U.S. patent application number 10/546939 was filed with the patent office on 2007-02-22 for nanopores, methods for using same, methods for making same and methods for characterizing biomolecules using same.
This patent application is currently assigned to Brown University. Invention is credited to Xinsheng S. Ling.
Application Number | 20070042366 10/546939 |
Document ID | / |
Family ID | 33102160 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042366 |
Kind Code |
A1 |
Ling; Xinsheng S. |
February 22, 2007 |
Nanopores, methods for using same, methods for making same and
methods for characterizing biomolecules using same
Abstract
Featured are devices and systems embodying one or more
solid-state nanopores useable for sensing and/or characterizing
single macromolecules as well as sequencing DNA or RNA and/or
determining RNA secondary structures. In once solid state nanopore
of the present invention the width and/or length of the nanopore is
defined or established by sharp edges of cleaved crystals that are
maintained in fixed relation during the formation of the insulating
member including the nanopore. In another aspect of the present
invention, there is featured a linear or 2-D electrically
addressable array of nanopores, where the nanopores are located at
points of intersections between grooves formed in an upper surface
of the insulating member and a groove formed in a lower surface of
the insulating member.
Inventors: |
Ling; Xinsheng S.; (East
Greenwich, RI) |
Correspondence
Address: |
WILMER CUTLER PICKERING HALE AND DORR LLP
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Brown University
1 Prospect Street Box 1949
Providence
RI
02912
|
Family ID: |
33102160 |
Appl. No.: |
10/546939 |
Filed: |
February 13, 2004 |
PCT Filed: |
February 13, 2004 |
PCT NO: |
PCT/US04/04138 |
371 Date: |
July 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60451088 |
Feb 28, 2003 |
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60507354 |
Sep 29, 2003 |
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60512140 |
Oct 16, 2003 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/6.1; 435/6.12; 977/924 |
Current CPC
Class: |
C12Q 1/6874 20130101;
C12Q 1/68 20130101; C12Q 1/6874 20130101; Y10S 977/72 20130101;
G01N 33/48721 20130101; B82Y 5/00 20130101; Y10S 977/932 20130101;
B82Y 15/00 20130101; C12Q 2565/631 20130101; C12Q 1/6869
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 977/924 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 3/00 20060101 C12M003/00 |
Claims
1. A device for characterizing biomolecules, comprising: an
insulating member, the insulating member including a through
aperture extending between opposing surfaces; and wherein a width
of the through aperture is established by a plurality of crystals
that are arranged in fixed relation with respect to each other
while forming the insulating member.
2. The device of claim 1, wherein edges of the plurality of
crystals cross each other at a predetermined angles and are
maintained in this arrangement while forming the insulating
member.
3. The device of claim 2, wherein the edges form or define an area
that is small enough that the molecules making up the insulating
member cannot enter into this area while forming the insulating
member.
4. The device of claim 2, wherein molecules of insulating material
are oriented with respect to the edges thereby forcing the
insulating material to form a contour around the crossing point
thereby defining the through aperture in the insulating member.
5. A method for characterizing a biomolecule comprising the steps
of: providing an insulating member, the insulating member including
a through aperture extending between opposing surfaces and wherein
a width of the through aperture is established by a plurality of
crystals that are arranged in fixed relation with respect to each
other while forming the insulating member; locating the insulating
member between ionic reservoirs at least one of which includes the
biomolecule to be characterized; and passing the biomolecule
through the through aperture of the provided insulating member.
6. The method of clam 5, further comprising the step of: detecting
an ionic current and changes in ionic current as the biomolecule is
passed through the through aperture; and characterizing the
biomolecule based on the detected ionic current and changes
thereto.
7. A method for determining RNA secondary structures comprising the
steps of: providing an insulating member, the insulating member
including a through aperture extending between opposing surfaces
and wherein a width of the through aperture is established by a
plurality of crystals that are arranged in fixed relation with
respect to each other while forming the insulating member; locating
the insulating member between ionic reservoirs at least one of
which includes the biomolecule to be characterized; operably
coupling coupling one end of the RNA to an optical tweezer; and
measuring a force at said one end as the RNA molecule is pulled
through the through aperture.
8. An electrically-addressable nanopore array comprising: an
insulating material member; wherein the insulating material layer
is configured and arranged so as to have a plurality or more of
grooves extending lengthwise in a first direction in a first
surface thereof; wherein the insulating material member is
configured and arranged so as to have a groove extending lengthwise
in a second direction in a second surface thereof, the first and
second surfaces being opposed to each other; wherein the second
direction is at an angle with respect to the first direction; and
wherein the grooves are formed in each of the first and second
surfaces so that at an intersection of each off the grooves in the
first surface and the groove in the second surface there is formed
an opening that comprises a nanopore.
9. The electrically-addressable nanopore array of claim 8, wherein
the insulating material members is formed from a first layer and a
second layer, where a surface of the first layer is the first
surface of the insulating material member, where the first layer
includes the plurality of grooves formed in the insulating member
first surface, where a surface of the second layer is the second
surface of the insulating material member and where the second
layer includes the groove formed in the insulating member second
surface.
10. The electrically-addressable nanopore array of claim 9, wherein
the first and second layers are one of bounded or secured to each
so as to form the insulating material member.
11. The electrically-addressable nanopore array of claim 8, wherein
the insulating material member is configured and arranged so as to
have a plurality of grooves extending lengthwise in a second
direction in the second surface thereof.
12. The electrically-addressable nanopore array of claim 11,
wherein the insulating material layer is configured and arranged so
as to have a plurality of sets of a plurality or more of grooves
extending lengthwise in a first direction in a first surface
thereof, where the grooves in each set are not connected to grooves
in another set.
13. The electrically-addressable nanopore array of claim 12,
wherein the grooves are formed in each of the first and second
surfaces so that at an intersection of each off the grooves in the
first surface of each set of grooves and one of the plurality of
grooves in the second surface there is formed an opening that
comprises a nanopore.
14. A method for characterizing a biomolecule comprising the steps
of: providing an electrically-addressable nanopore array including
an insulating material member, wherein the insulating material
member is configured and arranged so as to have a plurality or more
of grooves extending lengthwise in a first direction in a first
surface thereof and a groove extending lengthwise in a second
direction in a second surface thereof, the second direction is at
an angle with respect to the first direction, and wherein the
grooves are formed in each of the first and second surfaces so that
at an intersection of each off the grooves in the first surface and
the groove in the second surface there is formed an opening that
comprises a nanopore; locating the electrically-addressable
nanopore array between ionic reservoirs at least one of which
includes the biomolecule to be characterized; and passing the
biomolecule through one of the nanopores of the provided
electrically-addressable nanopore array.
15. The method of clam 14, further comprising the step of:
detecting an ionic current and changes in ionic current as the
biomolecule is passed through the one the nanopores; and
characterizing the biomolecule based on the detected ionic current
and changes thereto.
16. A method for determining RNA secondary structures comprising
the steps of: providing an electrically-addressable nanopore array
including an insulating material member, wherein the insulating
material member is configured and arranged so as to have a
plurality or more of grooves extending lengthwise in a first
direction in a first surface thereof and a groove extending
lengthwise in a second direction in a second surface thereof, the
second direction is at an angle with respect to the first
direction, and wherein the grooves are formed in each of the first
and second surfaces so that at an intersection of each off the
grooves in the first surface and the groove in the second surface
there is formed an opening that comprises a nanopore; locating the
electrically-addressable nanopore array between ionic reservoirs at
least one of which includes the biomolecule to be characterized;
operably coupling coupling one end of the RNA to an optical
tweezer; and measuring a force at said one end as said the RNA
molecule is pulled through one of the nanopores.
17. A nanopore comprising: an insulating member having opposed
surfaces; and a through aperture extending between said opposing
surfaces.
18. The nanopore of claim 17, wherein the width of said through
aperture is less than about 10 nm.
19. A nanopore array comprising a plurality of nanopores, wherein
each nanopore comprises: an insulating member having opposed
surface; and a through aperture extending between said opposing
surfaces.
20. The nanopore array of claim 19, wherein said through aperture
has a width that is less than about 10 nm.
21. The nanopore array of claim 19, wherein each of said nanopores
is electronically addressable.
22. A method for preparing a nanopore comprising an insulating
member having opposed surfaces and a through aperture extending
between said surfaces, the method comprising the steps of: forming
an aperture having a width that is established by fixing a
plurality of crystals fixed in position to one another; forming an
insulating member by casting an insulating material over said
plurality of crystals fixed in position relative to one another,
thereby forming the insulating material with said aperture
extending through opposing surfaces of said insulating material;
and removing said plurality of crystals, to thereby prepare a
nanopore comprising an insulating member having opposed surfaces
and a through aperture extending between said surfaces.
23. A nanopore comprising an insulating member having opposed
surfaces and a through aperture extending between said surfaces,
said nanopore having been prepared by a method comprising the steps
of: forming an aperture having a width that is established by
fixing a plurality of crystals fixed in position to one another;
forming an insulating member by casting an insulating material over
said plurality of crystals fixed in position relative to one
another, thereby forming the insulating material with said aperture
extending through opposing surfaces of said insulating material;
and removing said plurality of crystals, to thereby prepare a
nanopore comprising an insulating member having opposed surfaces
and a through aperture extending between said surfaces.
24. A method for preparing a nanopore array comprising an
insulating member having opposed first and seconds surfaces and one
or more through apertures extending between said surfaces, the
method comprising the steps of: forming one or more grooves in the
first surface that extend lengthwise in a first direction; forming
one or more grooves in the second surface that extend lengthwise in
a first direction, the second direction being at an angle with
respect to the first direction; removing material at intersections
of the one or more grooves in the first surface and the one or more
grooves in the second surface; to thereby prepare a nanopore
comprising an insulating member having opposed surfaces and a one
or more through aperture extending between said surfaces.
25. The device of claim 2, wherein the edges of the crystals are
spaced from each other a distance (Se) and the crystal distance
spacing is set so as to satisfy (Se/Tm).ltoreq.0.5 where Tm is a
thickness of a molecule of the material making up the insulating
member.
26. The device of claim 2, wherein the edges of the crystals are
spaced from each other a distance (Se) and the crystal distance
spacing is set so as to be in the range of from about 1 .ANG. to
about 10 .ANG..
27. The device of claim 1, wherein the through aperture has a
length less than or equal 20 .ANG..
28. The device of claim 1, wherein the through aperture has a
length (d) that satisfies the relation 2 .ANG..ltoreq.d.ltoreq.10
.ANG..
29. The method of claim 5, wherein the through aperture of the
provided insulating member has a length less than or equal 20
.ANG..
30. The method of claim 1, wherein the through aperture of the
provided insulating member has a length (d) that satisfies the
relation 2 .ANG..ltoreq.d.ltoreq.10 .ANG..
31. The method of claim 5, wherein the edges of the crystals are
spaced from each other a distance (Se) and the crystal distance
spacing is set so as to satisfy (Se/Tm).ltoreq.0.5 where Tm is a
thickness of a molecule of the material making up the insulating
member.
32. The method of claim 5, wherein the edges of the crystals are
spaced from each other a distance (Se) and the crystal distance
spacing is set so as to be in the range of from about 1 .ANG. to
about 10 .ANG..
33. The method of claim 7, wherein the through aperture of the
provided insulating member has a length less than or equal 20
.ANG..
34. The method of claim 7, wherein the through aperture of the
provided insulating member has a length (d) that satisfies the
relation 2 .ANG..ltoreq.d.ltoreq.10 .ANG..
35. The method of claim 7, wherein the edges of the crystals are
spaced from each other a distance (Se) and the crystal distance
spacing is set so as to satisfy (Se/Tm).ltoreq.0.5 where Tm is a
thickness of a molecule of the material making up the insulating
member.
36. The method of claim 7, wherein the edges of the crystals are
spaced from each other a distance (Se) and the crystal distance
spacing is set so as to be in the range of from about 1 .ANG. to
about 10 .ANG..
37. A method for preparing a two-dimensional nanopore array
comprising an insulating member having opposed surfaces and a
plurality of through aperture extending between said surfaces, the
method comprising the steps of: forming each of the plurality of
apertures having a width that is established by fixing a plurality
of crystals fixed in position to one another for each aperture;
forming an insulating member by casting an insulating material over
each of said plurality of crystals fixed in position relative to
one another for each aperture, thereby forming the insulating
material with said each aperture extending through opposing
surfaces of said insulating material; and removing said plurality
of crystals for each aperture, to thereby prepare a two-dimensional
nanopore array comprising an insulating member having opposed
surfaces and a plurality of through apertures extending between
said surfaces.
38. A method for preparing a nanopore array comprising the steps
of: providing a plurality of nanopore assemblies, each of the
plurality of nanopore assemblies including an insulating member
having opposed surfaces and a through aperture extending between
said surfaces, assembling the plurality of nanopore assemblies so
the through apertures form at least a one-dimensional array of
through apertures; and wherein said providing a plurality of
nanopore assemblies includes performing the steps of for each
nanopore assembly: forming an aperture having a width that is
established by fixing a plurality of crystals fixed in position to
one another, forming an insulating member by casting an insulating
material over said plurality of crystals fixed in position relative
to one another, thereby forming the insulating material with said
aperture extending through opposing surfaces of said insulating
material, and removing said plurality of crystals, to thereby
prepare a nanopore comprising an insulating member having opposed
surfaces and a through aperture extending between said
surfaces.
39. The method of claim 38, wherein said assembling the plurality
of nanopore assemblies includes assembling the plurality of
nanopore assemblies so the through apertures form a two-dimensional
array of through apertures.
40. The method of any of claims 38-39, wherein the through aperture
of each of the plurality of provided nanopore assemblies has a
length less than or equal 20 .ANG..
41. The method of any of claims 38-39, wherein the through aperture
of each of the plurality of provided nanopore assemblies has a
length (d) that satisfies the relation 2 .ANG..ltoreq.d.ltoreq.10
.ANG..
42. The method of any of claims 38-39, wherein said forming an
aperture includes spacing edges of the crystals from each other a
distance (Se), where the distance is set so as to satisfy the
relation (Se/Tm).ltoreq.0.5 where Tm is a thickness of a molecule
of the material making up the insulating member.
43. The method of any of claims 38-39, wherein said forming an
aperture includes spacing edges of the crystals from each other a
distance (Se), where the distance is set so as to be in the range
of from about 1 .ANG. to about 10 .ANG..
44. A method for sequencing DNA or RNA comprising the steps of:
providing an insulating member, the insulating member including a
through aperture extending between opposing surfaces and wherein a
width of the through aperture is established by a plurality of
crystals that are arranged in fixed relation with respect to each
other while forming the insulating member; coating at least inner
surfaces of the through aperture with a PNA including one of the A,
T, G or C bases that characterize DNA/ RNA; locating the insulating
member between ionic reservoirs at least one of which includes the
DNA/RNA to be sequenced; operably coupling coupling one end of the
DNA/RNA to an optical tweezer; measuring a force as a function of
time at said one end as said DNA/RNA is pulled through the through
aperture; and correlating the measured force as a function of time
to one of A, T, G, or C bases, to thereby prepare sequencing the
DNA or RNA.
45. A method for sequencing DNA or RNA comprising the steps of:
providing a plurality of nanopore assemblies, each of the plurality
of nanopore assemblies including an insulating member having
opposed surfaces and a through aperture extending between said
surfaces, assembling the plurality of nanopore assemblies so the
through apertures form at least a one-dimensional array of through
apertures; coating at least inner surfaces of the through aperture
of each of the plurality of nanopore assemblies with a PNA
including one of the A, T, G or C bases that characterize DNA/RNA;
locating the insulating member of each nanopore assembly so an
ionic reservoir which includes the DNA/RNA to be sequenced is
disposed on a side of at least one of the plurality of nanopore
assemblies; operably coupling coupling one end of the DNA/RNA for
the through aperture of said at least one nanopore assembly to an
optical tweezer; measuring a force as a function of time at said
one end as said DNA/RNA is pulled through the through aperture of
said at least one nanopore assembly; correlating the measured force
as a function of time to one of A, T, G, or C bases, to thereby
sequencing the DNA or RNA; and wherein said providing a plurality
of nanopore assemblies includes performing the steps of for each
nanopore assembly: forming an aperture having a width that is
established by fixing a plurality of crystals fixed in position to
one another, forming an insulating member by casting an insulating
material over said plurality of crystals fixed in position relative
to one another, thereby forming the insulating material with said
aperture extending through opposing surfaces of said insulating
material, removing said plurality of crystals, and coating at least
inner surfaces of the through aperture with a PNA including one of
the A, T, G or C bases that characterize DNA/RNA;
46. The device of claim 1, further comprising a coating of a PNA
including one of the A, T, G or C bases that characterize DNA/RNA;
wherein the coating is applied so as to coat at least inner
surfaces of the through aperture.
47. The electrically-addressable nanopore array of claim 8, further
comprising a coating of a PNA including one of the A, T, G or C
bases that characterize DNA/RNA; wherein the coating is applied so
as to coat at least inner surfaces of the opening formed at the
intersections of each of the grooves in the first and second
surfaces.
48. The nanopore of claim 17, further comprising a coating of a PNA
including one of the A, T, G or C bases that characterize DNA/RNA;
wherein the coating is applied so as to coat at least inner
surfaces of the through aperture.
49. The nanopore array of claim 19, further comprising a coating of
a PNA including one of the A, T, G or C bases that characterize
DNA/RNA; wherein the coating is applied so as to coat at least
inner surfaces of the through aperture for each of the plurality of
nanopores.
50. A method for sequencing DNA or RNA comprising the steps of:
providing any one of the device of claim 46, the
electrically-addressable nanopore array of claim 47, the nanopore
of claim 48 and the nanopore array of claim 49; locating said any
one of the device, the electrically-addressable nanopore array, the
nanopore and the nanopore array so as to be between ionic
reservoirs at least one of which includes the DNA/RNA to be
sequenced; operably coupling coupling one end of the DNA/RNA to an
optical tweezer; measuring a force as a function of time at said
one end as said DNA/RNA is pulled through the through aperture; and
correlating the measured force as a function of time to one of A,
T, G, or C bases, to thereby prepare sequencing the DNA or RNA.
51. The method for preparing a nanopore according to claim 22,
further comprising the step of coating at least inner surfaces of
the through aperture with a PNA including one of the A, T, G or C
bases that characterize DNA/RNA;
52. The method for preparing a nanopore array according to claim
24, further comprising the step of coating at least inner surfaces
of the one or more through apertures with a PNA including one of
the A, T, G or C bases that characterize DNA/RNA;
Description
FIELD OF INVENTION
[0001] The present invention relates to devices used for
characterizing biomolecules as well as sequencing DNA and RNA, and
more particularly to a nanopore and/ or an array of nanopores for
use in rapid characterization of biomolecules and high through-put
DNA sequencing.
BACKGROUND OF THE INVENTION
[0002] Transport of nucleic acids and other biological molecules
through membrane channels of nanometer scale is ubiquitous in
Nature. Examples include the movement of RNA molecules and
transcription factors across nuclear pores; viral DNA injected
through a bacterium membrane in phage infection; and the uptake of
specific oligonucleotides by membrane proteins. Understanding the
function of these nanoscale channels and how biomolecules move
through them is a major task of modem molecular biology and
biophysics. In recent years, a fascinating field of nanopore
biophysics has emerged in the interdisciplinary area of molecular
biology, nanotechnology, and single molecule biophysics. In 1994,
Berzrukov, Vodyanoy, and Parsegian showed that one can use a
biological nanopore as a Coulter counter to count individual
molecules. In 1996, in a landmark paper by Kasianowicz, Brandin,
Branton, and Deamer (KBBD), [Kasianowicz, L J, Brandin, E, Branton,
D. & Deamer, D. W. Characterization of individual
polynucleotide molecules using a membrane channel, Proc. Nat Acad.
Sci. USA 93, 13770-13773 (1996)] an ambitious idea was proposed for
ultrafast single-molecule sequencing of single-stranded (ss) DNA
molecules using nanopore ionic conductance as a sensing mechanism.
Since then several groups have explored the potential of
.alpha.-hemolysin protein pore as a possible candidate for
achieving this objective.
[0003] DNA sequencing using nanopore ionic conductance in
accordance with the KBBD method involves an insulating wall
separating two reservoirs of ionic solutions, where the wall
contains a hole so small that only one strand of a single-stranded
DNA molecule can fit through the pore. When a voltage is applied to
the two reservoirs, the electrical potential drop should almost
entirely occur at the nanopore. Similar to the case of point
contact resistance or conductance between two metals, the
electrical conductance or resistance between two ionic reservoirs
is also the conductance or resistance of the nanopore. Because of
the low mobility of the DNA molecule, the ionic conductance through
the nanopore is dominated by the flux of Na.sup.+ and Cl.sup.-
flushing through the nanopore. The ionic conductance drops when a
DNA molecule enters the nanopore.
[0004] The amount of conductance drop will be a measure of the
physical size of the part of the DNA molecule that is inside the
narrowest part of the channel. Because the chemical groups
(nucleotides) of a DNA molecule have slightly different sizes, one
should be able to measure the genetic information of the
nucleotides of a DNA molecule by measuring the time dependence of
the nanopore conductance.
[0005] The underlying assumption of the KBBD method is that the
four different nucleotides composing DNA or RNA molecules may have
different blockage effects in the ionic current when the molecule
moves through a nanopore, because they have different atomic
compositions. The basic concept of voltage driven DNA translocation
through a nanopore is illustrated in FIG. 1 that shows (a) a
schematic of a voltage-driven DNA translocation experiment using
.alpha.-hemolysin nanopore and (b) typical translocation events
using poly (dA) in buffer 1 M KCI, 1 mM Tris-EDTA (pH 8.5).
[0006] There have been encouraging preliminary data suggesting that
this idea may be feasible. For example, it has been shown that
homopolymers of poly-adenine (DNA) give rise to a slightly lower
blockage level than polycytosines, and are translocated in a few
.mu.sec per base rate, under room temperature conditions. These
findings are indeed very encouraging for the realization of a rapid
DNA sequencing method.
[0007] The KBBD methodology uses a .alpha.-hemolysin protein ion
channel embedded in a lipid bilayer membrane as a natural nanopore.
It has been demonstrated that a DNA polymer consisting of 70
cytosine nucleotides and 30 adenine nucleotides does give rise to a
measurable signal in the nanopore conductance. However, because the
natural nanopores are long channels, typically 30 nm in length,
many nucleotides are inside the channel at any time during
translocation and the effect of individual nucleotides on the ionic
conductance is lost. The fact that one can see a difference at all
between C and A nucleotides in the nanopore conductance is a sign
that one should be able to ultimately see the effect of a single
nucleotide on the nanopore conductance, if one can make a nanopore
with length comparable to that of a single nucleotide (.about.0.4
nm).
[0008] There also has been recently demonstrated (Golovchenko's
group in the Harvard Physics Department) a reliable nano sculpting
approach for making a single nanopore of 1.5 nm in diameter in a
silicon-nitride solid-state membrane. In this approach, the
processing steps that use focused ion beam lithography and low
energy sputtering with feedback monitoring are highly reproducible
and reliable, however these nanopores are still too long in length
(>10 nm) for measuring single nucleotides. J. Li, D Stein, C
McMullan, D Branton, M. J. Aziz, and J. A. Golovchenko; Ion-beam
sculpting at nanometer length scales, Nature 412, 166-169
(2001).
[0009] In a recent experiment by Meller et al. it was found that
the amount of current blockage and the time it takes for the DNA
molecule to go through the nanopore channel have large
fluctuations. A. Meller, L. Nivon, and D Branton, Voltage-driven
DNA translocations through a nanopore, Phys. Rev. Lett. 86,
3435-3438 (2001). There is shown in FIG. 2 histograms illustrating
the blockage current versus polymer length (left panel) and the
translocation velocity versus polymer length for the same polymer
(right panel). Consequently it can be seen that a good
understanding of the origin(s) of these fluctuations is needed in
order to observe individual bases in the standard DNA translocation
experiment. In fact, the physics of how a linear flexible molecule
(such as a DNA) threads through a nanopore under the influence of a
driving force is poorly understood, making it difficult to extract
size information from the ionic current data. There are several
theoretical papers addressing various aspects of this problem, such
as the effects of fluctuations of polymer ends (entropic barrier),
DNA-channel interactions, etc. From the experimental side, it is
highly desirable to measure the DNA translocation process at either
a constant velocity (at low velocity if possible) or constant
force. Previous temperature-dependent measurements of DNA
translocation through .alpha.-hemolysin pore suggest that thermal
motion of DNA is a source of noise in the blockage current. There
are ongoing efforts attempting to stop the translocation process
while the DNA is inside the .alpha.-hemolysin pore.
[0010] Ideally, one would like to measure the ionic current while
the DNA molecule is fully stretched inside the pore (e.g. using
optical tweezers (see FIG. 3)). In such an experiment, fluctuations
in the ionic current due to DNA thermal motion can be reduced or
avoided. Also, one can hold the molecule stationary and use signal
averaging to further reduce noise in the ionic current. To do this
however, one needs to (a) attach a single-stranded DNA to a
double-stranded DNA and then the latter to a bead (using for
example streptavidin-biotin linker) held by an optical tweezers
[see Introduction to Optical Tweezers;
www.nbi.dk/.about.tweezer/introduction.htm] and next (b) to drive
the single stranded DNA through the pore and catch it from the
other side using another bead (also with streptavidin-biotin
linker). All these processes except optical trapping are stochastic
and can be very time consuming. In order to make this idea
practical, one needs to use a high-throughput device such as an
array of nanopores and each pore in the array has to be addressable
electrically, and independently.
[0011] Currently there are two successful methods for fabricating
solid-state nanopores in insulating materials. One method involves
the use of ion-beam sculpting of silicon nitride and the other uses
a-beam lithography and wet etching in crystalline silicon followed
by oxidation. In general these techniques have been used to
validate that a nanopore can be used in the described fashion, but
have not been used to yield a solid state nanopore device having
high through-put.
[0012] U.S. Pat. No. 6,428,959 describes methods for determining
the presence of double stranded nucleic acids in a sample. In those
methods, nucleic acids present in a fluid sample are translocated
through a nanopore, e.g., by application of an electric field to
the fluid sample. The current amplitude through the nanopore is
monitored during the translocation process and changes in the
amplitude are related to the passage of single- or double-stranded
molecules through the nanopore. Those methods find use in a variety
of applications in which the detection of the presence of
double-stranded nucleic acids in a sample is desired, e.g., in
hybridization assays, such as Northern blot assays, Southern blot
assays, array based hybridization assays, etc.
[0013] It would be desirable to provide a new nanopore that can be
formed using synthetic material and methods for using and making
such a nanopore. It also would be desirable to provide a nanopore
.ltoreq.10 nm in length. It would be desirable to provide a device
and method that yields a linear array of nanopores or
two-dimensional array of nanopores. It also would be particularly
desirable to provide such linear and 2-dimensional nanopore arrays
where the nanopores are separately electrically addressable.
SUMMARY OF THE INVENTION
[0014] The present invention features devices and systems embodying
one or more solid-state nanopores that can be used to sense and/or
characterize single macromolecules as well as sequencing DNA or
RNA. Such devices and systems have a wide range of applications in
molecular biology and single-molecule biophysics. In addition to
being useful for rapid DNA sequencing, the devices and systems of
the present invention can be used in a variety of applications
involving, but not limited to single-molecule biophysics, molecular
biology, and biochemistry. For example, nanopore devices and
systems of the present invention are contemplated for use as a
molecular comb to probe the secondary structure of RNA molecules,
for use in detecting biological warfare agents, and
contaminants/pollutants in air and/or water.
[0015] According to one aspect of the present invention, there is
featured a device including a member of an insulating material,
wherein the insulating member is configured and arranged so as to
include a through aperture comprising a nanopore therein. The
through aperture comprises a plurality of crystals that have been
cleaved to form atomically sharp edges, which crystals are arranged
in fixed relation while forming the insulating material. In
particular embodiments, the crystal edges cross each other at a
predetermined angle, more particularly an angle of about 90
degrees. It is within the scope of the present invention, however,
for the crystal edges to be crossed each other at an angle of less
than or more than 90 degrees thereby forming nanopores having
different cross-sections or different cross-sectional shapes.
[0016] The crossing cleaved crystal edges essentially form or
define an area that is small enough that the molecules making up
the insulating material cannot enter into this area. In this way,
the molecules of the insulating material should be oriented with
respect to the cleaved edges and thus forced to make a contour
around the crossing point, thereby leaving a small hole comprising
the through aperture therein. In a particular embodiment, the
crystals are GaAs crystals that are well known in the art. However,
the present invention can be practiced using other crystals known
to those skilled in the art and having similar characteristics that
lend themselves to being cleaved and being held in a fixed
relation, while the insulating material comprising the insulating
member is formed, such as for example NaCl crystals.
[0017] In a particular embodiment, the insulating material is a
material that is characterized as being flowable and which
solidifies at the end of the process for forming the nanopore as
well as during normal operating conditions (e.g., room
temperature). In an illustrative embodiment, the insulating member
is made from a curable polymer such as liquid PDMS
(poly-dimethylsiloxane), polystyrene or PMMA and GaAs crystals are
used to form the nanopore through aperture. This technique yields a
nanopore having a length or channel length of 20 .ANG.
(Angstrom/10.sup.-10 m) or less, more particularly a channel length
of 20 .ANG. or less or more particularly a channel length of about
4 .ANG. and more specifically a channel length (d) that satisfies
one of the following relationships; 2 .ANG..ltoreq.d.ltoreq.10
.ANG. or 4 .ANG..ltoreq.d.ltoreq.10 .ANG..
[0018] In the illustrative example, GaAs crystals, common
semiconductor substrate materials, are cleaved to form atomically
sharp edges. The cleaved crystals are then arranged so two such
crystal edges are brought together to within a few angstroms
distance and held fixed using standard STM electronics and use
electron tunneling current as a feedback mechanism (this assumes
that the GaAs crystals are doped and have finite electrical
conductivity). In more particular embodiments, the spacing of the
crystal edges is in the range of from between 1 .ANG. and 10 .ANG.,
more specifically about 2 .ANG..
[0019] In further embodiments, the crystal edges are spaced from
each other so the distance between the crystal edges is less than
the thickness (Tm) of the molecule of the insulating material. In
more specific embodiments, the spacing (Se) between the crystal
edges is set so as to satisfy the following relationship
Se/Tm.ltoreq.0.5.
[0020] The curable polymer liquid PDMS (poly-dimethylsiloxane) is
then poured into the region of the cutting edges and cured while
the two crystal edges are held fixed. As indicated herein, at the
crossing point between the two edges, the distance between the
edges is so small that no molecules of the polymer can enter this
region. The molecules of the polymer also are advantageously
oriented parallel to the respective cleaved edges and be forced to
make a contour around the crossing point, leaving a small hole. In
such a methodology, the width and the length of the nanopore are
controlled by the distance between the two edges and the diameter
of polymers.
[0021] Following curing or formation of the insulating member in
its final form, the crystals are removed, e.g., by washing, In
another exemplary embodiment, the crystals used to form the through
aperture are NaCl crystals, which crystals are removed by washing
with H.sub.20.
[0022] Also featured are systems and methods utilizing the nanopore
device of the present invention to perform any of a number of
analytical processes including but not limited to characterizing
biomolecules, sequencing DNA and determining RNA secondary
structures. Such methods include providing an insulating member as
hereinabove described including a nanopore, wherein a diameter and
length of the nanopore are defined by the sharp edges of cleaved
crystals that are maintained in fixed relation during the formation
of the insulating member and locating the insulating member so as
to be disposed between two ionic reservoirs. Such methods further
include passing the biomolecules or DNA through the nanopore and
characterizing the biomolecule or the DNA based on changes in ionic
current or other physical parameter. As to the method for
determining RNA secondary structures, the method further includes
operably coupling one end of the RNA to an optical tweezer and
measuring a force at said one end as the RNA molecule is pulled
through the nanopore.
[0023] According to another aspect of the present invention there
is featured an electrically-addressable nanopore array that is
configured and arranged so as to allow high through-put analyses of
biomolecules as well as sequencing DNA. In one particular
embodiment, the electrically addressable nanopore array includes a
linear or one-dimensional array of nanopores. In another
embodiment, the electrically addressable nanopore array includes a
two-dimensional array of nanopores. In other embodiments, the
two-dimensional array is in the form of a plurality or more of
linear arrays. In further embodiments the linear and two
dimensional nanopore arrays are formed and arranged in the
insulating material so each of the nanopores can be addressed
independently using an electric current provided by, for example, a
standard patch-clamp arrangement.
[0024] In particular embodiments, the electrically addressable
nanopore array includes an insulating material layer. The
insulating material layer is configured and arranged so as to have
one or more of grooves extending lengthwise in a first direction in
a first surface thereof and being parallel to each other. The
insulating material layer is configured and arranged so a groove
also is formed in the insulating material layer second surface that
extends lengthwise in a second direction in a second surface
thereof, where the first and second surfaces are opposed to each
other. Also, the second direction is at an angle with respect to
the first direction, more particularly the first and second
directions are orthogonal to each other or at about a 90 deg. angle
with respect to each other.
[0025] The grooves also are formed in each of the first and second
surfaces so as to extend downwardly from one surface to the other
surface, so that at an intersection of each of the grooves in the
first surface and the groove in the second surface there is formed
an opening that comprises a nanopore. In this way a plurality of
nanopores are formed in a linear or one-dimensional array in the
insulating material at the intersection of each of the plurality of
grooves in the first surface and the groove formed in the second
surface.
[0026] As indicated above, in further embodiments the insulating
material layer is configured and arranged so as to have a plurality
or more of grooves extending lengthwise in the second direction in
the second first surface thereof and being parallel to each other.
The plurality of grooves also are formed in the second surface so
as to extend downwardly from one surface to the other surface such
that at an intersection of each of the grooves in the first surface
and each of the grooves in the second surface there is formed an
opening that comprises a nanopore. In this way a plurality of
nanopores are formed in a two-dimensional array in the insulating
material at the intersection of each of the plurality of grooves in
the first surface and the second surface. In more particular
embodiments, the grooves are V-shaped.
[0027] In exemplary embodiments, the insulating material layer is
formed from a first sub-layer and a second sub-layer that are
bonded or secured to each other using any of a number of techniques
known to those skilled in the art and appropriate for the materials
being used so as to form the insulating material layer. The first
sub-layer is configured and arranged so as to include a plurality
of grooves that extend between opposing surfaces, one of the
opposing surfaces comprising the first surface of the insulating
material surface. Similarly, the second sub-layer is configured and
arranged so as to include a groove that extends between opposing
surfaces, one of the opposing surfaces comprising the second
surface of the insulating material surface. In further embodiments,
and as indicated herein the second sub-layer can be configured and
arranged so as to include a plurality of grooves that each extend
between opposing surfaces, one of the opposing surfaces comprising
the second surface of the insulating material surface. When bonded
or secured to each other, the first and second-sub-layers are
oriented so that the first and second surfaces of the insulating
material layer are opposed to each other and so that the grooves in
the first sub-layer are at an angle with respect to the one or more
grooves in the second sub-layer, more particularly the first and
second sub-layers are oriented so that the grooves in the
respective sub-layers are orthogonal to each other or at about a 90
deg. angle with respect to each other.
[0028] In specific embodiments, at least portions of the tip of
each groove in the first and second sub-layer is configured and
arranged so as to be open and each groove is formed in each of the
first and second sub-layers so as to be separate from each other.
The open portions also are arranged such that at an intersection of
each of the grooves in the first sub-layer and each of the one or
more grooves in the second sub-layer, there is formed an opening
that comprises a nanopore. In this way a plurality of nanopores are
formed in a linear or one-dimensional array in the insulating
material at the intersection of each of the plurality of grooves in
the first sub-layer and each groove in the second sub-layer. In
more particular embodiments, the grooves are V-shaped and portions
of the tips of the V-shaped grooves are formed so as to be
opened.
[0029] In an exemplary, illustrative embodiment, the first and
second sub-layers comprise a silicon wafer and the grooves are
formed in the silicon wafer using an oxide or nitride mask in KOH
solutions. Such a technique allows grooves, more particularly
V-shaped grooves to be formed with high accuracy (e.g., within 20
nm) by controlling the etching rate and etching time. After the
grooves are etched in the surfaces of the silicon wafers, the first
and second sub-layers are bonded together. After bonding, the
assemblage is oxidized to form SiO.sub.2 to insulate all silicon
surfaces.
[0030] Also featured are systems and methods utilizing an
electrically-addressable nanopore array of the present invention to
perform any of a number of analytical processes including but not
limited to characterizing biomolecules, sequencing DNA and
determining RNA secondary structures. Such methods include
providing an linear or two-dimensional electrically-addressable
nanopore array such as that hereinabove described and locating the
insulating member so as to be disposed between two ionic
reservoirs. Such methods further include passing the biomolecules
or DNA through any one or more of the nanopores of the linear or
two-dimensional array and characterizing the biomolecule or the DNA
based on changes in ionic current or other physical parameter. As
to the method for determining RNA secondary structures, the method
further includes operably coupling one end of the RNA to an optical
tweezer and measuring a force at said one end as the RNA molecule
is pulled through any one of the nanopores comprising the linear or
two-dimensional array.
[0031] According to another aspect of the present invention, there
is featured a PNA functionalized nanopore device as well as methods
and systems related thereto. Such a PNA functionalized includes a
nanopore device including one or more nanopores as herein described
and a PNA coating that is applied so as to cover at least an
interior surface of the at least one or more nanopores. In more
particular embodiments, the PNA material is synthesized so as to be
characterized or constituted by one of the bases that make up a DNA
or RNA molecule. Related methods include operably coupling one end
of the DNA/RNA molecule to an optical tweezer and measuring a force
at said one end as the molecule is pulled through any one of the
nanopores comprising the linear or two-dimensional array. The
method further includes sequencing the DNA/RNA molecule by
determining the fluctuations in force as a function of time and
correlating the fluctuations to the bonding effect between the one
basis characterizing the PNA coating and any one of the number of
bases that appear in DNA or RNA.
[0032] Other aspects and embodiments of the invention are discussed
below.
BRIEF DESCRIPTION OF THE DRAWING
[0033] For a fuller understanding of the nature and desired objects
of the present invention, reference is made to the following
detailed description taken in conjunction with the accompanying
drawing figures wherein like reference character denote
corresponding parts throughout the several views and wherein:
[0034] FIG. 1 illustrates the basic concept of voltage driven DNA
translocation through a nanopore and more particularly (a) is a
schematic of a voltage-driven DNA translocation experiment using
.alpha.-hemolysin nanopore and (b) illustrates typical
translocation events using poly(dA) in buffer 1 M KCl, 1 mM
Tris-EDTA (pH 8.5);
[0035] FIGS. 2A,B provides histograms illustrating (FIG. 2A) the
blockage current versus polymer length and (FIG. 2B) the
translocation velocity versus polymer length for the same
polymer;
[0036] FIG. 3 is a diagrammatic view illustrating the cutting edges
mechanism for forming a nanopore using curable polymers;
[0037] FIG. 4 is a schematic view of an exemplary feedback
mechanism for controlling the distance between the two cutting
edges;
[0038] FIG. 5 is a three-dimensional view of a portion of the cured
polymer after the GaAs crystals are removed;
[0039] FIG. 6 is a perspective view of an array of
electrically-addressable nanopores according to an aspect of the
present invention;
[0040] FIG. 7 is a perspective view of an electrically-addressable
nanopore array device according to the present invention with a
linear array of nanopores with only one groove in the upper side of
the device for clarity;
[0041] FIG. 8 is a schematic view of an electrically-addressable
nanopore array according to the present invention with a
two-dimensional array of nanopores;
[0042] FIG. 8A is a top view with a partial cutaway of a
two-dimensional array comprised of a plurality or more of the
single nanopore assembly of the present invention;
[0043] FIG. 8B is a side view of the array of FIG. 8A through a
single nanopore assembly;
[0044] FIG. 9 is an illustrative view illustrating holding of a
molecule stationary across a nanopore using beads and optical
tweezers;
[0045] FIG. 10 is a block-diagram view of an illustrative optics
and video system and the general arrangement thereof with respect
to an electrically-addressable nanopore array device of the present
invention;
[0046] FIG. 11 is a schematic view illustrating the technique for
obtaining information relating to the secondary structure(s) of
RNA; and
[0047] FIGS. 12A,B are schematic views illustrating a sequencing
technique using PNA functionalized nanopores according to the
present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0048] Referring now to the various figures of the drawing wherein
like reference characters refer to like parts, there is shown in
FIGS. 3-5 various views to illustrate the process for making a
nanopore insulating member 10, that includes a nanopore 12,
according to one aspect of the present invention. More
particularly, FIG. 3 is a diagrammatic view illustrating the
cutting edges mechanism for forming the nanopore 12 of an
insulating member 10 of the present invention using curable
polymers; FIG. 4 is a schematic view of an exemplary feedback
mechanism for controlling the distance between the two cutting
edges of the crystals; and FIG. 5 is a three-dimensional view of a
portion of the insulating member 10 after the GaAs crystals are
removed.
[0049] According to one aspect of the present invention there is
provided a nanoprobe insulating member 10 that is particularly
configured and arranged so as to include a nanopore 12 therein that
extends across a thickness of the member. In more particular
embodiments, the insulating member is constructed of any of a
number of insulating materials known to those skilled in that art
and adaptable for use in the insulating member of the present
invention. In use, the insulating nanoprobe member 10 is arranged
so as to separate two reservoirs of ionic solutions, where the
insulating member contains a hole, i.e., the nanopore 12
dimensioned so that that preferably only one strand of a
single-stranded DNA molecule can pass there through. Also, when a
voltage is applied to the two reservoirs, the electrical potential
drop advantageously occurs entirely at the nanopore.
[0050] The following describes one technique for making the
insulating member 10 of the present invention. However, other
methods or techniques are contemplated that are adaptable to
maintain crystals in fixed relation so as to provide a mechanism
for defining a through aperture that comprises a nanopore. Such a
methodology advantageously yields an insulating member having a
nanopore whose length or channel length can be controlled so as to
be about 20 .ANG. (Angstrom/10.sup.-10 m) or less, more
particularly a channel length of about 10 .ANG. or less or more
particularly a channel length of about 4 .ANG. and more
specifically a channel length (d) that satisfies one of the
following relationships; 2 .ANG..ltoreq.d.ltoreq.10 .ANG. or 4
.ANG..ltoreq.d.ltoreq.10 .ANG.. As indicated herein, conventional
techniques for forming nanopores for such use, result in a
structure in which the nanopore has a length in excess of the
length of the DNA strand to be characterized which can lead to
inaccuracies in the characterization.
[0051] As illustration of this technique, crystals are cleaved to
form atomically sharp edges and two of these cleaved crystals are
brought together within a few angstroms distance of each other. In
addition, the crystals are arranged so the crystal edges are
cutting each other at an angle of about 90 degrees, as shown in the
diagram in FIG. 3. The two crystals are held in fixed relation
using standard STM (scanning tunneling microscope) electronics such
as that shown in FIG. 4 and using the electron tunneling current as
a feedback mechanism. At the crossing point between the two edges,
the distance between the edges is so small that no molecules larger
than a certain size can enter into this region. Stated another way,
the crossing point is established such that molecules having a
width greater than a desired width cannot enter into this region.
In more particular embodiments, the edges of the crystals are
spaced from each other so as be in the range of from between 1
.ANG. and 10 .ANG., more specifically about 2 .ANG.. In further
embodiments, the crystal edges are spaced from each other so the
distance between the crystal edges is less than the thickness (Tm)
of the molecule of the insulating material. In more specific
embodiments, the spacing (Se) between the crystal edges is set so
as to satisfy the relationship Se/Tm.ltoreq.0.5.
[0052] The crystals contemplated for use in the present invention
include a wide range of crystals and in a particular, exemplary
illustrative embodiment the crystals include GaAs crystals, a
common semiconductor material, and NaCl crystals. However, the
present invention can be practiced using other crystals known to
those skilled in the art and having similar characteristics that
lend themselves to being cleaved and being held in a fixed relation
while the insulating material comprising the insulating member is
being formed.
[0053] The crystals when so arranged use the crossing cleaved
crystal edges as a mold (e.g., nano-molding). After arranging the
crystals in fixed relation to one another an insulating material
(e.g., a flowable insulating material), such as a curable polymer
liquid PDMS (poly-dimethylsiloxane), is poured into the region of
the cutting edges and allowed to cure while the two crystal edges
are held fixed. The distance between the edges is set to be so
small that no molecules can enter this forbidden region. Thus,
molecules comprising the insulating material making up the
insulating member 10 are prevented from gaining access into the
so-called forbidden region. One possible molecular conformation of
the polymers near the crossing point also is shown in FIG. 3. The
molecules of the insulating material are advantageously oriented
parallel to the respective cleaved edges and are forced to make a
contour around the crossing point, leaving a small hole of a given
width and length. The width and the length of the small hole are
controlled by the distance between the two edges and the diameter
of the insulating material (e.g., polymers).
[0054] In further embodiments, and after the insulating member 10
is so-formed, the crystals that were used to controllably form the
nanopore or opening in the insulating member are removed using any
of a number of techniques known to those skilled in the art that do
not appreciably effect the insulating material about the formed
hole, e.g., in the case of NaCl crystals, by washing with water.
Following removal of the crystals, an opening is formed in the
member such as that illustrated in FIG. 5, having a desired width
and length. Because the chemical groups (nucleotides) of a DNA
molecule have slightly different sizes, one can identify the
nucleotides of a DNA molecule, and hence sequence the DNA molecule,
by measuring the time dependence of the nanopore conductance. In
this way, a nanopore is formed in the insulating material; having a
desired width and length for purposes of characterizing the
biomolecules, more particularly fast characterization of the
biomolecule. Such an insulating member 10 is thus of great utility
for a wide range of uses including forensics, rapid sequencing of
DNA and research. As indicated herein, the amount of the
conductance drop will be a measure of the physical size of the part
of the DNA molecule that is inside the narrowest part of the
channel.
[0055] Referring now to FIG. 6, there is shown a perspective view
of device 100 including an array of electrically-addressable
nanopores 112, more particularly a linear array of nanopores
according to another aspect of the present invention. The device
100 includes an insulating member 110 having a top surface 112 and
a bottom surface 114. A plurality of grooves 116 are formed in the
top surface using any of a number of techniques known to those
skilled in the art that are appropriate for the material comprising
the insulating member. The grooves 116 in the top surface 112 are
formed in the insulating member 110 so as to generally extend
downwardly towards the bottom surface 114 a predetermined distance
from the top surface. Correspondingly, the groove 118 in the bottom
surface 114 is formed in the insulating member 110 so as to
generally extend upwardly towards the top surface 112 a
predetermined distance from the bottom surface.
[0056] In addition, at the points of intersection between the
grooves 116 in the top surface 112 and the groove 118 in the bottom
surface 114, the insulating member is configured and arranged so an
opening or nanopore 122 is provided in the insulating member 110 so
as to fluidly couple each of the top surface grooves 116 with the
bottom surface groove 118. The formation of the grooves 116, 118
and the nanopores 122 are discussed in more detail hereinafter.
[0057] In more particular embodiments, the insulating member 110
includes a first layer 120a and a second layer 120b that are bonded
or otherwise secured to each other using any of a number of
techniques known to those skilled in the art appropriate for the
material being used to form the insulating member. In this
particular embodiment, the top surface grooves 116 are contained in
the first layer 120 and the bottom surface groove is contained in
the second layer.
[0058] The nanopores 122 are formed in the insulating member or
each of the first and second layers using any of a number of
techniques appropriate for the material being used and appropriate
for forming a nanopore having the desired width and length for
e.g., rapidly characterizing a biomolecule. In one particular
embodiment, the first and second layers 120a,b are bonded or
secured together and the nanopores 116 are formed at the
intersections of the top and bottom surface grooves 116, 118. In
another particular embodiment, portions of the tips of the top and
bottom surface grooves 116, 118 are formed so as to included an
opening in each groove proximal the point of intersection such that
when the first and second layers 120a,b are bonded or secured
together the sharp edge openings in the grooves are aligned so as
to form a nanopore.
[0059] In addition, in the illustrated embodiment, the grooves 116,
118 are formed in the top and bottom surfaces 112,114 so as to
provide deep V-shaped grooves. This shall not be limiting as other
shapes as are known to those skilled in the art that are otherwise
adaptable and consistent with the function of the grooves in the
present invention are contemplated for use in the present
invention.
[0060] In a particularly illustrative embodiment, the insulating
member 110 and the first and second layers are initially formed
from a silicon material, which is subsequently oxidized after
further processing of the insulating member to form SiO.sub.2, an
insulating material. Although, the starting material is silicon,
this shall not be considered limiting as other materials are
contemplated for use with the present invention that would allow
creation of the linear array of nanopores 122 as herein described
using techniques that are appropriate for the material of use.
[0061] More specifically, the silicon material is subjected to
micromachining techniques as are known to those skilled in the art
to form the grooves 116, 188 and the nanopores 122. In particular,
the grooves and the nanopores are etched in the silicon wafer using
an oxide or nitride mask, in KOH solutions with relatively high
accuracy, or by use of beam lithography and wet etching techniques.
It has been found that conventional or standard wet etching
procedures as known in the art can produce highly uniform grooves
as is illustrated in FIG. 6.
[0062] In further embodiments, the silicon wafers making up the
first and second layers 120a,b are bonded or secured together and
then material at the intersections of the top surface grooves 116
and the bottom surface groove 118 the tips of the grooves are
further etched using a similar process to that used to form the
grooves to form an opening there through. In yet another
embodiment, during the formation of the grooves 116, 118, portions
of the tips or valleys of the grooves, in particular the portions
proximal the intersections of the top and bottom surface grooves,
are further etched so as to form an opening in these portions of
the tips. The first and second layers are the bonded or secured to
each other and the sharp edges of the openings form the nanopores
at the intersections. In this way, "cutting-edges" pores are
yielded, one by bonding two silicon wafers with the sharp edges of
the grooves facing each other at 90.degree., and the other by
directly etching from one or both sides of the silicon wafers with
grooves at 90.degree. relative to each other. As is known to those
skilled in the art, the etching of the surfaces can be accurately
controlled by controlling the etching rate and etching time.
Following such etching and bonding of the silicon wafers or
material forming the first and second layers 120a,b, the assemblage
is then oxidized to form SiO2 to insulate all silicon surfaces,
thereby forming the insulating member in its operable form.
[0063] In further embodiments, and after making the insulating
member 110 so as to be in its operable form, the nanopores 122 are
further examined using for example an electron microscope to
determine if the nanopores that are formed have the desired width.
If not, and assuming that the width of a given nanopore is less
than a critical width, the nanopore is exposed to a high-energy
electron beam to modify the dimensions of the nanopore. As more
fully describe below, small holes shrink spontaneously due to
surface tension when exposed to the electron beam and when the
electron beam is switched off, the material quenches and retains
its shape.
[0064] The following further illustrates this modification process
using for example, a commercial transmission electron microscope
(TEM), operated at an accelerating voltage of 300 kV. It is well
known in electron microscopy that a high electron intensity can
damage or deform the specimen, and in general one tries to minimize
this effect. However, in the present invention this effect is used
to modify the dimensions of the silicon oxide nanopores in a
controlled fashion. It has been found that an electron intensity
around 10.sup.6 to 10.sup.7 A/m.sup.2 causes pores to shrink if the
pore has an initial diameter about 50 nm or lower.
[0065] The power of this technique lies in the possibility to
fine-tune the diameter of nanopores with unprecedented precision.
By lowering the beam intensity or blanking it, the shrinking
process can be stopped within seconds when the desired diameter has
been reached. In addition, changes in pore diameter can be
monitored in real time using the imaging mechanism of the
microscope. Rough shrinking can be done at least an order of
magnitude faster by increasing the electron intensity, and can
gradually be slowed down for ultimate control. The precision is
ultimately limited by the resolution of the microscope. In practice
the resolution is limited to about 1 nm due to the surface
roughness of the silicon oxide. The level of control offered by
this technique is at least an order of magnitude better than
conventional e-beam lithography, which has an ultimate resolution
of about 10 nm. As such, this use of this modification technique
provides a mechanism to reduce the required dimensional control in
the lithographic process for forming the grooves and nanopores,
because any pore with a diameter below 50 nm can be shrunk to a
nanometer-sized pore.
[0066] The physics attendant with the growing and shrinking that
has been observed using the above electron beam process is
determined by the surface tension of the viscous silicon oxide. In
this state, the structure will deform to find a configuration with
a lower free energy F. Simple free-energy consideration suggest
that the surface energy of pores with radius r<1/2h can be
lowered by reducing r, and that the surface energy for pores with
radius r>1/2h can be increased by increasing size. The "critical
diameter" discriminating the two cases is of order of the thickness
of the silicon, with the exact ratio depending on the geometry of
the pore. This scaling argument is valid at any scale, and explains
elegantly the observed dynamics in our pores.
[0067] The advantage of this technique of the present invention is
that nanometer-scale sample modifications are possible with
direct-visual feedback at sub-nanometer resolution. The process is
based on standard silicon processing and commercially available TEM
microscopy. A modest resolution of <50 nm is required in the
lithography defining the pore, as fine tuning in the electron beam
is done as a final step. Using the SOI based process, this
requirement is straightforward to obtain with e-beam lithography,
and should be attainable even with optical lithography alone. Other
advantages of the techniques of the present invention compared to
recently reported "ion beam sculpting" techniques are that the
technique of the present invention does not change the chemical
composition of the material surrounding the pore because the
electron beam softens the glassy silicon oxide, allowing it to
deform and be slowly driven by the surface tension. The electron
microscope also provides real time visual feedback, and when the
desired morphology has been obtained the electron beam intensity is
lowered and the SiO.sub.2 is quenched to its initial glassy
state.
[0068] Referring now to FIG. 8 there is shown a schematic view of
an electrically-addressable nanopore array 200 according to the
present invention configured with a two-dimensional array of
nanopores 116. In the illustrated embodiment shown in FIG. 6, the
two dimensional array is made up of two or more sets of linear
arrays that are formed on a single insulating member 110. In the
illustrated embodiment, the grooves 116a,b in the top surface are
formed so there is one set of grooves 116a for one of the linear
arrays and so there is another set of grooves 116b for each of the
other linear arrays. In further embodiments, the grooves 116a,b in
the top surface 112 or the first layer 120a are formed so the
grooves for each linear array are separate from or not connected to
the grooves of another linear array. For example, when the grooves
are being etched in the top surface of a silicon wafer, material is
not advantageously removed from the top surface between the ends of
the grooves.
[0069] The foregoing shall not be considered as limiting as it is
within the scope of the present invention for the grooves 116 in
the top surface of a two-dimensional array according to the present
invention to be configurable so that the grooves in the top surface
are interconnected, thereby forming a single set of top surface
grooves. For example, in cases where the ionic current is not
crucial to analysis or characterization, such as for analysis of
RNA secondary structures as described below, the two-dimensional
array can be configured so that the grooves in the top surface are
interconnected to form a single set of grooves.
[0070] Referring now to FIG. 7 there is shown a perspective view of
an electrically-addressable nanopore array device 300 according to
the present invention including an electrically-addressable
nanopore array 100 further including a linear array of nanopores
122. For clarity, only one top surface groove 116 is shown or
illustrated. However, this shall not be considered as limiting, as
the linear array 100 can include a plurality or more, for example
100 top surface grooves thereby forming 100 nanopores. Further, and
although a linear array is illustrated, it is within the scope of
the present invention for the device to include a two-dimensional
array 200 including the array shown in FIG. 8.
[0071] In the foregoing discussion, reference shall be made to the
FIGS. 6 and 8 that illustrate further details of the
electrically-addressable arrays 100, 200 contemplated for use with
an electrically-addressable nanopore array device 300 of the
present invention. Also, the electrodes and fluid ports are shown
for illustration purposes. In an actual device the electrodes and
the fluid ports are sealed.
[0072] An electrically-addressable nanopore array device 300
according to the present invention includes cover slips 302 as are
known to those skilled in the art, that seal the top and bottom
surfaces of the array. Following fabrication of the
electrically-addressable nanopore array 100, the cover slips 302
are secured or bonded to the top and bottom surfaces thereof using
any of a number of techniques known to those skilled in the art
appropriate for the materials being used. This is preferably done
after the electrically-addressable nanopore array 100, 200 is
determined to be in operable condition (e.g., nanopores of the
desired width and length appropriate for the particular analytical
technique). In addition, the electrically-addressable nanopore
array device 300 is appropriately arranged with pumping lines and
electrodes (e.g., Ag/AgCl wires) as is known to those skilled in
the art. As is known to those skilled in the art other devices,
apparatuses and systems (not shown) are interconnected to the
electrically-addressable nanopore array device 300 for purposes of
supplying samples for analysis, for collection of data and for the
analysis of the collected data.
[0073] Referring now to FIGS. 8A,B there is shown various views of
a two-dimensional nanopore array 500 according to another aspect of
the present invention. The two-dimensional array 500 according to
this aspect of the present invention, and with reference to FIG.
8A, comprises a plurality or more of single nanopore assemblies 510
of the present invention each including a nanopore 512 preferably
formed using one of the techniques described herein. There is more
particularly shown in FIG. 8A a top view, with a partial cutaway,
of a two-dimensional array 500 of this aspect of the present
invention and there is shown in FIG. 8B a side view of the array of
FIG. 8A through a single nanopore assembly. Reference shall be made
to the foregoing discussion regarding FIGS. 3-5 as to the
techniques for making a nanopore assembly 512 according to the
present invention having the desired characteristics. Reference
also should be made to the foregoing discussion for FIG. 7 as to
further details, features and characteristics of electrically
addressable nanopore arrays not described below. In addition to the
nanopore assemblies 510, the two-dimensional nanopore array 500
includes a top member 520 and a bottom member 530.
[0074] The nanopore assemblies 510 are configured and arranged in
the two-dimensional array 500 such that the first volume 514 for
each nanopore assembly is separate and electrically isolatable from
each other. The top member 520 is affixed or secured to atop
surface(s) of each of the nanopore assemblies 510 in such a fashion
so as to also maintain the first volume 514 of each nanopore
assembly separate and electrically isolatable from each other. The
top member 520 further includes a plurality or more of through
apertures 522 that are arranged in the top member so that there is
one aperture in fluid communication with the first volume 514 of
each nanopore assembly 510.
[0075] Similarly, the bottom member 530 is secured to a lower
surface(s) of each nanopore assembly 510 so a passage 516 in each
nanopore assembly and the bottom member defines one or more volumes
for receiving the material that has passed through a nanopore 512
such that the passed through material can be appropriately handled
or processed further.
[0076] The top and bottom members 520, 530 are made from any of a
number of materials known to those skilled in the art that are
appropriate for the intended use and being securable to the
surface(s) of the nanopore assemblies (e.g., such as the cover
slips 320 herein described). The through apertures 522 in the top
member 520 are formed therein using any of a number of techniques
known to those skilled in the art. The size and depth of such
through apertures 522 are such as to allow material to easily pass
there through into the first volume 514, and minimizing the
potential for damage to the material as it passes through the
through aperture. It should be recognized that the size or diameter
of the through apertures 522 need not be set so as to meet the same
desired characteristics for a nanopore 512.
[0077] Such an arrangement and configuration yields a
two-dimensional nanopore array 500 in which each nanopore 512 is
separately, electrically addressable from any other of the
nanopores making up the array. Thus, material to be analyzed,
processed and/or evaluated can be introduced into each of the
nanopores 512 so the material (e.g., DNA) passing though any one
nanopore can be uniquely or separately identified, characterized,
analyzed, processed or evaluated. Such a two-dimensional array 500
exhibits a high through put yet maintains the capability for
providing details, characteristics or features of the material
passing through each of the nanopores 512.
[0078] The nanopore assemblies 510 also are immobilized using any
of a number of techniques known to those skilled in the art,
including but not limited to mechanical and adhesive securing
techniques, so the assemblage of the nanopore assemblies in effect
forms a unitary structure. For example, the nanopore assemblies can
be secured to each other using an adhesive material. In more
particular embodiments, the top member 520 the bottom member 530
and the nanopore assemblies 510 are secured to each other so as to
form a unitary structure.
[0079] In the illustrated embodiment, the two-dimensional array 500
is made up of a 4.times.4 matrix of nanopores 512 or nanopore
assemblies 510. This shall not constitute a limitation, as it is
within the scope of the present invention for the array to be
composed of more or less nanopores or nanopore assemblies. For
example a two-dimensional array 500 according to the present
invention can be made up of 1000 or more nanopores 512 or a 1000 or
less nanopore 512 (e.g., an array comprising a matrix of
100.times.100 nanopores). Also while a square array is illustrated
(e.g., same number of nanopores 512 in x and y directions) this
shall not constitute a limitation as it is within the scope of the
present invention for the nanopore assemblies to be arranged so the
number of nanopores along one axis differs from the number of
nanopores along the other axis (e.g., 100.times.75 array of
nanopores). It also is contemplated that the nanopore assemblies
are arrangeable so the number of nanopores 512 varies along one
axis (e.g., varies between 100 and 70 nanopores) and so the number
of nanopores along the other axis generally differs from those
along the one axis (e.g., 75 nanopores).
[0080] Although the foregoing describes a two-dimensional array 500
comprising a plurality or more, more particularly a large number of
nanopore assemblies 512, it is within the scope of the present
invention using the herein described techniques for processing a
silicon wafer, to form a two-dimensional array of nanopores that
each are fluidly coupled to a first volume formed in the silicon
wafer and so that each first volume is separated from each other so
that each nanopore is separately, electrically addressable.
[0081] As indicated herein, a process for rapid DNA sequencing
using the ionic current have not been realized as yet, nor has this
process been useable to distinguish between adenine and guanine and
between cytosine and thiamine. Recent attempts using
.alpha.-hemolysin protein nanopore to discriminate the nucleotides
within the purine (adenine and guanine) and pyrimidine (cytosine,
thiamine, and uracil) groups also have not been successful.
However, the disadvantages of prior art processes are overcome by
the devices of the present invention. For example, in one
embodiment the electrically-addressable nanopore array device 300
of the present invention is further configured and arranged so as
to reduce signal noise believed to be attributable to thermal
motion of DNA as it passes through the nanopore. More particularly,
the electrically-addressable nanopore array device 300 is
configured and arranged with an objective lens 320 on either side
of the array device, so as to form two optical traps or two optical
tweezers to hold the DNA molecule stationary across a nanopore 122.
In this way, noise in the ionic current signal being caused by the
wiggling motion of the DNA molecule near and/or in the nanopore
should be greatly reduced. Also by holding the molecule stationary,
signal-averaging techniques can be used to further reduce noise in
the ionic current.
[0082] Holding the molecule stationary can be best understood from
the following illustrative example, although it well within the
skill of those in the art to apply and adapt other techniques that
are more appropriate for the items undergoing
analysis/characterization. A single-stranded DNA is attached to a
double stranded DNA and then the latter is attached to a bead 400
using a streptaviding-biotin linker, which bead is to be held by
optical tweezers. Next the single-stranded DNA is driven through
the nanopore 122 and caught from the other side using another bead
400 also with a streptaviding-biotin linker, which also is held by
optical tweezers. The holding of the molecule stationary across the
nanopore 122 using beads 400 is shown illustratively in FIG. 9. In
an illustrative embodiment, the beads 400 or microspheres are
polystyrene micropsheres or beads.
[0083] There is shown in FIG. 10 an illustrative optics and video
system 500 and the general arrangement of the components thereof
with respect to the electrically-addressable nanopore array device
300 (EANA in figure) of the present invention. Such an optical and
video system 500 is equipped with optical tweezers and a digital
video microscopy system that is based on a Zeiss Axiovert 135
inverted microscope. In further embodiments, the illustrated Argon
ion laser can be replaced by a semiconductor-based infra-red laser,
to minimize light damage to the biomolecules on the microspheres.
The system includes two objectives, and the condenser in the
standard optical microscope is replaced by a 100.times.
oil-immersion objective. Also, illumination of the sample can be
accomplished using a de-focused diode light source through the
objectives.
[0084] As is generally known to in the art, a subfield within laser
physics is optical trapping and an optical tweezer is an example of
an optical trap. A strongly focused laser beam has the ability to
catch and hold particles (e.g., particles of dielectric materials)
in a predetermined size range. This technique makes it possible to
study and manipulate particles like atoms and molecules (even
large) and small dielectric spheres. A review on uses of optical
tweezers can be found in A. Ashkin "Optical trapping and
manipulation of neutral particles using lasers", Proc. Natl. Acad.
Sci. USA, vol. 94, pp. 4853-4860, May 1997.
[0085] As is known in the art, a long (single-stranded) RNA
(ribonucleic acid) molecule can partially fold onto itself, forming
secondary structures, due to local pairing of complementary bases.
It is believed that these secondary structures of RNA are more
important for the properties of the molecules than their primary
sequences. For example, the diverse functions of RNA molecules in
cells, from messenger RNA (mRNA) that carries the genetic
information from DNA (deoxyribonucleic acid) to protein synthesis,
to transfer RNA (tRNA) that carries amino acids to ribosomes during
translation, arise largely from their secondary structures.
Developing a physical technique for fast determination of RNA
secondary structures is a major goal of the emerging field of
single molecule biophysics.
[0086] In the present invention, buffers containing RNA attached
microspheres or beads 400 are flushed through the lower surface
groove 118, while the nanopores 122 are biased by an electric
field. Occasionally an RNA molecule is pulled through a nanopore
122 by the electric field. One can identify this event by looking
for a localized microsphere or bead 400 near a pore. Then, the
electrical field is turned off to allow the RNA to form secondary
structures. Now, and also with reference to FIG. 11, the
microsphere or bead 400 is slowly pulled away using optical
tweezers and the force on the bead is measured as the RNA molecule
is pulled (upward) through the nanopore 122. The sequential
breakage of the RNA base pairs at the entrance of the nanopore 122
will appear as force fluctuations as a function of time, hence
revealing the information about the secondary structure of the test
molecule. It should be noted that this situation is quite different
from the previous RNA pulling experiments in which the RNA was
pulled from the two ends, where without the prior knowledge of the
secondary structure of an RNA, it would be difficult to assign the
features in a force vs. time trace. The present invention does not
require prior knowledge of the secondary structure of an RNA
molecule.
[0087] Referring now to FIGS. 12A,B there is shown schematically
another technique for characterizing (e.g., sequencing) DNA or RNA
using nanopores or nanopore arrays of the present invention. More
particularly these schematic views illustrate a sequencing
technique using PNA functionalized nanopores 600 according to the
present invention. Reference shall be made to the foregoing
discussion for the various techniques and arrangements for
providing a nanopore device, apparatus or assemblage according to
the present invention without PNA. For clarity, the following
discussion and figures illustrate the technique and device of the
present invention by referring to a single nanopore. It is within
the scope of the present invention, however, for the below
described technique and device to be adapted for use with a
plurality or more of nanopores that are arranged so as to form a
linear array or a two-dimensional array.
[0088] After forming a nanopore 602, peptide nucleic acid (PNA) is
applied so as to coat at least a portion of the nanopore, for
example at least the interior surface of the nanopore 602 so as to
form a PNA coating 604. The PNA coating 604 and the nanopore 602 so
coated is herein referred to as PNA functionalized nanopore 600.
The PNA coating 604 on the nanopore is exposed to any DNA/ RNA
passing through the opening 606 in the PNA functionalized nanopore
600. In particular embodiments, the PNA is synthesized so that it
is essentially constituted by one of the bases of DNA or RNA (e.g.,
adenosine, thymidine, guanine, cytosine, uracil). More specifically
the PNA is synthesized so that the one basis characterizing or
constituting the PNA is set so as to have a noticeable effect on
the passage of the DNA/RNA through the PNA functionalized nanopore
600, and more particularly different effects depending upon the
different bases that can make up the DNA/RNA.
[0089] As illustration and with particular reference to FIG. 12B,
the PNA coating 604 is such as to be constituted or characterized
as having essentially only adenosine bases (hereinafter referred to
as A-PNA). Consequently, the A-PNA will have differing effects
(e.g., hydrogen bonding) on the passage of a strand of DNA/RNA
through the functionalized nanopore 600 depending upon the
particular bases constituting the DNA and the sequence of the bases
in the DNA/RNA as well as the distance (e.g., bonding distance)
between the PNA coating 604 and the DNA/RNA strand. For example,
there is a strong bonding effect between A and T bases,
consequently a comparatively large force is applied upon the
DNA/RNA strand thereby retarding movement of the strand within the
functionalized nanopore 600.
[0090] Using techniques known to those skilled in the art such as
those described herein, the force retarding movement or other
related parameter (e.g., time to move a given distance) is
determined or detected and the detected/determined parameter is
evaluated to determine if the parameter corresponds to that which
would be exhibited if the A-PNA is bonding to a T, A, G or C bases
of the DNA. In one illustrative technique, an end of the DNA/RNA
strand is driven through the PNA functionalized nanopore 600 and
caught from the other side using a bead 400 with a
streptaviding-biotin linker. Optical tweezers as is known in the
art are used to hold the bead 400. In a further illustrative
embodiment, the beads 400 or microspheres are polystyrene
micropsheres or beads.
[0091] The microsphere or bead 400 is slowly pulled away from the
PNA functionalized nanopore 600 using optical tweezers and the
force on the bead is measured as the DNA/RNA molecule is pulled
through the functionalized nanopore. As indicated herein, the bases
making up the DNA/RNA molecule will cause differing forces to be
applied to the molecule affecting the movement of the molecule
through the fictionalized nanopore 600. These different effects
will appear as force fluctuations as a function of time, hence
revealing the information at least about the sequence of the A, T,
G, C bases making up the test molecule.
[0092] Although a preferred embodiment of the invention has been
described using specific terms, such description is for
illustrative purposes only, and it is to be understood that changes
and variations may be made without departing from the spirit or
scope of the following claims.
Incorporation by Reference
[0093] All patents, published patent applications and other
references disclosed herein are hereby expressly incorporated by
reference in their entireties by reference.
Equivalents
[0094] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *
References