U.S. patent application number 16/389859 was filed with the patent office on 2019-10-17 for devices and methods for target molecule characterization.
This patent application is currently assigned to Arizona Board of Regents on behalf of Arizona State University. The applicant listed for this patent is Arizona Board of Regents on behalf of Arizona State University. Invention is credited to Stuart LINDSAY, Peiming ZHANG.
Application Number | 20190317040 16/389859 |
Document ID | / |
Family ID | 39831560 |
Filed Date | 2019-10-17 |
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United States Patent
Application |
20190317040 |
Kind Code |
A1 |
LINDSAY; Stuart ; et
al. |
October 17, 2019 |
DEVICES AND METHODS FOR TARGET MOLECULE CHARACTERIZATION
Abstract
An system for recognition of a translocating polymeric target
molecule includes a device having at least one constriction that is
sized to permit translocation of only a single copy of the
molecule. A pair of spaced apart sensing electrodes border the
constriction, which may be a nanopore. The first electrode is
connected to a first affinity element and the second electrode is
connected to a second affinity element. Each affinity element may
be connected to its corresponding electrode via one or more
intermediary compounds, such as a linker molecule and/or an
electrode attachment molecule. The first and second affinity
elements are configured to temporarily form hydrogen bonds with
first and second portions of the target molecule as the latter
passes through the constriction. During translocation, the
electrodes, affinity elements and first and second portions of the
target molecule complete an electrical circuit and allow a
measurable electrical current to pass between the first and second
electrodes. The time-varying nature of this electrical current, and
the specific affinity elements employed, allow one to characterize
the target molecule.
Inventors: |
LINDSAY; Stuart; (Phoenix,
AZ) ; ZHANG; Peiming; (Gilbert, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arizona Board of Regents on behalf of Arizona State
University |
Scottsdale |
AZ |
US |
|
|
Assignee: |
Arizona Board of Regents on behalf
of Arizona State University
Scottsdale
AZ
|
Family ID: |
39831560 |
Appl. No.: |
16/389859 |
Filed: |
April 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15203383 |
Jul 6, 2016 |
10330632 |
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16389859 |
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12594366 |
Oct 2, 2009 |
9395352 |
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PCT/US2008/059602 |
Apr 7, 2008 |
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15203383 |
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61022155 |
Jan 18, 2008 |
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60989089 |
Nov 19, 2007 |
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60922288 |
Apr 6, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
G01N 33/48721 20130101; G01N 27/3278 20130101; G01N 27/3276
20130101; C12Q 1/6869 20130101; C12Q 2565/631 20130101; G01N 27/028
20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327; C12Q 1/6869 20060101 C12Q001/6869; G01N 27/02 20060101
G01N027/02; G01N 33/487 20060101 G01N033/487 |
Goverment Interests
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Nos. IR21 HG3061, IR21 HG004378-01 and GM 21966, awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
1-63. (canceled)
64. A method of identifying a particular portion of a target
molecule as the target molecule translocates through a
constriction, the method comprising: providing an apparatus
comprising a molecular recognition device, the device comprising a
partition having a first side and a second side, and at least one
constriction passing between the first and second sides, the
constriction being shaped and sized to permit translocation of only
a single copy of a target molecule therethough; first and second
sensing electrodes associated with the first side of the partition
and being separated from one another by a first gap; a first
affinity element connected to the first electrode; a second
affinity element connected to the second electrode and the device
being located in the apparatus such that a first chamber is located
on the first side of the device and a second chamber is located on
the second side of the device; introducing a target molecule into
the second chamber; translocating by electrophoresis the target
molecule so that it moves through the constriction; and detecting
an electrical current passing through the first electrode, the
first affinity element, a first site on a portion of the target, a
second site on a portion of the target molecule, the second
affinity element, and the second electrode.
65. The method according to claim 64, wherein the target molecule
has a leading end and a trailing end.
66. The method of claim 65, further comprising threading the
leading end of the target molecule through the constriction and
into the first chamber prior to detecting the electrical
current.
67. The method according to claim 65, further comprising affixing a
first magnetic bead proximate the leading end of the target
molecule to control translocation through the constriction.
68. The method according to claim 67, further comprising affixing a
second magnetic bead proximate the trailing end of the target
molecule.
69. The method according to claim 64, wherein the target molecule
is DNA and the first affinity element is a guanidinium moiety.
70. The method according to claim 69, wherein, the second affinity
element comprises one of the bases A, T, C or G, or a modified
version thereof, or a nucleoside analog thereof.
71. The method according to claim 64, further comprising recording
information reflective of the electrical current as a function of
time; and obtaining at least one parameter reflective of an
identification of the particular portion, from said recorded
information.
72. The method according to claim 71, wherein said at least one
parameter comprises one from the group consisting of charge,
duration of current signal, shape of current signal and decay of
current.
73. The method according to claim 72, further comprising comparing
said at least one parameter with a predetermined threshold to
determine whether said particular portion has been recognized.
74. The method according to claim 73, further comprising detecting
electrical current from a same portion of a predetermined number of
copies of said target molecule to thereby improve recognition
accuracy of said portion.
75. A device for the detection of a target molecule comprising; a
partition comprising one or more constrictions sized to permit
translocation of a target molecule through the one or more
constrictions; an electrode pair on the partition in communication
with the one or more constrictions; a first affinity element and a
second affinity element bound to one or both members of the
electrode pair, wherein the first affinity element is capable of
binding to a first site on the target molecule as it enters the one
or more constrictions; and wherein the second affinity element is
capable of binding to a second site on the target molecule as it
enters the one or more constrictions in the presence of the first
affinity element bound to the first site on the target molecule;
wherein the electrode pair is arranged so that binding of the
target molecule by the first affinity element bound to one member
of the electrode pair coupled with binding of the target molecule
by the second affinity element to the other member of the electrode
pair generates an electrical signal.
76. The device of claim 75, wherein the first and second affinity
elements are bound to one or both members of the electrode pair via
a linker.
77. The device of claim 76, wherein the electrodes in the electrode
pair are spaced from 0.5 to 10 nm apart.
78. The device of claim 76, wherein the target molecule is a
nucleic acid.
79. The device of claim 78, wherein the first affinity element is
capable of binding to phosphate groups in the nucleic acid backbone
and/or the second affinity element is capable of binding to
specific bases in the nucleic acid.
80. The device of claim 79, wherein the first affinity element
comprises a guanidinium moiety, such as
guanidinoethyldisulfide.
81. A method for detecting a target molecule comprising; applying a
force to translocate a target molecule though a constriction on a
partition, wherein the constriction is sized to permit passage of a
target molecule, wherein an electrode pair is in communication with
the constriction; wherein a first affinity element and a second
affinity element are bound to one or both members of the electrode
pair; wherein the first affinity element is capable of binding to a
first site on the target molecule as it enters the constriction;
wherein the second affinity element is capable of binding to a
second site on the target molecule as it enters the constriction in
the presence of the first affinity element bound to the target
molecule, and wherein the translocation is conducted under
conditions suitable to promote binding of target molecule to the
first affinity element and the second affinity element; measuring
an electrical signal generated by completion of an electrical
circuit formed by binding of the target molecule to the first
affinity element bound to one member of the electrode pair coupled
with binding of the target molecule to the second affinity element
to the other member of the electrode pair; and characterizing the
target molecule based on the measured electrical signal.
82. The method of claim 81, wherein applying a force comprises
applying a magnetic field or an electric field and/or an initial
electrophoretic threading of the target molecule into the
constriction.
83. The method of claim 81, wherein the electrodes in the electrode
pair are spaced from 0.5 to 10 nm apart.
84. The method of claim 81, wherein the target molecule is a
nucleic acid.
85. The method of claim 81, wherein the first affinity element is
capable of binding to phosphate groups in the nucleic acid backbone
of the nucleic acid and/or the second affinity element is capable
of binding to specific bases in the nucleic acid and/or the first
and second affinity elements are bound to one or both members of
the electrode pair via a linker, such as a flexible linker.
86. The method of claim 85, wherein the first affinity element
comprises a guanidinium moiety, such as
guanidinoethyldisulfide.
87. The method of claim 86, wherein the guanidinium moiety is
coupled via an amide linkage.
88. The method of claim 85, wherein the flexible linker comprises
an alkane chain of two or more methylene groups.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 15/203,383, filed on Jul. 6, 2016 and now
issued U.S. Pat. No. 10,330,632 which is a continuation of U.S.
application Ser. No. 12/594,366 filed Oct. 2, 2009 and now issued
as U.S. Pat. No. 9,395,352, which is a national stage filing of PCT
Application No. PCT/US2008/059602 filed Apr. 7, 2008 and claims
priority to U.S. Provisional Application Nos. 60/922,288 filed Apr.
6, 2007, 60/989,089 filed Nov. 19, 2007 and 61/022,155 filed Jan.
18, 2008. The contents of each of these applications are
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention is directed to systems, devices and
methods for identifying biopolymers, such as strands of DNA, as
they pass through a constriction, such as a nanopore. More
particularly, the invention is directed to such systems, devices
and methods in which the constriction is provided with a
functionalized unit which, together with a newly translocated
portion of the biopolymer, forms a temporary electrical circuit
that can be used to characterize that portion of the
biopolymer.
BACKGROUND OF THE INVENTION
[0004] One goal in the DNA sequencing industry is to reduce the
cost and increase the speed for sequencing an individual's genome.
A target accuracy of 99.99%.sup.1 is also important. Another vital
goal is long contiguous reads. Array technology is revealing a
remarkable long range complexity in the genome, some of which is
summarized in Table 1 below, taken from the review by Sharp et
al..sup.2. This complexity is difficult to retrieve through the
assembly of contigs much shorter than the size ranges referred to
in the table. Copy number variations are particularly vexing, yet
they have important phenotypes owing to gene-dose variations. These
clinically-important copy number variations are almost completely
uncorrelated with the SNPs extracted by conventional
re-sequencing..sup.3
TABLE-US-00001 TABLE 1 Long-range structures in the genome (from
Sharpe et al. Ann. Rev. Hum. Gen. 17 407 2006) Variation
Rearrangement type Size range.sup.a Single base-pair Single
nucleotide 1 bp changes polymorphisms, point mutations Small
insertions/ Binary insertion/deletion 1-50 bp deletions events of
short sequences (majority <10 bp in size) Short tandem repeats
Microsatellites and 1-500 bp other simple repeats Fine-scale
structural Deletions, duplications, 50 bp to 5 kb variation tandem
repeats, inversions Retroelement insertions SINEs, LINEs, LTRs, 300
bp to 10 kb ERVs.sup.b Intermediate-scale Deletions, duplications,
5 kb to 50 kb structural variation tandem repeats, inversions
Large-scale structural Deletions, duplications, 50 kb to 5 Mb.sup.
variation large tandem repeats, inversions Chromosomal variation
Euchromatic variants, large ~5 Mb to entire cytogenetically visible
chromosomes deletions, duplications, translocations, inversions,
and aneuploidy
[0005] Sequencing by extension relies on detection of a signal
owing to nucleotide addition by a polymerase and instruments using
many molecules in picoliter wells are already in commercial
use..sup.4 For Example, the 454 Life Sciences instrument exploits
energetic pyrophosphates to give label-free optical signals, but
the length of the read is limited by the dephasing problem as
individual clones in a well fall behind owing to errors in
nucleotide addition. Single molecule measurements present a way
around this problem.sup.5, 6 and this is the basis of the Helicos
system. However, the chemistry required to do this is far from
trivial and the use of non-natural nucleotides limits the length of
the extensions. An interesting variant is the proposal to sequence
by litigation (Xiaohua lab, UCSD.sup.7).
[0006] The use of the scanning tunneling microscope (STM) for
sequencing DNA (AFM and STM in novel approaches to sequencing) has
been studied in the past. DNA has been imaged at high resolution in
water.sup.8-10 but sequencing was not possible. In part, this is
because the magnitude of the tunneling current through DNA in terms
of base composition has not been easy to interpret. In addition,
the STM images themselves are not amenable to ready interpretation.
These images often reflect where the structure makes contact with
the underlying metal and not the real high points of the
molecule..sup.11 This complication may also affect the recent STM
imaging work of Oshiro and Umezawa..sup.12 This difficulty rules
out sequencing by imaging.
[0007] The nanopore approach.sup.13-25 has the great advantage of
only allowing one base to pass a particular point at a time (if the
orifice is small enough). It can also be highly precessive (moving
from one base to the next without "stuttering") if the driving
force is high enough. For instance, if one were to assume that an
allowable limit on stuttering misreads of 1 part in 10.sup.4
(99.99% accuracy), this would require a driving free-energy of 9.3
k.sub.BT. To achieve this requires either a voltage drop across a
base of 0.23V (a field of 4.times.10.sup.8 V/m) or a force of 55 pN
(where a base-to-base separation of 0.6 nm for stretched DNA was
used.sup.26). These conditions are readily achieved and
demonstrable.
[0008] Since Kasianowicz.sup.13 employed a biological nanopore, the
alpha-hemolysin ion channel, as a sensor to characterize DNA,
nanopore based biosensors have attracted much attention..sup.21,
27, 28 However, biological nanopores have several shortcomings as
they are unstable, fragile and fixed in size. Synthetic single
nanopores.sup.29-33 were developed as alternatives to biological
nanopores. These artificial single nanopores have been successfully
used to characterize DNA translocation, folding, and conformational
changes,.sup.34-37 the effects of high pH,.sup.38 low
temperatures,.sup.39 multiple DNA lengths,.sup.40 the effects of
electric field strength,.sup.41 the effects of surface modification
by atomic layer deposition (ALD).sup.42 and also the mimicking ion
channel activity..sup.43 Solid-state single nanopores now have a
real track record. They have the following advantages: (1) the pore
size is comparable to that of a single molecule; (2) the pore size
is tunable to fit a wide range of molecules; and (3) robust single
channels between cis and trans reservoirs are readily achieved.
They also have the following strengths as a manufacturable device:
(1) they are chemically and thermally stable; (2) their surfaces
are readily modified; (3) they are mechanically robust; and (4)
they are readily incorporated into integrated circuit (IC)
technology.
[0009] It was hoped originally that the DNA bases could be
identified directly via distinctive variations in the ionic current
through the nanopore as each base occluded the pore on transit of
the smallest part of the pore. However, the ionic current signal
has proved difficult to interpret. As a result, several new schemes
have been proposed. One method relies on electronic measurement of
tunnel current as the DNA bases pass through a tiny gap between a
pair of electrodes.sup.44-46 though there is some debate about the
feasibility of this approach..sup.47 Another proposes to exploit
the distinctive dipoles of the bases with measurement of dielectric
response on the molecular scale..sup.48 Yet another proposes to
measure the optical response as dye-labeled complimentary strands
are "peeled off" the main template strand by passage through the
nanopore..sup.49 A group associated with the present inventors has
focused on measuring the force associated with
translocation..sup.50 At the time of writing, there are no
published reports of a signal with single-base
resolution..sup.49
[0010] FIG. 2 shows the layout of a proposed sequence reader that
relies on differences in the tunnel-transport of electrons through
bases (taken from a recent review by Zwolak and Di Ventra.sup.49).
To understand the obstacles laying in the way of operating a reader
such as this one, it is useful to take a look at the history of
single molecule electronic measurements. When it comes to the
conductance of a molecule placed between metal electrodes,
published experimental data are all over the map.sup.51 and DNA is
a wonderful illustration of the problem. DNA has been reported to
be an insulator,.sup.52 semiconductor,.sup.53 conductor.sup.54 and
even superconductor.sup.55 (though the issue is now probably
resolved.sup.56). With the exception of one datum.sup.52 these
results are for conduction along the strand, but the problems
inherent in making these measurements are the same no matter what
the geometry is. Some of these problems include:
[0011] (1) Tunnel transport is exponentially sensitive to the
atomic arrangement of atoms in the tunneling path and even a bond
rotation can change transport by a significant amount..sup.57,
58
[0012] (2) Outside of ultrahigh vacuum, metal surfaces are covered
with (unknown amounts of) adventitious contamination leading to
dramatic variations in contact..sup.59
[0013] (3) Base sequence, fluctuations of structure, and very
importantly, counter ions, can dominate the electronic properties
of a polyelectrolyte like DNA..sup.60
[0014] FIG. 3 shows an experimental arrangement that captures some
of elements that would be required for any electronic sequencing of
DNA. The experimental arrangement permits reproducible
determination of single molecule conductance, albeit practicing the
art on a rather simple octane-dithiol molecule..sup.61 The target
molecule was chemically-contacted (to a planar bottom electrode and
a nanoparticle top electrode) using gold-thiol chemistry to form a
metal-chemical bond-molecule-chemical bond-metal sandwich. The
resulting reproducible data showed, very clearly, the effects of
having different numbers of molecules in the gap. The experiment
works because the tip is not well-connected to the surrounding
monolayer of octane monothiols, at best touching the terminal
methyl groups, but most likely interacting via a layer of
contaminant molecules. But the contamination is displaced by the
chemical bonding in the case of the desired thiolated top-contact.
FIG. 3A shows a single dithiolated octane molecule 31 inserted into
a defect in a monolayer 32 of octane-monothiol molecules on a gold
electrode 33 and a gold nanoparticle 34 is chemically attached as a
top electrode. A metal-coated AFM probe 35 makes contact with the
nanoparticle 34 to complete the circuit. As seen in FIG. 3B,
different contacts produce different current-voltage curves 36a-e,
but these are all integral multiples of each other, interpreted as
integer numbers of molecules (1, 2, 3 . . . ) in the gap. FIG. 3C
shows the superposition 37 that occurs when each curve is divided
at all points by the appropriate integer. FIG. 3D shows the
effective multiplier for thousand of curves--over 1000 contacts
fell in the `single molecule` bin.
[0015] FIG. 4 shows that the conductance through the desired path
44 is over a thousand times higher than the conductance through the
non-bonded path 46. The conductance measured for a single molecule
was quite close to what was predicted by a first principles theory
42 with no adjustable parameters.sup.61 (and the remaining small
discrepancy is now explained.sup.62). This is to be contrasted with
a "best case" agreement of a factor of 500 achieved in previous
reports of single molecule electronic properties..sup.51 Chemical
bonds, in and of themselves, only enhance tunnel current by a few
times..sup.63 The most important factor is probably the role that
bonding plays in displacing contamination from the tunnel junction,
as metal surfaces are invariably coated with hydrocarbons outside
of an ultra-clean, ultrahigh vacuum environment..sup.59 Subsequent
to the above-described experimental arrangements for measurement of
single molecule conductance, a variety of measurements and
techniques have evolved..sup.34, 51, 57, 58, 61, 64-82 83-86 In
addition, there have been very significant contributions to the
methods from the Tao group.sup.77 and the Columbia group..sup.87,
88 Many issues remain to be resolved, and it is important to point
out that, even with the best current methods, different atomic
arrangements of the contact at the electrode can lead to
differences in the measured conductance..sup.85, 86, 89
[0016] Hydrogen-bond mediated STM image-enhancement has been
reported..sup.12 In addition, it is now known that one may directly
measure hydrogen bond enhanced tunneling. Therefore, one area of
study is to build an electrical readout system that incorporates a
pair of electrodes in a nanopore. Nanopores with electrical
contacts are being constructed by several groups pursuing
sequencing by tunneling.sup.44-46 or capacitance
measurement..sup.48 An electrical readout would be difficult with
biomolecular nanopores.sup.13 and almost certainly requires the use
of a solid state nanopore. Registration of a pair of electrodes
with a small (about 2-3 nm diameter) nanopore is not an easy task.
Little material exists in the literature, but the two leading
groups working in this area are the Harvard Nanopore Sequencing
Group, where Golovchenko leads an effort towards solid state
nanopore sequencing.sup.29 and Timp's group at UIUC, which is
pursuing dielectric nanopore sequencing..sup.48 The Harvard group
is using a carbon nanotube placed across the nanopore that is
judiciously cut so that a nm-scale gap in the electrodes lies just
above the pore. The Timp group is working to build a layered
semiconductor capacitor, with conductive elements separated by a
sub-nm insulating spacer..sup.48, 49 The DNA would pass through a
nanopore drilled perpendicular to these layers. However, sequencing
of translocating DNA through such pores has proven to be
elusive.
SUMMARY
[0017] In one aspect, the present invention is directed to a
readout device and scheme for DNA sequencing through a
constriction, such as a nanopore. The scheme utilizes the electron
tunneling current mediated by specific hydrogen-bonding molecular
recognition events.
[0018] In another aspect, the present invention is directed to the
design and construction of a manufacturable instrument, constructed
so as to allow for parallel operation of many constrictions for
performing sequencing, such as of ssDNA or dsDNA.
[0019] The system employs at least one device having at least two
sensing electrodes spaced apart by a gap and positions on either
side of a constriction, such as a nanopore. The nanopore electrode
gap construction may be achieved by electrochemical assembly to
produce gaps that are reformable in-situ. Alignment of a nanogap
sensing electrode pair with a constriction is achieved by means of
novel `though-pore` plating process. Thereafter, active gap control
may be used to dynamically-control the gap. Since the natural DNA
bases frequently form mismatched basepairs, custom recognition
elements (referred to herein as "affinity elements") are used for
molecular recognition. Each constriction is functionalized with at
least one such custom affinity element. Electrophoresis, magnetic
bead technology and the signal from the pore itself can be used to
effect translocation through the constriction and characterization
of the molecule. The system is thus configured to acquire data
related to the locations of specific bases in a single strand of
DNA.
[0020] In the device, a pair of spaced apart sensing electrodes
border the constriction. The first sensing electrode is connected
to a first affinity element (e.g., a phosphate grabber when the
target molecule is ssDNA) while the second sensing electrode is
connected to a second affinity element. Each affinity element may
be connected to its corresponding electrode via one or more
intermediary compounds, such as a linker molecule, which itself
typically is connected to the electrode via an electrode attachment
molecule, such as a thiol. The first and second affinity elements
are configured to temporarily form hydrogen bonds with first and
second portions of the molecule as the latter passes through the
constriction. During translocation, the electrodes, affinity
elements and first and second portions of the target molecule
complete an electrical circuit and allow a measurable electrical
current to pass between the first and second electrodes. The
time-varying nature of this electrical current, and the specific
affinity elements employed, allow one to characterize the first and
second portions of the target molecule.
[0021] The present invention's approach to nanopore electrode
construction is directed to mimicking the scanning tunneling
microscopy that has proved effective and successful in experiments
with hydrogen-bond-based electronic recognition. Three elements of
this are: 1) self-aligned metal-gap-metal junctions capable of
being reformed in-situ; 2) active control of the tunnel gap; and 3)
manufacturability. The metal used in these junctions can be gold.
Trials with gold electrodes have indicated that the "blinking" of
contacts made to soft metals is not a significant
problem..sup.71
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] For a better understanding of the present invention and to
show how the same may be carried out in practice, reference will
now be made to the accompanying drawings, in which:
[0023] FIGS. 1 and 1A: Sequencing by recognition through affinity
elements, showing one of four types of reader (this one is for C),
A guanidinium ion tethered to one electrode via a flexible linker,
hydrogen bonds (yellow H-bonds) onto the nearest passing phosphate
on an ssDNA translocating a nanopore. If a flexibly tethered base
on a second electrode finds it's Watson-Crick complement on the
other side of the DNA (red H-bonds) a large current passes between
the two electrodes, signaling a base recognition event. The
components require an electrode gap of about 3 nm and an electrode
height of no more than 0.6 nm or 0.7 nm. The H-bonding also serves
to align the DNA in the device, while the flexible linkers provide
alignment tolerance. Translocation is controlled via
electrophoresis and magnetic beads (with net force F) an
arrangement compatible with a parallel assembly of many reading
heads.
[0024] FIG. 2: Proposed tunnel current readout system (From the
review by Zwolak and Di Ventra, 2007).
[0025] FIGS. 3A-3D: (FIG. 3A) Arrangement for contacting single
molecules. A single dithiolated octane molecule is (spontaneously)
inserted into a defect in a monolayer of octane-monothiol molecules
on a gold electrode and a gold nanoparticle is chemically attached
as a top electrode. A metal-coated AFM probe makes contact with the
nanoparticle to complete the circuit. (FIG. 3B) Different contacts
produce different current-voltage curves, but these are all
integral multiples of each other, interpreted as integer numbers of
molecules (1, 2, 3 . . . ) in the gap. (FIG. 3C) shows the
superposition that occurs when each curve is divided at all points
by the appropriate integer. (FIG. 3D) shows the effective
multiplier for thousand of curves--over 1000 contacts fell in the
`single molecule` bin.
[0026] FIG. 4: Current (log scale) vs. bias for the probe pushed
against an alkane monothiol monolayer ("Non-bonded"), bonded to a
gold nanoparticle ("Bonded") and, as calculated from
first-principles ("Theory").
[0027] FIG. 5: Shows how half of the nanopore measurement is
modeled in an STM measurement (a moving, functionalized, electrode
moves away from a fixed DNA base), whereas in FIG. 1 the DNA base
moves away from a fixed, functionalized, electrode.
[0028] FIGS. 6A-6D. FIG. 6A shows raw current vs. distance data for
a bare Au tip (green), thiophenol (Tp)-modified tip (orange) and
G-modified tip (black) on bare gold. FIG. 6C shows this on a
thymidine SAM and FIG. 6D shows this on a deoxycytidine SAM. FIG.
6B shows data taken with a C-functionalized probe on a thymidine
SAM (blue lines, green lines are data for the bare probe). Initial
conditions are i=3 nA, V=0.5V, retraction speed 133 nm/s. Data were
taken in trichlorobenzene.
[0029] FIGS. 7A-7B. Some typical curves for a G probe and a
deoxycytidine surface (a) or a thymidine surface (b) showing fits
to equations 1a and 1b. Arrows point to the transition between the
slow decay (.beta..sub.1) and the rapid decay (.beta..sub.1)
regions at a distance z.sub.c.
[0030] FIGS. 8A-8G. 8A-8C: Histograms (black bars) of the values of
the breakpoint, z.sub.c, for bare Au (a), a deoxycytidine SAM (b)
and a thymidine SAM (c) for the i-z curves shown in FIG. 1 (80% of
data are fitted). FIGS. 8D-8G show histograms of the charge
transferred in each retraction (all data are used here) for (8D)
bare gold, (8E) a deoxycytidine SAM, (8F) a thymidine SAM and (8G)
a SAM made with an equimolar mix of C and T. Black bars are for an
8-mercaptoguanine functionalized tip, orange bars are for a
thiophenol functionalized tip and green bars are for a bare tip.
The green shaded block indicates the region of unambiguous signals
for G-C basepairs.
[0031] FIGS. 9A-9C. Raw current vs. distance data taken in water.
Color coding for the curves and experimental conditions are
otherwise as in FIG. 6.
[0032] FIGS. 10A-10G. Analysis of decay curves taken in water.
Histograms of the charge transferred in each retraction are shown
in FIG. 10A (bare Au), FIG. 10B (deoxycytidine SAM) and FIG. 10C
(thymidine SAM). Black bars 102 are for an 8-mercaptoguanine
functionalized tip and green bars 101 are for a bare Au tip.
Typical i-z curves in water (FIGS. 10E & 10G) are compared with
their counterparts in trichlorobenzene (FIGS. 10D & 10F)
showing the complex structure of the data obtained in water (green
curves are control data obtained with bare tips).
[0033] FIGS. 11A-11D: Guanidinium monolayer on Au(111). FIG. 11A
shows monolayer-covered gold single-atom steps. FIG. 11B shows a
zoom-in showing the pits (some marked by arrows) that form owing to
the thiol-driven reconstruction of the Au surface. FIG. 11C shows
molecular resolution (some pits marked by arrows). FIGS. 11A-11C
were taken in tris-HCl. As seen in FIG. 11D, addition of phosphate
causes the surface to reconstruct.
[0034] FIGS. 12A-12B: FIG. 12A shows SPR data for adsorption of
dsDNA onto the guanidinium functionalized surface. The trace in
FIG. 12B shows that even acid treatments do not remove the DNA.
[0035] FIGS. 13A-13B: FIG. 13A shows 300 superimposed force curves
for a PEG-tethered 15 base DNA oligomer interacting with a
guanidinium functionalized surface. Most of the pulls indicate
adhesion at one point (sharp peaks) but an example of an "adhesion
plateau" is indicted. FIG. 13B is a scatter plot of peak force vs.
pulling distance. Note that most of the larger forces lie in the
PEG region and not the DNA region.
[0036] FIGS. 14A-14D: DNA adsorption onto the guanidinium
monolayer. FIGS. 14A and 14B show the same region of the substrate
before (FIG. 14A) and after (FIG. 14B) injection of DNA into the
STM imaging cell (data taken in tris-HCl). The pits (arrows, FIG.
14A) disappear after DNA adsorption and some reconstruction of the
underlying gold is evident in the shape changes of the small
islands. Close inspection reveals a highly ordered monolayer of
DNA. This is more obvious in the high contrast images shown in FIG.
14C of dsDNA and in FIG. 14D of ssDNA (Fourier transforms inset
upper right).
[0037] FIGS. 15A-15D show current-decay curves obtained with a bare
(green trace 151) or functionalized tip over various guanidinium
surfaces. FIG. 15A: a guanidinium-functionalized surface (G-tip,
pink traces 152); FIG. 15B: an oligo-T (T.sub.45) adsorbed onto the
guanidinium surface (C-tip, pink traces 153); FIG. 15C: an oligo-T
(T.sub.45) adsorbed onto the guanidinium surface (G-tip, orange
traces 154); FIG. 15D: an oligo-C (C.sub.45) adsorbed onto the
guanidinium surface (G-lip, black traces 155). Data were obtained
in Tris-HCl with a pH of 6.8.
[0038] FIGS. 16A-16C: Complex bandstructure estimate of the
electronic decay length, .beta., for G-C basepairs. FIG. 16A shows
part of the infinite "chain;" FIG. 16B shows the energy levels; and
FIG. 16C shows .beta. as a function of energy.
[0039] FIGS. 17A-17B; FIG. 17A shows a high-resolution imaging of a
prior art nanogap. FIG. 17B shows a prior art nanogap sculpted by
e-beam ablation (from Fischbein and Drndi , 2006 and 2007).
[0040] FIGS. 18A-18D: Testbed nanogap made by lithography and FIB.
FIG. 18A shows a schematic layout, including a covering layer of
SiO.sub.2. FIG. 18C shows a cross section of gap. FIG. 18B shows a
SEM image of a real device with another view into the nanogap shown
in FIG. 18D.
[0041] FIGS. 19A-19B: (19A) i-v plots for tunnel devices (as-made)
similar to that shown in FIG. 18. (19B) Current vs. time after
closing the gaps electrochemically and then stripping them open.
Quantum-conductance steps (indicated by arrows) are clearly
observed as Au is removed.
[0042] FIG. 20A-B: (20A) Electrodes on Si.sub.3N.sub.4 membrane
incorporated into a break junction apparatus (not to scale with
some dimensions exaggerated). This version is for trial gaps
without a nanopore. (20B) A buckling geometry that might prove
suitable for use with a nanopore (buckling exaggerated).
[0043] FIG. 21: Scheme for through-pore plating (showing a nanopore
made by TEM shrinkage as an inset, lower right). The key feature is
through-pore transport of Au.sup.+ ions, localizing deposition to
parts of the sensing electrodes (SE1, SE2) in close proximity to
the pore. Metal deposition and stripping is controlled by the built
in counter electrode (CE) using the built-in reference (RE) with
the sensing electrodes serving as working electrodes (operated at a
small potential difference, V.sub.t). V.sub.EC sets the potential
of the working electrodes. Measurements of pore current (I.sub.P)
and tunnel-current between the two working electrodes (I.sub.t) is
used as control parameters for final pore size and tunnel-gap size.
The two data sets together can be used to center the electrodes in
the pore.
[0044] FIG. 22A-C: Models for finite element analysis. 22A--2D
model of the electrodeposition setup. 22B--A close-up including the
double layer (EDL). 22C--Full 3D model of the electrodeposition
setup including EDL structure.
[0045] FIG. 23: Hydrogen bonding edges of DNA bases.
[0046] FIG. 24. Base pairing of the adenine reader (R.sub.A) with
natural DNA bases.
[0047] FIG. 25. Structure of a PNA trimer composed of modified
uracil and universal bases.
[0048] FIG. 26. Base pairing of the cytosine reader (R.sub.C) with
natural DNA bases.
[0049] FIG. 27. Proposed structures of modified guanines for
improving specificity of the C reader.
[0050] FIG. 28. Base pairing of the guanine reader (R.sub.G) with
natural DNA bases.
[0051] FIG. 29. Basepairing of the G-clamp with guanine.
[0052] FIG. 30. Base pairing of DAP with DNA bases and proposed
analogues of DAP as candidates for the T reader.
[0053] FIG. 31. A universal DNA base reader (R.sub.U): hydrogen
bonding schematic for
4-(mercaptomethyl)-1H-imidazole-2-carboxamide.
[0054] FIG. 32: Magnetic bead apparatus. The CCD can track a bead
being pulled into the nanopore to within 10 nm. Inset (upper right)
is the prototype laboratory apparatus.
[0055] FIG. 33A-C: (a) Forces on a molecule with bead stretching
and electrophoretic translocation. (b) Bead arrangement for
`flossing` experiment. (c) Magnetic force added to electrophoretic
force.
[0056] FIG. 34: I-Z curves for a guanine functionalized probe (A,
C, B) and a 2-aminoadenine functionalized probe (B, D, F) over a
bare guanidinium monolayer (A, B), a 45 base oligo T (C, D) and a
45 base oligo dC (E, F) adsorbed onto guanidinium. 3 H-bond
interactions give signals that extend significantly beyond 1 nm
(vertical gray lines). Initial set point is 0.4 nA at 0.4V, 133
nm/s retraction speed.
[0057] FIG. 35: Histograms of charge transfer for the I-Z curves.
A, C and E are for guanine functionalized probes and B, D and F are
for 2-aminoadenine functionalized probes. A and B (blue bars) show
data obtained on the guanidinium monolayer, C (orange bars) and D
(red bars) show data obtained with oligo T, E (brown bars) and F
(red bars) show data obtained with oligo dC. Charge
transfers>2.2 pC (blue shaded boxes) are unique to the
three-hydrogen bond interactions (E and D) and so serve to identify
the target base.
[0058] FIG. 36: Calculated current-voltage curve for
"guanidinium-phosphate-sugar-cytosine-guanine" on Au(111) with the
thiol positioned above the hollow site.
[0059] FIG. 37: Raw current vs. distance data for a bare tip (red)
vs tips modified with a modified guanine having two methylene units
attached to the N. Data were taken in 0.5 mM tris-HCl.
[0060] FIG. 38 shows an embodiment of a device in which the
constriction in found in a microfluidic channel formed on a surface
of the device.
[0061] FIGS. 39A and 39B show an embodiment of a device in which
the constriction is a pore through a substrate and the electrodes
comprise layers along the thickness of the pore.
[0062] FIGS. 40A and 40B show an electrodes comprise chemically
deposited layers of conducting metal.
[0063] FIG. 41 shows an exemplary electrical arrangement of a
device in accordance with one embodiment of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0064] FIG. 1 shows one embodiment of a device for sequencing
single-stranded. DNA (ssDNA) by hydrogen-bonding recognition, in
accordance with the present invention. In its simplest form, each
recognition-molecule (referred to as a `base-reader`) reads a
specific DNA base, the full sequence being assembled by juxtaposing
data from four different readers. As the ssDNA passes the
electrodes via a constriction (e.g., a nanopore), a guanidinium ion
grabs the nearest phosphate (depicted in FIG. 1 by the two yellow
hydrogen bonds), while a base reader recognizes its Watson-Crick
complement (depicted in FIG. 1 by the three red hydrogen bonds)
when it is present. So long as both molecular recognition events
overlap in time, a large current will flow, with the consequent
charge pulse signaling identification of the target base.
[0065] FIG. 1a shows the device 2 of FIG. 1 arranged in an
apparatus 100 configured to read bases in ssDNA, by measuring
tunneling current. A constriction in the form of a nanopore 1, is
formed in the device 2. In one embodiment, the device 2 is
comprises a chip 2 which, in turn, comprises a substrate provided
on a thin Si.sub.3N.sub.4 membrane. The top of the chip 2 seen in
FIG. 1A comprises a first side while the bottom of the chip 2,
which is hidden from view in FIG. 1A, comprises a second side.
Thus, the device 2 may be considered to comprise a partition having
a first side and a second side, and the ssDNA translocates from one
side of the partition to the other side of the partition, via the
constriction 1. First and second electrically conductive sensing
electrodes, 3, 4 which are spaced apart from one another by a gap,
are provided adjacent the nanopore 1 on the first side of the chip
2. In one embodiment, the gap between the first and second
electrodes is between 1.0-5.0 nm, though larger gaps may be
possible. The first and second sensing electrodes are preferably
formed of gold, though they may instead be formed of other
electrically conductive materials.
[0066] The chip 2 is mounted in the device 100 such that the chip's
first side (top) is exposed to a first fluid chamber 6 ("upper
chamber") and the chips second side (bottom) is exposed to a second
fluid chamber ("lower chamber"). As seen in FIG. 1A, the second
fluid chamber 6 contains the ssDNA 9 to be read, while the first
fluid chamber receives the ssDNA 9 translocating through the
nanopore 1 which forms a passage between the two fluid chambers 5,
6.
[0067] On the first side of the nanopore 1, a first affinity
element 8 is tethered to the first sensing electrode 3 via a first
flexible linker. Generally speaking, a "linker" is a chemical
designed so as to permit adequate motion of the affinity element to
self-assemble on the target while remaining in electrical
communication with an electrode. The first flexible linker itself
may be bonded to the first electrode via an electrode attachment
molecule, such as a thiol. In one embodiment, the first affinity
element 8 comprises guanidinium or a guanidinium derivative such as
guanidinoethyldisulfide. Guanidinium performs the function of
grabbing the phosphate backbone of the ssDNA 9 and thus serves as
"phosphate grabber."
[0068] On the second side of the nanopore, a second affinity
element 7 is tethered to the second sensing electrode 4 via a
second flexible linker. The second flexible linker itself may be
bonded to the second electrode via an electrode attachment
molecule, as described above. In one embodiment, the second
affinity element 7 comprises a base reader which is configured to
recognize one of the four bases on the ssDNA 9. In general, both
the phosphate grabber and the base reader form chemical bonds that
are readily broken at room temperature. Thus, the bonds formed
during translocation are made and broken on a timescale that
permits rapid binding and release of the target while still
allowing for detection and measurement of a tunneling current.
[0069] In one embodiment, the flexible linkers associated with
either or both sensing electrodes may comprise an alkane. A thiol
serves as the electrode attachment molecule, and so the combined
linker-electrode attachment molecule may comprise
--CH.sub.2--CH.sub.2--SH.
[0070] As also seen in the embodiment of FIG. 1A, a first magnetic
bead 10 may be affixed to a leading end of the ssDNA 9 and used to
pull the ssDNA 9 through the nanopore 1. Optical tracking of the
bead allows transit of the ssDNA to be followed to within 20 nm. It
is understood, however, that a second magnetic bead may be used on
the second side of the device (i.e., in second fluid chamber 5) to
help untangle the secondary structure of the DNA.
[0071] A pair of polarization electrodes 11 are used to polarize
the nanopore 1 for electrophoretic transport of the ssDNA 9. A
voltage bias 12 and a current monitor 13 are used to control the
electrophoretic transport.
[0072] The first and second sensing electrodes 3, 4 are connected
to a sensing electrode bias 14 and also to current measuring
circuitry 15 to gauge the tunneling current as each nucleotide is
detected during translocation of the ssDNA 9. It is from the
measured tunneling current at one or more nanopores that the
corresponding portion of the ssDNA can be identified.
[0073] The chip 2 of the device 100 seen in FIG. 1A may be
fabricated in a number of ways. In one embodiment, a number of such
chips 2 may be created at the same time using a single wafer in
conjunction with the following principal steps:
[0074] Step 1. Grow 100 nm Si.sub.3N.sub.4 on the top side of the
Si wafer.
[0075] Step 2. Photolithographically pattern sensing wires on top
of the Si.sub.3N.sub.4 using lift-off. The sensing wires will later
be cut into two electrodes for each chip.
[0076] Step 3. Photolithographically pattern a reference electrode
(RE) and a counter electrode (CE) on the underside, the RE and the
CE being brought to the edge of the windows (see, e.g., FIGS. 18
& 21).
[0077] Step 4. Grow 200 nm SiO.sub.x on both top and bottom to
insulate the various electrodes.
[0078] Step 5. Pattern and cut windows through SiOx and Si with HP
and KOH etches, exposing part of CE and RE on underside.
[0079] Step 6. On FIB, cut through SiO.sub.x and cut sensing wires
and shape ends. The gap between the electrodes is about 20 nm.
Exposed metal area should be less than a few square microns to
minimize leakage current from sensors.
[0080] Step 7. Turn chip and FIB mill nanopore through
Si.sub.3N.sub.4 centered on gap between electrodes. The thus-formed
pore is 5 to 10 nm at electrodes. Steps 5 and 6 can be done
automatically under computer control for many devices.
[0081] Step 8. Clean excess Ga ions from FIB milling with nitric
acid.
[0082] Step 9. Place gold plating solution below the chip (Si side)
and salt solution above it (Si.sub.3N.sub.4 side).
[0083] Step 10. Place Au onto sensing electrodes until a
predetermined tunnel current is obtained between the two sensing
electrodes. If this is coincident with a drop in the pore ionic
current (I.sub.P) then the electrodes are centered. The parameters
may be adjusted so that this process can be automated for
production.
[0084] Step 11. Open gap by stripping Au to achieve optimal
size.
[0085] Step 12. Rinse.
[0086] Step 13. Functionalize the chips by exposing them to
equimolar mix of phosphate grabber and base reader.
[0087] Step 14. If specific functionalization is necessary, hold
one electrode at >-1V Ag/AgCl and load a first recognition
reagent comprising the first affinity element. Rinse and then
expose to the second recognition reagent which comprises the second
affinity element. Rinse again.
[0088] Step 15. Mount chip in device so as to form the lower
chamber 5 and upper chamber 6.
[0089] It is understood that the wafer may have an large array of
such nanopores. In some embodiments, all the nanopores on a wafer
may be functionalized in the same exact manner. In other
embodiments, however, the nanopores on a wafer need not all be
functionalized with the same exact affinity elements.
[0090] In one embodiment, the wafer may be considered to comprise
an array of 2.times.2 sub-arrays. Each nanopore in a 2.times.2
subarray may then have a phosphate grabber (such as guanidinium) as
the first affinity element and a different one of the four base
readers as the second affinity element. This way, each 2.times.2
subarray comprises all four base readers for use in devices
configured for "parallel" DNA sequencing. The wafer may then be cut
into chips, each chip having a single 2.times.2. Alternatively, the
wafer may be cut into larger chips, each such chip comprising a
plurality of such 2.times.2 subarrays. This redundancy on a chip
can increase the certainty of recognition, as discussed further
below.
[0091] It is understood that multiple 1.times.4 sub-arrays may be
formed instead of 2.times.2 subarrays. In such case, the wafer may
be considered to comprise rows of nanopores whose members are
similarly functionalized. For instance, the wafer may comprise a
number of rows that is a multiple of four. Each nanopore in a given
row may then have a phosphate grabber as the first affinity
element, and the same base reader as the second affinity element.
Four rows that are adjacent to one another, may then have a
different base reader as the second affinity element in all their
nanopores. This allows one to cut up such a wafer into chips
comprising a single 1.times.4 subarray, or even into larger chips
comprising a plurality of such 1.times.4 subarrays.
[0092] Each nanopore 1 is functionalized by its associated second
affinity element 7 to recognize one of the four bases. Therefore,
to sequence DNA, it is understood that either: (a) a single copy of
the DNA must pass through a "gauntlet" comprising four differently
functionalized nanopores ("serial read"), or (b) four identical
copies of ssDNA must pass through four distinct, differently
functionalized nanopores ("parallel read").
[0093] When a single copy of DNA is used, the nanopores belong to
different chips and the DNA is threaded through the four chips.
Readouts of the electrical current detected from each of the four
nanopores can be aligned, using the known rate of translocation and
peak current values signifying a match to determine the DNA
sequence.
[0094] When four identical copies of DNA are used, it is desirable
that they translocate in synchrony. Readouts of the electrical
current detected from each of the four nanopores can then be
compared to look for peak values signifying a match.
[0095] Thus, in one embodiment, a device may be used to sequence
DNA by the following set of principal steps:
[0096] Step 1. A plurality of such nanopores, each functionalized
to recognize one of the four bases, should be provided. This can be
done using either serial reads or parallel reads, as described
above.
[0097] Step 2. Place DNA in lower chamber associated with each such
nanopore. Optionally modify the DNA so as to allow entry into the
pore from one direction only. In one embodiment, this may be done
by tethering the DNA to a bead.
[0098] Step 3. Electrophorese the DNA through the pore. if extra
pulling force is needed, functionalize the end that passes through
pore (after having been modified with e.g., biotin) and attach
magnetic bead.
[0099] Step 4. Pull DNA through by electrophoresis and/or magnetic
bead.
[0100] Step 5. Record current pulses (I.sub.t) as a function of
time.
[0101] Step 6. Align data from a plurality of reads for each type
of base reader.
[0102] Step 7. Align data from all 4 reads.
[0103] FIG. 4 illustrates that the current through a "good chemical
contact" can be thousands of time larger than current through a
simple physical contact. When the bonding in question is carried
out by means of hydrogen bonds, it is both (a) reversible and (b)
capable of molecular recognition.
[0104] The present invention utilizes the principle of hydrogen
bonding for molecular recognition. A number of measurements of
hydrogen-bond mediated tunneling using various combinations of
bases that form Watson-Crick or mismatch hydrogen bonding have
demonstrated the feasibility of this readout..sup.90
[0105] FIG. 5 shows the way in which STM measurements map onto a
preferred embodiment of nanopore measurement. Monolayers of bases
on gold using thiolated nucleosides are formed for the
purpose..sup.91-93 Thiolated nucleosides were chosen to overcome
the tendency of bases to lie flat on gold..sup.94 Both
5'-mercaptode oxycytidine (i--referred to as "C") and
5'-mercaptothymidine (ii--referred to as "T") have been shown to be
effective for this purpose. They form Watson-Crick (i) and G-T
wobble (ii) base pairs with guanine.sup.95.
[0106] STM images of monolayers of these nucleosides confirm the
phenomenon reported by Ohshiro and Umezawa:.sup.12 The contrast
obtained in images taken with a complementary base on the probe
(e.g., 8-mercaptoguanine imaging a 5'-thio-deoxycytidine monolayer)
was approximately double that obtained with a thio-phenol
functionalized probe used as a control (see the appendix.sup.90).
However, measurements of tunnel current as a function of
distance(as illustrated in FIG. 6) are much more striking and
relevant to the present invention.
[0107] FIGS. 6A-6D show data measurements of tunnel current as a
function of distance. In order to appreciate the significance of
these results, it must be understood that these are raw, unselected
data. Individual curves have simply been superimposed, with
different colors corresponding to different experimental
conditions. The separation of data from the various types of
measurement is immediately obvious even with this presentation of
overlapped, multiple traces. FIG. 6A shows the effect of the
controls (a bare Au tip 61, a thiophenol functionalized tip 62 or a
G-functionalized tip 63 on bare gold. All of these produce rapid
decays of the tunnel current, and the resulting curves overlap,
making them difficult to distinguish from one another. FIG. 6B
shows that data for a C-functionalized probe on a T monolayer
produces curves 65 that extend somewhat further in distance than
curves 61 from the bare Au. As seen in FIG. 6C, curves 65 for the
wobble-basepair (G-T) extend yet further. As seen in FIG. 6D, the
data for the Watson Crick G-C basepairs are distinguished by a
remarkable extension of the distance over which tunnel current is
sustained (upper traces 69). Thus, curves that correspond to the
Watson Crick basepairing are immediately identifiable in the raw
data. Initial conditions are i=3 nA, V=0.5V, retraction speed=133
nm/s. These data were taken in trichlorobenzene.
[0108] FIGS. 7A and 7B show a selection of just a few curves from
each of the G-C experiments (FIG. 7A) and G-T experiments (FIG. 7B)
to illustrate subsequent analysis. Curves can be fitted to two
separate regions of exponential decay:
a.sup.i=i.sup.0 .sup.exp-.beta..sup.1.sup.z 0<z<z.sub.c
(1a)
i=i(z.sub.c)exp-.beta..sub.2(z-z.sub.c) z.sub.c<z (1b)
[0109] The fits are shown by the solid lines in FIGS. 7A and 7B and
they lie on top of the experimental data. The important features of
this process are:
[0110] The slopes, characterized by .beta..sub.1 and .beta..sub.2
are not very different between G-C and G-T basepairs.
[0111] The values for .beta..sub.1 (about 0.1 .ANG..sup.-1) are
much too small to correspond to true electronic decay
constants.
[0112] The values for .beta..sub.2 (about 0.4 .ANG..sup.-1) are
typical of decay in the solvent alone and in line with previous
measurements of decay in a liquid medium..sup.96
[0113] The distance at which the decay transitions from behavior
characterized by .beta..sub.1 to behavior characterized by
.beta..sub.2 (z.sub.c) depends strongly on the type of basepair
(G-C vs. G-T). Arrows point to the transition between the slow
decay (.beta..sub.1) and the rapid decay (.beta..sub.1) regions at
a distance z.sub.c.
[0114] One interpretation of these data is that the first region
corresponds to stretching of the hydrogen bonded complex. The small
apparent decay length is readily explained if other components of
the system (e.g., bond rotations) are weaker than hydrogen bonds by
a factor of ten or so..sup.90 This is because the probe motion
corresponds to the strain of the complete system (including a
deformable STM probe.sup.97) while the tunneling is likely
controlled by the actual strain of the hydrogen bonds (see below)
which will be much less if they are "stiffer.".sup.98 Because the
decay in the second region is similar to that in solvent, it can be
ascribed to the response of the system after the hydrogen bonds
have broken. Therefore the breakpoints, z.sub.c, are a measure of
H-bond strength.
[0115] FIGS. 8A-8C show histograms of the values of z.sub.c for the
various experiments, using each of a G-Tip 81, a Tp-Tip 82 and a
Bare Tip 83. It is clearly an excellent parameter with which to
distinguish G-C from G-T basepairs in single molecule measurements.
Only about 80% of the curves are amenable to the analysis just
described. The remaining 20% are too noisy, or show no obvious
breakpoint. Nonetheless, as is clear from FIG. 6, all of the data
appear to be useful. A second, much simpler approach is to
integrate each curve. The horizontal axis corresponds to time, when
converted using the known retraction velocity of the probe. Thus,
the area under each curve (time.times.current) corresponds to the
total charge transferred in each interaction.
[0116] FIGS. 8D-8G histogram this total charge in picoCoulombs
(pC). In addition to control data with bare AU (FIG. 8D), C-G data
(FIG. 8E) and G-T data (FIG. 8F), also Shown is data taken from
monolayers made with an equimolar mix of C and T (FIG. 8G). The
green shading on the figure illustrates how values of charge
transfer above 15 pC uniquely identify G-C pairing. This occurs in
about half the reads.
[0117] These data have four qualitative characteristics.
[0118] First, these data appear to be robust even though tunneling
spectroscopy is known to be strongly affected by contamination and
geometry. This may be a consequence of the bonded nature of the
tunneling assembly. The H-bonds appear to work just like the
gold-thiol bonds that generated the data shown in FIGS. 3 and 4, as
explained herein.
[0119] Second, these results reflect single molecule interactions
and so equivalent geometry can also be realized using a nanopore
having translocated ssDNA passing therethrough. The reproducible
data shown in FIG. 6 is obtained using sharp probes, specifically
probes that are sharp enough to give single molecule resolution in
an STM image. In the nanopore, where electrodes would be densely
functionalized with, e.g., guanidinium or the base reader, the
presence of only one ssDNA strand will guarantee single molecule
interactions, with the caveat that the electrode gap and affinity
element reach be no more than the gap between two bases in
stretched DNA (ca 1.2 nm flanking the central base).
[0120] Third, the fidelity of the readout is adequate for detecting
individual nucleotides. The data presented in FIG. 8 show that an
unambiguous positive signal can be obtained in about half of the
reads. Since (0.5).sup.13<<10.sup.-4, the target of 99.99%
base calling accuracy could be obtained with as few as 13
independent reads. Even though the numbers may be somewhat less
favorable when all the types of cross reactions are measured (a
situation that might be mitigated with "designed base-readers"), a
small unambiguously positive signal can be handled with an adequate
number of independent reads.
[0121] Finally, the magnitude of the signal in these figures is not
extraordinary. The magnitudes show that the H-bond mediated
conductance is rather high. However, simulations show that the
magnitudes of these signals are on the order of magnitude to be
expected for H-bond mediated tunneling.
[0122] In the foregoing experiments, Tricholorobenzene (TCB) was
chosen as a medium since the hydrogen bonding interaction between
DNA bases and the base-reader would be too weak to be measured in
water, owing to competition from H-bonds with water molecules.
However, in a preferred embodiment water is the medium, since it is
most often used dissolving DNA. The novel technique works almost as
well in water as it does in TCB.
[0123] FIG. 9 presents raw current-distance data when water is used
as the medium. Once again, the signature of H-bonding is clear and
G-C pairs (black curves 92 FIG. 9C) are readily distinguished from
G-T pairs (black curves 92 FIG. 9Bb).
[0124] FIGS. 10A, 10B and 10C show histograms of the corresponding
charge transfer signals and contrast the results using a bare tip
101 and a G-tip 102. As seen in FIGS. 10D-10G, C-T base-pairs are
less readily distinguished in water than in TCB in these
conditions. This result in water may be explained by looking at
individual curves in detail, especially as seen in FIGS. 10E and
10G. The curves show features that suggest that the H-bonds do
indeed break sooner in water, but the current signal has a complex
shape and persists for a relatively long time. This may reflect the
formation of water bridges between the bases. This would account
for the increased signal from C-T pairs which are known to involve
the formation of water bridges.sup.99.
Forming Guanidinium Contacts to DNA
[0125] FIG. 11A shows a monolayer of guanidinium monolayer on
AU(111). 1,1'-(dithiodiethylene)diguanidine was synthesized by
reacting cystamine with N,N-bis(tert-butoxycarbonyl)thiourea in the
presence of 2-chloro-1-methylpyridiniumiodide (in dimethylformamide
at room temperature),.sup.100 followed by acid treatment. This
reagent was converted to .beta.-mercaptoethylguanidine (inset, FIG.
11A) on a TCEP (tris(2-carboxyethyl) phosphine)-immobilized gel
immediately before use. The guanidinium monolayer on Au was
verified by Fourier transform infrared reflectance (FTIR)
spectroscopy and ellipsometry. STM images of these monolayers are
shown in FIGS. 11A, 11B and 11C. The guanidinium molecule forms a
square lattice with a lattice constant of about 6 .ANG. (FIG. 11C).
These images were taken. in tris-HCl (pH 7) but the surface was
found to be very sensitive to the presence of other ions. In
particular, addition of phosphate buffer (pH 7) causes the surface
to reconstruct completely, removing the single atom deep "pits" in
the gold (arrows in FIGS. 11B and 11C) and forming ribbon-like
structures on the surface (FIG. 11D).
[0126] FIGS. 12A-12B illustrate surface plasmon resonance (SPR)
data, which shows that DNA is rapidly and irreversibly adsorbed
onto this surface. The adsorption is specific because it is blocked
in the presence of phosphate. Interestingly, the interaction of a
single DNA molecule with the surface is reversible. Single molecule
interaction was tested by tethering a 15 base oligomer to an AFM
probe with a polyethyleneglycol (PEG) linker and measuring the
adhesion as the probe was pushed into the surface and then
retracted. FIG. 12A shows SPR data for adsorption of dsDNA onto the
guanidinium functionalized surface. The trace in FIG. 12B shows
that even acid treatments do not remove the DNA.
[0127] FIG. 13A shows 300 superimposed force curves (A) for a
PEG-tethered 15 base DNA oligomer interacting with a guanidinium
functionalized surface. Most of the pulls indicate adhesion at one
point (sharp peaks) but an example of an "adhesion plateau" is
indicted. FIG. 13B is a scatter plot of peak force vs. pulling
distance. Note that most of the larger forces lie in the PEG region
and not the DNA region. Thus, while FIG. 13A shows an accumulation
of typical force curves, FIG. 13B shows a scatter plot of force
peak height vs. the distance at which the event occurred. Events in
the first 20 nm correspond to the PEG-tether length, while events
between 20 and 30 nm correspond to interactions with the DNA at the
end of the PEG (there are a small number of weak, non-specific
interactions at larger distances). The PEG adhesion is generally
larger than the DNA adhesion. The DNA features are generally
consistent with the formation of one, or a few H-bonds. Thus the
irreversible adsorption of DNA to the guanidinium surface is a
consequence of a cooperative interaction between the DNA molecules,
analogous to DNA condensation by multivalent ions..sup.101
[0128] FIGS. 14A-14D shows STM images of DNA adsorption onto the
guanidinium surface. The work of Ohshiro and Umezawa.sup.12 (who
used peptide nucleic acid, not DNA) and some recent studies in
vacuum.sup.102 notwithstanding, STM imaging of DNA is
controversial..sup.103 Therefore, FIG. 14 shows the results of
imaging the same region of the substrate before and after injection
of DNA into the liquid cell of the microscope. The results are
summarized in FIGS. 14A (pre-DNA injection) and 14B (post DNA
injection). Just as in the case of phosphate ion (FIG. 11D),
adsorption of DNA lifts the reconstruction of the gold, leading to
a loss of the "pits". The movement of the underlying gold is clear
from the shape changes in the small gold islands. Close inspection
of FIG. 14B shows that DNA strands appear to have been adsorbed in
a highly ordered monolayer in which the long axis of the strands
makes an angle of about 30.degree. with respect to the vertical
axis of the image. This spontaneous alignment does not occur for
short molecules, or mini-circles that can't pack side by side,
indicating further similarities with bulk DNA condensation by
multivalent ions..sup.101 Images of a flatter region show the DNA
structure much more clearly. FIG. 14C shows a region of dsDNA in
which the repeat of the double helix is quite clear (arrows and
note the 3.4 nm feature in the Fourier transform, inset). The
lateral packing distance, as evaluated by the Fourier transform
perpendicular to the long axis is about 2.5 nm. When an ssDNA is
used, the images are quite different, as shown in FIG. 14D. There
is no evidence of periodic structure along the chain direction (at
this resolution) and the lateral spacing falls to about 1 nm (see
the Fourier transform inset). It can be seen from these results
that DNA can be imaged with an STM on this surface, and thus, that
"good electrical contacts" are made by the phosphate-guanidinium
H-bonds. The specific role of guanidinium can be verified using an
amine-functionalized gold surface (of similar charge density to the
guanidinium surface) as a control. The formation of matched
hydrogen bonds is a key factor in enhanced electron transport.
Reading Base Composition from Adsorbed DNA
[0129] FIGS. 15A-15D show examples of current-distance curves
obtained over DNA adlayers on top of guanidinium in tris-HCl
buffer. These curves show current-decay curves obtained with a bare
(green trace 151) or functionalized tip over various guanidinium
surfaces. FIG. 15A: a guanidinium-functionalized surface (G-tip,
pink traces 152); FIG. 15B: an oligo-T (T45) adsorbed onto the
guanidinium surface (C-tip, pink traces 153); FIG. 15C: an oligo-T
(T45) adsorbed onto the guanidinium surface (G-tip, orange traces
154); FIG. 15D: an oligo-C (C45) adsorbed onto the guanidinium
surface (G-tip, black traces 155). Data were obtained in Tris-HCl
with a pH of 6.8. It can be seen from FIGS. 15A-15D that hydrogen
bond mediated tunnel current is capable of generating high level
signals (corresponding to a conductance on the order of 1 nS) that
yield single-base recognition signals of high fidelity and
contrast. Thus, one may likewise expect tunnel conductance of a
metal-linker-guanidinium-ssDNA-base reader-metal sandwich that is
on the order of 1 nS. This can provide signals compatible with CMOS
(the semiconductor technology used in most integrated circuits)
readout. Interestingly, the C-T mismatches produce less signal in
these conditions than they did in measurements made on
nucleosides..sup.90
Theoretical Confirmation of Experimental Results
[0130] One simple approach for calculating tunnel-conductance for a
complex system is to make an infinite chain of repeating elements
and use the methods of electronic bandstructure developed for solid
state physics. The usual output of bandstructure calculations is a
plot of energy vs. wavevector of the electrons, but, by solving for
complex wavevectors (which correspond to tunnel decay outside of
the allowed bands) the electronic decay constant, .beta., can be
estimated from the maximum value of this complex wavevector along
the lines that connect allowed states. This is called the method of
complex bandstructure..sup.57
[0131] FIG. 16A shows a portion of an infinite polymer of G-C
basepairs connected by Watson-Crick H-bonds formed by joining (in a
computer) C and G bases with a nitrogen atom. The length of the
"unit cell" in this polymer was 12.9 .ANG..
[0132] FIG. 16B shows a plot of the allowed energy levels for this
polymer, pursuant to using a plane wave basis to solve for the
electronic structure of this base-pair. The allowed energy levels
appear as flat lines (not dependent on k) because the states are
highly localized in this molecular system. The midpoint between the
highest molecular orbital (HOMO) and the lowest unoccupied
molecular orbital (LUMO) is the most likely energy at which the
electrons would be injected from a metal contact (the Fermi
level).
[0133] FIG. 16C shows curves representative of the complex
(forbidden) states for which the transport must be by tunneling,
using the same simulation. The semi-elliptical curve (red in the
figure) connecting the HOMO and LUMO is the most penetrating state
and the maximum value of .beta. is 0.74 .ANG..sup.-1. This means
the transmission probability through a single molecular unit
cytosine-guanine base-pair connected by a nitrogen) is
e.sup.-.beta.L=e.sup.-0.74.times.12.9=e.sup.-9.5. A simple estimate
for the conductance, G, of the cytosine-guanine basepair is
G.apprxeq.G.sub.0e.sup.-.beta.L=77 .mu.S.times.e.sup.-9.6=5.5 nS,
where G.sub.0 is the quantum of conductance. Thus the theoretical
estimate of the conductance of a base-pair is of the same order of
magnitude as the conductances used to obtain experimental data.
These data illustrate that, despite being thought of as "weak
bonds," the average electronic overlap near the HOMO-LUMO gap
including H-bonds can be quite strong. The average is as strong as
a sigma bond in terms of electron transport (though a sigma bond
is, of course, a much stronger bond in terms of bond energy).
Generation of Molecular Recognition Signals in a Tunnel Gap
Fabricated on a Chip; Optimization of Electrode Design and
Fabrication
[0134] he problem of how to make `molecular alligator clips` has
consumed the molecular electronics community for
decades..sup.104-106 The problem now appears to have been solved in
flexible junctions (like the STM where one electrode is controlled
with sub .ANG. precision).sup.107 but it is extremely difficult for
fixed junctions, at least in the case of single molecules. One
reason is that the outermost atoms of each electrode must be in
precisely the correct position to satisfy the bonding requirements
of the molecule that spans the gap. Some successful experiments
have been reported.sup.108-111 using electromigration, a technique
in which a nanogap is formed by "blowing" a fuse consisting of
nano-scale neck in a wire. But the same technique has been shown to
mimic molecular electronic effects in the absence of molecules
owing to the presence of complex metal structures in the
gap..sup.112 The requirements for atomic precision in bonding
molecules are mitigated in the embodiment of FIG. 1 because of the
use of flexible linkers as part of the tunnel junction. This is
possible because the embodiment of FIG. 1 depends on a binary
"signal-no signal" output rather than on an absolute conductance of
the gap. Thus, reliable manufacture of "clean" gaps of about 2 to 3
nm in size range and chemical functionalization of these gaps for
reliable readout, are both realizable.
[0135] FIGS. 17A-B shows the results of one prior art approach to
electrode design and the formation of gaps between electrodes. The
technology employed to manufacture and inspect such "clean gaps"
has undertaken a significant leap as a result of the work of Marija
Drndi at the University of Pennsylvania. By using electrodes placed
on very thin membranes (exactly as required for nanopores) Drndi 's
group has been able to image junctions using transmission electron
microscopy (TEM) to atomic precision..sup.113 Moreover, Drndi 's
group has shown that the lack of electron backscatter in thin-film
supports permits very high resolution electron-beam ablation of
metal structures..sup.114
[0136] Another approach to electrode design and the manufacture of
nanogaps is electrochemical deposition and stripping.
Electrochemical generation of nano-gaps has been in use for some
years.sup.115 but may sometimes be unreliable (see the
supplementary material in He et al..sup.79).
[0137] The present invention also takes advantage of a new approach
to electrochemical generation. In one embodiment of the present
invention, reference (RE) and counter (CE) electrodes are
incorporated into the chip itself, spaced a few microns from the
tunneling gap.
[0138] FIGS. 18A-18D shows a prototype testbed large-electrode
junction (i.e., large electrodes, small gap) in accordance with one
embodiment of the present invention. Gold electrodes that are 2
.mu.m wide by 30 nm high are patterned by lift-off onto a 200 nm
thick SiO.sub.2/Si.sub.3N.sub.4 substrate in a cruciform pattern.
One electrode is a continuous strip that is cut to form the two
sensing electrodes (SE1, SE2), which also serve as the working
electrodes for gold deposition and stripping. Two other electrodes
(RE, CE) are separated from the central wire by gaps of 3 .mu.m and
they serve as built-in counter- and reference-electrodes. The
electrodes are covered with a 200 nm thick layer of SiO.sub.2. The
wafer, containing 25 arrays, each of nine devices, is taken to the
focused ion beam mill (FIB) where a trench is cut across the
central wire to form the two sensing electrodes (SE1 and SE2). The
trench is widened and continued out to the RE and CE electrodes to
form microfluidic channels in communication with both sensing
electrodes and the CE and RE. As a consequence of the geometry of
the ion-beam milling, a 100 nm wide trench at the top of the
SiO.sub.2 corresponds to about one to two nm gap in the gold
electrodes. As a result, once the gap is chemically-cleaned of
excess Ga ions, stable and somewhat reproducible tunnel gaps are
formed.
[0139] FIG. 19A, which plots the log-current vs. bias voltage data
(and the linear data shown in the inset), confirms the presence of
such tunnel gaps. Fits of these data to the Simmons formula.sup.116
yield gap dimensions that are on the order of one nm. By placing a
drop of gold-plating solution in the gap and controlling the
deposition galvanostatically, while monitoring the tunnel current,
I.sub.t, it is possible to close the gap. Once closed (as detected
by current between SE1 and SE2) controlled stripping opens the gap
with atomic scale control.
[0140] FIG. 19BB, which plots conductance as a function of time
after the gaps are closed, shows quantum steps in conductance
(indicated by arrows) that characterize atom-sized filaments of
gold..sup.117 Thus atomic-scale control of gap size can be achieved
by electrochemical deposition and stripping.
[0141] Manufacture and testing of rim wide tunnel gaps. Using
electron ablation and TEM imaging on a 100 nm thick Si.sub.3N.sub.4
substrate (c.f. FIGS. 17A-17B) one may sculpt nm-wide tunnel gaps,
correlating gap geometry with measured tunnel-current response.
Optimal gap size for recognition (ca 2 nm to 3 nm) as measured by
reproducible electrical characteristics and chemical response (see
below) may thus be achieved.
[0142] Testing of gaps for recognition response. Gaps can be
functionalized, in the first instance, by exposure to an equimolar
solution of the guanidiniumthiolate and a mercapto-base. After
rinsing, recognition can be tested by exposing the junctions to
dilute solutions of nucleoside monophosphates and DNA dimers. The
goal is to get current signals that track the STM data, i.e., the
biggest signals occurring when Watson-Crick complementary
nucleoside monophosphates are injected into the gap, smaller
signals for mismatches and no signals at all for non-hydrogen
bonding nucleoside monophosphate analogs. In the event that just
one part of the gap dominates the tunneling (the desired result)
each type of interaction should be characterized by a somewhat
reproducible tunnel current. Though such testing is not the direct
equivalent of the STM or nanopore geometry, the lifetime of the
hydrogen bonding states generally correlate with bond stabilization
energy
( .varies. exp .DELTA. G k B T ) ##EQU00001##
so that the two sets of signals should correlate well. Electrode
design may be refined based on these data until the geometry is
optimized. Molecular-recognition signals from the "macro-scale"
testbed (FIG. 18) establish that the foregoing is achievable, even
though the signals are difficult to interpret because many
asperities contribute to tunneling.
[0143] Optimization of electrode functionalization. As seen in FIG.
1, the guanidinium phosphate-binder is formed on a first electrode
and a base-reader is formed on the opposite, second electrode. The
electric field in the gap is very large, so nucleotides may be
drawn into the gap and oriented. Accordingly, one may compare the
signals obtained from specifically functionalized electrodes
(guanidinium on one, base reader on the other) with those obtained
from electrodes randomly functionalized with a mixture of
guanidinium and base-reader. Specific functionalization of
nano-electrodes has been demonstrated by adsorbing thiolates from
an electrolyte with the electrodes held under potential control,
relying on the reductive cleavage of the Au--S bond at -1V vs.
Ag/AgCl to prevent adsorption onto one of the electrodes..sup.118
After rinsing, the previously uncovered electrode is functionalized
with a solution of the second reagent..sup.118 In these
experiments, one would expect to see signals at much lower
concentrations of nucleoside monophosphate if the specific
functionalization minimizes trapping of the target nucleoside
monophosphate on one electrode or the other. Given the
dielectrophoretic trapping capability of the gaps, one may obtain
statistical signals even down to dilutions estimated to contain
only one nucleoside monophosphate.
[0144] Optimization of electrochemical fabrication of electrodes. A
method for electrochemical growth-assisted formation of electrode
pairs can be developed. Such a method may be automated and suitable
for on-chip regeneration of the gaps. Lithographically defined
electrode pairs on a 100 nm Si.sub.3N.sub.4 substrate may be used
as a starting point. The electrode may then be used to assess
electrode growth and stripping as a function of the electrochemical
parameters (concentration, overpotential, starting electrode
geometry, deposition and stripping times). TEM images may be used
to analyze the resultant growth at close to atomic scale (c.f. FIG.
17A), and these results may be compared with predictions of
simulations. Quantitative agreement between simulation and
experiment validates the tools to be used in overall device
formation.
[0145] Characterization of electrode stability. Reproducibility of
the current-distance data indicates that the instability of
gold-thiol connections, as manifested in an electronic "blinking"
phenomenon.sup.71 is not a problem on a pointed electrode. However,
the long-term stability of fixed nanojunctions may still pose
problems. While the on-off times for the H-bonded nucleoside
monophosphates are on the order of milliseconds, "contact blinking"
may be marked by loss and return of signals on a time scale of tens
of seconds or more..sup.71 Such blinking, if it occurs, is not
fatal, but does need to be figured into the calculation of the
required reading redundancy.
[0146] Active control of the tunnel gap. Atomic scale control of
the initial construction of the gap is necessary, as is atomic
scale stability over long timescales. To help achieve the latter,
active control of the tunnel gap may be implemented. This
effectively mimics a scanning tunneling microscope on a chip. One
scheme for fine gap control comes from an apparatus originally
designed for controlled breaking of metal junctions ("break
junctions"), and this may be used as a way of controlling a
pre-fabricated gap.
[0147] FIG. 20A schematically illustrates such an apparatus 200 for
fine gap control. If a (originally flat) membrane 202, of thickness
(h), width (w) and length (L), is subject to a point three (F) at
its middle, two points on the upper surface move apart by an amount
.epsilon. owing to the induced curvature of the surface. Analysis
of bending moments yields the following result:.sup.119
= 3 FL 2 4 Ewh 2 ##EQU00002##
where E is the Young's modulus of the material (1.5.times.10.sup.11
N/m.sup.2 for Si.sub.3N.sub.4). In one embodiment, the target gap
size, .delta., is likely be no more than 5 nm. With h=100 nm, L=w=5
mm, a 10% adjustment of .delta. (.epsilon.=0.5 nm) would require a
force of 200 pN. Using the standard equation for a cantilever
spring.sup.120 shows that a motion (Z on FIG. 20A) of just 15.mu.
would be adequate to achieve this. These parameters are compatible
with MEMS fabrication processes, so that adjustable gaps could be
mass-produced eventually. Adjustment of the electrochemical gaps in
the present device may be accomplished by building a "break
junction" like apparatus, comparing the measurements of the gap
based on tunnel current to the predictions of finite element
elastic analysis of the junctions. The data obtained from this
exercise can be used to design a system integrated onto a chip
using standard MEMS procedures.
[0148] FIG. 20B shows a buckling geometry that may be implemented
to actively control a gap that incorporates a nanopore 208, such as
one that connects cis and trans fluid chambers.
Alignment of a Nanogap Electrode Pair with a Nanopore
[0149] Overview. Assembly and alignment of the reading head,
comprising a pore and electrodes can be achieved through
electrochemical self-assembly of electrode pairs. Electrochemical
deposition of electrodes minimizes the number of one-off
nanofabrication steps, resulting in devices that are easier to
manufacture. Furthermore an electrochemical approach makes it
possible to strip and reuse electrodes, a possible cure for failure
modes related to electrode geometry and functionalization. This
also reduces costs and enhances reliability.
[0150] FIG. 21 presents a strategy for controlled growth of
electrodes into the gap, in accordance with one embodiment of the
present invention. The deposition process is controlled from the
trans solution 210a compartment so that deposition is localized to
the region in the immediate vicinity of the pore 211. In addition
to the electrode starting geometry (optimized in specific aim 1),
other factors affecting this process are reagent concentrations,
electrode potentials and pore geometry. The process is complicated
by the high resistance of the pore. Simulations and TEM
measurements can be used to characterize real junctions to optimize
the process of forming the electrodes. Meller's TEM approach.sup.33
can be advantageously employed to produce small pores, but it may
also be possible to eliminate the TEM "filling" step. While the
electrode gap can be quite large, the nanopore 211 must be small
enough to permit only one DNA strand to pass at a time. Therefore,
alternatives to TEM could greatly simplify the production of
reading heads. Recent reports of controlled formation of pore as
small as 5 nm by FIB.sup.121 indicate that one may start with a 20
nm pore cut into a 20 nm constriction to form the two sensing
electrodes (the starting geometry shown in FIG. 22C). One can mill
through a thin Si.sub.3N.sub.4 from beneath the electrodes (which
are visible in the dual-beam FIB through the membrane). The gold
electrodes can then be electroplated out into the gap, narrowing
both the gap and the pore to the desired size (.about.2 to 3 nm).
Optimizing pore size can be advantageous given that smaller pores
result in greater DNA-pore interaction while distinct ssDNA
translocations have been observed in rather large pores..sup.38 In
the event that a small (<2 nm) pore is required to ensure
translocation of only single strands, or to remove secondary
structure, one may start with pores that have been "shrunk" on the
TEM..sup.33
[0151] Simulations. A computational approach can be used to
simulate the electrochemical processes in three stages: (1) 2D
modeling of the electrodeposition process ignoring double-layer
effects. (2) Subsequent inclusion of double layer effects. (3)
Finally, a full 3D model including the double layer.
[0152] 2D modeling (no double layer). FIG. 22A shows one embodiment
of a 2D model of the electrochemical system that can be used to
identify proper values for the processing parameters such as the
deposition potential and duration. The electrodeposition process
occurs via the reduction of Au+ ions at the surfaces of the two
sensing electrodes (SE1 222a and SE2 222b) held at potentials V1
and V2. These must differ by at least 50 mV for tunneling to be
detected, but this sensing potential could be switched during
deposition if uneven growth is a problem, The reduction of Au+ (in
the plating solution 226, e.g., KAu(CN)2) to Au causes a depletion
of Au+ ions near the electrode surface, and this in turn builds up
a concentration gradient driving more Au+ to the surface through
the nanopore 224 by diffusion. Thus the electrodeposition is a
process controlled by the reaction kinetics occurring at the
electrodes and the mass transport of Au+ in the solution through
the nanopore. To simulate this process, the combined problem of
electrokinetic flow and electrodeposition can be considered based
on the Butler-Volmer kinetic equation (which can take account of
the irreversible kinetics generally Observed at nanoelectrodes) and
diffusive treatment of mass transport. The rate of Au deposition is
defined as dh/dt=Je*M.sub.Au/d.sub.Au/F, where Je is the normal
electron flux across the electrodes, M.sub.Au and d.sub.Au are the
molecular weight and density of Au, respectively, and F is the
Faraday constant. Running the electrokinetic flow mode with the
"moving mesh" application in COMSOL Multiphysics, a finite-element
based modeling software, can solve this modeling problem. This
exercise provides initial values for the deposition potential and
duration.
[0153] 2D modeling of electrodeposition including double layer
effects. The description of electrodeposition on the nanoscale
requires explicit inclusion of the polarization that forms on the
electrode surface in contact with the electrolyte (the electrical
double layer, EDL). It has recently been reported that the EDL
structure outside nanoscale electrodes (having a critical
dimension<100 nm) affects not only the electron transfer across
the electrodes but also the ionic distribution in the
solution..sup.122
[0154] FIG. 22B shows a modification to the 2D model which includes
the EDL structure outside the electrodes 222a, 222b. Because of the
EDL, the Nernst-Planck equation can be used for the mass transport
phenomena due to the combined diffusion and electromigration of Au+
ions. Furthermore, in the presence of the EDL, ionic
electroneutrality does not hold inside the diffuse layer, thus an
electrostatic problem governed by the Poisson equation can be
solved at the same time. From this effort, proper values for the
deposition potential and duration can be determined. Since the rate
of Au deposition is related to the normal electron flux at the
electrode surfaces and the normal flux is affected by the shape and
curvature of the electrode surface, various types of surface shapes
and curvatures for the electrodes may be considered to identify a
proper electrode shape for achieving the best outcome for resizing
the nanopore without occluding the nanopore, shorting the sensing
electrodes in the electrodeposition process or having a gap height
that is too thick to sense a single base (base-base separation is
about 0.6 nm).
[0155] 3D modeling of electrodeposition including the double
layers. FIG. 22C shows a full 3D model of the deposition process.
The full 3D model evolves from the tools associated with the 2D
model and thus produces an optimized set of process parameters
including the electrodeposition potential, deposition duration, and
surface shape for the electrodes in a 3D setting.
[0156] Gap optimization. The use of a thin Si3N4 support allow TEM
examination of the gap, and independent monitoring of the orifice
size and tunnel gap comes from measurements of the pore ion current
and the tunnel current between sensing electrodes. The exact
geometry may not be critical, provided that (a) the electrode
height in the gap is <0.6 nm and (b) the widest passage(s)
through the structure are no more than 2 to 3 nm. The structure
proves to be somewhat tolerant of local details because (1) the
ionic current threads the DNA into the major part of the orifice,
(2) the (flexibly) tethered guanidinium and base-reader orients the
DNA by H-bonding. Devices can be tested both with nucleoside
monophosphates and with homopolymers (for all bases bar oligo-G
which is not well behaved). These tests determine whether or not
one needs to apply active control to the gap. While active control
of the gap is possible, it is complicated by the presence of both
cis and trans solution chambers in place. FIG. 20b shows one
candidate geometry (distortion greatly exaggerated) that one may
implement, pending finite element mechanical analysis.
Design and Synthesis of DNA Base-Readers
[0157] Elimination or reduction of base-pairing mismatches
simplifies the robustness of the sequencing. This can be realized
by using more specific DNA Base-Readers. Better affinity elements
based on chemical principles and theoretical modeling help with the
design of recognition reagents. Once synthesized, affinity elements
can be readily and rapidly characterized using STM methods, such as
those described herein.sup.90
[0158] General Considerations. As shown in FIG. 23, each of the
four bases has a distinguishable Watson-Crick edge, allowing one to
design a reader for each of them. Four separate readers are likely
to be required, although it may also be possible to employ a
"universal reader" having a particular structure. A set of
candidate structures can be screened to determine their specificity
and immunity to mispairing. A DNA base reader should have the
following chemical and structural features: (1) donor and acceptors
sites for formation of stable hydrogen bonds; (2) planar .pi.
system capable of stacking interactions and efficient mediation of
tunneling; (3) the molecules must be constructed such that the
Watson-Crick base pairing occurs with high specificity; (4) they
could incorporate steric obstruction of mismatches; and, (5) should
be stable to oxygen, light, water, and electrochemical reactions,
once coupled to the electrodes. It is best to reduce manipulations
of the target DNA (such as incorporation of modified nucleotides by
enzymes) to a minimum in view of the goal of reading long, native
DNA.
[0159] Electronic structure calculations can be carried out prior
to synthesis both to verify the proposed bonding, and to test the
effects of altering the structure of the heterocyclic rings on
electronic conductance.
The Adenine Reader (A Reader)
[0160] A commercially available 5-mercaptouracil may be used as a
candidate Adenine reader. FIG. 24 shows that 5-mercaptouracil can
form a Watson-Crick base pair with adenine, but it can also mispair
with other DNA bases C, G, and T..sup.123 Because each of these
mismatched base pairs has a similar hydrogen bonding pattern to the
Watson-Crick base pair, it may be very difficult to distinguish
them electronically, making adenine the most difficult base to
identify unambiguously. This problem is not necessarily fatal if
high fidelity data are available from the three other readers, but
a selective A-reader is highly desirable.
[0161] FIG. 25 shows a peptide nucleic acid (PNA) trimer comprising
one modified uracil flanked with universal bases, which may be
synthesized. Such a PNA may serve as a selective A-reader. PNA is a
structural mimic of DNA but it forms more stable DNA duplexes and
is more sensitive to mismatches than its DNA counterpart.sup.124 By
using a PNA trimer for recognition, one may convert a single base
pairing process into a DNA-PNA hybridization process. Thus, the
base pairing specificity of modified uracil relies not only on
hydrogen bonding but also on stacking with its nearest neighbors.
The additional stacking interaction promotes the pre-organization
of the base reader into the "right" conformation for Watson-Crick
base pairing. Universal bases form base pairs with normal DNA bases
indiscriminately.sup.125 so the PNA timer should have no
selectivity to the (n-1) and (n+1) flanking bases on the target
DNA. It has been demonstrated that a universal base can enhance the
mismatch discrimination in the DNA duplex
thermodynamically.sup.126, 127 and enzymatically..sup.128 In one
embodiment of the structure, a propargylthiol linker is attached at
5-position of uracil for connection to the electrode. In the event
that this linker is not long enough, another "molecular wire" may
be used. The PNA trimer can thus be tested against a series of
adenine centered DNA trimers with varied base contexts at their two
ends on gold substrates using the STM method..sup.90 Such a
strategy may also be applied to the design of other base
readers.
[0162] The PNA trimer can be synthesized manually or in an
automated peptide synthesizer. The synthesis of universal base PNA
monomer has been reported in the literature.sup.129, 130 The
modified uracil PNA monomer may be synthesized starting from
5-iodouracil-1-acetic acid..sup.131 The starting material reacts
with ethyl N-[2-Boc-aminoethyl]glycinate, providing a 5-iodouracil
PNA monomer that can be converted into the desired product through
the Sonogashira coupling.sup.132 with 3-benzoylthio-1-propyne
followed by treating with
di-tbutyl-1-(tbutylthio)hydrazine-1,2-dicarboxylate..sup.34
The Cytosine Reader (C Reader)
[0163] FIG. 26 illustrates that though 8-Mercaptoguanine can serve
as a C reader, guanine in general forms stable mismatched base
pairs such as G-G, G-A, and G-T.sup.99, 133, 134 Ideally, however,
these mismatches would be reduced. Sekine and coworkers have
demonstrated that 2-N-acetyl-3-deazaguanine (a2c3G) is more
selective to cytosine than guanine (see FIG. 27), and also
destabilizes the GA mismatch..sup.135 Compared to guanine, one of
the undesired hydrogen bond acceptors is removed and the rotation
of the NH.sub.2 group is constrained in a2c3G.
[0164] FIG. 27 shows a number of modified guanines which, based on
the work of Sekine, may serve as C readers. A common feature of
these molecules is that their Watson-Crick edge remains unchanged
and the undesired atoms are left out. S-acetylguanine is an
amine-acetylated derivative of 8-mercaptoguanine, which can be used
to determine how acetylation of the amine affects the specificity
of the guanine. With this control, S-deazaguanine, a deaza
derivative of S-acetylguanine (with the 3-nitrogen removed) should
reduce the sheared G-A mismatch..sup.136 S-aminopyridone is the
simplest candidate C reader and it should have the highest
specificity. A PNA trimer containing 3,7-dideazaguanine (S-deazaG)
is potentially useful for this purpose as well.
[0165] S-acetylguanine can be synthesized starting from
9-methyl-8-mercaptoguanine..sup.137 The thiol group is first
protected in a tbutyl disulfide form,34 and then the starting
material treated with acetyl chloride.sup.135 followed by
Al--NiCl2.about.THF..sup.138 The synthesis of S-deazaguanine is
straightforward using 3-deaza-9-methyl-guanine as the starting
material. S-aminopyridone can be synthesized starting from
4-iododiamonopyridine prepared according to the reported
procedure..sup.139 First, 4-iodo-6-acetylaminopyridone can then be
synthesized by adopting the method used by Sun et al,.sup.140 and
then converted to the desired product by treatment with thiourea.
The key step in synthesis of S-dideazaG PNA monomer is iodonation
of dideazaguanine. The approach developed by Ramzeva and
Seela.sup.141 can be employed for this. If such an approach is
found to have a selectivity problem, one may first prepare
7-iododidazaguanine using the regioselective reaction controlled by
a bulky group at 9-position of dideazaguanine and then convert it
to the desired product.
The Guanine Reader (G Reader)
[0166] In one embodiment, 6-Mercaptocytosine, a cytosine
derivative, can be used as a G reader. FIG. 28 shows that
6-Mercaptocytosine should form a more stable base pair with guanine
compared to the mismatches in neutral conditions. Other candidates
include 5-mercaptocytosine, 5-mercapto-1-methylcytosine,
6-mercapto-1-methylcytosine, and 1-(2-mercaptoethyl)cytosine, which
can easily be synthesized from commercially available starting
materials..sup.142 Studies of these molecules allow one to optimize
the G reader attachment and to determine how the N-1 methylation of
cytosine affects its specificity. The effects of pH on the
recognition of the G reader should also be taken into
consideration. It is known that protonation on DNA bases enhances
the stability of mismatched base pairs. Under slightly acidic
conditions, Cytosine forms stable hydrogen bonded base pairs with
protonated cytosine (C+) and adenine (A+)..sup.143, 144 The
protonation alters the electronic structure of DNA base pairs,
resulting in changes of their electronic properties..sup.145 Thus,
pH is a factor in achieving a high specificity. The electrode side
of the pore may be somewhat basic owing to the polarization of the
pore used to translocate the DNA into the cis chamber.
[0167] FIG. 29 shows the basepairing of a tricyclic cytosine
analogue (called a "G-clamp" with guanine. Lin and Mattecucci have
reported that such a G-clamp can simultaneously recognize both
Watson-Crick and Hoogsteen edges of a guanine when it was
incorporated into DNA (FIG. 29)..sup.146 The G-clamp has shown a
higher specificity than its counterpart, 5-methylcytosine. The
G-clamp may also be evaluated for its suitability as a G reader. A
thiolated G clamp can be synthesized based on a procedure published
by Gait and coworkers..sup.147
The Thymine Reader (T Reader)
[0168] In one embodiment, 2-amino-8-mercaptoadenine, a derivative
of 2-aminoadenine (DAP), can be used a T reader. FIG. 30 shows that
DAP forms a more stable Watson-Crick base pair with thymine due to
an additional N--H O hydrogen bond. However, it has been reported
that stability of the DAP-T base is sequence dependent in
DNA,.sup.148 which is attributed to varied base-stacking
interactions. Thus, a individual DAP coupled to an electrode should
recognize thymines in a single stranded DNA with high selectivity,
generating distinguishable electronic signals, DAP can form
mismatched base pairs with C and A..sup.148, 149 Other types of
mismatches, such as Hoogsteen base pairs, can also occur. As shown
in FIG. 30, one may employ three analogues of diaminopurine to
improve its specificity and affinity to the thymine base.
2,6-Diacetamido-4-mercaptopyridine, which can be synthesized by
treating 2,6-diacetamido-4-iodopyridine.sup.139 with sodium
hydrosulfide, is a simple DAP analogue which is more specific and
stable. In general, the DAP-T base pair is less stable than the G-C
base pair..sup.150 Recently, Brown and coworkers reported an
analogue of adenine, 7-aminopropargyl-7-deaza-2-aminoadenine, which
could form an "A:T" base pair with stability comparable to
G:C..sup.151 One may therefore test the base pairing specificity
and stability of its analogues 7-deaza-2-aminoadenine and
3,7-dideaza-2-aminoadenine by incorporating them into the PNA
trimer, respectively. The corresponding PNA monomers can be
synthesized from commercially available starting materials
6-Chloro-7-deazaguanine and
4,6-dichloro-1H-pyrrolo[3,2-c]pyridine.sup.152 using chemistries
described above.
A Universal Reader
[0169] One may attempt a universal reader capable of recognizing
the four natural DNA bases with distinguishable signatures.
4-(mercaptomethyl)-1H-imidazole-2-carboxamide is proposed as a
candidate universal reader. It includes two hydrogen bonding donors
and two hydrogen bonding acceptors, one half on the aromatic
imidazole ring and the other half on the amide side group. The
molecule can be attached to the electrode through the thiol
group.
[0170] FIG. 31 shows that the amide group is relatively free to
rotate around bond a, and the whole imidazolecarboxarnide can
freely rotate around bond b. In the solution, the molecule exists
in a mix of varied conformations. FIG. 31 also illustrates how this
molecule base-pairs with each of DNA bases in a different
conformation. Each of the conformations has a different energy due
to the asymmetric structure, so one may expect that each base pair
has a different, and distinct, free energy. Thus, it may be
possible to read unique signals out of the tunneling device.
[0171] The synthesis of
4-(mercaptomethyl)-1H-imidazole-2-carboxamide starts from
(1-trityl-1H-imidazole-5-yl)methanol. First, the hydroxyl group can
be converted to tbutyldisulfide as a latent thiol function,.sup.34,
153 and then a cyano group introduced to 2-position of the
imidazole ring,.sup.154 which can be hydrolyzed to
carboxamide..sup.1-55 Finally, the desired product can be obtained
by detritylation and reduction of the disulfide.
Theory and Modeling of Base Readers
[0172] The operation of the base-recognition head is an interesting
mix of mechanics (H-bond strength) and electronics (high
conductance when bonded). Modeling requires both
MD-simulation.sup.156, 157 and ab-initio quantum chemistry
methods..sup.158, 159 Such tools can be used to evaluate the
"base-readers" described above. Complete wires can be modeled with
pseudopotential density functional theory (DFT)..sup.160, 161
Complete circuits including the leads can be modeled with local
orbital DFT techniques..sup.162-164 One can also test for
unexpected outcomes. This can be done by using simpler empirical
potentials and constructing tight-binding electronic structure
models.sup.165 to search for alternate bonding schemes,
conformational isomers or tautomers.
Characterization and Control of DNA Translocation through a
Functionalized Pore
[0173] The operation of the sequencer depends on the speed and
controllability of translocation, the role of the sequence itself
in pore-friction and the degree to which secondary structures delay
transit. The many studies of DNA translocation through a nanopore
have generally focused on unfunctionalized nanopores (the exception
is Astier et al..sup.19). Translocation through a functionalized
nanopore is different. This should be evident given that
.lamda.-DNA translocates a approximately 6 nm diameter pore in a
few ms (at V=50 mV and 1M KCl),.sup.166 equivalent to a speed of 8
mm/s. It is has been measured that the H-bond lifetime is on the
order of a few ins, which corresponds to a "speed" of just microns
per second, on the assumption that each base is trapped in the
reader for a millisecond or so, since even with negative base
reads, the phosphate-guanidinium trapping still occurs. The force
generated in the STM pull is probably dominated by the softer
material in the gap, as disclosed in He et al..sup.90, but it is
surely quite large, as H-bonds require forces on the order of 100
pN to rupture at these pulling speeds (see FIG. 13 and Ashcroft et
al..sup.50). In one study of (cyclodextrin) functionalized
nanopores, nucleotides became trapped for significant times,
illustrating the large effect of pore functionalization..sup.19
[0174] In accordance with one embodiment of the present invention,
translocation of DNA through functionalized nanopores can be
accomplished using magnetic beads affixed to a leading end of the
DNA as the primary manipulation tool, because this technology is
compatible with parallel operation of many reading heads. This is
because one set of magnets can pull many beads. The force on a bead
of volume v and magnetization m in a field gradient
.differential. B .differential. z ##EQU00003##
is given by
F z = mv .differential. B .differential. z . ##EQU00004##
[0175] With a field gradient of 100 T/m (readily obtained with
permanent magnets) and 3 .mu.m superparamagnetic beads available
from Magsense (West Lafayette, Ind.), forces of up to 150 pN are
obtainable. This is comparable to optical tweezers.sup.167 and also
similar to the larger electrophoretic forces experienced in
nanopores..sup.166 A "magnetic tweezers" apparatus.sup.168 having a
high field gradient magnet stack.sup.169 can be used to study
translocation in functionalized nanopores.
[0176] FIG. 32 illustrates a device 320 that can track DNA transit
to within 10 nm by fitting the Airy-fringe pattern around the bead
when the objective is out of focus. The time resolution is limited
to the 50 Hz frame-grabbing rate of the camera interface, but this
is adequate with 1 ms transit times because the height resolution
is limited to around 20 bases (and 20 bases can transit in about 20
ms which is 1/50.sup.th of a second). The device 320 includes a
light some 331 which projects a light beam past magnets 332 towards
a DNA-tethered bead 333. A lens 334 amplifies the bead signal and
the resulting image is directed to a camera 335 via a mirror 336.
It is understood that other detection arrangements may also be
employed.
[0177] FIG. 33A illustrates some of the factors controlling
translocation. The electrophoretic force, F.sub.elec, is opposed by
H-bond friction 331 in the gap 332. The entry of the DNA 333 into
the pore 334 is typically opposed by entropy fluctuations, and,
more importantly, secondary structure 335. For a secondary
structure undergoing random thermal openings at a rate k.sub.0, the
opening rate on application of a force f is
k ( f ) = k 0 exp [ f x is k B T ] ##EQU00005##
where x.sub.is is the distance to the transition state from the
folded state along the direction in which the force is applied. The
smallest values of k.sub.0 for hairpins trapped in a nanopore is
about 1 s.sup.-1 which is really very slow. Based on measured
values for x.sub.is for a tight molecular nanopore.sup.50 (about
0.1 nm) an electrophoretic force of 100 pN would increase the
opening rate to about 10 s.sup.-1. Thus, secondary structure could
be a significant obstacle to fast reads. The ssDNA could be
pre-stretched using the magnetic bead 336 but this would reduce the
net force across the pore 334, increasing the rate of backwards
slippage.
[0178] As seen in FIG. 33B, pre-stretching would probably require a
bead 335a, 335b trapped at both ends to form a rotaxane with the
nanopore.sup.170.
[0179] As depicted in FIG. 33B, in yet another arrangement, one may
want to augment the electrophoretic force using a magnetic bead
335c.
[0180] In each of these experiments, one can measure the output of
the sensing electrodes using a DNA molecule of known sequence. This
allows one to correlate features in the gross transport (as
measured by bead movement and pore current) with local features (as
measured by the molecular recognition signal from the sensing
electrodes). One approach is to use the M13 genome as a source of
long ssDNA (6.5 kb). Cutting it requires hybridization with a short
helper strand in order to form a local dsDNA template for a
restriction enzyme. The short strand is easily removed by
filtration after denaturation. Next, splint-ligation may be used at
both ends, putting in a biotin at one end and a digoxigenin at the
other, with a two step affinity column purification of the long
product. Modification of .lamda.-DNA using incorporation of
modified dNTPs followed by magnetic extraction of the desired
strand at high pH may also be performed. The "flossing" experiment
(FIG. 33B) can be carried out by trapping the DNA-antiDIG bead from
the cis chamber using electrophoresis and then functionalizing the
DNA in the trans chamber with a strepavidin coated magnetic bead.
Finally, a novel "unstructured" DNA.sup.171, 172 may be available
for use (see the letter from Laderman). This farms Watson-Crick
basepairs with natural bases, but the modified bases will not pair
with each other. Presently, the modified nucleotides can be
incorporated in runs of up to 600 bases.
[0181] It should be evident to one skilled in the art that the
foregoing enables one improve upon the basis design and
methodology. More particularly, one may:
[0182] Measure the transit time of known oligomers through
nanoelectrode pores;
[0183] Re-measure transit times with functionalized pores. One can
thus test to see if the assymetry of the backbone (5'-3' vs. 3'-5')
affects readout fidelity and transit times, using bead
functionalization at one end or the other;
[0184] Measure transit times as a function of pH. Secondary
structure is removed at low pH.sup.38 but the same conditions that
remove secondary structure (pH>11.6) may also destroy
H-bonding.
[0185] Measure transit times through both functionalized and
unfunctionalized pores with unstructured DNA to measure the extent
to which secondary structure slows entry into the pore.
[0186] These measurements can determine the relative contributions
of secondary structure and H-bond friction in slowing transit.
Magnetic bead experiments may be designed to speed up or slow down
the translocation as needed. One may test these arrangements using
the functionalized, linearized M13 DNA, correlating the local
sequence data from the sensing electrodes with the progress of
translocation as measured optically.
Theory and Simulation
[0187] Simulations must include all the forces acting on the ssDNA
as it translocates.sup.17, 173-175--the magnetic force on the bead,
the electrophoresis force on the charged ssDNA, the hydrogen
bonding force of the guanidinium attempting to hold the DNA in
place, the hydrogen bonding force of the base-reader on the target
base, interactions of ssDNA with itself (secondary structure), the
viscous force of the water on the magnetic bead, and interactions
of the DNA with water and with the walls of the nanopore. The
length of the tether molecules is also critical. Varying it, even
slightly, may change the number of contacts and/or the probability
of simultaneous phosphate and base recognition. Modeling helps
guide the correct choice of parameters for successful sequencing.
Simulation strategy combines three main ingredients--molecular
dynamics (MD) simulations,.sup.157, 176 coarse grained
simulations.sup.177, 178 and analytic modeling at all levels.
Molecular dynamics can be used to pick out key molecular aspects of
the problem, specifically in the nanopore itself. Even within the
timescale-limitations of current MD techniques they provide a
picture of the mechanisms occurring within the active region of the
pore. The data produced from the MD simulations can be used to
build a coarse gained model of the process using a Brownian
dynamics algorithm'.sup.177 of a chain model for the ssDNA with a
magnetic bead in a viscous fluid..sup.179 Interactions observed in
the nanopore from the MD simulations can be included
parametrically. Free energy data for secondary structure can be
incorporated together with existing kinetic data..sup.18, 50
Characterization of Signals from Oligomers and Genomic DNA Using a
Set of Single Pores
[0188] The sequence-reconstruction problem has two inputs. One is
the optical tracking of transport which could give data at a
resolution that could be as high as 20 bases. The second is the
signals from the molecular reading heads themselves. Reading head
data of adequate quality could permit alignment of data from all
four reading heads with no other input. Data for each individual
base that is 99.99% accurate may be obtained by a combination of
improved affinity elements and multiple reads of the same
sequences. If the data from each head are of adequate quality, one
may record repeated runs for each type of base with high
fidelity.
[0189] When sequencing four copies of ssDNA using four nanopores,
each nanopore having a different base reader as the second affinity
element, four component sequence reads are created. Each sequence
read identifies, as a function of base location, the points at
which a nucleotide of a particular type has been detected. Since
there may be differences in the rates at which the four copies of
the ssDNA electrophoresce through their respective nanopores, there
may be an issue of aligning the four component sequence reads to
arrive at a final sequence read representing the sequenced ssDNA.
Blocks of a repeated base (e.g., 4.times. or 5.times.) are rare
enough that they can serve as good indices of position in the
genome, and yet frequent enough so that a significant number of
them occur in each read. Thus, upon obtaining a sequence of
component reads of ssDNA from each of four readers, one may align
the four sequences of component reads based on one or more
preselected blocks of a repeated nucleotide (which hopefully will
be present in at least one of the sequences of component reads.)
For example, positive reads of an A.sub.5 tract (A-A-A-A-A) would
be aligned with unique (or rare) gaps of null readings of 5 bases
in extent from the C, G and T readers. This is called the "framing
problem" in parallel transmission of digital data over noisy
channels..sup.180 The problem is greatly simplified if the
direction of the data stream (e.g., 3'-5') is fixed. Thus one can
develop protocols for preprocessing input DNA and ligating beads
(or even just form crosslinked dsDNA blockers) to control the entry
direction. Once any such needed alignment has been done, one may
then create a final sequence of reads representing the sequenced
ssDNA from the four component sequences of reads.
[0190] The optical tracking data can record each translocation to
within 20 bases at best, with maybe substantially poorer resolution
when entropy and secondary structure fluctuations are taken into
account. But it also serves as a check on the local alignment
algorithms, eliminating gross mistakes (i.e., juxtaposition errors
greater than the optical tracking resolution).
[0191] Quantitative data obtained from using the device of the
present invention may be used to develop data analysis tools for
rapid sequence recovery. Some of the issues that can be addressed
by such quantitative data (1) the transit times per base in the
read (base+phosphate H-bonds) vs. the no-read (phosphate H-bonds
only); the frequency with which a nucleotide is missed altogether;
(3) the fluctuations in average read speeds; (4) the role of
secondary structure; and (5) whether it would help if "stalling,"
owing to secondary structure, occurred predictably.
[0192] As discussed herein, one may construct a fixed-gap nanopore
sequence device capable of reading single bases with high fidelity.
Such a device may incorporate one or more of the following
features: electrochemically grown self-aligning electrodes, active
gap adjustment, and gold as the electrode material. In use, such a
device may be able to deal with the potential problems of secondary
and tertiary structures in long DNA transits. Furthermore, the
assembly of such devices may be facilitated and even automated for
consistency from unit to unit, thereby mitigating uncertainties in
the performance of one-off designs. The assembly and
functionalization methods allow for reforming and healing of
devices whose readers have been damaged or otherwise spent.
EXAMPLES
[0193] Example 1: Electrode-tethered guanidinium ions can be used
to complete an electrical circuit for "reading" bases in single
stranded DNA.
[0194] Hydrogen bonds are the molecular "Velcro" on Which much of
the reversible and chemically-specific bonding in biology is
based..sup.186 It has recently been shown that hydrogen bonds can
also enhance electron transport significantly,.sup.12, 90
observations that suggest a basis for a new type of electrical
biosensor. This sensor would use two sets of hydrogen bonds to
complete an electrical circuit by simultaneously binding two
independent sites on a target molecule.
[0195] Electrical contacts to DNA are currently made by means of
covalent modifications.sup.187 but it is possible to form "generic"
hydrogen bonds to DNA by means of an interaction between the
backbone phosphates and guanidinium ions..sup.188 This then allows
the construction of a "molecular sandwich" consisting of hydrogen
bonded contacts to DNA via the phosphates on one side and a base on
the other side as illustrated schematically in FIG. 1. The use of
different "Base Recognition" elements (FIG. 1) generates electrical
signals characteristic of a particular target base in the DNA.
[0196] A guanidinium monolayer was prepared by reacting
.beta.-mercaptoethylguanidine ("Phosphate Recognition" element)
with Au (111) surfaces and scanning tunneling microscope (STM)
images (taken in Tris buffer) are shown in FIGS. 11B and C. The
single-atom deep pits characteristic of thiol adsorption on
gold.sup.189 (arrows in FIG. 11B) proved to be sensitive to the
anions in solution and disappear in phosphate buffer.
[0197] DNA molecules in Tris buffer bind to these surfaces rapidly
and irreversibly, as verified by surface plasmon resonance and
Fourier transform infrared absorption spectroscopy. The adsorption
is specific because it is inhibited in phosphate buffer.
[0198] Electrical contact to the backbone phosphates was verified
by taking STM images of DNA adlayers on the
guanidinium-functionalized electrodes. STM of DNA.sup.190, 102, 8
has been the subject of controversy.sup.103 and the images of these
adlayers have unexpected properties, so the same region of the
guanidinium-treated substrate was imaged before and after injection
of a 2.8 kbp double stranded (ds) plasmid DNA (Litmus 28i from New
England Biolabs) into the sample cell of the microscope (FIGS. 14A
and B). The underlying gold reorganizes, causing the pits (arrows,
FIG. 14A) to disappear and a striped texture to appear on the
surface (white lines, FIG. 14B) when DNA is adsorbed. A
median-leveled image of a flatter region (FIG. 14C) shows the dsDNA
geometry quite clearly. The individual dsDNA molecules look like
rows of "beads" (arrows) with a helical repeat of 2.9 nm, close to
that of A-DNA (see the 2D-FFT, inset). The intermolecular packing
distance (white lines) is about 2.8 nm. The dsDNA packs into a
dense, highly organized layer, despite its nominally circular
structure. To test the interpretation of the "beads" as turns of
the double helix, long single-stranded (ss) loops of DNA (the 6.8
kb M13 genome) were imaged. A characteristic image is shown in FIG.
14D. The "beads" are absent and the features are narrower. The need
for specific hydrogen-bonds was tested by attempting to image DNA
on cystamine-treated gold electrodes (which interact with
phosphates less specifically) but images were unobtainable.
[0199] Images of small ssDNA oligomers (12 to 79 bases) appeared to
be extremely smooth, presumably because they were more perfectly
packed, but when molecular features were resolved, they
corresponded to the known dimensions of the oligomers. These
densely packed layers appear to require DNA-DNA interactions (much
like bulk DNA condensation.sup.101) because the shortest oligomers
and DNA minicircles (too small to bend) did not form ordered
layers. In turn, the ordering appears to be necessary to hold the
DNA in place under the sweeping motion of the STM probe, as atomic
force microscopy clearly shows that isolated molecules can adsorb
as well.
[0200] To test for the role of DNA-DNA interactions in the
reversibility of the absorption process, the adhesion of single
molecules using DNA tethered to an AFM probe was measured. The
force curves obtained on pulling the DNA away from the surface
showed features characteristic of the breaking of individual
hydrogen bonds, suggesting that additional DNA-DNA interactions are
required for the formation of the ordered monolayers.
[0201] The STM images demonstrate that charge can be transported
laterally through the tethered guanidinium and the ssDNA or dsDNA
molecules with a conductance on the order of a few tens of pS
(currents of ca. 10 pA at a bias of a fraction of a volt).
[0202] Example 2: A readable signal is obtainable if the circuit is
completed using a complementary DNA as second connector. Transport
measurements were carried out on the DNA adlayers using gold STM
probes insulated with polyethylene, functionalizing the exposed
gold apex with a thiolated nucleobase..sup.90 Either
8-mercaptoguanine (G) or 8-mercapto-2 aminoadenine (2AA) was used.
2AA was chosen because it can form three hydrogen bonds with
thymine..sup.181
[0203] Examples of conductance vs. retraction-distance (I-Z) curves
taken with these functionalized probes are given in FIGS. 34A-F,
with the vertical line in each figure denoted the 1 nm distance
mark. The green lower curves 340g in FIGS. 34C-34F are control data
taken using bare gold probes. All of the experimental data taken in
a successful run have been overlaid with no pre-selection of the
curves. When no DNA is present, G and 2AA probes yield decay curves
340b over guanidinium (all curves in FIGS. 34A and B) similar to
those obtained with bare probes (green lower curves 340g in FIGS.
34C-34F). With a 45 base oligo-T adsorbed onto the guanidinium, the
G and 2AA probes yield very different signals 340o, 340r,
respectively (FIGS. 34 C and D). With a G-probe, the signal 340o
has decayed after 1 nm (FIG. 34C), whereas the 2AA-probe yields
signals 340r out to nearly 2 nm (FIG. 34D). The response is
reversed with a 45 base oligo-dC adsorbed onto the guanidinium: the
G-probe signal 341w (see FIG. 34E) extends much further that the
2AA-probe signal 341r (FIG. 34F). Thus, the extended signals are
characteristic of triple hydrogen bonds.
[0204] The curves shown in FIG. 34A-F were quantified by
integrating them and forming corresponding histograms 350a, 350g,
350o, 350r, 351w and 351r. The integral is equal to the charge
transferred when the horizontal axis is converted to time using the
retraction speed of the probe, and the distribution is shown in
FIGS. 35A-F, respectively. Charge transfer in the blue-shaded
regions 350b (appearing, e.g., in FIGS. 35A & 35B) uniquely
identifies the triply hydrogen bonded interactions (G with C, 2AA
with T). With the charge transfer set to this threshold, there are
no false positive reads so only a small number of repeated reads
would identify a base with a high fidelity..sup.90
[0205] The high value of set-point conductance and the slow decay
of current do not reflect the intrinsic electronic properties of
the DNA because the tunnel junction is under significant
compressive stress, so the I-Z curve will be affected significantly
by elastic distortion of the material in the gap..sup.90 A better
estimate of the conductance of the DNA is given by the value in the
region of rapid decay just before the H-bonded junction
ruptures..sup.90 Inspection of the curves in FIGS. 34A-F suggests
that this conductance is on the order of tens of pS.
[0206] To determine whether this value of conductance can be
explained by a tunneling process, a theoretical investigation was
performed by evaluating the conductance from a current-voltage
(I-V) curve for "guanidinium-phosphate-sugar-cytosine-guanine"
connected to gold contacts. The tunneling current was computed
using a density functional theory (DFT) Green's function scattering
method.sup.67, 182 based on the Landauer approach.sup.183, 184 and
details are given in the supporting information. The calculated
conductance due to tunneling (FIG. 36) for
"guanidinium-phosphate-sugar-cytosine-guanine" (g-p-s-c-g) is
remarkably low, 9.27 fS, smaller than the experimental estimate by
several orders of magnitude.
[0207] To understand the origin of such low predicted conductance,
the complex bandstructure was investigated for each of the two
hydrogen-bonded components (a G-C basepair and
guanidinium-phosphate couple) separately, as well as for the
complete g-p-s-c-g assembly. The calculations yield an upper limit
on the inverse electronic decay length, .beta., assuming that the
Fermi level lies midway between the highest occupied and lowest
unoccupied molecular orbitals..sup.57 Table 2 lists the structures
investigated this way, together with values for .beta. and the
length, L, of the "unit cell" of the repeated structure. An order
of magnitude estimate (right column, Table 2) of the tunnel
conductance is obtained using G.apprxeq.G.sub.0e.sup.-.beta.L where
G.sub.0 is the quantum of conductance (77 .mu.S)..sup.57
[0208] Two striking features emerge from these calculations. First,
the conductance of the G-C basepair is remarkably high, on the
order of nS, a value one might expect from a sigma-bonded
system..sup.107 Second, the guanidinium-phosphate shows an
extremely low conductance, on the order of pS. As a result, the
conductance of the whole assembly remains very low (on the order of
fS). To improve the estimate of the conductance, G was recomputed
using .beta. at the Fermi level predicted by the DFT calculations.
At 1.32 fS it is still much lower than the experimental estimate.
Since tunneling calculations are generally in reasonable agreement
with the data for smaller molecule.sup.107 we concluded that some
mechanism other than tunneling operates in this system. Our
calculations do not include finite temperature or polarization
effects arising from the surrounding solvent, so that transport
mechanisms involving charge localization.sup.185 might come into
play.
[0209] FIG. 37 shows the (current distance) curves for the linker
shown. This linker has two methylene units in series attached to
the N of the guanine in the structure. The bundle of darker curves
370b on the right side of the plot are oligo-cytosine recognition
curves. The strands of light curves 370r on the left side of the
plot are controls taken with a bare tip. The signal levels are seen
in this figure to be relatively high with the indirect linkage of
the guanine not affecting the process.
[0210] In the embodiment of FIGS. 1 and 1A, the constriction is in
the form of a nanopore 1 which passes through a thickness of a
substrate. It is understood, however, that a constriction may take
on other forms and arrangements as well. Thus, in an alternate
embodiment, the constriction may comprise a narrowed portion of a
microfluidic channel formed on a surface of a substrate. In other
words, the constriction may lie on top of a device with the target
molecule passing from a first chamber on top of the device to a
second chamber on top of the same device. In such case, the
constriction connects the two chambers which are separated by a
surface partition.
[0211] FIG. 38 shows an exemplary arrangement, where the device 381
is placed in a microfluidic channel 382. The stream lines show the
fluid being diverted in order to pass through the constriction 383.
The electrode surfaces within the microfluidic channel are
insulated from the fluid in the channel by a protective layer of
insulation 384. Connections to the electrodes 385 exit the
structure outside of the fluid channel.
[0212] FIG. 39A shows an exemplary cross section of embodiment of a
device 391 through a fluid channel showing a planar electrode
arrangement. 391 is the top layer of insulation, 392 is a first
metal or doped semiconductor layer, 393 is a second layer of
insulation, 394 is a second metal or doped semiconductor layer and
395 is the insulating substrate on which the structure is formed.
The size of the constriction between the electrodes is determined
by the thickness of the second layer of insulation 393. The
structure is assembled by planar deposition of alternating
conducting and insulating layers on the substrate, followed by
formation of a channel, 396, through the entire structure.
[0213] FIG. 39B illustrates one embodiment of dimensions for
operation of the device seen in FIG. 39A. In this embodiment, the
spacing between the electrode pair, 392 and 394, is L and the
diameter of the channel 396 diameter is d. In order to obtain
adequate tunnel conductance through the target molecule, L lies in
the range from 0.5 to 10 nm. The channel diameter is constrained
only by the requirement that a molecule entering the channel touch
the sides, and hence the electrodes, during its transit through the
channel. If the speed of fluid flow through the channel is V meters
per second than the time spent between the electrode pair is:
t=V/L seconds
[0214] In this time the molecule must diffuse a lateral distance d,
given by
d= Dt
[0215] where D is the diffusion constant of the molecule. Thus the
maximum speed of transit of the sample passed the electrodes is
given by
VDL/d.sup.2.
Thus the speed with which fluid can be processed decreases rapidly
as the constriction size is increased. For example, with L=5 nm,
d=10 nm and D=100(m)2/s (typical of a small protein), V is
preferably less than 5 mm/s.
[0216] FIG. 40A shows a top view and FIG. 40B shows a cross section
of a further embodiment using opposing electrodes. 401 is a first
linear metal or doped polysilicon electrode. 402 is a second linear
metal or doped polysilicon electrode. 403 is a channel that has
been milled through the entire structure. 404 is a protective
insulating layer that covers the electrodes. 405 is the underlying
insulating substrate. 406A and 406B are electrodes, which may be
chemically deposited layers of conducting metal used to achieve a
small constriction between the opposing electrodes 401 and 402.
[0217] One exemplary manner of assembling the structure of FIGS.
40A and 40B is to make a stripe of doped polysilicon conductor on
the substrate 405, then coat over this stripe and the substrate
with an insulating layer of oxide, then use a focused ion beam mill
to cut a slot through the entire device, separating the stripe of
polysilicon into the two opposing electrodes 401 and 402. The
channel size resulting from focused ion beam milling is likely to
be about 30 nm, so the constriction is narrowed to the desired
nanometer dimension by, for example, electric chemical growth of a
metal such as gold on to the exposed conducting polysilicon
electrodes. This growth can be continued until the junction is
short-circuited, and then a small amount of gold removed
electrochemically, leaving a constriction of the desired size.
[0218] FIG. 41 provides an exemplary electrical arrangement
embodiment of an apparatus of the present invention. The apparatus
comprises a channel 412 formed through a chip 411. Two external
connections to the electrodes 413 and 414 are placed on a chip
carrier 415 that allows fluid access to the front and back of the
channel 412. One electrode 413 is grounded, while the other is
connected to the inverting terminal of a current to voltage
converter, 416. The non-inverting terminal is connected to a source
of bias, 417, so that feedback is applied through the current to
voltage conversion resistor R, the inverting terminal is held at a
potential of V volts with respect to ground, thus biasing the
non-grounded electrode. The output signal of the current to voltage
converter 416 is -iR volts where i is the tunnel current signal
generated by detection of one or more molecules in the device.
Typically V lies in the range from 10 mV to 1V, so that a 1 nS
tunnel conductance for a detected molecule would yield a current
between 10 pA and 1 nA. With R=1 G.OMEGA. this leads to output
voltages between 10 mV and 1V. The signal to noise of such a
detection system may be improved, for example, by using a larger
resistor R, but the response time (RCin, where Cin is the electrode
stray capacitance) becomes slower. With an R=1 G.OMEGA. resistor,
the shot noise is a tiny fraction of 1 pA, while a stray electrode
capacitance of 1 pF (possible with appropriate electrode and
instillation design) yields a response time of 1 ms.
[0219] In each of the embodiments seen in FIGS. 38-41, it is
understood that the various linkers, affinity elements, and the
like are connected to the various electrodes to create a
functioning apparatus in accordance with the present invention.
[0220] A device formed and operated in accordance with the present
invention may potentially provide a variety of desirable
capabilities.
[0221] Speed. Under certain conditions, the DNA can be pulled
through molecular nanopores.sup.50 at a speed of about 1 .mu.m/s
(.about.1600 bases/second at 0.6 nm per base for stretched DNA).
This corresponds to a dwell time of about 0.6 ms/base, well within
the range of current single molecule electronic measurements. At
this speed, 100 Mb per day could be sequenced in each reader.
However, the actual read-speed may be up to 10.times. slower than
this because of the intrinsic molecular friction in an H-bond
reader.
[0222] Accuracy. It is possible to make unique single molecule
identifications for a limited range of combinations of base
pairings. This pattern is expected to persist for other
combinations, provided that the appropriate affinity elements are
used. With a 50% hit rate, 13 independent reads would be needed to
reach 99.99% accuracy. On the other hand, a 10% unique hit rate
would require 90 reads.
[0223] Robustness. The STM measurements mimic the interaction of
the functionalized electrodes with the DNA. The STM probes have
operated successfully for thousands of measurements each. Spurious
(unwanted) adhesion is less with a functionalized probe than with a
bare probe.
[0224] Long Sequence Reads. Final sequence assembly is relatively
straightforward and the DNA can be tracked optically, allowing
perhaps 100 microns to be followed through the pore (i.e., >100
kb). The sequential nature of the readout should eliminate problems
associated with sequence repeats.
[0225] Simplicity The peat attraction of "sequencing by
recognition" is its simplicity (compared to other single-molecule
schemes). It can use just one measurement (charge transfer) with a
threshold to generate a binary output and it does not require
labeling DNA or nucleotides. The signal levels (0.5 nA, 0.5V) and
read speeds (ms per base) are compatible with CMOS.
[0226] Use of native DNA. Given high-fidelity "readers" for all
four bases, long genomic DNA could be read with no pre-processing,
saving the attachment of end-tethered beads for manipulating the
DNA (and even this modification may not be essential).
[0227] Cost. The basic reading head unit can be fabricated with
conventional lithography; a focused ion beam (FIB) milling step and
electrochemistry. Produced in quantity, the read heads should be
quite cheap, perhaps on the order of a hundred dollars. One machine
might comprise four blocks of 700 read heads, one block for each
base. If highly specific recognition chemistries could be found
(75% hit rate) each instrument might make 10 reads on 70 different
portions of the genome, yielding 4.2 Mega bases worth of 99.99%
quality sequence in 1 minute, completing an entire mammalian genome
in 17 hours. This short timescale is compatible with a cost goal of
$1000/genome.
[0228] Thus, in sum, the system described above is potentially able
to meet the cost target of $1000/genome, an accuracy of <1 error
per 10 kb, long contiguous reads on the order of tens of thousands
of bases, and minimal sample preparation, all while reading genomic
DNA.
[0229] Although the present invention has been described to a
certain degree of particularity, it should be understood that
various alterations and modifications could be made without
departing from the scope of the invention as hereinafter
claimed.
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GLOSSARY
[0420] Base-Reader (BR): A class of molecule, natural or manmade
that contains a predetermined pattern and spacing of Hydrogen bond
donors and acceptors fixed in space on a molecular scaffold to
allow the molecule to bond and recognize molecules with
complementary patterns and spacing of Hydrogen bond donors and
acceptors.
[0421] Universal-Base-Reader (UBR): A class of molecule, natural or
manmade that contains sufficient predetermined pattern and spacing
of Hydrogen bond donors and acceptors fixed in space on a molecular
scaffold to allow the molecule through conformational changes to
bond and recognize all molecules of interest with complementary
patterns and spacing of Hydrogen bond donors and acceptors.
[0422] Adenine-Base-Reader (ABR): A class of molecule, natural or
manmade that contains a predetermined pattern and spacing of
hydrogen bond donors and acceptors fixed in space on a molecular
scaffold to allow the Base-Reader to bond and recognize adenine
(abbreviated A). A few examples of an ABR class of molecule are
thymine (T), uracil (U) and Riboflavin. These molecules have
complementary patterns and spacing of Hydrogen bond donors and
acceptors to recognize adenine (A).
[0423] Cytosine-Base-Reader (CBR): A class of molecule, natural or
manmade that contains a predetermined pattern and spacing of
Hydrogen bond donors and acceptors fixed in space on a molecular
scaffold to allow the Base-Reader to bond and recognize cytosine
(abbreviated C). A few examples of a CBR class of molecule are
guanine (abbreviated G) and isoguanine. These molecules have
complementary patterns and spacing of Hydrogen bond donors and
acceptors to recognize cytosine (C).
[0424] Guanine-Base-Reader (GBR): A class of molecule, natural or
manmade that contains a predetermined pattern and spacing of
Hydrogen bond donors and acceptors fixed in space on a molecular
scaffold to allow the Base-Reader to bond and recognize guanine
(abbreviated G). A few examples of a GBR class of molecule are
cytosine (C) and 5-Methyleytosine. These molecules have
complementary patterns and spacing of Hydrogen bond donors and
acceptors to recognize guanine (G).
[0425] Thymine-Base-Reader (TBR): A class of molecule, natural or
manmade that contains a predetermined pattern and spacing of
Hydrogen bond donors and acceptors fixed in space on a molecular
scaffold to allow the Base-Reader to bond and recognize thymine
(abbreviated T) and or uracil (U). A few examples of a TBR class of
molecule are adenine (abbreviated A) and Coenzyme A. These
molecules have complementary patterns and spacing of Hydrogen bond
donors and acceptors to recognize thymine (T) and uracil (U).
[0426] Electrode Attachment Molecule (EAM): A class of molecule
that has the properties required to make a good electrical and
mechanical contact/bond with a conductive or semi-conductive
electrode. One exemplary example of such a molecule is an alkane
thiol on gold conductor. Sulfur has particular affinity for gold,
with a binding energy in the range of 20-35 kcal/mol (85-145
kJ/mol). An alkane with a thiol head group will stick to the gold
surface and can have a linker molecule covalently bonded to it and
a base reader or a phosphate group recognition molecule linked to
it.
[0427] Linker Molecule (LM): A class of molecule that has the
properties required to make a good electrical and mechanical
contact/bond with an Electrode Attachment Molecule and a Phosphate
Group Recognition Molecule or a Base Reader. One exemplary example
of such a molecule is a Poly (ethylene glycol) oligomer.
[0428] Phosphate Group Recognition Molecule (PGRM): A class of
molecule that will because of its favorable structural and chemical
properties form one or more hydrogen bonds with the phosphate
groups that comprise the backbone of a biopolymer. One of many
exemplary example of such a molecule is Guanidinium.
[0429] Guanidinium IUPAC name 1,1'-(dithiodiethylene)diguanidine.
Guanidinium is a chaotrope that causes molecular structure to be
disrupted; in particular, those formed by noncovalent forces such
as hydrogen bonding, Van der Waals interactions, and the
hydrophobic effect. It is also used as a general protein
denaturant. Guanidinium has the ability to hydrogen bond to the
phosphate groups attached to the sugar backbone of the DNA
molecule. The Hydrogen bonding occurs between the favorable spaced
atomic structure of the oxygen atoms of the phosphate groups and
the two free NH.sub.2 amine ends of the guanidinium. Guanidinium is
from the group of Guanidines which are a group of organic compounds
sharing a common functional group with the general structure
(R1R2N1)(R3R4N2)C.dbd.N3--R5. The central bond within this group is
that of an imine. In the simple case for Guanidinium the R's are
Hydrogen H so the structure looks like
(N1H.sub.2)(N2H.sub.2)C--N3--R5. The third Nitrogen may be replaced
by an oxygen or a carbon and still not affect the desired
functionality. Many molecules have the ability to hydrogen bond to
the phosphate groups and therefore can function as part of a DNA
reader. The important characteristics are the ability to
electrically conduct, the ability to hydrogen bond to the phosphate
groups and the ability to attach by covalent bond to electrically
conductive linker molecules.
[0430] Cystamine is an organic disulfide. IUPAC name
2,2'-Dithiobis(ethylamine). It is formed when cystine is heated,
the result of decarboxylation. Cystamine is an unstable liquid and
is generally handled as the dihydrochloride salt, C4H12N2S2.2HCl,
which is stable to 203-214.degree. C. at which point it decomposes.
Cystamine is toxic if swallowed or inhaled and potentially harmful
by contact.
[0431] N,N-bis(tert-butoxycarbonyl)thiourea guanylating reagent
used in making guanidinium Bernatowicz, M. S.; Wu, Y.; Matsueda, G.
R. Tetrahedron Lett. 1993, 34, 3389.
[0432] 2-chloro-1-methylpyridinium iodide reagent is used to make
guanidinium.
[0433] DMF (Dimethylformamide) is the organic compound with the
formula (CH.sub.3).sub.2NC(O)H. Commonly abbreviated DMF, this
colorless liquid is miscible with water and majority of organic
liquids. DMF is a common solvent for chemical reactions.
[0434] TCEP (tris(2-carboxyethyl)phosphine) is a reducing agent
frequently used in biochemistry and molecular biology applications.
It is often prepared and used as a hydrochloride salt. TCEP is also
available as a stabilized solution at neutral pH and immobilized
onto an agarose gel support to facilitate removal of the reducing
agent. TCEP is often used as a reducing agent to break disulfide
bonds within and between proteins as a preparatory step for gel
electrophoresis.
[0435] Tris is an abbreviation for (trishydroxymethylaminomethane)
which is also known by its IUPAC definition of
2-amino-2-hydroxymethyl-1,3-propanediol. It is widely used as a
component of buffer solutions, such as in TAE and TBE buffers used
in biochemistry, with an effective pH range between 7.0 and 9.2.
Tris is often used when working with nucleic acids. Tris is an
effective buffer for slightly basic solutions, which keeps DNA
deprotonated and soluble in water. Tris is commonly combined with
EDTA to make TB buffer for stabilization and storage of DNA. EDTA
binds to divalent cations, particularly magnesium (Mg2+). These
ions are necessary co-factors for many enzymes; Magnesium is a
co-factor for many DNA-modifying enzymes. Tris is toxic to
mammalian cells, and reacts strongly with pH electrodes. It is a
primary amine, and can thus react with aldehydes.
[0436] Tris-HCl is a solution frequently used in biochemistry made
from Tris base and concentrated hydrochloric acid (HClaq). To make
1 mol/L Tris-Cl dissolve 121.1 g of tris base in 700 ml of double
distilled water, bring to desired pH with concentrated HClaq
(usually 7.5 or 8.0), add double distilled water to 1 L, filter
with 0.5 .mu.m filter, autoclave, and store at room
temperature.
[0437] PEG (Polyethylene glycol) is a polymer comprising repeating
subunits of identical structure, called monomers, and is the most
commercially important polyether. Poly (ethylene glycol) refers to
an oligomer prepared by polymerization of ethylene oxide.
Derivatives of PEG are commonly used, the most common derivative
being the methyl ether methoxypoly (ethylene glycol)), abbreviated
mPEG.
[0438] The melting points of PEG and mPEG vary depending on the
formula weight of the polymer. PEG has the structure
HO--(CH2-CH2O)-n-H and is used as a linker molecule for its
flexibility and length.
[0439] STM (Scanning tunneling microscopy) is a powerful technique
for viewing surfaces at the atomic level. The STM is based on the
concept of quantum tunneling. When a conducting tip is brought very
near to a metallic or semi-conducting surface, a bias between the
two can allow electrons to tunnel through the vacuum between them.
Variations in current as the probe passes over the surface arc
translated into an image.
[0440] PNA (Peptide nucleic acid) is a chemical similar to DNA or
RNA. PNA does not occur naturally but is artificially synthesized.
DNA and RNA have a deoxyribose and ribose sugar backbone,
respectively, whereas PNA's backbone comprises repeating
N-(2-aminoethyl)-glycine units linked by peptide bonds. The various
purine and pyrimidine bases are linked to the backbone by methylene
carbonyl bonds. Since the backbone of PNA contains no charged
phosphate groups, the binding between PNA/DNA strands is stronger
than between. DNA/DNA strands due to the lack of electrostatic
repulsion. PNAs are not easily recognized by either nucleases or
proteases, making them resistant to enzyme degradation. PNAs are
also stable over a wide pH range.
[0441] TEM (Transmission electron microscopy) is an imaging
technique whereby a beam of electrons is transmitted through a
specimen. An image is formed, magnified and directed to appear
either on a fluorescent screen or on layer of photographic film or
is detected by a sensor such as a CCD camera.
[0442] Coenzyme A (CoA, CoASH, or HSCoA) is a coenzyme, notable for
its role in the synthesis and oxidization of fatty acids, and the
oxidation of pyruvate in the citric acid cycle. It is adapted from
cysteamine, pantothenate, and adenosine triphosphate.
* * * * *
References