U.S. patent number RE46,594 [Application Number 14/289,445] was granted by the patent office on 2017-10-31 for electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions.
This patent grant is currently assigned to LOS ALAMOS NATIONAL SECURITY, LLC. The grantee listed for this patent is Los Alamos National Security, LLC. Invention is credited to Elshan A. Akhadov, Mark A. Hoffbauer, Virginia Olazabal, Antonio Redondo, Jose-Maria Sansinena.
United States Patent |
RE46,594 |
Sansinena , et al. |
October 31, 2017 |
Electrochemical detection of single molecules using abiotic
nanopores having electrically tunable dimensions
Abstract
A barrier structure for use in an electrochemical stochastic
membrane sensor for single molecule detection. The sensor is based
upon inorganic nanopores having electrically tunable dimensions.
The inorganic nanopores are formed from inorganic materials and an
electrically conductive polymer. Methods of making the barrier
structure and sensing single molecules using the barrier structure
are also described.
Inventors: |
Sansinena; Jose-Maria (Los
Alamos, NM), Redondo; Antonio (Los Alamos, NM), Olazabal;
Virginia (Los Alamos, NM), Hoffbauer; Mark A. (Los
Alamos, NM), Akhadov; Elshan A. (Los Alamos, NM) |
Applicant: |
Name |
City |
State |
Country |
Type |
Los Alamos National Security, LLC |
Los Alamos |
NM |
US |
|
|
Assignee: |
LOS ALAMOS NATIONAL SECURITY,
LLC (Los Alamos, NM)
|
Family
ID: |
41315113 |
Appl.
No.: |
14/289,445 |
Filed: |
May 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13339010 |
Dec 28, 2011 |
|
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Reissue of: |
11525329 |
Sep 21, 2006 |
7638034 |
Dec 29, 2009 |
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Reissue of: |
11525329 |
Sep 21, 2006 |
7638034 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y
15/00 (20130101); G01N 33/48721 (20130101) |
Current International
Class: |
B81B
7/00 (20060101); G01N 27/26 (20060101); B82Y
15/00 (20110101); G01N 33/487 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
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.
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.
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on Micromachined Supports," Langmuir 2001, 17, 4, pp. 1240-1242.
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|
Primary Examiner: Jastrzab; Krisanne
Attorney, Agent or Firm: Pillsbury Winthrop Shaw Pittman,
LLP
Government Interests
STATEMENT REGARDING FEDERAL RIGHTS
This invention was made with government support under Contract No.
DE-AC 52-06 NA 25396, awarded by the U.S. Department of Energy. The
government has certain rights in the invention.
Parent Case Text
.Iadd.CROSS-REFERENCE TO RELATED APPLICATIONS.Iaddend.
.Iadd.More than one reissue application has been filed for the
reissue of U.S. Pat. No. 7,638,034. The reissue applications are
U.S. patent application Ser. No. 14/289,445 (the present
application), Ser. Nos. 14/289,388 and 13/339,010. This application
is a divisional reissue application of U.S. patent application Ser.
No. 13/339,010..Iaddend.
Claims
The invention claimed is:
.[.1. A barrier structure, the barrier structure comprising: a. a
first chamber; b. a second chamber; c. a barrier separating the
first chamber and the second chamber, wherein the barrier comprises
at least one electroactive nanopore structure joining the first
chamber and the second chamber, wherein the at least one
electroactive nanopore structure comprises: i. a wall defining a
electroactive nanopore connecting the first chamber and the second
chamber and having an electrically tunable diameter; ii. a first
electrode pair disposed in the wall, wherein electrodes of the
first electrode pair are disposed at opposite ends of the
electroactive nanopore, and wherein a first voltage across the
first electrode pair attracts a plurality molecules to the
electroactive nanopore and drives the plurality of molecules
through the electroactive nanopore; and iii. a second electrode
pair disposed in the wall between the first electrode pair; and iv.
a conductive polymer disposed over an electrode of the second
electrode pair, wherein the conductive polymer is responsive to a
second voltage across the second electrode pair and is capable of
expansion or contraction in response to the second voltage, and
wherein the expansion decreases the electrically tunable diameter
and the contraction increases the electrically tunable diameter;
and d. at least one power supply electrically coupled to the first
electrode pair and the second electrode pair, wherein the at least
one power supply provides the first voltage across the first
electrode pair and the second voltage across the second electrode
pair..].
.[.2. The barrier structure according to claim 1, further including
a current measuring device for measuring a current flowing between
the first electrode pair, wherein the current corresponds to a
predetermined molecular species..].
.[.3. The barrier structure according to claim 2, wherein the
barrier structure forms a portion of a sensor..].
.[.4. The barrier structure according to claim 1, wherein each of
the electrodes of the first electrode pair and the second electrode
pair comprises one of platinum, gold, graphite, a metal alloy, and
combinations thereof..].
.[.5. The barrier structure according to claim 1, wherein the first
electrode pair and the second electrode pair are separated by an
insulating material..].
.[.6. The barrier structure according to claim 1, wherein the
insulating material comprises at least one of a glass, a metal
oxide, a non-conductive polymer, and combinations thereof..].
.[.7. The barrier structure according to claim 1, wherein the
conductive polymer is one of polypyrrole, polyaniline, and
combinations thereof..].
.[.8. The barrier structure according to claim 1, wherein the
barrier structure forms a portion of one of a valve structure and a
membrane structure..].
.[.9. The barrier structure according to claim 1, wherein the at
least one power supply includes a DC power supply..].
.[.10. An electroactive nanopore structure, the electroactive
nanopore structure comprising: a. a wall defining a electroactive
nanopore having a first open end and a second open end and having a
electrically tunable diameter; b. a first electrode pair disposed
in the wall, wherein electrodes of the first electrode pair are
disposed at opposite ends of the electroactive nanopore, and
wherein a first voltage across the first electrode pair attracts a
plurality molecules to the electroactive nanopore and drives the
plurality of molecules through the electroactive nanopore; and c. a
second electrode pair disposed in the wall between the first
electrode pair; and d. a conductive polymer disposed over an
electrode of the second electrode pair, wherein the conductive
polymer is responsive to a second voltage across the second
electrode pair and is capable of expansion or contraction in
response to the second voltage, and wherein the expansion decreases
the electrically tunable diameter and the contraction increases the
electrically tunable diameter..].
.[.11. The electroactive nanopore according to claim 10, wherein
each of the electrodes of the first electrode pair and the second
electrode pair comprises one of platinum, gold, graphite, a metal
alloy, and combinations thereof..].
.[.12. The electroactive nanopore structure according to claim 10,
wherein the first electrode pair and the second electrode pair are
separated by an insulating material..].
.[.13. The electroactive nanopore structure according to claim 10,
wherein the insulating material comprises a glass, a metal oxide, a
non-conductive polymer, and combinations thereof..].
.[.14. The electroactive nanopore structure according to claim 10,
wherein the conductive polymer is one of polypyrrole, polyaniline,
and combinations thereof..].
.[.15. The electroactive nanopore structure according to claim 10,
wherein the electroactive nanopore structure forms a portion of one
of a valve structure, a sensor, and a membrane structure..].
.[.16. A stochastic sensor structure, the stochastic sensor
structure comprising: a. a first chamber; b. a second chamber; c. a
barrier separating the first chamber and the second chamber,
wherein the barrier comprises at least one electroactive nanopore
structure joining the first chamber and the second chamber, wherein
the at least one electroactive nanopore structure comprises: i. a
wall defining a electroactive nanopore connecting the first chamber
and the second chamber and having a electrically tunable diameter;
ii. a first electrode pair disposed in the wall, wherein electrodes
of the first electrode pair are disposed at opposite ends of the
electroactive nanopore, and wherein a first voltage across the
first electrode pair attracts a plurality molecules to the
electroactive nanopore and drives the plurality of molecules
through the electroactive nanopore; and iii. a second electrode
pair disposed in the wall between the first electrode pair; and iv.
a conductive polymer disposed over an electrode of the second
electrode pair, wherein the conductive polymer is responsive to a
second voltage across the second electrode pair and is capable of
expansion or contraction in response to the second voltage, and
wherein the expansion decreases the electrically tunable diameter
and the contraction increases the electrically tunable diameter; d.
at least one power supply electrically coupled to the first
electrode pair and the second electrode pair, wherein the at least
one power supply provides the first voltage across the first
electrode pair and the second voltage across the second electrode
pair; and e. a current measuring device for measuring a current
flowing between the first electrode pair, wherein the current
corresponds to a predetermined molecular species..].
.[.17. A method of making a electroactive nanopore structure,
wherein the electroactive nanopore structure comprises: a wall
defining a electroactive nanopore having a first open end and a
second open end and having a electrically tunable diameter; a first
electrode pair having electrodes disposed at opposite ends of the
electroactive nanopore; a second electrode pair comprising a second
anode and a second cathode disposed in the wall between the first
electrode pair; and a conductive polymer disposed over an electrode
of the second electrode pair; the method comprising the steps of:
a. providing a template comprising a strip of photocurable polymer;
b. depositing alternating layers of conductive material and
insulating material over the template, wherein the alternating
layers form the first electrode pair and the second electrode pair,
and wherein electrodes of the first electrode pair and the second
electrode pair are separated by at least one layer of insulating
material; c. removing the template to form the electroactive
nanopore; and d. depositing the conductive polymer on the electrode
of the second electrode pair to form the electrically tunable
diameter..].
.[.18. The method according to claim 17, wherein the step of
depositing alternating layers of conductive material and insulating
material over the template comprises: a. depositing a first
conductive layer over the template; b. depositing a first
insulating layer over the first conductive layer; c. depositing a
second conductive layer over the first insulating layer; d.
depositing a second insulating layer over the second conductive
layer; e. depositing a third conductive layer over the second
conductive layer, wherein the second conductive layer and the third
conductive layer form the second electrode pair; f. depositing a
third insulating layer over the third conductive layer; and g.
depositing a fourth conductive layer over the third conductive
layer, wherein the first conductive layer and the fourth conductive
layer form the first electrode pair..].
.[.19. The method according to claim 17, wherein at least one of
the first conductive layer, the first insulating layer, the second
conductive layer, the second insulating layer, the third conductive
layer, the third insulating layer, and the fourth conductive layer
is deposited by energetic neutral beam lithography/epitaxy..].
.[.20. The method according to claim 17, wherein the step of
depositing the conductive polymer comprises electrochemically
depositing the conductive polymer onto at least one of the second
anode and the second cathode..].
.[.21. The method according to claim 17, wherein the template
further comprises a cylinder having a diameter that is
substantially equal to the electrically tunable diameter of the
electroactive nanopore, wherein the cylinder comprises the
photcurable polymer..].
.[.22. The method according to claim 17, wherein the step of
removing the template to form the electroactive nanopore comprises
drilling through the alternating layers of conductive material and
insulating material with a focused ion beam to form the
electroactive nanopore..].
.[.23. A method of sensing the presence of an analyte molecule, the
method comprising the steps of: a. providing a sensor structure,
the sensor structure comprising a sampling chamber, a collection
chamber, and a separation structure separating the sampling chamber
and the collection chamber, wherein the separation structure
includes a electroactive nanopore structure comprising: a wall
defining a electroactive nanopore connecting the sampling chamber
and the collection chamber and having a electrically tunable
diameter; a first electrode pair having electrodes disposed at
opposite ends of the electroactive nanopore; a second electrode
pair disposed in the wall between the first electrode pair; and a
conductive polymer disposed over an electrode of the second
electrode pair; b. providing the analyte molecule to the sampling
chamber; c. passing the analyte molecule from the sampling chamber
into the electroactive nanopore; and d. measuring a current across
the first electrode pair, wherein the current is indicative of the
presence of the analyte molecule..].
.[.24. The method according to claim 23, wherein the step of
passing the analyte from the sampling chamber into the
electroactive nanopore comprises applying a first voltage across
the first electrode pair, wherein the first voltage is sufficient
to cause the analyte to migrate from the sampling chamber through
the electroactive nanopore structure to the collection
chamber..].
.[.25. The method according to claim 23, further including the step
of increasing or decreasing the electrically tunable diameter of
the electroactive nanopore..].
.[.26. The method according to claim 23, wherein the step of
increasing or decreasing the electrically tunable diameter of the
electroactive nanopore comprises applying a second voltage across
the second electrode pair, wherein the second electrode voltage
causes the conductive polymer to either expand or contract..].
.[.27. A method of controlling flow of a fluid between a first
chamber and a second chamber, the method comprising: a. providing a
barrier structure, wherein the barrier structure includes at least
one electroactive nanopore structure, wherein the at least one
electroactive nanopore structure comprises: a wall defining a
electroactive nanopore connecting the first chamber and the second
chamber and having a electrically tunable diameter; a first
electrode pair disposed in the wall and having electrodes disposed
at opposite ends of the electroactive nanopore; a second electrode
pair disposed in the wall between the first electrode pair; and a
conductive polymer disposed over an electrode of the second
electrode pair; b. providing the fluid to the first chamber; c.
passing the fluid from the first chamber into the electroactive
nanopore; and d. increasing or decreasing the electrically tunable
diameter of the electroactive nanopore to control the flow of the
fluid through the electroactive nanopore to the second
chamber..].
.[.28. The method according to claim 27, wherein passing the fluid
from the first chamber into the electroactive nanopore comprises
applying a first voltage across the first electrode pair, wherein
the first voltage is sufficient to cause the fluid to migrate from
the first chamber through the electroactive nanopore structure to
the second chamber..].
.[.29. The method according to claim 27, wherein the step of
increasing or decreasing the electrically tunable diameter of the
electroactive nanopore comprises applying a second voltage across
the second electrode pair, wherein the second electrode voltage
causes the conductive polymer to either expand or contract, and
wherein expansion of the conductive polymer increases the
electrically tunable diameter and contraction of the conductive
polymer decreases the electrically tunable diameter..].
.Iadd.30. A nanopore structure comprising a nanopore having an
opening and a wall defining the nanopore, wherein the opening has
an electrically tunable diameter, a first electrode pair disposed
in the wall, a second electrode pair disposed in the wall between
the first electrode pair, a polymer disposed over an electrode of
the second electrode pair, wherein the opening is an opening
through the polymer, wherein each of the electrodes of the first
electrode pair and the second electrode pair comprises one of
platinum, gold, graphite, a metal alloy, and combinations
thereof..Iaddend.
.Iadd.31. A nanopore structure comprising a nanopore having an
opening and a wall defining the nanopore, wherein the opening has
an electrically tunable diameter, a first electrode pair disposed
in the wall, a second electrode pair disposed in the wall between
the first electrode pair, a polymer disposed over an electrode of
the second electrode pair, wherein the opening is an opening
through the polymer, wherein the polymer comprises a conductive
polymer..Iaddend.
.Iadd.32. The nanopore structure of claim 31, wherein the
conductive polymer is one of polypyrrole, polyaniline, and
combinations thereof..Iaddend.
.Iadd.33. A nanopore structure comprising a nanopore having an
opening and a wall defining the nanopore, wherein the opening has
an electrically tunable diameter, a first electrode pair disposed
in the wall, a second electrode pair disposed in the wall between
the first electrode pair, a polymer disposed over an electrode of
the second electrode pair, wherein the opening is an opening
through the polymer, wherein the first electrode pair and the
second electrode pair are separated by an insulating
material..Iaddend.
.Iadd.34. The nanopore structure of claim 33, wherein the
insulating material comprises at least one of a glass, a metal
oxide, a non-conductive polymer, and combinations
thereof..Iaddend.
.Iadd.35. The nanopore structure of claim 31, wherein the
conductive polymer is capable of expansion or contraction in
response to a voltage..Iaddend.
.Iadd.36. The nanopore structure of claim 31, wherein the
conductive polymer is responsive to a voltage across the second
electrode pair..Iaddend.
.Iadd.37. The nanopore structure of claim 35, wherein the expansion
decreases the electrically tunable diameter and the contraction
increases the electrically tunable diameter..Iaddend.
.Iadd.38. A nanopore structure comprising a nanopore having an
opening and a wall defining the nanopore, wherein the opening has
an electrically tunable diameter, a first electrode pair disposed
in the wall, a second electrode pair disposed in the wall between
the first electrode pair, a polymer disposed over an electrode of
the second electrode pair, wherein the opening is an opening
through the polymer, wherein the polymer is configured to change at
least one dimension in response to an electrical stimulus..Iaddend.
Description
BACKGROUND OF INVENTION
The invention relates to barrier structures comprising nanopores.
More particularly, the invention relates to structures having
electroactive nanopores. Even more particularly, the invention
relates to electrochemical sensors having such structures.
The detection and identification of single molecules has received
increasing interest over the last few years, as there has been a
realization that this can be done by analyzing transport and
electrochemical phenomena through pores having nanoscale
dimensions. Measurements of the ionic current through a
single-protein channel incorporated into a freestanding lipid
bilayer membrane can form the basis of a new and versatile method
for single-molecule chemical and biological sensing, called
stochastic sensing. These sensors consist of a protein pore
embedded in an insulating membrane and operate by measuring the
characteristic current through the pore in the presence of
molecules of interest. The magnitude, duration, and rates of
occurrence of the current blockage allow rapid discrimination
between similar molecular species.
The main limitation in this nascent field is that the bulk of the
work has been focused on biologically-based stochastic sensors
using protein pores embedded in lipid bilayer membranes. The lipid
bilayer membrane into which the channel is immobilized is fragile
and unstable; such membranes have lifetimes on the order of a few
hours and, very rarely, exceed one day. These membranes are
extremely delicate and susceptible to breakage, requiring vibration
isolation tables, low acoustic noise environments, and special
solution handling. This is unacceptable for field-usable devices
and applications outside the laboratory. Furthermore, although a
range of membrane proteins, which can be modified as desired
through biochemistry or mutagenesis, may be exploited as sensors,
the availability of biological pores is still limited with respect
to having complete freedom in pore size, structure, and
composition. Attempts to fabricate solid-state nanopores that are
able to mimic the ion transport properties of protein ion channels
lack reproducible dimensional control at the nanometer scale.
Existing biologically-based stochastic membrane sensors are not
sufficiently robust for widespread use outside a controlled
laboratory setting. Therefore, what is needed is a stochastic
membrane sensor that is sufficiently robust to withstand use in
applications under normal conditions. What is also needed is a
membrane for a stochastic sensor that is not biologically-based.
What is further needed is a membrane for a stochastic sensor having
a diameter that is reproducibly controllable.
SUMMARY OF INVENTION
The present invention meets these and other needs by providing a
barrier structure for use in an electrochemical stochastic membrane
sensor for single molecule detection. The sensor is based upon
inorganic nanopores having electrically tunable dimensions. The
inorganic nanopores are formed from inorganic materials and an
electrically conductive polymer. Methods of making the barrier
structure and sensing single molecules using the barrier structure
are also described.
Accordingly, one aspect of the invention is to provide a barrier
structure. The barrier structure comprises: a first chamber; a
second chamber; a barrier separating the first chamber and the
second chamber. The barrier comprises at least one electroactive
nanopore structure joining the first chamber and the second
chamber. The at least one electroactive nanopore structure
comprises: a wall defining a electroactive nanopore connecting the
first chamber and the second chamber and having an electrically
tunable diameter; a first electrode pair disposed in the wall,
wherein electrodes of the first electrode pair are disposed at
opposite ends of the electroactive nanopore, and wherein a first
voltage across the first electrode pair attracts a plurality of
molecules to the electroactive nanopore and drives the plurality of
molecules through the electroactive nanopore; a second electrode
pair disposed in the wall between the first electrode pair; and a
conductive polymer disposed over an electrode of the second
electrode pair, wherein the conductive polymer is responsive to a
second voltage across the second electrode pair and is capable of
expansion or contraction in response to the second voltage, and
wherein the expansion decreases the electrically tunable diameter
and the contraction increases the electrically tunable diameter.
The barrier structure also comprises at least one power supply
electrically coupled to the first electrode pair and the second
electrode pair, wherein the at least one power supply provides the
first voltage across the first electrode pair and the second
voltage across the second electrode pair.
A second aspect of the invention is to provide an electroactive
nanopore structure. The electroactive nanopore structure comprises:
a wall defining a electroactive nanopore having a first open end
and a second open end and having a electrically tunable diameter; a
first electrode pair disposed in the wall, wherein electrodes of
the first electrode pair are disposed at opposite ends of the
electroactive nanopore, and wherein a first voltage across the
first electrode pair attracts a plurality molecules to the
electroactive nanopore and drives the plurality of molecules
through the electroactive nanopore; a second electrode pair
disposed in the wall between the first electrode pair; and a
conductive polymer disposed over an electrode of the second
electrode pair, wherein the conductive polymer is responsive to a
second voltage across the second electrode pair and is capable of
expansion or contraction in response to the second voltage, and
wherein the expansion decreases the electrically tunable diameter
and the contraction increases the electrically tunable
diameter.
A third aspect of the invention is to provide a stochastic sensor
structure. The stochastic sensor structure comprising: a first
chamber; a second chamber; a barrier separating the first chamber
and the second chamber, wherein the barrier comprises at least one
electroactive nanopore structure joining the first chamber and the
second chamber, wherein the at least one electroactive nanopore
structure comprises: a wall defining a electroactive nanopore
connecting the first chamber and the second chamber and having a
electrically tunable diameter; a first electrode pair disposed in
the wall, wherein electrodes of the first electrode pair are
disposed at opposite ends of the electroactive nanopore, and
wherein a first voltage across the first electrode pair attracts a
plurality molecules to the electroactive nanopore and drives the
plurality of molecules through the electroactive nanopore; and a
second electrode pair disposed in the wall between the first
electrode pair; and a conductive polymer disposed over an electrode
of the second electrode pair, wherein the conductive polymer is
responsive to a second voltage across the second electrode pair and
is capable of expansion or contraction in response to the second
voltage, and wherein the expansion decreases the electrically
tunable diameter and the contraction increases the electrically
tunable diameter; at least one power supply electrically coupled to
the first electrode pair and the second electrode pair, wherein the
at least one power supply provides the first voltage across the
first electrode pair and the second voltage across the second
electrode pair; and a current measuring device for measuring a
current flowing between the first electrode pair, wherein the
current corresponds to a predetermined molecular species.
A fourth aspect of the invention is to provide a method of making
an electroactive nanopore structure. The electroactive nanopore
structure comprises: a wall defining a electroactive nanopore
having a first open end and a second open end and having a
electrically tunable diameter; a first electrode pair having
electrodes disposed at opposite ends of the electroactive nanopore;
a second electrode pair comprising a second anode and a second
cathode disposed in the wall between the first electrode pair; and
a conductive polymer disposed over an electrode of the second
electrode pair. The method comprises the steps of: providing a
template comprising a strip of photocurable polymer; depositing
alternating layers of conductive material and insulating material
over the template, wherein the alternating layers form the first
electrode pair and the second electrode pair, and wherein
electrodes of the first electrode pair and the second electrode
pair are separated by at least one layer of insulating material;
removing the template to form the electroactive nanopore; and
depositing the conductive polymer on the electrode of the second
electrode pair.
A fifth aspect of the invention is to provide a method of sensing
the presence of an analyte molecule. The method comprises the steps
of: providing a sensor structure, the sensor structure comprising a
sampling chamber, a collection chamber, and a separation structure
separating the sampling chamber and the collection chamber, wherein
the separation structure includes a electroactive nanopore
structure comprising: a wall defining a electroactive nanopore
connecting the sampling chamber and the collection chamber and
having a electrically tunable diameter; a first electrode pair
having electrodes disposed at opposite ends of the electroactive
nanopore; a second electrode pair disposed in the wall between the
first electrode pair; and a conductive polymer disposed over an
electrode of the second electrode pair; providing the analyte to
the sampling chamber; passing the analyte molecule from the
sampling chamber into the electroactive nanopore; applying a first
voltage across the first electrode pair; and measuring a current
across the first electrode pair, wherein the current is indicative
of the presence of the analyte molecule.
A sixth aspect of the invention is to provide a method of
controlling flow of a fluid between a first chamber and a second
chamber. The method comprising the steps of: providing a barrier
structure, wherein the barrier structure includes at least one
electroactive nanopore structure, wherein the at least one
electroactive nanopore structure comprises: a wall defining a
electroactive nanopore connecting the first chamber and the second
chamber and having a electrically tunable diameter; a first
electrode pair disposed in the wall and having electrodes disposed
at opposite ends of the electroactive nanopore; a second electrode
pair disposed in the wall between the first electrode pair; and a
conductive polymer disposed over an electrode of the second
electrode pair; providing the fluid to the first chamber; passing
the fluid from the first chamber into the electroactive nanopore;
and increasing or decreasing the electrically tunable diameter of
the electroactive nanopore to control the flow of the fluid through
the electroactive nanopore to the second chamber.
These and other aspects, advantages, and salient features of the
present invention will become apparent from the following detailed
description, the accompanying drawings, and the appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of a barrier structure;
FIG. 2 is a flow chart for a method of sensing an analyte
molecule;
FIG. 3 is a flow chart for a method of making a barrier
structure;
FIG. 4a is a schematic representation showing the response of the
electrically tunable diameter of an electroactive nanopore to
voltage V.sub.Polymer;
FIG. 4b is a plot of V.sub.Polymer as a function of time;
FIG. 4c is a plot of current I.sub.Pore passing through the
electroactive nanopore, shown in FIG. 4a, as a function of
time;
FIG. 5 is a plot of V.sub.Polymer and molecular diameter showing
characteristic voltages V.sub.1 and V.sub.2 for molecules having
diameters d.sub.1 and d.sub.2, respectively; and
FIG. 6 is a schematic representation of the operation of a
stochastic sensor.
FIG. 7 is a flow chart for a method of controlling fluid.
DETAILED DESCRIPTION
In the following description, like reference characters designate
like or corresponding parts throughout the several views shown in
the figures. It is also understood that terms such as "top,"
"bottom," "outward," "inward," and the like are words of
convenience and are not to be construed as limiting terms. In
addition, whenever a group is described as either comprising or
consisting of at least one of a group of elements and combinations
thereof, it is understood that the group may comprise or consist of
any number of those elements recited, either individually or in
combination with each other.
Referring to the drawings in general and to FIG. 1 in particular,
it will be understood that the illustrations are for the purpose of
describing a particular embodiment of the invention and are not
intended to limit the invention thereto. Turning to FIG. 1, a
barrier structure of the present invention is shown. Barrier
structure 100 comprises a first chamber 110, a second chamber 120
and a barrier 130 separating first chamber 110 and second chamber
120.
First chamber 110 and second chamber 120 are adapted to contain a
fluid, and their dimensions and other characteristics depend on the
specific application in which barrier structure is used. In one
embodiment, for example, first chamber 110 may serve as a sampling
chamber for collecting a fluid for analysis, and second chamber 120
may serve as an analysis chamber. Alternatively, first chamber 110
and second chamber 120 may simply be reservoirs for containing a
fluid buffer, with barrier 140 limiting communication between the
reservoirs.
Barrier 140 comprises at least one electroactive nanopore 130
having a wall defining a solid-state electroactive nanopore 130 and
connecting first chamber 110 and second chamber 120. In one
embodiment, barrier 140 comprises an array of electroactive
nanopores 130. A first electrode pair 134, having two electrodes
disposed at opposite ends of electroactive nanopore 130, is
disposed in the nanopore wall at opposite ends of electroactive
nanopore 130. The two electrodes of first electrode pair 134 are
proximate to where wall 142 joins first chamber 110 and second
chamber 120, respectively. A first voltage V.sub.Pore, when applied
across first electrode pair 134, attracts a plurality of molecules
present in either first chamber or second chamber to electroactive
nanopore 130 and drives the plurality of molecules through
electroactive nanopore 130 and into the opposite chamber. First
electrode pair 134 may comprise any conductive material known in
the art such as, but not limited to, platinum, gold, graphite,
electrically conductive metal alloys, combinations thereof, and the
like.
A second electrode pair 132 comprising two electrodes is disposed
in wall 142 between the electrodes of first electrode pair 134.
Second electrode pair 132 may comprise any conductive material
known in the art such as, but not limited to, platinum, gold,
graphite, electrically conductive metal alloys, combinations
thereof, and the like.
The electrodes of first electrode pair 134 and second electrode
pair 132 are separated from each other by insulating material 144.
Insulating material 144 comprises at least one of a metal oxide
such as sapphire and silica (SiO.sub.2), glasses, nonconductive
polymers, silicon, and the like.
A conductive polymer 136 is disposed on the surface of wall 142
over an electrode of the second electrode pair 132. Conductive
polymer 136 has electrically tunable dimensions; i.e., it is
responsive to a second voltage V.sub.Polymer, applied across second
electrode pair 132, and is capable of expanding or contracting in
response to the second voltage. The presence of conductive polymer
136 on the surface of the wall of electroactive nanopore 130
provides the electroactive nanopore 130 with an electrically
tunable diameter 138 or cross-section. As conductive polymer 136
expands or contracts, its volume changes, causing the
cross-section, or diameter 138, of electroactive nanopore 130 to
correspondingly decrease and increase. Diameter 138 is also
reversibly tunable--i.e., it may be decreased and then increased,
or vice versa. Conductive polymer 136 comprises an ionically
conductive polymer such as, but not limited to, polypyrrole,
polyaniline, combinations thereof, and the like.
In the absence of a second voltage V.sub.Polymer, electroactive
nanopore 130 has a diameter 138 of up to about 50 nm. With the
application of the second voltage V.sub.Polymer, conductive polymer
136 may be expanded to decrease diameter 138 to a zero or near-zero
value, effectively closing electroactive nanopore 130.
The thickness of each of the electrodes 132, 134 in electroactive
nanopore 130 depends on the desired electrode size and the desired
spacing between electrodes. The thicknesses of the individual
layers of insulating material 140 must be sufficient to prevent
shorting or arcing between the electrode layers. The individual
electrodes and layers of insulating material 140 each have a
thickness of up to 5 nm. In one embodiment, the thickness is in a
range from about 1 nm to about 5 nm.
The length of electroactive nanopore 130 should be long enough to
accommodate a single analyte molecule of interest. As analyte
molecules of interest may vary from one application to another, the
length of electroactive nanopore 130 may be varied accordingly.
Electroactive nanopore 130 may, for example, have one length when
used to detect the presence of proteins, another length when
detecting polymers, and yet a third length when detecting DNA
molecules. Electroactive nanopore 130 may have a length in a range
from about 5 nm to about 50 nm. In one embodiment, electroactive
nanopore 130 has a length of up to 5 nm, which approximates the
length of protein pores that are used in stochastic sensors.
An example of how the current through electroactive nanopore 130 is
affected by applying second voltage V.sub.Polymer across second
electrode pair 132 and expanding conductive polymer 136 is
illustrated in FIGS. 4a, 4b, and 4c. With first voltage V.sub.Pore
across first electrode pair 134 held constant, V.sub.Polymer is
increased from a low value (A in FIG. 4b) to a medium value (B) to
a high value (C). Conductive polymer 136 correspondingly expands
(FIG. 4a), narrowing the diameter of the electroactive nanopore. As
diameter 138 decreases (and conductive polymer expands), the
current through electroactive nanopore 130 decreases as well (FIG.
4c).
A characteristic voltage corresponding to the second voltage (while
maintaining first voltage V.sub.Pore across first electrode pair
134 at a constant value) may be applied across second electrode
pair 132 to tune diameter 138 to the approximate size. The
application of characteristic voltages V.sub.1 and V.sub.2 for
molecules having sizes of d.sub.1 and d.sub.2, respectively, is
shown in FIG. 5. By applying voltage V.sub.1 across second
electrode pair 132, diameter 138 is tuned to the size of an analyte
molecule having diameter d.sub.1 while effectively preventing
larger molecules having diameter d.sub.2 from passing through
electroactive nanopore 130.
At least one power source (not shown) is electrically coupled to
first electrode pair 134 and second electrode pair 132, and
provides the first voltage across first electrode pair 134 and
second voltage across second electrode pair 132. The power source
may be either a DC power source or an AC power source.
In one embodiment, barrier structure 100 forms a portion of a
single-molecule--or stochastic--sensor that is adapted to detect
particular species of analyte molecules present in a fluid. Such a
sensor operates by measuring a characteristic current through
electroactive nanopore 130 in the presence of analyte molecules of
interest. The magnitude, duration, and rates of occurrence of the
current blockage by the analyte molecule allow rapid discrimination
between similar molecular species. Whereas previous stochastic
sensors formed using protein pores embedded in lipid membranes are
fragile and unstable, barrier structure 100 and electroactive
nanopore 130 are structurally stable, due to their construction
from inorganic materials and conductive polymers, and are capable
of repeated use.
The selectivity of the stochastic sensor is based on the
characteristic currents associated with the flow of different types
of molecules in an ionic aqueous solution. The molecules have
multiple measurable parameters that allow discrimination between
different--but similar--species. For a selected diameter 138, each
type of analyte molecule exhibits a different characteristic
current and noise signature.
The stochastic sensor includes two buffer reservoirs, which are
analogous to first chamber 110 and second chamber 120, joined by at
least one electroactive nanopore 130. To detect the analyte
molecule, first voltage V.sub.Pore is applied across first
electrode pair 134, driving molecules through electroactive
nanopore 130. The voltage applied between the second electrode pair
132 causes conductive polymer 136 to either expand or contract,
thus controlling the diameter 138 of electroactive nanopore
130.
The selectivity of the stochastic sensor is based on the
characteristic current signature associated with the flow of each
type of analyte molecule through electroactive nanopore 130. Small
ions flow through electroactive nanopore 130, producing a current
having a relatively high value. When an analyte molecule having a
diameter that is less than diameter 138 of electroactive nanopore
130 passes through the nanopore, the molecule partially occludes
the passage of ions, thereby causing the current to decrease. After
the analyte molecule traverses electroactive nanopore 130, normal
ion flow through the nanopore resumes and the current is restored
to its initial value. If an analyte molecule that is larger than
diameter 138 of electroactive nanopore 130 tries to traverse the
nanopore, the passage of ions through the nanopore is blocked and
the current drops to zero. The polarity of first electrode pair 134
must then be reversed to unblock the nanopore. When the inner
diameter 138 of electroactive nanopore 130 is increased by changing
V.sub.Polymer to allow the analyte molecule to pass through the
nanopore, the size of the molecule and its electrodynamic
interactions with the charges in conductive polymer 136 will
determine the current drop that is observed as the analyte molecule
traverses electroactive nanopore 130.
Electroactive nanopore 130 can be electrochemically characterized
by performing cyclic voltammetry between the electrodes of second
electrode pair 132 in the presence of a buffer while maintaining a
constant voltage across first electrode pair 134. The
electrochemical behavior of electroactive nanopore 130 can then be
characterized using the recorded cyclic voltammograms and the
current across electroactive nanopore 130. Electroactive nanopore
130 is then closed by applying the appropriate voltage
V.sub.Polymer across second electrode pair 132 while applying a
constant independent voltage V.sub.Pore across first electrode pair
134 and monitoring the current through the nanopore. This yields a
reference current for a state where substantially no molecules or
ions--or a minimum number of molecules or ions--are passing through
electroactive nanopore 130. Next, a sample containing a first
analyte molecular species is introduced into either first chamber
110 or second chamber 120, and voltage V.sub.Polymer across second
electrode pair 132 is decreased to slowly contract conductive
polymer 136 and open electroactive nanopore 130. The resulting
increase in diameter 138 of the nanopore results in a corresponding
increase in ionic current through the nanopore. The characteristic
voltage V.sub.Polymer associated with the first analyte molecular
species is the voltage associated with the passage of the first
analyte species through the nanopore.
FIG. 6 illustrates the principle of operation of the stochastic
sensor. Initially, only small ions flow through electroactive
nanopore 130, procuring a current having a relatively high value
((a) in FIG. 6). As one type of analyte molecule 160 that is
smaller than diameter 138 passes through electroactive nanopore
130, the analyte molecule 160 partially occludes the passage of
ions, causing the current to drop ((b) in FIG. 6). After the
molecule has traversed electroactive nanopore 130, the current is
restored to its original value, as shown in (b). In (c), a second
type of analyte molecule 162, larger than diameter 138, tries to
traverse electroactive nanopore 130. The passage of ions through
electroactive nanopore 130 is completely blocked and the current
goes to zero. Here, electroactive nanopore 130 may be unblocked by
reversing the first voltage V.sub.Pore. In (d), diameter 138 is
increased by changing V.sub.Polymer. The flow of ions--and the
current--through electroactive nanopore 130 then resumes. The size
of the second analyte molecule 162 and its electrodynamic
interactions with the charges in conductive polymer 136 will
determine the current drop when the molecule traverses
electroactive nanopore 130. Once the second analyte molecule 162
exits electroactive nanopore 130, the current returns to its
original value, as shown in (d).
The stochastic currents associated with molecules of the same
species passing through electroactive nanopore 130 are monitored
for later statistical analysis, which provides parameters, such as
blockage currents, blockage times, blockage frequencies, current
distribution, signal-to-noise ratios, and the like, that are used
together with the characteristic voltage for identification of the
analyte molecule.
If the analyte sample includes a mixture of molecules, random drops
in current to either positive values or the reference current may
occur, as some molecules pass through the electroactive nanopore
130 while others block the entrance to the nanopore. In such cases,
the characterization process is typically repeated, and the voltage
V.sub.Polymer across second electrode pair 132 is adjusted to the
characteristic voltage of each analyte molecular species. In
addition, the stochastic current is monitored for analytical
purposes.
The stochastic sensor described hereinabove incorporates for the
first time two important transport-selectivity capabilities into
the field of sensor development. First, because diameter 138 of
electroactive nanopore 130 can be modified in a controllable
manner, the sensor can be used to cleanly separate different
molecules on the basis of molecular size, ranging from simple ions
to complex compounds and even microorganisms. Second, because the
conductive polymer 136 can be charged in an ionic solution, the
stochastic sensor can discriminate between molecules of similar
size based on their different electrodynamic interactions with the
conducting polymer.
Furthermore, the use of solid-state electroactive nanopores such as
those described herein provides a significant advantage, as
fabrication of an array of several pores can be integrated with
electronics and on-chip computational hardware to provide a
portable device capable of performing multiple sensing functions.
Unlike sensors based on biological membranes and protein channels,
this robust sensor will be stable and functional over a wider range
of temperatures, solvents, voltages, and other potentially adverse
conditions.
This new technology not only could be used in sensing but also in
analytical chemistry, specifically in bio-separations,
electroanalytical chemistry, and in the development of new
approaches to DNA sequencing based on transport through the
electroactive nanopore.
In another embodiment, barrier structure 100 forms a portion of a
valve structure. Here, conductive polymer 136 may expand or
contract in response to changes in voltage V.sub.Polymer across
second electrode pair 132. As conductive polymer 136 expands or
contracts, the tunable diameter 138 of electroactive nanopore 130
either decreases or increases, thereby regulating flow between
first chamber 110 and second chamber 120.
In yet another embodiment, barrier structure 100 is a membrane that
separates first chamber 110 and second chamber 120. Here, barrier
structure 100 includes an array of electroactive nanopores 130.
Based on the characteristic voltage signature associated with the
flow of different types of molecules through electroactive nanopore
130, the membrane may be selectively tuned to allow certain
molecular species to pass from first chamber 110 to second chamber
120.
The invention also includes a method of sensing an analyte
molecule. A flow chart outlining the method is shown FIG. 2. In
Step 210, a sensor structure comprising a sampling chamber, a
collection chamber, and a separation structure is provided. The
separation structure includes at least one electroactive nanopore
130, described herein and shown in FIG. 1. An analyte molecule in a
buffer solution is provided to the sampling chamber (Step 220). The
analyte molecule then passes from the sampling chamber into the
electroactive nanopore in Step 230 by applying a first voltage
V.sub.Pore across first electrode pair 134. As previously described
herein, a current across first electrode pair 134 is generated by
ions in the buffer solution passing through electroactive nanopore
130. When an analyte molecule having a diameter that is less than
the diameter of electroactive nanopore 130 passes through the
nanopore, the molecule partially occludes the passage of ions,
thereby causing the current to decrease. After the analyte molecule
traverses electroactive nanopore 130, normal ion flow through the
nanopore resumes and the current is restored to its initial value.
The size of the analyte molecule and its electrodynamic
interactions with the charges in conductive polymer 136 will
determine the current drop that is observed as the analyte molecule
traverses electroactive nanopore 130. Accordingly, the current
across the first electrode pair 132 is measured in Step 240 to
determine whether the analyte molecule is present.
The invention also provides a method of making barrier structure
100 having electroactive nanopore 130. A flow chart of method 300
is shown in FIG. 3. In Step 310, a template is provided. The
template comprises a strip of a photocurable polymer such as a
polyimide or the like. The polymer strip, which is typically a few
centimeters in length and less than 1 mm wide, is deposited on an
insulating material such as sapphire, glass, or a silicon wafer. In
one embodiment, the template includes a polymeric cylinder
comprising the same photocurable polymer. The polymeric cylinder is
vertically placed on top of the polymer strip. The polymeric
cylinder has a diameter that is substantially equal to the desired
maximum diameter of the electroactive nanopore. In one embodiment,
the polymeric cylinder has a diameter of about 50 nm and a height
of about 200 nm. In another embodiment, the polymeric cylinder is
not provided.
In Step 320, alternating layers of conductive material and
insulating material are deposited over the template to form a
first--or outer--electrode pair and a second--or inner--electrode
pair separated by at least one layer of insulating material. In one
embodiment, the conductive material may comprise any conductive
material known in the art such as, but not limited to, platinum,
gold, graphite, conductive metal alloys known in the art,
combinations thereof, and the like. The insulating material
comprises at least one of a metal oxide, such as sapphire or silica
(SiO.sub.2), glasses, nonconductive polymers, silicon, or the
like.
The thicknesses of the individual layers of insulating material
must be sufficient to prevent shorting or arcing between the
electrode layers. The thickness of the individual layers of
conductive material depends on the desired electrode size and
distance between electrodes. The individual layers of conductive
and insulating material each have a thickness of up to 5 nm. In one
embodiment, the thickness is in a range from about 1 nm to about 5
nm.
In one embodiment, the conductive layers and insulating layers are
deposited using energetic neutral atom beam lithography/epitaxy
(also referred to herein as "ENABLE"), which is described in U.S.
Provisional Patent Application 60/738,624, filed on Nov. 21, 2005,
by Mark A. Hoffbauer et al., entitled "Method of Forming
Nanostructures on a Substrate," the contents of which are
incorporated herein in their entirety.
The template is then removed (Step 330), typically by dissolution
of the photocurable polymer. Where a polymer cylinder is provided,
dissolution of the template leaves a microfluidic channel--or
chamber--having a nanopore on top. In embodiments in which the
template does not include the polymeric cylinder described above,
the nanopore may be formed by drilling through the deposited
conductive and insulating layers using a focused ion beam. The
nanopore diameter reflects the size of the polymeric cylinder used
in the template, and is typically about 50 nm. A second
microfluidic channel or chamber is then formed from a polymeric
material such as polydimethyl siloxane or the like. The second
microfluidic chamber is then placed on top of the first
microfluidic chamber such that the electroactive nanopore is
enclosed between--and connects--the two chambers.
In Step 340, a conductive polymer film is electrochemically
deposited on one electrode of the second electrode pair. The
thickness of the conductive polymer film that is actually deposited
depends on the diameter of the nanopore, in one embodiment, the
thickness of the conductive polymer film is in a range from about
10 nm to about 50 nm. The conductive polymer comprises an ionic
conductive polymer such as, but not limited to, polypyrrole,
polyaniline, combinations thereof, and the like.
Method 300 can be optimized and updated for later fabrication of an
array of several electroactive nanopores. The nanopores can be
integrated with electronics and on-chip computational hardware to
do multiple sensing in a portable device.
The invention also provides a method of controlling fluid from a
first chamber to a second chamber. A flow chart for method 700 is
shown in FIG. 7. in step 710, a barrier structure, such as barrier
structure 100 including at least one electro active nanopore 130
described hereinabove, is provided. Fluid is provided to the first
chamber (Step 720) and is passed into the electroactive nanopore
(Step 730). Step 730 is accomplished by applying a first voltage
across first electrode pair 134 in electroactive nanopore 130. The
first voltage is sufficient to cause the fluid to migrate from the
first chamber through electroactive nanopore 130 to the second
chamber. In Step 740, the electrically tunable diameter 138 of
electroactive nanopore is either increased or decreased to control
the flow of the fluid through electroactive nanopore 130 to the
second chamber. The electrically tunable diameter may be either
decreased or increased by applying a second voltage across second
electrode pair 132 of electrode active nanopore 130. The second
electrode voltage causes conductive polymer 136 to either expand or
contract, which correspondingly causes electrically tunable
diameter 138 to either decrease or increase.
While typical embodiments have been set forth for the purpose of
illustration, the foregoing description should not be deemed to be
a limitation on the scope of the invention. Accordingly, various
modifications, adaptations, and alternatives may occur to one
skilled in the art without departing from the spirit and scope of
the present invention.
* * * * *