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 recovery of biomolecule-based information.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 287,458, filed December 8, 2021, and U.S. Provisional Patent Application No. 63 / 313,016, filed February 23, 2022, the contents of which are incorporated by reference herein. [Background technology]

[0002] Biomolecule-based (e.g., DNA-based) information storage systems have large storage capacity and stability over time, but 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] SUMMARY OF THE DISCLOSURE Provided herein are devices and methods for biomolecular detection.

[0004] Provided herein is a method for detecting a single molecule, the method comprising: (a) contacting a molecular sensor with at least one charge modulating element, the contact generating a current change; (b) measuring the current change; and (c) correlating the current change with the presence or absence of at least one charge modulating element, thereby detecting the single molecule. Further provided herein is a method in which the single molecule comprises a biomolecule. Further provided herein is a method in which the biomolecule comprises a nucleic acid. Further provided herein is a method in which the nucleic acid comprises DNA, RNA, or a mixture thereof. Further provided herein is a method in which the charge sensor comprises a graphene-enabled field effect transistor (GeFET) or a CMOS device. Further provided herein is a method in which the molecular sensor is in electrical communication with the charge sensor. Further provided herein is a method in which the molecular sensor comprises a polymerase. Further provided herein is a method in which the molecular sensor comprises an isothermal polymerase. Further provided herein is a method in which the molecular sensor comprises Phi29 polymerase or a variant thereof. Further provided herein are methods in which at least one charge modulating element comprises a negative charge or a positive charge. Further provided herein are methods in which at least one charge modulating element comprises a charge modulating nucleotide (CMN). Further provided herein are methods in which the contacting comprises incorporating a CMN into a polynucleotide primer. Further provided herein are methods in which the CMN comprises at least one modification to a canonical nucleotide. Further provided herein are methods in which the modification comprises a modification to a 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 in which the polyphosphate comprises at least 3, 4, 5, 6, 8 or 10 phosphates or variants thereof. Further provided herein are methods in which the modification comprises a modification to a 5'-terminal polyphosphate or chemical variant thereof. Further provided herein are methods in which the modification comprises a polymer. Further provided herein are methods in which 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 in which the nucleic acid chain comprises 25-5000 bases. Further provided herein are methods in which the nucleic acid chain is branched (dendrimer-like). Further provided herein are methods in which the nucleic acid chain comprises a secondary structure. Further provided herein are methods in which the secondary structure comprises one or more of a hairpin, a loop, a helix, a G-quadraplex and an I motif. Further provided herein are methods in which the nucleic acid chain comprises a single strand, a double strand or a triple strand. Further provided herein are methods in which the nucleic acid chain comprises at least one charge modulating chemical modification. Further provided herein is a method in which at least one charge modulating chemical modification increases the charge of CMN relative to an unmodified nucleotide. Further provided herein is a method in which at least one charge modulating chemical modification comprises one or more of amine, alkylamine, guanidinium, quaternary amine, imidazolium, pyridinium, and pyrrolidinium. Further provided herein is a method in which at least one charge modulating chemical modification decreases the charge of CMN relative to an unmodified nucleotide. Further provided herein is a method in which at least one charge modulating chemical modification comprises one or more of phosphate, phosphite, sulfonate, sulfite, carboxylate, xanthate, thiocarboxylate, boranophosphonate, and boric acid. Further provided herein is a method in which the nucleic acid strand comprises at least one sugar-modified nucleotide. Further provided herein is a method in which the sugar-modified nucleotide comprises a deoxynucleotide or a dideoxynucleotide. Further provided herein is a method in which the nucleic acid strand comprises a DNA-DNA, DNA-RNA, or DNA-PNA hybrid. Further provided herein is a method, wherein the nucleic acid strand comprises a phosphate modification.Further provided herein is a method wherein the phosphate modification comprises a hydrophobic group. Further provided herein is a method wherein the hydrophobic group comprises a linear or branched alkyl chain. Further provided herein is a method wherein the phosphate modification comprises a hydrophilic group. Further provided herein is a method wherein the hydrophilic group comprises polyethylene glycol. Further provided herein is a method wherein the polyethylene glycol comprises a molecular weight of 1000-100,000 daltons. Further provided herein is a method wherein the peptide chain is 1-100 amino acids in length. Further provided herein is a method wherein the CMN comprises a charged small molecule. Further provided herein is a method wherein the charged small molecule comprises one or more of a chelator, a dye, and a metal complex. Further provided herein is a method wherein the metal complex comprises ferrocene, Ru-dipy, and bis-cyclopentadienyl diiron. Further provided herein is a method wherein the current change is from 1 nanoamp to 100 picoamp. Further provided herein is a method wherein the current change is from 100 picoamp to 1 microamp. Further provided herein is a method, wherein the change in current is at least 1.01-3 times the background current. Further provided herein is a method, wherein steps a-c are repeated at least 50 times. Further provided herein is a method, wherein the method is configured to detect 1-200 biomolecules per second.

[0005] Further provided herein is a chemically sensitive field effect transistor device for single molecule sensing comprising a solid support, the solid support comprising a plurality of loci, each locus comprising a graphene layer, a gate electrode and a drain electrode in electrical communication through the graphene layer, and at least one insulating layer disposed between the gate electrode and the drain electrode, the loci having a pitch of 50-1000 nanometers. Further provided herein is a device further comprising at least one ground shield. Further provided herein is a device in which 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 in which 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 in which the graphene layer is about one atom thick. Further provided herein is a device comprising 100-1 billion loci. Further provided herein is a device in which each locus is 50-200 nm in size. Further provided herein are devices in which each location is a well, a channel or a substantially flat surface. 2 Further provided herein is a device having a 2 Further provided herein is a device having a 2 Further provided herein is a device having a 2 Further provided herein is a device,

[0006] Provided herein is a method for sequencing a single molecule polynucleotide, 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, the ternary complexes comprising a graphene binder; b) detecting one or more bases of the polynucleotides in real time, the detection occurring when the plurality of ternary complexes are bound to a graphene layer as 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 in which the graphene binder comprises an aromatic group. Further provided herein is a method in which the graphene binder comprises an aryl or heteroaryl group. Further provided herein is a method in which the graphene binder comprises a C6-C30 aryl or heteroaryl group. Further provided herein is a method in which 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 attached 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 attached via a linker. Further provided herein is a method in which the ternary complex is attached via a linker using a bond. Further provided herein is a method in which the attachment 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 in which the removal comprises contacting the surface with a solvent. Further provided herein is a method wherein the solvent comprises an organic solvent.Further provided herein is a method, wherein the organic solvent comprises MeCN, methanol, ethanol, 2-propanol, acetone, DMF, formamide, THF, or DMSO. Further provided herein is a method, wherein the organic solvent is heated. Further provided herein is a method, wherein the polymerase comprises Phi29 polymerase or a variant thereof. Further provided herein is a method, wherein the polymerase is configured to incorporate a charge-modified nucleotide as described herein. Further provided herein is a method, wherein the polymerase is bound to a surface in step a). Further provided herein is a method, wherein the polymerase is not bound to a surface in step a). Further provided herein is a method, wherein the plurality of polynucleotides comprises at least 100,000 unique nucleotides. Further provided herein is a method, wherein the plurality of polynucleotides is between 50 and 30,000 bases in length. Further provided herein is a method, wherein the detecting comprises contacting the ternary complex with at least one nucleotide. Further provided herein is a method, wherein the detecting comprises detecting a current change when CMN is incorporated. Further provided herein is a method, wherein the buried gate has a positive potential during step a). Further provided herein is a method wherein the buried gate has a positive or negative potential during step b).

[0007] Provided herein is a device for molecular sensing, comprising a first electrode including a neck region, a passivation layer, a second electrode, the first electrode and the second electrode disposed on a first base layer, and 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 the second portion of the first electrode and the second portion of the second electrode are separated by the passivation layer, and a first base layer and a second base layer, the first base layer disposed on a second base layer, and the first electrode and the second electrode disposed on a base layer. Further provided herein is a device having a nanogap of 1-50 nm. Further provided herein is a device having a nanogap of 10-30 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 surface layer of gold. Further provided herein is a device in which the gold layer has a thickness of 75 angstroms or less. 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 coupled to the first electrode and the second electrode. Further provided herein is a device in which the charge sensor comprises a polymer. Further provided herein is a device in which the polymer comprises at least one nucleic acid, amino acid, sugar or lipid. Further provided herein is a device in which the charge sensor comprises carbon. Further provided herein is a device in which the charge sensor is coupled to at least one of the first electrode or the second electrode via a sulfur-gold interaction.Further provided herein is a device in which the charge sensor is coupled to the molecular sensor via a tether. Further provided herein is a device in which the molecular sensor comprises an enzyme. Further provided herein is a device in which the molecular sensor comprises an antibody. Further provided herein is a device in which the enzyme comprises a polymerase. Further provided herein is a device in which the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein is a device in which the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein is a device in which at least one edge of the first electrode is undercut relative to the passivation layer. Further provided herein is a device in which the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode. Further provided herein is a device in which 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 in which the first electrode and the second electrode each comprise a gold surface layer. 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 width of the neck region is 200 nm or less.

[0008] Provided herein is a device for molecular sensing, comprising a first electrode disposed on a first base layer, a second electrode, the 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 the second portion of the first electrode and the second portion of the second electrode are separated by a passivation layer, and a first base layer and a second base layer, the first base layer disposed on a second base layer, and the first electrode and the second electrode disposed on a base layer. Further provided herein is a device configured to overlap a portion of the second electrode. Further provided herein is a device configured to passivate an electrode trace. Further provided herein is a device having a nanogap of 1-50 nm. Further provided herein is a device having a nanogap of 10-30 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 surface layer of gold. Further provided herein is a device in which the gold layer has a thickness of 75 angstroms or less. 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 coupled to the first electrode and the second electrode. Further provided herein is a device in which 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 in which the charge sensor comprises carbon. Further provided herein is a device in which the charge sensor is coupled to at least one of the first electrode or the second electrode via a sulfur-gold interaction. Further provided herein is a device in which the charge sensor is coupled to the molecular sensor via a tether. Further provided herein is a device in which the molecular sensor comprises an enzyme. Further provided herein is a device in which the molecular sensor comprises an antibody. Further provided herein is a device in which the enzyme comprises a polymerase. Further provided herein is a device in which the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein is a device in which the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein is a device in which at least one edge of the first electrode is undercut relative to the passivation layer. Further provided herein is a device in which the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode. Further provided herein is a device in which 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 in which the first electrode and the second electrode each comprise a gold surface layer. 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 width of the neck region is 200 nm or less.

[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 top of the first base layer, the second electrode being 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 are separated by the passivation layer, and a first base layer and a second base layer, the first base layer disposed on top of the second base layer, the first electrode and the second electrode disposed on top of the base layer. Further provided herein is a device having a nanogap of 1-50 nm. Further provided herein is a device having a nanogap between 10 and 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 surface layer of gold. Further provided herein is a device in which the gold layer has a thickness of 75 angstroms or less. 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 coupled 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 in which the charge sensor comprises carbon. Further provided herein is a device in which the charge sensor is coupled to at least one of the first electrode or the second electrode via a sulfur-gold interaction. Further provided herein is a device in which the charge sensor is coupled to the molecular sensor via a tether. Further provided herein is a device in which the molecular sensor comprises an enzyme. Further provided herein is a device in which the molecular sensor comprises an antibody. Further provided herein is a device in which the enzyme comprises a polymerase. Further provided herein is a device in which the longest linear dimension of the second electrode is perpendicular to the neck region. Further provided herein is a device in which the longest linear dimension of the second electrode is parallel to the neck region. Further provided herein is a device in which at least one edge of the first electrode is undercut relative to the passivation layer. Further provided herein is a device in which the surface area of ​​the first electrode is smaller than the surface area of ​​the second electrode. Further provided herein is a device in which 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 in which the first electrode and the second electrode each comprise a gold surface layer. 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 width of the neck region is 200 nm or less.

[0010] Provided herein is an array of any one of the devices described herein, in which at least a portion of the first and second electrodes are independently addressable. Further provided herein is an array in which the pitch distance of the nanogaps of at least some of the devices is 200 nanometers or less. Further provided herein is an array in which at least a portion of the first and second electrodes are independently addressable. Further provided herein is an array in which at least a portion of the first and second electrodes are independently addressable on the xy axis. Further provided herein is an array in which 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 in which 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 5000 devices of any one of the devices described herein. Further provided herein is an array in which at least a portion of the nanogaps of the devices is independently addressable on the z axis. Further provided herein is an array further comprising 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 further comprising a plurality of routing connections, the plurality of routing connections configured to addressably control respective devices of the array.

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

[0012] Provided herein is a method 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 the sensor; and c) measuring an electrical signal generated from the sensor. Provided herein is a method comprising: 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; and c) measuring an electrical signal generated from the polymerase. Provided herein is a method further comprising analyzing the electrical signal and confirming the identity of the at least one nucleotide triphosphate. Provided herein is a method further comprising: at least one nucleotide triphosphate comprising a non-canonical base. Provided herein is a method further comprising: at least one nucleotide triphosphate comprising a terminator configured to prevent chain extension. Provided herein is a method further comprising: repeating to confirm the identity of at least 20 bases. Provided herein is a method further comprising repeating to confirm the identity of at least 100 bases. Further provided herein is a method that is repeated to confirm the identity of at least 1000 bases.

[0013] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein 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. [Brief description of the drawings]

[0014] 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. [Figure 1]1 shows non-limiting examples of schemes for performing polynucleotide synthesis and sequencing according to some embodiments. [Diagram 2] 1 illustrates a non-limiting example of a graphene device described herein according to some embodiments. [Diagram 3] 1 shows an example of a charge-modulated nucleotide for use with the devices and methods described herein according to some embodiments. [Figure 4A] FIG. 1 shows a top view of a graphene device for polynucleotide sequencing with embedded gate and shield electrodes according to some embodiments. [Figure 4B] FIG. 1 shows a side view of a graphene device for polynucleotide sequencing with embedded gate and shield electrodes according to some embodiments. [Figure 5A] 13A-13C show zoom-ins of flexible structures with spots, channels or wells, respectively, according to some embodiments. [Figure 5B] 13A-13C show zoom-ins of flexible structures with spots, channels or wells, respectively, according to some embodiments. [Figure 5C] 13A-13C show zoom-ins of flexible structures with spots, channels or wells, respectively, according to some embodiments. [Figure 6A] 1 is a scheme of a solid support comprising an active area and a fluidic interface, according to some embodiments. [Figure 6B] FIG. 1 is a front view of an example of a solid support array, according to some embodiments, which in some examples may include thousands or more, or millions or more, of the polynucleotide synthesis devices described herein. [Figure 6C] FIG. 2 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 hundreds or more, or thousands or more, of solid support arrays. [Figure 7]1 illustrates an example of a 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) device, 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 illustrates a nanoelectrical device according to some embodiments, in which an analyte (a nucleic acid, shown as an example only) communicates with a molecular sensor connected to a charge sensor spanning the location. [Figure 12A] 1 illustrates a top 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 lines indicate a coating and a nanowire is shown bridging 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] 1 shows a top view of a cross finger with a mask device according to some embodiments. [Figure 14B] 13A-13C show side views of a cross finger with a mask device according to some embodiments. [Figure 15] 1 illustrates 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The exponential growth in the amount of information generated and stored necessitates a need for high-capacity storage systems. Biological molecules such as DNA molecules provide a suitable host for partial information storage due to their stability over time and capacity of 4-bit (or other) information coding, as opposed to traditional binary information coding. Provided herein are methods, devices and systems for real-time detection of single molecules (e.g., biological molecules). Provided herein are methods for improving nucleic acid sequencing by using 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 this invention belongs.

[0018] Numerical characteristics are expressed in range format throughout this disclosure. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, a description of a range should be considered to have all of the possible subranges specifically disclosed and each numerical value within the range to the nearest tenth of the lower limit, unless the context clearly dictates otherwise. For example, a description of a range such as 1-6 should be considered to have the specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and each value within the range such as 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of the ranges therebetween may be independently included in the smaller ranges, which are also included herein, subject to any limit specifically excluded in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included herein, 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 of the embodiments. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly dictates otherwise. It is further understood that the term "comprises" as used herein is intended to specify the presence of stated features, integers, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, terms such as "and / or" include any and all combinations of one or more of the associated listed items.

[0020] Unless otherwise stated or apparent from the context, as used herein, the term "about" in reference to a number or range of numbers should be understood to mean the stated number and + / - 10% of the number, or for values ​​recited for a range, less than 10% of the recited lower limit and more than 10% of the recited upper limit.

[0021] As used herein, the terms "preselected sequence", "predefined sequence" or "predetermined sequence" are used interchangeably. This term means that the sequences of the polymers are known and are selected prior to the synthesis or assembly of the polymers. In particular, although various aspects are described herein primarily with respect to the preparation of nucleic acid molecules, the sequences of the polynucleotides are known and are selected prior to the synthesis or assembly of the nucleic acid molecules.

[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 or string of symbols. In some examples, a series or string of symbols may include binary data.

[0023] The polynucleotide sequences described herein may include DNA, or RNA, or their analogs, or derivatives, unless otherwise specified. As used herein, the terms nucleic acid, polynucleotide, oligonucleotide, oligo, and oligonucleic acid are used synonymously throughout to refer to a polymer of nucleoside monomers. In some instances, the nucleic acid is linked via a phosphate or sulfur-containing bond. The nucleic acid includes, in some instances, DNA, RNA, non-canonical nucleic acid, non-natural nucleic acid, or other nucleosides. In some instances, the nucleotide includes a non-canonical base, sugar, or other moiety. In some instances, the nucleotide includes a terminator configured to prevent an extension reaction. In some instances, such a terminator is removed prior to the addition of a subsequent nucleotide 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 examples, includes detecting the presence, absence, concentration, and / or identity of a biomolecule or a portion of the biomolecule. In some examples, the biomolecule includes a polymer. In some examples, the devices, methods, compositions, and systems are used for sequencing (e.g., nucleic acids). In some examples, the solid support includes a surface. Further provided herein are multiple devices that are combined to form a larger array or chip. In some examples, voltage or current is measured to sense molecular changes associated with the charge sensor, molecular sensor, or a complex thereof. An array of such devices, in some examples, provides high-throughput reading of digital information encoded in nucleic acids. In some examples, the devices are arrayed on a solid support with each device or group of devices at an addressable location. A larger group of such devices, in some examples, is present in a serve rack unit.

[0026] The devices provided herein may be used for molecular sensing. The electrodes may be configured to detect a change in voltage, current, or resistance to detect a biomolecule. In some examples, the device for molecular sensing includes one or more electrodes. In some examples, the device for molecular sensing includes one, two, three, four, five, or more than five electrodes. In some examples, the device for molecular sensing includes a source electrode and a drain electrode. In some examples, an electrode of the one or more electrodes is configured as a source. In some examples, an electrode of the one or more electrodes is configured as a drain. In some examples, the 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, the 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, the one or more electrodes are in electrical communication. In some examples, the electrical communication between the 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 includes graphene. In some examples, the charge sensor includes a graphene-enabled field effect transistor (GeFET) or a CMOS device. In some examples, the charge sensor is coupled to a tether, such as those provided herein. In some examples, the tether is coupled to a charge sensor, a molecular sensor, or both. In some examples, the molecular sensor and the charge sensor are in electrical communication. In some examples, the molecular sensor includes 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 disposed on the one or more base layers. In some examples, the one or more base layers are a passivation layer or a gate layer.

[0027] A device 1100 (FIG. 11) for molecular sensing is provided herein. The device includes, in some examples, 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 in electrical communication with the first electrode 1105a and the second charge sensor coupling layer 1107b 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 coupled to a tether 1109. In some examples, the tether 1109 is coupled 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 disposed 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 / or the second electrode 1105b are separated by at least one passivation base layer (1104 or 1103). In some examples, the one or more base layers (e.g., 1103 and 1101) are separated by a gate layer 1102. In some examples, a gate layer 1102 is disposed on a base layer 1101. Such devices are in some examples combined into larger arrays. In some examples, the devices are integrated into electronic devices such as CMOS and connected 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 disposed on one or more base layers. In some examples, the first electrode 1202a and the second electrode 1202b are disposed 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 disposed on the second base layer 1204, and the first electrode 1202a and the second electrode 1203 are disposed on the base layers 1201 and 1204. In some examples, the devices described herein include a coating 1206 (FIG. 12C). 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 disposed 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 disposed on the second base layer 1302, and the first electrode 1302 and the second electrode 1303a are disposed on the 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 disposed 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 disposed on the second base layer 1402, and the first electrode 1402 and the second electrode 1403a are disposed on the base layer 1401. In some examples, the device further includes a second passivation layer 1406.

[0031] 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 further includes a passivation layer, such as 1605 and 1606. In some examples, the passivation layer 1605 / 1606 includes a channel or well. In some examples, a bottom 1607 of the well or channel (or trench) includes (or is exposed to) the 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 disposed 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 a passivation layer 1605. In some examples, the device includes a first base layer 1601 and a second base layer 1604. In some examples, the first base layer 1601 is disposed on the second base layer 1604, and the first electrode 1602a and the second electrode 1603 are disposed on the base layers 1601 / 1604. In some examples, the device stack is etched using the same hard mask such that the electrode edges are aligned and a deeper trench (channel or well) is etched underneath (FIGS. 16A-16B).

[0032] In some examples, the device includes a graphene layer configured to detect a change 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 device provided herein is 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 current change. In some examples, the charge sensor includes a graphene layer. In some examples, the information about the biomolecule includes information about a monomer of the biomolecule. For example, if the biomolecule is a polynucleotide, the information about the biomolecule includes information about the nucleotides. In some examples, the information about the nucleotides includes information about the 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 the amino acids.

[0033] Provided herein is a device including a solid support, a charge sensor, one or more electrodes, and at least one insulating layer. In some examples, the charge sensor includes a graphene layer. In some examples, the one or more electrodes include a gate electrode. In some examples, the one or more electrodes include 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, an insulating layer is disposed between the gate electrode and the drain electrode. In some examples, the solid support includes a plurality of locations. In some examples, the device further includes at least one ground shield. In some examples, the device further includes at least one recessed gate (electrode).

[0034] Provided herein is a fifth device 200 as shown in FIG. 2. 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 disposed 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.

[0035] 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 disposed 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 a current generated by a biomolecule interacting with a molecular sensor described herein.

[0036] Any of the above devices may be arranged on a solid support. In some examples, 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 examples, the devices 1500 are arranged on a solid support 1501 in a configuration. In some examples, the first electrode is placed on the x-axis and the second electrode 1502 is placed on the y-axis (FIG. 15). Any number of devices are present in an array in some examples. In some examples, the array includes between 10 and 1,000,000, 10 and 100,000, 10 and 10,000, 10 and 5,000, 50 and 1,000,000, 50 and 100,000, 50 and 10,000, 50 and 5,000, 100 and 1,000,000, 100 and 100,000, 100 and 5,000, or 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 such larger devices. In some examples, the density of the array can be as high as 2 to 1 billion different reaction sites (devices, or locations) 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 2In 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 The device, in some examples, further includes vias and / or other connections for electrical communication. In some examples, the device includes an electrode disposed on the z-axis.

[0037] 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, made from glass and with transparent windows for the array and fluidic 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 interface under the solid support area.

[0038] The solid supports described herein include an active area. In some examples, the active area includes an addressable solid support, area, or location for molecular sensing. In some examples, the active area includes an addressable area or location for nucleic acid storage. In some examples, the active area is in fluid communication with a solvent or other reagent. The active area includes a variety of 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 comprises a width of at least 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 5 mm, 5 mm, 10 mm, 12 mm, 14 mm, 16 mm, 18 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 60 mm, 70 mm, 80 mm, or greater than 80 mm, or about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 5 mm, about 5 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, or greater than about 80 mm. In some examples, the active area comprises a height of at least 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 5 mm, 5 mm, 10 mm, 12 mm, 14 mm, 16 mm, 18 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 60 mm, 70 mm, 80 mm, or greater than about 80 mm, or greater than about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 5 mm, about 5 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, or greater than about 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 area is at least 0.5 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 5 μm, 5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, or greater than about 80 μm, or greater than about 0 Including widths of about 0.5 μm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 5 μm, about 5 μm, about 10 μm, about 12 μm, about 14 μm, about 16 μm, about 18 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or greater than about 80 μm. In some examples, the active area is at least 0.5 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 5 μm, 5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, or greater than about 80 μm, or greater than about 0 In some examples, the active area is between 4 and 900 mm. 2 , 4~500mm 2 , 4~250mm 2 , 2~900mm 2 , 10~900mm 2 , 25~900mm 2 , 50~900mm 2 , 100~900mm 2 Or 400~900mm 2 In some examples, the active area and any passive areas are between 4 and 900 mm 2 , 4~500mm 2 , 4~250mm 2 , 2~900mm 2 , 10~900mm 2 , 25~900mm 2 , 50~900mm 2, 100~900mm 2 Or 400~900mm 2 In some examples, the active area is between 4 and 900 mm 2 , 4~500mm 2 , 4~250mm 2 , 2~900mm 2 , 10~900mm 2 , 25~900mm 2 , 50~900mm 2 , 100~900mm 2 Or 400~900mm 2 In some examples, the active area and any passive areas are between 4 and 900 mm on each side of the devices described herein. 2 , 4~500mm 2 , 4~250mm 2 , 2~900mm 2 , 10~900mm 2 , 25~900mm 2 , 50~900mm 2 , 100~900mm 2 Or 400~900mm 2 An exemplary active area within a solid support can be seen in Figure 6A. Package 607 includes active area 605 within solid support 603. Package 607 also includes fluidic interface 601.

[0039] Described herein are devices, compositions, systems and methods for molecular sensors. In some examples, a solid support has several sites or locations for molecular sensors. In some examples, the solid support includes up to 10,000 x 10,000 locations, or about 10,000 x 10,000 locations in area. In some examples, the solid support includes about 1000-20,000 x about 1000-20,000 locations in area. In some examples, the solid support comprises at least 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, 22,000, 24,000, 26,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 55,000, 56,000, 57,000, 58,000, 59,000, 60,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 1 0 position or about 10, about 30, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 12,000, about 14,000, about 16,000, about 18,000, about 20,000 pieces x at least 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions or about 10, about The area may include 30, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 12,000, about 14,000, about 16,000, about 18,000, about 20,000 locations. In some examples, the area is at most 0.25 square inches, 0.5 square inches, 0.75 square inches, 1.0 square inches, 1.25 square inches, 1.5 square inches, or 2.0 square inches.In some examples, the solid support has a diameter of at least 0.01 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.4 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, or greater than 10 μm, or about 0.01 μm, about 0.05 μm, or greater than 10 μm. The addressable locations may include a pitch of about 0.1 μm, about 0.1 μm, about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 2.5 μm, about 3.0 μm, about 3.5 μm, about 4.0 μm, about 4.5 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, or greater than 10 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.01 μm, about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 2.5 μm, about 3.0 μm, about 3.5 μm, about 4.0 μm, about 4.5 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, or less than about 10 μm. In some examples, the solid support comprises addressable locations having a pitch of about 5 μm. In some examples, the solid support comprises addressable locations having a pitch of about 2 μm. In some examples, the solid support comprises addressable locations having a pitch of about 1 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.01 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.02 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.05 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.08 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.1 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.2 μm.In some examples, the solid support comprises addressable locations 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 locations with a pitch of about 0.1 μm to about 3 μm. In some examples, the solid support has a diameter of at least 0.01 μm, 0.02 μm, 0.025 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.30 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, or greater than about 1 μm, or greater than about 0.01 μm, The solid support comprises addressable locations having a pitch of about 0.02 μm, about 0.025 μm, about 0.03 μm, about 0.04 μm, about 0.05 μm, about 0.1 μm, about 0.15 μm, about .02 μm, about 0.25 μm, about 0.30 μm, about 0.35 μm, about 0.4 μm, about 0.45 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, or greater than about 1 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.5 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.2 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.1 μm. In some examples, the solid support comprises addressable locations having a pitch of about 0.02 μm. In some examples, the solid support comprises addressable locations 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 locations with a pitch of about 0.01 μm to about 0.3 μm. In some examples, the solid support comprises addressable locations with a pitch of about 0.05 μm to about 1 μm.

[0040] 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 coupled to a charge sensor. In some examples, the electrodes on each side of the gap are coupled to at least one charge sensor. In some examples, the electrodes on each side of the gap are coupled to at least one shared charge sensor. In some examples, the length of the nanogap is about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, or about 50 nm. In some examples, the length of the nanogap is about 5-50 nm, about 5-25 nm, about 5-20 nm, about 10-50 nm, about 10-30 nm, about 15-25 nm, about 15-30 nm, about 20-40 nm, 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.

[0041] The devices described herein may include a neck region. In some examples, the electrode includes a 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.

[0042] In some examples, each location of the structure has a width of about 1 μm and a distance between the centers of each structure of about 2.1 μm. In some examples, each location of the structure has a width of about 0.5 μm and a distance between the centers of each structure of about 2 μm. In some examples, each location of the structure has a width of about 0.1 μm and a distance between the centers of each structure of about 0.2 μm. The locations 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 location for molecular sensing. In some examples, the rigid structure includes a substantially flat area, channel or well for molecular sensing.

[0043] Provided herein is a flexible structure having a surface with multiple locations for polynucleotide extension. Figures 5A-5C show zoom-ins of locations in a flexible structure. Each location in a portion of a flexible structure 501 can be a substantially flat spot 503 (e.g., flat), a channel 505, or a well 507. The locations 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 locations, channels, or wells for polynucleotide synthesis.

[0044] In some examples, the wells described herein have a ratio of width to depth (or height) between 1 and 0.01. In some examples, the width is a measurement of the width at the narrowest segment of the well. In some examples, the wells described herein have a ratio of width to depth (or height) between 0.5 and 0.01. In some examples, the width is a measurement of the width at the narrowest segment of the well. In some examples, the wells described herein have a ratio of width to depth (or height) 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 a plurality of distinct locations for molecular sensing. Exemplary structures for locations include, but are not limited to, substantially flat regions, channels, wells, or protrusions. The structures described herein may include a plurality of clusters, each cluster including a plurality of wells, locations, or channels. Alternatively, those described herein may include a uniform configuration of wells, locations, 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 height of the wells 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 height of the wells is about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, or more. In some examples, the height or depth of the wells is at least 10 nm, 25 nm, 50 nm, 75 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 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 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0045] 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 a measurement of the width 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 a measurement of the width 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, about 0.05, about 0.1, about 0.15, about 0.16, about 0.2, about 0.5, or about 1.

[0046] Described herein are structures for molecular sensing that include a plurality of distinct locations. 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 a plurality of channels, but the height or depth of the channels 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 height of the channels 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 nm, 25 nm, 50 nm, 75 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 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.

[0047] The width of a location on the surface of a structure for molecular sensing 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, such as about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, about 10 μm, about 5 μm, about 1 μm, or about 0.5 μm. In some examples, the width of a location is less than about 100 μm, less than about 90 μm, less than about 80 μm, less than about 70 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 20 μm, or less than about 10 μm. In some examples, the width of the location is at least 10 nm, 25 nm, 50 nm, 75 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or more than 1000 nm. In some examples, the width of the location 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 width of the well is in the range of about 50 nm to about 1000 nm. In some examples, the distance between the centers of two adjacent locations 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, for example, about 20 μm. In some examples, the total width of the location is about 5 μm, 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, or about 100 μm. In some examples, the total width of the location is about 1 μm to about 100 μm, about 30 μm to about 100 μm, or about 50 μm to 70 μm. In some examples, the distance between the centers of two adjacent locations 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 location is about 50 μm, about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, or about 1.5 μm. In some examples, the total width of the location is about 0.5 μm to 2 μm, about 0.75 μm to 1 μm, or about 0.9 μm to 2 μm.

[0048] In some examples, each location supports sensing of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another location. Provided herein are surfaces that include 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 include greater 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 locations. 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 positions. In some cases, each cluster includes 50-500, 50-200, 50-150, or 100-150 positions. In some cases, each cluster includes 100-150 positions. In some examples, each cluster includes 109, 121, 130, or 137 positions.

[0049] Provided herein are locations having a maximum segment width of 5-100 μm. In some cases, the locations have a maximum segment width of about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, or about 60 μm. In some cases, the locations are channels having multiple segments. In some examples, each segment has a center-to-center distance of 5-50 μm apart. In some cases, the center-to-center distance for each segment is about 5 μm, about 10 μm, about 15 μm, about 20 μm, or about 25 μm.

[0050] Provided herein are locations having a maximum segment width of 5 to 500 nm. In some cases, the locations have a maximum segment width of about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 80 μm, or about 100 nm. In some cases, the locations are channels having multiple segments. In some examples, each segment has a center-to-center distance of 5 to 50 nm apart. In some cases, the center-to-center distance for each segment is about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 50 nm, about 100 nm, or about 200 nm.

[0051] In some examples, the number of distinct polynucleotides synthesized on the surface of the structures described herein varies depending on the number of distinct locations available on the substrate. In some examples, the density of locations within a cluster of substrates can be in the range of 100 to 150 mm 2 At least one position per mm 2 At least or about 10 positions per mm 2 At least or about 25 positions per mm 2 At least or about 50 positions per mm 2 At least or approximately 65 positions per mm 2 At least or about 75 positions per mm 2 At least or about 100 positions per mm 2 At least or about 130 positions per mm 2 At least or about 150 positions per mm2 At least or approximately 175 positions per mm 2 At least or about 200 positions per mm 2 At least or about 300 positions per mm 2 At least or about 400 positions per mm 2 At least or about 500 positions per mm 2 At least or about 1,000 positions per mm 2 At least or about 10 per 4 position, mm 2 At least or about 10 per 5 position, mm 2 At least or about 10 per 6 In some cases, the substrate is 2 Approximately 10 positions per approx. ~ approx. 500 mm 2 , mm 2 Approximately 25 positions to approximately 400 mm 2 , mm 2 Approximately 50 positions to approximately 500 mm 2 , mm 2 Approximately 100 positions to approximately 500 mm 2 , mm 2 Approximately 150 to 500 mm per position 2 , mm 2 Approximately 10 positions ~ approximately 250 mm 2 , mm 2 Approximately 50 positions to approximately 250 mm 2 , mm 2 Approximately 10 positions ~ approximately 200 mm 2 , or mm 2 Approximately 50 positions to approximately 200 mm 2 In some cases, the substrate may include 2 Approximately 10 per 4 Position ~ approx. 10 5 mm 2 In some cases, the substrate may include 2 Approximately 10 per 5 Position ~ approx. 10 7 mm 2 In some cases, the substrate may include 2 At least 10 per 5 In some cases, the substrate may include a2 At least 10 per 6 In some cases, the substrate may include a 2 At least 10 per 7 In some cases, the substrate may include a 2 Approximately 10 per 4 Position ~ approx. 10 5 mm 2 In some examples, the density of locations in the clusters on the substrate is on the order of μm 2 At least one position per μm 2 At least or about 10 positions per μm 2 At least or about 25 positions per μm 2 At least or about 50 positions per μm 2 At least or about 65 positions per μm 2 At least or about 75 positions per μm 2 At least or about 100 positions per μm 2 At least or about 130 positions per μm 2 At least or about 150 positions per μm 2 At least or about 175 positions per μm 2 At least or about 200 positions per μm 2 At least or about 300 positions per μm 2 At least or about 400 positions per μm 2 At least or about 500 positions per μm 2 In some cases, the substrate has at least about 1,000 or more positions per μm. 2 Approximately 10 positions per ~ Approximately 500 μm 2 , μm 2 Approximately 25 positions per approx. ~ approx. 400 μm 2 , μm 2 Approximately 50 positions per unit to approximately 500 μm 2 , μm 2 Approximately 100 positions per unit to approximately 500 μm 2 , μm 2 Approximately 150 positions per unit to approximately 500 μm per unit 2 , μm 2 Approximately 10 positions per ~ Approximately 250 μm 2 , μm 2Approximately 50 positions per unit to approximately 250 μm 2 , μm 2 Approximately 10 positions per ~ Approximately 200 μm 2 , or μm 2 Approximately 50 positions to approximately 200 μm per 2 Includes.

[0052] In some examples, the distance between two adjacent centers in 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 locations is greater than 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, or about 100 μm. In some cases, the distance between the centers of two adjacent locations is less than about 200 μm, about 150 μm, about 100 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 cases, the distance between the centers of two adjacent locations is less than about 10,000 nm, about 8,000 nm, about 6,000 nm, about 4,000 nm, about 2,000 nm, about 1,000 nm, about 800 nm, about 600 nm, about 400 nm, about 200 nm, about 150 nm, about 100 nm, about 80 μm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm. In some examples, each square meter of the structures described herein is at least 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 In some examples, each position supports one polynucleotide. In some examples, 10 9 The polynucleotides are about 6 m 2 , about 5m 2 , about 4m 2 , about 3m 2 , about 2m 2 Or about 1m 2 is supported on a structure less than

[0053] In some examples, the structures described herein may be in the form of 2,000, 5,000, 10,000, 20,000, 30,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,800,000, 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, 1,400,000, 1,8 ... The present invention provides a support for sensing 00,000, 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, 1,0 ... A support for sensing 200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000 or more polynucleotides is provided, 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 growth of polynucleotides having at least 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more bases. In some configurations, the structures for molecular sensing described herein include sites for molecular sensing in a uniform configuration.

[0054] In some examples, polynucleotides are synthesized on separate locations of the structure. In some examples, each location supports sensing of a population of polynucleotides. In some cases, each location supports sensing of a population of polynucleotides having a different sequence than the population of polynucleotides grown on another location. In some examples, the locations of the structure are arranged within a plurality of 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 structures 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, 9,0 ...0, 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 locations. 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 positions. In some examples, each cluster includes 50-500, 100-150, or 100-200 positions.In some examples, each cluster comprises 109, 121, 130 or 137 positions. In some examples, each cluster comprises 5, 6, 7, 8, 9, 10, 11 or 12 positions. In some examples, polynucleotides from distinct positions within a cluster, when assembled, have a sequence that encodes a contiguous, longer polynucleotide of a given sequence. In some examples, each of the polynucleotides comprises a plurality of different nucleotide bases (e.g., A, T, C, G, etc.).

[0055] In some examples, the structures described herein are approximately the size of a plate (e.g., a chip), for example, about 40-120 mm by 25-100 mm. In some examples, the structures described herein have a diameter of about 1000 mm, about 500 mm, about 450 mm, about 400 mm, about 300 mm, about 250 mm, about 200 mm, about 150 mm, about 100 mm, or less than 50 mm. In some examples, the diameter of the substrate is about 25 mm to about 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, about 200 mm, about 150 mm, about 130 mm, about 100 mm, about 84 mm, about 76 mm, about 54 mm, about 51 mm, and about 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 2or more. In some examples, the thickness of the substrate is 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 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than 4.0 mm, or about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, or more than 4.0 mm. In some cases, the thickness varies with the 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, and the structure must be thick enough to support its own weight without cracking during handling.

[0056] Described herein are devices in which two or more solid supports are assembled. In some examples, the solid supports are linked together on a larger unit. The linkage may include exchange of fluids, electrical signals, or other exchange media between the solid supports. The unit may be capable of linking with several servers, computers, or network devices. For example, multiple solid supports are assembled or mounted on a rack unit, which can be easily inserted or removed from a server rack. The rack unit may include several solid supports. In some examples, the rack unit includes about 1, about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, about 10,000, about 20,000, about 50,000, about 100,000, or about 100,000 solid supports. In some examples, a rack unit includes at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, or more than 100,000 solid supports. In some examples, a rack unit includes 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 of a rack unit are in fluid communication, electrical communication, or both. In some examples, the server rack includes about 10, about 20, about 50, about 80, about 100, about 200, about 500, about 800, or about 1000 rack units. In some examples, the server rack includes at least about 10, about 20, about 50, about 80, about 100, about 200, about 500, about 800, or about 1000 rack units. In some examples, the server rack includes up to about 10, about 20, about 50, about 80, about 100, about 200, about 500, about 800, or about 1000 rack units.In some examples, all or a portion of the solid supports of a rack unit of a rack server are in fluid communication, electrical communication, or both. In some examples, two or more solid supports are not attached to each other. In some examples, two or more rack units including solid supports such as those described herein are stacked vertically. The fluid communication, electrical communication, or both may be formed using, as non-limiting examples, one or more tubes (e.g., microfluidic tubes), valves, actuators, robots, etc.

[0057] The nucleic acids present on the solid supports (and the information stored therein) can be accessed from the rack unit. See, for example, FIG. 6D. Accessing can include removal of the polynucleotides from the solid support, direct analysis of the polynucleotides on the solid support, or any other method that can manipulate or identify the information stored in the nucleic acid. The information can be accessed in some examples 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 can include interfacing the nucleic acid with additional devices such as a mass spectrometer, HPLC, sequencing instrument, PCR thermocycler, or other devices for manipulating nucleic acids. In some examples, accessing the nucleic acid information is achieved by cleavage of the polynucleotides from all or a portion of the solid support.

[0058] 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, for example, openings, openings adapted to support processing blades or other computer equipment. In some examples, a computer system such as those 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, the 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 examples, synthesis units, storage units, amplification units, and the like. The rack unit or server in some instances includes units for the inflow of reagents or the outflow of waste or synthesis products.

[0059] 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 for cleavage of the polynucleotide. In some examples, a lamp is used for cleavage of the polynucleotide, and a mask mediates the location of exposure of the UV light relative to the surface. In some examples, a laser is used for cleavage of the polynucleotide, and the open / closed state of a shutter controls the location of exposure of the UV light relative to the surface. In some examples, a computer system such as that provided herein directs the open / closed state of the shutter. In some examples, access to the 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.

[0060] 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 include single-walled carbon nanotube (SWNT)-based FETs, silicon nanowire (SiNW) FETs, graphene nanoribbon FETs (and MoS 2and related nanoribbon FETs, field effect transistors (FETs), tunnel FETs (TFETs), and steep subthreshold slope devices fabricated from 2D materials such as nanowires. In some examples, the nanowires include a polymer. In some examples, the polymer includes at least one nucleic acid, amino acid, sugar, or lipid. In some examples, the charge sensor includes carbon. In some examples, the charge sensor is coupled to at least one of the first electrode or the second electrode via sulfur-gold interactions. 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 includes a nucleic acid. In some examples, the charge sensor includes DNA or RNA. In some examples, the charge sensor includes 20-500, 50-500, 100-500, 150-1000, 150-500, 250-1000, 500-1000, or 600-1000 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, about 750, about 500, about 250, about 200, about 100, about 50, or about 20 nucleic acids. In some examples, the charge sensor comprises at least 1000, 750, 500, 250, 200, 100, 50, or 20 nucleic acids. In some examples, at least one nucleic acid is attached to a tether. In some examples, the polymer comprises 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 then about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, or about 40 nm. In some examples, the length of the charge sensor is then 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.

[0061] The device 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 through a tether. In some examples, the molecular sensor includes a polymerase. Some of the various polymerases are used in some examples in methods or compositions including protein-based enzymes and functional variants thereof isolated from biological systems. 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, E. coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus stearothermophilis Pol I. Family B DNA polymerases include eukaryotic DNA polymerase α, δ, and ε, DNA polymerase ζ, T4 DNA polymerase, Phi29 DNA polymerase, and RB69 bacteriophage DNA polymerase. Family C includes 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 S. 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.

[0062] The molecular sensor may be coupled to the charge sensor via a tether (or bond). In some examples, the molecular sensor is coupled 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 an antibody and an epitope, and the like. In some examples, a conductive tether is used to couple the molecular sensor to the charge sensor. Exemplary conductive tethers include those having structures including 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, electrostatic, 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 a bond. In some examples, the bond 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 the molecular sensor, the biomolecule, and the primer. In some examples, the ternary complex is bound to the charge sensor via the primer. In some examples, the ternary complex is bound to the charge sensor via the molecular sensor.In some instances, the ternary complex is coupled to the charge sensor via a biomolecule.

[0063] The molecular sensor may include a graphene binding moiety configured to couple the molecular sensor to a charge sensor including a graphene layer. Such a moiety may be coupled to other π-based charge sensors, such as a graphene layer. 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 C 6 -C 30 In some examples, the graphene binder comprises a C 6 -C 20 In some examples, the graphene binder comprises a C 6 -C 15 In some examples, the graphene binder comprises a C 6 -C 10 In some examples, the graphene binder comprises a C 10 -C 30 In some examples, the graphene binder comprises a C 15 -C 30 The graphene binder may include an aryl group or a heteroaryl group. In some examples, the graphene binder includes an aromatic hydrocarbon. In some examples, the graphene binder includes 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.

[0064] Nanoelectrical Device Manufacturing

[0065] 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 produce an electrode or a passivation layer. In some examples, the methods include patterning the electrode or the passivation layer. In some examples, the methods herein include etching. In some examples, the methods herein include isotropic or substantially isotropic etching. In some examples, the etching produces edges of the electrode that are undercut. In some examples, the methods include at least some of the following steps: a) providing one or more base layers, b) depositing material to produce a second electrode, c) patterning the second electrode, d) optionally planarizing, e) depositing material to produce a passivation layer, f) depositing material to produce a first electrode, g) patterning the first electrode, and h) isotropically etching the passivation layer, resulting in undercutting edges of the first electrode. 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.

[0066] Methods for supporting the passivation of biomolecules (such as nanowires) on a substrate are provided herein. In some examples, the surface of the structures described herein includes a material and / or is coated with a material that promotes a coupling reaction with the biomolecule for binding. To prepare a structure for passivating a biomolecule, surface modification may be utilized to chemically and / or physically alter the substrate surface by additive or subtractive processes that change one or more chemical and / or physical properties of the substrate surface or selected sites or regions of the surface. For example, surface modification includes (1) changing the wetting properties of the surface, (2) functionalizing the surface, e.g., providing, modifying or substituting surface functional groups, (3) defunctionalizing the surface, e.g., removing surface functional groups, (4) otherwise altering the chemical composition of the surface, e.g., by etching, (5) increasing or decreasing the surface roughness, (6) providing a coating on the surface, e.g., a coating that exhibits different wetting properties than the wetting properties of the surface, and / or (7) depositing particulates on the surface. In some examples, the surface of a structure is selectively functionalized to produce 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.

[0067] 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 active materials. In some examples, the passive regions are functionalized with passive materials. 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 passive materials that do not efficiently bind biomolecules. In some examples, the surfaces functionalized with passive materials prevent binding of biomolecules at sites to which passive functionalization agents are attached. In some cases, the surfaces include an active layer that defines only discrete locations for supporting biomolecules,

[0068] In some examples, the functionalization includes deposition of 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).

[0069] Any step or component in the following functionalization process may be omitted or modified depending on the desired properties of the final functionalized substrate. In some cases, additional components and / or process steps are added to the process workflow embodied herein. In some examples, the substrate is first cleaned, for example, using piranha solution. One example of a cleaning process involves washing the substrate with piranha solution (e.g., 90% H2O at elevated temperatures (e.g., 120° C.). 2 SO 4 , 10%H 2 O 2 ), rinsing (e.g., with water), and drying the substrate (e.g., with nitrogen gas). This process optionally includes immersing the piranha-treated substrate in a basic solution (e.g., NH 4 In some examples, the surface of the structure is piranha cleaned, optionally after piranha immersion and optional post-piranha treatment. An example of a plasma cleaning process includes oxygen plasma etching. In some examples, the surface is deposited with an active functionalizing agent after deposition. In some examples, the substrate is actively functionalized prior to cleaning, such as by piranha treatment and / or plasma cleaning.

[0070] The process for surface functionalization optionally includes resist coating and resist stripping. In some examples, after active surface functionalization, the substrate is spin-coated with a resist, such as SPR™ 3612 positive photoresist. The process for surface functionalization includes, in various examples, lithography with patterned functionalization. In some examples, photolithography is performed after resist coating. In some examples, after lithography, the surface is visually inspected for lithography defects. The process for surface functionalization includes, in some examples, a cleaning step, whereby substrate residues are removed, for example by plasma cleaning or etching. In some examples, a plasma cleaning step is performed some step after the lithography step.

[0071] In some examples, the resist-coated surface is treated to remove the resist, for example, after functionalization and / or after lithography. In some cases, the resist is removed using a solvent, for example, a stripping solution containing N-methyl-2-pyrrolidone. In some cases, resist stripping includes sonication or ultrasonic treatment. In some examples, the resist is coated and stripped, followed by active functionalization of the exposed areas to create the desired differential functionalization pattern.

[0072] In some examples, the methods and compositions described herein relate to the application of photoresist to generate modified surface properties in selected regions. In some examples, the application of photoresist is dependent on the fluid properties of the surface that 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 drawing the photoresist into small structures in a controlled manner before the resist solvent evaporates. In some examples, the contact points of the resist are pinned by sharp edges, thereby controlling the progression of the fluid. The underlying structures may be designed based on the desired flow patterns that are used to apply the photoresist during the fabrication and functionalization processes. The solid organic layer that is left behind after the solvent evaporates may be used to continue the subsequent steps of the fabrication process. The structures may be designed to control the flow of the fluid by promoting or inhibiting the capillary effect to adjacent fluid paths. For example, the structures are designed to avoid overlap between the top and bottom edges, which facilitates the retention of fluid in the upper structures that allow 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 substructures. An appropriate design may therefore be selected depending on the desired application of the resist.

[0073] In some examples, the structures described herein have a surface that includes a material that includes reactive groups capable of molecular sensing and 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.

[0074] 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 relative to 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 relative to the surface. The laser device may be used in conjunction with a moveable flexible structure. In such a configuration, the coordination of laser exposure and the movement of the flexible structure is used to generate a pattern of one or more agents having different nucleoside coupling capabilities.

[0075] Described herein are surfaces for molecular sensing that are reusable. After loading of charge sensors, the surface may be bathed, 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. Surfaces prepared for reuse are reused about 1 time, about 2 times, about 3 times, about 5 times, about 10 times, about 20 times, about 50 times, about 100 times, about 1,000 times, or more in some examples. Surfaces prepared for reuse are reused at least 1 time, 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 1,000 times, or more in some examples. In some examples, the remaining "life" or number of times the surface is suitable for reuse is measured or predicted.

[0076] 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, comprise metals, non-metals, mixed metal oxides, nitrides, carbides, silicon-based materials or other materials. In some examples, the metal oxide may be TiO 2 , Ta 2 O 5 , IrO 2 , RuO 2 , RhO 2 , Nb 2 O 5 , Al 2 O 3 ,BaO,Y 2 O 3 , HfO 2 , SrO, or other metal oxides known in the art. In some examples, metal carbides include TiC, WC, ThC 2 , ThC, V.C., W. 2 C, ZrC, HfC, NbC, TaC, Ta 2 C or other metal carbides known in the art. In some examples, metal nitrides include GaN, InN, BN, Be 3 N 2 , Cr 2 N, MoN, Si 3 N 4 ,TaN,Th 2 N2 , VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitrides known in the art. In some examples, the devices disclosed herein are fabricated using combinations of the materials listed herein or 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. In 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 direct thermal evaporation at a fixed angle. In some examples, the gold is deposited using electroplating. In some examples, the thickness of the gold layer is about 10, about 25, about 30, about 40, about 50, about 60, or about 75 angstroms. In some examples, the thickness of the gold layer is 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 angstroms or less, 30 angstroms or less, 40 angstroms or less, 50 angstroms or less, 60 angstroms or less, 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.

[0077] 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 (such as a nucleic acid) is mounted on the device surface. The mounting, in some examples, includes one or more of the steps of (a) applying a voltage to the electrodes to attract a nucleic acid strand to the electrodes, (b) monitoring a current path between the electrodes to determine whether the nanowire bridges between the two electrodes, and (c) when the current spikes (e.g., touches), the voltage is turned off so that no more DNA is attracted. In some examples, the applied voltage is about 10-100V, about 10-50V, about 10-25V, about 10-75V, or about 25-100V. In some examples, the loading voltage is about 10 V, about 15 V, about 20 V, about 25 V, about 30 V, about 40 V, about 50 V, about 60 V, about 70 V, about 75 V, about 80 V, about 90 V, or about 100 V. In some examples, the loading voltage is applied for about 0.01 seconds or less, about 0.05 seconds or less, about 1 second or less, about 2 seconds or less, about 3 seconds or less, about 4 seconds or less, about 5 seconds or less, about 6 seconds or less, about 7 seconds or less, about 8 seconds or less, about 9 seconds or less, about 10 seconds or less, about 15 seconds or less, about 20 seconds or less, about 25 seconds or less, about 30 seconds or less, about 40 seconds or less, about 50 seconds or less, about 60 seconds or less, about 90 seconds or less, or about 120 seconds or less.

[0078] Provided herein is a device that includes a surface. In some examples, the surface is modified to support molecular sensing at a predetermined location. In some examples, the surface of the device for molecular sensing provided herein is made from 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 a portion of the device. The device described herein may include a flexible material. Exemplary flexible materials include, but are not limited to, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. The device 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 combinations of the materials listed herein or any other suitable materials known in the art.

[0079] 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), polydimethylsiloxane (PDMS) (3.9-10.8 MPa). The solid supports described herein can have a tensile strength 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 a solid support for molecular sensing in the form of a flexible material that is storable in a continuous loop or reel, such as a tape or flexible sheet.

[0080] Young's modulus measures the resistance of a material 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), silicon (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10 GPa), 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. Since the relationship between flexibility and stiffness is inversely proportional to each other, a flexible material has a low Young's modulus and changes its shape considerably under load. In some examples, the solid supports described herein have a surface that has at least the flexibility of nylon.

[0081] 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.

[0082] The structures may be fabricated from a variety of materials that are suitable for the methods and compositions of the invention described herein. In examples, the materials from which the substrates / solid supports comprising the invention are fabricated exhibit low levels of polynucleotide binding. In some circumstances, materials that are transparent to visible and / or UV light may be utilized. Materials that are sufficiently conductive may be utilized, for example, materials that can form a uniform electric field across all or a portion of the substrates / solid supports described herein. In some examples, such materials may be connected to electrical ground. In some cases, the substrate or solid support may be thermally conductive or insulating. Materials may be chemically and thermally resistant to support chemical or biochemical reactions. For flexible materials, materials of interest may include nylon (both modified and unmodified), nitrocellulose, polypropylene.

[0083] 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 structures may be selected from the group of materials including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass. The substrates / solid supports of the microstructures, reactors therein, may be fabricated using the materials listed herein or in combination with any other suitable materials known in the art.

[0084] In some examples, the substrates disclosed herein include computer readable materials, including, but not limited to, magnetic media, reel-to-reel tape, cartridge tape, cassette tape, floppy disk, paper media, film, microfiche, continuous tape (e.g., belt), and any medium suitable for storing electronic instructions. In some cases, the substrate includes a magnetic reel-to-reel tape or a magnetic belt. In some examples, the substrate includes a flexible printed circuit board.

[0085] 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 thermally resistant to aid in chemical reactions. In some examples, the substrate is magnetic. In some examples, the structures include 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.

[0086] Molecular Sensing

[0087] The devices, systems, and methods described herein are useful for molecular sensing. In some examples, an analyte interacts with a molecular sensor that is in electrical communication with one or more electrodes. In some examples, interaction with the analyte results in a change in voltage, current, or resistance (signal or signature pattern) that is detectable in the device. In some examples, the analyte includes a nucleotide triphosphate. In some examples, one or more nucleotide triphosphates generate unique signals (that are distinguishable). In some examples, the various nucleotide triphosphates correlate with unique base identities. 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 the device described herein. In some examples, one or more nucleotide triphosphates include a non-canonical or non-natural amino acid. In some examples, the nucleotide triphosphates include non-natural or non-canonical bases. In some examples, the non-natural or non-canonical bases are configured to generate a unique signal or signal pattern. Such a signal, in some instances, is measured between one or more electrodes.

[0088] In an exemplary configuration, the method described herein is provided in FIG. 1. In some examples, the method includes any one of the steps of FIG. 1. A bound (and / or synthesized) polynucleotide 103 from a surface 101 is cleaved 102. A primer 104 is added 105, followed by a molecular sensor (e.g., a polymerase) bound to a sensor binding moiety 115 (graphene binder shown in FIG. 1, optionally via a linker) 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, shown as a base letter and an asterisk) 111 is added, which extends the primer of the ternary complex. A current change resulting from the incorporation of 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.

[0089] The method may include one or more of the following steps: (a) providing a polymerase coupled to a solid support charge sensor; (b) contacting the polymerase with a mixture of nucleotides; (c) detecting incorporation of the nucleotides by the charge sensor; (d) repeating steps (b)-(c) using a second mixture of the polymerase, template nucleic acid, and nucleotides; and (e) comparing the first signal pattern to the second signal pattern to determine the sequence of the template nucleic acid. In some examples, the mixture in (b) includes different types of nucleotides. In some examples, the first type of nucleotides is in a distinguishable state compared to other types of nucleotides in the mixture in (b). In some examples, the second type of nucleotides is in a non-distinguishable state compared to 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, the detection of incorporation of the nucleotides 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 in a distinguishable state 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 in a distinguishable state compared to other types of nucleotides in the second mixture, thereby obtaining a second signal pattern.

[0090] Also provided is a method for sequencing a nucleic acid, comprising one or more steps of: (a) providing a polymerase coupled to a solid support charge sensor; (b) contacting the polymerase with a mixture of nucleotides; (c) detecting incorporation of the nucleotides by the charge sensor; (d) repeating steps (b)-(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) comprises different types of nucleotides. In some examples, the first two types of nucleotides are in a first distinguishable state compared to 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 by the charge sensor comprises 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 of one of the first two types of nucleotides that is in a distinguishable state compared to the other first two types of nucleotides in the second mixture, thereby obtaining a second signal pattern.

[0091] 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 in addition, one or more non-natural nucleotides used in the mixture cause a delay in nucleotide incorporation or a slower rate of incorporation. Alternatively, or in addition, one or more non-natural nucleotides used in the method cause 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, the method of 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, the method of 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, the at least one nucleotide comprises a terminator configured to prevent chain extension. In some examples, the methods described herein are used to sequence at least 10 bases, 20 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 750 bases, 800 bases, 900 bases, 1000 bases, or more than 1000 bases. In some examples, the methods described herein are used to sequence about 10 bases, about 20 bases, about 50 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 750 bases, about 800 bases, about 900 bases, about 1000 bases, about 1500 bases, or about 2000 bases.In some examples, the methods described herein are used to sequence 10-1000 bases, 20-1000 bases, 50-1000 bases, 100-1000 bases, 50-2000 bases, 25-500 bases, 25-200 bases, 50-500 bases, or 50-750 bases. In some examples, the nucleotides comprise charge-modulated nucleotides.

[0092] The 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 characteristic unique to 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 nucleotide triphosphate types, 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 (e.g., a "high" abundance nucleotide). As a result, incorporation of the low abundance nucleotide is detectable as a relative delay or decrease in 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, some sequencing runs are completed on the same template, with each run being performed using a different nucleotide in the low state. In some examples, the signal patterns of the different runs are compared to determine the sequence of the template. In some examples, the method includes the use of a 3 high-1 low mixture of nucleotide triphosphates. Other ratios of high to low are also described herein, as are mixtures including, for example, a 1 high-3 low mixture, or a 2 high-2 low mixture. 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 repeated sequencing, since 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 that utilize a circular template, the polymerase contains a 5' exonuclease activity to digest the different nucleic acid strands that displace the circular template as the polymerase advances multiple times around the template. Whether the template is linear or circular, different primers are used in some examples for different sequencing runs performed on the same template. Different primers are designed in some examples to hybridize at different positions on the template. Thus, while each run by itself starts at a different position on the template, in some examples there is substantial overlap between the portions of the template that are 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 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 examples, each nucleotide triphosphate produces a distinguishable state. In some examples, the distinguishable state refers to a particular type of nucleotide triphosphate that has a unique characteristic or property that is uniquely exhibited under detection conditions compared to other nucleotide triphosphates. Exemplary distinguishable states include, but are not limited to, a state that 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 that 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 that has a chemical moiety or modification that is not present in other types of nucleotide triphosphates in the mixture, or a state that lacks a chemical moiety or modification that is present in other types of nucleotide triphosphates in the mixture. The distinguishable state may be exhibited when the nucleotide type interacts with the polymerase. In some examples, the signal is detected from a structural change, such as the appearance, disappearance, or change in a detectable signal from the molecule in response to a change in the structure, shape, or configuration of the molecular moiety.For example, a signal change can result from a change in the interaction of a label with a first portion of a molecule as it interacts with a second portion of the molecule.

[0093] The detectable signal may comprise a change in current, resistance or voltage, hi some instances, the detectable signal comprises a change in current. In some examples, the current change is between 1 nanoamp and 100 picoamp, between 1 nanoamp and 50 picoamp, between 1 nanoamp and 25 picoamp, between 1 nanoamp and 10 picoamp, between 1 nanoamp and 1 picoamp, between 1 nanoamp and 500 nanoamp, between 1 nanoamp and 250 nanoamp, between 1 nanoamp and 100 nanoamp, between 100 nanoamp and 100 picoamp, between 100 nanoamp and 1 picoamp, between 100 nanoamp and 500 nanoamp, between 500 nanoamp and 100 picoamp, between 500 nanoamp and 50 picoamp, between 500 nanoamp and 10 picoamp, between 1 picoamp and 100 picoamp, between 1 picoamp and 100 picoamp, between 250 nanoamp and 750 nanoamp. In some examples, the detectable signal is measured as a change in signal relative to a background signal (e.g., absence of analyte). In some examples, the current change 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.

[0094] The methods described herein may allow for rapid analysis of analytes. In some examples, 1-200 biomolecules, 1-500 biomolecules, 1-300 biomolecules, 1-150 biomolecules, 1-100 biomolecules, 10-500 biomolecules, 10-300 biomolecules, 50-300 biomolecules, 50-200 biomolecules, 100-200 biomolecules, or 150-400 biomolecules are analyzed per second. In some examples, at least 25 biomolecules, 50 biomolecules, 75 biomolecules, 100 biomolecules, 125 biomolecules, 150 biomolecules, 175 biomolecules, 200 biomolecules, 225 biomolecules, 250 biomolecules, 275 biomolecules, 300 biomolecules, 350 biomolecules, 400 biomolecules, 450 biomolecules, 500 biomolecules, or at least 600 biomolecules are analyzed per second. In some examples, the biomolecules include nucleotides or variants thereof. In some instances, the biomolecule comprises a charge-modulated nucleotide (CMN).

[0095] The analyte may include a nucleotide. In some examples, the analyte includes a charge-modulated nucleotide. In some examples, the modification includes a modification to a 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 includes a deaza or halogen modified base. In some examples, the modification includes a 7-deaza or 8-bromo modified base. In some examples, the CMN includes a modification to the 5' position. In some examples, the modification includes a modification to a 5' polyphosphate or a chemical variant thereof. In some examples, the modification includes a thiolated or brominated phosphate. In some examples, the polyphosphate includes at least 3, 4, 5, 6, 8, or 10 phosphates or variants thereof. In some examples, the modification includes a modification to a 5' terminal polyphosphate or a chemical variant thereof. In some examples, the modification includes a polymer. In some examples, the polymer includes 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 bases, 10 bases, 15 bases, 20 bases, 25 bases, 30 bases, 40 bases, 50 bases, 60 bases, 75 bases, 100 bases, 125 bases, 150 bases, 175 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 2000 bases, 2500 bases, 3000 bases or at least 5000 bases. In some examples, the nucleic acid strand comprises 5 bases or less, 10 bases or less, 15 bases or less, 20 bases or less, 25 bases or less, 30 bases or less, 40 bases or less, 50 bases or less, 60 bases or less, 75 bases or less, 100 bases or less, 125 bases or less, 150 bases or less, 175 bases or less, 200 bases or less, 300 bases or less, 400 bases or less, 500 bases or less, 600 bases or less, 700 bases or less, 800 bases or less, 900 bases or less, 1000 bases or less, 2000 bases or less, 2500 bases or less, 3000 bases or less, or 5000 bases or less.In some examples, the nucleic acid strand comprises about 5 bases, about 10 bases, about 15 bases, about 20 bases, about 25 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 75 bases, about 100 bases, about 125 bases, about 150 bases, about 175 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1000 bases, about 2000 bases, about 2500 bases, about 3000 bases or about 5000 bases. In some examples, the nucleic acid strand comprises 25-300 bases, 25-500 bases, 25-400 bases, 25-300 bases, 25-250 bases, 50-500 bases, 50-300 bases, 75-300 bases, 75-250 bases, 75-200 bases, 100-500 bases, 125-500 bases, 200-500 bases, 300-500 bases, 10-250 bases, 25-5000 bases, 50-5000 bases, 100-5000 bases, 200-5000 bases, 500-5000 bases, 1000-5000 bases, 2000-5000 bases, or 10-5000 bases.

[0096] 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 the 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 the CMN relative to an unmodified nucleotide. In some examples, the at least one charge-modulating chemical modification comprises one or more of a phosphate, a phosphite, a sulfonate, a sulfite, a carboxylate, a xanthate, a thiocarboxylate, a boranophosphonate, and a 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, a DNA-RNA, or a DNA-PNA hybrid.

[0097] The nucleic acid chain may include a phosphate modification. In some examples, the phosphate modification includes a hydrophobic group. In some examples, the hydrophobic group includes a straight or branched alkyl chain. In some examples, the hydrophobic group includes a C 5 -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 1000-100,000 daltons, 100-500,000 daltons, 100-250,000 daltons, 100-75,000 daltons, 100-50,000 daltons, 100-25,000 daltons, 100-10,000 daltons, 100-7500 daltons, 100-5000 daltons, 100-3000 daltons, or 100-2000 daltons. In some examples, the polyethylene glycol comprises a molecular weight of about 100 daltons, about 200 daltons, about 300 daltons, about 500 daltons, about 1000 daltons, about 2000 daltons, about 2500 daltons, about 3000 daltons, about 5000 daltons, about 7500 daltons, about 10,000 daltons, about 25,000 daltons, about 50,000 daltons, about 75,000 daltons, about 100,000 daltons, about 250,000 daltons or about 50,000 daltons. In some examples, the polyethylene glycol comprises a molecular weight of at least about 100 daltons, at least about 200 daltons, at least about 300 daltons, at least about 500 daltons, at least about 1000 daltons, at least about 2000 daltons, at least about 2500 daltons, at least about 3000 daltons, at least about 5000 daltons, at least about 7500 daltons, at least about 10,000 daltons, at least about 25,000 daltons, at least about 50,000 daltons, at least about 75,000 daltons, at least about 100,000 daltons, at least about 250,000 daltons or at least about 50,000 daltons.In some examples, the polyethylene glycol comprises a molecular weight of up to about 100 daltons, up to about 200 daltons, up to about 300 daltons, up to about 500 daltons, up to about 1000 daltons, up to about 2000 daltons, up to about 2500 daltons, up to about 3000 daltons, up to about 5000 daltons, up to about 7500 daltons, up to about 10,000 daltons, up to about 25,000 daltons, up to about 50,000 daltons, up to about 75,000 daltons, up to about 100,000 daltons, up to about 250,000 daltons, or up to about 50,000 daltons. In some examples, the polyethylene glycol comprises 10-600 monomers. In some examples, the polyethylene glycol comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, or about 600 monomers. In some examples, the polyethylene glycol comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 monomers. In some examples, the polyethylene glycol comprises up to about 10, up to about 20, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 200, up to about 300, up to about 400, up to about 500, or up to about 600 monomers. In some examples, the peptide chain is 1-100 amino acids long. In some examples, the peptide chain is about 1 amino acid long, about 2 amino acids long, about 3 amino acids long, about 4 amino acids long, about 5 amino acids long, about 10 amino acids long, about 20 amino acids long, about 30 amino acids long, about 40 amino acids long, about 50 amino acids long, about 60 amino acids long, about 70 amino acids long, about 80 amino acids long, about 90 amino acids long, or about 100 amino acids long.In some examples, the peptide chain is at least about 1 amino acid long, at least about 2 amino acids long, at least about 3 amino acids long, at least about 4 amino acids long, at least about 5 amino acids long, at least about 10 amino acids long, at least about 20 amino acids long, at least about 30 amino acids long, at least about 40 amino acids long, at least about 50 amino acids long, at least about 60 amino acids long, at least about 70 amino acids long, at least about 80 amino acids long, at least about 90 amino acids long, or at least about 100 amino acids long. In some examples, the peptide chain is up to about 1 amino acid long, up to about 2 amino acids long, up to about 3 amino acids long, up to about 4 amino acids long, up to about 5 amino acids long, up to about 10 amino acids long, up to about 20 amino acids long, up to about 30 amino acids long, up to about 40 amino acids long, up to about 50 amino acids long, up to about 60 amino acids long, up to about 70 amino acids long, up to about 80 amino acids long, up to about 90 amino acids long, or up to about 100 amino acids long. 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, hi some examples, the metal complex comprises ferrocene, Ru-dipy, and bis-cyclopentadienyl diiron.

[0098] The molecular sensor 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. The molecule is in some examples a polymerase, reverse transcriptase, exonuclease, or other nucleic acid enzyme. The portion of the molecule may be an atom that changes relative position, for example, by rotation of about one or more chemical bonds that occur in the molecular structure between the atoms. The portion of the molecule may be a domain of a polymer, such as a polymer commonly known in the relevant art. In some examples, a polymerase includes domains referred to as finger, palm, and thumb domains. In the case of a protein, the portion may be a region of secondary, tertiary, or quaternary structure. One or more labels may be attached to the molecule, for example, via a covalent bond. However, 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 a nucleic acid. In some examples, the molecular sensor includes a polymerase.

[0099] Methods for sequencing nucleic acids are provided herein. In some examples, the methods include one or more of: contacting a plurality of polynucleotides with at least one primer and at least one polymerase to form a plurality of ternary complexes, the ternary complexes comprising a graphene binder; detecting one or more bases of the polynucleotides, the detection occurring 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, about 50,000, about 100,000, about 250,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 750,000, about 800,000, about 900,000, about 1 million, about 10 million, about 100 million, about 200 million, about 500 million, or at least 750 million unique nucleotides. In some examples, the multiple polynucleotides are 50-30,000 bases in length, 50-10,000 bases in length, 50-1000 bases in length, 50-750 bases in length, 50-500 bases in length, 50-400 bases in length, 50-300 bases in length, 50-200 bases in length, 1000-30,000 bases in length, 1000-20,000 bases in length, 1000-10,000 bases in length, 2000-5000 bases in length, 2000-10,000 bases in length, 5000-30,000 bases in length, or 10,000-30,000 bases in length.In some examples, the plurality of polynucleotides is about 10 bases in length, about 50 bases in length, about 100 bases in length, about 200 bases in length, about 300 bases in length, about 400 bases in length, about 500 bases in length, about 750 bases in length, about 1000 bases in length, about 2000 bases in length, about 2500 bases in length, about 5000 bases in length, about 10,000 bases in length, about 20,000 bases in length, or about 30,000 bases in length.

[0100] Nucleic acid-based information

[0101] Devices, compositions, systems and methods for reading nucleic acid-based information (data) are provided herein. In a first step, a digital sequence is received that codes for an item of information (e.g., digital information in binary code for processing by a computer). 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., configuration spots) and reagents for nucleic acid synthesis are selected. The surface of the structure is prepared for nucleic acid synthesis. De novo polynucleotide synthesis is performed in some examples. The synthesized polynucleotides are stored and available in whole or in part for subsequent release. In some examples, a pool of predefined polynucleotides is assembled into a larger polynucleotide that represents the 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 sequence is 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.

[0102] 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 to correct 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 a location or address of the digital information encoded by the nucleic acid. In some examples, the index sequence defines a source of the digital information. The nucleic acid encoding the digital information, in some examples, includes overlaps 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.

[0103] The nucleic acids encoding the digital information may be stored in different media. In some examples, the nucleic acids are stored essentially as dry or lyophilized powders. In some examples, the nucleic acids are stored in a buffer. In some examples, the nucleic acids are stored on a chip, wafer or other silicon solid support. In some examples, the nucleic acids are stored within an organism (or population of organisms), such as a plasmid or genome.

[0104] Optionally, the initial stage of the data storage process disclosed herein includes obtaining 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, e-mails, recorded phone logs, Internet activity logs, drawings, paintings, prints, 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 encoded for an item of information in a digital format, or the amount of multiple files encoded for an item of information, can 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 gigabyte, 2 gigabytes, 3 gigabytes, 4 gigabytes, 5 gigabytes, 6 gigabytes, 7 gigabytes, 8 gigabytes, 9 gigabytes, 10 gigabytes, 20 gigabytes, 50 gigabytes, 100 gigabytes, 200 gigabytes, 300 gigabytes, 400 gigabytes, 500 gigabytes, 600 gigabytes, 700 gigabytes, 800 gigabytes, 900 gigabytes, 1000 gigabytes, 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 terabyte, 2 terabytes, 3 terabytes, 4 terabytes, 5 terabytes, 6 terabytes, 7 terabytes, 8 terabytes, 9 terabytes, 10 terabytes, 20 terabytes, 50 terabytes, 100 terabytes, 200 terabytes, 300 terabytes, 400 terabytes, 500 terabytes, 600 terabytes, 700 terabytes, 800 terabytes, 900 terabytes, 1000 terabytes, 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 petabyte, 2 petabytes, 3 petabytes, 4 petabytes, 5 petabytes, 6 petabytes, 7 petabytes, 8 petabytes, 9 petabytes, 10 petabytes, 20 petabytes, 50 petabytes, 100 petabytes, 200 petabytes, 300 petabytes, 400 petabytes, 500 petabytes, 600 petabytes, 700 petabytes, 800 petabytes, 900 petabytes, 1000 petabytes, or more than 1000 petabytes.

[0105] The solid support for molecular sensing as described herein has a high capacity for reading data. For example, the capacity of the solid support is at least 1 petabyte, 2 petabytes, 3 petabytes, 4 petabytes, 5 petabytes, 6 petabytes, 7 petabytes, 8 petabytes, 9 petabytes, 10 petabytes, 20 petabytes, 50 petabytes, 100 petabytes, 200 petabytes, 300 petabytes, 400 petabytes, 500 petabytes, 600 petabytes, 700 petabytes, 800 petabytes, 900 petabytes, 1000 petabytes, or more than 1000 petabytes, or About 1 petabyte, about 2 petabytes, about 3 petabytes, about 4 petabytes, about 5 petabytes, about 6 petabytes, about 7 petabytes, about 8 petabytes, about 9 petabytes, about 10 petabytes, about 20 petabytes, about 50 petabytes, about 100 petabytes, about 200 petabytes, about 300 petabytes, about 400 petabytes, about 500 petabytes, about 600 petabytes, about 700 petabytes, about 800 petabytes, about 900 petabytes, about 1000 petabytes, or more than about 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 comprises at least 1 gigabyte, 2 gigabytes, 3 gigabytes, 4 gigabytes, 5 gigabytes, 6 gigabytes, 7 gigabytes, 8 gigabytes, 9 gigabytes, 10 gigabytes, 20 gigabytes, 50 gigabytes, 100 gigabytes, 200 gigabytes or more, or about 1 gigabyte, about 2 gigabytes, about 3 gigabytes, about 4 gigabytes, about 5 gigabytes, about 6 gigabytes, about 7 gigabytes, about 8 gigabytes, about 9 gigabytes, about 10 gigabytes, about 20 gigabytes, about 50 gigabytes, about 100 gigabytes, about 200 gigabytes, or more than about 200 gigabytes of data.In some examples, the addressable array includes at least 1 terabyte, 2 terabytes, 3 terabytes, 4 terabytes, 5 terabytes, 6 terabytes, 7 terabytes, 8 terabytes, 9 terabytes, 10 terabytes, 20 terabytes, 50 terabytes, 100 terabytes, 200 terabytes or more, or about 1 terabyte, about 2 terabytes, about 3 terabytes, about 4 terabytes, about 5 terabytes, about 6 terabytes, about 7 terabytes, about 8 terabytes, about 9 terabytes, about 10 terabytes, about 20 terabytes, about 50 terabytes, about 100 terabytes, about 200 terabytes, or more than about 200 terabytes of data. In some examples, the item of information is stored in the background of the data. For example, the item of information encodes about 10 to about 100 megabytes of data and is stored in the background of 1 petabyte of data. In some examples, an item of information may be at least 1 megabyte, 10 megabytes, 20 megabytes, 30 megabytes, 40 megabytes, 50 megabytes, 60 megabytes, 70 megabytes, 80 megabytes, 90 megabytes, 100 megabytes, 150 megabytes, 200 megabytes, 300 megabytes, 400 megabytes, 500 megabytes, or more, or about 1 megabyte, about 10 megabytes, about 20 megabytes, about 30 megabytes, about 40 megabytes, about 50 megabytes, about 60 megabytes, about 70 megabytes, about 80 megabytes, about 90 megabytes, about 100 megabytes, or more. or encodes more than 1 megabyte, approximately 150 megabytes, approximately 200 megabytes, approximately 300 megabytes, approximately 400 megabytes, approximately 500 megabytes, or more than 500 petabytes of data and is stored in background data of 1 petabyte, 10 petabytes, 20 petabytes, 30 petabytes, 40 petabytes, 50 petabytes, 60 petabytes, 70 petabytes, 80 petabytes, 90 petabytes, 100 petabytes, 150 petabytes, 200 petabytes, 300 petabytes, 400 petabytes, 500 petabytes, or more than 500 petabytes.

[0106] Computer Systems

[0107] In various embodiments, any of the systems described herein are operably connected to a computer and optionally automated via the computer, either locally or remotely. In various examples, the methods and systems of the present invention further include software programs on the computer system and their use. Thus, computer control of the movement of the material deposition device, synchronization of dispense / vacuum / refill functions, such as coordination and synchronization of dispense and vacuum operations, etc., is within the scope of the present invention. In some examples, the computer system is programmed to interface between a user-specified base sequence and the location of the material deposition device to deliver precise reagents to specific areas of the 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 that includes an error-correcting code (e.g., Reed-Solomon (RS) code, low-density parity-check (LDPC) code, turbo code, etc.). In some examples, the computer system executes a program to convert data to a plurality of nucleic acid sequences, convert a plurality of nucleic acid sequences to 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).

[0108] The 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) to a nucleoside monomer added to a polynucleotide. In some cases, an algorithm including ML may be trained on training data to associate a signal (e.g., current / voltage) to 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 machine (SVM), or combinations thereof).

[0109] 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, an 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 the nodes may further include weights to adjust the input data to a given node (e.g., to activate the input data or to 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 variations thereof (e.g., adaptive moment estimation (Adam)). In further examples, the gradients 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.

[0110] In some cases, the 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, the neural network may include one or more convolutional layers. The inputs and outputs may be tensors that represent variables or attributes in a dataset (e.g., features) and 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, the 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.

[0111] The layers in the neural network may further include one or more pooling layers before or after the convolutional layer. The one or more pooling layers may reduce the dimensionality of the feature map using a filter that summarizes the 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 the region of the matrix, which helps to 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 inputs that pass onto the next layer. In some cases, the inputs may be flattened by reducing them 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 the nucleoside monomer added to the 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). The one or more dropout layers may randomly set certain weights as 0, which may set corresponding elements in the feature map as 0, so that the neural network may avoid overfitting. The neural network may further include one or more dense layers, which include a fully connected network. In the dense layer, 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, the errors may be backpropagated to improve the prediction. The 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.

[0112] The computer system 700 shown in FIG. 7 may be understood as a logical device that can read instructions from a medium 711 and / or a network port 705, which can be optionally connected to a server 709 having a fixed medium 712. The system can 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 can be accomplished through a communication medium directed to a server at a local or remote location. The communication medium can include any means of transmitting data and / or receiving data. For example, the communication medium can be a network connection, a wireless connection, or an Internet connection. Such a connection can be provided for communication via the World Wide Web. It is anticipated that data related to the present disclosure can be transmitted over such a network or connection for receipt and / or review by the parties 722.

[0113] FIG. 8 is a block diagram illustrating a first exemplary architecture of a computer system that may be connected in connection with an exemplary instance of the present invention. As shown in FIG. 8, the exemplary computer system may include a processor 802 for processing instructions. Non-limiting examples of processors include Intel Xeon™ processors, AMD Opteron™ processors, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processors, ARM Cortex-A8 Samsung S5PC100™ processors, ARM Cortex-A8 Apple A4™ processors, Marvell PXA 930™ processors. Multiple execution threads may be used for parallel processing. In some examples, multiple processors or processors with multiple cores may also be used, whether in a single computer system, in a cluster or distributed across a system over a network 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 north bridge 806 by a processor bus 808. The north bridge 806 is connected to a random access memory (RAM) 810 by a memory bus 812 and manages access to the RAM 810 by the processor 802. The north bridge 806 is also connected to a south bridge 814 by a chipset bus 816. Similarly, the south bridge 814 is connected to a peripheral bus 818. The peripheral bus may be, for example, a PCI, PCI-X, PCI Express or other peripheral bus. The north bridge and south bridge are often referred to as the processor chipset and manage data transfers between the processor, RAM and peripheral components on the peripheral bus 818.In some alternative architectures, the functionality of the northbridge 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 operations. For example, the accelerator may be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

[0114] Software and data can be stored in external storage 824 and loaded into RAM 810 and / or cache 804 for use by the processor. 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. System 800 also includes application software running on top of the operating system for managing data storage and optimization in accordance with an exemplary embodiment of the present invention. In this example, 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 hard disks (NAS) and other computer systems that can be used for distributed parallel processing.

[0115] FIG. 9 illustrates a network 900 with multiple computer systems 902a and 902b, multiple mobile phones and personal digital assistants 902c, and network attached hard disks (NAS) 904a and 904b. In an exemplary embodiment, systems 902a, 902b, and 902c can manage data storage and optimize data access for data stored on network attached hard disks (NAS) 904a and 904b. Mathematical models can be used on the data and evaluated using parallel processing distributed across computer systems 902a and 902b and mobile phone and personal digital assistant systems 902c. Computer systems 902a and 902b and mobile phone and personal digital assistant systems 902c can also provide parallel processing for adaptive data reconstruction of data stored on network attached hard disks (NAS) 904a and 904b. FIG. 9 illustrates 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. The 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).

[0116] In some exemplary embodiments, the 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 of the 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 in the memory subsystem 1004. Each MAP 1006a-f may include a 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, the MAPs may be used to evaluate algebraic expressions related to a data model and to implement adaptive data restructuring in an exemplary embodiment. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access the 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 execution in parallel.

[0117] 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, co-processors, 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 part 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 hard disks (NAS) and other local or distributed data storage devices and systems.

[0118] 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.

[0119] 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. EXAMPLES

[0120] Example 1: Fabrication of a graphene device

[0121] Any array of addressable devices as shown in FIG. 2 can be fabricated using the general method of graphene FET fabrication described in U.S. Pat. No. 9,859,394, the entirety of which is incorporated by reference, in any order, with dimensions ranging from 4 to 16 mm. 2 The chips are constructed with a pitch distance of 50 to 1000 nm.

[0122] Example 2: Sequencing using graphene devices

[0123] 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 that is bound to the graphene surface of the device, and a mixture of four nucleotides is contacted with the device and the template. The 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.

[0124] Example 3: Fabrication of a graphene device with buried gate and shield

[0125] Any array of addressable devices as 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. 2The chips are constructed with a pitch distance of 50 to 1000 nm.

[0126] Example 4: Sequencing using graphene devices with buried gates and shields

[0127] The device of Example 3 is used to sequence nucleic acids using a modified general method of Example 2. During loading of the ternary complex, a positive voltage is applied to the recessed gate. This attracts the negatively charged DNA (or other similar moieties) towards the surface and the graphene. A shielding layer at ground potential is configured with a small opening that allows the positive potential from the gate to leak out to a specific location on the chip surface. The localization of the potential causes the DNA (or similar moieties) to concentrate at the desired location on the graphene. This facilitates higher loading of the device. During sensor operation, the gate can be used to tune the graphene potential to maximize signal changes associated with molecular events occurring on or near the graphene layer.

[0128] Example 5: Fabrication of Edge Finger Devices

[0129] 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 produce a second electrode, c) patterning the second electrode, d) optionally planarizing, e) depositing material to produce a passivation layer, f) depositing material to produce a first electrode, g) patterning the first electrode, and h) isotropically etching the passivation layer, thereby undercutting the edge of the first electrode and the device comprising a nanogap of about 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 comprising nucleic acids, at least a portion of the nanowires bridging one or more of the first and second electrodes. Each nanowire includes at least one biotin functional handle (optionally connected via a tether). Nanowire loading involves (a) applying a voltage to the electrodes to attract the nucleic acid strand to the electrodes, (b) monitoring the current path between the electrodes to determine if the nanowire bridges the two electrodes, and (c) turning off the voltage when the current spikes (e.g., touches) so that no more DNA is attracted. The nanowire is then contacted with a polymerase bound to streptavidin (e.g., similar to FIG. 11) to facilitate the binding of the polymerase to the nanowire.

[0130] Example 6: Cross Finger Device

[0131] 13A-13B are fabricated according to the general procedure or Example 5. The devices are arranged according to the general configuration of FIG.

[0132] Example 7: Cross-finger device with oxide layer

[0133] Following the general procedure or Example 5, fabricate an array of the devices of Figures 14A-14B.

[0134] Example 8: Self-Aligned Finger Etching

[0135] Following the general procedure or Example 5, fabricate an array of the devices of Figures 16A-16B.

[0136] Example 9: Sequencing

[0137] The device of any of Examples 5 to 8 is used to sequence a nucleic acid. 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. 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.

[0138] 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 used in carrying out 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 are covered thereby.

Claims

1. 1. A method for single molecule sensing, comprising: forming a ternary complex by contacting a molecular sensor with at least one charge-modulating element, wherein contacting the molecular sensor with the at least one charge-modulating element produces a change in current; measuring the change in current; correlating said change in current with the presence or absence of said at least one charge-modulating element, thereby detecting said single molecule.

2. The method of claim 1, wherein the ternary complex comprises a graphene binding portion configured to attach the ternary complex to a charge sensor, and the charge sensor comprises a graphene layer.

3. The method described in claim 2, wherein the ternary complex is attached to the charge sensor via the molecular sensor and / or the at least one charge modulation element.

4. The method of claim 2, wherein the graphene layer is 1 to 5 atoms thick.

5. The method of claim 1, further comprising removing the ternary complex after detecting the single molecule.

6. The method of claim 4, wherein removing the ternary complex comprises washing with a solvent, the solvent comprising MeCN, methanol, ethanol, 2-propanol, acetone, DMF, formamide, THF, or DMSO.

7. The method of claim 4, wherein removing the ternary complex comprises washing with a solvent, the solvent being heated.

8. The method of claim 2, wherein the charge sensor comprises a graphene-enabled field effect transistor (GeFET) device.

9. The method of claim 2, wherein the charge sensor comprises a CMOS device.

10. The method of claim 1, wherein the molecule comprises a nucleic acid.

11. The method of claim 1, wherein the molecular sensor comprises a polymerase.

12. The method of claim 1, wherein the molecular sensor is conformationally labeled, and the conformational label comprises a label that responds to changes in the structure of a molecule, changes in the shape of a molecule, or changes in the configuration of a portion of a molecule.

13. The method of claim 1, wherein the at least one charge-modulating element comprises a charge-modulated nucleotide (CMN), the CMN comprising at least one modification to a canonical nucleotide.

14. The method of claim 13, wherein the at least one modification comprises a nucleic acid strand comprising 10 to 5000 bases.

15. The method of claim 13, wherein the at least one modification comprises a nucleic acid strand comprising a secondary structure, the secondary structure comprising one or more of a hairpin, a loop, a helix, a G-quadruplex, and an I-motif.

16. The method of claim 13, wherein the at least one modification comprises a nucleic acid strand comprising at least one charge-modulating chemical modification, the at least one charge-modulating chemical modification comprising one or more of an amine, an alkylamine, a guanidinium, a quaternary amine, an imidazolium, a pyridinium, and a pyrrolidinium.

17. The method of claim 1, wherein the ternary complex further comprises a polynucleotide primer, and contacting the molecular sensor with the at least one charge-modulating element comprises incorporating a charge-modulated nucleotide (CMN) into the polynucleotide primer.

18. The method of claim 1, wherein the current change is between 100 and 1000 picoamperes.

19. The method of claim 1, wherein the current change is at least 1.01 to 3 times the background current.

20. The method of claim 1, wherein 1 to 200 single molecules are detected per second.

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