U.S. patent application number 16/011065 was filed with the patent office on 2018-11-29 for biomolecular sensors and methods.
The applicant listed for this patent is Roswell Biotechnologies, Inc.. Invention is credited to Barry L. Merriman, Paul W. Mola.
Application Number | 20180340220 16/011065 |
Document ID | / |
Family ID | 57586591 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180340220 |
Kind Code |
A1 |
Merriman; Barry L. ; et
al. |
November 29, 2018 |
BIOMOLECULAR SENSORS AND METHODS
Abstract
Electronic sensors configured to detect single molecule targets
and methods of using and manufacturing such electronic sensors are
disclosed. A sensor may include a first electrode and a second
electrode separated by a sensor gap. The first and second
electrodes can be coupled by a sensor complex that can include a
biopolymer bridge molecule and a probe. The probe can interact with
a target molecule, and interaction of the probe and target molecule
can produce a signal suitable to provide detection of the target
molecule.
Inventors: |
Merriman; Barry L.; (San
Diego, CA) ; Mola; Paul W.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roswell Biotechnologies, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
57586591 |
Appl. No.: |
16/011065 |
Filed: |
June 18, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15336557 |
Oct 27, 2016 |
10036064 |
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16011065 |
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PCT/US16/39446 |
Jun 24, 2016 |
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15336557 |
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62184776 |
Jun 25, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
G01N 27/3278 20130101; B82Y 40/00 20130101; G01N 27/3276 20130101;
G01N 27/4145 20130101; G01N 27/3272 20130101; G01N 27/327 20130101;
B82Y 15/00 20130101; G01N 27/26 20130101 |
International
Class: |
C12Q 1/6869 20180101
C12Q001/6869; G01N 27/414 20060101 G01N027/414; G01N 27/327
20060101 G01N027/327; G01N 27/26 20060101 G01N027/26 |
Claims
1. A sensor device comprising: a first contact coupled to a first
electrode; a second contact coupled to a second electrode; a sensor
gap defined between one of the first contact and the first
electrode and one of the second contact and the second electrode;
and a bridge molecule comprising a first end and a second end;
wherein the bridge molecule comprises a biopolymer, and wherein the
bridge molecule is coupled to the first contact at the first end
and coupled to the second contact at the second end; and a probe
molecule coupled to the biopolymer to form a sensor complex,
wherein the probe molecule is coupled to the biopolymer by a
precisely positioned linker.
2. The sensor of claim 1, wherein the biopolymer bridge molecule
has an atomically precise attachment point for a probe
molecule.
3. The sensor device of claim 1, wherein the sensor gap has a
sensor gap dimension of between about 5 nm and about 30 nm.
4. The sensor device of claim 1, wherein the first end comprises a
first self-assembling anchor and the second end comprises a second
self-assembling anchor.
5. The sensor device of claim 1, wherein the biopolymer bridge
molecule comprises a linear biopolymer.
6. The sensor device of claim 5, wherein the linear biopolymer
comprises an end-to-end length that is less than a persistence
length of the linear biopolymer.
7. The sensor device of claim 5, wherein the linear biopolymer
comprises an end-to-end length configured to approximate a sensor
gap dimension of between about 5 nm and about 30 nm.
8. The sensor device of claim 1, further comprising a probe,
wherein the probe is attached to the biopolymer bridge molecule by
a precisely positioned linker of predetermined length, and whereby
the probe is configured to engage a target molecule.
9. The sensor device of claim 8, wherein the probe comprises an
enzyme configured to engage the target molecule.
10. The sensor device of claim 9, wherein the enzyme is a
polymerase or a reverse transcriptase.
11. A sensor device comprising: a first electrode overlying a
substrate surface; a second electrode overlying the substrate
surface; a sensor gap defined between the first electrode and the
second electrode; and a bridge molecule comprising a first end and
a second end; wherein the sensor gap comprises a sensor gap
dimension of between about 5 nm and about 30 nm; wherein the bridge
molecule comprises a biopolymer bridge molecule; and wherein the
bridge molecule is coupled to the first contact at the first end
and coupled to the second contact at the second end.
12. The sensor device of claim 11, wherein the biopolymer bridge
molecule has an atomically precise attachment point for a probe
molecule.
13. The sensor device of claim 11, wherein the first end comprises
a first self-assembling anchor and the second end comprises a
second self-assembling anchor.
14. The sensor device of claim 11, wherein the biopolymer bridge
molecule comprises a linear biopolymer.
15. The sensor device of claim 14, wherein the linear biopolymer
comprises an end-to-end length that is less than a persistence
length of the linear biopolymer.
16. The sensor device of claim 15, wherein the linear biopolymer
comprises an end-to-end length configured to approximate a sensor
gap dimension of between about 5 nm and about 30 nm.
17. The sensor device of claim 11, further comprising a probe,
wherein the probe is attached to the biopolymer bridge molecule by
a linker, and wherein the probe is configured to engage a target
molecule.
18. The sensor device of claim 17, wherein the probe comprises an
enzyme configured to engage the target molecule.
19. The sensor device of claim 18, wherein the enzyme is a
polymerase or a reverse transcriptase.
20. The sensor device of claim 11, wherein the biopolymer bridge
molecule comprises a nucleic acid duplex.
21. The sensor device of claim 20, wherein the nucleic acid duplex
comprises a DNA duplex, a DNA-RNA hybrid duplex, a DNA-PNA hybrid
duplex, a PNA-PNA duplex, or a DNA-LNA hybrid duplex.
22. The sensor device of claim 20, wherein the nucleic acid duplex
comprises a thiol-modified oligonucleotide.
23. The sensor device of claim 13, wherein one of the first
self-assembling anchor and the second self-assembling anchor
comprises a 5'-thiol modification
24. The sensor device of claim 20, wherein the nucleic acid duplex
further comprises an internal modification.
25. The sensor device of claim 13, wherein the biopolymer bridge
comprises a peptide sequence, and wherein one of the first
self-assembling anchor and the second self-assembling anchor
comprises a cysteine residue or a peptide configured to selectively
bind gold, aluminum, silicon dioxide, or other specific metals or
material contacts.
26. The sensor device of claim 17, wherein the probe is an enzyme
configured to engage the target molecule during a reaction in a
solution comprising a plurality of different target molecules,
wherein the reaction comprises a time period t, and wherein
contacting the target molecule produces a plurality of
conformational changes in the enzyme in response to the plurality
of target molecule features, wherein each of the plurality of
conformational changes modulates an electrical measurement in the
sensor to produce a signal feature.
27. The sensor device of claim 26, further comprising a signal
processing system configured to detect the signal feature.
28. The sensor device of claim 26, wherein the sensor device is
configured to produce a signal trace comprising a plurality of
signal features detected over time period t.
29. The sensor device of claim 11, further comprising a signal
interpretation device, wherein the signal interpretation device
comprises a signal interpretation map, and wherein the signal
interpretation map is calibrated against a signal trace from a
known target sequence.
30. A method of manufacturing a sensor device comprising: (a)
forming a first electrode and a second electrode on a substrate
surface, wherein the first electrode and the second electrode are
separated by a sensor gap; (b) placing a first contact on the first
electrode and a second contact on the second electrode, wherein the
first contact and the second contact are separated by a contact
gap; and (c) attaching a bridge molecule to the first contact and
the second contact.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation U.S. patent application
Ser. No. 15/336,557 filed on Oct. 27, 2016, entitled "BIOMOLECULAR
SENSORS AND METHODS," which is a continuation of International
Application No. PCT/US16/39446 filed on Jun. 24, 2016, entitled
"BIOMOLECULAR SENSORS AND METHODS" which claims priority to U.S.
Provisional Patent Application No. 62/184,776 filed on Jun. 25,
2015, entitled "METHODS, COMPOSITIONS, APPARATUS AND MANUFACTURING
METHODS OF MOLECULAR ELECTRONIC SENSORS," the disclosure of which
are incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 27, 2016, is named 68952_00193_SL.txt and is 4,467 bytes in
size.
FIELD
[0003] The present disclosure relates to electronic sensor devices.
In particular, the disclosure relates to electronic sensor devices
that comprise one or more biomolecule components in a measurement
circuit.
BACKGROUND
[0004] Measuring properties at the molecular scale presents
numerous challenges, due to the sensitivity required, and the
presence of many potential sources of noise. In describing sensors
for this purpose, it is therefore helpful to be clear about all
sources of measurement error. In general, for any system or object
that may be measured, a measured state, m, will only be an
approximation of the actual system state, a. This may be due to any
of a number of factors, such as imperfect signal interpretation
reflecting error due to the operation of the sensor, the readout
process, or the signal interpretation, and also because contacting
the sensor to the system in some cases may perturb the state of the
system. That the measured state m is different than the actual
state a reflects the measurement error of the combined sensor,
readout, and interpretation. Ideally, a sensor system will be
constructed to make this measurement error as small as
possible.
[0005] To measure states at a molecular scale, such as in the case
of sequencing a DNA molecule, various efforts have been directed to
creating sensor systems in which the sensor device has a "probe"
that contacts the molecules of interest, preferably on a
single-molecule scale, while other features of the sensor device
are on larger nano- or micro-scales for purposes of manufacturing
the sensor devices or integrating them into a signal transduction
system.
[0006] In particular, a biosensor is an analytical device that
functionally integrates a biological recognition component into a
signal transduction system, to measure properties of biologically
relevant molecules, such as DNA, RNA or proteins. That integration
provides rapid and convenient conversion of biological events to
detectable electrical signals. Of the various electrical biosensing
architectures that have been devised, systems based on field-effect
transistors (FETs) appear promising because they can directly
translate interactions between target molecules (e.g., biological
molecules) and the FET surface into detectable electrical signals.
In a typical FET device, current flows along a channel that is
connected to two electrodes (also referred to as the source and the
drain). The channel conductance between the source and the drain
can be modulated by a third electrode (also referred to as the
gate) that is capacitatively coupled to the channel through a thin
dielectric insulating layer. FETs can be used to detect target
chemicals and measure chemical concentrations for a wide range of
commercial applications. A classical and widely used example is a
FET-based pH sensor, used to measure hydrogen ion concentration.
This was introduced by Bergveld in the 1970's, and is used in
solid-state pH sensors. The general field of ion-sensitive FET
(ISFET) devices expands upon that concept for other chemical
concentration measurements.
[0007] A limitation of current FET-type biosensor systems is their
sensitivity. Current biosensor systems are unable to perform single
molecule detection and identification. Likewise, they are unable to
monitor single molecule reaction dynamics. These sensitivity
limitations of FET-type biosensors prevent their use as detectors
in important biochemical assays, such as in single molecule
sequencing reactions.
[0008] Some efforts to improve FET biosensor sensitivity have
focused on use of carbon nanostructures, such as carbon nanotubes,
to form the channel between electrodes. However, carbon
nanostructures pose various obstacles with respect to biosensor
functionalization. In particular, there is no way to engineer in
attachments sites at specific, desired atomic locations, for the
purpose of attaching functional or sensitizing probe molecules.
Additionally, present limits on precision, control, and scale of
the synthesis of carbon nanostructures pose further challenges with
respect to sensitivity and reliable production of individual
sensors, establishing high density scalable arrays of sensors, and
commercial viability of sensor manufacturing. Current carbon
nanotube synthesis methods typically produce structures on a scale
of around 100 nm or longer in length, a scale that is likely to
pose limitations with respect to sensitivity as well as sensor
density on a multi-sensor platform.
[0009] Thus, molecular-scale electronic biosensor devices with
architectures compatible with increased sensitivity and precision,
reliable engineering, and that are further compatible with
efficient and commercially-viable manufacturing methods for
achieving increased sensor density on a multi-sensor platform, are
desirable. Likewise, improved methods of manufacturing such sensor
devices are also desirable.
SUMMARY
[0010] The present disclosure generally relates to sensors, systems
including the sensors, and to methods of forming and using the
sensors and systems. Exemplary sensors can be used to, for example,
sequence molecules such as DNA, RNA, or other oligonucleotides.
While the ways in which various embodiments of the disclosure
address the drawbacks of the prior art sensors are discussed in
more detail below, in general, the disclosure provides sensors that
are relatively easy and inexpensive to manufacture.
[0011] In accordance with various embodiments of the disclosure, a
sensor includes a first contact coupled to a first electrode, a
second contact coupled to a second electrode, a sensor gap defined
between one of the first contact and the first electrode and one of
the second contact and the second electrode, and a bridge molecule
comprising a first end and a second end, wherein the bridge
molecule is coupled to the first contact at the first end and
coupled to the second contact at the second end. In accordance with
various aspects of these embodiments, the bridge molecule is a
biopolymer, or the bridge molecule is chemically synthesized. In
accordance with additional aspects, the sensor includes a third or
gate electrode. In these cases, the gate electrode can be used to
tune and/or activate the sensor device. In accordance with further
aspects, the sensor gap has a sensor gap dimension of between about
5 nm and about 30 nm. In accordance with additional aspects, the
first end or the bridge molecule comprises a first self-assembling
anchor; in accordance with further aspects, the second end
comprises a second self-assembling anchor. Exemplary bridge
molecules can include one or more of the following attributes: the
bridge molecule can be linear (e.g., a linear biopolymer), the
bridge molecule has an end-to-end length that is less than a
persistence length of the bridge molecule, and the bridge molecule
includes an end-to-end length configured to approximate the
dimension of the sensor gap. Exemplary sensors include a probe
attached to the bridge molecule. The probe can be configured to
engage a single target molecule. Exemplary probes can include or be
an enzyme configured to engage the target molecule during a
reaction in a solution.
[0012] In accordance with additional embodiments of the disclosure,
a sensor includes a first electrode overlying a substrate surface,
a second electrode overlying a substrate surface, a sensor gap
defined between the first electrode and the second electrode (or
between contacts attached to the electrodes), and a bridge molecule
comprising a first end and a second end, wherein the bridge
molecule is coupled to a first contact at the first end and coupled
to a second contact at the second end. The sensor gap can include a
sensor gap dimension of between about 5 nm and about 30 nm. In
accordance with various aspects of these embodiments, the bridge
molecule is a biopolymer, or the bridge molecule is chemically
synthesized. In accordance with additional aspects, the sensor
includes a third or gate electrode. In these cases, the gate
electrode can be used to tune and/or activate the sensor device. In
accordance with additional aspects, the first end or the bridge
molecule comprises a first self-assembling anchor; in accordance
with further aspects, the second end comprises a second
self-assembling anchor. Exemplary bridge molecules can include one
or more attributes noted herein. Exemplary sensors include a probe
attached to the bridge molecule. The probe can be configured to
engage a single target molecule. Exemplary probes can include or be
an enzyme configured to engage the target molecule during a
reaction in a solution.
[0013] In accordance with additional exemplary embodiments, a
system includes a sensor as described herein. The system can
additionally include one or more circuits, such as a circuit formed
using a substrate used to form the sensor or upon which the sensor
resides. Systems can additionally or alternatively include
additional circuits and/or devices to, for example, remove noise
from a signal and/or assist with interpretation of the signal.
[0014] In accordance with yet additional embodiments of the
disclosure, a method includes providing a sensor, such as a sensor
described herein; contacting a nucleic acid template with a
polymerase, wherein the polymerase is coupled to a bridge molecule
comprising a portion of a sensor; providing a nucleotide base mix;
performing, by the polymerase, an incorporation event comprising
incorporation of a nucleotide from the nucleotide base mix into a
synthesized nucleic acid; and detecting a signal produced by the
incorporation event. In accordance with various aspects of these
embodiments, a method can additionally include a step of applying
an electrical potential to the sensor--e.g., to tune or activate
the sensor. In accordance with further aspects, noise can be
removed from the signal.
[0015] In accordance with yet additional embodiments, a method of
manufacturing a biomolecular sensing device includes the steps of
forming a first electrode and a second electrode on a substrate
surface, wherein the first electrode and the second electrode are
separated by an electrode gap; placing a first contact on the first
electrode and a second contact on the second electrode, wherein the
first contact and the second contact are separated by a contact
gap; and attaching a bridge molecule to the first contact and the
second contact. Exemplary methods can further include the step of
contacting the bridge molecule with a probe to couple the probe to
the bridge molecule.
[0016] And, in accordance with further embodiments of the
disclosure, a method of sequencing an oligonucleotide comprises
using one or more sensors as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The subject matter of the present disclosure is particularly
pointed out and distinctly claimed in the concluding portion of the
specification. A more complete understanding of the present
disclosure, however, may best be obtained by referring to the
detailed description and claims when considered in connection with
the drawing figures.
[0018] FIG. 1 illustrates a schematic representation of a sensor in
accordance with various embodiments;
[0019] FIGS. 2A and 2B illustrate views of a sensor device in
accordance with various embodiments;
[0020] FIG. 3 illustrates a profile view of a portion of a sensor
in accordance with various embodiments;
[0021] FIG. 4 illustrates a sensor comprising a biopolymer bridge
molecule in accordance with various embodiments;
[0022] FIG. 5 illustrates a sensor comprising a biopolymer bridge
molecule in accordance with various embodiments;
[0023] FIGS. 6A and 6B illustrate views of a sensor device in
accordance with various embodiments;
[0024] FIG. 7 illustrates a signal trace before and after noise
removal in accordance with various embodiments;
[0025] FIG. 8 illustrates a process flow for a method of
fabricating electrodes using CMOS techniques in accordance with
various embodiments;
[0026] FIG. 9 illustrates a process flow for a method of
fabricating contacts using CMOS techniques in accordance with
various embodiments;
[0027] FIG. 10 illustrates a process flow for a method of
fabricating contacts using CMOS techniques and deposition of
preformed contact particles in accordance with various
embodiments;
[0028] FIGS. 11A-11C illustrate views of a sensor device fabricated
using CMOS techniques in accordance with various embodiments;
[0029] FIG. 12 illustrates a scanning electron micrograph of a
contact array following biopolymer bridge self-assembly in
accordance with various embodiments;
[0030] FIG. 13 illustrates a signal trace produced during a
biopolymer bridge self-assembly event for a sensor in accordance
with various embodiments;
[0031] FIG. 14 illustrates a signal trace produced during a process
of probe binding to a biopolymer bridge of a sensor in accordance
with various embodiments;
[0032] FIG. 15 illustrates a signal trace produced during template
binding to a probe in accordance with various embodiments;
[0033] FIG. 16 illustrates a signal trace produce during
template-dependent base incorporation by a probe in accordance with
various embodiments;
[0034] FIG. 17 illustrates a signal trace produced by a single
template-dependent base incorporation event by a sensor in
accordance with various embodiments;
[0035] FIG. 18 illustrates signal traces produced by a sensor in
accordance with various embodiments under various experimental
conditions;
[0036] FIG. 19 illustrates a signal traces produced by a sensor in
accordance with various embodiments under various conditions in
response to a target comprising unmodified and 5-methylcytosine
modified nucleotides;
[0037] FIG. 20 illustrates signal traces produced by a sensor in
accordance with various embodiments in response to a long template
sequence under various experimental conditions; and
[0038] FIG. 21 illustrates a chemically synthesized bridge molecule
in accordance with various embodiments.
DETAILED DESCRIPTION
[0039] The detailed description of exemplary embodiments herein
makes reference to the accompanying drawings, which show exemplary
embodiments by way of illustration and their best mode. While these
exemplary embodiments are described in sufficient detail to enable
those skilled in the art to practice the invention, it should be
understood that other embodiments may be realized and that logical,
chemical, and mechanical changes may be made without departing from
the spirit and scope of the inventions. Thus, the detailed
description herein is presented for purposes of illustration only
and not of limitation. For example, unless otherwise noted, the
steps recited in any of the method or process descriptions may be
executed in any order and are not necessarily limited to the order
presented. Furthermore, any reference to singular includes plural
embodiments, and any reference to more than one component or step
may include a singular embodiment or step. Also, any reference to
attached, fixed, connected or the like may include permanent,
removable, temporary, partial, full and/or any other possible
attachment option. Additionally, any reference to without contact
(or similar phrases) may also include reduced contact or minimal
contact.
[0040] In various embodiments, a single molecule biosensor device
can comprise a first electrode and a second electrode. The first
electrode and the second electrode are separated by a sensor gap
defined by the electrodes and/or contacts attached to the
electrodes. The first and second electrodes can be coupled by a
bridge molecule spanning the sensor gap. The bridge molecule can
comprise a biopolymer, such as nucleic acid or amino acid polymers.
The bridge may also comprise a chemically synthesized molecule,
which may include a synthetic organic molecule, a polymer
comprising synthetic analogs of biopolymer monomers, or other
wholly synthetic monomers not derived from a biological molecule. A
bridge molecule, whether comprised of a biopolymer or a synthetic
molecule, may have a known, atomically precise molecular structure.
The bridge molecule attachment to the electrodes may be mediated by
a contact. A probe molecule or molecular complex can be coupled to
the bridge molecule. The probe can be a biomolecule such as an
enzyme configured to interact with a single target molecule. In
various embodiments, a sensor device can comprise multiple single
molecule biosensors arrayed in parallel. Such multi-sensor devices
can be used to perform parallel detection, discrimination, and/or
characterization or identification of multiple individual target
molecules in a complex mixture of target and other molecules.
[0041] FIG. 1 illustrates a schematic representation of a sensor
device 100 comprising a sensor 101 in accordance with various
embodiments. Sensor 101 includes a first electrode 102 and a second
electrode 103. Sensor 101 may also include a gate 104, as described
in greater detail below. Sensor 101 can further comprise a sensor
complex 105 functionally coupled to the first electrode 102 and the
second electrode 103. In various embodiments, the sensor complex
may be coupled to the electrodes via first contact 106 and second
contact 107 attached to the respective electrodes. Sensor complex
105 can comprise multiple components, such as a bridge molecule and
a probe molecule, as described in greater detail below. Sensor
complex 105 can interact with the surrounding environment, thereby
enabling sensor 101 to perform a sensing function. For example, as
illustrated in FIG. 1, sensor complex 105 may interact with a
target molecule 108 such as a DNA molecule, and the sensor device
can be used to detect the presence of and/or properties of the
target molecule.
[0042] In various embodiments, sensor device 100 and sensor 101 may
be operatively connected to circuit 120 to detect a change of an
electrical property of sensor 101. Circuit 120 is preferably an
integrated circuit with micro-scale proximity to the sensor 101,
but circuit 120 could also be embodied as an external electrical
meter, such as a bench-top current meter. Sensor device 100 can
comprise a plurality of sensors 101. Integrated circuit 120 can
comprise a circuit architecture that may be fabricated using CMOS
fabrication methods. Integrated circuit 120 can comprise an
electronic measurement circuit for each sensor 101 that is
fabricated within the same chip that provides support for the
sensor. Expressed differently, a sensor device 100 can comprise a
sensor 101 and an integrated circuit 120 in an integrated
microcircuit. Integrated circuit 120 can further comprise readout
circuitry and input/output features for connection to an external
signal processing system 121.
[0043] In various embodiments, use of an integrated circuit 120
residing on a common semiconductor chip with sensor 101 can reduce
sources of electronic noise in readings that can be produced by
macroscopic, external circuit elements. For example, such a circuit
may be a mixed signal CMOS sensor, comprising a small number of
transistors, in the range of 1 to 200 depending on the performance
requirements for sensitivity and readout. Such a circuit can
function to measure current in a single sensor 101 in various
embodiments. Further, a sensor device 100 can comprise an
integrated circuit 120 comprising sensor/readout circuits for an
array of sensors 101 so as to support the simultaneous operation of
a large number of sensors in contact with the same sample.
[0044] In various embodiments, a sample contacted by a sensor 101
will comprise a liquid-phase sample. The solution comprising the
sample may be extremely dilute and at low ionic strength to reduce
the noise in electrical measurements performed using the sensor.
The acquired signal will typically be the current flowing between
electrodes 102 and 103 in the sensor, although it could be a
related observable electronic parameter such as the voltage between
electrodes, resistance/conductance between electrodes, or gate
voltage.
[0045] In various embodiments, the configuration of sensor 101 and
integrated circuit 120 in an integrated microchip chip format
amenable to fabrication using modern CMOS fabrication methods can
facilitate production of sensor devices with a highly compact
architecture. In various embodiments, the integrated circuit for a
sensor may be located within about 100 .mu.m of the sensor gap, or
within about 50 .mu.m of the sensor gap, or within about 20 .mu.m
of the sensor gap, or within about 10 .mu.m of the sensor gap, or
within about 5 .mu.m of the sensor gap, or within about 1 .mu.m of
the sensor gap. Moreover, in various embodiments, a sensor device
can comprise a plurality of sensors, each sensor having an
associated integrated circuit located within the parameters
specified above.
[0046] Signal processing system 121 can be configured to provide
electronic control of sensor device 100 and to receive, store, and
analyze signal received from the sensor device and each sensor 101
therein. Signal processing system 121 can comprise a computer
system with a processor and/or software configured to perform the
electronic control functions, including control of the voltage and
current applied to each sensor 101, and to perform the signal
processing functions for signal received from each sensor 101.
[0047] For example and as illustrated in FIG. 1, a sensor device
100 comprising a sensor 101 may be used to perform a nucleic acid
sequencing reaction. During operation of the device, a voltage may
be applied between the first electrode and the second electrode of
sensor 101, with interactions of the sensor with a target producing
modulation of current flow through a biopolymer bridge molecule
(see, e.g., 333, FIG. 3) that can be measured using integrated
circuit 120 and signal processing system 121. Sensor 101 may
produce a signal pattern 122 over time t with signal features 123
produced by the sensor in response to the sensor complex
interaction with features of target molecule 108. Signal processing
system 121 can receive and process the signal pattern and provide a
sequence output 124 in response to the signal pattern, which in
this context is the interpretation of the signal.
[0048] In various embodiments, a single molecule biosensor can take
the form of a transistor, such as a field effect transistor (FET),
with the attached bridge molecule and/or probe, and/or target
molecule and/or solution-phase molecules in close proximity to
these components, serving as a channel or conductive path in an
electrical circuit. In such an embodiment, a sensor complex
comprising a single probe molecule may be configured to bind or
interact with a single target molecule as explained in greater
detail below, thereby providing the biosensor with single molecule
sensitivity. Such a transistor embodiment may include a two or
three terminal transistor, or potentially more terminals, such as
in the case of multi-gate devices.
[0049] FIGS. 2A and 2B illustrate views of a sensor device 200 in
accordance with various embodiments. Sensor complexes are not shown
in the illustrated views of sensor device 200. Sensor device 200
comprises a plurality of sensors 201, with each sensor comprising a
first electrode 202 and a second electrode 203. Each sensor can
further comprise a sensor gap 239. In the illustrated embodiment,
each sensor comprises a first contact 206 attached to the first
electrode and a second contact 207 attached to the second
electrode. In various embodiments, the electrodes can be disposed
on a semiconductor substrate surface. For example, sensor device
200 can comprise a silicon nitride layer 260 overlying a silicon
dioxide layer 261. Sensor device 200 can further comprise buried
gate 204 underlying the semiconductor substrate layer(s) on which
the electrodes are disposed. The various components described above
can be fabricated on a support such as a silicon chip 263. As
illustrated schematically in FIG. 2A, each of the first electrode
201, the second electrode 202, and the gate 204 may be connected to
a signal processing system 221, which may be an external meter, as
depicted in the illustration, but which could alternatively be
integrated circuitry (details not shown).
[0050] With reference now to FIG. 3, a profile view of a portion of
a sensor 301 and sensor complex 305 are illustrated in greater
detail. Sensor 301 comprises first electrode 302 and second
electrode 303. First electrode 302 and second electrode 303 may be
disposed on a substrate 320. In various embodiments, sensor 301 can
further comprise a first contact 306 and a second contact 307
operatively coupled to first electrode 302 and second electrode
303, respectively. However, contacts are not strictly required, and
a sensor in accordance with the present disclosure need not
comprise a first and second contact. The ends of first electrode
302 and second electrode 303 define an electrode gap 330. Likewise,
for a sensor comprising contacts such as sensor 301, the distance
between first contact 306 and second contact 307 defines a contact
gap 331. The actual dimension of a contact gap for any given first
contact and second contact may vary dependent on the configuration
of the contact and the point of the contact used for reference. For
example, for the hemispherical first contact 306 and second contact
307 illustrated in FIG. 3, the dimension of contact gap 331 may be
measured between the nearest points of the contact or from center
to center. In various embodiments, one of the electrode gap and the
contact gap, or the gap defined collectively or by various
combinations of the electrodes and/or contacts, may be referred to
as a sensor gap.
[0051] With continued reference to FIG. 3, sensor 301 further
comprises sensor complex 305. In various embodiments, a sensor
complex 305 can comprise a bridge molecule 333 and a probe 334.
Probe 334 can be coupled to bridge molecule 333 via a linker 337,
which here is shown as a streptavidin-biotin complex, with the
biotin covalently incorporated into a nucleotide of the DNA bridge
333, and the streptavidin chemically, covalently cross-linked to
the polymerase 334. Each of the various components of sensor
complex 305 are described in greater detail below.
[0052] In various embodiments, a bridge molecule 333 can comprise a
chemically synthesized bridge molecule or a biopolymer bridge
molecule. A chemically synthesized bridge molecule or a biopolymer
bridge molecule may be configured to span a sensor gap both
structurally and functionally. For example, a chemically
synthesized molecule or biopolymer molecule may be configured
through selection and use of atomically precise molecular subunits
(e.g., monomeric units for incorporation into a polymeric bridge
molecule) that provide for construction of a bridge molecule with
known or predictable structural parameters, incorporation of
features that facilitate self-assembly to contact points and
self-assembly of a probe molecule to a bridge molecule, as well as
suitable electrochemical properties for electrical connection of
electrodes.
[0053] A chemically synthesized bridge molecule is a molecule that
can be assembled by a person of skill in the art of synthetic
organic chemistry. For example, a chemically synthesized molecule
can comprise a polypyrrole, polyaniline, or polythiophene backbone.
With reference briefly to FIG. 21, an example of a general
structure of a polythiophene-based chemically synthesized bridge
molecule 2100 is illustrated. Chemically synthesized bridge
molecule 2100 can comprise a chain of thiophene rings 2101 forming
the backbone of the bridge molecule, with n.sub.1 and n.sub.2
thiophene rings on either side of a probe support moiety 2102 that
may be configured at a specific location in the bridge molecule
2100. Since each thiophene ring 2101 is approximately 0.3 nm wide,
a chemically synthesized bridge molecule comprising about 10 to
about 100 rings could be constructed to span an about 3 nm to an
about 30 nm gap. The termini (e.g., A1 and A2) of a chemically
synthesized bridge molecule can comprise thiol or amine groups, or
other groups configured to bind to electrode or contact materials.
A chemically synthesized bridge molecule can also be configured
with a linker (e.g., L) suitable to provide attachment of a probe
molecule. Any other chemically synthesized bridge molecule
configuration, comprised of any suitable backbone moiety now known
to, or that may be hereinafter devised by, a person of ordinary
skill in the art, may be used in accordance with various
embodiments of the present disclosure.
[0054] As used herein, the term "biopolymer" can include any
molecule comprising at least one monomeric unit that can be
produced by a living organism, although the actual monomeric unit
comprising a biopolymer or the polymer itself need not be produced
by an organism and can be synthesized in vitro. Examples of
biopolymers include polynucleotides, polypeptides, and
polysaccharides, including well known forms of these such as DNA,
RNA and proteins. Bridge molecules that comprise a biopolymer can
include multi-chain polymeric proteins in a simple "coiled-coil"
configuration, as occurs in collagen proteins, or a more complex
folding of heavy and light chain polymeric proteins, such as in
immunoglobin molecules (e.g. IgG). Such complexes that comprise
biopolymers also include common nucleic acid duplex helices, such
as a DNA double helix, which is two DNA single strand molecules
bound into a helical double strand by hydrogen bonding, PNA-PNA
duplexes, as well as DNA-RNA, DNA-PNA, and DNA-LNA hybrid duplexes.
A biopolymer molecule need not be naturally occurring or produced
by an organism to be classified as a biopolymer. Instead, for
purposes of the present disclosure, the term "biopolymer" can
include molecules that are synthesized enzymatically as well as
non-enzymatically and can likewise include molecules comprising
synthetic analogues of naturally-occurring monomeric units. For
example, biopolymers can comprise peptide nucleic acids (PNAs) and
locked nucleic acids (LNAs), synthetic analogues of DNA and RNA
that have enhanced stability properties. In addition, a biopolymer
can comprise any of a variety of modifications that may be added to
a molecule. The use of biopolymer bridge molecules can provide
various benefits, including synthesis of precisely controlled
structures having suitable size and chemistry for sensor function,
they may be naturally compatible with the target molecules for the
sensor (e.g., compatible with the same liquid buffer medium), and
the biotech industry has developed extensive capabilities to
design, engineer and synthesize such molecules, and to manufacture
them economically and with high quality control.
[0055] A bridge molecule can be configured to span a sensor gap and
be coupled to an electrode and/or a contact on either side of the
sensor gap in a manner suitable to provide electronic communication
between the bridge molecule and the electrode and/or contact.
[0056] In various embodiments, a bridge molecule can comprise a
linear biopolymer such as a double-stranded DNA helix or an
.alpha.-helical polypeptide. As illustrated in FIG. 3, bridge
molecule 333 comprises a linear biopolymer double-stranded DNA
bridge molecule with a first end 334 coupled to first contact 306
and a second end 335 coupled to second contact 307.
[0057] In various embodiments, a rigid bridge structure may provide
advantages in terms of taking on a well-defined configuration
during and after assembly of the sensor complex. Without wishing to
be bound by theory, a linear biopolymer can comprise a
semi-flexible polymer that may be described by its bending
rigidity. On a short length scale, a linear biopolymer may behave
as a rigid polymer, requiring a strong force to bend the polymer,
while on a longer scale, the linear biopolymer may be bent or
curved more easily. The characteristic bending length measure
within which a linear biopolymer essentially behaves as a rigid
molecule in a certain set of environmental conditions is referred
to as the persistence length. The persistence length can depend on
the environmental conditions in which a bending force is exerted on
the polymer, with variables such as the temperature and ionic
conditions of the surrounding environment affecting the persistence
length. The persistence length of a linear biopolymer such as
double-stranded DNA may be estimated based on theoretical modeling
or it may be measured empirically for a set of environmental
conditions corresponding to a predetermined experimental condition
in which a device in accordance with various embodiments may be
used. For example, the persistence length of double-stranded DNA
has been calculated at about 30 nm to about 80 nm, and the
persistence length of an .alpha.-helical peptide calculated at
about 80 nm to about 100 nm in various conditions that may
approximate the conditions in which a sensor in accordance with
various embodiments of the present disclosure may be used. Thus, in
various embodiments, a double-stranded DNA molecule or an
.alpha.-helical peptide having an end-to-end length, as measured
along its major axis, of less than the respective persistence
length parameters described above may behave as an essentially
rigid polymer, thereby providing certain advantages or benefits
with respect to device assembly and performance.
[0058] In various embodiments, use of linear biopolymers comprised
of DNA or amino acids permits the straightforward construction of
nano-scale sensor components having a predetermined length based on
the monomeric composition (i.e., the primary structure) of the
biopolymer. Without wishing to be bound by theory, use of a linear
biopolymer with an end-to-end length of less than the persistence
length may enhance the efficiency of a self-assembly step during
construction of a biomolecular sensing device in accordance with
various embodiments. Use of such linear biopolymers provides an
ability to maintain the specifications of a biopolymer bridge
molecule within parameters in which their micromechanical
properties are more predictable than for longer linear biopolymers
that may bend or fold, thereby reducing the influence of
undesirable stochastic effects, for example, during bridge molecule
synthesis, handling, self-assembly, or sensor operation. Moreover,
the use of linear biopolymers permits precise specification of the
bridge molecule length to the sensor gap (i.e., the electrode gap
and/or contact gap dimension and architecture), providing a further
ability to readily test the performance of theoretical structural
models and device improvements and to make incremental,
well-controlled, and empirically-testable modifications. In various
embodiments, a linear biopolymer bridge molecule may be configured
to provide a reduced rate of miscoupling of both the first
self-assembling anchor at the first and the second self-assembling
anchor and the second end to one of the first contact and the
second contact due to the essentially rigid nature of the linear
biopolymer bridge molecule at the scale used in the sensor device
(e.g., an end-to-end length of between about 5 nm and about 30 nm).
Similarly, a biopolymer bridge molecule may be configured to
provide a reduced rate of single-end coupling. This may result when
the substantially rigid bridge molecule, once coupled at a first
contact, restricts the second end to spend more time in the
proximity of the desired second contact point, owing to the spacing
of contacts, thereby increasing the rate of the desired second
coupling reaction.
[0059] As mentioned above, a biopolymer bridge molecule can
comprise a double-stranded DNA molecule. In various embodiments, a
double-stranded DNA can comprise a thiol-modified oligo comprising
a thiol-modified nucleotide or base. A thiol-modified nucleotide
can comprise a self-assembling anchor configured to bind to a gold
nanobead or similar surface contact. In various embodiments, a
self-assembling anchor can comprise a 5'-thiol modified nucleotide,
which can be located at or near the 5' terminus of an
oligonucleotide. A double-stranded DNA molecule can comprise a
complementary pair of oligonucleotides, with each oligonucleotide
comprising a 5'-thiol modified nucleotide, such that the assembled
double-stranded DNA comprises a self-assembling anchor located at
both termini of a double-stranded DNA molecule. For example, in
various embodiments, a double-stranded DNA molecule can comprises
oligonucleotides with the following sequences:
TABLE-US-00001 (SEQ ID NO: 1) 5'-/5ThioMC6-D/TGC GTA CGT ATG TCA
TGA ATG GCG CAG ACT GAT GTC CTA TGA CGT CGC TAC TGC AGT ACT-3', and
(SEQ ID NO: 2) 5'-/5ThioMC6-D/AGT ACT GCA GTA GCG ACG TCA TAG GAC
A/iBiodT/C AGT CTG CGC CAT TCA TGA CAT ACG TAC GCA-3',
with the "/5ThioMC6-D/" denoting a 5'-thiol modifier and "/iBiodT/"
denoting an internal biotin-modified deoxythymidine nucleotide
(Integrated DNA Technologies, Inc., Coralville, Iowa). When
annealed to one another, these oligos provide a double-stranded DNA
molecule with a 5'-thiol modified nucleotide located at each end of
the molecule as the first and second self-assembling anchors.
[0060] A double-stranded DNA molecule bridge can also further
comprise a biotin linker component to facilitate linking a probe
molecule to the bridge with a complementary avidin-type linker
component. In various embodiments and as illustrated in the reverse
oligonucleotide sequence described above, a biotin-modified
oligonucleotide can be incorporated into one of the oligos of a
double-stranded DNA molecule bridge. In various embodiments, the
biotin-modified oligo is an internal modification, such as via a
modified thymidine residue (biotin-dT). A variety of biotin
modification configurations may be used, including attachment to
thymidine via a C6 spacer, attachment via a triethyleneglycol
spacer, attachment via a photocleavable spacer arm, dual biotin
modifications, desthiobiotin modifications, and biotin azide
modifications. Other modifications that are now known to a person
of skill in the art or may be hereinafter devised and may be made
to an oligonucleotide to facilitate linkage to a probe molecule are
within the scope of the present disclosure. Similarly, other common
small molecules with a protein binding partner, such digoxigenin,
can play a similar role to that of biotin for such purposes of
conjugation to probe molecules at precisely atomically specified
points in the bridge molecule.
[0061] In various embodiments, a peptide biopolymer bridge molecule
can comprise various configurations and/or features suitable to
provide various desirable bridge molecule structure and performance
characteristics, including electrode or contact binding
characteristics, structural characteristics, electrical performance
characteristics, and the like. For example, a peptide biopolymer
bridge can comprise an L-cysteine residue at one or both of the
amino terminus and the carboxyl terminus to serve as a
self-assembling anchor via thiol-metal binding to specific metal
contacts that engage in strong thiol binding, such as gold,
palladium or platinum. In other embodiments, a biopolymer bridge
molecule can comprise a peptide with the known capacity to
selectively and strongly bind gold contacts for purposes of
self-assembly and electro-mechanical connection into the circuit.
Specific such peptides include those with the following amino acid
sequences: MHGKTQATSGTIQS (SEQ ID NO: 3), VSGSSPDS (SEQ ID NO: 4),
and LKAHLPPSRLPS (SEQ ID NO: 5). Other peptides selected for such
properties can similarly bind other specific metal or material
contacts. For example, VPSSGPQDTRTT (SEQ ID NO: 6) is a known
aluminum binding peptide, and MSPHPHPRHHHT (SEQ ID NO: 7) is a
known silicon dioxide binding peptide. In various other
embodiments, a biopolymer bridge molecule can comprise a peptide
sequence that includes repetitions of an amino acid motif or motifs
selected from one of the following amino acid sequence motifs known
to favor the formation of stable alpha-helix conformations,
providing for a linear, rigid, conductive bridge: EAAAR (SEQ ID NO:
8), EAAAK (SEQ ID NO: 9), EEEERRRR (SEQ ID NO: 10), and EEEEKKKK
(SEQ ID NO: 11). Such a peptide biopolymer bridge molecule can also
comprise a modified amino acid consisting of a lysine residue with
a covalently attached biotin to provide a conjugation point at a
precisely atomically defined location for avidin-based conjugation
to probe molecule complexes. A modified lysine can replace a
standard lysine or arginine residue in such a peptide sequence
motif, to otherwise maintain or minimally alter the properties of
the alpha-helix.
[0062] In various embodiments, a biopolymer bridge molecule can
have other configurations. For example and as illustrated in FIG.
4, a biopolymer bridge molecule 433 can comprise a linear
biomolecule that is flexed, folded, or comprises a certain degree
of secondary structure. In various embodiments, a biopolymer bridge
molecule can further comprise molecules having tertiary and/or
quaternary structure, including globular proteins, antibodies, and
multi-subunit protein complexes. An example is illustrated in FIG.
5, in which the biopolymer bridge molecule 533 comprises an
immunoglobin G protein (IgG). In the illustrated embodiment, the
electrical contacts (506, 507) are gold nano-particles, and the IgG
has been established with a specific affinity to bind such
particles.
[0063] Similarly to sensor 301, the configurations illustrated in
FIG. 4 and FIG. 5 each comprise a probe (436 and 536, respectively)
coupled to the biopolymer bridge molecules via linkers (437 and
537, respectively). The illustrated embodiments are intended to
exemplify the range of possible biopolymer bridge molecule
configurations that may be couple to electrodes or contacts
comprising different materials and configurations, including
different metallic or non-metallic conducting or semiconducting
contacts in different structural configurations. In various
embodiments, electrodes or contacts may further be coated, treated,
or derivatized to facilitate bridge assembly and/or attachment
using products such as InnovaCoat GOLD nanoparticles (Innova
Biosciences).
[0064] A probe in accordance with various embodiments can comprise
any suitable molecule or multicomponent molecular complex. A probe
may be selected based on the molecule to be detected by the sensor
or the biochemical reaction to be monitored. Various examples of
probes include peptides, proteins, enzymes, nucleic acids,
ribozymes, catalytic DNAs, and the like. In various embodiments, an
enzyme can comprise a lysozyme, a kinase, or a polymerase. Any
molecule or complex that exhibits a specific change in physical,
chemical, or electronic configuration in response to binding or
processing of a substrate or target molecule may be used as a probe
in accordance with various embodiments of the present
disclosure.
[0065] In various embodiments, a probe can comprise an enzyme such
as polymerase or a reverse transcriptase suitable for interacting
with individual DNA or RNA target molecules. Enzymes that catalyze
the template-dependent incorporation of nucleotide bases into a
growing oligonucleotide strand undergo conformational changes in
response to sequentially encountering template strand nucleic acid
bases and/or incorporating template-specified natural or analog
bases (i.e., an incorporation event). Such conformational changes
can modulate electrical current through a bridge molecule to which
the probe is coupled, thereby provide a sequence-specific signal
pattern in a manner that is dependent on the template molecule. As
described above, the signal pattern may be detected by a signal
processing system and translated to a sequence data output.
Moreover, the presence of a modified nucleotide in a target nucleic
acid sequence may produce unique conformational changes and
corresponding signal features in a signal pattern that can enable a
sensor device and signal processing system to directly determine,
for example, methylation of bases in a target sequence on a
base-by-base basis. Such a label-free, direct sequencing method may
permit discrimination of a nucleotide-specific incorporation event
in a sequencing reaction using nucleotide base mix comprising a
mixture of natural and/or analog bases corresponding to all four
bases of DNA, although a sequencing process comprising sequentially
providing individual natural or analog bases in a serial and/or
cyclic fashion may also be used. The use of a reverse transcriptase
as the probe molecule can similarly enable the direct sequencing of
RNA molecules without the need for an intermediate cDNA conversion
step.
[0066] In various embodiments and as described briefly above, a
probe can be attached to the bridge molecule via a self-assembling
linker. A self-assembling linker can comprise any of a number of
structures suitable to attach a first biomolecule to a second
biomolecule. In various embodiments, a self-assembling linker can
comprise a first linker component and a second linker component
that is complementary to the first linker component. The first
linker component and the second linker component may be joined by
self-assembly to form an assembled linker based on an affinity of
the first linker component for the second linker component. A first
linker component can be associated, for example, with a bridge
molecule, and a second linker component can be associated with a
probe. A linker component associated with a bridge molecule can be
engineered to a specific site in the bridge molecule, such that
self-assembly of the probe to the bridge produces coupling of the
probe to the bridge molecule at a predetermined location on the
bridge molecule. A linker component selected for association with
the probe may be configured to minimize interference between the
probe and a target, both with respect to the size of the linker
component and the position at which it is conjugated to the probe.
In this manner, joining the complementary first and second linker
components can provide functional attachment of the probe to the
bridge molecule. A self-assembling linker can comprise a
biotin-avidin coupling mechanism, with an avidin (or other
avidin-like) protein first linker component and a biotin small
molecule second linker component, which components form a strong
non-covalent bond with one another. Other avidin-like proteins
include streptavidin, rhizavidin, bradavidin, NeutrAvidin, other
various amino-acid modified forms of avidin or streptavidin, as
well as divalent or monomeric derivatives of such avidins which
retain biotin-binding functionality. In various embodiments, for
example, a biotin may be conjugated to the bridge molecule and a
streptavidin conjugated to the probe molecule. A self-assembling
linker can also comprise the well-known "click-chemistry"
mechanisms for bioconjugation. A self-assembling linker can also
comprise an antigen-antibody coupling, for example with an antigen
present on the bridge molecule coupling to an antibody conjugated
to the probe molecule. A self-assembling linker can also comprise,
for example, a SpyCatcher peptide first linker component and a
SpyTag peptide second linker component, with the two components
binding to form an irreversible covalent bond. Any other
self-assembling linker system in any configuration now known to, or
that may be hereinafter devised by, a person of ordinary skill in
the art may be used to couple a probe to a bridge molecule.
[0067] In various embodiments, a sensor need not comprise a probe
molecule distinct from the bridge molecule. Instead, the bridge
molecule itself may be configured to be acted on by a target
molecule. For example, a bridge can comprise a protein binding
site, such as a kinase binding site, and be used to detect the
presence and/or activity of the corresponding protein in a sample
based on binding of the target protein to the bridge and/or
modification of the bridge by the target protein.
[0068] With reference now to FIGS. 6A and 6B, perspective views of
a partially-fabricated sensor device 600 with and without a sensor
enclosure are illustrated. Sensor device 600 is a three terminal
sensor device comprising a buried gate 640. Device 600 illustrated
in FIG. 6A comprises various features of a sensor device that may
be produced using CMOS fabrication techniques, such as gate 640
underlying substrate 641 and oxide 642, along with first electrodes
602 and second electrodes 603 separated by electrode gaps 630, and
each electrode having an attached contact 606/607. Attachment of
the various sensor complex components described above, including a
bridge molecule and probe, may be performed in downstream
self-assembly steps. In various embodiments and as illustrated in
FIG. 6B, sensor device 600 may first be configured with an
enclosure 643 configured to enclose or form a flow cell around
sensor gaps 630 prior to completing assembly of the sensors by
contacting the sensor with a solution comprising the bridge and/or
probe molecules. Likewise, enclosure 643 may also be used to
perform assays such as sequencing reactions. Enclosure 643 can be
separately formed and attached to a structure including device
600.
Biomolecule Detection and Nucleic Acid Base Discrimination
[0069] In various embodiments, a method for detecting the dynamics
and kinetics of a single molecule sensing device such as device 100
(FIG. 1) is provided. Any method for measuring changes in
electrical conductance of a sensor 101 comprising a bridge molecule
can be used to monitor a sensor device described herein. In various
embodiments, a voltage of less than about 10 V can be applied to a
sensor comprising a biomolecular bridge molecule, and in various
embodiments described in greater detail below, a voltage of about
0.5 V is applied. The current flowing through the sensor can be
measured as a function of time using integrated circuit 120. Target
binding and/or processing events by a probe (i.e., enzyme activity
in the case of an enzymatic probe) in sensor complex 105 can
produce changes to the conductivity of the sensor 101, modulating
the measured current to produce a signal pattern 122 over time t
comprising signal features 123. Such events, and the associated
conformational changes, including structural, chemical, and
electronic changes (i.e., charge distributions in an enzyme,
substrates, and surrounding solution) can comprise kinetic features
of target binding and processing, with the various events producing
current fluctuations comprising signal features 123 that can be
measured, recorded, discriminated, analyzed or stored using signal
processing techniques which are known in the art. The signal
features can comprise any of a range of possible forms, including
wavelets with shapes that are triangular, sinusoidal, or have any
number of Fourier components. For example, a polymerase used as a
probe in a sensor can provide a polymerase kinetic signature for
each discrete interaction with a template base (i.e., a target
molecule feature) and/or a template-dependent nucleotide
incorporation (i.e., the polymerase kinetic signature is template
base-dependent), with a nucleic acid template target comprising a
sequence of target molecule features at discrete positions in the
target molecule (i.e., first, second, and nth target molecule
features at first, second, and nth target molecule positions), each
target molecule feature producing a corresponding signal feature
during detection by a sensor in accordance with the present
disclosure. The n target molecule features can correspond to n
consecutive bases of a single stranded DNA template molecule (i.e.,
the target) which is processed by the polymerase enzyme to
sequentially incorporate complementary nucleotides at these n
target molecule features. The amplitudes, durations, and shapes of
a signal pattern comprising a series of signal features can encode
a target-specific sensor response that can be analyzed using signal
processing system 121 to compare the signal pattern to a signal
interpretation map to determine the identity of the target.
Increasing the time resolution of signal detection and analysis may
provide an ability to further resolve kinetic variability,
transitions, and intermediate states of a probe-target
interaction.
[0070] Since the fidelity of nucleotide incorporation is paramount
to accurate nucleic acid sequencing, in various embodiments, a
method of sequencing may rely on analog bases that increase the
conformational changes of template-based nucleotide incorporation,
thereby producing clearer signals, and/or otherwise provide an
enhanced ability to discriminate incorporation of the analog base,
thereby providing for enhanced sequencing accuracy. Non-labeled
analog bases that can be used to enhance the kinetic or dynamic
discrimination of template-dependent nucleotide incorporation are
well known and can include modifications of the purine and
pyrimidine bases and the deoxyribose or ribose and phosphate
portions of a nucleotide. In particular, this can include adding
additional groups to the gamma-phosphate of the nucleotide, which
accepts large and diverse molecular modifications that are cleaved
off during incorporation and therefore do not permanently impact
the growing strand and its interaction with the polymerase.
[0071] In various embodiments, a method can provide detection of
unmodified and modified nucleotide bases in a nucleic acid template
sequence. For example, a method may be suitable to distinguish a
modified template nucleotide, including N.sup.6-methyladenosine,
N.sup.4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine,
5-formylcytosine, and 5-carboxylcytosine bases, as well as damaged
template sequence positions such as abasic sites. Without wishing
to be bound by theory, a DNA polymerase catalyzing incorporation of
a nucleotide into a complementary nucleic acid strand during a
sequencing reaction may exhibit differential polymerase kinetics in
a manner dependent on the identity of the nucleotide in the
template strand. Using devices and methods in accordance with the
present disclosure, the identity of a nucleotide base in a nucleic
acid template may be determined in near real-time based on
detection of an electronic signature corresponding to the
incorporation event. Unlike other systems and methods that rely on
detection of a fluorescence signal associated with incorporation of
a fluorophore-labeled nucleotide, fluorescence-based detection
reagents and signal detection devices are not required, thereby
reducing cost and complexity of the process.
[0072] In various embodiments, a method can comprise removing noise
from a signal trace. Removing noise can comprise performing signal
processing, such as to remove 60 Hz line noise. Removing noise from
a signal trace can reduce the error of signal trace interpretation.
An example of a signal trace produced by sequencing a 12-base
nucleic acid template, before (upper signal trace) and after (lower
signal trace) removal of 60 Hz line noise from the signal, is
illustrated in FIG. 7. Various methods of noise removal may be
used, depending on the character of such noise, and such methods
are well known to a person of skill in the art in the field of
signal processing.
[0073] In various embodiments, signal processing to determine the
sequence of a target bound to a sensor may comprise a probabilistic
determination of the identity of the target, rather than an exact
determination of the sequence. The actual sequence of a target
molecule may be one of a number of possible unique sequences, each
possible unique sequence having a unique theoretical signal. A
determination of the sequence of the target molecule may require a
comparison of experimentally measured signal with a signal
interpretation map comprising a database of unique theoretical
signals. The signal interpretation map may be generated based on a
training data set or library produced using known target sequences,
signal processing based on positive and negative control
measurements to reduce signal artifact such as noise, blur, drift,
and the like, as well as application of machine learning and/or
statistical methods such as neural networks, clustering, curve
fitting, model fitting, Bayesian inference, etc.
Manufacturing and Assembly of a Sensor Device
[0074] In various embodiments of the present disclosure, a method
of producing a molecular biosensor device as described herein is
provided. A method of producing a molecular biosensor device can
comprise a combination of CMOS fabrication processes and molecular
biology methods. CMOS fabrications processes can comprise
high-resolution optical lithography methods that are well known in
the art and are suitable for commercial scale production of
integrated circuits, including devices such as FETs. In various
embodiments, CMOS fabrication processes can be used to produce
integrated circuits comprising individual sensors having a first
electrode and a second electrode deposited on a semiconductor base,
with the first electrode and the second electrode separated by a
precisely defined sensor gap. In a preferred embodiment, a
nano-electrode, gap and contact design would be chosen so as to be
manufacturable entirely within CMOS processes. In particular, if
specific simple geometries are chosen for these elements, they can
be fabricated using the high resolution optical lithography
methods, such as Extreme UV (EUV) and Deep UV (DUV) sources,
combined with phase-shifting masks, multiple-patterning, and other
techniques used to achieve highest resolution CMOS fabrication
nodes, including current and future 16 nm nodes, 14 nm nodes, 10 nm
nodes, 7 nm nodes and 5 nm nodes as embodied by specific
fabrication facilities, such as those at major foundry companies,
(e.g., TSMC or GlobalFoundries). Such processes have uniquely high
resolution for making certain specific pattern features, such as
straight line segments, straight line cuts, and circular spots. Use
of these process-specific geometric elements in the design of
nano-electrode, nano-contact, and/or gap geometries can facilitate
fabrication of a sensor device in accordance with various
embodiments in the associated CMOS process. However, in general the
manufacturing techniques employed may also comprise non-CMOS
process methods, such as e-beam lithography, nano-imprint
lithography, or milling and etching techniques such as focused ion
beam milling and plasma etching. Molecular biology fabrication
methods can comprise synthesis of the desired bridge molecules with
precise control over the atomic configuration, and delivery of
solutions of such biomolecules in a liquid phase under conditions
suitable to permit interaction and coupling of the biomolecules
with electronic sensor components produced in upstream CMOS or
other fabrication method process, and/or with other biomolecules,
in specifically designed self-assembly reaction processes.
[0075] In various embodiments, a method of manufacturing a sensor
device described herein can comprise steps that including:
manufacturing an integrated circuit microchip, fabrication of
sensor electrodes and/or contacts, synthesis of a bridge
biomolecule, assembling the bridge biomolecule to the electrodes
and/or contacts, coupling a probe to the bridge biomolecule, and
enclosing the sensor device in a flow cell. In various embodiments,
a sensor can comprise a two terminal circuit, or a sensor can
comprise a three terminal circuit with a gate. In various
embodiments, a gate may have a buried gate configuration; however,
lateral gate and other gate configurations, including finFET
structures, may also be used.
[0076] In various embodiments, an electrode, contact, and/or gate
may be comprised of conductive metal materials. For example, an
electrode, contact, and/or gate may comprise aluminum, titanium,
chromium, copper, gold, palladium, platinum, and the like. In
various embodiments, an electrode, contact, and/or gate may
comprise semiconductor materials, including doped semiconductor
materials that may be used to produce n-type and p-type
semiconductor electrodes. In various embodiments, an electrode and
a contact attached to the electrode can comprise the same material,
and in various other embodiments, a contact can comprise a material
that is different from an electrode to which it is attached.
[0077] In various embodiments, an electrode may have any suitable
structural configuration. For example, an electrode can comprise a
generally rectangular cross-section, although other geometric and
irregular cross-sectional profiles are possible and within the
scope of the present disclosure. In various embodiments, an
electrode can have a maximum cross-sectional dimension (i.e., the
maximum dimension of the electrode in a cross-section of the
electrode) of less than about 30 nm, or less than about 25 nm, or
less than about 20 nm, or less than about 15 nm, or less than about
14 nm, or less than about 13 nm, or less than about 12 nm, or less
than about 11 nm, or less than about 10 nm, or less than about 9
nm, or less than about 8 nm, or less than about 7 nm, or less than
about 6 nm, or less than about 5 nm, or less than about 4 nm, or
less than about 3 nm.
[0078] Similarly, in various embodiments, a contact may have any
suitable structural configuration. For example, a contact can
comprise a generally semi-spherical or hemi-spherical
cross-sectional profile, although other geometric and irregular
cross-sectional profiles are possible and within the scope of the
present disclosure. In various embodiments, a contact can have a
maximum cross-sectional dimension (i.e., the maximum dimension of
the contact in a cross-section of the contact) of less than about
20 nm, or less than about 15 nm, or less than about 14 nm, or less
than about 13 nm, or less than about 12 nm, or less than about 11
nm, or less than about 10 nm, or less than about 9 nm, or less than
about 8 nm, or less than about 7 nm, or less than about 6 nm, or
less than about 5 nm, or less than about 4 nm, or less than about 3
nm.
[0079] In various embodiments, the first electrode and the second
electrode may be alternately referred to as a source and/or drain,
and in various embodiments, a source and/or drain can comprise a
distinct structural component from an electrode.
[0080] A method of manufacturing can comprise using lithography
methods to define a first electrode location and a second electrode
location on the surface of a substrate. The first electrode
location and the second electrode location may be defined to
produce a precisely defined electrode gap between them upon
completion of electrode fabrication. Similarly, in various
embodiments, a method of manufacturing can comprise using
lithography methods to define a first contact position and a second
contact position. The first contact position and the second contact
position may be defined to produce a precisely defined contact gap
between them upon completion of contact fabrication. Likewise, a
contact can be configured with a defined structure. Various methods
that may be used to manufacture a biosensor are described in
greater detail below.
[0081] With reference now to FIG. 8, a lithographic method 800 for
fabricating electrodes is illustrated. In various embodiments, a
fabrication method may begin with a microchip substrate such as a
silicon substrate 880 overlayed with a silicon oxide layer 881 a
resist layer 882. The resist layer can comprise any suitable resist
material suitable, such as poly(methyl methacrylate). Adhesion
promoters may also be used in a fabrication process in accordance
with the present disclosure. In the illustrated embodiment, e-beam
lithography is used to expose the resist layer and to define a
first electrode track 883 and a second electrode track 884 in the
resist layer (step 810). Following the lithography step, the resist
is developed (step 820) to remove the resist in the areas defined
in the lithography step. Next, a deposition step (step 830) may be
performed to form a first electrode 802 and a second electrode 803
on the substrate surface. Any suitable material and deposition
method may be used, including, for example, metal sputter coating.
Likewise, any suitable substrate surface treatment, such as
application of an intermediate attachment layer to provide suitable
bonding between electrode and substrate, may be performed prior to
performing the deposition step. In various embodiments, the first
and second electrodes are fabricated from gold using a sputtering
deposition method. Following the deposition step, a lift-off step
(step 840) is performed to remove the remaining resist, leaving the
first electrode and the second electrode disposed on the surface of
the substrate.
[0082] In various embodiments, a lithographic method for
fabricating nano-electrodes such as method 800 can achieve highly
precise electrode configurations. For example, the electrodes can
be configured with consistent length, width, and thickness
specifications. In various embodiments, an electrode can have a
width of between about 10 nm and about 40 nm, such as a width of
about 20 nm. Likewise, the electrode gap defined by the first
electrode and the second electrode can be configured with a precise
electrode gap dimension. In various embodiments, the electrode gap
dimension may be between about 3 nm and about 30 nm. For example,
the electrode gap for a pair of electrodes in a sensor in
accordance with various embodiments can be between about 3 nm and
about 30 nm, or between about 4 nm and about 25 nm, or between
about 5 nm and about 20 nm, or between about 6 nm and about 17 nm,
or between about 7 nm and about 15 nm. In various embodiments, an
electrode gap can be fabricated with a dimension of about 3 nm,
about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9
nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14
nm, or about 15 nm. As will be evident to a person of ordinary
skill in the art, the various method steps described above can be
used to produce multiple pairs of electrodes in parallel at high
density and with highly precise physical specifications in a
process amenable to commercial-scale production of sensor devices
using CMOS fabrication and/or other microelectronic fabrication
methods.
[0083] Without wishing to be bound by theory, providing a sensor
having an electrode gap (or a sensor gap) with an electrode gap
dimension as described above may provide various advantages with
respect to sensor performance and/or fabrication. For example, for
an electrode gap having a dimension below about 3 nm, spurious
sources of current conduction through the solution (i.e., the
sample environment) and bulk will start to increase, creating added
noise. In addition, such gaps may not be large enough to
accommodate various probe molecules of interest, such as enzymes.
Moreover, such gaps are not compatible with current CMOS
manufacturing capabilities. The cost and complexity of
manufacturing bridge molecules with atomically precise
specifications for electrode gaps greater than about 30 nm, such as
by using biopolymers or chemically synthesized molecules, rises
substantially, and the rigidity various bridge molecules may
decrease with lengths beyond about 30 nm. Likewise, the
conductivity of many molecules drops substantially below useful
parameters beyond those lengths, and greater lengths also limit the
ability to closely pack sensors in high density arrays. Thus,
sensors with electrode gaps in the range of about 3 nm to 30 nm may
afford certain advantages with respect to the function,
manufacturability, scalability and economics of a sensor
device.
[0084] An example of a sensor device fabricated in accordance with
the method described above is illustrated in FIGS. 11A-11C, which
shows scanning electron micrographs of the surface of a sensor
device 1000 at 37-fold, 5000-fold, and 50,000-fold magnification,
respectively. Sensor device 1000 comprises nano-electrodes and
nano-contacts for 20 sensors, as well as leads and pads for
connection to an external current meter. Pads and leads located on
the surface of the substrate are clearly visible in FIG. 11A.
Sensor electrodes appear as a lighter vertical band in the center
of FIG. 11B. In FIG. 11C, first electrodes 1102 and second
electrodes 1103 can be seen clearly, along with electrode gap 1120
defined between the first and second electrodes.
[0085] In various embodiments and with reference now to FIG. 9, a
method of manufacturing a biomolecular sensing device can comprise
a lithographic method 900 for fabricating and/or determining the
location of a contact. In various embodiments, a fabrication method
may begin with a microchip comprising a substrate on which a first
electrode 802 and a second electrode 803 are disposed. The
microchip can comprise silicon substrate 880 overlayed with silicon
oxide layer 881 and a suitable resist layer 982. In the illustrated
embodiment, e-beam lithography is used to expose the resist layer
and to define a first contact position 985 and a second contact
position 986 in the resist layer (step 910). In various
embodiments, the location of the contact may be defined to overlay
one of the first electrode and the second electrode, such as near a
distal end of the electrode adjacent to the electrode gap. The size
and pattern defined for the contact may contribute to determining
the size and shape of the contact formed in later process steps, as
described below. Following the lithography step, the resist is
developed (step 920) to remove the resist in the contact positions
defined in the lithography step. Next, a deposition step (step 930)
may be performed to form a first contact 906 and a second contact
907 on the first and second electrode surfaces. As for the
electrodes, any suitable material and deposition method may be
used. Likewise, any suitable substrate surface treatment, such as
application of an intermediate attachment layer to provide suitable
bonding between electrode and substrate, may be performed prior to
performing the deposition step. The contacts can comprise a
different material from the electrodes, or the contacts can
comprise the same material used to fabricate the electrodes. In
various embodiments, the first and second contacts are fabricated
from gold using an electrochemical deposition method. Following the
deposition step, a lift-off step (step 940) is performed to remove
the remaining resist, leaving the first contact and the second
contact disposed on the surfaces of the first and second
electrodes.
[0086] Alternately, in various embodiments, a method for
fabricating a contact can comprise deposition of preformed contact
nanoparticles. Preformed contact nanoparticles can be deposited
into a void formed in a resist layer and configured to receive a
contact nanoparticle and position it at a contact position, or
contact nanoparticles can be deposited using a chemical
derivatization layer to achieve attachment at a contact
position.
[0087] As illustrated in FIG. 10, a method 1000 of depositing a
preformed contact particle into void formed in a resist layer can
comprise the same steps described above for method 900 with respect
to steps 910 and 920. Following creation of a void configured to
receive a preformed contact particle, a solution comprising a
plurality of preformed contact particles 1087 can be contacted with
the device (step 1030) and the particles deposited into the voids
using pressure, mixing, surface tension, buoyancy, centrifugal
force, or other methods to introduce a particle into a void.
Following deposition of the particles, excess solution and
particles may be removed. A lift-off step can be performed to
remove remaining resist as described above with respect to step
940, and the preformed contact particles can optionally be annealed
to the electrodes in a subsequent as necessary to form strongly
attached first and second contacts (1006, 1007).
[0088] Alternately, a method of depositing preformed contact
nanoparticles using a chemical derivatization treatment can
comprise steps similar to steps 910 and 920 described above with
respect to the method illustrated in FIG. 9. For example, one such
widely used surface derivatization compatible with a silicon
substrate surface is silanization, which can include coating a
substrate surface with molecules such as aminosilanes (for example,
APTES) or mercaptosilanes (for example, MPTES). These molecules
adhere to a silicon surface, and then their exposed ends readily
cross-link to other materials such as gold nanoparticles to bind
them to the surface. Then, in a step similar to step 930, such a
derivatization treatment can be applied rather than depositing a
contact metal or other material. After a lift-off step similar to
step 940 is performed, the first electrode and the second electrode
will comprise a surface derivatization at the locations intended
for attachment of the first contact and the second contact. The
device comprising the derivatized electrode surfaces can be
contacted with a solution comprising a plurality of preformed
contact particles. The particles may have a surface or coating that
is complimentary to or otherwise binds specifically to the
derivatized electrode surfaces, thereby localizing the contact
particles to the defined contact positions. The derivatization
treatment and any particle coating may be removed in a removal
step, as necessary, and the preformed contact particles annealed to
the electrodes as described above. An example of this approach is
the use of an APTES-coated silicon surface to specifically bind a
gold nanoparticle.
[0089] In various embodiments, contact structures can be created by
various direct means, such as positioning gold nanoparticle beads
on electrodes by use of atomic force microscopy (AFM), or by
deposition of excess beads followed by AFM removal of unwanted
beads.
[0090] In various other embodiments, contact structures and/or an
electrode gap can be formed in place via material removal, such as
by using focused ion beam (FIB) milling. For example, an electrode
gap can be carved into a previously established continuous metal
nanowire using FIB, thereby creating a first electrode and a second
electrode simultaneously with forming the electrode gap.
[0091] Following fabrication of the electrodes and contacts of a
sensor or array of sensors, the sensor(s) may be enclosed in a flow
cell or similar device suitable to permit controlled introduction
of a liquid solution to the sensor(s). Enclosing the sensor chip in
a flow cell is typically done by molding a flow cell from PDMS or
other polymer or plastic, and using this to encase the chip,
leaving the fabricated electrodes and contacts suitably exposed for
bridge and probe assembly as well as subsequent assays using the
completed sensor(s). In various embodiments, a surface passivation
treatment may be applied to the substrate surface and portions of
the exposed electrodes to reduce electrical noise that can occur
from contact with liquid samples. The passivation treatment can be
applied to leave the electrodes and/or contacts in the area of the
sensor gap untreated. For example in various embodiments, a 30 nm
wide area aligned with the sensor gap may be left untreated. The
passivation treatment may be performed prior to enclosing the
sensor chip with a flow cell. A sensor in accordance with various
embodiments can have electronic noise of less than about 1 pA, or
less than about 0.9 pA, or less than about 0.8 pA, or less than
about 0.7 pA, or less than about 0.6 pA, or less than about 0.5 pA,
or less than about 0.4 pA, or less than about 0.3 pA, or less than
about 0.2 pA, when a voltage of about 0.5 V is applied and the
sensor is immersed in a low ionic strength buffer solution
otherwise suitable to support activity of an enzyme, for example
DNA polymerase I enzyme.
[0092] Fabrication of a biopolymer bridge can be performed by any
of a variety of methods that may be used to synthesize biopolymer
molecules, including in vivo synthesis methods, in vitro enzymatic
synthesis methods, chemical synthesis methods, or any combination
thereof. Various methods for producing biopolymer molecules
suitable for use as a bridge molecule in accordance with the
present disclosure will be well known to a person of ordinary skill
in the art. Likewise, methods for derivatizing or modifying a
biopolymer bridge molecule to provide an anchor or a linker
component as described herein are likewise well known. The various
specific biopolymer bridge molecules described herein are provided
by way of example and should not be interpreted as limiting the
scope of the present disclosure, and synthetic bridge molecules may
be used in accordance with various embodiments of the present
disclosure.
[0093] In various embodiments, attachment of a biopolymer bridge
molecule to a probe may be performed by a self-assembly chemical
reaction. Likewise, attachment of a biopolymer bridge molecule to
electrodes or contacts may also be performed by a self-assembly
chemical reaction. Such self-assembly reactions may be performed by
putting the two components to be attached into contact with one
another via a solution comprising at least one of the components.
In various embodiments, attachment of a biopolymer bridge to a
probe can be performed before, after, or simultaneously with
attachment of the bridge to electrodes or contacts. Similar
considerations apply to a bridge molecule produced by synthetic
chemistry.
[0094] In various embodiments, a method of making a sensor device
includes assembling a biopolymer bridge molecule to the first
electrode and the second electrode. The bridge molecule assembly
step can comprise a self-assembly step. Self-assembly can be
performed by contacting the partially constructed sensor device
comprising the first and second electrode with a solution
comprising the bridge molecule. The bridge molecule can
self-assemble to the first electrode and the second electrode based
on an affinity between the first end and the second end of the
bridge molecule and the first electrode and the second electrode.
In various embodiments, self-assembly of the sensor components can
be monitored electronically by the sensor device, as described
below in Example 2 and with reference to FIGS. 13-15. Electronic
monitoring can provide a quality control function and serve to
identify sensors in a device that are properly assembled. Signal
from sensors circuits that do not provide assembly process signals
within predetermined parameters may be disregarded in downstream
analyses, such as sequencing analyses, performed with the
device.
Example 1
Biopolymer Bridge Self-Assembly
[0095] A double-stranded DNA biopolymer bridge molecule with an
end-to-end length of about 20 nm was constructed using the oligo
set forth in SEQ ID NO: 1 comprising a 5'-thiol modification and
the oligo set forth in SEQ ID NO.: 2 comprising a 5'-thiol
modification and an internal biotin modification. The bridge
molecules were labelled for visualization purposes using a
streptavidin-gold tag. A test array 1200 (FIG. 12) of gold
nanoparticle contacts was fabricated using e-beam lithography
techniques to deposit pairs of gold contacts, each pair of contacts
defining a contact gap of about 20 nm, center-to-center. A buffered
solution comprising the gold-labelled bridge molecules was placed
in contact with the test array of gold nanoparticle contacts.
Following a brief incubation period, excess solution was removed
and the array was washed and imaged by scanning electron microscopy
(SEM). An SEM image showing the arrangement of gold contacts 1270
and gold tags 1271 is illustrated in FIG. 12. For several contact
pairs (indicated with arrows), a gold tag 1271 can be seen disposed
between the contact pair, indicating successful self-assembly of
the biomolecular bridge molecule to the pair of contacts.
Example 2
Detection of Self-Assembly Steps
[0096] A sensor device with a single sensor comprising gold
contacts attached to electrodes with a contact gap of about 20 nm,
center-to-center, was fabricated using e-beam lithography
techniques. The sensor was enclosed with a PDMS flow cell
comprising a 1 mm wide by 0.4 mm high channel that was open on
either end to permit introduction of liquid into a first end of the
flow cell interior and displacement of liquid from the second end
of the flow cell, and solution the cell contacting the sensor. The
flow cell channel was oriented orthogonally to the direction of the
electrodes comprising the sensor, with the sensor located in
approximately the middle of the length of the flow cell channel. A
low ionic strength buffer solution was introduced into the flow
cell, and a 0.5 V potential was applied to the sensor throughout
subsequent serial steps of introduction and self-assembly of a
double-stranded DNA bridge molecule (as described above for Example
1, but without a gold tag) (FIG. 13), introduction and binding of a
streptavidin-tagged Klenow fragment (FIG. 14), introduction and
binding of a 50 base primed single-stranded DNA molecule (FIG. 15),
and introduction of a dNTP mix to initiate template-based synthesis
by the Klenow fragment (FIG. 16). The sequence of the DNA template
molecule include the following oligo sequence featuring a poly-A
region:
TABLE-US-00002 (SEQ ID NO: 14) 5'-cgc cgc gga gcc aag aaa aaa aaa
aaa aaa aaa aa ttgcatgtcctgtga-3' and the primer used was: (SEQ ID
NO: 15) 3'-aac gta cag gac act-5'
[0097] In addition, similar sequences with the poly-A tract
replaced by poly-C, G, and T tracts were used to investigate the
effect of different template bases.
[0098] As illustrated in FIG. 13, the measured current rises over a
three second period. The two inflections points (A and B) in the
signal trace are thought to correspond to binding of the
5'-thiol-modified terminal base anchors to the first and second
contact. The signal trace following introduction of a solution
comprising streptavidin-tagged Klenow fragment (FIG. 14) exhibits a
sharp increase in current at about 1.5 s that is likely to
correspond to a streptavidin linker component of a Klenow fragment
enzyme contacting and binding the biotin linker component of the
biopolymer bridge. In FIG. 15, a sharp signal peak is present in
the signal trace following introduction of the template strand to
the flow cell, with the peak interpreted to correspond to template
binding by the Klenow fragment. The signal trace measured following
introduction of a dNTP mix comprising all for DNA bases,
illustrated in FIG. 16, likewise exhibits a distinct signal feature
at about 1 s. This may represent dissociation of the synthesized
duplex from the polymerase enzyme, and the signal trace from about
0.7 s to about 0.95 s may correspond to the kinetic signature
produced by the sensor in response to nucleotide incorporation
based on the bound template DNA.
Example 3
Detection of Nucleotide Base Incorporations
[0099] A sensor device comprising a biopolymer bridge molecule and
Klenow fragment probe was fabricated and assembled as described
above in Example 2. The sensor device was used to produce signal
traces in response to DNA synthesis reactions performed using
single-stranded DNA templates of various lengths and sequence
compositions. FIG. 17 illustrates a signal trace for a template
sequence that provides for the incorporation of a single base. The
signal feature at 0.5 s is interpreted to correspond to the
template-dependent activity of the Klenow fragment and base
incorporation, and the much weaker signal features just after 0.6 s
are interpreted to correspond to some form of noise or spurious
signal in the system. FIG. 18 illustrates signal traces for various
template tracts. The template and primer described above in Example
2 were used for the illustrated reactions.
[0100] The top and bottom signal traces are control experiments in
which buffer without dNTPs is introduced to a sensor. The second,
third, and fourth signal traces (from top to bottom) were produced
in response to introducing dTTP into solution (expected to result
in 20 incorporation events directed by the 20 A bases of the
template), followed by addition of dCTP (expected to allow another
3 incorporations directed by the GAA triplet in the template, 3' to
5'), followed by the addition of dNTP (expected to polymerize as
directed remaining 12 bases of the template) so that the signals
produced result from 20, 3, and 12 incorporation events. The signal
trace comprising the signal features located between arrows for
each signal trace is interpreted to correspond to signal modulation
due to template-dependent enzyme activity. The relative durations
of these perturbed signal regions is in the expected proportion of
20:3:12, and the third such tract displays a clear spike that may
correspond to the 12 discrete incorporation events. FIG. 7
illustrates an additional example of a signal trace produced by a
DNA synthesis reaction performed using the device described above
and the template described above with 12 unpaired template bases.
These results demonstrate that a sensor in accordance with various
embodiments can produce a signal trace comprising signal features
in response to template-dependent DNA polymerase probe activity.
This also demonstrates the value of noise removal in clarifying the
signal: the upper panel in FIG. 7 is the raw measured signal, and
the lower panel has undergone signal processing to remove noise, in
this case specific 60 Hz line noise was eliminated with a bandpass
filter.
Example 4
Detection of Methylated Template Bases
[0101] A sensor device comprising a biopolymer bridge molecule and
Klenow fragment probe was fabricated and assembled as described
above in Example 2. The sensor device was used to produce signal
traces in response to DNA synthesis performed using a
single-stranded DNA template comprising both cytosine and
5-methylcytosine modified nucleotides. The template sequence
included unpaired base nucleotides having the sequence
5'-13x(N)-5x(GmC)-5x(GC)-G-3' (i.e., 5'-NNN NNN NNN NNN NGmC GmCG
mCGmC GmCG CGC GCG CGC G-3' (SEQ ID NO: 12), where N is any
standard nucleotide and where mC is 5-methylcytosine). This
template sequence was designed to produce a complementary
synthesized strand having the sequence 5'-C-5x(GC)-5x(GC)-13x(N)-3'
(i.e., 5'-CGC GCG CGC GCG CGC GCG CGC NNN NNN NNN NNN N-3' (SEQ ID
NO: 13)), with the underlined guanosine bases corresponding to the
positions of the 5-methylcytosine modified nucleotides in the
template strand. A 0.5 V was applied to the sensor, and current was
measured through the course of sequential introductions and
incubations with water, buffer, a buffered solution of dCTP, a
buffered solution of dGTP, and a buffered solution with a mix of
all four dNTP bases. The expected result of this would be a single
dCTP incorporation event, then 20 dGTP and dCTP incorporation
events, the first 10 of which are against the unmodified cytosine
bases, and the latter 10 against the 5-methylcytosine modified
nucleotides. Detection of methylation would show up as a different
character of signal in the second 10 of these 20 events.
[0102] Signal traces produced during incubation with each reagent
are illustrated in FIG. 19. Incubation with water and buffer
produced very low, baseline current with little variation. Addition
of a solution comprising dCTP produced a sharp sequence feature
corresponding to a single base incorporation of dCTP against the
template lead base G. Addition of dGTP, creating a solution
comprising both dCTP and dGTP, permitted synthesis through the 10
base incorporations corresponding to unmodified nucleotides
followed by synthesis through the 10 base incorporations
corresponding to the 5-methylcytosine bases in the template strand.
The signal trace produced in this incubation period shows signal
features with higher current from about 0.35 s to about 0.5 s,
followed by signal features with lower current from about 0.5 s to
about 0.65 s. This shift in signal amplitude is interpreted as a
distinct change in the sensor signal in response to the effect of
the methylation status of the template sequence on the polymerase
and resultant signal modulation. This evidence supports the ability
of a sensor in accordance with various embodiments of the present
disclosure to directly distinguish the presence of modified
nucleotides in a target sequence during a sequencing reaction.
Example 5
Detection of Signal Over Long DNA Strand Reads
[0103] A sensor device comprising a biopolymer bridge molecule and
Klenow fragment probe was fabricated and assembled as described
above in Example 2. The sensor device was used to produce signal
traces in response to DNA synthesis performed using a
single-stranded DNA template comprising an approximately 5400 bp
template sequence derived from the genome of phi X 174
bacteriophage. A dNTP mix was provided in the experimental
sequencing reaction, while a ddNTP (dideoxynucleotide triphosphate)
mix was provided for a control reaction. The ddNTP mix terminates
the polymerization process after one incorporation of such a
dideoxy terminator, and thus essentially no sequencing sensing
signal should result. The data was acquired at 20 ms time sampling
resolution, which is too coarse to observe individual incorporation
spikes, but allowed data collection for a timer period over 300
seconds, long enough to observe the entire polymerization process
at the expected enzyme rate of approximately 20 bases per
second.
[0104] FIG. 20 illustrates the signal trace produced by the
experimental reaction with a dNTP mix (upper signal trace) compared
to that for the control reaction using a ddNTP mix (lower trace).
The signal trace for the experimental sequencing run included a
number of distinct, gross signal features (noted with arrows)
lacking in the control reaction and also produced a higher current
than the control reaction. The signal trace produced over the 100
second period shown suggests that a sensor in accordance with
various embodiments of the present disclosure may be suitable to
produce a detectable signal in response to template-based
nucleotide incorporation activity of a DNA polymerase probe over
the course of an extended sequencing run for a long template
sequence. Thus, there is no immediate limitation on the length or
template such a sensor can process.
Additional Examples
[0105] Additional nonlimiting examples of the disclosure include
the following.
[0106] 1. A sensor comprising: [0107] a first contact coupled to a
first electrode; [0108] a second contact coupled to a second
electrode; a sensor gap defined between one of the first contact
and the first electrode and one of the second contact and the
second electrode; and [0109] a bridge molecule comprising a first
end and a second end; [0110] wherein the bridge molecule is a
biopolymer bridge molecule; and [0111] wherein the bridge molecule
is coupled to the first contact at the first end and coupled to the
second contact at the second end.
[0112] 2. A sensor comprising: [0113] a first electrode overlying a
substrate surface; [0114] a second electrode overlying the
substrate surface; [0115] a sensor gap defined between the first
electrode and the second electrode; and [0116] a bridge molecule
comprising a first end and a second end; [0117] wherein the sensor
gap comprises a sensor gap dimension of between about 5 nm and
about 30 nm; and [0118] wherein the bridge molecule is coupled to
the first contact at the first end and coupled to the second
contact at the second end.
[0119] 3. The sensor as in examples 1 or 2, further comprising a
gate electrode.
[0120] 4. The sensor of example 1, wherein the sensor gap has a
sensor gap dimension of between about 5 nm and about 30 nm.
[0121] 5. The sensor as in examples 1 or 2, wherein the first end
comprises a first self-assembling anchor and/or the second end
comprises a second self-assembling anchor.
[0122] 6. The sensor of example 2, wherein the bridge molecule
comprises a biopolymer bridge molecule.
[0123] 7. The sensor of any of examples 1-6, wherein the bridge
molecule comprises a chemically synthesized bridge molecule.
[0124] 8. The sensor of any of examples 1-7, wherein the bridge
molecule comprises a linear biopolymer.
[0125] 9. The sensor of any of examples 1-8, wherein the bridge
molecule comprises an end-to-end length of less than a persistence
length of the bridge molecule.
[0126] 10. The sensor of any of examples 1-9, wherein the bridge
molecule comprises an end-to-end length configured to approximate
the sensor gap dimension.
[0127] 11. The sensor as in any of examples 1-10, wherein the
bridge molecule comprises a nucleic acid duplex.
[0128] 12. The sensor of example 11, wherein the nucleic acid
duplex comprises one of a DNA duplex, a DNA-RNA hybrid duplex, a
DNA-PNA hybrid duplex, a PNA-PNA duplex, and a DNA-LNA hybrid
duplex.
[0129] 13. The sensor of example 11, wherein the nucleic acid
duplex comprises a thiol-modified oligo.
[0130] 14. The sensor of any of examples 6-13, wherein one of the
first self-assembling anchor and the second self-assembling anchor
comprises a 5'-thiol modified nucleotide.
[0131] 15. The sensor of example 11, wherein the nucleic acid
duplex further comprises an internal biotin-modified
nucleotide.
[0132] 16. The sensor of any of examples 1-15, wherein the bridge
molecule comprises a peptide sequence, and wherein one of the first
self-assembling anchor and the second self-assembling anchor
comprises an L-cysteine residue.
[0133] 17. The sensor as in any one of examples 1-16, wherein the
bridge molecule is configured to self-assemble to produce a bridge
molecule conformation when a fluid medium comprising the bridge
molecule is contacted with one of the first contact and the second
contact.
[0134] 18. The sensor of any one of examples 1-17, further
comprising a probe, wherein the probe is attached to the bridge
molecule.
[0135] 19. The sensor of any one of examples 1-18, further
comprising a linker attached to the bridge molecule.
[0136] 20. The sensor of any one of examples 18-19, wherein the
probe is configured to engage a single target molecule.
[0137] 21. The sensor of any of examples 1-20, wherein the
molecular bridge and/or probe comprises an enzyme.
[0138] 22. The sensor of example 21, wherein the enzyme is one of a
polymerase and a reverse transcriptase.
[0139] 23. The sensor of any of examples 20-22, wherein the target
molecule comprises a plurality of target molecules features, each
target molecule feature having a discrete position, including a
first target molecule feature at a first position, a second target
molecule feature at a second position, and an nth target molecule
feature at an nth position.
[0140] 24. The sensor of example 18, wherein the probe is an enzyme
configured to engage the target molecule during a reaction in a
solution comprising a plurality of different target molecules,
wherein the reaction comprises a time period t, and wherein
contacting the target molecule produces a plurality of conformation
changes in the enzyme in response to the plurality of target
molecule features, wherein each of the plurality of configuration
changes modulates an electrical current in the sensor to produce a
signal feature.
[0141] 25. A system comprising a sensor according to any of
examples 1-24.
[0142] 26. The system of claim 25, further comprising a signal
processing system coupled to the sensor and configured to detect
the signal feature.
[0143] 27. The system of any of examples 25-26 or the sensor
according to any of examples 1-24, wherein the sensor is configured
to produce a signal trace comprising a plurality of signal features
detected over time period t.
[0144] 28. The system of any of examples 25-27, further comprising
a signal interpretation device.
[0145] 29. The system of example 28, wherein the signal
interpretation device comprises a signal interpretation map.
[0146] 30. The system of any of examples 28-29, wherein the signal
interpretation map is calibrated against a signal trace from a
known target sequence.
[0147] 31. The system of any of examples 28-30, wherein the signal
interpretation device is configured to return a signal
interpretation in response to the signal trace produced by a target
sequence.
[0148] 32. The system of any of examples 28-31, wherein the signal
interpretation includes a probabilistic evaluation of a likelihood
that a signal trace interpretation matches a possible actual
sequence.
[0149] 33. A method comprising: [0150] providing a sensor according
to any of examples 1-24, 27; [0151] contacting a nucleic acid
template with a polymerase, wherein the polymerase is coupled to a
bridge molecule comprising a portion of a sensor; [0152] optionally
applying an electrical potential to the sensor; [0153] providing a
nucleotide base mix; [0154] performing, by the polymerase, an
incorporation event comprising incorporation of a nucleotide from
the nucleotide base mix into a synthesized nucleic acid; and [0155]
detecting a signal produced by the incorporation event.
[0156] 34. The method of example 33, further comprising a series of
incorporation events performed in a time period t, wherein the
series of incorporation events produces a signal trace comprising a
sequence of signal features.
[0157] 35. The method of example 34, wherein each signal feature
corresponds to one of the series of incorporation events.
[0158] 36. The method of any of examples 34-35, wherein the signal
trace further comprises noise, and wherein the method further
comprises removing the noise from the signal trace.
[0159] 37. The method of any of examples 34-36, wherein each
incorporation event produces polymerase kinetic signature that is
template base-dependent.
[0160] 38. The method of any of examples 34-37, wherein the
polymerase kinetic signature contributes to the signal feature.
[0161] 39. The method of any of examples 33-38, wherein the method
is suitable to distinguish a first signal feature produced in
response to an unmodified template nucleotide and a second signal
feature produced in response to a modified template nucleotide.
[0162] 40. The method of example 39, wherein the modified template
nucleotide is one of N.sup.6-methyladenosine,
N.sup.4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine,
5-formylcytosine, and 5-carboxylcytosine.
[0163] 41. The method of example 39, wherein the modified template
nucleotide is an abasic site.
[0164] 42. A method of manufacturing a biomolecular sensing device
comprising: [0165] forming a first electrode and a second electrode
on a substrate surface, wherein the first electrode and the second
electrode are separated by an electrode gap; [0166] placing a first
contact on the first electrode and a second contact on the second
electrode, wherein the first contact and the second contact are
separated by a contact gap; and [0167] attaching a bridge molecule
to the first contact and the second contact.
[0168] 43. The method of example 42, further comprising contacting
the bridge molecule with a probe to couple the probe to the bridge
molecule, wherein the probe is coupled to the bridge molecule by
self-assembly.
[0169] 44. The method of any of examples 42-43, wherein attaching
the bridge molecule to the first contact and the second contact
comprises a self-assembly step.
[0170] 45. The method of any of examples 42-44, wherein the
electrode gap and/or the contact gap is between about 5 nm and
about 30 nm.
[0171] 46. The method of any of examples 42-45, wherein the first
contact and/or the second contact comprise gold nanoparticles with
a diameter of about 5 nm.
[0172] 47. The method of any of examples 42-46, wherein a first
contact position and/or a second contact position is determined
using a lithography method.
[0173] 48. The method of any of examples 42-47; further comprising
placing a photoresist layer over the substrate surface comprising
the first electrode and the second electrode, and defining a first
contact position and a second contact position using a lithography
method.
[0174] 49. The method of any of examples 42-48, further comprising
applying a surface derivatization treatment to the substrate
surface at the first contact position and the second contact
position.
[0175] 50. The method of example 49, wherein the surface
derivatization treatment comprises silanization.
[0176] 51. The method of any of examples 42-50, further comprising
depositing a gold layer and performing a lift-off step to leave a
first gold contact disposed on the first electrode and/or a second
gold contact disposed on the second electrode.
[0177] 52. The method of any of examples 42-51, further comprising
contacting the device with a solution comprising a plurality of
gold nanoparticles and introducing a first gold nanoparticle at the
first contact position and/or a second gold particle at the second
contact position.
[0178] 53. The method of any of examples 42-52, wherein the bridge
molecule is attached to the first contact and the second contact by
self-assembly prior to contacting the bridge molecule with a/the
probe.
[0179] 54. The method of any of examples 42-53, wherein the bridge
molecule is contacted with the probe to produce a sensor complex by
self-assembly prior to attaching the bridge molecule to the first
contact and the second contact by self-assembly.
[0180] 55. The method of any of examples 42-54, further comprising
fabricating an integrated circuit electronically coupled to the
first electrode and the second electrode,
[0181] 56. The method of example 55, wherein the integrated
circuit, the first electrode, and the second electrode comprise a
mixed-signal integrated circuit.
[0182] 57. The method of example 56, wherein the integrated
circuit, the first electrode, and the second electrode are
fabricated using a CMOS fabrication method.
[0183] 58. The method of any of examples 42-57, wherein the first
and second contact are fabricated using a CMOS fabrication
method.
[0184] 59. The method of any of examples 55-58, wherein the
integrated circuit, the first electrode, and the second electrode
are fabricated using a fabrication method suitable to produce a
field effect transistor.
[0185] Benefits, other advantages, and solutions to problems have
been described herein with regard to specific embodiments.
Furthermore, the connecting lines shown in the various figures
contained herein are intended to represent exemplary functional
relationships and/or physical couplings between the various
elements. It should be noted that many alternative or additional
functional relationships or physical connections may be present in
a practical system. However, the benefits, advantages, solutions to
problems, and any elements that may cause any benefit, advantage,
or solution to occur or become more pronounced are not to be
construed as critical, required, or essential features or elements
of the inventions. The scope of the inventions is accordingly to be
limited by nothing other than the appended claims, in which
reference to an element in the singular is not intended to mean
"one and only one" unless explicitly so stated, but rather "one or
more." Moreover, where a phrase similar to "at least one of A, B,
or C" is used in the claims, it is intended that the phrase be
interpreted to mean that A alone may be present in an embodiment, B
alone may be present in an embodiment, C alone may be present in an
embodiment, or that any combination of the elements A, B and C may
be present in a single embodiment; for example, A and B, A and C, B
and C, or A and B and C. Different cross-hatching is used
throughout the figures to denote different parts but not
necessarily to denote the same or different materials.
[0186] Systems, methods and apparatus are provided herein. In the
detailed description herein, references to "one embodiment", "an
embodiment", "an example embodiment", etc., indicate that the
embodiment described may include a particular feature, structure,
or characteristic, but every embodiment may not necessarily include
the particular feature, structure, or characteristic. Moreover,
such phrases are not necessarily referring to the same embodiment.
Further, when a particular feature, structure, or characteristic is
described in connection with an embodiment, it is submitted that it
is within the knowledge of one skilled in the art to affect such
feature, structure, or characteristic in connection with other
embodiments whether or not explicitly described. Although the
various examples and embodiments described herein refer to methods
of signal detection in relation to nucleic acid targets, the
devices and methods of the present disclosure are in no way limited
to applications comprising detection and sequencing of nucleic
acids. Likewise, although the various examples and embodiments
described herein refer to sensors comprising biopolymer bridges
molecules, chemically synthesized bridge molecules are within the
scope of the present disclosure. After reading the description, it
will be apparent to one skilled in the relevant art(s) how to
implement the disclosure in alternative embodiments.
[0187] Furthermore, no element, component, or method step in the
present disclosure is intended to be dedicated to the public
regardless of whether the element, component, or method step is
explicitly recited in the claims. No claim element herein is to be
construed under the provisions of 35 U.S.C. 112(f), unless the
element is expressly recited using the phrase "means for." As used
herein, the terms "comprises", "comprising", or any other variation
thereof, are intended to cover a non-exclusive inclusion, such that
a process, method, article, or apparatus that comprises a list of
elements does not include only those elements but may include other
elements not expressly listed or inherent to such process, method,
article, or apparatus.
Sequence CWU 1
1
15160DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide5'-5ThioMC6-D (5'-thiol modifier)
1tgcgtacgta tgtcatgaat ggcgcagact gatgtcctat gacgtcgcta ctgcagtact
60260DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide5'-5ThioMC6-D (5'-thiol
modifier)modified_base(29)..(29)iBiodT (internal biotin-modified
deoxythymidine) 2agtactgcag tagcgacgtc ataggacatc agtctgcgcc
attcatgaca tacgtacgca 60314PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Met His Gly Lys Thr Gln Ala
Thr Ser Gly Thr Ile Gln Ser 1 5 10 48PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Val
Ser Gly Ser Ser Pro Asp Ser 1 5 512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Leu
Lys Ala His Leu Pro Pro Ser Arg Leu Pro Ser 1 5 10 612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Val
Pro Ser Ser Gly Pro Gln Asp Thr Arg Thr Thr 1 5 10 712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Met
Ser Pro His Pro His Pro Arg His His His Thr 1 5 10 85PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Glu
Ala Ala Ala Arg 1 5 95PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Glu Ala Ala Ala Lys 1 5
108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Glu Glu Glu Glu Arg Arg Arg Arg 1 5
118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Glu Glu Glu Glu Lys Lys Lys Lys 1 5
1234DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(13)a, c, t, g, unknown
or
othermodified_base(15)..(15)5-methylcytosinemodified_base(17)..(17)5-meth-
ylcytosinemodified_base(19)..(19)5-methylcytosinemodified_base(21)..(21)5--
methylcytosinemodified_base(23)..(23)5-methylcytosine 12nnnnnnnnnn
nnngcgcgcg cgcgcgcgcg cgcg 341334DNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(22)..(34)a, c, t, g, unknown or other
13cgcgcgcgcg cgcgcgcgcg cnnnnnnnnn nnnn 341450DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14cgccgcggag ccaagaaaaa aaaaaaaaaa aaaaattgca
tgtcctgtga 501515DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 15tcacaggaca tgcaa 15
* * * * *