U.S. patent application number 16/741971 was filed with the patent office on 2020-05-21 for nanometric material having a nanopore enabling high-sensitivity molecular detection and analysis.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Daniel Branton, Slaven Garaj, Jene A. Golovchenko.
Application Number | 20200158712 16/741971 |
Document ID | / |
Family ID | 43057332 |
Filed Date | 2020-05-21 |
United States Patent
Application |
20200158712 |
Kind Code |
A1 |
Branton; Daniel ; et
al. |
May 21, 2020 |
Nanometric Material Having A Nanopore Enabling High-Sensitivity
Molecular Detection and Analysis
Abstract
Provided herein is a nanopore sensor, including a self-supported
solid state material selected from hexagonal-BN, a mono-atomic
glass, MoS.sub.2, WS.sub.2, MoSe.sub.2, MoTe.sub.2, TaSe.sub.2,
NbSe.sub.2, NiTe.sub.2, Bi.sub.2Sr.sub.2CaCu.sub.2O.sub.x, and
Bi.sub.2Te.sub.3, having a thickness less than about 5 nm. A
nanopore extends through the material thickness. A connection from
the first material surface to a first reservoir provides, at the
first material surface, a species in an ionic solution from the
first reservoir to the nanopore, and a connection from the second
material surface to a second reservoir collects in the second
reservoir the species and ionic solution after translocation of the
species and ionic solution through the nanopore. An electrical
circuit is connected with the nanopore, through the material
thickness, from the first reservoir to the second reservoir, to
monitor translocation of species in the ionic solution through the
nanopore in the solid state material.
Inventors: |
Branton; Daniel; (Lexington,
MA) ; Garaj; Slaven; (Boston, MA) ;
Golovchenko; Jene A.; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
43057332 |
Appl. No.: |
16/741971 |
Filed: |
January 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13419383 |
Mar 13, 2012 |
10564144 |
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16741971 |
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PCT/US2010/049238 |
Sep 17, 2010 |
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13419383 |
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61243607 |
Sep 18, 2009 |
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61355528 |
Jun 16, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2015/0038 20130101;
G01N 15/1056 20130101; G01N 15/12 20130101; C12Q 2565/631 20130101;
B82Y 30/00 20130101; G01N 33/48721 20130101 |
International
Class: |
G01N 33/487 20060101
G01N033/487; G01N 15/12 20060101 G01N015/12; B82Y 30/00 20060101
B82Y030/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under
Contract No. 2R01HG003703-04 awarded by the NIH. The Government has
certain rights in the invention.
Claims
1. A nanopore sensor comprising: a self-supported solid state
material selected from hexagonal-BN, a mono-atomic glass,
MoS.sub.2, WS.sub.2, MoSe.sub.2, MoTe.sub.2, TaSe.sub.2,
NbSe.sub.2, NiTe.sub.2, Bi.sub.2Sr.sub.2CaCu.sub.2O.sub.x, and
Bi.sub.2Te.sub.3, the solid state material having a thickness,
between a first solid state material surface and a second solid
state material surface opposite the first solid state material
surface, that is less than about 5 nm; a nanopore having a nanopore
diameter extending through the solid state material thickness
between the first and second solid state material surfaces; a
connection from the first solid state material surface to a first
reservoir to provide, at the first solid state material surface, a
species in an ionic solution from the first reservoir to the
nanopore; a connection from the second solid state material surface
to a second reservoir to collect in the second reservoir the
species and ionic solution after translocation of the species and
ionic solution through the nanopore from the first solid state
material surface to the second solid state material surface; and an
electrical circuit connected with the nanopore, through the solid
state material thickness, from the first reservoir to the second
reservoir, to monitor translocation of the species in the ionic
solution through the nanopore in the solid state material.
2. The nanopore sensor of claim 1 wherein the nanopore diameter is
less than about 3 nm.
3. The nanopore sensor of claim 1 wherein the nanopore diameter is
greater than the solid state material thickness.
4. The nanopore sensor of claim 1 wherein the nanopore diameter is
less than about 2.5 nm.
5. The nanopore sensor of claim 1 wherein the nanopore diameter is
no more than about 5% larger than a diameter characteristic of the
species in the ionic solution translocating through the
nanopore.
6. The nanopore sensor of claim 1 wherein the electrical circuit is
connected between the first and second reservoirs to measure flow
of ionic current through the nanopore in the solid state
material.
7. The nanopore sensor of claim 1 wherein the electrical circuit
includes an electrical current monitor connected for measuring
time-dependent ionic current flow through the nanopore.
8. The nanopore sensor of claim 7 wherein the electrical current
monitor is connected for measuring time-dependent blockages of
ionic current flow through the nanopore, indicative of species
translocation through the nanopore.
9. The nanopore sensor of claim 1 further comprising an electrode
disposed in each of the first and second ionic solutions for
applying a voltage across the nanopore to electrophoretically cause
species translocation through the nanopore.
10. The nanopore sensor of claim 1 wherein the ionic solution is
characterized by a salt content that is greater than about 2 M.
11. The nanopore sensor of claim 1 wherein the ionic solution is
characterized by a pH that is greater than about 8.
12. The nanopore sensor of claim 1 wherein the ionic solution is
KCl.
13. The nanopore sensor of claim 1 wherein the nanopore diameter is
between about 1 nm and about 5 nm.
14. The nanopore sensor of claim 1 wherein the solid state material
thickness is less than about 1 nm.
15. The nanopore sensor of claim 1 wherein the solid state material
thickness is less than about 0.7 nm.
16. The nanopore sensor of claim 1 wherein the solid state material
is mechanically supported at edges of the solid state material by a
frame structure.
17. The nanopore sensor of claim 1 wherein the species in the ionic
solution to translocate through the nanopore comprises at least one
of biomolecules, DNA molecules, and RNA molecules.
18. The nanopore sensor of claim 1 wherein the species in the ionic
solution to translocate through the nanopore comprises at least one
of nucleotides and oligonucleotides.
19. The nanopore sensor of claim 1 wherein the species in the ionic
solution to translocate through the nanopore comprises a
protein.
20. The nanopore sensor of claim 1 wherein the species in the ionic
solution to translocate though the nanopore comprises a polymer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of copending U.S. application Ser. No.
13/419,383, filed Mar. 13, 2012, which is a continuation-in-part of
International Application PCT/US2010/049238, having an
international filing date of Sep. 17, 2010, and which claims the
benefit of U.S. Provisional Application No. 61/243,607, filed Sep.
18, 2009, and U.S. Provisional Application No. 61/335,528, filed
Jun. 16, 2010, the entirety of all of which are hereby incorporated
by reference.
BACKGROUND
[0003] This invention relates generally to molecular detection and
analysis, and more particularly relates to configurations for a
nanopore arranged to detect molecules translocating through the
nanopore.
[0004] The detection, characterization, identification, and
sequencing of molecules, including biomolecules, e.g.,
polynucleotides such as the biopolymer nucleic acid molecules DNA,
RNA, and peptide nucleic acid (PNA), as well as proteins, and other
biological molecules, is an important and expanding field of
research. There is currently a great need for processes that can
determine the hybridization state, configuration, monomer stacking,
and sequence of polymer molecules in a rapid, reliable, and
inexpensive manner. Advances in polymer synthesis and fabrication
and advances in biological development and medicine, particularly
in the area of gene therapy, development of new pharmaceuticals,
and matching of appropriate therapy to patient, are in large part
dependent on such processes.
[0005] In one process for molecular analysis, it has been shown
that molecules such as nucleic acids and proteins can be
transported through a natural or solid-state nano-scale pore, or
nanopore, and that characteristics of the molecule, including its
identification, its state of hybridization, its interaction with
other molecules, and its sequence, i.e., the linear order of the
monomers of which a polymer is composed, can be discerned by and
during transport through the nanopore. Transport of a molecule
through a nanopore can be accomplished by, e.g., electrophoresis,
or other translocation mechanism.
[0006] In one particularly popular configuration for molecular
analysis with a nanopore, the flow of ionic current through a
nanopore is monitored as a liquid ionic solution, and molecules to
be studied that are provided in the solution, traverse the
nanopore. As molecules in the ionic solution translocate through
the nanopore, the molecules at least partially block flow of the
liquid solution, and the ions in the solution, through the
nanopore. This blockage of ionic solution can be detected as a
reduction in measured ionic current through the nanopore. With a
configuration that imposes single-molecule traversal of the
nanopore, this ionic blockage measurement technique has been
demonstrated to successfully detect individual molecular nanopore
translocation events.
[0007] Ideally, this ionic blockage measurement technique for
molecular analysis, like others that have been proposed, should
enable molecular characterization with high sensitivity and
resolution on the scale of single monomer resolution. Unambiguous
resolution of individual monomer characteristics is critical for
reliable applications such as biomolecular sequencing applications.
But this capability has been difficult to achieve in practice,
particularly for solid-state nanopore configurations. It has been
found that the length of a solid state nanopore, determined by the
thickness of a material layer or layers in which the nanopore is
formed, impacts the nature of molecular traversal of the nanopore,
and directly limits the sensitivity and the resolution with which
molecules in the nanopore can be detected and analyzed.
SUMMARY OF THE INVENTION
[0008] There is provided a nanopore sensor that overcomes the
sensitivity and resolution limitations of conventional nanopore
sensors. The nanopore sensor includes a self-supported solid state
material selected from hexagonal-BN, a mono-atomic glass,
MoS.sub.2, WS.sub.2, MoSe.sub.2, MoTe.sub.2, TaSe.sub.2,
NbSe.sub.2, NiTe.sub.2, Bi.sub.2Sr.sub.2CaCu.sub.2O.sub.x, and
Bi.sub.2Te.sub.3, and has a thickness less than about 5 nm. A
nanopore, having a nanopore diameter, extends through the solid
state material thickness. A connection from the first solid state
material surface to a first reservoir provides, at the first solid
state material surface, a species in an ionic solution from the
first reservoir to the nanopore. A connection from the second solid
state material surface to a second reservoir is provided to collect
in the second reservoir the species and ionic solution after
translocation of the species and ionic solution through the
nanopore from the first solid state material surface to the second
solid state material surface An electrical circuit is connected
with the nanopore, through the material thickness, from the first
reservoir to the second reservoir, to monitor translocation of
species in the ionic solution through the nanopore in the solid
state material.
[0009] This nanopore sensor arrangement and the sensing methods
enabled therewith provide high-resolution, high-sensitivity
molecular detection and analysis, thereby achieving the detection,
sensing, and analysis of species such as closely-spaced monomers in
a polymer and accordingly, enable sequential resolution of
different ionic flow blockages caused by each monomer in, for
example, a strand of a DNA polymer.
[0010] Other features and advantages of the invention will be
apparent from the following description and accompanying figures,
and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic perspective view of an example
graphene nanopore device for detecting molecules by measurement of
ionic flow through the nanopore;
[0012] FIGS. 2A-2E are schematic side views of six theoretical
nanopores in membranes, each nanopore of 2.4 nm in diameter and
ranging in nanopore length from 0.6, nm, 1 nm, 2 nm, 5 nm and 10
nm, respectively, with the average ionic current density at various
regions through each nanopore represented the lengths of arrows
shown in the nanopores;
[0013] FIG. 3 is a plot of ionic current blockade, defined as the
absolute value of the difference between the ionic current through
an unblocked nanopore and the ionic current through the same
nanopore when blocked with a molecule of the indicated diameter,
for a 3M KCl ionic solution and a nanopore bias of 160 mV, for
nanopores having a 2.5 nm diameter and effective lengths of 0.6 nm,
2 nm, 5 nm, and 10 nm;
[0014] FIG. 4 is an X-ray diffraction image of an experimental
graphene membrane, displaying the requisite hexagonal pattern that
arises from the hexagonal packing of carbon atoms in a single
graphene layer;
[0015] FIG. 5 is a plot of Raman shift measurements for an
experimental graphene membrane indicating single-layer graphene for
the membrane;
[0016] FIG. 6 is a plot of experimentally-measured data of ionic
current as a function of the voltage bias applied between 3M KCl
ionic solutions on the cis and trans sides of an experimental
graphene membrane;
[0017] FIG. 7 shows the plot of FIG. 6 and a plot of ionic current
as a function of voltage for an experimental graphene membrane
including an 8 nm-wide nanopore;
[0018] FIG. 8 is a plot of ionic conductance as a function of
nanopore diameter for nanopores having a length of 0.6 nm, 2 nm,
and 10 nm;
[0019] FIG. 9 is a plot of measured ionic current as a function of
time for a 2.5 nm nanopore in an experimental graphene membrane as
DNA fragments translocate through the nanopore;
[0020] FIGS. 10A-10C are plots of measured ionic current as a
function of time taken from the plot of FIG. 9, showing in detail
the current profile for DNA nanopore translocation in single-file
fashion, in partially-folded fashion, and folded in half;
[0021] FIG. 11 is a plot of ionic current blockage as a function of
DNA translocation of a nanopore in a graphene membrane for 400
translocation events; and
[0022] FIG. 12 is a plot of the percentage change in ionic current
blockade as a function of distance through a nanopore, for a 0.6
nm-long nanopore and for a 1.5 nm-long nanopore.
DETAILED DESCRIPTION
[0023] FIG. 1 is a schematic perspective view of an example
graphene nanopore molecular characterization device 10. For clarity
of discussion, device features illustrated in FIG. 1 are not shown
to scale. As shown in FIG. 1, in the device there is provided a
nano-scale aperture, or nanopore 12, in a bare, single-layer
graphene membrane 14. The graphene membrane is self-supported,
meaning that there are no structures under the extent of the
membrane to support the membrane. At the edges of the membrane
there can be provided, e.g., a support frame 16, which in turn can
be provided on a support substrate or other structure 18. The
self-supported bare graphene membrane is configured in a fluidic
cell such that on the first, or cis, side of the graphene membrane
is a connection to a first liquid reservoir or liquid supply
containing a liquid solution including molecules 20 to be
characterized, and on the second, or trans, side of the graphene
membrane is a connection to a second liquid reservoir, into which
characterized molecules are transported by translocation through
the graphene nanopore 12.
[0024] In one application of the graphene nanopore, shown in the
figure, the molecules 20 to be characterized comprise
single-stranded DNA molecules (ssDNA) having a sequence of
nucleoside bases 22 to be characterized, for example, by
determining the identity of the sequence of bases along each ssDNA
backbone. For clarity of discussion this sequencing example will be
employed in the following description, but such is not the
exclusive application of the graphene nanopore characterization
device. In addition, the sequencing operation described below is
not limited to the example of DNA; the polynucleotide RNA can
similarly be characterized. The molecular characterization enabled
by the graphene nanopore device includes a wide range of analyses,
including, e.g., sequencing, hybridization detection, molecular
interaction detection and analysis, configuration detection, and
other molecular characterizations. The molecules 20 to be
characterized can include, in general, any molecule, including
polymers and biomolecules such as proteins, nucleic acids such as
the polynucleotides DNA and RNA, sugar polymers, and other
biomolecules. The discussion below is therefore not intended to be
limiting to a particular implementation, but provides details of
one example in a range of embodiments for molecular
characterization.
[0025] There is provided for the graphene nanopore of FIG. 1 an
arrangement of features for causing molecules 20 to traverse the
nanopore through the bare, self-supported, single-layer graphene
membrane. For example, there can be provided silver chloride
electrodes 24, 26 immersed in the solutions on either side of the
graphene membrane 14, for controlling the voltage of each solution
across the graphene membrane. Application of a voltage bias 24
between the electrodes in the two solutions on opposing sides of
the membrane causes molecules, e.g., ssDNA molecules, provided in
the solution on the first, or cis, side of the membrane, to be
electrophoretically driven into and through the nanopore 12 to the
solution on the second, or trans side of the membrane, because the
DNA backbone is negatively charged when in solution.
[0026] The inventors herein have made a surprising discovery that
the ionic resistivity perpendicular to the plane of a bare,
single-layer graphene membrane separating two ionic solution-filled
reservoirs is extremely large, making it possible to establish a
significant voltage bias across the graphene membrane, between the
two solutions, in the manner described above. As explained further
in the experimental discussion below, this discovery enables the
configuration of FIG. 1 in which electrical control of the
potential across a single layer of graphene can be maintained in a
manner required for molecular electrophoresis.
[0027] It is further discovered that a bare, single-layer graphene
membrane is sufficiently mechanically robust to operate as a
structural barrier between two solution-filled reservoirs whether
or not these reservoirs are in communication directly with each
other through a nanopore in the graphene membrane that is supported
only at its edges by a frame, i.e., that is self-supported across
its extent. As a result, a nanopore-articulated membrane of a
single bare graphene layer can operate to separate two ionic
solution-filled reservoirs, using methods known to those familiar
with the nanopore field, for application of a voltage bias between
the two ionic solutions on the cis and trans sides of the bare
graphene membrane to electrophoretically drive molecules through
the nanopore.
[0028] Other techniques and arrangements can be employed for
drawing molecules through the nanopore, and no particular technique
is required. Further details and examples for electrophoretic
driving of molecular translocation through a nanopore are provided
in "Molecular and Atomic Scale Evaluation of Biopolymers," U.S.
Pat. No. 6,627,067, to Branton et al., issued Sep. 30, 2003, the
entirety of which is hereby incorporated by reference.
[0029] As shown in FIG. 1, there can be provided circuitry 26, 28
for measuring changes in ionic current flow between the cis and
trans sides of the graphene membrane, through the nanopore 12. With
this configuration, translocation of molecules through the nanopore
12 can be detected and based on the detection, can be analyzed as
the molecules are driven through the nanopore. This molecular
detection technique is but one of a wide range of detection
techniques that can be employed with the graphene membrane and
nanopore. Tunneling current between electrodes, e.g., between
carbon nanotubes or other probes articulated at the nanopore,
conductance changes in probes or in the graphene membrane itself,
or other molecular detection technique can be employed, as
described, e.g., in "Molecular Characterization with Carbon
Nanotube Control," U.S. Pat. No. 7,468,271 by Golovchenko et al.,
issued Dec. 23, 2008, the entirety of which is hereby incorporated
by reference.
[0030] Considering specifically the technique of molecular
detection by ionic current flow measurement, the inventors herein
have made a surprising discovery that the ionic current through the
nanopore of the bare, single-layer graphene membrane, when empty of
a translocating species, and the ionic current flow through the
nanopore, when blocked by a molecule that is in the nanopore, are
both approximately 3 times greater than the ionic current flow
through a similar-diameter nanopore in any other known lipid or
solid state membrane interface. This significantly greater ionic
current flow through a nanopore in the bare, single-layer graphene
membrane, compared to a similar-diameter biopore or nanopore in
another solid state membrane, is understood by the inventors to be
due to the thinness of the graphene membrane and correspondingly,
the length of the nanopore through the membrane.
[0031] A bare graphene membrane is a single-atom layer of a
hexagonal carbon lattice that is therefore atomically thin, being
only about 0.3 nm thick. At this thickness, ionic flow through a
nanopore in the bare, single-layer graphene membrane can be
characterized in a regime in which the length of the nanopore is
much less than the diameter of the pore. In this regime, the ionic
conductance of the nanopore is proportional to the nanopore
diameter, d, and the ionic current density through the nanopore is
sharply peaked at the periphery of the nanopore, that is, at the
edge of the nanopore, compared with the current density at the
middle of the nanopore. In contrast, nanopores having a length that
is greater than the nanopore diameter are characterized by an ionic
conductance that is proportional to the nanopore area, and that is
homogeneous across the nanopore diameter, with ionic conductivity
uniformly flowing down through the middle of the nanopore as well
as at the periphery of the nanopore.
[0032] The clear distinction between nanopore conductances in these
two nanopore length regimes are illustrated in FIGS. 2A-2E.
Referring to those figures, there are shown representations of the
average current density at ten points across nanopores each having
a diameter of 2.4 nm and having a length of 0.6 nm, 1 nm, 2 nm, 5
nm, and 10 nm, respectively. The relative lengths of the arrows in
the figures indicate the relative average current density in the
region in a nanopore that is represented by the location of each
arrow. As shown in FIGS. 2A-2C, for nanopore lengths that are less
than the nanopore diameter of 2.4 nm, the current density is peaked
at the nanopore periphery. As the nanopore length approaches the
nanopore diameter, the conductance across the nanopore becomes more
uniform. When the nanopore length is greater than the nanopore
diameter, as in FIGS. 2D and 2E, the ionic conductance is uniformly
homogenous across the nanopore, with no preference for the nanopore
periphery. The local current density within different regions of
the nanopores becomes more and more homogeneous as the nanopore
length is increased.
[0033] As a consequence, a nanopore in a bare, self-supported
single-layer graphene membrane in which the nanopore diameter is
greater than the nanopore length exhibits a total ionic
conductance, in an unobstructed state, that is significantly
greater than the total conductance of a nanopore of equal diameter
in a membrane having a thickness greater than the nanopore
diameter. Other conditions being equal, the greater conductance
results in a significantly greater total ionic current through an
open nanopore of a given diameter in a membrane thinner than the
diameter than in an open nanopore of equal diameter in a membrane
thicker than the diameter. The larger ionic currents through the
graphene membrane facilitate high-accuracy measurement of ionic
current flow through the nanopore.
[0034] Because the ionic current through nanopores having a length
less than nanopore diameter is primarily at the nanopore periphery
rather than through the nanopore center axis, small changes in the
diameter of molecules centrally traversing the nanopore have an
enormous effect on the change in ionic current flow. This is due to
the fact that differences in the diameters of molecules are
manifested at the nanopore edge, where ionic current flow is
greatest for short-length nanopores, rather than in the nanopore
center, where for short-length nanopores ionic current is lower. As
a result, a bare, single-layer graphene nanopore having a length
less than the nanopore diameter is more sensitive to
molecularly-dimensioned particles or differences in
differently-dimensioned particles, molecules, or their components
than are nanopores having a length greater than the nanopore
diameter.
[0035] The consequence of this consideration is shown
quantitatively in FIG. 3, in which there is plotted the computed
ionic current blockage level in a nanopore as a function of the
diameter of polymer molecules centrally traversing nanopores having
a diameter of 2.5 nm and having effective lengths of 0.6 nm, 2 nm,
5 nm, and 10 nm. The computed current blockade is the absolute
value of the difference between the ionic current though an
unblocked nanopore, i.e., no polymer molecule in the nanopore, and
the ionic current through the same nanopore when blocked with a
polymer of the indicated diameter. The plots assume molecular
translocation with an ionic solution of 3M KCl and a voltage bias
of 160 mV between cis and trans sides of the nanopore. As shown in
the plots here, the ionic current through the nanopores
demonstrates increasing sensitivity to changes in diameter of
translocating molecules as the length of the nanopores is
decreased.
[0036] The inventors have further discovered that the sensitivity
in a nanopore's conductivity to changes in translocating molecules'
diameters is maximized when the nanopore diameter is set to be as
close as possible to the diameter of the translocating molecules.
This condition is true for nanopores of any length. For example, as
shown in the plots of FIG. 3, for nanopores of 2.5 nm in diameter,
as the translocating molecule diameter approaches the nanopore
diameter, the current blockage rises, even where the nanopore
length is greater than the nanopore diameter. But for nanopores in
which nanopore length is less than nanopore diameter, namely 2 nm
and 0.6 nm in the plotted data, it is shown that such short-length
nanopores are much more acutely sensitive to small changes in
translocating molecule diameter as the molecule diameter approaches
the nanopore diameter. For these nanopores the blockade currents
rise exponentially with increases in blocking molecule diameter.
For the 5 nm and 10 nm-long nanopores, which are larger than the
nanopore diameter, the blockade currents rise only in a near linear
manner, even as blocking molecules' diameters approach the nanopore
diameter.
[0037] Thus, the resolution of closely-spaced differences in
translocating molecules' diameters is preferably maximized by
providing in a single-layer graphene membrane a nanopore having a
diameter that is both greater than the membrane edge thickness but
not much greater than the diameter expected for molecules that are
translocating the nanopore, e.g., no more than 5% greater. To
determine this second condition for nanopore diameter for a given
application, there can be carried out an analysis like that
described in the Example below. Briefly, in such an analysis, there
is determined via, e.g., a Laplace equation, the ionic current
density of the ionic solution that will be used for molecular
translocation, the desired sensitivity of molecular translocation
detection is set, and the general requirements for what nanopore
diameter is feasible are determined. Based on these factors, and
the overriding constraint that the nanopore diameter is greater
than the membrane thickness, a nanopore diameter that optimizes all
of these factors can then be selected.
[0038] In conducting such a nanopore design analysis, the widest
feature of a molecular structure that exists along the length of a
molecule can be considered as setting the molecular diameter. The
nanopore diameter that optimizes the factors above can then be
specified, particularly to be no more than about 5% greater than
the widest molecular feature. Given that a nanopore may not be
optimally circular, the smallest extent across the expanse of a
nanopore can be considered the diameter of the nanopore for this
design purpose. This smallest nanopore extent is to be no more than
about 5% greater than the widest molecular feature of a molecular
structure to translocate the nanopore.
[0039] The inventors have further discovered that the electrical
noise from a bare, self-suspended single-layer graphene nanopore
separating two electrically-biased ionic solution-filled reservoirs
is proportionally no greater than the electrical noise from any
other solid state nanopore. As a result, given that the ionic
current change, i.e., ionic blockage, through a graphene nanopore
is greater during traversal of a molecule of any given diameter
than it is in other known nanopores having a length greater than
the nanopore diameter, a bare, single-layer graphene nanopore can
produce a better signal-to-noise ratio than other known nanopores
because the greater the number of ions counted per unit time, or
per traversing nucleobase, will be more precise than at a lesser
count rate. These discoveries, together with graphene's known
chemical inertness and exceptionally great strength, establish a
nanopore-articulated bare, single-layer graphene membrane as a
superior interface for molecular detection and
characterization.
[0040] As a result of these discoveries, it is preferred that the
membrane be provided as a single layer of graphene that is bare,
i.e., that is not coated on either side with any material layer or
species that adds to the graphene membrane thickness. In this
state, the thickness of the membrane is minimized and is safely in
the short-length nanopore regime in which peripheral ionic current
flow is maximized and in which the nanopore conductivity as a
function of changes in the analytes physical dimensions is
maximized. The very short nanopore length provided by the graphene
membrane also makes it possible for a graphene nanopore to sense
closely-spaced monomers in a polymer and thus to sequentially
resolve the different ionic blockages caused by each monomer in,
for example, a strand of a DNA polymer.
[0041] It is recognized that a single-layer graphene membrane has
an affinity for many molecules such as polymer molecules like DNA
and RNA. It can therefore be expected that DNA, RNA, and other like
molecules have a tendency to adsorb onto a bare graphene membrane
preferentially. It is preferred that the absorptive properties of
the graphene surface be at least partially inhibited with an
appropriate environment and/or surface treatment that maintains the
membrane in a bare state without added surface layers.
[0042] For example, there can be provided an ionic solution that is
characterized by a pH greater than about 8, e.g., between about 8.5
and 11 and that includes a relatively high salt concentration,
e.g., greater than about 2M and in the range from 2.1M to 5M. By
employing a basic solution of high ionic strength, adherence of
molecules to the surface of the bare graphene membrane is
minimized. Any suitable selected salt can be employed, e.g., KCl,
NaCl, LiCl, RbCl, MgCl.sub.2, or any readily soluble salt whose
interaction with the analyte molecule is not destructive.
[0043] In addition, as explained in detail below, during synthesis
and manipulation of the graphene membrane it is preferred that
extreme care be taken to maintain the membrane in a pristine
condition such that substantially no residues or other species,
which might attract molecules to the graphene surface, are present.
It is also recognized that in operation, the graphene membrane can
be electrically manipulated to repel molecules from the graphene
surface. For example, given translocation of negatively-charged DNA
molecules through a nanopore in a graphene membrane, a graphene
membrane can itself be electrically biased at a negative potential
that repels the negatively-charged DNA molecule. Here electrical
contact can be made to the graphene membrane in any suitable manner
that enables application of a selected voltage. In such a scenario,
the voltage between ionic solutions on either side of the graphene
membrane can be set sufficiently high to produce an electrophoretic
force which overcomes the repulsion at the graphene surface to
cause DNA translocation through the nanopore rather than adsorption
at the graphene surface.
[0044] Turning to methods for producing the graphene nanopore
device, a single layer of bare graphene can be synthesized by any
convenient and suitable technique, and no specific synthesis
technique is required. In general, atmospheric chemical vapor
deposition with methane gas on a catalyst material, e.g., a nickel
layer, can be employed to form the graphene layer. Raman
spectroscopy, transmission electron microscopy, and selected-area
diffraction studies can be employed to verify that a region of
synthesized graphene to be employed truly is single-layer in
nature.
[0045] The transfer of the graphene layer to a device structure for
arrangement as a graphene membrane can be conducted by any suitable
technique, but it is preferred that any materials employed in the
transfer do not corrupt the graphene surface. In one preferable
technique, a selected handle material is coated over the
synthesized graphene layer on the catalyst material and substrate.
For many applications, it can be preferred to employ a handle
material that is easily removed from the graphene surface once
handling of the graphene layer is complete. Methyl
methacrylite-methylacrylic acid co-polymer (MMA-MAA) can be a
particularly well-suited handle material. With a layer of MMA-MAA
in place on the graphene layer, the entire structure can be cut
into pieces.
[0046] The resulting pieces can then be processed to remove the
catalyst layer and substrate material underlying the graphene layer
while adhered to the handle layer. For example, given a catalyst
layer of Ni, an HCl solution can be employed to etch away the Ni
layer and free the graphene/MMA-MAA composite, with distilled water
employed to rinse. The graphene/MMA-MAA composite, floating on the
water, can then be captured by, e.g., a silicon wafer coated with a
SiN.sub.x layer. The central region of the silicon wafer can be
etched by KOH or other suitable etchant to produce a free-standing
SiN.sub.x membrane, e.g., of 50.times.50 .mu.m.sup.2 area. A
focused ion beam (FIB) or other process can then be employed to
drill a suitable hole through the SiN.sub.x membrane such that it
forms a frame for the graphene layer membrane. For example, a
square window of, e.g., 200 nm.times.200 nm can be formed in the
nitride membrane to produce a frame for the graphene membrane.
[0047] With this device configuration complete, the
graphene/MMA-MAA composite can be placed over the square window in
the graphene membrane, employing, e.g., nitrogen wind (a gentle jet
of nitrogen) to firmly press the graphene against the substrate.
The MMA-MAA can then be removed, e.g., under a slow drip of
acetone, followed by immersion in acetone, dichloroethane, and
isopropanol.
[0048] It is preferable to remove any residues from the graphene
film to reduce the tendency of species to adhere to the graphene
once configured as a membrane. For example, once the MMA-MAA is
removed, the resulting structure including a graphene membrane
outstretched across a nitride frame, as in FIG. 1, can be immersed
in, e.g., a solution of KOH at room temperature briefly, e.g., for
1 min, and then vigorously rinsed with, e.g., water, then
isopropanol, and finally ethanol. To avoid damage to the graphene
membrane, the structure can be critical-point dried. Finally, the
structure can be exposed to a selected environment, e.g., a rapid
thermal annealing process at about 450.degree. C. in a stream of
gas containing 4% H.sub.2 in He for, e.g., 20 minutes, to drive off
any remaining hydrocarbons. To avoid recontamination, the structure
preferably is then immediately loaded into, e.g., a TEM, for
further processing.
[0049] A nanopore can then be formed in the graphene membrane.
Focused electron beam or other process can be employed to form the
nanopore. The nanopore diameter preferably is greater than the
thickness of the graphene membrane, to obtain the benefits of the
unexpected discovery of increased peripheral ionic current flow and
increased sensitivity to change in molecular dimension as described
above. For translocation of ssDNA, a nanopore diameter of between
about 1 nm and about 20 nm can be preferred, with a diameter of
between about 1 nm and about 2 nm most preferred. For translocation
of dsDNA, a nanopore diameter of between about 2 nm and about 20 nm
can be preferred, with a diameter of between about 2 nm and about 4
nm most preferred. After nanopore formation, it is preferred to
keep the graphene structure under a clean environment, e.g., a
vacuum of .about.10.sup.-5 Torr.
[0050] To complete the nanopore molecular sensing device of FIG. 1,
the mounted graphene membrane can be inserted between two
half-cells in, e.g., a microfluidic cassette of
polyether-etherketone (PEEK) or other suitable material, sealed
with, e.g., polydimethylsiloxane (PDMS) gaskets. It can be
preferred that the gasket orifice be smaller than the dimensions of
the graphene membrane to completely seal off the edges of the
graphene membrane from the solutions.
Example I
[0051] This example describes an experimental demonstration of a
single-layer, bare graphene membrane. A graphene layer was
synthesized by CVD on a nickel surface. The nickel was provided as
a film by E-beam evaporation on a silicon substrate coated with a
layer of SiO.sub.2. The nickel layer was thermally annealed to
generate a Ni film microstructure with single-crystalline grains of
sizes between about a 1 .mu.m and 20 .mu.m. The surfaces of these
grains had atomically flat terraces and steps, similar to the
surface of single crystal substrates for epitaxial growth. With
this topology, the growth of graphene on Ni grains resembles the
growth of graphene on the surface of a single crystal
substrate.
[0052] In the CVD synthesis, the Ni layer was exposed to H.sub.2
and CH.sub.4 gases at a temperature of about 1000.degree. C. Raman
spectroscopy, transmission electron microscopy and selected area
diffraction studies showed the graphene film to be of excellent
quality and mostly (87%) a mixture of one and two layer thick
domains, with domain sizes of .about.10 .mu.m. Thicker regions of
three or more graphene layers, easily distinguished by color
contrast in an optical microscope, covered only a small fraction of
the total surface. If thicker regions or domain boundaries were
found, that area was discarded.
[0053] Graphene was transferred to a carrier Si/SiN.sub.x chip by
first coating the graphene with MMA-MAA copolymer (MMA(8.5)MAA EL9,
Microchem Corp.) and cut into 0.5 nm.times.0.5 mm pieces. These
pieces were immersed for .about.8 hr in 1N HCl solution to etch
away the Ni film and free the graphene/polymer membrane, which was
transferred to distilled water on which the graphene/polymer
floated, graphene-side down. Carrier Si chips coated with
.about.250 nm thick SiN.sub.x were used to scoop up of the floating
graphene/polymer film pieces, taking care that the graphene/polymer
films were each stretched over the central region of a chip. The
central region of the chip had been microfabricated using standard
anisotropic etch techniques to leave a .about.50.times.50
.mu.m.sup.2 area of the SiN.sub.x coating as a free-standing
SiN.sub.x membrane into which a square window, .about.200
nm.times.200 nm, had been drilled using a focused ion beam (FIB). A
nitrogen gas wind was used to firmly press the graphene against the
chip's surface. This led to expulsion of a small amount of liquid
from under the graphene, which adhered strongly and irreversibly to
the carrier chip's SiN.sub.x coating. The polymer on top of the
graphene was removed under a slow drip of acetone, followed by
subsequent immersions in acetone, dichloroethane, and finally
isopropanol.
[0054] To remove any residues from the graphene film, each chip was
subsequently immersed in 33 wt % solution of KOH at room
temperature for 1 min and then vigorously rinsed with isopropanol
and ethanol. To avoid damage to the suspended free-standing portion
of the graphene film, each chip was critical-point dried. Finally,
the chips were loaded into a rapid thermal annealer and heated to
450.degree. C. in a stream of gas containing 4% H.sub.2 in He for
20 minutes to drive off any remaining hydrocarbons. To avoid
recontamination, the chips were immediately loaded into a
transmission electron microscope for further processing.
[0055] There is shown in FIG. 4 an X-ray diffraction image of one
of the graphene membranes, displaying the requisite hexagonal
pattern that arises from the hexagonal packing of carbon atoms in a
single graphene layer. There is shown in FIG. 5 the Raman shift
measurements for the graphene layer. The very small G peak and very
sharp 2D peak, producing a G/2D ratio of less than 1, indicates a
single-layer membrane.
Example II
[0056] This example describes an experimental determination of the
conductance of the single-layer, bare graphene membrane of Example
I.
[0057] A chip-mounted single-layer graphene membrane from Example I
was inserted between the two half-cells of a custom-built
microfluidic cassette made of polyether-etherketone (PEEK). The two
sides of the chip were sealed with polydimethylsiloxane (PDMS)
gaskets. The opening of the gasket that pressed against the
graphene film on the Si/SiN.sub.x carrier chip had an inside
diameter of .about.100 .mu.m. Consequently, the gasket orifice was
smaller than the dimensions of the graphene membrane (0.5.times.0.5
mm.sup.2), and completely sealed off the graphene membrane edge
from the electrolyte. On the opposite side of the chip, the
electrolyte was in contact with the graphene membrane only through
the 200 nm wide square window in SiN.sub.x membrane. Note that with
this arrangement there was a large area difference between the two
graphene membrane faces in contact with the electrolyte (a circular
area of 100 .mu.m diameter vs. a square 200 nm.times.200 nm
area).
[0058] The two half-cells were first filled with ethanol to
facilitate wetting of the chip surface. The cell was then flushed
with deionized water, followed by 1M KCl salt solution with no
buffer. To avoid any potential interaction between the graphene
membrane and solutes which could affect experimental measurements,
all electrolytes used in the experiment were kept as simple as
possible and were unbuffered. All solution pHs ranged only 0.2 pH
units, from 5.09 to 5.29, as measured both before and after use in
the described experiments.
[0059] Ag/AgCl electrodes in each half-cell were used to apply an
electric potential across the graphene membrane and to measure
ionic currents. The current traces were acquired using an Axopatch
200B (Axon instruments) amplifier, which was connected to an
external 8-pole Bessel low-pass filter (type 90IP-L8L, Frequency
Devices, Inc.) operating at 50 kHz. The analog signal was digitized
using a NI PCI-6259 DAQ card (National Instruments) operating at
250 kHz sampling rate and 16-bit resolution. All experiments were
controlled through IGOR Pro software.
[0060] FIG. 6 is a plot of experimentally-measured data of ionic
current as a function of the voltage bias applied between 3 M KCl
ionic solutions on the cis and trans sides of the graphene
membrane. Applying Ohm's Law to this data, it is found that the
ionic current resistivity is well into the 3-4 G'.OMEGA. range
perpendicular to the plane of the graphene membrane. This
demonstrates one discovery of the invention that the ionic
resistivity perpendicular to the plane of a graphene membrane is
very large, and enables a configuration in which a significant
electrical bias can be maintained across a bare, single-layer
graphene membrane separating two voltage-biased ionic
solution-filled reservoirs.
[0061] With a 100 mV bias applied between the two Ag/AgCl
electrodes, ionic current measurements for a variety of chloride
electrolytes on the cis and trans sides of the graphene membrane
were conducted. Conductivities of the electrolytes were measured
using an Accumet Research AR50 conductivity meter, which had been
calibrated using conductivity standard solutions (Alfa Aesar,
product #43405, 42695, 42679). All the fluidic experiments were
performed under temperature controlled laboratory conditions, at
24.degree. C. Table 1 shows that the graphene membrane's
conductance is far below the nS level. The highest conductances
were observed for solutions with the largest atomic size cations,
Cs and Rb, correlated with a minimal hydration shell that mediates
their interaction with the graphene. This conductance was
attributed to ion transport through defect structures in the
free-standing graphene membrane.
TABLE-US-00001 TABLE I Graphene Sol. Conductivity Hydration energy
Solution Conductance (pS) (10.sup.-3Sm.sup.-1) (eV) CsCl 67 .+-. 2
1.42 3.1 RbCl 70 .+-. 3 1.42 3.4 KCl 64 .+-. 2 1.36 3.7 NaCl 42
.+-. 2 1.19 4.6 LiCl 27 .+-. 3 0.95 5.7
[0062] Contributions from electrochemical currents to and from the
graphene membrane were ruled out by a further experiment. Here, to
investigate the contribution from electrochemical (Faradic)
currents, a separate large-area graphene film (.about.2.times.4
mm.sup.2) was transferred to a glass slide and contacted at one end
with silver paint attached to a metallic clip over which wax
insulation was placed. The exposed end of the graphene film was
immersed in 1M KCl electrolyte with a Ag/AgCl counter electrode,
and the electrochemical I-V curves were measured in the same
voltage range as used in the trans-electrode experiments. After
normalizing for the surface area, it was concluded that any
electrochemical currents in the trans-electrode devices were three
orders of magnitude too small to account for the .about..mu.A
currents measured through the as-grown graphene membranes in Table
1. The observed conductances for different cations fall much faster
than the solution conductivities on going from CsCl to LiCl,
suggesting an influence of graphene-cation interactions.
Nevertheless, there cannot be completely ruled out ionic transport
through graphene that is in contact with the chip surface.
Example III
[0063] This example describes an experimental determination of the
conductance of the single-layer, bare graphene membrane of Example
I including a nanopore.
[0064] A single nanometer-sized nanopore was drilled through
several of the graphene membranes of Example I using a focused
electron beam in a JEOL 2010 FEG transmission electron microscope
operated at 200 kV acceleration voltage. The nanopore diameter was
determined by EM visualization in a well-spread electron beam so as
to keep the total electron exposure of the graphene membrane to a
minimum. A nanopore diameter of 8 nm was determined as the average
of 4 measurements along different nanopore axes, as determined from
calibrated TE micrographs using DigitalMicrograph software (Gatan,
Inc.). If the chip or TEM holder had any contaminating organic
residue, amorphous carbon was seen to visibly deposit under the
electron beam. Such devices were discarded. After drilling the
nanopore, the graphene nanopore chips that were not immediately
investigated were kept under a clean vacuum of .about.10.sup.-5
Torr.
[0065] FIG. 7 displays both a plot of ionic current as a function
of applied voltage as given above in Example II for a continuous
graphene membrane, as well as for a graphene membrane including an
8 nm-wide nanopore. These plots demonstrate that the ionic
conductivity of the graphene membrane is increased by orders of
magnitude by the nanopore.
[0066] It is found that experiments with known graphene nanopore
diameters and known ionic solution conductivities enable deduction
of the bare, single-layer graphene membrane's effective insulating
thickness. Ten separate graphene membranes from Example I were
processed to include nanopore diameters ranging from 5 to 23 nm.
Then the ionic conductance of each of the ten membranes was
measured with a 1 M KCl solution provided for both cis and trans
solution reservoirs, with a conductivity of 11 S m.sup.-1. FIG. 8
is a plot of the measured ionic conductance as a function of
nanopore diameter for the 10 membranes. The solid curve in the
figure is the modeled conductance of a 0.6 nm-thick insulating
membrane, which is the best fit to the experimentally measured
conductances. The modeled conductance for a 2 nm-thick membrane is
shown as a dotted line, and the modeled conductance for a 10
nm-thick membrane is shown as a dashed-dotted line, presented for
comparison.
[0067] The ionic conductance, G, of a nanopore of diameter, d, in
an infinitely thin insulating membrane is given by:
G.sub.thin=.sigma.d (1)
where .sigma.=F(.mu..sub.K+.mu..sub.Cl)c is the conductivity of the
ionic solution, F is the Faraday constant, c is ionic
concentration, and p, (c) is the mobility of potassium (i=K) and
chloride (i=Cl) ions used for a KCl ionic solution. The linear
dependence of conductance on diameter follows from the current
density being sharply peaked at the nanopore's perimeter for an
infinitely thin membrane, as described above. For membranes thicker
than the nanopore diameter the conductivity becomes proportional to
the nanopore area. For finite but small thicknesses of membrane,
computer calculations can predict the conductance.
[0068] As shown in the plot of FIG. 8, in agreement with Expression
(1), the conductivities of the single-layer bare graphene nanopores
with diameters ranging from 5 to 23 nanometers exhibited a
near-linear dependence on nanopore diameter. The modeled curve was
produced based on calculations of nanopore ionic conductivity in an
idealized uncharged, insulating membrane, as a function of nanopore
diameter and membrane thickness. Points on this curve were obtained
by numerically solving the Laplace equation for the ionic current
density, with appropriate solution conductivity and boundary
conditions, and integrating over the nanopore area to get the
conductivity. These numerical simulations were performed using the
COMSOL Multiphysics finite element solver in appropriate 3-D
geometry with cylindrical symmetry along the axis of the nanopore.
The full set of Poisson-Nerst-Planck equations was solved in the
steady-state regime. In the range of physical parameters of
interest, high salt concentration and small applied voltage, the
numerical simulation solution was found not to differ significantly
from the solution of the Laplace equation with fixed conductance,
which has significantly less computational penalty. The membrane
thickness, L, used in this idealized model is herein referred to as
graphene's Insulating Thickness, or L.sub.IT. The best fit to the
measured nanopore conductance data in FIG. 8 yields L.sub.GIT=0.6
(+0.9-0.6) nm, with the uncertainty determined from a least square
error analysis.
Example IV
[0069] This example describes experimental measurement of DNA
translocation through a nanopore in a single-layer, bare graphene
membrane of Example I.
[0070] The microfluidic cell of the examples above was flushed with
3M KCl salt solution at pH 10.5, containing 1 mM EDTA. As explained
above, high salt concentration and high pH were found to minimize
DNA-graphene interaction and thus these solution conditions can be
preferred. 10 kbp restriction fragments of double-stranded lambda
DNA molecules were introduced to the cis chamber of the system. The
negatively charged DNA molecules were electrophoretically drawn to
and driven through the nanopore by the applied electrophoretic
force of 160 mV. Each insulating molecule passing through the
nanopore transiently reduced, or blocked, the ionic conductivity of
the nanopore in a manner that reflects both polymer size and
conformation. As the DNA fragments traversed the nanopore due to
the applied electrophoretic force, the translocation events were
analyzed with MATLAB using a fitting function that consisted of
multiple square pulses convoluted with an appropriate Bessel filter
function to mimic the recording conditions.
[0071] FIG. 9 is a plot of measured ionic current through the
nanopore as a function of time for one minute from the time a
voltage bias was applied between the cis and trans reservoirs. Each
drop in measured current in the plot corresponds to a DNA
translocation through the nanopore, and enables characterization of
two parameters, namely, the average current drop, or blockade, and
the duration of the blockade, which is the time it takes for the
molecule to completely translocate through the nanopore. Note the
high number of translocation events for the bare graphene membrane
nanopore in the one minute time period, indicating successful
inhibition of DNA adherence to the bare graphene membrane surface
with the high pH salt solution and the careful cleaning and
handling of the graphene membrane during preparation for the DNA
translocation experiments.
[0072] FIGS. 10A, 10B, and 10C are plots of measured ionic current
through the nanopore for single translocation events. In FIG. 10A
there is demonstrated the ionic current flow blockage during a
translocation of DNA in single-file fashion. In FIG. 10B there is
demonstrated the ionic current flow blockage during translocation
of DNA that has been partially folded. Finally, in FIG. 10C there
is demonstrated the ionic current flow blockage during
translocation of DNA that has been folded in half. These three
experimental translocation events typify the possible ionic current
flow measurements that can occur during translocation of DNA
fragments, and demonstrates that DNA folding and conformation can
occur with the graphene nanopore as with thicker conventional solid
state nanopores.
[0073] A 5 nm-wide nanopore was formed in a separate graphene
membrane from Example I and double-stranded DNA translocation
experiments were conducted for the 3 M KCl solution of pH 10.4.
Each single molecule translocation event can be characterized by
two parameters: the average current drop, or blockade, and the
duration of the blockade, which is the time it takes for the
molecule to completely translocate through the pore. FIG. 11 is a
scatter plot showing the value of current drop and blockade
duration for each of 400 double stranded DNA single molecule
translocations through the graphene nanopore. The characteristic
shape of this data is similar to that obtained in silicon nitride
nanopore experiments where almost all the events, folded and
unfolded, fall near a line of constant electronic charge deficit
(ecd), i.e., regardless of how the otherwise identical molecules
are folded, each blocks the same amount of ionic charge movement
through the nanopore during the total time it takes each molecule
to move through the nanopore. Here as in the previous experiment it
is demonstrated that the double stranded DNA passed through the
nanopore uninhibited by sticking to the graphene surface. The few
events that are encircled in the plot do not satisfy this condition
and their long translocation times indicate graphene-DNA
interactions, which slow their translocation through the
nanopore.
[0074] In the plot of FIG. 11 the insets show two single-molecule
translocation events. In the right-hand event a molecule passed
through the nanopore in an unfolded linear fashion, as in the
example of FIG. 10A. In the left-hand event the molecule was folded
over on itself when it entered the nanopore as in the example of
FIG. 10B, increasing the current blockade for a short time.
[0075] Measurements of nanopore conductance during DNA
translocation events can be employed as an alternative method for
evaluating the effective insulting thickness of the graphene
membrane, L.sub.IT. The experimentally-determined open-nanopore and
DNA-blocked nanopore conductance was compared with that determined
by numerical solutions, where the membrane thickness and the
nanopore diameter are the fitting parameters. Here a DNA molecule
was modeled as a long, stiff, insulating rod of diameter 2 nm which
threads through the center of a nanopore. For lateral resolution
calculations, there was added a step of 2.2 nm in diameter to the
DNA model, and the change in the ionic current was calculated as
the discontinuity translocates through the center of the nanopore.
The total ionic current was calculated by integrating current
density across the diameter of the nanopore.
[0076] Using the observed mean current blockade,
.DELTA.I=1.24.+-.0.08 nA during translocation of unfolded
double-stranded DNA of diameter 2.0 nm, and the observed
conductance of the nanopore G=105.+-.1 nS absent DNA, the graphene
membrane insulating thickness was determined as L.sub.IT=0.6.+-.0.5
nm, in excellent agreement with the value deduced above from open
nanopore measurements alone, as discussed above. The nanopore
diameter d.sub.GIT=4.6.+-.0.4 nm deduced from these calculations
also agrees with the geometric diameter of 5.+-.0.5 nm obtained
from a TEM of the nanopore.
[0077] The best fit value L.sub.IT=0.6 nm from both experiments
agrees with molecular dynamics simulations showing the
graphene-water distance to be 0.31-0.34 nm on each side of the
membrane. L.sub.IT might also be influenced by the typical presence
of immobilized water molecules and adsorbed ions in the Stern
layer. On the other hand, theoretical studies argue against any
immobilized water layer on graphene, and experimental measurements
support an anomalously high slip between water and an internal
curved carbon nanotube surface. Although very little is actually
known about the surface chemistry of specifically adsorbed ions on
bare single graphene layers, measurements of the ionic current
through the inner volume of carbon nanotubes with diameters less
than 1 nm may indicate that ions are not immobilized on these
graphitic surfaces at all. The sub-nanometer values for L.sub.IT
determined here support this view.
[0078] The extremely small value for L.sub.IT obtained here
suggests that nanopores in single-layer, bare graphene membranes
are uniquely optimal for discerning spatial and/or chemical
molecular structure along the length of a molecule as it passes
through the nanopore. Numerical modeling of the molecular detection
resolution obtainable by such a nanopore can be accomplished based
on the determination of graphene membrane insulating thickness,
L.sub.IT.
[0079] In an example of such a model, there is specified a long,
insulating, 2.2 nm-diameter cylinder that symmetrically
translocates through the center of a 2.4 nm-diameter nanopore. At
one position along its length, the cylinder diameter changes
discontinuously from 2.2 nm to 2.0 nm. Solving for the conductance
for this geometry as the discontinuity passes through the pore,
there is obtained the data shown in the plot of FIG. 12. The
decreasing ionic current flow blockade, corresponding to increasing
nanopore conductance, is clearly seen as the large diameter portion
of the molecular cylinder exits the nanopore. The results of
calculations for two L.sub.IT values are shown. For the
conservative L.sub.IT=1.5 nm, the spatial resolution, defined as
the distance over which the conductivity changes from 75% of its
greatest value to 25% of that value, is given by
.delta.z.sub.GIT=7.5 .ANG., whereas the best-fit value
L.sub.GIT=0.6 nm leads to .delta.z.sub.GIT=3.5 .ANG..
[0080] There can be concluded both from the experiments detailed
above as well as from the modeling described above that a nanopore
in a bare single-layer graphene membrane is inherently capable of
probing molecules with sub-nanometer resolution. Functionalizing
the graphene nanopore boundary or observing its local in-plane
ionic conductivity during translocations can provide additional or
alternative means of further increasing the resolution of this
system.
[0081] From the description above, it has been demonstrated that an
atomically thin sheet of bare, single-layer graphene can be
fabricated into a self-supporting membrane including a nanopore, of
a diameter larger than the membrane thickness, for sensing
molecular translocation events through the nanopore. As a bare,
single layer, the thickness of the graphene membrane is minimized
and is safely in the short-length nanopore regime in which
peripheral ionic current flow is maximized and in which the
nanopore conductivity as a function of nanopore length is
maximized. The very short nanopore length provided by the graphene
membrane also makes it possible for a graphene nanopore to sense
closely-spaced monomers in a polymer and thus to sequentially
resolve the different ionic blockages caused by each monomer in,
for example, a strand of a DNA polymer.
[0082] Based on these considerations, it is recognized that if
technological advances enable such, a solid state membrane of an
alternative material can be substituted for the single-layer, bare
graphene membrane layer. Specifically, a solid state membrane
having a thickness that is less than about 1 nm and that can
mechanically support a nanopore extending through the membrane
thickness with a diameter that is greater than the membrane
thickness can be employed to obtain the molecular sensing
capability described above, and in particular, the DNA sensing
capability. It is to be understood that the requirements for
resistivity perpendicular to the plane of the membrane, the
mechanical integrity, and other characteristics described above can
be required to enable the arrangement of the membrane material
between cis and trans reservoirs for molecular translocation though
the membrane nanopore. Ionic current blockage measurement or other
electrical measurement can be employed as-suitable for a given
application and no particular measurement technique is
required.
[0083] For many molecular sensing applications, the solid state
membrane can be provided as a nanometric material that is less than
about 5 nanometers in thickness. Such nanometric materials include,
e.g., atomically-thin materials, which in general can be described
as materials having a thickness of an atomic monolayer or a few
atomic layers, such as a bilayer or trilayer of atoms. A
mono-atomically-thick material is herein defined as a material
which is one atom in thickness, but need not be atoms of just one
element. Atoms of a plurality of different elements can be included
in an atomic layer. The mono-atomically-thick layer can be
decorated at the layer top and/or bottom with heterogeneous atoms
and other species that do not lie in the plane of the atoms.
[0084] Such atomically-thin materials include, e.g.,
two-dimensional free-standing atomic crystals, and other structures
having a characteristic unit, like a lattice constant, that is
repeating in two dimensions but not the third. Atomically-thin
materials also include non-crystalline materials, such as glassy
materials for which a mono-atomic layer and few-atomic-layers can
be formed. Other example nanometric materials include materials
that are a single molecule in thickness, or that are two or three
molecules in thickness.
[0085] Beyond the example nanometric material graphene that has
been described above, other nanometric materials that can be
employed include fluorographene, graphane, graphene oxide,
hexagonal boron nitride (hexagonal-BN), mono-atomic glasses, and
other such materials. Other suitable nanometric materials include,
e.g., MoS.sub.2, WS.sub.2, MoSe.sub.2, MoTe.sub.2, TaSe.sub.2,
NbSe.sub.2, NiTe.sub.2, Bi.sub.2Sr.sub.2CaCu.sub.2O.sub.x, and
Bi.sub.2Te.sub.3. These are representative examples of suitable
nanometric solid state materials but are not limiting; any suitable
nanometric material in which or more nanopores can be formed for
molecular analysis in the configuration of FIG. 1 can be
employed.
[0086] Extending this understanding further, it is recognized that
alternative configurations to a nanopore can be employed. For
example, a membrane or other structure in which there can be
produced an aperture having a very sharp or pointed edge location
at which the aperture diameter is reduced to the nanometer scale,
and which is larger than the thickness of the location of the
diameter reduction, can also be employed. Thus, any solid state
structural configuration in which an aperture can be configured
meeting these requirements can be employed to achieve the
advantages for molecular sensing described above.
[0087] It is recognized, of course, that those skilled in the art
may make various modifications and additions to the embodiments
described above without departing from the spirit and scope of the
present contribution to the art. Accordingly, it is to be
understood that the protection sought to be afforded hereby should
be deemed to extend to the subject matter claims and all
equivalents thereof fairly within the scope of the invention.
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