which the first electrode and the second electrode comprise platinum, titanium nitride, or titanium. Further provided herein is a device in which the first electrode and the second electrode each comprise a gold surface layer. Further provided herein is a device in which the gold layer is 75 angstroms or less in thickness. Further provided herein is a device in which the gold surface layer is deposited on an adhesion layer. Further provided herein is a device in which the adhesion layer comprises titanium or chromium. Further provided herein is a device in which the first electrode and the second electrode each comprise gold nanoislands. Further provided herein is a device in which the first base layer comprises silicon oxide or silicon nitride. Further provided herein is a device in which the second base layer comprises silicon. Further provided herein is a device in which the device further comprises a charge sensor, the charge sensor being in electrical communication with the first electrode and the second electrode. Further provided herein is a device, wherein a charge sensor is attached to the first electrode and the second electrode.Further provided herein is a device wherein the charge sensor comprises a polymer. Further provided herein is a device wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid. Further provided herein is a device wherein the charge sensor comprises carbon. Further provided herein is a device wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interactions. Further provided herein is a device wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine. Further provided herein is a device wherein the charge sensor is further attached to the molecular sensor via a tether. Further provided herein is a device wherein the tether comprises one or more components. Further provided herein is a device wherein one or more components comprises a biotin-streptavidin construct. Further provided herein is a device wherein one or more components comprises SpyCatcher or SpyTag. Further provided herein is a device wherein one or more components comprises a peptide linker. Further provided herein is a device wherein one or more components comprises a protein. Further provided herein is a device wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein is a device wherein the molecular sensor comprises an enzyme. Further provided herein is a device wherein the molecular sensor comprises an antibody. Further provided herein is a device wherein the enzyme comprises a polymerase. Further provided herein is a device wherein the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein is a device wherein the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein is a device wherein at least one edge of the first electrode is undercut relative to the passivation layer. Further provided herein is a device wherein the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode. Further provided herein is a device wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region. Further provided herein is a device wherein each of the first electrode and the second electrode comprises a gold surface layer.Further provided herein is a device wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein is a device wherein the second base layer comprises silicon. Further provided herein is a device wherein the neck region is 200 nm or less in width.

[0009] Provided herein is a device for molecular sensing, comprising: a first electrode including a neck region; a passivation layer including a channel or well, the bottom of the well or channel including a first base layer; a second electrode, the first electrode and the second electrode disposed on the first base layer, the second electrode at least partially embedded in the passivation layer, a first portion of the neck region overlapping a first portion of the second electrode such that the first electrode and the second electrode are separated by a nanogap, and a second portion of the first electrode and a second portion of the second electrode separated by the passivation layer; and first and second base layers, the first base layer disposed on the second base layer, and the first and second electrodes disposed on the base layers. Further provided herein are devices having a nanogap of 1 to 50 nm. Further provided herein are devices having a nanogap of 10 to 30 nm. Further provided herein is a device having a nanogap of 50 nm or less. Further provided herein is a device wherein the passivation layer comprises an oxide. Further provided herein is a device wherein the oxide comprises silicon, nitride, or carbide. Further provided herein is a device wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium. Further provided herein is a device wherein the first electrode and the second electrode each comprise a gold surface layer. Further provided herein is a device wherein the gold layer is 75 angstroms or less in thickness. Further provided herein is a device wherein the gold surface layer is deposited on an adhesion layer. Further provided herein is a device wherein the adhesion layer comprises titanium or chromium. Further provided herein is a device wherein the first electrode and the second electrode each comprise gold nanoislands. Further provided herein is a device wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein is a device wherein the second base layer comprises silicon. Further provided herein is a device, wherein the device further comprises a charge sensor, the charge sensor being in electrical communication with the first electrode and the second electrode. Further provided herein is a device, wherein the charge sensor is attached to the first electrode and the second electrode.Further provided herein is a device wherein the charge sensor comprises a polymer. Further provided herein is a device wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid. Further provided herein is a device wherein the charge sensor comprises carbon. Further provided herein is a device wherein the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interactions. Further provided herein is a device wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine. Further provided herein is a device wherein the charge sensor is further attached to the molecular sensor via a tether. Further provided herein is a device wherein the tether comprises one or more components. Further provided herein is a device wherein one or more components comprises a biotin-streptavidin construct. Further provided herein is a device wherein one or more components comprises SpyCatcher or SpyTag. Further provided herein is a device wherein one or more components comprises a peptide linker. Further provided herein is a device wherein one or more components comprises a protein. Further provided herein is a device wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper. Further provided herein is a device wherein the molecular sensor comprises an enzyme. Further provided herein is a device wherein the molecular sensor comprises an antibody. Further provided herein is a device wherein the enzyme comprises a polymerase. Further provided herein is a device wherein the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein is a device wherein the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein is a device wherein at least one edge of the first electrode is undercut relative to the passivation layer. Further provided herein is a device wherein the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode. Further provided herein is a device wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region. Further provided herein is a device wherein each of the first electrode and the second electrode comprises a gold surface layer.Further provided herein is a device wherein the first base layer comprises silicon oxide or silicon nitride. Further provided herein is a device wherein the second base layer comprises silicon. Further provided herein is a device wherein the neck region is 200 nm or less in width.

[0010] Provided herein is an array of any one of the devices described herein, wherein at least a portion of the first and second electrodes are independently addressable. Further provided herein is an array wherein the pitch distance of nanogaps in at least some of the devices is 200 nanometers or less. Further provided herein is an array wherein at least a portion of the first and second electrodes are independently addressable. Further provided herein is an array wherein at least a portion of the first and second electrodes are independently addressable on the x- and y-axes. Further provided herein is an array wherein at least a portion of the first and second electrodes are independently addressable on the z-axis. Further provided herein is an array comprising at least 50 devices of any one of the devices described herein. Further provided herein is an array comprising at least 5,000 devices of any one of the devices described herein. Further provided herein is an array comprising at least 100,000 devices of any one of the devices described herein. Further provided herein is an array wherein the pitch distance of nanogaps in at least some of the devices is 2 microns or less. Further provided herein is an array, wherein the array further comprises a plurality of vias, the plurality of vias configured to connect to at least two vertical layers of devices. Further provided herein is an array, wherein the array further comprises a plurality of routing connections, the plurality of routing connections configured to addressably control each device in the array.

[0011] Provided herein are methods for fabricating any one of the devices described herein, the methods comprising: a) providing one or more base layers; b) depositing material to form a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to form a passivation layer; f) depositing material to form a first electrode; g) patterning the first electrode; and h) isotropically etching the passivation layer such that an edge of the first electrode is undercut. Further provided herein are methods further comprising depositing gold on a first top layer of the device. Further provided herein are methods wherein the one or more base layers comprise thermal oxide on silicon. Further provided herein are methods comprising etching or lithography. Further provided herein are methods comprising RIE (reactive ion etching). Further provided herein are methods wherein the patterning comprises lithography and / or RIE. Further provided herein are methods that do not include electron beam or DUV (deep ultraviolet) lithography. Further provided herein are methods that include depositing gold on the first electrode and the second electrode. Further provided herein are methods that the first electrode and the second electrode are separated by a nanogap. Further provided herein are methods that the nanogap is 1 to 50 nm. Further provided herein are methods that the nanogap is 10 to 30 nm. Further provided herein are methods that the nanogap is 50 nm or less. Further provided herein are methods that the passivation layer comprises an oxide. Further provided herein are methods that the oxide comprises silicon, nitride, or carbide. Further provided herein are methods that the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.

[0012] Provided herein are methods of using any one of the devices described herein for molecular sensing, comprising: a. providing an analyte; b. allowing the analyte to react, bind, or otherwise interact with a sensor; and c. measuring an electrical signal generated from the sensor. Further provided herein are methods comprising: a. providing at least one nucleotide triphosphate, at least one template, and at least one primer; b. extending the primer with the at least one nucleotide triphosphate; and c. measuring an electrical signal generated from the polymerase. Further provided herein are methods further comprising analyzing the electrical signal to confirm the identity of the at least one nucleotide triphosphate. Further provided herein are methods wherein at least one nucleotide triphosphate comprises a non-standard base. Further provided herein are methods wherein at least one nucleotide triphosphate comprises a terminator configured to prevent chain extension. Further provided herein are methods wherein the identity of at least 20 bases is confirmed. Further provided herein are methods wherein the identity of at least 100 bases is confirmed. Further provided herein is a method that is repeated to confirm the identity of at least 1000 bases. [Brief explanation of the drawings]

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0014] [Figure 1] 1 illustrates non-limiting examples of polynucleotide synthesis and sequencing schemes according to some embodiments. [Figure 2] 1 illustrates non-limiting examples of graphene devices described herein, according to some embodiments. [Figure 3]1 depicts examples of charge-modulated nucleotides for use with the devices and methods described herein, according to some embodiments. [Figure 4A] 1 depicts a plan view of a device for polynucleotide sequencing with recessed gate and shield electrodes, according to some embodiments. [Figure 4B] 1 depicts a side view of a device for polynucleotide sequencing with recessed gate and shield electrodes, according to some embodiments. [Figure 5A] 1 depicts a close-up view of a flexible structure having spots, channels, or wells, respectively, according to some embodiments. [Figure 5B] 1 depicts a close-up view of a flexible structure having spots, channels, or wells, respectively, according to some embodiments. [Figure 5C] 1 depicts a close-up view of a flexible structure having spots, channels, or wells, respectively, according to some embodiments. [Figure 6A] 1 is a schema of a solid support comprising an active area and a fluidics interface, according to some embodiments. [Figure 6B] 1 is a front view of an example of a solid support array, according to some embodiments, which may, in some instances, include thousands or more, or millions or more, of the polynucleotide synthesis devices described herein. [Figure 6C] FIG. 1 is a rear view of an example of a solid support array, according to some embodiments. [Figure 6D] 1 is an example of a rack instrument, according to some embodiments. Such an instrument may contain arrays of hundreds or more, or even thousands or more, of solid supports. [Figure 7] 1 illustrates an example computer system according to some embodiments. [Figure 8] FIG. 1 is a block diagram illustrating the architecture of a computer system according to some embodiments. [Figure 9]FIG. 1 illustrates a network configured to incorporate multiple computer systems, multiple mobile phones and personal digital assistants, and a network attached storage (NAS) according to some embodiments. [Figure 10] FIG. 1 is a block diagram of a multi-processor computer system using a shared virtual address memory space, according to some embodiments. [Figure 11] 1 depicts a nanoelectric device according to some embodiments: An analyte (a nucleic acid, shown by way of example only) is in communication with a molecular sensor connected to a locus-spanning charge sensor. [Figure 12A] 1 illustrates a plan view of an edge finger device according to some embodiments. [Figure 12B] 1 illustrates a side view of an edge finger device according to some embodiments. [Figure 12C] 1 shows a side view of an edge finger device according to some embodiments, where the dotted line indicates the coating and the nanowire is shown bridging the two electrodes. [Figure 13A] 1 illustrates a top view of a cross finger device according to some embodiments. [Figure 13B] 1 illustrates a side view of a cross finger device according to some embodiments. [Figure 14A] 10A-10C show a plan view of a cross finger with a mask device according to some embodiments. [Figure 14B] FIG. 10 shows a side view of a cross finger with a mask device according to some embodiments. [Figure 15] 1 depicts an array of multiple cross finger devices according to some embodiments. [Figure 16A] 1 illustrates a top view of a self-aligned finger etching device according to some embodiments. [Figure 16B] 1 illustrates a side view of a self-aligned finger etching device according to some embodiments. [Figure 17A]1 depicts a side view of a nanoelectric device including a metal cross without undercuts, according to some embodiments. [Figure 17B] 1 depicts a side view of a nanoelectric device including a metal cross with undercuts, according to some embodiments. [Figure 18A] 1 depicts a side view of a nanoelectrical device using adhesion layer-free gold deposition resulting in gold island growth, according to some embodiments. [Figure 18B] 1 depicts a photograph of a side view of a nanoelectrical device using gold deposition without an adhesion layer, according to some embodiments. [Figure 18C] 1 depicts a side view of a nanoelectrical device using gold deposition with an adhesion layer resulting in a continuous gold film, according to some embodiments. [Figure 19A] FIG. 1 depicts a side view of a nanoelectric device with gold islands having a biomolecule conjugated between two gold islands, according to some embodiments. [Figure 19B] FIG. 1 depicts a side view of a nanoelectric device with biomolecules conjugated directly onto the electrodes, according to some embodiments. [Figure 20] 1 depicts a side view of a nanoelectric device employing a dual layer passivation such as oxide and carbide / nitride, according to some embodiments. [Figure 21] 1 depicts an exemplary molecular wire construct, according to some embodiments. [Figure 22A] FIG. 1 depicts an exemplary schematic diagram for surface functionalization, according to some embodiments. [Figure 22B] 1 depicts a schematic diagram of an exemplary functionalization on a gold surface, according to some embodiments. [Figure 23A] FIG. 1 depicts an exemplary schematic for further surface functionalization, according to some embodiments. [Figure 23B] 1 depicts an exemplary schematic diagram of an enzyme linker structure, according to some embodiments. [Figure 23C] FIG. 1 depicts an exemplary schematic diagram of a map of dual-biotin-tagged P29 variant RPN, according to some embodiments. [Figure 24] 1 depicts an exemplary schematic diagram for assembling a molecular nanowire construct, according to some embodiments. [Figure 25] FIG. 1 depicts an exemplary schematic for characterization of tagged surfaces using gold nanoparticles, according to some embodiments. [Figure 26] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising gold nanoparticles, according to some embodiments. [Figure 27] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising biotin and monomeric streptavidin, according to some embodiments. [Figure 28] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising cysteamine, according to some embodiments. [Figure 29] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising biotin and streptavidin, according to some embodiments. [Figure 30] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising SpyCatcher and SpyTag, where SpyTag is conjugated to a polymerase, according to some embodiments. [Figure 31] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising SpyCatcher and SpyTag, according to some embodiments, wherein SpyCatcher is conjugated to a polymerase. [Figure 32] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising SpyCatcher and SpyTag, and a CTPR peptide, according to some embodiments. [Figure 33] 1 depicts an exemplary schematic illustrating a molecular wire construct comprising a linker comprising a peptide, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0015] As the amount of information generated and stored increases exponentially, there is a need for larger capacity storage systems.Biomolecules such as DNA molecules provide suitable hosts for partial information storage due to their stability over time and capacity of 4-bit (or other) information coding, in contrast to traditional binary information coding.Provided herein are methods, devices, and systems for real-time detection of single molecules (e.g., biomolecules).Provided herein are methods for improving nucleic acid sequencing through the use of nanoelectrical sensing devices.

[0016] definition

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these inventions belong.

[0018] Throughout this disclosure, numerical characteristics are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values ​​within that range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limits in the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly dictates otherwise.

[0019] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiments. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but are understood not to preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0020] Unless otherwise specified or clear from the context, as used herein, the term "about" in reference to a number or range of numbers is understood to mean the specified number and number + / - 10%, or 10% below the recited lower limit and 10% above the recited upper limit for the recited values ​​for a range.

[0021] As used herein, the terms "preselected sequence," "predefined sequence," or "predetermined sequence" are used interchangeably. These terms mean that the sequence of a polymer is known and is selected prior to the synthesis or assembly of the polymer. In particular, various embodiments are described herein primarily with respect to the preparation of nucleic acid molecules, where the sequence of a polynucleotide is known and selected prior to the synthesis or assembly of the nucleic acid molecule.

[0022] As used herein, terms such as "symbol" generally refer to a representation of a unit of digital information. Digital information may be divided or converted into one or more symbols. In one example, a symbol may be a bit, and a 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 series of symbols or a string of symbols. In some examples, a series of symbols or a string of symbols may include binary data.

[0023] The polynucleotide sequences described herein may include DNA or RNA, or analogs or derivatives thereof, unless otherwise specified. As used herein, the terms nucleic acid, polynucleotide, oligonucleotide, oligo, and oligonucleic acid are used synonymously throughout to refer to polymers of nucleoside monomers. In some instances, nucleic acids are linked via phosphate- or sulfur-containing linkages. Nucleic acids include, in some instances, DNA, RNA, non-standard nucleic acids, non-natural nucleic acids, or other nucleosides. In some instances, nucleotides include non-standard bases, sugars, or other moieties. In some instances, nucleotides include terminators configured to prevent extension reactions. In some instances, such terminators are removed before the addition of subsequent nucleotides to the growing chain.

[0024] Devices for molecular sensing

[0025] Provided herein are devices, methods, compositions, and systems useful for molecular sensing. Sensing, in some instances, includes detecting the presence, absence, concentration, and / or identity of a biomolecule or a portion thereof. In some instances, the biomolecule includes a polymer. In some instances, the devices, methods, compositions, and systems are used for sequencing (e.g., nucleic acids). In some instances, the solid support includes a surface. Further provided herein are multiple devices combined to form a larger array or chip. In some instances, voltage or current is measured to sense molecular changes associated with a charge sensor, molecular sensor, or complex thereof. Arrays of such devices, in some instances, provide high-throughput readout of digital information encoded in nucleic acids. In some instances, devices are arrayed on a solid support, with each device or group of devices at an addressable locus. Larger groups of such devices, in some instances, reside in a serve rack unit.

[0026] The devices provided herein may be used for molecular sensing. The electrodes may be configured to detect changes in voltage, current, or resistance to sense biomolecules. In some examples, a device for molecular sensing includes one or more electrodes. In some examples, a device for molecular sensing includes 1, 2, 3, 4, 5, or more than 5 electrodes. In some examples, a device for molecular sensing includes a source electrode and a drain electrode. In some examples, one or more of the electrodes are configured as a source. In some examples, one or more of the electrodes are configured as a drain. In some examples, a device for molecular sensing includes a passivation layer. In some examples, the passivation layer includes an oxide layer. In some examples, the passivation layer includes a nitride layer. In some examples, the passivation layer reduces the reactivity of the surface to which it is applied. In some examples, a device for molecular sensing includes a charge sensor coupling layer. In some examples, the charge sensor coupling layer is a metal such as gold. In some examples, one or more electrodes are in electrical communication. In some examples, the electrical communication between one or more electrodes forms a gap. In some examples, the gap is a nanogap. In some examples, the charge sensor spans the gap (e.g., a nanogap). In some examples, the charge sensor comprises graphene. In some examples, the charge sensor comprises a graphene-enabled field effect transistor (GeFET) or a CMOS device. In some examples, the charge sensor is attached to a tether such as those provided herein. In some examples, the tether is attached to the charge sensor, the molecular sensor, or both. In some examples, the molecular sensor and the charge sensor are in electrical communication. In some examples, the molecular sensor comprises a polymerase. In some examples, the polymerase is an isothermal polymerase, Phi29 polymerase, or a variant thereof. In some examples, the one or more electrodes are located on the one or more base layers. In some examples, the one or more base layers are passivation layers or gate layers.

[0027] A device (1100) for molecular sensing is provided herein (FIG. 11). The device, in some examples, includes a first electrode (1105a) and a second electrode (1105b). In some examples, a portion of the first electrode (1105a) and a portion of the second electrode (1105b) are partially covered with a passivation layer (1106a and 1106b, respectively). In some examples, a portion of the first electrode (1105a) and a portion of the second electrode (1105b) are partially covered with a charge sensor coupling layer (e.g., gold, 1107a and 1107b, respectively). In some examples, the distance between the first charge sensor coupling layer (1107a), which is in electrical communication with the first electrode (1105a), and the second charge sensor coupling layer (1107b), which is in electrical communication with the second electrode (1105b), forms a nanogap (1112). In some examples, a charge sensor (1108) spans the nanogap. In some examples, the first electrode (1105a) is configured as a source. In some examples, the first electrode (1105b) is configured as a drain. In some examples, the charge sensor (1108) is attached to a tether (1109). In some examples, the tether (1109) is attached to the charge sensor (1108) and the molecular sensor (1110). In some examples, the molecular sensor (1110) is configured to detect an analyte (1111). In some examples, the first electrode (1105a) and the second electrode (1105b) are located on one or more base layers. In some examples, the one or more base layers include a passivation layer or a gate layer. In some examples, the first electrode (1105a) and the second electrode (1105b) are separated by at least one passive base layer (1104 or 1103). In some examples, one or more base layers (e.g., 1103 and 1101) are separated by a gate layer (1102). In some examples, the gate layer (1102) is located above the base layer (1101). Such devices are combined into larger arrays. In some examples, the devices are integrated into electronic devices such as CMOS and interfaced to a computer.

[0028] A first device (1200) for molecular sensing is provided herein (FIGS. 12A-12B). In some examples, the device includes a first electrode (1202a). In some examples, the first electrode includes a neck region (1202b). In some examples, the device includes a second electrode (1203). In some examples, the first electrode (1202a) and the second electrode (1202b) are located on one or more base layers. In some examples, the first electrode (1202a) and the second electrode (1202b) are located on a first base layer (1201). In some examples, 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., a nanogap). In some examples, the nanogap is the smallest defining dimension of the device. In some examples, the second portion of the first electrode (1202a) and the second portion of the second electrode (1203) are separated by a passivation layer (1205). In some examples, the first base layer (1201) is located on the second base layer (1204), and the first electrode (1202a) and the second electrode (1203) are located on the base layers (1201 and 1204). In some examples, the devices described herein include a coating (1206) (FIG. 12). In some examples, the coating (1206) is configured to couple a charge sensor (1207). In some examples, the charge sensor (1207) includes a nanowire (1207). In some examples, the nanowire (1207) includes a nucleic acid.

[0029] A second device (1300) for molecular sensing is provided herein (FIGS. 13A-13B). In some examples, the device includes a first electrode (1302). In some examples, the first electrode is located on a first base layer (1301). In some examples, the device includes a second electrode (1303a). In some examples, the second electrode (1303a) includes a neck region (1303b). In some examples, 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). In some examples, a second portion of the first electrode (1302) and a second portion of the second electrode (1303a) are separated by a passivation layer (1305). In some examples, the device includes a first base layer (1301) and a second base layer (1304). In some examples, the first base layer (1301) is located on a second base layer (1302), and the first electrode (1302) and the second electrode (1303a) are located on the first base layer (1301). In some examples, the device further includes a second passivation layer (1406). In some examples, the second passivation layer (1406) is further configured to overlap a portion of the second electrode (1403) (FIGS. 14A-14B). In some examples, the second passivation layer (1406) passivates the electrode traces.

[0030] A third device (1400) for molecular sensing is provided herein (FIGS. 14A-14B). In some examples, the device includes a first electrode (1402). In some examples, the first electrode (1402) is located on a first base layer (1401). In some examples, the device includes a second electrode (1403a), the second electrode (1403a) including a neck region (1403b). In some examples, 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). In some examples, a second portion of the first electrode (1402) and a second portion of the second electrode (1403a) are separated by a passivation layer (1405). In some examples, the device includes a first base layer (1401) and a second base layer (1404). In some examples, the first base layer (1401) is located on a second base layer (1402), and the first electrode (1402) and the second electrode (1403a) are located on the first base layer (1401). In some examples, the device further includes a second passivation layer (1406).

[0031] Gold may be deposited on the first electrode, the second electrode, or both on the devices described herein. In a first approach, as shown in the schematic diagram of FIG. 17A, gold is deposited without undercutting. In this approach, in some instances, a short circuit path between the top and bottom electrodes may be formed through an overlying oxide, as illustrated in FIG. 17A. In such instances, a precise, ultra-thin gold film may be deposited to avoid the short circuit. In a second approach, gold is deposited with an undercut between the first electrode (top electrode) and the second electrode (bottom electrode). An exemplary schematic diagram is provided in FIG. 17B. In some instances, wet chemistry is used to achieve the oxide undercut. In some instances, metal electrodes with oxide undercutting can help prevent short circuits between the top and bottom electrodes after gold deposition. This allows for tailoring to the required gold thickness.

[0032] In some examples, an adhesion layer is deposited on the first electrode (top electrode), the second electrode (bottom electrode), or both. In some examples, an adhesion layer is not deposited on the first electrode (top electrode), the second electrode (bottom electrode), or both. In some examples, the adhesion layer comprises titanium (Ti), chromium (Cr), or both. In some examples, for example, as shown in Figures 18A-18B, gold is deposited directly on the electrodes (e.g., without an adhesion layer). As shown, gold nanoislands are formed in the absence of an adhesion layer. In some examples, gold deposition with an adhesion layer results in a continuous gold film, for example, as shown in Figure 18C.

[0033] Biomolecules can be conjugated on electrodes. In some examples, biomolecules are conjugated to gold deposited on the surface of an electrode. In some examples, biomolecules are conjugated to one or more surfaces on which gold is deposited on an electrode (e.g., extending between nanogaps). In some examples, biomolecules are conjugated to the surfaces of a first electrode (top electrode) and a second electrode (bottom electrode), both of which are deposited with gold. In some examples, biomolecules are conjugated to one or more gold islands (e.g., extending between nanogaps). An exemplary schematic is provided in FIG. 19A. In some examples, biomolecules are conjugated to gold islands on a first electrode (top electrode) and a gold island on a second electrode (bottom electrode). In some examples, biomolecules are conjugated directly to one or more electrodes, as illustrated, for example, in FIG. 19B. In some instances, gold nanoislands, such as those illustrated in Figure 19A, provide lower contact resistance and result in higher currents compared to direct conjugation of biomolecules on continuous metal electrodes, such as those illustrated in Figure 19B. In some instances, electrode surfaces exposed to the ambient environment (e.g., TiN) develop a thin oxide layer, resulting in higher contact resistance when biomolecules are directly conjugated in the absence of gold (e.g., Figure 19B). In some instances, the oxide layer inhibits direct conjugation of biomolecules on electrodes not made of noble metals.

[0034] In some examples, the device includes passivation around the device's metal cross. An exemplary schematic diagram is provided in Figure 20. For example, a passivation layer can be used to protect the fields and metal routing. When using an adhesion layer to form a continuous gold film (e.g., Figure 18C), the continuous gold film can increase the likelihood of shorting at the edges of the passivation openings in subsequent steps (not shown in Figure 20). To mitigate this, a dual-layer passivation, such as oxide and carbide / nitride, can be used. Different etch rates during wet etching can create undercuts, which prevent the deposition of a continuous gold layer at the edges of the passivation and thus avoid shorts when operating the sequencing chip.

[0035] A fourth device (1600) for molecular sensing is provided herein (FIGS. 16A-16B). In some examples, the device includes a first electrode (1602a). In some examples, the first electrode (1602a) includes a neck region (1602b). In some examples, the device includes a passivation layer such as (1605) or (1606). In some examples, the passivation (1605) / (1606) includes a channel or well. In some examples, the bottom (1607) of the well or channel (or trench) includes (or is exposed to) a first base layer (1601). In some examples, the device includes a second electrode (1603). In some examples, the first electrode (1602a) and the second electrode (1603) are located on the first base layer (1601), and the second electrode (1603) is at least partially embedded in the passivation layer (1605). In some examples, 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). In some examples, a second portion of the first electrode (1602a) and a second portion of the second electrode (1603) are separated by the passivation layer (1605). In some examples, the device includes a first base layer (1601) and a second base layer (1604). In some examples, a first base layer (1601) is located on top of a second base layer (1604), and a first electrode (1602a) and a second electrode (1603) are located on top of the base layers (1601) / (1604). In some examples, the device stack is etched using the same hard mask so that the electrode edges are aligned and deeper trenches (channels or wells) are etched underneath (Figures 16A-16B).

[0036] In some examples, the device includes a graphene layer configured to detect changes in local current. In some examples, such a layer is coupled to a polymerase that provides a unique signal (sensing) corresponding to a nucleotide incorporation event. In some examples, the devices provided herein are used to sequence nucleic acids. In some examples, the device includes one or more of a charge sensor and a molecular sensor. In some examples, the molecular sensor interacts with a biomolecule and transmits information about the biomolecule to the charge sensor via a change in current. In some examples, the charge sensor includes a graphene layer. In some examples, the information about the biomolecule includes information about monomers of the biomolecule. For example, if the biomolecule is a polynucleotide, the information about the biomolecule includes information about nucleotides. In some examples, the information about the nucleotides includes information about bases (e.g., A, T, C, and G). As another example, if the biomolecule is a peptide, the information about the peptide includes information about amino acids.

[0037] Provided herein is a device comprising a solid support, a charge sensor, one or more electrodes, and at least one insulating layer. In some examples, the charge sensor comprises a graphene layer. In some examples, the one or more electrodes comprise a gate electrode. In some examples, the one or more electrodes comprise a drain electrode. In some examples, the gate electrode and the drain electrode are in electrical communication through the charge sensor. In some examples, the gate electrode and the drain electrode are in electrical communication through the graphene layer. In some examples, the insulating layer is located between the gate electrode and the drain electrode. In some examples, the solid support comprises a plurality of sites. In some examples, the device further comprises at least one ground shield. In some examples, the device further comprises at least one buried gate (electrode).

[0038] A fifth device (200) as shown in FIG. 2 is provided herein. In some examples, the device includes a solid support (204), a graphene layer (201), a gate electrode (203a), a drain electrode (203b), and at least one insulating layer (202a) / (202b). In some examples, the insulating layer is located between the gate electrode (203a) and the drain electrode (203b). In some examples, the gate electrode (203a) and the drain electrode (203b) are in electrical communication through the graphene layer (201). In some examples, the gate electrode (203a) and the drain electrode (203b) comprise platinum. In some examples, the solid support (204) or the insulating layer (202a) / (202b) comprise silicon or silicon nitride.

[0039] A sixth device (400) is provided herein as shown in Figures 4A-4B. In some examples, the device includes a solid support (408), a graphene layer (401), a gate electrode (403a), a drain electrode (403b), and at least one insulating layer (409), at least one ground shield (407a) / (407b), and a buried gate (405). In some examples, the at least one ground shield (407a) / (407b) includes an opening that allows electrical contact between the graphene layer (401) and the buried gate (405). In some examples, the insulating layer (409) is located between two or more of the gate electrode (403a), the drain electrode (403b), the at least one ground shield (407a) / (407b), the buried gate (405), and the graphene layer (401). In some examples, the gate electrode (403a) and the drain electrode (403b) are in electrical communication through the graphene layer (401). In some examples, one or more of the gate electrode (403a), the drain electrode (403b), the buried gate (405), and the at least one ground shield (407a) / (407b) comprise platinum. In some examples, the solid support or insulating layer (409) comprises silicon or silicon nitride. In some examples, the buried gate (405) is charged with a voltage that attracts or repels molecules from the charge sensor (e.g., the graphene layer (401)). In some examples, the buried gate (405) is charged with a voltage that modulates the current generated by biomolecules interacting with the molecular sensors described herein.

[0040] Any of the above devices may be arranged on a solid support. In some instances, the devices are arranged on a solid support such that at least some of the devices, or a portion of the devices, are addressable. In some instances, the devices (1500) are arranged on a solid support (1501) in a configuration. In some instances, the first electrode is positioned on the x-axis and the second electrode (1502) is positioned on the y-axis (Figure 15). Any number of devices may be present in an array, in some instances. In some examples, the array comprises between 10 and 1,000,000, between 10 and 100,000, between 10 and 10,000, between 10 and 5,000, between 50 and 1,000,000, between 50 and 100,000, between 50 and 10,000, between 5,000, between 100 and 1,000,000, between 100 and 100,000, between 100 and 10,000, between 100 and 5,000, or between 500 and 1,00,000 devices. In some examples, the array includes 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. In some examples, the devices are arranged into larger devices, etc. In some examples, the density of the array can be as high as 2 to 1 billion different reaction sites (devices, or loci) per square cm. In some examples, the density of the array can be 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 In some examples, the density of the array is between 10,000 and 100,000 reaction sites / cm.2 , 100,000 to 500,000 reaction sites / cm 2 , 100,000 to 1,000,000 reaction sites / cm 2 , 10,000 to 1,000,000 reaction sites / cm 2 , or 100,000 to 1,000,000,000 reaction sites / cm 2 In some examples, the device further includes vias and / or other connections for electrical communication. In some examples, the device includes an electrode located in the z-axis.

[0041] The devices described herein may include a solid support. Exemplary solid supports can be seen in Figures 6B-6C. Figure 6B shows the front side of the solid support, which is made of glass and includes transparent windows for arrays and fluid ports. Figure 6C shows the back side of the solid support, which is a circuit with electrical contacts (e.g., LGA 1 mm pitch) and thermal interfaces below the solid support area.

[0042] The solid supports described herein include an active area. In some examples, the active area includes an addressable solid support, area, or locus for molecular sensing. In some examples, the active area includes an addressable area or locus for nucleic acid storage. In some examples, the active area is in fluid communication with a solvent or other reagent. The active area includes various dimensions. For example, the dimensions of the active area are between about 1 mm and about 50 mm by about 1 mm and about 50 mm. In some examples, the active area includes 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. In some examples, the active region includes a height of at least or greater than 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 80 mm. For example, the dimensions of the active region are between about 1 μm and about 50 μm by about 1 μm and about 50 μm. In some examples, the active region includes a width of at least or greater than 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 80 μm. In some examples, the active area comprises a height of at least 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 greater than 80 μm. In some examples, the active area comprises a height of 4-900, 4-500, 4-250, 2-900, 10-900, 25-900, 50-900, 100-900, or 400-900 mm. 2 In some examples, the active region and optional passive region are between 4 and 900 mm, between 4 and 500 mm, between 4 and 250 mm, between 2 and 900 mm, between 10 and 900 mm, between 25 and 900 mm, between 50 and 900 mm, between 100 and 900 mm, or between 400 and 900 mm. 2 In some examples, 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 2In some examples, the active region and any passive region may be between 4 and 900 mm, between 4 and 500 mm, between 4 and 250 mm, between 2 and 900 mm, between 10 and 900 mm, between 25 and 900 mm, between 50 and 900 mm, between 100 and 900 mm, or between 400 and 900 mm for each side of the devices described herein. 2 An exemplary active area within a solid support can be seen in Figure 6A. Package (607) contains active area (605) within solid support (603). Package (607) also contains fluidic interface (601).

[0043] Devices, compositions, systems, and methods for molecular sensors are described herein. In some examples, a solid support has several sites or positions for molecular sensors. In some examples, the solid support comprises up to 10,000 x 10,000 positions, or about 10,000 x 10,000 positions in area. In some examples, the solid support comprises about 1000-20,000 x about 1000-20,000 positions in area. In some examples, 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, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90, ... 0,000 locations by 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 locations. In some examples, the area is at most 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 square inches. In some examples, the solid support comprises addressable loci with a pitch of at least 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 greater than 10 μm. In some examples, the solid support comprises addressable loci with a pitch of 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 10 μm or less. In some examples, the solid support comprises addressable loci with a pitch of about 5 μm. In some examples, the solid support comprises addressable loci with a pitch of about 2 μm. In some examples, the solid support comprises addressable loci with a pitch of about 1 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.01 μm.In some examples, the solid support comprises addressable loci with a pitch of about 0.02 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.05 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.08 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.1 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.2 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.05 μm to about 10 μm, about 0.05 to about 1 μm, about 0.05 to about 1 μm, about 0.1 μm to about 1 μm, about 0.2 μm to about 0.8 μm, about 0.3 μm to about 0.5 μm, about 1 μm to about 3 μm, or about 0.05 μm to about 1 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.1 μm to about 3 μm. In some examples, the solid support comprises addressable loci with a pitch of at least about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or greater than about 1 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.5 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.2 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.1 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.02 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.02 μm to about 1 μm, about 0.02 to about 0.8 μm, about 0.05 to about 0.1 μm, about 0.1 μm to about 1 μm, about 0.2 μm to about 0.8 μm, about 0.3 μm to about 0.5 μm, about 0.1 μm to about 0.3 μm, or about 0.05 μm to about 0.3 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.01 μm to about 0.3 μm. In some examples, the solid support comprises addressable loci with a pitch of about 0.05 μm to about 1 μm.

[0044] The device may include gaps or distances between one or more electrodes of various lengths. In some examples, the electrodes on each side of the gap are attached to a charge sensor. In some examples, the electrodes on each side of the gap are attached to at least one charge sensor. In some examples, the electrodes on each side of the gap are attached to at least one shared charge sensor. In some examples, the length of the nanogap is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 nm. In some examples, the length of the nanogap is about 5-50, about 5-25, about 5-20, about 10-50, about 10-30, about 15-25, about 15-30, about 20-40, or about 25-50 nm. In some examples, the length of the nanogap is 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 35 nm or less, 40 nm or less, or 50 nm or less. In some examples, the length of the nanogap is at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, or at least 50 nm.

[0045] The devices described herein may include a neck region. In some examples, the electrode includes the neck region. In some examples, the neck region is proximate to the nanogap. In some examples, the neck region has a width that is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, or more than 95% shorter than the largest dimension of the electrode. In some examples, the neck region has a width that is at least 0.5 times, at least 1 time, at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or 100 times shorter than the largest dimension of the electrode. In some examples, the first electrode includes a neck region. In some examples, the second electrode includes a neck region. In some examples, the first electrode includes a neck region and the second electrode does not include a neck region. In some examples, the neck region has a width of 250 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 75 nm or less, or 50 nm or less. In some examples, the neck region has a width of about 250 nm, about 200 nm, about 150 nm, about 125 nm, about 110 nm, about 100 nm, about 90 nm, about 75 nm, or about 50 nm.

[0046] In some examples, each locus of the structure has a width of about 1 μm and a center-to-center distance of about 2.1 μm. In some examples, each locus of the structure has a width of about 0.5 μm and a center-to-center distance of about 2 μm. In some examples, each locus of the structure has a width of about 0.1 μm and a center-to-center distance of about 0.2 μm. The loci may include shapes including, but not limited to, circular, rectangular, tapered, or rounded shapes. Alternatively, or in combination, the structure is rigid. In some examples, the rigid structure includes a locus for molecular sensing. In some examples, the rigid structure includes a substantially flat region, channel, or well for molecular sensing.

[0047] Provided herein is a flexible structure having a surface with multiple loci for polynucleotide extension. Figures 5A-5C show zoom-in views of loci in the flexible structure. Each locus in a portion of the flexible structure (501) can be a substantially flat spot (503) (e.g., flat), a channel (505), or a well (507). The loci may include shapes including, but not limited to, circular, rectangular, tapered, or rounded shapes. Alternatively, or in combination, the structure is rigid. In some examples, the rigid structure includes loci, channels, or wells for polynucleotide synthesis.

[0048] In some examples, the wells described herein have a width to depth (or height) ratio of 1 to 0.01. In some examples, the width is measured at the narrowest segment of the well. In some examples, the wells described herein have a width to depth (or height) ratio of 0.5 to 0.01. In some examples, the width is measured at the narrowest segment of the well. In some examples, the wells described herein have a width to depth (or height) ratio of about 0.01, about 0.05, about 0.1, about 0.15, about 0.16, about 0.2, about 0.5, or about 1. Provided herein are structures for molecular sensing that include multiple distinct loci for molecular sensing. Exemplary structures for loci include, but are not limited to, substantially flat regions, channels, wells, or protrusions. The structures described herein may include multiple clusters, each cluster including multiple wells, loci, or channels. Alternatively, the structures described herein may include a uniform configuration of wells, loci, or channels. The structures provided herein may include wells having a height or depth of about 0.1 μm to about 5 μm, about 0.1 μm to about 400 μm, about 0.1 μm to about 300 μm, about 0.1 μm to about 200 μm, about 0.1 μm to about 100 μm, about 0.1 μm to about 0.5 μm, or about 0.01 μm to about 0.5 μm. In some examples, the well height is less than 0.10 μm, less than 0.08 μm, less than 0.6 μm, less than 0.40 μm, or less than 0.2 μm. In some examples, the well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nm or more. In some examples, the height or depth of the wells is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater than 1000 nm. In some examples, the height or depth of the wells is in the 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 nm. In some examples, the height or depth of the wells is in the range of about 50 nm to about 1 μm.In some examples, the height of the wells is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900, or about 1000 nm.

[0049] The structures for molecular sensing provided herein may include a channel. The channel may have a width to depth (or height) ratio of 1 to 0.01. In some examples, the width is measured at the narrowest segment of the microchannel. In some examples, the channels described herein have a width to depth (or height) ratio of 0.5 to 0.01. In some examples, the width is measured at the narrowest segment of the microchannel. In some examples, the channels described herein have 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.

[0050] Described herein are structures for molecular sensing that include multiple distinct loci. The structures include, but are not limited to, substantially flat regions, channels, protrusions, or wells for molecular sensing. In some examples, structures described herein are provided that include multiple channels, wherein the channel height or depth is about 5 μm to about 500 μm, about 5 μm to about 400 μm, about 5 μm to about 300 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 10 μm to about 50 μm. In some cases, the channel height is less than 100 μm, less than 80 μm, less than 60 μm, less than 40 μm, or less than 20 μm. In some cases, the channel height is about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, or more. In some examples, the channel height or depth is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some examples, the height or depth of the channels ranges from about 10 nm to about 1000 nm, from about 25 nm to about 900 nm, from about 50 nm to about 800 nm, from about 75 nm to about 700 nm, from about 100 nm to about 600 nm, or from about 200 nm to about 500 nm. The channels described herein may be arranged on a surface in clusters or as a uniform field.

[0051] The width of the locus on the surface of the molecular sensing structure described herein may be about 0.1 μm to about 500 μm, about 0.5 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to 100 μm, or about 0.1 μm to about 100 μm, e.g., about 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. In some examples, the width of the locus is less than about 100 μm, 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 10 μm. In some examples, the locus width is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater than 1000 nm. In some examples, the locus width is in the 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 nm. In some examples, the well width is in the range of about 50 nm to about 1000 nm. In some examples, the distance between the centers of two adjacent loci is about 0.1 μm to about 500 μm, about 0.5 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 5 μm to about 30 μm, e.g., about 20 μm. In some examples, the total width of the loci is about 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some examples, the total width of the loci is about 1 μm to 100 μm, 30 μm to 100 μm, or 50 μm to 70 μm. In some examples, the distance between the centers of two adjacent loci is about 0.5 μm to about 2 μm, about 0.5 μm to about 2 μm, about 0.75 μm to about 2 μm, about 1 μm to about 2 μm, about 0.2 μm to about 1 μm, about 0.5 μm to about 1.5 μm, about 0.5 μm to about 0.8 μm, or about 0.5 μm to about 1 μm, for example, about 1 μm.In some examples, the total width of the locus is about 50 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, or 1.5 μm. In some examples, the total width of the locus is about 0.5 μm to 2 μm, 0.75 μm to 1 μm, or 0.9 μm to 2 μm.

[0052] In some examples, each locus supports sensing of a population of polynucleotides having a sequence that differs from a population of polynucleotides propagated on another locus. Provided herein are surfaces comprising 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. Provided herein are surfaces that 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, 5,000,000, or 10,000,000 or more distinct loci. In some cases, 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 50-500, 50-200, 50-150, or 100-150 loci. In some cases, each cluster includes 100-150 loci. In some examples, each cluster includes 109, 121, 130, or 137 loci.

[0053] Provided herein are loci having a maximum segment width of 5 to 100 μm. In some cases, the loci have a maximum segment width of about 30, 35, 40, 45, 50, 55, or 60 μm. In some cases, the loci are channels having multiple segments. In some examples, each segment has a center-to-center distance of 5 to 50 μm apart. In some cases, the center-to-center distance for each segment is about 5, 10, 15, 20, or 25 μm apart.

[0054] Provided herein are loci having a maximum segment width of 5 to 500 nm. In some cases, the loci have a maximum segment width of about 30, 35, 40, 45, 50, 55, 60, 80, or 100 nm. In some cases, the loci are channels having multiple segments. In some examples, each segment has a center-to-center distance of 5 to 50 nm. In some cases, the center-to-center distance for each segment is about 5, 10, 15, 20, 25, 50, 100, or 200 nm.

[0055] In some instances, the number of distinct polynucleotides synthesized on the surface of a structure described herein varies depending on the number of distinct loci available on the substrate. In some instances, the density of loci within a cluster on the substrate is greater than or equal to 1000 mm 2 At least or about 1 locus per mm 2 At least or about 10 loci per mm 2 At least or about 25 seats per mm 2 At least or about 50 loci per mm 2 At least or about 65 seats per mm 2 At least or about 75 seats per mm 2 At least or about 100 loci per mm 2 At least or about 130 seats per mm 2 At least or about 150 seats per mm 2 At least or about 175 seats per mm 2 At least or about 200 loci per mm 2At least or about 300 loci per mm 2 At least or about 400 loci per mm 2 At least or about 500 loci per mm 2 At least or about 1,000 loci per mm 2 At least or about 10 per 4 seat, mm 2 At least or about 10 per 5 seat, mm 2 At least or about 10 per 6 In some cases, the substrate is 2 Approximately 10 seats per approx. 500mm 2 , mm 2 Approximately 25 seats per approx. 400mm 2 , mm 2 Approximately 50 to 500 mm per seat 2 , mm 2 Approximately 100 seats per approx. 500mm 2 , mm 2 Approximately 150 to 500 mm per seat 2 , mm 2 Approximately 10 seats per approx. ~ approx. 250mm 2 , mm 2 Approximately 50 to 250mm per seat 2 , mm 2 Approximately 10 seats per approx. 200mm 2 , or mm 2 Approximately 50 to 200mm per seat 2 In some cases, the substrate may include 2 Approximately 10 per 4 About 10 minutes from the seat 5 mm 2 In some cases, the substrate may include 2 Approximately 10 per 5 About 10 minutes from the seat 7 mm 2 In some cases, the substrate may include 2 At least 10 per 5 In some cases, the substrate includes a seat 2 At least 10 per 6 In some cases, the substrate includes a seat 2At least 10 per 5 In some cases, the substrate includes a seat 2 Approximately 10 per 4 About 10 minutes from the seat 5 mm 2 In some examples, the density of the sites in the clusters of the substrate is 2 At least one locus per μm 2 At least or about 10 loci per μm 2 At least or about 25 loci per μm 2 At least or about 50 loci per μm 2 At least or about 65 loci per μm 2 At least or about 75 loci per μm 2 At least or about 100 loci per μm 2 At least or about 130 loci per μm 2 At least or about 150 loci per μm 2 At least or about 175 loci per μm 2 At least or about 200 loci per μm 2 At least or about 300 loci per μm 2 At least or about 400 loci per μm 2 At least or about 500 loci per μm 2 In some cases, the substrate has at least about 1,000 or more loci per μm. 2 Approximately 10 sites per ~ 500 μm 2 , μm 2 Approximately 25 sites per ~ 400 μm 2 , μm 2 Approximately 50 sites per ~ approximately 500 μm 2 , μm 2 Approximately 100 sites per ~ 500 μm 2 , μm 2 Approximately 150 sites per ~ 500 μm 2 , μm 2 Approximately 10 sites per ~ approximately 250 μm 2 , μm 2 Approximately 50 sites per ~ approximately 250 μm 2 , μm 2 Approximately 10 sites per ~ approximately 200 μm 2, or μm 2 Approximately 50 sites per ~ approximately 200 μm 2 Includes:

[0056] In some examples, the distance between two adjacent centers within a cluster is about 10 μm to about 500 μm, about 10 μm to about 200 μm, or about 10 μm to about 100 μm. In some cases, the distance between the centers of two adjacent loci is greater than about 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the distance between the centers of two adjacent loci is less than about 200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In some cases, the distance between the centers of two adjacent loci is less than about 10,000 nm, 8,000 nm, 6,000 nm, 4,000 nm, 2,000 nm, 1,000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm, 100 nm, 80 μm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In some examples, each square meter of the structures described herein has at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 In some instances, each locus supports one polynucleotide. In some instances, 10 9 The polynucleotides may be about 6, 5, 4, 3, 2, or 1 m long, as described herein. 2 is supported on a structure less than

[0057] In some examples, the structures described herein may be 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, 2,000,000, 5,000,000, 6,000,000, 7,000,000, 800,000, 900,000, 1,000,000, 1,200,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 1,000,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 2,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 1,500,000, 1,600,000, 1,700,000, 2,0 The present invention provides a support for sensing 1,000, 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 non-identical polynucleotides. In some cases, the construct may include 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, or more fragments of a fragment containing ...300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or more fragments of a fragment containing 1,000, 1,200,000, or more fragments of a fragment containing 1,000, 1,200,000, or more fragments of a fragment containing 1,000, 1,200,000, or more fragments of a fragment containing 1,000, 1,200,0 Provided is a support for sensing 0,000, 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, in some examples, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some examples, the structures provide a surface environment for the propagation 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 or more bases. In some configurations, the structures for molecular sensing described herein comprise sites for molecular sensing of a uniform configuration.

[0058] In some examples, polynucleotides are synthesized on separate loci of the structure. In some examples, each locus supports sensing of a population of polynucleotides. In some cases, each locus supports sensing of a population of polynucleotides having a different sequence from the population of polynucleotides grown on another locus. In some examples, the loci of the structure are arranged within multiple clusters. In some examples, the structure includes 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. In some examples, the structure may be 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, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 21,000,000, 22,000,000, 23,000,000, 24,000,000, 25,000,000, 26,000,00 and containing more than 0,000, 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. In some cases, 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 examples, each cluster includes 50-500, 100-150, or 100-200 loci. In some examples, each cluster includes 109, 121, 130, or 137 loci. In some examples, each cluster includes 5, 6, 7, 8, 9, 10, 11, or 12 loci.In some instances, polynucleotides from distinct loci within a cluster, when assembled, have a sequence that encodes a contiguous, longer polynucleotide of a given sequence, in some instances, each of the polynucleotides comprises multiple different nucleotide bases (e.g., A, T, C, G, etc.).

[0059] In some examples, the structures described herein are approximately the size of a plate (e.g., a chip), e.g., about 40-120 mm by about 25-100 mm. In some examples, the structures described herein have diameters of about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 100 mm, or 50 mm or less. In some examples, the diameter of the substrate is about 25 mm to 1000 mm, about 25 mm to about 800 mm, about 25 mm to about 600 mm, about 25 mm to about 500 mm, about 25 mm to about 400 mm, about 25 mm to about 300 mm, or about 25 mm to about 200 mm. Non-limiting examples of substrate sizes include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 84 mm, 76 mm, 54 mm, 51 mm, and 25 mm. In some examples, the substrate is at least 100 mm 2 , 200mm 2 , 500mm 2 , 1,000mm 2 , 2,000mm 2 , 4,500mm 2 , 5,000mm 2 , 10,000mm 2 , 12,000mm 2 , 15,000mm 2 , 20,000mm 2 , 30,000mm 2 , 40,000mm 2 , 50,000mm 2The thickness may be about 50 mm to about 2000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 200 mm to about 1000 mm, or about 250 mm to about 1000 mm. Non-limiting example thicknesses include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm, and 925 mm. In some examples, the thickness is at least 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 greater than 4.0 mm. In some cases, the thickness varies with diameter and depends on the composition of the substrate. For example, a structure comprising a material other than silicon may have a different thickness than a silicon structure of the same diameter. The thickness of the structure may be determined by the mechanical strength of the material used; the structure must be thick enough to support its own weight without cracking during handling.

[0060] Described herein are devices in which two or more solid supports are assembled. In some examples, the solid supports are connected together on a larger unit. The connection may include the exchange of fluids, electrical signals, or other exchange media between the solid supports. The unit may be capable of connecting to several servers, computers, or network devices. For example, multiple solid supports are integrated into or mounted on a rack unit, which can be easily inserted or removed from a server rack. A rack unit may contain several solid supports. In some examples, a rack unit contains 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. In some examples, a rack unit contains at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, or 100,000 or more solid supports. In some examples, a rack unit contains up to 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 examples, all or a portion of the solid supports in a rack unit are in fluid communication, electrical communication, or both. In some examples, the server rack comprises about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some examples, the server rack comprises at least about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some examples, the server rack comprises up to about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some examples, all or a portion of the rack units of a rack server are in fluid communication, electrical communication, or both. In some examples, the two or more solid supports are not associated with one another.In some examples, two or more rack units containing solid supports such as those described herein are stacked vertically. Fluid communication, electrical communication, or both may be formed using, by way of non-limiting example, one or more tubes (e.g., microfluidic tubes), valves, actuators, robots, etc.

[0061] Nucleic acids present on solid supports (and the information stored therein) can be accessed from a rack unit. See, for example, Figure 6D. Accessing can include removing polynucleotides from the solid support, directly analyzing the polynucleotides on the solid support, or any other method that allows for manipulation or identification of the information stored in the nucleic acids. In some examples, the information can be accessed from multiple racks, a single rack, a single solid support in a rack, a portion of a solid support, or a single location on a solid support. In various examples, accessing includes interfacing the nucleic acids with an additional device, such as a mass spectrometer, HPLC, sequencing instrument, PCR thermocycler, or other device for manipulating nucleic acids. In some examples, accessing the nucleic acid information is achieved by cleaving the polynucleotides from all or a portion of the solid support.

[0062] In some examples, the rack units or rack servers are located in a data center. In some examples, the data center utilizes mechanical structures used to mount conventional computing and data storage resources in rack units, such as openings, processing blades, or openings adapted to support other computer equipment. In some examples, a computer system such as that provided herein is used to retrieve polynucleotides from one or more rack units on one or more rack servers. In some examples, a user (e.g., a technician, researcher, customer, etc.), a computer system, or both, supports the retrieval of one or more rack units on one or more rack servers. In some examples, rack units can be retrieved from the rack servers using a robotic system, such as a robotic arm. In some examples, the robotic system is in communication with the computer system. The robotic system may be used to interface any component of the data storage system with another component of the data storage system. In some examples, the interface includes transfer, storage, movement, processing, or retrieval. In some examples, the robotic system moves solid supports between components (e.g., units or chambers) of the data storage system. The components may include, by way of non-limiting example, a synthesis unit, a storage unit, an amplification unit, etc. The rack unit or server in some instances includes units for the inflow of reagents or the outflow of waste or synthesis products.

[0063] Cleavage, in some examples, involves exposure to chemical reagents (ammonia or other reagents), electrical potential, radiation, heat, light, sound, or other forms of energy capable of manipulating chemical bonds. In some examples, cleavage occurs by charging one or more electrodes in the vicinity of the polynucleotide. In some examples, electromagnetic radiation in the form of UV light is used to cleave the polynucleotide. In some examples, a lamp is used to cleave the polynucleotide, and a mask mediates the location of the UV light exposure relative to the surface. In some examples, a laser is used to cleave the polynucleotide, and the open / closed state of a shutter controls the location of the UV light exposure relative to the surface. In some examples, a computer system such as those provided herein directs the open / closed state of the shutter. In some examples, access to nucleic acid information (including removal / addition of racks, solid supports, reagents, nucleic acids, or other components) is fully automated (e.g., using a computer system provided herein). In some examples, the chip has one or more contacts. In some examples, the chip includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, or more than 200 contacts.

[0064] The devices herein may include a biomolecule coupled to one or more electrodes. In some examples, the biomolecule is a charge sensor. In some examples, the charge sensor bridges or spans at least two electrodes. In some examples, the charge sensor includes a nanowire. In some examples, the charge sensor includes a polymer. In some examples, the polymer includes a nucleic acid. In some examples, the charge sensor is configured to couple to one or more electrodes of a device described herein. In some examples, the charge sensor includes a detection device that converts a perturbation at its surface or an electric field around it into an electrical signal. For example, the charge sensor can convert the arrival or departure of a reaction component into an electrical signal. The charge sensor can also convert an interaction between two reaction components or a conformational change in a single reaction component into an electrical signal. In some examples, the charge sensor includes a nanowire. In some examples, the charge sensor includes a GeFET. Exemplary charge sensors are field effect transistors (FETs) and steep subthreshold slope devices, such as single-walled carbon nanotube (SWNT)-based FETs, silicon nanowire (SiNW) FETs, graphene nanoribbon FETs (and related nanoribbon FETs fabricated from 2D materials such as MoS2), tunneling FETs (TFETs), etc. In some examples, the nanowire comprises a polymer. In some examples, the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid. In some examples, the charge sensor comprises carbon. In some examples, the charge sensor is coupled to at least one of the first electrode or the second electrode via a sulfur-gold interaction. In some examples, the charge sensor is further coupled to a molecular sensor via a tether, such as those described herein. In some examples, the charge sensor comprises a nucleic acid. In some examples, the charge sensor comprises DNA or RNA. In some examples, the charge sensor includes 20 to 500, 50 to 500, 100 to 500, 150 to 1000, 150 to 500, 250 to 1000, 500 to 1000, or 600 to 1000 nucleic acids.In some examples, the charge sensor comprises 1000 or less, 750 or less, 500 or less, 250 or less, 200 or less, 100 or less, 50 or less, or 20 or less nucleic acids. In some examples, the charge sensor comprises about 1000, 750, 500, 250, 200, 100, 50, or about 20 nucleic acids. In some examples, the charge sensor comprises at least 1000, 750, 500, 250, 200, 100, 50, or at least 20 nucleic acids. In some examples, at least one nucleic acid is attached to a tether. In some examples, the polymer includes a moiety for attachment to one or more electrodes. In some examples, the charge sensor is attached to at least one of the first electrode or the second electrode via sulfur-gold interactions. In some examples, the length of the charge sensor is about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, or 40 nm. In some examples, the length of the charge sensor is 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 35 nm or less, or 40 nm or less. In some examples, at least 1, 2, 5, 10, 15, 20, 25, 50, 75, or 80% of the electrodes are coupled to molecular sensors.

[0065] The devices herein may include a molecular sensor. In some examples, the molecular sensor is configured to detect the presence or absence of an analyte. In some examples, the molecular sensor is in electrical communication with the charge sensor, optionally via a tether. In some examples, the molecular sensor includes a polymerase. Some of the various polymerases are used in methods or compositions, including protein-based enzymes isolated from biological systems and their functional variants. In some examples, the polymerase is configured to catalyze the polymerization of nucleic acids using an existing nucleic acid strand as a template. Polymerases include, but are not limited to, DNA polymerases and RNA polymerases. Exemplary DNA polymerases include those classified by structural homology into families identified as A, B, C, D, X, Y, and RT. Family A DNA polymerases include T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase γ, Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase I, and Geobacillus stearothermophilus DNA polymerase I. Family B DNA polymerases include eukaryotic DNA polymerases α, δ, and ε, DNA polymerase ζ, T4 DNA polymerase, Phi29 DNA polymerase, and RB69 bacteriophage DNA polymerase. Family C includes the E. coli DNA polymerase III α subunit. Family D includes polymerases from the euryarchaeal subdomain of archaea. Family X DNA polymerases include eukaryotic polymerases Pol β, Pol σ, Pol X, and Pol p, and Saccharomyces cerevisiae Pol 4. DNA polymerases in family Y include Pol η, Pol ι, Pol κ, E. coli Pol IV (DINB), and E. coli Pol V (UmuD'2C).The RT (reverse transcriptase) family of DNA polymerase includes retroviral reverse transcriptase and eukaryotic telomerase.Exemplary RNA polymerase includes but is not limited to viral RNA polymerase such as T7 RNA polymerase, eukaryotic RNA polymerase such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV and RNA polymerase V, and archaeal RNA polymerase.

[0066] The molecular sensor may be attached to the charge sensor via a tether (or bond). In some examples, the molecular sensor is attached to the charge sensor by a non-covalent bond, such as a bond formed between a receptor and a ligand. Bonds include, but are not limited to, bonds between streptavidin (or a variant or analog thereof) and biotin (or an analog thereof), bonds between complementary nucleic acids, bonds between antibodies and epitopes, and the like. In some examples, a conductive tether is used to attach the molecular sensor to the charge sensor. Exemplary conductive tethers include those having structures containing doped polythiophene, poly(3,4-ethylenedioxythiophene), polyacetylene, polypyrrole, polyaniline, polyfluorene, polyphenylene, polypyrene, polyazulene, polynaphthalene, polycarbazole, polyindole, or polyazepine. Charge doping of the tether structure is achieved, in some examples, by oxidation of the polymer. Exemplary conductive tethers and methods for their preparation are described in Vemitskaya et al. Russ. Chem. Rev. 66:44311 (1997); MacDiarmid, Angew. Chem., mt. Ed. 40:2581-2590 (2001); or McNeill et al., Aust. J. Chem. 16:1056-75 (1963). In some examples, the molecular sensor is a polymerase. In some examples, the charge sensor is a nanowire. In some examples, the molecular sensor is coupled to the charge sensor via π-bonding, electrostatics, FS interactions, or other monovalent non-covalent bonding modalities. In some examples, the molecular sensor is coupled to the charge sensor via a linker using conjugation. In some examples, the conjugation includes nucleophile / carbonyl, azide / phosphine, 1,4 Michael addition, 1,3-dipolar cycloaddition, inverse electron demand cycloaddition, olefin metathesis, or cross-coupling reactions. In some examples, a ternary complex is formed between a molecular sensor, a biomolecule, and a primer. In some examples, the ternary complex is attached to the charge sensor via a primer.In some examples, the ternary complex is coupled to the charge sensor via a molecular sensor. In some examples, the ternary complex is coupled to the charge sensor via a biomolecule.

[0067] The molecular sensor may include a graphene binding moiety configured to couple the molecular sensor to a charge sensor including a graphene layer. Such moieties may be coupled to other π-based charge sensors, such as graphene layers. In some examples, the graphene layer is 1-5 atoms thick. In some examples, the graphene layer is about 1 atom thick. In some examples, the graphene binder includes an aromatic group. In some examples, the graphene binder includes an aryl group or a heteroaryl group. In some examples, the graphene binder is a C6-C 30 In some examples, the graphene binder comprises a C6-C 20 In some examples, the graphene binder comprises a C6-C 15 In some examples, the graphene binder comprises a C6-C 10 In some instances, the graphene binder may comprise a C 10 -C 30 In some instances, the graphene binder may comprise a C 15 -C 30 The graphene binder comprises an aryl group or a heteroaryl group. In some examples, the graphene binder comprises an aromatic hydrocarbon. In some examples, 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. In some examples, the ternary complex (molecular sensor, biomolecule, and primer) is bound to the graphene binder via a primer, a polymerase, or a biomolecule.

[0068] An exemplary schematic diagram of a molecular wire construct is provided in FIG. 21. In some examples, the wire extends between two surfaces, such as the surface of a first electrode and the surface of a second electrode. The wire may extend between nanogaps in devices described herein. The molecular wire construct may include a molecular sensor (e.g., a polymerase) and a charge sensor (e.g., a nanowire). The molecular sensor and charge sensor may be in electrical communication as described herein. For example, the wire construct schematically illustrated in FIG. 21 may extend between the gaps shown in any one of the devices in FIGS. 11-20. In some examples, the wire extends from the surface of the first electrode (2105a) and the surface of the second electrode (2105b). In some examples, the surface of the first or second electrode is gold deposited as described herein. In some examples, the wire extends from the gold surface of the first electrode (2105a) and the gold surface of the second electrode (2105b). In some examples, one or both of the surfaces may include a tether (2125), a linking moiety (2110), or both. In some examples, the tether (2125) or the linking moiety (2110) is connected to a molecular wire construct. In some examples, the tether (2125) is biotin and the linking moiety (2110) is streptavidin. The molecular wire construct may include one or more linkers that can associate with the tether or linking moiety of the surface. In some examples, as shown, the molecular wire construct includes a molecular sensor (2115), such as a polymerase. In some examples, the molecular sensor may include one or more linkers (2120) that can associate with the tether (2125), the linking moiety (2110), or both. In some examples, the one or more linkers (2120) include a protein. In some examples, the one or more linkers (2120) include a peptide linker. In some examples, the one or more linkers (2120) include biotin. In some examples, one or more linkers (2120) comprise streptavidin. In some examples, one or more linkers (2120) comprise SpyCatcher. In some examples, one or more linkers (2120) comprise SpyTag.

[0069] The molecular wire constructs described herein can have one or more components, as illustrated by way of non-limiting example in Figure 21. The molecular wire constructs can have, by way of non-limiting example, a charge sensor (e.g., a polymerase), one or more linker constructs, such as a biotin-SA construct or a SpyCatcher and / or SpyTag construct, a gold nanoparticle, or a surface tether or linker (e.g., cysteamine, cysteine, thiol-biotin, protein, peptide, etc.). In some examples, the linker construct comprises a C1q / TNF-related protein (CTRP peptide). In some examples, the linker construct comprises a tryptophan-zipper pentamer. In some examples, the peptide linker comprises a five-stranded phenylalanine zipper.

[0070] In some examples, biotin-streptavidin (SA) constructs are used as molecular wire constructs. In some examples, DNA wire constructs are more resistive than biotin-SA constructs. In some examples, currents in the low nA range are achieved with biotin-SA wires. In some examples, high concentrations of thiol-DNA, long reaction times in buffer, or both may be required for Au conjugation. In some examples, Au conjugation involves the use of greater than about 100 μM thiol-DNA. In some examples, Au conjugation involves reaction times of about 48 hours or more. In some examples, biotin-SA wires can be easily assembled. In some examples, high concentrations of building blocks are readily available, allowing for shorter reaction conditions. In some examples, polymerase can be inserted into the biotin-SA wire, resulting in higher currents (e.g., higher signals) in the electric field.

[0071] In some examples, the molecular wire construct is about 5 nm to 20 nm in length. In some examples, the length of the molecular wire construct is adjusted by adjusting the length of one or more linkers (2120) or tethers (2125). The one or more linkers may be peptide linkers. In some examples, the molecular wire construct is about 5, 8, 10, 12, 15, 18, or 20 nm in length. In some examples, the molecular wire construct is at least about 5, 8, 10, 12, 15, or 18 nm in length. In some examples, the molecular wire construct is up to about 8, 10, 12, 15, 18, or 20 nm in length. In some examples, 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.

[0072] Figures 22A-22B provide a schematic diagram of surface functionalization. In some examples, surface (2205) comprises an electrode surface, which may comprise gold, as described herein. In some examples, the surface may be functionalized with a tether. In some examples, the tether comprises a first portion extending from the surface (2210) and a second portion (2215) available for association or conjugation with other molecules. In some examples, the first portion (2210) comprises cysteamine, and the second portion (2215) comprises biotin. A non-limiting example of a scheme for functionalizing a gold surface with cysteamine and biotin is provided in Figure 22B. For example, a gold surface can be functionalized with cysteamine by exposing the surface to 18 mM cysteamine in water for 4 hours. The surface can then be treated with biotin-NHS in DIPEA and BMF at 60°C for 4 hours.

[0073] In some examples, the surface is further functionalized with a linking moiety (2305) as described herein. An exemplary schematic is provided in Figure 23A. In some examples, the biotinylated surface is functionalized with SA. In some examples, the surface can be functionalized with SA by exposing the biotinylated surface to 100 μM SA in 0.1X PBS. In some examples, the biotinylated surface can be characterized using fluorescently labeled SA.

[0074] A molecular wire construct for association with a surface described herein is illustrated schematically in FIG. 23. The molecular wire can include a molecular sensor (2315). The molecular sensor can be in communication with a charge sensor as described herein. The molecular sensor, in some cases, can include a polymerase, such as those described herein. The molecular sensor can include one or more linkers (2320), such as a peptide linker. In some examples, the peptide linker includes a terminal portion (2310) for association with a functionalized surface. In some examples, the terminal portion includes biotin. Thus, in some cases, the molecular wire includes a bis-biotinylated polymerase at the C-terminus and N-terminus. The biotin on the molecular sensor can be used to link the molecular wire to a surface, such as an SA on a surface described herein. In some examples, the length of the linker (e.g., a peptide linker) is adjusted depending on the experiment. For example, the length of the linker is adjusted depending on empirical experiments based on nanoelectric devices, such as those described herein, without limitation. FIG. 23C provides a map illustrating a dual-biotin-tagged P29 variant RPN that can be used to generate bis-biotinylated polymerases.

[0075] An exemplary schematic of the assembly of a molecular wire is provided in Figure 24. As shown, a surface (e.g., gold) may be functionalized for association or conjugation with the molecular wire. In some examples, the molecular wire may contain DNA for sequencing. The molecular wire may be assembled for sequencing. In an exemplary embodiment, the molecular wire is assembled in 10 mM MOPS, 10 mM KOAc, and 5 mM Mg at pH 7.5. 2+ In some examples, the surface is functionalized with SA and the molecular wire comprises biotin to form a molecular wire construct. The molecular wire construct can be used to sequence DNA using the devices and methods described herein.

[0076] The molecular wire construct may include gold nanoparticles. In some examples, the gold nanoparticles can be used to characterize streptavidin or biotin binding. An exemplary schematic is provided in Figure 25. In some examples, the surface is functionalized with biotin and the gold nanoparticles are functionalized with SA, or vice versa. In some examples, the nanoparticles are 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 nm. In some examples, the nanoparticles are 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. In some examples, the nanoparticles are up to 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 examples, the nanoparticles are up to 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.

[0077] An example of gold nanoparticles in a molecular wire construct is illustrated in Figure 26. As shown, the molecular wire construct may be linked between gold-coated surfaces. The surfaces may be electrodes on the devices described herein. In some examples, the gold surface is functionalized with thiol-biotin. The thiol-biotin may be functionalized with streptavidin. In some examples, the streptavidin may be bound to biotin-labeled gold nanoparticles. The biotin-labeled gold nanoparticles may be further bound to monomeric streptavidin. The monomeric streptavidin may be linked to a polymerase via a peptide linker. However, in some examples, the molecular wire construct may not include gold nanoparticles. Referring to Figure 27, the gold-coated surface may be functionalized with monomeric streptavidin and thiol-biotin, which may be linked to a polymerase via a peptide linker. In some examples, as shown in Figure 29, a gold-coated surface is functionalized with monomeric streptavidin and thiol-biotin, which can be linked to a polymerase via a peptide linker (e.g., an AVI tag).

[0078] In a further example, a gold-coated surface can be functionalized with cysteamine, as shown, for example, in Figure 28. In some examples, the cysteamine can be functionalized with biotin-PEG aldehyde, which can be used to link monomeric streptavidin and a polymerase via a peptide linker, as described herein.

[0079] In some examples, the gold-coated surface may be functionalized with a terminal cysteine. A SpyCatcher moiety or SpyTag may be used to link the polymerase between the surfaces. In some examples, cysteines on the gold-coated surface are functionalized with SpyCatcher, as shown in FIG. 30. In such cases, SpyTag may be further used to link the polymerase. In some examples, cysteines on the gold-coated surface are functionalized with SpyTag, as shown in FIG. 31. In such cases, SpyCatcher moieties may be further used to link the polymerase. In some examples, the gold-coated surface is partially functionalized, for example, as shown in FIG. 30. In some examples, the gold-coated surface is fully functionalized, for example, as shown in FIG. 31.

[0080] In some examples, the linkage in the molecular wire construct further comprises a peptide. In some examples, the peptide comprises C1q / TNF-related protein (CTRP protein). A terminal cysteine ​​residue on the gold-coated surface can be functionalized with the CTRP peptide. In an exemplary embodiment, the CTRP peptide can be further linked to a SpyTag, which can be connected to a polymerase via SpyCatcher, as shown in Figure 32. In some examples, the peptide linker comprises a peptide zipper. The peptide zipper can comprise a tryptophan-zipper pentamer or a five-stranded phenylalanine zipper. In an exemplary embodiment, the gold-coated surface can be functionalized with a terminal cysteine. A peptide zipper, such as a tryptophan-zipper pentamer or a five-stranded phenylalanine zipper, or both, can be used to connect to a polymerase, as shown in Figure 33.

[0081] Nanoelectric Device Fabrication

[0082] Methods for fabricating devices and surfaces for molecular sensing are provided herein. In some examples, the methods herein include depositing material onto one or more base layers. In some examples, the methods include depositing material to create an electrode or a passivation layer. In some examples, the methods include patterning the electrode or the passivation layer. In some examples, the methods described herein include etching. In some examples, the methods described herein include isotropic or substantially isotropic etching. In some examples, the etching creates an undercut edge of the electrode. In some examples, the methods include at least some of the following steps: a) providing one or more base layers; b) depositing material to create a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to create a passivation layer; f) depositing material to create a first electrode; g) patterning the first electrode; and h) isotropically etching the passivation layer such that the edge of the first electrode is undercut. In some examples, the method further includes depositing a material configured to bond to the nanowires. In some examples, the method further includes depositing gold on a top layer of the device. In some examples, the method further includes depositing gold on one or more electrodes. In some examples, the method includes etching or lithography. In some examples, the method includes RIE (reactive ion etching). In some examples, the patterning includes lithography and / or RIE. In some examples, the method does not include e-beam or DUV (deep ultraviolet) lithography. In some examples, the method includes depositing gold on the first electrode and the second electrode.

[0083] Methods for supporting the immobilization of biomolecules (such as nanowires) on a substrate are provided herein. In some examples, the surfaces of the structures described herein include and / or are coated with a material that facilitates coupling reactions with biomolecules for attachment. To prepare structures for immobilizing biomolecules, surface modification may be employed, which chemically and / or physically alters the substrate surface through additive or subtractive processes that alter one or more chemical and / or physical properties of the substrate surface or selected sites or regions of the surface. For example, surface modification can include (1) changing the wetting properties of the surface; (2) functionalizing the surface, e.g., by providing, modifying, or substituting surface functional groups; (3) defunctionalizing the surface, e.g., by removing surface functional groups; (4) otherwise altering the chemical composition of the surface, e.g., by etching; (5) increasing or decreasing surface roughness; (6) providing a coating on the surface, e.g., a coating that exhibits wetting properties different from those of the surface; and / or (7) depositing particulates on the surface. In some examples, the surface of a structure is selectively functionalized to create two or more distinct regions on the structure, with at least one region having different surface or chemical properties than another region of the same structure, including, but not limited to, surface energy, chemical termination, surface concentration of chemical moieties, etc.

[0084] The surfaces provided herein can have active regions, passive regions, or both. The active regions may be referred to as actively functionalized surfaces. In some examples, the active regions are functionalized with an active material. In some examples, the passive regions are functionalized with a passive material. In some examples, the surfaces of the structures disclosed herein are modified to include one or more actively functionalized surfaces configured to bind to the surface of both the substrate and biomolecules, thereby supporting coupling reactions to the surface. In some examples, the surfaces are also functionalized with a passive material that does not efficiently bind biomolecules. In some examples, the surface functionalized with a passive material prevents binding of biomolecules at sites where the passive functionalizing agent is attached. In some cases, the surface includes an active layer that defines only discrete loci for supporting biomolecules.

[0085] In some examples, functionalization involves depositing a functionalizing agent onto the structure by any deposition technique, including 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, evaporation), and molecular layer deposition (MLD).

[0086] Optional steps or components in the following functionalization processes are omitted or modified according to the desired properties of the final functionalized substrate. In some cases, additional components and / or process steps are added to the process workflow implemented herein. In some examples, the substrate is first cleaned, for example, using piranha solution. One example of a cleaning process involves immersing the substrate in piranha solution (e.g., 90% H2SO4, 10% HO2) at elevated temperatures (e.g., 120°C), rinsing (e.g., with water), and drying the substrate (e.g., with nitrogen gas). This process optionally includes a post-piranha treatment, which involves immersing the piranha-treated substrate in a basic solution (e.g., NH4OH) followed by an aqueous rinse (e.g., with water). In some examples, the surface of the structure is optionally piranha cleaned after the piranha immersion and optional post-piranha treatment. One example of a plasma cleaning process includes oxygen plasma etching. In some examples, the surface is deposited with an active functionalizing agent after vapor deposition. In some instances, the substrate is actively functionalized prior to cleaning, such as by piranha treatment and / or plasma cleaning.

[0087] The process for surface functionalization optionally includes resist coating and resist stripping. In some examples, after the active surface functionalization, the substrate is spin-coated with a resist, such as SPR™ 3612 positive photoresist. In various examples, the process for surface functionalization includes lithography using patterned functionalization. In some examples, photolithography is performed after resist coating. In some examples, after lithography, the surface is visually inspected for lithography defects. In some examples, the process for surface functionalization includes a cleaning step, whereby substrate residues are removed, for example, by plasma cleaning or etching. In some examples, a plasma cleaning step is performed during some steps after the lithography step.

[0088] In some examples, resist-coated surfaces are treated to remove the resist, for example, after functionalization and / or after lithography. In some cases, the resist is removed using a solvent, such as a stripping solution containing N-methyl-2-pyrrolidone. In some cases, resist stripping involves sonication or ultrasonic treatment. In some examples, the resist is coated and stripped, followed by active functionalization of the exposed areas to create a desired, unique functionalized pattern.

[0089] In some examples, methods and compositions described herein relate to the application of photoresist to create modified surface properties in selected regions. In some examples, the application of photoresist varies depending on the surface fluid properties, which define the spatial distribution of the photoresist. Without being bound by theory, surface tension effects associated with the applied fluid may dictate the flow of the photoresist. For example, surface tension and / or capillary action effects may facilitate the drawing of the photoresist into small structures in a controlled manner before the resist solvent evaporates. In some examples, contact points of the resist are pinned by sharp edges, thereby controlling the advancement of the fluid. The underlying structures may be designed based on the desired flow pattern used to apply the photoresist during the fabrication and functionalization processes. The solid organic layer remaining after the solvent evaporates may be used to continue subsequent steps in the fabrication process. The structures may be designed to control fluid flow by promoting or inhibiting capillary effects to adjacent fluid paths. For example, the structures may be designed to avoid overlap between the top and bottom edges, which promotes the retention of fluid in the upper structures, allowing for specific placement of the resist. In an alternative example, the upper and lower edges overlap, thereby causing the applied fluid to wick into the underlying structures. An appropriate design may therefore be selected depending on the desired application of the resist.

[0090] In some examples, the structures described herein have a surface comprising a material that contains reactive groups capable of molecular sensing and that is at least 0.1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm, or 25 nm thick. Exemplary materials include, but are not limited to, gold, glass, and silicon, such as silicon oxide and silicon nitride. In some cases, exemplary surfaces include nylon and PMMA.

[0091] In some examples, electromagnetic radiation in the form of UV light is used to pattern the surface. In some examples, a lamp is used to pattern the surface, and a mask mediates the location of the UV light exposure on the surface. In some examples, a laser is used to pattern the surface, and the open / closed state of a shutter controls the location of the UV light exposure on the surface. The laser device may be used in conjunction with a movable flexible structure. In such a configuration, coordination of laser exposure and movement of the flexible structure is used to generate a pattern of one or more agents having different nucleoside coupling capabilities.

[0092] Described herein are reusable surfaces for molecular sensing. After loading with charge sensors, the surface may be bath-immersed, washed and cleaned, baked and etched, and otherwise functionally regenerated to a state suitable for subsequent molecular sensing. The number of times the surface is reused and the method for recycling / preparing the surface for reuse vary depending on the subsequent application. In some instances, surfaces prepared for reuse are reused approximately 1, 2, 3, 5, 10, 20, 50, 100, 1,000, or more times. In some instances, surfaces prepared for reuse are reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1,000, or more times. In some instances, the remaining "lifespan" or number of times the surface is suitable for reuse is measured or predicted.

[0093] In some examples, the layers of the device are incorporated into a solid support. In some examples, the layers comprise electrodes or are configured for use as electrodes. The devices, in some examples, comprise at least 2, 3, 4, 5, 6, 10, 20, or more electrodes per device. In some examples, the electrodes are configured as source, drain, or gate. In some examples, the layers comprise metal oxide layers. In some examples, the layers comprise metal oxide layers with a continuous metal layer underneath. The electrodes, in some examples, comprise at least one conductor and are fabricated from materials known in the art. In some examples, the electrodes comprise at least one conductor and one or more insulators or semiconductors. In some examples, the electrodes comprise platinum, titanium, or titanium nitride. In some examples, the 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. The materials, in some examples, include metals, non-metals, mixed metal oxides, nitrides, carbides, silicon-based materials, or other materials. In some examples, metal oxides include TiO2, Ta2O5, IrO2, RuO2, RhO2, Nb2O5, Al2O3, BaO, YO3, HfO2, SrO, or other metal oxides known in the art. In some examples, metal carbides include TiC, WC, ThC2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbides known in the art. In some examples, metal nitrides include GaN, InN, BN, Be3N2, Cr2N, MoN, Si3N4, TaN, Th2N2, VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitrides known in the art. In some examples, devices disclosed herein are fabricated using the materials listed herein or in combination with any other suitable materials known in the art. In some examples, the layers described herein are coated with an additional metal. In some examples, the layers described herein are coated with an additional material configured to attach nanowires, hi some examples, one or more layers are coated with gold.In some examples, one or more electrodes are coated with gold. In some examples, the gold is deposited using direct thermal evaporation. In some examples, the gold is deposited using fixed angle direct thermal evaporation. In some examples, the gold is deposited using electroplating. In some examples, the gold layer has a thickness of about 10, about 25, 30, 40, 50, 60, or about 75 angstroms. In some examples, the gold layer has a thickness of at least 10, 25, 30, 40, 50, 60, or at least 75 angstroms. In some examples, the gold layer has a thickness of 10 angstroms or less, 25, 30, 40, 50, 60, or 75 angstroms or less. In some examples, the gold layer is added to a layer comprising another metal. In some examples, the gold layer is added to a layer comprising titanium.

[0094] In some examples, the device is in contact with one or more charge sensors. In some examples, the charge sensor is attached to one or more electrodes. In some examples, the charge sensor spans at least two electrodes. Such a charge sensor is, in some examples, attached to a tether. In some examples, the tether is further coupled to a molecular sensor. In some examples, the charge sensor comprises a nanowire. In some examples, the nanowire comprises a nucleic acid. In some examples, the charge sensor (e.g., a nucleic acid) is mounted on the device surface. The mounting, in some examples, includes one or more of the following steps: (a) applying a voltage to the electrodes to attract a nucleic acid strand to the electrodes; (b) monitoring the current path between the electrodes to determine whether the nanowire bridges the two electrodes; and (c) turning off the voltage when the current spikes (e.g., touches) to prevent further DNA attraction. In some examples, the applied voltage is about 10-100 V, 10-50 V, 10-25 V, 10-75 V, or 25-100 V. In some examples, the load 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 examples, the load voltage is applied for about 0.01 seconds, 0.05 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 90 seconds, or 120 seconds or less.

[0095] Provided herein are devices including a surface. In some examples, the surface is modified to support molecular sensing at a predetermined location. In some examples, the surface of the molecular sensing device provided herein is made of various materials that can be modified to support molecular sensing. In some cases, the device is sufficiently conductive, for example, capable of forming a uniform electric field across all or part of the device. The devices described herein may include a flexible material. Exemplary flexible materials include, but are not limited to, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. The devices described herein may include a rigid material. Exemplary rigid materials include, but are not limited to, glass, fused silica, silicon, silicon oxide, silicon nitride, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof), and metals (e.g., gold, platinum). The devices disclosed herein may be fabricated from materials including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, the devices disclosed herein are fabricated using a combination of the materials listed herein or any other suitable materials known in the art.

[0096] The devices described herein may include materials having a range of tensile strengths. Exemplary materials having a range of tensile strengths include, but are not limited to, nylon (70 MPa), nitrocellulose (1.5 MPa), polypropylene (40 MPa), silicone (268 MPa), polystyrene (40 MPa), agarose (1-10 MPa), polyacrylamide (1-10 MPa), and polydimethylsiloxane (PDMS) (3.9-10.8 MPa). The solid supports described herein can have tensile strengths of 1-300, 1-40, 1-10, 1-5, or 3-11 MPa. The solid supports described herein can have a tensile strength of about 1 MPa, about 1.5 MPa, about 2 MPa, about 3 MPa, about 4 MPa, about 5 MPa, about 6 MPa, about 7 MPa, about 8 MPa, about 9 MPa, about 10 MPa, about 11 MPa, about 20 MPa, about 25 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 150 MPa, about 200 MPa, about 250 MPa, about 270 MPa, or more. In some examples, the devices described herein include solid supports for molecular sensing that are in the form of flexible materials that can be stored in a continuous loop or reel, such as a tape or flexible sheet.

[0097] Young's modulus measures a material's resistance to elastic (recoverable) deformation under load. Exemplary materials with a range of Young's modulus stiffness include, but are not limited to, nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicone (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10 GPa), and polydimethylsiloxane (PDMS) (1-10 GPa). The solid supports described herein can have a Young's modulus of 1-500 GPa, 1-40 GPa, 1-10 GPa, 1-5 GPa, or 3-11 GPa. The solid supports described herein can have a tensile strength of about 1 GPa, about 1.5 GPa, about 2 GPa, about 3 GPa, about 4 GPa, about 5 GPa, about 6 GPa, about 7 GPa, about 8 GPa, about 9 GPa, about 10 GPa, about 11 GPa, about 20 GPa, about 25 GPa, about 40 GPa, about 50 GPa, about 60 GPa, about 70 GPa, about 80 GPa, about 90 GPa, about 100 GPa, about 150 GPa, about 200 GPa, about 250 GPa, about 400 GPa, about 500 GPa, or more. Because flexibility and stiffness are inversely related to each other, a flexible material has a low Young's modulus and changes its shape significantly under load. In some examples, the solid supports described herein have a surface that is at least as flexible as nylon.

[0098] In some cases, the devices disclosed herein include a silicon oxide base and a silicon oxide surface layer. Alternatively, the devices may have a silicon oxide base. The surfaces of the devices provided herein may be textured, thereby increasing the total surface area for molecular sensing. The devices disclosed herein, in some examples, include at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. The devices disclosed herein, in some examples, are fabricated from silicon-on-insulator (SOI) wafers.

[0099] The structures may be fabricated from a variety of materials suitable for the methods and compositions of the present invention described herein. In some instances, the materials from which the substrates / solid supports comprising the present invention are fabricated exhibit low levels of polynucleotide binding. In some circumstances, materials that are transparent to visible and / or UV light can be utilized. Sufficiently conductive materials can be utilized, for example, materials capable of forming a uniform electric field across all or a portion of the substrates / solid supports described herein. In some instances, such materials may be connected to electrical ground. In some cases, the substrate or solid support can be thermally conductive or insulating. The material can be chemically and heat resistant to support chemical or biochemical reactions. For flexible materials, materials of interest can include nylon (both modified and unmodified), nitrocellulose, and polypropylene.

[0100] For rigid materials, particular materials of interest include glass, fused silica, silicon, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, etc.), and metals (e.g., gold, platinum, etc.). The structure may be selected from the group consisting of materials including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), and glass. The substrate / solid support of the microstructure, the reactor therein, may be fabricated using the materials listed herein or in combination with any other suitable material known in the art.

[0101] In some examples, the substrates disclosed herein comprise computer-readable material, including, but not limited to, magnetic media, reel-to-reel tape, cartridge tape, cassette tape, flexible disk, paper media, film, microfiche, continuous tape (e.g., belt), and any medium suitable for storing electronic instructions. In some cases, the substrate comprises magnetic reel-to-reel tape or a magnetic belt. In some examples, the substrate comprises a flexible printed circuit board.

[0102] The structures described herein may be transparent to visible and / or UV light. In some examples, the structures described herein are sufficiently electrically conductive to form a uniform electric field across all or a portion of the structure. In some examples, the structures described herein are thermally conductive or insulating. In some examples, the structures are chemically and heat resistant to support chemical reactions. In some examples, the substrate is magnetic. In some examples, the structures comprise a metal or metal alloy. The structures described herein may be incorporated into a rack, such as a rack unit of a rack server described herein.

[0103] Molecular Sensing

[0104] The devices, systems, and methods described herein are useful for molecular sensing. In some examples, an analyte interacts with a molecular sensor in electrical communication with one or more electrodes. In some examples, interaction with the analyte results in a detectable voltage, current, or resistance change (signal or signal pattern) in the device. In some examples, the analyte includes a nucleotide triphosphate. In some examples, one or more nucleotide triphosphates generate unique (distinguishable) signals. In some examples, each type of nucleotide triphosphate correlates with a unique base identity. By repeating such measurements to generate one or more signals, in some examples, the sequence or identity of a nucleoside in a nucleic acid is determined. In some examples, the molecular sensor includes a polymerase. In some examples, binding of the nucleotide triphosphate to the polymerase generates a measurable signal within a device described herein. In some examples, one or more nucleotide triphosphates include a non-standard or non-natural amino acid. In some examples, the nucleotide triphosphate includes a non-natural or non-standard base. In some examples, the non-natural or non-standard base is configured to generate a unique signal or signal pattern. Such signals are measured between one or more electrodes in some examples.

[0105] In an exemplary configuration, the method described herein is provided in FIG. 1. In some examples, the method includes any one of the steps in FIG. 1. A polynucleotide (103) bound (and / or synthesized) from a surface (101) is cleaved (102). A primer (104) is added (105), followed by binding of a molecular sensor (e.g., a polymerase) to a sensor binding moiety (115) (a graphene binder, optionally via a linker, as shown in FIG. 1) to form a ternary complex (108). The ternary complex (108) is then contacted (109) with a graphene device (110), binding the ternary complex (108) to the graphene layer. A charge-modulated nucleotide (CMN, indicated by a base letter and an asterisk) (111) is added, which extends the primer in the ternary complex. The change in current resulting from the incorporation of the CMN (111) generates a signal (114) that can be measured, thereby identifying the incorporated base. In some examples, the charge sensor includes a graphene layer and the molecular sensor includes a polymerase.

[0106] The method may include one or more of the following steps: (a) providing a polymerase bound to a solid support charge sensor; (b) contacting the polymerase with a mixture of nucleotides; (c) detecting nucleotide incorporation using the charge sensor; (d) repeating steps (b) and (c) using a second mixture of polymerase, template nucleic acid, and nucleotides; and (e) comparing the first signal pattern with the second signal pattern to determine the sequence of the template nucleic acid. In some examples, the mixture in (b) contains different types of nucleotides. In some examples, the first type of nucleotides are distinguishable from the other types of nucleotides in the mixture in (b). In some examples, the second type of nucleotides are not distinguishable from the other types of nucleotides in the mixture in (b). In some examples, the polymerase incorporates the nucleotides of the mixture in (b) into a nascent strand relative to the template nucleic acid strand. In some examples, detecting nucleotide incorporation by charge includes a first type of nucleotide that generates a signal that is unique compared to signals generated by other nucleotides in the mixture, thereby obtaining a first signal pattern. In some examples, repeating steps (b) and (c) using a polymerase includes a second type of nucleotide that is distinguishable compared to other types of nucleotides in the second mixture. In some examples, repeating steps (b) and (c) using a polymerase includes a first type of nucleotide that is not distinguishable compared to other types of nucleotides in the second mixture, thereby obtaining a second signal pattern.

[0107] Also provided is a method for sequencing a nucleic acid, the method comprising one or more of the following steps: (a) providing a polymerase bound to a solid support charge sensor; (b) contacting the polymerase with a mixture of nucleotides; (c) detecting incorporation of the nucleotides using the charge sensor; (d) repeating steps (b) and (c) using a second mixture of the polymerase, template nucleic acid, and nucleotides; and (e) comparing the first signal pattern with the second signal pattern to determine the sequence of the template nucleic acid. In some examples, the mixture in (b) contains different types of nucleotides. In some examples, the first two types of nucleotides are in a first distinguishable state from the second two types of nucleotides in the mixture in (b). In some examples, the polymerase incorporates the nucleotides of the mixture in (b) into a nascent strand relative to the template nucleic acid strand. In some examples, the detection of incorporation of the nucleotides using the charge sensor includes the first two types of nucleotides generating a signal that is distinguishable from the signal generated by the second two types of nucleotides in the mixture, thereby obtaining a first signal pattern. In some examples, repeating step (b) and step (c) uses a polymerase for one of the first two types of nucleotides that is distinguishable compared to the other first two types of nucleotides in the second mixture, thereby obtaining a second signal pattern.

[0108] In some examples, one or more non-natural nucleotides present in the mixture generate a signal change with an inverted polarity compared to other nucleotides in the mixture. Alternatively, or additionally, one or more non-natural nucleotides used in the mixture cause a delay in nucleotide incorporation or a reduced incorporation rate. Alternatively, or additionally, one or more non-natural nucleotides used in the method generate a significantly altered signal height. These signal parameters can be detected to distinguish the nucleotide in the template nucleic acid to which the non-natural nucleotide complements during polymerase activity. In some examples, a method using the device described herein includes one or more of the following steps: providing an analyte; reacting, binding, or interacting the analyte with a sensor; and measuring an electrical signal generated from the sensor. In some examples, a method using the device described herein includes one or more of the following steps: providing at least one nucleotide, at least one template (nucleic acid), and at least one primer; extending the primer with at least one nucleotide; and measuring an electrical signal generated from the polymerase. In some examples, the electrical signal is analyzed to confirm the identity of at least one nucleotide incorporated by the polymerase. In some examples, at least one nucleotide comprises a terminator configured to prevent chain elongation. In some examples, the 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 examples, the 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 examples, the methods described herein are used to sequence 10 to 1000, 20 to 1000, 50 to 1000, 100 to 1000, 50 to 2000, 25 to 500, 25 to 200, 50 to 500, or 50 to 750 bases. In some examples, the nucleotides comprise charge-modulated nucleotides.

[0109] Interaction between the molecular sensor and the analyte (e.g., a polymerase and a nucleotide triphosphate) can generate a detectable signal. In some examples, the analyte comprises a charge-modulated nucleotide. In some examples, the non-natural moiety or modification present in the non-natural nucleotide generates a change in the polymerase structure (compared to the structure generated by a nucleotide lacking the moiety or modification), thereby generating a unique characteristic in one or more signal parameters detected by the charge sensor to which the polymerase is bound. 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. In some examples, the 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., a "low" abundance nucleotide) compared to the other three types (e.g., a "high" abundance nucleotide). As a result, incorporation of the low-abundance nucleotide is detectable as a relative delay or decreased incorporation rate. In some examples, this characteristic is utilized to identify the location in the template of the nucleotide complementary to the low-abundance nucleotide. In some examples, several sequencing runs are completed on the same template, with each run performed using a different nucleotide at a low level. In some examples, the signal patterns of the different runs are compared to determine the sequence of the template. In some examples, the method involves using a 3-high-1-low mixture of nucleotide triphosphates. Other ratios of high to low, as well as mixtures including, for example, a 1-high-3-low mixture or a 2-high-2-low mixture, are also described herein. Further useful mixture configurations using different concentrations of nucleotides are described in U.S. Pat. No. 7,556,922, which is incorporated herein by reference. In certain embodiments, the template nucleic acid is circular. In some examples, the use of a circular template provides a convenient format for performing iterative sequencing, as the polymerase does not need to be replaced and can instead be run multiple times around the template (each revolution effectively sequencing the template repeatedly).In some examples utilizing a circular template, the polymerase contains 5' exonuclease activity to digest different nucleic acid strands that displace the circular template as the polymerase progresses around the template multiple times. Whether the template is linear or circular, different primers are used in different sequencing runs performed on the same template in some examples. In some examples, different primers are designed to hybridize at different positions on the template. Thus, while each run begins at a different position on the template, in some examples, there is substantial overlap between the portions of the template sequenced in each run. In some examples, the signal patterns resulting from each run are aligned based on the expected start site for each run to aid in sequence calling and error checking. In some examples, the charge sensor used in the methods described herein detects nucleotide incorporation by the polymerase via the field effect using a SWST, FET, nanowire FET, FinFET, tri-gate FET, tunnel FET, or another field-sensitive device. In some examples, the sensor is magnetic, electrochemical, or nanoelectromechanical. In some instances, each nucleotide triphosphate generates a distinguishable state. In some instances, a distinguishable state refers to a specific type of nucleotide triphosphate that has a unique characteristic or property that is exhibited under detection conditions compared to other nucleotide triphosphates. Exemplary distinguishable states include, but are not limited to, a state in which the nucleotide triphosphate is present in an amount or concentration substantially less than the amount or concentration of other types of nucleotide triphosphates in the mixture, a state in which the nucleotide triphosphate is present in an amount or concentration substantially greater than the amount or concentration of other types of nucleotide triphosphates in the mixture, a state with a chemical moiety or modification not present in other types of nucleotide triphosphates in the mixture, or a state lacking a chemical moiety or modification present in other types of nucleotide triphosphates in the mixture. A distinguishable state can be exhibited when a nucleotide type interacts with a polymerase.In some instances, the signal is detected from a structural change, such as the appearance, disappearance, or change in a detectable signal from a molecule in response to a change in the structure, shape, or configuration of the molecular moiety. For example, the signal change can result from a change in the interaction of a label with a first portion of the molecule as it interacts with a second portion of the molecule.

[0110] The detectable signal may comprise a change in current, resistance, or voltage. In some examples, the detectable signal comprises a change in current. In some examples, the change in current is between 1 nanoamp and 100 picoamps, between 1 nanoamp and 50 picoamps, between 1 nanoamp and 25 picoamps, between 1 nanoamp and 10 picoamps, between 1 nanoamp and 1 picoamp, between 1 nanoamp and 500 nanoamps, between 1 nanoamp and 250 nanoamps, between 1 nanoamp and 100 nanoamps, between 100 nanoamps and 100 picoamps, between 1 nanoamp ... 00 nanoamperes to 10 picoamperes, 100 nanoamperes to 1 picoampere, 100 nanoamperes to 500 nanoamperes, 500 nanoamperes to 100 picoamperes, 500 nanoamperes to 50 picoamperes, 500 nanoamperes to 10 picoamperes, 1 picoamperes to 100 picoamperes, 10 picoamperes to 100 picoamperes, or 250 nanoamperes to 750 nanoamperes. In some examples, the detectable signal is measured as a change in signal relative to a background signal (e.g., in the absence of analyte). In some examples, 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 the background signal.

[0111] The methods described herein may enable rapid analysis of analytes. In some examples, 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. In some examples, 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. In some examples, the biomolecules include nucleotides or variants thereof. In some examples, the biomolecules include charge-modulated nucleotides (CMNs).

[0112] The analyte may comprise a nucleotide. In some examples, the analyte comprises a charge-modulated nucleotide. In some examples, the modification comprises a modification to the base (nucleobase) of the CMN, such as a modification to a C, T, G, or C base. Exemplary CMNs are shown in FIG. 3. In some examples, the modification comprises a deaza- or halogen-modified base. In some examples, the modification comprises a 7-deaza- or 8-bromo-modified base. In some examples, the CMN comprises a modification to the 5'-position. In some examples, the modification comprises a modification to a 5'-polyphosphate or a chemical variant thereof. In some examples, the modification comprises a thiolated or brominated phosphate. In some examples, the polyphosphate comprises at least 3, 4, 5, 6, 8, or 10 phosphates or variants thereof. In some examples, the modification comprises a modification to a 5'-terminal polyphosphate or a chemical variant thereof. In some examples, the modification comprises a polymer. In some examples, the polymer comprises one or more of a nucleic acid chain, a peptide chain, a polysaccharide, a lipid, a synthetic polymer, and a dendrimer. In some examples, the nucleic acid strand 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 examples, the nucleic acid strand 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. In some examples, the nucleic acid strand 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.In some examples, the nucleic acid strand comprises 25 to 300, 25 to 500, 25 to 400, 25 to 300, 25 to 250, 50 to 500, 50 to 300, 75 to 300, 75 to 250, 75 to 200, 100 to 500, 125 to 500, 200 to 500, 300 to 500, 10 to 250, 25 to 5000, 50 to 5000, 100 to 5000, 200 to 5000, 500 to 5000, 1000 to 5000, 2000 to 5000, or 10 to 5000 bases.

[0113] In some examples, the nucleic acid strand is branched (dendrimer). In some examples, the nucleic acid strand comprises a secondary structure. In some examples, the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadruplex, and an I-motif. In some examples, the nucleic acid strand comprises a single strand, a double strand, or a triplex. In some examples, the nucleic acid strand comprises at least one charge-modulating chemical modification. In some examples, the at least one charge-modulating chemical modification increases the charge of CMN relative to an unmodified nucleotide. In some examples, 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 examples, the at least one charge-modulating chemical modification decreases the charge of CMN relative to an unmodified nucleotide. In some examples, the at least one charge-modulating chemical modification comprises one or more of phosphate, phosphite, sulfonate, sulfite, carboxylate, xanthate, thiocarboxylic acid, boranophosphonate, and boric acid. In some examples, the nucleic acid strand comprises at least one sugar-modified nucleotide. In some examples, the sugar-modified nucleotide comprises a deoxynucleotide or a dideoxynucleotide. In some examples, the nucleic acid strand comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid.

[0114] The nucleic acid strand may include a phosphate modification. In some examples, the phosphate modification includes a hydrophobic group. In some examples, the hydrophobic group includes a linear or branched alkyl chain. In some examples, the hydrophobic group includes a C5-C 50It comprises an aliphatic chain. In some examples, the phosphate modification comprises a hydrophilic group. In some examples, the hydrophilic group comprises polyethylene glycol (PEG). In some examples, the polyethylene glycol comprises a molecular weight of 1,000 to 100,000, 100 to 500,000, 100 to 250,000 daltons, 100 to 75,000, 100 to 50,000, 100 to 25,000, 100 to 10,000, 100 to 7,500, 100 to 5,000, 100 to 3,000, or 100 to 2,000 daltons. In some examples, 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 examples, 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. In some examples, the polyethylene glycol comprises a molecular weight of up to 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 examples, the polyethylene glycol comprises 10 to 600 monomers. In some examples, the polyethylene glycol comprises about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 monomers. In some examples, the polyethylene glycol comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 monomers. In some examples, the polyethylene glycol comprises up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 monomers. In some examples, the peptide chain is 1-100 amino acids in length.In some examples, 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 examples, 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 examples, 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. In some examples, the CMN comprises a charged small molecule. In some examples, the charged small molecule comprises one or more of a chelator, a dye, and a metal complex. In some examples, the metal complex comprises ferrocene, Ru-dipy, and bis-cyclopentadienyl diiron.

[0115] The molecular sensors described herein may be conformationally labeled. In some examples, the conformational label includes at least one label that responds to a change in the structure of the molecule, a change in the shape of the molecule, or a change in the configuration of a portion of the molecule. In some examples, the molecule is a polymerase, reverse transcriptase, exonuclease, or other nucleic acid enzyme. A portion of a molecule can be, for example, an atom that changes relative position due to the rotation of about one or more chemical bonds that occur in the molecular structure between atoms. A portion of a molecule can be a domain of a polymer, such as a polymer commonly known in the relevant art. In some examples, polymerases include domains referred to as finger, palm, and thumb domains. In the case of proteins, a portion can be a region of secondary structure, tertiary structure, or quaternary structure. One or more labels can be attached to a molecule, for example, via a covalent bond. However, the one or more labels need not be attached to a molecule that is, for example, located in close proximity to the molecule. In certain embodiments, the label is not attached to a reactant or product of the molecule, such as a nucleotide or nucleic acid. In some examples, the molecular sensor includes a polymerase.

[0116] Methods for sequencing nucleic acids are provided herein. In some examples, the methods include one or more of the following steps: 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 complexes comprise a graphene binder; detecting one or more bases of the polynucleotides, wherein the detection occurs when the plurality of ternary complexes are bound to the graphene layer; removing the ternary complexes from the surface; and repeating the previous steps to sequence the polynucleotides. In some examples, the ternary complexes are attracted to the graphene layer by applying a positive charge to one or more electrodes embedded under 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 nucleotides. 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 nucleotides. In some examples, the plurality of polynucleotides is 50 to 30,000, 50 to 10,000, 50 to 1000, 50 to 750, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 1000 to 30,000, 1000 to 20,000, 1000 to 10,000, 2000 to 5000, 2000 to 10,000, 5000 to 30,000, or 10,000 to 30,000 bases in length. In some examples, the plurality of polynucleotides is about 10, 50, 100, 200, 300, 400, 500, 750, 1000, 2000, 2500, 5000, 10,000, 20,000, or 30,000 bases in length.

[0117] Nucleic acid-based information

[0118] Devices, compositions, systems, and methods for reading nucleic acid-based information (data) are provided herein. In the first step, a digital sequence encoding an item of information (e.g., binary-coded digital information for computer processing) is received. An encryption method is applied to convert the digital sequence from one or more symbols (e.g., binary code) into a nucleic acid sequence. A surface material for nucleic acid extension, a design of locations for nucleic acid extension (e.g., constituent spots), and reagents for nucleic acid synthesis are selected. The surface of the structure is prepared for nucleic acid synthesis. In some instances, de novo polynucleotide synthesis is performed. The synthesized polynucleotides are stored and available, in whole or in part, for subsequent release. In some instances, a pool of predetermined polynucleotides is assembled into larger polynucleotides representing digital information. Once released, the polynucleotides are sequenced, in whole or in part, using the devices, systems, and methods described herein and subjected to decryption to convert the nucleic acid sequence back into a digital sequence. The digital sequences are then assembled to obtain an alignment encoding for the original information item. In some examples, the polynucleotides are sequenced using the methods and devices described herein.

[0119] The digital information encoded by the nucleic acid may include an error correction component. In some examples, the error correction component includes an error correction code, such as a Reed-Solomon (RS) code, an LDPC code, a Polar code, or a Turbo code. In some examples, the error correction code spreads the stored digital data across many polynucleotides. In some examples, spreading the data across multiple polynucleotides builds in redundancy for correcting deletions (e.g., missing oligos). In some examples, the digital information can be restored in the presence of errors. In some examples, the error correction component includes a parity base. In some examples, the error correction component includes an index sequence. In some examples, the index sequence defines the location or address of the digital information encoded in the nucleic acid. In some examples, the index sequence defines the source of the digital information. The nucleic acid encoding the digital information, in some examples, includes overlap with one or more nucleic acids in the same library or set. In some examples, the error correction component includes regions of overlap or redundancy. In some examples, an algorithm is applied to the sequenced nucleic acid to reduce errors. In some examples, the error correction algorithm includes consensus sequencing, HEDGE (Hash Encoded, Decoded by Greedy Exhaustive Search), or other methods.

[0120] Nucleic acids encoding digital information may be stored on different media. In some instances, the nucleic acids are stored essentially as dry or lyophilized powders. In some instances, the nucleic acids are stored in a buffer solution. In some instances, the nucleic acids are stored on a chip, wafer, or other silicon solid support. In some instances, the nucleic acids are stored within an organism (or population of organisms), such as a plasmid or genome.

[0121] Optionally, the initial stage of the data storage process disclosed herein involves acquiring or receiving one or more items of information in the form of initial code. Items of information include, but are not limited to, text, audio, and visual information. Exemplary sources for items of information include, but are not limited to, books, periodicals, electronic databases, medical records, printed matter, forms, audio recordings, animal records, biological profiles, broadcasts, films, short videos, emails, recorded phone logs, internet activity logs, drawings, paintings, printouts, photographs, pixelated graphics, and software code. Exemplary sources of biological profiles for items of information include, but are not limited to, gene libraries, genomes, gene expression data, and protein activity data. Exemplary formats for items of information include, but are not limited to, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls, .xlsx, .rtf, .jpg, .gif, .psd, .bmp, .tiff, .png, and .mpeg. The amount of individual file size encoding an item of information, or the amount of multiple files encoding an item of information, in digital format may include, but is not limited to, up to 1024 bytes (equal to 1 KB), 1024 KB (equal to 1 MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1 TB), 1024 TB (equal to 1 PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyte, or more. In some examples, the amount of digital information is at least 1 gigabyte (GB). In some examples, 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 examples, the amount of digital information is at least 1 terabyte (TB).In some examples, 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 examples, the amount of digital information is at least 1 petabyte (PB). In some examples, 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.

[0122] The solid support for molecular sensing as described herein has a high capacity for data readout. For example, the capacity of the solid support is at least or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1000 petabytes. In some examples, the capacity of the solid support is about 1 to about 10 petabytes, or about 1 to about 100 petabytes. In some examples, the capacity of the solid support is about 100 petabytes. In some examples, the data is stored as an array of packets addressable as droplets. In some examples, the data is stored as an array of packets addressable as spots. In some examples, the data is stored as an array of packets addressable as dry wells. In some examples, the addressable array contains 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. In some examples, the addressable array contains 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. In some examples, the item of information is stored in a background of data. For example, the item of information encodes about 10 to about 100 megabytes of data and is stored in a background of 1 petabyte of data. In some examples, the item of information encodes at least or more than about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or 500 megabytes of data, and is stored with more than 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or 500 petabytes of background data.

[0123] Computer Systems

[0124] In various embodiments, any of the systems described herein are operably connected to a computer and, optionally, automated via a computer, either locally or remotely. In various examples, the methods and systems of the present invention further include software programs on a computer system and their use. Thus, computer control of material deposition device movement, dispense operations, and synchronization of dispense / vacuum / refill functions, such as coordination and synchronization of vacuum operations, is within the scope of the present invention. In some examples, a computer system is programmed to interface between a user-specified base sequence and the position of a material deposition device to deliver precise reagents to specific regions of a substrate. As an example, a computer system, such as the system shown in FIG. 7 or FIG. 8, may be used to encode data represented as a set of symbols into another set of symbols. For example, data may be represented as numeric symbols, such as binary values ​​of "0" and "1," and the computer system may execute a program including error-correcting codes (e.g., Reed-Solomon (RS) codes, low-density parity-check (LDPC) codes, turbo codes, etc.). In some examples, the computer system executes a program to convert data into a plurality of nucleic acid sequences, convert a plurality of nucleic acid sequences into data, or both. In some examples, the program may be a machine learning algorithm. In some examples, the machine learning algorithm may determine a nucleotide base based on a signal (e.g., an electrical signal such as a current or voltage).

[0125] A program may be executed on the computer system provided herein. In some examples, the program includes a statistical algorithm or a machine learning algorithm. In some examples, an algorithm including machine learning (ML) is used to associate a signal (e.g., current / voltage) with a nucleoside monomer added to a polynucleotide. In some cases, an algorithm including ML may be trained with training data to associate a signal (e.g., current / voltage) with a nucleoside monomer added to a polynucleotide. In some cases, the algorithm includes a classical ML algorithm for classification and / or clustering (e.g., K-means, mean-shift clustering, density-based spatial clustering of applications with noise (DBSCAN), expectation-maximization (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).

[0126] In some cases, the algorithm includes a learning algorithm that includes one or more layers, such as a neural network. A neural network may include connected nodes in the network, which may perform functions such as transforming or altering input data. In some examples, the output from a given node may be passed as an input to another node. In some embodiments, a node in the network may include an input unit, a hidden unit, an output unit, or a combination thereof. In some cases, an input node may be connected to one or more hidden units. In some cases, one or more hidden units may be connected to an output unit. A node may receive an input and generate an output based on an activation function. In some embodiments, the input or output may be a tensor, a matrix, a vector, an array, or a scalar. In some embodiments, the activation function may be a rectified linear unit (ReLu) activation function, a sigmoid activation function, or a hyperbolic tangent function. In some embodiments, the normalization function may be a softmax activation function. The connections between nodes may further include weights for adjusting input data to a given node (e.g., to activate the input data or inactivate the input data). In some embodiments, the weights may be learned by a neural network. In some embodiments, the neural network may be trained using gradient-based optimization. In some cases, the gradient-based optimization may be composed of one or more loss functions. In some examples, the gradient-based optimization may be conjugate gradient descent, stochastic gradient descent, or variants thereof (e.g., adaptive moment estimation (Adam)). In further examples, the gradient in the gradient-based optimization may be calculated using backpropagation. In some embodiments, the nodes may be organized into a graph to generate a network (e.g., a graph neural network).In some embodiments, the nodes may be organized into one or more layers to generate a network (e.g., a feedforward network, a convolutional neural network (CNN), a recurrent neural network (RNN), etc.). In some cases, the neural network may be a deep neural network that includes two or more layers.

[0127] In some cases, a neural network may include one or more recurrent layers. In some examples, the one or more recurrent layers may be one or more long short-term memory (LSTM) layers or gated recurrent units (GRUs), which may perform sequential data classification and clustering. In some embodiments, a neural network may include one or more convolutional layers. The inputs and outputs may be tensors representing variables or attributes in a dataset (e.g., features), which may be referred to as feature maps (or activation maps). In some cases, the convolutions may be one-dimensional (1D) convolutions, two-dimensional (2D) convolutions, three-dimensional (3D) convolutions, or any combination thereof. In further cases, the convolutions may be 1D transposed convolutions, 2D transposed convolutions, 3D transposed convolutions, or any combination thereof. In some examples, one-dimensional convolutional layers may be suitable for time series data because they can classify time series through parallel convolutions. In some examples, the convolution layer may be used to analyze signals or patterns in signals (e.g., current / voltage) for nucleoside monomers added to a polynucleotide.

[0128] Layers in a neural network may further include one or more pooling layers before or after the convolutional layer. One or more pooling layers may reduce the dimensionality of the feature map using a filter that summarizes a region of the matrix. This may downsample the number of outputs, thereby reducing the parameters and computational resources required for the neural network. In some embodiments, the one or more pooling layers may be max pooling, min pooling, average pooling, global pooling, norm pooling, or a combination thereof. Max pooling can reduce the dimensionality of the data by taking only the maximum value in a region of the matrix, which helps capture important features. In some embodiments, the one or more pooling layers may be one-dimensional (1D), two-dimensional (2D), three-dimensional (3D), or any combination thereof. The neural network may further comprise one or more flattening layers, which can flatten the input passing onto the next layer. In some cases, the input may be flattened by reducing it to a one-dimensional array. The flattened input may be used to output a classification of the object (e.g., a classification of the signal (e.g., current / voltage) for a nucleoside monomer added to a polynucleotide, etc.). The neural network may further include one or more dropout layers. The dropout layers may be used during training of the neural network (e.g., to perform binary or multi-class classification). One or more dropout layers may randomly set certain weights as 0, which may set corresponding elements in the feature map as 0, so that the neural network may avoid overfitting. The neural network may further include one or more dense layers, which include fully connected networks. In the dense layers, information may be passed through the fully connected network to generate a predicted classification of the object, and errors may be calculated. In some embodiments, errors may be backpropagated to improve the prediction. One or more dense layers may include a softmax activation function, which may convert a vector of numbers into a vector of probabilities.These probabilities may be subsequently used in classifications, such as classification of signals (eg, current and / or voltage) for nucleoside monomers added to a polynucleotide.

[0129] The computer system (700) shown in FIG. 7 may be understood as a logical device capable of reading instructions from a medium (711) and / or a network port (705), which may optionally be connected to a server (709) having a fixed medium (712). The system may include a CPU (701), a disk drive (703), optional input devices such as a keyboard (715) and / or a mouse (716), and an optional monitor (707). Data communication may be achieved through a communication medium directed to a server at a local or remote location. The communication medium may include any means of transmitting and / or receiving data. For example, the communication medium may be a network connection, a wireless connection, or an Internet connection. Such a connection may provide for communication via the World Wide Web. It is anticipated that data related to the present disclosure may be transmitted over such a network or connection for receipt and / or review by a party (722).

[0130] FIG. 8 is a block diagram illustrating a first exemplary architecture of a computer system that can be connected in connection with an exemplary instance of the present invention. As shown in FIG. 8, the exemplary computer system can include a processor 802 for processing instructions. Non-limiting examples of processors include the Intel Xeon™ processor, the AMD Opteron™ processor, the Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, the ARM Cortex-A8 Samsung S5PC100™ processor, the ARM Cortex-A8 Apple A4™ processor, and the Marvell PXA 930™ processor. Multiple execution threads can be used for parallel processing. In some examples, multiple processors or processors with multiple cores can also be used, whether within a single computer system, in a cluster, or distributed across a networked system including multiple computers, mobile phones, and / or personal digital assistant devices. As shown in FIG. 8, a high-speed cache (804) may be connected to or incorporated within the processor (802) to provide high-speed memory for instructions or data recently or frequently used by the processor (802). The processor (802) is connected to a northbridge (806) by a processor bus (808). The northbridge (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). The northbridge (806) is also connected to a southbridge (814) by a chipset bus (816). The southbridge (814) is similarly connected to a peripheral bus (818). The peripheral bus may be, for example, a PCI, PCI-X, PCI Express, or other peripheral bus.The northbridge and southbridge, often referred to as the processor chipset, manage data transfers between the processor, RAM, and peripheral components on the peripheral bus (818). In some alternative architectures, the northbridge functions may be incorporated into the processor instead of using a separate northbridge chip. In some examples, the system (800) may include an accelerator card (822) attached to the peripheral bus (818). The accelerator may include a field programmable gate array (FPGA) or other hardware for accelerating certain processes. For example, the accelerator may be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

[0131] Software and data can be stored in external storage (824) and 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 which include Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally equivalent operating systems, and application software running on top of the operating system for managing data storage and optimization in accordance with exemplary embodiments of the present invention. In this example, the system (800) also includes network interface cards (NICs) (820 and 821) connected to a peripheral bus for providing a network interface to external storage, such as network-attached storage (NAS) and other computer systems, that can be used for distributed parallel processing.

[0132] 9 illustrates a network 900 including multiple computer systems 902a and 902b, multiple mobile phones and personal digital assistants 902c, and network-attached storage systems (NAS) 904a and 904b. In an exemplary embodiment, systems 902a, 902b, and 902c manage data storage and optimize data access for data stored on the network-attached storage systems (NAS) 904a and 904b. Mathematical models can be applied to the data and evaluated using parallel processing distributed across the computer systems 902a and 902b and the mobile phone and personal digital assistant system 902c. The computer systems 902a and 902b and the mobile phone and personal digital assistant system 902c can also provide parallel processing for adaptive data reconstruction of the data stored on the network-attached storage systems (NAS) 904a and 904b. 9 shows only one example, and a wide variety of other computer architectures and systems can be used with various embodiments of the present invention. For example, blade servers can be used to provide parallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected to the backplane or through a separate network interface as a network-attached hard disk (NAS).

[0133] In some exemplary embodiments, processors may maintain separate memory spaces and transmit data through a network interface, backplane, or other connector for parallel processing by other processors. In other embodiments, some or all processors may 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, according to an exemplary embodiment. The system includes multiple processors (1002a-f) that can access a shared memory subsystem (1004). The system incorporates multiple programmable hardware memory algorithm processors (MAPs) (1006a-f) within the memory subsystem (1004). Each MAP (1006a-f) may include memory (1008a-f) and one or more field programmable gate arrays (FPGAs) (1010a-f). The MAPs provide configurable functional units, and specific algorithms or portions of algorithms may be provided to the FPGAs (1010a-f) for processing in close coordination with the respective processors. For example, MAPs may be used to evaluate algebraic expressions over a data model and, in an exemplary embodiment, to perform adaptive data restructuring. In this example, each MAP is globally accessible by all processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access its associated memory (1008a-f), allowing it to perform tasks independently and asynchronously with its respective microprocessor (1002a-f). In this configuration, a MAP can pipeline algorithms and feed results directly to another MAP for parallel execution.

[0134] The above computer architectures and systems are merely examples, and a wide variety of other computer, mobile phone and personal digital assistant architectures and systems can be used in connection with the exemplary embodiments, including systems using any combination of general-purpose processors, coprocessors, FPGAs and other programmable logic devices, systems on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some embodiments, all or portions of the computer system can be implemented in software or hardware. Any of a variety of data storage media can be used in connection with the exemplary embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, network-attached storage (NAS), and other local or distributed data storage devices and systems.

[0135] In an exemplary embodiment, the computer system may be implemented using software modules executing on any of the above or other computer architectures and systems. In other embodiments, the system's functionality may be implemented partially or fully in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), systems on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the set processor and optimizer may be implemented with hardware acceleration through the use of hardware accelerator cards.

[0136] The following examples are presented to more clearly illustrate to those skilled in the art the principles and practice of the embodiments disclosed herein, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise specified, all parts and percentages are by weight. [Example]

[0137] Example 1: Fabrication of graphene devices

[0138] Any array of addressable devices shown in FIG. 2 can be fabricated using the general method for graphene FET fabrication described in U.S. Pat. No. 9,859,394, which is incorporated by reference in its entirety, and can be fabricated in arrays ranging in size from 4 to 16 mm. 2 and build chips with pitch distances of 50 to 1000 nm.

[0139] Example 2: Sequencing using graphene devices

[0140] The device of Example 1 is used to sequence nucleic acids. The device is contacted with a nucleic acid template, at least one primer configured to bind to the nucleic acid template, and a polymerase. The polymerase includes a pyrene moiety bound to Phi29 polymerase. The primer, sensor, and nucleic acid template form a ternary complex bound to the graphene surface of the device. A mixture of four nucleotides is contacted with the device and the template, and at least one nucleic acid generates a uniquely detectable signal from the device upon interaction with the polymerase. Optionally, after extension of the at least one primer with a nucleotide triphosphate and measurement of a signal corresponding to one of the nucleotide triphosphates, the terminator is removed from the incorporated nucleotide. The process is repeated to confirm the identity of each added base, thereby determining the sequence of the nucleic acid template. Optionally, the ternary complex is washed from the device, and the device is reused.

[0141] Example 3: Fabrication of a graphene device with buried gate and shield

[0142] Any array of addressable devices shown in Figures 4A-4B can be fabricated using the general method of graphene FET fabrication described in Example 1, with dimensions ranging from 4 to 16 mm. 2and build chips with pitch distances of 50 to 1000 nm.

[0143] Example 4: Sequencing using graphene devices with buried gates and shields

[0144] The device of Example 3 is used to perform nucleic acid sequencing using a modified version of the general method of Example 2. During loading of the ternary complex, a positive voltage is applied to the recessed gate. This attracts negatively charged DNA (or other similar moieties) toward the surface and graphene. A shielding layer at ground potential is configured with small openings that allow the positive potential from the gate to leak to specific locations on the chip surface. This localization of the potential causes the DNA (or similar moieties) to concentrate at desired locations on the graphene. This facilitates higher loading of the device. During sensor operation, the gate can be used to adjust the graphene potential to maximize signal changes associated with molecular events occurring on or near the graphene layer.

[0145] Example 5: Fabrication of Edge Finger Devices

[0146] An array of devices having the general structure of Figures 12A-12C is fabricated by: a) providing one or more base layers; b) depositing material to create a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing material to create a passivation layer; f) depositing material to create a first electrode; g) patterning the first electrode; and h) isotropically etching the passivation layer so that the edges of the first electrode are undercut and the device contains a nanogap of approximately 20 nm. Each electrode is fabricated from one or more of titanium, platinum, and titanium nitride. The passivation layer comprises an oxide. A layer of gold is then deposited on the electrode. After fabrication, the device is contacted with one or more nanowires containing nucleic acids, at least a portion of which bridges one or more of the first and second electrodes. Each nanowire contains at least one biotin-functionalized handle (optionally connected via a tether). Nanowire loading involves (a) applying a voltage to the electrodes to attract nucleic acid strands to the electrodes, (b) monitoring the current path between the electrodes to determine whether the nanowire bridges the two electrodes, and (c) turning off the voltage when the current spikes (i.e., touches) to prevent further DNA attraction. The nanowire is then contacted with streptavidin-bound polymerase (e.g., as in Figure 11) to facilitate polymerase binding to the nanowire.

[0147] Example 6: Cross Finger Device

[0148] 13A-13B are fabricated according to the general procedure or Example 5. The devices are arranged according to the general configuration of FIG.

[0149] Example 7: Cross-finger device with oxide layer

[0150] Fabricate an array of the devices of Figures 14A-14B according to the general procedure or Example 5.

[0151] Example 8: Self-Aligned Finger Etching

[0152] Fabricate an array of the devices of Figures 16A-16B according to the general procedure or Example 5.

[0153] Example 9: Sequencing

[0154] Nucleic acid sequencing is performed using any of the devices described in Examples 5 to 8. The device is contacted with a nucleic acid template, at least one primer configured to bind to the nucleic acid template, and a mixture of four nucleotide triphosphates (and / or CMN) is contacted with the device and the template, and the at least one nucleic acid generates a uniquely detectable signal from the device upon interaction with a polymerase. After extension of the at least one primer with the nucleotide triphosphate and measurement of a signal corresponding to one of the nucleotide triphosphates, the terminator is removed from the incorporated nucleotide. The process is repeated to confirm the identity of each added base (e.g., A, T, C, or G), thereby determining the sequence of the nucleic acid template.

[0155] Example 10: Gold deposition on devices

[0156] Gold is deposited onto the electrodes of a device fabricated according to the general procedure of Example 6. The growth of gold islands is shown schematically in Figure 18A and observed without an adhesion layer, as depicted in Figure 18B. Gold deposition after forming an adhesion layer on the electrode results in a continuous gold layer, as shown schematically in Figure 18C.

[0157] Biomolecules are conjugated at the nanogaps of the device between gold islands on the electrode surface, as illustrated in Figure 19, for sequencing following the general procedure of Example 9. Conjugation to gold nanoislands provides lower contact resistance compared to direct binding (e.g., Figure 19B). As described herein, passivation is further utilized to protect the fields and metal routing, as shown in Figure 20.

[0158] Example 11: Molecular wire construct with biotin-streptavidin

[0159] Molecular wire constructs are assembled across nanogaps in devices with gold-coated electrodes as generally described in Example 10. The molecular wire constructs contain a biotin-streptavidin (SA) construct (e.g., Figure 21) that can be used to link a polymerase between the electrode surfaces.

[0160] The gold-coated surface is functionalized by exposing it to 18 mM cysteamine in water for 4 hours. Once functionalized with cysteamine, the surface is exposed to biotin-NHS in DIPEA and DMF at 60 °C for 4 hours to obtain a biotin-functionalized gold-coated surface (e.g., Figures 22A-22B). The surface is further exposed to streptavidin to create a surface with accessible streptavidin (e.g., Figure 23A). A bis-biotinylated polymerase (e.g., Figure 23B) is constructed using a dual-biotin-tagged P29 mutant RPN (Figure 23C). The bis-biotinylated polymerase assembles to form a molecular wire construct across the nanogap of the device under the following conditions: 10 mM MOPS, pH 7.5, 10 mM KOAc, and 5 mM Mg. 2+ (Figure 24).

[0161] The molecular wire construct is used for sequencing as generally described in Example 9.

[0162] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0163] The present disclosure is further illustrated by the following non-limiting sections.

[0164] Item 1. A method for single molecule sensing, comprising: a. contacting the molecular sensor with at least one charge modulator, wherein the contact results in a change in current; b. measuring the change in current; c. sensing a single molecule by correlating the change in current with the presence or absence of at least one charge modulator; A method comprising:

[0165] Item 2. The method of Item 1, wherein the single molecule comprises a biomolecule.

[0166] Item 3. The method according to Item 2, wherein the biological molecule comprises a nucleic acid.

[0167] Item 4. The method according to any one of Items 1 to 3, wherein the nucleic acid comprises DNA, RNA, or a mixture thereof.

[0168] Item 5. The method of any one of items 1 to 4, wherein the current sensor comprises a graphene-enabled field-effect transistor (GeFET) or a CMOS device.

[0169] Item 6. The method of any one of items 1 to 5, wherein the molecular sensor is in electrical communication with the charge sensor.

[0170] Item 7. The method according to any one of items 1 to 6, wherein the molecular sensor comprises a polymerase.

[0171] Item 8. The method of item 7, wherein the molecular sensor comprises an isothermal polymerase.

[0172] Item 9. The method of item 7, wherein the molecular sensor comprises Phi29 polymerase or a variant thereof.

[0173] Item 10. The method of any one of items 6 to 9, wherein the charge sensor comprises a nanowire.

[0174] Item 11. The method of any one of items 6 to 10, wherein the charge sensor fills a gap between the first electrode and the second electrode.

[0175] 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 a sulfur-gold interaction.

[0176] Item 13. The method according to item 11 or 12, wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine.

[0177] Item 14. The method according to any one of Items 10 to 13, wherein the charge sensor comprises a linker between the surface of the first electrode or the second electrode and the polymerase.

[0178] Item 15. The method of item 14, wherein the linker comprises one or more moieties.

[0179] Item 16. The method of item 15, wherein the one or more components include a biotin-streptavidin construct.

[0180] Item 17. The method of item 15, wherein the one or more components include SpyCatcher or SpyTag.

[0181] Item 18. The method of item 15, wherein one or more components comprises a peptide linker.

[0182] Item 19. The method of Item 15, wherein the one or more components comprise a protein.

[0183] Item 20. The method of item 19, wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.

[0184] Item 21. The method of any one of items 1 to 20, wherein at least one charge modulator comprises a negative or positive charge.

[0185] Item 22. The method of any one of items 1 to 21, wherein at least one charge modulator comprises a charge-modulated nucleotide (CMN).

[0186] Item 23. The method according to any one of items 1 to 22, wherein the contacting step comprises incorporating CMN into a polynucleotide primer.

[0187] Item 24. The method according to Item 22 or 23, wherein the CMN comprises at least one modification relative to a standard nucleotide.

[0188] Item 25. The method of Item 24, wherein the at least one modification comprises a modification to a CMN base.

[0189] Item 26. The method according to Item 25, wherein at least one modification comprises a 7-deaza or 8-bromo modified base.

[0190] Item 27. The method according to any one of Items 24 to 26, wherein the CMN comprises a modification at the 5' position.

[0191] Item 28. The method of Item 27, wherein the at least one modification comprises a modification to a 5' polyphosphate or a chemical variant thereof.

[0192] Item 29. The method according to any one of Items 24 to 27, wherein at least one modification comprises a thiolated or brominated phosphate.

[0193] Item 30. The method of Item 28 or 29, wherein the polyphosphate salt comprises at least 3, 4, 5, 6, 8, or 10 phosphate salts or variants thereof.

[0194] Item 31. The method according to any one of Items 24 to 27, wherein at least one modification comprises a modification to a terminal 5' polyphosphate or a chemical variant thereof.

[0195] Item 32. The method according to any one of Items 24 to 31, wherein at least one modification comprises a polymer.

[0196] Item 33. The method according to 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.

[0197] Item 34. The method according to Item 33, wherein the nucleic acid strand comprises 25 to 5,000 bases.

[0198] Item 35. The method according to Item 33 or 34, wherein the nucleic acid chain is branched (dendrimer).

[0199] Item 36. The method according to any one of Items 33 to 35, wherein the nucleic acid strand comprises a secondary structure.

[0200] Item 37. The method of item 36, wherein the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadruplex, and an I-motif.

[0201] Item 38. The method according to any one of Items 27 to 37, wherein the nucleic acid strand comprises a single strand, a double strand, or a triple strand.

[0202] Item 39. The method according to any one of Items 27 to 38, wherein the nucleic acid strand comprises at least one charge-modulating chemical modification.

[0203] Item 40. The method of Item 39, wherein at least one charge-modulating chemical modification increases the charge of CMN relative to an unmodified nucleotide.

[0204] 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.

[0205] Item 42. The method of Item 39, wherein at least one charge-modulating chemical modification reduces the charge of CMN relative to an unmodified nucleotide.

[0206] Item 43. The method of item 39, wherein the at least one charge-modulating chemical modification comprises one or more of phosphate, phosphite, sulfonate, sulfite, carboxylate, xanthogenate, thiocarboxylic acid, boranophosphonate, and boric acid.

[0207] Item 44. The method according to any one of Items 27 to 43, wherein the nucleic acid strand comprises at least one sugar-modified nucleotide.

[0208] Item 45. The method of Item 44, wherein the sugar-modified nucleotide comprises a deoxynucleotide or a dideoxynucleotide.

[0209] Item 46. The method according to any one of items 27 to 45, wherein the nucleic acid strand comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid.

[0210] Item 47. The method according to any one of Items 27 to 46, wherein the nucleic acid strand comprises a phosphate modification.

[0211] Item 48. The method of Item 47, wherein the phosphate modification comprises a hydrophobic group.

[0212] Item 49. The method of item 48, wherein the hydrophobic group comprises a straight chain alkyl or a branched chain alkyl.

[0213] Item 50. The method of Item 47, wherein the phosphate modification comprises a hydrophilic group.

[0214] Item 51. The method of Item 50, wherein the hydrophilic group comprises polyethylene glycol.

[0215] Item 52. The method according to Item 51, wherein the polyethylene glycol has a molecular weight of 1,000 to 100,000 daltons.

[0216] Item 53. The method according to Item 33, wherein the peptide chain is 1 to 100 amino acids in length.

[0217] Item 54. The method of Item 11, wherein the CMN comprises a charged small molecule.

[0218] 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.

[0219] Item 56. The method according to Item 55, wherein the metal complex comprises ferrocene, Ru-dipy, and bis-cyclopentadienyldiirone.

[0220] Item 57. The method according to any one of Items 1 to 56, wherein the change in current is between 1 nanoampere and 100 picoamperes.

[0221] Item 58. The method according to any one of Items 1 to 56, wherein the change in current is between 100 picoamperes and 1 microampere.

[0222] Item 59. The method according to any one of items 1 to 56, wherein the change in current is at least 1.01 to 3 times the background current.

[0223] Item 60. The method according to any one of Items 1 to 56, wherein steps a to c are repeated at least 50 times.

[0224] Item 61. The method of any one of items 1 to 56, configured to detect 1 to 200 single molecules per second.

[0225] Item 62. a solid support comprising a plurality of loci, each locus comprising a graphene layer; a gate electrode and a drain electrode in electrical communication with each other via the graphene layer; at least one insulating layer located between the gate electrode and the drain electrode; A chemically sensitive field effect transistor device comprising: A chemically sensitive field effect transistor device, the substrate having a pitch of 50 to 1000 nanometers.

[0226] Item 63. The device of Item 62, further comprising at least one ground shield.

[0227] Item 64. The device of Item 63, wherein at least one ground shield is at ground potential.

[0228] Item 65. The device of any one of items 62 to 64, further comprising at least one buried gate.

[0229] Item 66. The device of any one of items 63 to 65, wherein at least one ground shield comprises an opening that allows electrical communication between the graphene layer and the at least one buried gate.

[0230] Item 67. The device of any one of items 62 to 66, wherein the graphene layer is approximately one atom thick.

[0231] Item 68. The device according to any one of items 62 to 67, wherein the device comprises 100 to 1 billion loci.

[0232] Item 69. The device of any one of items 62 to 68, wherein each locus is 50 to 200 nm in size.

[0233] Item 70. The device of any one of items 62 to 69, wherein each locus is a well, a channel, or is substantially planar.

[0234] Item 71. Device is 4 to 900 mm 2 71. The device according to any one of items 62 to 70,

[0235] Item 72. Device is 4-16 mm 2 71. The device according to any one of items 62 to 70,

[0236] Item 73. Device is 200-2000mm 2 71. The device according to any one of 62 to 70,

[0237] Item 74. A method for sequencing a single molecule polynucleotide, 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 complexes comprise a graphene binder; b) detecting one or more bases of a polynucleotide in real time, wherein the detection occurs when a plurality of ternary complexes bind to the graphene layer according to any one of items 1 to 3; c) removing the ternary complex from the surface; d) repeating steps a to c to sequence the polynucleotide; A method comprising:

[0238] Item 75. The method of item 74, wherein the graphene binder comprises an aromatic group.

[0239] Item 76. The method according to item 74 or 75, wherein the graphene binder comprises an aryl group or a heteroaryl group.

[0240] Item 77. Graphene binder is C6-C 3077. The method according to any one of items 74 to 76, comprising an aryl group or a heteroaryl group.

[0241] Item 78. The method according to any one of Items 74 to 77, wherein the graphene binder comprises an aromatic hydrocarbon.

[0242] Item 79. The method of any one of items 74 to 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 benzo[c]fluorene.

[0243] Item 80. The method according to any one of Items 74 to 79, wherein step c) includes cleaning the surface.

[0244] Item 81. The method according to items 74 to 80, wherein the ternary complex is bound to the graphene binder via a primer, a polymerase, or a polynucleotide library.

[0245] Item 82. The method according to items 74 to 81, wherein the ternary complex is linked via a linker.

[0246] Item 83. The method according to items 74 to 82, wherein the ternary complex is linked via a linker using conjugation.

[0247] Item 84. The method of item 83, wherein the conjugation comprises a nucleophile / carbonyl, azide / phosphine, 1,4 Michael addition, 1,3-dipolar cycloaddition, inverse electron demand cycloaddition, olefin metathesis, or cross-coupling reaction.

[0248] Item 85. The method of any one of items 74 to 84, wherein the removing step comprises contacting the surface with a solvent.

[0249] Item 86. The method according to Item 85, wherein the solvent comprises an organic solvent.

[0250] Item 87. The method according to Item 86, wherein the organic solvent comprises MeCN, methanol, ethanol, 2-propanol, acetone, DMF, formamide, THF, or DMSO.

[0251] Item 88. The method according to Item 86 or 87, wherein the organic solvent is heated.

[0252] Item 89. The method of any one of items 74 to 88, wherein the polymerase comprises Phi29 polymerase or a variant thereof.

[0253] Item 90. The method of any one of items 74 to 89, wherein the polymerase is configured to incorporate a charge-modified nucleotide according to any one of items 22 to 56.

[0254] Item 91. The method according to any one of Items 74 to 90, wherein the polymerase is bound to a surface in step a).

[0255] Item 92. The method according to any one of Items 74 to 90, wherein the polymerase is not bound to a surface in step a).

[0256] Item 93. The method of any one of items 74 to 92, wherein the plurality of polynucleotides comprises at least 100,000 unique polynucleotides.

[0257] Item 94. The method according to any one of Items 74 to 93, wherein the plurality of polynucleotides is 50 to 30,000 bases in length.

[0258] Item 95. The method of any one of items 74 to 94, wherein the detecting step comprises contacting the ternary complex with at least one nucleotide.

[0259] Item 96. The method of any one of items 74 to 95, wherein the detecting step includes measuring a change in current when the CMN is incorporated.

[0260] Item 97. The method of any one of items 74 to 96, wherein the buried gate has a positive potential during step (a).

[0261] Item 98. The method of any one of items 74 to 97, wherein during step (b) the buried gate has a positive or negative potential.

[0262] Item 99. a first electrode including a neck region; a passivation layer; a second electrode, wherein the first electrode and the second electrode are disposed on the first base layer, a first portion of the neck region overlapping a first portion of the second electrode such that the first electrode and the second electrode are separated by a nanogap, and a second portion of the first electrode and a second portion of the second electrode are separated by a passivation layer; a first base layer and a second base layer, the first base layer being disposed on the second base layer, and the first electrode and the second electrode being disposed on the base layers; A device for molecular sensing comprising:

[0263] Item 100. The device according to Item 99, wherein the nanogap is 1 to 50 nm.

[0264] Item 101. The device according to Item 99, wherein the nanogap is 10 to 30 nm.

[0265] Item 102. The device according to item 99, wherein the nanogap is 50 nm or less.

[0266] Item 103. The device of any one of items 99 to 102, wherein the passivation layer comprises an oxide.

[0267] Item 104. The device of item 103, wherein the oxide comprises silicon, nitride, or carbide.

[0268] Item 105. The device of any one of items 99 to 104, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.

[0269] Item 106. The device of any one of items 99 to 105, wherein each of the first electrode and the second electrode comprises a gold surface layer.

[0270] Item 107. The device according to item 106, wherein the gold layer is 75 angstroms or less in thickness.

[0271] Item 108. The device according to item 106, wherein a surface layer of gold is deposited on the adhesion layer.

[0272] Item 109. The device of item 108, wherein the adhesion layer comprises titanium or chromium.

[0273] Item 110. The device of any one of items 99 to 109, wherein each of the first electrode and the second electrode comprises gold nanoislands.

[0274] Item 111. The device of any one of items 99 to 107, wherein the first base layer comprises silicon oxide or silicon nitride.

[0275] Item 112. The device of any one of items 99 to 111, wherein the second base layer comprises silicon.

[0276] Item 113. The device of any one of items 99 to 112, wherein the device further comprises a charge sensor, the charge sensor being in electrical communication with the first electrode and the second electrode.

[0277] Item 114. The device of item 113, wherein the charge sensor is attached to the first electrode and the second electrode.

[0278] Item 115. The device of any one of items 113-114, wherein the charge sensor comprises a polymer.

[0279] Item 116. The device of item 115, wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.

[0280] Item 117. The device of any one of items 113 to 116, wherein the charge sensor comprises carbon.

[0281] Item 118. A device described in any one of items 113 to 117, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via a sulfur-gold interaction.

[0282] Item 119. The device according to item 118, wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine.

[0283] Item 120. The device of any one of items 113 to 118, wherein the charge sensor is further attached to the molecular sensor via a tether.

[0284] Item 121. The device of item 120, wherein the tether comprises one or more moieties.

[0285] Item 122. The device of item 121, wherein one or more components include a biotin-streptavidin construct.

[0286] Item 123. The device of item 122, wherein the one or more components include SpyCatcher or SpyTag.

[0287] Item 124. The device of item 122, wherein one or more components comprise a peptide linker.

[0288] Item 125. The device of item 122, wherein one or more components comprise a protein.

[0289] Item 126. The device of item 125, wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.

[0290] Item 127. The device of item 120, wherein the molecular sensor comprises an enzyme.

[0291] Item 128. The device of item 120, wherein the molecular sensor comprises an antibody.

[0292] Item 129. The device of item 127, wherein the enzyme comprises a polymerase.

[0293] Item 130. The device of any one of items 99 to 129, wherein the longest linear dimension of the second electrode is perpendicular to the neck region.

[0294] Item 131. The device of any one of items 99 to 129, wherein the longest linear dimension of the second electrode is parallel to the neck region.

[0295] Item 132. A device according to any one of items 99 to 131, wherein at least one edge of the first electrode is undercut relative to the passivation layer.

[0296] Item 133. The device of any one of items 99 to 132, wherein the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode.

[0297] Item 134. A device according to any one of items 99 to 133, wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region.

[0298] Item 135. The device of any one of items 99 to 134, wherein each of the first electrode and the second electrode comprises a gold surface layer.

[0299] Item 136. The device according to item 135, wherein the gold surface layer is deposited with an adhesion layer.

[0300] Item 137. The device according to item 136, wherein the adhesion layer comprises titanium or chromium.

[0301] Item 138. The device of any one of items 99 to 137, wherein each of the first electrode and the second electrode comprises gold nanoislands.

[0302] Item 139. The device of any one of items 99 to 138, wherein the first base layer comprises silicon oxide or silicon nitride.

[0303] Item 140. The device of any one of items 99 to 139, wherein the second base layer comprises silicon.

[0304] Item 141. The device of any one of items 99 to 140, wherein the neck region is 200 nm or less in width.

[0305] Item 142. a first electrode disposed on the first base layer; a second electrode including a neck region, a first portion of the neck region overlapping a first portion of the first electrode such that the first electrode and the second electrode are separated by a nanogap, and a second portion of the first electrode and a second portion of the second electrode are separated by a passivation layer; a first base layer and a second base layer, the first base layer being disposed on the second base layer, and the first electrode and the second electrode being disposed on the base layers; A device for molecular sensing comprising:

[0306] Item 143. The device of item 142, wherein the passivation layer is configured to overlap a portion of the second electrode.

[0307] Item 144. The device of item 142, wherein the passivation layer is configured to passivate the electrode traces.

[0308] Item 145. The device according to any one of items 142 to 144, wherein the nanogap is 1 to 50 nm.

[0309] Item 146. A device according to any one of items 142 to 144, wherein the nanogap is 10 to 30 nm.

[0310] Item 147. The device according to any one of items 142 to 144, wherein the nanogap is 50 nm or less.

[0311] Item 148. The device of any one of items 142 to 147, wherein the passivation layer comprises an oxide.

[0312] Item 149. The device of item 148, wherein the oxide comprises silicon, nitride, or carbide.

[0313] Item 150. The device of any one of items 142 to 149, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.

[0314] Item 151. The device of any one of items 142 to 150, wherein each of the first electrode and the second electrode comprises a gold surface layer.

[0315] Item 152. The device according to item 151, wherein the gold surface layer is 75 angstroms or less in thickness.

[0316] Item 153. The device according to item 151, wherein a surface layer of gold is deposited on the adhesion layer.

[0317] Item 154. The device according to item 152, wherein the adhesion layer comprises titanium or chromium.

[0318] Item 155. The device of any one of items 142 to 150, wherein each of the first electrode and the second electrode comprises gold nanoislands.

[0319] Item 156. The device of any one of items 142 to 152, wherein the first base layer comprises silicon oxide or silicon nitride.

[0320] Item 157. The device of any one of items 142 to 156, wherein the second base layer comprises silicon.

[0321] Item 158. A device according to any one of items 142 to 157, wherein the device further comprises a charge sensor, the charge sensor being in electrical communication with the first electrode and the second electrode.

[0322] Item 159. The device of item 158, wherein the charge sensor is attached to the first electrode and the second electrode.

[0323] Item 160. The device of any one of items 158-159, wherein the charge sensor comprises a polymer.

[0324] Item 161. The device of item 160, wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.

[0325] Item 162. The device of any one of items 158 to 161, wherein the charge sensor comprises carbon.

[0326] Item 163. A device according to any one of items 158 to 162, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via a sulfur-gold interaction.

[0327] Item 164. The device according to item 163, wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine.

[0328] Item 165. A device according to any one of items 158 to 163, wherein the charge sensor is further attached to the molecular sensor via a tether.

[0329] Item 166. The device according to item 165, wherein the tether comprises one or more moieties.

[0330] Item 167. The device of item 166, wherein one or more components comprise a biotin-streptavidin construct.

[0331] Item 168. The device of item 167, wherein one or more components include SpyCatcher or SpyTag.

[0332] Item 169. The device of item 167, wherein one or more components include a peptide linker.

[0333] Item 170. The device of item 167, wherein one or more components comprise a protein.

[0334] Item 171. The device of item 170, wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.

[0335] Item 172. The device of item 165, wherein the molecular sensor comprises an enzyme.

[0336] Item 173. The device of item 165, wherein the molecular sensor comprises an antibody.

[0337] Item 174. The device of item 172, wherein the enzyme comprises a polymerase.

[0338] Item 175. The device of any one of items 142 to 174, wherein the longest linear dimension of the second electrode is perpendicular to the neck region.

[0339] Item 176. The device of any one of items 142 to 174, wherein the longest linear dimension of the second electrode is parallel to the neck region.

[0340] Item 177. A device according to any one of items 142 to 176, wherein at least one edge of the first electrode is undercut relative to the passivation layer.

[0341] Item 178. A device according to any one of items 142 to 177, wherein the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode.

[0342] Item 179. A device according to any one of items 142 to 178, wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region.

[0343] Item 180. The device of any one of items 142 to 179, wherein each of the first electrode and the second electrode comprises a gold surface layer.

[0344] Item 181. The device of any one of items 142 to 180, wherein the first base layer comprises silicon oxide or silicon nitride.

[0345] Item 182. The device of any one of items 142 to 181, wherein the second base layer comprises silicon.

[0346] Item 183. The device of any one of items 142 to 182, wherein the neck region is 200 nm or less in width.

[0347] Item 184. a first electrode including a neck region; a passivation layer including a channel or well, the bottom of the well or channel including the first base layer; a second electrode, wherein the first electrode and the second electrode are disposed on the first base layer, the second electrode is at least partially embedded in the passivation layer, 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, and a second portion of the first electrode and a second portion of the second electrode are separated by the passivation layer; a first base layer and a second base layer, the first base layer being disposed on the second base layer, and the first electrode and the second electrode being disposed on the base layers; A device for molecular sensing comprising:

[0348] Item 185. The device according to item 184, wherein the nanogap is 1 to 50 nm.

[0349] Item 186. The device according to item 184, wherein the nanogap is 10 to 30 nm.

[0350] Item 187. The device according to item 184, wherein the nanogap is 50 nm or less.

[0351] Item 188. The device of any one of items 184 to 187, wherein the passivation layer comprises an oxide.

[0352] Item 189. The device of item 188, wherein the oxide comprises silicon, nitride, or carbide.

[0353] Item 190. The device of any one of items 184 to 189, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.

[0354] Item 191. The device of any one of items 184 to 190, wherein each of the first electrode and the second electrode comprises a gold surface layer.

[0355] Item 192. The device according to item 191, wherein the gold layer is 75 angstroms or less in thickness.

[0356] Item 193. The device according to item 191, wherein a surface layer of gold is deposited on the adhesion layer.

[0357] Item 194. The device according to item 192, wherein the adhesion layer comprises titanium or chromium.

[0358] Item 195. The device of any one of items 184 to 190, wherein each of the first electrode and the second electrode comprises gold nanoislands.

[0359] Item 196. The device of any one of items 184 to 192, wherein the first base layer comprises silicon oxide or silicon nitride.

[0360] Item 197. The device of any one of items 184 to 196, wherein the second base layer comprises silicon.

[0361] Item 198. The device of any one of items 184 to 197, wherein the device further comprises a charge sensor, the charge sensor being in electrical communication with the first electrode and the second electrode.

[0362] Item 199. The device of item 198, wherein the charge sensor is attached to the first electrode and the second electrode.

[0363] Item 200. The device of any one of items 198-199, wherein the charge sensor comprises a polymer.

[0364] Item 201. The device of Item 200, wherein the polymer comprises at least one nucleic acid, amino acid, sugar, or lipid.

[0365] Item 202. The device of any one of items 198 to 201, wherein the charge sensor comprises carbon.

[0366] Item 203. A device described in any one of items 198 to 202, wherein the charge sensor is attached to at least one of the first electrode or the second electrode via a sulfur-gold interaction.

[0367] Item 204. The device according to item 203, wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine.

[0368] Item 205. A device according to any one of items 198 to 203, wherein the charge sensor is further attached to the molecular sensor via a tether.

[0369] Item 206. The device of item 205, wherein the tether comprises one or more moieties.

[0370] Item 207. The device of item 206, wherein one or more components include a biotin-streptavidin construct.

[0371] Item 208. The device of item 206, wherein the one or more components include SpyCatcher or SpyTag.

[0372] Item 209. The device of item 206, wherein one or more components include a peptide linker.

[0373] Item 210. The device of item 206, wherein one or more components comprise a protein.

[0374] Item 211. The device of item 210, wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.

[0375] Item 212. The device of item 205, wherein the molecular sensor comprises an enzyme.

[0376] Item 213. The device of item 205, wherein the molecular sensor comprises an antibody.

[0377] Item 214. The device of item 212, wherein the enzyme comprises a polymerase.

[0378] Item 215. The device of any one of items 184 to 214, wherein the longest linear dimension of the second electrode is perpendicular to the neck region.

[0379] Item 216. A device according to any one of items 184 to 214, wherein the longest linear dimension of the second electrode is parallel to the neck region.

[0380] Item 217. A device according to any one of items 184 to 216, wherein at least one edge of the first electrode is undercut relative to the passivation layer.

[0381] Item 218. A device according to any one of items 184 to 217, wherein the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode.

[0382] Item 219. A device described in any one of items 184 to 218, wherein the longest linear dimension of the first electrode is perpendicular to the longest linear dimension of the neck region.

[0383] Item 220. The device of any one of items 184 to 219, wherein each of the first electrode and the second electrode comprises a gold surface layer.

[0384] Item 221. The device of any one of items 184 to 220, wherein the first base layer comprises silicon oxide or silicon nitride.

[0385] Item 222. The device of any one of items 184 to 221, wherein the second base layer comprises silicon.

[0386] Item 223. The device of any one of items 184 to 222, wherein the neck region is 200 nm or less in width.

[0387] Item 224. The array of any one of devices 99-223, wherein at least a portion of the first electrodes and second electrodes are independently addressable.

[0388] Item 225. The array of item 224, wherein the pitch distance of the nanogaps of at least some of the devices is 200 nanometers or less.

[0389] Item 226. The array of any one of items 224-225, wherein at least a portion of the first electrodes and second electrodes are independently addressable.

[0390] Item 227. The array of any one of items 224 to 226, wherein at least a portion of the first electrodes and second electrodes are independently addressable on the x- and y-axes.

[0391] Item 228. The array of any one of items 224 to 226, wherein at least a portion of the first electrodes and second electrodes are independently addressable on the z-axis.

[0392] Item 229. An array according to any one of items 224 to 228, comprising at least 50 devices according to any one of items 99 to 223.

[0393] Item 230. The array according to any one of items 224 to 229, comprising at least 5000 devices according to any one of items 99 to 223.

[0394] Item 231. The array according to any one of items 224 to 230, comprising at least 100,000 devices according to any one of items 99 to 223.

[0395] Item 232. The array of any one of items 224 to 231, wherein the pitch distance of the nanogaps in at least some of the devices is 2 microns or less.

[0396] Item 233. An array according to any one of items 224 to 232, wherein the array further comprises a plurality of vias, the plurality of vias being configured to connect at least two vertical layers of the device.

[0397] Item 234. An array described in any one of items 224 to 233, wherein the array further includes a plurality of routing connections, the plurality of routing connections configured to addressably control each device of the array.

[0398] Item 235. A method for manufacturing a device according to any one of Items 99 to 223, comprising: a) providing one or more base layers; b) depositing a material to create a second electrode; c) patterning the second electrode; d) optionally planarizing; e) depositing a material to create a passivation layer; f) depositing a material to produce a first electrode; g) patterning the first electrode; h) isotropically etching the passivation layer such that the edges of the first electrode are undercut; A method comprising:

[0399] Item 236. The method of Item 235, further comprising depositing gold on the first top layer of the device.

[0400] Item 237. The method of any one of items 235-236, wherein the one or more base layers comprise thermal oxide on silicon.

[0401] Item 238. The method according to any one of items 235 to 237, comprising etching or lithography.

[0402] Item 239. The method according to any one of Items 235 to 238, comprising RIE (reactive ion etching).

[0403] Item 240. The method of any one of Items 235 to 239, wherein the patterning step includes lithography and / or RIE.

[0404] Item 241. The method according to any one of items 235 to 240, which does not include electron beam or DUV (deep ultraviolet) lithography.

[0405] Item 242. The method of any one of items 235 to 241, comprising depositing gold on the first electrode and the second electrode.

[0406] Item 243. The method of any one of Items 235 to 242, wherein the first electrode and the second electrode are separated by a nanogap.

[0407] Item 244. The method according to Item 243, wherein the nanogap is 1 to 50 nm.

[0408] Item 245. The method according to Item 243, wherein the nanogap is 10 to 30 nm.

[0409] Item 246. The method according to Item 243, wherein the nanogap is 50 nm or less.

[0410] Item 247. The method of any one of Items 235 to 246, wherein the passivation layer comprises an oxide.

[0411] Item 248. The method of item 247, wherein the oxide comprises silicon, nitride, or carbide.

[0412] Item 249. The method of any one of items 235 to 248, wherein the first electrode and the second electrode comprise platinum, titanium nitride, or titanium.

[0413] Item 250. A method for using the device according to any one of items 99 to 223 for molecular sensing, a. providing an analyte; b. allowing the analyte to react, bind, or otherwise interact with the sensor; c. measuring the electrical signal generated by the sensor; A method comprising:

[0414] Item 251. a. providing at least one nucleotide triphosphate, at least one template, and at least one primer; b. extending the primer with at least one nucleotide triphosphate; c. measuring the electrical signal generated by the polymerase; 251. The method of claim 250, comprising:

[0415] Item 252. The method of Item 251, further comprising analyzing the electrical signal to confirm the identity of at least one nucleotide triphosphate.

[0416] Item 253. The method according to any one of Items 251 to 252, wherein at least one nucleotide triphosphate comprises a non-standard base.

[0417] Item 254. The method of any one of items 251 to 253, wherein at least one nucleotide triphosphate comprises a terminator configured to prevent chain elongation.

[0418] Item 255. The method according to any one of Items 251 to 254, repeated to confirm the identity of at least 20 bases.

[0419] Item 256. The method according to any one of Items 251 to 255, repeated to confirm the identity of at least 100 bases.

[0420] Item 257. The method according to any one of Items 251 to 256, repeated to confirm the identity of at least 1000 bases.

Claims

1. 1. A method for single molecule sensing, comprising: a. contacting a molecular sensor with at least one charge modulator, wherein the contact results in a change in current; b. measuring the change in the current; c. sensing the single molecule by correlating the change in current with the presence or absence of the at least one charge modulator; A method comprising:

2. The method of claim 1 , wherein the single molecule comprises a biomolecule.

3. The method of claim 2 , wherein the biomolecule comprises a nucleic acid.

4. The method of claim 3 , wherein the nucleic acid comprises DNA, RNA, or a mixture thereof.

5. The method of any one of claims 1 to 4, wherein the charge sensor comprises a graphene-enabled field effect transistor (GeFET) or a CMOS device.

6. The method of any one of claims 1 to 5, wherein the molecular sensor is in electrical communication with a charge sensor.

7. The method of any one of claims 1 to 6, wherein the molecular sensor comprises a polymerase.

8. The method of claim 7 , wherein the molecular sensor comprises an isothermal polymerase.

9. The method of claim 7 , wherein the molecular sensor comprises Phi29 polymerase or a variant thereof.

10. The method of any one of claims 6 to 9, wherein the charge sensor comprises a nanowire.

11. The method of any one of claims 6 to 10, wherein the charge sensor fills the gap between the first electrode and the second electrode.

12. The method of claim 11 , wherein the charge sensor is attached to at least one of the first electrode or the second electrode via a sulfur-gold interaction.

13. 13. The method of claim 11 or 12, wherein the surface of the first electrode or the second electrode is functionalized with thiol-biotin, terminal cysteine, or cysteamine.

14. The method of any one of claims 10 to 13, wherein the charge sensor comprises a linker between the surface of the first electrode or the second electrode and the polymerase.

15. The method of claim 14 , wherein the linker comprises one or more moieties.

16. The method of claim 15, wherein the one or more components comprises a biotin-streptavidin construct.

17. 16. The method of claim 15, wherein the one or more components include SpyCatcher or SpyTag.

18. 16. The method of claim 15, wherein the one or more components comprises a peptide linker.

19. The method of claim 15 , wherein the one or more components comprises a protein.

20. 20. The method of claim 19, wherein the protein comprises a C1q / TNF-related protein, a tryptophan-zipper pentamer, or a five-stranded phenylalanine zipper.

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