U.S. patent application number 15/160697 was filed with the patent office on 2016-09-08 for target detection with nanopore.
The applicant listed for this patent is Target Detection with Nanopore. Invention is credited to William B. Dunbar, Daniel Alexander Heller, Trevor J. Morin.
Application Number | 20160258939 15/160697 |
Document ID | / |
Family ID | 56690322 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160258939 |
Kind Code |
A1 |
Morin; Trevor J. ; et
al. |
September 8, 2016 |
Target Detection with Nanopore
Abstract
Provided are methods for detecting a target molecule or particle
suspected to be present in a sample, comprising (a) contacting the
sample with (i) a fusion molecule comprising a ligand capable of
binding to the target molecule or particle and a binding domain,
and (ii) a polymer scaffold comprising at least one binding motif
to which the binding domain is capable of binding, under conditions
that allow the target molecule or particle to bind to the ligand
and the binding domain to bind to the binding motif; (b) loading
the polymer into a device comprising a pore comprising an opening
in a structure that separates an interior space of the device into
two volumes, and configuring the device to pass the polymer through
the pore from one volume to the other volume, wherein the device
further comprises a sensor configured to identify objects passing
through the pore; and (c) determining, with the sensor, whether the
fusion molecule or particle bound to the binding motif is bound to
the target molecule or particle, thereby detecting the presence of
the target molecule or particle in the sample.
Inventors: |
Morin; Trevor J.; (Santa
Cruz, CA) ; Heller; Daniel Alexander; (Santa Cruz,
CA) ; Dunbar; William B.; (Santa Cruz, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Target Detection with Nanopore |
Santa Cruz |
CA |
US |
|
|
Family ID: |
56690322 |
Appl. No.: |
15/160697 |
Filed: |
May 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2014/046397 |
Jul 11, 2014 |
|
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15160697 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6804 20130101;
G01N 33/537 20130101; C12Q 1/6869 20130101; B82Y 15/00 20130101;
G01N 33/48721 20130101; G01N 33/54306 20130101; G01N 33/54366
20130101; C12Q 1/6825 20130101; G01N 33/6872 20130101; C12Q 1/6825
20130101; C12Q 2522/101 20130101; C12Q 2565/631 20130101; C12Q
1/6869 20130101; C12Q 2522/101 20130101; C12Q 2565/631 20130101;
C12Q 1/6804 20130101; C12Q 2522/101 20130101; C12Q 2565/631
20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. A method for detecting a target molecule or particle suspected
to be present in a sample, comprising: contacting the sample with
(i) a fusion molecule comprising a ligand capable of binding to the
target molecule or particle and a binding domain, and (ii) a
polymer scaffold comprising at least one binding motif to which the
binding domain is capable of binding, under conditions that allow
the target molecule or particle to bind to the ligand and the
binding domain to bind to the binding motif; loading the polymer
into a device comprising a pore that separates an interior space of
the device into two volumes, and configuring the device to pass the
polymer through the pore from one volume to the other volume,
wherein the device comprises a sensor configured to identify
objects passing through the pore; and determining, with the sensor,
whether the fusion molecule bound to the binding motif is bound to
the target molecule or particle, thereby detecting the presence of
the target molecule or particle in the sample.
2. The method of claim 1, wherein the target molecule is selected
from the group consisting of a protein, a peptide, a nucleic acid,
a chemical compound, an ion, and an element.
3. The method of claim 1, wherein the target particle is selected
from the group consisting of a protein complex or aggregate, a
protein/nucleic acid complex, a fragmented or fully assembled
virus, a bacterium, a cell, and a cellular aggregate.
4. The method of claim 1, wherein step (a) is performed prior to
step (b).
5. The method of claim 1, wherein step (b) is performed prior to
step (a).
6. The method of claim 1, further comprising applying a condition
suspected to alter the binding between the target molecule or
particle and the ligand, and carrying out the determination
again.
7. The method of claim 6, wherein the condition is selected from
the group consisting of removing the target molecule or particle
from the sample, adding an agent that competes with the target
molecule or particle, or the ligand for binding, and changing the
pH, salt, or temperature.
8. The method of claim 1, wherein the binding motif comprises a
chemical modification for binding to the binding domain.
9. The method of claim 8, wherein the chemical modification is
selected from the group consisting of acetylation, methylation,
summolation, glycosylation, phosphorylation, and oxidation.
10. The method of claim 1, wherein the polymer is at least one of a
deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide
nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.
11. The method of claim 1, wherein the binding domain is selected
from the group consisting of a helix-turn-helix, a zinc finger, a
leucine zipper, a winged helix, a winged helix turn helix, a
helix-loop-helix, and a high mobility group box (HMG-box).
12. The method of claim 1, wherein the binding domain is selected
from the group consisting of locked nucleic acids (LNAs), peptide
nucleic acids (PNAs), transcription activator-like effector
nucleases (TALENs), clustered regularly interspaced short
palindromic repeats (CRISPRs), dendrimers, peptides, and
aptamers.
13. The method of claim 1, wherein the ligand is selected from the
group consisting of an antibody, an antibody fragment, an epitope,
a hormone, a neurotransmitter, a cytokine, a growth factor, a cell
recognition molecule, a nucleic acid, a peptide, an aptamer, and a
receptor.
14. The method of claim 1, wherein the binding domain and the
ligand are linked via an interaction selected from the group
consisting of a covalent bond, a hydrogen bond, an ionic bond, a
metallic bond, a van der Walls force, a hydrophobic interaction,
and a planar stacking interaction, or are translated as a
continuous polypeptide, to form the fusion molecule.
15. The method of claim 1, further comprising contacting the sample
with a detectable label capable of binding to the target molecule
or particle, or the target molecule or particle/ligand complex.
16. The method of claim 1, wherein the polymer comprises at least
two units of the binding motif.
17. The method of claim 1, wherein the polymer comprises at least
two different binding motifs; the sample is in contact with at
least two fusion molecules each comprising a different binding
domain capable of binding to a different one of the at least two
different binding motifs and a different ligand capable of binding
to a different target molecule or particle; and the sensor is
configured to identify whether the fusion molecule bound to each
binding motif is bound to a target molecule or particle.
18. The method of claim 1, wherein the sensor comprises electrodes
further configured to apply a voltage differential between the two
volumes and measure current flow through the pore.
19. The method of claim 1, wherein the device comprises an upper
chamber, a middle chamber and a lower chamber, wherein the upper
chamber is in communication with the middle chamber through a first
pore, and the middle chamber is in communication with the lower
chamber through a second pore; wherein the first pore and second
pore are about 1 nm to about 100 nm in diameter, and are about 10
nm to about 1000 nm apart from each other; and wherein each of the
chambers comprises an electrode for connecting to a power
supply.
20. The method of claim 1, further comprising moving the polymer in
a reverse direction after the binding motif passes through the
pore, such as to identify, again, whether the fusion molecule bound
to each binding motif is bound to a target molecule or
particle.
21. A kit, package or mixture for detecting the presence of a
target molecule or particle, comprising: a fusion molecule
comprising a ligand capable of binding to the target molecule or
particle and a binding domain; a polymer scaffold comprising at
least one binding motif to which the binding domain is capable of
binding; and a device comprising a pore that separates an interior
space of the device into two volumes, wherein the device is
configured to allow the polymer to pass through the pore from one
volume to the other volume, and wherein the device further
comprises a sensor adjacent to the pore configured to identify
whether the binding motif is (i) bound to the fusion molecule while
the ligand is bound to the target molecule or particle, (ii) bound
to the fusion molecule while the ligand is not bound to the target
molecule or particle, or (iii) not bound to the fusion
molecule.
22. The kit, package or mixture of claim 21, further comprising a
sample suspected of containing the target molecule or particle.
23. The kit, package or mixture of claim 22, wherein the sample
further comprises a detectable label capable of binding to the
target molecule or particle, or the target molecule or
particle/ligand complex.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT Application No.
PCT/US2014/046397, filed Jul. 11, 2014, which claims the benefit of
PCT Application No. PCT/US2014/036861, filed May 5, 2014, the
contents of which are each incorporated by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 20, 2016, is named 34004US_CRF_sequencelisting.txt and is
563 bytes in size.
BACKGROUND
[0003] Detection of nano-scale and micro-scale particles, such as
circulating tumor cells, bacteria and viruses, has immense clinical
utility. Currently available methods include immunohistochemistry
and nucleic acid-based detection, and cell proliferation is
typically required before a sensitive detection can be carried
out.
[0004] Molecular detection and quantitation are also important, and
can be carried out with various methods depending on the type of
the molecule. For instance, a nucleotide sequence can be detected
by virtue of its sequence complementarity to a probe or primer,
through hybridization and/or amplification, or in fewer occasions,
with a protein that recognizes the sequence. A protein, on the
other hand, is commonly detected with an antibody that specifically
recognizes and binds the protein. An enzyme-linked immuno sorbent
assay (ELISA), in this respect, is highly commercialized and
commonly used.
[0005] Methods also exist for detecting or quantitating various
other large or small molecules, such as carbohydrates, chemical
compounds, ions, and elements.
[0006] Methods and systems for highly sensitive detection of
molecules as well as particles, such as tumor cells and pathogenic
organisms, have broad applications, in particular, clinically, for
pathogen detection and disease diagnosis, for instance.
Additionally, such detection may: allow for the personalization of
medical treatments and health programs; facilitate the search for
effective pharmaceutical drug compounds and biotherapeutics; and
enable clinicians to identify abnormal hormones, ions, proteins, or
other molecules produced by a patient's body and/or identify the
presence of poisons, illegal drugs, or other harmful chemicals
ingested or injected into a patient.
[0007] Currently available techniques for the detection of
molecules and particles are generally expensive, labor-intensive,
skill-intensive, and/or time-intensive. A need exists for improved
detection techniques, which produce accurate results quickly,
cheaply, and easily.
SUMMARY
[0008] Various aspects disclosed herein may fulfill one or more of
the above-mentioned needs. The systems and methods described herein
each have several aspects, no single one of which is solely
responsible for its desirable attributes. Without limiting the
scope of this disclosure as expressed by the claims that follow,
the more prominent features will now be discussed briefly. After
considering this discussion, and particularly after reading the
section entitled "Detailed Description," one will understand how
the sample features described herein provide for improved systems
and methods.
[0009] In one embodiment, the present disclosure provides a method
for assaying whether a target molecule or particle is present in a
sample, the method comprising: (a) contacting the sample with (i) a
fusion molecule comprising a ligand capable of binding to the
target molecule or particle and a binding domain, and (ii) a
polymer scaffold comprising at least one binding motif to which the
binding domain of the fusion molecule is capable of binding, under
conditions that allow the target molecule or particle to bind to
the ligand and the binding domain to bind to the binding motif; (b)
loading the polymer into a device comprising a pore that separates
an interior space of the device into two volumes, and configuring
the device to pass the polymer through the pore from one volume to
the other volume, wherein the device comprises a sensor configured
to identify objects passing through the pore; and (c) determining,
with the sensor, whether the fusion molecule bound to the binding
motif is bound to the target molecule or particle, thereby
detecting the presence or absence of the target molecule or
particle in the sample.
[0010] In some aspects, the target molecule is selected from the
group consisting of a protein, a peptide, a nucleic acid, a
chemical compound, a lipid, a receptor, an ion, and an element.
[0011] In some aspects, the target particle is selected from the
group consisting of protein complexes and protein aggregates,
peptide aggregates, protein/nucleic acid complexes, fragmented or
fully assembled viruses, bacteria, cells, and cellular
aggregates.
[0012] In some aspects, step (a) of the method for assaying whether
a target molecule or particle is present in a sample is performed
prior to step (b). In some aspects, step (b) is performed prior to
step (a).
[0013] In some aspects, the method further comprises applying a
condition suspected to alter the binding between the target
molecule or particle and the ligand, and carrying out the
determination again. In some aspects, the condition is selected
from the group consisting of removing the target molecule or
particle from the sample, adding an agent that competes with the
target molecule or particle or the ligand for binding, and changing
the pH, salt concentration, or temperature.
[0014] In some aspects, the binding motif comprises a chemical
modification for binding to the binding domain. In some aspects,
the chemical modification is selected from the group consisting of
acetylation, methylation, summolation, glycosylation,
phosphorylation, and oxidation.
[0015] In some aspects, the polymer comprises a deoxyribonucleic
acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA),
a DNA/RNA hybrid, or a polypeptide. In some aspects, the polymer is
a synthetic scaffold.
[0016] In some aspects, the binding domain is selected from the
group consisting of a helix-turn-helix, a zinc finger, a leucine
zipper, a winged helix, a winged helix turn helix, a
helix-loop-helix and an HMG-box.
[0017] In some aspects, the binding domain is selected from the
group consisting of locked nucleic acids (LNAs), PNAs,
transcription activator-like effector nucleases (TALENs), clustered
regularly interspaced short palindromic repeats (CRISPRs),
peptides, dendrimers, and aptamers (DNA and/or protein).
[0018] In some aspects, the ligand is a protein. In some aspects,
the ligand is selected from the group consisting of an antibody, an
antibody fragment, an epitope, a hormone, a neurotransmitter, a
cytokine, a growth factor, a cell recognition molecule, a nucleic
acid, a peptide, and a receptor. In some aspects, the ligand is an
aptamer (e.g., DNA, protein, or DNA/protein). In some aspects, the
ligand is a small molecule compound.
[0019] In some aspects, the binding domain and the ligand are
linked via a covalent bond, a hydrogen bond, an ionic bond, a
metallic bond, van der Walls force, a hydrophobic interaction, or a
planar stacking interaction, or are translated as a continuous
polypeptide, to form the fusion molecule.
[0020] In some aspects, the method further comprises contacting the
sample with a detectable label capable of binding to the target
molecule, target particle or target/ligand complex.
[0021] In some aspects, the polymer comprises at least two units of
the binding motif.
[0022] In some aspects, the polymer comprises at least two
different binding motifs. In such aspects, the sample is in contact
with at least two fusion molecules, each of which comprises a
different binding domain capable of binding to a different one of
the at least two different binding motifs, and a different ligand
capable of binding to a different target molecule or particle; and
the sensor is configured to identify whether the fusion molecule
bound to each binding motif is bound to a target molecule or
particle.
[0023] In some aspects, the sensor comprises electrodes further
configured to apply a voltage across the two volumes.
[0024] In some aspects, the device comprises an upper chamber, a
middle chamber and a lower chamber, wherein the upper chamber is in
communication with the middle chamber through a first pore, and the
middle chamber is in communication with the lower chamber through a
second pore.
[0025] In one aspect, the first pore and second pore are about 1 nm
to about 100 nm in diameter. Such pores can be suitable for
detecting molecules such as proteins and nucleic acids. In one
aspect, the first pore and second pore are as large as about 50,000
nm in diameter, which can be suitable for detecting larger
particles such as tumor and bacterial cells.
[0026] In some aspects, the pores are about 10 nm to about 1000 nm
apart from each other. In some such aspects, the distance between
the pores is sized such that the polymer scaffold may
simultaneously extend through both the first and second pores. In
other aspects, the pores are more than 1000 nm apart from each
other.
[0027] In some aspects, each of the chambers comprises an electrode
for connecting to a power supply.
[0028] In some aspects, the method further comprises moving the
polymer in a reverse direction after the binding motif passes
through at least one pore, such as to identify, again, whether the
fusion molecule bound to each binding motif is bound to a target
molecule or particle.
[0029] Also provided are kits, packages or mixtures that detect the
presence of a target molecule or particle. In some aspects, the
kit, package or mixture is comprised of (a) a fusion molecule,
which itself is a ligand capable of binding to the target molecule
or particle and a binding domain, (b) a polymer scaffold, which is
comprised of at least one binding motif to which the binding domain
is capable of binding, (c) a device, which is comprised of a pore
that separates an interior space of the device into two volumes,
wherein the device is configured to allow the polymer to pass
through the pore from one volume to the other volume, and wherein
the device is further comprised of a sensor configured to identify
whether the binding motif is (i) bound to the fusion molecule while
the ligand is bound to the target molecule or particle, (ii) bound
to the fusion molecule while the ligand is not bound to the target
molecule or particle, or (iii) not bound to the fusion molecule. In
some aspects, the device is further comprised of a second pore that
further separates the interior space of the device such that three
volumes: an upper chamber, a middle chamber, and a lower chamber,
are present.
[0030] In some aspects, the kit, package or mixture further
comprises a sample suspected of containing the target molecule or
particle. In some aspects, the sample further comprises a
detectable label capable of binding to the target molecule,
particle, ligand/target complex, or ligand/particle complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Provided as embodiments of this disclosure are drawings that
illustrate features by exemplification only, and not
limitation.
[0032] FIG. 1 illustrates the detection of a target molecule or
particle with one embodiment of the presently disclosed method.
[0033] FIG. 2 provides the illustration of a more specific example,
where a double-stranded DNA is used as the polymer scaffold, and a
human immunodeficiency virus (HIV) envelope protein is used as the
ligand. The combination is used to detect an anti-HIV antibody.
[0034] FIG. 3A, FIG. 3B, and FIG. 3C show representative and
idealized current profiles of three example molecules,
demonstrating that binding between a target molecule (or particle)
and a fusion molecule can be detected when passing through a
nanopore, since it has a different current profile, compared to
that of the fusion molecule alone or the DNA alone. Specifically,
FIG. 3A shows current profiles consistent with higher salt
concentrations (>0.4 M KCl, for example at 1M KCl) in the
experimental buffer and a positive applied voltage, generating a
positive current flow through the pore. By another example, FIG. 3B
shows current profiles consistent with lower salt concentrations
(>0.4 M KCl, for example at 100 mM KCl) in the experimental
buffer and again at a positive applied voltage. By another example,
FIG. 3C shows current profiles consistent with lower salt
concentrations (>0.4 M KCl, for example at 100 mM KCl) in the
experimental buffer and a negative applied voltage.
[0035] FIG. 4 illustrates the multiplexing capability of the
present technology by including different binding motifs in the
polymer scaffold. Such multiplexing can be accomplished with one
nanopore or more than one nanopore.
[0036] FIG. 5A, FIG. 5B, and FIG. 5C illustrate a nanopore device
with at least two pores separating multiple chambers.
[0037] Specifically, FIG. 5A is a schematic of a dual-pore chip and
a dual-amplifier electronics configuration for independent voltage
control (V.sub.1 or V.sub.2) and current measurement (l.sub.1, or
l.sub.2) of each pore. Three chambers, A-C, are shown and are
volumetrically separated except by common pores.
[0038] FIG. 5B is a schematic where electrically, V.sub.1 and
V.sub.2 are principally applied across the resistance of each
nanopore by constructing a device that minimizes all access
resistances to effectively decouple l.sub.2 and l.sub.2.
[0039] FIG. 5C depicts a schematic in which competing voltages are
used for control, with arrows showing the direction of each voltage
force.
[0040] FIGS. 6A, 6B, and 6C illustrate a nanopore device having one
pore connecting two chambers and example results from its use.
Specifically, FIG. 6A depicts a schematic diagram of the nanopore
device. FIG. 6B depicts a representative current trace showing a
blockade event resulting from the passage of a double-stranded DNA
passing through the pore. The current amplitude shift amount
(.DELTA.l=l.sub.0-l.sub.B) and duration to are used to quantify the
passage event. FIG. 6C depicts a scatter plot showing the change in
current amount (.DELTA.l) vs. translocation time (t.sub.D) for all
blockade events recorded over 16 minutes.
[0041] FIG. 7A and FIG. 7B depict current traces measured within
one embodiment of a nanopore device fabricated in accordance with
the present invention. The provided current traces show that
unbound dsDNA causes current enhancement events at KCl
concentrations below 0.4 M. Current enhancements appeared as
downward shifts in the provided experiment, since the voltage and
current are both negative (as in FIG. 3C). Specifically, in DNA
alone control experiments using a 10-11 nm diameter pore in 0.1M
KCl at -200 mV, 5.6 kb dsDNA scaffold (FIG. 7A) causes brief
current enhancement events that are 50-70 pA in amplitude and
10-200 microseconds in duration. Likewise, 48 kb Lambda DNA (FIG.
7B) causes current enhancement events 50-70 pA in amplitude and
50-2000 microseconds in duration.
[0042] FIG. 8 depicts a schematic diagram of a polymer scaffold.
Specifically, FIG. 8 shows a 5,631 by dsDNA scaffold and the
location of 10 total VspR binding sites. Of the 10 VspR binding
sites, 5 are of one 14 base-pair sequence, 3 of a different 18 base
pair sequence, and 2 are of a 27 base pair sequence. Also shown are
the distances (in base pairs) between the binding sites.
[0043] FIG. 9A and FIG. 9B each show schematic representations of
embodiments of a nanopore with a scaffold passing therethrough.
Each also shows a resultant current profile associated with the
scaffold passage as measured by one embodiment of the disclosed
nanopore device. In particular, FIG. 9A and FIG. 9B compare events
with DNA scaffold alone (FIG. 9A) and VspR-bound DNA (FIG. 9B).
Specifically, FIG. 9A shows a graphic depicting the 5,631 by dsDNA
scaffold passing through the pore, and a representative current
enhancement event (downward 50 pA shift lasting 100 microseconds)
when the scaffold passes through the pore. FIG. 9B shows a graphic
depicting multiple VspR bound to a dsDNA scaffold that is passing
through the pore, and a representative current attenuation event
(upward 150 pA shift lasting 1.1 milliseconds) when the VspR-bound
scaffold passes through the pore. At an applied voltage of -100 mV,
the open channel current is negative, so downward events correspond
to current enhancement events, and upward events correspond to
current attenuation events (as in FIG. 3C). The shift direction is
preserved, even though the baseline is zeroed for display
purposes.
[0044] FIG. 10 shows ten more representative current attenuation
events depicted in a current profile consistent with the VspR-bound
scaffold passing through the pore. All shifts are consistent with
current attenuations; the baseline is zeroed for display
purposes.
[0045] FIG. 11 shows two representative current events depicted in
a current profile captured in an experiment with 5.6 kb dsDNA
scaffold and RecA protein at 180 mV and 1M KCl using a 16-18 nm
diameter nanopore. The first event is consistent with an unbound
dsDNA or possibly a free RecA (or multiple associated RecA
proteins) passing through the pore, at 280 pA mean current
attenuation lasting 70 microseconds. The second event is consistent
with RecA-bound scaffold passing through the pore, at 1.1 nA mean
current attenuation lasting 2.7 milliseconds. RecA-bound events
commonly display deeper blockades with longer duration.
[0046] FIG. 12 depicts four more current profiles, each showing a
representative current event consistent with RecA-bound scaffold
passing through the pore.
[0047] FIG. 13 shows scatter plots and histograms depicting all
1385 events recorded over 10 minutes in one experiment conducted
using embodiments of methods described herein. In the depicted
graphs, one data point is provided for each event. In particular,
the depicted graphs show: (a) maximum conductance in nS (maximum
current shift in pA divided by voltage in mV) vs. time duration in
seconds, with time duration on a log-scale; (b) a probability
histogram of the maximum conductance shift values; (c) mean
conductance (mean current shift divided by voltage) vs. time
duration, with time duration on a log-scale; (d) a probability
histogram of the mean conductance values; and (e) a probability
histogram of the time duration on a log-scale.
[0048] FIG. 14A, FIG. 14B, and FIG. 14C illustrate results from a
nanopore device detecting DNA/RecA complexes and RecA-antibody on
DNA/RecA complexes, and the results differentiating these complexes
from unbound DNA and also from free RecA.
[0049] Specifically, FIG. 14A is a gel shift assay. Specifically,
the DNA/RecA/mAb ARM191 Gel Shift Experiments (EMSA) have lanes: 1)
Ladder, top rung 5000 bp; 2) Scaffold DNA only in RecA labeling
buffer; 3) DNA/RecA complex, 1:1 RecA protein to theoretical RecA
binding sites; 4) DNA/RecA/Ab complex, DNA/Rec incubated with a
1:2000 dilution of monoclonal Ab ARM191; 5) Scaffold DNA only in Ab
labeling buffer; and 6) Scaffold DNA mixed with mAb (ARM191).
[0050] FIG. 14B shows representative events for DNA (230 pA, 0.1
ms), DNA/RecA (390 pA, 1.1 ms), and probable DNA/RecA/Ab (860 pA,
1.5 ms). RecA-bound DNA event amplitudes are uniformly smaller than
in earlier figures (FIGS. 11-13) since the pore used to measure
these events is considerably larger (27-29 nm in diameter).
[0051] FIG. 14C depicts a (i) Scatter plot of l.DELTA./l vs.
t.sub.D and (ii) horizontal probability histogram of l.DELTA./l for
two separate experiments overlaid. In a RecA alone control
experiment, 0.5 uM RecA (*) was measured at 180 mV in 1M KCl with a
20 nm diameter pore, generating 767 events over 10 min. Note that
only 0.6% of RecA events exceed a criteria of (600 pA, 0.2 ms)
under these conditions. In another experiment, three reagents were
added in sequence in 1M LiCl. First, 0.1 uM DNA (.quadrature.) was
measured at 200 mV with a 20 nm diameter pore, generating 402
events at 0.1 events/sec. After the pore enlarged to 27 nm, 1.25 nM
DNA/RecA (.cndot.) was added, generating 3387 events at 1.44
events/sec. Lastly, 1.25 nM DNA/RecA/Ab (.largecircle.) was added
generating 4953 events at 4.49 events/sec. Events exceeding the
(600 pA, 0.2 ms) criteria grew monotonically from 0% with DNA
alone, to 5.2% (176) with DNA/RecA added, and up to 9.8% (485) with
DNA/RecA/Ab added. While RecA could have increased event durations
in LiCl, as shown for DNA, event amplitudes are unlikely to shift
significantly toward the (600 pA, 0.2 ms) criteria.
[0052] FIG. 15A and FIG. 15B illustrates schematic diagrams of
polymer scaffolds consistent with embodiments of the present
disclosure. Specifically, FIG. 15A shows a 5.6 kb dsDNA scaffold
used in various experiments, the scaffold having been engineered to
bind 12-mer peptide-nucleic-acid (PNA) molecules, with each PNA
having 3 biotinylated sites for binding avidin (e.g., neutravidin,
and or monovalent streptavidin). Also shown is FIG. 15B identifying
the 25 distinct PNA binding sites on the scaffold that localize up
to 75 avidin biomarker binding sites.
[0053] FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D illustrate: (FIG.
16A) a schematic of the 5.6 kb dsDNA scaffold passing through a
nanopore; (FIG. 16B) a schematic of a free neutravidin passing
through a nanopore; (FIG. 16C) a schematic of the dsDNA labeled
with a PNA passing through a nanopore, the PNA having all three
biotin sites bound by Neutravidin; and (FIG. 16D) corresponding
current traces as measured in the chamber above the pore in a
nanopore device fabricated in accordance with the present
disclosure. In FIG. 16D, the current traces depict representative
translocation events in the recorded current from separate nanopore
experiments with DNA alone, Neutravidin alone, and
DNA/PNA/Neutravidin complexes. As detailed in the examples, the
deeper and longer event pattern in the D/P/N experiment is
identified as a DNA/PNA/Neutravidin event and is clearly
distinguished from DNA alone or Neutravidin alone events.
[0054] FIG. 17A, FIG. 17B, and FIG. 17C illustrate: (FIG. 17A) a
scatter plot of the current shift vs. the duration (l.DELTA./l vs.
t.sub.D) of all recorded events for three separate experiments at
200 mV with 1011 nm diameter pores, the experiments including:
(D)--5.6 kb dsDNA alone at 1 nM yielding 1301 events over 16
minutes; (N) Neutravidin alone at 80 nM yielding 2589 events over
11 minutes; and (D/P/N) DNA/PNA/Neut complexes at 60 pM yielding
4198 events over 7.3 minutes. D/P/N subpopulations overlap with the
N and D control experiment populations, with most events in the DPN
experiment matching unbound N event characteristics; (FIG. 17B) a
horizontal probability histogram of l.DELTA./l for the three data
sets. The inset shows a histogram for a subset of 578 DPN events
with t.sub.D>0.08 ms, which attempts to trim out non-DNA events
from the D/P/N data set (from controls, 8% of N events and 54% of D
events have to >0.08 ms). Such DPN events have significant
spread in l.DELTA./l, with 252 (6% of the total) of these
longer-duration events above 2.4 nA, whereas from controls, only 18
(0.7%) N events and 33 (2.5%) D events have (l.DELTA./l,
t.sub.D)>(2.4 nA, 0.08 ms); and (FIG. 17C) DNA/PNA/Neutravidin
Gel Shift Experiments (EMSA) with lanes: 1) sizing Ladder, top rung
5000 bp; 2) DNA only in labeling buffer; 3) DNA/PNA+3.times. excess
Neut to biotin; 4) DNA/PNA+7.times. excess Neut to biotin; 5)
DNA/PNA+16.times. excess Neut to biotin; 6) DNA/PNA+36.times.
excess Neut to biotin; and 7) DNA/PNA in labeling buffer.
[0055] Some or all of the figures are schematic representations for
exemplification; hence, they do not necessarily depict the actual
relative sizes or locations of the elements shown. The figures are
presented for the purpose of illustrating one or more embodiments
with the explicit understanding that they will not be used to limit
the scope or the meaning of the claims that follow below.
DETAILED DESCRIPTION
[0056] Throughout this application, the text refers to various
embodiments of the present devices, compositions, systems, and
methods. The various embodiments described are meant to provide a
variety of illustrative examples and should not be construed as
descriptions of alternative species. Rather, it should be noted
that the descriptions of various embodiments provided herein may be
of overlapping scope. The embodiments discussed herein are merely
illustrative and are not meant to limit the scope of the present
invention.
[0057] Also throughout this disclosure, various publications,
patents and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure in their entireties.
[0058] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "an electrode"
includes a plurality of electrodes, including mixtures thereof.
[0059] As used herein, the term "comprising" is intended to mean
that the systems, devices, and methods include the recited
components or steps, but not excluding others. "Consisting
essentially of" when used to define systems, devices, and methods,
shall mean excluding other components or steps of any essential
significance to the combination. "Consisting of" shall mean
excluding other components or steps. Embodiments defined by each of
these transition terms are within the scope of this invention.
[0060] All numerical designations, e.g., distance, size,
temperature, time, voltage and concentration, including ranges, are
approximations which are varied (+) or (-) by increments of 0.1. It
is to be understood, although not always explicitly stated that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
components described herein are merely exemplary, and that
equivalents of such are known in the art.
[0061] As used herein, "a device comprising a pore that separates
an interior space" shall refer to a device having a pore that
comprises an opening within a structure, the structure separating
an interior space into more than one volume or chamber.
Molecular Detection
[0062] The present disclosure provides methods and systems for
molecular detection and quantitation. In addition, the methods and
systems can also be configured to measure the affinity of a
molecule binding with another molecule. Further, such detection,
quantitation, and measurement can be carried out in a multiplexed
manner, greatly increasing its efficiency.
[0063] FIG. 1 provides an illustration of one embodiment of the
disclosed methods and systems. More specifically, the system
includes a ligand 104 that is capable of binding to a target
molecule 105 to be detected or quantitated. The ligand 104 can be
part of, or be linked to, a binding moiety (referred to as "binding
domain") 103 that is capable of binding to a specific binding motif
101 on a polymer scaffold 109. Together, the ligand 104 and the
binding domain 103 form a fusion molecule 102. In various
embodiments, both components of the fusion molecule 102 (i.e., both
the ligand 104 and the binding domain 103) bind to their respective
targets (e.g., target molecule 105 and binding motif 101,
respectively) with high affinity and specificity.
[0064] Therefore, if all are present in a solution, the fusion
molecule 102 binds, on one end, to a polymer scaffold (or simply,
"polymer") 109 through the specific recognition and binding between
the binding motif 101 and the binding domain 103, and on the other
end, to the target molecule 105 by virtue of the interaction
between the ligand 104 and the target molecule 105. Such bindings
cause the formation of a complex that includes the polymer 109, the
fusion molecule 102 and the target molecule 105.
[0065] The formed complex can be detected using a that includes a
nanopore (or simply, pore) 107, and a sensor. The pore 107 is a
nano-scale or micro-scale opening in a structure separating two
volumes. The sensor 107 may be positioned within or adjacent the
pore 107 or elsewhere within the two volumes. The sensor is
configured to identify objects passing through the pore 107. For
example, in some embodiments, the sensor identifies objects passing
through the pore 107 by detecting a change in a measurable
parameter, wherein the change is indicative of an object passing
through the pore 107. This device is referred throughout as a
"nanopore device". In some embodiments, the nanopore device 108
includes means, such as electrodes connected to power sources, for
moving the polymer 109 from one volume to another, across the pore
107. As the polymer 109 can be charged or be modified to contain
charges, one example of such means generates a potential or voltage
across the pore 107 to facilitate and control the movement of the
polymer 109. In a preferred embodiment, the sensor comprises a pair
of electrodes, which are configured to both detect the passage of
objects, and provide a voltage, across the pore 107. In this
embodiment, a voltage-clamp or a patch-clamp is used to
simultaneously supply a voltage across the pore and measure the
current through the pore.
[0066] When a sample that includes the formed complex is loaded in
the nanopore device 108, the nanopore device 108 can be configured
to pass the polymer 109 through the pore 107. When the binding
motif 101 is within the pore or adjacent to the pore 107, the
binding status of the motif 101 can be detected by the sensor.
[0067] The "binding status" of a binding motif, as used herein,
refers to whether the binding motif is bound to a fusion molecule
with a corresponding binding domain, and whether the fusion
molecule is also bound to a target molecule. Essentially, the
binding status can be one of three potential statuses: (i) the
binding motif is free and not bound to a fusion molecule (see 305
in FIG. 3A); (ii) the binding motif is bound to a fusion molecule
that does not bind to a target molecule (see 306 in FIG. 3A); or
(iii) the binding motif is bound to a fusion molecule that is bound
to a target molecule (see 307 in FIG. 3A).
[0068] Detection of the binding status of a binding motif can be
carried out by various methods. In one aspect, by virtue of the
different sizes of the binding motif at each status, when the
binding motif passes through the pore, the different sizes result
in different electrical currents across the pore. In one aspect, as
shown in FIG. 3A, with a positive voltage applied and KCl
concentrations greater than 0.4 M in the experiment buffer, the
measured current signals 301, when 305, 306, and 307 pass through
the pore, are signals 302, 303, and 304, respectively. All three
event types are subjected to current attenuation when KCl
concentrations are greater than 0.4 M, causing a reduction in the
positive current flow. The three signals 302, 303, and 304 can be
differentiated from one another by the amount of the current shift
(height) and/or the duration of the current shift (width), or by
any other feature in the signal that differentiates the three event
types. It may also be that 304 is commonly different than 302 and
303, but that 302 and 303 are not commonly different from each
other, in which case, robust detection of the biomarker bound to
the passing molecule can still be accomplished. In another aspect,
as shown in FIG. 3B, with a positive voltage applied and KCl
concentrations less than 0.4 M in the experiment buffer, the
measured current signals 308, when 312, 313, and 314 pass through
the pore, are signals 309, 310, and 311, respectively. Passage of
dsDNA alone causes current enhancement events (309) at KCl
concentrations less than 0.4 M. This was shown in the published
research by Smeets, Ralph M M, et al. "Salt dependence of ion
transport and DNA translocation through solid-state nanopores."
Nano Letters 6.1 (2006): 89-95. Hence, the signal 309 can be
differentiated from 310 and 311 by the event amplitude direction
(polarity) relative to the open channel baseline current level
(308), in addition to the three signals commonly having different
amounts of the current shift (height) and/or the duration of the
current shift (width), or by any other feature in the signal that
differentiates the three event types. In another aspect, as shown
in FIG. 3C, with a negative voltage applied and KCl concentrations
less than 0.4 M in the experiment buffer, the negative measured
current signals 315, when 319, 320, and 321 pass through the pore,
are signals 316, 317, and 318, respectively. Compared to signals
309, 310, and 311 with a positive voltage, the signals 316, 317,
and 318 have the opposite polarity since the applied voltage has
the opposite (negative) polarity. In all aspects of the FIG. 3A,
FIG. 3B, and FIG. 3C embodiments, the sensor comprises electrodes,
which are connected to power sources and can detect the current.
Either one or both of the electrodes, therefore, serve as a
"sensor." In this embodiment, a voltage-clamp or a patch-clamp is
used to simultaneously supply a voltage across the pore and measure
the current through the pore.
[0069] In some aspects, an agent 106 as shown in FIG. 1 is added to
the complex to aid detection. This agent is capable of binding to
the target molecule or the ligand/target molecule complex. In one
aspect, the agent includes a charge, either negative or positive,
to facilitate detection. In another aspect, the agent adds size to
facilitate detection. In another aspect, the agent includes a
detectable label, such as a fluorophore.
[0070] In this context, an identification of status (iii) indicates
that a polymer-fusion molecule-target molecule complex has formed.
In other words, the target molecule is detected.
Particle Detection
[0071] The present disclosure also provides, in some aspects,
methods and systems for detecting, quantitating, and measuring
particles such as proteins, protein aggregates, oligomers, or
protein/DNA complexes, or cells and microorganisms, including
viruses, bacteria, and cellular aggregates.
[0072] In some aspects, the pore within the structure that
separates the device into two volumes has a size that allows
particles, such as viruses, bacteria, cells, or cellular
aggregates, to pass through. A ligand that is capable of binding to
a target particle to be detected or quantitated can be included in
the solution in the nanopore device such that the ligand can bind
to the unique target particle and the polymer scaffold through a
binding domain and a binding motif to form a complex. Many such
particles have unique markers on their surfaces that can be
specifically recognized by a ligand. For instance, tumor cells can
have tumor antigens expressed on the cell surface, and bacterial
cells can have endotoxins attached on the cell membrane.
[0073] When the formed complex in a solution loaded into the
nanopore device is moved along with the polymer scaffold to pass
through the pore, the binding status of the complex within or
adjacent to the pore can be detected such that the target
microorganisms bound to the ligands can be identified using methods
similar to the molecular detection methods described elsewhere in
the disclosure.
Polymer Scaffold
[0074] A polymer scaffold suitable for use in the present
technology is a scaffold that can be loaded into a nanopore device
and passed through the pore from one end to the other.
[0075] Non-limiting examples of polymers include nucleic acids,
such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or
peptide nucleic acid (PNA), dendrimers, and linearized proteins or
peptides. In some aspects, the DNA or RNA can be single-stranded or
double-stranded, or can be a DNA/RNA hybrid molecule.
[0076] In one aspect, the polymer is synthetic or chemically
modified. Chemical modification can help to stabilize the polymer,
add charges to the polymer to increase mobility, maintain
linearity, or add or modify the binding specificity. In some
aspects, the chemical modification is acetylation, methylation,
summolation, oxidation, phosphorylation, glycosylation, or the
addition of biotin.
[0077] In some aspects, the polymer is electrically charged. DNA,
RNA, PNA and proteins are typically charged under physiological
conditions. Such polymers can be further modified to increase or
decrease the carried charge. Other polymers can be modified to
introduce charges. Charges on the polymer can be useful for driving
the polymer to pass through the pore of a nanopore device. For
instance, a charged polymer can move across the pore by virtue of
an application of voltage across the pore.
[0078] In some aspects, when charges are introduced to the polymer,
the charges can be added at the ends of the polymer. In some
aspects, the charges are spread over the polymer.
[0079] In one embodiment, each unit of the charged polymer is
charged at the pH selected. In another embodiment, the charged
polymer includes sufficient charged units to be pulled into and
through the pore by electrostatic forces. For example, a peptide
containing sufficient entities can be charged at a selected pH
(lysine, aspartic acid, glutamic acid, etc.) so as to be used in
the devices and methods described herein. Likewise, a co-polymer
comprising methacrylic acid and ethylene is a charged polymer for
the purposes of this invention if there is sufficient charged
carboxylate groups of the methacrylic acid residue to be used in
the devices and methods described herein. In one embodiment, the
charged polymer includes one or more charged units at or close to
one terminus of the polymer. In another embodiment, the charged
polymer includes one or more charged units at or close to both
termini of the polymer. One co-polymer example is a DNA wrapped
around protein (e.g. DNA/nucleosome). Another example of a
co-polymer is a linearized protein conjugated to DNA at the N- and
C-terminus.
Binding Motifs and Binding Domains
[0080] For nucleic acids and polypeptides such as the polymer
scaffold, a binding motif can be a nucleotide or peptide sequence
that is recognizable by a binding domain. In some embodiments, the
binding domain is a peptide sequence forming a functional portion
of a protein, although the binding domain does not have to be a
protein. For nucleic acids, for instance, there are proteins that
specifically recognize and bind to sequences (motifs) such as
promoters, enhancers, thymine-thymine dimers, and certain secondary
structures such as bent nucleotide and sequences with single-strand
breakage.
[0081] In some aspects, the binding motif includes a chemical
modification that causes or facilitates recognition and binding by
a binding domain. For example, methylated DNA sequences can be
recognized by transcription factors, DNA methyltransferases or
methylation repair enzymes. In other embodiments, biotin may be
incorporated into, and recognized by, avidin family members. In
such embodiments, biotin forms the binding motif and avidin or an
avidin family member is the binding domain.
[0082] Molecules, in particular proteins, that are capable of
specifically recognizing nucleotide binding motifs are known in the
art. For instance, protein domains such as helix-turn-helix, a zinc
finger, a leucine zipper, a winged helix, a winged helix turn
helix, a helix-loop-helix and an HMG-box, are known to be able to
bind to nucleotide sequences.
[0083] In some aspects, the binding domains can be locked nucleic
acids (LNAs), Protein Nucleic Acids of all types (e.g. bisPNAs,
gamma-PNAs), transcription activator-like effector nucleases
(TALENs), clustered regularly interspaced short palindromic repeats
(CRISPRs), or aptamers (e.g., DNA, RNA, protein, or combinations
thereof).
[0084] In some aspects, the binding domains are one or more of DNA
binding proteins (e.g., zinc finger proteins), antibody fragments
(Fab), chemically synthesized binders (e.g., PNA, LNA, TALENS, or
CRISPR), or a chemical modification (i.e., reactive moieties) in
the synthetic polymer (e.g., thiolate, biotin, amines,
carboxylates).
Target Molecule/Particles and Ligands
[0085] In the present technology, a target molecule or particle is
detected or quantitated by virtue of its binding to a ligand in a
fusion molecule that binds to a polymer scaffold. A target molecule
or particle and a corresponding binding ligand can recognize and
bind each other. For a particle, there can be surface molecules or
markers suitable for a ligand to bind (therefore the marker and the
ligand form a binding pair).
[0086] Examples of binding pairs that enable binding between a
target molecule or a molecule on a particle include, but are not
limited to, antigen/antibody (or antibody fragment); hormone,
neurotransmitter, cytokine, growth factor or cell recognition
molecule/receptor; and ion or element/chelate agent or ion binding
protein, such as a calmodulin. The binding pairs can also be
single-stranded nucleic acids having complementary sequences,
enzymes and substrates, members of protein complex that bind each
other, enzymes and cofactors, enzymes and one or more of their
inhibitors (allosteric or otherwise), nucleic acid/protein, or
cells or proteins detectable by cysteine-constrained peptides.
[0087] In some embodiments, the ligand is a protein, protein
scaffold, peptide, aptamer (DNA or protein), nucleic acid (DNA or
RNA), antibody fragment (Fab), chemically synthesized molecule,
chemically reactive functional group or any other suitable
structure that forms a binding pair with a target molecule.
[0088] Therefore, any target molecule in need of detection or
quantitation, such as proteins, peptides, nucleic acids, chemical
compounds, ions, and elements, can find a corresponding binding
ligand. For the majority of proteins and nucleic acids, an antibody
or a complementary sequence, or an aptamer can be readily
prepared.
[0089] Likewise, binding ligands (such as antibodies and aptamers)
can be readily found or prepared for particles, such as protein
complexes and protein aggregates, protein/nucleic acid complexes,
fragmented or fully assembled viruses, bacteria, cells, and
cellular aggregates.
Fusion Molecule
[0090] A "fusion molecule" is intended to mean a molecule or
complex that contains two functional regions, a binding domain and
a ligand. The binding domain is capable of binding to a binding
motif on a polymer scaffold, and the ligand is capable of binding
to a target molecule.
[0091] In some aspects, the fusion molecule is prepared by linking
the two regions with a bond or force. Such a bond and force can be,
for instance, a covalent bond, a hydrogen bond, an ionic bond, a
metallic bond, van der Walls force, hydrophobic interaction, or
planar stacking interaction.
[0092] In some aspects, the fusion molecule, such as a fusion
protein, can be expressed as a single molecule from a recombinant
coding nucleotide. In some aspects, the fusion molecule is a
natural molecule having a binding domain and a ligand suitable for
use in the present technology.
[0093] Many options exist for connecting the binding domain with
the ligand to form the fusion molecule. For example, the components
may be connected via chemical coupling through functionalized
linkers such as free amine, carboxylate coupling, thiolate,
hydrazide, or azide (click) chemistry or the binding domain and the
ligand may form one continuous transcript.
[0094] FIG. 2 illustrates a more specific embodiment of the system
shown in FIG. 1. In FIG. 2, the fusion molecule is a chimeric
protein that includes a zinc finger protein or domain 202 and a
human immunodeficiency virus (HIV) envelop protein 203. The zinc
finger protein 202 can bind to a suitable nucleotide sequence on
the polymer scaffold, a double-stranded DNA 201; the HIV envelop
protein 203 can bind to an anti-HIV antibody 204 which can be
present in a biological sample (e.g., a blood sample from a
patient) for detection.
[0095] When the double-stranded DNA 201 passes through a pore 205
of a nanopore device 206, the nanopore device 206 can detect
whether a fusion molecule is bound to the DNA 201 and whether the
bound fusion molecule binds to an anti-HIV antibody 204.
Measurement of Affinity of Binding
[0096] The present technology can be used also for measuring the
binding affinity between two molecules and to determine other
binding dynamics. For instance, after the binding motif passes
through the pore of a nanopore device, the device can be
reconfigured to reverse the moving direction of the polymer
scaffold (as described below) such that the binding motif can pass
through the pore again.
[0097] Before the binding motif enters the pore again, one can
change the conditions in the sample that is loaded into the
nanopore device. For instance, changing the condition can be one or
more of removing the target molecule from the sample, adding an
agent that competes with the target molecule or the ligand for
binding, and changing the pH, salt, or temperature.
[0098] Under the changed conditions, the binding motif may be
passed through the pore again. Therefore, whether the target
molecule is still bound to the fusion molecule can be detected to
determine how the changed conditions impact the binding.
[0099] In some aspects, once the binding motif is in the pore, it
is retained there while the conditions are changed, and thus the
impact of the changed conditions can be measured in situ.
[0100] Alternatively or in addition, the polymer scaffold can
include multiple binding motifs and each of the binding motifs can
bind to a fusion molecule that can bind to one or more specific a
target molecule(s) or particle(s). While each binding motif passes
through the pore, the conditions of the sample can be changed,
allowing detection of changed binding between the ligand and the
target molecule or particle on a continued basis.
Multiplexing
[0101] In some aspects, rather than including multiple binding
motifs of the same kind as described above, a polymer scaffold can
include multiple types of binding motifs, each having different
corresponding binding domains. In such embodiments, a sample can
include multiple types of fusion molecules, each type including one
of the different corresponding binding domains and a ligand for a
different target molecule or target microorganism.
[0102] An additional method of multiplexing includes assaying a
collection of different scaffold molecules during a test, with each
different scaffold associating with different fusion molecule(s).
To determine what target molecules are in solution, scaffolds of
the same type are labeled such that the sensor can identify what
fusion molecule will bind to that particular scaffold. This can be
accomplished, for example, by barcoding each type of scaffold with
polyethylene glycol molecules of varying lengths or sizes.
[0103] With such a setting, a single polymer scaffold can be used
to detect multiple types of target molecules or target
microorganisms (e.g. bacterium or virus), or target cells (e.g.
circulating tumor cells). FIG. 4 illustrates such a method. Here, a
double-stranded DNA 403 is used as the polymer scaffold, the
double-stranded DNA 403 including multiple binding motifs: two
copies of a first binding motif 404, two copies of a second binding
motif 405, and one copy of a third binding motif 406.
[0104] When the DNA passes through a nanopore device 407 that has
two coaxial pores, 401 and 402, the binding status of each of the
binding motifs is detected, in which both copies of binding motif
404 bind to a corresponding target molecule, both copies of binding
motif 405 bind to a corresponding target molecule; and the fusion
molecule bound to binding motif 406 does not bind to a
corresponding target molecule.
[0105] This way, with a single polymer and a single nanopore
device, the present technology can simultaneously detect multiple
different target molecules or target microstructure (e.g.,
aggregates, microorganisms, or cells). Further, by determining how
many copies of binding motifs are bound to the target molecules or
target microorganisms, and by tuning conditions that impact the
bindings, the system can obtain more detailed binding dynamic
information.
Nanopore Devices
[0106] A nanopore device, as provided, includes at least a pore
that forms an opening in a structure separating an interior space
of the device into two volumes, and at least a sensor configured to
identify objects (for example, by detecting changes in parameters
indicative of objects) passing through the pore.
[0107] The pore(s) in the nanopore device are of a nano scale or
micro scale. In one aspect, each pore has a size that allows a
small or large molecule or microorganism to pass. In one aspect,
each pore is at least about 1 nm in diameter. Alternatively, each
pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9
nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm,
19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70
nm, 80 nm, 90 nm, or 100 nm in diameter.
[0108] In one aspect, the pore is no more than about 100 nm in
diameter. Alternatively, the pore is no more than about 95 nm, 90
nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm,
40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.
[0109] In some aspects, each pore is at least about 100 nm, 200 nm,
500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or
30000 nm in diameter. In one aspect, the pore is no more than about
100000 nm in diameter. Alternatively, the pore is no more than
about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm,
8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or
1000 nm in diameter.
[0110] In one aspect, the pore has a diameter that is between about
1 nm and about 100 nm, or alternatively between about 2 nm and
about 80 nm, or between about 3 nm and about 70 nm, or between
about 4 nm and about 60 nm, or between about 5 nm and about 50 nm,
or between about 10 nm and about 40 nm, or between about 15 nm and
about 30 nm.
[0111] In some aspects, the pore(s) in the nanopore device are of a
larger scale for detecting large microorganisms or cells. In one
aspect, each pore has a size that allows a large cell or
microorganism to pass. In one aspect, each pore is at least about
100 nm in diameter. Alternatively, each pore is at least about 200
nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000
nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm,
1800 nm, 1900 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500
nm, or 5000 nm in diameter.
[0112] In one aspect, the pore is no more than about 100,000 nm in
diameter. Alternatively, the pore is no more than about 90,000 nm,
80,000 nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm,
20,000 nm, 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm,
4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.
[0113] In one aspect, the pore has a diameter that is between about
100 nm and about 10000 nm, or alternatively between about 200 nm
and about 9000 nm, or between about 300 nm and about 8000 nm, or
between about 400 nm and about 7000 nm, or between about 500 nm and
about 6000 nm, or between about 1000 nm and about 5000 nm, or
between about 1500 nm and about 3000 nm.
[0114] In some aspects, the nanopore device further includes means
to move a polymer scaffold across the pore and/or means to identify
objects that pass through the pore. Further details are provided
below, described in the context of a two-pore device.
[0115] Compared to a single-pore nanopore device, a two-pore device
can be more easily configured to provide good control of speed and
direction of the movement of the polymer across the pores.
[0116] In one embodiment, the nanopore device includes a plurality
of chambers, each chamber in communication with an adjacent chamber
through at least one pore. Among these pores, two pores, namely a
first pore and a second pore, are placed so as to allow at least a
portion of a polymer to move out of the first pore and into the
second pore. Further, the device includes a sensor capable of
identifying the polymer during the movement. In one aspect, the
identification entails identifying individual components of the
polymer. In another aspect, the identification entails identifying
fusion molecules and/or target molecules bound to the polymer. When
a single sensor is employed, the single sensor may include two
electrodes placed at both ends of a pore to measure an ionic
current across the pore. In another embodiment, the single sensor
comprises a component other than electrodes.
[0117] In one aspect, the device includes three chambers connected
through two pores. Devices with more than three chambers can be
readily designed to include one or more additional chambers on
either side of a three-chamber device, or between any two of the
three chambers. Likewise, more than two pores can be included in
the device to connect the chambers.
[0118] In one aspect, there can be two or more pores between two
adjacent chambers, to allow multiple polymers to move from one
chamber to the next simultaneously. Such a multi-pore design can
enhance throughput of polymer analysis in the device.
[0119] In some aspects, the device further includes means to move a
polymer from one chamber to another. In one aspect, the movement
results in loading the polymer across both the first pore and the
second pore at the same time. In another aspect, the means further
enables the movement of the polymer, through both pores, in the
same direction.
[0120] For instance, in a three-chamber two-pore device (a
"two-pore" device), each of the chambers can contain an electrode
for connecting to a power supply so that a separate voltage can be
applied across each of the pores between the chambers.
[0121] In accordance with one embodiment of the present disclosure,
provided is a device comprising an upper chamber, a middle chamber
and a lower chamber, wherein the upper chamber is in communication
with the middle chamber through a first pore, and the middle
chamber is in communication with the lower chamber through a second
pore. Such a device may have any of the dimensions or other
characteristics previously disclosed in U.S. Publ. No.
2013-0233709, entitled Dual-Pore Device, which is herein
incorporated by reference in its entirety.
[0122] In some embodiments as shown in FIG. 5A, the device includes
an upper chamber 505 (Chamber A), a middle chamber 504 (Chamber B),
and a lower chamber 503 (Chamber C). The chambers are separated by
two separating layers or membranes (501 and 502) each having a
separate pore (511 or 512). Further, each chamber contains an
electrode (521, 522 or 523) for connecting to a power supply. The
annotation of upper, middle and lower chamber is in relative terms
and does not indicate that, for instance, the upper chamber is
placed above the middle or lower chamber relative to the ground, or
vice versa.
[0123] Each of the pores 511 and 512 independently has a size that
allows a small or large molecule or microorganism to pass. In one
aspect, each pore is at least about 1 nm in diameter.
Alternatively, each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm,
6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm,
16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45
nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.
[0124] In one aspect, the pore is no more than about 100 nm in
diameter. Alternatively, the pore is no more than about 95 nm, 90
nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm,
40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.
[0125] In one aspect, the pore has a diameter that is between about
1 nm and about 100 nm, or alternatively between about 2 nm and
about 80 nm, or between about 3 nm and about 70 nm, or between
about 4 nm and about 60 nm, or between about 5 nm and about 50 nm,
or between about 10 nm and about 40 nm, or between about 15 nm and
about 30 nm.
[0126] In other aspects, each pore is at least about 100 nm, 200
nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm,
or 30000 nm in diameter. In one aspect, each pore is 50,000 nm to
100,000 nm in diameter. In one aspect, the pore is no more than
about 100000 nm in diameter. Alternatively, the pore is no more
than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000
nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm,
or 1000 nm in diameter.
[0127] In some aspects, the pore has a substantially round shape.
"Substantially round", as used here, refers to a shape that is at
least about 80 or 90% in the form of a cylinder. In some
embodiments, the pore is square, rectangular, triangular, oval, or
hexangular in shape.
[0128] Each of the pores 511 and 512 independently has a depth
(i.e., a length of the pore extending between two adjacent
volumes). In one aspect, each pore has a depth that is least about
0.3 nm. Alternatively, each pore has a depth that is at least about
0.6 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10
nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm,
20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80
nm, or 90 nm.
[0129] In one aspect, each pore has a depth that is no more than
about 100 nm. Alternatively, the depth is no more than about 95 nm,
90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45
nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm.
[0130] In one aspect, the pore has a depth that is between about 1
nm and about 100 nm, or alternatively, between about 2 nm and about
80 nm, or between about 3 nm and about 70 nm, or between about 4 nm
and about 60 nm, or between about 5 nm and about 50 nm, or between
about 10 nm and about 40 nm, or between about 15 nm and about 30
nm.
[0131] In some aspects, the nanopore extends through a membrane.
For example, the pore may be a protein channel inserted in a lipid
bilayer membrane or it may be engineered by drilling, etching, or
otherwise forming the pore through a solid-state substrate such as
silicon dioxide, silicon nitride, grapheme, or layers formed of
combinations of these or other materials. In some aspects, the
length or depth of the nanopore is sufficiently large so as to form
a channel connecting two otherwise separate volumes. In some such
aspects, the depth of each pore is greater than 100 nm, 200 nm, 300
nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. In some
aspects, the depth of each pore is no more than 2000 nm or 1000
nm.
[0132] In one aspect, the pores are spaced apart at a distance that
is between about 10 nm and about 1000 nm. In some aspects, the
distance between the pores is greater than 1000 nm, 2000 nm, 3000
nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, or 9000 nm. In
some aspects, the pores are spaced no more than 30000 nm, 20000 nm,
or 10000 nm apart. In one aspect, the distance is at least about 10
nm, or alternatively, at least about 20 nm, 30 nm, 40 nm, 50 nm, 60
nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm.
In another aspect, the distance is no more than about 1000 nm, 900
nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm,
150 nm, or 100 nm.
[0133] In yet another aspect, the distance between the pores is
between about 20 nm and about 800 nm, between about 30 nm and about
700 nm, between about 40 nm and about 500 nm, or between about 50
nm and about 300 nm.
[0134] The two pores can be arranged in any position so long as
they allow fluid communication between the chambers and have the
prescribed size and distance between them. In one aspect, the pores
are placed so that there is no direct blockage between them. Still,
in one aspect, the pores are substantially coaxial, as illustrated
in FIG. 5A.
[0135] In one aspect, as shown in FIG. 5A, the device, through the
electrodes 521, 522, and 523 in the chambers 503, 504, and 505,
respectively, is connected to one or more power supplies. In some
aspects, the power supply includes a voltage-clamp or a
patch-clamp, which can supply a voltage across each pore and
measure the current through each pore independently. In this
respect, the power supply and the electrode configuration can set
the middle chamber to a common ground for both power supplies. In
one aspect, the power supply or supplies are configured to apply a
first voltage V.sub.1 between the upper chamber 505 (Chamber A) and
the middle chamber 504 (Chamber B), and a second voltage V.sub.2
between the middle chamber 504 and the lower chamber 503 (Chamber
C).
[0136] In some aspects, the first voltage V.sub.1 and the second
voltage V.sub.2 are independently adjustable. In one aspect, the
middle chamber is adjusted to be a ground relative to the two
voltages. In one aspect, the middle chamber comprises a medium for
providing conductance between each of the pores and the electrode
in the middle chamber. In one aspect, the middle chamber includes a
medium for providing a resistance between each of the pores and the
electrode in the middle chamber. Keeping such a resistance
sufficiently small relative to the nanopore resistances is useful
for decoupling the two voltages and currents across the pores,
which is helpful for the independent adjustment of the
voltages.
[0137] Adjustment of the voltages can be used to control the
movement of charged particles in the chambers. For instance, when
both voltages are set in the same polarity, a properly charged
particle can be moved from the upper chamber to the middle chamber
and to the lower chamber, or the other way around, sequentially. In
some aspects, when the two voltages are set to opposite polarity, a
charged particle can be moved from either the upper or the lower
chamber to the middle chamber and kept there.
[0138] The adjustment of the voltages in the device can be
particularly useful for controlling the movement of a large
molecule, such as a charged polymer, that is long enough to cross
both pores at the same time. In such an aspect, the direction and
the speed of the movement of the molecule can be controlled by the
relative magnitude and polarity of the voltages as described
below.
[0139] The device can contain materials suitable for holding liquid
samples, in particular, biological samples, and/or materials
suitable for nanofabrication. In one aspect, such materials include
dielectric materials such as, but not limited to, silicon, silicon
nitride, silicon dioxide, graphene, carbon nanotubes, TiO.sub.2,
HfO.sub.2, Al.sub.2O.sub.3, or other metallic layers, or any
combination of these materials. In some aspects, for example, a
single sheet of graphene membrane of about 0.3 nm thick can be used
as the pore-bearing membrane.
[0140] Devices that are microfluidic and that house two-pore
microfluidic chip implementations can be made by a variety of means
and methods. For a microfluidic chip comprised of two parallel
membranes, both membranes can be simultaneously drilled by a single
beam to form two concentric pores, though using different beams on
each side of the membranes is also possible in concert with any
suitable alignment technique. In general terms, the housing ensures
sealed separation of Chambers A-C. In one aspect as shown in FIG.
5B, the housing would provide minimal access resistance between the
voltage electrodes 521, 522, and 523 and the nanopores 511 and 512,
to ensure that each voltage is applied principally across each
pore.
[0141] In one aspect, the device includes a microfluidic chip
(labeled as "Dual-core chip") is comprised of two parallel
membranes connected by spacers. Each membrane contains a pore
drilled by a single beam through the center of the membrane.
Further, the device preferably has a Teflon.RTM. housing for the
chip. The housing ensures sealed separation of Chambers A-C and
provides minimal access resistance for the electrode to ensure that
each voltage is applied principally across each pore.
[0142] More specifically, the pore-bearing membranes can be made
with transmission electron microscopy (TEM) grids with a 5-100 nm
thick silicon, silicon nitride, or silicon dioxide windows. Spacers
can be used to separate the membranes, using an insulator, such as
SU-8, photoresist, PECVD oxide, ALD oxide, ALD alumina, or an
evaporated metal material, such as Ag, Au, or Pt, and occupying a
small volume within the otherwise aqueous portion of Chamber B
between the membranes. A holder is seated in an aqueous bath that
is comprised of the largest volumetric fraction of Chamber B.
Chambers A and C are accessible by larger diameter channels (for
low access resistance) that lead to the membrane seals.
[0143] A focused electron or ion beam can be used to drill pores
through the membranes, naturally aligning them. The pores can also
be sculpted (shrunk) to smaller sizes by applying a correct beam
focusing to each layer. Any single nanopore drilling method can
also be used to drill the pair of pores in the two membranes, with
consideration to the drill depth possible for a given method and
the thickness of the membranes. Predrilling a micro-pore to a
prescribed depth and then a nanopore through the remainder of the
membranes is also possible to further refine the membrane
thickness.
[0144] In another aspect, the insertion of biological nanopores
into solid-state nanopores to form a hybrid pore can be used in
either or both pores in the two-pore method. The biological pore
can increase the sensitivity of the ionic current measurements, and
is useful when only single-stranded polynucleotides are to be
captured and controlled in the two-pore device, e.g., for
sequencing.
[0145] By virtue of the voltages present at the pores of the
device, charged molecules can be moved through the pores between
chambers. Speed and direction of the movement can be controlled by
the magnitude and polarity of the voltages. Further, because each
of the two voltages can be independently adjusted, the direction
and speed of the movement of a charged molecule can be finely
controlled in each chamber.
[0146] One example concerns a charged polymer scaffold, such as a
DNA, having a length that is longer than the combined distance that
includes the depth of both pores plus the distance between the two
pores. For example, a 1000 by dsDNA is about 340 nm in length, and
would be substantially longer than the 40 nm spanned by two 10
nm-deep pores separated by 20 nm. In a first step, the
polynucleotide is loaded into either the upper or the lower
chamber. By virtue of its negative charge under a physiological
condition at a pH of about 7.4, the polynucleotide can be moved
across a pore on which a voltage is applied. Therefore, in a second
step, two voltages, in the same polarity and at the same or similar
magnitudes, are applied to the pores to move the polynucleotide
across both pores sequentially.
[0147] At about the time when the polynucleotide reaches the second
pore, one or both of the voltages can be changed. Since the
distance between the two pores is selected to be shorter than the
length of the polynucleotide, when the polynucleotide reaches the
second pore, it is also in the first pore. A prompt change of
polarity of the voltage at the first pore, therefore, will generate
a force that pulls the polynucleotide away from the second pore as
illustrated in FIG. 5C.
[0148] Assuming that the two pores have identical voltage-force
influence and |V.sub.1|=|V.sub.2|+.delta.V, the value .delta.V>0
(or <0) can be adjusted for tunable motion in the V.sub.1| (or
V.sub.2) direction. In practice, although the voltage-induced force
at each pore will not be identical with V.sub.1=V.sub.2,
calibration experiments can identify the appropriate bias voltage
that will result in equal pulling forces for a given two-pore chip;
and variations around that bias voltage can then be used for
directional control.
[0149] If, at this point, the magnitude of the voltage-induced
force at the first pore is less than that of the voltage-induced
force at the second pore, then the polynucleotide will continue
crossing both pores towards the second pore, but at a lower speed.
In this respect, it is readily appreciated that the speed and
direction of the movement of the polynucleotide can be controlled
by the polarities and magnitudes of both voltages. As will be
further described below, such a fine control of movement has broad
applications.
[0150] Accordingly, in one aspect, provided is a method for
controlling the movement of a charged polymer through a nanopore
device. The method entails (a) loading a sample comprising a
charged polymer in one of the upper chamber, middle chamber or
lower chamber of the device of any of the above embodiments,
wherein the device is connected to one or more power supplies for
providing a first voltage between the upper chamber and the middle
chamber, and a second voltage between the middle chamber and the
lower chamber; (b) setting an initial first voltage and an initial
second voltage so that the polymer moves between the chambers,
thereby locating the polymer across both the first and second
pores; and (c) adjusting the first voltage and the second voltage
so that both voltages generate force to pull the charged polymer
away from the middle chamber (voltage-competition mode), wherein
the two voltages are different in magnitude, under controlled
conditions, so that the charged polymer moves across both pores in
either direction and in a controlled manner.
[0151] To establish the voltage-competition mode in step (c), the
relative force exerted by each voltage at each pore is to be
determined for each two-pore device used, and this can be done with
calibration experiments by observing the influence of different
voltage values on the motion of the polynucleotide, which can be
measured by sensing known-location and detectable features in the
polynucleotide, with examples of such features detailed later in
this disclosure. If the forces are equivalent at each common
voltage, for example, then using the same voltage value at each
pore (with common polarity in upper and lower chambers relative to
grounded middle chamber) creates a zero net motion in the absence
of thermal agitation (the presence and influence of Brownian motion
is discussed below). If the forces are not equivalent at each
common voltage, achieving equal forces involves the identification
and use of a larger voltage at the pore that experiences a weaker
force at the common voltage. Calibration for voltage-competition
mode can be done for each two-pore device, and for specific charged
polymers or molecules whose features influence the force when
passing through each pore.
[0152] In one aspect, the sample containing the charged polymer is
loaded into the upper chamber and the initial first voltage is set
to pull the charged polymer from the upper chamber to the middle
chamber and the initial second voltage is set to pull the polymer
from the middle chamber to the lower chamber. Likewise, the sample
can be initially loaded into the lower chamber, and the charged
polymer can be pulled to the middle and the upper chambers.
[0153] In another aspect, the sample containing the charged polymer
is loaded into the middle chamber; the initial first voltage is set
to pull the charged polymer from the middle chamber to the upper
chamber; and the initial second voltage is set to pull the charged
polymer from the middle chamber to the lower chamber.
[0154] In one aspect, the adjusted first voltage and second voltage
at step (c) are about 10 times to about 10,000 times as high, in
magnitude, as the difference/differential between the two voltages.
For instance, the two voltages can be 90 mV and 100 mV,
respectively. The magnitude of the two voltages, about 100 mV, is
about 10 times of the difference/differential between them, 10 mV.
In some aspects, the magnitude of the voltages is at least about 15
times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times,
100 times, 150 times, 200 times, 250 times, 300 times, 400 times,
500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000
times, 6000 times, 7000 times, 8000 times or 9000 times as high as
the difference/differential between them. In some aspects, the
magnitude of the voltages is no more than about 10000 times, 9000
times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times,
3000 times, 2000 times, 1000 times, 500 times, 400 times, 300
times, 200 times, or 100 times as high as the
difference/differential between them.
[0155] In one aspect, real-time or on-line adjustments to the first
voltage and the second voltage at step (c) are performed by active
control or feedback control using dedicated hardware and software,
at clock rates up to hundreds of megahertz. Automated control of
the first or second or both voltages is based on feedback of the
first or second or both ionic current measurements.
Sensors
[0156] As discussed above, in various aspects, the nanopore device
further includes one or more sensors to carry out the
identification of the binding status of the binding motifs.
[0157] The sensors used in the device can be any sensor suitable
for identifying a molecule or particle, such as a polymer. For
instance, a sensor can be configured to identify the polymer by
measuring a current, a voltage, a pH value, an optical feature, or
residence time associated with the polymer. In other aspects, the
sensor may be configured to identify one or more individual
components of the polymer or one or more components bound to the
polymer. The sensor may be formed of any component configured to
detect a change in a measurable parameter where the change is
indicative of the polymer, a component of the polymer, or
preferably, a component bound to the polymer. In one aspect, the
sensor includes a pair of electrodes placed at two sides of a pore
to measure an ionic current across the pore when a molecule or
particle, in particular a polymer, moves through the pore. In
certain aspects, the ionic current across the pore changes
measurably when a polymer segment passing through the pore is bound
to a fusion molecule and/or fusion molecule-target molecule
complex. Such changes in current may vary in predictable,
measurable ways corresponding with, for example, the presence,
absence, and/or size of the fusion molecules and target molecules
present.
[0158] In one embodiment, the sensor measures an optical feature of
the polymer, a component (or unit) of the polymer, or a component
bound to the polymer. One example of such measurement includes the
identification of an absorption band unique to a particular unit by
infrared (or ultraviolet) spectroscopy.
[0159] When residence time measurements are used, the size of the
component can be correlated to the specific component based on the
length of time it takes to pass through the sensing device.
[0160] Still further, in embodiments directed towards detecting
units of the polymer, the sensor can include an enzyme distal to
the sensing device, where the enzyme is capable of separating the
terminal unit of the polymer from the penultimate unit, thereby
providing for a single molecular unit of the polymer. The single
molecule, such as a single nucleotide or an amino acid, can then
translocate through the pore and may or may not be detected.
However, when the enzyme encounters a bound target molecule, the
enzyme will not be able to cleave the penultimate unit, and
therefore will become stalled or will skip to the next available
cleavage sites, thus releasing a fragment that has a comparable
size difference from a single unit and is thus detectable.
Detection can be done with sensors as described in this application
or detected with methods such as mass spectrometry. Methods for
measuring such units are known in the art and include those
developed by Cal Tech (see, e.g.,
spectrum.ieee.org/tech-tallVat-work/test-and-measurement/a-scale-for-weig-
hing-single-molecules). The results of such analysis can be
compared to those of the sensing device to confirm the correctness
of the analysis.
[0161] In some embodiments, the sensor is functionalized with
reagents that form distinct non-covalent bonds with each
association site or each associated target molecule. In this
respect, the gap is large enough to allow effective measuring. For
instance, when a sensor is functionalized with reagents to detect a
feature on DNA that is 5 nm on a dsDNA scaffold, a 7.5 nm gap can
be used, because DNA is 2.5 nm wide.
[0162] Tunnel sensing with a functionalized sensor is termed
"recognition tunneling." Using current technology, a Scanning
Tunneling Microscope (STM) with recognition tunneling identifies a
DNA base flanked by other bases in a short DNA oligomer. As has
been described, recognition tunneling can provide a "universal
reader" designed to hydrogen-bond in a unique orientation to
molecules that a user desires to be detected. Most reported is the
identification of nucleic acids; however, it is herein modified to
be employed to detect target molecules on a scaffold.
[0163] A limitation with the conventional recognition tunneling is
that it can detect only freely diffusing molecules that randomly
bind in the gap, or that happen to be in the gap during microscope
motion, with no method of explicit capture in the gap. However, the
collective drawbacks of the STM setup can be eliminated by
incorporating the recognition reagent, optimized for sensitivity,
within an electrode tunneling gap in a nanopore channel.
[0164] Accordingly, in one embodiment, the sensor includes surface
modification by a reagent. In one aspect, the reagent is capable of
forming a non-covalent bond with an association site or an attached
target molecule. In a particular aspect, the bond is a hydrogen
bond. Non-limiting examples of the reagent include
4-mercaptobenzamide and 1-H-Imidazole-2-carboxamide.
[0165] Furthermore, the methods of the present technology can
provide DNA delivery rate control for one or more recognition
tunneling sites, each positioned in one or both of the nanopore
channels, and voltage control can ensure that each target molecule
resides in each site for a sufficient duration for robust
identification.
[0166] Sensors in the devices and methods of the present disclosure
can comprise gold, platinum, graphene, or carbon, or other suitable
materials. In a particular aspect, the sensor includes parts made
of graphene. Graphene can act as a conductor and an insulator, thus
tunneling currents through the graphene and across the nanopore can
sequence the translocating DNA.
[0167] In some embodiments, the tunnel gap has a width from about 1
nm to about 20 nm. In one aspect, the width of the gap is at least
about 1 nm, or alternatively, at least about 1.5, 2, 2.5, 3, 3.5,
4, 4.5, 5, 6, 7, 8, 9, 10, 12, or 15 nm. In another aspect, the
width of the gap is not greater than about 20 nm, or alternatively,
not greater than about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, 4, 3, or 2 nm. In some aspects, the width is between
about 1 nm and about 15 nm, between about 1 nm and about 10 nm,
between about 2 nm and about 10 nm, between about 2.5 nm and about
10 nm, or between about 2.5 nm and about 5 nm.
[0168] In other embodiments, the tunnel gap is suitable for
detecting micro-sized particles (e.g., viruses, bacteria, and/or
cells) and has a width from about 1000 nm to about 100,000 nm. In
some embodiments, the width of the gap is between about 10,000 nm
and 80,000 nm or between about 20,000 nm and 50,000 nm. In another
embodiment, the width of the gap is between about 50,000 nm and
100,000 nm. In some embodiments, the width of the gap is not
greater than about 100,000 nm, 90,000 nm, 80,000 nm, 70,000 nm,
60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000 nm,
9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000
nm, or 1000 nm.
[0169] In some embodiments, the sensor is an electric sensor. In
some embodiments, the sensor detects a fluorescent detection means
when the target molecule or the detectable label passing through
has a unique fluorescent signature. A radiation source at the
outlet of the pore can be used to detect that signature.
EXAMPLES
[0170] The present technology is further defined by reference to
the following example and experiments. It will be apparent to those
skilled in the art that many modifications may be practiced without
departing from the scope of the current invention
[0171] The example section begins by first pointing out principal
reasons to use a polymer scaffold and fusion molecules in biomarker
detection. A primary reason is that a biomarker alone, below a
certain size threshold, is undetectable with a nanopore, as shown
for proteins of varying sizes in Calin Plesa, Stefan W. Kowalczyk,
Ruben Zinsmeester, Alexander Y. Grosberg, Yitzhak Rabin, and Cees
Dekker. "Fast translocation of proteins through solid state
nanopores." Nano letters 13, no. 2 (2013): 658-663. Moreover, even
those biomarkers that are detectable may not be distinguishable. A
biomarker will yield the same nanopore signature as all other
molecules of comparable size/charge, preventing discrimination. By
using a scaffold and fusion molecules, we can avoid both of these
problems. In particular, we show by the examples that detection of
representative fusion molecules on scaffolds can be demonstrated,
and further that detection of target molecules to fusion molecules
on the scaffold can also be detected. With this capability,
discrimination can be achieved by appropriate engineering of the
ligand domain of the fusion molecule, to achieve specificity for
the target molecule of interest.
Example 1
DNA Alone in Solid-State Nanopore Experiment
[0172] Nanopore instruments use a sensitive voltage-clamp amplifier
to apply a voltage V across the pore while measuring the ionic
current l.sub.0 through the open pore (FIG. 6A). When a single
charged molecule such as a double-stranded DNA (dsDNA) is captured
and driven through the pore by electrophoresis (FIG. 6B), the
measured current shifts from l.sub.0 to l.sub.B, and the shift
amount .DELTA.l=l.sub.0-l.sub.B and duration t.sub.D are used to
characterize the event. After recording many events during an
experiment, distributions of the events (FIG. 6C) are analyzed to
characterize the corresponding molecule. In this way, nanopores
provide a simple, label-free, purely electrical single-molecule
method for biomolecular sensing.
[0173] In the DNA experiment shown in FIG. 6A, FIG. 6B, and FIG.
6C, the single nanopore fabricated in silicon nitride (SiN)
substrate is a 40 nm diameter pore in 100 nm thick SiN membrane
(FIG. 6A). In FIG. 6B, the representative current trace shows a
blockade event caused by a 5.6 kb dsDNA passing in a single file
manner (unfolded) through an 11 nm diameter nanopore in 10 nm thick
SiN at 200 mV and 1M KCl. The mean open channel current is
l.sub.0=9.6 nA, with mean event amplitude l.sub.B=9.1 nA, and
duration t.sub.D=0.064 ms. The amplitude shift is
.DELTA.l=l.sub.0-l.sub.B=0.5 nA. In FIG. 6C, the scatter plot shows
l.DELTA./l vs. t.sub.D for all 1301 events recorded over 16
minutes.
[0174] In the DNA experiment shown in FIG. 7A, dsDNA alone causes
current enhancement events at 100 mM KCl. This was shown in the
published research of Smeets, Ralph M M, et al. "Salt dependence of
ion transport and DNA translocation through solid-state nanopores."
Nano Letters 6.1 (2006): 89-95). The study showed that, while the
amplitude shift .DELTA.l=l.sub.0-l.sub.B>0 for KCl concentration
above 0.4 M, the shift has opposite polarity (.DELTA.l<0) for
KCl concentration below 0.4 M. As this is a negative voltage
experiment (-200 mV) with KCl concentration below 0.4 M, we see
that the DNA event has the same polarity (316) relative to the
baseline (315) as shown in FIG. 3C.
Example 2
VspR Protein Binding to DNA Scaffold and Nanopore Detection
[0175] The VspR protein is a 90 kDa protein from V. cholerae that
binds directly to dsDNA with high micromolar affinity (see
reference: Yildiz, Fitnat H., Nadia A. Dolganov, and Gary K.
Schoolnik. "VpsR, a Member of the Response Regulators of the
Two-Component Regulatory Systems, Is Required for Expression of
Biosynthesis Genes and EPSETr-Associated Phenotypes in Vibrio
cholerae 01 El Tor." Journal of bacteriology 183, no. 5 (2001):
1716-1726). In this example of target detection using nanopore
technology, VspR acts as the fusion molecule with a site-specific
DNA binding domain, and a ligand specific binding site that can be
engineered for the purpose of detecting a variety of targets,
including antibodies or sugars. In this demonstration, we show
detection of the VspR on the DNA scaffold as a model fusion
molecule. The scaffold contains 10 VspR specific binding sites
(FIG. 8). To preserve affinity of VspR for dsDNA binding, we use
0.1 M KCl, a salt concentration in which DNA alone translocations
cause current enhancements, as shown in Example 1 and FIG. 7A. The
5.631 kb DNA scaffold contains 10 total VspR binding sites: 5 of
one sequence (14 base pairs), 3 of a different sequence (18 base
pairs), and 2 of a third sequence (27 bp). The three different
sequences may not bind VspR with equal affinity. In experiments,
VspR protein concentration is 18 nM in the recording buffer, and
180 nM during labeling (binding step). This results in 18.times.
excess of VspR protein to binding sites on DNA. The experiment was
run at pH 8.0 (pl of VspR protein is 5.8). Taking Kd and DNA
concentration into account, only 0.1-1% of DNA should be fully
occupied by VspR, with a larger percentage partially occupied, and
some unknown remaining percentage of DNA entirely unbound. There is
also free VspR protein in solution during the nanopore
experiment.
[0176] Two representative events are shown in FIG. 9A and FIG. 9B.
In the experiments with VspR, VspR concentration was 18 nM (1.6
mg/L), 10 nM binding sites. The scaffold concentration was 1 nM
resulting in capture every 6.6 seconds. From this, the theoretical
sensitivity using a 10 uI sample is 116 pM (0.01 mg/ml). The pore
size is 15 nm in diameter and length. The voltage is -100 mV, and
note that negative voltages create negative currents, so upward
shifts correspond to attenuation events, as shown for the
VspR-bound DNA event (FIG. 9B), whereas downward shifts create
positive shifts as shown for the unbound DNA scaffold event (FIG.
9A). This is consistent with the idealized signal patterns and
conditions in FIG. 3C, with the DNA event (316) having faster
duration and the opposite polarity compared to the fusion
molecule-bound DNA event (320). Thus, the key observation from this
figure is that VspR-bound events have the opposite signal polarity
compared to unbound DNA events. FIG. 10 shows ten more
representative current attenuation events consistent with the
VspR-bound scaffold passing through the pore. There were 90 such
events over 10 minutes of recording, corresponding to 1 VspR-bound
event every 6.6 seconds. Events were attenuations of 50 to 150 pA
in amplitude and 0.2 to 2 milliseconds in duration. As stated,
downward events correspond to current enhancement events and upward
events correspond to current attenuation events in FIG. 9A, FIG.
9B, and FIG. 10, and this shift direction is preserved even though
the baseline is zeroed for display purposes.
Example 3
RecA Protein Binding to DNA Scaffold and Nanopore Detection
[0177] RecA comprises the elements of a fusion molecule, and this
example demonstrates the ability to use these elements to detect a
target biomarker. Specifically, the fusion molecule consists of the
portion of RecA that binds DNA (i.e. the DNA binding domain) and
the portion of RecA (epitope) that baits the biomarker (anti-RecA
antibody). DNA and RecA experiments were performed first in the
absence and then in the presence of anti-RecA antibody.
[0178] Reagent DNA/RecA consists of the 5.6 kb dsDNA scaffold
molecule coated in RecA. RecA is a 38 kDa bacterial protein
involved in DNA repair, which is capable of polymerizing along
dsDNA (see [C Bell. Structure and mechanism of Escherichia coli
RecA ATPase. Molecular microbiology, 58(2):358-366, January 2005]).
This reagent is created by incubating 60 nM scaffold with 112 uM
RecA protein in 10 mM gamma-S-ATP, 70 mM Tris pH 7.6, 10 mM MgCl,
and 5 mM DTT (New England Biolabs). Gamma-S-ATP is included since
RecA binds to dsDNA with greater affinity if the RecA has ATP
bound. Since RecA can hydrolyze ATP to ADP, thereby reducing its
affinity for DNA, the non-hydrolyzable gamma-S ATP analog prevents
this transition to ADP and thus the higher affinity state is
maintained. Even though the ratio of RecA to DNA is one RecA
molecule for every possible 3-bp binding site, we expect that not
all the RecA protein is binding and thus there is free RecA in
solution, as observed in other nanopore studies (see Smeets, R. M.
M., S. W. Kowalczyk, A. R. Hall, N. H. Dekker, and C. Dekker.
"Translocation of RecA coated double-stranded DNA through
solid-state nanopores." Nano letters 9, no. 9 (2008): 3089-3095,
and Kowalczyk, Stefan W., Adam R. Hall, and Cees Dekker. "Detection
of local protein structures along DNA using solid-state nanopores."
Nano letters 10, no. 1 (2009): 324-328). DNA/RecA samples are then
adjusted to 1M KCl or LiCl, 10 mM EDTA and tested in a nanopore
experiment or excess RecA protein is removed using gel filtration
(ThermoScientific Spin Columns).
[0179] In one set of experiments, we used a 16-18 nm diameter pore
formed in a 30 nm thick SiN membrane, applying 180 mV in 1M KCl at
pH 8. In separate control experiments, unbound 5.6 kb dsDNA
scaffold generates 95% of events in the range of 2100-400 pA and
530-500 microseconds. Also, free RecA events are 2100-600 pA,
20-200 usec. Finally, RecA-bound DNA events are typically much
deeper blockades, in the range 0.51-3 nA, and with longer duration
(0.200-3 milliseconds). Representative events for RecA-bound DNA
are shown in FIG. 11 and FIG. 12. These events have interesting
patterns, which in the paper by Kowalczyk et al. ["Detection of
local protein structures along DNA using solid-state nanopores."
Nano letters 10, no. 1 (2009): 324-328)] the authors attempt to
infer the location and length of RecA filaments that are bound to
each DNA; however, this is speculative, since it assumes a uniform
passage rate through the pore even though another study showed that
dsDNA does not pass through a pore at a uniform rate [Lu, Bo, et
al. "Origins and consequences of velocity fluctuations during DNA
passage through a nanopore." Biophysical journal 101.1 (2011):
70-79]. FIG. 13 part (a) and (c) show on the vertical axis the
maximum and mean current shift, respectively, normalized by
voltage, and the event duration on the horizontal axis. Both event
plots have all 1385 events recorded over 10 minutes. The amplitude
is normalized by voltage to give event conductance shift values,
which is common in nanopore research papers. For example, a mean
conductance of 14 nS at 200 mV is equivalent to a mean current
amplitude of 2.8 nA. There are two observable sub-populations in
amplitude (or equivalently, conductance) and duration, with the
deeper and longer duration events attributable to RecA-bound DNA
and the faster shallower events attributable to free RecA in
solution. We verified the identity of the faster, shallower
subpopulation as free RecA by running RecA alone control
experiments. This was also verified in the earlier study [Smeets,
R. M. M., S. W. Kowalczyk, A. R. Hall, N. H. Dekker, and C. Dekker.
"Translocation of RecA coated double-stranded DNA through
solid-state nanopores." Nano letters 9, no. 9 (2008): 3089-3095].
Looking at the maximum current shift value (FIG. 13, parts (a) and
(b)) instead of the mean (FIG. 13, parts (c) and (d)) makes the
subpopulations events more distinct. Note that RecA-bound DNA vs.
unbound DNA event patterns are consistent with the model signal
patterns in FIG. 3A.
[0180] In separate experiments, to demonstrate detection of a
target antibody, RecA antibody was used. The DNA/RecA reagent binds
an antibody biomarker creating a DNA/RecA/Ab complex by incubating
one nanomolar DNA/RecA for 30 mins with either an anti-RecA
monoclonal antibody (ARM191, Fisher Scientific) or polyclonal RecA
anti-serum (gift from Prof. Ken Knight, Ph. D., UMass Medical
School), at a 1:10000 dilution. Electrophoretic mobility shift
assays, 5% TBE polyacrylamide gel in 1.times.TBE buffer, are used
to test the DNA/RecA and DNA/RecA/Ab complexes by comparing
migration of complexes to DNA only or the proper controls.
[0181] The nanopore experiments were run at 200 mV in 1M LiCl with
a pore that varied in diameter: 20 nm during the DNA alone control,
and then enlarged to 27 nm after RecA-bound DNA complexes were
added. In a gel shift experiment, FIG. 14A shows a clear shift for
DNA/RecA/mAb above DNA/RecA, which is in turn well above the
unbound 5.6 kb dsDNA scaffold. This complex was tested
experimentally with a nanopore. Specifically, 0.1 nM DNA was added
to the chamber above the pore, and after 10 minutes of recording,
1.25 nM DNA/RecA was added. After another period of recording, 1.25
nM DNA/RecA/mAb was added. With the AB-bound complexes in solution,
a new multi-level event type was observed (FIG. 14B) that did not
match event patterns characteristic of the other two complex types
(DNA, DNA/RecA). The .DELTA.l vs. t.sub.D distributions of events
recorded during each phase of the experiment (FIG. 14C) show that
RecA-bound DNA events have longer durations t.sub.D, and 3 times as
many events had a mean amplitude shift .DELTA.l greater than 0.6 nA
after DNA/RecA/mAb was added. A simple criteria for tagging events
in this data set as also being Ab-bound is (.DELTA.l, t.sub.D) (0.6
nA, 0.2 ms). Identifying a best signature that is almost absent in
unbound DNA events, but is present in a significant fraction of
RecA-bound events (with or without antibody also bound to
DNA/RecA), is useful for detection of the presence of RecA-bound
DNA complexes in solution above the nanopore. For the purpose of
antibody detection, we take this a step further, and aim to
identify a best signature that is almost absent in unbound DNA and
RecA-bound DNA event types, but is present in a significant
fraction of RecA-bound events with antibody also bound to DNA/RecA.
This provides a criterion for detection of the presence of
RecA-bound DNA complexes in solution above the nanopore. As these
DNA and RecA and RecA-antibody experiments are done with a positive
voltage with KCl concentration above 0.4 M, we see that the event
patterns in FIG. 14B are comparable to the idealized patterns in
FIG. 3A.
Example 4
Fusion Molecules Comprising PNA and Biotin for Target Protein
Detection
[0182] The previous example explores detection of RecA-antibody
bound to RecA-coated dsDNA complexes. Since RecA binds 3 by regions
non-specifically, and thus RecA-antibody could bind to any RecA on
dsDNA, it is also desirable to demonstrate an approach that permits
target binding to specific sites. In particular, we use a 5.6 kb
dsDNA scaffold that is engineered to bind 12-mer
peptide-nucleic-acid (PNA) molecules, with each PNA having 3
biotinylated sites for binding an avidin family member (e.g.,
neutravidin, and or monovalent streptavidin) (FIG. 15A). The
scaffold has 25 distinct sites that together localize up to 75
avidin biomarker binding sites (FIG. 15B). Our data (FIG. 16D)
shows that the DNA/PNA/Neut complexes cause event signatures that
are detectable above other background event types (unbound DNA
alone, Neutravidin alone, PNA/Neutravidin alone) and can therefore
be tagged as fully assembled (i.e. DNA/PNA/Neutravidin) events. In
this setup, it is the fully assembled DNA/PNA/Neutravidin complex
that acts as the scaffold+fusion molecule. In the remainder of the
example, we provide sufficient detail to show that
DNA/PNA/Neutravidin complexes can be detected with a nanopore.
[0183] In this setup, the fusion molecule contains two separate
domains, one that binds a unique DNA sequence and another that
binds to an anti-Neutravidin antibody. The DNA binding domain is a
protein nucleic acid molecule (PNA) that binds to the unique
sequence (GAAAGTGAAAGT, uSeq1) that is repeated 25 times throughout
the scaffold (FIG. 15B). PNA molecules are similar to
oligonucleotides having A, T, C, G bases, which are capable of
pairing with their complementary sequences, but instead of a
phosphate backbone like typical oligonucleotides, the backbone is
protein. This eliminates the negative charge provided by the
phosphate backbone, and thus, PNA molecules can incorporate into
dsDNA by displacing the complementary DNA strand, making a new
DNA/PNA hybrid for the short stretch that encompasses the PNA
molecule. The PNA used in the experiment had the sequence
GAA*AGT*GAA*AGT where the * indicates that a biotin was
incorporated into the PNA backbone at the gamma position by
coupling to a Lysine amino acid, and thus, each PNA has three
biotin molecules (PNABio). To create the fusion molecule bound
scaffold, a 60 nM scaffold is heated to 95 C for 2 minutes, cooled
to 60 C and incubated with a 10.times. excess of PNA to possible
binding sites in 15 mM NaCl for 1 hr and then cooled to 4 C. The
excess PNA is dialyzed out (20k MWCO, Thermo Scientific) for 2 hrs
against 10 mM Tris pH 8.0. This DNA/PNA complex is then labeled
with a 10 fold excess Neutravidin protein (Pierce/Thermo
Scientific) to possible biotin sites (assuming a 60% reduction of
PNA during dialysis). The reaction is electrophoresed as described
above to assess purity, concentration, and potential aggregation.
This reagent, DNA/PNA/Neutravidin (D/P/N), is stored at -20 C until
use.
[0184] FIG. 17A and FIG. 17B show data comparing .DELTA.l vs. tD
distributions from three separate experiments: DNA alone,
Neutravidin alone, and D/P/N reagents. The largest 1All events in
the D/P/N experiment are most likely attributed to D/P/N complexes
(FIG. 16D), providing a simple criteria for tagging events as
fusion molecule bound (i.e., scaffold with PNA and Neutravidin
bound). Specifically, we can flag an event as being
"fusion-molecule bound" if |.DELTA..sup.l|>4 nA for that event.
For the data sets in FIG. 17A, 9.3% (390) of events in the D/P/N
experiment have |.DELTA..sup.l|>4 nA, with only 0.46% of D and
0.16% of N events in controls exceeding 4 nA. In a separate
experiment (data not shown) with a 7 nm diameter pore at 1M KCl and
200 mV applied, in a control with only PNA and Neutravidin at 0.4
nM concentration, no events (0%) exceeded 4 nA. Applying our
mathematical criteria, the random variable Q={Fraction of flagged
events} has a binomial distribution, and using this and other
statistical modeling tools, we can compute the 99% confidence
interval for this data set as Q=9.29.+-.1.15%. Since 9.29%>0.46%
(the max false-positive %) is satisfied well within the 99%
confidence interval for Q, we have a positive test result, and in
under 8 minutes of data gathering. In fact the same 99% confidence
is achieved for this data set with only the first 60 seconds of the
data. The gel shift (FIG. 17C) shows that scaffold DNA migration is
retarded in a Neutravidin dependent manner; this guided us to using
the 10.times. concentration in this preliminary experiment, as it
appeared all DNA is labeled and a nearly homogenous population is
created. We do not see a shift with the DNA/PNA complex, though a
shift was observed in another DNA/PNA nanopore study using shorter
DNA (Alon Singer, Meni Wanunu, Will Morrison, Heiko Kuhn, Maxim
Frank-Kamenetskii, and Amit Meller." Nanopore based sequence
specific detection of duplex DNA for genomic profiling." Nano
letters 10, no. 2 (2010): 738-742.).
[0185] It is to be understood that while the invention has been
described in conjunction with the above embodiments, the foregoing
description and examples are intended to illustrate and not limit
the scope of the invention. Other aspects, advantages and
modifications within the scope of the invention will be apparent to
those skilled in the art to which the invention pertains.
Sequence CWU 1
1
1112DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 1gaaagtgaaa gt 12
* * * * *