U.S. patent application number 16/174933 was filed with the patent office on 2019-08-08 for digital protein sensing chip and methods for detection of low concentrations of molecules.
The applicant listed for this patent is ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY. Invention is credited to Stuart LINDSAY, Pei PANG, Peiming ZHANG.
Application Number | 20190242885 16/174933 |
Document ID | / |
Family ID | 54324585 |
Filed Date | 2019-08-08 |
View All Diagrams
United States Patent
Application |
20190242885 |
Kind Code |
A1 |
LINDSAY; Stuart ; et
al. |
August 8, 2019 |
DIGITAL PROTEIN SENSING CHIP AND METHODS FOR DETECTION OF LOW
CONCENTRATIONS OF MOLECULES
Abstract
A sensing device is provided that includes a tunnel junction
created by forming a hole in a layered tunnel junction (for
example). A chemically, well-defined surface may be formed by
coupling affinity reagents to the electrodes, which, by these
means, the surface may be configured to be selective for a
particular analyte.
Inventors: |
LINDSAY; Stuart; (Phoenix,
AZ) ; ZHANG; Peiming; (Gilbert, AZ) ; PANG;
Pei; (Tempe, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE
UNIVERSITY |
Scottsdale |
AZ |
US |
|
|
Family ID: |
54324585 |
Appl. No.: |
16/174933 |
Filed: |
October 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15303960 |
Oct 13, 2016 |
10145846 |
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PCT/US2015/026241 |
Apr 16, 2015 |
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16174933 |
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61980317 |
Apr 16, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0887 20130101;
B01L 2300/0645 20130101; G01N 33/48721 20130101; B01L 2200/10
20130101; G01N 33/5438 20130101; B01L 3/502761 20130101; B01L
3/502715 20130101; B01L 2300/0861 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/487 20060101 G01N033/487; B01L 3/00 20060101
B01L003/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under ROI
HG006323 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A device for detecting the binding of a molecule to a cognate
ligand, comprising: a first planar electrode having a length, a
width and a thickness; an insulating layer having a length, a width
and a thickness; a second planar electrode having a length, a width
and a thickness; an opening between the electrodes configured to
expose the gap between them and establishing an
electrodes-insulating layer junction, wherein the opening is spaced
away from the perimeter of each electrode; one or more recognition
molecules comprising a cognate ligand; a chemical group configured
to couple the cognate ligand to at least one of the electrodes; and
a reference electrode in communication with at least one of the
electrodes.
2. The device of claim 1, wherein the reference electrode is
configured with a substantially constant potential difference with
respect to at least one of the electrodes.
3. The device of claim 1, wherein the cognate ligand comprises at
least one of an antibody, a Fab fragment, an aptamer and a peptide
configured to bind to one or more protein targets.
4. The device of claim 1, wherein the device is configured to
detect single molecular binding events corresponding to low
concentration sample solutions.
5. The device of claim 1, wherein the device is configured for
sequencing a peptide chain.
6. A system for detecting the binding of a molecule to a cognate
ligand, comprising: the device of claim 1; and purification means
for purifying a patient serum sample for obtaining one or more
target proteins.
7. The system of claim 6, wherein the purification means comprises
a lab-on-a-chip.
8. The system of claim 6, further comprising a pair of fluid
chambers, the device formed between the pair of fluid chambers.
9. The system of claim 8, wherein the pair of fluid chamber are in
fluid communication via the opening in the device.
10. A method for detecting at least one binding event between a
recognition molecule and a target molecule, comprising: providing
the device of claim 1; providing a substantially fixed bias between
the electrodes; flowing a sample solution adjacent the junction;
recording current signals generated as a result of one or more
binding events between one or more molecules in the sample and
corresponding recognition molecules; and determining at least one
of a number and type of molecules present in a sample solution
based on the characteristics of the signal generated as each type
of molecule binds.
11. The method according to claim 10, further comprising recording
at least one of the number and type of molecules determined.
12. The method according to claim 10, wherein the characteristics
comprise at least one of baseline current, peak current above a
baseline, peak width, peak shape as encoded by Fourier, wavelet or
Cepstrum component amplitudes, and flatness of the peak top
expressed as root mean square signal variation.
13. The method according to claim 10, further comprising at least
one of recording and counting signals generated by single molecule
binding events of a plurality of protein variants contained in the
sample solution.
14. The method according to claim 10, further comprising providing
the target molecule, including: providing a sample to a first
channel of a processing device, the sample including cells
containing the target molecule, the first channel including
antibodies for capturing the cells in the sample; lysing the
captured cells to generate a lysate including the target molecule;
eluting the lysate into a second channel including antibodies for
binding the target molecule; generating fragments of the target
molecule from the bound target molecule; and providing the
fragments of the target molecule as the target molecule to the
device.
15. The method according to claim 14, wherein the sample includes
blood cells, wherein the target molecule includes a protein, and
wherein the fragments of the target molecule includes peptides of
the protein.
16. The method according to claim 14, the generating the fragments
further including capturing a portion of the fragment of the target
molecule, the providing the fragments including providing the
portion of the fragment of the target molecule to the device.
17. The method according to claim 14, herein the fragments of the
target molecule includes peptides of the protein, further
comprising, prior to providing the fragments, modifying the
fragments to ligate a charged peptide to the N terminus of the
peptides of the protein, the providing including providing the
modified fragments as the target molecule to the device.
18-28. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/303,960, filed Oct. 13, 2016, which is the U.S. national
phase of PCT/US2015/026241, filed Apr. 16, 2015 which claims
priority to U.S. provisional application No. 61/980,317 titled
DIGITAL PROTEIN SENSING CHIP AND METHODS FOR DETECTION OF LOW
CONCENTRATIONS OF MOLECULES", filed Apr. 16, 2014, the entire
disclosures of which are incorporated herein by reference.
BACKGROUND
[0003] FIG. 1 shows a plot of the concentration of known proteins
in cells versus the total number of known proteins (Archakov, A.
I., Y. D. Ivanov, A. V. Lisitsa, and V. G. Zgoda, Afin Fishing
Nanotechnology Is the Way to Reverse the Avogadro Number in
Proteomics. Proteomics, 2007. 7: p. 4-9). Extrapolation of the
known data to low concentrations suggests that there are many more
unknown proteins present at concentrations well below the current
detection limit. This limit is set by the smallest dissociation
constant of affinity reagents used to collect proteins from cell
extract or serum, and these are typically nM at best. In order to
collect proteins at much smaller concentrations, large amounts of
sample are generally required, together with a multitude of
affinity reagents, so that binding by a very small fraction (e.g.,
when the concentration of sample is much less than K.sub.d)
provides a useable amount of sample.
[0004] Digital detection may be used to individually count captured
molecules resulting in increased sensitivity. This has been
demonstrated by Mok et al. (Mok, J., M. N. Mindrinos, R. W. Davis,
M. Javanmard, Digital Microfluidic Assay for Protein Detection.
Proc Natl Acad Sci U S A, 2014. 111: p. 1323998111) via a
"lab-on-a-chip" in which captured analytes are each tethered to a
bead, where each bead is subsequently detected individually via a
corresponding electrical signal in a narrow channel.
SUMMARY OF THE DISCLOSURE
[0005] Accordingly, some of the embodiments of the present
disclosure can be configured to detect binding events of a minute
fraction of a population of affinity reagents. In some such
embodiments, this may be accomplished by, for example, direct
electrical detection of individual molecules, and may also be done
in a time resolved manner (for example). Thus, in some embodiments,
even if only one affinity reagent among a large population is
bound, the event can be detected, and in some embodiments, it may
be detected even if bound for just a short amount of time.
[0006] In some embodiments, digital detection may be provided which
may be used to lower the concentration detection limit down to a
minute fraction of K.sub.d. Furthermore, because detection in some
embodiments is carried out via a nanoscale device at a single
molecule level, the total amount of sample required may be greatly
reduced as compared to conventional approaches (e.g., ELISA, mass
spectrometry). Moreover, because a binding event(s) generates a
direct electrical signal characteristic of the analyte, in some
embodiments, labeling or secondary probes are not required.
[0007] In some embodiments, a sensing device is provided that
includes a tunnel junction created by forming a hole in a layered
tunnel junction (for example). A chemically, well-defined surface
may be formed by coupling affinity reagents to the electrodes,
which, by these means, the surface may be configured to be
selective for a particular analyte.
[0008] Some embodiments of the present disclosure provide a readily
manufacturable platform for monitoring, for example, molecule
binding events between an affinity reagent and a target molecule
(and in some embodiments, a single binding event). Dynamic
information obtained from such embodiments may be used to extend
detection capability down to concentrations that may be orders of
magnitude below the K.sub.d of the affinity reagent.
[0009] In some embodiments, a device for detecting the binding of a
molecule to a cognate ligand is provided which may comprise a first
planar electrode having a length, a width and a thickness, an
insulating layer having a length, a width and a thickness, the
insulating layer covering a substantial portion of the first
electrode, and a second planar electrode having a length, a width
and a thickness, and arranged adjacent to the insulating layer. The
insulating layer may be configured to be sandwiched between the
first and second electrodes, and the second electrode may be
configured with a width that is less than the width of the first
electrode. The device may also include an opening through the
electrodes configured to expose the gap between them and
establishing an electrodes-insulating layer junction, where the
opening is spaced away from the perimeter of each electrode, one or
more recognition molecules comprising a cognate ligand, a chemical
group configured to couple the cognate ligand to at least one of
the electrodes, and a reference electrode in communication with at
least one of the electrodes.
[0010] In some embodiments, the reference electrode is configured
with a substantially constant potential difference with respect to
at least one of the electrodes. In addition, the cognate ligand may
comprise at least one of an antibody, a Fab fragment, an aptamer
and a peptide configured to bind to one or more protein
targets.
[0011] In some embodiments, the device is configured to detect
single molecular binding events corresponding to low concentration
sample solutions, and may also be configured for sequencing a
peptide chain.
[0012] In some embodiments, a system for detecting the binding of a
molecule to a cognate ligand is provided and may comprise one
and/or another of devices for detecting binding events as disclosed
herein, and purification means for purifying a patient serum sample
for obtaining one or more target proteins. Such purification means
may comprise, for example, a lab-on-a-chip (e.g., as disclosed
herein).
[0013] In some embodiments, a method for detecting at least one
binding event between a recognition molecule and a target molecule
is provided, which may comprise providing one and/or another of
detecting devices disclosed in the present disclosure, providing a
substantially fixed bias between the electrodes, flowing a sample
solution adjacent to the junction, recording current signals
generated as a result of one or more binding events between one or
more molecules in the sample and corresponding recognition
molecules, and determining at least one of a number and type of
molecules present in a sample solution based on the characteristics
of the signal generated as each type of molecule binds.
[0014] The characteristics of the signal may comprise at least one
of baseline current, peak current above a baseline, peak width,
peak shape as encoded by Fourier, wavelet or Cepstrum component
amplitudes, and flatness of the peak top expressed as root mean
square signal variation.
[0015] Some method embodiments may further include recording at
least one of the number and type of molecules determined.
[0016] Some method embodiments may further comprise at least one of
recording and counting signals generated by single molecule binding
events of a plurality of protein variants contained in the sample
solution.
[0017] At least some of the embodiments provide a platform which
may be readily configured to sequence proteins, for example, using
methods outlined in international patent application no.
PCT/US2013/024130, entitled, "System apparatuses and methods for
reading an amino acid sequence," hereby incorporated by
reference.
[0018] In some embodiments, a system for generating a target
molecule includes a first channel configured to receive a sample.
The sample includes cells, and the cells contain a biomolecule. The
first channel includes antibodies for capturing the cells in the
sample, and the first channel is further configured to receive a
lysis buffer for lysing the captures cells to generate a lysate.
The system also includes a second channel fluidly coupled to the
first channel. The second channel is configured to receive the
lysate and is functionalized with antibodies for binding the
biomolecule in the lysate. The second channel is further configured
to receive an eluting solution to generate an elution buffer
including the biomolecule. The system also includes a third channel
fluidly coupled to the second channel. The third channel is
configured to receive the elution buffer including the biomolecule
and is further configured to fragment the biomolecule to generate a
solution including the target molecules.
[0019] In some embodiments, a system includes a first recognition
tunneling electrode configured to interface a first reservoir
during use. The system also includes a second recognition tunneling
electrode coupled to the first recognition tunneling electrode. The
system also includes a dielectric substrate coupled to the second
recognition tunneling electrode. The dielectric substrate is
configured to interface a second reservoir during use. A nanopore
is formed through the first recognition tunneling electrode, the
second recognition tunneling electrode, and the dielectric
substrate, and is configured to fluidly couple the first reservoir
and the second reservoir. The system also includes a bias voltage
source configured to establish a voltage bias between the first
recognition tunneling electrode and the second recognition
tunneling electrode. The system also includes a current monitor
configured to generate a recognition tunneling signal when a
molecule passes adjacent to or through the nanopore.
[0020] In some embodiments, a system includes a first microfluidic
device, the first microfluidic device including a first
microfluidic channel. The first microfluidic channel including a
first inlet port and a first outlet port. The system also includes
a first recognition tunneling electrode configured to interface the
first microfluidic device. The system also includes a second
recognition tunneling electrode coupled to the first recognition
tunneling electrode. The system also includes a second microfluidic
device, the second microfluidic device including a second
microfluidic channel. The second microfluidic channel includes a
second inlet port and a second outlet port. A nanopore is formed
through the first microfluidic channel, the first recognition
tunneling electrode, the second recognition tunneling electrode,
and the second microfluidic channel. The nanopore is configured to
fluidly couple the first microfluidic channel and the second
microfluidic channel. The system also includes a bias voltage
source configured to establish a voltage bias between the first
recognition tunneling electrode and the second recognition
tunneling electrode. The system also includes a current monitor
configured to generate a recognition tunneling signal when a
molecule passes adjacent to or through the nanopore.
[0021] These and other embodiments of the present disclosure, as
well as objects and advantages of one or more thereof, will become
event more evident with reference to the attached drawings and
detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 illustrates a plot of the concentration of proteins
in vivo versus the number of protein species at a given
concentration.
[0023] FIG. 2 illustrates a prior art device of Pishrody et al.
(Pishrody, S. M., S. Kunwar, and G. T. Mathai, Electronic Detection
of Biological Molecules Using Thin Layers, U.S. Pat. No. 6,824,974
B2 Nov. 30, 2004).
[0024] FIG. 3 illustrates a device according to some of the
embodiments of the present disclosure.
[0025] FIG. 4 illustrates a system according to some embodiments,
which shows connection of biasing components and a reference
electrode(s), as well as illustrating the surface of the electrodes
functionalized with the ICA reader molecules (for example).
[0026] FIG. 5, main panel: a scanning-electron microscope (SEM)
image of a top view of a device according to some embodiments;
insert (e) illustrates a trench cut into the center region where
electrodes intersect (according to some embodiments).
[0027] FIG. 6 illustrates a tunneling-electron microscope (TEM)
cross section of a tunnel junction edge according to some
embodiments, which shows a 2 nm (for example) sensing gap (a slice
of the junction was lifted out, e.g., using focused ion beam
milling, to take this image).
[0028] FIG. 7 illustrates collected signals without (a, b) and with
(c, d) a reference electrode connected to a device according to
some embodiments.
[0029] FIG. 8 illustrates a method for forming a quasi-reference,
according to some embodiments, by extending the lower electrode so
that it contacts the fluid.
[0030] FIG. 9 illustrates recognition signals at low concentration
of analyte, according to some embodiments; current spikes that go
up to 90 pA correspond to molecules binding a pair of recognition
molecules such that the tunnel gap is bridged, while the background
(30 pA) current comes from binding events to just one
electrode.
[0031] FIG. 10 illustrates distributions of current peak values for
dGMP, dCMP, dmCMP (5-methyl cytosine monophosphate) and a basic
nucleotide at low (10 nM) concentrations, according to some
embodiments of the present disclosure.
[0032] FIG. 11 illustrates a plot of the distribution of start
times of current spikes at low concentrations, as detected by some
embodiments, illustrating an exponential distribution for single
molecule events.
[0033] FIG. 12 illustrates a signal train from a detection device
according to some embodiments as the four DNA nucleotides are
flushed through with rinses in between; the signal comprises a
baseline current (BL) and noise spikes, both of which are
chemically-sensitive.
[0034] FIG. 13 illustrates baseline current vs. analyte
concentration, with respect to some embodiments of the present
disclosure, with the solid line being a fit to the Hill-Langmuir
isotherm (equation 1).
[0035] FIG. 14 is a plot of current spike height (.DELTA.I) and
baseline current (BL) vs. bias for a sample of 10 nM dAMP as
determined by some embodiments of the present disclosure. Controls
using the buffer solution alone are shown in the lower two curves.
Conductance of the device increases abruptly at 350 mV bias which
is 450 mV vs Ag/AgCl at the top electrode 103, given that the
bottom electrode 101 (see FIG. 3) was held at +100 mV vs Ag/AgCl.
The scale directly beneath the plot indicates the bias across the
device, while the lower scale indicates the potential of the top
electrode with respect to the Ag/AgCl reference.
[0036] FIG. 15 illustrates types of binding that generate the
signal spikes and the background current, according to some
embodiments of the disclosure.
[0037] FIG. 16 illustrates a current-signal train generated by 1
.mu.M concentration of the Abltide peptide (with a phosphorylated
tyrosine in this case), with respect to some embodiments, where the
upper trace shows the control signal in buffer solution alone.
[0038] FIG. 17 illustrates signals being different for the
phosphorylated and unphosphorylated versions of the Abltide
peptide, as shown by a scatter plot of the value of two variables,
the cluster amplitude and the ratio of high to low frequency
amplitudes of the FFT of the cluster signals.
[0039] FIG. 18 is a schematic illustration of a lab-on-a-chip
configuration for isolating peptides from blood cells, according to
some embodiments. Enrichment of phosphorylated tyrosine containing
peptides can be added if needed, and reaction with the azidoacetic
anhydride and polyion solutions can be added when nanopore based
devices are used to further extend the sensitivity.
[0040] FIG. 19 illustrates a device according to some embodiments
for at least one of sequencing a polymer, and for electrophoretic
concentration of peptides ligated to charged tails, comprising a
device like that shown in FIG. 3, with a further orifice drilled
through the lower supporting membrane adjacent to the tunnel
junction.
[0041] FIG. 20a illustrates an RT junction with a nanopore that
allows transit of peptides from one side (Trans) of the chip to the
other (Cis), according to some embodiments, where coupling of
peptides to charged polymers (shown as d(T).sub.3 here) generates a
multitude of counts/second at sub pM concentrations in low salt
electrolytes.
[0042] FIG. 20b illustrates a microfluidic device for nanopore
translocation according to some embodiments, where a recognition
tunneling chip (dark slab) is sandwiched between upper and lower
chambers.
[0043] FIG. 20c illustrates a 10 nm diameter nanopore drilled
through an RT device, according to some embodiments, using helium
ion FIB.
DETAILED DESCRIPTION OF SOME OF THE EMBODIMENTS
[0044] FIG. 3 illustrates a device according to some of the
embodiments of the present disclosure. An example of the biasing
and reference electrode configuration and connection, which may be
used with such disclosed devices, is illustrated in FIG. 4.
[0045] Referring to FIG. 3, a device according to some embodiments
of the disclosure (which may also be referred to as a tunneling
device), may be constructed by deposition of a layer of Pd 101, or
similar noble metal, of about 10 nm thickness over/on an adhesion
layer, which may be about 1 nm Ti on at least one of a silicon or
silicon nitride support 100. This layer can cover an area of many
microns (e.g., about 1.times.1 micron up to about 100.times.100
microns) on each side, though, in some embodiments, the particular
dimensions may not be important in the plane of the device. In some
embodiments, the electrodes can be greater than 10 nm in thickness
(portions of the electrode spaced away from the junction by more
than 1 to 100 nm may not contribute to the signal), with some
embodiments including a thickness of less than about 500 nm may be
used.
[0046] The support may comprise any generally flat support
surface/substrate, including a semiconductor wafer surface or a
glass surface, or a mica surface. In some embodiments, a first
electrode (which may also be referred to as a lower metal
electrode) may comprise a strip of about 5 microns in width,
contacted at each end by, for example, gold wires (A1 and A2 in
FIG. 5), although any metallic material/contact to connect with the
electrode may be used.
[0047] In some embodiments, a majority of the lower portion of the
device may be covered with a dielectric material (i.e., insulating)
layer 102 of between about 2 nm to about 3 nm thick. The dielectric
material may be deposited by, for example, atomic layer deposition.
In some embodiments, the entire lower portion of the device may be
completely covered with the dielectric. Any dielectric material may
be used, including oxides of silicon and other semiconductors and
oxides of metals, including Hafnium Oxide. In some embodiments,
dielectric layers thicker than 2 to 3 nm may be used when seeking
larger target molecules (example embodiments may include a
dielectric layer of between about 1 nm to about 50 nm.
[0048] On the surface of the deposited layer of Al.sub.2O.sub.3, a
Pd (or similar metal) wire 103 of less than about 100 nm width, may
be deposited. While any metal will perform the required function of
the wire 103 the noble metals, Au, Pt, Pd, Rd, Ag, Os, Ir are
preferred. In some embodiments, this top wire is configured with
the noted range of width, for at least one of the following
reasons. Firstly, the edges do not contribute signals because the
edges of the top 103 and bottom 101 electrodes are well separated
by the film of Al.sub.2O.sub.3 or other dielectric. Thus, signals
can be limited to an area of the device that is opened deliberately
as described below. Secondly, tunnel junctions can be produced at
high yield, since a narrow top electrode may be less likely to
contain an area with pinholes through the dielectric layer 102.
Accordingly, in some embodiments, a wire of less than about 500 nm
in width may be preferable. The quality of the tunnel junction
formed between the electrodes 101 and 103 may be evidenced by
measuring current 106 when a voltage 105 is applied across the
device.
[0049] In some embodiments, in order to expose the edges of the
electrodes, which may be referred to as the tunneling junction, to
the solution containing analyte molecules, a hole 104 is cut
through the structure using, for example, reactive ion etching
(RIE). In some embodiments, this may be carried out using Cl.sub.2
gas to etch the palladium and BCl.sub.3 gas to etch the
Al.sub.2O.sub.3. A TEM cross section through the edge of the
exposed tunnel junction, taken by milling out a slice with a
focused ion beam, is shown in FIG. 6, which clearly illustrates a 2
nm separation of the electrodes at the edge of the junction
(according to some embodiments). A top down view of the region cut
away by RIE is shown in the inset on the upper right (e) in FIG.
5.
[0050] In some embodiments, the device may be covered with a layer
of PMMA, which is opened (e.g., via lithography) over the hole and
a microfluidic channel may be positioned such that analyte can be
delivered to the tunnel junction.
[0051] Referring to FIG. 4, the lower electrode 101 may be
connected to a reference electrode (shown here as Ag/AgCl
reference, 203) which may be held at a bias V.sub.ref 202 with
respect to lower electrode 101. Thus, the top electrode 103 may be
at a potential of V.sub.ref+V.sub.bias with respect to the
reference electrode. By holding electrode 103 positive of zero
volts versus Ag/AgCl, the lower electrode 101 may be kept away from
instabilities cause by adsorption of negative charges which could
move the electrode into a region where hydrogen evolution occurs.
Values of V.sub.ref for Pd electrodes, in some embodiments, may be
between 0 to +200 mV, with 100 mV being commonly used. The effect
of the reference electrode, in some embodiments, is to stabilize
the device, and example of which is illustrated in FIG. 7. As
shown, panels a and b show signals taken with DNA nucleotides dAMP
(a) and dGMP (b) without a reference electrode. Accordingly, large
swings in current, and regions where no signal occurs, are
consequences of the electrochemical instability of the device.
However, in some embodiments, upon connecting a reference electrode
to the device (e.g., to the top electrode 103), panels d and e, the
device is stable, and can produce trains of reproducible signal for
times that usually exceed an extensive period of time (e.g., 5
hours).
[0052] The reference may be any of the standard reference
electrodes placed in contact with the solution that carries the
molecules to the junction. Examples include, for example, Ag/AgCl
as shown in FIG. 4, a saturated calomel electrode, and a hydrogen
electrode. Such electrodes have an advantage that the
electrochemical processes (e.g., for devices according to some
embodiments) can be related to a standard potential scale, useful
in interpreting signals from the device. However, it is not
essential for stable operation of the device. All that is required,
in some embodiments, is a connection to an electrode of relatively
constant polarization (e.g., a "quasi reference") where the
relationship of the electrode potential to standard scales (such as
the normal hydrogen scale) is not known. However, a quasi-reference
is easier to implement. A quasi reference may be any large
electrode area in contact with the solution. Accordingly, if the
interfacial capacitance is very large compared to that of the
device electrodes (e.g., pF or more), than the quasi reference may
not change potential significantly as molecules are adsorbed from
the solution. FIG. 8 shows one way to implement this on the device
itself. As shown, portions of the device away from the tunnel
junction may be passsivated with a layer of insulator 404,
typically PMMA, that has been patterned to expose the junction.
Lower electrode 101 is shown extending out of the insulated region
404 to leave a large area 405 exposed to contact the electrolyte
carrying the target molecules. In some embodiments, the quasi
reference may be made by extending the lower Pd electrode, but any
suitable metal could used for this function, including Ag, Au, Pt,
Ir, Rd (for example).
[0053] Current spikes are shown in FIG. 7 for an exemplary device
and target molecules/analytes, according to some embodiments, which
may be produced when the electrodes are functionalized with a layer
of recognition molecules (201 in FIG. 4). In the noted example, the
recognition molecules are
4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide (ICA). When the
analyte concentration is reduced to a low enough value (e.g., about
10 nM for the DNA nucleotide dAMP), the complex signals resolve
into reproducible spikes of current that sit on top of a background
current signal. An example is given in FIG. 9, in this case for 10
nM solution of dAMP in 1 nM phosphate buffer, pH=7.0. The current
background is about 30 pA and the spikes positioned on top of
background reach about 90 pA. The distribution of signals may be
narrow, as shown for dGMP, dCMP, d(5-methylCMP) and an a basic
nucleotide in FIG. 10. Furthermore, according to such embodiments,
the interval between the arrival of successive peaks, is
exponentially distributed (FIG. 11), which is illustrative of
single-molecule origin of these spikes.
[0054] The baseline current signal may also contain chemical
information. FIG. 12 shows a current trace for micromolar
concentrations of the four nucleotides, dGMP, dAMP, dCMP and dTMP
upon being flowed through a device according to some embodiments.
As shown, at each rinse with buffer alone, the baseline signal
disappears, and the baseline is different for each analyte. The
baseline signal increases with analyte concentration (FIG. 13),
thus representing specific chemical effects on the tunnel junction
current caused by adsorption of the analyte molecules.
[0055] In some embodiments, the signals, which may include both the
current spikes and the baseline, have a non-linear dependence on
V.sub.bias as shown in FIG. 14. As shown, both the baseline signal
(BL) and the spikes (AI) increase in amplitude more rapidly with
voltage when the potential of the top electrode (103) exceeds 450
mV vs. Ag/AgCl. Thus, the same electrochemical change may affect
both the process that gives the spikes and the process that gives
the baseline. Cyclic voltammetry shows that this potential on the
Ag/AgCl scale corresponds to a reversible oxidation of the ICA
reader molecules. Thus, the current path in both cases (baseline
signals and signal spikes on top of the baseline) is via the ICA
reader molecules.
[0056] Inspection of the background current at low concentration of
analyte is illustrated in FIG. 9, which shows discrete steps in the
signal (enlarged in the inset), which provides support that, in
some embodiments, the background may be the sum of many single
molecule processes. In these embodiments, the baseline current
increases with adsorption of analyte molecules as the concentration
is increased (FIG. 13), and also when the reader molecule (201 in
FIG. 4) is oxidized, providing support that the signal corresponds
to the attachment of analyte molecules to the reader molecule.
Referring to FIG. 15, 300 shows a reader molecule and 301 shows a
reader molecule with an attachment to an analyte molecule 302. On
occasion, a reader molecule is aligned on each electrode such that
an analyte molecule can be bound in a manner (303) that provides a
conducting path between the two electrodes 101 and 103.
Accordingly, quantum mechanical calculations provide evidence that
the type of connection shown in 303 is responsible for the larger
current spikes in FIG. 8 (of 90 pA amplitude). The multitude of
other types of binding, like those shown as 301, combine to provide
the background signal. It is a feature according to some such
embodiments, that the background signal may be, itself, chemically
sensitive and generates signals of high frequency with much smaller
concentrations than those that give the bigger spikes generated by
the rarer attachments like those illustrated as 303.
[0057] For example, in some embodiments, increased sensitivity of
detecting binding events may be illustrated by providing a device
having two electrodes, each of 10 nm height and 50 nm width, the
total electrode area presented to the solution is 1000 (nm).sup.2.
Each reader molecule occupies about 1 (nm).sup.2, thus providing
about 1000 reader molecules on the electrodes. Clearly, in such
embodiments, the binding of just one molecule may be readily
detectable. Thus, it is straightforward to configure a device
according to some embodiment to detect the binding of about 0.1% of
the available sites. Furthermore, in some embodiments, even if the
site is occupied for just a fraction of time, electrical signals
are detected. For example, an event of 1 ms duration is readily
detectable, as shown by the signals in FIG. 9. In such embodiments,
assuming that the device is operated for 1 second, it may therefore
be possible to detect binding that occurs for only 1 part in 1000
of the observation time.
[0058] One of skill in the art will appreciate that according to
such embodiments, when taken together with the ability to observe
events like this from just 1 in 10.sup.3 molecules, a binding
fraction of 1 part in 10.sup.6 generates a readily detectable
signal. As an example, suppose that the reader molecule is an
affinity element, e.g., an antibody, Fab fragment, apatmer or
peptide, with a K.sub.d of 1 nm. This would correspond to a K.sub.d
value for a particularly good antibody, although a poor cognate
ligand will have a K.sub.d of 100 nM or better. Fractional coverage
.theta. of the electrode surface is given by the Hill-Langmuir
isotherm:
.theta. = C / K d 1 + C / K d ( 1 ) ##EQU00001##
where C is the concentration of analyte molecules. In equation (1),
.theta. implies that, with K.sub.d.about.1 nM, a concentration as
small as 10.sup.-15 M would yield one 1 ms event per second. A
K.sub.d of 100 nM would yield one event every 100 s. This
represents an enormous increase in sensitivity compared to current
techniques, where nM concentrations are typically the lower
limit.
[0059] Using a STM, it has been shown that peptides and amino acids
can be identified by means of characteristic features of the
recognition tunneling signal (Zhao, Y., B. Ashcroft, P. Zhang, H.
Liu, S. Sen, W. Song, J. Im, B. Gyarfas, S. Manna, S. Biswas, C.
Borges, and S. Lindsay, Single Molecule Spectroscopy of Amino Acids
and Peptides by Recognition Tunneling. Nature Nanotechnology 9,
466-473 (2014)). Accordingly, FIG. 16 shows recognition tunneling
signals generated by a chip according to some embodiments of the
present disclosure, functionalized with ICA molecules. The top
trace is a control sample in buffer alone, the bottom trace is a
series of signals produced when a 1 .mu.M solution of a peptide
(the Abltide sequence, a target for the Abl tyrosine kinase) is
added. FIG. 17 shows an example of two characteristics of the
signal spike generated by such embodiments, the maximum amplitude
in a cluster of signals, and the ratio of the high to low
amplitudes in the Fourier transform of a signal cluster, readily
separate spikes from the phosphorylated and unphosphorylated
peptides. In a 2D probability density map of recorded events, data
points from the phosphorylated and unphosphorylated peptides fall
into different regions of the plot (there is some overlap in the
region marked with an ellipse). Since many drugs target peptides
sites for phosphorylation and dephosphorylation, this information
may be of clinical significance. As shown above, an analysis like
this can be carried out at sample concentrations that may be as low
as 10.sup.-15M. In some embodiments, since, the device uses a
sample volume of only about 10 ul, amounts of sample as low as
10.sup.-20 Moles (or just 6000 molecules) may be readily
detected.
[0060] In clinical applications, such measurements may be limited
by the dynamic range of concentrations of analytes present in serum
or urine. Therefore, some pre-filtering and selection of targets
for analysis may be required. Accordingly, FIG. 18 illustrates a
lab-on-a-chip, according to some embodiments, which may be
configured to process a clinical sample for presentation a
recognition tunneling chip according to some embodiments. In this
figure, the design is configured for the example of phosphorylation
of the JAK2 kinase using whole blood as the input, but it will be
recognized that similar arrangements can be used for many other
specific clinical problems. As shown, whole blood is input at 601
to a channel that uses antibodies in a channel 604 to capture blood
cells. The captured cells may be lysed with a lysis buffer 602
introduced by means of valves 603 provided the on-chip. Waste may
be eluted via 605. The lysate may be eluted via port 606 into, for
example, a protein capture channel 608 which may be functionalized
with antibodies for the target (shown here as JAK2). Waste may be
eluted through port 609. Elution buffer may be used to transfer the
isolated proteins via 610 to a channel containing trypsinized beads
611, which generates peptide fragments of the protein which can
then be passed out 613 to be sent to the tunnel junction of devices
according to some embodiments for analysis.
[0061] In some embodiments, in the event that the concentration of
modified (i.e. phosphorylated) peptide is too small for detection,
the output of the trypsin column 611 can be passed to a further
selection channel 614 where antibodies for, e.g., phosphorylated
tyrosine, capture the phosphorylated fraction. Waste may be eluted
through 616 after which the captured peptides are eluted 619 for
transfer to the tunnel junction for counting.
[0062] In co-pending U.S. provisional patent application No.
61/826,855, entitled, "Improved Chemistry For Translocation Of A
Polymer Through A Nanopore" a method is described for concentrating
peptides by adding a charged tail to them using azidoacetic
anhydride to ligate a charged peptide to the N terminus of the
target peptide, and them using electrophoresis to draw the
molecules into the tunnel gap. If such further concentration is
desired, and additional stage is added to the chip according to
some embodiments of the present disclosure. For such a case,
peptides may be passed from 614 (or from 613 if enrichment of a
modified species is not required or desired) to reaction channel
618 where azidoaceticanhydride 617 is added. The reactants are
passed to a buffer exchange column 621 and waste products eluted at
620. The modified peptide can now be reacted with the charged tail
(polyion solution) which is added at 622 and the reaction allowed
to occur in the channel 623. The eluted complex of peptide and
charged tail is collected at 624 to be passed to the tunnel
junction of the device according to some embodiments of the present
disclosure. One method to implement electrophoretic detection of
the target molecule is to incorporate a nanopore into the tunnel
junction, as shown in FIG. 19.
[0063] Operation of some of the embodiments of the present
disclosure can be used with ICA molecules (developed for reading
DNA bases), as well as with an argenine-glycine-aspartic acid (RGD)
peptide terminated in a cysteine (--SH to bind the metal). In the
later, very small concentrations of integrin, the protein that
binds the RGD sequence, can be detected. Clearly, any cognate
ligand containing a residue or chemical terminus that allows it to
be attached to the electrodes can be used in this application.
[0064] The device according to some embodiments of the present
disclosure may be readily adaptable to strand sequencing of
peptides as disclosed in co-pending international application no.
PCT/US2013/024130. For such application, the peptide chain is
passed by the electrode junction sequentially, such that, a small
pore is arranged in close proximity to the electrodes, so that a
peptide, pulled through the pore by a charged tail pass each
residue by the electrodes. Once the underlying substrate is exposed
by RIE (as described above), a small opening 501 can be cut through
the remainder of the substrate, as illustrated in FIG. 19. In some
embodiments, an opening, placed within a few nm of the electrodes,
can be drilled using an electron beam or a helium ion beam without
damaging the electrodes (for example).
[0065] In some embodiments, systems are presented for utilizing a
device according to some embodiments, which involves sandwiching
the device between two fluid chambers, in fluid communication with
each other via a nanopore in the tunnel junction, and providing a
biasing components) configured to apply an electric field across
the nanopore (for example). One such configuration is shown
schematically in FIG. 20a. FIG. 20a shows an overview of a scheme
for drawing peptides ligated with a charged tail. A fluid, 201, is
in communication with a nanopore 202, an opening connecting the
fluid reservoir 201 on the trans side (also referred to as a "trans
reservoir") to a second fluid reservoir 202 on the cis side (also
referred to as a "cis reservoir"). A first reference electrode 203
is placed in the trans reservoir 201, and second reference
electrode 204 is placed in the cis reservoir 202. A bias voltage
205 is applied between the reservoirs 201, 202. When a peptide 206
is tethered to a negatively charged molecule (e.g., such as DNA
207) the reference electrode 204 is made positive with respect to
the reference electrode 203 so as to draw the negatively charged
molecule 207 into the pore 202 and through the pore, pulling the
peptide 206 behind it. This arrangement/approach is reversed for
positively charged molecules. The voltage V can be between about 1
mV and about 10V, and can be preferably between about 20 mV and
about 500 mV.
[0066] The nanopore 202, shown cut open in FIG. 20a, is cut through
two palladium electrodes 210, 211 (though other noble metals such
as Pt and Au could be used) each of about 1 nm to about 20 nm
thickness, and preferably of about 10 nm thickness. The nanopore
202 can be between about 1 nm and about 100 nm in diameter, with
about 2 to about 10 nm preferred. The metal layers are separated by
a thin dielectric layer 212, made (in some embodiments) from
aluminum oxide, though other oxides such as hafnium oxide and/or
magnesium oxide can be used. The thickness of this dielectric layer
212 is between about 0.1 to about 100 nm, and preferably about 1 to
about 4 nm. The nanopore 202 continues through a dielectric
substrate 213 which can be an oxide of silicon, a layer of graphene
or MoS.sub.2, or, in the preferred embodiment Silicon Nitride. The
thickness of this substrate can be about 1 nm to about 100 nm, with
about 10 nm to about 20 nm preferred.
[0067] A bias 215 is also applied between the two electrodes 210
and 211, and a current to voltage converter device 216 (which may
also be referred to as a current monitor) can be used to generate
the recognition tunneling signals. The voltage 215 can be between
about 1 mV and about 10 V, with about 20 mV to about 800 mV
preferred. Either one of the electrodes 210, 211 can be connected
to a reference electrode 219 placed into either one of the
reservoirs 201, 202. The reference electrode 219 can be biased with
a voltage 218 that can be between about 0V and about .+-.1 V.
[0068] An arrangement for creating the two fluid reservoirs 201,
202 is shown in FIG. 20b. The recognition tunneling device 300
(consisting at least of the layers 210, 211, 212 and 213 in FIG.
20a) is sandwiched between two microfluidic devices 301, 302. The
microfluidic devices 301, 302 can be made from any elastomer but
silicone rubber is preferred. The microfluidic devices 301, 302
contain channels 303, 304 formed so as to direct fluid injected
into ports 305 and 306 to contact the upper or lower side of the
device 300. Reference electrodes 203 and 204 are embedded in the
channels 303, 304. Exit ports 307 and 308 permit fluid to be
withdrawn from the channels. In order to achieve a well controlled
potential drop across the nanopore 202 formed in the device 300 it
is desirable to have the channel be at least about 0.1 mm by about
0.1 mm in cross section and, in some embodiments, no longer than
about 10 cm in length.
[0069] Any and all references to publications or other documents,
including but not limited to, patents, patent applications,
articles, webpages, books, etc., presented anywhere in the present
application, are herein incorporated by reference in their
entirety. However, mention of any reference, article, publication,
patent, patent publication, and patent application cited herein is
not, and should not be taken as an acknowledgment or any form of
suggestion that they constitute valid prior art or form part of the
common general knowledge in any country in the world.
[0070] Although a few variations have been described in detail
above, other modifications are possible. For example, any logic
flows depicted in the accompanying figures and/or described herein
do not require the particular order shown, or sequential order, to
achieve desirable results. Other implementations may be within the
scope of at least some of the following exemplary claims.
[0071] As noted elsewhere, these embodiments have been described
for illustrative purposes only and are not limiting. Other
embodiments are possible and are covered by the disclosure, which
will be apparent from the teachings contained herein. Thus, the
breadth and scope of the disclosure should not be limited by any of
the above-described embodiments but should be defined only in
accordance with claims supported by the present disclosure and
their equivalents. Moreover, embodiments of the subject disclosure
may include methods, systems and apparatuses/devices which may
further include any and all elements from any other disclosed
methods, systems, and devices, including any and all elements
corresponding to binding event determinative systems, devices and
methods. In other words, elements from one or another disclosed
embodiments may be interchangeable with, or additions to or
deletions of, elements from other disclosed embodiments. In
addition, one or more features/elements of disclosed embodiments
may be removed and still result in patentable subject matter (and
thus, resulting in yet more embodiments of the subject disclosure).
Also, some embodiments correspond to systems, devices and methods
which specifically lack one and/or another element, structure,
and/or steps (as applicable), as compared to teachings of the prior
art, and therefore represent patentable subject matter and are
distinguishable, and thus patentable, therefrom (i.e. claims
directed to such embodiments may contain negative limitations to
note the lack of one or more features prior art teachings).
* * * * *