Methods and devices for molecular characterization

Abstract

Disclosed herein include sensors, sensor devices, and related systems and methods of using the same to characterize a biomolecule, as well as methods for making devices that can be used in the methods and systems provided herein. The sensor described herein can comprise at least two closely spaced electrode sheets separated by at least one insulating layer. The sensor can further comprise a sensor edge capable of interacting with a molecule, thereby modulating a tunneling current between the two electrode sheets. In some embodiments, the sensors and devices described herein can be used for nucleic acid sequencing.

Description

METHODS AND DEVICES FOR MOLECULAR CHARACTERIZATIONBACKGROUNDField

[0001] The present disclosure relates generally to the fields of biology and chemistry, and in particular molecular analysis.Description of the Related Art

[0002] A number of conventional techniques are available for characterizing biomolecules, such as nanopore sequencing, which is used to analyze biological molecules by observing a signal related to molecular passage through the nanopore. Such devices and methods however suffer from signal noise, insufficient translocation speed control, and other shortcomings. There exists a need for developing systems and methods capable of exerting precise control over biomolecule translocation speed and direction and exhibiting sufficient specificity of measurement.SUMMARY

[0003] Disclosed herein include sensors for characterizing a stranded molecule. In some embodiments, the sensor comprises: at least one first electrode sheet, at least one second electrode sheet, and at least one first insulating layer disposed between the at least one first electrode sheet and the at least one second electrode sheet, wherein the sensor comprises a sensor edge capable of interacting with a stranded molecule, thereby modulating a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet.

[0004] In some embodiments, the at least one first electrode sheet and / or the at least one second electrode sheet is atomically thin. In some embodiments, the at least one first electrode sheet and / or the at least one second electrode sheet comprises a two- dimensional electrically conductive material. In some embodiments, the two-dimensional electrically conductive material comprises gold, titanium nitride (TiN), poly (3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), molybdenum disulfide(M0S2), copper, platinum, nickel, or a combination thereof. In some embodiments, the at least one first electrode sheet and the at least one second electrode sheet comprise a same electrically conductive material or different electrically conductive materials. In some embodiments, the at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer have adjacent exposed edge portions which collectively form the sensor edge. In some embodiments, the at least one first electrode sheet comprises one, two, three, four, or five sheets of electrodes, the at least one second electrode sheet comprises one, two, three, four, or five sheets of electrodes, or both. In some embodiments, the at least one first electrode sheet and / or the at least one second electrode sheet comprises graphene. In some embodiments, the at least one first electrode sheet comprises graphene and the at least one second electrode sheet does not comprise graphene.

[0005] In some embodiments, the at least one first insulating layer comprises one, two, three, four, or five first insulating layers. In some embodiments, the at least one insulting layer comprises ceramics, diamond, two-dimensional polymers, self-assembled monolayers or bilayers, and / or other electrically insulating organic and inorganic moieties. In some embodiments, the ceramics comprises SiaN4, AI2O3, HfCh. or a combination thereof. In some embodiments, the at least one first insulating layer comprises one or more atomically thin sheets. In some embodiments, the at least one first insulating layer comprises boron nitride. In some embodiments, the at least one first insulating layer comprises hexagonal boron nitride (hBN). In some embodiments, the at least one first insulating layer comprises three sheets of boron nitride or hBN. In some embodiments, the at least one first electrode sheet and the at least one second electrode sheet each comprises a single graphene sheet, and the at least one first insulating layer comprises three sheets of hBN.

[0006] In some embodiments, the sensor edge is configured to partially or fully surround the stranded molecule. In some embodiments, the sensor edge is in a closed-loop geometry or a linear geometry. In some embodiments, the sensor edge is in a linear geometry and has a width of about 5-10 nm. In some embodiments, the sensor edge is in a closed-loop geometry having a cross-sectional dimension of about 2-3 nm. In some embodiments, the sensor edge is functionalized with one or more chemical group(s). In some embodiments, the functionalization comprises hydrogenation, hydroxylation, carboxylation, carbonylation, amination, or a combination thereof. In some embodiments, the sensor edge is configured tominimize the tunneling electrical resistance between the sensor edge and the stranded molecule, e.g., configured with a charge-injecting group. In some embodiments, the sensor edge is configured to enhance the tunneling electrical interaction between the sensor edge and the stranded molecule. In some embodiments, the configuration enhances binding with the stranded molecule through hydrogen bond interaction, electrostatic interaction, hydrophobic / intercalating interaction, biomolecular binding, or a combination thereof.

[0007] In some embodiments, the sensor edge comprises a functional group selected from the group consisting of: carbonyl, carboxyl, hydroxyl, aldehyde, carboxylate, ester, or amine functional group, or a combination thereof. In some embodiments, the sensor edge is functionalized with a p-phenylenediamine moiety. In some embodiments, the edge portions of the at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer are functionalized with a same functional group or different functional groups. In some embodiments, a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet is capable of being measured when the stranded molecule is adhered to the sensor edge and a voltage is applied across the at least one first electrode sheet and the at least one second electrode sheet. In some embodiments, the sensor comprises: at least one second insulating layer located on a surface of the at least one first electrode sheet or the at least one second electrode sheet, and at least one third electrode sheet located on a surface of the at least one second insulating layers. In some embodiments, the at least one first electrode sheet and the at least one second electrode sheet comprise graphene and the at least one third electrode sheet does not comprise graphene.

[0008] Disclosed herein include sensor devices for characterizing a stranded molecule. In some embodiments, the sensor device comprises: a fluidic passage disposed within a support structure (e.g., a support structure comprising a semiconductor material), two or more global electrodes capable of providing a voltage to translocate the stranded molecule through the fluidic passage, at least one sensor provided herein disposed in the support structure with the sensor edge exposed to the fluidic passage; and a detector configured to detect a current between the at least one first electrode sheet and the at least one second electrode sheet as the stranded molecule is adhered to the sensor edge.

[0009] In some embodiments, the fluidic passage comprises a vertical fluidic passage section and the at least one sensor is disposed in the support structure with the sensor edge exposed to the vertical fluidic passage section. In some embodiments, the sensor edge is in a closed-shape geometry or a linear geometry forming one or more apertures in the support structure. In some embodiments, the fluidic passage comprises a lateral fluidic passage section in fluidic connection with the vertical fluidic passage section and wherein the at least one sensor is disposed in a bottom of the lateral fluidic passage section with the sensor edge exposed to the vertical fluidic passage section. In some embodiments, the lateral fluidic passage section is about 50-500 nm in width, 1-15 mm in length, and / or 1-100 nm in height. In some embodiments, the lateral fluidic passage section has an aspect ratio of height to length in a range from about 1: 10 to about 1: 1000, e.g., from about 1:50 to about 1:500. In some embodiments, the lateral fluidic passage section has a cross-sectional area of about tens to hundreds of nm2. In some embodiments, the lateral fluidic passage section and / or the vertical fluidic passage section has a dimension configured to accommodate no more than one stranded molecule.

[0010] In some embodiments, the lateral fluidic passage section has a wedge shape. In some embodiments, an opening of the lateral fluidic passage section to the vertical fluidic passage section is about 5-10 nm in width. In some embodiments, the lateral fluidic passage section and / or the vertical fluidic passage section is in nanometer or micrometer scale. In some embodiments, the lateral fluidic passage section and / or the vertical fluidic passage section has a chemically modified inner surface. In some embodiments, the inner surface is chemically modified to carry a charge. In some embodiments, the inner surface comprises glass, a self-assembled monolayer, an organic molecule, aluminum oxide, a lipid bilayer, or a combination thereof. In some embodiments, the lateral and / or vertical fluidic passage section is filled with hydrogel. In some embodiments, the hydrogel is naturally occurring or synthetic. In some embodiments, the hydrogel is agarose, polyacrylamide, PVA, or a combination thereof. In some embodiments, the lateral and / or vertical fluidic passage section comprises nanoparticle matrices. In some embodiments, the sensor device is a sensor chip. In some embodiments, the sensor device comprises: at least two, three, four, or five sensors. In some embodiments, each sensor is exposed to a distinct fluidic passage. In some embodiments, the sensor device comprises: a loading well and a fluidic reservoir both influidic connection to the fluidic passage, wherein the loading well provides an inlet to the fluidic passage and the fluidic reservoir provides an outlet from the fluidic passage. In some embodiments, (a) multiple electrodes and / or multiple insulating layers stacked on top of each other; (b) multiple sensors stacked on top of each other; and / or (c) multiple devices stacked on top of each other. In some embodiments, said configuration improves signal.

[0011] Disclosed herein include sensor devices for characterizing a stranded molecule. In some embodiments, the sensor device comprises: a fluidic passage disposed in a support structure (e.g., a support structure comprising a semiconductor material), wherein the fluidic passage comprises a lateral fluidic passage and a vertical fluidic passage, wherein the lateral fluidic passage is in fluidic connection with the vertical fluidic passage, two or more global electrodes capable of providing a voltage to translocate the stranded molecule through the lateral and vertical fluidic passages, at least one sensor disposed in a bottom of the lateral fluidic passage, wherein the at least one sensor comprises at least one first graphene sheet, at least one second graphene sheet, and at least one first insulating layer disposed between the at least one first graphene sheet and the at least one second graphene sheet, and wherein the at least one sensor comprises a sensor edge capable of interacting with the stranded molecule, thereby modulating a tunneling current between the at least one first graphene sheet and the at least one second graphene sheet; and a detector configured to detect an electrical signal between the at least one first graphene sheet and the at least one second graphene sheet when the stranded molecule is adhered to the sensor edge.

[0012] Disclosed herein include methods of characterizing a stranded molecule. In some embodiments, the method comprises: providing a sensor device disclosed herein; applying a voltage across the fluidic passage to produce a current flow through the fluidic passage, causing a single stranded molecule to translocate through the fluidic passage at a time and allowing said single stranded molecule to interact with the sensor edge of the at least one sensor; detecting a first electrical signal between the at least one first electrode sheet and the at least one second electrode sheet of the sensor edge when a first portion of the stranded molecule is adhered to the edge portions of the at least one first electrode sheet and the at least one second electrode sheet of the at least one sensor; and determining a characteristic of the first portion of the stranded molecule based on the detected first electrical signal.

[0013] In some embodiments, the method comprises: translocating the stranded molecule across the sensor edge; and measuring a second electrical signal through the edges when a second portion of the strand molecule is adhered to both edge portions of the at least one first electrode sheet and the at least one second electrode sheet, following the translocation of the stranded molecule. In some embodiments, the method comprises: linearizing the stranded molecule along the fluidic passage, e.g., by adjusting the magnitude and direction of the voltage across the fluidic passage.

[0014] In some embodiments, the first portion of the stranded molecule is in a stretched conformation when the first portion of the stranded molecule is adhered to both edge portions of the at least one first electrode sheet and the at least one second electrode sheet of the at least one sensor. In some embodiments, the sensor edge of the at least one sensor is functionalized with one or more chemical group(s) and the adhesion between the stranded molecule and the sensor edge of the at least one sensor occurs through electrostatic interaction, hydrogen bonding, hydrophobic interaction, intercalating interaction, ionic interaction, or a combination thereof between the stranded molecule and the chemical group(s).

[0015] In some embodiments, detecting the first electrical signal and / or the second electrical signal comprises applying a voltage between the at least one first electrode sheet and the at least one second electrode sheet and measuring a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet. In some embodiments, detecting the first electrical signal and / or the second electrical signal comprises applying a voltage across the global electrodes and measuring a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet. In some embodiments, detecting the first electrical signal and / or the second electrical signal comprises measuring an ionic current flowing through the edge portions of the at least one first electrode sheet and the at least one second electrode sheet. In some embodiments, the method comprises: measuring an in-plane tunneling conductance across the first portion of the stranded molecule. In some embodiments, the stranded molecule is fully enclosed by the sensor edge.

[0016] In some embodiments, translocating the stranded molecule comprises: dissociating the first portion of the stranded molecule from the edge portions of the at leastone first electrode sheet and the at least one second electrode sheet, and adhering the second portion of the stranded molecule to the edge portions of the at least one first electrode sheet or the at least one second electrode sheet. In some embodiments, dissociating the first portion of the stranded molecule from the edge portions comprises dissociating the first portion of the stranded molecule from the edge portion of the at least one first electrode, followed by dissociating the first portion of the stranded molecule from the edge portion of the at least one second electrode. In some embodiments, the stranded molecule is in a relaxed state during said dissociating. In some embodiments, the stranded molecule remains adhered to at least one edge portion of the at least one first electrode sheet or the at least one second electrode sheet. In some embodiments, dissociating the first portion of the stranded molecule and adhering the second portion of the stranded molecule comprises (1) adjusting the magnitude and / or direction of the voltage between a global electrode and the at least one first electrode sheet or the at least one second electrode sheet, (2) adjusting the interaction between the edge portions and the stranded molecule, or both. In some embodiments, translocating the stranded molecule moves the stranded molecule across the sensor edge by a portion or a unit of the stranded molecule. In some embodiments, the stranded molecule is partially or fully surrounded by the sensor edge.

[0017] In some embodiments, the characteristic of the first portion of the stranded molecule comprises polynucleotide sequence, polynucleotide methylation, polypeptide sequence, protein glycosylation, protein-polynucleotide binding event, or a combination thereof. In some embodiments, the stranded molecule is a nucleic acid molecule and determining the characteristic of the first portion of the stranded molecule comprises determining a sequence information of a nucleic acid base. In some embodiments, the method comprises: depositing a sample comprising the stranded molecule in a loading well of the sensor device. In some embodiments, the stranded molecule comprises a polymer, a polynucleotide, an oligonucleotide, a polysaccharide, a polypeptide, or a combination thereof.

[0018] Disclosed herein include systems for characterizing a stranded molecule. In some embodiments, the system comprises: one or more sensor devices disclosed herein; an electronic control system electrically connected to the one or more sensor devices, the electronic control system configured to apply an input electrical signal to the one or moresensor devices and to receive an electrical signal from the one or more sensor devices when a stranded molecule is adhered to the at least one sensor edge of the one or more sensor device; and a computer control system for analyzing the output electrical signal from the one or more devices to determine a characteristic of the stranded molecule.

[0019] In some embodiments, the input electrical signal applies a voltage across the two or more global electrodes, a voltage across the at least one first electrode sheet and the at least one second electrode sheet, or both. In some embodiments, the input electrical signal comprises an alternating current voltage, a direct current voltage, an electrical impedance, or a combination thereof. In some embodiments, the output electrical signal comprises a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet, a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet, an ionic current flowing through the edge portions of the at least one first electrode sheet and the at least one second electrode sheet, an in-plane tunneling conductance across the stranded molecule, or a combination thereof.

[0020] Disclosed herein include methods of making a sensor device. In some embodiments, the method comprises: depositing and patterning at least one first electrode sheet, at least one first insulating layer, and at least one second electrode sheet on a substrate; depositing one or more dielectric layer to the substrate and patterning the one or more dielectric layer to form a lateral fluidic passage; and etching a vertical fluidic passage, the vertical fluidic passage in fluidic connection with the lateral fluidic passage.

[0021] In some embodiments, depositing and patterning the at least one first electrode sheet, the at least one first insulating layer, and the at least one second electrode sheet on a substrate comprises depositing the at least one first electrode sheet, patterning the at least one first electrode sheet to a wedge shape, depositing the at least one first insulating layer and the at least one second electrode sheet on the at least one first electrode sheet, and patterning the at least one first insulating layer and the at least one second electrode sheet to a wedge shape. In some embodiments, the method comprises: depositing a first conductor layer on the substrate structure prior to depositing the at least one second electrode sheet; and depositing a second conductor layer on the at least one first electrode sheet following depositing the at least one first electrode sheet.

[0022] In some embodiments, the first conductor layer and / or the second conductor layer comprises a metal pad. In some embodiments, the metal pad is a Ti / Au pad or a Cr / Au pad. In some embodiments, depositing one or more dielectric layer comprises sequentially depositing a first dielectric layer and a second dielectric layer. In some embodiments, the first and second dielectric layers comprise a same dielectric material or different dielectric materials. In some embodiments, the first and second dielectric layers are different in thickness. In some embodiments, the method comprises: applying a capping layer and global electrodes on the top of the one or more dielectric layer. In some embodiments, the capping layer is made of glass and comprises a prepatterned opening for loading a molecule.

[0023] Disclosed herein include methods of making a sensor device. In some embodiments, the method comprises: providing a stack film comprising at least one first electrode sheet, at least one first insulating layer and at least one second electrode sheet, the stack being deposited with a metal film and with a substrate comprising one or more microwells in connection with the metal film; anodizing the metal film in an electrolyte solution, thereby converting the metal to metal oxide and forming a plurality of nanochannels in the metal film; and forming an aperture through the stack film in a microwell by applying a voltage across the stack film.

[0024] In some embodiments, the metal film comprises aluminum, magnesium, titanium, or a combination thereof In some embodiments, the metal film is an aluminum film. In some embodiments, the electrolyte solution is an oxalic acid. In some embodiments, the density and / or dimension of the plurality of nanochannels are controlled by adjusting the voltage and temperature of the anodization and / or the concentration of the electrolyte solution. In some embodiments, the method comprises: positioning a global electrode in each of the one or more microwells. In some embodiments, the aperture has a dimension of about lnm-5nm. In some embodiments, the plurality of nanochannels have a dimension of 20nm- lOOnm.

[0025] Details of one or more implementations of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, thedrawings, and the claims. Neither this summary nor the following detailed description purports to define or limit the scope of the inventive subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a schematic illustration of a non-limiting exemplary EdgeRead sensor geometry.

[0027] FIG. 2 depicts a non-limiting exemplary embodiment of an edge configuration.

[0028] FIG. 3 depicts a non-limiting exemplary embodiment of an EdgeRead sensor setup.

[0029] FIG. 4 depicts a non-limiting exemplary embodiment of “Caterpillar” DNA motion control.

[0030] FIG. 5A illustrates an exemplary embodiment of tunneling conductance measurement across parallel plane electrodes in a nucleic acid molecule. FIG. 5B illustrates an exemplary embodiment of in-plane tunneling conductance measurement in a nucleic acid molecule.

[0031] FIG. 6 depicts a non-limiting exemplary embodiment of a close-loop EdgeRead combined measurement.

[0032] FIGS. 7A-7H illustrate a non-limiting exemplary workflow for sequencing a nucleic acid molecule using the sensor device disclosed herein.

[0033] FIG. 8 depicts a non-limiting exemplary embodiment of wafer-scale EdgeRead fabrication.

[0034] FIG. 9 depicts a non-limiting exemplary embodiment of lithography-free channel / Edge fabrication.

[0035] Throughout the drawings, reference numbers may be re-used to indicate correspondence between referenced elements. The drawings are provided to illustrate example embodiments described herein and are not intended to limit the scope of the disclosure.DETAILED DESCRIPTION

[0036] In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0037] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0038] Commercially available nanopore sequencing devices rely on a protein to comprise the nanopore and in some cases to regulate the speed of translocation through the nanopore. This results in a long stretch (e.g., 8+) of nucleotides or other monomers being present in the nanopore and contributing to the signal at any time. Further, the motive force which drives the passage is constant and distant from the nucleic acid, resulting in stochastic rates of passage through the nanopore. These two fundamental problems combined result in a signal that requires substantial deconvolution and with that, errors in the determination of the nucleic acid, amino acid, or other linear polymer sequence.

[0039] Disclosed herein includes a two-dimensional (2-D) material-based molecular characterization element and device, also referred to as “the Edge” or “EdgeRead”. The thinness of the Edge design, facilitated by the mono-layer fabrication, allows interrogation of a single or a small number of polymer units (e.g., bases, amino acids). This can minimize the deconvolution required to accurately determine the polymer sequence.

[0040] Adding control to the speed of translocation (including reversibility) across an edge formed by the 2-D electrode material (e.g., graphene) disclosed herein or across the device can allow the device to dwell on a particular sequence until a level of certainty of the identity of a molecular unit (e.g., a base) in achieved or more simply to control the translocation speed to a speed which is optimal for monomer or multimer identification and / or instrument throughput.

[0041] The non-biologic nature of disclosed approaches allows greater flexibility in the aspect ratios of the device as it they are unconstrained by the structure of the protein component. This enables flexibility in the device to allow the direct characterization of polymers, such as sequencing RNA without prior reverse transcription into DNA. This approach also enables the direct characterization / sequencing of proteins. Additionally, because biological molecules are sensitive to post-synthesis modification, a non-biological approach as used herein facilitates the characterization of both modified and unmodified molecules and their discrimination. For example, the sequencing of methylated DNA through a thin-layer electronic sensor provided herein can allow the detection of methylated DNA bases by detecting changes in the electric current flowing through the base under detection. Similarly, non-limiting post-translational modification to proteins, such as phosphorylation, glycosylation, SUMOylation, ubiquitination, etc. can be detected.

[0042] All sequencing techniques presently available require purification of the biomolecule to be sequenced in some fashion prior to sequencing. The single molecule approach of the Edge can produce a signal with sufficient specificity such that, with sufficient training, it can distinguish the biomolecule passing across the Edge to a sufficient degree to assign its identity to a particular class of biomolecules and then proceed with identification of the sequence of that biomolecule in spite of being a DNA, RNA, protein, or other biomolecule. Thus, a cell lysate or other biospecimen (e.g., plasma) can be loaded directly onto the device and generate sequence of the protein, nucleic acid, and other components.

[0043] The device can also be pre-loaded with components to enhance the entry of a particular class or subset of biomolecules across the Edge, such as, for example a complementary oligonucleotide or an antibody, or to deplete a certain class of biomolecules, such as, for example, by adding proteinase K to digest proteins or an oligonucleotide distant from the sensor to deplete ribosomal RNAs.Definitions

[0044] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0045] As used herein, the term “biomolecule” broadly refers to a molecule that is relevant in biological systems. The term “a stranded molecule” refers to a molecule or biomolecule comprising a plurality of repeating structural units, typically at least three, linked together via covalent bonds. A stranded molecule can include, for example, polymers, polynucleotides, DNA, RNA, polypeptides, proteins, polysaccharides, and combinations thereof. The stranded molecule can be naturally occurring or synthetic.

[0046] As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. A polynucleotide can be single-, double-, or multi - stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids / triple helices, or a polymer including purine and pyrimidine bases (e.g., the five biologically occurring bases adenine, guanine, thymine, cytosine and uracil) or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In some embodiments, a nucleic acid or polynucleotide can refer to any nucleic acid, whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages.

[0047] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expressionmodifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0048] As used herein, the term “polysaccharides” refers to carbohydrates which may be hydrolyzed to two or more monosaccharide molecules. The polysaccharides may contain a backbone of repeating carbohydrates, i.e., sugar units, bound together by glycosidic linkages. Examples of polysaccharides include, but are not limited to, alginate, agarose, chitosan, dextran, starch, and gellan gum. Glycosaminoglycans are polysaccharides containing amino sugars as a component. Examples of glycosaminoglycans include, but are not limited to, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratin sulfate, dextran sulfate, heparin sulfate, heparin, glucuronic acid, iduronic acid, galactose, galactosamine, and glucosamine.EdgeRead Overview

[0049] Provided herein includes sensors, devices, and related methods for characterizing a biomolecule by monitoring an electrical signal (e.g., electric tunneling current and / or ionic conductivity) across a portion of a biomolecule as the biomolecule translocates across an exposed edge portion of the sensor. Provided herein include, in some embodiments, a two-dimensional (2-D) material-based device, also referred to as “EdgeRead”, for molecule characterization (e.g., nucleic acid sequencing). The device takes advantage of the ultimate atomic thinness of the 2-D materials and unprecedented control over vertical device resolution on wafer scale. In some embodiments, the thickness of each 2D-material layer is equal to a single nucleic acid base pair length (e.g., 0.34 nm) for a total thickness of about 1.6 nm. For example, two graphene electrodes in the device can be separated by the controlled number of dielectric layers (e.g., hBN layers) forming an open circuit without nucleic acid present. When a molecule (e.g., a nucleic acid molecule) is in contact with the Edge, the graphene edge, being a conductor, closes the circuit and allows current to run with a resistance that depends on the characteristics of the molecule (e.g., basepair sequence) forming the circuit. The edge of the graphene electrodes (and / or the dielectric layers, e.g., the hBN layers) can be functionalized with moieties forming hydrogen / ionic / pi- stacking bonds with the molecule such that it remains in contact with the edge. The graphene edge moieties can be conjugated and provide direct charge transfer from the graphene electrode into the molecule while also providing a prescribed number of degrees of freedom for the translocation of the molecule. The edge of the insulating layers (e.g., hBN layers) can be functionalized with aliphatic moieties that restrict electron tunneling while supporting the prescribed molecule retention. The energetics of the molecule (e.g., nucleic acid molecule) retention is such that it allows axial translocation of the molecule during electrophoresis. Such architecture can ensure that the variable resistance arm of the electrical circuit (i.e., the molecule in contact with the edge) is composed of only one to a few monomers (e.g., 1-3 base pairs) ensuring high accuracy. The number of monomers can be predetermined. The wedge-shaped geometry and the dimensions of the channel of the device ensure only a single molecule (e.g., a single nucleic acid molecule) is read at any given time. FIG. 1 is a schematic illustration of a non-limiting exemplary EdgeRead sensor geometry. FIG. 2 is a schematic illustration of a non-limiting exemplary edge configuration.

[0050] The methods, sensors, and devices described herein have many advantages over the prior technologies. For example, the multi-modal simultaneous sequencing approach can sequence nucleic acids by directly recording the tunneling conductance of nucleic acid bases along the molecular axis via parallel plane electrodes and across a single base or basepair of a nucleic acid molecule via in-plane electrodes. The methods, sensors and devices described herein can sequence a nucleic acid molecule by also recording the ionic conductivity of the buffer solution around the nucleic acid molecule constrained by the edge. The detection of both the tunneling conductance of a nucleic acid base and the ionic conductivity of the buffer solution around the nucleic acid base can be coupled to optical detection. This multi-modal approach can allow the identification of post-synthesis modifications or other macromolecule modifications, including, for example, protein glycosylation, methylation, phosphorylation, and ubiquitination, by combining the molecular conductivity and ionic exclusion volume data. Due to the atomic thinness and the electric properties of the layers in the edge sensor, it can reduce the momentarily sampled base-pair or other monomer of the multimeric species number to as few as one, thus increasing thesequencing resolution. The closely positioned electrode array at the edge, supplemented by the global electrophoresis electrodes in the buffer volume, can assist in controlling the nucleic acid translocation speed and direction, potentially with single base-pair resolution, by proscribed alternating current modulation of the electrode array. The edge geometric parameters (e.g., -lOnm long) overcome the lithographic challenge of creating sub 3nm nanopores and can ensure at-scale fabrication using state-of-the-art semiconductor manufacturing practices. The electronic control of nucleic acid translocation by the edge can be performed at -KHz - MHz frequencies thus allowing sequencing at ps / bp rates. Translocation directionality can also be controlled electronically via such modulation thus allowing to “re-wind” and re-sequence DNA as desired. The methods, sensors and devices described herein overcome the issue of manufacturability of nanoscopic (sub-5nm) features, e g., nanopores at scale.Edge Sensor

[0051] Disclosed herein includes an electronic molecule sensor, also referred to as “EdgeRead”, comprising alternating electrode sheets separated by alternating insulating layers. The sensor typically comprises an electrode-dielectric stack including a plurality of layers / sheets with at least one insulating layer disposed between at least two electrode sheets. The electrode sheet can be atomically thin and electrically conducting. In some embodiments, the molecule sensor comprises a sandwich structure with graphene on the top and bottom and insulating sheets between the at least two graphene sheets. The insulating sheets can be atomically thin sheets. In some embodiments, the insulating sheets can be boron nitride or hexagonal boron nitride (hBN). The electrodes and the insulating layers can both comprise one, two, three, or more sheets. In some embodiments, the layers are in sheet form, which may comprise a single sheet or multiple sheets of 2-D conductive or dielectric material. The molecule sensor can comprise an edge exposed to a solution with analyte (e.g., a target molecule). The edge used herein refers to a lateral or an end portion of the electrode sheets and / or the insulating layer. A target molecule to be characterized can be partially surrounded or fully enclosed by the edge. The length of the exposed edge can be on the nanometer scale. The edges of the electrode sheets (e.g., graphene sheets), the insulating sheets, or both, can be chemically modified by etching the electrodes and / or the insulatinglayers at an edge portion and introducing a functional group at the etched edge. The edges of the electrode sheets and the insulating sheets can be modified with a same chemical group or different chemical groups, or a same biological group or different biological group (e.g., a single stranded DNA binding protein or domain). Voltage (e.g., DC current, AC current, other waveforms, or electrical impedance) can be applied across the two electrode layers (e.g., graphene sheets) such that there is a voltage drop across the exposed electrode (e.g., graphene) edges when a target molecule is adhered to the sensor edge. The presence and identity of an analyte can be detected by a change in tunneling current caused by the position of the analyte with respect to the electrode edges.

[0052] In some embodiments, the sensor disclosed herein comprises at least one first electrode sheet, at least one second electrode sheet, and at least one first insulating layer disposed between the at least one first electrode sheet and the at least one second electrode sheet, wherein the sensor comprises a sensor edge capable of interacting with a stranded molecule, thereby modulating a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet. The at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer can have adjacent exposed edge portions which collectively form the sensor edge. The at least one first electrode sheet and the at least one second electrode sheet can comprise a same electrically conductive material or different electrically conductive materials.

[0053] The EdgeRead can be designed to directly measure the conductance of a target biomolecule under investigation to elucidate its primary structure - subunit / monomer composition / sequence. At the core of the technology is the ability to inject, extract, and measure electrical current across a small and well-defined portion of the investigated molecule. For example, in the context of nucleic acid sequencing, each base residing near an electrode layer can lead to different tunneling current across two closely spaced electrodes (e.g., graphene electrodes) because of the different electronic level structure of the bases. The EdgeRead can therefore characterize each nucleotide base by measuring the tunneling current across the two closely spaced electrodes as the nucleic acid translates across the electrode edges.

[0054] The sensor can be used to characterize any stranded molecule or biomolecule composed of multiple units and monomers. In some embodiments, a targetmolecule can be a biomolecule, including for example polymers, polynucleotides, oligonucleotides, polypeptides, polysaccharides, or combinations thereof.Electric and dielectric layers

[0055] The sensor can comprise at least a first electrode sheet, at least a second electrode sheet, and at least one insulating layer disposed between the at least the first electrode sheet and the at least the second electrode sheet. The at least one first electrode sheet can comprise one, two, three, four, or five sheets of electrodes, the at least one second electrode sheet can comprise one, two, three, four, or five sheets of electrodes, or both. In some embodiments, the at least one first electrode sheet and / or the at least one second electrode sheet comprises graphene. In some embodiments, the EdgeRead can be composed of two single-sheet graphene electrodes with three sheets of hBN in-between from the materials, fabrication, and performance considerations. In some embodiments, increasing the number of insulating layers (e.g., hBN layers) and / or electrode layers can mitigate potential defects leading to current leakage or dielectric breakdown. For example, in some embodiments, the sensor can further comprise at least one second insulating layer located on a surface of the at least one first electrode sheet or the at least one second electrode sheet, and at least one third electrode sheet located on a surface of the at least one second insulating layers (see, for example, FIG. 4). The second insulating layer can contain a dielectric material same as or different from the first insulating layer. The at least one third electrode sheet can contain an electric material same as or different from the first electrode sheet and / or the second electrode sheet. In some embodiments, the at least one first electrode sheet and the at least one second electrode sheet comprise graphene and the at least one third electrode sheet does not comprise graphene.

[0056] In some embodiments, the molecule sensor can comprise multiple (e.g., one, two, three, four, five or more) individually addressable graphene electrodes separated by dielectric layers. Such arrangement in the “read-mode” can mean double-reading a segment of molecule (e.g., a segment of a nucleic acid sequence) on one pass as well as providing enhanced translocation control.Edge geometry

[0057] The sensor further comprises a sensor edge capable of interacting with a target molecule, thereby modulating a tunneling current between the at least one firstelectrode sheet and the at least one second electrode sheet. The at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer can have adjacent exposed edge portions which collectively form the sensor edge. The geometry of the sensor edge can be linear (with “sandwich” structure only on one side of the channel that DNA flows through) or closed-loop (where the edge encloses a molecule axis on all sides irrespective of the perimeter geometry). A target molecule to be characterized can be partially (e.g., with a linear edge) or fully surrounded (e.g., with a closed-loop edge) by the sensor edge. The sensor edge of a molecule sensor can have a circular geometry, a spherical geometry, an ellipse geometry, a triangle geometry, a square geometry, or a rectangular geometry in a horizontal cross-sectional view, in which the sensor edge encloses a molecular of interest.

[0058] In some embodiments, the sensor edge is in a linear geometry. A linear edge can increase the critical dimension that needs to be lithographed due to the current nanotechnology bottleneck. For example, a nanopore 3 nm in diameter is more challenging to pattern at scale and with precision than a linear edge feature of 10 nm using current production lithography tools, even though the edge length for both geometries is roughly the same: ~10 nm. This consideration lifts one of the most challenging aspects for adopting solid-state nanopore technology - sub 5nm planar dimensional control. A linear edge can also allow for enhanced electrostatic molecule translocation control. In an enclosed geometry such as a nanopore, the electrostatic field flux equals zero anywhere within the perimeter of the electrode loop. In some embodiments, that means that the molecule (e.g., DNA) is experiencing zero net electrostatic force within the plane and inside the enclosed electrode loop, which would potentially result in it freely oscillating within the pore. When the geometry of the Edge electrode, however, is linear, the electric field flux cross product is a non-zero vector which results in either an attractive or repulsive force on the negatively charged DNA molecule depending on the electrode bias sign.

[0059] The width or length of a sensor edge can vary in different embodiments depending on the size of the molecule to be characterized. In some embodiments, and without being bound by any particular theory, the width or length of an edge is as small as possible to minimize the number of degrees of freedom (DOF) of molecule / Edge interactions. The width of a linear edge can be about 5-20 nm, for example, 5 nm, 6 nm, 7nm, 8 nm, 9 nm, 10 nm, 11 nm 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, or a number or a range between any two of these values. In some embodiments, the sensor edge has a width of about 5-10 nm, which represents the current at-scale lithography limitation. In some embodiments, the width of about 5-10 nm is sufficient for the adequate performance of nucleic acid sequencing. Considering the double stranded DNA diameter of ~2 nm, a 5-10 nm edge length would contain a single DNA molecule within the desired DOF parameter field. In some embodiments, the dimension of the edge can be adjusted to accommodate the molecule to be characterized.

[0060] In some embodiments, the sensor edge forms a close-loop shape. The closed-loop is an alternate edge geometry that can also allow for the tunneling current measurement. With the critical dimension of this geometry being the loop diameter, the Edge length of any closed-loop geometry is at least 3.14 (n) times greater. Accordingly, in some embodiments, the diameter dimension of a close-loop edge is about 2 nm to 3 nm. In some embodiments, the dimension of a closed-loop edge can be greater than 3 nm to accommodate the molecule to be characterized.Electrodes

[0061] The edge of the sensor described herein comprises a sandwich structure with electrodes on the top (e.g., one or more electrode sheets) and bottom (e.g., one or more electrode sheets) and one or more insulating sheets between the electrodes. The material for the electrodes can be electrically conductive and vertically definable at the atomic scale. The electrode sheets can be composed of thin-layer metals (e.g., Au), aluminum nitride or other electrically conductive materials. Exemplary materials suitable for the electrode sheets include, but are not limited to, graphene, gold, titanium nitride (TiN), poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), molybdenum disulfide (M0S2), copper, platinum, nickel, or a combination thereof. The at least one first electrode sheet and the at least one second electrode sheet can comprise a same electrically conductive material or different electrically conductive materials. In some embodiments, both the at least one first electrode sheet and the at least one second electrode sheet comprise graphene. In some embodiments, the at least one first electrode sheet comprises graphene and the at least one second electrode sheet does not comprise graphene.

[0062] In some embodiments, the electrode sheets comprise one or more graphene sheets. A graphene sheet has two types of electro transfer sites - edge and basal. The graphene edge plane atoms have been reported to possess significantly higher electron transfer rates and reactivity compared to basal planes in electrochemical studies. There are several benefits of employing graphene as opposed to any other conductive thin-layer material. First, the graphene sheet has a single-atom thickness (3.4A), which defines the molecule / electrode interaction DOF parameter field in the vertical dimension. Additionally, this thickness corresponds to the single nucleic acid base-pair planar dimension on the subAngstrom level, which adds to the enhanced measurement resolution. Second, graphene has unsurpassed current-carrying capacity overall and high current injection density at the edge in particular. Third, the edge of the graphene has the ability to be functionalized with desired chemical functionality.Insulating layer

[0063] An insulating layer is composed of non-conducting dielectric material. The dielectric material in the insulating layer can possess one or more of the following characteristics: (1) being thickness-definable on the atomic level (at least sub -nanometer); (2) capable of maintaining sufficient dielectric properties at a defined thickness; and (3) ideally capable of being synthesized on graphene. In some embodiments, the insulating layer comprises boron nitride, such as hexagonal boron-nitride (hBN). Similar to graphene, hBN has a thickness of about 3.4 A, with the optimal crystallographic match to graphene as well as high energy of adhesion to graphene. Other dielectric materials that can be used in the insulating layer include, but are not limited to, ceramics (SisN4, AI2O3, HfCh, Z1O2, HfSiO / i, ZrSiO.1, or any combination thereof), diamond-like carbon, carbon boron nitride, 2D polymers, self-assembled monolayers or bilayers and other electrically insulating organic and inorganic moieties. The appropriate dielectric material or a combination thereof can be selected for the sandwich structure based on layer thickness, dielectric properties, fabrication appropriateness, as well as chemical properties with respect to edge functionalization.

[0064] The at least one first insulating layer can comprise one, two, three, four, or five first insulating layers. The number of insulating layers can comprise a same dielectric material or different dielectric materials. In some embodiments, at least one first insulating layer comprises boron nitride, optionally the at least one first insulating layer compriseshexagonal boron nitride (hBN). For example, the insulating layer can comprise three sheets of boron nitride or hBN. The thickness of the insulating layer(s) can vary from single-atom thick (e.g., hBN sheet) to hundreds of microns and even millimeter (e.g., silicon substrate and polymers, respectively). In some embodiments, it may be necessary to functionalize graphene electrodes with a certain moiety while leaving hBN edge bare or have two distinct functionalizations on electrode and dielectric edges.Edge functionalization

[0065] The sensor edge including the exposed edge of the electrode sheet and / or the insulating layer can be chemically modified or functionalized with a functional group. The terms “functionalized” or “functionalization” can refer to attaching, conjugating or grafting a moiety to a substrate (e.g., a graphene edge) with a functional group that is capable of reacting with an analyte. Functionalization of the edge with chemical moieties or enzymes that interact with specific target molecules (e.g., nucleotides or amino acids) can further enhance molecular interaction, periodically slow transit speed, and / or modulating the monitored electrical parameters as a biomolecule passes across the edge. For example, to enhance nucleotide-specific and / or amino acid-specific interactions, enzymes (can be, e.g., wildtype enzymes or modified enzymes) such as polymerases, exonuclease, proteases, helicases, or other chemical moieties can be introduced to the edge to specifically bind individual nucleotide or amino acid types or short sequences of polynucleotides or amino acids. In some embodiments, to contain the molecule / edge interaction DOF parameter field and increase control over molecule / edge interactions, a certain electrode edge functionalization can be beneficial. Introduced chemical groups can influence the affinity of the molecule (e.g., nucleic acid affinity) to the Edge, charge transfer and extraction to / from the molecule (e.g., nucleic acid molecule), as well as contain the molecule in the optimal position with respect to the edge during measurement and translocation. Upon graphene lattice disturbance - breaking carbon-carbon bonds and exposing those unsaturated bonds to the environment (creating the Edge via any means) - the exposed carbon atoms can inevitably become functionalized.

[0066] Various chemical edge modifications are suitable, including, for example, hydrogenation, hydroxylation, carboxylation, carbonylation, amination, and others identifiable to a person skilled in the art. Accordingly, the sensor edge (e.g., graphene edge)can be modified with a carbonyl, carboxyl, hydroxy!, aldehyde, carboxylate, ester, or amine functional group, or a combination thereof The hydroxyl group can be a primary', secondary or tertiary' hydroxyl group. The amine group can be a primary amine, secondary amine or tertiary' amine. In some embodiments, hydrogenation of the edge of graphene electrodes can improve their sensitivity for nucleic acid sequencing purposes. Hydrogenation can introduce a hydrogen bond that can form between the hydrogen atom at the graphene electrode edges and atoms carrying a partial negative charge on the target molecule. The H-bonds between graphene electrodes and the translocating molecules (e g., nucleic acid bases) can enhance the coupling between them and thus substantially increase the magnitudes of transverse tunneling currents. As a result, the current measurability and the speed with which the nucleic acid sequence can be read can be greatly improved. In some embodiments, the functionalization can cause an attractive force so that it can slow down the translocation of the molecule through the channel, providing more time for the transverse conductance measurement of each molecular unit located between the graphene edges.

[0067] These functional groups possess varying chemical and physical properties with respect to molecular interaction and thus, in some embodiments, it is important to control the graphene edge chemistry with respect to utilities such as charge insertion / extraction, steric interaction, electrostatic binding, hydrogen binding, hydrophobic / intercalating binding, metal modification, and biomolecule binding (e.g., enzyme binding) as described below. In an exemplary embodiment, a p-phenylenediamine moiety functionalized to the graphene edge can be charge-conducting (conjugated), bear a slightly positive charge at neutral pH (base, pKa 6.2 - binding the DNA phosphate group) and experience angular motion subject to electrical modulation (gating) by the other electrode potentially commensurate with single-base step translocation control.

[0068] Charge insertion / extraction. In some embodiments, edges of a sensor, including electrode edges and / or edges of insulating layers, can be modified with certain charge-injecting groups to minimize the tunneling electrical resistance between the graphene edge and target molecule. Hydrogenation of the graphene Edge is geometrically the smallest possible molecular functionalization (least effect on the interaction DOF field). It possesses good charge transfer properties, as well as the ability for hydrogen bonding due to a positive dipole moment. The Edge can be modified with an organic molecule or an inorganicmolecule. In some embodiments, and without being bound by any particular theory, if an organic molecule is selected for Edge functionalization, it should be as electrically conductive as possible. Conjugated moieties can be strong candidates where the charge is conducted through their hybridized 7i-electron orbitals.

[0069] Functional group steric interaction. Molecular edge modification can positively affect the Edge / target molecule interaction DOF field. For example, such a moiety can possess a certain length / rotational angle such that it is free (DNA-interacting) end motion with respect to the DNA axis, would be co-planar, as well as have the absolute value around 3.4A - the inter-base distance. In such an arrangement, the nucleic acid can be translocated across the Edge one base-pair per electrical modulation cycle of the graphene Edge electrodes.

[0070] Electrostatic molecular binding. In some embodiments, the backbone of a target molecule can be positively or negatively charged. For example, the nucleic acid phosphate backbone is negatively charged at neutral pH and can be electrostatically attracted and bound by positively charged moieties around neutral pH such as amines, imine, and imidazolium salts. Therefore, in some embodiments, the edges can be modified with positively charged moieties such as amines, imine or imidazolium for nucleic acid sequencing. Phosphate backbone binding can be the preferred binding mode for EdgeRead due to its nondiscriminatory (base-wise) nature, its outward positioning with respect to the molecular axis, as well as offering purely electronic binding control (electrode voltage modulation). One potential challenge is that the charge, if injected into the phosphate group, must tunnel through the ribose unit prior to entering the delocalized 7t-electron cloud of the bases. This can be mitigated by increasing the tunneling bias as well as adding functionalizing moieties that would inject charge directly into the groove (major or minor for dsDNA) or couple to bases via hydrogen bonding described below.

[0071] Hydrogen bond binding. In some embodiments, the target molecule can be bound to the edge structure at positions / sites other than phosphate backbone. Hydrogen bonding can also be utilized for measuring single- stranded DNA or even double-stranded DNA at some degree of intercalation.

[0072] Combined binding. The edge structure can be modified in a fashion that the electrostatic binding to the backbone (e g., the phosphate group in a nucleic acidmolecule) is coupled with additional hydrogen binding of conjugated moieties to the bases. In such way, the strong Coulombic interaction between the phosphate of nucleic acid molecule and hydrogenated or amminated Edge can hold the nucleic acid molecule in the optimal position, while the conjugated moiety of appropriate geometry injects / extracts charge to / from the nucleic acid bases.

[0073] Hydrophobic / intercalating binding. In some embodiments, the edge structure can be functionalized with intercalating agents such as DNA intercalating agents. DNA intercalating agents are generally conjugated hydrophobic heterocyclic ring molecules that resemble the ring structure of base pairs. Exemplary DNA intercalating agents include, for example, amonafide, SYBR Green, ethidium bromide, acridine orange, and actinomycin D. The DNA intercalating agents can intercalate between base-pairs via hydrophobic interactions and potentially provide greater tunneling conductivity from the electrode directly through the base 7r-stack than through the phosphate backbone (and ribose).

[0074] Metal functionalization. The edge can be functionalized with metal atoms / ions that can provide charge injection / extraction, nucleic acid affinity as well as a degree of non-voltage modulated electrostatic nucleic acid affinity in their ionic state. Exemplary metal atom / ion include, for example, Ca, Ni, Cu, Zn, Mg, K, and others identifiable to a person skilled in the art.

[0075] The one or more sensor edges described herein, including the edges of the at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer can be functionalized with a same functional group or different functional groups. For example, the edges of the at least one first electrode sheet and the at least one second electrode sheet can be functionalized with a same functional group or different functional groups. In some other embodiments, the edges of the insulating layers are functionalized with a same functional group as the electrode sheet. In some embodiments, the edges of the insulating layers are functionalized with a functional group different from the electrode sheet. In some embodiments, the edges of the insulating layers are not functionalized. In some embodiments, the one or more sensor edges described herein, including the edges of the at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer can be functionalized with a same enzyme or different enzymes.Sensor Device

[0076] Provided herein also includes a sensor device for characterizing a stranded molecule. In some embodiments, the sensor device can comprise a fluidic passage disposed within a support structure, two or more global electrodes capable of providing a voltage to translocate the stranded molecule through the fluidic passage, at least one sensor described herein disposed in the support structure with the sensor edge exposed to the fluidic passage, and a detector configured to detect a current between the at least one first electrode sheet and the at least one second electrode sheet as the stranded molecule is adhered to the sensor edge. The adhesion of the stranded molecule to the sensor edge can occur through physical and / or chemical interactions between the stranded molecule and the sensor edge such as the functional groups of the sensor edge.

[0077] The sensor device can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sensors described herein. In some embodiments, the number of sensors in a sensor device can be about, at least, at least about, at most, or at most about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 3,000,000, 5,000,000, 8,000,000, 10,000,000, or a number or a range between any two of these values. The multiple sensors can be exposed to a distinct fluidic passage.

[0078] The support structure can comprise a conductor material, semiconductor material, a dielectric material, glass, plastic, polymeric material, ceramic material, or others commonly used in fabrication of fluidic devices. In some embodiments, the support structure comprises a semiconductor material. The term “semiconductor material” refers to any material common in semiconductor industry. Examples of semiconductor materials that can be employed as the substrate include, but are not limited to, Si, SiGe, SiGeC, SiC, Ge alloys, or others identifiable to a person skilled in the art. In some embodiments, the support structure is composed of a Si-containing semiconductor material, such as silicon.

[0079] The fluidic passage in the sensor device can comprise a vertical fluidic passage section and at least one sensor is disposed in the support structure with at least one sensor edge exposed to the vertical fluidic passage section such that a stranded molecule can interact with the sensor edge as it translocates through the vertical fluidic passage. The sensoredge(s) can be in a closed-shape geometry or a linear geometry forming one or more apertures in the support structure.

[0080] The fluidic passage can further comprise a lateral fluidic passage section in fluidic connection with the vertical fluidic passage section and the at least one sensor is disposed in a bottom of the lateral fluidic passage section with the sensor edge exposed to the vertical fluidic passage section (see, for example, FIG. 3). A lateral channel can be formed on the top surface above the sensor. The channel can be wedge shaped with the narrow portion of the wedge at the sensing element. The lateral channel can extend down the vertical face to accommodate the translocated molecule and leads to a buffer reservoir containing a global electrode. An opening of the lateral fluidic passage section to the vertical fluidic passage section can be about 5-10 nm in width. The dimension of the fluidic passage can vary in different embodiments. In general, the lateral fluidic passage section and / or the vertical fluidic passage section is in nanometer or micrometer scale. In some embodiments, the lateral fluidic passage section and / or the vertical fluidic passage section has a dimension configured to accommodate no more than one stranded molecule. For example, the lateral fluidic passage section can be about 50-500 nm in width, 1-30 pm in length, and / or 1-100 nm in height. The lateral fluidic passage section can have an aspect ratio of height to length in a range from about 1 :10 to about 1 : 1000, optionally from about 1 :50 to about 1 :500. In some embodiments, the lateral fluidic passage section can have a cross-sectional area of about tens to hundreds of nm2.

[0081] The sensor device can further comprise a loading well and a fluidic reservoir both in fluidic connection to the fluidic passage, in which the loading well provides an inlet to the fluidic passage and the fluidic reservoir provides an outlet from the fluidic passage.

[0082] In some embodiments, the sensor device described herein is configured to allow a single stranded molecule to flow through the fluidic passage at any given time. Several strategies can be used to achieve this goal, including, for example, geometric constrain, intermolecular repulsion / hindrance, and / or surface charge constraints. For example, a micro / nanoscopic channel with high-aspect-ratio leading to the edge portion can ensure multi-strand molecule exclusion. The small cross-sectional area of the channel (e.g., tens to hundreds of nm2) coupled with its high aspect ratio can make entry of more than onenucleic acid strand into the channel electrostatically and entropically unfavorable. In some instances, the geometry of the EdgeRead channel bears a wedge shape of high aspect ratio with its acute angle bearing a 5-10nm wide opening (see, for example, FIG. 1). Intermolecular repulsion can also be used to hinder the entry of more than one stranded molecule into the channel. For example, in solutions with low osmolarity, the negatively charged nucleic acid molecules take linear configurations. Within a nanoscopic channel, such linear charge distribution ensures electrostatic DNA / DNA repulsion and thus exclusion of all but one DNA molecule from the channel. In some embodiments, the interior surface of the channel can be composed of or chemically modified to compose of materials bearing negative charge (e.g., glass, or self-assembled monolayer with exposed phosphate group) to further promote exclusion of negatively charged molecules from the channel to the degree necessary to ensure single molecule interrogation requirement. For example, in the case of nucleic acid sequencing, the interior surface of the channel can be chemically modified with negatively charged molecules or chemical groups to ensure a single nucleic acid strand in the channel at any given time. Similarly, the interior surface of the channel can also be chemically modified with positively charged molecules or chemical groups to promote exclusion of positively charged molecules.

[0083] A notable advantage of some embodiments of the EdgeRead technology provided herein is in electronic control of the speed and direction of molecular translocation. For example, an array of individually controlled electrodes (e.g., two buffer-biasing electrodes and two / three / more graphene Edge DNA-biasing electrodes) when voltage- modulated in certain sequence, can ensure base-by-base nucleic acid translocation with prescribed frequency. The translocation directionality can be controlled electronically via such voltage modulation thus allowing to “re-wind” and re-sequence the nucleic acid molecule as desired.

[0084] One of the major current solid-state nanopore nucleic acid sequencing challenges is the fast nucleic acid translocation speed through the active region of the device. Most often, the nucleic acid translocation speed is controlled enzymatically, e.g., DNA is “fed” via an enzyme with precise translocation step-size and controllable step frequency. In the devices described herein, certain materials having high nucleic acid affinity due to their surface or bulk properties can be used to slow the translocation of the nucleic acids. Thesurface of the channel leading to the edge (e.g., the lateral channel) can be modified such that is can either attract or repel nucleic acid molecules. For example, the interior surface of the lateral channel can be made of glass for nucleic acid repulsion or aluminum oxide for nucleic acid affinity. The interior surface of the channel can also be chemically modified to comprise organic molecules (SAMs, lipid bilayers, etc.) such as phenyl or amino groups. The interior surface of the channel can also be chemically modified with a biomolecule.

[0085] In some embodiments, to further slow down a nucleic acid translocation, the channel can be filled with a hydrogel such as agarose, polyacrylamide, PVA, or others identifiable to a skilled person. The hydrogel can typically slow down DNA by orders of magnitude depending on the electrical bias, DNA length and the gel density. The hydrogel can be naturally occurring or synthetic. For example, unmodified 5% agarose gel has a pore size on the order of 30 nm, while 1% agarose gel has a pore size of about 200-300 nm. By adjusting the gel density, the translocation speed of a biomolecule can be controlled. Gels of certain density can be further chemically modified with affinity sites such as phenyl and / or amino groups to further slow down molecular movement. In some embodiments, the channel can be filled up with nanoparticle matrices which can slow down nucleic acid translocation with similar velocity effects.

[0086] The global electrodes, also referred to as buffer-biasing electrodes, can provide an electrical voltage bias across the fluidic passage. The global electrodes can control the global electrophoretic force applied on the stranded molecule by applying a prescribed bias across the entire liquid volume in the device. A voltage can be adjusted to apply a force to the translated molecule such that the translation direction and rate through the fluidic passage can be controlled as desired. The voltage can be adjusted to produce an electrophoretic force along the lateral fluidic passage (wedge channel in FIG. 3) to facilitate the translocation of a stranded molecule from the loading well to the opening near the top of the vertical edge. Once the molecule enters the vertical channel, the voltage can be adjusted to produce an electrophoretic force along the vertical channel. Under a biased voltage, electrolytes in the channel can move through the edge electrophoretically, thereby generating an ionic current signal. The edge electrodes (e.g., the graphene electrodes) can also be used to manipulate molecular movement on macro- and nanoscale. When the target molecule is in physical contact with the sensor edge, the tunneling conductance between the electrodes canbe measured. The graphene electrodes can therefore induce controlled molecular motion at the nanoscale in the vicinity of the edge.[00871 In a non-limiting exemplary embodiment, a sensor device can comprise a fluidic passage disposed in a support structure, wherein the fluidic passage comprises a lateral fluidic passage and a vertical fluidic passage, wherein the lateral fluidic passage is in fluidic connection with the vertical fluidic passage, optionally the support structure comprises a semiconductor material, two or more global electrodes capable of providing a voltage to translocate the stranded molecule through the lateral and vertical fluidic passages, at least one sensor disposed in a bottom of the lateral fluidic passage, wherein the sensor comprises at least one first graphene sheet, at least one second graphene sheet, and at least one first insulating layer disposed between the at least one first graphene sheet and the at least one second graphene sheet, and wherein the sensor comprises a sensor edge capable of interacting with the stranded molecule, thereby modulating a tunneling current between the at least one first graphene sheet and the at least one second graphene sheet, and a detector configured to detect an electrical signal between the at least one first graphene sheet and the at least one second graphene sheet when the stranded molecule is adhered to the sensor edge.

[0088] Provided herein also include systems for characterizing a molecule using one or more senor device described herein. The system can comprise one or more sensor devices / chips described herein, means for providing input electrical signals, means for reading out the electrical signals from the chips, and means for analyzing the electrical signals to identify the analytes, e.g., base calling in the nucleic acid sequencing. In some embodiments, a system can comprise one or more sensor devices described herein, an electronic control system electrically connected to the one or more sensor device, the electronic control system configured to apply an input electrical control signal to the one or more sensor devices and to receive an output electrical signal from the one or more sensor devices when a stranded molecule is adhered to the at least one sensor edge of the one or more sensor device, and a computer control system for analyzing the output electrical signal from the one or more devices to determine a characteristic of the stranded molecule. The input electrical control signal can apply a voltage across the two or more global electrodes, a voltage across the at least one first electrode sheet and the at least one second electrode sheet, or both. The input electrical control signal can comprise an alternating current voltage, adirect current voltage, an electrical impedance, or a combination thereof. The output electrical signal can comprise a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet, a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet, and / or an ionic current flowing through the edge portions of the at least one first electrode sheet and the at least one second electrode sheet. The computer control system can analyze the output electrical signal over time and use such information to determine a characteristic of the stranded molecule, for example, to identify the sequence of a nucleic acid molecule. The computer control system can also include components for computational data analysis. In the embodiments of nucleic acid sequencing, the electronic signals from the chips processing nucleic acid molecules represent raw data attributed to the conductivity and exclusion volume of nucleic acid bases. Each datapoint can represent a signal originating from one or more bases positioned between the edge electrodes. The computational data analysis component of the computer control system can convert raw data attributed to the conductivity and exclusion volume of the nucleic acid bases to the identity of the nucleic acid bases. The computer can also control the performance of the one or more sensor devices, for example, by providing a sequence of input electrical signals to the global electrodes and / or the first and second electrode sheets in the sensor device. The systems described herein can be used in connection with various operating systems to execute algorithms or computer-implemented instructions designed to translocate the molecule in a controlled manner and to identify the characteristic of individual monomers in a stranded monomer such as to identify the sequence on individual bases in a nucleic acid molecule.Methods for Molecular Characterization

[0089] Disclosed herein also include methods of characterizing a stranded molecule. In some embodiments, the method can comprise providing a sensor device described herein, applying a voltage across the fluidic passage to produce a current flow through the fluidic passage, causing a single stranded molecule to translocate through the fluidic passage at a time and allowing said single stranded molecule to interact with the sensor edge of the sensor, detecting a first electrical signal between the at least one first electrode sheet and the at least one second electrode sheet of a sensor edge when a firstportion of the stranded molecule is adhered to the edge portions of the at least one first electrode sheet and the at least one second electrode sheet of a sensor, and determining a characteristic of the first portion of the stranded molecule based on the detected first electrical signal. The stranded molecule can be partially or fully surrounded by the sensor edge.

[0090] The method and device described herein can be compatible with a range of biomolecules that are polymeric in nature with unit repeat structures. A stranded molecule to be characterized can be a polymer, a polynucleotide, a polysaccharide, a polypeptide, or a combination thereof. In some embodiments, the stranded molecule is a polynucleotide such as DNA or RNA. The polynucleotide can be naturally occurring or synthetic, single-stranded or double-stranded.

[0091] The method and device described herein are capable of determining a range of characteristic of a biomolecule, so long as the characteristic of a biomolecule affects the electrical signal (e.g., tunneling current, ionic current, resistance, impedance, voltage) between the two closely spaced electrode sheets when the biomolecule is in close vicinity of or adhered to the edges of the electrode sheets. For example, a characteristic of a portion or a chemical unit of a stranded molecule can include, e.g., nucleic acid sequence, chemical modification such as methylation, phosphorylation, glycosylation, ubiquitination, lipidation, proteolysis, PEGylation, amino acid sequence, biomolecule secondary structure, a binding event such as a protein-polynucleotide binding event, or others identifiable to a person skilled in the art. In cases the stranded molecule is a nucleic acid molecule (e.g., DNA or RNA), determining the characteristic of the first portion of the stranded molecule can comprise determining a sequence information of a nucleic acid base.

[0092] The method can further comprise translocating the stranded molecule across the sensor edge, and measuring a second electrical signal through the edges when a second portion of the stranded molecule is adhered to both edge portions of the at least one first electrode sheet and the at least one second electrode sheet, following the translocation of the stranded molecule. Each portion of the stranded molecule can correspond to a single chemical unit (e.g., one base of a nucleic acid molecule or one amino acid of a polypeptide). In some embodiments, each portion of the stranded molecule can comprise more than one chemical unit (e.g., one, two, three, four, five or more units). The number of units to becharacterized in an individual detection depends on the thickness of the edge geometry. For example, five bases can fit at the edge composed of graphene / 3-layer hBN / graphene. As the stranded molecule translocates through the fluidic passage, additional electrical signals can be measured as each portion of the stranded molecule (e.g., each base of a nucleic acid molecule) sequentially adheres to the edges of the two closely spaced electrode sheets. Therefore, the method can further comprise measuring a third electrical signal through the edges when a third portion of the stranded molecule is adhered to both edge portions of the at least one first graphene sheet and the at least one second graphene sheet. The method can further comprise measuring a fourth electrical signal through the edges when a fourth portion of the stranded molecule is adhered to both edge portions of the at least one first graphene sheet and the at least one second graphene sheet. The measurement can be continued till the terminal unit of the stranded molecule adheres to the electrode edges.

[0093] The method can further comprise linearizing the stranded molecule as the molecule translocates through the fluidic passage. The stranded molecule can be linearized by adjusting the magnitude and direction of the voltage across the fluidic passage, such as by adjusting the magnitude and direction of the voltage across the global electrodes and / or the two closely spaced electrode sheets (e.g., graphene sheets). Other methods and structures can also be used to facilitate the linearization of the molecule, including for example chemical modification of the interior surface of the fluidic passage, or using a variety of obstacles or other structural confinements.

[0094] The stranded molecule can be in a stretched conformation or in a relaxed, flexible conformation as the molecule translocates through the channel. For example, the first portion of the stranded molecule can be in a stretched conformation when the first portion of the stranded molecule is adhered to both edge portions of the at least one first electrode sheet and the at least one second electrode sheet of the sensor. The adhesion of the stranded molecule to the sensor edge can occur through physical and / or chemical interactions between the stranded molecule and the sensor edge. The sensor edge can be functionalized with one or more chemical group(s) or functional group(s) and the adhesion between the stranded molecule and the sensor edge can occur through electrostatic interaction, hydrogen bonding, hydrophobic interaction, intercalating interaction, ionic interaction, or a combination thereof between the stranded molecule and the chemical group(s). For example, the exposed edges ofgraphene sheets can be functionalized by attaching a chemical moiety to the edges which has an affinity to a portion of the biomolecule.

[0095] Detection of an electrical signal can take place when the stranded molecule is in close vicinity of and / or adhered to the sensor edge. The particular electrical signal depends on the context in which the method and device are employed as well as the device configuration. The electrical signal to be detected can include, for example, a tunneling current across the two closely spaced electrode sheets, a voltage drop between the two closely spaced electrode sheets, an ionic current flowing through the sensor edge, resistance, impedance, electric potential, translocation time or transit speed of the molecule through the channel.

[0096] In some embodiments, detecting an electrical signal comprises measuring an ionic current flowing through the edge portions of the two closely spaced electrode sheets. As a molecule is driven across the sensor edge by an electric field, it excludes ions in the opening around the sensor edge, resulting in a temporal decrease in the ionic current. The magnitude and the duration of the current blockade provide information on the diameter and length of the molecule, respectively. For sequencing, each nucleotide blocks the ionic current in a unique way that is dependent on its molecular size and shape.

[0097] In some embodiments, detecting an electrical signal comprises measuring a tunneling current between the two closely spaced electrode sheets. The two closely spaced electrode sheets can be two parallel plane electrodes vertically aligned such that one electrode sheet is positioned on the top of the other. FIG. 5A illustrates an exemplary embodiment of tunneling conductance measurement in a nucleic acid molecule along the molecular axis. Once the nucleic acid molecule is in contact with the edge electrodes in either stretched or relaxed state and is bound to the edge moieties, its electrical properties are measured. Graphene electrodes are voltage biased (e.g., 0.1V) and charge is injected into the nucleic acid molecule via edge moieties (e.g., phenylenediamine) at the top graphene electrode. The electrons tunnel along the n-stacking of the nucleic acid bases (e.g., AAC) and are ejected into bottom graphene electrode. The tunneling current is recorded for the AAC base sequence. After the nucleic acid molecule is translocated along the edge by one base pair the tunneling current measurement is repeated. In this position, the tunneling conductivity of TAA base sequence is recorded. Computational algorithms can be used tocompare the tunneling conductance data obtained from the sequentially translocated nucleic acid molecule and to elucidate its base pair sequence. In some embodiments, detection of an electrical signal is a combined measurement of excluded volume of ions in the opening around the molecule and a tunneling conductance of units between the two closely spaced electrodes (see, for example, FIG. 6). In some of these embodiments, the molecule can be surrounded by the sensor edge in all directions.

[0098] In some embodiments, detecting an electrical signal can further comprise detecting an in-plane tunneling conductance across a molecule or a portion thereof. FIG. 5B illustrates an exemplary embodiment of in-plane tunneling conductance measurement in a nucleic acid molecule. Variations in the in-plane current through a graphene electrode due to the traversal of a molecule can be measured. The aperture or gap in the graphene electrode is small enough to ensure that the nucleotide under interrogation can bridge the two electrical contacts. In some embodiments, the nucleic acid molecule under interrogation is fully enclosed by the edge of the graphene electrode. In some embodiments, the aperture or gap can be a nanopore through which a molecule can translocate.

[0099] In some embodiments, detecting the first electrical signal and / or the second electrical signal comprises applying a voltage between the at least one first electrode sheet and the at least one second electrode sheet and measuring a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet. In some embodiments, detecting the first electrical signal and / or the second electrical signal can comprise applying a voltage across the global electrodes and measuring a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet. In some embodiments, detecting the first electrical signal and / or the second electrical signal comprises measuring an ionic current flowing through the edge portions of the at least one first electrode sheet and the at least one second electrode sheet.

[0100] Following the detection, the stranded molecule can advance along the passage by a segment or a portion of the molecule which can comprise one or more chemical units. Therefore, translocating the stranded molecule can move the stranded molecule across the sensor edge by a portion or a unit of the stranded molecule (e.g., a base). Translocating the stranded molecule can comprise dissociating the first portion of the stranded molecule from the edge portions of the at least one first electrode sheet and the at least one secondelectrode sheet, and adhering the second portion of the stranded molecule to the edge portions of the at least one first electrode sheet or the at least one second electrode sheet. Dissociating the first portion of the stranded molecule from the edge portions can comprise dissociating the first portion of the stranded molecule from the edge portion of the at least one first electrode, followed by dissociating the first portion of the stranded molecule from the edge portion of the at least one second electrode. During the dissociation step, the stranded molecule can adopt a relaxed, flexible conformation, and the stranded molecule can remain adhered to at least one edge portion of the at least one first electrode sheet or the at least one second electrode sheet. Dissociating the first portion of the stranded molecule and adhering the second portion of the stranded molecule can be achieved by (1) adjusting the magnitude and / or direction of the voltage between a global electrode and the at least one first electrode sheet or the at least one second electrode sheet, (2) adjusting the interaction between the edge portions and the stranded molecule, or both. For example, dissociating the first portion of the stranded molecule and adhering the second portion of the stranded molecule can comprise independently energizing the at least one first electrode sheet or the at least one second electrode sheet.

[0101] FIG. 7A-7H illustrate a non-limiting, exemplary workflow of sequencing a nucleic acid molecule using the method and device described herein. In FIG. 7A, a prepared sample is deposited into the loading well. The electrophoretic movement of a nucleic acid molecule through the wedge channel is encouraged by energizing electrode El vs G2(+) with appropriate voltage bias (e.g., 0.5V). The wedge channel is configured such that only one molecule is allowed into the channel due to electronic, entropic, and steric intermolecular competitive interactions. As the molecule enters the channel, the molecule stretches linearly and translocates along the channel towards the edge bottom electrode (G2) and through the nanoscopic channel opening. The leading end of the molecule is electrostatically attracted to and adheres to G2 via edge moiety interaction aided by an adhesion force that can attribute to various physical and / or chemical interactions between the molecule and functional groups on the G2 edge (FIG. 7B). Electrostatic stretching of the nucleic acid molecule can be achieved by energizing electrodes El(+) vs E2(-) and Gl(-) with appropriate voltage bias (e.g., 0.2 V) (FIG. 7C). The selected voltage applies an electrostatic force onto the nucleic acid molecule with a magnitude less than the adhesion force at the bottom electrode sheet (G2). The nucleicacid molecule remains adhered at the leading agent to G2 and stretches across the sensor edge towards the loading well (FIG. 7C). Once the nucleic acid molecule is in a stretched conformation, the voltage bias at G1 is released and the molecule adheres to G1 moieties with an adhesion force that can attribute to various physical and / or chemical interactions between the molecule and functional groups on the G1 edge (FIG. 7D). It is noted that in a relaxed state, the number of bases between G1 and G2 is n, which is determined by dielectric thickness and moiety identity, while in a stretched state the number of bases between G1 and G2 is n-1.

[0102] Measurement of DNA tunneling conductance between G1 and G2 can thus be performed and base-sequence conductance profde can be recorded. Either 2-point measurement or 4-point measurement can be performed. For the 2-point measurement, a voltage bias between G1 and G2 is introduced (e.g., 0.1V) and the resulting current is measured. For the 4-point measurement, a voltage bias between El and E2 is introduced (e.g., 0.1V) and the voltage drop between G1 and G2 is measured. The voltage drop essentially records the resistivity profile of base-sequence between the edge electrodes. In some embodiments, a 4-point probe measurement can be preferrable as it is not influenced by the contact resistance. In such cases, there is no charge transfer between the electrodes and the DNA molecule and only the voltage drop is measured. In addition to the tunneling conductance measurement, measurement of the ionic flow around the Edge can also be performed and the ionic exclusion volume profile of the nucleic bases at the sensor edge can be recorded. A combined tunneling and ionic flow measurement is thus performed.

[0103] Once the conductance and / or resistance profile is recorded, the nucleic acid molecule can be translocated across the sensor edge by one base. To achieve this goal, the tail end of the nucleic acid molecule is decoupled from the G1 moieties by energizing Gl(-) vs E2(+) with a voltage bias such that the adhesion force imposed at G1 is overcome, and the nucleic acid tail end relaxes while being anchored at G2 via the adhesion force (FIG. 7E). Once the molecule is in the relaxed state, a positive voltage bias at G1 is introduced and the molecule adheres to G1 moieties with an adhesion force (FIG. 7F). A conductance and / or resistance measurement can be performed at this stage to confirm the state of the nucleic acid molecule. The nucleic acid molecule can be stretched out towards the E2 electrode by a voltage bias between E2(+) and G2(-) (FIG. 7G). Once the molecule is stretched out, thevoltage bias at G2 is reversed and the molecule adheres to G2 moieties with an adhesion force (FIG. 7H) and the molecule is advanced by one base as compared to in FIG. 7D. The stretching motion mechanism disclosed herein can also be used to reverse the molecule translocation at any point during sequencing. To sequence the entire molecule, the above steps can be repeated as needed until the last base reaches Gl. Once the entire molecule is sequenced, the molecule is detached from the edge electrodes via strong negative bias between Gl(-), G2 (-) and E2(+) and exits through the vertical channel. Another molecule can at this point enter the channel to be sequenced. The described modulation should be performed at frequencies and waveforms commensurate with nucleic acid translocation velocity, moiety interaction dynamics and electric field strength distribution as will be apparent to a person skilled in the art.

[0104] In some embodiments, it can be beneficial for better translocation control and measurement accuracy to introduce a third electrode sheet and a second insulating layer into the stack (see, for example, FIG. 4). The third electrode sheet can comprise any atomically thin, electrically conductive material described herein or known in the art. In some embodiments, the first and second electrode sheets comprise graphene and the third electrode sheet does not comprise graphene. In some embodiments, all the edge electrode sheets are graphene sheets.

[0105] At high modulation frequencies (e.g., MHz) the global electrodes alone may be inadequate due to buffer-related capacitive effects, whereas within the nanoscopic geometry of the edge stack (well within the Debye length of expected working ionic strength buffers) such high frequency modulations should be allowable. With three edge electrodes 1 nm apart, a nucleic acid molecule can be moved locally, relying solely on the edge electrodes in the “caterpillar” fashion (see, for example, FIG. 4). In some embodiments, the degree of control of such system can be significantly higher than the one also involving liquid-biasing electrodes for nucleic acid “stepping”. In some embodiments, and without being bound by any particular theory, this is because the electrostatic interaction force between the nucleic acid molecule and the edge electrodes is independent of the degree of advancement of the nucleic acid molecule with respect to the sensor edge towards either El or E2. The edge electrodes can provide local, nanoscopic electric field gradient acting on individual phosphate backbone units in the vicinity of the sensor edge. While the electrostatic forceimposed of the nucleic acid molecule by the global El and E2 electrodes can change depending on the degree of advancement / translocation across the sensor edge and thus the degree of advancement will have to be accounted for during translocation control of this kind.Sensor Device Fabrication

[0106] One of the benefits of the disclosed EdgeRead technology is manufacturability of nanoscop ic (1-10 nm) features required for nucleic acid sequencing. The disclosed technologies allow fabrication of highly dense EdgeRead sensor arrays on waferscale with features bearing atomic-precision geometry control in both vertical and horizontal dimensions of the device.

[0107] Features of the EdgeRead can be manufactured using various fabrication techniques known in the art. In some embodiments, the sensors and devices described herein can be fabricated using the current semiconductor fabrication processes where all deposition, patterning, and etching occur on wafer substrates of standard dimensions and compositions. Sub-5 nm features that are required for nucleic acid sequencing using solid-state devices can be produced using current technologies. However, to achieve such resolutions, serial lithography techniques are used (e.g., electron-beam, focused-ion beam lithography), where each feature is produced serially. Such processes are expensive and slow - hours per cm2wafer areas. Truly wafer-scale photolithography is currently limited in resolution to ~30 nm features - insufficiently small for DNA sequencing features (nanopores, molecular-scale electrodes, etc.). The EdgeRead technology described herein circumvents the current lithography limitations by producing nanoscopic (1-10 nm) features combined with alternative molecular identification techniques (e.g., tunneling conductance).

[0108] Disclosed herein also include methods of manufacturing the sensor devices described herein. In some embodiments, the method can comprise depositing and patterning at least one first electrode sheet, at least one first insulating layer, and at least one second electrode sheet on a substrate, depositing one or more dielectric layer to the substrate and patterning the one or more dielectric layer to form a lateral fluidic passage, and etching a vertical fluidic passage in the substrate, the vertical fluidic passage in fluidic connection with the lateral fluidic passage.

[0109] In some embodiments, depositing and patterning the at least one first electrode sheet, the at least one first insulating layer, and the at least one second electrode sheet on a substrate comprises depositing the at least one first electrode sheet, patterning the at least one first electrode sheet to a wedge shape, depositing the at least one first insulating layer and the at least one second electrode sheet on the at least one first electrode sheet, and patterning the at least one first insulating layer and the at least one second electrode sheet to a wedge shape.

[0110] In some embodiments, the method can further comprise depositing a first conductor layer on the substrate structure prior to depositing the at least one second electrode sheet and depositing a second conductor layer on the at least one first electrode sheet (e.g., following depositing the at least one first electrode sheet). The first conductor layer and / or the second conductor layer comprises a metal pad, optionally the metal pad is a Ti / Au pad or a Cr / Au pad. The first and second conductor layers are positioned to ensure an electrical contact (e.g., ohmic contact) with the two electrode sheets. The first conductor layer can be embedded between the substrate and the at least one second electrode sheet and the second conductor layer can be embedded between the at least one first electrode sheet and a dielectric layer such that the electrode sheets are in electrical contact with the conductor layers.

[0111] Depositing one or more dielectric layer can comprise depositing a first dielectric layer and a second dielectric layer. The first and second dielectric layer can comprise a same dielectric material or different dielectric materials. The first and second dielectric layers can be different in thickness. For example, one dielectric layer may be thicker than the other.

[0112] The method can further comprise applying (e.g., via wafer bonding) a capping layer (e.g., glass) and global electrodes on the top of the one or more dielectric layer. The capping layer can have a pre-patterned opening such as a loading well for loading a molecule.

[0113] An exemplary, non-limiting fabrication procedure on a wafer scale is illustrated in FIG. 8. A carrier substrate (e.g., silicon wafer with oxide layer) is deposited with an array of metal electrodes / traces for contacting the bottom Edge electrode. A layer of Edge bottom electrode material (e.g., monolayer graphene) is then deposited over the entirecarrier surface including metal electrodes / traces such that graphene makes electrical contact with the metal electrodes / traces. The monolayer material can be patterned into a desired shape (e.g., wedge). A stack of Edge monolayer materials (e.g., 3-layer hBN / graphene) is deposited on top of the carrier covering the entire carrier surface, including patterned graphene. The top layer graphene can be patterned into a desired shape (e.g., wedge). Metal electrodes / traces are deposited on top of graphene and make electrical contact with the top Edge electrode. Dielectric layer is deposited over the entire carrier surface (e.g., lOnm SiN) - to passivate graphene. Thicker dielectric layer is deposited on top (e.g., lOOnm SiCE). The thick dielectric layer is patterned into a horizontal channel of high aspect ratio (e.g., wedge channel, FIG. 8). A vertical channel is etched through all the layers including the substrate such that it intersects the profile of the horizontal channel at its closed end such that an opening forms in the horizontal channel and both channels are conjoint. Etching the vertical channel can also expose the Edge electrodes just under the nanoscopic opening in the horizontal channel, thus allowing electrical manipulation and investigation of the nature of a molecule translocating through the nanoscopic opening (see, for example, FIG. 2). A capping layer, e.g., 100-micrometer thick glass with prepattemed openings (e.g., loading wells) and metal electrodes (global electrophoresis electrodes), are wafer-bonded on top of the entire carrier such that the loading wells are aligned with the wide end of the horizontal channel (see, for example, FIG. 1).

[0114] In some embodiments, the wedge-shaped horizontal channel has a channel width of 100 nm at its starting point (near the loading well) and length of 10 micrometers with an aspect ratio of 1:100 and has an acute angle of about 0.57° at the end where the horizontal channel and the vertical channel intersect. At this aspect ratio, patterning a via through the channel that crosscuts the wedge 100 nm from the “close point” end can generate the edge width of 1 nm. Offsetting the via by 200 nm towards the open end of the wedge generates the edge width of 2 nm. For a 10 nm edge width, the via must be offset by 1 micron. Thus, the accuracy of the edge width control depends on the mask aligner / stepper accuracy. With a typical Nikon i-Line stepper accuracy of ~25 nm, the edge length accuracy should be -2.5A.

[0115] The sensors and devices described herein can also be manufactured using other lithography-free approaches, such as controlled breakdown of dielectric (CBD)approach. Nanoscopic aperture through the edge stack films can also be opened via controlled dielectric breakdown process. The process is stochastic and relies on creating a voltage across the edge stack membrane to the point where the dielectric material in the membrane experiences electric breakdown and the charge flows across the membrane along the breakdown path creating the aperture in the membrane in the breakdown point and growing the aperture as the charges progress through it over time in some embodiments. The aperture size can thus be controlled via controlling the pulse current and frequency parameters in some embodiments. The apertures created this way can be as small at Inm in diameter and can extend through membranes that are hundreds of nanometers thick in some embodiments.

[0116] In some embodiments, a method of making a sensor device comprises providing a stack film comprising at least one first electrode sheet, at least one first insulating layer and at least one second electrode sheet, the stack being deposited with a metal film and with a substrate comprising one or more microwells in connection with the metal film (e.g., aluminum film), anodizing the metal film in an electrolyte solution (e.g., an oxalic acid), thereby converting the metal to metal oxide and forming a plurality of nanochannels in the metal film, and forming an aperture through the stack film in a microwell of the one or more microwells by applying a voltage across the stack film. In some embodiments, only one nanochannel of the plurality of nanochannels in each microwell comprises an aperture formed at the bottom, through which a molecule can translocate. The metal film can comprise aluminum, magnesium, titanium, or a combination thereof. In some embodiments, the metal film is an aluminum film, and the anodization converts aluminum to aluminum oxide. The density and / or dimension of the plurality of nanochannels can be controlled by adjusting the voltage and temperature of the anodization and / or the concentration of the electrolyte solution. The aperture can have a dimension of less than 5 nm (e.g., 1, 2, 3, 4 or 5 nm), while the plurality of nanochannels can have a dimension of about 20-100 nm (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100 nm, or a number or a range between any two of these values).

[0117] Provided below is an exemplary, non-limiting method of creating a sensor device with a closed-loop sensor edge via the dielectric breakdown method within a nanoscopic channel fabricated in thin-film aluminum via the anodic aluminum oxidation (AAO) process (FIG. 9). For example, a channel having a dimension of 20 nm in diameterand 50 nm to microns in length can satisfy the single-nucleic acid molecule interrogation requirement by imposing similar conditions onto nucleic acid molecules as the wedge channel would via entropic, electrostatic, and steric intermolecular competition. This fabrication process can rely on stochastic physical processes without the requirement of lithography for the formation of both the molecule exclusion nano-channel and the edge aperture on sub-5 nm scale.

[0118] As illustrated in FIG. 9, to fabricate a sensor device described herein, edge stack materials (e.g., graphene / 3 -layer hBN / graphene) can be supported with a thin film of aluminum (e.g., about 200 nanometers thick). The aluminum can be deposited onto the edge materials stack that has previously been transferred onto the receiving substrate. Alternatively, the aluminum can be deposited onto the edge materials stack while still on graphene / hbn / graphene synthesis substrate (e.g., copper, wafer, etc.). Metal assisted exfoliation (MAE) can then be used to remove the edge stack materials from the synthesis substrate and onto a receiving substrate. Additional information about metal assisted exfoliation process is described, for example, in U.S. Patent No. 9,840,024, the content of which is incorporated herein by reference in its entirety. The receiving substrate can be a thin sheet material (e.g., glass, polymer, ceramic, metal, with a thickness of about 10 micrometers to millimeters) with small openings (e.g., microwells) similar to the loading well in the wedge format, in a diameter of about 10 micrometers. The edge stack materials can be patterned into desired electrode shapes and addressed with metal electrodes / traces prior to aluminum deposition. The edge stack / aluminum / perforated sheet support is then anodized in oxalic acid with the aluminum being the anode. Upon oxidation, the aluminum converts to aluminum oxide and a hexagonal array of nanochannels forms open on the side opposite to the edge stack materials (see FIG. 9, step 3). The anodization only occurs within the perimeter of the microwell where oxalic acid is in contact with the aluminum film and forms the channels open into the microwell. The channel density can be controllable with AAO parameters including for example temperature, voltage, reagent concentration, and others identifiable to a person skilled in the art. The channel diameter is between 20 nm and several hundred nanometers and can also be controlled. There is a layer of aluminum oxide 30 nm thick that forms on the edge materials stack interface. The closed-loop edge opening can then be created through the edge stack supported with the 300 nm aluminum oxide via dielectricbreakdown process. The dielectric breakdown process is stochastic and forms only one opening per microwell regardless of the number of nanochannels interfacing the edge membrane. Each microwell can have an individual liquid-biasing electrode in it (similar to El in the wedge format, see FIG. 3) such that the voltage bias across the membrane in all wells individually can be controlled (which is not required for anodization but required for the breakdown process). The electrode can be patterned on either side of the microwell sheet (around the perimeter of the well, for example) or be located inside the microwell. The density of edge sensors in this case will be determined by the density of the microwells.Terminology

[0119] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0120] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0121] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absenceof such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g, the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B ”

[0122] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0123] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0124] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A sensor for characterizing a stranded molecule, comprising: at least one first electrode sheet, at least one second electrode sheet, and at least one first insulating layer disposed between the at least one first electrode sheet and the at least one second electrode sheet, wherein the sensor comprises a sensor edge capable of interacting with a stranded molecule, thereby modulating a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet.

2. The sensor of claim 1, wherein the at least one first electrode sheet and / or the at least one second electrode sheet is atomically thin.

3. The sensor of claim 1 or 2, wherein the at least one first electrode sheet and / or the at least one second electrode sheet comprises a two-dimensional electrically conductive material, optionally the two-dimensional electrically conductive material comprises gold, titanium nitride (TiN), poly (3, 4-ethylenedi oxy thiophene) polystyrene sulfonate (PEDOT:PSS), molybdenum disulfide (M0S2), copper, platinum, nickel, or a combination thereof.

4. The sensor of any one of claims 1-3, wherein the at least one first electrode sheet and the at least one second electrode sheet comprise a same electrically conductive material or different electrically conductive materials.

5. The sensor of any one of claims 1-4, wherein the at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer have adjacent exposed edge portions which collectively form the sensor edge.

6. The sensor of any one of claims 1-5, wherein the at least one first electrode sheet comprises one, two, three, four, or five sheets of electrodes, the at least one second electrode sheet comprises one, two, three, four, or five sheets of electrodes, or both.

7. The sensor of any one of claims 1-6, wherein the at least one first electrode sheet and / or the at least one second electrode sheet comprises graphene.

8. The sensor of any one of claims 1-7, wherein the at least one first electrode sheet comprises graphene and the at least one second electrode sheet does not comprise graphene.

9. The sensor of any one of claims 1-8, wherein the at least one first insulating layer comprises one, two, three, four, or five first insulating layers.

10. The sensor of any one of claims 1-9, wherein the at least one insulting layer comprises ceramics, diamond, two-dimensional polymers, self-assembled monolayers or bilayers, and / or other electrically insulating organic and inorganic moieties, optionally, the ceramics comprises SisN4, AI2O3, HfCh. or a combination thereof.

11. The sensor of any one of claims 1-10, wherein the at least one first insulating layer comprises one or more atomically thin sheets.

12. The sensor of any one of claims 1-11, wherein the at least one first insulating layer comprises boron nitride, optionally the at least one first insulating layer comprises hexagonal boron nitride (hBN).

13. The sensor of any one of claims 1-12, wherein the at least one first insulating layer comprises three sheets of boron nitride or hBN.

14. The sensor of any one of claims 1-13, wherein the at least one first electrode sheet and the at least one second electrode sheet each comprises a single graphene sheet, and the at least one first insulating layer comprises three sheets of hBN.

15. The sensor of any one of claims 1-14, wherein the sensor edge is configured to partially or fully surround the stranded molecule.

16. The sensor of any one of claims 1-15, wherein the sensor edge is in a closed- loop geometry or a linear geometry.

17. The sensor any one of claims 1-16, wherein the sensor edge is in a linear geometry and has a width of about 5-10 nm.

18. The sensor of any one of claims 1-17, wherein the sensor edge is in a closed- loop geometry having a cross-sectional dimension of about 2-3 nm.

19. The sensor of any one of claims 1-18, wherein the sensor edge is functionalized with one or more chemical group(s), optionally the functionalization comprises hydrogenation, hydroxylation, carboxylation, carbonylation, amination, or a combination thereof.

20. The sensor of any one of claims 1-19, wherein the sensor edge is configured to minimize the tunneling electrical resistance between the sensor edge and the stranded molecule, optionally configured with a charge-injecting group.

21. The sensor of any one of claims 1-20, wherein the sensor edge is configured to enhance the tunneling electrical interaction between the sensor edge and the strandedmolecule, optionally the configuration enhances binding with the stranded molecule through hydrogen bond interaction, electrostatic interaction, hydrophobic / intercalating interaction, biomolecular binding, or a combination thereof.

22. The sensor of any one of claims 1-21, wherein the sensor edge comprises a functional group selected from the group consisting of: carbonyl, carboxyl, hydroxyl, aldehyde, carboxylate, ester, or amine functional group, or a combination thereof.

23. The sensor of any one of claims 1-22, wherein the sensor edge is functionalized with a p-phenylenediamine moiety.

24. The sensor of any one of claims 1-23, wherein the edge portions of the at least one first electrode sheet, the at least one second electrode sheet, and the at least one first insulating layer are functionalized with a same functional group or different functional groups.

25. The sensor of any one of claims 1-24, wherein a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet is capable of being measured when the stranded molecule is adhered to the sensor edge and a voltage is applied across the at least one first electrode sheet and the at least one second electrode sheet.

26. The sensor of any one of claims 1-25, further comprising at least one second insulating layer located on a surface of the at least one first electrode sheet or the at least one second electrode sheet, and at least one third electrode sheet located on a surface of the at least one second insulating layers.

27. The sensor of claim 26, wherein the at least one first electrode sheet and the at least one second electrode sheet comprise graphene and the at least one third electrode sheet does not comprise graphene.

28. A sensor device for characterizing a stranded molecule, comprising: a fluidic passage disposed within a support structure, optionally the support structure comprises a semiconductor material, two or more global electrodes capable of providing a voltage to translocate the stranded molecule through the fluidic passage, at least one sensor of any one of claims 1-27 disposed in the support structure with the sensor edge exposed to the fluidic passage; anda detector configured to detect a current between the at least one first electrode sheet and the at least one second electrode sheet as the stranded molecule is adhered to the sensor edge.

29. The sensor device of claim 28, wherein the fluidic passage comprises a vertical fluidic passage section and the at least one sensor is disposed in the support structure with the sensor edge exposed to the vertical fluidic passage section.

30. The sensor device of claim 29, wherein the sensor edge is in a closed-shape geometry or a linear geometry forming one or more apertures in the support structure.

31. The sensor device of any one of claims 28-30, wherein the fluidic passage comprises a lateral fluidic passage section in fluidic connection with the vertical fluidic passage section and wherein the at least one sensor is disposed in a bottom of the lateral fluidic passage section with the sensor edge exposed to the vertical fluidic passage section.

32. The sensor device of any one of claims 28-31, wherein the lateral fluidic passage section is about 50-500 nm in width, 1-15 mm in length, and / or 1-100 nm in height.

33. The sensor device of any one of claims 28-32, wherein the lateral fluidic passage section has an aspect ratio of height to length in a range from about 1 : 10 to about 1 : 1000, optionally from about 1 : 50 to about 1 :500.

34. The sensor device of any one of claims 28-33, wherein the lateral fluidic passage section has a cross-sectional area of about tens to hundreds of nm2.

35. The sensor device of any one of claims 28-34, wherein the lateral fluidic passage section and / or the vertical fluidic passage section has a dimension configured to accommodate no more than one stranded molecule.

36. The sensor device of any one of claims 28-35, wherein the lateral fluidic passage section has a wedge shape, optionally an opening of the lateral fluidic passage section to the vertical fluidic passage section is about 5-10 nm in width.

37. The sensor device of any one of claims 28-36, wherein the lateral fluidic passage section and / or the vertical fluidic passage section is in nanometer or micrometer scale.

38. The sensor device of any one of claims 28-37, wherein the lateral fluidic passage section and / or the vertical fluidic passage section has a chemically modified inner surface, optionally the inner surface is chemically modified to carry a charge.

39. The sensor device of claim 38, wherein the inner surface comprises glass, a self-assembled monolayer, an organic molecule, aluminum oxide, a lipid bilayer, or a combination thereof.

40. The sensor device of any one of claims 28-39, wherein the lateral and / or vertical fluidic passage section is fdled with hydrogel, optionally the hydrogel is naturally occurring or synthetic.

41. The sensor device of claim 40, wherein the hydrogel is agarose, polyacrylamide, PVA, or a combination thereof.

42. The sensor device of any one of claims 28-41, wherein the lateral and / or vertical fluidic passage section comprises nanoparticle matrices.

43. The sensor device of any one of claims 28-42, wherein the sensor device is a sensor chip.

44. The sensor device of any one of claims 28-43, comprising at least two, three, four, or five sensors, optionally each sensor is exposed to a distinct fluidic passage.

45. The sensor device of any one of claims 28-44, further comprising a loading well and a fluidic reservoir both in fluidic connection to the fluidic passage, wherein the loading well provides an inlet to the fluidic passage and the fluidic reservoir provides an outlet from the fluidic passage.

46. The sensor device of any one of claims 28-45, further comprising: (a) multiple electrodes and / or multiple insulating layers stacked on top of each other; (b) multiple sensors stacked on top of each other; and / or (c) multiple devices stacked on top of each other, optionally said configuration improves signal.

47. A sensor device for characterizing a stranded molecule, comprising: a fluidic passage disposed in a support structure, wherein the fluidic passage comprises a lateral fluidic passage and a vertical fluidic passage, wherein the lateral fluidic passage is in fluidic connection with the vertical fluidic passage, optionally the support structure comprises a semiconductor material, two or more global electrodes capable of providing a voltage to translocate the stranded molecule through the lateral and vertical fluidic passages, at least one sensor disposed in a bottom of the lateral fluidic passage, wherein the at least one sensor comprises at least one first graphene sheet, at least one second graphenesheet, and at least one first insulating layer disposed between the at least one first graphene sheet and the at least one second graphene sheet, and wherein the at least one sensor comprises a sensor edge capable of interacting with the stranded molecule, thereby modulating a tunneling current between the at least one first graphene sheet and the at least one second graphene sheet; and a detector configured to detect an electrical signal between the at least one first graphene sheet and the at least one second graphene sheet when the stranded molecule is adhered to the sensor edge.

48. A method of characterizing a stranded molecule, comprising: providing the sensor device of any one of claims 28-47; applying a voltage across the fluidic passage to produce a current flow through the fluidic passage, causing a single stranded molecule to translocate through the fluidic passage at a time and allowing said single stranded molecule to interact with the sensor edge of the at least one sensor; detecting a first electrical signal between the at least one first electrode sheet and the at least one second electrode sheet of the sensor edge when a first portion of the stranded molecule is adhered to the edge portions of the at least one first electrode sheet and the at least one second electrode sheet of the at least one sensor; and determining a characteristic of the first portion of the stranded molecule based on the detected first electrical signal.

49. The method of claim 48, further comprising: translocating the stranded molecule across the sensor edge; and measuring a second electrical signal through the edges when a second portion of the strand molecule is adhered to both edge portions of the at least one first electrode sheet and the at least one second electrode sheet, following the translocation of the stranded molecule.

50. The method of claim 48 or 49, further comprising linearizing the stranded molecule along the fluidic passage, optionally by adjusting the magnitude and direction of the voltage across the fluidic passage.

51. The method of any one of claims 48-50, wherein the first portion of the stranded molecule is in a stretched conformation when the first portion of the strandedmolecule is adhered to both edge portions of the at least one first electrode sheet and the at least one second electrode sheet of the at least one sensor.

52. The method of any one of claims 48-51, wherein the sensor edge of the at least one sensor is functionalized with one or more chemical group(s) and the adhesion between the stranded molecule and the sensor edge of the at least one sensor occurs through electrostatic interaction, hydrogen bonding, hydrophobic interaction, intercalating interaction, ionic interaction, or a combination thereof between the stranded molecule and the chemical group(s).

53. The method of any one of claims 48-52, wherein detecting the first electrical signal and / or the second electrical signal comprises applying a voltage between the at least one first electrode sheet and the at least one second electrode sheet and measuring a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet.

54. The method of any one of claims 48-53, wherein detecting the first electrical signal and / or the second electrical signal comprises applying a voltage across the global electrodes and measuring a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet.

55. The method of any one of claims 48-54, wherein detecting the first electrical signal and / or the second electrical signal comprises measuring an ionic current flowing through the edge portions of the at least one first electrode sheet and the at least one second electrode sheet.

56. The method of any one of claims 48-55, further comprising measuring an inplane tunneling conductance across the first portion of the stranded molecule.

57. The method of claim 56, wherein the stranded molecule is fully enclosed by the sensor edge.

58. The method of any one of claims 48-57, wherein translocating the stranded molecule comprises: dissociating the first portion of the stranded molecule from the edge portions of the at least one first electrode sheet and the at least one second electrode sheet, and adhering the second portion of the stranded molecule to the edge portions of the at least one first electrode sheet or the at least one second electrode sheet.

59. The method of claim 58, wherein dissociating the first portion of the stranded molecule from the edge portions comprises dissociating the first portion of the stranded molecule from the edge portion of the at least one first electrode, followed by dissociating the first portion of the stranded molecule from the edge portion of the at least one second electrode.

60. The method of claim 59, wherein the stranded molecule is in a relaxed state during said dissociating.

61. The method of any one of claims 56-60, wherein the stranded molecule remains adhered to at least one edge portion of the at least one first electrode sheet or the at least one second electrode sheet.

62. The method of any one of claims 56-61, wherein dissociating the first portion of the stranded molecule and adhering the second portion of the stranded molecule comprises (1) adjusting the magnitude and / or direction of the voltage between a global electrode and the at least one first electrode sheet or the at least one second electrode sheet, (2) adjusting the interaction between the edge portions and the stranded molecule, or both.

63. The method of any one of claims 48-62, wherein translocating the stranded molecule moves the stranded molecule across the sensor edge by a portion or a unit of the stranded molecule.

64. The method of any one of claims 48-63, wherein the stranded molecule is partially or fully surrounded by the sensor edge.

65. The method of any one of claims 48-64, wherein the characteristic of the first portion of the stranded molecule comprises polynucleotide sequence, polynucleotide methylation, polypeptide sequence, protein glycosylation, protein-polynucleotide binding event, or a combination thereof.

66. The method of any one of claims 48-65, wherein the stranded molecule is a nucleic acid molecule and determining the characteristic of the first portion of the stranded molecule comprises determining a sequence information of a nucleic acid base.

67. The method of any one of claims 48-66, further comprising depositing a sample comprising the stranded molecule in a loading well of the sensor device.

68. The method any one of claims 48-67, wherein the stranded molecule comprises a polymer, a polynucleotide, an oligonucleotide, a polysaccharide, a polypeptide, or a combination thereof.

69. A system for characterizing a stranded molecule, comprising: one or more sensor devices of any one of claims 28-47; an electronic control system electrically connected to the one or more sensor devices, the electronic control system configured to apply an input electrical signal to the one or more sensor devices and to receive an electrical signal from the one or more sensor devices when a stranded molecule is adhered to the at least one sensor edge of the one or more sensor device; and a computer control system for analyzing the output electrical signal from the one or more devices to determine a characteristic of the stranded molecule.

70. The system of claim 69, wherein the input electrical signal applies a voltage across the two or more global electrodes, a voltage across the at least one first electrode sheet and the at least one second electrode sheet, or both.

71. The system of claim 69 or 70, wherein the input electrical signal comprises an alternating current voltage, a direct current voltage, an electrical impedance, or a combination thereof.

72. The system of any one of claims 69-71, wherein the output electrical signal comprises a tunneling current between the at least one first electrode sheet and the at least one second electrode sheet, a voltage drop between the at least one first electrode sheet and the at least one second electrode sheet, an ionic current flowing through the edge portions of the at least one first electrode sheet and the at least one second electrode sheet, an in-plane tunneling conductance across the stranded molecule, or a combination thereof.

73. A method of making a sensor device, comprising: depositing and patterning at least one first electrode sheet, at least one first insulating layer, and at least one second electrode sheet on a substrate; depositing one or more dielectric layer to the substrate and patterning the one or more dielectric layer to form a lateral fluidic passage; and etching a vertical fluidic passage, the vertical fluidic passage in fluidic connection with the lateral fluidic passage.

74. The method of claim 73, wherein depositing and patterning the at least one first electrode sheet, the at least one first insulating layer, and the at least one second electrode sheet on a substrate comprises depositing the at least one first electrode sheet, patterning the at least one first electrode sheet to a wedge shape, depositing the at least one first insulating layer and the at least one second electrode sheet on the at least one first electrode sheet, and patterning the at least one first insulating layer and the at least one second electrode sheet to a wedge shape.

75. The method of claim 73 or 74, further comprising: depositing a first conductor layer on the substrate structure prior to depositing the at least one second electrode sheet; and depositing a second conductor layer on the at least one first electrode sheet following depositing the at least one first electrode sheet.

76. The method of claim 75, wherein the first conductor layer and / or the second conductor layer comprises a metal pad, optionally the metal pad is a Ti / Au pad or a Cr / Au pad.

77. The method of any one of claims 73-76, wherein depositing one or more dielectric layer comprises sequentially depositing a first dielectric layer and a second dielectric layer.

78. The method of claim 77, wherein the first and second dielectric layers comprise a same dielectric material or different dielectric materials.

79. The method of claim 77 or 78, wherein the first and second dielectric layers are different in thickness.

80. The method of any one of claims 73-79, further comprising applying a capping layer and global electrodes on the top of the one or more dielectric layer, optionally the capping layer is made of glass and comprises a prepatterned opening for loading a molecule.

81. A method of making a sensor device, comprising: providing a stack film comprising at least one first electrode sheet, at least one first insulating layer and at least one second electrode sheet, the stack being deposited with a metal film and with a substrate comprising one or more microwells in connection with the metal film;anodizing the metal film in an electrolyte solution, thereby converting the metal to metal oxide and forming a plurality of nanochannels in the metal film; and forming an aperture through the stack film in a microwell by applying a voltage across the stack film.

82. The method of claim 81, wherein the metal film comprises aluminum, magnesium, titanium, or a combination thereof, optionally the metal film is an aluminum film.

83. The method of claim 81 or 82, wherein the electrolyte solution is an oxalic acid.

84. The method of any one of claims 81-83, wherein the density and / or dimension of the plurality of nanochannels are controlled by adjusting the voltage and temperature of the anodization and / or the concentration of the electrolyte solution.

85. The method of any one of claims 81-84, further comprising positioning a global electrode in each of the one or more microwells.

86. The method of any one of claims 81-85, wherein the aperture has a dimension of about lnm-5nm.

87. The method of any one of claims 81-86, wherein the plurality of nanochannels have a dimension of 20nm-100nm.

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