U.S. patent application number 11/107540 was filed with the patent office on 2005-08-25 for nanopore device and methods of fabricating and using the same.
Invention is credited to Barth, Phillip W..
Application Number | 20050186629 11/107540 |
Document ID | / |
Family ID | 34394583 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186629 |
Kind Code |
A1 |
Barth, Phillip W. |
August 25, 2005 |
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
Inventors: |
Barth, Phillip W.; (Portola
Valley, CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.
INTELLECTUAL PROPERTY ADMINISTRATION, LEGAL DEPT.
P.O. BOX 7599
M/S DL429
LOVELAND
CO
80537-0599
US
|
Family ID: |
34394583 |
Appl. No.: |
11/107540 |
Filed: |
April 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11107540 |
Apr 15, 2005 |
|
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10693064 |
Oct 23, 2003 |
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Current U.S.
Class: |
435/6.19 ;
204/450 |
Current CPC
Class: |
B81C 1/00087 20130101;
B81B 2203/0127 20130101; G01N 33/48721 20130101; B81B 2201/058
20130101; B81B 2203/0338 20130101 |
Class at
Publication: |
435/006 ;
204/450 |
International
Class: |
C12Q 001/68 |
Claims
That which is claimed is:
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 .mu.m to about
10 .mu.m.
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.
Description
CROSS-REFERENCE
[0001] 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
[0002] 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).
[0003] 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.
[0004] 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
[0005] 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.
[0006] 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 .mu.m to about 4
.mu.m.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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,
.beta.-galactosidase, luciferase, and the like.
[0016] 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.
[0017] 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.
[0018] 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 .mu.m.
[0019] 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.
[0020] Resonant tunneling comprises two effects, one called
"matched level resonance" and one called "matched barrier
resonance."
[0021] 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.
[0022] 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.
[0023] A biopolymer that is "in", "within" or moving through a
nanopore means that the entire biopolymer any portion thereof, may
located within the nanopore.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] "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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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
[0038] 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:
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] FIG. 3A shows a step of an embodiment of the fabrication
method of the present invention.
[0045] FIG. 3B shows a step of an embodiment of the fabrication
method of the present invention.
[0046] FIG. 3C shows a step of an embodiment of the fabrication
method of the present invention.
[0047] FIG. 3D shows a step of an embodiment of the fabrication
method of the present invention.
[0048] FIG. 3E shows a step of an embodiment of the fabrication
method of the present invention.
[0049] FIG. 3F shows a step of an embodiment of the fabrication
method of the present invention.
[0050] FIG. 3G shows a step of an embodiment of the fabrication
method of the present invention.
[0051] FIG. 3H shows a step of an embodiment of the fabrication
method of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] The Subject Devices
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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 .alpha.-hemolysin, particularly
.alpha.-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.
[0067] 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.
[0068] 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.
[0069] 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 .mu.m to about 10 .mu.m, including about 1 .mu.m to about
9 .mu.m, such as from about 2 .mu.m to about 8 .mu.m, from about 3
.mu.m to about 7 .mu.m, from about 4 .mu.m to about 6 .mu.m. In
representative embodiments, the microfluidic introduction member
has an inner diameter ranging from about 1 .mu.m to about 4
.mu.m.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] Microfluidic introduction members 24, such as microfluidic
channels, are situated within microfluidic leads 22. Third cavity
26, being typically about 3 .mu.m to about 15 .mu.m in diameter,
including about 5 .mu.m in diameter, and fourth cavity 28, being
typically about 50 .mu.m to about 70 .mu.m in diameter, including
about 60 .mu.m 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.
[0076] 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.
[0077] 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/NR-
2000/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.
[0078] 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/cau-
stic-etching.pdf) to form channels 24. Parylene.RTM. 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.RTM., a silicone polymer, silicon dioxide, silicon
nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS
oxide, plasma nitride, an insulator, a semiconductor, and a
metal.
[0083] 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.RTM., a silicone polymer, silicon dioxide, silicon
nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS
oxide, plasma nitride, an insulator, a semiconductor, and a
metal.
[0084] 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.RTM., a
silicone polymer, silicon dioxide, silicon nitride, silicon
oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma
nitride, an insulator, a semiconductor, and a metal.
[0085] 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.
[0086] 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.RTM., a silicone polymer, silicon dioxide, silicon
nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS
oxide, plasma nitride, an insulator, a semiconductor, and a
metal.
[0087] 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.RTM., a silicone polymer, silicon dioxide,
silicon nitride, silicon oxynitride, silicon-rich silicon nitride,
TEOS oxide, plasma nitride, an insulator, a semiconductor, and a
metal.
[0088] 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.RTM., a
silicone polymer, silicon dioxide, silicon nitride, silicon
oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma
nitride, an insulator, a semiconductor, and a metal.
[0089] 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.RTM., a
silicone polymer, silicon dioxide, silicon nitride, silicon
oxynitride, silicon-rich silicon nitride, TEOS oxide, plasma
nitride, an insulator, a semiconductor, and a metal.
[0090] 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.RTM., a silicone polymer, silicon dioxide, silicon
nitride, silicon oxynitride, silicon-rich silicon nitride, TEOS
oxide, plasma nitride, an insulator, a semiconductor, and a
metal.
[0091] Region 54 may comprise one or more of a group including but
not limited to a polymer, photoresist, SU8 photoresist, epoxy,
polyimide, Parylene.RTM., a silicone polymer, silicon dioxide,
silicon nitride, silicon oxynitride, silicon-rich silicon nitride,
TEOS oxide, plasma nitride, an insulator, a semiconductor, and a
metal.
[0092] 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.
[0093] 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.
[0094] 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).
[0095] 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.
[0096] Fabrication of the Subject Devices
[0097] 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.
[0098] 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.
[0099] 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 .mu.m to 15 .mu.m,
including about 10 .mu.m.
[0100] 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.
[0101] 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.
[0102] 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.RTM., 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.RTM., 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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/N- R2000/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.
[0108] 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.
[0109] Layer 56, comprising, for example, polyimide or silicon
oxynitride, and ranging in thickness from about 1 .mu.m to about 5
.mu.m, including about 2 .mu.m, 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.RTM., 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.
[0110] The method continues as shown in FIG. 3G. A layer 60,
comprising, for example polyimide, ranging in thickness from about
15 .mu.m to about 30 .mu.m, including about 25 .mu.m, 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] Methods of Using the Subject Devices
[0117] 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.
[0118] Utility
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
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