WO2017087908A1

WO2017087908A1 - Nanochannel devices and methods for analysis of molecules

Info

Publication number: WO2017087908A1
Application number: PCT/US2016/062950
Authority: WO
Inventors: Aaron Tynes HAMMACK; Bruno Marchon; Anika A. KINKHABWALA; Ping-Chuan Christina TSAI
Original assignee: Epibiome Inc
Current assignee: Epibiome Inc
Priority date: 2015-11-20
Filing date: 2016-11-18
Publication date: 2017-05-26
Anticipated expiration: 2018-05-20

Abstract

The present invention provides devices and methods for analyzing a target analyte based on simultaneous measurements of transverse tunneling signals. The device comprises a nanochannel, a plurality of electrodes longitudinally disposed along the nanochannel and forming a plurality of tunneling gaps. As the target analyte translocates through the nanochannel, electrical signals measured at the plurality of tunneling gaps allow characterization of the target analyte. The device may comprise a plurality of nanochannels connected by a plurality of reservoirs, wherein the plurality of reservoirs can be coupled to driving systems for translocating the target analyte. Nanochannels with bends that apply mechanical tension on polymers are further disclosed.

Description

NANOCHANNEL DEVICES AND METHODS FOR ANALYSIS OF MOLECULES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Patent Application No. 62/258,413 filed on November 20, 2015, the contents of which are incorporated herein by reference in their entirety.

FILED OF THE INVENTION

[0001] The present invention relates to devices, systems, and methods for analysis of molecules, such as biopolymers, by simultaneously measuring multiple transverse tunneling signals as the molecules translocate through a nanochannel.

BACKGROUND OF THE INVENTION

[0002] The sequence of a biopolymer, such as a DNA, RNA or protein, directly determines its structure and function. Methods for determining the sequences of biopolymers rapidly, accurately, and cheaply have remained to be in broad demand, and promise to have major impacts in diverse fields, including healthcare, biotechnology, agriculture, food safety, and security. Despite recent developments in the next-generation nucleic acid sequencing technologies, current DNA sequencing runs can take hours or days before sequencing data become available, and are cost-prohibitive for a lot of applications and consumer groups.

[0003] While most current nucleic acid sequencing methods require manipulation steps, such as amplification or labeling of the nucleic acids, nanopore -based methods provide a direct, single -molecule sequencing approach. Conventional nanopore DNA sequencers measure the current flow through a nanopore that is obstructed by a single DNA molecule as the DNA passes through the nanopore. As each nucleobase causes a slightly different blockage for current flowing through the nanopore, the changing current through the nanopore can be used to read the DNA sequence for a single molecule of DNA. Current implementations of the nanopore- based sequencing methods are slow, and suffer from high error rates.

[0004] Theoretical and experimental studies have demonstrated that electrons tunneling across a DNA molecule can be used to identify single nucleotides (See, for example, M. Zwolak, M. Di Ventra, Nano Lett. 5, 421 (2005) and US Patent No. 6,905,586). Nanopore-based sequencing devices characterizing DNA by measuring the tunneling currents or capacitance have been designed (See, for example, WO2008124706A2). However, to extract sequence information, the electrodes probing the tunneling current have to be functionalized with reagents designed to hydrogen bond specifically to one nucleobase or another to enhance the differential signals of the four nucleobases.

[0005] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0006] The present application provides devices, systems and methods for analyzing a target analyte by simultaneous measurements of transverse tunneling signals as the target analyte translocates along a nanochannel.

[0007] In one aspect of the present application, there is provided a device for analyzing a target analyte, comprising: a) a nanochannel; b) a plurality of detection electrodes disposed longitudinally along the nanochannel; and c) one or more reference electrodes disposed longitudinally along the nanochannel; wherein the target analyte translocates through the nanochannel; and wherein each detection electrode forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes. In some

embodiments, the nanochannel is dimensioned to allow translocation of only a single copy of the target analyte in an unfolded fashion.

[0008] In some embodiments according to any one of the devices described above, the plurality of detection electrodes are disposed longitudinally along a first side of the nanochannel, and the one or more reference electrodes are disposed longitudinally along a side opposite to the first side of the nanochannel.

[0009] In some embodiments according to any one of the devices described above, the plurality of detection electrodes and/or the one or more reference electrodes are disposed directly along the nanochannel.

[0010] In some embodiments according to any one of the devices described above, the nanochannel has a cross-section, and the cross-section is rectangular.

[0011] In some embodiments according to any one of the devices described above, the width of each tunneling gap is about 1 nm to about 4 nm (such as about 2 nm).

[0012] In some embodiments according to any one of the devices described above, the length of the nanochannel is between about 50 nm and about 5μπι.

[0013] In some embodiments according to any one of the devices described above, the nanochannel comprises a bend that applies a mechanical tension on the target analyte when the target analyte passes around the bend. In some embodiments, the plurality of detection electrodes are disposed in proximity to the bend. In some embodiments, the nanochannel is serpentine shaped.

[0014] In some embodiments according to any one of the devices described above, the plurality of detection electrodes are unevenly spaced. In some embodiments, the plurality of detection electrodes are randomly spaced.

[0015] In some embodiments according to any one of the devices described above, wherein the target analyte is a polymer comprising monomelic units each having a unit length, the spacing between any two adjacent detection electrodes is not an integer multiple of the unit length.

[0016] In some embodiments according to any one of the devices described above, the device further comprises a driving system for translocating the target analyte through the nanochannel. In some embodiments, the driving system comprises a DC power supply configured to generate an electrophoretic bias longitudinally across the nanochannel. In some embodiments, the driving system further comprises an AC power supply configured to apply an alternating electrophoretic bias longitudinally across the nanochannel, wherein the altering electrophoretic bias allows reversal of the direction in which the target analyte translocates. In some embodiments, the driving system comprises a thermal element configured to generate a thermal gradient longitudinally across the nanochannel. In some embodiments, the driving system comprises a magnetic field capable of pulling a magnetic bead attached to one end of the target analyte. In some embodiments, the driving system comprises an optical field capable of pulling a dielectric bead attached to one end of the target analyte.

[0017] In some embodiments according to any one of the devices described above, the nanochannel has a first open end and a second open end, and the first open end is connected to a first reservoir on and the second open end of the nanochannel is connected to a second reservoir. In some embodiments, the driving system translocates the target analyte from the first reservoir to the second reservoir through the nanochannel.

[0018] In some embodiments according to any one of the devices described above, the nanochannel comprises a wall comprising an insulating layer. In some embodiments, the wall further comprises an interior coating that facilitates absorption of the medium on the interior surface of the wall. In some embodiments, the wall further comprises a protective layer that reduces dielectric breakdown of the insulating layer. In some embodiments, the wall comprises a plurality of insulating layers and a plurality of protective layers, and wherein the plurality of insulating layers and the plurality of protective layers form an alternating structure. In some embodiments, the wall is fabricated by atomic layer deposition.

[0019] In some embodiments according to any one of the devices described above, the nanochannel is filled with a medium comprising a solvent compatible with the target analyte. In some embodiments, the medium further comprises an electrolyte. In some embodiments, the medium further comprises a reducing agent. In some embodiments, the medium further comprises a denaturing agent. In some embodiments, the medium is adjusted to provide a dielectric constant suitable for probing a tunneling resonance level of the target analyte. In some embodiments, wherein the solvent is hydrophilic, the interior coating comprises a hydrophilic material, such as a hydrophilic material selected from the group consisting of polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), and polyvinyl alcohol (PVA). In some embodiments, wherein the solvent is lipophilic, the interior coating comprises a lipophilic material.

[0020] In some embodiments according to any one of the devices described above, the device comprises a tuning element operable to adjust one or more of the plurality of tunneling gaps to allow probing of a tunneling resonance level of the target analyte that passes through the one or more tunneling gaps. In some embodiments, the tuning element is a tunable power supply configured to generate a voltage bias across the one or more tunneling gaps. In some embodiments, the tuning element is a constriction element operable to reversibly adjust the width of the one or more tunneling gaps. In some embodiments, the constriction element is selected from a thermal actuator, a piezoelectric actuator, and an acoustic actuator.

[0021] In some embodiments according to any one of the devices described above, the plurality of detection electrodes comprise one or more metallic electrodes.

[0022] In some embodiments according to any one of the devices described above, the plurality of detection electrodes comprise one or more non-metallic or semi-metallic electrodes. In some embodiments, the plurality of detection electrodes comprise one or more graphene sheets.

[0023] In some embodiments according to any one of the devices described above, the plurality of detection electrodes comprise one or more triangular electrodes.

[0024] In some embodiments according to any one of the devices described above, each of the plurality of detection electrodes is connected to an electrical detector, wherein the electrical detector measures an electrical signal when the target analyte passes through the tunneling gap associated with the detection electrode. In some embodiments, the plurality of detection electrodes comprise one or more plasmonically active electrodes to plasmonically enhance the electrical signals when the target analyte passes through the one or more tunneling gaps associated therewith.

[0025] In some embodiments according to any one of the devices described above, the plurality of detection electrodes comprise one or more spin-value electrodes that can spin- polarize electrons tunneling through the tunneling gaps. In some embodiments, the target analyte is magnetically labeled to enhance the electrical signals detected by the electrical detectors connected to the one or more spin-value electrodes.

[0026] In some embodiments according to any one of the devices described above, the nanochannel is transparent.

[0027] In some embodiments according to any one of the devices described above, the device further comprises an optical detector for simultaneous measurement of an optical signal when the target analyte passes through one or more of the plurality of tunneling gaps. In some embodiments, the optical detector is a microscope. In some embodiments, the optical signal is one or more selected from the group consisting of electroluminescence, fluorescence, Raman scattering, bioluminescence, and chemiluminescence associated with the target analyte. In some embodiments, the device further comprises a plasmonically enhanced electrode that plasmonically enhances the optical signal. In some embodiments, each of the plurality of detection electrodes is a plasmonically enhanced electrode.

[0028] In some embodiments according to any one of the devices described above, the device further comprises a biological nanopore disposed within one of the plurality of tunneling gaps.

[0029] In some embodiments according to any one of the devices described above, the device comprises a plurality of the nanochannels, wherein the nanochannels are connected to each other via a plurality of reservoirs. In some embodiments, each of the plurality of the nanochannel has a plurality of detection electrodes disposed longitudinally along the nanochannel, each of the plurality of the nanochannel has and one or more reference electrodes disposed longitudinally along the nanochannel, and each detection electrode forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes. In some

embodiments, the plurality of nanochannels are connected to each other via the plurality of reservoirs to provide a loop through which the target analyte translocates.

[0030] In some embodiments according to any one of the devices described above, the device further comprises a loading reservoir fluidically connected to the nanochannel, wherein the target analyte is loaded to the loading reservoir in order to be exposed to the nanochannel. In some embodiments, the loading reservoir is connected to the nanochannel via a funnel-shaped port.

[0031] In some embodiments according to any one of the devices described above, the target analyte is a non-polymeric molecule.

[0032] In some embodiments according to any one of the devices described above, the target analyte is a non-biological polymer.

[0033] In some embodiments according to any one of the devices described above, the target analyte is a biopolymer, such as a DNA, RNA or polypeptide. In some embodiments, the device further comprises an unfolding compartment that unfolds the biopolymer. In some embodiments, the unfolding compartment comprises a reducing agent. In some embodiments, the unfolding compartment comprises a denaturing agent. In some embodiments, wherein the target analyte is a polypeptide, the unfolding compartment comprises a protease that cleaves the polypeptide into a plurality of polypeptide fragments, wherein each polypeptide fragment translocates through the nanochannel.

[0034] In some embodiments according to any one of the devices described above, wherein the target analyte is a DNA, or an RNA, the device further comprises a polymerase attached to one end of the nanochannel to control the speed at which the target analyte translocates through the nanochannel.

[0035] One aspect of the present invention provides a method of analyzing a target analyte, comprising: a) applying a force to translocate the target analyte in a first direction through a nanochannel; wherein a plurality of detection electrodes are disposed longitudinally along the nanochannel; wherein one or more reference electrodes are disposed longitudinally along the nanochannel; and wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes; b) simultaneously measuring a plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and c) characterizing the target analyte based on the plurality of electrical signals. In some embodiments, said applying a force comprises applying an electric field longitudinally across the nanochannel. In some embodiments, said applying a force comprises applying a magnetic field longitudinally across the nanochannel, and wherein the target analyte is attached to a magnetic bead. In some embodiments, said applying a force comprises applying a magnetic field gradient transversely across the nanochannel, and wherein the target analyte is attached to a magnetic bead. In some embodiments, said applying a force comprises applying an optical field longitudinally across the nanochannel, and wherein the target analyte is attached to a dielectric bead. In some embodiments, the steps a)-c) are repeated for a desired number of times.

[0036] In some embodiments according to any one of the methods of analyzing described above, the method further comprises: d) applying a second force to translocate the target analyte in the reverse direction with respect to the first direction through the nanochannel; e) simultaneously measuring a second plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and f) characterizing the target analyte based on the second plurality of electrical signals. In some embodiments, steps a)-f) are repeated more than once.

[0037] In some embodiments according to any one of the methods of analyzing described above, the method further comprises recording a signal profile (including profiles of multiple signals, or multiple modalities of signals) comprising the plurality of electrical signals as a function of time. In some embodiments, said characterizing comprises differential analysis of the signal profile (including profiles of multiple signals, or multiple modalities of signals) to obtain at least one parameter reflective of an identification of the target analyte.

[0038] In some embodiments according to any one of the methods of analyzing described above, the method further comprises unfolding the target analyte prior to translocating the target analyte through the nanochannel.

[0039] In some embodiments according to any one of the methods of analyzing described above, only one copy of the target analyte translocates through the nanochannel.

[0040] In some embodiments according to any one of the methods of analyzing described above, the target analyte is a non-polymeric molecule.

[0041] In some embodiments according to any one of the methods of analyzing described above, the target analyte is a polymer. In some embodiments, wherein the polymer comprises monomeric units each having a unit length, the spacing between any two adjacent detection electrodes is not an integer multiple of the unit length. In some embodiments, the target analyte is a non-biological polymer. In some embodiments, the target analyte is a biopolymer, such as a DNA, an RNA or a protein.

[0042] In some embodiments according to any one of the methods of analyzing described above, wherein the target analyte is a DNA or an RNA, the method further comprises binding the target analyte to a polymerase.

[0043] In some embodiments according to any one of the methods of analyzing described above, wherein the target analyte is a polypeptide, the method further comprises cleaving the polypeptide with a protease. In some embodiments, the method further comprises treating the polypeptide with a reducing agent that reduces the disulfide bonds of the polypeptides. In some embodiments, the method further comprises treating the polypeptide with a denaturing agent.

[0044] In some embodiments according to any one of the methods of analyzing described above, said characterizing comprises determining the length of the target analyte.

[0045] In some embodiments according to any one of the methods of analyzing described above, said characterizing comprises determining the sequence of the target analyte.

[0046] In some embodiments according to any one of the methods of analyzing described above, said characterizing comprises determining the vibrational energy spectrum of the target analyte.

[0047] Another aspect of the present invention provides a method of fabricating a nanochannel device, comprising: a) disposing a plurality of detection electrodes longitudinally along a nanochannel; and b) disposing one or more reference electrodes longitudinally along the nanochannel; wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes. In some embodiments, the method further comprises etching a sacrificial layer to form the nanochannel.

[0048] In some embodiments according to any one of the methods of fabricating as described above, the method further comprises connecting the nanochannel device to a constriction element operable to reversibly adjust the width of one or more of the plurality of tunneling gaps. In some embodiments, the constriction element comprises a thermal actuator, piezoelectric actuator, or an acoustic actuator.

[0049] Further provided in one aspect of the present invention is a device for analysis of a polymeric target analyte, comprising a nanochannel comprising a bend, wherein the polymeric target analyte translocates through the nanochannel, and wherein the bend applies a mechanical tension on the polymeric target analyte.

[0050] These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 depicts a cross-section schematic of an exemplary nanochannel device whereby multiple electrical measurements across a target analyte can be simultaneously measured. [0052] FIG. 2 depicts a top view schematic of an exemplary nanochannel device that shows how multiple simultaneous measurements can be made on the same polymeric target analyte.

[0053] FIG. 3 depicts a side view schematic showing spacing among the detection electrodes and general fabrication geometry of an exemplary nanochannel device.

[0054] FIG. 4 depicts a band diagram for tunneling conductance across an empty tunneling gap (top) and through a target analyte present in the tunneling gap (bottom).

[0055] FIG. 5 depicts a top view schematic of an exemplary nanochannel device comprising a plurality of nanochannels that are interconnected via a plurality of reservoirs to form a loop through which a target analyte translocates.

[0056] FIG. 6 depicts close-up views of an exemplary nanochannel and tunneling electrodes. Detail B shows a single nanochannel with measurement tunnel electrodes, and Section BB-BB shows an individual tunneling electrode with the nanochannel located in the tunneling gap. Units of marked dimensions are in micrometers.

[0057] FIG. 7 depicts close-up views of an exemplary multi-nanochannel device comprising a plurality of nanochannels that are interconnected via a plurality of reservoirs. The multi- nanochannel device allows target analytes to circulate through the nanochannels and reservoirs. Section AA-AA shows two of the reservoirs with tunneling electrodes arrayed between them and electrophoretic electrodes on top of the reservoirs. Units of marked dimensions are in micrometers.

[0058] FIG. 8 depicts an array of exemplary multi-nanochannel devices, which may be used as an array of nano-sequencers.

[0059] FIG. 9 shows a cartoon representation of idealized tunneling current time traces for a short polymer measured across each electrode along an exemplary nanochannel. Only when the target analyte enters the gap between two electrodes, a signal that varies above the background signal is expected. The signal is repeated for every electrode, but the measurements are offset in time because of the location of the electrode within the nanochannel.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present application provides devices and methods for analyzing a target analyte by simultaneously measuring multiple transverse tunneling signals across a plurality of tunneling gaps along a nanochannel as the target analyte translocates through the nanochannel. One major distinction of the disclosed devices and methods over the current nanopore-based DNA sequencing devices comes from the use of nanochannels instead of nanopores. Use of the nanochannels allows highly precise, rapid, and cost-effective characterization of a variety of target analytes, including, but not limited to, biopolymers, such as DNA, RNA and polypeptides. The nanochannel devices and methods described herein allow each monomeric unit of a polymeric target analyte to be measured multiple times, rather than just once, greatly improving the precision of the measurements. Moreover, transverse tunneling measurements have a higher signal-to-noise ratio per measurement because of the nanometer-sized tunneling gap between the electrodes, which makes measurements of tunneling current across each monomeric unit of a polymeric target analyte possible. Moreover, transverse tunneling measurements can measure the vibrational energy spectrum of the target analyte, which is chemically specific. Additionally, since the tunneling current measurement is decoupled from the driving circuit that translocates the target analyte, the speed and direction at which the target analyte moves through the channel can be controlled independently from the measurements, enabling the molecule to be translocated back and forth ("flossed") within the nanochannel, and measured until the output (such as sequence) is as accurate as is required for the application. The present devices can be fabricated using well-known nanofabrication techniques, resulting in devices that are reliable, durable and relatively cheap. The devices may also be fabricated with materials that are compatible with cleaning agents that can unclog the nanochannel or remove target analytes to allow reuse. The devices and methods are useful for determining the length and sequence information of the target analyte, promising wide applications in industries, such as infectious disease epidemiology, biodiversity discovery and conservation, cancer genomics, paleontology, oceanography, microbiome characterization and manipulation, pharmacogenomics, and social science research.

I. Definition

[0061] As used herein, "tunneling signal" refers to any detectable signal associated with an electron tunneling through a tunneling gap. The tunneling signal can be electrical signal that is measurable as "tunneling current" or "tunneling conductance." "Tunneling current" as used herein is also commonly known as the "transverse tunneling current."

[0062] As used herein, "resonance level," and "tunneling resonance level" are used interchangeably to refer to energy levels of an electron tunneling through the resonance cavity across a tunneling gap. "Molecular resonance level" refers to energy levels of an electron tunneling through a molecule.

[0063] As used herein, "target analyte" may refer to any molecule of interest capable of being detected and analyzed by the devices, systems and methods described herein, including biological molecules and non-biological molecules. "Target analyte" is used herein in various contexts to refer to either the whole molecule of interest, or segments of the molecule of interest. "Target analyte" may also include modified, such as labeled, cleaved, chemically or

enzymatically treated versions of a molecule of interest.

[0064] "Biological molecules" are molecules present in living organisms or derivatives thereof, including, but not limited to, biopolymers, such as polypeptides (such as protein), lipids, carbohydrates (such as oligosaccharide and polysaccharide), and nucleic acids (such as DNA, RNA, oligonucleotides), as well as small molecules such as amino acids, deoxyribonucleotides, ribonucleotides, other primary metabolites, secondary metabolites, and natural products.

[0065] "Polypeptide" refers to a single chain of peptide comprising amino acids linked covalently by peptide bonds. Polypeptides include short and long peptide chains, as well as single-chain protein, and any one chain of a multi-chain protein.

[0066] "Non-biological molecules" are molecules not related to any biological molecule, including organic polymers and non-polymeric molecules.

[0067] As used herein, "unfolded" refers to a conformation state of a molecule that is fully extended due to disruption of interactions among non-adjacent subunits (such as monomelic units, or chemical groups) of the molecule that are not directly linked to each other via chemical bonds. For example, an unfolded polypeptide has essentially lost its secondary and tertiary structures.

[0068] It is understood that embodiments of the invention described herein include

"consisting" and/or "consisting essentially of embodiments.

[0069] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0070] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0071] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0072] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

II. Nanochannel Device

[0073] The present invention discloses nanochannel devices for analyzing a target analyte. The nanochannel devices of the present invention comprise one or more nanochannels, wherein each nanochannel has a plurality of detection electrodes for simultaneous measurements of tunneling signals (such as tunneling current or conductance) of a target analyte (such as a polymer, for example, a biopolymer), when the target analyte passes through a plurality of tunneling gaps during translocation of the target analyte through each nanochannel. As the tunneling signals are sensitive to tunneling mediated by single electrons and orbitals (such as atomic and bond orbitals, molecular orbitals, and vibrational spectra) of the target analyte or segment thereof that is present between each tunneling gap, the tunneling signal measured at the tunneling gap can be used to characterize the target analyte or segment thereof, for example, to determine chemical identity or sequence information of the target analyte.

[0074] Accordingly, in one aspect, there is provided a device for analyzing a target analyte, comprising a) a nanochannel, b) a plurality of detection electrodes disposed longitudinally along the nanochannel, and c) one or more reference electrodes disposed longitudinally along the nanochannel, wherein the target analyte translocates through the nanochannel, and wherein each detection electrode forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes. In some embodiments, a single reference electrode is coupled to the plurality of detection electrodes. In some embodiments, the device comprises at least two reference electrodes. In some embodiments, at least two detection electrodes are coupled to one reference electrode. In some embodiments, each of the plurality of detection electrodes is independently coupled with one reference electrode. Independent pairs of one detection electrode coupled to one reference electrode across the tunneling gaps may allow precise measurement and control (such as adjustment or signal enhancement) across the tunneling gaps, and reduce interference among the tunneling gaps.

[0075] The plurality of detection electrodes may be disposed in a non-conductive matrix or substrate. One or more matrix layers may be disposed between the detection electrodes and the nanochannel. Similarly, the one or more reference electrodes may be disposed in a non- conductive matrix or a substrate. One or more matrix layers may be disposed between the reference electrodes and the nanochannel. In some embodiments, the plurality of detection electrodes are disposed directly along the nanochannel. In some embodiments, the one or more reference electrodes are disposed directly along the nanochannel. In some embodiments, the plurality of detection electrodes and the one or more reference electrodes are disposed directly along the nanochannel.

[0076] FIG. 1 shows a cross-section view of a tunneling gap across an exemplary nanochannel that can be used in the device of the present invention. Substrate layer 100 comprises a metal or a semiconductor material, and can serve as a reference electrode. On the substrate are one or more nanochannels through which target analytes translocate. A single nanochannel 120 is shown in FIG. 1. Devices of the present invention can have any suitable number of

nanochannels to facilitate multiplex analysis, for example, about any one of 1, 2-10, 10-100, 100-1000, or more than 1000 nanochannels. The nanochannel 120 has a nanometer-sized rectangular cross-section, and comprises a wall surrounding a central cavity 125. The center cavity 125 can be filled with a medium that is compatible with the target analyte. For example, the target analyte shown in FIG. 1 is a DNA 128, and the medium comprises an aqueous solution. The wall of the nanochannel comprises an insulating layer 122 and a hydrophilic coating 124 that is on the interior surface of the insulating layer 122 facing the central cavity 125. A detection electrode 130 is disposed directly on the side of the nanochannel that is opposite to the side contacting the substrate 100. The detection electrode 130 can be a metal electrode. The gap between the detection electrode 130 and the substrate 100 is the tunneling gap 135. The detection electrode 130 has a fine electrode 132 with nanometer dimensions in direct contact with the nanochannel 120 to allow measurement of a tunneling signal across a single nucleotide or a few (such as no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 20) nucleotides. The fine electrode 132 is connected to a microelectrode 134. A circuit having a tunable AC power supply 150 and an electrical detector 160 is connected between the detection electrode 130 and the substrate 100. The tunable AC power supply 150 can apply a voltage bias across the tunneling gap 135. The electrical detector 160 can measure tunneling conductance through the target analyte at the tunneling gap 135. The detection electrode 130 and the nanochannel 120 are embedded in a supporting matrix 140 that is disposed on top of the substrate 100. The dimensions depicted in FIG. 1 are not drawn to scale.

Spacing of tunneling gaps

[0077] Each detection electrode coupled to a reference electrode can provide a tunneling gap, or a measurement point along the nanochannel. The number and positions of the tunneling gaps along the nanochannel are determined by the number and positions of the detection electrodes. A distinctive feature of the present invention is the multiple detection electrodes that provide multiple tunneling gaps along the nanochannel, which allows simultaneous measurements of tunneling signals across multiple target analytes or across multiple segments (such as monomeric units) of a polymeric target analyte translocating through the nanochannel. [0078] In some embodiments, in order to maximize resolution, it is desirable to have the width of the detection electrode to be as small as the fabrication methods can reproducibly make, so that each detection electrode can measure tunneling signals through a sufficiently small segment of the target analyte, such as a single monomelic unit or a few (for example no more than about any of 2, 3, 4, 5, or 10) monomeric units of a polymeric target analyte. In some embodiments, the detection electrode comprises a graphene sheet to provide a subnanometer-sized detection electrode.

[0079] The spacing between adjacent detection electrodes can be as close as limited by the fabrication methods without causing interference among the electrodes. A spacing of at least about any of 2.5, 3, 3.5, 4, 5, or more times the width of the detection electrode may be adopted between adjacent detection electrodes. For example, current electron beam lithography methods can be used to fabricate an electrode as thin as about 5 nm. In such case, a spacing of at least about 15 nm may be adopted between adjacent detection electrodes to avoid interference. In some embodiments, the spacing between adjacent detection electrodes is long enough so that the number of "squares" of aqueous solution between electrodes is sufficiently large to be at least 10 times the electrical impedance of the tunnel junctions, i.e. low leakage currents, this includes the final path to ground in the reservoirs and the electrodes driving the fluidic current. The spacing of the electrodes also depends on the operating salt concentration. As apparent to those who are skilled in the art, the electrodes are electrically isolated from each other by an impedance larger than the tunnel junction impedance.

[0080] Any number of detection electrodes can be disposed along the nanochannel, including, but not limited to, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 60, 70, 80, 90, 100, or more detection electrodes. The number of detection electrodes is based on the size of the detection electrode, spacing between adjacent detection electrodes, the length of the nanochannel, and the fundamental periodicity (i.e. unit length) of the target analyte, such as the length of a monomeric unit of a polymeric target analyte. In some embodiments, the total number of detection electrodes in the device, such as along a single nanochannel, or along a plurality of nanochannels in a multi-nanochannel device, is designed based on the Nyquist- Shannon sampling theorem.

[0081] In some embodiments, the plurality of detection electrodes are unevenly spaced. In some embodiments, the plurality of detection electrodes are randomly spaced. In some embodiments, the spacing between two adjacent detection electrodes is not an integer multiple of (i.e. "non-commensurate to") the unit length of a polymeric target analyte. The uneven and/or non-commensurate spacing of the detection electrodes can improve sampling accuracy and avoid systematic errors.

[0082] Various designs for the positions of the detection electrodes and the reference electrodes along the nanochannel can be adopted. In some embodiments, the plurality of detection electrodes are disposed longitudinally along a first side of the nanochannel, and the one or more reference electrodes are disposed longitudinally along a side opposite to the first side of the nanochannel. In some embodiments, the plurality of detection electrodes are not disposed along the same side of the nanochannel.

[0083] FIG. 2 shows a top view of an exemplary device comprising a single nanochannel with a plurality of tunneling gaps that allow simultaneous measurements of tunneling signals across multiple segments of a polymeric analyte translocating through the nanochannel. A supporting matrix 210 is disposed on top of a substrate 200 that serves as a reference electrode. The supporting matrix can be isolated from the global chip "ground" to achieve sufficient electrical isolation, and the walls of the nanochannel can be chosen to form a conducting or insulating barrier connected to the electrophoretic ground. Within the supporting matrix 210 are a nanochannel 220 and a plurality of detection electrodes 240, 245, 250, 255, 260, 265, 270, 275 and 280. One side of the nanochannel 220 is in direct contact with the substrate 200, while the opposite side of the nanochannel 220 is in direct contact with the plurality of detection electrodes 240, 245, 250, 255, 260, 265, 270, 275 and 280. A plurality of tunneling gaps form between the plurality of detection electrodes 240, 245, 250, 255, 260, 265, 270, 275 and 280 and the substrate 200. A polymeric target analyte (such as DNA) 230 translocates through the nanochannel 220 in an unfolded fashion, thereby segments of the polymeric target analyte pass through the plurality of the tunneling gaps. Although FIG. 2 shows only 9 detection electrodes, it can be readily appreciated by one skilled in the art that other suitable numbers of detection electrodes could be disposed along the nanochannel in a similar fashion. A circuit 242 is connected between the substrate 200 and the detection electrode 240. A circuit 247 is connected between the substrate 200 and the detection electrode 245. Similarly, each of detection electrodes 250, 255, 260, 265, 270, 275 and 280 is connected to the substrate 200 via a similar circuit, which is omitted in this schematic. The circuit (e.g. 242, 200) comprises a tunable AC power supply and an electrical detector for measuring tunneling conductance. The spacing between adjacent detection electrodes (and thus tunneling gaps) is uneven and non- commensurate to the length of the monomelic unit of the polymeric target analyte 230. The non- commensurate spacing of the tunneling gaps can increase the resolution of sampling of the monomelic units in the polymeric target analyte 230.

[0084] FIG. 3 shows a side view of the exemplary device illustrated in FIG. 2. In this view, a nanochannel 310 is disposed directly on a substrate 300. The nanochannel 310 has a wall 312 comprising an insulating layer with a hydrophilic coating on the interior surface. The central cavity 314 of the nanochannel 310 is fluidically connected to a first reservoir 330 on a first open end 316 of the nanochannel 310, and a second reservoir 340 on a second open end 318 of the nanochannel 310. The connections between the first reservoir 330 and the first open end 316 of the nanochannel 310, and between the second reservoir 340 on the second open end 318 of the nanochannel 310 can be funnel-shaped. Two target ananlytes 320 and 325, for example, DNA molecules, translocate along the nanochannel 310, one molecule at a time, inside the central cavity 314. The central cavity 314 may be filled with an aqueous solution when DNA molecules are translocated. The regions of the target analytes 320 and 325 inside the central cavity 314 are unfolded, while regions of the target analytes 320 and 325 outside the nanochannel 310 are still partially folded. A plurality of detection electrodes 352, 354, 356, 358, 360, 362, 364, and 366 are disposed directly along the side of the nanochannel 310 that is opposite to the side that contacts the substrate 310, providing a plurality of tunneling gaps 372, 374, 376, 378, 380, 382, 384, and 386 along the nanochannel 310, and a plurality of measuring points along the target analytes 320 and 325. The spacings among the detection electrodes 352, 354, 356, 358, 360, 362, and 364 are marked as λΐ, λ2, λ3, λ4, λ5, λ6 and λ7, respectively. The spacings are not equal to each other, and not equal to an integer multiple of the length between two adjacent DNA nucleotides (λ_ΠΝΑ)· A circuit 390 comprising a DC power supply connects the first reservoir 330 and the second reservoir 340, applying an electrophoretic bias (V_transport) across the nanochannel 310. For example, in the configuration of FIG. 3, the second reservoir 340 has a positive electrical potential with respect to the first reservoir 330. As DNA has a negatively charged backbone, the DNA molecules 320 and 325 translocate in the direction from the first reservoir 330 to the second reservoir 340. The DC power supply can easily be configured to apply an electrophoretic bias in the reverse direction to translocate the DNA molecules in the reverse direction. The exemplary device shown in FIG. 2 may also be used to analyze other charged polymeric molecules or non-polymeric molecules, such as RNA, polypeptides, nucleotides, or charged non-biological polymers. Nanochannel

[0085] The nanochannel is typically a tube or a groove, comprising a wall surrounding a central cavity. The target analyte translocates through the central cavity. The cross-section of the nanochannel can be of any shape. In some embodiments, the cross-section of the nanochannel is rectangular, or square. In some embodiments, the cross-section is round or elliptical. The dimension of the central cavity of the nanochannel is nanometer-sized, for example, no more than about any one of 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. In the case of a cylindrical nanochannel, the dimension corresponds to the diameter of the cross-section. In the case of a rectangular nanochannel, the width of the nanochannel refers to the dimension spanning across the tunneling gap, while the height of the nanochannel refers to the second dimension of the cross-section. The shape and dimension(s) of the cross-section of the nanochannel or the central cavity may help to maintain the target analyte, especially polymeric target analyte, such as biopolymers, in an unfolded conformation.

[0086] The width of the tunneling gap is defined by the distance between the detection electrode and the reference electrode. In the cases where the detection electrode and the reference electrode are directly disposed on a rectangular nanochannel, the width of the tunneling gap is equal to the width of the nanochannel. The width of the rectangular nanochannel is the sum of the thickness of the two opposing sides of the wall (i.e. material layer(s)) of the nanochannel across the tunneling gap, and the width of the central cavity of the nanochannel through which the target analyte translocates. The width of the central cavity should be large enough to allow one copy of the target analyte to pass in an unfolded fashion. In some embodiments, the width of the central cavity of the nanochannel is sufficiently small to allow only one copy of the target analyte to pass in an unfolded fashion. In some embodiments, the width of the central cavity of the nanochannel restricts the thermal motion of the target analyte to reduce distribution of the tunneling signals through the target analyte.

[0087] The length of the nanochannel may be comparable to the length of the target analyte. As used herein, "length of the target analyte" refers to the length of the largest dimension of the target analyte in the unfolded conformation. In some embodiments, the length of the nanochannel is longer than the length of the target analyte. In some embodiments, the length of the nanochannel is shorter than the length of the target analyte. In some embodiments, the length of the nanochannel is at least about any one of 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μτη, 2 μιη, 3 μιη, 4 μιη, 5 μιη, 6 μιη, 7 μιη, 8 μιη, 9 μιη, 10 μιη, or more. In some embodiments, the length of the nanochannel is about any one of lOnm to 50 nm, 50 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μηι, 1 μηι to 5 μηι, 5 μηι to 10 μηι, 50 nm to 500 nm, or 50 nm to 1 μιη, or 50 nm to 5 μιη. A desirable feature of the nanochannel device is the high aspect ratio (length to cross-section dimension ratio) of the nanochannel, which allows placement of multiple tunneling gaps along the nanochannel. In such nanochannels, a nonpolymeric analyte or a monomelic unit of a polymeric analyte may be measured multiple times as it passes through the nanochannel, improving the accuracy of the measurements. A polymeric analyte may also be extended over a single channel, while the tunneling signals of multiple segments (such as monomeric units) of the polymeric analyte may be measured simultaneously, increasing the speed at which sufficient signals can be collected to characterize the full length of the polymeric analyte. In some embodiments, the device comprises a plurality of nanochannels, and the total length of the plurality of the nanochannels is comparable to the length of the target analyte. In some embodiments, the total length of the plurality of the nanochannels is longer than the length of the target analyte. In some embodiments, the total length of the plurality of the nanochannels is shorter than the length of the target analyte.

[0088] The nanochannel can be straight or curved. In some embodiments, the nanochannel comprises a bend. In some embodiments, the nanochannel comprises more than one (such as at least about any one of 2, 3, 4, 5, 6, 7, or more) bends. In some embodiments, the nanochannel is serpentine shaped. In some embodiments, the nanochannel is meander or out-of-plane, for example, three dimensional shaped. In some embodiments, the bend is useful for applying mechanical tension on a polymeric target analyte that is extended over the bend. Such mechanical tension may help to straighten the target analyte, thereby reducing the number of monomers in the measurement volume and increasing precision of the measurements of tunneling signals and optionally other signals (such as optical or plasmonic signals) detected in proximity to the bend. The bend may have any angle or curvature that result in the desirable amount of mechanical tension without breaking the target analyte. In some embodiments, the angle of the bend is about 90° to 180°, such as about any one of 120 °-130 °, 130 °-140 °, 140⁰ - 150 °, 150 °-160 °, 160 °-170 °, 120 °-160 °, or 150 °-175 °. In some embodiments, one or more (including all) of the plurality of detection electrodes are disposed in proximity to the bend. "In proximity to the bend" refers to the region of a bent nanochannel adjacent to the bend, and in such region, the target analyte experiences a mechanical tension and is fully extended. For example, the detection electrodes may be disposed within no more than about any one of 1/10, 1/8, 1/6, ¼, 1/3, ½, or 1 time the radius of the curvature of the bend. Other electrodes, such as plasmonically enhanced electrodes, may also be disposed in proximity to the bend for enhanced signal detection.

[0089] The nanochannel can have any structure or surface functionalization that can modulate the bending energy and/or friction experienced by the analyte. In some embodiments, one or more constriction defects (such as bulges) are placed within the nanochannel. The constriction defects can partition the length of a polymeric analyte, induce non-uniform stretching, and/or selectively trap or pin molecules at specific points in the nanochannel. See, for example, Reccius C. H. , et al, Phys Rev Lett, 95, 268101.

[0090] The nanochannel has a first open end and a second open end, through which the target analyte can enter or exit the nanochannel. The target analyte may be translocated through the nanochannel in either direction, i.e. from the first open end to the second open end, or from the second open end to the first open end. In some embodiments, the first open end of the nanochannel is connected to a first reservoir, and the second open end of the nanochannel is connected to a second reservoir. The target analyte or a fraction thereof may be stored in the first reservoir or the second reservoir. For example, a number of copies of the target analyte may be stored in the first reservoir, translocated through the nanochannel from the first reservoir to the second reservoir, and exit into the second reservoir; the target analyte may also be translocated in the reverse direction. In some embodiments, the target analyte spans partial or the entire length of the nanochannel, and wherein the target analyte is translocated in the direction from the first reservoir to the second reservoir, from the second reservoir to the first reservoir, or in a back-and-forth fashion. Regions of the target analyte may be retained in the first reservoir and/or the second reservoir during the translocation. Repeated back-and-forth translocation of the target analyte is also envisioned, and when the target analyte is translocated back-and-forth repetitively around a small range of positions with respect to the nanochannel, such motion is referred to as "flossing." The dimensions of the first reservoir and the second reservoir may be in nanometers to micrometers. One or more copies of the target analyte may be present in the first reservoir and/or the second reservoir. Additionally, the target analyte may be allowed to partially fold inside the first reservoir and/or the second reservoir. In some embodiments, a funnel-shaped port connects the first reservoir to the first open end of the nanochannel, and/or a funnel-shaped port connects the first reservoir to the first open end of the nanochannel. The funnel-shaped port can trap target analytes and facilitate entrance of the target analytes into the nanochannels. [0091] The device may be a microfluidic device. The nanochannel may be filled with a medium comprising a solvent compatible with the target analyte. In some embodiments, the nanochannel is fluidically connected to the first reservoir and the second reservoir via the medium. In some embodiments, the first reservoir and the second reservoir contain the same medium as the nanochannel. Choice of the ingredients in the medium depends on the chemical and physical nature of the target analyte. For example, a hydrophilic solvent may be chosen to carry a hydrophilic target analyte, such as DNA, R A, or polypeptide; a lipophilic solvent may be chosen to carry a lipophilic target analyte, such as a lipid or a hydrophobic organic polymer. The medium may be buffered at a suitable pH and contain a suitable amount of salt to enhance the stability of the target analyte. In some embodiments, the medium is conductive. For example, the medium may comprise an electrolyte. The medium may further comprise one or more ingredients that keep the target analyte in an unfolded conformation. In some

embodiments, the medium comprises a reducing agent. For example, wherein the target analyte is a polypeptide, such as a protein having one or more disulfide bonds, suitable reducing agents include, but are not limited to, 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), and Tris (2-carboxyethyl) phosphine hydrochloride (TCEP). In some embodiments, the medium comprises a denaturing agent. For example, wherein the target analyte is a polypeptide, the denaturing agent may be a detergent, such as sodium dodecyl sulfate (SDS).

[0092] In some embodiments, the nanochannel is a solid state nanochannel. The nanochannel may comprise any material suitable for measuring tunneling signals and for nanofabrication. In some embodiments, the nanochannel comprises transparent materials to facilitate optical measurements. In some embodiments, the wall of the nanochannel comprises an insulating layer. The insulating layer may comprise a dielectric material with a suitable dielectric constant for measurement of the tunneling signals. Depending on the different wetting characteristics of the medium (or solvent in the medium) and the wettability of the insulating layer, the interior surface of the wall may be chemically treated to facilitate absorption of the medium on the interior surface and to avoid trapping bubbles in the nanochannel. In some embodiments, the wall further comprises an interior coating, such as on the interior surface of the insulating layer, to facilitate absorption of the medium on the interior surface of the wall. For example, wherein the solvent of the medium is hydrophilic, the interior coating may comprise a hydrophilic material. Suitable hydrophilic materials include, but are not limited to, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), and polyvinyl alcohol (PVA). In the cases wherein the solvent is lipophilic, the interior coating may comprise a lipophilic material. In some embodiments, the interior coating reduces non-specific binding to non-target molecules in the sample. To protect electrochemistry happening at the electrodes, which would lead to their breakdown at the tunneling gaps, the wall may further comprise a protective layer or patches on the outside surface of the insulating layer. In some embodiments, the wall comprises a plurality of insulating layers and a plurality of protective layers, wherein the plurality of insulating layers and the plurality of protective layers form an alternating structure. All material layers (such as insulating layer, interior coating, and protective layer) of the wall are very thin, such as of subnanometer size. Atomic layer deposition methods known in the art can be used to fabricate any one or more of the material layers of the wall. In some embodiments, the material layers comprise materials that are resistant to cleaning agents that can degrade and/or remove target analytes.

[0093] In some embodiments, the nanochannel is a hybrid nanochannel comprising one or more biological nanopores, such as a protein, for example, alpha-hemolysin or MspA, which can be modified or unmodified. In some embodiments, a biological nanopore is disposed inside a tunneling gap. In some embodiments, each tunneling gap has a biological nanopore inside. For example, the biological nanopore may be attached to the interior of a solid state nanochannel across a tunneling gap. Alternatively, for example, the detection electrode and the reference electrode across a tunneling gap may be attached to a biological nanopore, and the tunneling gap having the biological nanopore is disposed between solid state segments of the nanochannel. A lipid bilayer may further be disposed between the biological nanopore and the wall of the nanochannel, or between the biological nanopore and the detection/reference electrodes.

Driving system for translocation

[0094] To translocate the target analyte through the nanochannel, the device may further comprise a driving system. In some embodiments, the driving system is directly coupled to the nanochannel, for example, to the first reservoir and/or the second reservoir. Any known driving system in the art may be used singly or in combination to translocate molecules through a nanochannel, and the choice of a suitable driving system depends on the chemical and physical nature of the target analyte. For example, for charged target analytes, such as nucleotides, nucleic acids, and polypeptides in SDS buffer, the device may comprise a power supply configured to generate an electrophoretic bias longitudinally across the nanochannel. In some embodiments, the power supply is connected between a first driving electrode attached to the first open end of the nanochannel, and a second driving electrode attached to the second open end of the nanochannel. In some embodiments, the first driving electrode is connected to the first reservoir and the second driving electrode is connected to the second reservoir. In some embodiments, the power supply is a DC power supply that can translocate the target analyte in a single direction. In some embodiments, the power supply is an AC power supply configured to apply an alternating electrophoretic bias longitudinally across the nanochannel, wherein the altering electrophoretic bias allows reversal of the direction in which the target analyte translocates. In some embodiments, the direction of the electrophoretic bias is reversed repetitively to translocate a charged target analyte in a back-and-forth fashion. In some embodiments, the repetitive reversal of the direction of the electrophoretic bias is rapid, resulting in a "flossing" motion, allowing repetitive sampling and measurements around the same region of the target analyte.

[0095] The electrophoretic bias in some embodiments may have a finite frequency chosen outside of the measurement regime as known to those skilled in the art. The electrophoretic bias can also be modified along the nanochannel by direct inject of salt from a local reservoir along the nanochannel.

[0096] In some embodiments, the driving system comprises a thermal element configured to generate a thermal gradient longitudinally across the nanochannel. In some embodiments, the thermal element is configured to generate a thermal gradient in alternating directions longitudinally across the nanochannel, in order to translocate the target analyte back and forth through the nanochannel.

[0097] In some embodiments, the target analyte is modified with a translocation handle, such as a magnetic bead or a dielectric bead, in order to be translocated by the driving system. In some embodiments, the driving system comprises a magnetic field capable of pulling a magnetic bead attached to one end (such as the leading end) of the target analyte. In some embodiments, the driving system comprises an optical field capable of pulling a dielectric bead attached to one end (such as the leading end) of the target analyte. In some embodiments, the target analyte is modified with more than one translocation handles. For example, the target analyte may be modified with a first translocation handle on one end, and a second translocation handle on the other end of the target analyte. The driving system may be capable of pulling either one of the two translocation handles at a time to translocate the target analyte from the first open end of the nanochannel to the second open end, from the second open end of the nanochannel to the first open end, or in a back-and-forth fashion.

[0098] Any one of the driving systems described herein is decoupled from the detection electrodes that detect the tunneling signals, which allows independent manipulation of the translocation direction of the target analyte while the tunneling signals are measured using detectors connected to the detection electrodes. This design is in contrast to the traditional nanopore-based sequencing systems, in which the measurable blockade current and the driving system for translocating the target analyte are in the same direction, and thereby cannot be independently manipulated. With the driving system that can alternate the direction in which the target analyte translocates, the tunneling signal of a segment (such as a monomelic unit) of the target analyte may be measured multiple times while the target analyte is translocated in a back- and-forth fashion or in a flossing motion through the nanochannel. Repeated measurements enable higher signal to noise ratio and higher accuracy compared to single measurements for each segment of the target analyte in the traditional nanopore-based systems.

[0099] The translocation speed of the target analyte may be adjusted based on the quality of the tunneling signal. A fast translocation speed allows rapid analysis of a target analyte, while a slow translocation speed allows multiple measurements of tunneling signals around the same segment of the target analyte during the time the segment passes through a tunneling gap. A variety of methods may be used to control the translocation speed of the target analyte. For example, increasing the electrophoretic bias (voltage) across the nanochannel can increase the translocation speed of a charged target analyte, and decreasing the electrophoretic bias (voltage) across the nanochannel can decrease the translocation speed of a charged target analyte. The viscosity of the medium in the central cavity of the nanochannel may also be increased to slow down the translocation speed of the target analyte.

[0100] In some embodiments, proteins (such as enzymes) are used to control the translocation speed of a polymeric target analyte. Any known protein or enzyme that can control the translocation speed of a biopolymer through a solid state nanopore may be incorporated in the nanochannel device of the present invention. See, for example, WO20101 17470A2. For example, suitable proteins include, but are not limited to, DNA or RNA metabolizing enzymes, DNA or RNA translocating enzymes (e.g. helicase), DNA or RNA binding proteins. In some embodiments, a DNA-dependent DNA or RNA polymerase is used to control the translocation speed of a DNA target analyte. In some embodiments, an RNA-dependent DNA or RNA polymerase is used to control the translocation speed of an RNA target analyte. The polymerase may either be included in the reservoir containing the DNA or RNA target analyte prior to the target analyte enters the nanochannel. In some embodiments, the polymerase is attached to one end of the nanochannel (e.g. the first open end or the second open end) to control the translocation speed of the target analyte, wherein the target analyte is a DNA or an RNA. The nucleic acid strand translocating through the nanochannel may comprise either the template strand or a nascent strand synthesized by the polymerase.

Resonance tuning

[0101] The present device may have features that allow tuning of the tunneling gaps to the molecular resonance level of the target analyte. The tunneling gap provides a resonance cavity for an electron to tunnel through substances present in the tunneling gap, including any material layer of the nanochannel, any dielectric medium in the cavity of the nanochannel, and any target analyte that passes through the nanochannel, in particular the segment of the target analyte, such as a monomelic unit of a polymeric target analyte, that is present between the tunneling gap. FIG. 4 shows exemplary band diagrams of an electron tunneling through an empty tunneling gap (top) and through a target analyte (such as a nucleotide of a DNA) present in the tunneling gap. As shown in the top figure of FIG. 4, the width of the tunneling gap as determined by the width of the nanochannel, including the thickness of the material layer of the nanochannel (e.g.

insulating layer shown as dins) and the width of the cavity of the nanochannel (shown as d_Channei), along with the dielectric constants and atomic arrangements of the substances in the tunneling gap (e.g. material layer of the nanochannel and any medium in the cavity of the nanochannel), can determine the various energy levels (e.g. E₀, Ei, E₂, E₃) of an electron tunneling through the tunneling gap. However, as shown in the bottom band diagram of FIG. 4, each target analyte has a characteristic band diagram with multiple molecular resonance levels owing to the unique atomic and bond orbitals in the target analyte. In order to optimize the tunneling signal detected at the tunneling gap, the resonance cavity between the tunneling gap should be tuned to the resonance spectrum of the target analyte, i.e. , one of the resonance energy levels of the "empty" tunneling gap (with no target analyte present) should match with one of the molecular resonance levels of the target analyte to provide the maximum measurable signal when the target analyte passes through the tunneling gap.

[0102] Adjustment and optimization of conditions associated with the tunneling gap for each type of target analyte may be important in obtaining reliable tunneling signals. A suitable width of the tunneling gap, suitable material layer (e.g. insulating layer) of the nanochannel, and suitable medium that fills the nanochannel and carries the target analyte, may be chosen for probing a resonance level of the target analyte, and/or to maximize the separation of resonance levels of different segments (e.g. monomelic units) of a polymeric target analyte. In some embodiments, the insulating layer of the nanochannel has a suitable dielectric constant for probing a resonance level of the target analyte. In some embodiments, the thickness of the insulating layer of the nanochannel is suitable for probing a resonance level of the target analyte. In some embodiments, the medium is adjusted to provide a dielectric constant suitable for probing a resonance level of the target analyte. In some embodiments, a solvent with a suitable dielectric constant is chosen for use in the medium. In some embodiments, the medium is adjusted by adjusting the amount of electrolyte in the medium.

[0103] In general, a nanometer-sized tunneling gap is preferred for probing the tunneling resonance levels of the target analyte, and to maximize the strength of the tunneling signals. In some embodiments, the width of each tunneling gap is about any of 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, or more. In some embodiments, the width of each tunneling gap is about any one of 1-2 nm, 2-3 nm, 3-4 nm, 4-5 nm, 5-10 nm, 1-3 nm, 3-5 nm, 2- 4 nm, 1-4 nm, or 1-5 nm. In some embodiments, the width of each tunneling gap is about 1 nm to about 4 nm. In some embodiments, the width of each tunneling gap is about 2 nm. In some embodiments, the width of each tunneling gap is fixed. In some embodiments, the width of each tunneling gap is adjustable (such as dynamically adjustable). In some embodiments, the width of each tunneling gap is independently adjustable.

[0104] In some embodiments, the device further comprises a tuning element operable to adjust one or more of the plurality of tunneling gaps to allow probing of a tunneling resonance level of the target analyte that passes through the one or more tunneling gaps. In some embodiments, the adjustment maximized signal to noise ratio. In some embodiments, the adjustment increases the tunneling signal strength to a desirable value. In some embodiments, the adjustment decreases the tunneling signal strength to a desirable value. In some embodiments, the adjustment allows separation of tunneling signals associated with different segments (such as monomelic units) of the target analyte. In some embodiments, the tuning element is connected to a feedback element (e.g. a feedback circuit) that allows adjustment of the width of a tunneling gap based on the quality of the tunneling signal (for example, strength of the signal, signal to noise ratio, distribution of the signal, separation of differential signals, etc.) detected at the tunneling gap. The tunneling gaps may be adjusted individually or in groups, and via a single tuning element or via a combination of tuning elements.

[0105] In some embodiments, the tuning element can adjust the width of the tunneling gap. Various methods can be used to adjust the width of the tunneling gap. For example, the width of the tunneling gap can be adjusted by adjusting the width of the nanochannel. In some embodiments, the device comprises one or more constriction elements operable to reversibly adjust the width of the one or more tunneling gaps. In some embodiments, the one or more constriction elements can reversibly adjust the width of the nanochannel or a fraction thereof, thereby adjusting the width of the tunneling gaps associated with the nanochannel or fraction thereof. In some embodiments, each tunneling gap is independently adjusted by a constriction element. Any known constriction element in the art may be used, including, but not limited to, thermal actuators, piezoelectric actuators, or acoustic actuators. In some embodiments, the constriction element is a thermal actuator. In some embodiments, the constriction element is a piezoelectric actuator. In some embodiments, the constriction element is an acoustic actuator.

[0106] In some embodiments, the device comprises a tunable power supply configured to generate a voltage bias across one or more tunneling gaps. In some embodiments, a range of voltage bias is screened to choose a suitable voltage across one or more tunneling gaps based on the quality of the tunneling signals detected at the tunneling gap. In some embodiments, each detection electrode is independently connected to a tunable power supply configured to generate a voltage bias across each tunneling gap.

Electrodes

[0107] The detection electrodes and the reference electrodes can be made of any conductive material, such as a metal, a metal alloy, gold, platinum, a gold alloy, a platinum alloy, carbon, carbon nanotubes, graphene, or titanium nitride. In some embodiments, one or more detection electrodes and/or reference electrodes are made of metal. In some embodiments, one or more detection electrodes and/or reference electrodes are made of a non-metal or semi -metal material. In some embodiments, the reference electrode is a substrate, such as a doped polysilicon substrate. The electrodes may be formed on or in between or be partially insulated with any suitable inorganic or organic insulating material, such as inorganic materials including silicon oxide, silicon nitride, metal oxides, or organic materials, including polymers such as polyethylene, polystyrene, polymethylmethacrylate and others known in the art. The insulating material may be configured to prevent background noise from the electrode when a current is flowing. For example, an electrode may be completely covered with HDPE except for a small tip or apex.

[0108] The detection electrodes and reference electrodes can be of any shape. In some embodiments, one or more detection electrodes and/or reference electrodes are metallic lines. In some embodiments, one or more detection electrodes and/or reference electrodes are triangular. Triangular electrodes may have reduced junction resistance and enhanced electrical and mechanical robustness. In some embodiments, each detection electrode is further connected to a microelectrode. [0109] Each detection electrode and the reference electrode may be connected independently to a circuit. The circuit may comprise a power supply (such as a tunable AC power supply, optionally with an offset power supply), and an electrical detector that can measure an electrical signal when the target analyte passes through the tunneling gap between the detection electrode and the reference electrode. The electrical signal can be the tunneling signal, such as tunneling current, tunneling conductance, or tunneling capacitance.

[0110] The tunneling signals can be enhanced in a variety of ways. For example, one or more of the detection electrodes and/or reference electrodes may be spin-value electrodes that can spin-polarize electrons tunneling through the tunneling gaps. When the target analyte is magnetically labeled, spin-polarization of the electrons can enhance the tunneling signal (such as tunneling current) detected by the electrical detectors connected to the spin-value electrodes. In some embodiments, each type of monomelic unit of target analyte is independently labeled with a different magnetic label to enhance the differential tunneling signals of the different monomeric units. Ferromagnetic electrodes (such as spin valves) may be used as the detection electrodes and the reference electrodes to provide magnetic tunnel junction for spin-polarization of the tunneling electrons.

[0111] The electrical tunneling signal may also be enhanced using plasmonically active electrodes. For example, a nano-antenna comprising two metallic end portions, such as the nano- antenna described in U.S. Patent No. 9,075,010, may be used as or attached to a pair of detection electrode and reference electrode to provide plasmonically active electrodes.

Combination with other signals

[0112] The devices of the present invention may further be capable of detecting one or more signals other than transverse tunneling signals, which may facilitate analysis of the target analyte. Such signals may also be characteristic of the chemical structure of the target analyte, and thus can be combined with the transverse tunneling signals to determine the chemical identity (such as sequence) of the target analyte. Alternatively, such signals may provide information of the location of the target analyte in the nanochannel, or simply be used to determine whether the target analyte is present in the nanochannel. Useful signals may include, but are not limited to, plasmonic signals, optical signals, and axial current through the nanochannel (e.g., blockade current as the target analyte translocates through the nanochannel).

[0113] In some embodiments, the device further comprises an optical detector for

simultaneous measurement of an optical signal when the target analyte passes through one or more of the plurality of tunneling gaps. To facilitate the optical measurement, the nanochannel may comprise transparent materials. Sources of the optical signal include, but are not limited to, electroluminescence, fluorescence, Raman scattering, bioluminescence, and chemiluminescence associated with the target analyte. For example, electroluminescence may be associated with the tunneling electrons through the target analyte. Fluorescence, bioluminescence and

chemiluminescence may be due to inherent properties of the target analyte, or may be associated with an optical label (such as fluorescent, bioluminescent, or chemiluminescent label respectively) attached to the target analyte. In some embodiments, the optical detector is an optical microscope.

[0114] In some embodiments, the device comprises one or more plasmonically enhanced electrodes that plasmonically enhance the optical signal. In some embodiments, the

plasmonically enhanced electrodes are independent from the detection electrodes and/or the reference electrodes. In some embodiments, at least one pair of plasmonically enhanced electrodes is disposed across a tunneling gap along the nanochannel. In some embodiments, the plasmonically enhanced electrodes are disposed in proximity to a bend of the nanochannel, wherein the bend applies a mechanical tension on the target analyte, thereby straightening the target analyte and reducing the number of monomer units in the measurement volume.

Multi-nanochannel device

[0115] The devices of the present invention may comprise a single nanochannel or a plurality of nanochannels (referred herein as "multi-nanochannel network" or "multi-nanochannel device"). Any of the nanochannels and the other components, such as electrodes, driving systems, tuning elements, and detectors, described above may be incorporated in a device comprising a plurality of nanochannels. The target analyte may translocate through any one or combination of the plurality of the nanochannels. In some embodiments, the plurality of nanochannels are connected to each other to provide a pre-determined microfluidic structure, such as a loop. The device may have any suitable number of interconnected nanochannels, including, but are not limited to, about any of 1-5, 5-10, 10-20, 20-50, 50- 100, 1-10, 1-50, 1- 100, or more than 100 nanochannels.

[0116] In some embodiments, each of the plurality of nanochannels has a plurality of detection electrodes disposed longitudinally along the nanochannel, and each of the plurality of nanochannels has one or more reference electrodes disposed longitudinally along the nanochannel, wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes. [0117] In a multi-nanochannel device, a large number of detection electrodes may fit along the nanochannels in a multi-channel device to provide a large number of measurement points of tunneling signals. Additionally, as each nanochannel may have a bend, multiple bends in a multi-nanochannel device may be more effective in keeping a long polymeric target analyte straight. The multi-nanochannel devices may be especially suitable for analyzing long polymeric target analytes, such as chromosomal DNA or large protein molecules.

[0118] In some embodiments, the plurality of nanochannels are connected to each other via a plurality of reservoirs. The reservoirs may be used to temporarily store regions of the target analyte. In some embodiments, the plurality of nanochannels are connected to each other via the plurality of reservoirs to provide a loop through which the target analyte translocates. The target analyte may translate through the loop multiple times for repeated measurements of tunneling signals. For a target analyte longer than the length of the loop, regions of the target analyte may be partially folded in the reservoirs, but the regions of the target analyte threaded through the nanochannels are kept unfolded. While the target analyte translocates through the loop, different regions of the target analyte become unfolded and available for measurements of the tunneling signals. In some embodiments, the plurality of nanochannels are connected to the plurality of electrolyte reservoirs in a manner to drive different or similar electrolyte concentrations in portions or across the plurality of nanochannels.

[0119] Many configurations among the nanochannels and the reservoirs may be envisioned and suitable for different types of analysis. In some embodiments, each nanochannel is connected to a first reservoir via the first open end of the nanochannel, and a second reservoir via the second open end of the nanochannel. Each reservoir may be connected to two nanochannels or more than two (such as about 3, 4, 5, 6, or more) nanochannels. In some embodiments, each pair of nanochannels are connected via a reservoir. The reservoirs may have nanometer-sized dimensions (referred to as "nanoreservoirs") or micrometer-sized dimensions (referred to as "microreservoir"). Typically, reservoirs in the middle of the translocation route of a multi-nanochannel network are nanoreservoirs, and the dimensions of the nanoreservoirs can by about any of 10 nm to 50 nm, 50 nm to 100 nm, or 100 nm to 500 nm.

[0120] In some embodiments, the multi-nanochannel device comprises a plurality of driving systems to guide the translocation of the target analyte through each nanochannel, and/or from one reservoir to another reservoir. The driving systems may be configured to apply an electrophoretic bias or a magnetic field across each nanochannel. In some embodiments, each reservoir is coupled to a driving electrode to allow translocation of a charged target analyte via electrophoresis through the nanochannels disposed in between the reservoirs.

[0121] FIG. 5 depicts a top view of an exemplary multi-nanochannel device. Five

nanochannels 510, 515, 520, 525, and 530 are shown in the figure, but any number of nanochannels may be connected to provide a multi-nanochannel network in the device.

Nanochannels 510, 515, 520, 525, and 530 are fluidically connected to each other. Nanochannel 510 is connected on a first open end to a first microreservoir 540 via a funnel shaped port 545. The second open end of nanochannel 510 is connected to a nanoreservoir 550, which is further connected to nanochannels 515 and 520. Nanochannel 515 is further connected to a

nanoreservoir 555, which is connected to nanochannel 525. Nanochannel 520 is further connected to a nanoreservoir 560, which is connected to nanochannel 525. Thereby, nanochannels 515, 520 and 525 are connected via nanoreservoirs 550, 555, and 560 to provide a loop structure through which the target analyte may translocate for multiple times.

Nanoreservoir 560 is further connected to nanochannel 530, which is connected on the second open end to a microreservoir 570. Each of the nanoreservoirs 550, 555, and 560 is connected to a driving electrode (552, 557, and 562 respectively). The driving electrodes provide electrophoretic bias across the nanochannels 515, 520 and 525 to drive translocation of the target analyte through the loop structure. Each of the nanochannels 510, 515, 520, 525, and 530 has a bend (512, 517, 522, 527, and 532 respectively) to apply mechanical tension on the region of the target analyte passing around the bend to keep the region straight. The nanochannels may have more than one bend in practice. A set of detection electrodes (513, 518, 523, 528, and 533 respectively) are disposed directly on each nanochannel (510, 515, 520, 525, and 530 respectively) in proximity to the bend (512, 517, 522, 527, and 532 respectively) to allow simultaneous measurements of tunneling signals across the tunneling gaps between the detection electrodes (513, 518, 523, 528, and 533 respectively) and the substrate 500. Five detection electrodes for each set are shown as an example in the figure, but each set of detection electrodes may have fewer or more detection electrodes. A plasmonically enhanced antenna shown as a bowtie structure (514, 519, 524, 529, and 534 respectively) is independently disposed in proximity to the bend (512, 517, 522, 527, and 532 respectively) of the nanochannel (510, 515, 520, 525, and 530 respectively) to enable simultaneous measurements of optical signals associated with the target analyte. The nanochannels 510, 515, 520, 525, and 530, nanoreservoirs 550, 555, and 560, sets of detection electrodes 513, 518, 523, 528, and 533, driving electrodes 552, 557, and 562, and plasmonically enhanced antennas 514, 519, 524, 529, and 534 are all embedded in a supporting matrix 580 disposed on top of the substrate 500.

[0122] The device may comprise a loading reservoir fluidically connected to the nanochannel (or the multi -nanochannel network), wherein the target analyte is loaded to the loading reservoir in order to be exposed to the nanochannel (or the multi -nanochannel network). The device may also comprise a disposal reservoir fluidically connected to the nanochannel (or the multi- nanochannel network), wherein the target analyte exits into the disposal reservoir after the target analyte translocates through all nanochannel(s) in the device. In some embodiments, the loading reservoir is connected to the nanochannel (or multi-nanochannel network) via a funnel-shaped port. The funnel-shaped port may facilitate attraction of the target analyte into the nanochannel (or multi-nanochannel network).

[0123] FIGs. 6-8 show an exemplary multi-nanochannel device, which may be used as an array of nano-sequencers for target analytes. FIG. 6 shows a section of a single nanochannel around a bent with a plurality of tunneling gaps that allow simultaneous measurements of tunneling signals across multiple segments of a target analyte translocating through the nanochannel. The width of the tunneling gap in this exemplary device is 2 nm (0.002 μιη) between the top and bottom electrodes. The nanochannel wall is lined with an insulating layer, which prevents breakdown of the device and electrochemistry, as well as a hydrophilic layer, which may ensure proper wetting of the nanochannel. FIG. 7 shows how an array of electrodes may be used to measure the target analyte as it traverses between reservoirs in an interconnected nanochannel network that forms a loop. Section AA-AA of FIG. 7 shows a cross-section through one nanochannel. The reservoirs can be taller than the nanochannels to provide sufficient volume for intermediate storage of the target analytes in between nanochannel transits. Each network of nanochannels and reservoirs can be used as a nano-sequencer. Conventional microfluidic channels can be used to route the target analytes into the nanochannel network. An array of nanochannel networks is shown in FIG. 8. Six input and output microfluidic channels routing to six separate nanochannel networks are illustrated. Electrophoretic transport shuttles molecules from large reservoirs on the sides into the nanochannel network reservoirs at the center. In principle, arbitrary networks of nanochannel networks are possible.

Target analytes

[0124] The devices disclosed herein can be used to characterize a variety of target analytes, including non-polymeric molecules, and polymeric molecules. In some embodiments, the target analyte is an unbranched molecule. In some embodiments, the target analyte is a branched molecule that has been cleaved into unbranched fragments. The electron tunneling through a tunneling gap is very sensitive to the atomic arrangement of atoms in the tunneling gap.

Therefore, molecules with different chemical structures are expected to have distinct electron tunneling signatures.

[0125] In some embodiments, the target analyte is a non-polymeric molecule, such as a monomer (for example, nucleotide, amino acid, monosaccharide, and non-biological monomer), a metabolite, a ligand, a pollutant, or a small organic molecule.

[0126] In some embodiments, the target analyte is a polymeric molecule. In some

embodiments, the target analyte is a homopolymer. In some embodiments, the target analyte is a heteropolymer having two least two types of monomeric units. In some embodiments, different types of monomeric units in the heteropolymer have the same unit length. In some

embodiments, different types of monomeric units in the heteropolymer have different unit lengths. In some embodiments, the target analyte is a non-biologic polymer, such as polyester. In some embodiments, the target analyte is a biopolymer, including, but not limited to, nucleic acids, polypeptides, polysaccharides, lipids, and glycoproteins. The biopolymer may be derived from any organism, including, but not limited to, bacteria, viruses, yeasts, plants (such as agriculture crops) and animals such as invertebrates (e.g., flies, worms), mammals (e.g., mice, rats, fish, primates, and human).

[0127] In some embodiments, the target analyte is a nucleic acid, including, but not limited to, DNA, RNA, DNA/RNA hybrid, oligonucleotide, and PNA. In some embodiments, the nucleic acid is single stranded. In some embodiments, the nucleic acid is double stranded. In some embodiments, the DNA is genomic DNA. In some embodiments, the DNA is epigenetically modified. In some embodiments, the DNA is PCR-amplified DNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is a non-coding RNA, such as miRNA, siRNA and lincRNA. In some embodiments, the RNA is a viral RNA. Nucleic acids, such as RNA, may have secondary structures, such as hairpin, which may be unfolded prior to translocating through the nanochannel. Double -stranded nucleic acids may be separated into individual strands to facilitate analysis in the nanochannel. Any known denaturing methods in the art may be used to unfold nucleic acids, and/or separating the two strands in a double-stranded nucleic acid, for example, heating and rapid cooling of the nucleic acids. It might also be beneficial to cleave long nucleic acids, such as genomic DNA, using nucleases or restriction enzymes prior to translocating the DNA through the nanochannel, and/or to isolate regions of interest for analysis. [0128] In some embodiments, the target analyte is a polypeptide. In some embodiments, the polypeptide is a protein. The protein may be unfolded prior to translocation through the nanochannel. Any known protein denaturing methods in the art may be used to unfold the polypeptide. For example, the polypeptide may be treated with a reducing agent, such as 2- mercaptoethanol, DTT, or TCEP, to reduce the disulfide bonds in the polypeptide. In some embodiments, the polypeptide (or reduced polypeptide) is treated with iodoacetic acid to protect the free cysteines to avoid formation of disulfide bonds. In some embodiments, the polypeptide is treated with a denaturing agent, such as SDS. In some embodiments, the polypeptide is cleaved by a protease (such as trypsin) to provide short polypeptide fragments prior to translocation through the nanochannel. In some embodiments, the short polypeptide fragments may be subject to gel electrophoresis to pool fragments by size, and fragments of interest may be isolated from the gel and subsequently translocated through the nanochannel.

[0129] The device may further comprise an unfolding compartment for unfolding and/or pre- treating polymeric target analytes prior to translocating the target analyte through the nanochannel. The unfolding compartment may comprise medium and reagents useful for unfolding the target analyte, for example, reducing agents, denaturing agents, enzymes (e.g., protease, nuclease, restriction enzyme, etc.), and combinations thereof. The unfolding compartment may also comprise a heating and/or cooling system to unfold the polymer.

III. Nanochannel Systems

[0130] The present invention further provides apparatus and systems for analyzing a target analyte comprising any of the devices disclosed herein. In some embodiments, the nanochannel system comprises an array of the device (such as multi-nanochannel device) for multiplexed analysis of target analytes. The system may further comprise one or more elements selected from the group consisting of signal processing unit, computer processor (such as CPU), visualization unit, and a user interface. The system may be portable or mobile.

[0131] In some embodiments, the system comprises a signal processor for characterizing the target analyte based on the measured tunneling signals. The signal processor may comprise a computer processor that can dynamically control the circuits and detectors in the device, record signals, analyze signals, and optionally provide feedback to the device, for example, to tune the tunneling gaps based on the quality of the signals. The computer processor may also comprise databases for storing signature signal data for a plurality of molecules to be characterized.

[0132] In some embodiments, there is provided a computer system for analyzing a target analyte using any of the devices described herein, where the system comprises at least one processor, and where the processor includes computer instructions operating thereon for performing any of the methods described in the present disclosure.

[0133] Various implementations of the embodiments disclosed above, in particular at least some of the methods/processes disclosed, may be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and/or combinations thereof. These various implementations may include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0134] Such computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, for example, and may be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language. As used herein, the term "machine-readable medium" refers to any computer program product, apparatus and/or device (e.g., magnetic discs, optical disks, memory, Programmable Logic Devices (PLDs)) used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term "machine -readable signal" refers to any signal used to provide machine instructions and/or data to a programmable processor.

[0135] To provide for interaction with a user, some of the subject matter described herein may be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor and the like) for displaying information to the user and a keyboard and/or a pointing device (e.g., a mouse or a trackball) by which the user may provide input to the computer. For example, this program can be stored, executed and operated by the dispensing unit, remote control, PC, laptop, smart-phone, media player or personal data assistant ("PDA"). Other kinds of devices may be used to provide for interaction with a user as well; for example, feedback provided to the user may be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user may be received in any form, including acoustic, speech, or tactile input.

[0136] Certain embodiments of the subject matter described herein may be implemented in a computing system and/or devices that includes a back-end component (e.g., as a data server), or that includes a middleware component (e.g., an application server), or that includes a front- end component (e.g., a client computer having a graphical user interface or a Web browser through which a user may interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, or front-end components. The components of the system may be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include a local area network ("LAN"), a wide area network ("WAN"), and the Internet.

[0137] The computing system according to some such embodiments described above may include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other client-based system, in which the client computers are in

communication with databases, and the like. The client computers may communicate with the server via a network (e.g., intranet, internet, VPN).

IV. Methods of Fabricating Nanochannel Devices

[0138] The present invention also relates to methods of fabricating a nanochannel device useful for characterizing a target analyte. The methods may be used to fabricate any of the devices described herein.

[0139] One aspect of the present invention provides a method of fabricating a nanochannel device, comprising: disposing a plurality of detection electrodes longitudinally along a nanochannel; and disposing one or more reference electrodes longitudinally along the nanochannel; wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes.

[0140] In some embodiments, there is provided a method of fabricating a nanochannel device, comprising: forming a nanochannel; forming a plurality of detection electrodes; forming one or more reference electrodes, whereby the plurality of detection electrodes are disposed longitudinally along the nanochannel; whereby the one or more reference electrodes are disposed longitudinally along the nanochannel; and wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes. The nanochannel, the plurality of detection electrodes, and the one or more reference electrodes may be formed on a substrate. The various elements may be formed individually or simultaneously.

[0141] Any nanoscale fabrication method known in the art for may be used in the method for fabricating the nanochannel device. Suitable methods include, but are not limited to, nanoimprint lithography, electron beam lithography, and UV lithography. The detection electrodes (such as metal electrodes) may be disposed or formed along the nanochannel by any method known in the art, such as Focused Ion Beam chemical vapor deposition, atomic layer deposition, or chemical vapor deposition. The nanochannel and the detection electrodes may be disposed on a substrate, such as a silicon membrane. The substrate may be doped to serve as the one or more reference electrodes. Alternatively, the substrate may be removed after the plurality of detection electrodes are disposed or formed along the nanochannel, and the one or more reference electrodes may be disposed or formed along the side of the nanochannel that is originally in contact with the substrate.

[0142] In some embodiments, the method further comprises etching a sacrificial layer to form the nanochannel. For example, atomic layer deposition (ALD) methods may be used to obtain a nanometer-sized solid structure, such as a long, narrow rectangular prism, comprising an insulating layer wrapping around a sacrificial layer. ALD techniques may also be used to provide thin conformal coatings on the insulating layer, such as a protective layer to prevent the dielectric breakdown across the tunneling gaps. Current ALD techniques enable deposition of material layers with a thickness of subnanometer to a few nanometers. The sacrificial layer is consequently etched away to provide a central cavity in the nanometer-sized solid structure, thereby providing the nanochannel with a suitable dimension for measuring the tunneling signals across tunneling gaps along the nanochannel.

[0143] In some embodiments, the innermost layer of the nanochannel has a hydrophilic coating. In some embodiments, the hydrophilicity of the coating is tuned using a monolayer deposited by chemical vapor deposition.

[0144] The small dimension of the central cavity and the thin wall of the nanochannel may be important for allowing electrical measurements across the tunneling gaps, as the detection electrodes and reference electrodes may be located as close to the target analyte as possible in such situations. A suitable dimension for the central cavity is about 1 nm to about 4 nm, such as about 2 nm. An exemplary rectangular nanochannel device fabricated with the methods described herein has a dimension of about 2 nm x about 10 nm x about 300 nm.

[0145] In some embodiments, the method comprises first forming a nanochannel having one or more tunneling gaps with widths larger than the suitable dimension (such as about 1 nm to about 4 nm, for example about 2 nm), and subsequently adjusting the width of the tunneling gaps by contracting the nanochannel. Suitable methods for contracting the nanochannel include, but are not limited to, thermal expansion, piezoelectric contraction, acoustic modulation, and combinations thereof.

[0146] In some embodiments, the fabrication method further comprises connecting the nanochannel device to a constriction element operable to reversibly adjust the width of one or more of the plurality of tunneling gaps. The constriction element allows dynamic adjustment of the width of the one or more of the plurality of tunneling gaps by reversibly adjusting either the cross-section dimension (such as width of a rectangular nanochannel) of the entire nanochannel, or local segments of the nanochannel spanning the tunneling gaps. The constriction element can be selected from a thermal actuator, a piezoelectric actuator, an acoustic actuator, or combinations thereof.

[0147] Methods of nanometer-scale active control using thermal expansion have been described. In some embodiments, a resistor is fabricated near the nanochannel that heats the material surrounding the nanochannel and causes it to change in size because of mismatched thermal expansion coefficients. This design is analogous to the resistive heating design used in hard drive heads that controls the fly height. See, for example, US Patent No. 7,133,254.

[0148] In some embodiments, the method is repeated to dispose a plurality of nanochannels according to a pre-determined structure, and the method further comprises disposing a plurality of reservoirs to connect the plurality of nanochannels to provide a multi-nanochannel device.

[0149] In some embodiments, the method is repeated to form a plurality of the nanochannels, and the method further comprises forming a plurality of reservoirs, whereby the plurality of the nanochannels are connected to each other via the plurality of reservoirs according to a predetermined structure to provide a multi-nanochannel device.

[0150] The reservoirs may be disposed or formed using known methods in the art, such as UV or electron beam lithography. In some embodiments, the pre-determined structure is a loop. In some embodiments, each reservoir connects a pair of the nanochannels.

V. Methods of Analyzing Target Analytes

[0151] The present invention also provides methods for analyzing a target analyte by simultaneously measuring multiple tunneling signals along a nanochannel through which the target analyte translocates. Any of the devices described herein may be used in the methods.

[0152] One aspect of the present invention provides a method of analyzing a target analyte, comprising: a) applying a force to translocate the target analyte in a first direction through a nanochannel; wherein a plurality of detection electrodes are disposed longitudinally along the nanochannel; wherein one or more reference electrodes are disposed longitudinally along the nanochannel; and wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes; b) simultaneously measuring a plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and c) characterizing the target analyte based on the plurality of electrical signals. The electrical signals may include tunneling current or tunneling conductance.

[0153] Any suitable driving force, such as electrophoretic bias, magnetic field, or optical field, as discussed in the "Driving system for translocation" section above may be used in step (a) to translocate the target analyte through the nanochannel. In some embodiments, step a) comprises applying an electric field longitudinally across the nanochannel to allow translocation of a charged target analyte by electrophoresis. In some embodiments, the target analyte is magnetic or attached to one or more magnetic beads, wherein step a) comprises applying a magnetic field longitudinally or a magnetic field gradient transversely across the nanochannel to translocate the target analyte by pulling on the magnetic bead(s). In some embodiments, the target analyte is attached to one or more dielectric beads, wherein step a) comprises applying an optical field longitudinally across the nanochannel to translocate the target analyte by pulling on the dielectric bead(s).

[0154] In some embodiments, step b) comprises applying a voltage bias across the tunneling gaps. A suitable initial voltage bias may first be set across all tunneling gaps based on the molecular resonance level of the target analyte. In some embodiments, the voltage bias is adjusted until the desired set-point electrical signal is achieved, and/or separation of signals of different monomelic units of a polymeric target analyte is achieved. In some embodiments, the width of one or more tunneling gaps may be dynamically adjusted to tune to a resonance level of the target analyte.

[0155] In some embodiments, a single set of measurements provides enough information for the characterization of the target analyte. For example, the tunneling signals may be used to determine whether the target analyte is present in the nanochannel, how many copies of the target analyte are present in the nanochannel, and the location of the target analyte within the nanochannel based on the magnitude of the tunneling signals at the various tunneling gaps along the nanochannel. Additionally, if the translocation speed of the target analyte is known or determined, the length of the target analyte may be readily calculated based on the location of the target analyte within the signal if the length of the target analyte is shorter than the nanochannel. [0156] In some embodiments, steps a)-c) are repeated for a desired number of times. For example, sets of tunneling signals may be measured at a variety of positions of the target analyte with respect to the nanochannel. A tunneling signal profile may be measured over time as the target analyte translocates through the nanochannel to provide repeated measurements of the same segments of the target analyte at different tunneling gaps. A target analyte may be continuously measured over the entire course or over a partial course of its translocation along the nanochannel.

[0157] The target analyte may be translocated in a reserve direction with respect to the first direction to allow repeated measurements of a region in the target analyte. The target analyte may be translocated in a back-and-forth fashion repetitively and rapidly (i.e. "flossing") to allow multiple rounds of repeated measurements of the same region of the target analyte, until satisfactory quality of the tunneling signals is achieved. Any reverse translocation methods and back-and-forth translocation methods described in the "Driving system for translocation" section can be used. The repeated measurement by reverse translocation can be applied to every region of the target analyte or a particular region of interest, such as a difficult to analyze region, for example, repeat sequences.

[0158] In some embodiments, the method further comprises the steps of: d) applying a second force to translocate the target analyte in the reverse direction with respect to the first direction through the nanochannel; e) simultaneously measuring a second plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and f) characterizing the target analyte based on the second plurality of electrical signals.

[0159] In some embodiments, the method comprises the steps of a) applying a force to translocate the target analyte in a first direction through a nanochannel; wherein a plurality of detection electrodes are disposed longitudinally along the nanochannel; wherein one or more reference electrodes are disposed longitudinally along the nanochannel; and wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes; b) simultaneously measuring a plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; c) characterizing the target analyte based on the plurality of electrical signals; d) applying a second force to translocate the target analyte in the reverse direction with respect to the first direction through the nanochannel; e) simultaneously measuring a second plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and f) characterizing the target analyte based on the second plurality of electrical signals. In some embodiments, steps a)-c), steps d)-f), or steps a)-f) are repeated more than once.

[0160] In some embodiments, there is provided a method of analyzing a target analyte, comprising: a) applying force to translocate the target analyte through a multi -nanochannel device comprising a plurality of nanochannels connected via a plurality of reservoirs to provide a nanochannel loop; wherein a plurality of detection electrodes are disposed longitudinally along each nanochannel; wherein one or more reference electrodes are disposed longitudinally along each nanochannel; and wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes; b) simultaneously measuring a plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps in the nanochannel loop; c) characterizing the target analyte based on the plurality of electrical signals. With the nanochannel loop, any of the devices described above comprising a nanochannel loop, repeated measurements of the target analyte may be achieved by repeating steps a)-b) while translocating the target analyte through the loop for a desired number of times in a single direction.

[0161] Characterization of the target analyte based on the plurality of electrical signals may comprise any signal processing technique known in the art. For example, differential analysis may be applied to the signal profile (including profiles of multiple signals, or multiple modalities of signals) of the target analyte to obtain at least one parameter reflective of an identification of the target analyte. For polymeric target analytes, signal signatures of individual types of monomelic units or oligomeric units (such as dimer, trimer, etc.) may be first obtained to allow comparison to the signal profile (including profiles of multiple signals, or multiple modalities of signals) of the target analyte, and determination of the sequence of the polymeric analyte by matching the signal profile (including profiles of multiple signals, or multiple modalities of signals) to the signal signatures. FIG. 9 shows an exemplary idealized time trace of a short polymer, such as a short oligonucleotide, along different detection electrodes in an exemplary nanochannel. Repeated measurements can be averaged and used to reduce noise. Other signal processing steps may include, but are not limited to, rejection of fast data spikes, automatic peak detection, and reduction of background signal. Additional signals, such as optical signals and plasmonic signals may be combined with the electrical signal for data analysis.

[0162] Characterization of the target analyte may include determining the length or chemical identity (such as sequence) of the target analyte. For polymeric target analytes, the tunneling signal profile may be used to estimate the translocation speed of the target analyte through the nanochannel or the nanochannel loop. The entire target analyte need to be translocated through the nanochannel or the nanochannel loop once in order to determine the full length or the full sequence of the target analyte.

[0163] The methods described herein are applicable for any target analyte described in the "Nanochannel Device" section. For polymeric target analytes, the method may further comprise unfolding of the polymeric target analytes. For example, a polypeptide may be treated with a reducing agent (such as beta-mercaptoethanol, DTT, or TCEP) and/or a denaturing agent (such as SDS) prior to subjecting the polypeptide to the method for analysis. Free cysteines in a reduced polypeptide may further be blocked by treatment with iodoacetic acid to prevent the formation of disulfide bonds. The polypeptide may also be kept in a denaturing solution such as SDS solution while translocating through the nanochannel. In some embodiments, long target analyte may be cleaved chemically or by an enzyme prior to subjecting the target analyte to the method of analysis. For example, nucleic acids may be cleaved into small fragments by nuclease or restriction enzymes. Polypeptides may be pre-treated with proteases (such as trypsin) prior to the analysis.

[0164] In some embodiments, the method further comprises contacting the nanochannel or multi-nanochannel device with a cleaning agent, and reusing the nanochannel or multi- nanochannel device to analyze a second target analyte. The cleaning agent is any agent that may degrade and/or remove the target analyte and is compatible with the material layers of the nanochannel(s) and reservoir(s). In some embodiments, wherein the target analyte is a nucleic acid, the cleaning agent comprises a nuclease. In some embodiments, wherein the target analyte is a protein, the cleaning agent comprises a protease. In some embodiments, the cleaning agent comprises a strong acid or a strong base. In some embodiments, the nanochannel or multi- nanochannel device is washed with the medium prior to the reuse. In some embodiments, the nanochannel or multi-nanochannel device is reused for at least about any one of 1, 5, 10, 20, 50, 100, or more times.

[0165] The nanochannel-based methods and devices of the present invention are useful in a variety of applications. For example, the length and chemical identity of a target analyte may be determined using any of the nanochannel devices and/or the analysis methods as described above. Exemplary applications of the methods and devices are discussed below.

[0166] In some embodiments, the method is used to determine the length of the target analyte. In some embodiments, the target analyte is a polymer, such as a biological polymer or a non- biological polymer. In some embodiments, a population of polymeric analytes are analyzed by the method, wherein each copy of the polymeric analyte is individually subjected to the method to determine its length, thereby providing a length distribution of the population of the polymeric analytes. Compared to traditional methods of length distribution analysis, such as using gel electrophoresis or chromatography, the methods based on the nanochannel device are rapid, have single-molecule sensitivity, and require smaller amount of sample.

[0167] In some embodiments, the method is used to determine the chemical identity of the target analyte, for example, by matching the electrical signals or signal profile (including profiles of multiple signals, or multiple modalities of signals) of the target analyte against a database of tunneling signal signatures of chemical compounds. In some embodiments, the method is used to determine the sequence of a polymeric target analyte. In some embodiments, the method is used to determine the vibrational energy spectrum of the target analyte.

[0168] In some embodiments, the method is used for sequencing a biopolymer. In some embodiments, the method is used for nucleic acid sequencing, such as DNA sequencing or RNA sequencing. In some embodiments, the method is used for sequencing genomic DNA. In some embodiments, the method is used for detecting epigenetic modifications. In some embodiments, the method is used for sequencing mRNA. In some embodiments, the method is used for sequencing viral RNA. A population of nucleic acid molecules can be subjected to the method, wherein individual sequences of the nucleic acids in the population are determined. For example, a total RNA sample or a small RNA extract from a cell may be subjected to the method. In some embodiments, a mixture of DNA and RNA molecules are subjected to the method without first separating the DNA molecules from the RNA molecules. In some embodiments, the method is used for polypeptide sequencing. In some embodiments, a population of polypeptides is subjected to the method. Copy numbers of biopolymers in a population sharing the same sequence may be determined using the method, thereby enabling determination of expression levels of the biopolymer. In some embodiments, the method is used for copy number analysis of a genetic allele. In some embodiments, the method is used for RNA expression analysis. In some embodiments, the method is used for protein expression analysis. The nucleic acid sequencing and polypeptide sequencing methods using the nanochannel device are widely useful in a variety of fields, including, but not limited to, diagnosis of a disease, single-cell sequencing, forensic analysis, metagenomics analysis, detection of virus and pathogens, genetic testing, personalized medicine, food safety testing, biotechnology and molecular biology research. VI. Exemplary Embodiments

[0169] The exemplary embodiments below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way.

[0170] Embodiment 1. In some embodiments, there is provided a device for analyzing a target analyte, comprising: a) a nanochannel; b) a plurality of detection electrodes disposed longitudinally along the nanochannel; and c) one or more reference electrodes disposed longitudinally along the nanochannel; wherein the target analyte translocates through the nanochannel; and wherein each detection electrode forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes.

[0171] Embodiment 2. In some further embodiments of embodiment 1, the nanochannel is dimensioned to allow translocation of only a single copy of the target analyte in an unfolded fashion.

[0172] Embodiment 3. In some further embodiments of embodiment 1 or embodiment 2, the plurality of detection electrodes are disposed longitudinally along a first side of the nanochannel, and wherein the one or more reference electrodes are disposed longitudinally along a side opposite to the first side of the nanochannel.

[0173] Embodiment 4. In some further embodiments of any one of embodiments 1-3, the plurality of detection electrodes are disposed directly along the nanochannel.

[0174] Embodiment 5. In some further embodiments of any one of embodiments 1-4, the one or more reference electrodes are disposed directly along the nanochannel.

[0175] Embodiment 6. In some further embodiments of any one of embodiments 1-5, the nanochannel has a cross-section, and wherein the cross-section is rectangular.

[0176] Embodiment 7. In some further embodiments of any one of embodiments 1-6, the width of each tunneling gap is about 1 nm to about 4 nm.

[0177] Embodiment 8. In some further embodiments of any one of embodiments 1-7, the length of the nanochannel is between about 50 nm and about 5μιη.

[0178] Embodiment 9. In some further embodiments of any one of embodiments 1-8, the nanochannel comprises a bend that applies a mechanical tension on the target analyte when the target analyte passes around the bend.

[0179] Embodiment 10. In some further embodiments of embodiment 9, the plurality of detection electrodes are disposed in proximity to the bend.

[0180] Embodiment 1 1. In some further embodiments of embodiment 9 or embodiment 10, the nanochannel is serpentine shaped. [0181] Embodiment 12. In some further embodiments of any one of embodiments 1-1 1, the plurality of detection electrodes are unevenly spaced.

[0182] Embodiment 13. In some further embodiments of embodiment 12, the plurality of detection electrodes are randomly spaced.

[0183] Embodiment 14. In some further embodiments of any one of embodiments 1-13, the target analyte is a polymer comprising monomeric units each having a unit length, and wherein the spacing between any two adjacent detection electrodes is not an integer multiple of the unit length.

[0184] Embodiment 15. In some further embodiments of any one of embodiments 1-14, the device further comprises a driving system for translocating the target analyte through the nanochannel.

[0185] Embodiment 16. In some further embodiments of embodiment 15, the driving system comprises a DC power supply configured to generate an electrophoretic bias longitudinally across the nanochannel.

[0186] Embodiment 17. In some further embodiments of embodiment 16, the driving system further comprises an AC power supply configured to apply an alternating electrophoretic bias longitudinally across the nanochannel, wherein the altering electrophoretic bias allows reversal of the direction in which the target analyte translocates.

[0187] Embodiment 18. In some further embodiments of embodiment 15, the driving system comprises a thermal element configured to generate a thermal gradient longitudinally across the nanochannel.

[0188] Embodiment 19. In some further embodiments of embodiment 15, the driving system comprises a magnetic field capable of pulling a magnetic bead attached to one end of the target analyte.

[0189] Embodiment 20. In some further embodiments of embodiment 15, the driving system comprises an optical field capable of pulling a dielectric bead attached to one end of the target analyte.

[0190] Embodiment 21. In some further embodiments of any one of embodiments 1-20, the nanochannel has a first open end and a second open end, and wherein the first open end is connected to a first reservoir and the second open end of the nanochannel is connected to a second reservoir. [0191] Embodiment 22. In some further embodiments of embodiment 21, the driving system translocates the target analyte from the first reservoir to the second reservoir through the nanochannel.

[0192] Embodiment 23. In some further embodiments of any one of embodiments 1-22, the nanochannel comprises a wall comprising an insulating layer.

[0193] Embodiment 24. In some further embodiments of any one of embodiments 1-23, the nanochannel is filled with a medium comprising a solvent compatible with the target analyte.

[0194] Embodiment 25. In some further embodiments of embodiment 24, wherein the medium further comprises an electrolyte.

[0195] Embodiment 26. In some further embodiments of embodiment 24 or embodiment 25, the medium further comprises a reducing agent.

[0196] Embodiment 27. In some further embodiments of any one of embodiments 24-26, the medium further comprises a denaturing agent.

[0197] Embodiment 28. In some further embodiments of any one of embodiments 24-27, the medium is adjusted to provide a dielectric constant suitable for probing a tunneling resonance level of the target analyte.

[0198] Embodiment 29. In some further embodiments of any one of embodiments 24-28, the wall further comprises an interior coating that facilitates absorption of the medium on the interior surface of the wall.

[0199] Embodiment 30. In some further embodiments of embodiment 29, the solvent is hydrophilic, and wherein the interior coating comprises a hydrophilic material.

[0200] Embodiment 31. In some further embodiments of embodiment 30, the hydrophilic material is selected from the group consisting of polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), and polyvinyl alcohol (PVA).

[0201] Embodiment 32. In some further embodiments of embodiment 29, the solvent is lipophilic, and wherein the interior coating comprises a lipophilic material.

[0202] Embodiment 33. In some further embodiments of any one of embodiments 23-32, the wall further comprises a protective layer that reduces dielectric breakdown of the insulating layer.

[0203] Embodiment 34. In some further embodiments of embodiment 33, the wall comprises a plurality of insulating layers and a plurality of protective layers, and wherein the plurality of insulating layers and the plurality of protective layers form an alternating structure. [0204] Embodiment 35. In some further embodiments of any one of embodiments 23-34, the wall is fabricated by atomic layer deposition.

[0205] Embodiment 36. In some further embodiments of any one of embodiments 1-35, the device further comprises a tuning element operable to adjust one or more of the plurality of tunneling gaps to allow probing of a tunneling resonance level of the target analyte that passes through the one or more tunneling gaps.

[0206] Embodiment 37. In some further embodiments of embodiment 36, the tuning element is a tunable power supply configured to generate a voltage bias across the one or more tunneling gaps.

[0207] Embodiment 38. In some further embodiments of embodiment 36, the tuning element is a constriction element operable to reversibly adjust the width of the one or more tunneling gaps.

[0208] Embodiment 39. In some further embodiments of embodiment 38, the constriction element is selected from a thermal actuator, a piezoelectric actuator, and an acoustic actuator.

[0209] Embodiment 40. In some further embodiments of any one of embodiments 1-39, the plurality of detection electrodes comprise one or more metallic electrodes.

[0210] Embodiment 41. In some further embodiments of any one of embodiments 1-39, the plurality of detection electrodes comprise one or more non-metallic or semi-metallic electrodes.

[0211] Embodiment 42. In some further embodiments of embodiment 41, the plurality of detection electrodes comprise one or more graphene sheets.

[0212] Embodiment 43. In some further embodiments of any one of embodiments 1-42, the plurality of detection electrodes comprise one or more triangular electrodes.

[0213] Embodiment 44. In some further embodiments of any one of embodiments 1-43, each of the plurality of detection electrodes is connected to an electrical detector, wherein the electrical detector measures an electrical signal when the target analyte passes through the tunneling gap associated with the detection electrode.

[0214] Embodiment 45. In some further embodiments of any one of embodiments 1-44, the plurality of detection electrodes comprise one or more spin-value electrodes that can spin- polarize electrons tunneling through the tunneling gaps.

[0215] Embodiment 46. In some further embodiments of embodiment 45, the target analyte is magnetically labeled to enhance the electrical signals detected by the electrical detectors connected to the one or more spin- value electrodes. [0216] Embodiment 47. In some further embodiments of any one of embodiments 44-46, the plurality of detection electrodes comprise one or more plasmonically active electrodes to plasmonically enhance the electrical signals when the target analyte passes through the one or more tunneling gaps associated therewith.

[0217] Embodiment 48. In some further embodiments of any one of embodiments 1-47, the nanochannel is transparent.

[0218] Embodiment 49. In some further embodiments of any one of embodiments 1-48, the device further comprises an optical detector for simultaneous measurement of an optical signal when the target analyte passes through one or more of the plurality of tunneling gaps.

[0219] Embodiment 50. In some further embodiments of embodiment 49, the optical detector is a microscope.

[0220] Embodiment 51. In some further embodiments of embodiment 49 or embodiment 50, the optical signal is one or more selected from the group consisting of electroluminescence, fluorescence, Raman scattering, bioluminescence, and chemiluminescence associated with the target analyte.

[0221] Embodiment 52. In some further embodiments of any one of embodiments 49-51, the device further comprises a plasmonically enhanced electrode that plasmonically enhances the optical signal.

[0222] Embodiment 53. In some further embodiments of embodiment 52, each of the plurality of detection electrodes is a plasmonically enhanced electrode.

[0223] Embodiment 54. In some further embodiments of any one of embodiments 1-53, the device further comprises a biological nanopore disposed within one of the plurality of tunneling gaps.

[0224] Embodiment 55. In some further embodiments of any one of embodiments 1-54, wherein the device comprises a plurality of the nanochannels, wherein the nanochannels are connected to each other via a plurality of reservoirs.

[0225] Embodiment 56. In some further embodiments of embodiment 55, the plurality of nanochannels are connected to each other via the plurality of reservoirs to provide a loop through which the target analyte translocates.

[0226] Embodiment 57. In some further embodiments of any one of embodiments 1-56, the device further comprises a loading reservoir fluidically connected to the nanochannel, wherein the target analyte is loaded to the loading reservoir in order to be exposed to the nanochannel. [0227] Embodiment 58. In some further embodiments of embodiment 57, the loading reservoir is connected to the nanochannel via a funnel-shaped port.

[0228] Embodiment 59. In some further embodiments of any one of embodiments 1-58, the target analyte is a non-polymeric molecule.

[0229] Embodiment 60. In some further embodiments of any one of embodiments 1-58, the target analyte is a non-biological polymer.

[0230] Embodiment 61. In some further embodiments of any one of embodiments 1-58, the target analyte is a biopolymer.

[0231] Embodiment 62. In some further embodiments of embodiment 61, the target analyte is a DNA.

[0232] Embodiment 63. In some further embodiments of embodiment 61, the target analyte is an R A.

[0233] Embodiment 64. In some further embodiments of embodiment 62 or embodiment 63, the device further comprises a polymerase attached to one end of the nanochannel to control the speed at which the target analyte translocates through the nanochannel.

[0234] Embodiment 65. In some further embodiments of embodiment 61, the target analyte is a polypeptide.

[0235] Embodiment 66. In some further embodiments of any one of embodiments 61-65, the device further comprises an unfolding compartment that unfolds the biopolymer.

[0236] Embodiment 67. In some further embodiments of embodiment 66, the unfolding compartment comprises a reducing agent.

[0237] Embodiment 68. In some further embodiments of embodiment 66 or embodiment 67, the unfolding compartment comprises a denaturing agent.

[0238] Embodiment 69. In some further embodiments of embodiment 65, the device comprises an unfolding compartment comprising a protease that cleaves the polypeptide into a plurality of polypeptide fragments, wherein each polypeptide fragment translocates through the nanochannel.

[0239] Embodiment 70. In some embodiments, there is provided a method of analyzing a target analyte, comprising: a) applying a force to translocate the target analyte in a first direction through a nanochannel; wherein a plurality of detection electrodes are disposed longitudinally along the nanochannel; wherein one or more reference electrodes are disposed longitudinally along the nanochannel; and wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes; b) simultaneously measuring a plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and c) characterizing the target analyte based on the plurality of electrical signals.

[0240] Embodiment 71. In some further embodiments of embodiment 70, said applying a force comprises applying an electric field longitudinally across the nanochannel.

[0241] Embodiment 72. In some further embodiments of embodiment 70, said applying a force comprises applying a magnetic field longitudinally or a magnetic field gradient transversely across the nanochannel, and wherein the target analyte is attached to a magnetic bead.

[0242] Embodiment 73. In some further embodiments of embodiment 70, said applying a force comprises applying an optical field longitudinally across the nanochannel, and wherein the target analyte is attached to a dielectric bead.

[0243] Embodiment 74. In some further embodiments of any one of embodiments 70-73, the steps a)-c) are repeated for a desired number of times.

[0244] Embodiment 75. In some further embodiments of any one of embodiments 70-74, the method further comprises: d) applying a second force to translocate the target analyte in the reverse direction with respect to the first direction through the nanochannel; e) simultaneously measuring a second plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and f) characterizing the target analyte based on the second plurality of electrical signals.

[0245] Embodiment 76. In some further embodiments of embodiment 75, steps a)-f) are repeated more than once.

[0246] Embodiment 77. In some further embodiments of any one of embodiments 74-76, the method further comprises recording a signal profile comprising the plurality of electrical signals as a function of time.

[0247] Embodiment 78. In some further embodiments of embodiment 77, said characterizing comprises differential analysis of the signal profile to obtain at least one parameter reflective of an identification of the target analyte.

[0248] Embodiment 79. In some further embodiments of any one of embodiments 70-78, the method further comprises unfolding the target analyte prior to translocating the target analyte through the nanochannel.

[0249] Embodiment 80. In some further embodiments of any one of embodiments 70-79, only one copy of the target analyte translocates through the nanochannel. [0250] Embodiment 81. In some further embodiments of any one of embodiments 70-80, the target analyte is a non-polymeric molecule.

[0251] Embodiment 82. In some further embodiments of any one of embodiments 70-80, the target analyte is a polymer.

[0252] Embodiment 83. In some further embodiments of embodiment 82, the polymer comprises monomelic units each having a unit length, and wherein the spacing between any two adjacent detection electrodes is not an integer multiple of the unit length.

[0253] Embodiment 84. In some further embodiments of embodiment 82 or embodiment 83, the target analyte is a non-biological polymer.

[0254] Embodiment 85. In some further embodiments of embodiment 82 or embodiment 83, the target analyte is a biopolymer.

[0255] Embodiment 86. In some further embodiments of embodiment 85, the target analyte is a DNA.

[0256] Embodiment 87. In some further embodiments of embodiment 85, the target analyte is an R A.

[0257] Embodiment 88. In some further embodiments of embodiment 86 or embodiment 87, the method further comprises binding the target analyte to a polymerase.

[0258] Embodiment 89. In some further embodiments of embodiment 85, the target analyte is a polypeptide.

[0259] Embodiment 90. In some further embodiments of embodiment 89, the method further comprises cleaving the polypeptide with a protease.

[0260] Embodiment 91. In some further embodiments of embodiment 89 or embodiment 90, the method further comprises treating the polypeptide with a reducing agent that reduces the disulfide bonds of the polypeptides.

[0261] Embodiment 92. In some further embodiments of any one of embodiments 89-91, the method further comprises treating the polypeptide with a denaturing agent.

[0262] Embodiment 93. In some further embodiments of any one of embodiments 70-92, said characterizing comprises determining the length of the target analyte.

[0263] Embodiment 94. In some further embodiments of any one of embodiments 70-93, said characterizing comprises determining the sequence of the target analyte.

[0264] Embodiment 95. In some further embodiments of any one of embodiments 70-94, said characterizing comprises determining the vibrational energy spectrum of the target analyte. [0265] Embodiment 96. In some embodiments, there is provided a method of fabricating a nanochannel device, comprising: a) disposing a plurality of detection electrodes longitudinally along a nanochannel; and b) disposing one or more reference electrodes longitudinally along the nanochannel; wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes.

[0266] Embodiment 97. In some further embodiments of embodiment 96, the method further comprises etching a sacrificial layer to form the nanochannel.

[0267] Embodiment 98. In some further embodiments of embodiment 96 or embodiment 97, the method further comprises connecting the nanochannel device to a constriction element operable to reversibly adjust the width of one or more of the plurality of tunneling gaps.

[0268] Embodiment 99. In some further embodiments of embodiment 98, the constriction element comprises a thermal actuator, piezoelectric actuator, or an acoustic actuator.

[0269] Embodiment 100. In some embodiments, there is provided a device for analysis of a polymeric target analyte, comprising a nanochannel comprising a bend, wherein the polymeric target analyte translocates through the nanochannel, and wherein the bend applies a mechanical tension on the polymeric target analyte.

Claims

CLAIMS What is claimed is:

1. A device for analyzing a target analyte, comprising:

a) a nanochannel;

b) a plurality of detection electrodes disposed longitudinally along the nanochannel; and c) one or more reference electrodes disposed longitudinally along the nanochannel;

wherein the target analyte translocates through the nanochannel; and

wherein each detection electrode forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes.

2. The device of claim 1, wherein the nanochannel is dimensioned to allow translocation of only a single copy of the target analyte in an unfolded fashion.

3. The device of claim 1 or claim 2, wherein the plurality of detection electrodes are disposed longitudinally along a first side of the nanochannel, and wherein the one or more reference electrodes are disposed longitudinally along a side opposite to the first side of the nanochannel.

4. The device of any one of claims 1-3, wherein the width of each tunneling gap is about 1 nm to about 4 nm.

5. The device of any one of claims 1-4, wherein the nanochannel comprises a bend that applies a mechanical tension on the target analyte when the target analyte passes around the bend.

6. The device of any one of claims 1-5, wherein the plurality of detection electrodes are unevenly spaced.

7. The device of any one of claims 1-6, wherein the target analyte is a polymer comprising monomeric units each having a unit length, and wherein the spacing between any two adjacent detection electrodes is not an integer multiple of the unit length.

8. The device of any one of claims 1-7, further comprising a driving system for translocating the target analyte through the nanochannel.

9. The device of any one of claims 1-8, wherein the nanochannel has a first open end and a second open end, and wherein the first open end is connected to a first reservoir and the second open end of the nanochannel is connected to a second reservoir.

10. The device of any one of claims 1-9, wherein the nanochannel comprises a wall comprising an insulating layer.

11. The device of any one of claims 1-10, wherein the nanochannel is filled with a medium comprising a solvent compatible with the target analyte.

12. The device of claim 11, wherein the wall further comprises an interior coating that facilitates absorption of the medium on the interior surface of the wall.

13. The device of any one of claim 10-12, wherein the wall further comprises a protective layer that reduces dielectric breakdown of the insulating layer.

14. The device of any one of claims 1-13, further comprising a tuning element operable to adjust one or more of the plurality of tunneling gaps to allow probing of a tunneling resonance level of the target analyte that passes through the one or more tunneling gaps.

15. The device of any one of claims 1-14, wherein each of the plurality of detection electrodes is connected to an electrical detector, wherein the electrical detector measures an electrical signal when the target analyte passes through the tunneling gap associated with the detection electrode.

16. The device of any one of claims 1-15, wherein the plurality of detection electrodes comprise one or more spin-value electrodes that can spin-polarize electrons tunneling through the tunneling gaps.

17. The device of any one of claims 1-16, wherein the nanochannel is transparent.

18. The device of any one of claims 1-17, further comprising an optical detector for simultaneous measurement of an optical signal when the target analyte passes through one or more of the plurality of tunneling gaps.

19. The device of any one of claims 1-18, further comprising a biological nanopore disposed within one of the plurality of tunneling gaps.

20. The device of any one of claims 1-19, comprising a plurality of the nanochannels, wherein the nanochannels are connected to each other via a plurality of reservoirs.

21. The device of claim 20, wherein the plurality of nanochannels are connected to each other via the plurality of reservoirs to provide a loop through which the target analyte translocates.

22. The device of any one of claims 1-21, further comprising a loading reservoir fluidically connected to the nanochannel, wherein the target analyte is loaded to the loading reservoir in order to be exposed to the nanochannel.

23. The device of any one of claims 1-22, wherein the target analyte is a non-polymeric molecule.

24. The device of any one of claims 1-22, the target analyte is a non-biological polymer.

25. The device of any one of claims 1-22, wherein the target analyte is a biopolymer.

26. The device of claim 25, further comprising an unfolding compartment that unfolds the biopolymer.

27. A method of analyzing a target analyte, comprising:

a) applying a force to translocate the target analyte in a first direction through a nanochannel; wherein a plurality of detection electrodes are disposed longitudinally along the nanochannel; wherein one or more reference electrodes are disposed longitudinally along the nanochannel; and

wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes;

b) simultaneously measuring a plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and

c) characterizing the target analyte based on the plurality of electrical signals.

28. The method of claim 27, wherein said applying a force comprises applying an electric field longitudinally across the nanochannel.

29. The method of claim 27 or claim 28, wherein the steps a)-c) are repeated for a desired number of times.

30. The method of any one of claims 27-29, further comprising:

d) applying a second force to translocate the target analyte in the reverse direction with respect to the first direction through the nanochannel;

e) simultaneously measuring a second plurality of electrical signals generated as the target analyte passes through the plurality of tunneling gaps; and

f) characterizing the target analyte based on the second plurality of electrical signals.

31. The method of claim 30, wherein steps a)-f) are repeated more than once.

32. The method of any one of claims 29-31, further comprising recording a signal profile comprising the plurality of electrical signals as a function of time.

33. The method of claim 32, wherein said characterizing comprises differential analysis of the signal profile to obtain at least one parameter reflective of an identification of the target analyte.

34. The method of any one of claims 27-33, further comprising unfolding the target analyte prior to translocating the target analyte through the nanochannel.

35. The method of any one of claims 27-34, wherein the target analyte is a non-polymeric molecule.

36. The method of claim 27-34, wherein the target analyte is a non-biological polymer.

37. The method of claim 27-34, wherein the target analyte is a biopolymer.

38. The method of any one of claims 27-37, wherein said characterizing comprises determining the length of the target analyte.

39. The method of any one of claims 27-38, wherein said characterizing comprises determining the sequence of the target analyte.

40. The method of any one of claims 27-39, wherein said characterizing comprises determining the vibrational energy spectrum of the target analyte.

41. A method of fabricating a nanochannel device, comprising:

a) disposing a plurality of detection electrodes longitudinally along a nanochannel; and b) disposing one or more reference electrodes longitudinally along the nanochannel;

wherein each of the plurality of detection electrodes forms a tunneling gap to provide a plurality of tunneling gaps when coupled with the one or more reference electrodes.

42. A device for analysis of a polymeric target analyte, comprising a nanochannel comprising a bend, wherein the polymeric target analyte translocates through the nanochannel, and wherein the bend applies a mechanical tension on the polymeric target analyte.