High-density multiplexed nanopore devices with lateral tunnel junctions for biomolecular detection and sequencing

Abstract

Systems and methods are disclosed for delivering and / or linking molecules such as DNA between tunable metallic nanogaps and measuring electrical and / or optical properties. As an example, a high-density multiplexed chip is disclosed having multiple groups, each group containing multiple nanodevices, and the chip can be coupled to one or more multi-well structures for delivering samples to each individual group. In this manner, the chip can be used for high-throughput analysis of molecules such as DNA.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 161,681, filed March 16, 2021, entitled "HIGH DENSITY AND MULTIPLEXED NANOPORE DEVICES WITH TRANSVERSE TUNNELING JUNCTION FOR BIOMOLECULE DETECTION AND SEQUENCING," the entire contents of which are incorporated herein by reference.

[0002] Government Licensing Rights This invention was made with government support under FA9550-16-1-0052 awarded by the Air Force Office of Scientific Research. The government has certain rights in this invention.

[0003] The present disclosure relates to single molecule detection, and in particular to systems and methods for making and using nanopore devices having lateral tunnel junctions. [Background technology]

[0004] Nanopore sequencing has shown promising promise for rapid single-molecule sequencing and characterization. Biological nanopores based on membrane protein channels and their bioengineered variants offer atomic-level reproducibility in shape and size, allowing them to be engineered to match the diameter of biomolecules (e.g., DNA) for slower translocation speeds and higher contrast in ionic currents. However, challenges remain in terms of signal quality and resolution. These challenges have led to the development of quantum tunneling-based detection methods. This method requires the integration of solid-state nanopores and tunnel junctions. However, existing approaches lack precision and reproducibility and have met with little success. Therefore, to improve quantum tunneling-based detection technologies and realize the potential for high-throughput analysis, it is necessary to develop fabrication methods that enable the high density of solid-state nanopores on a single chip, enabling parallel processing of samples. Summary of the Invention

[0005] Disclosed herein are devices, systems, fabrication methods, and their uses for measuring the electrical and / or optical properties of single molecules using tunable metallic nanogaps embedded in nanofluidic systems. The disclosed device fabrication methods, as described herein, enable the fabrication of a single chip with a high-density array of nanodevices (also called electronic devices or electronic nanodevices). The nanodevices can be grouped into distinct groups on a single chip, thereby providing the ability to effectively present different samples to different groups for high-throughput analysis of the electrical and / or optical properties of molecules corresponding to the different samples. In embodiments, translocation and / or mounting / linking events between a molecule and an electrode containing a tunable metallic nanogap can be characterized based on ionic current traces between an upper and lower chamber with the nanogap disposed therebetween, based on tunneling current measured by the nanogap electrode, and / or based on the optical properties of the molecule.

[0006] Thus, in one embodiment, an electronic device comprises an upper fluidic channel / chamber and a lower fluidic channel / chamber fabricated on a planar substrate, the upper fluidic channel / chamber including a first open window at the top of the electronic device but sealed at the bottom of the electronic device, and the lower fluidic channel / chamber including a second open window at the bottom of the electronic device but sealed at the top of the electronic device. The electronic device further includes a first electrode and a second electrode disposed in a space of the electronic device between the upper and lower fluidic channel / chambers. The electronic device further includes a nanogap having a dimension determined in part by the distance between the first and second electrodes, the distance being defined by electrochemically depositing the first and second electrodes under feedback control with one or more metallic materials in the upper and / or lower fluidic channel / chamber, thereby forming an electronic device with a single pathway for molecules to move from the upper to the lower fluidic channel / chamber, or vice versa. The distance between the first and second electrodes is between about 1 and 100 nm, and the nanogap is self-aligned, with the narrowest bottleneck in a single path between the upper fluidic channel / chamber and the lower fluidic channel.

[0007] In one embodiment, a method for measuring electronic and / or optical properties from a single molecule comprises using an electronic device as disclosed herein to detect individual mounting and / or rearrangement events of a single molecule by correlated ionic currents between an upper fluidic channel / chamber and a lower fluidic channel / chamber, and tunneling currents between a first electrode and a second electrode through a nanogap, and to perform electrical and / or optical characterization.

[0008] In one embodiment, performing electrical and / or optical characterization includes performing Raman spectroscopy through a transparent substrate or from the top side of a non-transparent substrate of the electronic device to characterize the dynamic structure of a single molecule.

[0009] In one embodiment, performing the electrical and / or optical characterization includes determining a sequence corresponding to a single molecule.

[0010] In one embodiment, a chip for measuring electrical and / or optical properties from single molecules comprises a plurality of electronic devices as disclosed herein, which may be between 2 and 1000, or between 1000 and 10,000, and in some examples may be greater than 10,000.

[0011] In embodiments, the plurality of electronic devices are divided into a predetermined number of different groups. In some examples, one or more multiwell structures may be coupled to the chip, with individual wells corresponding to each of the predetermined number of different groups. In some examples, the one or more multiwell structures comprise: a first multiwell structure coupled to an upper side of the chip for delivering sample to an upper fluidic channel / chamber of each of the plurality of electronic devices; and, optionally, a second multiwell structure coupled to a lower side of the chip for receiving the sample initially delivered to the upper fluidic channel / chamber after migration of analytes in each sample to the lower fluidic channel / chamber.

[0012] In an embodiment, the chip may include one or more multiplexers associated with each group to simultaneously collect signals from the electronic devices corresponding to that particular group.

[0013] In one embodiment, a system for high-throughput analysis of single molecules includes a chip as disclosed herein comprising any number of electronic nanodevices as disclosed herein and a fluidic device capable of providing individual samples to each of a plurality of distinct groups. The system further includes a controller storing instructions in a non-transitory memory that, when executed, directs the fluidic device to provide individual samples to one or more of the plurality of distinct groups, and after providing, records data including one or more of an ionic current between an upper fluidic channel / chamber and a lower fluidic channel / chamber, a tunneling current, and / or an optical signal from each electronic device corresponding to one or more of the plurality of distinct groups, the data corresponding to individual mounting / linking and / or translocation events of the individual molecules within each electronic device.

[0014] In one embodiment, a method for fabricating an electronic device including a nanopore and a tunnel junction includes depositing a first sacrificial layer that defines a final cavity for electrochemical deposition and depositing a second outer sacrificial layer on a substrate layer that defines a final nanofluidic space connecting the nanopore with an upper fluidic channel / chamber and a lower fluidic channel / chamber; disposing a pair of electrodes on top of the first sacrificial layer at a spacing of about 600 nm to 2 μm; depositing a passivation layer on top of the pair of electrodes, the first sacrificial layer, the second outer sacrificial layer, and the planar substrate; and dry etching. performing a process to remove a first portion of the passivation layer and a second portion of the substrate layer from a lower side of the substrate layer, thereby providing a first window in the second outer sacrificial layer corresponding to the upper fluid channel / chamber and a second window in the second outer sacrificial layer corresponding to the lower fluid channel / chamber; chemically etching the first and second sacrificial layers to construct a final nanofluidic space; and narrowing the gap between the pair of electrodes to form a nanopore and a tunnel junction by a controlled electrochemical deposition process of metal onto the pair of electrodes.

[0015] The foregoing and other features of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. [Brief explanation of the drawings]

[0016] Embodiments will be readily understood from the following detailed description taken in conjunction with the accompanying drawings and appended claims, which are presented by way of example and not limitation in the accompanying drawings. [Figure 1A] Figure 1A shows a high-level diagram of a biomolecular translocation detection circuit. In such a circuit, a biomolecule (e.g., DNA, protein, peptide, small molecule, RNA, mRNA, etc.) is moved from the cis chamber to the trans chamber by a bias (e.g., a negative bias for DNA), and the ionic current (I) is recorded. Simultaneously, a small bias is applied between transverse electrodes, and the current flowing through the tunneling junction (ITunneling) is recorded to track the same translocation event. A pair of metal electrodes forms a nanogap through which the biomolecule is driven. [Figure 1B] Figure 1B shows that nearly 100% correlated signals from both the ionic and tunneling current channels can identify the same DNA translocation event. Figure 1B shows a single representative event showing the correlation in time and polarity between the tunneling and ionic currents. [Figure 1C] Figure 1C shows that nearly 100% correlated signals from both the ionic and tunneling current channels can identify the same DNA translocation event. Figure 1C shows a longer time record of translocation occurring in both directions, depicted by the polarity of the signal. [Figure 1D]Figure ID shows that nearly 100% correlated signals from both the ionic and tunneling current channels can identify the same DNA translocation event. Figure ID shows a longer recording of translocation occurring in both directions, indicated by the polarity of the signal. The signals in Figures 1B-1D are illustrative of the types of signals that can be recorded using devices of the type shown in Figure 1A and electronic devices and chips incorporating electronic devices of the present disclosure. [Figure 2A] FIG. 2A shows a representative example of the initial structure (left) from which the fabrication of a nanodevice of the present disclosure begins, and the final structure after narrowing the gap between the electrodes. [Figure 2B] FIG. 2B is a schematic diagram illustrating various stages of fabrication of the disclosed nanodevice according to embodiments disclosed herein. [Figure 3A] FIG. 3A illustrates how reversible pulse electrochemical deposition can be used to precisely control the dimensions of nanopores and tunnel junctions. [Figure 3B] FIG. 3B illustrates how reversible pulse electrochemical deposition can be used to precisely control the dimensions of the nanopore and tunnel junction. [Figure 3C] FIG. 3C illustrates how reversible pulsed electrochemical deposition can be used to precisely control the dimensions of nanopores and tunnel junctions. [Figure 3D] FIG. 3D illustrates how reversible pulsed electrochemical deposition can be used to precisely control the dimensions of nanopores and tunnel junctions. [Figure 3E] FIG. 3E illustrates how reversible pulsed electrochemical deposition can be used to precisely control the dimensions of nanopores and tunnel junctions. [Figure 4A] Figure 4A shows selected frames of a time-lapse recording during electrode metal deposition with different deposition pulses. [Figure 4B] Figure 4B shows selected frames of a time-lapse recording during electrode metal deposition with different deposition pulses. [Figure 4C]FIG. 4C exemplarily shows options for influencing / optimizing the nanopore shape during the metal electrodeposition process onto the tunnel junction electrodes. [Figure 4D] FIG. 4D exemplarily shows options for influencing / optimizing the nanopore shape during the metal electrodeposition process onto the tunnel junction electrodes. [Figure 5A] FIG. 5A shows a top view of a nanodevice according to an embodiment disclosed herein, exemplarily illustrating a DNA translocation pathway through the nanogap. [Figure 5B] FIG. 5B shows a cross-sectional view of a nanodevice according to embodiments disclosed herein, exemplarily illustrating a DNA translocation pathway through the nanogap. [Figure 6A] FIG. 6A is a three-dimensional view of a nanodevice according to an embodiment disclosed herein. [Figure 6B] Figure 6B is a three-dimensional view of a nanodevice according to an embodiment disclosed herein. Figure 6B is a transparent rendering of the nanodevice depicted in Figure 6A. [Figure 7] FIG. 7 illustrates a schematic diagram of a single chip including multiple nanodevices according to embodiments disclosed herein, the multiple nanodevices being arranged in an array of defined groups. [Figure 8] FIG. 8 shows a high level diagram of a single chip of the present disclosure with multiple electronic circuits integrated into the substrate of the chip, with respect to upper and lower multi-well structures. [Figure 9] FIG. 9 illustrates a high-level exemplary system for automated use of nanodevices and / or chips of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] In the following detailed description, references are made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration embodiments that may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope. Therefore, the following detailed description is not to be taken in a limiting sense.

[0018] Although various steps may be described in sequence as multiple separate steps in a manner that is useful in understanding the embodiments, the order of description should not be construed to imply that these steps are order dependent.

[0019] This specification may use perspective descriptions such as top / bottom, back / front, upper / lower, etc. Such descriptions are used merely to facilitate discussion and are not intended to limit the application of the disclosed embodiments.

[0020] The terms "coupled" and "coupled," along with their derivatives, may be used. It should be understood that these terms are not intended as synonyms for each other. Rather, in particular embodiments, "coupled" may be used to indicate that two or more elements are in direct physical or electrical contact with each other. "Coupled" may mean that two or more elements are in direct physical or electrical contact. However, "coupled" may also mean that two or more elements are not in direct contact with each other, but yet still cooperate or interact with each other.

[0021] As used herein, a phrase in the form "A / B" or "A and / or B" means (A), (B), or (A and B). As used herein, a phrase in the form "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C). As used herein, a phrase in the form "(A)B" means (B) or (AB), i.e., A is an optional element.

[0022] The terms "embodiment" or "embodiments" may be used herein, and may each refer to one or more of the same or different embodiments. Furthermore, terms such as "comprising," "having," and the like, when used with respect to embodiments, are intended to be synonymous and generally "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 "is not limited to," etc.).

[0023] With respect to the use of any plural and / or singular terms herein, those skilled in the art will be able to convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0024] The terms "a" or "an" may mean one or more than one item.

[0025] The term "about" means within plus or minus 10% of the stated value. For example, "about 100" refers to any number between 90 and 110. Furthermore, the recitation of numerical ranges includes any number subsumed within that range and / or any range of numbers subsumed within that range. For example, a numerical range of 1 to 10 encompasses that range and further includes individual numbers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and ranges within that numerical range (e.g., 1-2, 1-4, 2-5, 3-7, 4-9, 5-10, etc.).

[0026] Unless otherwise noted, technical terms are used according to conventional usage. Methods and materials suitable for practicing or testing the present disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods known in the art relevant to the present disclosure are described in various general references.

[0027] I. Terminology

[0028] To facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0029] Analyte: A substance whose chemical constituents are identified and measured. In some examples, analytes include DNA, proteins, enzymes, RNA, small molecules, peptides, and / or other biomolecules.

[0030] Binding or stable binding: A bond between two substances or molecules, such as the binding of an antibody to an antigen. Binding can be detected by any procedure known to those skilled in the art, such as the physical or functional properties of the complex formed.

[0031] Biomolecule: A molecule produced by a living organism and / or that has some activity in the body. For example, a biomolecule is an organic molecule, particularly a biological macromolecule (such as a protein or nucleic acid). In some instances, a biomolecule is interchangeable with an "analyte molecule." In some instances, therapeutic biomolecules can be artificially produced for use in the body.

[0032] Chemical Modification: Many different processes involving a change in the chemical makeup or structure of a molecule. As an example, a chemically modified electrode is one whose surface has been chemically altered to change its physical, chemical, electrochemical, optical, electrical, and / or transport properties.

[0033] Top chamber and bottom chamber: A "top chamber" is a first chamber, and a "bottom chamber" is a second chamber opposite the top chamber, such as the opposite side of the top chamber. As used herein, a top chamber opens to the top side of a nanodevice, and a bottom chamber opens to the bottom side of a nanodevice. In embodiments, the top chamber has a negative electrode, and the bottom chamber opposite the nanogap has a positive electrode, such that negatively charged molecules in the top chamber can be guided through the nanogap to the bottom chamber by a driving bias. The top chamber is also referred to as a "cis chamber," and the bottom chamber is also referred to as a "trans chamber." In other examples, the potentials applied to the top and bottom chambers can be reversed in polarity. That is, applying a negative potential to the top chamber and a positive potential to the bottom chamber can reverse the movement of molecules through the channel.

[0034] Contacting: To be placed in direct physical relationship, including both solids and liquids.

[0035] Deposit: A collection or layer of deposited or layered solid material, cohesive or non-cohesive.

[0036] Dielectric: A dielectric material is a type of insulator that polarizes when exposed to an electric field. When a dielectric is placed in an electric field, virtually no current flows through it. This is because, unlike metals, dielectrics do not have loosely bound or free electrons that can drift through the material. Instead, electric polarization occurs.

[0037] Electrochemical Deposition: A process for depositing a thin, tightly adherent coating of a desired metal, oxide, or salt on the surface of a conductive substrate by simple electrolysis of a solution containing the desired metal ions or their chemical complexes. Electrochemical deposition uses an electric field to transport metal ions in solution and coat the substrate surface. Electrochemical deposition is an efficient procedure for preparing metal nanoparticles. This process may be reversible. For example, the term "reversible pulse electrochemical deposition" discussed herein refers to a process in which the desired coating can be both deposited and removed any number of times.

[0038] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified from the cells of the organism in which it naturally occurs, or from other biological components of the organism itself (such as other chromosomal and extrachromosomal DNA, RNA, proteins, and cells). "Isolated" nucleic acid molecules and proteins can be understood to have been purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0039] Label: A detectable agent, such as a label, can be attached (indirectly or directly) to a nucleic acid molecule or protein, thereby allowing the detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent agents, fluorescent agents, haptens, enzymes, and combinations thereof. Guidance on labeling methods and the selection of suitable labels for various purposes is discussed, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0040] Linked or linker: The term "linked" means directly or indirectly attached. For example, a first moiety can be covalently or non-covalently (e.g., electrostatically) linked to a second moiety. This includes, but is not limited to, covalently binding one molecule to another, non-covalently binding one molecule to another (e.g., electrostatically), non-covalently binding one molecule to another through hydrogen bonding, non-covalently binding one molecule to another through van der Waals forces, and any combination of such bonds. Indirect binding is also possible, such as through the use of a "linker" (a molecule or group of atoms positioned between two moieties).

[0041] In some embodiments, linked components are associated by chemical or physical means such that the components cannot freely disperse from one another, e.g., two components can be covalently bonded to one another such that they cannot disperse or diffuse separately.

[0042] Microfluidics: As discussed herein, "microfluidics" refers to both the behavior of fluids through microchannels and the fabrication technology of ultra-small devices containing chambers or tunnels through which fluids flow or are confined. Microfluidics pertains to small volumes of fluid, down to femtoliters. As discussed herein, "microfluidic device" or "microfluidic system" refers to a fabricated structure (e.g., a microfluidic chip) having any number of microchannels etched or molded into a material (e.g., glass, silicon, PDMS, etc.). "Microfluidic system" refers to a microfluidic device that incorporates other aspects, including, but not limited to, pumps, pressure regulators, etc. Microfluidic devices / systems are encompassed by the terms "fluidic device" and "fluidic system" as disclosed herein, although "fluidic device / system" may additionally or alternatively refer to a larger-scale device that operates to flow fluids from one location to another. In an example, the fluidic devices / systems of the present disclosure can be automated.

[0043] Multiplexing Electronics: The term "multiplexing electronics" refers to electronic equipment capable of multiplexing, combining multiple analog or digital signals into a single signal over a shared medium. A "multiplexer" refers to a combinational logic circuit that functions as a switcher, switching multiple inputs to a single common output line. Also called a "MUX," it transmits digital or analog signals at high speed over a single line and a shared device, restoring the individual signals at the receiving end.

[0044] Nanogap: The term "nanogap" refers to a pathway of nanoscale dimensions through which a biomolecule travels during its journey from the upper chamber to the lower chamber (or vice versa) of a nanopore device as disclosed herein. The nanogap is formed by the space between two lateral electrodes, and the dimensions (e.g., width) of the nanogap can be altered by controlled electrochemical deposition, e.g., reversible pulse electrochemical deposition / dissolution. Herein, a nanogap is also referred to as a "nanopore."

[0045] Nucleic Acid: A deoxyribonucleotide or ribonucleotide polymer that may contain analogs of natural nucleotides that hybridize to a nucleic acid molecule in a manner similar to naturally occurring nucleotides. In a specific example, a nucleic acid molecule is a single-stranded (ss) DNA or RNA molecule, such as a probe or primer. In another specific example, a nucleic acid molecule is a double-stranded (ds) nucleic acid, such as a target nucleic acid. The term "nucleotide" refers to a base-sugar-phosphate combination and includes ribonucleoside triphosphates ATP, UTP, CTG, GTP, and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.

[0046] Optional: "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0047] Protein: The terms "protein," "peptide," and "polypeptide" interchangeably refer to a polymer of amino acids and / or amino acid analogs joined by peptide bonds or peptide bond mimetics. The 20 naturally occurring amino acids and their one-letter and three-letter names are as follows: alanine (A) Ala, cysteine ​​(C) Cys, aspartic acid (D) Asp, glutamic acid (E) Glu, phenylalanine (F) Phe, glycine (G) Gly, histidine (H) His, isoleucine (Ile), lysine (K) Lys, leucine (L) Leu, methionine (M) Met, asparagine (N) Asn, proline (P) Pro, glutamine (Q) Gln, arginine (R) Arg, serine (S) Ser, threonine (T) Thr, valine (V) Val, tryptophan (W) Trp, and tyrosine (Y) Tyr. In one embodiment, the protein / peptide / polypeptide is an antibody or a fragment or portion thereof. In some embodiments, the protein / peptide / polypeptide may be post-translationally modified.

[0048] Raman spectroscopy: A spectroscopic technique typically used to determine the vibrational modes of molecules, although rotational and other low-frequency modes of the system may also be observed. Raman spectroscopy is used in chemistry to obtain structural fingerprints for molecular identification.

[0049] Sample: A mixture of molecules, including at least analyte molecules, that is subjected to manipulation according to the nanodevices, chips, systems and / or methods of the present disclosure.

[0050] Translocation: Change in position. As used herein, a translocation event refers to the movement of a biomolecule (e.g., DNA) through a nanogap.

[0051] Transparent Substrate: A material composed of components with a uniform refractive index. A transparent material exhibits one color overall, or a vibrant spectrum of colors in any combination, and appears transparent; light can pass through the substrate without significant scattering. The opposite of translucent is opaque or non-transparent. Examples of transparent substrates include, but are not limited to, glass and quartz.

[0052] Under sufficient conditions: A phrase used to describe any environment that allows for a desired activity.

[0053] II. Overview

[0054] Many challenges remain to be overcome before the full potential of nanodevices integrated on a chip can be realized. One problem with current approaches is that the microfluidic interface with the nanodevices occupies a large area relative to the total chip size, significantly reducing the density of nanodevices that can be included on a single chip. Delivering different samples to different groups of nanodevices can be tedious and challenging. For example, this requires multiple channels, complicating the overall assembly process and potentially significantly reducing chip yield. Of course, a reduced nanodevice density also reduces the opportunities for high-throughput analysis. Another issue is that currently, the width of the barrier between adjacent microfluidic channels must be large enough to compensate for alignment errors and overflow of adhesive structures, which can significantly increase etching times due to increased diffusion lengths. Therefore, the travel distance to reach the tunnel junction is longer, potentially leading to clogging and reduced detection rates.

[0055] It is noted herein that the above-mentioned problems can be at least partially addressed by a nanodevice fabrication method that includes disposing / fabricating cis and trans chambers around a tunnel junction structure (e.g., a nanogap) in a manner that utilizes both sides of a substrate, as opposed to fabrication methods that utilize only one side of the substrate. By utilizing both sides of the substrate for fabrication, the chambers disposed / fabricated around the tunnel junction substantially correspond to an upper chamber (cis chamber) and a lower chamber (trans chamber), as opposed to the cis and trans chambers being each located on a specific side (e.g., top) of the substrate. More specifically, in the fabrication approach outlined herein, the resulting upper chamber is accessible (e.g., open) from the top of the nanodevice, but the lower chamber is inaccessible from the top of the nanodevice because it is sealed at the top. Meanwhile, the lower chamber is accessible (e.g., open) from the bottom of the nanodevice, but is inaccessible from the bottom of the nanodevice because it is sealed at the bottom.

[0056] The nanodevice architecture disclosed herein is advantageous in that it allows for dramatically increased density of nanodevices on a single chip (e.g., footprints of 10 μm × 10 μm to 100 μm × 100 μm compared to millimeter dimensions). Using the nanodevice fabrication approach disclosed herein, nanodevices can be easily integrated into arrays on a single chip and grouped into distinct groups. The structural design disclosed herein allows for the integration of multiple electronics within the substrate, advantageously improving signal quality and enabling simultaneous electrodeposition and sample analysis. Furthermore, the structural design of the nanodevice disclosed herein can simplify sample delivery. For example, a multiwell structure covering the top and, optionally, bottom, allows for easy delivery of different samples to different device groups on a single chip, allowing for analysis of such samples in parallel or sequentially, e.g., in a predefined order. This multiwell approach can reduce or completely avoid issues associated with microfluidic channels, such as leakage. Furthermore, due to the structural design of the nanodevices disclosed herein, the ionic current of each group of nanodevices on a single chip can capture dislocation events from all nanodevices corresponding to a particular group, while the tunneling signal from each nanodevice can be correlated to the total ionic current for the group. For example, for each group, the ionic current corresponding to each nanodevice can be integrated to obtain the total ionic current associated with dislocation events, and the tunneling signal from each nanodevice can be correlated to the integrated total ionic current. The integrated ionic current can provide an indication of the total number of dislocation events that have occurred within a particular group, reducing the demands on the electronics associated with the chips and nanodevices disclosed herein.

[0057] Manufacturing Procedures According to One or More Exemplary Embodiments Next, an exemplary procedure for fabricating a nanodevice of the present disclosure will be described with reference to Figure 2A and steps 1 through 8 in Figure 2B. Referring to Figure 2A, the left side shows an initial structure that serves as the starting point for a nanodevice of the present disclosure, in which a pair of electrodes is sandwiched between dielectric passivation layers to form a confined cavity. The initial distance between the electrodes can be between 200 nm and 5 μm, e.g., between 500 nm and 2 μm, e.g., between 600 nm and 2 μm, e.g., about 1 μm. The cavity thickness can be approximately 10-20 nm, which can be easily fabricated using top-down lithography protocols. The gap between the electrodes can then be finely reduced in a reversible and controllable manner (e.g., controlled electrochemical deposition) to construct a tunnel junction and simultaneously form a nanopore device at its narrowest point (see the right side of Figure 2A). The electrodes and dielectric layer separate the chip space into two chambers (e.g., upper and lower), between which the nanopore and tunnel junction are self-aligned, so that molecules moving through the nanopore from one chamber to the other must pass between the electrodes.

[0058] Turning now to Figure 2B, steps 1-6 illustrate exemplary fabrication steps for a nanopore device, including construction of an initial device (e.g., similar to that depicted in Figure 2A), and steps 7-8 illustrate final preparation. The drawing in Figure 2B is not to scale for clarity. The dotted lines in the top view indicate the location of the cross section.

[0059] Sacrificial Structure Layout

[0060] The first step is to define an inner sacrificial layer 102 on a substrate 101. The substrate 101 may comprise one or more of silicon with a SiO2 and / or Si3N4 coating, sapphire, quartz, or other materials compatible with top-down lithography. The inner sacrificial layer 102 may comprise one or more of chromium (Cr), nickel (Ni), magnesium (Mg), and / or aluminum (Al). The inner sacrificial layer has a thickness of 1-100 nm and dimensions of 10 μm x 10 μm.

[0061] In a second step, outer trenches and outer sacrificial layers 103 are defined on both sides of the inner sacrificial layer, with overlapping edges. For the second step, etching of the trenches into the substrate 101 can be performed, for example, by reactive ion etching (RIE). The etching depth can be between about 100 and 500 nm. The trenches can be filled with one or more of Al, Mg, Cr, and / or Ni. The thickness of the metal corresponding to the outer sacrificial layer 103 can be at least somewhat thicker than the RIE etching depth. For example, the metal thickness can be 20-500 nm thicker, e.g., 20-200 nm thicker, e.g., 200-300 nm thicker. As a result of this increased thickness, the metal corresponding to the outer sacrificial layer 103 is slightly higher than the original top surface of the substrate 101. The outer sacrificial layer 103 may extend 10-120 μm, e.g., 20-100 μm, away from the center of the inner sacrificial layer 102.

[0062] 1. Construction of Initial Electrodes and Fluidic Channels / Chambers

[0063] In the third step, a lateral electrode 104 is defined at the center of the inner sacrificial layer 102. A pair of lateral electrodes 104 can be fabricated with their tips aligned with the centers of the inner sacrificial layer 102 and the outer sacrificial layer 103. As described above, the initial gap distance between the electrodes can be between 200 nm and 5 μm, for example, between 500 nm and 2 μm, for example, between 600 nm and 2 μm, for example, about 1 μm. The initial gap distance can be determined by the resolution of photolithography. The pair of lateral electrodes 104 can include one or more of gold (Au), platinum (Pt), and / or palladium (Pd), or other metals or alloys compatible with top-down lithography and subsequent electrochemical deposition. As used herein, the term "other metals" includes ruthenium (Ru), rhodium (Rh), palladium (Pd), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), silver (Ag), copper (Cu), and rhenium (Re). The thickness of the metals corresponding to the first set of lateral electrodes 104 may be between 20 and 100 nm.

[0064] In the fourth step, the top of the electrode is sealed with a dielectric layer 105. Specifically, the entire upper (top) surface of the chip is fully passivated with HfO2, ZrO2, SiO2, Si3N4, or a combination of multiple dielectric layers. This can be done by atomic layer deposition (ALD) or plasma-enhanced chemical vapor deposition (PECVD). In some examples, one or more additional layers of SU-8 polymer (or parylene or other polymer dielectric) can be added to aid in sealing. The thickness of the dielectric layer 105 can be between about 100 nm and about 100 μm. In some examples, before the ALD or PECVD coating step, 0.5 to 2 nm of Cr or Ti or other metal is sputtered onto the electrode surface and optionally oxygen plasma treated to promote better adhesion and sealing of the dielectric layer deposited by ALD or PECVD on the electrode and substrate, especially in the region around the electrode's side edges.

[0065] In the fifth and sixth steps, a masked RIE process is used to selectively open a first window 106 and a second window 107 to expose the outer sacrificial layer 103 from the top side (window 106) and the bottom side (window 107) of the device. Thus, the window 106 is formed by etching the dielectric layer 105 from the top side of the device, and the window 107 is formed by etching the substrate 101 from the bottom side of the device.

[0066] Once steps 1-6 have been performed, the initial device can be stored in an ambient environment for later use.

[0067] Preparation of nanopore and tunneling gap before recording

[0068] The initial device must be prepared with appropriate dimensions immediately before use. Therefore, in step 7, the inner sacrificial layer (102) and outer sacrificial layer (103) are removed by wet chemical etching to form an upper chamber 108 and a lower chamber 109 on either side of the central nanoscale channel. The upper chamber 108 and lower chamber 109 can be between 1 and 100 μm in diameter. In one embodiment, by appropriately supplying a first etchant (e.g., Al etchant) and then a second etchant (e.g., Cr etchant) through the fluidic channel, the sacrificial layer is first removed, forming a central nanoscale cavity around the electrode 104 and creating a continuous space where the nanofluidic channels on both sides are connected to the upper chamber (108) and the lower chamber (109). The channel can be washed before and after each etching step using phosphate buffer (PB) containing 5 mM KH2PO4 and 5 mM NaHPO4 (pH 7.26), allowing ionic conductance between the chambers to be measured. Typical conductance before the final chemical etch is complete may be less than about 20-30 pS, as determined by the electronics baseline, and may be about 2-5 nS after the inner sacrificial layer 102 has been completely removed.

[0069] Next, in step 8, we use a feedback control technique adapted from previous studies (Wang, Y., Sadar, J., Tsao, C.W., Mukherjee, S., Qing, Q., "Nanopore chip with self-aligned transverse tunneling junction for DNA detection," Biosens Bioelectron 2021, 193:113552; Sadar, J.; Wang, Y.; Qing, Q., "Confined Electrochemical Deposition in Sub-15 nm Space for Preparing Nanogap Electrodes," ECS Trans 2017, 77(7), 65-72; Qing, Q.; Chen, F.; Li, P.; Tang, W.; Wu, Z.; Liu, Z., "Financial tuning metallic nanogap size with electrodeposition by utilizing high frequency impedance in feedback," Angew Chem Int Ed Engl 2005, 44(47), 7771-5), the entire contents of which are incorporated herein by reference, the channel is filled with an electrolyte containing a metal (e.g., Au, Pt, Pd, Ni, Co, or other metal compatible with the electrochemical deposition process), and the conductance between the lateral electrodes is monitored in real time to control the electrochemical deposition of the metal onto the existing electrode 104. In one example, the electrolyte contains 18.5 mM KAu(CN)2 and 180 mM potassium citrate, and the conductance between the lateral electrodes is monitored in real time to control the electrochemical deposition of Au onto the existing electrode 104 with Ag / AgCl as the counter electrode.In embodiments, the final dimensions of the nanopore and tunnel junction 110 can be finely and reproducibly tuned by a reversible pulse deposition method, as described below. The final electrode cap size can be defined by the thickness of the original inner sacrificial layer 102. It should be appreciated that a feedback-controlled electrochemical deposition process within the confined cavity formed by etching the inner sacrificial layer 102 closes the initial gap between the electrodes 104, thereby simultaneously forming the nanopore and lateral tunnel junction 110. In embodiments, the gap between the electrodes may be between about 1 nm and 100 nm, between about 10 and 50 nm, between about 1 and 20 nm, between about 20 and 60 nm, Examples of gap sizes include, but are not limited to, 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nm. In embodiments, the minimum gap size corresponds to the diameter of a single biopolymer. For example, the diameter of a single-stranded DNA molecule is approximately 1.0 nm. In embodiments, the maximum size of the gap corresponds to the diameter of the three-dimensional size of the molecule, such as a protein molecule, to be characterized, e.g., on the order of about 5 nm to 10 nm, and larger, approaching 100 nm.

[0070] Figures 3A-3D show reversible pulsed electrochemical deposition for precise control of nanopore and tunnel junction dimensions. Figures 3A-3B show optical images of the initial device (Figure 3A) and the final tunnel junction (Figure 3B). Figures 3A-3B show optical images from the backside of the coverslip of the initial lateral electrodes (Figure 3A) and after electrochemical deposition, where the gap has been closed under precise control (Figure 3B). As electrochemical deposition progresses, a decrease in ionic conductance between the upper and lower chambers is first observed, followed by an increase in conductance between the lateral electrodes, indicating a short circuit (Figure 3C). Specifically, Figure 3C shows the current (I) between the upper and lower chambers under a bias of 50 mV during deposition, where the electrodes are shortening. ionic ) and the current between the lateral electrodes under a bias of 1.45 mV (I tunneling ) are simultaneously recorded. It can be seen that, at a constant deposition bias, significant lateral growth occurs on the electrode, and it takes a very long time to short the electrode. Without being bound by theory, this is due to the formation of metal ions (e.g., Au(CN)2) near the tip of the electrode within the confined nanoscale cavity. - This is due to the rapid consumption of ions. Deposition mostly occurs at the outer edge of the electrode, where there is easy access to the external ion supply. Therefore, the pulse deposition method can be used to more effectively deposit at the tip and close the gap. That is, by applying a deposition potential for a short time and then leaving the system at a potential where no redox reactions occur (a "resting" potential), the ion concentration in the central cavity recovers by diffusion. There is a clear correlation between lateral growth on the electrode and the duration of the deposition pulse (Figures 4A-4B). A systematic comparison revealed that the optimal pulse width for effective tip growth is 2-50 ms and the resting potential duration is ~2 s.

[0071] Briefly, Figures 4A-4B show specific frames from a time-lapse recording during metal deposition with various deposition pulses. Figure 4A shows the tip morphology before and after deposition with a 400 ms, 950 mV square wave followed by a 1.6 s, 0 V rest potential. Figure 4B shows the tip morphology with a 2 ms, 950 mV square wave followed by a 1.998 s, 500 mV rest potential. Scale bars are 20 µm. Due to the short deposition pulse width of 950 mV and the high rest potential, the final deposited tip exhibits less lateral growth and forms quantum contacts in a shorter time.

[0072] A particular advantage of using electrochemistry to tailor the tunnel junction is that the process is fully reversible. At the end of each period of pulse deposition, a control program was created that actively tracks the conductance between the lateral electrodes. If the conductance exceeds a threshold, the program reduces the bias V dep By reversing the polarity of the electrode, the electrode is switched from deposition mode to elution mode, so that the metal (e.g., Au) is removed in controlled steps. Figure 3D shows an example where the transition occurs at 342 seconds, when the conductance reaches above one conductance quantum, G. More specifically, Figure 3D shows the controlled metal (e.g., Au) deposition (V dep >0) and elution (V dep <0) the potential V applied to the Ag / AgCl counter electrode dep and the corresponding amplitude of the AC conductance between the transverse electrodes recorded at 10.13 kHz (conductance quantum G0 = 2·e 2 The graph shows the recording of the conductance (units of / h=77.5 μS) and phase. An automatic control program monitors the conductance during deposition. When the conductance reaches a threshold above 1 G0, the program starts V dep The polarity of the electrode is switched (342 seconds) and the metal (e.g., Au) is removed from the electrode with a negative pulse.

[0073] G0 was used as a typical threshold because it serves as a calibration for the zero distance between the electrodes when they first make atomic contact. By repeating this process with high reproducibility, we can open and close the tunneling gap and form a stable junction as defined by the final tunneling conductance (Figure 3E). Specifically, Figure 3E shows the conductance between the lateral electrodes, indicating multiple controlled, reversible gap openings and closings.

[0074] Note that the conductance between the lateral electrodes can be tracked using an AC signal ranging from 1 Hz to ~10 kHz with a lock-in amplifier as a feedback control signal. For example, if a higher frequency is used, the conductance may begin to show clear changes at longer distances, on the order of tens of nanometers, due to the capacitive component, thereby widening the distance control range. This is also demonstrated in Figure 3D, where the recorded phase of the AC conductance using a 10.13 kHz reference signal shows an early transition in phase from highly capacitive to highly resistive, before a later large change in amplitude occurs. Nevertheless, in this disclosure, we focus on dimensions that best match the diameter of DNA molecules, and therefore used a low-frequency AC signal (5 Hz) to monitor and control the junction size, which is most sensitive in the tunneling region. For devices used in DNA translocation devices, the disclosed control program can be used to stabilize the junction conductance at a predetermined set point, e.g., ~1 nS, which corresponds to a gap distance between approximately 3 and 4 nm, e.g., approximately 3.4 nm. In one embodiment, a DC bias can be used to monitor conductance and control the nanogap size with the most sensitivity in the ∼1 nm tunneling regime.

[0075] Therefore, the construction of the nanopore device disclosed herein using feedback-controlled electrodeposition of metal onto a pair of initial electrodes sandwiched between two dielectric passivation layers can be used to shrink the initial gap / cavity so that the nanopore device and the tunnel junction are formed simultaneously and self-alignedly. This refers to how a nanogap is created using electrodeposition of metal onto a pair of initial electrodes between two dielectric passivation layers. As used herein, a "self-aligned" nanogap is created by a process in which the nanogap is formed at the point where the electrodes contact the fluid along the length of the fluid channel. It is understood herein that in some instances, the size and sharpness of the tunneling electrodes may deteriorate (e.g., become dull) over a period of time due to rapid depletion of metal ions in the confined space. It is recognized herein that during feedback-controlled electrodeposition of metal onto a pair of initial electrodes, metal deposition tends to occur more at the ends of the electrodes, where access to metal ions is better, than at the tip region, where depletion occurs more rapidly. Furthermore, because metal is preferentially deposited at the ends of the electrodes, the deposition time until the tunnel junction reaches the desired size (for example, on the order of one to several nanometers) can be quite long.

[0076] Accordingly, several approaches are disclosed herein that improve various aspects of nanopore device construction procedures. Specifically, methodologies are disclosed herein that can improve at least 1) the deposition rate of the final device, 2) the sharpness of the tips corresponding to the electrodes comprising the tunnel junction, and 3) the stability of the junctions ultimately formed.

[0077] In one example, high-speed pulse electrodeposition was compared with slow-speed pulse or continuous deposition methods. In a typical experiment, the optimal deposition time was found to be less than 50 milliseconds, during which the electrode is held at a reducing potential, allowing metal ions to be reduced and deposited on the electrode, followed by a rest period of 500 milliseconds to approximately 2 seconds (2000 milliseconds). During this rest period, the electrode is held at a potential where there is no faradaic current, allowing diffusion processes to replenish metal ions consumed near the electrode tip. In the current setup, deposition times of 2 to 5 ms (e.g., 2 ms, 3 ms, 4 ms, 5 ms) have been found to be optimal. However, shorter deposition times can be used without departing from the scope of this disclosure. For example, shorter deposition times, including those between 10 μs and 1 ms, can be used if the equipment bandwidth allows. More specifically, the deposition time may be between 10 and 500 μs, or between 500 μs and 1 second, or between 10 and 450 μs, or between 10 and 400 μs, or between 10 and 350 μs, or between 10 and 300 μs, or between 10 and 250 μs, or between 10 and 200 μs, or between 10 and 150 μs, or between 10 and 100 μs, or between 10 and 90 μs, or between 10 and 80 μs, or between 10 and 70 μs, or between 10 and 60 μs, or between 10 and 50 μs, or between 10 and 40 μs, or between 10 and 30 μs, or between 10 and 20 μs. In some examples, the deposition time can be even shorter, such as less than 10 μs, e.g., less than 1 μs. It will be appreciated herein that shorter deposition times may in turn allow for shorter dwell times, thereby improving (e.g., shortening) the overall timeframe for construction of the nanodevices of the present disclosure.

[0078] In another example, it is disclosed herein that a small bias of approximately 50-100 mV between two chambers (e.g., cis and trans) can promote electrophoretic and electroosmotic flow between the chambers without affecting the faradaic process at the electrodes. This has the effect of promoting rapid compensation of metal ions to the deposition region, which, as mentioned above, can allow for shorter dwell times and therefore shorter device construction times.

[0079] In another example, it is recognized herein that an asymmetric setup can result in asymmetric deposition, resulting in sharper tips corresponding to each of the tunneling electrodes. Turning to FIG. 4C, an exemplary diagram is shown illustrating that when metal ions (shown as spheres) are allowed to diffuse uniformly into the electrode, the overall radius of the electrode increases. Specifically, with reference to FIG. 4C, the dashed line indicates the electrode shape before the uniform deposition process, and the arrows indicate how the electrode size increases uniformly as a result of the uniform deposition process. Alternatively, turning to FIG. 4D, when metal ions (shown as spheres) are supplied to only one of the two chambers, asymmetric deposition can enable a sharper electrode shape near the tip, as shown in the bottom half of FIG. 4D.

[0080] It is further recognized herein that a newly fabricated tunnel junction that has reached the desired distance, as indicated by the conductance between the electrodes, may become unstable over a period of time. A solution to this problem is to exploit the reversibility of the electrochemical process to repeatedly build the gap any number of times. That is, the junction can be enlarged by metal oxidation (by switching the polarity of the potential applied to the metal electrodes), followed by removal of the newly deposited metal and a subsequent re-deposition step to reach the desired gap size again. The purpose of this oxidation-reduction cycle would be to fill the most active sites on the electrodes (e.g., those most susceptible to deformation / reshaping over time under ambient conditions) and then remove the most active and mobile atoms in the reverse process. Repeating this process any number of times (e.g., 10–1000 times) sacrifices the surface sites most likely to undergo reshaping, leading to an unstable gap shape, resulting in a “retarded” surface and, therefore, a stable gap.

[0081] Nanodevice Processing According to Embodiments

[0082] 5A-5B, top views (FIG. 5A) and cross-sectional views (FIG. 5B) of a nanodevice fabricated in accordance with embodiments disclosed herein are shown, illustrating how a biomolecule (in this case, DNA) moves through the nanodevice. In one embodiment, molecules are supplied to the upper chamber 108 and can move under a driving bias applied between the upper chamber 108 and the lower chamber 109, passing through the central tunnel junction / nanopore and exiting the lower chamber 109. The ionic current between the upper chamber 108 and the lower chamber 109 and the tunneling current between the lateral electrodes can be used to analyze the biomolecule. In another example, by changing the polarity of the potential applied between the upper and lower chambers, the movement of the biomolecule can be reversed, allowing the same biomolecule to be controlled to pass through the tunnel junction / nanopore multiple times in different directions for repeated analysis.

[0083] Also within the scope of the present disclosure is the use of optical signals in addition to, or instead of, relying on one or more of ionic currents and tunneling currents to analyze biomolecules. For example, in embodiments, the nanodevice of the present disclosure can be mounted on a microscope with an objective lens that can focus on the nanogap through a transparent substrate or from the top side of a non-transparent substrate, and the fluorescent signal can be used to track the movement and migration of molecules within the nanogap. Additionally or alternatively, the lens can be used to detect Raman signals from the gap as the molecules pass through the electrodes (e.g., via Raman microscopy). The electrodes are connected to a voltage source and an ammeter to record the tunneling signal across the gap. Two electrodes can be used between the upper and lower chambers to apply a bias potential of 10-1000 mV to drive the molecules through the gap.

[0084] In embodiments, when a molecular analyte is supplied to the upper chamber on one side of the nanogap, the molecule can be guided through the nanogap by a bias, e.g., between 10 and 1000 mV, applied between the upper and lower chambers. It is contemplated that the magnitude of the bias can be adjusted to achieve a desired translocation rate. In some embodiments, it is advantageous to attach the molecule to a metal electrode for at least a predetermined time period. This includes, but is not limited to, (1) simple blocking due to the size of the molecule relative to the nanogap dimensions; (2) modifying the molecule to have binding sites that can form chemical bonds to the metal, such as thiol groups that can bind to a gold surface; or (3) modifying the surface of the metal electrode to have specific binding sites along the ends or sides of the molecule that recognize specific functional groups on the molecule. In these configurations, the binding sites can be valence chemical bonds, such as S-Au / Pt / Pd, when a sulfhydryl group is contacted with a metal such as Au, Pt, or Pd. For example, lysines in protein molecules can be modified with a thiolation reagent to generate free sulfhydryl groups that can form valence chemical bonds with the metal surface. Binding sites can also be designed to be specific, high-affinity interactions between proteins and functional groups, such as avidin-biotin interactions. For example, thiolated biotin can be included on the substrate, and the metal surface can be modified with S-Au / Pt / Pd bonds to expose the biotin group to the medium. Protein molecules can then be linked with streptavidin, which will bind to the biotin group when they reach the nanogap. Other interactions, such as hydrogen bonds and π-π interactions, can also be used.

[0085] It is contemplated that mounting events can be detected by both the ionic current between the upper and lower chambers and the tunneling current between the metal electrodes, and that the molecular conductance can be evaluated when such events are detected. Furthermore, optical characterization, such as tip-enhanced Raman spectroscopy (TERS), can be performed through the transparent substrate or from the top side of a non-transparent substrate to understand the dynamic structure of the molecule.

[0086] In some embodiments, the present disclosure provides a method for sampling / characterizing small samples of molecules, small particles, or materials, comprising delivering a sample, such as a molecular analyte, to an upper chamber on one side of a nanogap and inducing the sample to migrate through the nanogap by a bias applied between the upper and lower chambers, e.g., a bias of between about 1 and about 1000 mV.

[0087] In some embodiments, the present disclosure provides a measurement device for analyzing samples consisting of single molecules, small particles, or small amounts of material. The molecular measurement device includes at least one nanofluidic channel through which a solution containing the sample to be analyzed can flow, and a pair of electrodes defining a nanogap across the nanofluidic channel through which the sample passes. In embodiments, the size of the nanogap is selected based on the molecular size of the sample material to be observed by the device, thereby allowing only a single sample material to pass through the nanogap at a time. In embodiments, the distance between the electrodes ranges from 1 to 100 nm, forming a self-aligned nanogap with the narrowest bottleneck in the path between the two channels / chambers. It is contemplated that the nanogap can be adjusted during the electrochemical deposition process to address the different properties of molecules of different sizes and shapes. In embodiments, the sample includes DNA, such as single-stranded DNA and / or double-stranded DNA. In embodiments, the sample includes RNA. In embodiments, the sample includes a protein. Other embodiments include small molecules, any number of different polymers, and the like.

[0088] The nanogap provides an output that represents environmental conditions within the nanogap that change in response to the presence of a sample within the nanogap. These environmental properties can be electrical or optical.

[0089] In further embodiments, the present disclosure provides methods for measuring optical properties such as conductance and tip-enhanced Raman spectra from single molecules by detecting individual mounting events of single molecules via correlated ionic currents between the channels / chambers and tunneling currents between the metal electrodes (e.g., through defined nanogaps), and then performing electrical and / or optical characterization.

[0090] Turning now to FIGS. 6A-6B, two three-dimensional views of a portion of a nanodevice of the present disclosure are shown. FIG. 6A is depicted opaque, while FIG. 6B is depicted transparently to provide an internal view of a portion of such a nanodevice. For the portion of the nanodevice shown in FIGS. 6A-6B, the nanodevice is configured to have an upper portion 605 and a lower portion 606. Also shown are the substrate 101, lateral electrodes 104, dielectric layer 105, and a schematic vicinity of the upper and lower chambers 108 and 109. Also shown are through-holes 610 and trenches 615. The trenches 615 are formed to guide analytes in the upper chamber 108 through sealed portions of the nanodevice structure (e.g., sealed at the top and bottom) on their way through the nanogap located between the lateral electrodes 104 and from the through-holes 610 to the lower chamber 109.

[0091] As discussed above, an advantage of fabricating nanodevices with top and bottom chambers as disclosed herein is that this design allows for a significant reduction in the area occupied by the microfluidic interface on one side (e.g., the top) of the device. Thus, by using the fabrication methods disclosed herein, the density of nanodevices on a single chip can be substantially increased, as opposed to similar designs in which the microfluidic interfaces corresponding to the cis and trans chambers are associated with only one side (e.g., the top) of the nanodevice.

[0092] Thus, the fabrication methods disclosed herein allow for the integration of numerous nanodevices onto a single chip. As a representative example, a single chip may correspond to a 170 μm thick, 1-inch quartz coverslip (Electron Microscopy Sciences, Item No. 72256-02), a 500 μm thick, 4-inch quartz wafer (University wafer, Item No. 518), or a single-crystal silicon wafer, or other similar such starting substrate. In such examples, thousands (e.g., over 1,000, tens of thousands, or more, and any number in between) may be integrated onto a single chip. It is within the scope of this disclosure to group predetermined numbers (e.g., tens, hundreds, or even thousands) of nanodevices into distinct regions of such a chip to allow for simultaneous delivery of various samples to different groups.

[0093] Turning to FIG. 7 , a single chip 705 is illustrated with a plurality of different nanodevices 710 integrated onto the single chip 705. Additionally, a plurality of different groups 715 of nanodevices 710 are illustrated. In this exemplary illustration, each group 715 includes eight nanodevices; however, as noted above, it is within the scope of this disclosure for each group 715 to include tens, hundreds, or, in some instances, thousands of individual nanodevices 710. Additionally, just four groups 715 are shown: a first group 720, a second group 721, a third group 722, and a fourth group 723. However, it is within the scope of this disclosure that there may be tens, hundreds, or, in some instances, even thousands of different groups. The footprint of a single nanodevice 710 is on the order of 10 μm×10 μm to approximately 100 μm×100 μm.

[0094] Thus, fabricating nanodevices as disclosed herein with an upper chamber (e.g., upper chamber 108) and a lower chamber (e.g., lower chamber 109) allows for the integration of numerous nanodevices into a single chip, and further allows for the grouping of multiple nanodevices into individual groups. This significantly reduces the complexity associated with delivering samples to nanodevices and groups of nanodevices. For example, multi-well structures located on the upper and lower surfaces of a chip containing the disclosed nanodevices can be used to easily deliver samples to various device groups. Furthermore, multiplexed electronics can be integrated into the substrate (e.g., substrate 101), improving signal quality and enabling simultaneous electrodeposition and sample analysis.

[0095] As an example, a short pulse is followed by a rest period, as described above for the pulsed electrochemical deposition process. It is recognized herein that, for a chip 705 such as that shown in FIG. 7 containing multiple nanodevices 710 in groups 715, multiplexed electronics can be used to sequentially perform the pulsed electrochemical deposition process on all or a selected number of nanodevices on the chip. For example, depending on the number of nanodevices for which a pulsed deposition process is required, a pulse can be applied to a first nanodevice, then a second nanodevice, then a third nanodevice, then a fourth nanodevice, and so on. Because the pulses are of short duration (e.g., 2-5 milliseconds or less, e.g., in the microsecond range) compared to the rest periods (e.g., 500 milliseconds to 2 seconds), a large number (e.g., all) of the nanodevices on a chip can be pulsed sequentially, followed by a rest period, and the process can be repeated again until all nanodevices have been pulsed. In other words, sequential pulsing of nanodevices allows each nanodevice to be pulsed for a time span equal to or less than the rest period of the first nanodevice pulsed. The sequential nature of the pulse / pause sequence can be electronically controlled as a function of pulse time, pause time, and the number of nanodevices requiring pulsed electrochemical deposition, optimizing electronic resources, reducing the time to fabricate the disclosed chips, and improving the reproducibility of nanodevice fabrication. It is also within the scope of the present disclosure to perform pulsed electrochemical deposition steps on certain nanodevices and groups of nanodevices while providing samples to other nanodevices / groups of nanodevices for analysis thereof.

[0096] 8, there is shown a high-level schematic diagram of a chip 805 combined with an upper multiwell structure 810 and a lower multiwell structure 815. For reference, arrow 801 points toward the top of chip 805 and thus points in the general direction of the upper chamber (not shown in FIG. 8). Alternatively, arrow 802 points toward the bottom of chip 805 and therefore points in the general direction of the lower chamber (not shown in FIG. 8). Although not explicitly shown, it will be understood that chip 805 includes multiple different nanodevices.

[0097] The upper multiwell structure 810 is disposed on the top of the chip 805 and, although not explicitly shown, may be understood to include multiple wells. In an example, the number of wells corresponds to the number of groups of nanodevices. Similarly, the lower multiwell structure 815 contacts the bottom of the chip 805, and the number of wells associated with the lower multiwell structure 815 may also correspond to the number of groups of nanodevices integrated into the chip 805. Thus, in an example, the upper multiwell structure 810 and the lower multiwell structure 815 may include the same number of wells. However, in some examples, the upper and / or lower multiwell structures may have a number of wells different from the number of groups of nanodevices without departing from the scope of the present disclosure. In some examples, the lower structure may not include individual wells but may instead include a single large area. In this manner, contacting a sample with nanodevices integrated on the chip 805 may include providing the sample to the appropriate wells corresponding to the nanodevices with which the sample is intended to contact. In this manner, different nanodevices can be easily contacted with different samples, enabling parallel analysis of various samples.

[0098] For example, it is within the scope of this disclosure that the sample be provided by a multichannel pipette and introduced manually, robotically, and / or microfluidically. The sample can be introduced into the appropriate wells of the upper multiwell structure 810, where analytes present in the sample are driven by a bias to migrate the nanodevices in their corresponding groups. After migration from the upper chamber (e.g., upper chamber 108) to the lower chamber (e.g., lower chamber 109), the analytes ultimately reach the lower multiwell structure 815. In some embodiments, it is within the scope of this disclosure that the sample in the lower multiwell structure 815 may be reanalyzed in a similar manner, analyzed in some other manner to obtain further information about the analytes in the analyzed sample, or stored for possible future use. Alternatively, the analyzed sample may be discarded.

[0099] The substrate (e.g., substrate 101) of chip 805 may, in some embodiments, include integrated multiplexing electronics 820, as described above. In one example, the multiplexing electronics includes one or more of the following components: (1) a module (e.g., a potentiostat) capable of varying and holding the potential of individual devices; (2) a module (e.g., a transimpedance amplifier) ​​capable of detecting the tunneling current across individual tunnel junctions; (3) a module (e.g., a reader / writer) capable of sending target potentials to selected devices and reading tunnel junction signals from selected devices at specific times; (4) a module (e.g., a multiplexer) capable of switching connections between the reader / writer and individual devices with programmable timing and sequence; and (5) a module that converts digital and analog signals between external control electronics and the multiplexer so that individual devices can be addressed, and the target potentials can be sent to and the tunneling currents can be read from specific devices. This information can be controlled by an external controller and program to coordinate the deposition process and collect the tunneling current from all devices in a multiplexed manner. In some embodiments, any electronic components utilized to interact with (e.g., control or receive information from) at least one nanodevice can be integrated into (e.g., fabricated on a substrate of) chip 805. Any or all of the electronic components and multiplexing electronics integrated with chip 805 can be fabricated on or within chip 805 (e.g., on a substrate of chip 805 or within chip 805) before or after at least one (e.g., all) of the nanodevices are fabricated.

[0100] 9, an exemplary system 900 of the present disclosure is illustrated. System 900 may be part of a control system 14. Control system 14 is an interconnection of components that form a system configuration that provides a desired process response. In this embodiment, control system 14 includes a controller 12 that provides logic and control instructions for the process, one or more sensors 18 that measure various physical properties, and one or more actuators 21 that change environmental conditions. Control system 14 may also include signaling means (not shown) that converts measurements from sensors 18 and / or instructions generated by controller 12 into one or more signals that are then transmitted to other elements / components of the system. For example, controller 12 may receive input data from one or more sensors 18, process the input data, and trigger actuators 21 in response to the processed input data based on instructions or code programmed therein that correspond to one or more routines, procedures, functions, methods, etc. Control system 14 may operate according to an open-loop system, a closed-loop system, a sequential control system, and / or a batch control system.

[0101] The controller 12 may comprise circuitry including, for example, one or more central processing units (CPUs) including one or more processor cores, a graphics processing unit (GPU), a programmable logic controller (PLC), a microprocessor, a digital signal processor (DSP), one or more field programmable gate arrays (FPGAs), an application-specific integrated circuit (ASIC), or any suitable combination thereof. In some embodiments, an FPGA is utilized to provide real-time control of the movement of molecules or proteins through the nanogap. For example, an FPGA can be utilized to shuttle molecules or proteins through the nanogap one or more times. The circuitry of the controller 12 may be coupled to or include memory / storage and may be configured to execute instructions stored in the memory / storage to perform various applications, logic, etc. on the controller 12 and / or other elements of the control system 14. In some embodiments, the circuitry of the controller 12 may be a dedicated processor / controller for operating in accordance with various embodiments herein.

[0102] Sensors 18 include devices, modules, or subsystems designed to detect events or changes in the environment and transmit information about the detected events (e.g., sensor data) to other devices, modules, subsystems, etc., such as controller 12. Examples of such sensors 18 include, among others, inertial measurement units (IMUs) including accelerometers, gyroscopes, and / or magnetometers, and microelectromechanical systems (MEMS) or nanoelectromechanical systems (NEMS) including three-axis accelerometers, three-axis gyroscopes, and / or magnetometers, level sensors, flow sensors, temperature sensors (e.g., thermistors), pressure sensors, barometric pressure sensors, weigh scales, altimeters, image capture devices (e.g., cameras), light detection and ranging (LiDAR) sensors, proximity sensors (e.g., infrared detectors, etc.), depth sensors, ambient light sensors, ultrasonic transceivers, microphones, etc. In some embodiments, the electronic nanodevices / chips of the present disclosure can be considered sensors in that they are designed to sense biomolecular translocation / mounting / linking events, and such information can be relayed to the controller 12 and / or other devices, modules, subsystems, etc.

[0103] An actuator 21 is a device, module, or subsystem that changes the state, position, and / or direction of, or moves or controls, an external mechanism / system or a mechanism or system including the actuator 21 itself. The actuator 21 includes electrical and / or mechanical elements that convert energy (e.g., electrical current, moving air and / or liquid, etc.) into some kind of motion. The actuator 21 may be or include one or more electronic (or electrochemical) devices, such as piezoelectric biomorphs, solid-state actuators, solid-state relays (SSRs), shape memory alloy-based actuators, electroactive polymer-based actuators, relay driver integrated circuits (ICs), and / or the like. The actuator 21 may be or include one or more electromechanical devices, such as pneumatic actuators, hydraulic actuators, electrostatic actuators (EHAs), electromechanical switches including electromechanical relays (EMRs), motors (e.g., DC motors, stepper motors, servomechanisms, linear motors, linear drives, etc.). The actuator 21 may be connected to and control the movement of other devices such as valves, vacuum generators (e.g., venturi-based ejectors, blowers, etc.), pumps (e.g., vacuum pumps, suction pumps, hydraulic pumps, compressors, etc.), gears, wheels, thrusters, propellers, claws, clamps, hooks, audible sound generators, and / or the like.

[0104] The signaling means (not shown) can include any element or combination of elements for communicating information / commands to components of the control system 14. In some embodiments, the signaling means is or includes a suitable bus or interconnect (IX) technology, such as Peripheral Component Interconnect (PCI), PCI Express (PCIe), Industry Standard Architecture (ISA), Universal Serial Bus (USB), HyperTransport Interconnect, Time Trigger Protocol (TTP), Fieldbus (e.g., IEC 61158)-based IX, such as PROFIBUS, Modbus, Common Industrial Protocol (CIP) IX, Ethernet Industrial Protocol (EtherNetIP), etc. In some embodiments, the signaling means is or includes one or more network interface controllers that connect the controller 12 to other components / devices using a physical connection, which may be electrical (e.g., "copper interconnect") or optical, and which operates according to a wired network protocol, such as Ethernet, Industrial Ethernet, Ethernet over USB, Controller Area Network (CAN), Local Interconnect Network (LIN), Profinet, etc. In some embodiments, the signaling means is or includes a radio frequency transmitter (and receiver) or transceiver configured to enable communication with or via a wireless network using modulated electromagnetic radiation over a non-solid medium (e.g., over an air interface).

[0105] The embodiment shown in FIG. 9 also includes a robotic system 20 and an electronic nanodevice and / or chip 25 of the present disclosure. In embodiments, the robotic system 20 includes or can include a fluidic device / system, such as a microfluidic device / system. Generally, the robotic system 20 may include machinery capable of delivering one or more samples to, for example, wells of a multiwell structure (e.g., upper multiwell structure 810 in FIG. 8 ) in an automated manner. For example, the robotic system 20 may include one or more multichannel pipettes, tubes, vacuum and / or pressure sources, movable arms, microfluidic delivery channels, etc., to enable retrieval of a sample from one location and delivery / delivery to one or more wells associated with a multiwell structure, as disclosed herein. In some examples, the robotic system 20 can manipulate the electronic devices and / or chips of the present disclosure, for example, to position the electronic devices and / or chips at desired locations for subsequent use, to couple or detach multiwell structures (e.g., upper multiwell structure 810 and / or lower multiwell structure 815) from corresponding chips, etc.

[0106] In embodiments, the robotic system 20 may additionally or alternatively include other components, such as a microscope that is automatically adjustable in response to commands received from the controller 12. In embodiments, such a microscope is a TERS microscope.

[0107] In embodiments, controller 12 can send and receive electrical and / or optical signals to and from electronic nanodevices and / or chips 25. For example, the controller may be capable of sending and receiving signals for feedback control of electrochemical deposition of electrodes associated with the electronic nanodevices and / or chips 25, and may additionally or alternatively be capable of acquiring data related to individual molecular translocation and / or mounting / linking events corresponding to any number of electronic nanodevices included as part of the electronic nanodevice / chip 25. For example, controller 12 may acquire data corresponding to ionic conductance and / or tunneling conductance associated with single-molecule translocation / linking / mounting events. In the case of optical signals, it will be appreciated that in some embodiments, robotic system 20 may be configured to collect optical data via a microscope associated with the robotic system and transmit the data to controller 12 for further processing.

[0108] Thus, generally, the controller 12 may be configured to send and receive information (e.g., electrical signals) to and from the robotic system 20, the robotic system 20 may be capable of sending and receiving information to and from the controller 12 and / or to and from the electronic nanodevice / chip 25, and the electronic nanodevice / chip 25 may be capable of sending and receiving information to and from the robotic system 20 and / or to and from the controller 12. This is exemplarily shown by arrow 910.

[0109] In some embodiments of the present disclosure, an electronic device is provided, comprising: an upper fluidic channel / chamber and a lower fluidic channel / chamber fabricated in a planar substrate, wherein the upper fluidic channel / chamber is sealed at a lower portion of the electronic device and includes a first open window at the upper portion of the electronic device, and the lower fluidic channel / chamber is sealed at the upper portion of the electronic device and includes a second open window at the lower portion of the electronic device; first and second electrodes disposed in a space of the electronic device between the upper and lower fluidic channel / chambers; and a nanogap having a dimension defined in part by the distance between the first and second electrodes, wherein the nanogap has a single pathway for molecules to move from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber, the distance being between about 1 and 100 nm, and the nanogap is self-aligned and has a narrowest bottleneck in the single pathway between the upper and lower fluidic channel / chamber.

[0110] In some embodiments, the distance is defined by the first and second electrodes being electrochemically deposited under feedback control using one or more metallic materials in the upper and / or lower fluidic channels / chambers, thereby forming a single pathway.

[0111] In some embodiments, the planar substrate is a transparent substrate.

[0112] In some embodiments, the transparent substrate is glass or quartz.

[0113] In some embodiments, the planar substrate is a non-transparent substrate.

[0114] In some embodiments, the planar substrate is silicon coated with SiO2 and / or Si3N4.

[0115] In some embodiments, the first and second electrodes are formed of gold, palladium, platinum, or other metals or alloys compatible with top-down lithography and electrochemical deposition, or combinations thereof.

[0116] In some embodiments, the space in which the first electrode and the second electrode are disposed is at least partially formed by one or more dielectric layers.

[0117] In some embodiments, the one or more dielectric layers are HfO2, ZrO2, SiO2, Si3N4, or a combination thereof.

[0118] In some embodiments, the electronic device further comprises an additional polymer dielectric layer for encapsulation.

[0119] In some embodiments, the additional polymer dielectric layer comprises one or more of an SU-8 polymer and parylene.

[0120] In some embodiments, the dielectric layer or layers, with or without an additional polymer dielectric layer, are from about 100 nm to about 100 μm.

[0121] In some embodiments, the one or more metallic materials are Ni, Co, gold, palladium, platinum, iridium, alloys thereof, or combinations thereof.

[0122] In some embodiments, the first and second electrodes electrochemically deposited with one or more metallic materials under feedback control further include a pulsed electrochemical deposition process having a pulse width of 50 ms or less and a rest period of about 500 ms to 2 seconds between pulses.

[0123] In some embodiments, the molecule is DNA.

[0124] In some embodiments of the present disclosure, a method for measuring electronic and / or optical properties from one or more molecules is provided, the method comprising: detecting individual mounting and / or rearrangement events of one or more molecules by correlating, with an electronic device, an ionic current between an upper fluidic channel / chamber and a lower fluidic channel / chamber and a tunneling current between a first electrode and a second electrode through a nanogap; and performing at least one electrical and / or optical characterization of the one or more molecules.

[0125] In some embodiments, the planar substrate is a transparent substrate, and performing the electrical and / or optical characterization includes performing Raman spectroscopy by performing tip-enhanced Raman spectroscopy through the transparent substrate to characterize the dynamic structure of at least one of the one or more molecules.

[0126] In some embodiments, performing the electrical and / or optical characterization includes determining a sequence corresponding to at least one of the one or more molecules.

[0127] In some embodiments of the present disclosure, a chip for measuring electrical and / or optical properties from one or more molecules is provided, the chip including a plurality of electronic devices.

[0128] In some embodiments, the number of the plurality of electronic devices is between 2 and 1000.

[0129] In some embodiments, the number of the plurality of electronic devices is between 1,000 and 10,000.

[0130] In some embodiments, the number of the plurality of electronic devices is greater than 10,000.

[0131] In some embodiments, the plurality of electronic devices is divided into a predetermined number of different groups.

[0132] In some embodiments, the chip further comprises one or more multiwell structures coupled to the chip, wherein individual wells of the one or more multiwell structures correspond to each of a predetermined number of different groups.

[0133] In some embodiments, the chip further includes one or more multiplexers for collecting signals from electronic devices corresponding to each of a predetermined number of different groups.

[0134] In some embodiments, the one or more multiwell structures further include a first multiwell structure coupled to the upper side of the chip for providing a sample to each upper fluidic channel / chamber of the plurality of electronic devices, and / or a second multiwell structure coupled to the lower side of the chip for receiving a sample provided to the upper fluidic channel / chamber after migration of analytes in each sample to the lower fluidic channel / chamber.

[0135] In some embodiments of the present disclosure, a system for high-throughput analysis of single molecules is provided, comprising: a chip; a fluidic device capable of providing an individual sample to each of a set number of different groups; and a controller for storing in a non-transitory memory instructions that, when executed, cause the controller to direct the fluidic device to provide the individual sample to one or more of the predetermined number of different groups, and record, after providing the individual sample, data including one or more of an ionic current between an upper fluidic channel / chamber and a lower fluidic channel / chamber, a tunneling current between a first electrode and a second electrode, and an optical signal from each electronic device of the predetermined number of different groups, wherein the data corresponds to individual mounting and / or translocation events of the individual molecules within the electronic devices of the predetermined number of different groups.

[0136] In some embodiments of the present disclosure, a method for fabricating an electronic device including a nanopore and a tunnel junction is provided, the method including: depositing a first sacrificial layer on a substrate layer to define a final cavity for electrochemical deposition; depositing a second outer sacrificial layer on the substrate layer to define an upper fluid channel / chamber and a lower fluid channel / chamber on two sides of the final cavity; disposing a pair of electrodes on top of the first sacrificial layer with an interval of about 200 nm to 2 μm; depositing a passivation layer on top of the pair of electrodes, the first sacrificial layer, the second outer sacrificial layer, and a substrate layer; and performing a dry etching process to remove a portion of the passivation layer and a portion of the substrate layer from a lower side of the substrate layer to provide a first window in the second outer sacrificial layer corresponding to the upper fluid channel / chamber and a second window in the second outer sacrificial layer corresponding to the lower fluid channel / chamber, respectively.

[0137] In some embodiments, the method further includes chemically etching the first and second sacrificial layers to provide the final cavity, the upper fluidic channel / chamber, and the lower fluidic channel / chamber, and narrowing the gap between the pair of electrodes by a controlled electrochemical deposition process of metal onto the pair of electrodes to form the nanopore and the tunnel junction.

[0138] In some embodiments, the first sacrificial layer comprises one or more of chromium, nickel, magnesium, and aluminum.

[0139] In some embodiments, the first sacrificial layer has a thickness of 1 to 100 nm.

[0140] In some embodiments, the first sacrificial layer has dimensions equivalent to about 10 μm×10 μm.

[0141] In some embodiments, the second outer sacrificial layer comprises one or more of aluminum, magnesium, chromium, and nickel.

[0142] In some embodiments, the second outer sacrificial layer has a thickness of about 100 to 500 nm.

[0143] In some embodiments, the controlled electrodeposition process further comprises performing a pulsed electrochemical deposition process comprising a pulse width of 50 ms or less and a rest period between pulses of between about 500 ms and 2 seconds.

[0144] In some embodiments, the pulse width is between 1 ms and 5 ms.

[0145] In some embodiments, the pulse width is between 1 μs and 500 μs.

[0146] In some embodiments, the pair of electrodes comprises gold, platinum, palladium, or other noble metals or alloys compatible with top-down lithography and electrochemical deposition.

[0147] In some embodiments, the metal used to reduce the gap between a pair of electrodes is nickel, cobalt, gold, palladium, platinum, iridium, alloys thereof, or combinations thereof.

[0148] In some embodiments, reducing the spacing between the pair of electrodes by a controlled electrodeposition process further includes providing metal to only one of the upper and lower fluid channels / chambers.

[0149] In some embodiments, narrowing the gap between the pair of electrodes by a controlled electrodeposition process further comprises repeatedly narrowing and widening the gap between the pair of electrodes a predetermined number of times by repeatedly reversing the polarity of the pair of electrodes.

[0150] In some embodiments, narrowing the gap between a pair of electrodes by a controlled electrodeposition process is used to achieve a final gap between a pair of electrodes of 1 nm to 100 nm.

[0151] In some embodiments, the final spacing is between 1 nm and 20 nm.

[0152] In some embodiments, the final spacing is 1-2 nm.

[0153] In some embodiments, the passivation layer comprises one or more dielectric layers, each comprising one or more of HfO2, ZrO2, SiO2, and Si3N4. In some embodiments, the passivation layer is further encapsulated with one or more additional layers of SU-8 polymer, parylene, and other polymer dielectrics.

[0154] While specific embodiments have been illustrated and described herein, those skilled in the art will recognize that a wide variety of alternative and / or equivalent embodiments or implementations calculated to achieve the same purpose may be substituted for the illustrated and described embodiments without departing from their scope. Those skilled in the art will readily appreciate that the embodiments may be implemented in a wide variety of ways. This application is intended to cover any adaptations or variations of the embodiments discussed herein. It is manifestly intended, therefore, that the embodiments be limited only by the claims and the equivalents thereof. item Some embodiments of the present disclosure have the features described in the following items. [Item 1] 1. An electronic device comprising: an upper fluid channel / chamber and a lower fluid channel / chamber fabricated in a planar substrate, the upper fluid channel / chamber sealed at a lower portion of the electronic device and including a first open window at a top portion of the electronic device, and the lower fluid channel / chamber sealed at the upper portion of the electronic device and including a second open window at the bottom portion of the electronic device; a first electrode and a second electrode disposed in a space of the electronic device between the upper fluid channel / chamber and the lower fluid channel / chamber; a nanogap having a dimension defined in part by the distance between the first electrode and the second electrode, the nanogap having a single path for molecules to travel from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber; Equipped with the distance is between about 1 and 100 nm, the nanogap is self-aligned, and has the narrowest bottleneck in the single path between the upper fluid channel / chamber and the lower fluid channel; Electronic devices. [Item 2] Item 10. The electronic device of item 1, wherein the distance is defined by the first and second electrodes electrochemically deposited under feedback control using one or more metallic materials in the upper fluid channel / chamber and / or the lower fluid channel / chamber, thereby forming the single path. [Item 3] Item 3. The electronic device according to item 2, wherein the planar substrate is a transparent substrate. [Item 4] Item 4. The electronic device according to item 3, wherein the transparent substrate is glass or quartz. [Item 5] Item 3. The electronic device of item 2, wherein the planar substrate is a non-transparent substrate. [Item 6] Item 6. The electronic device of item 5, wherein the planar substrate is silicon coated with SiO2 and / or Si3N4. [Item 7] 7. The device of any one of items 2 to 6, wherein the first electrode and the second electrode are formed of gold, palladium, platinum, or other metals or alloys compatible with top-down lithography and electrochemical deposition, or combinations thereof. [Item 8] 8. The electronic device according to any one of items 2 to 7, wherein the space in which the first electrode and the second electrode are arranged is at least partially formed by one or more dielectric layers. [Item 9] Item 9. The electronic device of item 8, wherein the one or more dielectric layers are HfO2, ZrO2, SiO2, Si3N4, or a combination thereof. [Item 10] 10. The electronic device of claim 8 or 9, further comprising an additional polymer dielectric layer for encapsulation. [Item 11] Item 11. The electronic device of item 10, wherein the additional polymer dielectric layer comprises one or more of SU-8 polymer and parylene. [Item 12] Item 12. The electronic device of any one of items 8 to 11, wherein the one or more dielectric layers, with or without the additional polymer dielectric layer, are about 100 nm to about 100 μm. [Item 13] 13. The electronic device according to any one of items 2 to 12, wherein the one or more metal materials are Ni, Co, gold, palladium, platinum, iridium, an alloy thereof, or a combination thereof. [Item 14] Item 14. The electronic device of item 13, wherein the first and second electrodes electrochemically deposited with the one or more metallic materials under feedback control further comprises a pulsed electrochemical deposition operation having a pulse width of 50 ms or less and a rest period between pulses of between about 500 ms and 2 seconds. [Item 15] 15. The electronic device according to any one of items 2 to 14, wherein the molecule is DNA. [Item 16] 1. A method for measuring electronic and / or optical properties from one or more molecules, comprising: detecting individual mounting and / or translocation events of the one or more molecules using the electronic device according to any one of items 2 to 15 by correlating the ionic current between the upper fluidic channel / chamber and the lower fluidic channel / chamber and the tunneling current between the first electrode and the second electrode through the nanogap; performing an electrical and / or optical characterization of at least one of the one or more molecules; A method comprising: [Item 17] the planar substrate is a transparent substrate, 17. The method of claim 16, wherein performing electrical and / or optical characterization comprises performing Raman spectroscopy by performing tip-enhanced Raman spectroscopy through the transparent substrate to characterize the dynamic structure of the at least one of the one or more molecules. [Item 18] 17. The method of claim 16, wherein said electrical and / or optical characterization comprises determining a sequence corresponding to said at least one of said one or more molecules. [Item 19] A chip for measuring electrical and / or optical properties from one or more molecules, comprising: A chip comprising a plurality of the electronic devices, each of which is the electronic device according to any one of items 2 to 15. [Item 20] 20. The chip according to item 19, wherein the number of the plurality of electronic devices is between 2 and 1000. [Item 21] Item 20. The chip according to item 19, wherein the number of the plurality of electronic devices is between 1,000 and 10,000. [Item 22] 20. The chip of claim 19, wherein the number of electronic devices is greater than 10,000. [Item 23] 23. The chip according to any one of items 19 to 22, wherein the plurality of electronic devices are divided into a predetermined number of different groups. [Item 24] 24. The chip of claim 23, further comprising one or more multiwell structures coupled to the chip, the individual wells of the one or more multiwell structures corresponding to each of the predetermined number of different groups. [Item 25] 25. The chip of any one of items 23 to 24, further comprising one or more multiplexers for collecting signals from electronic devices corresponding to each of the predetermined number of different groups. [Item 26] The one or more multiwell structures include: a first multiwell structure coupled to the top side of the chip for supplying sample to the upper fluidic channels / chambers of each of the plurality of electronic devices; and / or a second multiwell structure coupled to the lower side of the chip for receiving the sample provided to the upper fluidic channel / chamber after transfer of each analyte of the sample to the lower fluidic channel / chamber; 26. The chip according to any one of Items 23 to 25, comprising: [Item 27] 1. A system for high-throughput analysis of single molecules, comprising: The chip according to any one of Items 23 to 26, a fluidic device capable of delivering an individual sample to each of said predetermined number of different groups; A controller, which when executed causes the controller to: instructing the fluidic device to deliver individual samples to one or more of the predetermined number of different groups; after each sample supply, recording data including one or more of an ion current between the upper fluidic channel / chamber and the lower fluidic channel / chamber, a tunneling current between the first electrode and the second electrode, and an optical signal from each electronic device of the predetermined number of different groups, wherein the data corresponds to each mounting and / or rearrangement event of each molecule within the electronic devices of the predetermined number of different groups; a controller for storing instructions in a non-transitory memory to cause the A system including: [Item 28] 1. A method of fabricating an electronic device comprising a nanopore and a tunnel junction, comprising: depositing a first sacrificial layer on a substrate layer to define a final cavity for electrochemical deposition; depositing a second outer sacrificial layer on the substrate layer to define upper and lower fluid channels / chambers on two sides of the final cavity; disposing a pair of electrodes on the first sacrificial layer with a spacing of about 200 nm to 2 μm; depositing a passivation layer on top of the pair of electrodes, the first sacrificial layer, the second outer sacrificial layer, and the substrate layer; performing a dry etching process to remove a portion of the passivation layer and a portion of the substrate layer from a lower side of the substrate layer, thereby providing a first window in the second outer sacrificial layer corresponding to the upper fluid channel / chamber and a second window in the second outer sacrificial layer corresponding to the lower fluid channel / chamber; A method comprising: [Item 29] chemically etching the first and second sacrificial layers to provide the final cavity, the upper fluid channel / chamber, and the lower fluid channel / chamber; narrowing the gap between the pair of electrodes by a controlled electrochemical deposition process of metal onto the pair of electrodes to form the nanopore and the tunnel junction; 29. The method of claim 28, further comprising: [Item 30] 30. The method of claim 29, wherein the first sacrificial layer comprises one or more of chromium, nickel, magnesium, and aluminum. [Item 31] 31. The method according to any one of items 28 to 30, wherein the first sacrificial layer is between 1 and 100 nm thick. [Item 32] 32. The method according to any one of items 28 to 31, wherein the first sacrificial layer has dimensions equivalent to about 10 μm×10 μm. [Item 33] 33. The method of any one of items 28 to 32, wherein the second outer sacrificial layer comprises one or more of aluminum, magnesium, chromium, and nickel. [Item 34] 34. The method according to any one of items 28 to 33, wherein the second outer sacrificial layer has a thickness of about 100 to 500 nm. [Item 35] The controlled electrodeposition process comprises: performing a pulsed electrochemical deposition process comprising a pulse width of 50 ms or less and a rest period between pulses of between about 500 ms and 2 seconds; The method according to any one of Items 29 to 34. [Item 36] Item 36. The method of item 35, wherein the pulse width is between 1 ms and 5 ms. [Item 37] 36. The method of claim 35, wherein the pulse width is between 1 μs and 500 μs. [Item 38] 38. The method according to any one of items 28 to 37, wherein the pair of electrodes comprises gold, platinum, palladium, or other noble metal or alloy compatible with top-down lithography and electrochemical deposition. [Item 39] 39. The method according to any one of items 28 to 38, wherein the metal used to narrow the gap between the pair of electrodes is nickel, cobalt, gold, palladium, platinum, iridium, an alloy thereof, or a combination thereof. [Item 40] Narrowing the gap between the pair of electrodes by the controlled electrodeposition process includes: 40. The method of any one of items 29 to 39, further comprising providing the metal in only one of the upper fluid channel / chamber and the lower fluid channel / chamber. [Item 41] Narrowing the gap between the pair of electrodes by the controlled electrodeposition process includes: 41. The method according to any one of items 29 to 40, further comprising repeatedly narrowing and widening the gap between the pair of electrodes a predetermined number of times by repeatedly reversing the polarity of an electrochemical potential applied to the pair of electrodes. [Item 42] 42. The method according to any one of items 29 to 41, wherein the narrowing of the gap between the pair of electrodes by the controlled electrodeposition process is used to make the final gap between the pair of electrodes between 1 nm and 100 nm. [Item 43] Item 43. The method of item 42, wherein the final spacing is between 1 nm and 20 nm. [Item 44] Item 44. The method of item 43, wherein the final spacing is between 1 and 2 nm. [Item 45] 45. The method of any one of items 28 to 44, wherein the passivation layer comprises one or more dielectric layers, each comprising one or more of HfO2, ZrO2, SiO2, and Si3N4. [Item 46] Item 46. The method of item 45, wherein the passivation layer is further encapsulated with one or more additional layers of SU-8 polymer, parylene, and other polymer dielectrics. [Item 47] A device, a system including the device, a method of making the device, a method of making the system, a method of using the device, or a method of using the system, wherein the device is for delivering molecules across tunable metallic nanogaps, linking molecules across the tunable metallic nanogaps, and / or measuring electrical and optical properties that enable single molecule detection, substantially as disclosed and described in the specification and drawings herein. [Configuration 1] an upper fluid channel / chamber and a lower fluid channel / chamber fabricated in a planar substrate, the upper fluid channel / chamber sealed at a lower portion of an electronic device and including a first open window at a top portion of the electronic device, and the lower fluid channel / chamber sealed at the upper portion of the electronic device and including a second open window at the bottom portion of the electronic device; a first electrode and a second electrode disposed in a space of the electronic device between the upper fluid channel / chamber and the lower fluid channel / chamber; a nanogap having a dimension defined in part by a distance between the first electrode and the second electrode, the nanogap having a single path for molecules to travel from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber; the distance is between about 1 and 100 nm, and has the narrowest bottleneck in the single path between the upper fluid channel / chamber and the lower fluid channel; Electronic devices. [Configuration 2] The electronic device of configuration 1, wherein the distance is defined by electrochemically depositing the first electrode and the second electrode under feedback control using one or more metal materials in the upper fluid channel / chamber and / or the lower fluid channel / chamber, thereby forming the single path. [Configuration 3] 3. The electronic device of claim 2, wherein the planar substrate is a transparent substrate. [Configuration 4] 4. The electronic device of claim 3, wherein the transparent substrate is glass or quartz. [Configuration 5] 3. The electronic device of claim 2, wherein the planar substrate is a non-transparent substrate. [Configuration 6] The planar substrate is made of SiO 2 and / or Si 3 N 4 6. The electronic device of claim 5, wherein the electronic device is silicon coated with [Configuration 7] 3. The electronic device of claim 2, wherein the first electrode and the second electrode are formed of gold, palladium, platinum, or other noble metals or alloys compatible with top-down lithography and electrochemical deposition, or combinations thereof. [Configuration 8] 3. The electronic device of claim 2, wherein the space in which the first electrode and the second electrode are disposed is at least partially formed by one or more dielectric layers. [Configuration 9] The one or more dielectric layers may be HfO 2 , ZrO 2 , SiO 2 , Si 3 N 4 9. The electronic device of claim 8, wherein the electronic device is a [Configuration 10] 9. The electronic device of embodiment 8, further comprising an additional polymer dielectric layer for encapsulation. [Configuration 11] 11. The electronic device of claim 10, wherein the additional polymer dielectric layer comprises one or more of an SU-8 polymer and a parylene. [Configuration 12] 12. The electronic device of claim 11, wherein the one or more dielectric layers, with or without the additional polymer dielectric layer, are about 100 nm to about 100 μm. [Configuration 13] 3. The electronic device of claim 2, wherein the one or more metallic materials are Ni, Co, gold, palladium, platinum, iridium, alloys thereof, or combinations thereof. [Configuration 14] 14. The electronic device of claim 13, further comprising a pulsed electrochemical deposition process in which the first and second electrodes electrochemically deposited with the one or more metallic materials under feedback control have a pulse width of 50 ms or less and a rest period between pulses of between about 500 ms and 2 seconds. [Configuration 15] 3. The electronic device of claim 2, wherein the molecule is DNA. [Configuration 16] 10. The electronic device of claim 1, wherein the nanogap is self-aligned. [Configuration 17] 1. A method for measuring electrical and / or optical properties from one or more molecules, comprising: Detecting individual mounting and / or rearrangement events of the one or more molecules by correlating ionic currents between the upper and lower fluidic channels / chambers and tunneling currents between the first and second electrodes through the nanogap using the electronic device of any one of configurations 1 to 16; performing at least one electrical and / or optical characterization of said one or more molecules; A method comprising: [Configuration 18] the planar substrate is a transparent substrate, wherein said performing electrical and / or optical characterization comprises performing Raman spectroscopy by performing tip-enhanced Raman spectroscopy through said transparent substrate to characterize the dynamic structure of said at least one of said one or more molecules. 18. The method according to claim 17. [Configuration 19] 18. The method of claim 17, wherein said performing electrical and / or optical characterization comprises determining a sequence corresponding to said at least one of said one or more molecules. [Configuration 20] A chip for measuring electrical and / or optical properties from one or more molecules, comprising: a plurality of electronic devices, each of which is the electronic device according to any one of configurations 1 to 16; Tips. [Configuration 21] 21. The chip according to claim 20, wherein the number of the plurality of electronic devices is 2 to 1000. [Configuration 22] 21. The chip of claim 20, wherein the number of the plurality of electronic devices is 1,000 to 10,000. [Configuration 23] 21. The chip of embodiment 20, wherein the number of electronic devices is greater than 10,000. [Configuration 24] 21. The chip of claim 20, wherein the plurality of electronic devices are divided into a predetermined number of different groups. [Configuration 25] The chip of configuration 24, further comprising one or more multiwell structures coupled to the chip, wherein individual wells of the one or more multiwell structures correspond to each of the predetermined number of different groups. [Configuration 26] 25. The chip of configuration 24, further comprising one or more multiplexers for collecting signals from electronic devices corresponding to each of the predetermined number of different groups. [Configuration 27] the one or more multiwell structures include a first multiwell structure coupled to the top side of the chip for supplying sample to the upper fluidic channels / chambers of each of the plurality of electronic devices; and / or a second multiwell structure coupled to a lower side of the chip for receiving the samples provided to the upper fluidic channels / chambers after analytes in each of the samples have migrated to the lower fluidic channels / chambers; 26. The chip of claim 25, comprising: [Configuration 28] 1. A system for high-throughput analysis of single molecules, comprising: a fluidic device capable of providing an individual sample to each of said predetermined number of different groups; When executed, the controller will directing the fluidic device to deliver individual samples to one or more of the predetermined number of different groups; after said supplying of each sample, recording data including one or more of an ionic current between said upper fluidic channel / chamber and said lower fluidic channel / chamber, a tunneling current between said first electrode and said second electrode, and an optical signal from each electronic device of said predetermined number of different groups, said data corresponding to individual mounting and / or rearrangement events of individual molecules within said electronic devices of said predetermined number of different groups; a controller for storing instructions in a non-transitory memory to cause the 25. The chip of claim 24, comprising: [Configuration 29] 1. A method of fabricating an electronic device comprising a nanopore and a tunnel junction, comprising: depositing a first sacrificial layer on the substrate layer to define a final cavity for electrochemical deposition; depositing a second outer sacrificial layer on the substrate layer to define upper and lower fluid channels / chambers on two sides of the final cavity; disposing a pair of electrodes on the first sacrificial layer with a spacing of about 200 nm to 2 μm; depositing a passivation layer on top of the pair of electrodes, the first sacrificial layer, the second outer sacrificial layer, and the substrate layer; performing a dry etching process to remove a portion of the passivation layer and a portion of the substrate layer from a lower side of the substrate layer, thereby providing a first window in the second outer sacrificial layer corresponding to the upper fluid channel / chamber and a second window in the second outer sacrificial layer corresponding to the lower fluid channel / chamber, respectively; A method comprising: [Configuration 30] chemically etching the first and second sacrificial layers to provide the final cavity, the upper fluid channel / chamber, and the lower fluid channel / chamber; narrowing the gap between the pair of electrodes by a controlled electrochemical deposition process of metal onto the pair of electrodes to form the nanopore and the tunnel junction; 30. The method of claim 29, further comprising: [Configuration 31] 31. The method of claim 30, wherein the first sacrificial layer comprises one or more of chromium, nickel, magnesium, and aluminum. [Configuration 32] 30. The method of claim 29, wherein the first sacrificial layer is between 1 and 100 nm thick. [Configuration 33] 30. The method of claim 29, wherein the first sacrificial layer has dimensions equivalent to approximately 10 μm×10 μm. [Configuration 34] 30. The method of claim 29, wherein the second outer sacrificial layer comprises one or more of aluminum, magnesium, chromium, and nickel. [Configuration 35] 30. The method of claim 29, wherein the second outer sacrificial layer has a thickness of about 100-500 nm. [Configuration 36] The controlled electrochemical deposition process comprises: performing a pulsed electrochemical deposition process having a pulse width of 50 ms or less and a rest period between pulses of about 500 ms to 2 seconds; The method according to any one of aspects 30 to 35. [Configuration 37] 37. The method of claim 36, wherein the pulse width is between 1 ms and 5 ms. [Configuration 38] 37. The method of claim 36, wherein the pulse width is between 1 μs and 500 μs. [Configuration 39] 39. The method of any one of aspects 29-35, 37 and 38, wherein the pair of electrodes comprises gold, platinum, palladium, or other metal or alloy compatible with top-down lithography and electrochemical deposition. [Configuration 40] 39. The method of any one of claims 29-35, 37, and 38, wherein the metal used to narrow the gap between the pair of electrodes is nickel, cobalt, gold, palladium, platinum, iridium, alloys thereof, or combinations thereof. [Configuration 41] Narrowing the gap between the pair of electrodes by the controlled electrochemical deposition process includes: providing the metal to only one of the upper fluid channel / chamber and the lower fluid channel / chamber. A method according to any one of aspects 30 to 35, 37, and 38. [Configuration 42] Narrowing the gap between the pair of electrodes by the controlled electrochemical deposition process includes: and repeatedly narrowing and widening the gap between the pair of electrodes a predetermined number of times by repeatedly reversing the polarity of an electrochemical potential applied to the pair of electrodes. A method according to any one of aspects 30 to 35, 37, and 38. [Configuration 43] 39. The method of any one of aspects 30-35, 37 and 38, wherein the controlled electrochemical deposition process is used to narrow the gap between the pair of electrodes to a final gap between the pair of electrodes of 1 nm to 100 nm. [Configuration 44] 44. The method of claim 43, wherein the final spacing is between 1 nm and 20 nm. [Configuration 45] 45. The method of claim 44, wherein the final spacing is 1 to 2 nm. [Configuration 46] The passivation layers each comprise HfO 2 , ZrO 2 , SiO 2 , and Si 3 N 4 The method of any one of configurations 29-35, 37, 38, 44, and 45, comprising one or more layers of a dielectric comprising one or more of: [Configuration 47] 47. The method of embodiment 46, wherein the passivation layer is further encapsulated with one or more additional layers of SU-8 polymer, parylene, and other polymer dielectrics.

Claims

1. A chip for measuring electrical and / or optical properties from one or more molecules, comprising: a plurality of electronic devices, each of the plurality of electronic devices comprising: an upper fluid channel / chamber and a lower fluid channel / chamber fabricated in a planar substrate, the upper fluid channel / chamber sealed at a lower portion of an electronic device and including a first open window at a top portion of the electronic device, and the lower fluid channel / chamber sealed at the upper portion of the electronic device and including a second open window at the bottom portion of the electronic device; a first electrode and a second electrode disposed in a space of the electronic device between the upper fluid channel / chamber and the lower fluid channel / chamber; a nanogap having a dimension defined in part by a distance between the first electrode and the second electrode, the nanogap having a single path for molecules to travel from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber; a plurality of electronic devices, the distance is between about 1 and 100 nm, and the nanogap comprises the narrowest bottleneck in the single path between the upper fluidic channel / chamber and the lower fluidic channel; the plurality of electronic devices is divided into a predetermined number of different groups; the chip further includes one or more multiwell structures coupled to the chip, wherein individual wells of the one or more multiwell structures correspond to each of the predetermined number of different groups; Tips.

2. 2. The chip of claim 1, wherein for a first electronic device of the plurality of electronic devices, the distance is defined by electrochemically depositing the first electrode and the second electrode under feedback control using one or more metallic materials within the upper fluidic channel / chamber and / or the lower fluidic channel / chamber, thereby forming the single path.

3. The chip of claim 2 , wherein for the first electronic device, the planar substrate is a transparent substrate.

4. The chip of claim 3 , wherein for the first electronic device, the transparent substrate is glass or quartz.

5. The chip of claim 2 , wherein for the first electronic device, the planar substrate is a non-transparent substrate.

6. For the first electronic device, the planar substrate is made of SiO ２ and / or Si ３ N ４ 6. The chip of claim 5, wherein the chip is silicon coated with

7. 3. The chip of claim 2, wherein for the first electronic device, the first electrode and the second electrode are formed of gold, palladium, platinum, or other noble metals or alloys compatible with top-down lithography and electrochemical deposition, or combinations thereof.

8. The chip of claim 2 , wherein for the first electronic device, a space in which the first electrode and the second electrode are disposed is at least partially formed by one or more dielectric layers.

9. For the first electronic device, the one or more dielectric layers are HfO ２ , ZrO ２ , SiO ２ , Si ３ N ４ or a combination thereof.

10. The chip of claim 8 further comprising an additional polymer dielectric layer for encapsulation.

11. The chip of claim 10 , wherein the first electronic device further comprises one or more of the additional polymer dielectric layer: SU-8 polymer and parylene.

12. The chip of claim 11 , wherein for the first electronic device, the one or more dielectric layers, with or without the additional polymer dielectric layer, are from about 100 nm to about 100 μm.

13. The chip of claim 2 , wherein for the first electronic device, the one or more metallic materials are Ni, Co, gold, palladium, platinum, iridium, alloys thereof, or combinations thereof.

14. 14. The chip of claim 13, wherein the first and second electrodes electrochemically deposited with the one or more metallic materials under feedback control for the first electronic device further comprises a pulsed electrochemical deposition process having a pulse width of 50 ms or less and a rest period between pulses of between about 500 ms and 2 seconds.

15. The chip of claim 2 , wherein the molecule is DNA.

16. The chip of claim 1 , wherein for a first electronic device of the plurality of electronic devices, the nanogap is self-aligned.

17. 1. A method for measuring electrical and / or optical properties from one or more molecules using an electronic device, said electronic device comprising: an upper fluid channel / chamber and a lower fluid channel / chamber fabricated in a planar substrate, the upper fluid channel / chamber sealed at a lower portion of an electronic device and including a first open window at a top portion of the electronic device, and the lower fluid channel / chamber sealed at the upper portion of the electronic device and including a second open window at the bottom portion of the electronic device; a first electrode and a second electrode disposed in a space of the electronic device between the upper fluid channel / chamber and the lower fluid channel / chamber; a nanogap having a dimension defined in part by a distance between the first electrode and the second electrode, the nanogap having a single path for molecules to travel from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber; Including, the distance is between about 1 and 100 nm, and the nanogap comprises the narrowest bottleneck in the single path between the upper fluidic channel / chamber and the lower fluidic channel; The method comprises: Detecting individual mounting and / or translocation events of the one or more molecules using the chip of claim 1 by correlating ionic currents between the upper and lower fluidic channels / chambers and tunneling currents between the first and second electrodes through the nanogap; performing at least one electrical and / or optical characterization of the one or more molecules; Including, the planar substrate is a transparent substrate, performing the electrical and / or optical characterization includes performing Raman spectroscopy by performing tip-enhanced Raman spectroscopy through the transparent substrate to characterize the dynamic structure of at least one of the one or more molecules; method.

18. 20. The method of claim 17, wherein said electrical and / or optical characterization comprises determining a sequence corresponding to said at least one of said one or more molecules.

19. The chip according to claim 1 , wherein the number of the plurality of electronic devices is between 2 and 1000.

20. The chip of claim 1 , wherein the number of the plurality of electronic devices is between 1000 and 10,000.

21. The chip of claim 1 , wherein the number of electronic devices in the plurality is greater than 10,000.

22. A chip for measuring electrical and / or optical properties from one or more molecules, comprising: a plurality of electronic devices, each of the plurality of electronic devices comprising: an upper fluid channel / chamber and a lower fluid channel / chamber fabricated in a planar substrate, the upper fluid channel / chamber sealed at a lower portion of an electronic device and including a first open window at a top portion of the electronic device, and the lower fluid channel / chamber sealed at the upper portion of the electronic device and including a second open window at the bottom portion of the electronic device; a first electrode and a second electrode disposed in a space of the electronic device between the upper fluid channel / chamber and the lower fluid channel / chamber; a nanogap having a dimension defined in part by a distance between the first electrode and the second electrode, the nanogap having a single path for molecules to travel from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber; a plurality of electronic devices, the distance is between about 1 and 100 nm, and the nanogap comprises the narrowest bottleneck in the single path between the upper fluidic channel / chamber and the lower fluidic channel; the plurality of electronic devices is divided into a predetermined number of different groups; the chip further comprises one or more multiplexers for collecting signals from electronic devices corresponding to each of the predetermined number of different groups; Tips.

23. the one or more multiwell structures include a first multiwell structure coupled to the top side of the chip for supplying sample to the upper fluidic channels / chambers of each of the plurality of electronic devices; and / or a second multiwell structure coupled to the lower side of the chip for receiving the samples provided to the upper fluidic channels / chambers after analytes in each of the samples have migrated to the lower fluidic channels / chambers; The chip of claim 1 , comprising:

24. 1. A system for high-throughput analysis of single molecules, comprising: A chip for measuring electrical and / or optical properties from one or more molecules, comprising: the chip includes a plurality of electronic devices; Each of the plurality of electronic devices an upper fluid channel / chamber and a lower fluid channel / chamber fabricated in a planar substrate, the upper fluid channel / chamber sealed at a lower portion of an electronic device and including a first open window at a top portion of the electronic device, and the lower fluid channel / chamber sealed at the upper portion of the electronic device and including a second open window at the bottom portion of the electronic device; a first electrode and a second electrode disposed in a space of the electronic device between the upper fluid channel / chamber and the lower fluid channel / chamber; a nanogap having a dimension defined in part by a distance between the first electrode and the second electrode, the nanogap having a single path for molecules to travel from the upper fluidic channel / chamber to the lower fluidic channel / chamber and / or from the lower fluidic channel / chamber to the upper fluidic channel / chamber; Including, the distance is between about 1 and 100 nm, and the nanogap comprises the narrowest bottleneck in the single path between the upper fluidic channel / chamber and the lower fluidic channel; the plurality of electronic devices are divided into a predetermined number of different groups; a fluidic device capable of providing an individual sample to each of said predetermined number of different groups; When executed, the controller will directing the fluidic device to deliver individual samples to one or more of the predetermined number of different groups; after said supply of each sample, recording data including one or more of an ionic current between the upper fluidic channel / chamber and the lower fluidic channel / chamber, a tunneling current between the first electrode and the second electrode, and an optical signal from each electronic device of said predetermined number of different groups, said data corresponding to individual mounting and / or rearrangement events of individual molecules within said electronic devices of said predetermined number of different groups; a controller for storing instructions in a non-transitory memory to cause the A system comprising:

25. 18. The method of claim 17, wherein the distance is defined by electrochemically depositing the first and second electrodes in the upper fluid channel / chamber and / or the lower fluid channel / chamber using one or more metallic materials under feedback control, thereby forming the single path.

26. 23. The chip of claim 22, wherein for a first electronic device of the plurality of electronic devices, the distance is defined by electrochemically depositing the first and second electrodes in the upper fluidic channel / chamber and / or the lower fluidic channel / chamber using one or more metallic materials under feedback control, thereby forming the single path.

27. 25. The system of claim 24, wherein for a first electronic device of the plurality of electronic devices, the distance is defined by electrochemically depositing the first and second electrodes in the upper fluidic channel / chamber and / or the lower fluidic channel / chamber using one or more metallic materials under feedback control, thereby forming the single pathway.

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