U.S. patent application number 16/537232 was filed with the patent office on 2020-01-30 for system and method for single molecule detection.
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, Yanan Zhao.
Application Number | 20200033320 16/537232 |
Document ID | / |
Family ID | 59019845 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200033320 |
Kind Code |
A1 |
Lindsay; Stuart ; et
al. |
January 30, 2020 |
SYSTEM AND METHOD FOR SINGLE MOLECULE DETECTION
Abstract
A single molecule sensing or detecting device includes a first
electrode and a second electrode separated from the first electrode
by a gap. The first electrode and the second electrode have an
opening formed therethrough. At least one of the first electrode
and the second electrode is functionalized with a recognition
molecule. The recognition molecule has an effective length L1 and
is configured to selectively bind to a target molecule having an
effective length L2. The size of the gap is configured to be
greater than L2, but less than or equal to the sum of L1 and
L2.
Inventors: |
Lindsay; Stuart; (Phoenix,
AZ) ; Zhang; Peiming; (Gilbert, AZ) ; Zhao;
Yanan; (Tempe, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIZONA BOARD OF REGENTS on behalf of ARIZONA STATE
UNIVERSITY |
Scottsdale |
AZ |
US |
|
|
Family ID: |
59019845 |
Appl. No.: |
16/537232 |
Filed: |
August 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15375901 |
Dec 12, 2016 |
10379102 |
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16537232 |
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62266282 |
Dec 11, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/48721
20130101 |
International
Class: |
G01N 33/487 20060101
G01N033/487 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under R01
HG006323 awarded by The National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A sensing device for sensing protein fluctuations comprising: a
first electrode; a second electrode separated from the first
electrode by a gap, a first recognition molecule having a first end
and a second end, the first end being connected to the first
electrode and the second end being connected to a protein at a
first point; a second recognition molecule having a first end and a
second end, the first end being connected to the second electrode
and the second end being connected to the protein at a second
point; a means for applying a voltage bias; and a detector
configured to detect fluctuations in current.
2. The sensing device of claim 1, wherein the first and the second
recognition molecules are identical.
3. The device of claim 1, wherein the size of the gap is between
about 2 nm to about 15 nm.
4. The device of claim 1, wherein the size of the gap is between
about 2 nm to about 10 nm.
5. The device of claim 1, wherein the size of the gap is between
about 5 nm to about 15 nm.
6. The device of claim 1, wherein the first and second recognition
molecules comprise a peptide.
7. A method for detecting protein fluctuations, the method
comprising: applying a voltage bias across a first electrode to a
second electrode of a sensing device, the sensing device
comprising: the first electrode; the second electrode separated
from the first electrode by a gap, a first recognition molecule
having a first end and a second end, the first end being connected
to the first electrode and the second end being connected to a
protein at a first point; a second recognition molecule having a
first end and a second end, the first end being connected to the
second electrode and the second end being connected to the protein
at a second point; monitoring current generated between the first
and second electrode over time; wherein protein fluctuation is
detected if the current generated fluctuates.
8. The method of claim 7, wherein the first and the second
recognition molecules are identical.
9. The method of claim 7, wherein the first and the second
recognition molecules are identical.
10. The method of claim 7, wherein the size of the gap is between
about 2 nm to about 15 nm.
11. The method of claim 7, wherein the size of the gap is between
about 2 nm to about 10 nm.
12. The method of claim 7, wherein the size of the gap is between
about 5 nm to about 15 nm.
13. The method of claim 7, wherein the first and second recognition
molecules comprise a peptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 15/375,901, filed on Dec. 12, 2016, which
claims priority to and the benefits of U.S. Provisional Application
No. 62/266,282, filed on Dec. 11, 2015, the contents of each of
which are incorporated herein by reference in their entireties.
ABSTRACT OF THE DISCLOSURE
[0003] The present disclosure presents systems, methods and devices
for detecting single molecules by direct electronic measurement as
they bind a cognate ligand. In some embodiments, high contrast
signals are produced with no labels and sample concentrations in
the femtomolar range.
BACKGROUND
[0004] Electron tunneling is, in principle, sensitive to the
presence of a molecule in a tunnel gap formed between two closely
spaced metal electrodes (Zwolak and Di Ventra 2005). However, in
practice, tunnel gaps are quite insensitive to molecules that may
be trapped between the electrodes because the inevitable
hydrocarbon contamination of metal electrodes outside of an
ultrahigh vacuum clean environment makes for a poor contact between
the electrodes and the molecules.
[0005] It has been shown that reproducible and characteristic
electrical signals can be obtained if molecules are chemically
attached to each electrode forming a tunnel junction, by, for
example, sulfur-metal bonds (Cui, Primak et al. 2001). Such
permanent connections, however, do not make for versatile detectors
because the molecule that bridges the gap must be modified at two
sites with groups such as thiols. Pishrody et al. (Pishrody, Kunwar
et al. 2004), proposed a solution in which electrode pairs were
functionalized with molecules that did not, by themselves bridge
the gap, but rather, formed a bridged structure when a target
molecule became bound. This prior art is illustrated in FIG. 1. As
shown, a first metal electrode 10 and a second metal electrode 12
are separated by a dielectric layer 16 with the electrode gap
exposed at the edge of the layered device. A first recognition
molecule 14a and a second recognition molecule 14b are chemically
tethered to the electrodes by reactive groups 18. The molecules 14a
and 14b are chosen so as to bind a target molecule 20 in such a way
as to form a bridge across the gap between the electrodes when 20
binds both 14a and 14b. For example 14a and 14b may be composed of
DNA oligomers chosen so have a sequence that, taken together, is
complementary to a target DNA molecule 20. However, the simple
device of FIG. 1 cannot be used as a single molecule detector, but
rather, only as a system of a large number of such devices
functionalized with many pairs of recognition molecules. In this
way, the presence of certain molecules in a sample could be
determined upon the measurement of current from many binding
events.
[0006] U.S. publication no. 2010/0084276 (Lindsay et al.) discloses
a device designed for sequencing polymers, such as DNA. In some
embodiments of this prior art, as illustrated in FIG. 2, two
closely spaced electrodes 30, 31 are separated by dielectric layer
33. A nanopore 34 is then drilled through the structure and the
exposed electrodes functionalized with recognition molecules 35.
The molecules bind to a target analyte 36 at two separate sites.
Thus, once an analyte molecule enters the pore, it brings together
the recognition molecules to form a connected pathway across the
gap. The approach of such embodiments differ from that of Pishrody
at least because (a) the nanopore of Lindsay et al. permits only
one analyte to enter at a time (e.g., so that a polymer may be
sequenced, as each chemical unit of the polymer enters the pore and
generates a characteristic signal), and (b) the gap between the
electrodes 30 and 31 is sized such that that single molecule
binding event generates a large current.
SUMMARY OF SOME OF THE EMBODIMENTS OF THE DISCLOSURE
[0007] It is an object of at least some of the embodiments of the
present disclosure to provide a device that detects single molecule
binding events by, for example, direct electronic detection of
binding on only a single ligand, e.g., such as an antibody.
[0008] It is another object of at least some of the embodiments of
the present disclosure to provide a device with a large exposed
junction area configured for sensing low concentrations of samples
rapidly. For example, in some embodiments, such junction areas
correspond to junction gaps of from 0.1 to 100 nm, with the lateral
extent of the junctions ranging from 1 nm to 100 microns. Sample
concentrations can be as low as one femtomolar, or even lower. A
large junction area can be configured to collect molecules from a
large sample volume, so that the time for molecules to diffuse into
the junction can be small. For example, for a junction of a few
microns in lateral extent, and a gap size of 4 nm, exposure to a
concentration of 100 femtomoles results in generation of signals in
about 10 s.
[0009] In some embodiments, a device for sensing molecules in
solution is provided which includes a first electrode and a second
electrode separated from the first electrode by a gap. One or more
of the electrodes are functionalized with one or more recognition
molecules having an effective length L1 and configured to
selectively bind to a target molecule having an effective length
L2. The gap is configured to be greater than L2, but less than or
equal to the total of L1 and L2.
[0010] In some embodiments, a method for sensing molecules in
solution is provided, which includes providing the device according
to some embodiments of the disclosure (e.g., the device embodiment
above), applying a voltage bias across the electrodes, providing a
sample to the device, monitoring current over time to determine at
least one of the features thereof of a background and noise spikes,
and determining, based on at least one of the background and noise
spikes, determining one or more of: the presence of the target
molecule; and a number of non-target molecules adsorbed on the
first electrode and/or on the second electrode.
[0011] In some embodiments, a device includes a first electrode and
a second electrode separated from the first electrode by a gap. At
least one of the first electrode and the second electrode is
functionalized with a recognition molecule. The recognition
molecule has an effective length L1 and is configured to
selectively bind to a target molecule having an effective length
L2. The gap is configured to be greater than L2 in thickness, but
less than or equal to the sum of L1 and L2.
[0012] In some embodiments, a method includes applying a voltage
bias across a first electrode and a second electrode of a device.
The second electrode is separated from the first electrode by a
gap. At least one of the first electrode and the second electrode
is functionalized with a recognition molecule that has an effective
length L1 and is configured to selectively bind to a target
molecule having an effective length L2. The method also includes
contacting the first electrode and the second electrode with a
solution containing the target molecule in a concentration from
about 10 fM to about 10 pM. The method also includes monitoring
current generated between the first electrode and the second
electrode over time. The method also includes determining one or
more of: based on a fluctuating portion of the current, the
presence of the target molecule; and based on a background portion
of the current, a number of non-target molecules adsorbed on the
first electrode and on the second electrode.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 illustrates a bridged electrode pair according to the
prior art.
[0014] FIG. 2 illustrates a bridged electrode pair within a
nanopore according to the prior art.
[0015] FIG. 3 illustrates a model of .alpha.v.beta..sub.3
integrin.
[0016] FIGS. 4a-b illustrate (a) Cyclic RGD peptide. (b) Binding
site of RGD peptide at the junction of the .alpha. and .beta.
subunits of integrin.
[0017] FIG. 5 illustrates (a) Scanning tunneling microscope
experiment to demonstrate capture of .alpha.v.beta..sub.3 integrin
with functionalization of just one electrode. (b) Typical current
trace when an integrin is captured as the probe is withdrawn.
[0018] FIG. 6 illustrates (a) histogram of current peak values for
integrin capture (current is shown increasing negative to the left
here). (b) Distribution of withdrawal distances to the peak signal
(the starting gap, 0 on this plot, is 2.7 nm).
[0019] FIG. 7, illustrates a cross-sectional view of a tunnel
junction edge of a device as exposed by an opening, according to
some embodiments of the present disclosure.
[0020] FIG. 8 illustrates current data recorded for two
concentrations of .alpha..sub.4.beta..sub.1 integrin, a molecule
not bound by the RGD peptide with a device according to some
embodiments.
[0021] FIG. 9 illustrates current data from a device according to
some embodiments, in buffer solution (a) and then after adding 1 pM
(b), and then 10 pM (c) of .alpha.v.beta..sub.3 integrin.
[0022] FIG. 10 illustrates control signals (a, buffer, b,
.alpha..sub.4.beta..sub.1integrin) for a nanopore device according
to some embodiments of the present disclosure into which only a
single molecule can be received.
[0023] FIG. 11 illustrates a signal obtained when 1 nM
.alpha.v.beta..sub.3 integrin was placed in contact with the
nanopore device according to some embodiments of the
disclosure.
[0024] FIG. 12 illustrates cyclic voltammetry of a palladium
electrode of a device according to some embodiments of the present
disclosure functionalized with cyclic RGD peptide and exposed to a
.alpha.v.beta..sub.3 integrin solution. Scale is millivolts
relative to a silver wire quasi reference.
[0025] FIG. 13 illustrates output of a device according to some
embodiments with a large (micron sized) junction length exposed to
a 10 pM solution of .alpha..sub.4.beta..sub.1 integrin (lower
trace) followed by a 10 femtomolar solution of .alpha.v.beta..sub.3
integrin (upper trace).
DESCRIPTION OF THE PRESENT INVENTION IN ITS PREFERRED
EMBODIMENT
[0026] A single molecule sensing or detecting device includes a
first electrode and a second electrode separated from the first
electrode by a gap. The first electrode and the second electrode
have an opening formed therethrough. At least one of the first
electrode and the second electrode is functionalized with a
recognition molecule. The recognition molecule has an effective
length L1 and is configured to selectively bind to a target
molecule having an effective length L2. The size of the gap is
configured to be greater than L2, but less than or equal to the sum
of L1 and L2.
[0027] In some embodiments, the device further includes an
insulating layer disposed in the gap, wherein a thickness of the
insulating layer is less than or equal to the sum of L1 and L2. In
some embodiments, the size of the gap is at least twice the
effective length L1. In some embodiments, the size of the gap is
equal to the sum of L1 and L2. In some embodiments, the size of the
gap is between about 2 nm to about 15 nm. In some embodiments, the
size of the gap is between about 2 nm to about 10 nm. In some
embodiments, the size of the gap is between about 5 nm to about 15
nm. In some embodiments, the recognition molecule includes any
suitable peptide such as, for example, a cyclic RGD peptide. In
some embodiments, the size of the opening is between 0.1 nm and 100
microns in a linear dimension.
[0028] In some embodiments, the first electrode and/or the second
electrode are configured to generate a current upon binding of the
target molecule. and the current includes a fluctuating portion
and/or a background portion. In some embodiments, the background
portion of the current is based on a number of non-target molecules
adsorbed on the first electrode and/or on the second electrode. In
some embodiments, the fluctuating portion is based on a
concentration of the target molecule in a solution containing the
target molecule, the solution in contact with the first electrode
and the second electrode, and the concentration of the target
molecule in the solution is from about 10 fM to about 1 .mu.M.
[0029] In some embodiments, a method for sensing or detecting a
target molecule includes applying a voltage bias across a first
electrode and a second electrode of a molecular sensing or
detecting device. The first electrode and second electrode
collectively have an opening formed therethrough. The second
electrode separated from the first electrode by a gap, and at least
one of the first electrode and the second electrode is
functionalized with a recognition molecule. The recognition
molecule includes an effective length L1 and is configured to
selectively bind to a target molecule having an effective length
L2. The method also includes contacting the first electrode and the
second electrode with a solution containing the target molecule in
a concentration from about 10 fM to about 1 .mu.M. The method also
includes monitoring current generated between the first electrode
and the second electrode over time. The method also includes
determining one or more of: the presence of the target molecule;
and a number of non-target molecules adsorbed on the first
electrode and/or on the second electrode.
[0030] In some embodiments, determining the presence of the target
molecule is based on a fluctuating portion of the current. In some
embodiments, determining a number of non-target molecules adsorbed
on the first electrode and/or on the second electrode is based on a
background portion of the current. In some embodiments, the device
further includes an insulating layer disposed in the gap, and a
thickness of the insulating layer is less than or equal to the sum
of L1 and L2. In some embodiments, the gap is at least twice the
effective length L1 in thickness. In some embodiments, the size of
the gap is equal to the sum of L1 and L2. In some embodiments, the
size of the gap is between about 2 nm to about 15 nm. In some
embodiments, the size of the gap is between about 2 nm to about 10
nm. In some embodiments, the size of the gap is between about 5 nm
to about 15 nm. In some embodiments, the recognition molecule
includes a peptide. In some embodiments, the peptide is a cyclic
RGD peptide.
[0031] It is commonly assumed that proteins are excellent
insulators. Direct measurements of the conductance of small
peptides (i.e., short protein fragments) in their linear form shows
that current decays very rapidly with an increase in the length
(i.e., number of amino acid residues) of the peptide (Xiao, Xu et
al. 2004). However, scanning-tunneling microscope studies of
electron-transfer proteins (Ulstrup 1979, Artes, Diez-Perez et al.
2012), can show remarkably large conductance values. While these
values are impossible to reconcile with the short electronic decay
lengths measured in peptides, it has recently been suggested that
many proteins, in their three dimensional, folded form, are poised
in a critical state between being a bulk conductor (metal-like) and
an insulator, such that local fluctuations can drive proteins into
states that are transiently conductive (Vattay, Salahub et al.
2015). Accordingly, some embodiments of the present disclosure are
disclosed which enable proteins to form highly conductive bridges
across gaps between electrodes that are much larger than could
possibly support electron tunneling currents. Even with the most
favorable electronic properties of a molecule in a tunnel junction,
tunnel conductances drop below femtoseimens for distances of 3 to 4
nm. Such large gaps provide, in at least some embodiments, a large
current signal, even when the target protein is bound to only one
electrode by a recognition reagent, with currents corresponding to
nanoseimens of conductance.
EXAMPLES
[0032] To illustrate the process we use the example of
.alpha.v.beta..sub.3 integrin, which comprises two subunits (the
.alpha. and .beta. chains) that meet at the apex of pyramidal shape
that is about (in some embodiments) 9 nm high (FIG. 3). This
protein is strongly bound by a cyclic RGB peptide (FIG. 4a) at a
unique site near the apex of the pyramidal shape (FIG. 4b). In FIG.
4b, 41 is the junction between the .alpha. and .beta. chains and 40
is the cyclic RGD peptide (Choi, Kim et al. 2010). The peptide is
relatively small being about 1 nm across its widest folded
dimension.
[0033] Accordingly, in some embodiments, functionalizing just one
of a pair of electrodes generates a unique electrical recognition
signal for a corresponding molecule(s). To do this, a scanning
tunneling microscope (STM) was used (see STM, FIG. 5a), where a
gold probe 51 was functionalized with the RGD peptide 53 via the
chemical interaction between the cysteine residue and the gold. The
probe was positioned at a set point bias (V) and current (I) such
that the apex of the probe was held approximately 2.7 nm above a
bare gold substrate, 52. As the probe was pulled away an extra
distance .DELTA.Z from the surface, a decaying current could be
observed during some of the experiments (e.g., feature 57 in FIG.
5b). However, in the absence of the .alpha.v.beta..sub.3 integrin,
no other features were observed, even if fairly high concentrations
(e.g., 100 nM) of a protein such as BSA were added to the solution
in the STM. Once 100 nM .alpha.v.beta..sub.3 integrin 56 was added
to the solution, a new feature appeared as a current peak away from
the origin 58 in FIG. 5b.
[0034] A statistical analysis of the distribution of features in
terms of the peak current (FIG. 6a) and distance above
approximately 2.7 nm for which a peak occurs (.DELTA.Z in FIG. 6b)
shows that signals of many tens of picoamps are generated at
distances of about 3 nm to about 6 nm overall (2.7 nm +.DELTA.Z).
In contrast to conventional tunneling signals, these signals peak
when the probe is some distance away from the surface, signifying
that the probe has captured a conductive particle. Importantly, no
such features were seen in the absence of the .alpha.v.beta..sub.3
integrin, or in the presence of a protein (BSA) that does not bind
the cyclic RGD peptide.
[0035] FIG. 7 illustrates a cross-sectional view of a tunnel
junction edge (i.e., the edge of an opening) of the device
according to some embodiments of the present disclosure which
includes a first metal electrode 71 (e.g., palladium, gold and/or
platinum) onto which is deposited a layer of an insulating
dielectric such as alumina 72 (for example). A second metal
electrode 73 is then deposited, typically using one of the metals
used for the first electrode. In some embodiments (not shown), the
first electrode 71 and the second electrode 73 A cut is then made
in the structure to expose the edge of these layers (fabrication of
this type of device is described in detail in co-pending, published
WO2015/161119, and also in Pang, Ashcroft et al. 2014) to form the
opening/nanopore 78, shown here as a plane adjacent to the
elctrodes 71, 73. Accordingly, the first electrode 71 and the
second electrode 73 can have an opening formed therethrough such
as, for example, a nanopore. Said another way, the first electrode
71 and the second electrode 73 can be said to be arranged within or
adjacent to an opening, or have a nanopore formed therethrough.
[0036] In some such embodiments (of those illustrated in, e.g.,
FIG. 7), it is a particular feature of such embodiments in the use
of, depending upon the embodiments, either or both of (a)
recognition molecules that bind a target at only one site, so that
the geometric constraints of forming a chemical bridge do not
apply, and (b) a choice of dimensions that gives a very high
signal-to-background ratio. Specifically, for example, the
insulating layer 72 is deposited with a thickness d that is chosen
to be greater than the longest linear dimension of the recognition
molecules 74 (L.sub.1, referred to as its effective length).
However, according to some embodiments, it is chosen to be less
than the combined overall length of the largest dimension of a
target molecule 75 (L.sub.2, also referred to as its effective
length) bound to the recognition molecule 74 (L.sub.1+L.sub.2). In
the case of the integrin-RGD pair (FIGS. 3 and 4) the RGD molecule
is about 1 nm at its longest, while the integrin is 9 nm, for a
total of about 10 nm. This is a distance over which electron
tunneling currents generally do not flow because the tunneling
probability would be infinitesimal. However, a large particle that
fluctuates into a highly conductive state could be used to mediate
current flow (Vattay, Salahub et al. 2015). In some embodiments,
the gap need only be made substantially larger than twice the
largest dimension of the recognition molecules (i.e.,
>2L.sub.1). Thus, for example, if a recognition molecule is 1 nm
at its longest dimension, then the gap is configured to be greater
than 2 nm, preferably by about 10%, to accommodate variations in
the junction geometry (larger than 2.2 nm in this example). In some
embodiments, a gap size can be from the noted minimum up to the
size of one recognition molecule plus the size of the target
protein. For example, if the largest dimension of the protein is 9
nm, then the gap in this case can be as big 9 nm plus the size of
one of the recognition molecules (1 nm in this example), thus, a
gap of 10 nm. In experiments, devices functionalized with the
cyclic RGD peptide and fabricated with a gap d of 3.5 to 4 nm show
no background current, which continues to be the case even when the
junctions are exposed to a homologous protein
(.alpha..sub.4.beta..sub.1 integrin) that does not bind the RGD
peptide. FIG. 8 shows current-vs time traces for devices in contact
with 10 pM (a) and 100 pM (b) solutions of
.alpha..sub.4.beta..sub.1 integrin.
[0037] However, when the junctions are exposed to the target
protein (.alpha..sub.v.beta..sub.3 integrin) signals appear
immediately. FIG. 9 shows (a) the signal in 1 mM phosphate buffer
(pH 7.0) just before the addition of a 1 pM solution of
.alpha..sub.v.beta..sub.3 integrin in the same buffer solution (b).
A clear signal is immediately generated which includes two (2)
features as marked: a background current (of about 0.5 nA in this
case) and noise spikes of 0.5 to 1 nA superimposed on top. On
increasing the concentration of .alpha..sub.v.beta..sub.3 integrin
to 10 pM, the background current increases by nearly an order of
magnitude (while the fluctuations remain generally constant).
[0038] Accordingly, in some embodiments, the background signal
corresponds to the number of molecules adsorbed on the electrodes.
This can be substantiated by collecting signals from a device small
enough to allow only one integrin molecule to be trapped. In such a
device, experiments were performed where the electrode edges were
exposed by drilling a nanopore of approximately 12 nm diameter
through the junction device. The electrodes were functionalized
again with the cyclic RGD peptide. FIG. 10 shows that in phosphate
buffer (a) or in the presence of a 1 nM solution of the non-binding
control (.alpha..sub.4.beta..sub.1 integrin) no signals are
generated. However, when 1 nM .alpha.v.beta..sub.3 integrin is
added a signal is generated (FIG. 11). Note that even though the
concentration of the protein is 100.times. that used to generate
the signals shown in FIG. 9b, there is essentially no background
current, only the fluctuating current component (of about 0.2 nA in
this case). This is because there is room for only one molecule at
a time in the device, and this confirms that, in some embodiments,
the background current arises from adsorption of many
molecules.
[0039] Stable operation of the device requires control of the
operating potential as described for similar devices in PCT
publication no. WO2015/130781, entitled, "Methods, Apparatuses and
Systems for Stabilizing Nano-Electronic Devices in Contact with
Solutions", the entire disclosure of which is herein incorporated
by reference. FIG. 12 shows cyclic voltamograms taken with an RGD
functionalized palladium electrode in the presence of a solution of
.alpha.v.beta..sub.3 integrin. As shown, Faradaic current begins to
rise above about 400 mV (with respect to a silver wire quasi
reference electrode). Since, this is the upper limit of the bias
applied to the across the electrode gap, the device operates stably
if one electrode is connected to a silver wire (or Ag/AgCl)
reference and the other electrode is kept below +400 mV with
respect to the reference.
[0040] In experiments, the concentration used to obtain signals
with the single molecule capture device had to be quite high (i.e.,
nanomolar or higher) in order for the probability of capturing a
single molecule in a reasonable time to be significant. In some
embodiments, this probability is proportional to the volume from
which molecules can be captured in a reasonable time. For example,
if the molecules diffuse freely with a diffusion constant D (e.g.,
about 10.sup.-11m.sup.2/s), then the volume from which molecules
can be collected in a time t, over a linear junction length L, is
given approximately by .pi.r.sup.2L where r.sup.2=Dt. Taking t=60 s
and L=36 nm (approximately the length of the junction around the
edge of a 12 nm diameter pore), about 40 molecules would be present
at 1 nM concentration in the resulting volume of
6.5.times.10.sup.-17 m.sup.3 (=6.5.times.10.sup.-14 liters).
Referring to FIG. 7, if the junction length, X, is greatly
increased (over the value of L given for the perimeter of a
nanopore earlier, L=36 nm in the example given) then
correspondingly, the sensitivity of the device will also increase.
Thus, for a device with X=10 .mu.m, the capture volume in 1 minute
capture time becomes 10.sup.-14 m.sup.3 or almost 100.times.
greater. Thus, signals are readily obtained at 1 pM concentrations
as shown in FIG. 9b. In fact, upon the solution being flowed over
the device, the effective capture length is orders of magnitude
greater. For the cyclic RGD peptide, capturing .alpha.v.beta..sub.3
integrin, the binding process appears to be almost irreversible, so
essentially all of the molecules within a capture radius can be
swept up. Thus, if about a linear cm of fluid is flowed past a
junction slowly enough that each volume of length equal to the
junction length spends about a minute over the junction, then
concentrations as small as a femtomole will yield a signal.
[0041] FIG. 13 shows an experiment in which a 10 pM solution of
.alpha..sub.4.beta..sub.1 integrin was flowed over large (X=0.1
.mu.m junction) for several minutes with no signal being produced.
When 10 fM of .alpha.v.beta..sub.3 integrin was introduced, a large
signal appeared after a few minutes, which substantially exceeds
the sensitivity estimated above (where much longer exposure times
would be required for the even larger (X=10 .mu.m) junction
geometry.
[0042] One of skill in the art recognizes that the specific
dimensions given here are exemplary only. For example, a much
larger gap (e.g., 5 to 15 nm), can be used if the recognition
molecules (cognate ligands) are full sized antibodies (e.g., about
10 nm in extent), so the gap size (d in FIG. 7) would be, for
example, 20 nm. An alternative to antibodies could be single-domain
antibodies such as those produced by Abcore Inc. (for example).
Such single-domain antibodies include molecular weights of 50 kD
and linear dimensions of around 2.5 nm, so gaps of 5 nm would be
appropriate.
[0043] Any and all references to publications or other documents,
including but not limited to, patents, patent applications,
articles, webpages, books, etc., presented anywhere in the present
application, are herein incorporated by reference in their
entirety.
[0044] As noted elsewhere, the disclosed embodiments have been
presented for illustrative purposes only and are not limiting.
Other embodiments are possible and are covered by the disclosure,
which will be apparent from the teachings contained herein. Thus,
the breadth and scope of the disclosure should not be limited by
any of the above-described embodiments but should be defined only
in accordance with claims supported by the present disclosure and
their equivalents. Moreover, embodiments of the subject disclosure
may include methods, compositions, systems and apparatuses/devices
which may further include any and all elements from any other
disclosed methods, compositions, systems, and devices, including
any and all elements corresponding to detecting one or more target
molecules (e.g., DNA, proteins, and/or components thereof). In
other words, elements from one or another disclosed embodiments may
be interchangeable with elements from other disclosed embodiments.
Moreover, some further embodiments may be realized by combining one
and/or another feature disclosed herein with methods, compositions,
systems and devices, and one or more features thereof, disclosed in
materials incorporated by reference. In addition, one or more
features/elements of disclosed embodiments may be removed and still
result in patentable subject matter (and thus, resulting in yet
more embodiments of the subject disclosure). Furthermore, some
embodiments correspond to methods, compositions, systems, and
devices which specifically lack one and/or another element,
structure, and/or steps (as applicable), as compared to teachings
of the prior art, and therefore represent patentable subject matter
and are distinguishable therefrom (i.e. claims directed to such
embodiments may contain negative limitations to note the lack of
one or more features prior art teachings).
[0045] Also, while some of the embodiments disclosed are directed
to detection of a protein molecule, within the scope of some of the
embodiments of the disclosure is the ability to detect other types
of molecules.
[0046] When describing the molecular detecting methods, systems and
devices, terms such as linked, bound, connect, attach, interact,
and so forth should be understood as referring to linkages that
result in the joining of the elements being referred to, whether
such joining is permanent or potentially reversible. These terms
should not be read as requiring a specific bond type except as
expressly stated.
[0047] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0048] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0049] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0050] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of" or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of" "only one of"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0051] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0052] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
LITERATURE CITED
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