Nanopore device and methods of fabricating and using the same

Abstract

Nanopore devices including microfluidic introduction members and methods of using the same are provided. The subject devices include first and second fluid containment members separated by a fluid barrier having a single nanopore therein providing fluid communication between the first and second fluid containment members, and a microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore. Also provided are methods of fabricating such a device and methods of using such a device for improved detection and characterization of a sample

Description

CROSS-REFERENCE

This application is a continuation-in-part application of Ser. No. 10/693,064 (Agilent Docket No. 10021090-1), filed Oct. 23, 2003, which is incorporated herein by reference in its entirety.

BACKGROUND

Manipulating matter at the nanometer scale is important for many electronic, chemical and biological advances (See Li et al., “Ion beam sculpting at nanometer length scales”, Nature, 412: 166-169, 2001). Such techniques as “ion beam sculpting have shown promise in fabricating molecule scale holes and nanopores in thin insulating membranes. These pores have also been effective in localizing molecular-scale electrical junctions and switches (See Li et al., “Ion beam sculpting at nanometer length scales”, Nature, 412: 166-169, 2001).

Artificial nanopores have been fabricated by a variety of research groups with a number of materials. Generally, the approach is to fabricate these nanopores in a solid-state material or a thin freestanding diaphragm of material supported on a frame of thick silicon to form a nanopore chip. Some materials that have been used to date for the diaphragm material include silicon nitride and silicon dioxide.

Because of the potential applicability of nanopore devices for a variety of different applications, there is continued interest in the development of new nanopore device structures and methods of using the same.

SUMMARY OF THE INVENTION

Nanopore devices including microfluidic introduction members and methods of using the same are provided. The subject devices include first and second fluid containment members separated by a fluid barrier having a single nanopore therein providing fluid communication between the first and second fluid containment members, and a microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore. Also provided are methods of fabricating such a device and methods of using such a device for improved detection and characterization of a sample.

A feature of the present invention provides a device including first and second fluid containment members separated by a fluid barrier having a single nanopore therein providing fluid communication between the first and second fluid containment members; and a microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore. In some embodiments, the device includes two or more microfluidic introduction members for conveying a sample to the nanopore. In some embodiments, the nanopore has a diameter ranging from 1 nm to about 100 nm. In certain embodiments, the microfluidic introduction member is a microfluidic channel. In some embodiments, the microfluidic channel has an inner diameter ranging from about 1 μm to about 4 μm.

In some embodiments, the device further includes first and second electrodes positioned on opposite sites of the nanopore. In some embodiments, the device further includes a first voltage element for applying a first electrical voltage between the first and second electrodes. In some embodiments, the device further includes a first current measurement element for measuring an electrical current between the first and second electrodes.

In some embodiments, the device further includes third and fourth electrodes positioned about the perimeter of the nanopore. In some embodiments, the device further includes a second voltage element for applying a second electrical voltage between the third and fourth electrodes. In some embodiments, the device further includes a second current measurement element for measuring an electrical current between the third and fourth electrodes.

Another feature of the invention provides a method for fabricating a nanopore in a solid substrate, including producing a nanodimensioned passageway through a planar solid substrate; and producing a microfluidic introduction member on the planar solid substrate, wherein a terminal end of the microfluidic introduction member is positioned proximal to the nanodimensioned passageway. In some embodiments, the nanodimensioned passageway is produced in the planar solid substrate using a focused ion beam protocol. In some embodiments, the microfluidic introduction member is produced in the planar solid substrate using a low-temperature plasma-deposited silicon oxynitride protocol.

Yet another feature of the invention provides a method including applying an electrical voltage between first and second perimeter electrodes of a device including first and second fluid containment members separated by a fluid barrier having a single nanopore therein providing fluid communication between the first and second fluid containment members; and a microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore, and monitoring the electrical current between the first and the second perimeter electrodes.

In some embodiments, the method further includes providing a sample in the microfluidic introduction member prior to the monitoring. In further embodiments, the sample further includes a polymeric compound, such as nucleic acid. In some embodiments, the monitoring is performed over a period of time. In some embodiments, the method is a method of characterizing a polymeric compound, such as sequencing a nucleic acid.

Definitions

A “biopolymer” is a polymer of one or more types of repeating units, regardless of the source (e.g., biological (e.g.,. naturally-occurring, obtained from a cell-based recombinant expression system, and the like) or synthetic). Biopolymers may be found in biological systems and particularly include polypeptides, polynucleotides, proteoglycans, sphingoedgeids, etc., including compounds containing amino acids, nucleotides, or a mixture thereof.

The terms “polypeptide” and “protein” are used interchangeably throughout the application and mean at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. A polypeptide may be made up of naturally occurring amino acids and peptide bonds, synthetic peptidomimetic structures, or a mixture thereof. Thus “amino acid”, or “peptide residue”, as used herein encompasses both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the D- or the L-configuration.

In general, biopolymers, e.g., polypeptides or polynucleotides, may be of any length, e.g., greater than 2 monomers, greater than 4 monomers, greater than about 10 monomers, greater than about 20 monomers, greater than about 50 monomers, greater than about 100 monomers, greater than about 300 monomers, usually up to about 500, 1000 or 10,000 or more monomers in length. “Peptides” and “oligonucleotides” are generally greater than 2 monomers, greater than 4 monomers, greater than about 10 monomers, greater than about 20 monomers, usually up to about 10, 20, 30, 40, 50 or 100 monomers in length. In certain embodiments, peptides and oligonucleotides are between 5 and 30 amino acids in length.

The terms “polypeptide” and “protein” are used interchangeably herein. The term “polypeptide” includes polypeptides in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones, and peptides in which one or more of the conventional amino acids have been replaced with one or more non-naturally occurring or synthetic amino acids. The term “fusion protein” or grammatical equivalents thereof references a protein composed of a plurality of polypeptide components, that while typically not attached in their native state, typically are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins. The term polypeptide includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase, and the like.

A “monomeric residue” of a biopolymer is a subunit, i.e., monomeric unit, of a biopolymer. Nucleotides are monomeric residues of polynucleotides and amino acids are monomeric residues of polypeptides.

A “substrate” refers to any surface that may or may not be solid and which is capable of holding, embedding, attaching or which may comprise the whole or portions of an excitable molecule.

The term “nanopore” refers to a pore or hole having a minimum diameter on the order of nanometers and extending through a thin substrate. Nanopores can vary in size and can range from 1 nm to around 300 nm in diameter. Most effective nanopores have been roughly around 1.5 nm to 30 nm, e.g., 3 nm-20 nm in diameter. The thickness of the substrate through which the nanopore extends can range from 1 nm to around 10 μm.

The term “resonant tunneling” refers to the tunneling of a particle, typically an electron, from one location to another through two or more energy barriers enclosing one or more quantum well states situated between the locations. The one location and another typically comprise electrodes.

Resonant tunneling comprises two effects, one called “matched level resonance” and one called “matched barrier resonance.”

Matched level resonance may be detected as enhanced conduction between two electrodes as seen in a plot of the differential of current with respect to voltage when plotted versus applied voltage, i.e., a peak in dI/dV versus V, where I is current, V is applied voltage, and dI/dV is the differential of current with respect to voltage.

Matched barrier resonance may be detected, when the condition of matched level resonance is also present, as greatly enhanced signal-to-noise ratios for the differential conductance peaks generated by the matched level resonance effect.

A biopolymer that is “in”, “within” or moving through a nanopore means that the entire biopolymer any portion thereof, may located within the nanopore.

The terms “translocation” and “translocate” refer to movement through a nanopore from one side of the substrate to the other, the movement occurring in a defined direction.

The terms “portion” and “portion of a biopolymer” refer to a part, subunit, monomeric unit, portion of a monomeric unit, atom, portion of an atom, cluster of atoms, charge or charged unit.

In many embodiments, the methods are coded onto a computer-readable medium in the form of “programming”, where the term “computer readable medium” as used herein refers to any storage or transmission medium that participates in providing instructions and/or data to a computer for execution and/or processing. Examples of storage media include floppy disks, magnetic tape, CD-ROM, a hard disk drive, a ROM or integrated circuit, a magneto-optical disk, or a computer readable card such as a PCMCIA card and the like, whether or not such devices are internal or external to the computer. A file containing information may be “stored” on computer readable medium, where “storing” means recording information such that it is accessible and retrievable at a later date by a computer.

With respect to computer readable media, “permanent memory” refers to memory that is permanent. Permanent memory is not erased by termination of the electrical supply to a computer or processor. Computer hard-drive ROM (i.e. ROM not used as virtual memory), CD-ROM, floppy disk and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non-permanent memory. A file in permanent memory may be editable and re-writable.

A “computer-based system” refers to the hardware means, software means, and data storage means used to analyze the information of the present invention. The minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention. The data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.

To “record” data, programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.

A “processor” references any hardware and/or software combination that will perform the functions required of it. For example, any processor herein may be a programmable digital microprocessor such as available in the form of an electronic controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.

“Communicating” information means transmitting the data representing that information as electrical signals over a suitable communication channel (for example, a private or public network). “Forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. The data may be transmitted to the remote location for further evaluation and/or use. Any convenient telecommunications means may be employed for transmitting the data, e.g., facsimile, modem, internet, etc.

The term “adjacent” refers to anything that is near, next to or adjoining. For instance, a nanopore referred to as “adjacent to an excitable molecule” may be near an excitable molecule, it may be next to the excitable molecule, it may pass through an excitable molecule or it may be adjoining the excitable molecule. “Adjacent” can refer to spacing in linear, two-dimensional and three-dimensional space. In general, if a quenchable excitable molecule is adjacent to a nanopore, it is sufficiently close to the edge of the opening of the nanopore to be quenched by a biopolymer passing through the nanopore. Similarly, electrodes that are positions adjacent to a nanopore are positioned such that resonance tunneling occurs a biopolymer passes through the nanopore. Compositions that are adjacent may or may not be in direct contact.

The term “substantially flat” refers to a surface that is nearly flat or planar. In most cases, this term should be interpreted to be nearly or approximately uniformly flat.

The term “lateral extent” refers to a direction or directions lying substantially parallel to the substantially flat major surfaces of a component of a diaphragm, diaphragm component, or entire device. Thus, for example, a long thin finger of material meandering along a surface has a lateral extent that is small in relation to its overall length in a direction perpendicular to that length, and a lateral extent that is long in the direction of its length. Again, for example, an area of circular shape has a lateral extent that is uniform in all directions parallel to the major surface in which it lies.

If one composition is “bound” to another composition, the compositions do not have to be in direct contact with each other. In other words, bonding may be direct or indirect, and, as such, if two compositions (e.g., a substrate and a nanostructure layer) are bound to each other, there may be at least one other composition (e.g., another layer) between to those compositions. Binding between any two compositions described herein may be covalent or non-covalent.

The term “assessing” includes any form of measurement, and includes determining if an element is present or not. The terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably and may include quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, and/or determining whether it is present or absent.

It is to be understood that the terms used in the descriptive language below, and in the claims below, are described using the adjectives “first,” “second,” “third,” “fourth,” “fifth,” “sixth,” and the like, in order to describe the device and method of the present invention in a manner consistent with a clear description of the device, but not necessarily in an order related to the sequence of steps in the method of fabricating the device. In particular, the use of these adjectives herein does not imply a numerical ordering herein, but is merely used as a verbal method of grouping; for example, the use of the word “third” to describe a particular feature does not necessarily imply the existence of a corresponding “second” feature. The use of such adjectives is consistent between the description of the device, the description of the method, and the claims.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

FIG. 1A shows a plan view of a first embodiment of the device 10 of the present invention, the viewpoint being at surface 1-1 shown in FIG. 3A.

FIG. 1B shows a plan view of a first embodiment of the device 10 of the present invention, the viewpoint being near plane 2-2 shown in FIG. 3A.

FIG. 2A shows a cross sectional view of a first embodiment of the device 10 of the present invention, the viewpoint being at plane 3-3 shown in FIG. 1A.

FIG. 2B shows a cross sectional view of a second embodiment of the device 10 of the present invention, the viewpoint being at plane 3-3 shown in FIG. 1A.

FIG. 2C shows a cross sectional view of a third embodiment of the device 10 of the present invention, the viewpoint being at plane 3-3 shown in FIG. 1A.

FIG. 3A shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3B shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3C shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3D shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3E shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3F shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3G shows a step of an embodiment of the fabrication method of the present invention.

FIG. 3H shows a step of an embodiment of the fabrication method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Nanopore devices including microfluidic introduction members and methods of using the same are provided. The subject devices include first and second fluid containment members separated by a fluid barrier having a single nanopore therein providing fluid communication between the first and second fluid containment members, and a microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore. Also provided are methods of fabricating such a device and methods of using such a device for improved detection and characterization of a sample.

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions, method steps, or equipment, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Methods recited herein may be carried out in any order of the recited events that is logically possible, as well as the recited order of events.

Unless defined otherwise below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Still, certain elements are defined herein for the sake of clarity. In the event that terms in this application are in conflict with the usage of ordinary skill in the art, the usage herein shall be controlling.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits; ranges excluding either or both of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recited events that is logically possible, as well as the recited order of events.

It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an,” “the,” and “one of” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

The term “about” refers to being closely or approximate to, but not exactly. A small margin of error is present. This margin of error would not exceed plus or minus the same integer value. For instance, about 0.1 micrometers would mean no lower than 0 but no higher than 0.2.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention.

The Subject Devices

The present invention provides a nanopore device including microfluidic introduction members. In general, the subject devices include a first and second fluid containment members separated by a fluid barrier having a single nanopore therein providing fluid communication between the first and second fluid containment members. The subject devices also include a microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore.

A variety of nanopore configurations are well known in the art and are suitable for use with the subject devices. Suitable nanopores may be natural, such as a protein channel, or the nanopores may be artificial. As used herein, the terms “nanopore” and “channel” are used interchangeably to refer to structures having a nanoscale passageway through which ionic current can flow. The inner diameter of the nanopore may vary considerably depending on the intended use of the device. Typically, the channel or nanopore will have an inner diameter of at least about 0.5 nm, usually at least about 1 nm and more usually at least about 1.5 nm, where the diameter may be as great as 50 nm or longer, including 100 nm or 300 nm, but in many embodiments will usually not exceed about 10 nm, and usually will not exceed about 5 nm.

In those embodiments in which the subject device is designed to characterize polymeric molecules the inner diameter of the nanopore may be sufficient to allow translocation of singled stranded, but not double stranded, nucleic acids. As such, in these embodiments, the inner-diameter will be at least about 1 nm, usually at least about 1.5 nm and more usually at least about 2 nm, but will usually not exceed about 3 nm, and more usually will not exceed about 5 nm.

The nanopore should allow a sufficiently large ionic current under an applied electric field to provide for adequate measurement of current fluctuations. As such, under an applied electric field of 120 mV in the presence of pH 7.5 buffered solution, the open (i.e. unobstructed) nanopore should provide for an ionic current that is at least about 1 pA, usually at least about 10 pA and more usually at least about 100 pA. Typically, the ionic current under these conditions will not exceed about 5 nA and more usually will not exceed about 20 nA. In addition, the channel should provide for a stable ionic current over a relatively long period of time. Generally, channels finding use in the subject devices provide for accurate measurement of ionic current for at least about 1 min, usually at least about 10 min and more usually at least about 1 hour, where they may provide for a stable current for as long as 24 hours or longer.

In some embodiments, the nanopore will be a naturally occurring or synthetic nanopore. In certain embodiments, the nanopore will be a proteinaceous material, by which is meant that it is made up of one or more, usually a plurality, of different proteins associated with each other to produce a channel having an inner diameter of appropriate dimensions, as described above. Suitable channels or nanopores include porins, gramicidins, and synthetic peptides. Of particular interest is the heptameric nanopore or channel produced from α-hemolysin, particularly α-hemolysin from Staphylococcus aureus, where the channel is preferably rectified, by which is meant that the amplitude of the current flowing in one direction through the channel exceeds the amplitude of the current flowing through the channel in the opposite direction.

The above description of various nanopore configurations is merely representative of the types of nanopore structures that may be present in the subject devices. Representative nanopore devices are further described in U.S. Pat. Nos. 5,795,782, 6,015,714, 6,362,002, 6,464,842, 6,627,067, 6,673, 615, 6,674,594, 6,706,203, 6,706,204, 6,783,643, 6,267,872, U.S. Patent Publication Nos. 2003/0044816, 2003/0066749, 2003/0104428, WO 00/34527; the disclosures of which are herein incorporated by reference.

Aspects of the subject devices include the presence of at least one microfluidic introduction member for conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore. The microfluidic introduction member provides for movement a sample by one of or a combination of pressure driven flow, electroosmotic flow, and electrophoretic flow, capillary flow, etc., thus placing the sample in close proximity to a nanopore much more quickly than could be achieved by diffusion of the sample to the nanopore from a more distant introduction point within either the package cavity or the substrate cavity.

The subject device may include a single microfluidic introduction member, or may include a plurality of microfluidic introduction members, such as about 2, 3, 4, 5, and up to 10, 15, and 25 of such microfluidic introduction members. The microfluidic introduction member may be made of a variety of shapes and sizes that allow a sample to be conveyed from a first site distal from the nanopore to a second site proximal to the nanopore. However, the microfluidic introduction member must have an inner diameter of sufficient width to be capable of conveying a sample that includes polymeric compounds. In representative embodiments, the microfluidic introduction member has an inner diameter ranging from about 0.5 μm to about 10 μm, including about 1 μm to about 9 μm, such as from about 2 μm to about 8 μm, from about 3 μm to about 7 μm, from about 4 μm to about 6 μm. In representative embodiments, the microfluidic introduction member has an inner diameter ranging from about 1 μm to about 4 μm.

The microfluidic introduction member may be positioned anywhere on the subject device. The microfluidic introduction member may be positioned atop the device or below further layers of materials. However, the positioning must be capable of conveying a fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore. Accordingly, one end of the microfluidic introduction member must be positioned in close proximity to the nanopore. The microfluidic introduction member may be fabricated by a variety of different methods and material well known in the art. For example, the method of fabricating a microfluidic introduction member as described in detail in U.S. Pat. No. 6,096,656, the disclosure of which is incorporated herein in its entirety.

The invention having will now be further described in terms of various representative embodiments as depicted in the figures. FIGS. 1 and 2 show various embodiments of the device 10 of the present invention. FIG. 1A is a plan view of one embodiment of the device 10 at surface 1-1 as shown in FIG. 2A, and FIG. 1B is a plan view of the device 10 at plane 2-2 as shown in FIG. 2A. Surface 1-1 is not a plane but has an upward jog in the center of FIG. 2A in order to display features of the device in a clear manner in FIG. 1A. Surface 1-1 is rotationally symmetric about a vertical centerline, not shown, in FIG. 2A. FIG. 2A is a cross section view at plane 3-3 shown in FIG. 1A. The figures are not to scale, and some features are greatly exaggerated for purposes of description.

A nanopore 12 comprising a first hole extending through a diaphragm 14 is generally depicted in the figures, the diaphragm 14 being supported by a rigid frame comprising a semiconductor chip 18. The diaphragm 14 may range in lateral extent from 5 to at least 100 micrometers. The diaphragm 14 comprises a first insulator material, and typically comprises silicon nitride 200 nm thick. The diaphragm 14 may comprise an additional material or materials, not shown. The dimensions described here are for illustrative purposes only and should not be interpreted to limit the scope of invention.

A variety of embodiments of the subject device 10 of the invention are provided in FIGS. 2A to 2C. It will be appreciated by one skilled in the art that the nanopore 12 may consist of any configuration that allows for the necessary properties, as described in greater detail above. Accordingly, the nanopore 12 may take the form of a frustum, cylinder, rectangle, cube, and the like. In some embodiments, the nanopore 12 will be in the form of a frustum, as shown in FIG. 2C. In other embodiments, the nanopore 12 will be in the form of a cylinder, as shown in FIGS. 2A and 2B.

A detailed description of the device 10 of the invention is as follows, with reference to FIGS. 1-2. Nanopore 12 comprising a first hole is situated in diaphragm 14. Diaphragm 14 comprises a window comprising a first insulating material, and is supported within a collar comprising a second region 16 comprising a second insulating material. Second region 16 is supported in substrate 18 comprising a semiconductor, typically comprising silicon. A fifth insulator region, not shown in FIGS. 1-2 but shown in FIG. 3E as feature 54, may optionally comprise an additional part of the diaphragm. Optional sixth insulator regions 19 lie atop substrate 18 and beneath one of electrical leads 20 and microfluidic leads 22, as shown in FIG. 2A.

Microfluidic introduction members 24, such as microfluidic channels, are situated within microfluidic leads 22. Third cavity 26, being typically about 3 μm to about 15 μm in diameter, including about 5 μm in diameter, and fourth cavity 28, being typically about 50 μm to about 70 μm in diameter, including about 60 μm in diameter, penetrate third region 30 and fourth region 32 respectively. Third region 30 comprises a third insulator material and is disposed atop features 14, 16, 18, 19, 20, and 24. Fourth region 32 comprises a fourth insulator material and is disposed atop region 30. Feature 34 comprises an external package applied atop the device 10. An optional O-ring 36 sits in gland 38 and defines the area of contact of a liquid solution, not shown, disposed within package cavity 40 and making contact with regions 32, region 30, diaphragm 14, and nanopore 12. A liquid solution, not shown, also is disposed within substrate cavity 41 beneath diaphragm 14 and makes contact with diaphragm 14 and with nanopore 12. As shown, substrate cavity 41 extends entirely through substrate 18, but this is not a necessity of the invention. As shown, the lateral extent of the substrate cavity 41 near its top coincides with a portion of region 16, but this is not important and the lateral extent of the substrate cavity 41 near its top may instead or in addition coincide with a portion of diaphragm 14.

Electrical leads 20 closely approach nanopore 12 and in some embodiments can make contact with the nanopore, for example, to form tunneling electrodes as described in detail in patent application Ser. No. 10/462,216, Filed on Jun. 12, 2003, NANOPORE WITH RESONANT TUNNELING ELECTRODES. That application describes both the structure and method of fabrication of resonant tunneling electrodes associated with a nanopore, and it will be appreciated, based on that description and on the description of device 10 herein, that such resonant tunneling electrodes can be incorporated into device 10 as shown in FIG. 2C. In FIG. 2C, electrical lead 20(310) comprises a tunneling electrode and corresponds to tunneling electrode feature 310 of patent application Ser. No. 10/462,216, while electrical lead 20(314) comprises a tunneling electrode and corresponds to tunneling electrode feature 314 of patent application Ser. No. 10/462,216.

Microfluidic introduction members, such as microfluidic channels 24 are disposed within microfluidic leads 22, and microfluidic channels 24 closely approach nanopore 12. Microfluidic leads 22 and microfluidic channels 24 can be fabricated by methods known to those skilled in microstructure fabrication. For example, microfluidic leads 22 can comprise oxynitride deposited at a temperature of 90 C atop preformed mandrel regions comprising positive photoresist, the mandrel regions later being removed by dissolution in acetone to form channels 24. The technique is detailed on the worldwide websitesandia.gov/media/NewsRel/NR2000/canals.htm. The technique is also described in greater detail in U.S. Pat. No. 6,096,656, the disclosure of which is incorporated herein in its entirety.

Alternatively, microfluidic leads 22 can comprise silicon dioxide deposited at a temperature of 250 C atop preformed mandrel regions comprising polyimide, the polyimide later being removed by high-density oxygen plasma etching (see for example “Polyimide sacrificial layer and novel materials for post-processing surface micromachining,” A Bagolini, et al., (J. Micromech. Microeng. 12 (2002) 385-389) or by caustic etching (see, for example, the worldwide website of dupont.con/kapton/general/caustic-etching.pdf) to form channels 24. Parylene® may be used to form microfluidic leads 22 in a manner similar to that which has been reported in the literature, e.g. “Polymer-Based Electrospray Chips for Mass Spectrometry,” Xuan-Qi Wang, et al., Proceedings, IEEE 12th International Micro Electro Mechanical Systems Conference (MEMS'99), Orlando, Fla., pp. 523-528, Jan 17-21, 1999. Other variations on the materials and methods used to fabricate microfluidic leads 22 and microfluidic introduction members 24 will occur to those skilled in microstructure fabrication.

A combination of a hydraulic pressure drop and a voltage gradient along the length of microfluidic introduction members 24 can advantageously quickly move polymeric molecules, such as DNA, in solution within a liquid filling microfluidic introduction members 24 from a first site distal from the nanopore 12 to a second site proximal to nanopore 12. The movement of such molecules can occur by a combination of pressure driven flow, electroosmotic flow, and electrophoretic flow, thus placing such molecules in close proximity to nanopore 12 much more quickly than could be achieved by diffusion of such molecules to the nanopore 12 from a more distant introduction point within either package cavity 40 or substrate cavity 41.

Region 30 comprises a third region comprising a third insulator material. Region 30 is depicted laying atop features 14, 16, 18, 19, 20, and 24. It should be appreciated that in some instances it may be desirable that the third insulator material comprise the walls of microfluidic leads 22, in which case the microfluidic leads 22 and the region 30 can comprise a unitary structure. Cavity 26 comprises a third cavity penetrating region 30 and providing an opening atop diaphragm 14 and atop nanopore 12.

Region 32 comprises a fourth region comprising a fourth insulator material. Cavity 28 comprises a fourth cavity penetrating region 32 and providing an opening atop region 30.

The choice of materials for all regions of the device 10 depends on process compatibility considerations, electrical characteristics including permittivity and resistivity, and compatibility with use of the device during measurement and cleaning. Diaphragm 14, typically comprising silicon nitride, may comprise one or more of one of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Region 16, typically comprising silicon dioxide, may comprise one or more of one of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Region 18, typically comprising silicon, may comprise a semiconductor from a group including but not limited to silicon, germanium, and gallium arsenide, and may also comprise one or more of one of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Electrical leads 20, typically comprising aluminum, comprise a conducting material which may comprise one of a metal, a silicide, an organic conductor and a superconductor, including but not limited to aluminum, gold, platinum, palladium, iridium, copper, chromium, and nickel.

Microfluidic leads 22, typically comprising oxynitride, may comprise one or more of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Microfluidic introduction members 24 may be formed using mandrel materials comprising one or more of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Region 30, typically comprising polyimide, may comprise one or more of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Region 32, typically comprising polyimide, may comprise one or more of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Regions 19, typically comprising silicon dioxide, may comprise one or more of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Region 54 may comprise one or more of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma nitride, an insulator, a semiconductor, and a metal.

Thus the device 10 of the invention can be optimized by choices of materials, region thicknesses, region lateral extents, and cavity lateral extents to provide desired minimal capacitances across the diaphragm 14 and from leads 20 and 22 to one or more of cavities 40 and 41.

It will be appreciated that, while electrical leads 20 and microfluidic leads 22 have been presented in FIGS. 1-2 and in the above description in positions immediately beneath the third region 30, this is not a necessity of the invention. Alternatively or in addition, electrodes 20 or microfluidic leads 22 may be placed at locations including but not limited to atop the third region 30, atop the fourth region 32, in a recessed region of the substrate beneath the plane of the bottom surface of layer 30, and within the substrate cavity 41.

In some embodiments, the device 10 also includes a first voltage element, not shown, for applying a first electrical voltage between regions 40 and 41 including the nanopore 12 present therein. The electric field applying element is typically capable of generating a voltage of at least about 10 mV, usually at least about 50 mV and more usually at least about 100 mV. In some embodiments, as represented in FIG. 2B, the first electrical voltage is applied between first electrode 212 and second electrode 214 comprising silver/silver chloride electrodes positioned on opposite sides of the nanopore 12. In other embodiments, as represented in FIG. 2C, third electrode 20(310) and fourth electrode 20(314) are positioned about the perimeter of the nanopore 12 producing a resonant tunneling sensor as further described above and in U.S. patent application Ser. No. 10/462,216, the disclosure of which is incorporated herein by reference. In some embodiments the device 10 also includes a second voltage element, not shown, for applying a second electrical voltage between third electrode 20(310) and fourth electrode 20(314).

In some embodiments, the device 10 further includes a first current measurement element, not shown, for monitoring the current flow between first electrode 212 and second electrode 214 and processing the observed current flow to produce a usable output. In some embodiments, the device 10 further includes a second current measurement element, not shown, for monitoring the current flow between third electrode 20(310) and fourth electrode 20(314) and processing the observed current flow to produce a usable output. Typically, such first and second current monitoring elements include a very low noise amplifier and current injector, and an analog to digital (A/D) converter. The device may further include other elements of the output generating system, including data acquisition software, an electronic storage medium, etc.

Fabrication of the Subject Devices

Having described the device of the invention, a description of the method of fabrication of the invention is now in order. A non-limiting exemplary method of fabricating an embodiment of the subject device 10 is provided in FIGS. 3A to 3H.

The exemplary method begins as shown in FIG. 3A by providing a substrate 18 and forming a masking layer 42 comprising, for example, silicon dioxide, formed atop substrate 18.

The method continues as shown in FIG. 3B wherein layer 42 is defined, for example via lithography and etching, into masking regions 44. Cavity regions 46 are formed, for example by etching in a hot aqueous caustic solution of potassium hydroxide in water, and are etched to a depth of, for example, about 5 μm to 15 μm, including about 10 μm.

The method continues as shown in FIG. 3C wherein cavity regions 46 are filled, typically by a deposition method such as TEOS oxide deposition, with the second insulator material forming insulator regions 48. It will be appreciated that between the structure shown in FIG. 3B and that shown in FIG. 3C, planarization of the upper surface of substrate 18 will in some embodiments occur via chemomechanical polishing (CMP). Regions 49 comprise fifth and sixth insulator materials, comprising for example thermally grown silicon dioxide, which is typically formed after CMP. It will be appreciated that the structure illustrated in FIG. 3C may be formed by use of the SUMMIT V fabrication process developed at Sandia Laboratories and commercialized by MEMX (see, for example, the worldwide website of memx.com/technology.htm), or by other fabrication processes, and that at the same time one of active electrical circuitry and microstructures, not shown, may be fabricated one of in or on substrate 18 by use of the SUMMIT V process, or by other fabrication processes. At the same time regions 49 are formed, a layer of insulator, not shown, may be formed on the lower surface of substrate 18.

The method continues as shown in FIG. 3D wherein layer 50, including the first insulating material, typically comprising silicon nitride, is formed atop regions 48 and 49, typically by means of low pressure chemical vapor deposition. Advantageously, at the same time as layer 50 is formed, a layer 52 is formed on the lower surface of substrate 18, or upon the exposed lower surface of any layers or structures which happen to be on the lower surface of substrate 18.

The method continues as shown in FIG. 3E wherein layer 52, no longer shown, has been formed by lithograph and plasma etching, into a masking region for the subsequent etching of substrate 18, in some embodiments, in a hot aqueous caustic solution of potassium hydroxide, to form substrate cavity 41. Fifth region 54 comprises a fifth insulator material, comprising one or more of one of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, and plasma nitride. The lower surface of region 54, which was one of regions 49 in FIG. 3D, is exposed by the process of etching, while other parts of regions 49 now form regions 19 comprising sixth insulator regions as shown in FIG. 2B. The sixth insulator regions comprise one or more of one of a group including but not limited to a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, and plasma nitride. Regions 48 now form region 16 as shown in FIGS. 1-2, and layer 50 has now been formed by the etching process into diaphragm 14 as shown in FIGS. 1-2.

In some embodiments, region 54 may be left in place beneath diaphragm 14, region 54 and diaphragm 14 thereby forming a composite diaphragm, and such a composite diaphragm may be formed into a tensile diaphragm having a compressive region as described in U.S. patent application Ser. No. 10/670,554, Filed on Sep. 25, 2003—APPARATUS AND METHOD FOR MAKING A TENSILE DIAPHRAGM WITH A COMPRESSIVE REGION, the disclosure of which is incorporated herein by reference in its entirety, the tensile diaphragm having a compressive region being used in subsequent steps of the present method in a manner not explicitly shown in the present embodiment of the fabrication process.

Alternatively in some embodiments, region 54 may be left in place beneath diaphragm 14, region 54 and diaphragm 14 thereby forming a composite diaphragm, and such a composite diaphragm may be formed into a tensile diaphragm having an insert as detailed in U.S. patent application Ser. No. 10/670,551, Filed on Sep. 25, 2003 APPARATUS AND METHOD FOR MAKING A TENSILE DIAPHRAGM WITH AN INSERT, the disclosure of which is incorporated herein by reference in its entirety, the tensile diaphragm having an insert being used in subsequent steps of the present method in a manner not explicitly shown in the present embodiment of the fabrication process, and region 54 being removed at some later point in the fabrication process.

At the point in the fabrication process shown in FIG. 3E the nanopore 12 may be fabricated, then covered by later layers formed in the process and re-exposed near the end of the process. The nanopore 12 may be positioned anywhere in or through a substrate. The nanopore may be established using any methods well known in the art. For example, the nanopore may be sculpted in the substrate by means of a low-energy argon ion beam sculpting of an initially larger hole formed by etching or focused ion beam machining, or by sputtering, etching, photolithography, or other methods and techniques well known in the art.

If electrodes associated with a nanopore are to be included in the device of the invention as shown in FIG. 2C, and as described in (Ser. No. 10/462,216, Filed on Jun. 12, 2003—NANOPORE WITH RESONANT TUNNELING ELECTRODES), then at this point in the method the nanopore may be fabricated and the electrodes 20(310) and 20(314) and associated insulators 312 and 316 may be fabricated. For simplicity of description, the fabrication of the nanopore, electrodes, and insulators are not described explicitly herein, but it will be appreciated that such fabrication can be performed in a manner described in U.S. patent application Ser. No. 10/426,216, Filed on Jun. 12, 2003—NANOPORE WITH RESONANT TUNNELING ELECTRODES, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the fabrication of the nanopore, electrodes, and insulators can be started at this point in the fabrication process and completed later in the fabrication process. In other embodiments, and as illustrated in FIGS. 3F-3H, the fabrication of the nanopore can be delayed to later in the fabrication process.

The exemplified method continues as shown in FIG. 3F wherein region 54 is removed for purposes of simple description of the fabrication method. Electrical leads 20 and microfluidic leads 22 having microfluidic introduction members 24 are fabricated using techniques available to those skilled in the art. For example, microfluidic leads 22 can comprise oxynitride deposited at a temperature of 90 C atop preformed mandrel regions comprising positive photoresist, the mandrel regions later being removed by dissolution in acetone to form microfluidic channels 24. The technique is detailed on the worldwide website sandia.gov/media/NewsRel/NR2000/canals.htm. The technique is also described in greater detail in U.S. Pat. No. 6,096,656, the disclosure of which is incorporated herein in its entirety.

In particular, microfluidic introduction members 24 may be opened by use of a clearing technique as discussed above such as acetone dissolving, oxygen plasma etching, or caustic etching, before subsequent layers are deposited above microfluidic introduction members 24, but in the local area of microfluidic introduction members 24 beneath region 57 there may advantageously be no openings into which fluid can intrude. This lack of opening in the area beneath region 57 can be achieved by having the horizontal one of microfluidic introduction members 24 as shown in FIG. 1A connect with the vertical one of microfluidic introduction members 24 as shown in FIG. 1A at the stage of the fabrication process shown in FIG. 3F. On the nanopore chip in regions far from the nanopore the unseen ends of microfluidic introduction members 24, to which external fluidic connections may later be made, can be occluded by one or more of various methods to prevent later deposited layers from intruding into them.

Layer 56, comprising, for example, polyimide or silicon oxynitride, and ranging in thickness from about 1 μm to about 5 μm, including about 2 μm, comprises the third insulator material, and is later to be shaped to form region 30. Region 57 is a portion of layer 56 later to be removed to form cavity 26 shown in FIGS. 1-2. Layer 58, comprising, for example, aluminum, is a masking material which will later serve as an etch-stop layer during fabrication of cavity 28 shown in FIGS. 1-2. Layer 58 may comprise one of a group including but not limited to a metal, a polymer, photoresist, SU8 photoresist, epoxy, polyimide, Parylene®, a silicone polymer, silicon dioxide, silicon nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS oxide, and plasma nitride. Advantageously, in some embodiments layer 56 may comprise a photosensitive material, so that region 57 may be defined by a photolithographic exposure, without at this time performing a developing step, leaving a region to be developed away later, before layer 58 is deposited. In such embodiments layer 58 can advantageously comprise an opaque material, for instance a metal such as aluminum. In some embodiments, a hole, not shown, may be formed in layer 58 over region 57 to allow later etching of region 57.

The method continues as shown in FIG. 3G. A layer 60, comprising, for example polyimide, ranging in thickness from about 15 μm to about 30 μm, including about 25 μm, comprising a fourth insulator material has been formed on top of layer 58, and fourth cavity 28 has been formed in layer 58 to define region 32. Layer 58 at the bottom of fourth cavity 28 serves as an etch stop layer.

The exemplified method continues to a final form of device 10 as shown in FIG. 3H. If a hole, not shown, has been previously formed in region 58 over region 57 as discussed above, and if the third insulator material comprising layer 56 has similar etching characteristics to the material of layer 60, then etching can simply continue through the hole in layer 58 to remove region 57, forming cavity 26 comprising a third cavity. The portion of layer 58 at the bottom of cavity 28 is then etched away to edge 62. The microfluidic leads 22 in the region beneath cavity 26 are then etched, opening microfluidic introduction members 24 in the region beneath cavity 26 and leaving the opened local ends of microfluidic introduction members 24 self aligned with the edges of cavity 26. Those portions of layer 58 not etched away remain in place, but are not explicitly shown in FIGS. 1-2. Nanopore 12 is then formed, for example by focused ion beam machining followed by argon ion beam sculpting, producing an embodiment of the final device structure as exemplified in FIG. 3H.

If no hole had been formed in layer 58 before etching of cavity 28, but a previous photolithographic exposure had been performed of region 57, then after cavity 28 is formed the portion of layer 58 at the bottom of cavity 28 is etched away. The photolithographically exposed region 57 is then developed away, and the microfluidic leads 22 in the region beneath cavity 26 are then etched, opening microfluidic introduction members 24 in the region beneath cavity 26 and leaving the opened local ends of microfluidic introduction members 24 self aligned with the edges of cavity 26. Again, those portions of layer 58 not etched away remain in place, but are not explicitly shown in FIGS. 1-2. Nanopore 12 is then formed, for example by focused ion beam machining followed by argon ion beam sculpting, producing an embodiment of the final device structure as exemplified in FIG. 3H.

It will be appreciated that the fabrication sequence described above is by way of example only, and that there are others techniques well known to those skilled in the art which may be used to arrive at the same final structure. It will be appreciated also that the use of known adhesion promoter techniques between various layers will improve the yield of the fabrication process and the quality of the finished nanopore chip, and the use of such adhesion promoter techniques is assumed during the fabrication process even where not explicitly described.

It will be appreciated that the fabrication of a nanopore may be accomplished by means other than focused ion beam drilling and argon ion beam sculpting. For example, other known means of fabricating a nanopore include masking with a nanoparticle followed by layer evaporation around the masking nanoparticle, next followed by removal of the nanoparticle and etching within the hole that had been masked by the nanoparticle. Such techniques, both known and unknown may be used to fabricate nanopores in accordance with the present invention.

It will be appreciated that, while the present invention is aimed toward utility in nanopore structures, it may prove to have utility for other devices both known and unknown. Such devices include devices with microscale and nanoscale dimensions. Microscale dimensions are defined to include dimensions from 100 nm to 1 mm, and nanoscale dimensions are defined to include dimension from 0.1 nm to 1 um.

Methods of Using the Subject Devices

In general, the method of using the subject device 10 of the present invention includes applying an electrical voltage between electrodes 212 and 214 of the device and monitoring one of the electrical current through the nanopore and the electrical current between electrodes 20(310) and 20(314). In certain embodiments, the current flowing through the nanopore between electrodes 212 and 214 is monitored and recorded over a period of time, and in some embodiments the current flowing between electrodes 20(310) and 20(314) is monitored and recorded over a period of time. The monitoring of electrical current provides information on the properties of molecular moieties situated within the nanopore or translocating through the nanopore. In certain embodiments, when the device 10 is in use, package cavity 40 and substrate cavity 41 are filled with a conductive ionic aqueous solution. In certain embodiments, the monitoring of the electrical current may be performed while a compound is translocating the nanopore. In such embodiments, the fluid sample may be placed in the microfluidic introduction member. The microfluidic introduction member provides for conveying the fluid sample from a first site distal from the nanopore to a second site proximal to the nanopore.

Utility

The subject devices find use in a variety of different applications in which the ionic current through a nanopore is monitored or resonant tunneling current through a sample is monitored. Representative applications in which the subject devices find use include, for example, separation of molecules, capturing of molecules, characterization of polymeric compounds, e.g., the determining of the base sequence of a nucleic acid; and the like.

In some embodiments, the subject device 10 of the present invention is useful in characterizing a polymeric compound, such as DNA. In such embodiments, a polymeric compound, such as DNA, present in a fluid sample is placed in the microfluidic introduction member, such as microfluidic channel, which member coveys the fluid sample containing the polymeric compound from a first site distal from the nanopore to a second site proximal to the nanopore.

Once the fluid sample containing the polymeric compound is in close proximity to the nanopore, for example, is present in region 26 of the device 10, the polymeric compound present in the fluid sample can diffuse quickly to an even closer proximity to the nanopore, in which closer proximity one of the electric field extending through the nanopore, or the spatial gradient of the electric field extending through the nanopore, acts to pull the polymeric compound into and through the nanopore, thus causing translocation of the polymeric compound through the nanopore. For example, a single stranded nucleic acid may be translocated through the nanopore and the current flowing through the nanopore between electrodes 212 and 214 may be monitored and recorded over a period of time, thereby providing a range of values representing the fluctuation of the current flowing through the nanopore as the polymeric compound translocates through the nanopore. Such a fluctuation of current over time may then be analyzed to determine the characteristics of the polymeric compound.

In some other embodiments, for example, a single stranded nucleic acid may be translocated through the nanopore, and a time-varying second voltage may be applied between electrodes 20(310) and 20(314), and the current flowing between electrodes 20(310) and 20(314) may be monitored and recorded.

During use, the second voltage element may be ramped, i.e., by application of a time-varying second voltage between electrodes 20(310) and 20(314), in order to provide a resonant tunneling current through a sample. The general principle is to ramp the tunneling voltage across the electrodes over the energy spectrum of the translocating polymeric compound. At specific voltages the incident energy will sequentially match the internal nucleotide energy levels, giving rise to a detectable change, e.g., an increase in the observed current, an increase in the slope of the current versus voltage, or both. In representative embodiments, the ramp-time of the applied voltage is short compared to the nucleotide translocation time through the nanopore. For example, in certain embodiments, the applied tunneling voltage frequency may be in excess of about 10 MHz.

In certain embodiments, wherein the electrodes 20(310) and 20(314) are positioned about the nanopore to provide peripheral electrodes as demonstrated in FIG. 2C, as a monomer translocates through the nanopore and between the two perimeter electrodes and as the second voltage is at a given voltage level, the monomer will inevitably reach a position in space where the two quantum mechanical tunneling barriers separating it from each the two perimeter electrodes are equal in magnitude, regardless of the presence of barrier asymmetry for other spatial positions of the monomer or for other voltage levels. At this position, there will be a detectable resonant tunneling current increase compared to other positions, and as the second voltage is ramped it, in effect, scans the internal energy spectrum of the individual monomer. The record of fluctuation in tunneling current between electrodes 20(310) and 20(314) thus comprises a resonant tunneling spectrum of the monomer. The applied voltage and tunneling current can thus be seen to produce a defined signal that is indicative of the portion of the biopolymer that is proximal to the third electrode 20(310) and fourth electrode 20(314). Each monomeric unit of the polymeric compound will produce a differing signal in the tunneling current over time as the varying voltage is applied. These differing signals provide sequential resonant tunneling spectra of each sequential portion of the polymeric compound that is sequentially positioned proximal to the “sweet spot” near electrodes 20(310) and 20(314) wherein the two quantum mechanical tunneling barriers separating it from each the two perimeter electrodes are equal in magnitude. These spectra can then be compared by computer to previous spectra or “finger prints” of nucleotides or portions of the polymeric compound that have already been recorded, i.e., a reference or control. The residue of the polymeric compound can then be determined by comparison to this reference or control, e.g., that may be in the form of a database. This data and information can then be stored and supplied as output data of a final sequence.

From the resultant recorded current fluctuations, the base sequence of the nucleic acid can be determined. Methods of characterizing polymeric molecules in this manner are further described in application Ser. No. 08/405,735 and entitled Characterization of Individual Polymer Molecules Based on Monomer-interface Interactions, the disclosure of which is herein incorporated by reference.

Results obtained from such methods may be raw results, such as signal lines for the signal producing system of the device. In the alternative, the results may be processed results, such as those obtained by subtracting a background measurement, or an indication of the identity of a particular residue of a polymeric compound (for example an indication of a particular nucleotide or amino acid.

Representative applications for use of nanopore devices are further described in U.S. Pat. Nos. 5,795,782, 6,015,714, 6,362,002, 6,464,842, 6,627,067, 6,673, 615, 6,674,594, 6,706,203, 6,706,204, 6,783,643, 6,267,872, U.S. Patent Publication Nos. 2003/0044816, 2003/0066749, 2003/0104428, WO 00/34527; the disclosures of which are herein incorporated by reference.

In certain embodiments, the subject methods also include a step of transmitting data or results from the monitoring step, as described above, to a remote location. By “remote location” is meant a location other than the location at which the array is present and hybridization occur. For example, a remote location could be another location (e.g. office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. As such, when one item is indicated as being “remote” from another, what is meant is that the two items are at least in different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart.

The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

1. A device comprising:

a first fluid containment member;

a second fluid containment member;

a fluid barrier separating said first and second fluid containment members;

a nanopore present in said fluid barrier; and

a microfluidic introduction member for conveying a fluid sample from a first site distal from said nanopore to a second site proximal to said nanopore.

2. The device according to claim 1, wherein said nanopore has a diameter length ranging from about 1 nm to about 100 nm.

3. The device according to claim 1, wherein said device comprises two or more microfluidic introduction members for conveying a sample to said nanopore.

4. The device according to claim 1, wherein said microfluidic introduction member is a microfluidic channel.

5. The device according to claim 4, wherein said microfluidic channel has an inner diameter ranging from about 1 μm to about 10 μm.

6. The device according to claim 1, wherein said device further comprises first and second electrodes positioned on opposite sides of said nanopore.

7. The device according to claim 6, wherein said device further comprises an element for applying a voltage between said first and second electrodes.

8. The device according to claim 6, wherein said device further comprises an element for measuring an electrical current between said first and second electrodes.

9. A method comprising:

applying a voltage between first and second electrodes of a device comprising first and second fluid containment members separated by a fluid barrier having a single nanopore therein; and a microfluidic introduction member for conveying a fluid sample from a first site distal from said nanopore to a second site proximal to said nanopore, and

monitoring electrical current through said said nanopore or between said electrodes.

10. The method according to claim 9, further comprising:

providing a sample in the microfluidic introduction member prior to said monitoring.

11. The method according to claim 10, wherein same sample comprises a polymeric compound.

12. The method according to claim 11, wherein said polymeric compound is nucleic acid.

13. The method according to claim 9, wherein said monitoring is performed over a period of time.

14. The method according to claim 9, wherein said method is a method of characterizing a polymeric compound.

15. The method according to claim 14, wherein said method of characterizing is a method of sequencing a nucleic acid.