Related Patent Application This patent application claims the benefit of U.S. provisional patent application no. 61/323,778 filed on April 13, 2010, entitled SYNTHETIC HYBRID NANOPORE DEVICES AND USES THEREOF, naming Eric Ervin as inventor and designated by Attorney Docket No. EBS-1001 -PV. The entire content of the foregoing patent application is incorporated herein by reference, including, without limitation, all text, tables and drawings.

Field

The technology relates in part to nanopore devices. Such devices are useful for sensing target molecules and sequencing biopolymers, for example.

Background

Devices having relatively small diameter channels can be used to detect small molecules and can be used to sequence biopolymers (e.g., DNA, RNA, peptides, polypeptides). Certain devices have channels with diameters in the nanometer range, and sometimes are referred to as "nanopore" devices. Such devices often are constructed from a non-conductive material such as glass, quartz, sapphire, silicon nitride, silicon dioxide, or graphene for example.

A molecule having an outer diameter less than the diameter of a channel in a nanopore device can be mounted within the channel. For example, a channel of a device may be loaded with a lipid bilayer and a membrane protein having a relatively small pore can be inserted in the bilayer. An example of such a membrane protein is alpha-hemolysin. Hence, a channel of such a device may be "filled" with another molecule having a relatively small pore, thereby reducing the effective diameter of an opening in the channel.

Summary

Described herein are synthetic hybrid nanopore (SHN) devices, and methods of manufacturing and using them. In some embodiments, provided are nanopore devices including: a solid support that includes a first surface, an opposing second surface, and a wall or walls between the first surface and the second surface, a channel in the solid support that includes a proximal opening at the first surface, a distal opening at the second surface and an interior sidewall surface, and a cyclic molecule attached effectively by a covalent linkage to a zone on the interior sidewall surface of the channel, whereby the cyclic molecule forms a pore in the channel.

In certain embodiments provided also are nanopore devices including: a membrane having a thickness with a first, exterior, side and a second, interior, side, the first side being opposite the second side, at least one nanopore extending through the membrane, thus forming at least one channel connecting the first side and the second side of the membrane, wherein the nanopore has a first opening, a second opening, a depth, an upper portion or zone on the interior sidewall surface within the at least one channel and a lower portion or zone on the interior sidewall surface within the at least one channel, wherein the first opening opens to the first side of the membrane, and the second opening opens to the second side of the membrane, wherein the surface properties of the upper portion within the at least one nanopore are chemically modified with a first set of modifying molecules, the first set of modifying molecules are covalently attached to at least one cyclic molecule, wherein the diameter of the nanopore device is determined by the internal diameter of the cyclic molecule. In some embodiments, the surface properties of the first, exterior, side are chemically modified with a second set of modifying molecules. In certain embodiments, the surface properties of the lower portion within the at least one nanopore are chemically modified with a third set of modifying molecules. In certain embodiments, the second set of modifying molecules and the third set of modifying molecules are the same molecules.

Also provided in some embodiments are processes for preparing a nanopore device that include: providing a solid support that includes a first surface, an opposing second surface, and a wall or walls between the first surface and the second surface, a channel in the solid support that includes a proximal opening at the first surface, a distal opening at the second surface and an interior sidewall surface; and a plug within the channel that includes a proximal end and a distal end; chemically protecting the first surface, the second surface, and/or the wall or walls; removing a portion of the proximal end of the plug, thereby producing a shortened plug and revealing a first zone on the interior sidewall of the channel in proximity to the proximal opening; contacting the first zone with a cyclic molecule under attachment conditions, whereby the cyclic molecule is attached effectively by a covalent linkage to the interior surface of the channel in the zone, and whereby the cyclic molecule forms a pore in the channel; and removing the shortened plug. In some embodiments, provided also are methods of fabricating a nanopore device that include: producing at least one nanopore extending through a membrane that has a thickness with a first, exterior, side and a second, interior, side, the first side being opposite the second side, thus forming at least one channel connecting the first side and the second side of the membrane, wherein the nanopore has a first opening, a second opening, a depth, an upper portion or zone on the interior sidewall surface within the at least one channel and a lower portion or zone on the interior sidewall surface within the at least one channel, wherein the first opening opens to the first side of the membrane, and the second opening opens to the second side of the membrane, measuring the size of the first opening of the nanopore, modifying the first, exterior side of the membrane with a first set of modifying molecules, modifying the upper portion within the at least one nanopore with a second set of modifying molecules, wherein the second set of modifying molecules are chosen based on the measured size of the nanopore and the outer diameter of a cyclic molecule to be bound to the second set of modifying molecules, modifying the lower portion within the at least one nanopore with a third set of modifying molecules, and delivering the cyclic adapter to the second set of modifying molecules and covalently binding the cyclic adapter to the second set of modifying molecules. In certain embodiments, the first set of modifying molecules and the third set of modifying molecules are the same molecules. In certain embodiments, also provided is a kit, including a nanopore device as described herein and directions for detecting an analyte using the nanopore device.

Provided also in certain embodiments are methods that include: contacting a nanopore device with a composition that includes or does not include an analyte, in which the nanopore device includes a solid support that includes a first surface, an opposing second surface, and a wall or walls between the first surface and the second surface; a channel in the solid support that includes a proximal opening at the first surface, a distal opening at the second surface and an interior sidewall surface; and a cyclic molecule attached effectively by a covalent linkage to a zone on the interior sidewall surface of the channel, whereby the cyclic molecule forms a pore in the channel; measuring an electric current applied to the nanopore device, thereby determining an electric current measurement; and detecting the presence, absence or amount of the analyte based on the electric current measurement. In some embodiments, the analyte is selected from small organic molecules, organic molecule enantiomers, small biological molecules, solid particles, nucleosides, nucleotide monophosphates, chemical and biological warfare agents, toxins, small molecules, and the like and combinations thereof. In certain embodiments, the analyte is DNA and/or RNA. In some embodiments, detecting the analyte determines a nucleotide sequence.

In some embodiments, a nanopore device comprises a coating between the interior sidewall surface and the cyclic molecule. The coating sometimes is (i) associated with the interior sidewall surface by covalent attachment and (ii) joined to the cyclic molecule. In certain embodiments, the coating is joined to the cyclic molecule by a covalent attachment. In some embodiments, the coating is of a thickness substantially equal to thickness theta, where theta = (C _D - CM ₀ D)/2, C _d is the diameter of the channel at the zone, and CM ₀ D is the outer diameter of the cyclic molecule. In certain embodiments, the density of the coating is sufficient to substantially prevent significant ion current flow around the cyclic molecule. In some embodiments, the zone is in proximity to the proximal opening of the channel. In certain embodiments, the diameter of the pore in the channel is substantially equal to the inner diameter of the cyclic molecule. In some embodiments, the solid support includes glass. In certain embodiments, the glass is chosen from fused silica glass, ninety-six percent silica glass, soda-lime silica glass, borosilicate glass, aluminosilicate glass, lead glass, doped glass comprising desired additives, functionalized glass comprising desired reactive groups, the like and combinations thereof. In some

embodiments, the solid support includes a mineral. In certain embodiments, the mineral is quartz. In some embodiments, the quartz is chosen from quartz, tridymite, cristobalite, coesite, lechatelierite, stishovite. In some embodiments, the solid support includes sapphire, silicon nitride, silicon dioxide, or graphene.

In certain embodiments, one or more of the first surface, second surface, wall or walls comprise a chemical protecting agent attached thereto in order to prevent adsorption of the cyclic molecule and non-specific absorption of other molecules. In some embodiments, the chemical protecting agent is a non-reactive chemical group. In certain embodiments, the non-reactive chemical group comprises a silane. In some embodiments, the non-reactive chemical group includes a terminal cyano group or a terminal methyl group.

In certain embodiments, the cyclic molecule is comprised of one cyclic molecule. In certain embodiments, the cyclic molecule includes two or more cyclic molecules. In some embodiments, the two or more cyclic molecules include a vertical stacking arrangement. In certain embodiments, the cyclic molecule is selected from cyclodextrins (alpha, beta, gamma), cyclic peptides, crown ethers, porphyrins, cycloalkanes, carbon nanotubes, calixarenes, organic and/or non-organic cyclic chemical structures, biological cyclic structures (e.g., protein pores, ion channels), the like and combinations thereof. In some embodiments, the coating includes silanes, thiols, reactive groups added to or present on the solid support, or compounds having a length that can be chemically controlled and having ends covalently bound to the cyclic molecule on one end and the solid support on the other. In certain embodiments, the silane has a chain length of between about 0.1 nanometers and about 50 nanometers (e.g., about 0.5 nanometers to 1 .2 nanometers in length). In some embodiments, the channel in the solid support has a diameter between about 1 nanometer and about 50 nanometers. In certain embodiments, the nanopore generated by the attachment of the cyclic molecule to the zone on the interior sidewall surface of the channel has a diameter between about 0.1 nanometers and about 20 nanometers. In some embodiments, the coating is attached by an irreversible covalent linkage to the channel. In some embodiments, the coating is attached by a reversible covalent linkage to the channel. In certain embodiments, the coating is attached by a reversible covalent linkage to the cyclic molecule. In some embodiments, the reversible covalent linkage is generated by a reaction selected from an amine reacting with a N-Hydroxysuccinimide (NHS) ester, an imidoester, a pentafluorophenyl (PFP) ester, a hydroxymethyl phosphine, carbonyl compounds; a carboxyl reacting with a carbodiimide; a sulfhydryl reacting with a maleimide, a haloacetyl, a pyridyldisulfide, and/or a vinyl sulfone; an aldehyde reacting with a hydrazine; any non-selective group reacting with diazirine and/or aryl azide; a hydroxyl reacting with isocyanate; a hydroxylamine reacting with a carbonyl compound, the like and combinations thereof. In certain embodiments, the cyclic molecule is directly attached to the zone on the interior sidewall surface of the channel by a reversible covalent attachment.

In some embodiments, a nanopore device further includes a second zone to which one or more activities carried out by one or more functional molecules is covalently attached. In certain embodiments, the one or more activities is an enzymatic activity. In some embodiments, the one or more functional molecules is an enzyme. In certain embodiments, the one or more activities is selected from a polymerase activity, a helicase activity, an endonuclease activity, an exonuclease activity, a nickase activity, and combinations thereof. In some embodiments, the one or more functional molecules is selected from a polymerase enzyme, a helicase enzyme, an endonuclease enzyme, an exonuclease enzyme, a nicking enzyme and combinations thereof. In certain embodiments, the one or more activities are functionally coupled (e.g., chimeric molecule having multiple activities; each activity is provided by a separate molecule, and the separate molecules are linked to the nanopore device in a particular zone). In some embodiments, the second zone is proximal to (on either side of) the zone to which the cyclic molecule is effectively linked by reversible covalent attachment.

Certain embodiments are described further in the following description, examples, claims and drawings.

Brief Description of the Drawings

The drawings illustrate embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

FIG 1 shows a synthetic hybrid nanopore (SHN) that includes at least one single cyclic molecule covalently attached to the small opening in a conical-shaped glass nano-dimension pore. FIG 2 shows a cross-sectional view of a SHN containing two different covalently attached cyclic molecules.

FIG 3 shows a schematic drawing of fabrication steps that can be used to produce a SHN embodiment. Covalent silane linkers are depicted in the top portion (proximal portion) of the channel, and a non-reactive silane layer is depicted on the outer surfaces of the device and within the channel at a lower portion (distal portion of the channel). An internal non-reactive silane layer is not shown in step 4.

FIG 4 shows a cross-section view of a SHN containing two different covalently attached molecules, one a cyclic molecule and one a biomolecule with a functional activity (e.g., an enzyme with enzyme activity). An example of the embodiment shown in FIG 4 is described in the section entitled "Synthetic Hybrid Nanopore Devices with Functional Molecules". Detailed Description

A non-limiting embodiment of a synthetic hybrid nanopore device is shown in FIG 1. A synthetic hybrid nanopore device 10 (SHN device, e.g., depicted in FIG 1 ) can include at least one cyclic molecule 14 covalently (reversibly or irreversibly) attached to and spanning the aperture 15 (e.g., proximal opening) of a conical-shaped channel 18 penetrating a solid support 1 1 (e.g., thin membrane). Conical-shaped channel 18 includes distal opening 19. The void of cyclic molecule 14, forms pore 16. A solid support 1 1 (e.g., thin membrane) can include a highly resistive material, including, but not limited to, glass, quartz, diamond, sapphire, silicon oxide, silicon nitride, aluminum oxide (alumina), Teflon, polycarbonate, or other polymer film. A solid support 1 1 may be chemically derivatized (e.g., derivatized without a coating), and include free amine, carboxy, thiol reactive groups and the like. A solid support 1 1 can be of any useful shape and the cross section of a channel can be of any suitable shape (e.g., conical, inverse cone, frustrum, cylinder and the like). The diameter of a channel opening in a solid support sometimes is about 1 nanometer to about 50 nanometers (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45 nanometers), as shown in FIG 1.

A SHN can be chemically modified such that it includes an analyte specific binding or "sensing" site. A SHN often can be provided in substantial dry form, and often such a SHN is rehydratable. A SHN device provides several advantages detailed in the table hereafter.

An effective pore size of a SHN often is determined by the internal diameter of a covalently attached cyclic molecule 14. A cyclic molecule 14 can be any small cyclic molecule that allows for ionic flow from one side of the molecule, through the molecule itself, to the other side of the molecule, in an electrically conductive solution. Non-limiting examples of cyclic molecules 14 include cyclodextrins (alpha, beta and gamma), cyclic peptides, crown ethers, porphyrins, cycloalkanes, carbon nanotubes, calixarenes, and all other organic and/or non-organic cyclic chemical structures, in addition to biological cyclic structures, such as protein pores and ion channels. A cyclic structure that includes chemical specificity on its outer edge to allow it to be covalently attached to the interior sidewall 20 or zone 13 of the channel within the solid support 1 1 can be selected. The outer diameter of a cyclic molecule sometimes is about 0.1 nanometers to about 20 nanometers (e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2.0, 3.0, 4.0, 5.0 nanometers). The inner diameter of a cyclic molecule is about 0.1 nanometer to about 19 nanometers (e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 nanometers).

A cyclic molecule 14 sometimes is attached directly by covalent linkage to a zone 13 (e.g., proximal portion of interior sidewall surface 20, near proximal opening) in the channel. In cases where a channel diameter approximately matches the outer diameter of the cyclic molecule, a tether 12 (described herein) sometimes is not utilized. In certain embodiments, a cyclic molecule analog that allows for covalent attachment of the cyclic molecule to the interior sidewall surface 20 of the thin membrane sometimes is selected. In some embodiments, a solid support 1 1 having a derivitized surface (e.g., derivitized glass) sometimes is provided and the cyclic molecule 14 is chemically reacted and attached directly to the channel of the solid support.

In some embodiments, attachment of the cyclic molecule 14 to the thin membrane 1 1 occurs via a chemical tether 12. A chemical tether 12 can be applied in a coating in the channel of the solid support 1 1 , in certain embodiments. A chemical tether 12 often is chosen from a group of silanes of the appropriate length. Silanes generally bind to a glass surface via a silicon-oxygen covalent bond. A silane may include one or more chemical substituents other than hydrogen, in some embodiments, including, but not limited to alkyl, alkoxy, cyano, carboxy, ester, amino and the like. In certain embodiments, a silane has a structure according to Formula I:

R Formula I or a salt or isomer thereof, where:

each R ¹ , R ² and R ³ independently is hydrogen, -OH, C1 -C20 alkyl, substituted C1 -C20 alkyl, C1 -C20 alkoxy, substituted C1-C20 alkoxy, -C(O)- C1 -C20 alkyl (alkanoyl), substituted -C(O)- C1-C20 alkyl, -C(O)- C6-C10 aryl (aroyl), substituted -C(O)- C6-C10 aryl, -C(0)OH (carboxyl), - C(0)0- C1-C20 alkyl (alkoxycarbonyl), substituted -C(0)0- C1-C20 alkyl, -NR ^a R ^b , -C(0)NR ^a R ^b (carbamoyl), substituted C(0)NR ^a R ^b , halo, nitro, or cyano,

the substituents on the alkyl, aryl or heterocyclic groups are hydroxy, C1 -C10 alkyl, hydroxyl C1 -C10 alkylene, C1 -C6 alkoxy, C3-C6 cycloalkyl, C1 -6 alkoxy C1-6 akylene, amino, cyano, halogen or aryl;

each R ^a and R ^b independently is hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, halo C1 -C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkanoyl, hydroxy C1-C6 alkyl, aryl, aryl C1 -C6 alkyl, Het, Het C1 - C6 alkyl, or C1-C6 alkoxycarbonyl; and

X is a leaving group (e.g., hydroxyl, halogen (e.g., bromine, chlorine, fluorine, iodine)). In certain embodiments a silane is 3-cyanopropyldimethylchlorosilane.

A tether 12 can provide a mechanism for covalently attaching a cyclic molecule 14, and can also decrease the channel size in the solid support 1 1. In some embodiments, a tether 12 is selected in part on length, where the length sometimes is about equal to about half of the subtraction result of inner diameter of the channel in the solid support 1 1 less the outer diameter of the cyclic molecule 14. In the latter embodiments, selection of such a tether may prevent significant ion current flow around the cyclic molecule. When fabricating a SHN device 10 out of glass, non-limiting examples of tethers 12 include silanes, thiols, or any other linear or branch chain chemical compound having a length that can be chemically controlled and having bifunctional ends (e.g., ends that can be covalently bound to the cyclic molecule on one end and the solid support on the other). The outer diameter or effective length of a tether molecule sometimes is about 0.1 nanometer to about 20 nanometers (e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10 nanometers). Methods of attaching a chemical tether 12 (e.g. silanes) to a cyclic molecule 14 and/or a channel, which can be referred to as "crosslinking," can occur between a reactive group at the end of the tether 12 and a complementary reactive group on (i) on the outside of the cyclic molecule 14, and/or (ii) channel solid support 1 1. This attachment, which results in the covalent attachment of the cyclic molecule 14 (e.g. direct or indirect attachment to the solid support), can be reversible, meaning that that the covalent bond formed can be broken by a secondary chemical reaction, or irreversible. Methods of forming the covalent attachment of the cyclic molecule at the orifice of the solid support includes, but are not limited to, an amine reacting with a N-Hydroxysuccinimide (NHS) ester, an imidoester, a pentafluorophenyl (PFP) ester, a hydroxymethyl phosphine, an oxiran or any other carbonyl compound; a carboxyl reacting with a carbodiimide; a sulfhydryl reacting with a maleimide, a haloacetyl, a pyridyldisulfide, and/or a vinyl sulfone; an aldehyde reacting with a hydrazine; any non-selective group reacting with diazirine and/or aryl azide; a hydroxyl reacting with isocyanate; a hydroxylamine reacting with a carbonyl compound and the like.

A SHN device may include one or more zones arranged on the interior sidewall of the channel between the distal opening and proximal opening of the channel to which one or more types of molecules can be effectively conjugated. In some embodiments, a SHN device comprises one zone to which a cyclic molecule is effectively conjugated. In the latter embodiments, the zone often is configured around the entire circumference of the inner surface of the channel, and the length of the zone is a portion of the channel length. There sometimes is no other zone to which another type of molecule is effectively attached. In certain embodiments, a SHN device comprises two or more zones in the channel. In the latter embodiments, each zone often is configured around the entire circumference of the inner surface of the channel, the length of each zone often is a portion of the channel length, two zones sometimes are contiguous or non-contiguous with one another, and one zone often is located proximal to another zone. In some embodiments, a cyclic molecule may be in effective connection with a first zone, and a another type of molecule (e.g., a functional molecule) may be in effective connection with a second zone. In some embodiments, the first zone may be located proximal to the second zone in the channel, and in certain embodiments, the second may be located proximal to the first zone in the channel.

Fabricating Synthetic Hybrid Nanopore Devices

In some embodiments fabrication of a SHN device 10 can include some or all of the following general steps. A nanopore often is synthesized within a solid support 1 1 (e.g., thin membrane). The channel 18 generally is conical in cross section, although other geometries also are acceptable.

In some embodiments, a nanopore or channel is generated within a thin membrane 1 1 by sealing an etched metal tip into the membrane, as shown in FIG 3 and described in Example 1. In certain embodiments, the inside of the nanopore is filled via a plug or other like device or is chemically altered with a molecule that prohibits modification of the inside of the pore. In some embodiments, a pore is filled with an etched tip prior to modifying the outside surface, or would be plugged prior to coating to prevent the coating applied to the outside from entering the pore. The outside surface of the membrane often is chemically protected, leaving the inside of the pore substantially unmodified, in some embodiments. This protection can be carried out by coating the membrane with a non reactive chemical group, such as a silane terminating in a cyano or methyl group. This protection also can prohibit functionalized tethers, introduced later, from coating the outside of the membrane.

The nanopore plug often is removed. The plug (e.g., metal electrode, gel, chemical coating, polymer, and the like) can be removed by a suitable method (e.g., electrochemically, chemically, mechanically, and the like), leaving the unmodified nanopore. In many cases only the top portion of the plug will be removed initially, thus allowing only chemical modification of only a portion of the nanopore aperture.

The diameter of the nanopore often is measured. The diameter of the nanopore can be measured by any appropriate procedure, including but not limited to electrochemical and/or microscopy methods, and the like.

An appropriate length for optional tether 12 molecules (e.g., coating) is chosen in some embodiments. The length of the tether 12 (i.e. silane, thiol, and the like) sometimes is chosen to decrease the pore size to approximately match the outer diameter of the cyclic molecule that eventually is attached to the tethers and prevent significant ion current flow around the molecule. After one or more tethers are selected, the inside aperture of the nanopore often is modified with a selected tether(s) 12 of choice in certain embodiments.

The outside of a nanopore often is initially modified with a non-reactive chemical group that prevents the cyclic molecule from eventually binding to anywhere but the interior sidewall of the channel 20 or a zone 13. The inside of the pore often is modified by placing the membrane in a solution containing the chemical agent. Sonication, pressure, temperature, initially filling the pore with an alcohol and the like can be used to aid pore wetting. In some embodiments, these steps can be repeated to provide two or more zones for attachment. In some embodiments, these steps can be repeated to provide a mechanism for two or more types of cyclic molecules 14 and 14' to be attached, as shown in FIG 2. In some embodiments with two or more cyclic molecules, cyclic molecules 14 and 14' are the same type of molecule. In some embodiments, cyclic molecules 14 and 14' are different molecules. In some embodiments, cyclic molecule 14' is attached with tether 12' molecules. In some embodiments, tether 12' molecules and tether 12 molecules are the same molecules. In some embodiments, tether 12' molecules and tether 12 molecules are different molecules.

The rest of the nanopore plug often is removed as depicted in FIG 3 and described in Example 1.

The rest of the pore often is chemically protected. The internal portion of the nanopore, below the aperture, and/or the other side of the membrane can be chemically protected, as described in Example 1 and depicted in FIG 3. Synthetic Hybrid Nanopore Devices with Functional Molecules

In some embodiments, a nanopore device comprises a cyclic molecule 14 effectively attached to an inner surface of the channel (e.g., effective covalent attachment to the channel), often via conjugation to a functional molecule 22. In some embodiments, a nanopore device further includes a second zone 23 to which one or more activities carried out by one or more functional molecules 22, which functional molecules often are covalently attached (e.g., effectively attached or directly attached), as depicted in FIG 4 for example. In certain embodiments, the one or more activities is an enzymatic activity. In some embodiments, the one or more functional molecules 22 is an enzyme. In certain embodiments, the one or more activities is selected from a polymerase activity, a helicase activity, an endonuclease activity, an exonuclease activity, a nickase activity, ligase activity, phospatase activity, transferase activity and combinations thereof. In some embodiments, the one or more functional molecules is selected from a polymerase enzyme, a helicase enzyme, an endonuclease enzyme, an exonuclease enzyme, a nicking enzyme, ligase enzyme, phosphatase enzyme, transferase activity, the like and combinations thereof. In certain embodiments, the one or more activities are functionally coupled (e.g., chimeric molecule having multiple activities; each activity is provided by a separate molecule, and the separate molecules are linked to the nanopore device in a particular zone). In some embodiments, the second zone 23 is proximal to the zone to which the cyclic molecule 14 is linked by reversible or non-reversible covalent attachment. In some embodiments, the second zone 23 is distal to the zone 13 to which the cyclic molecule 14 is linked by reversible or non-reversible covalent attachment.

In some embodiments a channel is generated using a method described herein for the synthetic hybrid nanopore containing one cyclic molecule. In some embodiments the channel comprises two covalent attachment zones 13 and 23 at its proximal opening. In zone 13 a cyclic molecule 14 can be attached. In some embodiments, cyclic molecule 14 can be attached via tether 12 molecules. In zone 23 an enzyme 22, as defined above, can be attached. In some embodiments, enzyme 22 can be attached via tether 21 molecules. In some embodiments a cyclic molecule 14 can be attached proximal to the enzyme. In some embodiments, the enzyme is an exonuclease that interacts with a single-stranded DNA (ss-DNA) and systematically cleaves off single nucleosides monophosphates. In the latter embodiments the enzyme, or exonuclease, interacts with the single ss-DNA and cleaves off a base that then is detected by the cyclic molecule.

In some embodiments the enzyme 22 can be a molecule that ratchets DNA in a specific direction. As an example, an enzyme 22 located directly above or below the cyclic molecule 14 interact with nucleic acid (e.g., DNA or RNA) and slowly pull or push a nucleic acid strand into or out of the cyclic molecule 14 in a controlled manner.

The enzyme 22 can be any molecule that manipulates another analyte molecule in a specific manner, by chemically modifying it or moving it, for example, such that the analyte molecule can then interact with the cyclic molecule 14. The movement of the analyte molecule by the enzyme 22 can be consistent or stochastic in terms of movement length and time of movement. In some embodiments, the enzyme 22 interacts with the analyte molecule prior to being introduced to the cyclic molecule 14 and in certain embodiments the enzyme interacts with the analyte molecule as it, or after it, leaves the cyclic molecule 14.

Methods for attaching tether 23 molecules (e.g. silanes, thiol, or polyethylen glycol (PEG)) to an enzyme 22 or multiple enzymes, which can be referred to as "crosslinking," can occur between a reactive group at the end of the tether and a complementary reactive group on the enzyme 22. This attachment, which results in the covalent attachment of the enzyme 22 (e.g. direct or indirect attachment to the solid support), can be reversible, meaning that that the covalent bond formed can be broken by a secondary chemical reaction, or irreversible. Methods of forming the covalent attachment of the enzyme to zone 23 include, but are not limited to, standard bioconjugate techniques, a biotin group at the end of the tether attaching to an avidin-labeled enzyme, a avidin group at the end of the tether attaching to a biotin labeled enzyme, short urea linkage, thiol chemistry, an amine reacting with a N-Hydroxysuccinimide (NHS) ester, an imidoester, a pentafluorophenyl (PFP) ester, a hydroxymethyl phosphine, an oxiran or any other carbonyl compound; a carboxyl reacting with a carbodiimide; a sulfhydryl reacting with a maleimide, a haloacetyl, a pyridyldisulfide, and/or a vinyl sulfone; an aldehyde reacting with a hydrazine; any non-selective group reacting with diazirine and/or aryl azide; a hydroxyl reacting with isocyanate; a hydroxylamine reacting with a carbonyl compound and the like.

In some embodiments, two attachment zones 13 and 23, differing in chemistry can be generated inside of a channel. Under aqueous conditions, in the presence of different or varying electrolyte and buffer conditions, a cyclic molecule 14 can be added to the channel so that the cyclic molecule 14 can bind to attachment zone 13. The remaining cyclic molecules in solution then can be rinsed away. Following attachment of the cyclic molecule 14 under aqueous conditions, in the presence of different or varying electrolyte and buffer conditions enzyme 22 then can be added to the channel so that the enzyme can bind to attachment zone 23. After attachment, the remaining enzymes in solution can be rinsed away. Temperature, pressure, voltage, and concentration gradients can be used to aid in the attachment of each of the cyclic molecule 14 and the enzyme 22 to its respective attachment zone. In some embodiments, the enzyme 22 can be attached inside of the channel before attaching the cyclic molecule 14, and in some embodiments, the enzyme 22 can be attached after attaching the cyclic molecule 14 inside the channel. In some embodiments, the enzyme 22 can be attached to an attachment zone 23 without attaching a cyclic molecule 14.

Synthetic Hybrid Nanopore Device Applications Synthetic hybrid nanopore devices can be used for detection, differentiation and sequencing applications for example. Such applications can occur as an analyte molecule of interest interacts with or translocates through the nanopore orifice.

A nanopore can be used as an analyte sensor in resistive-pulse method or stochastic sensing applications. A nanopore sensor often utilizes a direct current (DC) potential across a membrane which contains a nanopore, and separates two electrolyte solutions. The DC potential produces an ionic current flow through the nanopore, the magnitude of this current being determined by the geometry of the nanopore, the internal charge characteristics of the nanopore, and the conductivity of the electrolyte solution within the nanopore. When an analyte molecule, which is contained in the electrolyte solution, blocks or translocates the nanopore, the conductance of the nanopore decreases, resulting in a decrease in the DC current response. The frequency of

blocking/translocating events yields the analyte concentration, while the magnitude and duration of the blocking/translocating events provide information about the identity of the analyte. In some embodiments a SHN is used in an aqueous medium. Electrodes placed on both sides of the nanopore orifice are used to hold either a direct current (DC) voltage or an alternating current (AC) voltage across the nanopore. Analyte detection, differentiation, sequencing, and the like is determined by monitoring and analyzing the fluctuations in the time varying current response. A SHN can be used as a nanopore sensor element for numerous applications including, but not limited to, the detection and discrimination of small organic molecules, organic molecule enantiomers, small biological molecules, solid particles, nucleosides, nucleotide monophosphates, chemical and biological warfare agents, toxins, small molecules, and the like. In addition, it can be used to directly sequence DNA, ss-DNA, RNA, peptides, and proteins. In some embodiments, detection of the analyte determines a nucleotide sequence.

In the application of a SHN, a conductive solution or electrolyte often fills, or is in contact with, the interior and exterior of a SHN. The electrolyte sometimes is composed of ionic salts dissolved in a liquid. Ionic salts include, but are not limited to, sodium chloride (NaCI), potassium chloride (KCI), lithium chloride (LiCI), sodium bromide (NaBr), and the like. The liquid component of the electrolyte often is water but may be any other suitable liquid in which a salt is soluble. The electrolyte may also be composed of an ionic liquid. Examples of ionic liquids include, but are not limited to, 1-Butyl-3-methylimidazolium hexafluorophosphate, 1 -butyl-3,5-dimethylpyridinium bromide, and the like.

To measure the conductivity across a SHN, electrodes are placed interior to and exterior to a SHN. The electrodes are in contact with the conductive liquid on the interior and exterior sides. Typical electrode materials include but are not limited to Ag/AgCI, platinum, gold, carbon, and the like. An AC or DC voltage bias is applied across the two electrodes and the resulting current through a SHN is measured.

A non-limiting example of a nanopore commonly used in non-SHN device sensing experiments includes the transmembrane protein alpha hemolysin (alpha-HL). Sensing experiments can be performed by inserting an alpha-HL channel into a synthetic planar lipid bilayer (PLB) suspended over the orifice of a synthetic support, and measuring the conductance of the channel. These alpha-HL protein channels have been shown to detect metal ions, DNA segments, proteins of various sizes, and various organic molecules. Due to the reproducibility in pore size and the ability to chemically or biologically engineer the pore such that it contains an analyte specific binding site, these experiments can be used for comparison to SHN devices. Examples

The examples set forth below illustrate certain embodiments and do not limit the technology. Example 1: Preparation of Synthetic Hybrid Nanopore Devices

A detailed step-by-step method for fabricating a SHN, starting with a glass nanopore electrode is described within this section and shown in FIG 3. Fabrication of nanopore electrodes are known and fabrication of an ultra small (<10 nm radii) ultra shallow (<50 nm depths) nanopore electrode is described below.

1. A Pt wire is electrochemically etched, in a NaCN solution in order to produce a sharp conical- shaped tip. 2. The sharpened Pt tip is etched a second time in a dilute H ₂ S0 ₄ solution, in order to produce an ultra sharp (with radii of curvature below 5 nm) Pt tip, free of an oxide layer.

3. The sharpened Pt wire is inserted into a glass capillary (quartz and sapphire capillaries may also be used).

4. The end of the capillary containing the sharpened Pt tip is flame sealed using an H ₂ flame, H ₂ /0 ₂ flame, laser, or other heating source.

5. The sealed glass bulb is polished down to the Pt wire, exposing a very small Pt disk electrode.

6. The outside glass surface is modified, via silane chemistry. Typically, a silane that terminates in a CN functional group is used, but any silane that terminates in a hydrophobic group can be used. This silane is known for its non-reactive nature and ability to protect glass surfaces from

nonspecific absorption. Silanizing the outer surface of the nanodisk electrode, prior to etching the Pt nanodisk electrode, will prevent the cross linking silane that will be placed inside nanopore, from coating the outer surface.

7. The Pt nanodisk electrode is etched to form a shallow, nanopore electrode. Here, an etching procedure developed by, Sun and Mirkin for removing a very thin (> 1 nm thick) layer of Pt is used. This is accomplished by using an etching solution containing 60% (by volume) distilled water, 30% 5 M CaCI ₂ , and 10% HCI is used in combination with a 1.5 V amplitude, 2 MHz frequency AC waveform, applied for various amount of time, to etch very shallow recessed Pt electrodes. Once the small shallow nanopore electrodes are fabricated, they can then be converted into SHNs, as depicted in FIG 3. These SHN will contain at least a single cyclic molecule (cyclodextrin (CD)) covalently bound to the opening aperture of a nanopore. The covalent bonds formed can be irreversible or reversible depending on the application. The inside of the shallow glass pore is silanized using a cross linker terminated silane (terminating in -NH ₂ , -ONH ₂ , -SH, or other chemical crosslinking group). The length of silane tether is pore diameter specific, meaning that the silane length is chosen so that the CD/cross linker extends across the nanopore opening. This dense internal silane layer not only provides the mechanism for covalently attaching the CD, but also decreases the pore size to approximately match that of the CD outer diameter and prevent significant ion current flow around the CD.

After the internal walls of the shallow support structure are silanized, the rest of the internal Pt is removed via electrochemical etching and mechanical methods. The unmodified interior surface of the nanopore membrane can then be chemically protected with a cyano silane, known for its non- reactive nature and ability to protect glass surfaces from nonspecific absorption. This prevents the cyclodextrin from binding in unwanted areas. Other silanes or surface treatments may also be used or the surface may remain unmodified.

Once the site specific chemically modified nanopore membrane, FIG 3 (step 3), is fabricated, a cyclic molecule containing one or more covalent linkers on its outer edge, will be covalently attached to the inside of the chemically modified nanopore membrane, producing a SHN. In some embodiments, the attachment will typically occur under aqueous conditions with the CD typically being placed inside of the chemically modified nanopore membrane. Voltage, pressure, and electrolyte gradients will be used to drive the CD to the opening aperture of the GNM and control the attachment. CD attachment at the pore aperture is monitored by measuring ionic current flow across the chemically modified GNM orifice. Trapping the CD, meaning one or more cross linking tethers on the CD have reacted with their chemical targets, can take anywhere from 10 minutes to 4 hours, depending on the cross linker used. Covalent attachment is determined by the result of a steady-state current that is independent of voltage and internal pressure. The entire fabrication process of a SHN, starting with an ultra small ultra shallow glass nanopores electrode is depicted in FIG 3.

Example 2: Examples of Embodiments

Provided hereafter are certain non-limiting embodiments of the technology.

A1. A nanopore device, comprising:

a solid support that includes a first surface, an opposing second surface, and a wall or walls between the first surface and the second surface;

a channel in the solid support that includes a proximal opening at the first surface, a distal opening at the second surface and an interior sidewall surface; and

a cyclic molecule attached effectively by a covalent linkage to a zone on the interior sidewall surface of the channel, whereby the cyclic molecule forms a pore in the channel.

A2. The nanopore device of embodiment A1 , comprising a coating between the interior sidewall surface and the cyclic molecule, which coating is (i) associated with the interior sidewall surface by covalent attachment and (ii) joined to the cyclic molecule. A3. The nanopore device of embodiment A2, wherein the coating is joined to the cyclic molecule by a covalent attachment.

A4. The nanopore device of embodiment A2 or A3, wherein the coating is of a thickness substantially equal to thickness theta, wherein:

theta = (C _D - CM _OD )/2,

C _D is the diameter of the channel at the zone, and

CMOD is the outer diameter of the cyclic molecule.

A5. The nanopore device of any one of embodiments A2 to A4, wherein the density of the coating is sufficient to substantially prevent significant ion current flow around the cyclic molecule.

A6. The nanopore device of any one of embodiments A1 to A5, wherein the zone is in proximity to the proximal opening of the channel. A7. The nanopore device of any one of embodiments A1 to A6, wherein the diameter of the pore in the channel is substantially equal to the inner diameter of the cyclic molecule.

A8. The nanopore device of any one of embodiments A1 to A7 wherein the solid support comprises glass.

A9. The nanopore device of embodiment A8, wherein the glass is chosen from fused silica glass, ninety-six percent silica glass, soda-lime silica glass, borosilicate glass, aluminosilicate glass, lead glass, doped glass comprising desired additives, functionalized glass comprising desired reactive groups, the like and combinations thereof.

A10. The nanopore device of any one of embodiments A1 to A6, wherein the solid support comprises a mineral. A1 1. The nanopore device of embodiment A10, wherein the mineral is quartz.

A12. The nanopore device of embodiment A1 1 , wherein the quartz is chosen from quartz, tridymite, cristobalite, coesite, lechatelierite, stishovite A13. The nanopore device of any one of embodiments A1 to A12, wherein one or more of the first surface, second surface, wall or walls comprise a chemical protecting agent attached thereto.

A14. The nanopore device of embodiment A13, wherein the chemical protecting agent is a non- reactive chemical group.

A15. The nanopore device of embodiment A14, wherein the non-reactive chemical group comprises a silane.

A16. The nanopore device of embodiment A15, wherein the non-reactive chemical group comprises a terminal cyano group or a terminal methyl group.

A17. The nanopore device of any one of embodiments A1 to A16, wherein the cyclic molecule comprises two or more cyclic molecules. A18. The nanopore device of embodiment A17, wherein the two or more cyclic molecules comprise a vertical stacking arrangement.

A19. The nanopore device of any one of embodiments A1 to A18, wherein the cyclic molecule is selected from a cyclodextrin (α, β, or y), cyclic peptide, crown ether, porphyrin, cycloalkane, carbon nanotube, calixarene, organic and/or non-organic cyclic chemical structure, biological cyclic structure (e.g., protein pore, ion channel), and the like and combinations thereof.

A20. The nanopore device of any one of embodiments A1 to A18, wherein the cyclic molecule is a cyclodextrin (α, β, or y).

A21. The nanopore device of any one of embodiments A1 to A20, wherein the coating comprises a silane, thiol, reactive group added to or present on the solid support, or a compound having a length that can be chemically controlled and having ends covalently bond to the cyclic molecule on one end and the solid support on the other.

A22. The nanopore device of any one of embodiments A1 to A20, wherein the coating comprises silane. A23. The nanopore device of embodiment A22, wherein the silane has a chain length of between about 0.1 and about 50 nanometers.

A24. The nanopore device of any one of embodiments A1 to A23, wherein the coating is attached by a reversible covalent linkage to the channel.

A25. The nanopore device of any one of embodiments A 1 to A24, wherein the coating is attached by a reversible covalent linkage to the cyclic molecule.

A25. The nanopore device of embodiment A24 or A25, wherein the reversible covalent linkage is generated by a reaction selected from an amine reacting with a N-Hydroxysuccinimide (NHS) ester, an imidoester, a pentafluorophenyl (PFP) ester, a hydroxymethyl phosphine, carbonyl compounds; a carboxyl reacting with a carbodiimide; a sulfhydryl reacting with a maleimide, a haloacetyl, a pyridyldisulfide, and/or a vinyl sulfone; an aldehyde reacting with a hydrazine; any non-selective group reacting with diazirine and/or aryl azide; a hydroxyl reacting with isocyanate; a hydroxylamine reacting with a carbonyl compound, and the like and combinations thereof.

A26. The nanopore device of any one of embodiments A1 to A22, wherein the cyclic molecule is directly attached to the zone on the interior sidewall surface of the channel by a reversible covalent attachment.

A27. The nanopore device of any one of embodiments A1 to A26, further comprising a second zone to which one or more functional molecules each having one or more activities is covalently attached.

A28. The nanopore device of embodiment A27, wherein the one or more activities is an enzymatic activity. A29. The nanopore device of embodiment A27, wherein the one or more functional molecules is an enzyme.

A30. The nanopore device of embodiment A27 or A28, wherein the one or more activities is selected from a polymerase activity, a helicase activity, an endonuclease activity, an exonuclease activity, a nickase activity, and combinations thereof.

A31. The nanopore device of embodiment A27 or A29, wherein the one or more functional molecules is selected from a polymerase enzyme, a helicase enzyme, an endonuclease enzyme, an exonuclease enzyme, a nicking enzyme and combinations thereof.

A32. The nanopore device of any one of embodiments A27 to A31 , wherein the one or more activities are functionally coupled.

A33. The nanopore device of any one of embodiments A1 to A32, wherein the second zone is proximal to the zone to which the cyclic molecule is effectively linked by reversible covalent attachment.

A34. The nanopore device of any one of embodiments A1 to A33, wherein the channel in the solid support has a diameter between about 1 nanometer and about 50 nanometers. A35. The nanopore device of any one of embodiments A1 to A34, wherein the nanopore generated by the attachment of the cyclic molecule to the zone on the interior sidewall surface of the channel has a diameter between about 0.1 nanometers and about 20 nanometers.

B1. A process for preparing a nanopore device, comprising:

providing a solid support that includes:

a first surface, an opposing second surface, and a wall or walls between the first surface and the second surface,

a channel in the solid support that includes a proximal opening at the first surface, a distal opening at the second surface and an interior sidewall surface; and

a plug within the channel that includes a proximal end and a distal end; chemically protecting the first surface;

removing a portion of the proximal end of the plug, thereby producing a shortened plug and revealing a first zone on the interior sidewall of the channel in proximity to the proximal opening; contacting the first zone with a cyclic molecule under attachment conditions, whereby the cyclic molecule is attached effectively by a covalent linkage to the interior surface of the channel in the zone, and whereby the cyclic molecule forms a pore in the channel; and

removing the shortened plug.

B2. The process of embodiment B1 , which comprises chemically protecting a second zone on the interior sidewall of the channel revealed by removing the shortened plug.

B3. The process of embodiment B1 or B2, which comprises contacting the first zone with a coating under attachment conditions, which coating is (i) associated with the interior sidewall surface by covalent attachment and (ii) joined to the cyclic molecule.

B4. The process of embodiment B3, wherein the coating is joined to the cyclic molecule by a covalent attachment.

B5. The process of embodiment B3 or B4, wherein the coating is of a thickness substantially equal to thickness theta, wherein: theta = (C _D - CM _OD )/2,

C _D is the diameter of the channel at the zone, and

CMOD is the outer diameter of the cyclic molecule. B6. The process of any one of embodiments B3 to B5, wherein the density of the coating is sufficient to substantially prevent significant ion current flow around the cyclic molecule.

B7. The process of any one of embodiments B1 to B6, wherein the coating comprises silane. B8. The process of embodiment B7, wherein the silane has a chain length of between about 0.1 nanometers and about 50 nanometers.

C1 . A method, comprising:

contacting a nanopore device with a composition that includes or does not include an analyte, which nanopore device comprises:

a solid support that includes a first surface, an opposing second surface, and a wall or walls between the first surface and the second surface;

a channel in the solid support that includes a proximal opening at the first surface, a distal opening at the second surface and an interior sidewall surface; and a cyclic molecule attached effectively by a covalent linkage to a zone on the interior sidewall surface of the channel, whereby the cyclic molecule forms a pore in the channel;

measuring an electric current applied to the nanopore device, thereby determining an electric current measurement; and

detecting the presence, absence or amount of the analyte based on the electric current measurement.

C2. The method of embodiment C1 , wherein the analyte is selected from small organic molecules, organic molecule enantiomers, small biological molecules, solid particles, nucleosides, nucleotide monophosphates, chemical and biological warfare agents, toxins, small molecules, and the like and combinations thereof.

C3. The method of embodiment C2, wherein the analyte is DNA and/or RNA. C4. The method of C3, wherein detecting the analyte determines a nucleotide sequence.

D1 . A kit, comprising a nanopore device of any one of embodiments A1 to A7 and directions for detecting an analyte using the nanopore device.

E1. A nanopore device comprising:

a membrane having a thickness with a first, exterior, side and a second, interior, side, the first side being opposite the second side,

at least one nanopore extending through the membrane, thus forming at least one channel connecting the first side and the second side of the membrane, wherein the nanopore has a first opening, a second opening, a depth, an upper portion within the at least one channel and a lower portion within the at least one channel, wherein the first opening opens to the first side of the membrane, and the second opening opens to the second side of the membrane, wherein the surface properties of the upper portion within the at least one nanopore are chemically modified with a first set of modifying molecules,

the second set of modifying molecules are covalently attached to at least one cyclic molecule, wherein the diameter of the nanopore device is determined by the internal diameter of the cyclic molecule. E2. The device in embodiment E1 , wherein the surface properties of the first, exterior, side are chemically modified with a second set of modifying molecules.

E3. The device in embodiment E1 , and wherein the surface properties of the lower portion within the at least one nanopore are chemically modified with a third set of modifying molecules.

E4. The device in embodiment E1 , wherein the at least one channel is between about 1 nanometers and about 50 nanometers in diameter.

E5. The in embodiment E1 , wherein the diameter of the nanopore determined by the internal diameter of the cyclic molecule is between about 0.1 nanometer and about 20 nanometers.

F1. A method of fabricating a nanopore device comprising:

producing at least one nanopore extending through a membrane that has a thickness with a first, exterior, side and a second, interior, side, the first side being opposite the second side, thus forming at least one channel connecting the first side and the second side of the membrane, wherein the nanopore has a first opening, a second opening, a depth, an upper portion within the at least one channel and a lower portion within the at least one channel, wherein the first opening opens to the first side of the membrane, and the second opening opens to the second side of the membrane,

measuring the size of the first opening of the nanopore,

modifying the first, exterior side of the membrane with a first set of modifying molecules, modifying the upper portion within the at least one nanopore with a second set of modifying molecules, wherein the second set of modifying molecules are chosen based on the measured size of the nanopore and the outer diameter of a cyclic molecule to be bound to the second set of modifying molecules,

modifying the lower portion within the at least one nanopore with a third set of modifying molecules, and

delivering the cyclic adapter to the second set of modifying molecules and covalently binding the cyclic adapter to the second set of modifying molecules.

The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary 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 technology.

The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term "a" or "an" can refer to one of or a plurality of the elements it modifies (e.g., "a reagent" can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term "about" as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term "about" at the beginning of a string of values modifies each of the values (i.e., "about 1 , 2 and 3" refers to about 1 , about 2 and about 3). For example, a weight of "about 100 grams" can include weights between 90 grams and 1 10 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative

embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.

Certain embodiments of the technology are set forth in the claim(s) that follow(s).