WO2013056182A1 - Piezoelectric devices and methods - Google Patents

Abstract

Piezoelectric devices are provided for use in conjunction with a nanochannel for measuring desired characteristics of a charged polymer. Related methods of use and kits are also provided. Systems, methods, and kits for analyzing polymers utilizing Hall-effect imaging are also provided.

Description

PIEZOELECTRIC DEVICES AND METHODS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit and priority under 35 U.S. C. 119(e) of United States Provisional Application No. 61/546,060, filed October 12, 2011, and United States Provisional Application No. 61/584,819, filed January 10, 2012, the disclosure of each of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Inexpensive biopolymer characterization in a manner that is fast, accurate, and low-cost has become an area of increased importance to the medical community and the population at large. A variety of approaches have attempted to address this need. Nanopore sequencing approaches have been proposed, however, single nucleotide resolution of extended DNA molecules has not yet been achieved.

[0003] One major disadvantage of current and prior nanopore analysis techniques is controlling the rate at which the translocating polynucleotide is analyzed. For example, the translocation rate is nucleotide composition dependent and can range between 10⁵ to 10⁷ nucleotides per second under the measurement conditions outlined by Kasianowicz et al, Proc. Nat'l. Acad. Sci., USA, 1996, 93: 13770-3. Other estimates put translocation rates through nanopores at about 1 bp/10 μ8 for a-hemolysin and about 1 bp/10 ns for a solid-state pore. See, e.g., U.S. Pat. App. Pub. No. 201 10226623.

[0004] Therefore, the correlation between any given polynucleotide's length and its translocation time is, at the very least, not straightforward. For example, due to electronic limitations, existing detection techniques cannot resolve individual current fluctuations at the rapid rate necessary to resolve individual nucleotides. Another disadvantage of previous nanopore analysis techniques is that each individual polymer typically passes through the detection zone only once.

[0005] Theoretical suggestions have been made to address the translocation rate quandary, though each has major drawbacks and limitations. For example, these suggestions include the use of nanosteppers, chemical modification of pore surfaces, modification of amino acid sequences of naturally-occurring nanopores, attachment of polynucleotide processive enzymes to pore openings to ratchet target polynucleotide sequences, the use of molecular adaptors positioned within nanopores, electrostatic positioning systems, and nano-vice-grip-type assemblies. Each suggestion attempts to slow translocation rates and, if successful, would result in unpredictable and varying translocation rates and overall slowing the rate of base calling. Moreover, these proposed methods do not directly address the problem of poor or inaccurate individual base resolutions, and many rely on complicated chemistries, detailed pre-sequencing sample processing, and/or the coordinated functioning of multiple biological or chemical variables. It is therefore desirable to address these and other limitations in existing polymer characterization approaches. The present invention addresses these and other needs in the art.

[0006] None of the references described or referred to herein are admitted to be prior art to the claimed invention.

SUMMARY OF THE INVENTION

[0007] A piezoelectric device is provided for use in conjunction with a nanochannel for measuring desired characteristics of a charged polymer, wherein the charged polymer is translocated through the nanochannel. Also provided is a device comprising: (a) a piezoelectric module; (b) a nanochannel comprising an inner surface having a width and a depth; and (c)an electric circuit spanning the width of the nanochannel for evaluating the dielectric or charge density properties of a moiety presented within the inner surface of the nanochannel, wherein the piezoelectric module is provided in electronic communication within the circuit.

[0008] In occasional embodiments the piezoelectric module comprises a piezoelectric transducer, and wherein the electric circuit comprises an electric signal pathway, the device further comprising a static magnetic field source capable of producing a magnetic field, wherein the pathway for the charged polymer, the magnetic field, and the electric signal pathway intersect one-another, and are situated at right angles to one-another in a three dimensional orientation at a discrete examination location. Most frequently in such embodiments the device comprises a Hall- effect imaging module.

[0009] Often the charged polymer comprises a nucleic acid molecule, a natural or synthetic oligonucleotide, a natural or synthetic polynucleotide, DNA, RNA, PNA, a protein, a peptide, a chimeric amino acid molecule, and/or a chimeric nucleic acid molecule. Often the nanochannel is a nanopore or a carbon nanotube and often the nanochannel, or inner surface of the nanochannel has a width or diameter of about 3 nm or less. Nanochannels of the present disclosure have an inner channel area having a cross-sectional area designed to permit passage of a polymer; this inner area may be referred to as a channel width and is often referred to in terms of width (i.e., distance across the area) or diameter (same). In frequent embodiments the desired characteristics, which often include identification of a monomer such as a nucleotide within the charged polymer are measured while the charged polymer is located within the nanochannel.

[00010] In certain embodiments electrophoretic forces drive the translocation of the charged polymer. In other embodiments magnetic forces drive the translocation. In still other embodiments a combination of electrophoretic forces and magnetic forces drive the

translocation.

[00011] In other frequent embodiments a piezoelectric analysis device is provided for use in analyzing a polymer, comprising: (a) a first electrode and a second electrode comprising an electric circuit; (b) a piezioelectric actuator in electronic communication with the electric circuit; (c) a nanochannel having a proximal and distal portion that provides fluid communication between a proximal fluid reservoir and a distal fluid reservoir; and (d) a piezoelectric actuator deflection measuring device, wherein the first and second electrodes are positioned on opposing faces of the nanochannel, and wherein the piezioelectric actuator is operably connected to the piezoelectric actuator deflection measuring device to permit measurement of physical deflections of the piezoelectric actuator in response to electronic signals from the electric circuit.

[00012] In certain embodiments the nanochannel comprises an elongate orientation having a channel width or diameter of at least about 1.5nm.

[00013] In certain embodiments the nanochannel comprises a stepped nanochannel comprising one or more stepped portions. The stepped portion is often a feature within the channel provided to induce a localized stretch or bend in a translocating or static polymer positioned in the nanochannel. Such stepped portions often comprise a natural or synthetic bridge helix, a natural or synthetic trigger loop, a combination of a bridge helix and trigger loop, a fullerene molecule, a carbon molecule, or a physical feature within, dividing, or distinguishable from though positioned within, the nanochannel, or other site. Frequently, when a stepped nanochannel is provided the first and second electrodes comprise, or are positioned adjacent to, the stepped portion of the nanochannel. In occasional embodiments the stepped portion is provided in conjunction with a conventional nanochannel lacking a piezoelectric recognition element.

[00014] In certain embodiments a magnetic element is positioned in contact with, or proximal to the proximal fluid reservoir, wherein the magnetic element provides a selectable magnetic field extending through at least a portion of the nanochannel.

[00015] In certain embodiments the device further comprises electrophoretic electrodes comprising a first electrode positioned in contact with the proximal fluid reservoir and a second electrode positioned in contact with the distal fluid reservoir.

[00016] In frequent embodiments the piezoelectric actuator is positioned in a cantilevered manner. In certain embodiments the piezoelectric actuator comprises an electrostrictive actuator, a bimorph actuator, a multimorph actuator, a shear actuator, or a combination thereof. In certain frequent embodiments, the piezoelectric actuator deflection measuring device comprises a capacitive position sensor, a strain gauge, a vibrometer, an accelerometer, an interferometer, a spectrum analyzer, and/or a tunnel gap modulation spectroscopy device.

[00017] In certain embodiments the piezoelectric actuator has a resonant frequency ranging between 10 kHz to 3 GHz. In frequent embodiments a charged polymer translocates through the nanochannel at a translocation rate defined as the rate at which any particular monomer in the polymer passes a defined point in the nanochannel, and the piezoelectric actuator has a resonant frequency to translocation rate ratio of between 1 : 1 to 1 : 100,000. Occasionally the rate is above 1 : 100,000, e.g., up to about 1 : 1,000,000, or more. In other embodiments, the piezoelectric actuator has a resonant frequency to translocation rate ratio of between 100,000: 1 to 1 : 1. Often the resonant frequency of the piezoelectric actuator is 3.3MHz, 6.6, MHz, 9,9 MHz, 13.2 MHz, or 16.5 MHz. [00018] In certain frequent embodiments a device is provided, comprising: (a) a nanochannel defining a flow path having a proximal and a distal end opening and an examination location positioned between the proximal and distal end openings; and (b) a piezoelectric actuator, or a cantilevered extension thereof, positioned in the examination location, wherein the piezoelectric actuator or cantilevered extension thereof comprises at least a portion of the flow path, and wherein the piezioelectric actuator is operably connected to a piezoelectric actuator deflection measuring device.

[00019] The present description also provides methods of identifying the sequence of a charged polymer, comprising introducing the charged polymer to the piezoelectric device described herein, causing the translocation of the charged polymer between a pair of electrodes, passing an electric signal through each monomer of the charged polymer, and analyzing a physical deflection of the piezoelectric actuator to determine the identity of each monomer. In certain embodiments, the identity of each monomer is determined with reference to reference data generated utilizing one or more control polynucelotides.

[00020] Kits comprising one or more piezoelectric devices are also provided.

[00021] In frequent embodiments, a method is provided for identifying the sequence of a charged polymer, comprising introducing the charged polymer to the device of any preceding claim, causing the translocation of the charged polymer between a pair of electrodes, passing an electric signal through each monomer of the charged polymer while in the presence of a static magnetic field situated transverse to the orientation of the electric signal and the polymer, and analyzing resulting ultrasonic pulses unique to each monomer within the polymer to determine the identity of each monomer.

[00022] In frequent embodiments, a system for analyzing a polymer is provided, comprising: (a) a nanochannel defining a pathway for a charged polymer; (b) a static magnetic field source capable of producing a magnetic field; and (c) an electric signal pathway, wherein the pathway for the charged polymer, the magnetic field, and the electric signal pathway are situated at right angles to one-another in a three dimensional orientation at a discrete examination location. Often, the system further comprises a transducer, such as a piezoelectric transducer, for producing or detecting radio sound waves such as ultrasound waves. Frequently, the system further comprises one or more detector(s) capable of detecting ultrasonic pulses produced in the examination location, often comprising a piezoelectric transducer. Often an array of a plurality of piezoelectric transducers is provided in the system.

[00023] Often the nanochannel comprises a graphene nanoribbon, a nanopore, or carbon nanotube. When the nanochannel comprises a nanoribbon, the system often further comprises one or more detector(s) capable of detecting ultrasonic pulses produced by, or physical deflections caused within, the nanoribbon. In such embodiments, the magnetic field is often produced by a magnetic field source having a magnetic strength of about 0.7 Tesla or greater, or at least about 1 Tesla, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17 Tesla, or higher. Though the magnetic field is often referred to herein with regard to magnet strength, however, the effective magnetic field strength (e.g., T/m²) may also be utilized to describe the magnetic field and/or the magnets producing the magnetic field. For example, one exemplary magnetic field strength contemplated herein is about 400 T/m², though numerous other magnetic field strengths are contemplated corresponding to a particularly identified magnet strength.

[00024] General methods of identifying the sequence of a charged polymer comprising the use of Hall-effect imaging. In certain embodiments, methods of identifying the dielectric or charge density properties of a charged polymer are also provided, comprising introducing the charged polymer to the above system employing a statis magnetic field, presenting at least a portion of the charged polymer within discrete examination location and conducting Hall-effect imaging of the charged polymer to thereby identify the dielectric or charge density properties of the charged polymer. Frequently the identified dielectric or charge density properties of the charged polymer provide information identifying each monomer of the polymer present in the discrete examination location. Also frequently, the charged polymer comprises a nucleic acid molecule, a natural or synthetic oligonucleotide, a natural or synthetic polynucleotide, DNA, RNA, PNA, a protein, a peptide, a chimeric amino acid molecule, and/or a chimeric nucleic acid molecule.

[00025] Kits are also provided for use with a strong static magnetic field source to provide for Hall-effect imaging of a polynucleotide, comprising a nanochannel defining a pathway for a charged polymer and an electric signal pathway, wherein the pathway for the charged polymer intersects the electric signal pathway at a right angle, and wherein the strong magnetic field source, when in use, produces a magnetic field that intersects the intersection of the electric signal pathway and the pathway for the charged polymer at a right angle to the electric signal pathway and the pathway for the charged polymer. Such kits can be provided in cartridge form, or another form, for use in an instrument that employs one or more strong magnetic field sources and one or more means for monitoring ultrasonic signals. Often the instrument employs an array of means for monitoring ultrasonic signals.

[00026] These and other features, aspects, and advantages of the present invention will become apparent to those skilled in the art after considering the following detailed description, appended claims and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[00027] FIG. la and lb depict exemplary cantilevered orientations of representative piezoelectric actuators. FIG. lc depicts a basic exemplary electrical circuit diagram including a piezoelectric device.

[00028] FIGS. 2a-2d depict various arrangements of nanochannels of the present invention.

[00029] FIGS. 3a-3b depict various arrangements of nanochannels of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[00030] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

Definitions

[00031] Unless defined otherwise, all terms of art, notations and other scientific terms or terminology used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional

methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted. All patents, applications, published applications and other publications referred to herein are hereby incorporated by reference for at least the reasons for which they are cited. If a definition set forth in this application is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this application prevails over the definition that may be incorporated by reference.

[00032] As used herein, "a" or "an" means "at least one" or "one or more."

[00033] As used herein, the term "nanochannel" encompasses naturally-occurring, natural, and synthetic "nanopores," "nanotubes" (including carbon nanotubes), pores, holes, or apertures in surfaces (e.g., graphene layers or graphene nanoribbons) having nanometer dimensions, in addition to channels etched or otherwise formed in a substrate. Nanochannels of the present description are not limited to any specific geometry, aspect ratio, size, shape, cross-sectional profile, or other physical aspect, except that the geometry of a particular nanochannel having a length along the central line having the narrowest cross-section ranging from about 1 to about 1,000 nm. In one embodiment, the cross-sectional width or diameter of the nanopore is the same or greater than the diameter of a non-stretched single-stranded nucleic acid.

[00034] Nanochannels can have one open end or opposing open ends and are composed of one or more materials or comprised of an arrangement of layers with each layer comprising of one or more materials. Any particular layer can be as a single atom (e.g., a graphene nanoribon) of about 3.4A, have uniform or non-uniform thickness and have planar or non-planar geometry. The electrically conductive material can comprise the surfaces of the nanochannel (e.g., graphene material), or can be can be flush with respect to the local nanopore sidewall geometry (e.g., electrodes), can protrude toward the central axis of the pore, or can be undercut in the peripheral direction. Moreover, where electrodes are utilized, only a portion of the electrically conductive material in such embodiments may be exposed inside the nanopore. Multiple separate electrically conductive materials can be located on the same layer and individual portions exposed separately on the surface of the nanopore. As noted, in certain embodiments the electrically conductive material, for example a graphene layer, comprises a single layer material nanochannel, optionally including one or more supports or support layers. The graphene layer often in such circumstances comprises a metallic graphene nanoribbon, a metallic graphene nanoribbon with zigzag edges, chiral graphene nanoribbons, or two- dimensional topological insulators. In such circumstances, the electrically conductive material, which may be one or more atoms thick, often incorporates a hole or pore therethrough, thereby forming the nanochannel.

[00035] As used herein "electrically conductive material," refers to a conductor, which may be a carbon nanotube, graphene layer, nanowire, InSnO, noble metal, or noble metal alloy, used as an electrode, electrode layer, or comprising a circuit or portion thereof such as in a field effect transistor configuration. The electrode or electrode layer can, for example, be electrically connected to a voltage source and an applied potential can be used that causes the electrode to act as an anode. The change in current or voltage can then be detected and analyzed to identify a characteristic of a charged polymer, e.g., a monomer such as a nucleotide in a specific position on the polymer. A change in electrical signal, for example, a DC signal, an AC signal, or both, can be detected, which results from a variation of any of a variety of electrically transducatable properties, for example, resistance, capacitance, inductance, polarization moment, tunneling current, and the like. In certain embodiments, the electrically conductive material comprises a nano-field effect transistor device (nano-FET).

[00036] As used herein "examination location" refers to a "detection zone" such as a discrete area where a target is interrogated and examined. The terms "examination location" and "detection zone" are used interchangeably herein. Frequently, the examination location comprises a particular region in a nanochannel between tunneling electrodes. Also frequently, the examination location comprises the area (or a portion thereof) of a pore in a surface, such as a graphene layer or nanoribbon. In certain embodiments, the examination location may comprise or consist of a region that does not include a target polymer or pathway (e.g., a nanochannel, pore, hole, aperture, etc.) for a target polymer. For example, in certain Hall-effect based examination schemes, the examination location may comprise a portion of a graphene layer adjacent to or surrounding an aperture extending through the layer.

[00037] As used here the term "stepped portion" or "stepped portions" refers to a step feature for inducing a localized stretch in a translocating or static polymer. In the context of a nanochannel, a stepped portion often comprises a natural or synthetic bridge helix, a natural or synthetic trigger loop, a fullerene molecule, a carbon molecule, a physical feature within, dividing, or

distinguishable from though positioned within, the nanochannel, or other site. The term "localized" refers to a discreet area in close proximity with an examination zone. Most frequently, the term "localized" when used in conjunction with the term "stepped" refers to an area comprising the examination zone.

[00038] As used herein the term "operably connected," "operable connection," "operable connections," or "operable communication" refers to electric continuity between two or more elements. Frequently, these terms refer to electronic communication between two or more physical systems, for example between an electronic circuit and a processor. Often these terms refer to electronic communication between an electric system or circuit and a piezoelectric device. Often these terms refer to an electric system or circuit, a piezoelectric device, actuator, or module, and a device for evaluating the piezoelectric device. In the most frequent embodiments, operable connection is not established when particular elements of an electronic system are connected only by virtue of the fact that they are each connected to the same processor or processing element. For example, for purposes of the present disclosure, an element provided to adjust the positioning of a polymer within a nanochannel (e.g., piezoelectric device, an electric circuit, magnetic element, or other device or feature) is most often not provided in electronic communication with the electronic circuit provided to analyze the polymer. In certain embodiments, an element provided to adjust the positioning of a polymer within a nanochannel is provided in communication with the electronic circuit provided to analyze the polymer only by virtue of the fact that they are connected to the same processor; in the most frequent embodiments this communication not considered operable communication as used herein. Though examination and movement systems may be operable as a chorus within the system as a whole, operable connection refers to connection between particularly identified discreet elements within the overall system unless otherwise specified.

[00039] As used herein, a "charged polymer" refers to any natural or synthetic nucleic acid or nucleic acid analog, including DNA or RNA. The following exemplary molecules are also included, such as amino acids, proteins, saccharides, polysaccharides, PNA, synthetically produced nucleic acids, synthetically produced amino acids or proteins, synthetically produced saccharides or polysaccharides; other charged biomolecules comprised of combinations of nucleic acids, amino acids, or saccharides; or the same or other molecules used as or bound to detectable labels. Such charged polymers have distinctive dielectric and/or charge density properties attributable to the nature of the monomer units (e.g., a nucleotide) present in the polymer that are individually distinguishable or distinguishable as a group of two or more units using the present devices, systems, and methods.

Piezoelectricity

[00040] The active element of a piezoelectric sensor basically comprises a polarized material with electrodes attached to two of its opposite faces. When an electric field is applied across the material, the polarized molecules will align themselves with the electric field, resulting in induced dipoles within the molecular or crystal structure of the material. This alignment of molecules causes the material to change dimensions or deform. This phenomenon is known as electrostriction. A permanently -polarized material such as quartz (S1O2), barium titanate (BaTiOs), among a variety of other materials produces an electric field when the material changes dimensions as a result of an imposed mechanical force, and vice-versa. This phenomenon is known as the piezoelectric effect, which was discovered by Pierre Curie and Jacques Curie in 1880.

[00041] Piezoelectricity is the combined effect of the electrical behavior of the material:

D= sE

where D is the electric charge density displacement (electric displacement), ε is permittivity and E is electric field strength, and Hooke's Law:

S = sT

where S is strain, s is compliance and T is stress. These may be combined into so-called coupled equations, of which the strain-charge form is:

{S} = [s^E]{T} + [d^t]{E}

{D} = [d]{T} + [ ¹]{E}

where [d] is the matrix for the direct piezoelectric effect and [d'] is the matrix for the converse piezoelectric effect. The superscript E indicates a zero, or constant, electric field; the superscript T indicates a zero, or constant, stress field; and the superscript t stands for transposition of a matrix.

[00042] The strain-charge for a material of the 4mm (C_4v) crystal class (such as a poled piezoelectric ceramic such as tetragonal PZT or BaTiOs) as well as the 6mm crystal class may also be written as (ANSI IEEE 176):

where the first equation represents the relationship for the converse piezoelectric effect and the latter for the direct piezoelectric effect. See, e.g., Damjanovic, Reports on Progress in Physics 61 : 1267-1324 (1998). D and E are vectors, i.e., Cartesian tensor of rank- 1 ; and permittivity ε is Cartesian tensor of rank 2. Strain and stress are, in principle, also rank-2 tensors. But conventionally, because strain and stress are symmetric tensors, the subscript of strain and stress can be re-labeled in the following fashion: 11→ 1; 22→ 2; 33→ 3; 23→ 4; 13→ 5; 12→ 6 (alternative reference conventions are also occasionally used in the art, e.g., where 12→ 4; 23

→5; 31→6).

[00043] In total, there are 4 piezoelectric coefficients, < ¾/, gy, and hy defined as follows:

where the first set of 4 terms correspond to the direct piezoelectric effect and the second set of 4 terms correspond to the converse piezoelectric effect. See, e.g., Kochervinskii, Crystallography Reports 48 ( coefficients dy for crystals having a crystal-

field induced type of polarization can be calculated from electrostatic lattice constants or higher- order Madelung constants. Birkholz, Z. Phys. B 96: 333-340 (1995).

Piezoelectric materials

[00044] Twenty known crystal classes exhibit direct piezoelectricity (i.e., 1, 2, m, 222, mm2, 4, 4, 422, 4 mm, 42m, 3, 32, 3m, 6, 6, 622, 6 mm, 62m, 23, 43m). Ten of these represent the polar crystal classes, which exhibit spontaneous polarization without mechanical stress due to a non- vanishing electric dipole moment associated with their unit cell, and which exhibit

pyroelectricity. If the dipole moment is reversed by the application of an electric field, the material is said to be ferroelectric.

[00045] Naturally-occurring piezoelectric crystals include Berlinite (A1P0₄), sucrose (table sugar), quartz, Rochelle salt, topaz, and tourmaline-group minerals.

[00046] Synthetic crystals exhibiting piezioelectricity effects include Gallium orthophosphate (GaP0₄) and Langasite (La₃Ga₅SiOi₄). Certain synthetic ceramics are well known piezioelectric materials. These ceramics include, for example, lead titanate (PbTi0₃), ceramics including perovskite or tungsten-bronze structures, barium titanate (BaTi0₃), lead zirconate titanate (Pb[Zr_xTii-J0₃ 0< <l) (PZT), potassium niobate (KNb0₃), lithium niobate (LiNb0₃), lithium tantalite (LiTa0₃), sodium tungstate (Na₂W0₃), Ba₂ a b₅0₅, Pb₂K bsOi₅, sodium potassium niobate ( aKNb), bismuth ferrite (BiFeOs), Sodium niobate (NaNbC ), and lead magnesium niobate-lead titanate (PMN-PT).. Ceramic materials, in general, have a piezoelectric constant/sensitivity that is roughly two times larger than that of natural single crystal materials and can be inexpensively produced. Moreover, polymers such as Polyvinylidene fluoride (PVDF) exhibit piezoelectricity several times greater than quartz.

[00047] The high modulus of elasticity of many piezoelectric materials is comparable to that of many metals and goes up to 10⁶ N/m². Even though piezoelectric sensors are electromechanical systems that react to compression, the sensing elements show almost zero deflection. As such, piezoelectric sensors are rugged, have an extremely high natural frequency and exhibit excellent linearity over a wide amplitude range. Moreover, piezoelectric materials and their effects are insensitive to electromagnetic fields and radiation, and are generally stable at a variety of temperatures, which enables measurements under difficult and complex conditions.

[00048] Depending on how a piezoelectric material is cut, three main modes of operation can be distinguished: transverse, longitudinal, and shear. In the transverse effect a force is applied along a neutral axis (y) and the charges are generated along the (x) direction, perpendicular to the line of force. The amount of charge depends on the geometrical dimensions of the respective piezoelectric actuator. When dimensions a,b,c apply:

C_x = d_xyF_yb I a, where a is the dimension in line with the neutral axis, b is in line with the charge generating axis and d is the corresponding piezoelectric coefficient. See e.g., Gautschi, PIEZOELECTRIC

SENSORICS: FORCE, STRAIN, PRESSURE, ACCELERATION AND ACOUSTIC EMISSION SENSORS, MATERIALS AND AMPLIFIERS (Springer 2002).

[00049] In the longitudinal effect the amount of charge produced is proportional to the applied force, but is independent of size and shape of the piezoelectric actuator. Using several actuators that are mechanically in series and electrically in parallel is generally the way to increase the charge output. The resulting charge is:

C_x d_xxF_xn,

where άχχ is the piezoelectric coefficient for a charge in x-direction released by forces applied along x-direction (in pC/N). F_x is the applied Force in x-direction [N] and n corresponds to the number of stacked actuators.

Piezoelectric Actuators

[00050] Provided that a controller can deliver the requisite impulse (e.g., current and slew rate), a piezoelectric actuator (100, 101) can reach its nominal displacement in approximately 1/3 of the period of its resonant frequency. This phenomenon, which describes the minimum rise time of a piezoelectric actuator, is explained by the following equation:

Where:

T_min = time [s]

fo = resonant frequency [Hz]

[00051] For example, a piezoelectric transducer having a 10 kHz resonant frequency reaches its nominal displacement within 30 μ8. Similarly, a piezoelectric transducer having a 33,333 Hz resonant frequency will reach its nominal displacement within 10 μ8, and a piezoelectric transducer having a 3.3 MHz resonant frequency will reach its nominal displacement within 10 ns. Piezoelectric sensors having resonant frequencies in these ranges, and higher, are known in the art. See, e.g., Giurgiutiu & Lyshevski, "Piezoelectric Wafer Active Sensors," in

MlCROMECHATRONICS: MODELING, ANALYSIS, AND DESIGN WITH MATLAB® Ch. 1 1 (CRC

Press 2003). In fact, broadband transducers with frequencies up to 150 MHz are commercially available.

[00052] A transducer's frequency is a representation of its central frequency, it will respond to frequencies above and below the central frequency. In general, the broader the frequency range, the larger the resolving power of the transducer. Lower frequency (e.g., 0.5MHz-2.25MHz) piezo actuators associated with a transducer provide greater energy and penetration in a material, while high frequency piezo actuators (e.g., 15.0MHz-25.0MHz) provide reduced penetration but greater sensitivity to minor discontinuities. It is understood in the art that high frequency transducers can provide excellent flaw resolution and measurement capabilities.

[00053] In a frequent embodiment a piezoelectric material is chosen or designed that has a resonant frequency that corresponds to the translocation rate of a charged polymer. For example, for a charged polymer containing multiple charged monomers/units, a piezoelectric material is chosen that has a resonant frequency that corresponds to the rate at which each monomer/unit passes the piezoelectric material, or a portion thereof, within a nanochannel. This resonant frequency vs. translocation correlation is often a 1 : 1, 1 :2, 1 :3, 1 :4., 1 :5, 2: 1, 3 : 1, 4:, 1, or 5: 1 relationship, but can be a different correlation relationship that is larger or smaller, depending on the resolution desired, type of polymer being analyzed, and/or analysis goal. For example, the relationship can be about 1 : 10, 1 : 100, 1 : 1000, 1 : 10,000, 1 : 100,000, 1 : 1,000,000, 10: 1, 100: 1, 1000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1, or another ratio in between, above or below. Though not bound by any particular theory, the larger the resonant frequency is versus the translocation rate, the higher the sampling rate and sensitivity of the system. As used herein "piezoelectric actuator" refers to the cantilevered piezoelectric element (e.g., PZT with electrodes) in addition to any requisite components necessary for drive signals (amplified, filtered, or otherwise treated) to be provided to the element to attain certain resonant frequencies, such as a piezoelectric transducer and/or controller. [00054] Thus, for a nanochannel system that provides a translocation rate of 1 base per 10ns for a polynucleotide, a piezoelectric material having a 3.3 MHz resonant frequency may be chosen for the analysis to provide a 1 : 1 relationship. Alternatively, the piezoelectric material can be provided that has a 6.6 MHz, 9.9 MHz, 13.2 MHz, or 16.5 MHz resonant frequency (to provide, for example, 1 :2, 1 :3, 1 :4, or 1 :5 relationships), or other resonant frequencies, for analysis of charged polymers translocating a particular point in a nanochannel at about 1 base per 10ns. Similar calculations can be made in connection with other translocation rates, e.g., 1 base per 10

[00055] Notch filters and/or rNPUTSHAPING® (Convolve, Inc., Armonk, NY) is frequently utilized to compensate for rapid expansion of the piezoelectric translator to avoid overshooting the target frequency range. rNPUTSHAPING® provides a real-time, feedforward technology that nullifies resonances both inside and outside the servo-loop and virtually eliminates the settling phase (see, e.g., U.S. Pat. Nos. 7,483,232, 6,314,473). Though not wishing to be bound by any particular theory, the INPUTSHAPING® procedure requires determination of critical resonant frequencies in the system. A non-contact instrument like a polytec laser doppler vibrometer is often utilized for such measurements. The values, most importantly the resonant frequency of the sample, are then fed into the INPUTSHAPING® signal processor. Signal processing algorithms remove undesired resonances in the system and avoid excitation of auxiliary components. Because the processor is outside the servo-loop, it works in open-loop operation as well. This results in the fastest possible motion, with settling within a time equal one period of the lowest resonant frequency.

[00056] Signal preshaping is also often utilized to reduce rolloff, phase error and hysteresis in applications with repetitive inputs. The result is to improve the effective bandwidth, especially for tracking applications such as out-of-round turning of precision mechanical or optical parts. Signal preshaping is implemented in object code, based on an analytical approach in which the complex transfer function of the system is calculated. Signal preshaping is, in general, more effective than simple phase-shifting approaches and can improve the effective bandwidth by a factor of 10 in multi-frequency applications.

[00057] Frequency response and harmonics (caused by nonlinearity of the piezo-effect) are determined in two steps using Fast Fourier Transformation (FFT), and the results are often used to calculate the new control profile for the trajectory. The new control signal compensates for any existing or developed system non-linearities. For example, it is possible to increase the command rate from 20 Hz to 200 Hz for a piezo system with a resonant frequency of 400 Hz without compromising stability. At the same time, the tracking error is reduced by a factor of about 50.

[00058] Dynamic Digital Linearization (DDL) is similar in performance to Input Preshaping, but is often easier to use. This technique, for example, can optimize multi-axis motion such as a raster scan or tracing an ellipse and generally does not require external metrology or signal processing. Commercially available digital controllers often integrate this technique to their operating systems (e.g., Physik Instrumente, model E-710 and E-711 digital controllers). DDL uses position information from capacitive sensors integrated in the piezo mechanics to calculate an optimum control signal. Similar to signal preshaping, improved linearity and tracking accuracy of up to 3 orders of magnitude is achieved. To reduce tracking error this improved accuracy can be applied in a feed-forward manner.

Nanochannel Fabrication

[00059] Nanochannels can be formed in a substrate by, e.g., lithographic and etching steps. The substrate may be, e.g., a silicon-on-insulator wafer, with, for example, a Si surface, a Si wafer, or a fused silica substrate. Lithography in the sub- 100 nanometer regime may be performed by various techniques, for example, including: electron beam lithography (EBL), nanoimprint lithography (NIL), or deep ultraviolet optical lithography. See, e.g., Liang et al, Nano Lett. 7:3774-3780 (2007); Austin et al, App. Phys. Lett. 84:5299-5301 (2004); and Guo, J. Phys. D: Appl. Phys. 37:R123-R141 (2004). Optical lithography has been successfully used in the mass production of devices with critical dimensions as small as 32 nm. EBL and NIL are used extensively due to their versatility and capability of reliably producing sub- 10 nm features. Any of these methods may be used to pattern the nanochannels described herein.

[00060] The removal of material for the formation of nanochannels can be performed by, e.g., etching. Wet etching generally includes the immersion of material in a solution capable of selective removal. Dry etching, i.e., reactive ion etching (RIE), involves the exposure of a surface to charged plasma. For the resolution and control required of nanoscale fabrication, RIE is often preferred due to its consistency, controllability, and efficiency. Microfluidic channels or reservoirs leading to nanochannels can be etched using wet or dry methods.

[00061] The length of the nanochannel can be chosen with regard to the length of the charged polymer. For example, a randomly coiled polymer (e.g., DNA) will generally elongate when introduced into a confined space such as a nanochannel, such that when the confinement space becomes smaller the extent of elongation becomes greater.

[00062] Sensing electrodes can be fabricated before, during, or after nanochannel formation. Similar to etching and lithography, numerous metal deposition techniques are known and suitable for fabrication of sensing electrodes according to the present invention. Exemplary techniques include electron beam evaporation, thermal evaporation, chemical deposition, and sputtering. These and other techniques are detailed, for example, in U.S. Pat. App. Pub. No. 20110168562. The sensing electrodes frequently have thicknesses ranging from 1 nm to 50 nm at the point where the sensing electrodes intersect a nanochannel. The sensing electrodes may be wider and/or thicker in regions distal to the fluidic channels and approaching contact pads disposed at the perimeter of the device.

[00063] To complete the device, a cover may be introduced to prevent evaporation of liquid from the nanochannel. The cover may be formed over the nanochannel pathways or over the entirety of the fluidic channels, e.g., on a wafer, if multiple channels are present on a substrate. The cover often contains holes or ports to permit introduction of fluid and/or sample to the nanochannels. The cover may be made of a glass plate such as borosilicate glass,

phosphosilicate glass, quartz, fused silica, fused quartz, a silicon wafer or other suitable substrates. Various techniques are suitable for accomplishing this step including anodic bonding. In anodic bonding, an underlying silicon wafer and a glass substrate are pressed together and heated while a large electric field is applied across the interface. Direct silicon bonding has also been used to join two silicon wafers. Other bonding methods use, for example, an adhesive layer to bond the cap to the substrate.

[00064] One exemplary fabrication process for defining the sensing electrode involves utilizing a conventional (100) p-type silicon wafer, which is thermally oxidized in a hydrated atmosphere to grow a thick (e.g., >1 μιη) silicon-dioxide (S1O2) layer. This S1O2 layer often serves as insulation between subsequently formed adjacent metal sensing electrodes, and also often reduces overall device capacitance.

[00065] Using high resolution optical lithography, the pattern of the nanochannel is introduced to a first photoresist masking layer. RTE with an anisotropic etch species, such as CI2, is often used to transfer the pattern into the S1O2 layer. After completing the dry etch procedure, residual resist is removed and the substrate is cleaned. The preferred width and depth of the channel is determined by the requirements for the device sensitivity. Increased sensitivity is, in general, obtained by decreasing the volume of the channel between sensing electrodes. Channel size, width, and depth, is often dictated by the size or properties of the charged polymer analyte. It is occasionally desired to fabricate the nanochannel with dimensions that extend the DNA strand within the channel. For example, for double-stranded DNA (dsDNA), it has been found that the use of channels with dimensions of 100 nm or less are able to extend the biopolymer. See, e.g., Tegenfeldt et al, Proc. Nat'l. Acad. Sci. USA 101 : 10979-10983 (2004).

[00066] In certain embodiments, after etching of a nanochannel, embedded sensing electrodes are fabricated. High resolution optical lithography is often used to transfer an electrode pattern to a second photoresist masking layer. RIE with an anisotropic etch species, such as CI2, is often used to transfer the pattern into the S1O2 layer. Upon completion of pattern transfer to the S1O2 layer, a thin metal adhesion promotion layer is often deposited. Tantalum, for example, provides a suitable layer having a thickness of about 30-50 A, deposited via electron beam evaporation. Next, the sensing electrode material is deposited, generally without exposing the substrate to atmosphere. Platinum, gold, chrome, titanium, silver chloride, silver, and graphene are some exemplary metals for the sensing electrodes, which are often deposited via electron beam evaporation. The thickness of the metal is frequently dictated by the depth of the etched trenches, such that the resulting metal trace is approximately planar with a top surface of the S1O2 layer. Upon completion of the metal deposition, the substrate is immersed in a photoresist solvent that removes excess metal from the surface and the substrate is cleaned. Chemical- mechanical polishing (CMP) is often used to remove excess metal on the top surface and creating a planar top surface.

[00067] To complete the fabrication of the sensor, a cover is frequently incorporated to provide a leak-free seal, enabling fluidic conduction. Frequent cap materials include borosilicate glass, fused silica, fused quartz, quartz, or phosphosilicate glass. Holes are occasionally incorporated in the cap layer to provide access to, for example, if present, a fluidic inlet, a fluid outlet and/or the sensing electrode(s). Ultrasonic etching is frequently used to introduce holes or ports in the cover material. Anodic bonding, for example, is then frequently used to bond the cover layer to the underlying substrate, e.g., silicon wafer. Anodic bonding of these layers, in general, provides a durable and leak-free seal.

[00068] When the nanochannel takes the form of a nanopore any of a variety of known fabrication methods may be employed, e.g., methods, techniques, and/or materials described in, for example, U.S. Pat. Nos. 6,696,022, 6,413,792, 6,200,893, 7,625,840, 8,206,568; U.S. Pat. App. Pub. Nos. 2011 155574, 201 10226623, and 20080311375; Andreozzi et al, Nanotechnology, 201 1, 22(33):335303; Yang et al, Nanotechnology 22(28):285310 (201 1); Ayub et al., J. Phys.

Condens. Matter., 2010, 22(45):454128; and van den Hout et al, Nanotechnology, 2010, 21(1 1): 115304. As one example, nanopores with diameters of between two and three nm can be fabricated by using materials such as S1₃N4 or S1O2. See, e.g., U.S. Pat. Nos. 6,627,067 and 7,238,485. Such nanopores can comprise silicon nitride membranes produced by depositing an LPCVD S1₃N4 film, ranging from 30 nm to 200 nm thick, on the top of a 300 μιη thick Si handle wafer, and having a polyimide photoresist. Nanometer-size pores can be created in membranes using a tightly focused (e.g., 1.6 nm spot-size) cone angle (e.g., 9°a), high energy (e.g., 200 kV) electron beam emanating from a transmission electron microscope (TEM) (e.g., JEM-2010F, JEOL-2200F, etc., JEOL Ltd., Tokyo, Japan) operating in convergent beam diffraction mode. Pores larger or smaller can be consistently obtained, as desired based on the ultimate end application. By stringently controlling the beam conditions and membrane thickness (e.g., by electron energy loss spectroscopy) pores with virtually identical geometry can be consistently produced with sub-nanometer precision.

[00069] Electrodes can be fabricated on or within the nanochannel using micromachining techniques or etched after metal deposition and e-beam lithography. These and other techniques are detailed, for example, in U.S. Pat. App. Pub. No. 20110168562. Multiple sets of electrodes can be included to permit detection at different points of the nanochannel, thus providing averaged data due to multiple reads of a single polynucelotide. Some examples of electrodes include those described, for example, in U.S. Pat. Nos. 7,619,290, 7,595,260, 7,500,213, 7,385,267, and 7,301, 199.

[00070] In addition, a nanopore can be formed in a substrate that comprises a plurality of spaced apart electrode layers (e.g., by way of an insulating layer, a dielectric material, semiconducting material, or other materials or combinations thereof) each comprising a noble metal or an alloy thereof. Each electrode can, in turn, independently comprise a metal oxide, for example, indium-tin oxide (ITO), AI2O₃, Ta₂0₅, ^Os, Zr0₂, Ti0₂, or combinations or alloys thereof. Graphene can be used, for example, for on-chip integration of molecular sensing and signal processing electronics. See, e.g., Lin et al, Nano Lett, 2009, 9(1):422^126; Hollander et al, Nano Lett, 201 1, l l(9):3601-3607.

[00071] Nanotubes, such as carbon nanotubes may also form nanochannels of the present invention. The present piezoelectric devices and methods can be readily adapted to the use of nanotubes for confining, translocating, and examining charged polymers, due at least to certain electrical advantages these materials provide. Methods of nanotube generation and use to translocate charged polymers are described, for example, in U.S. Pat. App. Pub. No.

20110168562.

[00072] Certain contemplated embodiments utilize nanochannels adapted to contain recognition elements that bind nucleotides in a single stranded polynucleotide and complete to complete a circuit, for example, as described in U.S. Pat. App. Pub. No. 20110168562. In such embodiments it is advantageous to alter the path of the translocating polynucleotide at the point of the recognition element utilizing an active site molecule, for example, a synthesized polypeptide having the amino acid sequence of a polymerase bridge helix, a synthesized polypeptide having the amino acid sequence of a polymerase trigger loop, a fullerene molecule, a carbon nanotube, or a combination thereof. Here the translocating polynucleotide is guided over the active site molecule, which manipulates and bends the polynucleotide in a manner similar to the active site in a polymerase enzyme. See, e.g., Kornberg, Proc. Nat'l Acad. Sci. USA, 2007,

104(32): 12955-12961 review article and cited references. In order to obtain the requisite polynucleotide manipulation it is advantageous to incorporate the use of an electrostatic element having a positive charge immediately after the recognition element, on the opposite side of the translocation path from the recognition element. The electrostatic element is activated at a very low level, below that of the electrophoretic force pulling the polynucleotide through the nanochannel, but enough to attract the phosphate backbone to the element in a manner sufficient to cause the requisite physical manipulation of the polynucleotide without stopping or significantly slowing its electrophoretic progress through the reading circuit. The

polynucleotide will exit a portion of the nanochannel, pass over the active site molecule, and become subject to electrostatic attraction forces at the point of the recognition element. This physical manipulation has the advantage of flexing the polynucleotide chain in a manner that permits a higher fidelity interaction with the recognition element and a consequently stronger electronic signal.

[00073] Nanochannels of the present invention are often formed in graphene nanoribbons, including graphene nanoribbons with zigzag edges, graphene nanoribbons with armchair edges, chiral graphene nanoribbons, or two-dimensional topological insulators. Graphene is essentially a two-dimensional allotrope of carbon having tightly packed carbon atoms into a honeycomb lattice formation. Though nanochannels of the present invention may be formed in multiple graphene layers having a thickness of more than a single atom, nanoribbons, which have a thickness of a single atom are often preferred. Similar to pores in multilayer graphene, pores, holes or apertures (all referring to the same general idea) having nanometer dimensions (e.g., greater than about 1 nm in diameter) can be employed in graphene nanoribbons, which provides certain advantages in the present invention. For example, due to the single-atom thickness, at any point during translocation of a biopolymer such as a polynucleotide through a pore in a nanoribbon, only a single monomer within the polymer is located within the pore between the surrounding edges of the nanoribbon (i.e., the detection zone). This permits isolation of conductance differences between monomers in the polymer down to a single signal or range of signals for each monomer. The electrical field differences due to the presence of each individual charged monomer can be monitored via means described herein (e.g., exemplary piezoelectric devices described herein). In such embodiments, similar to other schemes described herein, it is advantageous to employ an electrical circuit (e.g., a field effect transistor configurations optionally employing nanowires) that includes the pore/hole/aperture within the circuit. In practice, current passes through the circuit and across the pore, and fluctuations in the current or electric field due to the presence of each individual charged monomer (e.g., nucleotide) are monitored. In addition, such nanoribbons can be employed with multiple pores, e.g., an array of pores, permitting the concurrent interrogation of multiple polymers. In such embodiments, it is often advantageous to employ a single circuit, as noted above, for each of the multiple pores in the nanoribbon to provide for efficient signal monitoring for each polymer as it passes through each pore. In embodiments employing graphene layers or graphene nanoribbons, translocation of the test polymer through the pore in the graphene can be via electrophoretic force, magnetic force, electrostatic positioning, combinations thereof, or other means known in the art.

[00074] Graphene, and graphene nanoribbons often render the need for tunneling electrodes (e.g., comprised of a carbon nanotube, a graphene layer, InSnO, a noble metal, a noble metal alloy, etc.) unnecessary. Though not wishing to be bound by any particular conductance theory in such embodiments, carbon atoms present at the pore edges readily interact with available bond partners present in a monomer present within the pore enhancing conductance and the ability to monitor changes in conductance due to the presence of each unique monomer. In the case of a polynucleotide, each nucleotide present in the pore of the presently described graphene nanoribbons affects the charge density around the pore in a manner that is unique to each nucleotide, causing a change in the edge conduction currents that is monitored according to the present methods. In frequent embodiments, Green functions are utilized to decode the conduction current changes. Often charge density functional theory is coupled with a Green function to decode signals. In particularly preferred embodiments, the presently described nanoribbons are employed in the signal monitoring schemes described herein such as via the use of piezoelectric -based or Hall-effect-based monitoring schemes. Piezo Motion Measurement

[00075] As described, the piezoelectric actuator deflects (107) in response to the unique electrical signals coming from the charged polymer as it translocates past the electrodes and the circuitry connecting piezoelectric actuator and its associates electronics. Due to the piezoelectric effect, the amplitude and/or frequency of this deflection corresponds to the unique electric signal for each individual monomer (i.e., nucleotide base) on the charged polymer. Piezoelectric bender actuators, for example, such as serial and parallel bimorphs can be utilized to provide unique deflection trajectories. Use of bender piezo actuators such as electrostrictive, bimorphs, multimorphs, and shear actuators are contemplated to provide unique measureable deflections, with or without the use of lever motion amplifiers. Generally, the piezoelectric actuator exhibits a unique deflection and an immediate return to a pre-deflection physical state. In certain embodiments, the piezoelectric actuator exhibits a unique deflection and in its subsequent return to a pre-deflection state, it overshoots an initial physical state. The presently described techniques account and adapt for this potential overshoot issue, for example, through the use of techniques such as rNPUTSHAPI G®, signal preshaping, FFT, DDL and other similar techniques.

[00076] Frequent embodiments of the present invention involve measurement of this piezoelectric actuator deflection response. Frequently, this deflection comprises bending of the piezoelectric device, which when viewed over time and over multiple incoming unique electronic signals can be viewed as a vibratory deflection of the device. In certain embodiments, the deflection comprises lengthening or shortening of a certain pre-determined aspect of the piezoelectric device in response to the incoming unique electronic signals. In certain embodiments involving measurement piezoelectric actuator deflection response, deflection measurement is accomplished through the use of any one of a variety of techniques, utilizing specialized apparatuses. For example, capacitive position sensors, strain gauges, vibrometers, accelerometers, interferometers, spectrum analyzers, and/or tunnel gap modulation spectroscopy devices comprise certain contemplated measurement tools.

[00077] The deflecting peizo actuator is frequently in the nanometer or larger scale, which permits a variety of detection techniques having a variety of resolution levels. Nevertheless, contemplated measurement techniques are capable of nanometer and sub-nanometer resolution levels. In certain embodiments a capacitive position sensor is utilized to measure piezo actuator deflection (e.g., model D-015, Physik Instrumente; D509 or D-510 PISeca™ non-contact capacitive sensors, Physik Instrumente), together with suitable sensor electronics (e.g., Model No. E-852.10, Physik Instrumente).

[00078] Another exemplary apparatus comprises a vibrometer controller equipped with a displacement encoder having nanometer or sub-nanometer resolution (e.g., model OFV-5000 HF or UHF-120 vibrometer controller and DD-300 or DD500 displacement decoder (0.015 nm resolution), Polytec PI, Inc., Tustin, CA). The UHF-120 system, in particular, consists of a heterodyne interferometer having a controller. In this instrument a heterodyne detector signal is provided by the optical head, which is acquired with a digital oscilloscope, permitting out-of- plane vibration frequencies up to 1.2 GHz. The digitized detector signal is transferred to a PC where the heterodyne carrier is demodulated by a new software module in a VibSoft (Polytec PI, Inc., Tustin, CA) software package. In addition, laser doppler vibrometry has been known to provide detection zone of around 60nm. See, e.g., Biedermann, "Vibrational Spectra of

Nanowires Measured Using Laser Doppler Vibrometry and STM Studies of Epitaxial Graphene: An LDRD Fellowship Report," Sandia Report p. 53 (Sandia National Laboratories, 2009). Another exemplary setup comprises an interferometer (e.g., model A- 150, PbMo04, 80-MHz, Schott North America, Inc., Elmsford, NY), digital oscilloscope (e.g., model TDS 540D, 500 MHz, Tektronix, Beaverton, OR), and an rf spectrum analyzer (e.g., model 3026, 3 GHz, Tektronix, Beaverton, OR). With regard to tunnel gap modulation spectroscopy (TGMS), this is a scanning tunneling microscopy (STM)-derived technique that utilizes exponential dependence of tunnel current to gap distance to measure the frequency of vibrations at the nanoscale. Moreover, TGMS can span the MHz to GHz range. See Biedermann (cited above) at p. 137.

Polymer analysis

[00079] Frequency response and harmonics (caused by nonlinearity of the piezo-effect) are determined in two steps using Fast Fourier Transformation (FFT), and the results are often used to calculate the new control profile for the trajectory. The new control signal compensates for any existing or developed system non-linearities. For example, it is possible to increase the command rate from 20 Hz to 200 Hz for a piezo system with a resonant frequency of 400 Hz without compromising stability. At the same time, the tracking error is reduced by a factor of about 50.

[00080] It is well established that DNA in solution carries negative electrical charges on phosphate groups on the backbone of each strand of the molecule. As such, single stranded DNA similarly carries negative electrical charges on its phosphate backbone. As described herein, nanochannels can be used to exploit the negatively charged DNA polynucleotide backbone to pull the DNA through the pore in a linear fashion using an (induced or natural) ionic current. Nanochannels are also useful, for example, because they provide a discrete area having a designed electrical signature in which to observe a passing polynucleotide.

[00081] There are four types of nucleotide bases in DNA (A, C, T, G) and RNA (A, C, U, G), each has a unique electrical signal while incorporated in a polynucleotide chain. See, e.g., Sigalov et al, Neno Lett., 2008, 8:56-63; Gierhart et al., Sens Actuators B. Chem., 2008, 132:593-600; Zwolak et al, Nano Lett, 2005, 5:421-424; U.S. Pat. App. Pub. No. 201 10168562. Examining this electrical signature using tunneling current between atomic-scale electrodes and tying the signature to a particular location on the polynucleotide as it passes through the nanochannel provides a means for analyzing the polynucleotide sequence. As such, the entire sequence of even very large DNA sequences can be identified quickly, in a single run without amplification, through the nanochannel. [00082] In direct contrast to current attempts at nanopore-based DNA sequencing, the present devices and methods provide the capability of reliably and accurately sequencing a

polynucleotide without resorting to trapping, electrostatic positioning, enzyme-based ratcheting, nanochannel inner wall surface treatment or modification, chemical-recognition moieties, or other means to slow the passage of the polynucleotide through the nanopore or nanochannel. Though these and similar means can be readily employed in the present invention, they are not necessary. Rather, the present devices and methods are capable of continuously resolving single bases at translocation rates of 1 base/10 μ8, 1 base/10ns, or faster, without the use of

complicated optics, enzymes, reagents, or chemistries. The presently described devices and methods, therefore, are capable of sequencing an entire human genome of about 3 billion base pairs faster than currently available or known developing technologies. With the enhanced resolution rates provided by the present invention, it is contemplated that the present methods can highly accurately sequence a human genome in a matter of hours, minutes, or less.

[00083] Cantilevered orientations of representative piezoelectric actuators (100, 101) are depicted in FIG. 1. In FIG. la a single layer of piezoelectric material (127) (e.g., PZT) is sandwiched between two electrodes (102, 103), anchored in a cantilevered orientation by a solid support 108, and connected to an electrical circuit (109, 110). FIG. lb depicts a multiple layer piezoelectric actuator containing two levels of piezoelectric material (127) sandwiched between electrodes (104, 105, 106), anchored in a cantilevered orientation by a solid support 108, and connected to an electrical circuit 109, 1 10). These are merely representative arrangements of piezoelectric materials, electrodes, and electronics; any of a variety of orientations and materials are contemplated for use in the piezoelectric actuators of the description contemplated herein.

[00084] An electrical circuit diagram is depicted in FIG. lc, including sensing electrodes (111, 112) separated by a detection zone (113), connected to a piezoelectric device and associated detection instrumentation (114). The piezoelectric device (114) is depicted simply in the diagram but is intended to include any and all of a variety of components necessary to translate the voltage differential noted between electrodes (111, 112) into physical deflection and the associated monitoring of this deflection. For example, inverters, amplifiers, controllers, and sensors, among other equipment necessary to resolve and interpret discreet individual electronic events using the piezoelectric effect, are included in the piezoelectric device (114).

[00085] FIGS. 2a-2d depict various arrangements of nanochannels of the present invention.

Although the electrodes (111, 112) are predicted as discrete elements, they can form entire layers of a multilayer substrate, they can be deposited through techniques described elsewhere herein, or any other orientation permitting tunneling currents. Moreover, although electrodes (111, 112) are depicted as being flush with the nanochannel surface, they may be inset or extend out from one or more inner nanochannel surfaces. Additionally, electrodes (111, 112) may be covered by a protective material to protect the sensing surfaces. Two or more sets of electrodes (111, 112) can be situated within any particular nanochannel to provide multiple detection zones (113). [00086] FIG. 2a depicts a nanopore (118) within substrate (115), containing sensing electrodes (111, 112) separated by a detection zone (113) defined by the width of the nanopore (118). The electrodes are in electric communication with a piezoelectric device (114). The nanopore (118) connects fluid reservoirs (116, 117). Proximal fluid reservoir (116) incorporates a negative electrophoretic electrode (not depicted) and distal fluid reservoir (117) incorporates positive electrophoretic electrode (not depicted) to create a electrophoretic potential to move charged polymers through the nanopore (118).

[00087] FIG. 2b depicts a nanopore (119) within substrate (115), containing sensing electrodes (111, 112) separated by a detection zone (113) defined by the width of the narrowest portion of the nanopore (119). The electrodes are in electric communication with a piezoelectric device (114). The nanopore (119) connects fluid reservoirs (116, 117). Proximal fluid reservoir (116) incorporates a negative electrophoretic electrode (not depicted) and distal fluid reservoir (117) incorporates positive electrophoretic electrode (not depicted) to create an electrophoretic potential to move charged polymers through the nanopore (119).

[00088] FIG. 2c depicts a nanopore (120) within substrates (115, 122), containing sensing electrodes (111, 112) separated by a detection zone (113) defined by the width of the narrowest portion of the nanopore (119). The electrodes are in electric communication with a piezoelectric device (114). The nanopore (119) connects fluid reservoirs (116, 117). Proximal fluid reservoir (116) incorporates a negative electrophoretic electrode (not depicted) and distal fluid reservoir (117) incorporates positive electrophoretic electrode (not depicted) to create a electrophoretic potential to move charged polymers through the nanopore (119). Nanopore (120) includes a step feature (126) designed to induce a localized stretch in polymers translocating through the nanopore (126), which will be required to snake through the step feature. The localized stretch occurs within detection zone (113), which may result in an increased inspection time for monomers occurring on the polymer. Though only a single step feature is depicted here, two or more step features, having the same or different orientations, can be incorporated in any particular nanochannel of the present description. To ease fabrication, electrodes (111, 112) are depicted as a discrete deposited layer in FIG. 2c, situated between two substrates (115, 122). Similar arrangements can be provided to incorporate multiple electrode sets in any particular nanochannel.

[00089] FIG. 2c depicts a nanotube defining channel (121), containing sensing electrodes (111, 112) separated by a detection zone (113). The electrodes are in electric communication with a piezoelectric device (114). The nanotube connects fluid reservoirs (116, 117). Proximal fluid reservoir (116) incorporates a negative electrophoretic electrode (not depicted) and distal fluid reservoir (117) incorporates positive electrophoretic electrode (not depicted) to create a

electrophoretic potential to move charged polymers through the nanotube. Though depicted as extending through outer (125) and inner (124) walls of the nanotube, electrodes (111, 112) can be situated within walls (124, 125), between walls (124, 125), or on an inner wall (124) of the nanotube. In particular embodiments, slits are cut in the nanotube to permit positioning/deposition of the electrodes.

[00090] FIG. 3 a is similar to FIG. 2a, but also includes a magnetic device (202) and detection electrodes (i.e., tunneling electrodes) (203, 204). FIG. 3a also depicts one exemplary orientation of electrophoretic electrodes (201, 202). In practice, detection electrodes (203, 204) are in

communication with the magnetic device to detect when a charged polymer has entered the detection zone (206) in order to properly time the induction of a magnetic field sufficient to slow, stop, or reverse the translocation of a charged polymer, labeled with a magnetic or paramagnetic label or particle, within the nanochannel (205). Electrophoretic forces act as the primary impetus to move the charged polymer from the proximal to the distal end of the nanochannel. These forces are, in certain embodiments, larger than the force exerted on the charged polymer by the magnetic field.

[00091] FIG. 3b is similar to FIG. 3 a, but instead of electrophoretic electrodes, the nanochannel incorporates a proximal magnetic device (202) and a distal magnetic device (207). Similar to electrophoretic forces described above, the magnetic devices here act on a charged polymer, labeled with a magnetic or paramagnetic label or particle, in a manner sufficient to guide the charged polymer into and through the nanochannel. In certain embodiments, the charged polymer is labeled with a magnetic or paramagnetic label or particle on its N- and C- termini. These labels may be the same or different labels or particles having the same or different sizes and magnetic properties.

[00092] As described above, in certain embodiments the nanochannel is comprised in a nanoribbon, such as a graphene nanoribbon. Translocation of polymers through these nanoribbons can be via any of the methods contemplated herein. Such graphene nanoribbons can be employed in a stand-alone fashion or used in conjunction with other nanochannel schemes described herein. For example, in certain embodiments a separate nanochannel is utilized to effect electrostatic positioning of the polymer, providing for a manipulatable rate of progress of the polymer through the detection zone comprised in the nanoribbon. In such emobodiments the separate nanochannel is positioned distal of the nanoribbon, where the polymer is run through the nanoribbon pore initially, then into a spatially distinct nanochannel that utilizes electrostatic positioning mechanisms or magnetic forces to control the rate of translocation of the polymer though the spatially distinct nanochannel as well as through the nanoribbon. In such embodiments, often the polymer is prepared to incorporate a known lead sequence of monomers, or other pre-determined sequence, that pass into the spatially distinct nanochannel prior to formal interrogation of the polymer in the detection zone. In alternative embodiments, formal interrogation begins as the polymer passes through the detection zone in the nanoribbon. One of skill in the art will appreciate that the reference to "spatially distinct nanochannel" in connection with this embodiment refers to nanochannels that are either entirely separate, or connected (e.g., in a stacked/layered configuration) to the nanoribbon. In any event, the spatially distinct nanochannel and the nanoribbon generally comprise discrete zones through which the polymer translocates. [00093] Similar concepts regarding current fluctuations and electric signal monitoring apply to the use of a nanoribbon in the present methods. For example, monitoring electric signal fluctuations resulting from the passage of each monomer of a polymer through the detection zone can be via the piezoelectric-based or Hall-effect-based monitoring schemes contemplated herein. In the case of piezoelectric-based monitoring schemes, the piezoelectric device is placed in electric

communication with the current passing through the nanoribbon and detection zone contained within the nanoribbon. As described elsewhere herein the piezoelectric device is provided with a resonant frequency that generally corresponds to the translocation rate of the polymer through the detection zone (i.e., in the KHz, Mhz or GHz ranges).

[00094] With regard to Hall-effect-based monitoring schemes, a variety of configurations may be employed since although interrogation of the polymer in such schemes depends, in-part, on electric signal fluctuations, the detector(s) need not be positioned in electric communication with the current passing through the nanoribbon. For example, certain monitoring schemes involve focusing interrogation on the polymer passing through the detection zone and ultrasonic pulses resulting from the different conductivities of a monomer within the polymer, the surrounding medium, and optionally the nanoribbon comprising the electric circuit or pathway. In alternative examples, interrogation is focused on the nanoribbon at a location adjacent to, surrounding, or at the edge of the pore within the nanoribbon. In such examples, the Lorentz force acts on the nanoribbon, causing physical deflections that depend on the identity of the monomer (i.e., which nucleotide is within the detection zone) within the pore. In such embodiments ultrasonic pulses resulting from such physical deflections, or the actual physical deflections of the nanoribbon, can be monitored, for example via the use of a laser interferometer or piezoelectric device or other sensor modality. Arrays of sensors, as described herein, can be utilized to provide enhanced resolution.

[00095] A frequent embodiment of the present invention includes one or more control units. A control unit may include, for example, a computer that connects to a specialized board with an application-specific integrated circuit, wherein the board connects to the device. A control unit may also, for example, be integrated with the device by way of a nano-electro-mechanical system, where nanofluidics (e.g., a well containing a charged polymer such as D A) is combined with electronics (e.g., a control unit). A control unit implements the step of applying time-dependent voltages to the electrophoretic electrodes to attract a linear charged polymer from a proximal well to a distal well, as well as the step of applying voltage to each positioning electrode, if present, to control the path of the travelling charged polymer.

[00096] Characterization activities may include, for example, DNA sequencing, identifying polymers having a particular characteristic (i.e., a polymorphism, insertion, deletion, etc.) that are present in a test sample, counting the number of monomers in each polymer, as well as separating two or more polymers according to one or more characteristics. Other

characterization activities may include detecting chemical modification of the charged polymer such as methylation content, methylation pattern, or methylation content and pattern. [00097] In the absence of a polymer such as DNA, the nanochannel contains ionic solution and typically has a baseline potential difference measured between sensing electrodes. As the exemplary DNA polymer enters the fluidic channel, the potential measured between the sensing electrodes changes because the DNA has a conductivity that differs from that of the ionic solution. For example, when DNA enters the nanochannel, the conductivity in the channel between sensing electrodes will typically be reduced as DNA is less conductive than the buffer solution (see, e.g., de Pablo et al, Phys. Rev. Lett., 2000, 85:4992-4995). More frequently, however, each nucleotide base in the polynucleotide is identified based on unique electric signal fluctuations caused within the circuit between the sensing electrodes due to the presence of each nucleotide.

[00098] The phenomenon of polynucleotide capture by, and translocation through, a nanopore immersed in an electrolyte have been the subject of recent research. See, e.g., U.S. Pat. App. Pub. No. 201 10226623; Timp et al, IEEE Trans. Nanotechnol, 2012, 9(3):281-294;

Aksimentiev et al, IEEE Nanotechnol. Mag., 2012, 3(l):20-28; Heng et al, Nano Lett., 2005, 5(10): 1883-1888; Iqbal & Bashir, NANOPORES : SENSING AND FUNDAMENTAL BIOLOGICAL INTERACTIONS (Springer Science 201 1). In general, a polynucleotide must diffuse within the electric field range of a pore to be pulled through it by the field. Once the polynucleotide is inside the pore, there are three main forces that affect the DNA. See, e.g., Iqbal & Bashir, cited above, at p. 297-98. The electric field, which acts primarily on the negatively charged polynucelotide phosphate backbone, electrostatic and/or nonpolar interaction with walls of the pore, and electrophoretic force resulting from the DNA movement through solution.

Accounting for these forces is useful in calculating polynucleotide translocation rates and the effects of these forces on polynucleotide stretching within the pore (see, e.g., US Patent Application Publication No. 20110226623).

[00099] In addition, methods of improving linear translocation of a polynucleotide through the nanochannel can be facilitated by, for example, attaching charged or neutral nanoparticles, nanospheres, or other moieties or macromolecules to the target polynucleotide prior to passage through the nanochannel. These particles can be attached, for example, to one end of polynucleotide (e.g., a single stranded DNA), resulting in an enhanced hydrodynamic electrophoretic force in a direction opposite the electrophoretic force. Exemplary

macromolecules that can be used for this purpose include those described, for example, in U.S. Pat. App. Pub. No. 20080241950 and U.S. Pat. No. 8, 1 14,599.

[000100] Alternatively, the nanoparticle, nanosphere, or other moieties or macromolecules may comprise a magnetic particle that is attached to one end of the target polynucleotide.

Magnetic nanoparticles may be generally of the type described, for example, in U.S. Pat. Nos. 7,906,345, 7,682,838, and 8,247,025. When the particle is attached to the end of the polynucleotide desired to enter the nanochannel first, the device can incorporate a magnet or magnetic field producing element, e.g., magnetic tweezers, distal to the proximal entry point of the nanochannel. The magnet or magnetic field-producing element thus operates to pull the polynucleotide through the nanochannel by acting upon on the attached magnetic particle. In certain embodiments this method of effecting translocation of the polynucleotide through the nanochannel is utilized in lieu of electrophoretic forces. In other embodiments the

electrophoretic and magnetic translocation means are utilized together. In these embodiments it is often important to account for magnetoelectrochemical and magnetohydrodynamic solution, ion, and particulate flow effects due to the presence of magnetic fields in an electrolyte solution. See Alemany & Chopart, "An Outline of Magnetoelectrochemistry," Fluid Mechanics and Its Applications, 2007, 80(Part IV):391-407; Davidson, AN INTRODUCTION TO

MAGNETOHYDRODYNAMICS (Cambridge University Press, 2001); West et al, "Application of magnetohydrodynamic actuation to continuous flow chemistry," Lab Chip, 2002, 2:224-230.

[000101] When the particle is attached to the end of the polynucleotide desired to enter the nanochannel after the polynucleotide to which it is attached enters the nanochannel, the device can incorporate a magnet or magnetic field producing element, e.g., magnetic tweezers, proximal to the nanochannel entry point. In such an embodiment the polynucleotide will become subject to the electrophoretic forces through the nanopore by the electrophoretic electrodes. Countering magnetic forces act in the opposite direction on the attached magnetic particle, thus slowing the translocation of the polynucleotide through the nanochannel.

Generally, with such embodiments the magnetic particle and/or the magnet or magnetic field producing element is chosen that produces a magnetic field having a resulting force on the magnetic particle that is smaller than the electrophoretic forces acting on the target

polynucleotide by the electrophoretic electrodes. In particular embodiments, the target polynucleotide is permitted to enter the nanochannel prior to activating the counter magnetic force. Often the target polynucleotide is completely held within the nanochannel prior to activating the counter magnetic force. For example, in certain embodiments the distal portion of the polynucleotide (i.e., the portion of the polynucleotide passing first through the nanochannel) is detected at a particular point in the nanochannel and the counter magnetic force is activated. A dedicated set of presence-sensing electrodes can be positioned in the nanochannel (for example electrodes of the type described in U.S. Pat. App. Pub. No. 20100310421) for the purpose of identifying the location of the polynucleotide within the channel for the purpose of activating the counter magnetic force. In certain embodiments the presence-sensing electrodes are in electronic communication with the magnetic force producing element such that it is automatically activated when the distal portion of the polynucleotide is detected in the nanochannel. Frequently, the presence-sensing electrodes are placed proximal to the piezoelectric detection zone, or zones (if multiple detection zones are incorporated), of the nanochannel, though the presence-sensing electrodes can be placed distal to this area, or alternatively, the detection electrodes can be used to independently detect polynucleotide presence. Though this set of embodiments is described herein together with the specific piezoelectric -based detection scheme described throughout the present description, it can be readily adapted in other existing or to be developed sequencing technologies, and particularly nanochannel- or nanopore-based polynucleotide analysis schemes. As noted above,

manipulating/slowing polynucleotide translocation rates remains a major existing challenge in the art. Though the piezoelectric -based analysis scheme described herein advantageously does not require translocation rate slowing strategies, such strategies can be employed.

[000102] The devices and methods of the present disclosure can be effectively used to identify the nucleotide sequence of a polynucleotide of interest with the help of reference data showing the characteristic electric signal associated with, and/or the masses of, the different types of individual nucleotide bases. In a frequently preferred embodiment, reference data is gathered under similar or the same experimental conditions (i.e., ionic strength of the fluid medium, temperature, pH, background noise, etc.), and using similar or the same

nanopore/detection configurations, as the ultimate test data. The control polynucelotide from which reference data can be generated may consist of the same type of nucleotide base (e.g., AAAA . . . , TTTT . . . , GGGG . . . , or CCCC . . .). In addition, reference data can be generated using a control polynucleotide having long stretches of the same type of nucleotide base followed by a stretch of another type of nucleotide base (e.g., . . . AAAA . . . TTTT . . .

GGGG . . . CCCC . . .). Based on other reference data, fine-tuned reference data can also be generated, if desired, utilizing alternating types of nucleotide bases in a single control polynucleotide strand (e.g., ATATATATATAT . . . , AGAGAGAGAG . . ., ACACACAC TCTCTCTCT . . ., GCGCGCGCG . . . , etc.). Fine-tuned reference data may occasionally be useful to assist with the resolution of particular nucleotide bases in a polynucleotide as they occur in sequence with other nucleotides. Due to the rapid translocation rates, characteristic signal bleed between nucleotides may be detected. The signal bleed in these circumstances can be identified as characteristic of particular contiguous nucleotide bases, for example, between A- T, A-C, A-G, T-A, T-C, T-G, C-T, C-A, C-G, G-T, G-A, G-C. Characteristic signals between three or more particular nucleotide bases can also be identified in this manner. Overall, this reference data is compared or correlated with measured or detected data to decode the nucleotide base sequence of a polynucleotide of interest.

[000103] An exemplary device containing a nanochannel and nanoscale sensing electrodes is illustrated in FIGs. 2 & 3. Electric current is transferred in the form of ionic flow in an electrolyte solution confined in the nanochannel (1 18, 119, 120, 121, 205). The role of the electrolyte is, in general, to maintain a uniformly distributed electric field in the fluidic channel. Typical electrolyte solutions are known, for example, in the use of electrophoresis. Exemplary electrolytes include Tris boric acid EDTA (TBE) and tris acetate EDTA (TAE). See, e.g., Sambrook & Russell, MOLECULAR CLONING: A LABORATORY MANUAL (3d ed. Cold Spring Harbor Press, 2001). However, any conductive medium that does not adversely affect the structural integrity of the charged polymer may be used. [000104] During operation, a current is supplied by applying a potential to a pair of electrodes, e.g., electrophoretic electrodes (200, 201) disposed at proximal and distal ends of the nanochannel (205) and in contact with the electrolytic solution. The electrophoretic electrodes are, in general, in electrical communication with wires leading to the proximal (1 16) and distal (1 17) ends of the nanochannel (205). In practice, a potential is applied along the nanochannel (205) to generate an electrophoretic force to pull the charged polymer (not depicted) from the proximal (1 16) to the distal (117) end of the nanochannel (205). The electrophoretic electrodes frequently generate a constant or an oscillating electrophoretic force in the nanochannel (205) for translocation of the charged polymer. The voltage between the electromotive electrodes may be constant or it may be changed over the course of a measurement. For instance, it is occasionally desirable, though frequently not required, to reduce the voltage when a charged polymer has entered the nanochannel (205), before it reaches the sensing electrodes.

[000105] The voltage across sensing electrodes (1 11, 1 12) is proportional to the local impedance in the nanochannel (205) between sensing electrodes (11 1, 1 12). The spacing of the electrodes is determined by multiple factors. The smaller the distance between electrodes in a sensing pair, all other factors being constant, the smaller the particle that can be detected by the sensing pair. However, fabrication limits occasionally introduce minor complexities in electrode placement. Thus, in a frequent embodiment, the selected distance involves a balance between fabrication reproducibility and device sensitivity.

[000106] The resulting sensing electrode arrangement provides a means to separate the current and voltage analyses. In one embodiment, the electrophoretic electrodes (200, 201) at the proximal and distal ends of the nanochannel (205) provide a current while the sensing electrodes (11 1, 112) disposed across the nanochannel (205) are used to measure voltage. The voltage electrodes often have an output impedance that is larger than the impedance of the volume being measured.

[000107] The fluidic channel may be, for example, subject to a constant electric field equal to the potential difference along the length of the channel divided by the length of the channel, i.e., 100 mV applied longitudinally to a 10 μιη long fluidic channel results in a field of 100 mV/10 μιη = 10 mV/μιη or 0.01 mV/nm. The potential difference between the electrophoretic electrodes separated by 10 nm is then the product of the distance between electrodes and the electric field or: 10 nm x 0.01 mV/nm=0.1 mV.

[000108] This potential, and similar potentials, are readily detectable with conventional electronic measurement tools. When a charged polymer such as a DNA molecule passes between a pair of sensing electrodes, the impedance between the sensing electrodes changes due to a resistivity difference between the electrolyte and the molecule. The resulting transient change in the potential is measured, while maintaining a constant current.

[000109] In use, the voltage between a pair of sensing electrodes is optionally monitored by a measurement tool, e.g., a voltmeter, configured to measure the potential difference between the sensing electrodes. In such an embodiment, the voltmeter is in electrical communication with each of the sensing electrodes via metal contact pads connected to nanowires leading to the sensing electrodes.

[000110] In practice, a sensing device comprising one or more nanochannels connecting one or more microfluidic wells, a piezoelectric sensor, electrophoretic electrodes, and sensing electrodes disposed along the length of each nanochannel, is filled with an ionic fluid. Multiple copies of a charged polymer such as a polynucelotide such as DNA or RNA are introduced into the microfluidic well. Electrophoretic electrodes are used to pull the polynucelotide from the well into the one or more nanochannels. As the polynucelotide enters the nanochannel, it assumes a linear conformation. As noted above, the degree to which it is linearized depends on a number of factors, for example, the length of the DNA strand, temperature, ionic conditions, and width and depth of the fluidic channel (all of which affect the forces acting on the polynucleotide).

[000111] The potential applied by the electrophoretic electrodes causes the polynucelotide to progress through the nanochannel, where it passes through a volume between the sensing electrodes. When the leading edge of the DNA enters a volume between the sensing electrodes, a change in an electrical characteristic such as cross channel current or potential between the sensing electrodes occurs. This electrical characteristic is unique for each species of nucleotide base. A piezoelectric device is situated in electrical communication (e.g., by way of direct contact, wired connection, circuit, transistor, memristor, memristor hybrid, or another means) with one or more of the electrodes such that the change in electrical characteristic is imparted to the piezoelectric device. The piezoelectric device responds to the unique electrical characteristic of each nucleotide base by exhibiting a corresponding unique physical deformation (e.g., lengthening, shortening, bending, etc.). This deformation is monitored by a detection device (e.g., a vibrometer, interferometer, or accelerometer), thereby determining the identity of each nucleotide base in the polynucleotide in real time as it passes the electrodes and piezoelectric device within the nanochannel. Additional data, such as timing, rate, frequency, raw data related to the changing electrical signal, among other characteristics may also be collected during detection. Moreover, information related to background electrical signals and vibrations are often monitored and accounted for during detection. Electronic storage devices and computer- implemented algorithms are generally utilized to process real-time raw data. Memristors or memristor hybrids are particularly useful in the present real-time polynucleotide sequencing methods and devices. Details regarding memristors and memristor hybrids and their use can be found, for example, in Strukov et al, Nature 453 :80-83 (2008); Williams, IEEE Spectrum 45(12) (December 2008); Eid et al, Science 323(5910): 133-138 (2009); and U.S. Pat. App. Pub. No. 20110236984.

[000112] In certain embodiments, the detection devices are configured to detect any natural or synthetic nucleic acid or nucleic acid analog, including DNA or RNA. In other embodiments, the detection devices are configured to detect amino acids, proteins, saccharides,

polysaccharides, PNA, synthetically produced nucleic acids, synthetically produced amino acids or proteins, synthetically produced saccharides or polysaccharides; other biomolecules comprised of combinations of nucleic acids, amino acids, or saccharides; or the same or other molecules used as or bound to detectable labels.

Hall-Effect Imaging

[000113] When an electric current is induced in a conductor and a magnetic field is applied perpendicular to the current, a voltage difference is produced in the conductor that is transverse to the current and magnetic field. This phenomenon is known as the Hall-effect. In this phenomenon, charge carriers passing through the conductor experience a force, called the Lorentz force, due to the magnetic field that is perpendicular to the current. The Lorentz force (F) is equal to - q(E + v x B). In this equation, q (1.602xl0^~19 C) is the elementary charge, E is the electric field, v is the particle velocity, and B is the magnetic field. This force separates positive and negative charges to opposite faces of the conductor, producing an asymmetric distribution of charge density. This charge asymmetry produces an electric field across the conductor (perpendicular to the current flowing through the conductor) described by the Hall coefficient. This charge separation produces an externally detectable voltage, the Hall voltage. The Hall coefficient represents the ratio of the induced electric field to the product of the current density and the applied magnetic field, defined as R = E/jB. In this equation, j is the current density of the electrons, E is the induced electric field, and B is the magnetic field. The magnitude of the Hall voltage (Vh) is equal to IB/qnd, where / is the current, B is the magnetic field, d is the sample thickness, and q (1.602 x 10^"19 C) is the elementary charge.

[000114] The Hall voltage amplitude is determined by the strength of the Lorentz force, the charge density, and charge mobility. The Lorentz force, therefore, is proportional to the magnetic field B and the velocity of motion v, while the charge density and mobility are characterized by the overall conductivity σ of the object, including any dielectric contribution.

[000115] Spatial information, in general, is encoded in V i to produce an image based on the Hail effect. Wen et al, IEEE Trans. Biomed. Eng., 1998, 45(1): 1 19- 124. One method of generating such images is through the use of an ultrasound pulse to localize motion to particular portions of a sample. This pulse produces acoustic vibration that passes through the sample medium, creating a Hall voltage that follows the path of the vibration in the sample. In the present methods, this Hall-effect voltage course is utilized to generate an image of the vibration path. This image contains information on the conductivity σ of the sample and acoustic parameters affecting Vh. The image in these embodiments is encoded in a time domain.

Therefore, reconstruction of the imaged sample is analogous to ultrasound imaging, though on a different scale, utilizing materials and methods described herein.

[000116] Imaging based on the Hall-effect in the presently described embodiments is based on the measurement of an interaction between a static magnetic field and an externally applied radiofrequency current when a uniquely charged molecular species is presented in a detection zone. It has been determined in the present invention that piezoelectric transducers are useful in this method because of their tuneability and ultra- fine sensitivity and since they can be fabricated to have spatial arrangements to specifically capture the Hall-effect signals produced in the present methods. One example includes the use of one, or an array of two or more, piezoelectric transducer(s).

[000117] Imaging based on measurement of the presently described Hall effects is often carried out in two alternative formats, a forward mode where electrical signals produced by a strong ultrasonic pulse are received (discussed above), and a reverse mode where ultrasonic signals generated by an electrical excitation pulse are received. Frequently the reverse mode is preferred in the present methods. In the reverse mode a voltage is applied in a fluid medium between two or more electrodes or through a pore in a graphene nanoribbon to establish an electric field in the fluid medium. In the fluid medium the local electric field at any point is proportional to the local apparent conductivity of the medium, including its components. At positions where there is a change in conductivity, for example, at an interface of two materials or fluids having different conductivities, the current density becomes discontinuous. Lorentz forces acting on the current at these locations are also discontinuous. These Lorentz force discontinuities produce detectable ultrasonic pulses, providing lateral spatial resolution of the interfaces. Axial spatial resolution is obtained, for example, by monitoring the timing of the arrival of the pulses and/or methods described in Roth & Schalte, Med. Biol. Eng. Comput, 2009, 47(6):475-577.

[000118] In certain embodiments an ionic current comprises the electrical current source, wherein a magnetic field is applied perpendicularly to the ionic current to induce the Hall-effect in the fluid medium. For ionic solutions, a model for the current flux comprises the Nernst- Planck equations for the ionic species present in the solution. For example, the ionic flux density of species k is:

k=uc_k-D_k c_k - z_k(D_k/RT)Fc_k( V+u x B), (k=l, . . . , N) where c_k is the molar concentration, D_k is the diffusion coefficient, z_k is the valance of the k^th ionic species, F is Faraday's constant (i.e., 96484.6 C/mol), R is the universal gas constant, T is the absolute temperature of the ionic solution, N is the total number of species present in the ionic solution, and u x B is the induction term. Under steady-state conditions and the current flux {J=F∑ z_kN_k} the potential in the ionic solution is governed by the local electroneutrality condition {∑ z_kN_k = 0}. See, e.g., Qian & Bau, Mech. Res. Commun., 2009, 36(1): 10-21.

[000119] The Lorentz force experienced in the ionic flow is a product of the current and the magnetic field strength. This force is often relatively constant at any particular point in a homogenous ionic fluid, but the force changes when another charged species, such as single stranded or double stranded DNA or RNA species, enters the ionic flow. This change can be detected at the detection zone. For example, the charged species, which represents a conductivity discontinuity in the fluid medium, experiences a different Lorentz force versus the surrounding fluid medium, and thus produces an ultrasonic signal, that is characteristic of the physical and electrical properties of the charged species. Each type of nucleoside triphosphate or nucleotide within an oligonucleotide or polynucleotide has a unique dielectric property and charge density. See, e.g., Xu et al., Small, 2007, 3(9): 1539-43; Zwolak & Di Ventra, Nano Lett, 2005, 5(3):421 -4; Wammu, Nat NanotechnoL, 2010, 5(1 1):807-814; Tsutsui et al., Nat Nanotechnol., 2010, 5(4):286-90; Chang et al., Nano Lett., 2010 10(3): 1070-5; Ivanov et al, Nano Lett., 201 1, 10: 1070-75; Nelson et al, Nano Lett., 2010, 10(9):3237-42. Thus, each type of nucleoside triphosphate or nucleotide produces a unique detectable ultrasonic signal according to the present methods and devices. These unique charge properties, encoded within corresponding unique ultrasonic signals, are identified and differentiated by the presently disclosed methods and devices such thai any particular nucleotide within a polynucleotide or oligonucleotide can be identified singularly, or within a longer chain of nucleotides when presented to the presently identified detection zones. Cleavage-based (e.g., exonuciease, chemical cleavage, etc.) or intact nucleotide strand-based methods of presenting nucleotides to the detection zone are thus contemplated by the present methods.

[000120] It is particularly useful to construct a detection zone to focus on the detection of Hall-effect induced ultrasonic pulses in a particular physical area. Though this technique can be used to image portions of a polynucleotide comprising two or more contiguous nucleotides, imaging of single nucleotides in a polynucleotide sequence, or alone, is frequently preferable. Construction of such detection zones, for example, can be though the use of focused arrays that detect ultrasonic pulses attributable to particular nucleotides in a stationary or moving nucleotide chain. Arrays can take any configuration known in the art, including one or more laterally arranged or circular array(s), or another configuration. In certain embodiments the nucleotide chain is held temporarily in a stationary position, e.g., for at or near one second or less, at or near one microsecond or less, at or near one millisecond or less, at or near one nanosecond or less, etc., via a magnetic or electrostatic means such that the chain can be read in a step-wise manner. Frequently, however, the nucleotide chain is read in a continuously moving manner such that the chain can pass through the detection zone without employing a means for slowing or stopping progression for any period of time. Piezoelectric probes, for example, probes of the type described herein Wen et al, Ultrason. Imaging., 1998, 20(3): 206-220, can be utilized in such analysis schemes. See also Wen et al, IEEE Trans. Biomed. Eng., 1998, 45(1): 119-124; Roth, IEEE Trans Biomed Eng., 1998, 45(10): 1294-6; Montalibet et al, Ultrason Imaging., 2001, 23(2): 1 17-32, Montalibet et al, Med Biol Eng Comput, 2001, 39(1): 15-20. However, other measurement techniques are useful and may, if desired, provide enhanced resolution and sensitivity. For example, optical ultrasonic sensors (e.g., interferometry sensors such as laser interferometry sensors) and optical-fiber-based sensors are useful to detect ultrasonic vibrations in the present methods. See, e.g., Monchalin, Appl. Phys. Lett. 1985, 47: 14-16; Huber & Green, Mat. Eval, 1991, 49:613-618; Hoyes et al, J. Meas. Sci. Tech., 1991, 2:628-634; Hamilton et al, IEEE Ultrason. Symp., 1997, 753-756; Coleman et al, Ultrasound Med. Biol, 1998, 24: 143-151 ; and Wen et al, Ultrasound Imaging, 1998, 20: 103-1 12.

[000121] Since the electric current in the fluid medium is preferably at a level below that which is sufficient to induce electrolysis of the medium, there is occasionally a maximum preferred ceiling for the current level aspect of the Hall coefficient. In occasions where such enhanced currents may induce localized electrolysis of the fluid medium, any potential effect on detection sensitivity or resolution is often minimized through appropriate apparatus or detection zone design modifications described herein.

[000122] The applied magnetic field is often a strong magnetic field, for example, above 1 Tesla, to enhance detection sensitivity. Frequently, such strong magnetic fields are produced by magnets having a strength of at or above 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or more Tesla. In frequent embodiments the magnetic strength ranges between about 1 Tesla to about 17 Tesla to enhance detection sensitivity. Occasionally, the applied magnetic field is provided by a magnet having a strength above 17 Tesla. Frequently, it is preferable to apply a strong magnetic field without regard to the use of electric currents that may induce localized medium electrolysis. Production of such strong magnetic fields can be by any known method, for example, by the use of electromagnetic or solid-state magnetic field sources. Such magnets are commercially available, though modifications noted herein are often necessary to adapt the magnet to apply consistent focused (e.g., micro or nano range) fields. Often it is not necessary to focus the magnetic field smaller than within the micro-range to apply the necessary field strength and consistency. Small magnets, such as Nd-Fe-B magnets (NEOMAX, Hitachi Metals, Japan) can also be utilized to produce strong magnetic fields, e.g., 400 T²/m. See, e.g., Watarai & Namba, Anal. Sci., 2001 , 17: 1233-36. Nanomagnets also comprise suitable sources for the magnetic fields of the present Hall-effect related analysis methods.

[000123] Electrolysis conditions of the fluid medium resulting from the use of large potential differences between electrodes can be overcome by design. For example, electrodes are positioned in conduits that are distinct from, though in fluid communication with, the main nanochannel(s). See, e.g., Qian & Bau, Mech. Res. Commun., 2009, 36(1): 10-21. In such circumstances it is occasionally advantageous to utilize increased potential differences between electrodes, thus permitting the use of correspondingly decreased magnetic field strengths, while maintaining the same or similar detection sensitivities and resolutions.

[000124] As exemplified in the diagram below, in one embodiment the electrophoretic electrodes provide an electric potential within an ionic solution in direction X. Concurrently, a magnetic field source is oriented to apply a magnetic field running perpendicular to the electric current, in direction Y. The ultrasonic detection mechanism, e.g., a piezoelectric probe and/or laser interferometer based detection zone, is positioned in a third direction Z, focused in the nanochannel at a right angle to the intersection of the electric and magnetic fields at a detection zone.

[000125] At this detection zone, ions within the solution are permitted to flow in direction X, thus generating a background ultrasonic signal detected by the Z-direction oriented detection mechanism. A polynucleotide strand is introduced to the nanochannel, where it flows through the nanochannel into the detection zone. At the point where the first nucleotide in the polynucleotide strand enters the detection zone, unique ultrasonic pulses are produced, reflecting the conductivity discontinuities between the ionic fluid medium and the nucleotide in the strand positioned in the detection zone. One, 2, or 3 dimensional characteristics of the nucleotide/ionic medium interface, which produces conductivity discontinuities, are obtained by measuring the characteristics and/or timing of the ultrasonic pulses, thus identifying the nucleotide in the detection zone. See, e.g., Roth & Schalte, Med. Biol. Eng. Comput., 2009, 47(6):475-577. This process proceeds for each nucleotide as it enters and passes through the detection zone to thereby identify characteristics about the polynucleotide such as the identity of each nucleotide in the polynucleotide sequence.

[000126] Thus, according to the present methods, one-, two-, or three-dimensional interrogations of a molecular species, e.g., a nucleotide, amino acid, polynucleotide, or polypeptide, are possible. For example, according to the present methods, one can obtain a detailed view of the molecular species to identify particular monomers within a polymer chain. Detector array design and configurations (e.g., a circular array) permits one of skill in the art to obtain multiple dimension resolution, if desired.

[000127] In one embodiment, ultrasonic signals from two or more nucleotides in the polynucleotide strand are produced and detected. The present methods and devices have the capability of not only identifying each nucleotide in such combined signals, but also differentiating the correct order of each nucleotide in the strand by virtue of its order of presentation into the detection zone. The timing of the receipt of such ultrasonic signals by the detection mechanism provides information important to elucidating the proper order of the nucleotides in the sequence. Piezoelectric probes and arrays are useful to obtain accurate timing of such ultrasonic pulse information. Accordingly, although it is advantageous to utilize micro- or nano-proportioned detection mechanisms, this is occasionally not necessary to effectively interrogate nanometer species such as individual nucleotides in a polynucleotide. Moreover, utilizing detection zone focusing arrangements are occasionally advantageous to reduce the detection zone to the nanometer range, while making use of micro-range or larger detectors. It is important, however, in such circumstances to manipulate, for example, potential differences and magnetic field strengths, as taught herein, to maintain sensitivity and to effectively discern signals attributable to a nucleotide species from background noise levels.

Applications

[000128] The presently described methods and devices provide a variety of diagnostic uses currently contemplated for existing and developing sequencing technologies. For example, the presently described methods and devices are useful for de novo sequencing, re-sequencing, polymoφhism/deletion/mutation/fusion/rearrangement identification and analysis, genetic testing, microbial identification, viral identification, methylation analysis, forensic analysis, general medical diagnostics, companion diagnostics, industrial applications, food analysis, air quality analysis, drug discovery or validation, personalized medicine, among a variety of other uses.

Example I

[000129] In accordance with the techniques of U.S. Pat. App. Pub. No. 2011226623, for example, silicon nitride membranes are produced by depositing an LPCVD Si3N4 film, ranging from 30 nm to 200 nm thick (nominally), on the top of a 300 μιη thick (float-zone) Si handle wafer. The amount of oxygen, silicon and nitride in the film is adjusted to control

hydrophobicity. To reduce the thickness, either the nitride membrane is sputtered in a 5 μιη x 5 μιη area using focused-ion beam milling or it is uniformly etched in 20: 1 H20:49% HF for 30-40 min at room temperature. Then, a polyimide photoresist with thickness of 3.6+-0.6 μιη is spin deposited on top of the chip, and a 5 μιη window is opened over the membrane using UV lithography. The polyimide is used primarily to reduce the parasitic substrate capacitance.

[000130] A nanometer-size pore is then sputtered into membranes like these using a tightly focused (1.6 nm spot-size) 9°a (cone angle), high energy (200 kV) electron beam emanating from a JEM-2010F transmission electron microscope (TEM) operating in convergent beam diffraction mode. Using TEM images taken at different tilt angles, the pore geometry is modeled as two intersecting cones (bi-conical) each with >20° cone angle. By stringently controlling the beam conditions and membrane thickness (guaranteed by Electron Energy Loss Spectroscopy) it is possible to produce pores with practically the same geometry with sub- nanometer precision.

[000131] A JEOL-2200F is utilized to impart precise control over the sample position, and an aberration probe corrector is utilized that allows for increased (8*) brightness with a smaller probe. This corrector enables sputterring with a smaller (<1.6 nm) spot which, in combination with the piezo-stage, provides more precise control over the pore geometry. This system facilitates the production of various nanometer-sized passages, such as a nanopassage having a 2.0 x 1.0 nm nano-slit with a 1 nm beam.

Example II

[000132] In accordance with the techniques of U.S. Pat. App. Pub. No. 2011 155574, for example, a support structure is provided, e.g., a silicon nitride membrane of about between about 200-1000 nm in thickness, for example as taught in U.S. Pat. No. 7, 118,657 or another conventionally known method. The membrane is provided with a starting aperture by, e.g., electron beam etching, ion beam milling, wet etching, plasma etching, ion beam sculpting, or other suitable process.

[000133] The starting aperture is generally circular, having a diameter of between about, e.g., 20 nm and 100 nm. The support structure is provided with an upper trench, or groove in the top surface of the structure and a lower trench in the bottom surface of the structure.

Occasionally material is removed from the top and/or bottom of the support structure to thin it out, whereby trenches are created in the thinned out portion. The upper and lower trenches are orthogonal to each other, and the aperture is formed at the intersection of the two trenches. This orientation enables self-alignment for the nanotube probe positioning, as explained below.

[000134] The upper and lower trenches can be produced by using a focused ion beam or by conventional masking and etching procedures. The depth of the trench that produces the desired diameter starting nanochannel, e.g., between about 20 nm and about 100 nm, can then be used for additional fabrications.

[000135] A nanotube is then positioned or synthesized in the upper trench, across the aperture. In situ synthesis of the nanotube can be carried out in the manner described above, e.g., with a catalyst deposited and patterned in the upper trench followed by CVD nanotube synthesis. Alternatively, pre-synthesized nanotubes can be dispensed onto the surface of the support structure and mechanically transported to the trench, for example using an atomic force microscopy tip to roll a nanotube in a trench to the location of the aperture.

[000136] The selected nanotube can be electrically contacted by contact pads formed prior to synthesis, as described above, or subsequently electrically contacted by forming, e.g., palladium contact pads that are in turn connected to larger gold contact pads that connect to off- chip circuitry by conventional methods, e.g., as in Javey et al., Nature, 2003, 424:654 and Javey et al, Nano Letters, 2004, 4:447. Through this process, palladium can be evaporated onto nanotubes, through a mask, at the desired location.

[000137] Next, a selected coating is deposited on the nanotube-support structure assembly, for example, using an atomic layer deposition (ALD) process. A pre-selected number of ALD cycles are carried out, depositing material on all surfaces of the support structure including the walls of the aperture and the trenches. As the material deposition is continued, the build up of deposited material at the aperture reduces the extent of the aperture. Accordingly, the deposition process is continued until a selected final nanochannel diameter is produced, e.g., a diameter of between about 1 nm and about 10 nm. Since ALD is a precise process, the material thickness produced by each ALD cycle can be characterized for a given support structure and nanotube arrangement and dimensions, which can then be controlled to achieve a selected final nanochannel diameter with the upper side of the nanotube coated. For example, with a starting aperture of 50 nm, 220 ALD cycles, each adding a layer 1 A-thick, would produce a nanochannel of 6 nm in diameter. Additional ALD cycles, and/or utilizing a smaller initial aperture size, can be utilized to further adjust down the final diameter of the nanochannel.

[000138] Once a particular nanochannel diameter is formed, a nanotube probe

configuration is produced at the perimeter of the nanochannel. This configuration is provided by cutting through the nanotube that lies exposed across the final nanochannel to produce two nanotube ends that abut on the nanochannel perimeter. In one example nanotube cutting technique, a high-energy electron beam is directed through the nanochannel, from the bottom or top of the support structure. The beam removes the exposed unprotected nanotube material from the nanochannel, while the aluminum oxide (or other ALD coating) protects the ALD covered regions of the nanotube and support structure from the beam. Once the bare nanotube is removed from the final nanochannel, a functional nanochannel device is produced, having the ends of nanotube probes abutting a nanochannel perimeter.

[000139] In a further fabrication sequence, a support structure such as a silicon nitride membrane is provided with an aperture in the manner discussed above in this Example.

Orthogonal trenches, are provided in the top and bottom surfaces, respectively, of the support structure, with the aperture located at the intersection of the trenches as discussed above in this Example. With this configuration, a nanotube is positioned in the lower trench in the manner discussed above in this Example.

[000140] Next, an ion beam is then directed from the upper side of the membrane through the aperture at an angle selected to cut the nanotube at the position that will leave it abutting the final nanochannel perimeter at the end of the fabrication sequence. This angled beam impinges and removes that portion of the nanotube in the aperture that is in the path of the beam. The portion of the nanotube in the aperture that was not in the path of the ion beam protrudes into the aperture.

[000141] Next, a second nanotube is provided in the upper trench such that it extends off- center across the aperture. This second nanotube can be synthesized in situ at the site of the trench or mechanically positioned in the trench.

[000142] With the second nanotube in position a selected material is deposited on the support structure, e.g., by ALD. As above, the deposited material forms a layer on all of the surfaces except those of the unsupported nanotube. Material deposition is continued until a selected nanochannel diameter is achieved, with the edge of the protruding nanotube portion and the second nanotube located at the final nanochannel perimeter. With this deposition complete, a functional nanochannel device is produced.

Example III

[000143] In general accordance with the techniques of U.S. Pat. App. Pub. No.

20110168562, for example, a nanochannel device having trans-base pair readers is built using lithography. Commercially-available multi-walled carbon nanotubes (MWCNTS) (4 to 5 nm diameter) are spread on a silicon wafer, located relative to marks on the chip using low- voltage scanning electron microscope, then covered in 700 nm of polymethyl methacrylate (PMMA). E- beam lithography is used to create a series of wells in the PMMA lying on the path of a carbon nanotube (CNT). The exposed regions of the CNT in the wells are removed with oxygen plasma, leaving CNT segments that connect adjacent wells. These CNTs are functionalized according to known procedures. The device is completed with a molded polydimethyl siloxane (PDMS) microfluidic cover that permits injection of fluids into and out of the reservoir wells. Single walled carbon nanotubes (SWCNT) (<1 to 2 nm diameter) devices are made by chemical vapor deposition (CVD) growth from Co nanoparticles followed by the same set of lithographic steps. Example IV

[000144] Graphene field-effect transistors are prepared according to the methods of Lin et al, Nano Lett., 2009, 9(l):422-426, for example, with probe pads designed for high-frequency measurements. Graphene is prepared by mechanical exfoliation on a high-resistivity Si substrate (>10 kH-cm) covered by a layer of 300 nm thermal S1O₂, and Raman spectroscopy is employed to count the number of graphene layers. Source and drain electrodes made of 1 nm Ti as the adhesion layer and 50 nm thick Pd are defined by e-beam lithography and lift-off. A 12 nm thick AI2O3 layer is then deposited by atomic layer deposition (ALD) at 250 °C as the gate insulator. In order to form a uniform coating of oxide on graphene, a functionalization layer consisting of 50 cycles of N02-TMA (trimethylaluminum) is deposited prior to the growth of gate oxide. This N02-TMA functionalization layer is important for the ALD process to achieve thin (<10 nm) gate dielectrics on graphene without producing pinholes that cause gate leakage. In addition, 10 nm/50 nm Pd/Au is deposited and patterned to form the top gate. The source electrodes are designed to overlap the graphene. The distance between the source and drain electrodes is 500 nm, and the top gate underlaps the source-drain gap with a gate length of 360 nm. The total gate width (or channel width), including both channels, is ~40 μπι.

[000145] The field-effect mobility eff can be calculated using the relation Δσ = q An ^ueff, where σ is the 2D conductivity, q is the electron charge, and n is the 2D carrier density that is controlled by the gate voltage. For this graphene device, eff is estimated to be 400 cm²/(V-s). Example V

[000146] A protocol is implemented providing piezoelectric measurement of a Methicillin- resistant Staphylococcus aureus polynucleotide (GenBank Accession Nos. D86934 and

ABO 14440) test sample in the nanochannels of Examples I-IV. In particular, a piezoelectric device having a bandwidth of 16.5 MHz is integrated into the electric circuit comprising the electrodes or graphene field-effect transistor, including a capacitive position sensor (model D- 510 PISeca™, Physik Instrumente), together with sensor electronics (Model No. E-852.10, Physik Instrumente). DDL is utilized to optimize multi-axis motion utilizing a digital controller (Physik Instrumente, model E-71 1). INPUTSHAPING® (Convolve, Inc., Armonk, NY) is optionally utilized to compensate for rapid expansion of the piezoelectric translator to avoid overshooting the target frequency range.

[000147] An alternative protocol is designed providing piezoelectric measurement of a Humulus lupulus cultivar Cascade valerophenone synthase gene (GenBank Accession No. EU685789) test sample in the nanochannels of Examples I-IV. In particular, a piezoelectric device having a bandwidth of 16.5 MHz is integrated into the electric circuit comprising the electrodes or graphene field-effect transistor, including a vibrometer controller equipped with a displacement encoder having nanometer or sub-nanometer resolution (e.g., model UHF-120 vibrometer controller and DD500 displacement decoder, Polytec PI, Inc., Tustin, CA). In this instrument a heterodyne detector signal is provided by the optical head, which is acquired with a digital oscilloscope, permitting out-of-plane vibration frequencies up to 1.2 GHz. The digitized detector signal is transferred to a PC where the heterodyne carrier is demodulated by the software module in VibSoft (Polytec PI, Inc., Tustin, CA). rNPUTSHAPING® (Convolve, Inc., Armonk, NY) is optionally utilized to compensate for rapid expansion of the piezoelectric translator to avoid overshooting the target frequency range.

[000148] An alternative protocol is designed providing piezoelectric measurement of a Saccharomyces cerevisiae polynucleotide (GenBank Accession No. HV760955) test sample in the nanochannels of Examples I-IV. In particular, a piezoelectric device having a bandwidth of 16.5 MHz is integrated into the electric circuit comprising the electrodes or graphene field-effect transistor, including an interferometer (e.g., model A-150, PbMo04, 80-MHz, Schott North America, Inc., Elmsford, NY), a digital oscilloscope (e.g., model TDS 540D, 500 MHz,

Tektronix, Beaverton, OR), and an rf spectrum analyzer (e.g., model 3026, 3 GHz, Tektronix, Beaverton, OR). rNPUTSHAPING® (Convolve, Inc., Armonk, NY) is optionally utilized to compensate for rapid expansion of the piezoelectric translator to avoid overshooting the target frequency range.

Example VI

[000149] To demonstrate the feasibility of Hall-effect imaging, a device is constructed to form cross-sectional images of a Humulus lupulus cultivar Cascade valerophenone synthase gene (GenBank Accession No. EU685789) suspended a chamber of electrolyte buffer solution (e.g., Krasnigi & Lee, Metallomics, 2012, 4(6):539-544, Singer et al, J. Phys. Condens. Matter, 2010, 22:4541 11, Sambrook & Green, Molecular Cloning: A Laboratory Manual (4d ed. 2012), etc.), placed in the field of a pair of .04 T Nd-Fe-B magnets (Sumitomo Special Materials, Japan, NEOMAX) in general view of the methods outlined in Wen et al, IEEE Trans. Biom. Eng., 1998, 45(1): 1 19-124, and Wen et al, Ultrason. Imaging, 1998, 20(3):206-220. The magnetic field, which is estimated to be about 400 T²/m, BO is in the "Y" direction. A piezoelectric transducer (TRS Technologies) emits longitudinal ultrasound waves, with both the wave vector and the physical vibration in the "Z" direction. The transducer is focused, with a nominal element size of 1mm and a bandwidth of 16.5 MHz. The transducer is driven with a unipolar pulse (Panametric pulser model 5073PR). The Lorentz force from the ultrasonic vibration was in the "X" direction, and the resulting Hall voltage is detected with electrodes placed in the chamber. Preamplification is realized with a broadband low-noise preamplifier. After a passive bandpass filter and another 30-dB gain, the signal is recorded with a PC-based digital oscilloscope (GaGe CobraMax).

[000150] Immediately after the onset of the ultrasound pulse, the Hall voltage is recorded for the time required for the ultrasound wave packet to traverse the chamber. The Hall voltage is proportional to the magnetic-field strength. A 2-D image is formed with the line-scan method by moving the polymer across the chamber, while recording the time course of the Hall voltage at each position.

[000151] This method comprises a voltage detection method of Hall-effect imaging. Based on the reciprocity relation of a linear electro-mechanical system, Hall-effect imaging of the exemplary polynucleotide is carried out in the reverse mode. In the reverse mode, or ultrasound detection mode, the pulser that is used to drive the ultrasound transducer is now connected to the pair of electrodes that were used to detect the Hall voltage in the forward mode, and the signal- sensing electronics are connected to the transducer. When a voltage pulse is generated across the electrodes, an electric field is setup in the chamber. Any location in the chamber responds to the local electric field with a current density proportional to the local apparent conductivity. As indicated above, at interfaces of changing conductivity the current density becomes

discontinuous, and so are the Lorentz forces on the currents. The discontinuities of the Lorentz forces result in ultrasound pulses from these interfaces. These pulses are then received by the ultrasound, thereby providing lateral spatial resolution. Axial spatial resolution is encoded within the times of arrival of the pulses. The driving electric field between the electrodes in the reverse mode corresponds, in general, to the sensitivity profile of these electrodes as Hall voltage detectors in the forward mode. The currents in the chamber giving rise to ultrasound pulses in the reverse mode correspond to the conversion from ultrasound vibration to Hall currents in the forward mode. In addition, the propagation of ultrasound pulses to the transducer in the reverse mode corresponds to the propagation of the driving ultrasound pulse from the transducer into the chamber in the forward mode.

[000152] Denote the ultrasound pressure wave as p(z, t). Using the linear inviscid force equation (a)

Sviz, t) piz, 0

piz)

dt ^' dz 40 the Hall volta e in (2) can be expressed as b) ....... ....... dr

Integration by parts yields (c)

M{z H p(— - ) Miz,

where (d)

is the ultrasound momentum transmitted across position z at time t. Practical ultrasound transducers emit little energy in the audio and DC frequency range. The lack of a DC component means that the net momentum of the wave packet is zero. Under this condition, it can be shown that the surface term in (c) is zero during the time the wave packet is somewhere within the ultrasound path. Thus, the Hall voltage can be expressed as in (e):

This expression shows that a nonzero Hall voltage only comes from positions where a gradient of σ/ρ exists. This point can be visualized by observing the total Hall-effect current, while following the progression of the ultrasound wave packet. When the wave packet is in a homogeneous region, the total current is proportional to the average vibration velocity in the packet, which is zero due to the absence of a DC component. When the wave packet passes an interface of different conductivities, the portion inside the high σ region contributes more current with the same velocity; thus, the integral in (f) is no longer zero.

/V^{i !}:ϊ: H ' ¾ f _s , : *Μ¾· * A

When the wave packet passes an interface of different mass densities, but no change in conductivity, the portion in the low-density region has higher vibration velocities, therefore, the integral in (f) is also nonzero. In both cases the total Hall-effect current becomes nonzero and the resulting Hall voltage marks the presence of the interface.

[000153] Lorentz vibration noise is the part of the coherent noise that only occurs in the static magnetic field. For example, the excitation pulse applied to the sample also produces radio frequency (RF) electric and magnetic fields in the vicinity of the piezoelectric probe. These RF fields induce eddy currents in the metallic components of the probe. In the presence of the static magnetic field, the Lorentz forces on the eddy currents cause vibrations in these components. These vibrations either directly enter the piezoelectric element or propagate into the chamber and create echoes. Both result in coherent noise, which is referred to as the Lorentz vibration noise. A waveguide, active compensation, and piezo array, as generally described in Wen et al, Ultrason. Imaging, 1998, 20(3):206-220 are optionally utilized to reduce the effect of noise in the system and to enhance resolution.

Example VII

[000154] A protocol is implemented expanding on the Hall-effect imaging methods of Example VI, expanded in view of the general experimental design and methods of Montalibet et al, Ultrason. Imaging., 2001, 23(2): 117-32, Montalibet et al, Med. Biol. Eng. Comput, 2001, 39(1): 15-20, and Roth & Schalte, Med. Biol. Eng. Comput., 2009, 47(6):475-577 to provide for enhanced resolution, and 2-dimensional and 3-dimenstional Hall effect imaging of a Methicillin- resistant Staphylococcus aureus polynucleotide (GenBank Accession Nos. D86934 and

ABO 14440) test sample.

Example VIII

[000155] A protocol is implemented expanding on the Hall-effect imaging methods of Examples VI and VII, and applying these methods to analyze a Saccharomyces cerevisiae polynucleotide (GenBank Accession No. HV760955) test sample in the nanochannels of Examples I-IV.

[000156] Numerous modifications may be made to the foregoing systems without departing from the basic teachings thereof. Although the present invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow.

Claims

The following is claimed:

1. A device comprising:

(a) a piezoelectric module;

(b) a nanochannel comprising an inner surface having a width and a depth; and

(c) an electric circuit spanning the width of the nanochannel for evaluating the dielectric or charge density properties of a moiety presented within the inner surface of the nanochannel,

wherein the piezoelectric module is provided in electronic communication within the circuit.

3. The device of any preceding claim, wherein the nanochannel is a nanopore, a carbon nanotube, or graphene nanoribbon.

4. The device of any preceding claim, wherein the width of the inner surface of the nanochannel is about 3 nm or less.

5. The device of any preceding claim, wherein the piezoelectric module comprises a piezoelectric transducer, and wherein the electric circuit comprises an electric signal pathway, the device further comprising a static magnetic field source capable of producing a magnetic field, wherein the pathway for the charged polymer, the magnetic field, and the electric signal pathway intersect one-another, and are situated at right angles to one-another in a three dimensional orientation at a discrete examination location.

6. The device of claim 5, wherein the device comprises a Hall-effect imaging module.

7. A piezoelectric analysis device for use in analyzing a polymer, comprising:

(a) a first electrode and a second electrode comprising an electric circuit;

(b) a piezioelectric actuator in electronic communication with the electric circuit;

(c) a nanochannel having a proximal and distal portion that provides fluid communication between a proximal fluid reservoir and a distal fluid reservoir; and

(d) a piezoelectric actuator deflection measuring device,

wherein the first and second electrodes are positioned on opposing faces of the nanochannel, and wherein the piezioelectric actuator is operably connected to the piezoelectric actuator deflection measuring device to permit measurement of physical deflections of the piezoelectric actuator in response to electronic signals from the electric circuit.

8. The device of claim 7, wherein the nanochannel comprises an elongate orientation having a width or diameter of at least about 1.5nm.

9. The device of any one of claims 1-8, wherein the nanochannel comprises a stepped nanochannel comprising one or more stepped portion(s).

10. The device of claim 9, wherein the first and second electrodes comprise or are positioned adjacent to the stepped portion of the nanochannel.

11. The device of any one of claims 7-10, wherein a magnetic element is positioned in contact with, or proximal to the proximal fluid reservoir, wherein the magnetic element provides a selectable magnetic field extending through at least a portion of the nanochannel.

12. The piezoelectric analysis device of any one of claims 7-11, further comprising electrophoretic electrodes comprising a first electrode positioned in contact with the proximal fluid reservoir and a second electrode positioned in contact with the distal fluid reservoir.

13. The piezoelectric analysis device of any one of claims 7-1 1, wherein the piezoelectric actuator has a resonant frequency ranging between 10 kHz to 3 GHz.

14. The piezoelectric analysis device of any one of claims 7-12, wherein a charged polymer translocates through the nanochannel at a translocation rate defined as the rate at which any particular monomer in the polymer passes a defined point in the nanochannel, and the piezoelectric actuator has a resonant frequency to translocation rate ratio of between 1 : 1 to 1 : 10,000.

15. The piezoelectric analysis device of any one of claims 7-14, wherein the resonant frequency of the piezoelectric actuator is 3.3MHz, 6.6, MHz, 9,9 MHz, 13.2 MHz, or 16.5 MHz.

16. A device, comprising:

(a) a nanochannel defining a flow path having a proximal and a distal end opening and an examination location positioned between the proximal and distal end openings; and

(b) a piezoelectric actuator, or a cantilevered extension thereof, positioned in the examination location, wherein the piezoelectric actuator or cantilevered extension thereof comprises at least a portion of the flow path, and wherein the piezioelectric actuator is operably connected to a piezoelectric actuator deflection measuring device.

17. A method of identifying the sequence of a charged polymer, comprising introducing the charged polymer to the device of any one of claims 1-4 or 7-16, causing the translocation of the charged polymer between a pair of electrodes, passing an electric signal through each monomer of the charged polymer, and analyzing a physical deflection of the piezoelectric actuator to determine the identity of each monomer.

18. A method of identifying the sequence of a charged polymer, comprising introducing the charged polymer to the device of any one of claims 1-4 or 7-16, causing the translocation of the charged polymer between a pair of electrodes, passing an electric signal through each monomer of the charged polymer while in the presence of a static magnetic field situated transverse to the orientation of the electric signal and the polymer, and analyzing resulting ultrasonic pulses unique to each monomer within the polymer to determine the identity of each monomer.

19. A kit comprising one or more devices of any one of claims 1-16.

20. A system for analyzing a polymer, comprising:

(a) a nanochannel defining a pathway for a charged polymer;

(b) a static magnetic field source capable of producing a magnetic field; and

(c) an electric signal pathway,

wherein the pathway for the charged polymer, the magnetic field, and the electric signal pathway intersect one-another, and are situated at right angles to one-another in a three dimensional orientation at a discrete examination location.

21. The device or system of any one of claims 5, 6, or 18, further comprising one or more detector(s) capable of detecting ultrasonic pulses produced in the examination location.

22. The device or system of any one of claim s 5, 6, or 18, further comprising an element capable of producing an ultrasonic pulse that passes through the examination location.

23. The device or system of claim 21, wherein the detector comprises a piezoelectric transducer.

24. The device or system of claim 22, wherein the element capable of producing an ultrasonic pulse comprises a piezoelectric transducer.

25. The device or system of any one of claims 20-24, wherein the nanochannel comprises a graphene nanoribbon, a nanopore, or carbon nanotube.

26. The device or system of any one of claims 5, 6, or 25, wherein the nanochannel comprises a graphene nanoribbon, further comprising one or more detector(s) capable of detecting ultrasonic pulses produced by, or physical deflections caused within, the nanoribbon.

27. The device or system of any one of claims 20 to 26, wherein the magnetic field source has a magnetic strength of at least 0.7 Tesla.

28. The device or system of claim 27, wherein the magnetic field source has a magnetic strength of at least 7 Tesla.

29. The device or system of claim 27, wherein the magnetic field source has a magnetic strength of between about 7 Tesla and about 17 Tesla.

30. A kit for use with a strong static magnetic field source to provide for Hall-effect imaging of a polynucleotide, comprising a nanochannel defining a pathway for a charged polymer and an electric signal pathway, wherein the pathway for the charged polymer intersects the electric signal pathway at a right angle, and wherein the strong magnetic field source, when in use, produces a magnetic field that intersects the intersection of the electric signal pathway and the pathway for the charged polymer at a right angle to the electric signal pathway and the pathway for the charged polymer.

31. A method of identifying the dielectric or charge density properties of a charged polymer, comprising introducing the charged polymer to the system of any one of claims 5, 6, or 20-29, presenting at least a portion of the charged polymer within discrete examination location and conducting Hall-effect imaging of the charged polymer to thereby identify the dielectric or charge density properties of the charged polymer.

32. The method of claim 31, wherein the identified dielectric or charge density properties of the charged polymer provide information identifying each monomer of the polymer present in the discrete examination location.

33. A method of identifying the sequence of a charged polymer comprising the use of Hall- effect imaging.