U.S. patent application number 16/247949 was filed with the patent office on 2019-05-16 for super resolution imaging.
The applicant listed for this patent is Illumina, Inc.. Invention is credited to Kevin L. Gunderson, Robert C. Kain, Tarun Khurana, Yir-Shyuan Wu.
Application Number | 20190144939 16/247949 |
Document ID | / |
Family ID | 51529811 |
Filed Date | 2019-05-16 |
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United States Patent
Application |
20190144939 |
Kind Code |
A1 |
Kain; Robert C. ; et
al. |
May 16, 2019 |
SUPER RESOLUTION IMAGING
Abstract
A detection apparatus that includes (a) an array of responsive
pads on a substrate surface; (b) an array of pixels, wherein each
pixel in the array has a detection zone on the surface that
includes a subset of at least two of the pads; and (c) an
activation circuit to apply a force at a first and second pad in
the subset, wherein the activation circuit is configured to apply a
different force at the first pad compared to the second pad, and
wherein the activation circuit has a switch to selectively alter
the force at the first pad and the second pad.
Inventors: |
Kain; Robert C.; (San Diego,
CA) ; Khurana; Tarun; (Fremont, CA) ;
Gunderson; Kevin L.; (Encinitas, CA) ; Wu;
Yir-Shyuan; (Albany, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Illumina, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
51529811 |
Appl. No.: |
16/247949 |
Filed: |
January 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14213340 |
Mar 14, 2014 |
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16247949 |
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13835492 |
Mar 15, 2013 |
9193998 |
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14213340 |
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61788954 |
Mar 15, 2013 |
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Current U.S.
Class: |
506/3 ; 435/6.1;
506/16; 506/9 |
Current CPC
Class: |
G01N 21/6454 20130101;
C12Q 1/6825 20130101; C12Q 1/6874 20130101; C12Q 1/6837 20130101;
C12Q 1/6825 20130101; C12Q 2563/103 20130101; C12Q 2563/113
20130101; C12Q 2563/179 20130101; C12Q 2565/101 20130101; C12Q
2565/101 20130101; C12Q 2565/607 20130101; C12Q 1/6837 20130101;
C12Q 2563/103 20130101; C12Q 2563/113 20130101; C12Q 2563/179
20130101; C12Q 2565/101 20130101; C12Q 2565/101 20130101; C12Q
2565/607 20130101; C12Q 1/6874 20130101; C12Q 2563/103 20130101;
C12Q 2563/113 20130101; C12Q 2563/179 20130101; C12Q 2565/101
20130101; C12Q 2565/101 20130101; C12Q 2565/607 20130101 |
International
Class: |
C12Q 1/6874 20060101
C12Q001/6874; G01N 21/64 20060101 G01N021/64; C12Q 1/6825 20060101
C12Q001/6825; C12Q 1/6837 20060101 C12Q001/6837 |
Claims
1. A detection apparatus comprising: an array of electrically
responsive pads on a substrate surface; an array of detection
pixels, wherein each individual detection pixel in the array has a
detection zone on the surface, each detection pixel configured to
simultaneously observe a subset of at least two of the electrically
responsive pads; and an activation circuit to apply electric field
at a first pad in the subset and a second pad in the subset,
wherein the activation circuit is configured to apply different
electric field at the first pad compared to the second pad, and
wherein the activation circuit comprises a switch to selectively
alter the electric field at the first pad compared to the second
pad, wherein the pads comprise target analytes to be detected.
2. The apparatus of claim 1, wherein each detection zone is square
and each pad occurs in a corner of four of the detection zones.
3. The apparatus of claim 1, wherein the target analytes comprise
at least two nucleic acid clusters that are included in the
detection zone for a single detection pixel, each of the clusters
comprises a different nucleotide sequence from the other
cluster.
4. The apparatus of claim 3, wherein each pad comprises a plurality
of nucleic acid clusters and each cluster is included in the
detection zone for a single detection pixel.
5. The apparatus of claim 3, wherein each pad comprises a single
nucleic acid cluster and the cluster is included in the detection
zones for at least two of the detection pixels.
6. The apparatus of claim 3, wherein the target analytes comprise
fluorescent moieties and the detection pixels are configured to
detect emission from the fluorescent moieties.
7. The apparatus of claim 6, wherein the activation circuit applies
a different electric field at the first pad compared to the second
pad, wherein the first pad comprises a fluorescence quencher at a
higher concentration than at the second pad.
8. The apparatus of claim 6, wherein the activation circuit applies
a different electric field at the first pad compared to the second
pad, wherein the first pad comprises a fluorescent probe at a
higher concentration than at the second pad.
9. The apparatus of claim 6, wherein the pads further comprise
electrochemical luminescence labels.
10. The apparatus of claim 9, wherein the activation circuit
applies a different electric field at the first pad compared to the
second pad, thereby producing more photons at the first pad than at
the second pad.
11. The apparatus of claim 1, wherein the activation circuit is
configured to apply an electric field at the first pad while no
field is applied at the second pad, and the switch is configured to
turn off the electric field at the first pad while applying an
electric field at the second pad.
12. The apparatus of claim 1, wherein the activation circuit is
configured to apply a positive electric field at the first pad
while applying negative electric field at the second pad, and the
switch is configured to apply negative electric field at the first
pad while applying positive electric field at the second pad.
13. A method of detecting analytes, comprising: providing the
detection apparatus of claim 1, wherein the two pads comprise
different target analytes, respectively; acquiring signals from
each of the detection pixels while selectively applying an electric
field at a first of the two pads to preferentially produce signal
from a first of the different target analytes compared to a second
of the target analytes, thereby preferentially acquiring signals
from the first of the target analytes compared to the second of the
target analytes; and acquiring signals from each of the detection
pixels while selectively applying an electric field at the second
of the two pads to preferentially produce signal from the second of
the different target analytes compared to the first of the target
analytes, thereby preferentially acquiring signals from the second
of the target analytes compared to the first of the target
analytes.
14. The apparatus of claim 1, wherein the array of pads has the
same pitch as the pitch for the array of pixels.
15. A method comprising: providing a detection apparatus of claim
1, simultaneously observing a first pad and a second pad using a
detection pixel; applying a first electrical field at the first
pad; and applying a second electrical field at the second pad, the
first electric field different than the second electric field, the
first pad and the second pad comprising target analytes to be
detected.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/213,340, filed on Mar. 14, 2014, which is a
continuation-in-part of U.S. application Ser. No. 13/835,492, filed
Mar. 15, 2013, now U.S. Pat. No. 9,193,998, which is based on, and
claims the benefit of, U.S. Provisional Application No. 61/788,954,
filed Mar. 15, 2013, now expired, each of which is incorporated
herein by reference in its entirety.
BACKGROUND
[0002] Embodiments of the present disclosure relate generally to
biological or chemical analysis and more particularly to systems
and methods using detection devices for biological or chemical
analysis.
[0003] Various protocols in biological or chemical research involve
performing a large number of controlled reactions on local support
surfaces or within predefined reaction chambers. The desired
reactions may then be observed or detected and subsequent analysis
may help identify or reveal properties of chemicals involved in the
reaction. For example, in some multiplex assays, unknown analytes
having identifiable labels (e.g., fluorescent labels) may be
exposed to thousands of known probes under controlled conditions.
Each known probe may be deposited into a corresponding location on
a surface. Observing any chemical reactions that occur between the
known probes and the unknown analyte on the surface may help
identify or reveal properties of the analyte. Other examples of
such protocols include known DNA sequencing processes, such as
sequencing-by-synthesis (SBS) or cyclic-array sequencing.
[0004] In some conventional fluorescent-detection protocols, an
optical system is used to direct an excitation light onto
fluorescently-labeled analytes and to also detect the fluorescent
signals that may emit from the analytes. The resolution of standard
imaging techniques is constrained by the number of pixels available
in the detection device, among other things. As such, these optical
systems can be relatively expensive and require a relatively large
bench-top footprint when detecting surfaces having large
collections of analytes. For example, nucleic acid arrays used in
genotyping, expression, or sequencing analyses can require
detection of millions of different sites on the array per square
centimeter. Limits in resolution increase cost and decrease
accuracy of these analyses
[0005] Thus, there exists a need for higher resolution apparatus
and methods, for example, to detect nucleic acid arrays. The
present disclosure addresses this need and provides other
advantages as well.
BRIEF SUMMARY
[0006] The present disclosure provides a detection apparatus that
includes (a) an array of responsive pads on a substrate surface;
(b) an array of pixels, wherein each pixel in the array has a
detection zone on the surface that includes a subset of at least
two of the responsive pads; and (c) an activation module to alter a
characteristic of a first pad in the subset and of a second pad in
the subset, wherein the activation module is configured to apply a
different characteristic at the first pad compared to the second
pad, and wherein the activation module has a switch to selectively
alter the characteristic at the first pad compared to the second
pad.
[0007] In particular embodiments, a detection apparatus can include
(a) an array of electrically responsive pads on a substrate
surface; (b) an array of pixels, wherein each pixel in the array
has a detection zone on the surface that includes a subset of at
least two of the electrically responsive pads; and (c) an
activation circuit to apply an electric field at a first pad in the
subset and a second pad in the subset, wherein the activation
circuit is configured to apply a different electric field at the
first pad compared to the second pad, and wherein the activation
circuit has a switch to selectively alter the electric field at the
first pad compared to the second pad.
[0008] The disclosure also provides a nucleic acid sequencing
system. The system can include (a) a detection apparatus having (i)
an array of responsive pads on a substrate surface; (ii) an array
of pixels, wherein each pixel in the array has a detection zone on
the surface that includes a subset of pads; and (iii) an activation
module to alter a characteristic of the pads in the subset
individually, wherein the activation module is configured to apply
a different characteristic to a first pad of the subset compared to
the other pads of the subset; (b) a readout circuit to acquire
signals from the array of pixels; (c) a control module that directs
the readout circuit to acquire signals from each of the pixels
during a sensing period and that directs the activation module to
sequentially apply different characteristic at the pads during the
sensing period; and (c) a processing module that correlates (i) the
signals acquired from the pixels during the sensing period and (ii)
the sequential application of the different characteristics at the
pads during the sensing period, in order to distinguish a sequence
of signals for each of the pads.
[0009] In particular embodiments, a nucleic acid sequencing system
can include (a) a detection apparatus having (i) an array of
electrically responsive pads on a substrate surface; (ii) an array
of pixels, wherein each pixel in the array has a detection zone on
the surface that includes a subset of four of the pads; and (iii)
an activation circuit to apply an electric field to the pads in the
subset individually, wherein the activation circuit is configured
to apply a different electric field at a first pad of the subset
compared to the other pads of the subset; (b) a readout circuit to
acquire signals from the array of pixels; (c) a control module that
directs the readout circuit to acquire signals from each of the
pixels during a sensing period and that directs the activation
circuit to sequentially apply different electric fields at the four
pads during the sensing period; and (c) a processing module that
correlates (i) the signals acquired from the pixels during the
sensing period and (ii) the sequential application of the different
electric fields at the four pads during the sensing period, in
order to distinguish a sequence of signals for each of the
pads.
[0010] The disclosure further provides a method of detecting
analytes. The method can include the steps of (a) providing a
detection apparatus having an array of responsive pads and an array
of pixels, wherein each pixel in the array has a detection zone
that includes a subset of at least two of the responsive pads,
wherein the two pads include different target analytes,
respectively; (b) acquiring signals from each of the pixels while
selectively applying a unique characteristic at a first of the two
pads to preferentially produce signal from a first of the different
target analytes compared to a second of the target analytes,
thereby preferentially acquiring signals from the first of the
target analytes compared to the second of the target analytes; and
(c) acquiring signals from each of the pixels while selectively
applying the unique characteristic at the second of the two pads to
preferentially produce signal from the second of the different
target analytes compared to the first of the target analytes,
thereby preferentially acquiring signals from the second of the
target analytes compared to the first of the target analytes.
[0011] In particular embodiments, a method of detecting analytes
can include the steps of (a) providing a detection apparatus having
an array of electrically responsive pads and an array of pixels,
wherein each pixel in the array has a detection zone that includes
a subset of at least two of the electrically responsive pads,
wherein the two pads include different target analytes,
respectively; (b) acquiring signals from each of the pixels while
selectively applying an electric field at a first of the two pads
to preferentially produce signal from a first of the different
target analytes compared to a second of the target analytes,
thereby preferentially acquiring signals from the first of the
target analytes compared to the second of the target analytes; and
(c) acquiring signals from each of the pixels while selectively
applying an electric field at the second of the two pads to
preferentially produce signal from the second of the different
target analytes compared to the first of the target analytes,
thereby preferentially acquiring signals from the second of the
target analytes compared to the first of the target analytes.
[0012] The present disclosure also provides a detection apparatus
that includes (a) an array of responsive pads on a substrate
surface, wherein each responsive pad includes a nucleic acid
feature of a plurality of nucleic acid features in the array,
wherein a first subset of nucleic acid features in the plurality of
nucleic acid features have a first universal sequence and different
target sequences, wherein a second subset of nucleic acid features
in the plurality of nucleic acid features have a second universal
sequence and different target sequences, wherein the first
universal sequence is different from the second universal sequence;
(b) an array of pixels, wherein each pixel in the array has a
detection zone on the surface that includes at least two nucleic
acid features of the plurality of nucleic acid features, the at
least two nucleic acid features including a nucleic acid from the
first subset of nucleic acid features and a nucleic acid from the
second subset of nucleic acid features; and (c) an activation
module to alter a characteristic of a pad in the first subset and
of a pad in the second subset, wherein the activation module is
configured to apply a different characteristic at the pad in the
first subset compared to the pad in the second subset, and wherein
the activation module has a switch to selectively alter the
characteristic at the pads in the first and second subsets.
Optionally, the detection apparatus can be included in a nucleic
acid sequencing system that also includes (I) a readout circuit to
acquire signals from the array of pixels; (II) a control module
that directs the readout circuit to acquire signals from each of
the pixels during a sensing period and that optionally directs the
activation circuit to sequentially actuate different responsive
pads in each of the detection zones during the sensing period; and
(III) a processing module that optionally correlates (i) the
signals acquired from the pixels during the sensing period and (ii)
the sequential actuation of the different responsive pads during
the sensing period, in order to distinguish a sequence of signals
for each of the pads.
[0013] Also provided is a method of detecting target nucleic acids,
including the steps of (a) providing a substrate comprising an
array of pads, the array of pads including a first subset of the
pads and a second subset of the pads; (b) delivering a first
solution to the substrate, wherein the first solution includes a
first plurality of different target nucleic acids that selectively
attach to the first subset of pads compared to the second subset of
pads; (c) delivering a second solution to the substrate, wherein
the second solution includes a second plurality of different target
nucleic acids that selectively attach to the second subset of pads
compared to the first subset of pads; and (d) detecting the
substrate using an apparatus having an array of pixels, wherein
each pixel in the array has a detection zone that includes (i) at
least one of the target nucleic acids that is attached to a pad of
the first subset of pads, and (ii) at least one of the target
nucleic acids that is attached to a pad of the second subset of
pads.
[0014] In some embodiments, a method of detecting nucleic acids can
include the steps of (a) providing a substrate comprising an array
of pads, the array of pads including a first subset of the pads and
a second subset of the pads; (b) contacting a solution of adapter
nucleic acids with the substrate while selectively actuating one or
both of the subsets of responsive pads, wherein the adapter nucleic
acids attach to responsive pads of the one or both subsets that are
selectively actuated; (c) contacting a first solution with the
substrate, wherein the first plurality of different target nucleic
acids attach to responsive pads of the first subset; (d) contacting
a second solution with the substrate, wherein the second plurality
of different target nucleic acids attach to responsive pads of the
second subset, wherein the responsive pads of one or both of the
first and second subset are attached to the adapter nucleic acids;
and (e) detecting the substrate using an apparatus having an array
of pixels, wherein each pixel in the array has a detection zone
that includes (i) at least one of the target nucleic acids that is
attached to a pad of the first subset of pads, and (ii) at least
one of the target nucleic acids that is attached to a pad of the
second subset of pads.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a diagrammatic view of a detection apparatus
having an array of pixels and an array of electrically responsive
pads.
[0016] FIG. 2 shows a diagrammatic view of a detection apparatus
having an array of pixels and an array of electrically responsive
pads, wherein the pads each have multiple target analyte
features.
[0017] FIG. 3 shows side views of (A) an array of pixels and an
array of electrically responsive pads integrated into a substrate,
and (B) an array of pixels in a detection unit positioned to detect
electrically responsive pads on a substrate that is separate from
the detection unit.
[0018] FIG. 4 shows a diagram of a detection system.
[0019] FIG. 5 shows a diagram of a detection apparatus that
differentially detects fluorescent analytes from two pads in the
detection zone of a single pixel by selectively attracting a
fluorescence quencher to one of the pads.
[0020] FIG. 6 shows fluorescence intensity modulation when applying
multiple cycles of charge on an electrically responsive pad having
a fluorescently labeled nucleic acid attached to the pad via a
gel.
[0021] FIG. 7 shows (A) a template nucleic acid hybridized to a
primer having a fluorophore at the 3' end and a quencher tethered
to the 5' end via a linker arm; (B) the quencher moiety drawn
toward the fluorophore by a positive electric field and (C) the
quencher moiety repelled away from the fluorophore by a negative
electric field.
DETAILED DESCRIPTION
[0022] This disclosure provides apparatus and methods for super
resolution imaging. The resolution of standard imaging techniques
is constrained by the number of pixels available in the detection
device, among other things. Standard imaging techniques provide, at
best, a single pixel for detection of each feature in an object.
Often several pixels must be used to collect signal from each
feature in order to obtain sufficient signal to noise. In contrast,
the super resolution imaging methods provided by the current
disclosure break the one-pixel-per-feature barrier and allow
several features to be distinguished by a single pixel. This leads
to advantages of reducing the optics hardware required to detect an
object of a given size and complexity. This can also increase the
resolution of a detection apparatus beyond the resolution of the
optics module being used. Thus, costs incurred in manufacturing and
using optics hardware can be reduced substantially.
[0023] In embodiments set forth herein, two features can be
resolved with a single pixel by differential treatment of the
features to render one of the features detectable in a first state
and the other feature detectable in another state. By extension,
several features can be resolved by a single pixel by sequentially
actuating individual features to a detectable state while the other
features are in an undetectable state.
[0024] A particularly useful application of the present apparatus
and methods is the detection of analytes, such as nucleic acids, on
solid supports, such as arrays of features to which the analytes
are attached. In particular embodiments, the solid support has
several responsive pads that are in the detection zone of a
particular pixel and each pad has a different target analyte
attached. The pads can be switchable between different states such
that a single pad is in a first state that produces signal from the
respective analyte; meanwhile the other pads are in a different
state such that the analytes at these other pads do not produce a
detectable signal. For example, the single pad may have an electric
field that attracts a detectable label (e.g. a fluorophore),
removes a detection inhibitor (e.g. a fluorescence quencher) or
induces electrochemical luminescence. By sequentially switching the
states (e.g. strength, polarity or presence of an electric field)
at the pads that are in the detection zone, the different analytes
can be individually induced to produce signal. By detecting the
pads in the different states the pixel can achieve super resolution
detection of several different analytes in its detection zone. An
accounting of which pads were actuated at different times during
acquisition of signal by the pixel will allow the respective
analytes to be distinguished.
[0025] Alternatively or additionally, to differential treatment of
two features during detection events, two features can be
distinguished by one pixel based on physical or chemical
differences that were imparted to the features prior to the
detection events. Thus, the two features need not be switched
between different states during or between detection steps. For
example, each of the two different target nucleic acid features
within the detection zone of the same pixel can have different
priming sequences such that they can be distinguished by
hybridizing different primers to each feature and/or extending
different primers at each feature. The hybridization and/or
extension can occur at the respective features at different times.
In multiplex formats, the priming sites can be universal sequences
such that a first subset of features in the array share a first
universal priming site sequence and a second subset of features in
the array share a second universal priming site sequence. In this
multiplex format, individual pixels can have a detection zone that
includes a feature from each subset. Thus each pixel can be capable
of super resolution detection based on differential primer
hybridization. For example, different primers can be delivered
sequentially for separate detection of features at different
times.
[0026] By way of further illustrative example, two pads that are in
the detection zone of the same pixel can differentially capture
target nucleic acids. Differential capture can be facilitated by
use of specific capture probes that are attached at respective pads
and that are selective for different target nucleic acids.
Nanofabrication methods, such as those used in the manufacture of
nucleic acid microarrays, can be used to place different capture
probes at separate features within the detection zone of the same
pixel. Alternatively, the same capture probes can be present at
both pads in the detection zone, but the pads can be differentially
activated in the presence of different adapter nucleic acids such
that the capture probes on the two different pads hybridize to
different adapter nucleic acids. The different adapter nucleic
acids can include a common sequence that is complementary to the
capture probes and different selective capture sequences that are
complementary to different target nucleic acids. As such, these
adapters can convert a universal surface to one that is capable of
selective target nucleic acid capture.
[0027] Similarly, different nucleic acids present on two pads can
serve as hybridization sites for different amplification primers or
different detection primers. Different amplification primers can be
used for differential amplification of target nucleic acids before
or during a detection event. In this example a first amplification
primer can amplify a subset of target nucleic acid templates that
have a first universal priming site that is complementary to the
first amplification primer. A second amplification primer can
amplify a subset of target nucleic acid templates that have a
second universal priming site that is complementary to the second
amplification primer. The different amplification primers can be
delivered to an array of pads at different times to create colonies
at a first subset of the pads for a first sensing period followed
by creation of colonies at a second subset of pads for a second
detection event. Different extension primers can be used for
differential detection of target nucleic acids for example in
techniques such as sequencing-by-synthesis, sequencing-by-ligation,
single base extension, oligonucleotide ligation, allele specific
primer extension or the like. In this example a first extension
primer can be used to detect a subset of target nucleic acid
templates that have a first universal priming site that is
complementary to the first extension primer and a second extension
primer can detect a subset of target nucleic acid templates that
have a second universal priming site that is complementary to the
second extension primer.
[0028] As exemplified in further detail below, super resolution
imaging can be achieved using electrically responsive pads, for
example, to achieve electric field assisted transport of charged
species to or from the pad. Other types of responsive pads can be
used similarly in an apparatus or method set forth herein. For
example, a responsive pad can alter other forces to create
non-diffusive forces on target analytes, probes or other materials
of interest (whether charged or not). Non-diffusive forces can be
provided by an external source such as those that produce an
electrical or magnetic field, or an agent that imposes molecular
crowding or chemical gradients within a reaction volume. For
example, magnetic or optical forces can be used to increase the
local concentration of desired materials at a pad in an array of
pads or to decrease the local concentration of the materials at a
particular pad. In such cases, the materials can include a magnetic
tag or optical tag that can be manipulated by such forces. Other
useful responsive pads can alter detection properties of an analyte
at the pad, for example, by inducing chemical changes at or near
the pad that activate, inhibit, destroy or create a detectable
label. Such pads can be used to replace electrically responsive
pads exemplified in several embodiments of the apparatus and
methods set forth herein.
[0029] Particular embodiments of the super resolution apparatus and
methods set forth herein provide the advantage of reducing the
number of different excitation lines and/or fluorophore labels
required to observe an analyte. In many fluorescent detection
systems, analytes are labeled with multiple different fluorophore
labels, respectively, and the labels are distinguished by use of
multiple different excitation wavelengths. Using the super
resolution apparatus and methods of the present disclosure,
responsive pads (e.g. electrically responsive pads) can remove the
necessity for multiple fluorophores and multiple excitation lines.
For example, instead of using two different fluorophores and two
different lasers to distinguish two analytes in a detection area,
two responsive pads can occur in the detection area. The pads,
although having different analytes, can be labeled with the same
fluorophore and excited with the same excitation laser. However, it
will be understood that in some embodiments multiple excitation
lines can be used in combination with super resolution to further
expand the number of different analytes detected by a given pixel.
More specifically, multiple features in the detection zone of a
pixel can be individually actuated to produce or inhibit signals,
and features that are active can be excited sequentially with
different radiation lines. As such, a pixel can distinguish a
number of analytes that is equivalent to the mathematical product
of the number of responsive pads in the detection zone of the pixel
multiplied by the number of excitation lines that produce emission
at each of the pads.
[0030] An alternative to actuated pads is to use pads that are
manufactured to have different physical or chemical properties.
Taking as an example, nucleic acid-based techniques, two pads that
are, or will be, present in the detection zone of the same pixel
can be nanofabricated to include different capture nucleic acids. A
first feature can have a first capture sequence and a second
feature can have a second capture sequence. In array formats, a
first subset of features can have capture probes with a first
universal sequence and a second subset of features can have capture
probes with a second universal sequence.
[0031] The present disclosure provides a detection apparatus that
includes (a) an array of electrically responsive pads on a
substrate surface; (b) an array of pixels, wherein each pixel in
the array has a detection zone on the surface that includes a
subset of at least two of the electrically responsive pads; and (c)
an activation circuit to apply an electric field at a first pad in
the subset and a second pad in the subset, wherein the activation
circuit is configured to apply a different electric field at the
first pad compared to the second pad, and wherein the activation
circuit has a switch to selectively alter the electric field at the
first pad compared to the second pad.
[0032] As used herein, the term "electrically responsive pad" means
an area on the surface of a substrate that produces an electric
field. The area on the surface can form an interface between the
substrate and a fluid that is in contact with the substrate. Thus,
the electric field can attract electrically charged species from
the fluid to the pad or the field can repel electrically charged
species from the pad. The direction of active transport to or from
the pad will depend upon the charge of the species in solution and
the charge of the field at or near the pad. Specifically,
positively charged species, such as positively charged fluorophores
or other optical labels, receptors, ligands, quenchers, labeled
probes or the like, can be attracted to the pad by inducing a
negative charge at or near the pad. Conversely, negatively charged
species, such as nucleic acids, nucleotides, or negatively charged
fluorophores, optical labels, receptors, ligands, quenchers,
labeled probes or the like, can be attracted to the pad by inducing
a positive charge at or near the pad.
[0033] As used herein, the term "electric field," when used in
reference to an electrically responsive pad, means the effect
produced by the existence of an electric charge on the pad or in
the volume of a medium that surrounds the pad. A charge placed in
the volume of a medium has a force exerted on it. Electric fields
are created by differences in voltage: the higher the voltage, the
stronger will be the resultant field. In contrast, magnetic fields
are created when electric current flows: the greater the current,
the stronger the magnetic field. An electric field will exist
whether or not current is flowing. Electric fields can be measured
in Volts per meter (V/m) or similar units. Electric field strength
of about 5 V/cm or higher, up to practical limits of Joule heating
and dielectric breakdown limits, are particularly useful to cause
movement of charged particles and species in the present methods
and apparatus. In particular embodiments, the maximum upper value
for the field strength is about 1000 V/cm.
[0034] Electrical potential greater than the redox potential of
water, roughly 1.23 V, will cause electrolysis of water. In some
embodiments, such as those using aqueous fluids, the applied
voltages can be in the range of -1 V to +1 V. In some embodiments,
it may be beneficial to employ a common ground counter electrode
separating the pads to minimize diffusion of labels, target
analytes or other substances from one pad to another.
[0035] Particular embodiments use an AC electric field with DC bias
to attract or repel charged species. Exemplary configurations for
applying an AC electric field with DC bias are set forth in U.S.
Pat. No. 8,277,628, which is incorporated herein by reference.
[0036] As used herein, the term "different electric field," when
used with respect to a reference electric field, includes, for
example, a field having opposite charge compared to the reference
field, no charge compared to the reference field, greater or lesser
charge compared to the reference field, a DC induced charge compare
to an AC induced charge at the reference field, or an AC induced
charge compared to a DC induced charge at the reference field.
[0037] The conductive surfaces of a pad can be metallic (e.g. gold,
titanium, indium tin oxide) or semiconducting in nature. In some
embodiments, it may be desirable to use an electrical conductor
that is transparent to radiation used in an optical detection step.
Examples of optically transparent electrode materials include, but
are not limited to metal oxides such as indium tin oxide, antimony
doped tin oxide, and cadmium tin oxide. This is particularly useful
in configurations where the pad occurs between a target analyte and
a pixel that will detect the analyte and/or when the pad occurs
between a target analyte and an excitation source used in a
fluorescence technique.
[0038] In particular embodiments, electrically responsive pads can
be electrically coupled to a power source to produce an electric
charge that attracts target nucleic acids or other substances. In
one configuration, a positive charge at the pad can attract nucleic
acids via the negatively charged sugar-phosphate backbone.
Exemplary methods and apparatus for using e-field assist to attract
nucleic acids or other substances to sites of an array are
described in U.S. Pat. No. 8,277,628, which is incorporated herein
by reference. Alternatively, pads of an array can be electrically
coupled to a power source to produce an electric charge that
inhibits binding of or removes target nucleic acids or other
substances from the pads. In one configuration, a negative charge
at the pads can repel nucleic acids via the negatively charged
sugar-phosphate backbone.
[0039] A low conductivity and low ionic buffers, such as 10-100 mM
histidine, can be used to promote electrophoretic transport of a
label, target analyte or other substance to or from an electrically
responsive pad. A low ionic strength buffer also provides a benefit
of increasing the Debye length, effectively increasing the spatial
extent of the electric field in the solution above the activated
pad.
[0040] Although several methods and apparatus of the present
disclosure are exemplified with regard to electrically responsive
pads, it will be understood that other responsive pads can be used
in place of these. As used herein, the term "responsive pad" means
an area on the surface of a substrate that can be physically or
chemically manipulated to alter a surface characteristic. The area
on the surface can form an interface between the substrate and a
fluid that is in contact with the substrate. A change in a surface
characteristic of the pad can induce a change in the fluid that
contacts the pad. Exemplary characteristic that can be altered
include, but are not limited to, electric field, electric current,
temperature, magnetic field, or a chemical property such as pH,
redox potential, hydrophobicity, hydrophilicity or chemical
reactivity. A characteristic of a responsive pad can be altered to
change the direction of transport of a material or substance to or
from the pad. A characteristic of a responsive pad can also be
altered to change the chemical composition or structural integrity
of a material or substance at the pad.
[0041] As used herein, the term "electrowetting control pad" refers
to a pad or area comprising an electrode covered by a hydrophobic
layer. The hydrophobic layer becomes hydrophilic upon activation of
the electrowetting control pad. The size of the electrowetting
control pad is generally approximately equivalent to the size of
the electrode. In particular embodiments, two or more electrodes
can be configured to be in the detection zone of the same pixel.
More particularly, features located on two or more different
electrowetting control pads can be located within the detection
zone of the same pixel.
[0042] As used herein, the term "transport" refers to movement of a
molecule through a fluid. The term can include passive transport
such as movement of molecules along their concentration gradient
(e.g. passive diffusion). The term can also include active
transport whereby molecules can move against their concentration
gradient or at an increased rate of passage along their
concentration gradient. Thus, transport can include applying energy
to move one or more molecule in a desired direction or to a desired
location such as a pad in an array of pads. Active transport can be
provided by an external source such as those that produce electric
or magnetic fields, or an agent that imposes molecular crowding or
chemical gradients within a reaction volume. For example, magnetic
or optical forces can be used to increase the local concentration
of amplification reagents. In such cases, one or more amplification
reagents can include a magnetic tag or optical tag that can be
manipulated by such forces.
[0043] An array of pixels can be configured such that each pixel in
the array has a detection zone that includes a subset of at least
two pads or other features to be detected. As used herein, the term
"detection zone," when used in reference to a pixel, means a
location that is simultaneously observed by the pixel. The location
can be, for example, a volume of space or area on a surface. For
example, a detection zone can include an area on the surface of an
array of pads that includes a subset of the pads. For example, the
detection zone of an individual pixel can include at least 2, 3, 4,
5, 10 or more pads or features. The number of pads or features in a
detection zone can be selected to suit the size of the pixel, the
size of the detection zone for the pixel (for example, as
influenced by optics between the pixel and the features or pads
observed), the size of the features or pads, or the size of any
analytes to be detected by the pixel. In particular embodiments, a
maximum number of pads or features in a detection area can be 10,
5, 4, 3 or 2.
[0044] As used herein, the term "each," when used in reference to a
collection of items, is intended to identify an individual item in
the collection but does not necessarily refer to every item in the
collection. Exceptions can occur if explicit disclosure or context
clearly dictates otherwise. Thus, reference to each pixel in an
array having a detection zone that includes a subset of at least
two pads, means that at least one pixel in the array has a
detection zone that includes the subset of pads. Although all of
the pixels in the array may have a similarly configured detection
zone, not all of the pixels in the array need to be so configured.
Rather some pixels may not have any pads in their respective
detection zone or some pixels may have only one pad in the
respective detection zone.
[0045] A diagrammatic representation of a relationship between an
array of pixel detection zones and an array of responsive pads that
provides super resolution imaging is shown in FIG. 1. Detection
apparatus 10 has a 5.times.5 array of pixel detection zones 1 and a
4.times.4 array of responsive pads 2. The arrays are offset such
that each pixel detection zone 1 includes four different pads 2.
For example pixel detection zone 1a includes pads 2a, 2b, 2c and
2d. In this exemplary configuration, each pad 2 occurs in the
detection zone of four different pixels 1. For example, pad 2a is
in the detection zone of pixels 1a, 1b, 1c and 1d. In the
exemplified configuration of FIG. 1, each detection zone is square
and each pad occurs in a corner of four of the detection zones.
Furthermore each pad is exemplified as being square and each
detection zone includes a corner of four of the pads. As used
herein, the term "in a corner" means at or near the intersection of
two vertices. For example, an object that occurs in all or part of
one quadrant of a square area can be considered to be in the corner
of the square area.
[0046] In the exemplary configuration of FIG. 1, the array of pads
has roughly the same pitch (center to center spacing for the pads)
as the pitch for the array of pixels. Also the pads have an area
that is roughly equivalent to the area of each pixel's detection
zone (although the areas for any given pad and detection zone only
partially overlap due to the offset between the two arrays). Also,
the pads are adjacent to each other and the pixel detection zones
are adjacent to each other. Thus, the pads and pixels have the same
spacing. This configuration is exemplary as the pitch, areas and
spacing can differ for the two arrays.
[0047] In some embodiments, the pads can have a pitch that is less
than the pitch for the detection zones. This can allow greater than
four pads per detection zone in the rectilinear configuration such
as the one exemplified in FIG. 1. Depending upon the desired use,
the array of pads on a surface can have a pitch that is no greater
than the pitch of the detection areas, no greater than half the
pitch of the detection areas, no greater than a quarter of the
pitch of the detection areas or no greater than a tenth of the
pitch of the detection areas, or smaller pitch.
[0048] The areas for individual pads can be substantially smaller
than the area of each detection zone. In particular embodiments
this can allow each detection zone to include several pads while
each pad is present in only a single detection zone (in contrast to
the example of FIG. 1 where each pad is present in four detection
zones). The area for individual pads on a surface can be at most
the same as the detection area, at most 75% of the detection area,
at most 50% of the detection area, at most 25% of the detection
area or at most 10% of the detection area or smaller.
[0049] The pads are exemplified as being juxtaposed to each other
and pixel detection zones are also exemplified as being juxtaposed
to each other in FIG. 1. In alternative embodiments, spacing can
occur between pads or between pixel detection zones. The spacing
for one of the arrays can be equivalent or different compared to
the other array. For example, the spacing between the pads on a
surface can be greater or smaller than the spacing between the
pixel detection areas on the surface.
[0050] Although detection areas and pads are exemplified above as
being square, it will be understood that reactive pads on a surface
and/or pixel detection areas on the surface can have other shapes
including, but not limited to, rectangular, circular, oval,
hexagonal, triangular, polygonal or the like. A particularly
illustrative example is a hexagonally packed array. Hexagonal
packing is a particularly useful configuration for close packing of
round pads or round detection areas. In an exemplary hexagonal
arrangement, each pixel in an array can have a detection area that
is round and includes a subset of seven responsive pads on the
surface of a substrate. Furthermore in this packing, each pad can
be included in the detection areas for three of the pixels. Thus
each detection area can be considered as approximating a hexagon
and each pad can be considered to cross into three detection zones
at a corner of each hexagon. Alternatively, each pad can have an
area that is round and can be included in seven detection areas on
the surface of a substrate. Furthermore in this packing, each pixel
in the array can have a detection area on the surface that includes
three of the pads. Thus, each pad can be considered as
approximating a hexagon and each detection area can be considered
to cross into three detection zones at a corner of each
hexagon.
[0051] An apparatus or system of the present disclosure can include
an array of pixels that are in a complementary metal oxide
semiconductor (CMOS) sensor, charge coupled device (CCD) sensor or
other digital cameras. In some embodiments the pixels are used to
detect chemiluminescence, electrochemical luminescence or other
optical signals that do not require excitatory radiation. However,
many embodiments utilize fluorescence based detection. In these
cases, an apparatus or system of this disclosure can include an
optical excitation assembly. Radiation can be provided from a
laser, light emitting diode (LED), or other appropriate radiation
source. Furthermore the radiation can be conditioned by optics to
reflect, filter, shape, direct or otherwise manipulate the
excitation radiation.
[0052] Embodiments described herein may utilize a step-and-shoot
procedure in which different portions of an array of pads are
individually detected or imaged between (or after) relative
movements of the detector and sample. For example, each area of the
array can be excited with a laser or other appropriate radiation
source and emission can be detected using an array of pixels
configured for super resolution imaging. Examples of step-and-shoot
optical components that can be modified for super resolution
imaging in accordance with the present disclosure are set forth in
US Pat. App. Pub. No. 2012/0270305 A1, which is incorporated herein
by reference. Embodiments described herein may utilize a scanning
procedure in which different portions of an array of pads are
detected or imaged during movement between the pads and optical
components. In some embodiments, the imaging assembly includes a
scanning time-delay integration (TDI) system. Furthermore, the
imaging sessions may include line-scanning one or more samples such
that a linear focal region of light is scanned across the array of
pads. Some methods of line-scanning that can be modified for super
resolution imaging in accordance with the present disclosure are
described, for example, in U.S. Pat. No. 7,329,860 and U.S. Pat.
Pub. No. 2009/0272914, each of which is incorporated herein by
reference. Scanning may also include moving a point focal region of
light in a raster pattern across the array of pads. Whether using
step-and-shoot, scanning, static image collection or other
configurations, embodiments can be configured for epi-fluorescent
imaging or total-internal-reflectance-fluorescence (TIRF) imaging.
Exemplary optical components and arrangements that can be modified
for super resolution imaging in this regard are set forth in U.S.
patent application Ser. No. 13/766,413; US Pat. App. Pub. Nos.
2010/0111768 A1 and 2012/0270305 A1; and U.S. Pat. Nos. 7,329,860
and 8,241,573, each of which is incorporated herein by
reference.
[0053] Certain embodiments include objective lenses having high
numerical aperture (NA) values. Exemplary high NA ranges for which
embodiments may be particularly useful include NA values of at
least about 0.6. For example, the NA may be at least about 0.65,
0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher. Those skilled in the
art will appreciate that NA, being dependent upon the index of
refraction of the medium in which the lens is working, may be
higher including, for example, up to 1.0 for air, 1.33 for pure
water, or higher for other media such as oils. However, other
embodiments may have lower NA values than the examples listed
above. Image data obtained by the optical assembly may have a
resolution that is between 0.1 and 50 microns or, more
particularly, between 0.1 and 10 microns. Optical assemblies may
have a resolution that is sufficient to individually resolve the
features or sites that are separated by a distance of less than 15
.mu.m, 10 .mu.m, 5 .mu.m, 2 .mu.m, 1 .mu.m, 0.5 .mu.m, or less.
[0054] In general, the NA value of an objective lens is a measure
of the breadth of angles for which the objective lens may receive
light. The higher the NA value, the more light that may be
collected by the objective lens for a given fixed magnification.
This is because the collection efficiency and the resolution
increase. As a result, multiple pads or features may be
distinguished more readily when using objectives lenses with higher
NA values. Therefore, in general, a higher NA value for the
objective lens may be beneficial for imaging.
[0055] The size of the pads (or target analyte features) and/or
spacing between the pads (or target analyte features) can vary such
that arrays can be high density, medium density or low density.
High density arrays are characterized as having pads (or features)
separated by less than about 15 .mu.m. Medium density arrays have
pads (or features) separated by about 15 to 30 .mu.m, while low
density arrays have pads (or features) separated by greater than 30
.mu.m. An array useful in some embodiments can have pads (or
features) that are separated by less than 100 .mu.m, 50 .mu.m, 10
.mu.m, 5 .mu.m, 1 .mu.m, or 0.5 .mu.m. An apparatus or method of
the present disclosure can be used to image an array of pads or
features at a resolution sufficient to distinguish pads or features
at the above densities or density ranges. In particular embodiments
the size of the responsive pads can be smaller than the resolution
limit of the detector component that is used. In this way, the
super resolution methods and apparatus of the present disclosure
can distinguish target analyte features at a resolution beyond the
limits of the detector component.
[0056] FIG. 2 shows a diagrammatic representation of an array of
pixel detection zones 1, an array of responsive pads 2 and an array
of target analyte features 3 that are configured to provide super
resolution imaging of the features 3. Each target analyte feature 3
is shown as being in the corner of a pad 2 that occurs in a
detection zone of a pixel 1. For example pixel detection zone 1a
includes a target analyte feature 3 that is present on each of pads
2a, 2b, 2c and 2d, respectively. In this exemplary configuration
(i) each target analyte feature occurs in a single detection zone,
(ii) each detection zone includes four different target analyte
features and each target analyte feature is present on a different
responsive pad than the other target analyte features in the
respective detection zone. Thus, each pixel can distinguish the
four target analytes based on differential actuation of the four
pads, respectively.
[0057] In an alternative embodiment to that exemplified in FIG. 2,
a target analyte can occur in multiple detection zones. For
example, one can envision a situation where the four target analyte
features 3 on pad 2a are replaced with a single feature that
occupies the entire surface area of pad 2a. As such the target
analyte feature will occur in the four detection zones 1a, 1b, 1c
and 1d. In this situation activation of signal at pad 2a will
result in detection by all four of the pixels. However, in this
configuration each pixel will still include four pads in its
detection zone.
[0058] A detection zone can include multiple target analyte
features, each on a separate responsive pad from others in the
detection zone. For example, the detection zone of an individual
pixel can include at least 2, 3, 4, 5, 10 or more target analyte
features, each on separate responsive pads. The number of target
analyte features in a detection zone can be selected to suit the
size of the pixel, the size of the detection zone for the pixel
(for example, as influenced by optics between the pixel and the
features or pads observed), the size of the pads and the size of
the target analyte features. In particular embodiments, a maximum
number of target analyte features in a detection area can be 10, 5,
4, 3 or 2.
[0059] Any of a variety of target analytes that are to be detected,
characterized, or identified can be used in an apparatus, system or
method set forth herein. Exemplary analytes include, but are not
limited to, nucleic acids (e.g. DNA, RNA or analogs thereof),
proteins, polysaccharides, antibodies, epitopes, receptors,
ligands, enzymes (e.g. kinases, phosphatases or polymerases), small
molecule drug candidates, cells, viruses, organisms, or the like.
An array of pads can include multiple different species from a
library of analytes. For example, the species can be different
antibodies from an antibody library, nucleic acids having different
sequences from a library of nucleic acids, proteins having
different structure and/or function from a library of proteins,
drug candidates from a combinatorial library of small molecules,
cells from a culture, tissue or organism, etc.
[0060] In some embodiments, analytes can be distributed on pads
such that they are individually resolvable. For example, a single
molecule of each analyte can be present at each pad. Alternatively,
analytes can be present as colonies or populations such that
individual molecules or cells are not necessarily resolved. The
colonies or populations can be homogenous with respect to
containing only a single species of analyte (albeit in multiple
copies). Taking nucleic acids as an example, each pad in an array
of pads can include a colony or population of nucleic acids and
every nucleic acid in the colony or population can have the same
nucleotide sequence (either single stranded or double stranded).
Colonies of nucleic acids can also be referred to as `nucleic acid
clusters`. Nucleic acid colonies can optionally be created by
cluster amplification or bridge amplification techniques as set
forth in further detail elsewhere herein. Multiple repeats of a
target sequence can be present in a single nucleic acid molecule,
such as a concatamer created using a rolling circle amplification
procedure. Thus, a responsive pad can contain multiple copies of a
single species of an analyte. Alternatively, a colony or population
of analytes that are at a pad can include two or more different
species. For example, one or more pads in an array of pads can each
contain a mixed colony having two or more different nucleic acid
species (i.e. nucleic acid molecules with different sequences). The
two or more nucleic acid species in a mixed colony can be present
in non-negligible amounts, for example, allowing more than one
nucleic acid to be detected in the mixed colony.
[0061] As set forth above for target analyte features in general,
each pad in an array of pads can include a plurality of nucleic
acid clusters and each cluster can occur in the detection zone for
a single pixel. Alternatively, each cluster can occur in the
detection zones for several pixels. For example, each pad can
include only a single nucleic acid cluster and the cluster can be
included in the detection zones for at least two pixels.
[0062] In some embodiments, at least two nucleic acid clusters are
included in the detection zone for a single pixel. Each of the
clusters can be homogenous with respect to the nucleotide sequence
present and the two clusters can have different nucleotide
sequences compared to each other. The clusters can be labeled, for
example, by a labeled nucleotide that has been incorporated in the
course of a sequencing-by-synthesis (SBS) technique. Other labels
are possible too such as those set forth elsewhere herein. The two
clusters can have the same label as each other or a different label
can be present at each of the two clusters. For example, in an SBS
technique the identity of the labels will depend upon the sequences
of the two clusters at the position of nucleotide incorporation.
This example can be extended to formats where 3 or 4 or more
nucleic acid clusters are included in the detection zone for a
single pixel. Accordingly, multiple clusters can have different
nucleotide sequences from each other and can be labeled with probes
that are the same or different.
[0063] As used herein, the term "different", when used in reference
to nucleic acids, means that the nucleic acids have nucleotide
sequences that are not the same as each other. Two or more
different nucleic acids can have nucleotide sequences that are
different along their entire length. Alternatively, two or more
different nucleic acids can have nucleotide sequences that are
different along a substantial portion of their length. For example,
two or more different nucleic acids can have target nucleotide
sequence portions that are different from each other while also
having a universal sequence region that is the same for both (or
all).
[0064] In particular embodiments, two clusters that are present in
the detection zone for the same pixel can be distinguished from
each other due to the presence of different priming sequences. A
first of the two clusters can be selectively hybridized to a first
primer that has specificity for a first priming sequence present
the first cluster compared to a second priming sequence that is
present on the second cluster. The first cluster can be detected or
sequenced based on hybridization of the first primer, for example,
if the first primer has a detectable label or if the first primer
undergoes a reaction, such as nucleotide extension, oligonucleotide
ligation, sequencing by synthesis or other technique, that recruits
label(s) to the cluster. In turn, the second cluster can be
detected based on specificity for a second primer to the second
priming sequence compared to the first priming sequence. The second
cluster can be selectively detected or sequenced using the
techniques exemplified above for the first cluster.
[0065] Analytes can be attached to a responsive pad. The attachment
can be covalent or non-covalent. In some embodiments, the
attachment can be mediated by a gel material. The analytes can be
nucleic acids that are attached to a gel material. Exemplary
methods and reactants for attaching nucleic acids to gels are
described, for example, in US Pat. App. Pub. No. 2011/0059865 A1,
or U.S. patent application Ser. No. 13/784,368, each of which is
incorporated herein by reference. Nucleic acids can be attached to
the gel or to the surface of a pad via their 3' oxygen, 5' oxygen,
or at other locations along their length such as via a base moiety
of the 3' terminal nucleotide, a base moiety of the 5' nucleotide,
and/or one or more base moieties elsewhere in the molecule.
Non-covalent modes of attachment include, for example, ionic
interactions between nucleic acid and a surface (or gel),
entrapment of nucleic acid within pores of a gel, protein-protein
interactions, binding between receptors and ligands and/or nucleic
acid, and other known modes.
[0066] An apparatus of the present disclosure can include a flow
cell. Exemplary flow cells, methods for their manufacture and
methods for their use are described in US Pat. App. Publ. Nos.
2010/0111768 A1 or 2012-0270305 A1; or WO 05/065814, each of which
is incorporated herein by reference. Flow cells provide a
convenient format for housing an array of responsive pads and that
is subjected to a sequencing-by-synthesis (SBS) reaction or other
technique that involves repeated delivery of reagents in cycles
(e.g. synthesis techniques or detection techniques having
repetitive or cyclic steps).
[0067] Any of a variety of labels or moieties can be present at a
responsive pad. Exemplary labels and moieties include, but are not
limited to fluorophores, chromophores, chemiluminescent species,
electrochemical luminescence species, fluorescence quenchers,
donors and/or acceptors for fluorescence resonance energy transfer
(FRET), nanocrystals and the like. Fluorophores that may be useful
include, for example, fluorescent lanthanide complexes, including
those of Europium and Terbium, fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene,
Lucifer Yellow, Cascade Blue, Texas Red, Alexa dyes, phycoerythin,
and others known in the art such as those described in Haugland,
Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; The
Synthegen catalog (Houston, Tex.), Lakowicz, Principles of
Fluorescence Spectroscopy, 2.sup.nd Ed., Plenum Press New York
(1999), or WO 98/59066, each of which is hereby incorporated by
reference. Exemplary quenchers include, but are not limited to,
DACYL(4-(4'-dimethylaminophenylazo)benzoic acid), Black Hole
Quenchers (Biosearch Technologies, Novato, Calif.), Qxl quenchers
(Anaspec, Freemont, Calif.), Iowa black quenchers, DABCYL, BHQ1,
BHQ2, QSY7, QSY9, QSY21, QSY35, BHQO, BHQ1, BHQ2, QXL680, ATTO540Q,
ATTO580Q, ATTO612Q, DYQ660, DYQ661 and IR Dye QC-1 quenchers.
Chemiluminescent species include, for example, luminal, reagents
used for detection in pyrosequencing, aequorin and other species
known in the art. Exemplary electrochemical luminescence species
include, but are not limited to Ru(bpy.sub.3).sup.2+, Bodipy dyes,
luminal derivatives, acridine esters and others known in the
art.
[0068] A label or moiety can be selected to suit a particular
application of an apparatus, system or method set forth herein. For
example, the label or moiety can be associated with a target
analyte that is present at the pad and detected using a technique
set forth below. The label or moiety can be in a detectable state
or a non-detectable state, for example, as influenced by a
characteristic of a responsive pad at which the label or moiety is
present. Thus, a probe or moiety can be located in a detection zone
of a pixel and the probe or moiety can optionally be in a state
that produces a signal that is detected by the pixel.
[0069] In particular embodiments, target analytes include
fluorescent moieties and the pixels are configured to detect
emission from the fluorescent moieties. An apparatus that includes
the fluorescent moieties can further include an excitation assembly
as set forth herein or otherwise known in the art. An apparatus
with an excitation assembly is also useful when using fluorescence
quenchers, donors and/or acceptors for fluorescence resonance
energy transfer (FRET) or nanocrystals. Responsive pads where
target analytes occur can be selectively placed in a state to
preferentially produce fluorescent signals. For example, a
responsive pad can be electrically activated to create an electric
field that selectively attracts a fluorescent label, donor,
acceptor or nanocrystal to the pad, thereby producing a fluorescent
signal at the pad. Alternatively, a responsive pad can be
electrically activated to create an electric field that selectively
repels or degrades a fluorescence quencher, thereby preferentially
producing a fluorescent signal from a label or moiety that was
previously quenched. In yet another example, a responsive pad can
be electrically activated to create an electric field that
selectively repels or degrades a fluorescent label, donor, acceptor
or nanocrystal, thereby inhibiting fluorescent signals from the
pad. Such inhibition can also result by electrically activating a
pad to create an electric field that selectively attracts a
fluorescence quencher. It is also possible to place a responsive
pad in a state (e.g. a neutral state) where the above reagents are
allowed to diffuse away. Differential activation of responsive pads
in such ways can be used to achieve super resolution detection of
fluorescent target analytes present at multiple responsive pads in
the detection zone of a single pixel.
[0070] Similarly, responsive pads having chemiluminescent moieties
or electrochemical luminescence moieties can be placed in different
states to achieve super resolution imaging. An apparatus used for
chemiluminescent or electrochemical luminescence detection can be
configured similar to that exemplified above for use of fluorescent
moieties, except that an excitation assembly is not necessary.
Rather, signal generation can be achieved by activating a
responsive pad to create an electric field that selectively
attracts a chemical capable of generating chemiluminescence, and
signal can be inhibited by activating the pad to create an electric
field that repels or degrades the chemical. Electrochemical
luminescence signal generation can be achieved by activating a
responsive pad to carry out a redox reaction that produces the
signal, and switching the pad can inhibit signal due to the pad
being in a state where the redox reaction does not occur.
Conversely, a responsive pad can be actuated to carry out a redox
reaction that inhibits signal and a switch can alter the pad to a
state where the redox reaction does not occur so that signal can be
generated.
[0071] Several embodiments set forth herein illustrate
differentiation of two or more features in the detection zone of
the same pixel using differential activation of responsive pads to
which the features are attached (or otherwise functionally
associated) during a detection step, between detection steps or
after a detection step. Alternatively or additionally, two features
that are present in the detection zone of the same pixel can be
distinguished based on a chemical or physical distinction that is
introduced prior to a particular detection step. Taking as an
example nucleic acid features, two or more pads can be
differentially actuated during one or more of target nucleic acid
capture, target nucleic acid amplification or hybridization of a
primer used for detection of the nucleic acids. For example, a
first library of target nucleic acids that includes members having
a first universal priming sequence can be captured on a first
subset of responsive pads and/or amplified on the first subset of
pads. A second library of target nucleic acids that includes
members having a second universal priming sequence can be captured
on a second subset of responsive pads and/or amplified on the
second subset of pads. The pixels and features can be arranged such
that a feature from the first subset and a feature from the second
subset are both located in the detection zone of a particular
pixel. These two features can be distinguished without necessarily
applying differential actuation during a detection step, after a
detection step or between detection steps. Rather, the features can
be distinguished based on nucleic acid hybridization specificity
whereby a first primer selectively hybridizes to the first
universal priming sequence and a second primer selectively
hybridizes to the second universal priming sequence.
[0072] In the above examples, it will be understood that changing
the state of responsive pads relative to each other can result in
filling a pad to capacity with a particular reagent (e.g. label or
probe), removing all of a particular reagent from a pad, degrading
all of a particular reagent at a pad or modifying all of a
particular reagent at a pad. However, in most embodiments it will
be sufficient and in some cases even desirable that differential
actuation of responsive pads results in higher relative
concentrations of a particular reagent at one pad compared to
another. For example an attractive electric field applied at a
first pad can create a relatively higher concentration of a
fluorophores, chromophores, chemiluminescent species,
electrochemical luminescence species, fluorescence quenchers,
donors and/or acceptors for fluorescence resonance energy transfer
(FRET), nanocrystals or other reagents at the first pad compared to
at a second pad where the field is not applied or where a different
field is applied. Similarly, a repellant or destructive field
applied at a first pad can create a relatively lower concentration
of these or other reagents at the first pad compared to at a second
pad where the field is not applied or where a different field is
applied.
[0073] FIG. 3 shows side profile views of two detection apparatus
that have an array of pixels and an array of responsive pads
configured as diagrammed in FIG. 2. Panel A shows an integrated
apparatus 200 wherein the array of pixels and the array of
responsive pads are fixed on a substrate. The pixels are optically
coupled to detection zones via light pipes in the substrate. For
example, signal from nucleic acid clusters 3a and 3b in detection
zone 1a passes through light pipe 12 to reach pixel 13. The light
pipe is bounded by vertical curtains 11a and 11b that prevent cross
talk of optical signals between pixels. Nucleic acid cluster 3a is
attached to responsive pad 2a and cluster 3b is attached to pad 2b.
Pads 2a and 2b can be independently actuated to allow super
resolution imaging of clusters 3a and 3b. As shown in Panel A, at
least a portion of each pad is in the detection zones for two
pixels. For embodiments that use fluorescence detection, light pipe
12 can include an optical filter that blocks excitation radiation
from reaching the pixel 13. An advantage of the configuration shown
in FIG. 3, Panel A is that the array of pixels and array of
responsive pads are positionally fixed relative to each other. This
design does not require alignment and focusing devices used in
multi-component designs where the two arrays can move relative to
each other.
[0074] Panel B of FIG. 3 shows a detection apparatus 300 having a
camera component 15 that is separated in space from the substrate
that is to be detected 16. In such a configuration the camera
component 15 can be moved relative to the array of responsive pads
that is located on substrate 16, for example, to achieve a desired
focus, resolution or alignment. Pixel 13 is directed to substrate
16 and has a detection area 1a that includes two nucleic acid
clusters 3a and 3b on separate responsive pads 2a and 2b,
respectively. The camera in Panel B is configured for
epifluorescent detection when an excitation assembly is present,
for example, at a position that excites the nucleic acid clusters
from the same side of substrate 16 that is observed by the pixels.
Again, pads 2a and 2b can be independently actuated to allow super
resolution imaging of clusters 3a and 3b. At least a portion of
each of the pads is in the detection zones for two pixels.
[0075] An apparatus of the present disclosure can include an
activation circuit to actuate changes in the characteristics of
responsive pads in an array of pads. FIG. 4 shows a diagrammatic
representation of a detection apparatus 100 including a detection
module 10 (including an array of reactive pads 2 and an array of
pixel detection areas 1), an activation circuit 20 configured to
actuate changes in each of the responsive pads 2 and, optionally,
to receive feedback regarding the state of each pad 2. As set forth
previously herein, the activation circuit can be configured to
individually address and actuate each of the responsive pads. The
activation circuit can produce changes in any of a variety of
characteristics at or near the responsive pads including, but not
limited to, presence or absence of electric charge; positive,
negative or neutral polarity of electric charge; strength of
electric field; shape of electric field; presence or absence of
electric current; direction of electric current; strength of
electric current; type of electric current (e.g. DC and/or AC);
shape for AC current waveform; frequency or magnitude of AC
current; magnetic state; presence or absence of a magnetic dipole;
chemical properties at the surface such as pH, redox potential,
hydrophobicity, hydrophilicity, presence of a reactive species or
absence of a reactive species; and the like.
[0076] Thus, an activation circuit can place responsive pads into
different states. As used herein, the term "different" or
"differential," when used in reference to a responsive pad, means
that the pad has at least one characteristic that is absent at
another pad or that is not present to the same degree as at another
pad. The characteristic is typically responsive to an activation
circuit or other device that actuates change at the pad. For
example, pads can be different with respect to changes in the
characteristics set forth herein, for example, with regard to an
activation circuit. It will be understood that the phrase "a
different electric field" can be used to refer to the presence or
absence of an electric field, such that a pad that has no electric
field can be considered to have a different electric field from a
pad that has an electric field unless explicitly stated to the
contrary. Other different states include, for example, changes in
hydrophobicity and hydrophilicity. Such changes in state can occur
due to electrowetting and other techniques known in the art for
manipulating droplets examples of which are set forth in US Pat.
App. Pub. No. 2013/0116128 A1, which is incorporated herein by
reference.
[0077] In particular embodiments an activation circuit is
configured to apply an electric field at first and second pads in
an array of pads (for example at two pads in the detection zone of
a single pixel). The activation circuit can be configured to apply
a different electric field at the first pad compared to the second
pad, and the activation circuit can have a switch to selectively
alter the electric field at the first pad compared to the second
pad.
[0078] As used herein, the term "selectively alter" means to alter
one thing (e.g. a first pad) to a greater degree than another thing
(e.g. a second pad). In some cases selective alteration can be
achieved by turning one thing on and another thing off. However, it
is also possible to make a selective alteration without turning one
of the things on and instead reducing or increasing an actuatable
characteristic of one of the things relative to the other thing.
Selective alteration can result in changes for any of the
characteristics set forth herein, for example, above with regard to
an activation circuit. For example, an activation circuit can be
configured to apply (or relatively increase) electric field at a
first pad while preventing (or relatively decreasing) electric
field at a second pad, and a switch can be configured to prevent
(or relatively decrease) electric field at the first pad while
applying (or relatively increasing) electric field at the second
pad. Similarly, an activation circuit can be configured to apply
positive electric field at a first pad while applying negative
electric field at a second pad, and a switch can be configured to
apply negative electric field at the first pad while applying
positive electric field at the second pad. As a further example, a
pad can be actuated to attract or repel an aqueous droplet or the
contents of an aqueous droplet. In particular embodiments a pad is
actuated by electrowetting to attract or repel a droplet.
[0079] An apparatus of the present disclosure can further include a
readout circuit to acquire signals from an array of pixels. The
detection apparatus 100 diagrammed in FIG. 4 includes a detection
module 10 (including an array of reactive pads 2 and an array of
pixel detection areas 1), an activation circuit 20 and a readout
circuit 30. The readout circuit 30 can be configured to obtain
signal information from the pixel detection areas 1 via the array
of pixels. The readout circuit can optionally be configured to
alter the gain of individual pixels or to turn pixels on and off in
response to the amount of signal received by one or more pixels in
the array of pixels.
[0080] An apparatus of the present disclosure can be included in a
detection system 500 as diagrammed in FIG. 4. The system can
include detection apparatus 100 fluidically coupled to a fluidic
system 70 and in operative communication with a control module 50
and a processing module 60. The fluidic system 70 can be configured
to deliver fluid reagents used in a detection method that occurs in
the detection apparatus 10. In some embodiments, such as those
using sequencing-by-synthesis as set forth below, the fluidic
system can deliver reagents in repeated cycles. Repeated cycles of
fluid delivery can also be useful for other applications where
polymeric molecules are synthesized or sequenced, or for other
applications. Alternatively, a fluidic system can be configured for
non-cyclic delivery of reagents to a given array of pads or at
least for a given sample present on the array of pads. Examples of
fluid systems that are coupled to biological arrays and that can be
readily adapted to deliver fluids to an array of pads set forth
herein are described in US Pat. App. Pub. No. US 2010/0009871 A1;
US Pat. App. Pub. No. 2012/0270305 A1 and U.S. patent application
Ser. No. 13/766,413, (published as US Pat. App. No. 2013/0260372
A1) each of which is incorporated herein by reference. Particularly
useful fluidic systems are those that move fluid droplets to and
from detection areas via electrowetting or other techniques as
described, for example, in U.S. patent application Ser. No.
13/670,318 (published as US Pat. App. Pub. No. 2013/0116128 A1),
which is incorporated herein by reference.
[0081] The control module 50 can be configured to direct the
readout circuit 30 to acquire signals from each of the pixel
detection areas 1 during a sensing period. The control module 50
can also communicate with the activation circuit 20 to direct
actuation of responsive pads during the sensing period. The
communication from the control module 50 can direct the activation
circuit to switch the actuation at the responsive pads. In an
exemplary embodiment of the system diagrammed in FIG. 4, the
control module 50 can direct the readout circuit 30 to acquire
signals from each of the pixels during a sensing period, direct the
activation circuit 20 to apply a different electric field at the
first pad compared to the second pad, during the sensing period,
and direct the activation circuit to switch to selectively alter
the electric field at the first pad compared to the second pad,
during the sensing period.
[0082] As used herein, the term "sensing period" means a time
frame, whether continuous or discontinuous, during which signal is
collected. Accordingly, a control module can direct a readout
circuit to acquire signals from a pixel continuously during a
sensing period, or alternatively, the control module can direct the
readout circuit to toggle the pixel between an on-state and an
off-state during a sensing period. Similarly, the gain at a pixel
can be increased or decreased during a sensing period.
[0083] Control module 50 can be further configured to communicate
with the fluidic system 70. The control module 50 can provide
instructions to the fluidic system to direct reagent delivery from
a particular reservoir, or other fluidic component, to the
detection apparatus 100. When multiple fluids are to be delivered,
for example as is the case for a sequencing-by-synthesis protocol,
the control module 50 can direct the sequence of fluid components
delivered to the detection apparatus 100. Generally, control module
50 can also direct the amount of fluid delivered at a particular
step of a protocol, the duration of delivery of a particular fluid,
the temperature for a particular fluid, the rate of fluid delivery
(e.g. via changes in fluid pressure), and the like. The control
module 50, being in communication with the fluidic system 70, the
readout circuit 30, the detection module 10, and the activation
circuit 20 can coordinate super resolution detection of a plurality
of target analytes that are chemically manipulated in an
array-based platform. An example of such a platform is one using a
sequencing-by-synthesis technique.
[0084] In particular embodiments, the control module 50 can also be
configured to receive feedback from the activation circuit 20,
readout circuit 30, fluidic system 70 or detection module 10.
Feedback from one or more of these components can be used to modify
directions sent to the component(s) from which feedback was
received or another component of the system. Thus, the control
module can assess the overall condition of the system and modify
function to achieve desired output or activity. Exemplary
algorithms and configurations for assessing and modifying function
of an array-based detection system are described in U.S. Pat. No.
8,244,479, which is incorporated herein by reference. In particular
embodiments, the control module 50 can also be in communication
with the processing module 60, to send directions or receive
feedback pertaining to the functions of the processing module
60.
[0085] A processing module 60 that is included in a system 500 of
the present disclosure can be in communication with a readout
circuit 30. The processing module 60 can receive signals from the
readout circuit 30 and modify the signals to create data in a
desired format. Taking an SBS system as an example, the processing
module 60 can determine the identity of a nucleotide that is
incorporated at a particular nucleic acid cluster from electrical
signals obtained by a pixel during a sensing period and from a
schedule of actuation periods for two or more pads that were in the
detection zone of the pixel during the sensing period. The
processing module 60 can further include algorithms to manipulate
data to provide a desired output that can be communicated to a
user. For example, data can be used to determine presence or
absence of a target analyte (e.g. presence of a nucleic acid
sequence or presence of a single nucleotide polymorphism in a
sequence), amount of a target analyte (e.g. ploidy level for a gene
sequence or expression level for an RNA sequence), structure of a
target analyte (e.g. nucleotide sequence of a nucleic acid),
chemical reactivity of a target analyte (e.g. binding affinity
between receptor and ligand or kinetics of reaction for an enzyme)
or the like.
[0086] Processing module 60 can also be configured to send
directions to other components of the system based on data obtained
or processed. For example, processing module 60 can determine when
enough information has been obtained from a sample that further
manipulation and/or observation of the sample can be stopped.
Directions can then be sent to the detection apparatus, for example
via the control module, to cease or pause data acquisition.
[0087] The various components of system 500 or other system of the
present disclosure can be present in a single unit, for example,
having a relatively small footprint. Alternatively, the components
can be distributed, for example, in a network that includes data
connections and in some cases fluidic connections. In some
embodiments, information processing modules can be distributed in a
computer network that is connected to other components of the
system. In some cases one or more of the processing modules can be
cloud-based. Exemplary, cloud-based systems for processing
sequencing data and that can be adapted for use in a system or
method of the present disclosure are described in U.S. patent
application Ser. No. 13/790,596 (published as US Pat. App. Pub. No.
2013/0275486 A1) and Ser. No. 13/790,623 (published as US Pat. App.
Pub. No. 2013/0274148 A1), each of which is incorporated herein by
reference.
[0088] A system provided by the present disclosure can be used for
sequencing nucleic acids. The system can include (a) a detection
apparatus having (i) an array of electrically responsive pads on a
substrate surface; (ii) an array of pixels, wherein each pixel in
the array has a detection zone on the surface that includes a
subset of four of the pads; and (iii) an activation circuit to
apply an electric field to the pads in the subset individually,
wherein the activation circuit is configured to apply a different
electric field at a first pad of the subset compared to the other
pads of the subset; (b) a readout circuit to acquire signals from
the array of pixels; (c) a control module that directs the readout
circuit to acquire signals from each of the pixels during a sensing
period and that directs the activation circuit to sequentially
apply different electric fields at the four pads during the sensing
period; and (d) a processing module that correlates (i) the signals
acquired from the pixels during the sensing period and (ii) the
sequential application of the different electric fields at the four
pads during the sensing period, in order to distinguish a sequence
of signals for each of the pads.
[0089] The control module and processing module of a nucleic acid
sequencing system can be configured to carry out a
sequencing-by-synthesis (SBS) protocol. In SBS, extension of a
nucleic acid primer along a nucleic acid template (e.g. a target
nucleic acid or amplicon thereof) is monitored to determine the
sequence of nucleotides in the template. The underlying chemical
process can be polymerization (e.g. as catalyzed by a polymerase
enzyme). In a particular polymerase-based SBS embodiment,
fluorescently labeled nucleotides are added to a primer (thereby
extending the primer) in a template dependent fashion such that
detection of the order and type of nucleotides added to the primer
can be used to determine the sequence of the template. A plurality
of different templates at different features on an array of
responsive pads set forth herein can be subjected to an SBS
technique under conditions where events occurring for different
templates can be distinguished using super resolution imaging.
[0090] Flow cells provide a convenient format for housing an array
of nucleic acid clusters located on responsive pads that are
subjected to an SBS technique that involves repeated delivery of
reagents in cycles. Exemplary flow cells are set forth above and in
references cited above. To initiate a first SBS cycle, one or more
labeled nucleotides, DNA polymerase, etc., can be flowed
into/through a flow cell that houses an array of nucleic acid
clusters that have been hybridized to a sequencing primer. Those
sites of an array where primer extension causes a labeled
nucleotide to be incorporated can be detected. Optionally, the
nucleotides can further include a reversible termination property
that terminates further primer extension once a nucleotide has been
added to a primer. For example, the labeled nucleotide that is
contacted with the nucleic acid clusters can have a reversible
terminator moiety that gets added to a primer such that subsequent
extension cannot occur until a deblocking agent is delivered to
remove the moiety. Thus, for embodiments that use reversible
termination, a deblocking reagent can be delivered to the flow cell
(before or after detection occurs). Washes can be carried out
between the various delivery steps. The cycle can then be repeated
n times to extend the primer by n nucleotides, thereby detecting a
sequence of length n. Exemplary SBS procedures, fluidic systems and
detection system components that can be readily adapted for use in
a system of method of the present disclosure are described, for
example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497;
U.S. Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos.
7,329,492; 7,211,414; 7,315,019; 7,405,281, and US 2008/0108082,
each of which is incorporated herein by reference.
[0091] Other sequencing procedures that use cyclic reactions can be
used, such as pyrosequencing. Pyrosequencing detects the release of
inorganic pyrophosphate (PPi) as particular nucleotides are
incorporated into a nascent nucleic acid strand (Ronaghi, et al.,
Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res.
11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998);
U.S. Pat. Nos. 6,210,891; 6,258,568 and U.S. Pat. No. 6,274,320,
each of which is incorporated herein by reference). In
pyrosequencing, released PPi can be detected by being immediately
converted to adenosine triphosphate (ATP) by ATP sulfurylase, and
the level of ATP generated can be detected via luciferase-produced
photons. Thus, the sequencing reaction can be monitored by
detecting photons released during this chemiluminescent reaction.
Accordingly, excitation radiation sources used for fluorescence
based detection systems are not necessary for pyrosequencing
procedures. Useful fluidic systems, detectors and procedures that
can be used for application of pyrosequencing to arrays of the
present disclosure are described, for example, in WIPO Pat. App.
Ser. No. PCT/US11/57111, US 2005/0191698 A1, U.S. Pat. Nos.
7,595,883, and 7,244,559, each of which is incorporated herein by
reference.
[0092] Sequencing-by-ligation reactions are also useful including,
for example, those described in Shendure et al. Science
309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each
of which is incorporated herein by reference. Some embodiments can
include sequencing-by-hybridization procedures as described, for
example, in Bains et al., Journal of Theoretical Biology 135(3),
303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58
(1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO
1989/10977, each of which is incorporated herein by reference. In
both sequencing-by-ligation and sequencing-by-hybridization
procedures, target nucleic acids (or amplicons thereof) that are
present at sites of an array are subjected to repeated cycles of
oligonucleotide delivery and detection. Typically, the
oligonucleotides are fluorescently labeled and can be detected
using fluorescence detectors similar to those described with regard
to SBS procedures herein or in references cited herein.
[0093] A fluidic system used in a system or method set forth herein
can store and deliver one or more of the reagents or fluid
components set forth above in the context of target nucleic acid
capture, amplification or nucleotide sequencing protocols. For
example, the reagents can be stored in appropriate reservoirs prior
to delivery to a flow cell or substrate such as one already having
an array of nucleic acids. Furthermore, a detection apparatus can
include a nucleic acid feature (e.g. a nucleic acid cluster) having
an intermediate species produced by one of the steps (e.g. a primer
attached to a labeled and reversibly terminated nucleotide, or a
primer attached to a labeled nucleotide that lacks a terminator, or
a nucleic acid attached to a polymerase and/or nucleotide). The
nucleic acid feature can in turn be attached to a responsive pad
that is in one of the states exemplified herein for producing a
signal to be detected or in a state for inhibiting a signal from
being detected at the nucleic acid feature. Different features can
have differential properties such as different priming sites or
different primers hybridized to the features.
[0094] A processing module 60 that is included in a sequencing
system can be configured to correlate (i) the signals acquired from
the pixels during the sensing period, (ii) the sequential
activation of different pads during the sensing period, and (iii)
the sequence of reagents delivered to the substrate in order to
distinguish a sequence of signals for each of the pads and to
distinguish a sequence of reagents that produce the signals at each
of the pads. The nucleotide sequence of nucleic acid features at
each pad can in turn be determined from this correlation. The
nucleotide sequences or the signal data can be exported to another
processing device for sequence analysis.
[0095] The present disclosure also provides a detection apparatus
that includes (a) an array of responsive pads on a substrate
surface, wherein each responsive pad includes a nucleic acid
feature of a plurality of nucleic acid features in the array,
wherein a first subset of nucleic acid features in the plurality of
nucleic acid features have a first universal sequence and different
target sequences, wherein a second subset of nucleic acid features
in the plurality of nucleic acid features have a second universal
sequence and different target sequences, wherein the first
universal sequence is different from the second universal sequence;
(b) an array of pixels, wherein each pixel in the array has a
detection zone on the surface that includes at least two nucleic
acid features of the plurality of nucleic acid features, the at
least two nucleic acid features including a nucleic acid from the
first subset of nucleic acid features and a nucleic acid from the
second subset of nucleic acid features; and (c) an activation
module to alter a characteristic of a pad in the first subset and
of a pad in the second subset, wherein the activation module is
configured to apply a different characteristic at the pad in the
first subset compared to the pad in the second subset, and wherein
the activation module has a switch to selectively alter the
characteristic at the pads in the first and second subsets.
[0096] Optionally, the detection apparatus can be included in a
nucleic acid sequencing system that also includes (I) a readout
circuit to acquire signals from the array of pixels; (II) a control
module that directs the readout circuit to acquire signals from
each of the pixels during a sensing period and that optionally
directs the activation circuit to sequentially actuate different
responsive pads in each of the detection zones during the sensing
period; and (III) a processing module that optionally correlates
(i) the signals acquired from the pixels during the sensing period
and (ii) the sequential actuation of the different responsive pads
during the sensing period, in order to distinguish a sequence of
signals for each of the pads.
[0097] The present disclosure further provides a method of
detecting analytes. The method can include the steps of (a)
providing a detection apparatus having an array of electrically
responsive pads and an array of pixels, wherein each pixel in the
array has a detection zone that includes a subset of at least two
of the electrically responsive pads, wherein the two pads include
different target analytes, respectively; (b) acquiring signals from
each of the pixels while selectively applying an electric field at
a first of the two pads to preferentially produce signal from a
first of the different target analytes compared to a second of the
target analytes, thereby preferentially acquiring signals from the
first of the target analytes compared to the second of the target
analytes; and (c) acquiring signals from each of the pixels while
selectively applying an electric field at the second of the two
pads to preferentially produce signal from the second of the
different target analytes compared to the first of the target
analytes, thereby preferentially acquiring signals from the second
of the target analytes compared to the first of the target
analytes.
[0098] As used herein, the term "preferentially acquire," when used
in reference to a signal, means to detect more signal from one
source (e.g. from a first target analyte at a first pad) than from
another source (e.g. a second target at a second pad). In some
cases preferential acquisition can be achieved by detecting signal
only from one source and not from another. However, it is also
possible to detect more of a signal or a different type of a
signal, from one source compared to another when preferentially
acquiring signal.
[0099] A method set forth herein can be carried out using an
apparatus or system exemplified herein. However, other suitable
apparatus or systems can be used as desired for a particular
application of the methods. In particular embodiments, a detection
method can include a step of acquiring signals from a pixel while
selectively actuating a first of at least two pads that are in the
detection zone of the pixel. This can allow signal to be
preferentially produced from a first target analyte that is at one
of the pads compared to a second target analyte that is at the
other pad. Thus, signals can be preferentially acquired from the
first target analytes even when the second target analytes are in
the field of view of the pixel.
[0100] In one exemplary embodiment, the target analytes can produce
signal when the pads to which they are attached are actuated to
produce an electric field. This can be demonstrated for a pad that
attracts a label to the target analyte when the electric field is
turned on. In this case, a target analyte that is present at a pad
that is not activated to produce the electric field (or at a pad
that produces a substantially weaker field or at a pad that is
activated to produce a field of opposite polarity) will not produce
signal and will not be detected. The electric fields at the two
pads can be switched to change the pad from which signal is
detected by the pixel. As such, acquiring signal from the pixel and
accounting for the schedule of states for the pads can be used to
achieve super resolution detection of the different target analytes
that are simultaneously in the detection zone of the pixel. Thus,
different target analytes that are simultaneously present in the
detection zone of a single pixel can be distinguished by selective
spatial activation (or inhibition) of signal generation during
detection. Any of a variety of labels and detectable moieties
exemplified elsewhere herein can be used similarly.
[0101] In an alternative embodiment, the target analytes can be
prevented or inhibited from producing signal when the pads to which
they are attached are actuated to produce an electric field. This
can be demonstrated for a pad that has a label present and that
attracts a quencher when the electric field is turned on. FIG. 5
provides a diagrammatic representation of a detection apparatus in
three different states. In a first state (Panel A) neither of the
two pads is charged and the quencher (e.g. black hole quencher) is
not attracted to fluorescently labeled clusters that are present at
either of the pads. Thus, clusters at both pads are fully capable
of producing fluorescent signals. Panel B shows the state where the
pad at the left side of each detection zone is positively charged,
thereby creating an electric field that attracts the negatively
charged black hole quencher to the cluster. As such, the nucleic
acid clusters at the left side pads will be quenched and the pixel
will detect little to no signal from these clusters. However, the
right side pads will produce "unquenched" signal that is detected
by the pixel. As demonstrated by Panel C, the electric fields at
the two pads can be switched to change the cluster from which
signal is detected by each pixel, thereby allowing super resolution
imaging. In this case, the pads on the right attract quencher,
decreasing or preventing signal detection, and the pads on the left
release quencher to produce fluorescent signal that is detectable
by the pixel. Again, any of a variety of labels and detectable
moieties exemplified elsewhere herein can be used similarly.
[0102] Further by way of example, selectively applying an electric
field at the first of two pads in a method of the present
disclosure can attract a fluorescence quencher to the first pad to
preferentially quench fluorescence at the first pad, thereby
preferentially producing signal at the second of the two pads. In
this example a switch can be used to selectively apply the electric
field at the second pad to attract the fluorescence quencher to the
second pad to preferentially quench fluorescence at the second pad,
thereby preferentially producing signal at the first pad.
[0103] In a second example, selectively applying an electric field
at the first pad in a method of the present disclosure can attract
a fluorescent label to the first pad to preferentially produce
fluorescence at the first pad compared to the second pad. In this
second example, a switch can be used to selectively apply the
electric field at the second pad to attract the fluorescent label
to the second pad to preferentially produce fluorescence at the
second pad compared to the first pad.
[0104] In a third example, selectively applying an electric field
at the first pad can induce luminescence from electrochemical
luminescence labels at the first pad to preferentially produce
luminescence at the first pad compared to the second pad. In this
third example, a switch can be used to selectively apply the
electric field at the second pad to induce luminescence from
electrochemical luminescence labels at the second pad to
preferentially produce luminescence at the second pad pads compared
to the first pad.
[0105] Selective activation that allows super-resolution imaging
need not occur during a detection step or even during a sensing
period. For example, prior to the sensing period of a solid-phase
SBS protocol, subsets of target nucleic acids in a library can be
selectively captured at pads of a solid support, selectively
amplified to form features on pads of a solid support or
selectively hybridized to an SBS primer.
[0106] Accordingly, provided herein is a method of detecting target
nucleic acids, including the steps of (a) providing a substrate
comprising an array of pads, the array of pads including a first
subset of the pads and a second subset of the pads; (b) delivering
a first solution to the substrate, wherein the first solution
includes a first plurality of different target nucleic acids that
selectively attach to the first subset of pads compared to the
second subset of pads; (c) delivering a second solution to the
substrate, wherein the second solution includes a second plurality
of different target nucleic acids that selectively attach to the
second subset of pads compared to the first subset of pads; and (d)
detecting the substrate using an apparatus having an array of
pixels, wherein each pixel in the array has a detection zone that
includes (i) at least one of the target nucleic acids that is
attached to a pad of the first subset of pads, and (ii) at least
one of the target nucleic acids that is attached to a pad of the
second subset of pads.
[0107] For embodiments that include delivery of a first solution
and a second solution to a substrate, it will be understood that
the solutions can be delivered simultaneously as a preformed single
solution or they can be delivered in a way that the solutions mix
to form a single solution in the presence of the substrate.
Alternatively, the first and second solutions can be separate
solutions that are delivered to a substrate sequentially. The first
solution can be removed from contact with the substrate prior to
delivery of the second solution. Optionally, the substrate can be
treated with one or more wash solutions between delivery of the
first solution and delivery of the second solution.
[0108] A first plurality of nucleic acids and second plurality of
nucleic acids can be differentially captured at two or more pads on
a substrate based on differential properties of the nucleic acids,
differential properties of the pads or both. An exemplary property
of the nucleic acids that can be exploited for differential capture
include, but are not limited to, presence of particular sequence
regions (e.g. a priming sequence, capture sequence, or the like).
These sequence regions can facilitate capture via hybridization to
complementary sequences present on capture probes that are located
at each pad. Other exemplary properties include, but are not
limited to, presence of a binding moiety (e.g. a ligand or
receptor), charge, mass, length (i.e. number of nucleotides in the
sequence), secondary structure (e.g. presence of single stranded or
double stranded domains), or the like. Differential properties of
the pads can include actuated or activated properties such as those
set forth elsewhere herein, the sequence of attached nucleic acid
capture probes, presence or absence of nucleic acid adapters,
presence or absence of a ligand or receptor, etc.
[0109] In some embodiments, the first plurality of nucleic acids
may have a first universal sequence that is different from a second
universal sequence that is present in the nucleic acids of the
second plurality of nucleic acids. Subsets of pads having capture
probes that are complementary to the respective universal sequences
will provide differential capture.
[0110] Alternatively, subsets of unique capture probes need not be
used. Rather, differential capture can be achieved by actuating a
particular subset of pads for capture in the presence of a first
solution of nucleic acids and against capture in the presence of a
second solution of nucleic acids. Thus, selective capture can be
achieved by sequential treatment of an array of responsive pads
with different nucleic acid solutions under controlled actuation of
the pads.
[0111] In a further embodiment, differential capture of target
nucleic acids can be achieved by differential pretreatment of an
array of responsive pads prior to contacting the array with target
nucleic acids. An exemplary pretreatment is the modification of
different responsive pads to attach adapter nucleic acids. This can
be achieved by actuating a particular subset of pads for
modification in the presence of a first solution of adapters and
against modification in the presence of a second solution of
adapters. Thus, selective modification can be achieved by
sequential treatment of an array of responsive pads with different
adapter solutions under controlled actuation of the pads. The
adapters can be, for example, nucleic acids having a first sequence
region that is complementary to capture probes on the pads and a
second sequence region that is complementary to a universal
sequence on a particular population of target nucleic acids. Thus,
in the example above, the adapters in the first solution can have a
capture probe complement sequence and a first universal sequence
complement sequence while the adapters in the second solution have
the same capture probe complement sequence and a second universal
sequence complement. Following differential treatment of the
responsive pads to form adapter-modified pads, the adapter modified
pads can be treated with a first and second plurality of target
nucleic acids having first and second universal sequences,
respectively, to thereby achieve differential capture of the target
nucleic acids.
[0112] Exemplary reagents and techniques for modifying surfaces
with adapters and using the modified surfaces to attach target
nucleic acids are set forth in U.S. Pat. App. No. 61/928,368, which
is incorporated herein by reference. Such reagents and methods can
be used in a method or composition set forth herein.
[0113] Although hybridization based capture is exemplified above,
it will be understood that capture can be mediated by other
physical and chemical interactions between nucleic acids and pads
including, but not limited to, receptor-ligand interactions,
chemical crosslinking, covalent bonds, ionic interactions, magnetic
interactions (e.g. Nucleic acids can be attached to beads that are
held to pads via magnetism), or the like.
[0114] An array of features having two different features in the
detection zone of the same pixel, and wherein the different
features have nucleic acids with different universal sequences, can
be detected by sequentially (a) hybridizing first primers to the
first universal sequence of the target nucleic acids that are
attached to a first subset of pads; (b) extending the first primers
by addition of at least one nucleotide; (c) hybridizing second
primers to the second universal sequence of the target nucleic
acids that are attached to a second subset of pads; and (d)
extending the second primers by addition of at least one
nucleotide, whereby signals are detected from the target nucleic
acids that are attached to the first subset of responsive pads at a
different time than when signals are detected from the target
nucleic acids that are attached to the second subset of responsive
pads.
[0115] Optionally, a method of the present disclosure can include a
step of amplifying first target nucleic acids that are attached to
pads of a first subset of pads using primers that are complementary
to a first universal sequence that is present on the first target
nucleic acids, and amplifying second target nucleic acids that are
attached to pads of the second subset of pads using primers that
are complementary to a second universal sequence that is present on
the second target nucleic acids. Amplification can be carried out
by solid phase amplification methods set forth herein such as
bridge amplification or solid-phase PCR. Accordingly, amplification
can be selectively achieved when the first and second universal
sequences are complementary to different amplifications
primers.
[0116] Selective amplification at different features can be
facilitated by the presence of different priming sequences at each
feature, for example as set forth above, and/or by selective
actuation of pads to attract or repel reagents used for
amplification. For example, selective actuation can be used to
selectively hybridize amplification primers to target nucleic acids
that are attached to a first subset of responsive pads compared to
target nucleic acids at other pads. A method set forth herein can
include a step of contacting a solution of first amplification
primers with a substrate while selectively actuating a first subset
of responsive pads, wherein the first amplification primers
selectively hybridize to a first plurality of different target
nucleic acids attached to the first subset of responsive pads
compared to a second plurality of different target nucleic acids
attached to a second subset of pads. Continuing with the example,
the method can optionally include a step of contacting a solution
of second amplification primers with the substrate while
selectively actuating the second subset of responsive pads in the
array, wherein the second amplification primers selectively
hybridize to the second plurality of different target nucleic acids
compared to the first plurality of different target nucleic acids.
The first amplification primers can have the same sequence as the
second amplification primers. Alternatively, the first
amplification primers can have a different sequence compared to the
second amplification primers.
[0117] Selective amplification can facilitate selective detection
when a first target nucleic acid is selectively amplified to allow
detection in the zone of a pixel followed by selective
amplification of the a second target nucleic acid that is detected
by the same pixel in the same detection zone.
[0118] In particular embodiments, a method of detecting nucleic
acids can include the steps of (a) providing a substrate comprising
an array of pads, the array of pads including a first subset of the
pads and a second subset of the pads; (b) contacting a first
solution with the substrate while selectively actuating the first
subset of responsive pads in the array, wherein the first plurality
of different target nucleic acids attach to responsive pads of the
first subset that are selectively actuated; (c) contacting a second
solution with the substrate while selectively actuating the second
subset of responsive pads in the array, wherein the second
plurality of different target nucleic acids attach to responsive
pads of the second subset that are selectively actuated; and (d)
detecting the substrate using an apparatus having an array of
pixels, wherein each pixel in the array has a detection zone that
includes (i) at least one of the target nucleic acids that is
attached to a pad of the first subset of pads, and (ii) at least
one of the target nucleic acids that is attached to a pad of the
second subset of pads. Optionally, the target nucleic acids include
a universal sequence that is the same for nucleic acids of the
first and second plurality. The universal sequence of the target
nucleic acids can, in some embodiments, hybridize to capture probes
attached to the first and second subset of pads.
[0119] In some embodiments, a method of detecting nucleic acids can
include the steps of (a) providing a substrate comprising an array
of pads, the array of pads including a first subset of the pads and
a second subset of the pads; (b) contacting a solution of adapter
nucleic acids with the substrate while selectively actuating one or
both of the subsets of responsive pads, wherein the adapter nucleic
acids attach to responsive pads of the one or both subsets that are
selectively actuated; (c) contacting a first solution with the
substrate, wherein the first plurality of different target nucleic
acids attach to responsive pads of the first subset; (d) contacting
a second solution with the substrate, wherein the second plurality
of different target nucleic acids attach to responsive pads of the
second subset, wherein the responsive pads of one or both of the
first and second subset are attached to the adapter nucleic acids;
and (e) detecting the substrate using an apparatus having an array
of pixels, wherein each pixel in the array has a detection zone
that includes (i) at least one of the target nucleic acids that is
attached to a pad of the first subset of pads, and (ii) at least
one of the target nucleic acids that is attached to a pad of the
second subset of pads.
[0120] One or more of the adapters used in a method set forth
herein can include a universal sequence complement that is in turn
useful for hybridizing to a universal sequence of one or more
target nucleic acids. Thus, an adapter can serve to mediate capture
of target nucleic acids to desired pads or features of an array set
forth herein. Taking the embodiment set forth above as an example,
the target nucleic acids in the first plurality can have a first
universal sequence and the target nucleic acids in the second
plurality can have a second universal sequence that is different
from the first universal sequence. The first target nucleic acids
can be selectively attached to the first subset of pads due to the
first universal sequences hybridizing to the first adapter nucleic
acids that are attached to the first subset of pads. Similarly, the
selective attachment of the second target nucleic acids to the
second subset of pads can involve the second universal sequences
hybridizing to the second adapter nucleic acids that are attached
to the second subset of pads, wherein the first adapter nucleic
acids have a different sequence from the second adapter nucleic
acids.
[0121] As set forth previously herein, a method of the present
disclosure can be used to determine the nucleotide sequences of a
plurality of different nucleic acids. Another useful application is
gene expression analysis. Gene expression can be detected or
quantified using RNA sequencing techniques, such as those, referred
to as digital RNA sequencing. RNA sequencing techniques can be
carried out using sequencing methodologies known in the art such as
those set forth above. Gene expression can also be detected or
quantified using hybridization techniques carried out by direct
hybridization to an array of probe features, for example, on pads
in an apparatus set forth herein. The methods set forth herein can
also be used to determine genotypes for a genomic DNA sample from
one or more individual. Exemplary assays for array-based expression
and genotyping analysis that can be modified for use in a method
set forth herein are described in U.S. Pat. Nos. 7,582,420;
6,890,741; 6,913,884 or 6,355,431 or US Pat. Pub. Nos. 2005/0053980
A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which is
incorporated herein by reference.
Example I
Fluorescence Quenching Using Direct Electrode Energy Transfer
[0122] This example shows that selectively applying an electric
field at an electrically responsive pad can quench fluorescent
labels or moieties at the pad. To achieve super resolution imaging,
a switch can be used to alternately apply electric field at two
pads that are in the detection zone of a single pixel. Application
of the field can quench fluorescence, for example, via energy
transfer between a dipole emitter (e.g. a fluorophore) and a
metallic surface. A fluorescently labeled nucleic acid molecule is
particularly useful due to the flexibility in the molecule which
allows the fluorophore to be pulled closer to the surface (by the
field) where quenching is greatest. Other flexible target analytes
or those having flexible linkers can be used similarly. Nucleic
acids or other fluorescently labeled target analytes can be
attached to a surface and surface quenching can be carried out, for
example, as described in Rant et al. Nano Lett. 4:2441-2445 (2004),
which is incorporated herein by reference.
[0123] As an alternative to direct surface attachment, a nucleic
acid or other target analyte can be attached to a gel material that
is present at an electrically responsive pad. Exemplary methods and
reactants for attaching nucleic acids to gels are described, for
example, in US Pat. App. Pub. No. 2011/0059865 A1, or U.S. patent
application Ser. No. 13/784,368, each of which is incorporated
herein by reference.
[0124] FIG. 6 shows fluorescence intensity modulation after
applying multiple cycles of +/-0.4 V on an electrically responsive
pad coated with silane free acrylamide that was grafted to P5 and
P7 primers (SFA and P5/P7 primers are described in US Pat. App.
Pub. No. 2011/0059865 A1, which is incorporated herein by
reference). The P5 and P7 primers were hybridized with HEX dye
labeled fluorescent complementary primers. The voltage was pulsed
every 2 sec for 0.5 sec and the intensity dipped when the voltage
was applied. The intensity modulations appear to be due to
fluorescence quenching of the HEX dye label at the electrode
surface. In contrast an inactive electrode did not show the
intensity fluctuations in fluorescence signal.
Example II
Fluorescence Quenching Using Energy Transfer to Electrically
Conductive Polymers
[0125] This example shows that applying an electric field at an
electrically responsive pad that contains electrically conductive
polymers in a gel can quench fluorescent labels or moieties that
are in the gel.
[0126] Transparent conductive polymers, such as
poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate)
(PEDOT:PSS), polyacetylenes or polyphenylenes, can be embedded in a
gel, such as SFA, during the polymerization process. The
gel-polymer mix can be polymerized on the responsive pads to
provide a porous electrode using conditions previously described
for polymerization of SFA (see, US Pat. App. Pub. No. 2011/0059865
A1, which is incorporated herein by reference). A target analyte
such as fluorescently labeled nucleic acid can be embedded in the
gel. For example, DNA clusters can be grown in the gel-polymer
matrix using methods set forth previously herein. Application of an
electric field can quench fluorescence, for example, via energy
transfer between the fluorophore and the electrically conductive
polymers. The energy transfer to the fluorophores can be enhanced,
compared to the surface quenching methods of Example I, due to
greater proximity between the fluorophores and the conductive
polymer. This can in turn lead to more efficient quenching in a
conductive polymer-gel compared to on a conductive surface.
[0127] To achieve super resolution imaging, a switch can be used to
alternately apply electric field at two pads that are in the
detection zone of a single pixel. Pads having a fluorescently
labeled target analytes in the presence of conductive polymer-gel
will be alternately quenched allowing the target analytes to be
distinguished by a single pixel having a detection zone that
includes the two pads.
Example III
Fluorescence Intensity Modulation with Voltage Sensitive Dyes
[0128] This example shows applying an electric field at an
electrically responsive pad that contains a target analyte (e.g. a
nucleic acid) labeled with a voltage sensitive dye. Applying an
electric field can modulate signal from the dye.
[0129] An electrically responsive pad is coated with SFA that is
grafted to P5 and P7 primers as described in Example I. The P5/P7
primers are hybridized with complementary primers labeled with
voltage sensitive dyes such as di-8-ANEPPS (Life Technologies,
Carlsbad, Calif.), or ANNINE-6plus (Fromherz et al., Eur. Biophys.
J. 37: 509-514 (2008)). Application of an electric field to the pad
results in increased fluorescence intensity at the pad. Using
voltage sensitive dyes, the fluorescence intensity of nucleic acid
features clusters can be directly modulated by applying an electric
field.
Example IV
Fluorescence Intensity Modulation with Quenchers Tethered to
Sequencing Primers
[0130] This example shows that applying an electric field at an
electrically responsive pad that contains a probe having a quencher
moiety tethered to a fluorescent moiety can quench fluorescent
signal.
[0131] The strength of quenching is dependent, at least in part, on
the intermolecular distance between the quencher and the
fluorophore to be quenched. There are multiple approaches to
electrically modulate the inter-molecule distance between a
quencher and a fluorophore that are bound to the same DNA strand.
An example is shown in FIG. 7. Here a quencher is tethered to the
5' end of a sequencing primer using a linker arm. The 3' end of the
primer is hybridized to a target sequence and a fluorophore is
attached at the 3' terminus, for example, as a result of
incorporation of a fluorescently labeled nucleotide in an SBS
reaction. A quencher moiety that is appropriately charged at
working pH, can be electrically repelled or attracted towards the
electrodes by a field of opposite polarity. This results in local
increase or decrease, respectively, for the quencher in the
vicinity of the fluorophore. To achieve super resolution imaging, a
switch can be used to alternately apply electric field at two pads
that are in the detection zone of a single pixel. Fluorophores at
the pads will be alternately quenched allowing the respective
target analytes to be distinguished by a single pixel having a
detection zone that includes the two pads.
[0132] Throughout this application various publications, patents
and patent applications have been referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0133] The term "comprising" is intended herein to be open-ended,
including not only the recited elements, but further encompassing
any additional elements.
[0134] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the invention.
Accordingly, the invention is limited only by the claims.
* * * * *