U.S. patent application number 17/218027 was filed with the patent office on 2021-12-16 for flow cell systems and devices.
The applicant listed for this patent is Element Biosciences, Inc.. Invention is credited to Sinan ARSLAN, Minghao GUO, Molly HE, Matthew KELLINGER, Huizhen MAH, Michael PREVITE, Lei SUN, Leon Zilun ZHANG, Chunhong ZHOU.
Application Number | 20210387184 17/218027 |
Document ID | / |
Family ID | 1000005997183 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210387184 |
Kind Code |
A1 |
GUO; Minghao ; et
al. |
December 16, 2021 |
FLOW CELL SYSTEMS AND DEVICES
Abstract
Flow cell devices, cartridges, and systems are described that
provide reduced manufacturing complexity, lowered consumable costs,
and flexible system throughput for nucleic acid sequencing and
other chemical or biological analysis applications. The flow cell
device can include a capillary flow cell device or a microfluidic
flow cell device.
Inventors: |
GUO; Minghao; (San Diego,
CA) ; ZHANG; Leon Zilun; (San Diego, CA) ;
ZHOU; Chunhong; (San Diego, CA) ; KELLINGER;
Matthew; (San Diego, CA) ; PREVITE; Michael;
(San Diego, CA) ; ARSLAN; Sinan; (San Diego,
CA) ; HE; Molly; (San Diego, CA) ; MAH;
Huizhen; (San Diego, CA) ; SUN; Lei; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Element Biosciences, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000005997183 |
Appl. No.: |
17/218027 |
Filed: |
March 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
63037558 |
Jun 10, 2020 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2400/0644 20130101;
B01L 3/502738 20130101; C12Q 1/6874 20130101; B01L 2400/0478
20130101; G01N 21/6428 20130101; B01L 3/502715 20130101; C12N
15/1003 20130101; C12Q 1/6806 20130101; B01L 2300/0816 20130101;
G01N 2021/6439 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12Q 1/6874 20060101 C12Q001/6874; C12N 15/10 20060101
C12N015/10; C12Q 1/6806 20060101 C12Q001/6806; G01N 21/64 20060101
G01N021/64 |
Claims
1. A method of processing a sample of a subject having or suspected
of having Coronavirus Disease 2019 (COVID-19) caused by a severe
respiratory syndrome 2 (SARS-CoV-2) virus, said method comprising:
(a) providing a nucleic acid sequencing flow cell comprising two or
more microfluidic channels, wherein at least one of said two or
more microfluidic channels comprises a surface comprising a nucleic
acid sequence immobilized thereto, wherein said nucleic acid
sequence is derived from said sample of said subject having or
suspected of having said COVID-19; (b) contacting said nucleic acid
sequence with a primer having complementarity with said nucleic
acid sequence, to yield a primed nucleic acid sequence; (c) using
at least (i) a polymerizing enzyme and (ii) plurality of
nucleobases having a plurality of detectable labels to subject said
primed nucleic acid sequence to a primer extension reaction; (d)
while performing said primer extension reaction, using a detector
to detect a plurality of signals from at least a subset of said
plurality of detectable labels; and (e) using said plurality of
signals detected in (d) to generate sequencing data; (f) using one
or more logic circuits to process said sequencing data in parallel
to identify said nucleic acid sequence; and (g) using said nucleic
acid sequence identified in (f) to determine that said sample
contains a messenger ribonucleic acid (mRNA) molecule encoding said
SARS-CoV-2.
2. The method of claim 1, wherein said plurality of detectable
labels comprises a plurality of fluorophores.
3. The method of claim 1, wherein said using said plurality of
signals detected in (d) to identify said nucleic acid sequence
comprises imaging a first surface and an axially-displaced second
surface of said two or more microfluidic channels using a
fluorescent imaging system.
4. The method of claim 1, further comprising, before (a): (h)
introducing a fluid composition comprising said nucleic acid
sequence at a concentration of between about 0.5 nanomolar (nM) and
900 nM to said nucleic acid sequencing flow cell.
5. The method of claim 1, further comprising, before (a): (i)
hybridizing at least a portion of said nucleic acid sequence to at
least a portion of oligonucleotides grafted to said surface.
6. The method of claim 4, wherein said nucleic acid sequencing flow
cell is loaded with said fluid composition prior to (c).
7. The method of claim 1, wherein said nucleic acid sequence is
present at said surface at a surface density comprising at least
4,000 molecules per square micrometer (.mu.2).
8. The method of claim 5, wherein said surface is salinized and
said oligonucleotides are covalently grafted to said surface.
9. The method of claim 1, wherein at least one of said two or more
microfluidic channels is configured to hold a fluid volume between
about 10 to about 300 microliters.
10. The method of claim 1, wherein a fluorescent image of said
surface exhibits a contrast-to-noise ratio of at least or equal to
about 5 when said fluorescent image of said surface is obtained
with an inverted fluorescence microscope and a camera under
non-signal saturating conditions while said surface is immersed in
a buffer, and said plurality of detectable labels comprise Cyanine
dye-3.
11. The method of claim 1, wherein said nucleic acid sequence has a
length comprising more than or equal to about 100 base pairs.
12. The method of claim 1, wherein said nucleic acid sequencing
flow cell comprises a flow cell cartridge that mates with a
microfluidic chip.
13. The method of claim 1, further comprising, before (a): (j)
extracting said mRNA from said sample; and (k) converting said mRNA
to complementary DNA comprising said nucleic acid sequence.
14. The method of claim 1, wherein said sample comprises epithelial
cells.
15. The method of claim 1, wherein said one or more logic circuits
comprises a field programmable gate array (FPGA).
16. The method of claim 1, wherein said sequencing data is further
analyzed using a central processing unit (CPU).
17. The method of claim 1, said parallel processing is performed is
Cloud-based computing.
18. The method of claim 1, wherein said two or more microfluidic
channels comprises four microfluidic channels.
19. The method of claim 1, further comprising amplifying said
nucleic acid sequence prior to (a) or (b) using polymerase chain
reaction (PCR).
20. The method of claim 1, wherein said performing said primer
extension reaction comprises performing a sequencing-by-synthesis
reaction.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 63/037,558, filed Jun. 10, 2020, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] Flow-cell devices are widely used in chemistry and
biotechnology applications. Particularly in next-generation
sequencing (NGS) systems, such devices are used to immobilize
template nucleic acid molecules derived from biological samples and
then introduce a repetitive flow of sequencing-by-synthesis
reagents to attach labeled nucleotides to specific positions in the
template sequences. A series of label signals are detected and
decoded to reveal the nucleotide sequences of the template
molecules, e.g., immobilized and/or amplified nucleic acid template
molecules attached to an internal surface of the flow cell.
[0003] Typical NGS flow cells are multi-layer structures fabricated
from planar surface substrates and other flow cell components (see,
for example, U.S. Patent Application Publication No. 2018/0178215
A1), which are then bonded through mechanical, chemical, or laser
bonding techniques to form fluid flow channels. Such flow cells
typically require costly multi-step, precision fabrication
techniques to achieve the required design specifications. On the
other hand, inexpensive and off-the-shelf, single lumen (flow
channel) capillaries are available in a variety of sizes and shapes
but are generally not suited for ease of handling and compatibility
with the repetitive switching between reagents that are required
for application such as NGS.
SUMMARY
[0004] Described herein are novel flow cell devices and systems for
sequencing nucleic acids. The devices and systems described herein
can achieve a more efficient use of the reagents help reduce the
cost and time of the DNA sequencing process. The devices and
systems can utilize a commercially-available, off-the-shelf
capillaries or a micro or nano scale fluidic chip with a selected
pattern of channels. The flow cell devices and systems described
herein are suitable for rapid DNA sequencing and can help achieve
more efficient use of expensive reagents and reduce the amount of
time required for sample pre-treatment and replication compared to
other DNA sequencing techniques. The result is a much faster and
cost-effective sequencing method.
[0005] Some embodiments related to methods of processing a sample
of a subject having or suspected of having Coronavirus Disease 2019
(COVID-19) caused by a severe respiratory syndrome 2 (SARS-CoV-2)
virus, said method comprising: (a) providing a nucleic acid
sequencing flow cell comprising two or more microfluidic channels,
wherein at least one of said two or more microfluidic channels
comprises a surface comprising a nucleic acid sequence immobilized
thereto, wherein said nucleic acid sequence is derived from said
sample of said subject having or suspected of having said COVID-19;
(b) contacting said nucleic acid sequence with a primer having
complementarity with said nucleic acid sequence, to yield a primed
nucleic acid sequence; (c) using at least (i) a polymerizing enzyme
and (ii) plurality of nucleobases having a plurality of detectable
labels to subject said primed nucleic acid sequence to a primer
extension reaction; (d) while performing said primer extension
reaction, using a detector to detect a plurality of signals from at
least a subset of said plurality of detectable labels; and (e)
using said plurality of signals detected in (d) to generate
sequencing data; (f) processing said sequencing data using parallel
processing using one or more logic circuits to identify said
nucleic acid sequence; (g) using said nucleic acid sequence
identified in (f) to determine that said sample contains a
messenger ribonucleic acid (mRNA) molecule encoding said
SARS-CoV-2. In some embodiments, said plurality of detectable
labels comprises a plurality of fluorophores. In some embodiments,
said using said plurality of signals detected in (d) to identify
said nucleic acid sequence comprises imaging a first surface and an
axially-displaced second surface of said two or more microfluidic
channels using a fluorescent imaging system. In some embodiments,
methods further comprising, before (a): (h) introducing a fluid
composition comprising said nucleic acid sequence at a
concentration of between about 0.5 nanomolar (nM) and 900 nm to
said nucleic acid sequencing flow cell. In some embodiments,
methods further comprise, before (a): (i) hybridizing at least a
portion of said nucleic acid sequence to at least a portion of
oligonucleotides grafted to said surface. In some embodiments, said
nucleic acid sequencing flow cell is loaded with said fluid
composition prior to (c). In some embodiments, said nucleic acid
sequences are present at said surface at a surface density
comprising at least 4,000 molecules per square micrometer
(.mu..sup.2). In some embodiments, said surface is salinized and
said capture oligonucleotides are covalently grafted to said
surface. In some embodiments, said at least one of said two or more
microfluidic channels is configured to hold a fluid volume between
about 10 to about 300 microliters. In some embodiments, a
fluorescent image of said surface exhibits a contrast-to-noise
ratio of at least or equal to about 5 when said fluorescent image
of said surface is obtained with an inverted fluorescence
microscope and a camera under non-signal saturating conditions
while said surface is immersed in a buffer, and said plurality of
detectable labels comprise Cyanine dye-3. In some embodiments, said
nucleic acid sequence has a length comprising more than or equal to
about 100 base pairs. In some embodiments, said nucleic acid
sequencing flow cell comprises a flow cell cartridge that mates
with said microfluidic chip. In some embodiments, methods further
comprise, before (a): (j) extracting said mRNA from said sample;
and (k) converting said mRNA to complementary DNA comprising said
nucleic acid sequence. In some embodiments, said sample comprises
epithelial cells. In some embodiments, said one or more logic
circuits comprises a field programmable gate array (FPGA). In some
embodiments, said sequencing data is further analyzed using a
central processing unit (CPU). In some embodiments, said parallel
processing is performed is Cloud-based computing. In some
embodiments, said two or more microfluidic channels comprises four
microfluidic channels. In some embodiments, methods further
comprise amplifying said nucleic acid sequence prior to (a) or (b)
using polymerase chain reaction (PCR). In some embodiments, said
performing said primer extension reaction comprises performing a
sequencing-by-synthesis reaction.
[0006] Some embodiments relate to a flow cell device, comprising: a
first reservoir housing a first solution and having an inlet end
and an outlet end, wherein the first agent flows from the inlet end
to the outlet end in the first reservoir; a second reservoir
housing a second solution and having an inlet end and an outlet
end, wherein the second agent flows from the inlet end to the
outlet end in the second reservoir; a central region having an
inlet end fluidically coupled to the outlet end of the first
reservoir and the outlet end of the second reservoir through at
least one valve; wherein the volume of the first solution flowing
from the outlet of the first reservoir to the inlet of the central
region is less than the volume of the second solution flowing from
the outlet of the second reservoir to the inlet of the central
region.
[0007] Some embodiments relate to a flow cell device comprising: a
framework; a plurality of reservoirs harboring reagents common to a
plurality of reactions compatible with the flow cell; a single
reservoir harboring a reaction-specific reagent; a removable
capillary having 1) a first diaphragm valve gating intake of a
plurality of nonspecific reagents from the plurality of reservoirs,
and 2) a second diaphragm valve gating intake of a single reagent
from a source reservoir in close proximity to the second diaphragm
valve.
[0008] Some embodiments relate to a flow cell device comprising: a
flame work; a plurality of reservoirs harboring reagents common to
a plurality of reactions compatible with the flow cell; a single
reservoir harboring a reaction-specific reagent; a removable or
non-removable capillary having 1) a first diaphragm valve gating
intake of a plurality of nonspecific reagents from the plurality of
reservoirs, and 2) a second diaphragm valve gating intake of a
single reagent from a source reservoir in close proximity to the
second diaphragm valve. 3) optionally, a mounting embodiment
whereby capillaries are affixed/mounted to glass substrate via an
index mounting media.
[0009] Some embodiments relate to a flow cell device comprising: a)
one or more capillaries, wherein the one or more capillaries are
replaceable; b) two or more fluidic adaptors attached to the one or
more capillaries and configured to mate with tubing that provides
fluid communication between each of the one or more capillaries and
a fluid control system that is external to the flow cell device;
and c) optionally, a cartridge configured to mate with the one or
more capillaries such that the one or more capillaries are held in
a fixed orientation relative to the cartridge, and wherein the two
or more fluidic adaptors are integrated with the cartridge,
optionally, a mounting embodiment whereby capillaries are
affixed/mounted to glass substrate via an index mounting media.
[0010] Some embodiments relate to a method of sequencing a nucleic
acid sample and a second nucleic acid sample, comprising:
delivering a plurality of oligonucleotides to an interior surface
of an at least partially transparent chamber; delivering a first
nucleic acid sample to the interior surface; delivering a plurality
of nonspecific reagents through a first channel to the interior
surface; delivering a specific reagent through a second channel to
the interior surface, wherein the second channel has a lower volume
than the first channel; visualizing a sequencing reaction on the
interior surface of the at least partially transparent chamber; and
replacing the at least partially transparent chamber prior to a
second sequencing reaction.
[0011] Some embodiments relate to a method of reducing a reagent
used in a sequencing reaction, comprising: providing a first
reagent in a first reservoir; providing a second reagent in a first
second reservoir, wherein each of the first reservoir and the
second reservoir are fluidically coupled to a central region, and
wherein the central region comprises a surface for the sequencing
reaction; and sequentially introducing the first reagent and the
second reagent into a central region of the flow cell device,
wherein the volume of the first reagent flowing from the first
reservoir to the inlet of the central region is less than the
volume of the second reagent flowing from the second reservoir to
the central region.
[0012] Some embodiments relate to a method of increasing the
efficient use of a regent in a sequencing reaction, comprising:
providing a first reagent in a first reservoir; providing a second
reagent in a first second reservoir, wherein each of the first
reservoir and the second reservoir are fluidically coupled to a
central region, and wherein the central region comprises a surface
for the sequencing reaction; and maintaining the volume of the
first reagent flowing from the first reservoir to the inlet of the
central region to be less than the volume of the second reagent
flowing from the second reservoir to the central region.
[0013] Some embodiments relate to a method wherein the first
reagent is more expensive than the second agent. Other embodiments
relate to a method wherein the first reagent is selected from the
group consisting of a polymerase, a nucleotide, and a nucleotide
analog.
[0014] Certain circumstances relate to a method of detecting a
pathogen, comprising contacting a sample with a one or more binding
moieties wherein said binding moieties are immobilized on one or
more surfaces of a flow cell, said surfaces comprising one or more
layers of a hydrophilic coating, thereby immobilizing one or more
nucleic acid components of said sample for analysis. Other
circumstances relate to a method wherein one or more nucleic acid
components comprise a viral nucleic acid.
[0015] Some embodiments relate to a method of detecting a pathogen,
comprising contacting a sample with a one or more oligonucleotides
tethered to the interior surface of a flow cell comprising one or
more layers of a hydrophilic coating, and wherein the one or more
oligonucleotides exhibit a segment that specifically hybridizes to
a viral nucleic acid segment. Further, some embodiments, relate to
a method comprising one or more nucleic acid sequencing steps,
wherein said sequencing step comprises a sequencing by binding step
or wherein said sequencing step comprises a sequencing by synthesis
step.
[0016] Certain instances relate to a method comprising a nucleic
acid amplification step, wherein said amplification step is carried
out on the surface of one or more flow cells.
[0017] Some embodiments relate to a method comprising hybridizing a
tethered oligonucleotide to a viral nucleic acid, the method
further comprising a detection step, wherein the detection step is
a fluorescence detection step.
[0018] Certain circumstances comprise contacting a nucleic acid
with a fluorescent label.
INCORPORATION BY REFERENCE
[0019] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference in their
entirety to the same extent as if each individual publication,
patent, or patent application was specifically and individually
indicated to be incorporated by reference in its entirety. In the
event of a conflict between a term herein and a term in an
incorporated reference, the term herein controls.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0021] Some novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0022] FIG. 1 illustrates one embodiment of a single capillary flow
cell having 2 fluidic adaptors.
[0023] FIG. 2 illustrates one embodiment of a flow cell cartridge
comprising a chassis, fluidic adapters, and two capillaries.
[0024] FIG. 3 illustrates one embodiment of a system comprising a
single capillary flow cell connected to various fluid flow control
components, where the single capillary is compatible with mounting
on a microscope stage or in a custom imaging instrument for use in
various imaging applications.
[0025] FIG. 4 illustrates one embodiment of a system that comprises
a capillary flow cell cartridge having integrated diaphragm valves
to minimize dead volume and conserve certain key reagents.
[0026] FIG. 5 illustrates one embodiment of a system that comprises
a capillary flow cell, a microscope setup, and a temperature
control mechanism.
[0027] FIG. 6 illustrates one non-limiting example for temperature
control of the capillary flow cells through the use of a metal
plate that is placed in contact with the flow cell cartridge.
[0028] FIG. 7 illustrates one non-limiting approach for temperature
control of the capillary flow cells that comprises a non-contact
thermal control mechanism.
[0029] FIG. 8 illustrates visualization of cluster amplification in
a capillary lumen.
[0030] FIG. 9A-9C illustrates non-limiting examples of flow cell
device preparation: FIG. 9A shows the preparation of one-piece
glass flow cell; FIG. 9B shows the preparation of two-piece glass
flow cell; and FIG. 9C shows the preparation of three-piece glass
flow cell.
[0031] FIG. 10A-10C illustrates non-limiting examples of glass flow
cell designs: FIG. 10A shows an one-piece glass flow cell design;
FIG. 10B shows a two-piece glass flow cell design; and FIG. 10C
shows a three-piece glass flow cell design.
DETAILED DESCRIPTION
[0032] Described herein are systems and devices to analyze a large
number of different nucleic acid sequences from e.g., amplified
nucleic acid arrays in flow cells or from an array of immobilized
nucleic acids. The systems and devices described herein can also be
useful in, e.g., sequencing for comparative genomics, tracking gene
expression, micro RNA sequence analysis, epigenomics, and aptamer
and phage display library characterization, and other sequencing
applications. The systems and devices herein comprise various
combinations of optical, mechanical, fluidic, thermal, electrical,
and computing devices/aspects. The advantages conferred by the
disclosed flow cell devices, cartridges, and systems include, but
are not limited to: (i) reduced device and system manufacturing
complexity and cost, (ii) significantly lower consumable costs
(e.g., as compared to those for currently available nucleic acid
sequencing systems), (iii) compatibility with typical flow cell
surface functionalization methods, (iv) flexible flow control when
combined with microfluidic components, e.g., syringe pumps and
diaphragm valves, etc., and (v) flexible system throughput.
[0033] Described herein are capillary flow-cell devices and
capillary flow cell cartridges that are constructed from
off-the-shelf, disposable, single lumen (e.g., single fluid flow
channel) capillaries that may also comprise fluidic adaptors,
cartridge chassis, one or more integrated fluid flow control
components, or any combination thereof. Also disclosed herein are
capillary flow cell-based systems that may comprise one or more
capillary flow cell devices, one or more capillary flow cell
cartridges, fluid flow controller modules, temperature control
modules, imaging modules, or any combination thereof.
[0034] The design features of some disclosed capillary flow cell
devices, cartridges, and systems include, but are not limited to,
(i) unitary flow channel construction, (ii) sealed, reliable, and
repetitive switching between reagent flows that can be implemented
with a simple load/unload mechanism such that fluidic interfaces
between the system and capillaries are reliably sealed,
facilitating capillary replacement and system reuse, and enabling
precise control of reaction conditions such as temperature and pH,
(iii) replaceable single fluid flow channel devices or capillary
flow cell cartridges comprising multiple flow channels that can be
used interchangeably to provide flexible system throughput, and
(iv) compatibility with a wide variety of detection methods such as
fluorescence imaging.
[0035] Although the disclosed single flow cell devices and systems,
capillary flow cell cartridges, capillary flow cell-based systems,
microfluidic chip flow cell device, and microfluidic chip flow cell
systems, are described primarily in the context of their use for
nucleic acid sequencing applications, various aspects of the
disclosed devices and systems may be applied not only to nucleic
acid sequencing but also to any other type of chemical analysis,
biochemical analysis, nucleic acid analysis, cell analysis, or
tissue analysis application. It shall be understood that different
aspects of the disclosed devices and systems can be appreciated
individually, collectively, or in combination with each other.
[0036] Definitions: Unless otherwise defined, all of the technical
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art in the field to which this
disclosure belongs.
[0037] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Any reference to
"or" herein is intended to encompass "and/or" unless otherwise
stated.
[0038] As used herein, the term `about` a number refers to that
number plus or minus 10% of that number. The term `about` when used
in the context of a range refers to that range minus 10% of its
lowest value and plus 10% of its greatest value.
[0039] As used herein, the phrase `at least one of` in the context
of a series encompasses lists including a single member of the
series, two members of the series, up to and including all members
of the series, alone or in some cases in combination with unlisted
components.
[0040] As used herein, fluorescence is `specific` if it arises from
fluorophores that are annealed or otherwise tethered to the
surface, such as through a nucleic acid having a region of reverse
complementarity to a corresponding segment of an oligo on the
surface and annealed to said corresponding segment. This
fluorescence is contrasted with fluorescence arising from
fluorophores not tethered to the surface through such an annealing
process, or in some cases to background florescence of the
surface.
[0041] Nucleic acids: As used herein, a "nucleic acid" (also
referred to as a "polynucleotide", "oligonucleotide", ribonucleic
acid (RNA), or deoxyribonucleic acid (DNA)) is a linear polymer of
two or more nucleotides joined by covalent internucleosidic
linkages, or variants or functional fragments thereof. In naturally
occurring examples of nucleic acids, the internucleoside linkage is
typically a phosphodiester bond. However, other examples optionally
comprise other internucleoside linkages, such as phosphorothiolate
linkages and may or may not comprise a phosphate group. Nucleic
acids include double- and single-stranded DNA, as well as double-
and single-stranded RNA, DNA/RNA hybrids, peptide-nucleic acids
(PNAs), hybrids between PNAs and DNA or RNA, and may also include
other types of nucleic acid modifications. Also, as used herein,
nucleic acids may comprise nucleic acid portions of human, animal,
or plant pathogens, such as fungi, bacteria, archaea, eukaryotic
parasites, protozoa, or viruses, including but not limited to,
filoviruses, coronaviruses, adenoviruses, retroviruses, and the
like. Exemplary viruses having nucleic acid components contemplated
in this disclosure include but are not limited to ebola virus,
Marburg virus other filoviruses, alpha coronaviruses (such as 229E
and NL63), beta coronaviruses (such as OC43 and HKU1), other
coronaviruses, such as MERS-CoV, SARS-COV, 2019-nCoV and
SARS-CoV-2), retroviruses (such as the Human Immunodeficiency Virus
and Feline Immunodeficiency Virus), Adenoviruses, Influenza Viruses
(including H1N1 and H5N1 subtypes, but contemplating all subtypes
and combinations of influenza viruses), poxviruses, herpesviruses,
and the like. Additional viral nucleic acids contemplated herein
include but are not limited to nucleic acid components of vegetable
mosaic viruses (tomato mosaic virus, tobacco mosaic virus, cucumber
mosaic virus), and viruses related to common animal diseases,
including rabies virus. Similarly contemplated as nucleic acids
within the current disclosure are viroids and subviral pathogens
such as hepatitis delta RNA, Citrus exocortis viroid, Columnea
latent viroid, Pepper chat fruit viroid, Potato spindle tuber
viroid, Tomato chlorotic dwarf viroid, coconut cadang-cadang
viroid, and Tomato apical stunt viroid, and the like.
[0042] As used herein, a "nucleotide" refers to a nucleotide,
nucleoside, or analog thereof. In some cases, the nucleotide is an
N- or C-glycoside of a purine or pyrimidine base (e.g., a
deoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleoside
containing D-ribose). Examples of other nucleotide analogs include,
but are not limited to, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, and the like.
[0043] Nucleic acids may optionally be attached to one or more
non-nucleotide moieties such as labels and other small molecules,
large molecules (such as proteins, lipids, sugars, etc.), and solid
or semi-solid supports, for example through covalent or
non-covalent linkages with either the 5' or 3' end of the nucleic
acid. Labels include any moiety that is detectable using any of a
variety of detection methods known to those of skill in the art,
and thus renders the attached oligonucleotide or nucleic acid
similarly detectable. Some labels emit electromagnetic radiation
that is optically detectable or visible. Alternately or in
combination, some labels comprise a mass tag that renders the
labeled oligonucleotide or nucleic acid visible in mass spectral
data, or a redox tag that renders the labeled oligonucleotide or
nucleic acid detectable by amperometry or voltametry. Some labels
comprise a magnetic tag that facilitates separation and/or
purification of the labeled oligonucleotide or nucleic acid. The
nucleotide or polynucleotide is often not attached to a label, and
the presence of the oligonucleotide or nucleic acid is directly
detected.
[0044] Flow Cell Devices: Disclosed herein are flow devices that
include a first reservoir housing a first solution and having an
inlet end and an outlet end, wherein the first agent flows from the
inlet end to the outlet end in the first reservoir; a second
reservoir housing a second solution and having an inlet end and an
outlet end, wherein the second agent flows from the inlet end to
the outlet end in the second reservoir; a central region having an
inlet end fluidically coupled to the outlet end of the first
reservoir and the outlet end of the second reservoir through at
least one valve. In the flow cell device, the volume of the first
solution flowing from the outlet of the first reservoir to the
inlet of the central region is less than the volume of the second
solution flowing from the outlet of the second reservoir to the
inlet of the central region.
[0045] The reservoirs described in the device can be used to house
different reagents. In some aspects, the first solution housed in
the first reservoir is different from the second solution that is
housed in the second reservoir. The second solution comprises at
least one reagent common to a plurality of reactions occurring in
the central region. In some aspects, the second solution comprises
at least one reagent selected from the list consisting of a
solvent, a polymerase, and a dNTP. In some aspects, the second
solution comprise low cost reagents. In some aspects, the first
reservoir is fluidically coupled to the central region through a
first valve and the second reservoir is fluidically coupled to the
central region through a second valve. The valve can be a diaphragm
valve or other suitable valves.
[0046] The design of the flow cell device can achieve a more
efficient use of the reaction reagents than other sequencing
device, particularly for costly reagents used in a variety of
sequencing steps. In some aspects, the first solution comprises a
reagent and the second solution comprises a reagent and the reagent
in the first solution is more expensive than the reagent in the
second solution. In some aspects, the first solution comprises a
reaction-specific reagent and the second solution comprises
nonspecific reagent common to all reaction occurring in the central
region, and wherein the reaction specific reagent is more expensive
than the nonspecific reagent. In some aspects, the first reservoir
is positioned in close proximity to the inlet of the central region
to reduce dead volume for delivery of the first solutions. In some
aspects, the first reservoir is places closer to the inlet of the
central region than the second reservoir. In some aspects, the
reaction-specific reagent is configured in close proximity to the
second diaphragm valve so as to reduce dead volume relative to
delivery of the plurality of nonspecific reagents from the
plurality of reservoirs to the first diaphragm valve.
[0047] Central Region: The central region can include a capillary
tube or microfluidic chip having one or more microfluidic channels.
In some embodiments, the capillary tube is an off-shelf product.
The capillary tube or the microfluidic chip can also be removable
from the device. In some embodiments, the capillary tube or
microfluidic channel comprises an oligonucleotide population
directed to sequence a eukaryotic genome. In some embodiments, the
capillary tube or microfluidic channel in the central region can be
removable.
[0048] Capillary flow cell devices: Disclosed herein are single
capillary flow cell devices that comprise a single capillary and
one or two fluidic adapters affixed to one or both ends of the
capillary, where the capillary provides a fluid flow channel of
specified cross-sectional area and length, and where the fluidic
adapters are configured to mate with standard tubing to provide for
convenient, interchangeable fluid connections with an external
fluid flow control system.
[0049] FIG. 1 illustrates one non-limiting example of a single
glass capillary flow cell device that comprises two fluidic
adaptors--one affixed to each end of the piece of glass
capillary--that are designed to mate with standard OD fluidic
tubing. The fluidic adaptors can be attached to the capillary using
any of a variety of techniques known to those of skill in the art
including, but not limited to, press fit, adhesive bonding, solvent
bonding, laser welding, etc., or any combination thereof.
[0050] In general, the capillary used in the disclosed flow cell
devices (and flow cell cartridges to be described below) will have
at least one internal, axially-aligned fluid flow channel (or
"lumen") that runs the full length of the capillary. In some
aspects, the capillary may have two, three, four, five, or more
than five internal, axially-aligned fluid flow channels (or
"lumen").
[0051] A number specified cross-sectional geometries for a single
capillary (or lumen thereof) are consistent with the disclosure
herein, including, but not limited to, circular, elliptical,
square, rectangular, triangular, rounded square, rounded
rectangular, or rounded triangular cross-sectional geometries. In
some aspects, the single capillary (or lumen thereof) may have any
specified cross-sectional dimension or set of dimensions. For
example, in some aspects the largest cross-sectional dimension of
the capillary lumen (e.g. the diameter if the lumen is circular in
shape or the diagonal if the lumen is square or rectangular in
shape) may range from about 10 .mu.m to about 10 mm. In some
aspects, the largest cross-sectional dimension of the capillary
lumen may be at least 10 .mu.m, at least 25 .mu.m, at least 50
.mu.m, at least 75 .mu.m, at least 100 .mu.m, at least 200 .mu.m,
at least 300 .mu.m, at least 400 .mu.m, at least 500 .mu.m, at
least 600 .mu.m, at least 700 .mu.m, at least 800 .mu.m, at least
900 .mu.m, at least 1 mm, at least 2 mm, at least 3 mm, at least 4
mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at
least 9 mm, or at least 10 mm. In some aspects, the largest
cross-sectional dimension of the capillary lumen may be at most 10
mm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm, at most
5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, at
most 900 .mu.m, at most 800 .mu.m, at most 700 .mu.m, at most 600
.mu.m, at most 500 .mu.m, at most 400 .mu.m, at most 300 .mu.m, at
most 200 .mu.m, at most 100 .mu.m, at most 75 .mu.m, at most 50
.mu.m, at most 25 .mu.m, or at most 10 .mu.m. Any of the lower and
upper values described in this paragraph may be combined to form a
range included within the present disclosure, for example, in some
aspects the largest cross-sectional dimension of the capillary
lumen may range from about 100 .mu.m to about 500 .mu.m. Those of
skill in the art will recognize that the largest cross-sectional
dimension of the capillary lumen may have any value within this
range, e.g., about 124 .mu.m.
[0052] The length of the one or more capillaries used to fabricate
the disclosed single capillary flow cell devices or flow cell
cartridges may range from about 5 mm to about 5 cm or greater. In
some instances, the length of the one or more capillaries may be
less than 5 mm, at least 5 mm, at least 1 cm, at least 1.5 cm, at
least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at
least 4 cm, at least 4.5 cm, or at least 5 cm. In some instances,
the length of the one or more capillaries may be at most 5 cm, at
most 4.5 cm, at most 4 cm, at most 3.5 cm, at most 3 cm, at most
2.5 cm, at most 2 cm, at most 1.5 cm, at most 1 cm, or at most 5
mm. Any of the lower and upper values described in this paragraph
may be combined to form a range included within the present
disclosure, for example, in some instances the length of the one or
more capillaries may range from about 1.5 cm to about 2.5 cm. Those
of skill in the art will recognize that the length of the one or
more capillaries may have any value within this range, e.g., about
1.85 cm. In some instances, devices or cartridges may comprise a
plurality of two or more capillaries that are the same length. In
some instances, devices or cartridges may comprise a plurality of
two or more capillaries that are of different lengths.
[0053] Capillaries in some cases have a gap height of about or
exactly 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350,
400, or 500 um, or any value falling within the range defined
thereby. Some preferred embodiments have gap heights of about 50
um-200 um, 50 um to 150 um, or comparable gap heights. The
capillaries used for constructing the disclosed single capillary
flow cell devices or capillary flow cell cartridges may be
fabricated from any of a variety of materials known to those of
skill in the art including, but not limited to, glass (e.g.,
borosilicate glass, soda lime glass, etc.), fused silica (quartz),
polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS),
polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene
(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic
olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene
terephthalate (PET), polydimethylsiloxane (PDMS), etc.),
polyetherimide (PEI) and perfluoroelastomer (FFKM) as more
chemically inert alternatives. PEI is somewhere between
polycarbonate and PEEK in terms of both cost and compatibility.
FFKM is also known as Kalrez or any combination thereof.
[0054] The capillaries used for constructing the disclosed single
capillary flow cell devices or capillary flow cell cartridges may
be fabricated using any of a variety of techniques known to those
of skill in the art, where the choice of fabrication technique is
often dependent on the choice of material used, and vice versa.
Examples of suitable capillary fabrication techniques include, but
are not limited to, extrusion, drawing, precision computer
numerical control (CNC) machining and boring, laser photoablation,
and the like. Devices can be pour molded or injection molded to
fabricate any three dimension structure for adapting to single
piece flow cell.
[0055] Examples of commercial vendors that provide precision
capillary tubing include Accu-Glass (St. Louis, Mo.; precision
glass capillary tubing), Polymicro Technologies (Phoenix, Ariz.;
precision glass and fused-silica capillary tubing), Friedrich &
Dimmock, Inc. (Millville, N.J.; custom precision glass capillary
tubing), and Drummond Scientific (Broomall, Pa.; OEM glass and
plastic capillary tubing).
[0056] Microfluidic chip flow cell devices: Disclosed herein also
include flow cell devices that comprise one or more microfluidic
chips and one or two fluidic adapters affixed to one or both ends
of the microfluidic chips, where the microfluidic chip provides one
or more fluid flow channels of specified cross-sectional area and
length, and where the fluidic adapters are configured to mate with
the microfluidic chip to provide for convenient, interchangeable
fluid connections with an external fluid flow control system.
[0057] A non-limiting example of a microfluidic chip flow cell
device that comprises two fluidic adaptors--one affixed to each end
of the microfluidic chip (e.g., the inlet of the microfluidic
channels). The fluidic adaptors can be attached to the chip or
channel using any of a variety of techniques known to those of
skill in the art including, but not limited to, press fit, adhesive
bonding, solvent bonding, laser welding, etc., or any combination
thereof. In some instances, the inlet and/or outlet of the
microfluidic channels on the chip are apertures on the top surface
of the chip, and the fluidic adaptors can be attached or coupled to
the inlet and outlet of the microfluidic chips.
[0058] When the central region comprises a microfluidic chip, the
chip microfluidic chip used in the disclosed flow cell deices will
have at least a single layer having one or more channels. In some
aspects, the microfluidic chip has two layers bonded together to
form one or more channels. In some aspects, the microfluidic chip
can include three layers bonded together to form one or more
channels. In some embodiments, the microfluidic channel has an open
top. In some embodiments, the microfluidic channel is positioned
between a top layer and a bottom layer.
[0059] In general, the microfluidic chip used in the disclosed flow
cell devices (and flow cell cartridges to be described below) will
have at least one internal, axially-aligned fluid flow channel (or
"lumen") that runs the full length or a partial length of the chip.
In some aspects, the microfluidic chip may have two, three, four,
five, or more than five internal, axially-aligned microfluidic
channels (or "lumen"). The microfluidic channel can be divided into
a plurality of frames.
[0060] A number specified cross-sectional geometries for a single
channels are consistent with the disclosure herein, including, but
not limited to, circular, elliptical, square, rectangular,
triangular, rounded square, rounded rectangular, or rounded
triangular cross-sectional geometries. In some aspects, the channel
may have any specified cross-sectional dimension or set of
dimensions.
[0061] The microfluidic chip used for constructing the disclosed
flow cell devices or flow cell cartridges may be fabricated from
any of a variety of materials known to those of skill in the art
including, but not limited to, glass (e.g., borosilicate glass,
soda lime glass, etc.), quartz, polymer (e.g., polystyrene (PS),
macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),
polycarbonate (PC), polypropylene (PP), polyethylene (PE), high
density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic
olefin copolymers (COC), polyethylene terephthalate (PET),
polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI) and
perfluoroelastomer (FFKM) as more chemically inert alternatives. In
some embodiments, the microfluidic chip comprises quartz. In some
embodiments, the microfluidic chip comprises borosilicate
glass.
[0062] The microfluidic chips used for constructing the described
flow cell devices or flow cell cartridges may be fabricated using
any of a variety of techniques known to those of skill in the art,
where the choice of fabrication technique is often dependent on the
choice of material used, and vice versa. The microfluidic channels
on the chip can be constructed using techniques suitable for
forming micro-structure or micro-pattern on the surface. In some
aspects, the channel is formed by laser irradiation. In some
aspects, the microfluidic channel is formed by focused femtosecond
laser radiation. In some aspects, the microfluidic channel is
formed by etching, including but not limited to chemical or laser
etching.
[0063] When the microfluidic channels are formed on the
microfluidic chip through etching, the microfluidic chip will
comprise at least one etched layer. In some aspects, the
microfluidic chip can include comprise one non-etched layer, and
one non-etched layer, with the etched layer being bonded to the
non-etched layer such that the non-etched layer forms a bottom
layer or a cover layer for the channels. In some aspects, the
microfluidic chip can include comprise one non-etched layer, and
two non-etched layers, and wherein the etched layer is positioned
between the two non-etched layers.
[0064] The chip described herein includes one or more microfluidic
channels etched on the surface of the chip. The microfluidic
channels are defined as fluid conduits with at least one minimum
dimension from <1 nm to 1000 .mu.m. The microfluidic channels
can be fabricated through several different methods, such as laser
radiation (e.g., femtosecond laser radiation), lithography,
chemical etching, and any other suitable methods Channels on the
chip surface can be created by selective patterning and plasma or
chemical etching. The channels can be open, or they can be sealed
by a conformal deposited film or layer on top to create subsurface
or buried channels in the chip. In some embodiments, the channels
are created from the removal of a sacrificial layer on the chip.
This method does not require the bulk wafer to be etched away.
Instead, the channel is located on the surface of the wafer.
Examples of direct lithography include electron beam direct-write
and focused ion beam milling.
[0065] The microfluidic channel system is coupled with an imaging
system to capture or detect signals of DNA bases. The microfluidic
channel system, fabricated on either a glass or silicon substrate,
has channel heights and widths on the order of <1 nm to 1000
.mu.m. For example, in some embodiments a channel may have a depth
of 1-50 .mu.m, 1-100 .mu.m, 1-150 .mu.m, 1-200 .mu.m, 1-250 .mu.m,
1-300 .mu.m, 50-100 .mu.m, 50-200 .mu.m, or 50-300 .mu.m, or
greater than 300 .mu.m, or a range defined by any two of these
values. In some embodiments, a channel may have a depth of 3 mm or
more. In some embodiments, a channel may have a depth of 30 mm or
more. In some embodiments, a channel may have a length of less than
0.1 mm, between 0.1 mm and 0.5 mm, between 0.1 mm and 1 mm, between
0.1 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 25
mm, between 0.1 mm and 50 mm, between 0.1 mm and 100 mm, between
0.1 mm and 150 mm, between 0.1 mm and 200 mm, between 0.1 mm and
250 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm
and 25 mm, between 1 mm and 50 mm, between 1 mm and 100 mm, between
1 mm and 150 mm, between 1 mm and 200 mm, between 1 mm and 250 mm,
between 5 mm and 10 mm, between 5 mm and 25 mm, between 5 mm and 50
mm, between 5 mm and 100 mm, between 5 mm and 150 mm, between 5 mm
and 200 mm, between 1 mm and 250 mm, or greater than 250 mm, or a
range defined by any two of these values. In some embodiments, a
channel may have a length of 2 m or more. In some embodiments, a
channel may have a length of 20 m or more. In some embodiments, a
channel may have a width of less than 0.1 mm, between 0.1 mm and
0.5 mm, between 0.1 mm and 1 mm, between 0.1 mm and 5 mm, between
0.1 mm and 10 mm, between 0.1 mm and 15 mm, between 0.1 mm and 20
mm, between 0.1 mm and 25 mm, between 0.1 mm and 30 mm, between 0.1
mm and 50 mm, or greater than 50 mm, or a range defined by any two
of these values. In some embodiments, a channel may have a width of
500 mm or more. In some embodiments, a channel may have a width of
5 m or more. The channel length can be in the micrometer range.
[0066] The one or more materials used to fabricate the capillaries
or microfluidic chips for the disclosed devices are often optically
transparent to facilitate use with spectroscopic or imaging-based
detection techniques. The entire capillary will be optically
transparent. Alternately, only a portion of the capillary (e.g., an
optically transparent "window") will be optically transparent. In
some instances, the entire microfluidic chip will be optically
transparent. In some instances, only a portion of the microfluidic
chip (e.g., an optically transparent "window") will be optically
transparent.
[0067] As noted above, the fluidic adapters that are attached to
the capillaries or microfluidic channels of the flow cell devices
and cartridges disclosed herein are designed to mate with standard
OD polymer or glass fluidic tubing or microfluidic channel. As
illustrated in FIG. 1, one end of the fluidic adapter may be
designed to mate to capillary having specific dimensions and
cross-sectional geometry, while the other end may be designed to
mate with fluidic tubing having the same or different dimensions
and cross-sectional geometry. The adapters may be fabricated using
any of a variety of suitable techniques (e.g., extrusion molding,
injection molding, compression molding, precision CNC machining,
etc.) and materials (e.g., glass, fused-silica, ceramic, metal,
polydimethylsiloxane, polystyrene (PS), macroporous polystyrene
(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC),
polypropylene (PP), polyethylene (PE), high density polyethylene
(HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers
(COC), polyethylene terephthalate (PET), etc.), where the choice of
fabrication technique is often dependent on the choice of material
used, and vice versa.
[0068] Surface coatings: An interior surface (or surface of a
capillary lumen) of one or more capillaries or the channel on the
microfluidic chip is often coated using any of a variety of surface
modification techniques or polymer coatings known to those of skill
in the art.
[0069] Examples of suitable surface modification or coating
techniques include, but are not limited to, the use of silane
chemistries (e.g., aminopropyltrimethoxysilane (APTMS),
aminopropyltriethoxysilane (APTES), triethoxysilane,
diethoxydimethylsilane, and other linear, branched, or cyclic
silanes) for covalent attachment of functional groups or molecules
to capillary lumen surfaces, covalently or non-covalently attached
polymer layers (e.g., layers of streptavidin, polyacrilamide,
polyester, dextran, poly-lysine, polyacrylamide/poly-lysine
copolymers, polyethylene glycol (PEG), poly (n-isopropylacrylamide)
(PNIPAM), poly(2-hydroxyethyl methacrylate), (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA),
polyacrylic acid (PAA), poly(vinylpyridine), poly(vinylimidazole)
and poly-lysine copolymers), or any combination thereof.
[0070] Examples of conjugation chemistries that may be used to
graft one or more layers of material (e.g. polymer layers) to the
support surface and/or to cross-link the layers to each other
include, but are not limited to, biotin-streptavidin interactions
(or variations thereof), his tag--Ni/NTA conjugation chemistries,
methoxy ether conjugation chemistries, carboxylate conjugation
chemistries, amine conjugation chemistries, NHS esters, maleimides,
thiol, epoxy, azide, hydrazide, alkyne, isocyanate, and silane
chemistries.
[0071] The number of layers of polymer or other chemical layers on
the interior or lumen surface may range from 1 to about 10 or
greater than 10. In some instances, the number of layers is at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, or at least 10. In some
instances, the number of layers may be at most 10, at most 9, at
most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at
most 2, or at most 1. Any of the lower and upper values described
in this paragraph may be combined to form a range included within
the present disclosure, for example, in some instances the number
of layers may range from about 2 to about 4. In some instances, all
of the layers may comprise the same material. In some instances,
each layer may comprise a different material. In some instances,
the plurality of layers may comprise a plurality of materials.
[0072] In a preferred aspect, one or more layers of a coating
material may be applied to the capillary lumen surface or the
interior surface of the channel on the microfluidic chip, where the
number of layers and/or the material composition of each layer is
chosen to adjust one or more surface properties of the capillary or
channel lumen, as noted in U.S. patent application Ser. No.
16/363,842.
[0073] Examples of surface properties that may be adjusted include,
but are not limited to, surface hydrophilicity/hydrophobicity,
overall coating thickness, the surface density of
chemically-reactive functional groups, the surface density of
grafted linker molecules or oligonucleotide primers, etc. In some
preferred applications, one or more surface properties of the
capillary or channel lumen are adjusted to, for example, (i)
provide for very low non-specific binding of proteins,
oligonucleotides, fluorophores, and other molecular components of
chemical or biological analysis applications, including solid-phase
nucleic acid amplification and/or sequencing applications, (ii)
provide for improved solid-phase nucleic acid hybridization
specificity and efficiency, and (iii) provide for improved
solid-phase nucleic acid amplification rate, specificity, and
efficiency.
[0074] One or more surface modification and/or polymer layers may
be applied by flowing one or more appropriate chemical coupling or
coating reagents through the capillaries or channel prior to use
for their intended application. One or more coating reagents may be
added to a buffer used, e.g., a nucleic acid hybridization,
amplification reaction, and/or sequencing reaction to provide for
dynamic coating of the capillary lumen surface.
[0075] Low non-specific binding surface: The interior surface of
the channel and capillary tube described herein can be grafted or
coated with a composition comprising low non-specific binding
surface compositions that enable improved nucleic acid
hybridization and amplification performance.
[0076] In some instances, fluorescence images of the disclosed low
non-specific binding surfaces when used in nucleic acid
hybridization or amplification applications to create clusters of
hybridized or clonally-amplified nucleic acid molecules (e.g., that
have been directly or indirectly labeled with a fluorophore)
exhibit contrast-to-noise ratios (CNRs) of at least 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 20, 210, 220, 230, 240, 250, or greater than 250.
[0077] In order to scale primer surface density and add additional
dimensionality to hydrophilic or amphoteric surfaces, substrates
comprising multi-layer coatings of PEG and other hydrophilic
polymers have been developed. By using hydrophilic and amphoteric
surface layering approaches that include, but are not limited to,
the polymer/co-polymer materials described below, it is possible to
increase primer loading density on the surface significantly.
Traditional PEG coating approaches use monolayer primer deposition,
which have been generally reported for single molecule
applications, but do not yield high copy numbers for nucleic acid
amplification applications. As described herein "layering" can be
accomplished using traditional crosslinking approaches with any
compatible polymer or monomer subunits such that a surface
comprising two or more highly crosslinked layers can be built
sequentially. Examples of suitable polymers include, but are not
limited to, streptavidin, poly acrylamide, polyester, dextran,
poly-lysine, and copolymers of poly-lysine and PEG. In some
instances, the different layers may be attached to each other
through any of a variety of conjugation reactions including, but
not limited to, biotin-streptavidin binding, azide-alkyne click
reaction, amine-NHS ester reaction, thiol-maleimide reaction, and
ionic interactions between positively charged polymer and
negatively charged polymer. In some instances, high primer density
materials may be constructed in solution and subsequently layered
onto the surface in multiple steps.
[0078] Those of skill in the art will realize that a given
hydrophilic, low-binding support surface of the present disclosure
may exhibit a water contact angle having a value of anywhere less
than 50 degrees.
[0079] The disclosed interior surface of the channel and capillary
may comprise a substrate (or support structure), one or more layers
of a covalently or non-covalently attached low-binding, chemical
modification layers, e.g., silane layers, polymer films, and one or
more covalently or non-covalently attached primer sequences that
may be used for tethering single-stranded template oligonucleotides
to the support surface. In some instances, the formulation of the
surface, e.g., the chemical composition of one or more layers, the
coupling chemistry used to cross-link the one or more layers to the
support surface and/or to each other, and the total number of
layers, may be varied such that non-specific binding of proteins,
nucleic acid molecules, and other hybridization and amplification
reaction components to the support surface is minimized or reduced
relative to a comparable monolayer. Often, the formulation of the
surface may be varied such that non-specific hybridization on the
support surface is minimized or reduced relative to a comparable
monolayer. The formulation of the surface may be varied such that
non-specific amplification on the support surface is minimized or
reduced relative to a comparable monolayer. The formulation of the
surface may be varied such that specific amplification rates and/or
yields on the support surface are maximized. Amplification levels
suitable for detection are achieved in no more than 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles
in some cases disclosed herein.
[0080] Examples of materials from which the substrate or support
structure may be fabricated include, but are not limited to, glass,
fused-silica, silicon, a polymer (e.g., polystyrene (PS),
macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),
polycarbonate (PC), polypropylene (PP), polyethylene (PE), high
density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic
olefin copolymers (COC), polyethylene terephthalate (PET)), or any
combination thereof. Various compositions of both glass and plastic
substrates are contemplated.
[0081] The substrate or support structure may be rendered in any of
a variety of geometries and dimensions known to those of skill in
the art, and may comprise any of a variety of materials known to
those of skill in the art. For example, in some instances the
substrate or support structure may be locally planar (e.g.,
comprising a microscope slide or the surface of a microscope
slide). Globally, the substrate or support structure may be
cylindrical (e.g., comprising a capillary or the interior surface
of a capillary), spherical (e.g., comprising the outer surface of a
non-porous bead), or irregular (e.g., comprising the outer surface
of an irregularly-shaped, non-porous bead or particle). In some
instances, the surface of the substrate or support structure used
for nucleic acid hybridization and amplification may be a solid,
non-porous surface. In some instances, the surface of the substrate
or support structure used for nucleic acid hybridization and
amplification may be porous, such that the coatings described
herein penetrate the porous surface, and nucleic acid hybridization
and amplification reactions performed thereon may occur within the
pores.
[0082] The substrate or support structure that comprises the one or
more chemically-modified layers, e.g., layers of a low non-specific
binding polymer, may be independent or integrated into another
structure or assembly. For example, in some instances, the
substrate or support structure may comprise one or more surfaces
within an integrated or assembled microfluidic flow cell. The
substrate or support structure may comprise one or more surfaces
within a microplate format, e.g., the bottom surface of the wells
in a microplate. As noted above, in some preferred embodiments, the
substrate or support structure comprises the interior surface (such
as the lumen surface) of a capillary. In alternate preferred
embodiments the substrate or support structure comprises the
interior surface (such as the lumen surface) of a capillary etched
into a planar chip.
[0083] The chemical modification layers may be applied uniformly
across the surface of the substrate or support structure.
Alternately, the surface of the substrate or support structure may
be non-uniformly distributed or patterned, such that the chemical
modification layers are confined to one or more discrete regions of
the substrate. For example, the substrate surface may be patterned
using photolithographic techniques to create an ordered array or
random pattern of chemically-modified regions on the surface.
Alternately or in combination, the substrate surface may be
patterned using, e.g., contact printing and/or ink jet printing
techniques. In some instances, an ordered array or random patter of
chemically-modified discrete regions may comprise at least 1, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000, or 10,000 or more discrete regions, or any intermediate
number spanned by the range herein.
[0084] In order to achieve low nonspecific binding surfaces (also
referred to herein as "low binding" or "passivated" surfaces),
hydrophilic polymers may be nonspecifically adsorbed or covalently
grafted to the substrate or support surface. Typically, passivation
is performed utilizing poly(ethylene glycol) (PEG, also known as
polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl alcohol)
(PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP),
poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
dextran, or other hydrophilic polymers with different molecular
weights and end groups that are linked to a surface using, for
example, silane chemistry. The end groups distal from the surface
can include, but are not limited to, biotin, methoxy ether,
carboxylate, amine, NHS ester, maleimide, and bis-silane. In some
instances, two or more layers of a hydrophilic polymer, e.g., a
linear polymer, branched polymer, or multi-branched polymer, may be
deposited on the surface. In some instances, two or more layers may
be covalently coupled to each other or internally cross-linked to
improve the stability of the resulting surface. In some instances,
oligonucleotide primers with different base sequences and base
modifications (or other biomolecules, e.g., enzymes or antibodies)
may be tethered to the resulting surface layer at various surface
densities. In some instances, for example, both surface functional
group density and oligonucleotide concentration may be varied to
target a certain primer density range. Additionally, primer density
can be controlled by diluting oligonucleotide with other molecules
that carry the same functional group. For example, amine-labeled
oligonucleotide can be diluted with amine-labeled polyethylene
glycol in a reaction with an NHS-ester coated surface to reduce the
final primer density. Primers with different lengths of linker
between the hybridization region and the surface attachment
functional group can also be applied to control surface density.
Example of suitable linkers include poly-T and poly-A strands at
the 5' end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g.,
3 to 20 monomer units), and carbon-chain (e.g., C6, C12, C18,
etc.). To measure the primer density, fluorescently-labeled primers
may be tethered to the surface and a fluorescence reading then
compared with that for a dye solution of known concentration.
[0085] In some embodiments, the hydrophilic polymer can be a cross
linked polymer. In some embodiments, the cross-linked polymer can
include one type of polymer cross linked with another type of
polymer. Examples of the crossed-linked polymer can include
poly(ethylene glycol) cross-linked with another polymer selected
from polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl
alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP), poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
dextran, or other hydrophilic polymers. In some embodiments, the
cross-linked polymer can be a poly(ethylene glycol) cross-linked
with polyacrylamide.
[0086] The interior surface of one or more capillaries or the
channels on the microfluidic chip or wall of the capillary can
exhibit low non-specific binding of proteins and other
amplification reaction reagents or components, and improved
stability to repetitive exposure to different solvents, changes in
temperature, chemical affronts such as low pH, or long term
storage.
[0087] The disclosed low non-specific binding supports comprising
one or more polymer coatings, e.g., PEG polymer films, that
minimize non-specific binding of protein and labeled nucleotides to
the solid support. The subsequent demonstration of improved nucleic
acid hybridization and amplification rates and specificity may be
achieved through one or more of the following additional aspects of
the present disclosure: (i) primer design (sequence and/or
modifications), (ii) control of tethered primer density on the
solid support, (iii) the surface composition of the solid support,
(iv) the surface polymer density of the solid support, (v) the use
of improved hybridization conditions before and during
amplification, and/or (vi) the use of improved amplification
formulations that decrease non-specific primer amplification or
increase template amplification efficiency.
[0088] The advantages of the disclosed low non-specific binding
supports and associated hybridization and amplification methods
confer one or more of the following additional advantages for any
sequencing system: (i) decreased fluidic wash times (due to reduced
non-specific binding, and thus faster sequencing cycle times), (ii)
decreased imaging times (and thus faster turnaround times for assay
readout and sequencing cycles), (iii) decreased overall work flow
time requirements (due to decreased cycle times), (iv) decreased
detection instrumentation costs (due to the improvements in CNR),
(v) improved readout (base-calling) accuracy (due to improvements
in CNR), (vi) improved reagent stability and decreased reagent
usage requirements (and thus reduced reagents costs), and (vii)
fewer run-time failures due to nucleic acid amplification
failures.
[0089] The low binding hydrophilic surfaces (multilayer and/or
monolayer) for surface bioassays, e.g., genotyping and sequencing
assays, are created by using any combination of the following. In
some embodiments, genotyping assays are performed. In these
embodiments, the interior surface of the flow cell device is coated
with at least one oligonucleotide or nucleic acid molecule type. In
certain instances, a plurality of each oligonucleotide molecule
type is located within at least one discrete and spatially indexed
region of the surface, where it is immobilized. Each
oligonucleotide molecule type, or probe type, may comprise a
locus-specific primer or an allele specific primer. In certain
instances, the array of oligonucleotide molecule types, each at
discrete and spatially indexed regions is contacted with a mixture
comprising different nucleic acid template types, also known as
targets, and a polymerase to form a complex. In some instances, the
nucleic acid template is associated with a fluorescent label. In
some instances, the fluorescent label is conjugated to a polymeric
core, wherein the labeled core can be associated with a nucleic
acid template type. A non-covalent bond may be used to associate
the labeled polymeric core to the nucleic acid template. After
contacting the surface, the mixture is washed away, replaced by
another solution and imaged with a fluorescence imager. When
positive matches are made between the surface immobilized locus or
allele containing probes and the complimentary nucleic acid
template, the discrete region may exhibit a fluorescence signal
above the background. In certain instances, each discrete region
exhibiting a probe-target match is identified by the emission of
fluorescence and compared to the background. In certain instances,
the contrast to noise ratio (CNR) achieves values greater than 20.
Positive matches between the discrete and spatially indexed
locations may therefore identify the presence of specific loci or
alleles present in the multiplexed sample from which the nucleic
acid template is generated.
[0090] Polar protic, polar aprotic and/or nonpolar solvents for
depositing and/or coupling linear or multi-branched hydrophilic
polymer subunits on a substrate surface. Some multi-branched
hydrophilic polymer subunits may contain functional end groups to
promote covalent coupling or non-covalent binding interactions with
other polymer subunits. Examples of suitable functional end groups
include biotin, methoxy ether, carboxylate, amine, ester compounds,
azide, alkyne, maleimide, thiol, and silane groups.
[0091] Any combination of linear, branched, or multi-branched
polymer subunits coupled through subsequent layered addition via
modified coupling chemistry/solvent/buffering systems that may
include individual subunits with orthogonal end coupling
chemistries or any of the respective combinations, such that
resultant surface is hydrophilic and exhibits low nonspecific
binding of proteins and other molecular assay components. In some
instances, the hydrophilic, functionalized substrate surfaces of
the present disclosure exhibit contact angle measurements that do
not exceed 35 degrees.
[0092] Subsequent biomolecule attachment (e.g., of proteins,
peptides, nucleic acids, oligonucleotides, or cells) on the low
binding/hydrophilic substrates via any of a variety of individual
conjugation chemistries to be described below, or any combination
thereof. Layer deposition and/or conjugation reactions may be
performed using solvent mixtures which may contain any ratio of the
following components: ethanol, methanol, acetonitrile, acetone,
DMSO, DMF, H.sub.2O, and the like. In addition, compatible
buffering systems in the desirable pH range of 5-10 may be used for
controlling the rate and efficiency of deposition and coupling,
whereby coupling rates is excess of >5.times. of those for
conventional aqueous buffer-based methods may be achieved.
[0093] The disclosed low non-specific binding supports and
associated nucleic acid hybridization and amplification methods may
be used for the analysis of nucleic acid molecules derived from any
of a variety of different cell, tissue, or sample types known to
those of skill in the art. For example, nucleic acids may be
extracted from cells, or tissue samples comprising one or more
types of cells, derived from eukaryotes (such as animals, plants,
fungi, protista), archaebacteria, or eubacteria. In some cases,
nucleic acids may be extracted from prokaryotic or eukaryotic
cells, such as adherent or non-adherent eukaryotic cells. Nucleic
acids are variously extracted from, for example, primary or
immortalized rodent, porcine, feline, canine, bovine, equine,
primate, or human cell lines. Nucleic acids may be extracted from
any of a variety of different cell, organ, or tissue types (e.g.,
white blood cells, red blood cells, platelets, epithelial cells,
endothelial cells, neurons, glial cells, astrocytes, fibroblasts,
skeletal muscle cells, smooth muscle cells, gametes, or cells from
the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus,
bladder, stomach, colon, or small intestine). Nucleic acids may be
extracted from normal or healthy cells. Alternately or in
combination, acids are extracted from diseased cells, such as
cancerous cells, or from pathogenic cells that are infecting a
host. Some nucleic acids may be extracted from a distinct subset of
cell types, e.g., immune cells (such as T cells, cytotoxic (killer)
T cells, helper T cells, alpha beta T cells, gamma delta T cells, T
cell progenitors, B cells, B-cell progenitors, lymphoid stem cells,
myeloid progenitor cells, lymphocytes, granulocytes, Natural Killer
cells, plasma cells, memory cells, neutrophils, eosinophils,
basophils, mast cells, monocytes, dendritic cells, and/or
macrophages, or any combination thereof), undifferentiated human
stem cells, human stem cells that have been induced to
differentiate, rare cells (e.g., circulating tumor cells (CTCs),
circulating epithelial cells, circulating endothelial cells,
circulating endometrial cells, bone marrow cells, progenitor cells,
foam cells, mesenchymal cells, or trophoblasts). Other cells are
contemplated and consistent with the disclosure herein.
[0094] As a result of the surface passivation techniques disclosed
herein, proteins, nucleic acids, and other biomolecules do not
"stick" to the substrates, that is, they exhibit low nonspecific
binding (NSB). Examples are shown below using standard monolayer
surface preparations with varying glass preparation conditions.
Hydrophilic surface that have been passivated to achieve ultra-low
NSB for proteins and nucleic acids require novel reaction
conditions to improve primer deposition reaction efficiencies,
hybridization performance, and induce effective amplification. All
of these processes require oligonucleotide attachment and
subsequent protein binding and delivery to a low binding surface.
As described below, the combination of a new primer surface
conjugation formulation (Cy3 oligonucleotide graft titration) and
resulting ultra-low non-specific background (NSB functional tests
performed using red and green fluorescent dyes) yielded results
that demonstrate the viability of the disclosed approaches. Some
surfaces disclosed herein exhibit a ratio of specific (e.g.,
hybridization to a tethered primer or probe) to nonspecific binding
(e.g., B.sub.inter) of a fluorophore such as Cy3 of at least 2:1,
3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,
15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1,
75:1, 100:1, or greater than 100:1, or any intermediate value
spanned by the range herein. Some surfaces disclosed herein exhibit
a ratio of specific to nonspecific fluorescence signal (e.g., for
specifically-hybridized to nonspecifically bound labeled
oligonucleotides, or for specifically-amplified to
nonspecifically-bound (B.sub.inter) or non-specifically amplified
(Bra) labeled oligonucleotides or a combination thereof
(B.sub.inter+B.sub.intra)) for a fluorophore such as Cy3 of at
least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,
13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,
40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate
value spanned by the range herein.
[0095] Grafting low non-specific binding layer: The attachment
chemistry used to graft a first chemically-modified layer to an
interior surface of the flow cell (capillary or channel) will
generally be dependent on both the material from which the support
is fabricated and the chemical nature of the layer. In some
instances, the first layer may be covalently attached to the
support surface. In some instances, the first layer may be
non-covalently attached, e.g., adsorbed to the surface through
non-covalent interactions such as electrostatic interactions,
hydrogen bonding, or van der Waals interactions between the surface
and the molecular components of the first layer. In either case,
the substrate surface may be treated prior to attachment or
deposition of the first layer. Any of a variety of surface
preparation techniques known to those of skill in the art may be
used to clean or treat the support surface. For example, glass or
silicon surfaces may be acid-washed using a Piranha solution (a
mixture of sulfuric acid (H.sub.2SO.sub.4) and hydrogen peroxide
(H.sub.2O.sub.2)) and/or cleaned using an oxygen plasma treatment
method.
[0096] Silane chemistries constitute one non-limiting approach for
covalently modifying the silanol groups on glass or silicon
surfaces to attach more reactive functional groups (e.g., amines or
carboxyl groups), which may then be used in coupling linker
molecules (e.g., linear hydrocarbon molecules of various lengths,
such as C6, C12, C18 hydrocarbons, or linear polyethylene glycol
(PEG) molecules) or layer molecules (e.g., branched PEG molecules
or other polymers) to the surface. Examples of suitable silanes
that may be used in creating any of the disclosed low binding
support surfaces include, but are not limited to, (3-Aminopropyl)
trimethoxysilane (APTMS), (3-Aminopropyl) triethoxysilane (APTES),
any of a variety of PEG-silanes (e.g., comprising molecular weights
of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silane (i.e., comprising
a free amino functional group), maleimide-PEG silane, biotin-PEG
silane, and the like.
[0097] Any of a variety of molecules known to those of skill in the
art including, but not limited to, amino acids, peptides,
nucleotides, oligonucleotides, other monomers or polymers, or
combinations thereof may be used in creating the one or more
chemically-modified layers on the support surface, where the choice
of components used may be varied to alter one or more properties of
the support surface, e.g., the surface density of functional groups
and/or tethered oligonucleotide primers, the
hydrophilicity/hydrophobicity of the support surface, or the three
three-dimensional nature (i.e., "thickness") of the support
surface. Examples of preferred polymers that may be used to create
one or more layers of low non-specific binding material in any of
the disclosed support surfaces include, but are not limited to,
polyethylene glycol (PEG) of various molecular weights and
branching structures, streptavidin, polyacrylamide, polyester,
dextran, poly-lysine, and poly-lysine copolymers, or any
combination thereof. Examples of conjugation chemistries that may
be used to graft one or more layers of material (e.g. polymer
layers) to the support surface and/or to cross-link the layers to
each other include, but are not limited to, biotin-streptavidin
interactions (or variations thereof), his tag--Ni/NTA conjugation
chemistries, methoxy ether conjugation chemistries, carboxylate
conjugation chemistries, amine conjugation chemistries, NHS esters,
maleimides, thiol, epoxy, azide, hydrazide, alkyne, isocyanate, and
silane.
[0098] One or more layers of a multi-layered surface may comprise a
branched polymer or may be linear. Examples of suitable branched
polymers include, but are not limited to, branched PEG, branched
poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine),
branched poly(vinyl pyrrolidone) (branched PVP), branched),
poly(acrylic acid) (branched PAA), branched polyacrylamide,
branched poly(N-isopropylacrylamide) (branched PNIPAM), branched
poly(methyl methacrylate) (branched PMA), branched
poly(2-hydroxylethyl methacrylate) (branced PHEMA), branched
poly(oligo(ethylene glycol) methyl ether methacrylate) (branched
POEGMA), branched polyglutamic acid (branched PGA), branched
poly-lysine, branched poly-glucoside, and dextran.
[0099] In some instances, the branched polymers used to create one
or more layers of any of the multi-layered surfaces disclosed
herein may comprise at least 4 branches, at least 5 branches, at
least 6 branches, at least 7 branches, at least 8 branches, at
least 9 branches, at least 10 branches, at least 12 branches, at
least 14 branches, at least 16 branches, at least 18 branches, at
least 20 branches, at least 22 branches, at least 24 branches, at
least 26 branches, at least 28 branches, at least 30 branches, at
least 32 branches, at least 34 branches, at least 36 branches, at
least 38 branches, or at least 40 branches. Molecules often exhibit
a `power of 2` number of branches, such as 2, 4, 8, 16, 32, 64, or
128 branches.
[0100] Exemplary PEG multilayers include PEG (8 arm, 16 arm, 8 arm)
on PEG-amine-APTES. Similar concentrations were observed for
3-layer multi-arm PEG (8 arm, 16 arm, 8 arm) and (8 arm, 64 arm, 8
arm) on PEG-amine-APTES exposed to 8 uM primer, and 3-layer
multi-arm PEG (8 arm, 8 arm, 8 arm) using star-shape PEG-amine to
replace 16 arm and 64 arm. PEG multilayers having comparable first,
second and third PEG layers are also contemplated.
[0101] Linear, branched, or multi-branched polymers used to create
one or more layers of any of the multi-layered surfaces disclosed
herein may have a molecular weight of at least 500, at least 1,000,
at least 1,500, at least 2,000, at least 2,500, at least 3,000, at
least 3,500, at least 4,000, at least 4,500, at least 5,000, at
least 7,500, at least 10,000, at least 12,500, at least 15,000, at
least 17,500, at least 20,000, at least 25,000, at least 30,000, at
least 35,000, at least 40,000, at least 45,000, or at least 50,000
Daltons. In some instances, the linear, branched, or multi-branched
polymers used to create one or more layers of any of the
multi-layered surfaces disclosed herein may have a molecular weight
of at most 50,000, at most 45,000, at most 40,000, at most 35,000,
at most 30,000, at most 25,000, at most 20,000, at most 17,500, at
most 15,000, at most 12,500, at most 10,000, at most 7,500, at most
5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000,
at most 2,500, at most 2,000, at most 1,500, at most 1,000, or at
most 500 Daltons. Any of the lower and upper values described in
this paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the molecular
weight of linear, branched, or multi-branched polymers used to
create one or more layers of any of the multi-layered surfaces
disclosed herein may range from about 1,500 to about 20,000
Daltons. Those of skill in the art will recognize that the
molecular weight of linear, branched, or multi-branched polymers
used to create one or more layers of any of the multi-layered
surfaces disclosed herein may have any value within this range,
e.g., about 1,260 Daltons.
[0102] In some instances, e.g., wherein at least one layer of a
multi-layered surface comprises a branched polymer, the number of
covalent bonds between a branched polymer molecule of the layer
being deposited and molecules of the previous layer may range from
about one covalent linkages per molecule and about 32 covalent
linkages per molecule. In some instances, the number of covalent
bonds between a branched polymer molecule of the new layer and
molecules of the previous layer may be at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 12, at least 14, at least 16,
at least 18, at least 20, at least 22, at least 24, at least 26, at
least 28, at least 30, or at least 32, or more than 32 covalent
linkages per molecule. In some instances, the number of covalent
bonds between a branched polymer molecule of the new layer and
molecules of the previous layer may be at most 32, at most 30, at
most 28, at most 26, at most 24, at most 22, at most 20, at most
18, at most 16, at most 14, at most 12, at most 10, at most 9, at
most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at
most 2, or at most 1. Any of the lower and upper values described
in this paragraph may be combined to form a range included within
the present disclosure, for example, in some instances the number
of covalent bonds between a branched polymer molecule of the new
layer and molecules of the previous layer may range from about 4 to
about 16. Those of skill in the art will recognize that the number
of covalent bonds between a branched polymer molecule of the new
layer and molecules of the previous layer may have any value within
this range, e.g., about 11 in some instances, or an average number
of about 4.6 in other instances.
[0103] Any reactive functional groups that remain following the
coupling of a material layer to the support surface may optionally
be blocked by coupling a small, inert molecule using a high yield
coupling chemistry. For example, in the case that amine coupling
chemistry is used to attach a new material layer to the previous
one, any residual amine groups may subsequently be acetylated or
deactivated by coupling with a small amino acid such as
glycine.
[0104] The number of layers of low non-specific binding material,
e.g., a hydrophilic polymer material, deposited on the surface of
the disclosed low binding supports may range from 1 to about 10. In
some instances, the number of layers is at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, or at least 10. In some instances, the number of
layers may be at most 10, at most 9, at most 8, at most 7, at most
6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of
the lower and upper values described in this paragraph may be
combined to form a range included within the present disclosure,
for example, in some instances the number of layers may range from
about 2 to about 4. In some instances, all of the layers may
comprise the same material. In some instances, each layer may
comprise a different material. In some instances, the plurality of
layers may comprise a plurality of materials. In some instances at
least one layer may comprise a branched polymer. In some instance,
all of the layers may comprise a branched polymer.
[0105] One or more layers of low non-specific binding material may
in some cases be deposited on and/or conjugated to the substrate
surface using a polar protic solvent, a polar aprotic solvent, a
nonpolar solvent, or any combination thereof. In some instances the
solvent used for layer deposition and/or coupling may comprise an
alcohol (e.g., methanol, ethanol, propanol, etc.), another organic
solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethyl
formamide (DMF), etc.), water, an aqueous buffer solution (e.g.,
phosphate buffer, phosphate buffered saline,
3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any
combination thereof. In some instances, an organic component of the
solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, or 99% of the total, or any percentage spanned or
adjacent to the range herein, with the balance made up of water or
an aqueous buffer solution. In some instances, an aqueous component
of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, or 99% of the total, or any percentage spanned or
adjacent to the range herein, with the balance made up of an
organic solvent. The pH of the solvent mixture used may be less
than 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or greater
than 10, or any value spanned or adjacent to the range described
herein.
[0106] In some instances, one or more layers of low non-specific
binding material may be deposited on and/or conjugated to the
substrate surface using a mixture of organic solvents, wherein the
dielectric constant of at least once component is less than 40 and
constitutes at least 50% of the total mixture by volume. In some
instances, the dielectric constant of the at least one component
may be less than 10, less than 20, less than 30, less than 40. In
some instances, the at least one component constitutes at least
20%, at least 30%, at least 40%, at least 50%, at least 50%, at
least 60%, at least 70%, or at least 80% of the total mixture by
volume.
[0107] As noted, the low non-specific binding supports of the
present disclosure exhibit reduced non-specific binding of
proteins, nucleic acids, and other components of the hybridization
and/or amplification formulation used for solid-phase nucleic acid
amplification. The degree of non-specific binding exhibited by a
given support surface may be assessed either qualitatively or
quantitatively. For example, in some instances, exposure of the
surface to fluorescent dyes (e.g., Cy3, Cy5, etc.),
fluorescently-labeled nucleotides, fluorescently-labeled
oligonucleotides, and/or fluorescently-labeled proteins (e.g.
polymerases) under a standardized set of conditions, followed by a
specified rinse protocol and fluorescence imaging may be used as a
qualitative tool for comparison of non-specific binding on supports
comprising different surface formulations. In some instances,
exposure of the surface to fluorescent dyes, fluorescently-labeled
nucleotides, fluorescently-labeled oligonucleotides, and/or
fluorescently-labeled proteins (e.g. polymerases) under a
standardized set of conditions, followed by a specified rinse
protocol and fluorescence imaging may be used as a quantitative
tool for comparison of non-specific binding on supports comprising
different surface formulations--provided that care has been taken
to ensure that the fluorescence imaging is performed under
conditions where fluorescence signal is linearly related (or
related in a predictable manner) to the number of fluorophores on
the support surface (e.g., under conditions where signal saturation
and/or self-quenching of the fluorophore is not an issue) and
suitable calibration standards are used. In some instances, other
techniques known to those of skill in the art, for example,
radioisotope labeling and counting methods may be used for
quantitative assessment of the degree to which non-specific binding
is exhibited by the different support surface formulations of the
present disclosure.
[0108] Some surfaces disclosed herein exhibit a ratio of specific
to nonspecific binding of a fluorophore such as Cy3 of at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 50, 75, 100, or greater than 100, or any
intermediate value spanned by the range herein. Some surfaces
disclosed herein exhibit a ratio of specific to nonspecific
fluorescence of a fluorophore such as Cy3 of at least 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 50, 75, 100, or greater than 100, or any intermediate value
spanned by the range herein.
[0109] As noted, in some instances, the degree of non-specific
binding exhibited by the disclosed low-binding supports may be
assessed using a standardized protocol for contacting the surface
with a labeled protein (e.g., bovine serum albumin (BSA),
streptavidin, a DNA polymerase, a reverse transcriptase, a
helicase, a single-stranded binding protein (SSB), etc., or any
combination thereof), a labeled nucleotide, a labeled
oligonucleotide, etc., under a standardized set of incubation and
rinse conditions, followed be detection of the amount of label
remaining on the surface and comparison of the signal resulting
therefrom to an appropriate calibration standard. In some
instances, the label may comprise a fluorescent label. In some
instances, the label may comprise a radioisotope. In some
instances, the label may comprise any other detectable label known
to one of skill in the art. In some instances, the degree of
non-specific binding exhibited by a given support surface
formulation may thus be assessed in terms of the number of
non-specifically bound protein molecules (or other molecules) per
unit area. In some instances, the low-binding supports of the
present disclosure may exhibit non-specific protein binding (or
non-specific binding of other specified molecules, e.g., Cy3 dye)
of less than 0.001 molecule per .mu.m.sup.2, less than 0.01
molecule per .mu.m.sup.2, less than 0.1 molecule per .mu.m.sup.2,
less than 0.25 molecule per .mu.m.sup.2, less than 0.5 molecule per
.mu.m.sup.2, less than 1 molecule per .mu.m.sup.2, less than 10
molecules per .mu.m.sup.2, less than 100 molecules per .mu.m.sup.2,
or less than 1,000 molecules per .mu.m.sup.2. Those of skill in the
art will realize that a given support surface of the present
disclosure may exhibit non-specific binding falling anywhere within
this range, for example, of less than 86 molecules per .mu.m.sup.2.
For example, some modified surfaces disclosed herein exhibit
nonspecific protein binding of less than 0.5 molecule/um.sup.2
following contact with a 1 uM solution of Cy3 labeled streptavidin
(GE Amersham) in phosphate buffered saline (PBS) buffer for 15
minutes, followed by 3 rinses with deionized water. Some modified
surfaces disclosed herein exhibit nonspecific binding of Cy3 dye
molecules of less than 2 molecules per um.sup.2. In independent
nonspecific binding assays, 1 uM labeled Cy3 SA (ThermoFisher), 1
uM Cy5 SA dye (ThermoFisher), 10 uM Aminoallyl-dUTP--ATTO-647N
(Jena Biosciences), 10 uM Aminoallyl-dUTP--ATTO-Rho11 (Jena
Biosciences), 10 uM Aminoallyl-dUTP--ATTO-Rho11 (Jena Biosciences),
10 uM 7-Propargylamino-7-deaza-dGTP--Cy5 (Jena Biosciences, and 10
uM 7-Propargylamino-7-deaza-dGTP--Cy3 (Jena Biosciences) were
incubated on the low binding substrates at 37.degree. C. for 15
minutes in a 384 well plate format. Each well was rinsed 2-3.times.
with 50 ul deionized RNase/DNase Free water and 2-3.times. with 25
mM ACES buffer pH 7.4. The 384 well plates were imaged on a GE
Typhoon (GE Healthcare Lifesciences, Pittsburgh, Pa.) instrument
using the Cy3, AF555, or Cy5 filter sets (according to dye test
performed) as specified by the manufacturer at a PMT gain setting
of 800 and resolution of 50-100 .mu.m. For higher resolution
imaging, images were collected on an Olympus IX83 microscope
(Olympus Corp., Center Valley, Pa.) with a total internal
reflectance fluorescence (TIRF) objective (20.times., 0.75 NA or
100.times., 1.5 NA, Olympus), an sCMOS Andor camera (Zyla 4.2.
Dichroic mirrors were purchased from Semrock (IDEX Health &
Science, LLC, Rochester, N.Y.), e.g., 405, 488, 532, or 633 nm
dichroic reflectors/beamsplitters, and band pass filters were
chosen as 532 LP or 645 LP concordant with the appropriate
excitation wavelength. Some modified surfaces disclosed herein
exhibit nonspecific binding of dye molecules of less than 0.25
molecules per .mu.m.sup.2.
[0110] In some instances, the surfaces disclosed herein exhibit a
ratio of specific to nonspecific binding of a fluorophore such as
Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100,
or any intermediate value spanned by the range herein. In some
instances, the surfaces disclosed herein exhibit a ratio of
specific to nonspecific fluorescence signals for a fluorophore such
as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than
100, or any intermediate value spanned by the range herein.
[0111] The low-background surfaces consistent with the disclosure
herein may exhibit specific dye attachment (e.g., Cy3 attachment)
to non-specific dye adsorption (e.g., Cy3 dye adsorption) ratios of
at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1,
40:1, 50:1, or more than 50 specific dye molecules attached per
molecule nonspecifically adsorbed. Similarly, when subjected to an
excitation energy, low-background surfaces consistent with the
disclosure herein to which fluorophores, e.g., Cy3, have been
attached may exhibit ratios of specific fluorescence signal (e.g.,
arising from Cy3-labeled oligonucleotides attached to the surface)
to non-specific adsorbed dye fluorescence signals of at least 3:1,
4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1,
or more than 50:1.
[0112] In some instances, the degree of hydrophilicity (or
"wettability" with aqueous solutions) of the disclosed support
surfaces may be assessed, for example, through the measurement of
water contact angles in which a small droplet of water is placed on
the surface and its angle of contact with the surface is measured
using, e.g., an optical tensiometer. In some instances, a static
contact angle may be determined. In some instances, an advancing or
receding contact angle may be determined. In some instances, the
water contact angle for the hydrophilic, low-binding support
surfaced disclosed herein may range from about 0 degrees to about
50 degrees. In some instances, the water contact angle for the
hydrophilic, low-binding support surfaced disclosed herein may no
more than 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30
degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14
degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2
degrees, or 1 degree. In many cases the contact angle is no more
than any value within this range, e.g., no more than 40 degrees.
Those of skill in the art will realize that a given hydrophilic,
low-binding support surface of the present disclosure may exhibit a
water contact angle having a value of anywhere within this range,
e.g., about 27 degrees.
[0113] In some instances, the hydrophilic surfaces disclosed herein
facilitate reduced wash times for bioassays, often due to reduced
nonspecific binding of biomolecules to the low-binding surfaces. In
some instances, adequate wash steps may be performed in less than
60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example,
in some instances adequate wash steps may be performed in less than
30 seconds.
[0114] Oligonucleotide primers and adapter sequences: In general,
at least one layer of the one or more layers of low non-specific
binding material may comprise functional groups for covalently or
non-covalently attaching oligonucleotide molecules, e.g., adapter
or primer sequences, or the at least one layer may already comprise
covalently or non-covalently attached oligonucleotide adapter or
primer sequences at the time that it is deposited on the support
surface. In some instances, the oligonucleotides tethered to the
polymer molecules of at least one third layer may be distributed at
a plurality of depths throughout the layer.
[0115] In some instances, the oligonucleotide adapter or primer
molecules are covalently coupled to the polymer in solution, i.e.,
prior to coupling or depositing the polymer on the surface. In some
instances, the oligonucleotide adapter or primer molecules are
covalently coupled to the polymer after it has been coupled to or
deposited on the surface. In some instances, at least one
hydrophilic polymer layer comprises a plurality of
covalently-attached oligonucleotide adapter or primer molecules. In
some instances, at least two, at least three, at least four, or at
least five layers of hydrophilic polymer comprise a plurality of
covalently-attached adapter or primer molecules.
[0116] In some instances, the oligonucleotide adapter or primer
molecules may be coupled to the one or more layers of hydrophilic
polymer using any of a variety of suitable conjugation chemistries
known to those of skill in the art. For example, the
oligonucleotide adapter or primer sequences may comprise moieties
that are reactive with amine groups, carboxyl groups, thiol groups,
and the like. Examples of suitable amine-reactive conjugation
chemistries that may be used include, but are not limited to,
reactions involving isothiocyanate, isocyanate, acyl azide, NHS
ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane,
carbonate, aryl halide, imidoester, carbodiimide, anhydride, and
fluorophenyl ester groups. Examples of suitable carboxyl-reactive
conjugation chemistries include, but are not limited to, reactions
involving carbodiimide compounds, e.g., water soluble EDC
(1-ethyl-3-(3-dime thylaminopropyl)carbodiimide--HCL). Examples of
suitable sulfydryl-reactive conjugation chemistries include
maleimides, haloacetyls and pyridyl disulfides.
[0117] One or more types of oligonucleotide molecules may be
attached or tethered to the support surface. In some instances, the
one or more types of oligonucleotide adapters or primers may
comprise spacer sequences, adapter sequences for hybridization to
adapter-ligated template library nucleic acid sequences, forward
amplification primers, reverse amplification primers, sequencing
primers, and/or molecular barcoding sequences, or any combination
thereof. In some instances, 1 primer or adapter sequence may be
tethered to at least one layer of the surface. In some instances,
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different
primer or adapter sequences may be tethered to at least one layer
of the surface.
[0118] In some instances, the tethered oligonucleotide adapter
and/or primer sequences may range in length from about 10
nucleotides to about 100 nucleotides. In some instances, the
tethered oligonucleotide adapter and/or primer sequences may be at
least 10, at least 20, at least 30, at least 40, at least 50, at
least 60, at least 70, at least 80, at least 90, or at least 100
nucleotides in length. In some instances, the tethered
oligonucleotide adapter and/or primer sequences may be at most 100,
at most 90, at most 80, at most 70, at most 60, at most 50, at most
40, at most 30, at most 20, or at most 10 nucleotides in length.
Any of the lower and upper values described in this paragraph may
be combined to form a range included within the present disclosure,
for example, in some instances the length of the tethered
oligonucleotide adapter and/or primer sequences may range from
about 20 nucleotides to about 80 nucleotides. Those of skill in the
art will recognize that the length of the tethered oligonucleotide
adapter and/or primer sequences may have any value within this
range, e.g., about 24 nucleotides.
[0119] In some instances, the tethered adapter or primer sequences
may comprise modifications designed to facilitate the specificity
and efficiency of nucleic acid amplification as performed on the
low-binding supports. For example, in some instances the primer may
comprise polymerase stop points such that the stretch of primer
sequence between the surface conjugation point and the modification
site is always in single-stranded form and functions as a loading
site for 5' to 3' helicases in some helicase-dependent isothermal
amplification methods. Other examples of primer modifications that
may be used to create polymerase stop points include, but are not
limited to, an insertion of a PEG chain into the backbone of the
primer between two nucleotides towards the 5' end, insertion of an
abasic nucleotide (i.e., a nucleotide that has neither a purine nor
a pyrimidine base), or a lesion site which can be bypassed by the
helicase.
[0120] As will be discussed further in the examples below, it may
be desirable to vary the surface density of tethered
oligonucleotide adapters or primers on the support surface and/or
the spacing of the tethered adapter or primers away from the
support surface (e.g., by varying the length of a linker molecule
used to tether the adapter or primers to the surface) in order to
"tune" the support for optimal performance when using a given
amplification method. As noted below, adjusting the surface density
of tethered oligonucleotide adapters or primers may impact the
level of specific and/or non-specific amplification observed on the
support in a manner that varies according to the amplification
method selected. In some instances, the surface density of tethered
oligonucleotide adapters or primers may be varied by adjusting the
ratio of molecular components used to create the support surface.
For example, in the case that an oligonucleotide primer--PEG
conjugate is used to create the final layer of a low-binding
support, the ratio of the oligonucleotide primer--PEG conjugate to
a non-conjugated PEG molecule may be varied. The resulting surface
density of tethered primer molecules may then be estimated or
measured using any of a variety of techniques known to those of
skill in the art. Examples include, but are not limited to, the use
of radioisotope labeling and counting methods, covalent coupling of
a cleavable molecule that comprises an optically-detectable tag
(e.g., a fluorescent tag) that may be cleaved from a support
surface of defined area, collected in a fixed volume of an
appropriate solvent, and then quantified by comparison of
fluorescence signals to that for a calibration solution of known
optical tag concentration, or using fluorescence imaging techniques
provided that care has been taken with the labeling reaction
conditions and image acquisition settings to ensure that the
fluorescence signals are linearly related to the number of
fluorophores on the surface (e.g., that there is no significant
self-quenching of the fluorophores on the surface).
[0121] In some instances, the resultant surface density of
oligonucleotide adapters or primers on the low binding support
surfaces of the present disclosure may range from about 100 primer
molecules per .mu.m.sup.2 to about 1,000,000 primer molecules per
.mu.m.sup.2. In some instances, the surface density of
oligonucleotide adapters or primers may be at least 100, at least
200, at least 300, at least 400, at least 500, at least 600, at
least 700, at least 800, at least 900, at least 1,000, at least
1,500, at least 2,000, at least 2,500, at least 3,000, at least
3,500, at least 4,000, at least 4,500, at least 5,000, at least
5,500, at least 6,000, at least 6,500, at least 7,000, at least
7,500, at least 8,000, at least 8,500, at least 9,000, at least
9,500, at least 10,000, at least 15,000, at least 20,000, at least
25,000, at least 30,000, at least 35,000, at least 40,000, at least
45,000, at least 50,000, at least 55,000, at least 60,000, at least
65,000, at least 70,000, at least 75,000, at least 80,000, at least
85,000, at least 90,000, at least 95,000, at least 100,000, at
least 150,000, at least 200,000, at least 250,000, at least
300,000, at least 350,000, at least 400,000, at least 450,000, at
least 500,000, at least 550,000, at least 600,000, at least
650,000, at least 700,000, at least 750,000, at least 800,000, at
least 850,000, at least 900,000, at least 950,000, or at least
1,000,000 molecules per .mu.m.sup.2. In some instances, the surface
density of oligonucleotide adapters or primers may be at most
1,000,000, at most 950,000, at most 900,000, at most 850,000, at
most 800,000, at most 750,000, at most 700,000, at most 650,000, at
most 600,000, at most 550,000, at most 500,000, at most 450,000, at
most 400,000, at most 350,000, at most 300,000, at most 250,000, at
most 200,000, at most 150,000, at most 100,000, at most 95,000, at
most 90,000, at most 85,000, at most 80,000, at most 75,000, at
most 70,000, at most 65,000, at most 60,000, at most 55,000, at
most 50,000, at most 45,000, at most 40,000, at most 35,000, at
most 30,000, at most 25,000, at most 20,000, at most 15,000, at
most 10,000, at most 9,500, at most 9,000, at most 8,500, at most
8,000, at most 7,500, at most 7,000, at most 6,500, at most 6,000,
at most 5,500, at most 5,000, at most 4,500, at most 4,000, at most
3,500, at most 3,000, at most 2,500, at most 2,000, at most 1,500,
at most 1,000, at most 900, at most 800, at most 700, at most 600,
at most 500, at most 400, at most 300, at most 200, or at most 100
molecules per .mu.m.sup.2. Any of the lower and upper values
described in this paragraph may be combined to form a range
included within the present disclosure, for example, in some
instances the surface density of adapters or primers may range from
about 10,000 molecules per .mu.m.sup.2 to about 100,000 molecules
per .mu.m.sup.2. Those of skill in the art will recognize that the
surface density of adapter or primer molecules may have any value
within this range, e.g., about 3,800 molecules per .mu.m.sup.2 in
some instances, or about 455,000 molecules per .mu.m.sup.2 in other
instances. In some instances, as will be discussed further below,
the surface density of template library nucleic acid sequences
(e.g., sample DNA molecules) initially hybridized to adapter or
primer sequences on the support surface may be less than or equal
to that indicated for the surface density of tethered
oligonucleotide primers. In some instances, as will also be
discussed further below, the surface density of clonally-amplified
template library nucleic acid sequences hybridized to adapter or
primer sequences on the support surface may span the same range or
a different range as that indicated for the surface density of
tethered oligonucleotide adapters or primers.
[0122] Local surface densities of adapter or primer molecules as
listed above do not preclude variation in density across a surface,
such that a surface may comprise a region having an oligo density
of, for example, 500,000/um.sup.2, while also comprising at least a
second region having a substantially different local density.
[0123] Hybridization of nucleic acid molecules to low-binding
supports: In some aspects of the present disclosure, hybridization
buffer formulations are described which, in combination with the
disclosed low-binding supports, provide for improved hybridization
rates, hybridization specificity (or stringency), and hybridization
efficiency (or yield). As used herein, hybridization specificity is
a measure of the ability of tethered adapter sequences, primer
sequences, or oligonucleotide sequences in general to correctly
hybridize only to completely complementary sequences, while
hybridization efficiency is a measure of the percentage of total
available tethered adapter sequences, primer sequences, or
oligonucleotide sequences in general that are hybridized to
complementary sequences.
[0124] Improved hybridization specificity and/or efficiency may be
achieved through optimization of the hybridization buffer
formulation used with the disclosed low-binding surfaces, and will
be discussed in more detail in the examples below. Examples of
hybridization buffer components that may be adjusted to achieve
improved performance include, but are not limited to, buffer type,
organic solvent mixtures, buffer pH, buffer viscosity, detergents
and zwitterionic components, ionic strength (including adjustment
of both monovalent and divalent ion concentrations), antioxidants
and reducing agents, carbohydrates, BSA, polyethylene glycol,
dextran sulfate, betaine, other additives, and the like.
[0125] By way of non-limiting example, suitable buffers for use in
formulating a hybridization buffer may include, but are not limited
to, phosphate buffered saline (PBS), succinate, citrate, histidine,
acetate, Tris, TAPS, MOPS, PIPES, HEPES, MES, and the like. The
choice of appropriate buffer will generally be dependent on the
target pH of the hybridization buffer solution. In general, the
desired pH of the buffer solution will range from about pH 4 to
about pH 8.4. In some embodiments, the buffer pH may be at least
4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at
least 6.2, at least 6.4, at least 6.6, at least 6.8, at least 7.0,
at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least
8.0, at least 8.2, or at least 8.4. In some embodiments, the buffer
pH may be at most 8.4, at most 8.2, at most 8.0, at most 7.8, at
most 7.6, at most 7.4, at most 7.2, at most 7.0, at most 6.8, at
most 6.6, at most 6.4, at most 6.2, at most 6.0, at most 5.5, at
most 5.0, at most 4.5, or at most 4.0. Any of the lower and upper
values described in this paragraph may be combined to form a range
included within the present disclosure, for example, in some
instances, the desired pH may range from about 6.4 to about 7.2.
Those of skill in the art will recognize that the buffer pH may
have any value within this range, for example, about 7.25.
[0126] Suitable detergents for use in hybridization buffer
formulation include, but are not limited to, zitterionic detergents
(e.g., 1-Dodecanoyl-sn-glycero-3-phosphocholine,
3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,
3-(N,N-Dimethylmyristylammonio)propanesulfonate,
3-(N,NDimethylmyristylammonio) propanesulfonate, ASB-C80, C7BzO,
CHAPS, CHAPS hydrate, CHAPSO, DDMAB, Dimethylethylammoniumpropane
sulfonate, N,N-Dimethyldodecylamine Noxide,
N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, or
N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and anionic,
cationic, and non-ionic detergents. Examples of nonionic detergents
include poly(oxyethylene) ethers and related polymers (e.g.
Brij.RTM., TWEEN.RTM., TRITON.RTM., TRITON X-100 and IGEPAL.RTM.
CA-630), bile salts, and glycosidic detergents.
[0127] The use of the disclosed low-binding supports either alone
or in combination with optimized buffer formulations may yield
relative hybridization rates that range from about 2.times. to
about 20.times. faster than that for a conventional hybridization
protocol. In some instances, the relative hybridization rate may be
at least 2.times., at least 3.times., at least 4.times., at least
5.times., at least 6.times., at least 7.times., at least 8.times.,
at least 9.times., at least 10.times., at least 12.times., at least
14.times., at least 16.times., at least 18.times., at least
20.times., at least 25.times., at least 30.times., or at least
40.times. that for a conventional hybridization protocol.
[0128] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized buffer formulations
may yield total hybridization reaction times (i.e., the time
required to reach 90%, 95%, 98%, or 99% completion of the
hybridization reaction) of less than 60 minutes, 50 minutes, 40
minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5
minutes for any of these completion metrics.
[0129] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized buffer formulations
may yield improved hybridization specificity compared to that for a
conventional hybridization protocol. In some instances, the
hybridization specificity that may be achieved is better than 1
base mismatch in 10 hybridization events, 1 base mismatch in 20
hybridization events, 1 base mismatch in 30 hybridization events, 1
base mismatch in 40 hybridization events, 1 base mismatch in 50
hybridization events, 1 base mismatch in 75 hybridization events, 1
base mismatch in 100 hybridization events, 1 base mismatch in 200
hybridization events, 1 base mismatch in 300 hybridization events,
1 base mismatch in 400 hybridization events, 1 base mismatch in 500
hybridization events, 1 base mismatch in 600 hybridization events,
1 base mismatch in 700 hybridization events, 1 base mismatch in 800
hybridization events, 1 base mismatch in 900 hybridization events,
1 base mismatch in 1,000 hybridization events, 1 base mismatch in
2,000 hybridization events, 1 base mismatch in 3,000 hybridization
events, 1 base mismatch in 4,000 hybridization events, 1 base
mismatch in 5,000 hybridization events, 1 base mismatch in 6,000
hybridization events, 1 base mismatch in 7,000 hybridization
events, 1 base mismatch in 8,000 hybridization events, 1 base
mismatch in 9,000 hybridization events, or 1 base mismatch in
10,000 hybridization events.
[0130] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized buffer formulations
may yield improved hybridization efficiency (e.g., the fraction of
available oligonucleotide primers on the support surface that are
successfully hybridized with target oligonucleotide sequences)
compared to that for a conventional hybridization protocol. In some
instances, the hybridization efficiency that may be achieved is
better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% for any
of the input target oligonucleotide concentrations specified below
and in any of the hybridization reaction times specified above. In
some instances, e.g., wherein the hybridization efficiency is less
than 100%, the resulting surface density of target nucleic acid
sequences hybridized to the support surface may be less than the
surface density of oligonucleotide adapter or primer sequences on
the surface.
[0131] In some instances, use of the disclosed low-binding supports
for nucleic acid hybridization (or amplification) applications
using conventional hybridization (or amplification) protocols, or
optimized hybridization (or amplification) protocols may lead to a
reduced requirement for the input concentration of target (or
sample) nucleic acid molecules contacted with the support surface.
For example, in some instances, the target (or sample) nucleic acid
molecules may be contacted with the support surface at a
concentration ranging from about 10 pM to about 1 .mu.M (i.e.,
prior to annealing or amplification). In some instances, the target
(or sample) nucleic acid molecules may be administered at a
concentration of at least 10 pM, at least 20 pM, at least 30 pM, at
least 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at
least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at
least 700 pM, at least 800 pM, at least 900 pM, at least 1 nM, at
least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at
least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at
least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at
least 400 nM, at least 500 nM, at least 600 nM, at leasy 700 nM, at
least 800 nM, at least 900 nM, or at least 1 .mu.M. In some
instances, the target (or sample) nucleic acid molecules may be
administered at a concentration of at most 1 .mu.M, at most 900 nM,
at most 800 nm, at most 700 nM, at most 600 nM, at most 500 nM, at
most 400 nM, at most 300 nM, at most 200 nM, at most 100 nM, at
most 90 nM, at most 80 nM, at most 70 nM, at most 60 nM, at most 50
nM, at most 40 nM, at most 30 nM, at most 20 nM, at most 10 nM, at
most 1 nM, at most 900 pM, at most 800 pM, at most 700 pM, at most
600 pM, at most 500 pM, at most 400 pM, at most 300 pM, at most 200
pM, at most 100 pM, at most 90 pM, at most 80 pM, at most 70 pM, at
most 60 pM, at most 50 pM, at most 40 pM, at most 30 pM, at most 20
pM, or at most 10 pM. Any of the lower and upper values described
in this paragraph may be combined to form a range included within
the present disclosure, for example, in some instances the target
(or sample) nucleic acid molecules may be administered at a
concentration ranging from about 90 pM to about 200 nM. Those of
skill in the art will recognize that the target (or sample) nucleic
acid molecules may be administered at a concentration having any
value within this range, e.g., about 855 nM.
[0132] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized hybridization
buffer formulations may result in a surface density of hybridized
target (or sample) oligonucleotide molecules (i.e., prior to
performing any subsequent solid-phase or clonal amplification
reaction) ranging from about from about 0.0001 target
oligonucleotide molecules per .mu.m.sup.2 to about 1,000,000 target
oligonucleotide molecules per .mu.m.sup.2. In some instances, the
surface density of hybridized target oligonucleotide molecules may
be at least 0.0001, at least 0.0005, at least 0.001, at least
0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at
least 1, at least 5, at least 10, at least 20, at least 30, at
least 40, at least 50, at least 60, at least 70, at least 80, at
least 90, at least 100, at least 200, at least 300, at least 400,
at least 500, at least 600, at least 700, at least 800, at least
900, at least 1,000, at least 1,500, at least 2,000, at least
2,500, at least 3,000, at least 3,500, at least 4,000, at least
4,500, at least 5,000, at least 5,500, at least 6,000, at least
6,500, at least 7,000, at least 7,500, at least 8,000, at least
8,500, at least 9,000, at least 9,500, at least 10,000, at least
15,000, at least 20,000, at least 25,000, at least 30,000, at least
35,000, at least 40,000, at least 45,000, at least 50,000, at least
55,000, at least 60,000, at least 65,000, at least 70,000, at least
75,000, at least 80,000, at least 85,000, at least 90,000, at least
95,000, at least 100,000, at least 150,000, at least 200,000, at
least 250,000, at least 300,000, at least 350,000, at least
400,000, at least 450,000, at least 500,000, at least 550,000, at
least 600,000, at least 650,000, at least 700,000, at least
750,000, at least 800,000, at least 850,000, at least 900,000, at
least 950,000, or at least 1,000,000 molecules per .mu.m.sup.2. In
some instances, the surface density of hybridized target
oligonucleotide molecules may be at most 1,000,000, at most
950,000, at most 900,000, at most 850,000, at most 800,000, at most
750,000, at most 700,000, at most 650,000, at most 600,000, at most
550,000, at most 500,000, at most 450,000, at most 400,000, at most
350,000, at most 300,000, at most 250,000, at most 200,000, at most
150,000, at most 100,000, at most 95,000, at most 90,000, at most
85,000, at most 80,000, at most 75,000, at most 70,000, at most
65,000, at most 60,000, at most 55,000, at most 50,000, at most
45,000, at most 40,000, at most 35,000, at most 30,000, at most
25,000, at most 20,000, at most 15,000, at most 10,000, at most
9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500,
at most 7,000, at most 6,500, at most 6,000, at most 5,500, at most
5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000,
at most 2,500, at most 2,000, at most 1,500, at most 1,000, at most
900, at most 800, at most 700, at most 600, at most 500, at most
400, at most 300, at most 200, at most 100, at most 90, at most 80,
at most 70, at most 60, at most 50, at most 40, at most 30, at most
20, at most 10, at most 5, at most 1, at most 0.5, at most 0.1, at
most 0.05, at most 0.01, at most 0.005, at most 0.001, at most
0.0005, or at most 0.0001 molecules per .mu.m.sup.2. Any of the
lower and upper values described in this paragraph may be combined
to form a range included within the present disclosure, for
example, in some instances the surface density of hybridized target
oligonucleotide molecules may range from about 3,000 molecules per
.mu.m.sup.2 to about 20,000 molecules per .mu.m.sup.2. Those of
skill in the art will recognize that the surface density of
hybridized target oligonucleotide molecules may have any value
within this range, e.g., about 2,700 molecules per .mu.m.sup.2.
[0133] Stated differently, in some instances the use of the
disclosed low-binding supports alone or in combination with
optimized hybridization buffer formulations may result in a surface
density of hybridized target (or sample) oligonucleotide molecules
(i.e., prior to performing any subsequent solid-phase or clonal
amplification reaction) ranging from about 100 hybridized target
oligonucleotide molecules per mm.sup.2 to about 1.times.10.sup.7
oligonucleotide molecules per mm.sup.2 or from about 100 hybridized
target oligonucleotide molecules per mm.sup.2 to about
1.times.10.sup.12 hybridized target oligonucleotide molecules per
mm.sup.2. In some instances, the surface density of hybridized
target oligonucleotide molecules may be at least 100, at least 500,
at least 1,000, at least 4,000, at least 5,000, at least 6,000, at
least 10,000, at least 15,000, at least 20,000, at least 25,000, at
least 30,000, at least 35,000, at least 40,000, at least 45,000, at
least 50,000, at least 55,000, at least 60,000, at least 65,000, at
least 70,000, at least 75,000, at least 80,000, at least 85,000, at
least 90,000, at least 95,000, at least 100,000, at least 150,000,
at least 200,000, at least 250,000, at least 300,000, at least
350,000, at least 400,000, at least 450,000, at least 500,000, at
least 550,000, at least 600,000, at least 650,000, at least
700,000, at least 750,000, at least 800,000, at least 850,000, at
least 900,000, at least 950,000, at least 1,000,000, at least
5,000,000, at least 1.times.10.sup.7, at least 5.times.10.sup.7, at
least 1.times.10.sup.8, at least 5.times.10.sup.8, at least
1.times.10.sup.9, at least 5.times.10.sup.9, at least
1.times.10.sup.10, at least 5.times.10.sup.10, at least
1.times.10.sup.11, at least 5.times.10.sup.11, or at least
1.times.10.sup.12 molecules per mm.sup.2. In some instances, the
surface density of hybridized target oligonucleotide molecules may
be at most 1.times.10.sup.12, at most 5.times.10.sup.11, at most
1.times.10.sup.11, at most 5.times.10.sup.10, at most
1.times.10.sup.10, at most 5.times.10.sup.9, at most
1.times.10.sup.9, at most 5.times.10.sup.8, at most
1.times.10.sup.8, at most 5.times.10.sup.7, at most
1.times.10.sup.7, at most 5,000,000, at most 1,000,000, at most
950,000, at most 900,000, at most 850,000, at most 800,000, at most
750,000, at most 700,000, at most 650,000, at most 600,000, at most
550,000, at most 500,000, at most 450,000, at most 400,000, at most
350,000, at most 300,000, at most 250,000, at most 200,000, at most
150,000, at most 100,000, at most 95,000, at most 90,000, at most
85,000, at most 80,000, at most 75,000, at most 70,000, at most
65,000, at most 60,000, at most 55,000, at most 50,000, at most
45,000, at most 40,000, at most 35,000, at most 30,000, at most
25,000, at most 20,000, at most 15,000, at most 10,000, at most
5,000, at most 1,000, at most 500, or at most 100 molecules per
mm.sup.2. Any of the lower and upper values described in this
paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of hybridized target oligonucleotide molecules may range
from about 5,000 molecules per mm.sup.2 to about 50,000 molecules
per mm.sup.2. Those of skill in the art will recognize that the
surface density of hybridized target oligonucleotide molecules may
have any value within this range, e.g., about 50,700 molecules per
mm.sup.2.
[0134] In some instances, the target (or sample) oligonucleotide
molecules (or nucleic acid molecules) hybridized to the
oligonucleotide adapter or primer molecules attached to the
low-binding support surface may range in length from about 0.02
kilobases (kb) to about 20 kb or from about 0.1 kilobases (kb) to
about 20 kb. In some instances, the target oligonucleotide
molecules may be at least 0.001 kb, at least 0.005 kb, at least
0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb in
length, at least 0.2 kb in length, at least 0.3 kb in length, at
least 0.4 kb in length, at least 0.5 kb in length, at least 0.6 kb
in length, at least 0.7 kb in length, at least 0.8 kb in length, at
least 0.9 kb in length, at least 1 kb in length, at least 2 kb in
length, at least 3 kb in length, at least 4 kb in length, at least
5 kb in length, at least 6 kb in length, at least 7 kb in length,
at least 8 kb in length, at least 9 kb in length, at least 10 kb in
length, at least 15 kb in length, at least 20 kb in length, at
least 30 kb in length, or at least 40 kb in length, or any
intermediate value spanned by the range described herein, e.g., at
least 0.85 kb in length.
[0135] In some instances, the target (or sample) oligonucleotide
molecules (or nucleic acid molecules) may comprise single-stranded
or double-stranded, multimeric nucleic acid molecules further
comprising repeats of a regularly occurring monomer unit. In some
instances, the single-stranded or double-stranded, multimeric
nucleic acid molecules may be at least 0.001 kb, at least 0.005 kb,
at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1
kb in length, at least 0.2 kb in length, at least 0.3 kb in length,
at least 0.4 kb in length, at least 0.5 kb in length, at least 1 kb
in length, at least 2 kb in length, at least 3 kb in length, at
least 4 kb in length, at least 5 kb in length, at least 6 kb in
length, at least 7 kb in length, at least 8 kb in length, at least
9 kb in length, at least 10 kb in length, at least 15 kb in length,
or at least 20 kb in length, at least 30 kb in length, or at least
40 kb in length, or any intermediate value spanned by the range
described herein, e.g., about 2.45 kb in length.
[0136] In some instances, the target (or sample) oligonucleotide
molecules (or nucleic acid molecules) may comprise single-stranded
or double-stranded multimeric nucleic acid molecules comprising
from about 2 to about 100 copies of a regularly repeating monomer
unit. In some instances, the number of copies of the regularly
repeating monomer unit may be at least 2, at least 3, at least 4,
at least 5, at least 10, at least 15, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least 50, at
least 55, at least 60, at least 65, at least 70, at least 75, at
least 80, at least 85, at least 90, at least 95, and at least 100.
In some instances, the number of copies of the regularly repeating
monomer unit may be at most 100, at most 95, at most 90, at most
85, at most 80, at most 75, at most 70, at most 65, at most 60, at
most 55, at most 50, at most 45, at most 40, at most 35, at most
30, at most 25, at most 20, at most 15, at most 10, at most 5, at
most 4, at most 3, or at most 2. Any of the lower and upper values
described in this paragraph may be combined to form a range
included within the present disclosure, for example, in some
instances the number of copies of the regularly repeating monomer
unit may range from about 4 to about 60. Those of skill in the art
will recognize that the number of copies of the regularly repeating
monomer unit may have any value within this range, e.g., about 17.
Thus, in some instances, the surface density of hybridized target
sequences in terms of the number of copies of a target sequence per
unit area of the support surface may exceed the surface density of
oligonucleotide primers even if the hybridization efficiency is
less than 100%.
[0137] Nucleic acid surface amplification (NASA): As used herein,
the phrase "nucleic acid surface amplification" (NASA) is used
interchangeably with the phrase "solid-phase nucleic acid
amplification" (or simply "solid-phase amplification"). In some
aspects of the present disclosure, nucleic acid amplification
formulations are described which, in combination with the disclosed
low-binding supports, provide for improved amplification rates,
amplification specificity, and amplification efficiency. As used
herein, specific amplification refers to amplification of template
library oligonucleotide strands that have been tethered to the
solid support either covalently or non-covalently. As used herein,
non-specific amplification refers to amplification of primer-dimers
or other non-template nucleic acids. As used herein, amplification
efficiency is a measure of the percentage of tethered
oligonucleotides on the support surface that are successfully
amplified during a given amplification cycle or amplification
reaction. Nucleic acid amplification performed on surfaces
disclosed herein may obtain amplification efficiencies of at least
50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%, such as 98% or
99%.
[0138] Any of a variety of thermal cycling or isothermal nucleic
acid amplification schemes may be used with the disclosed
low-binding supports. Examples of nucleic acid amplification
methods that may be utilized with the disclosed low-binding
supports include, but are not limited to, polymerase chain reaction
(PCR), multiple displacement amplification (MDA),
transcription-mediated amplification (TMA), nucleic acid
sequence-based amplification (NASBA), strand displacement
amplification (SDA), real-time SDA, bridge amplification,
isothermal bridge amplification, rolling circle amplification,
circle-to-circle amplification, helicase-dependent amplification,
recombinase-dependent amplification, or single-stranded binding
(SSB) protein-dependent amplification.
[0139] Often, improvements in amplification rate, amplification
specificity, and amplification efficiency may be achieved using the
disclosed low-binding supports alone or in combination with
formulations of the amplification reaction components. In addition
to inclusion of nucleotides, one or more polymerases, helicases,
single-stranded binding proteins, etc. (or any combination
thereof), the amplification reaction mixture may be adjusted in a
variety of ways to achieve improved performance including, but are
not limited to, choice of buffer type, buffer pH, organic solvent
mixtures, buffer viscosity, detergents and zwitterionic components,
ionic strength (including adjustment of both monovalent and
divalent ion concentrations), antioxidants and reducing agents,
carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine,
other additives, and the like.
[0140] The use of the disclosed low-binding supports alone or in
combination with optimized amplification reaction formulations may
yield increased amplification rates compared to those obtained
using conventional supports and amplification protocols. In some
instances, the relative amplification rates that may be achieved
may be at least 2.times., at least 3.times., at least 4.times., at
least 5.times., at least 6.times., at least 7.times., at least
8.times., at least 9.times., at least 10.times., at least
12.times., at least 14.times., at least 16.times., at least
18.times., or at least 20.times. that for use of conventional
supports and amplification protocols for any of the amplification
methods described above.
[0141] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized buffer formulations
may yield total amplification reaction times (i.e., the time
required to reach 90%, 95%, 98%, or 99% completion of the
amplification reaction) of less than 180 mins, 120 mins, 90 min, 60
minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15
minutes, 10 minutes, 5 minutes, 3 minutes, 1 minute, 50 s, 40 s, 30
s, 20 s, or 10 s for any of these completion metrics.
[0142] Some low-binding support surfaces disclosed herein exhibit a
ratio of specific binding to nonspecific binding of a fluorophore
such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,
10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,
25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1,
or any intermediate value spanned by the range herein. Some
surfaces disclosed herein exhibit a ratio of specific to
nonspecific fluorescence signal for a fluorophore such as Cy3 of at
least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,
13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,
40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate
value spanned by the range herein.
[0143] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
buffer formulations may enable faster amplification reaction times
(i.e., the times required to reach 90%, 95%, 98%, or 99% completion
of the amplification reaction) of no more than 60 minutes, 50
minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes.
Similarly, use of the disclosed low-binding supports alone or in
combination with optimized buffer formulations may enable
amplification reactions to be completed in some cases in no more
than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or no more than 30 cycles.
[0144] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
reaction formulations may yield increased specific amplification
and/or decreased non-specific amplification compared to that
obtained using conventional supports and amplification protocols.
In some instances, the resulting ratio of specific
amplification-to-non-specific amplification that may be achieved is
at least 4:1 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1,
60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1,
700:1, 800:1, 900:1, or 1,000:1.
[0145] In some instances, the use of the low-binding supports alone
or in combination with optimized amplification reaction
formulations may yield increased amplification efficiency compared
to that obtained using conventional supports and amplification
protocols. In some instances, the amplification efficiency that may
be achieved is better than 50%, 60%, 70% 80%, 85%, 90%, 95%, 98%,
or 99% in any of the amplification reaction times specified
above.
[0146] In some instances, the clonally-amplified target (or sample)
oligonucleotide molecules (or nucleic acid molecules) hybridized to
the oligonucleotide adapter or primer molecules attached to the
low-binding support surface may range in length from about 0.02
kilobases (kb) to about 20 kb or from about 0.1 kilobases (kb) to
about 20 kb. In some instances, the clonally-amplified target
oligonucleotide molecules may be at least 0.001 kb, at least 0.005
kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least
0.1 kb in length, at least 0.2 kb in length, at least 0.3 kb in
length, at least 0.4 kb in length, at least 0.5 kb in length, at
least 1 kb in length, at least 2 kb in length, at least 3 kb in
length, at least 4 kb in length, at least 5 kb in length, at least
6 kb in length, at least 7 kb in length, at least 8 kb in length,
at least 9 kb in length, at least 10 kb in length, at least 15 kb
in length, or at least 20 kb in length, or any intermediate value
spanned by the range described herein, e.g., at least 0.85 kb in
length.
[0147] In some instances, the clonally-amplified target (or sample)
oligonucleotide molecules (or nucleic acid molecules) may comprise
single-stranded or double-stranded, multimeric nucleic acid
molecules further comprising repeats of a regularly occurring
monomer unit. In some instances, the clonally-amplified
single-stranded or double-stranded, multimeric nucleic acid
molecules may be at least 0.1 kb in length, at least 0.2 kb in
length, at least 0.3 kb in length, at least 0.4 kb in length, at
least 0.5 kb in length, at least 1 kb in length, at least 2 kb in
length, at least 3 kb in length, at least 4 kb in length, at least
5 kb in length, at least 6 kb in length, at least 7 kb in length,
at least 8 kb in length, at least 9 kb in length, at least 10 kb in
length, at least 15 kb in length, or at least 20 kb in length, or
any intermediate value spanned by the range described herein, e.g.,
about 2.45 kb in length.
[0148] In some instances, the clonally-amplified target (or sample)
oligonucleotide molecules (or nucleic acid molecules) may comprise
single-stranded or double-stranded multimeric nucleic acid
molecules comprising from about 2 to about 100 copies of a
regularly repeating monomer unit. In some instances, the number of
copies of the regularly repeating monomer unit may be at least 2,
at least 3, at least 4, at least 5, at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, at least 55, at least 60, at least 65, at
least 70, at least 75, at least 80, at least 85, at least 90, at
least 95, and at least 100. In some instances, the number of copies
of the regularly repeating monomer unit may be at most 100, at most
95, at most 90, at most 85, at most 80, at most 75, at most 70, at
most 65, at most 60, at most 55, at most 50, at most 45, at most
40, at most 35, at most 30, at most 25, at most 20, at most 15, at
most 10, at most 5, at most 4, at most 3, or at most 2. Any of the
lower and upper values described in this paragraph may be combined
to form a range included within the present disclosure, for
example, in some instances the number of copies of the regularly
repeating monomer unit may range from about 4 to about 60. Those of
skill in the art will recognize that the number of copies of the
regularly repeating monomer unit may have any value within this
range, e.g., about 12. Thus, in some instances, the surface density
of clonally-amplified target sequences in terms of the number of
copies of a target sequence per unit area of the support surface
may exceed the surface density of oligonucleotide primers even if
the hybridization and/or amplification efficiencies are less than
100%.
[0149] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
reaction formulations may yield increased clonal copy number
compared to that obtained using conventional supports and
amplification protocols. In some instances, e.g., wherein the
clonally-amplified target (or sample) oligonucleotide molecules
comprise concatenated, multimeric repeats of a monomeric target
sequence, the clonal copy number may be substantially smaller than
compared to that obtained using conventional supports and
amplification protocols. Thus, in some instances, the clonal copy
number may range from about 1 molecule to about 100,000 molecules
(e.g., target sequence molecules) per amplified colony. In some
instances, the clonal copy number may be at least 1, at least 5, at
least 10, at least 50, at least 100, at least 500, at least 1,000,
at least 2,000, at least 3,000, at least 4,000, at least 5,000, at
least 6,000, at least 7,000, at least 8,000, at least 9,000, at
least 10,000, at least 15,000, at least 20,000, at least 25,000, at
least 30,000, at least 35,000, at least 40,000, at least 45,000, at
least 50,000, at least 55,000, at least 60,000, at least 65,000, at
least 70,000, at least 75,000, at least 80,000, at least 85,000, at
least 90,000, at least 95,000, or at least 100,000 molecules per
amplified colony. In some instances, the clonal copy number may be
at most 100,000, at most 95,000, at most 90,000, at most 85,000, at
most 80,000, at most 75,000, at most 70,000, at most 65,000, at
most 60,000, at most 55,000, at most 50,000, at most 45,000, at
most 40,000, at most 35,000, at most 30,000, at most 25,000, at
most 20,000, at most 15,000, at most 10,000, at most 9,000, at most
8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000,
at most 3,000, at most 2,000, at most 1,000, at most 500, at most
100, at most 50, at most 10, at most 5, or at most 1 molecule per
amplified colony. Any of the lower and upper values described in
this paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the clonal copy
number may range from about 2,000 molecules to about 9,000
molecules. Those of skill in the art will recognize that the clonal
copy number may have any value within this range, e.g., about 2,220
molecules in some instances, or about 2 molecules in others.
[0150] As noted above, in some instances the amplified target (or
sample) oligonucleotide molecules (or nucleic acid molecules) may
comprise concatenated, multimeric repeats of a monomeric target
sequence. In some instances, the amplified target (or sample)
oligonucleotide molecules (or nucleic acid molecules) may comprise
a plurality of molecules each of which comprises a single monomeric
target sequence. Thus, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
reaction formulations may result in a surface density of target
sequence copies that ranges from about 100 target sequence copies
per mm.sup.2 to about 1.times.10.sup.12 target sequence copies per
mm.sup.2. In some instances, the surface density of target sequence
copies may be at least 100, at least 500, at least 1,000, at least
5,000, at least 10,000, at least 15,000, at least 20,000, at least
25,000, at least 30,000, at least 35,000, at least 40,000, at least
45,000, at least 50,000, at least 55,000, at least 60,000, at least
65,000, at least 70,000, at least 75,000, at least 80,000, at least
85,000, at least 90,000, at least 95,000, at least 100,000, at
least 150,000, at least 200,000, at least 250,000, at least
300,000, at least 350,000, at least 400,000, at least 450,000, at
least 500,000, at least 550,000, at least 600,000, at least
650,000, at least 700,000, at least 750,000, at least 800,000, at
least 850,000, at least 900,000, at least 950,000, at least
1,000,000, at least 5,000,000, at least 1.times.10.sup.7, at least
5.times.10.sup.7, at least 1.times.10.sup.8, at least
5.times.10.sup.8, at least 1.times.10.sup.9, at least
5.times.10.sup.9, at least 1.times.10.sup.10, at least
5.times.10.sup.10, at least 1.times.10.sup.11, at least
5.times.10.sup.11, or at least 1.times.10.sup.12 of clonally
amplified target sequence molecules per mm.sup.2. In some
instances, the surface density of target sequence copies may be at
most 1.times.10.sup.12, at most 5.times.10.sup.11, at most
1.times.10.sup.11, at most 5.times.10.sup.10, at most
1.times.10.sup.10, at most 5.times.10.sup.9, at most
1.times.10.sup.9, at most 5.times.10.sup.8, at most
1.times.10.sup.8, at most 5.times.10.sup.7, at most
1.times.10.sup.7, at most 5,000,000, at most 1,000,000, at most
950,000, at most 900,000, at most 850,000, at most 800,000, at most
750,000, at most 700,000, at most 650,000, at most 600,000, at most
550,000, at most 500,000, at most 450,000, at most 400,000, at most
350,000, at most 300,000, at most 250,000, at most 200,000, at most
150,000, at most 100,000, at most 95,000, at most 90,000, at most
85,000, at most 80,000, at most 75,000, at most 70,000, at most
65,000, at most 60,000, at most 55,000, at most 50,000, at most
45,000, at most 40,000, at most 35,000, at most 30,000, at most
25,000, at most 20,000, at most 15,000, at most 10,000, at most
5,000, at most 1,000, at most 500, or at most 100 target sequence
copies per mm.sup.2. Any of the lower and upper values described in
this paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of target sequence copies may range from about 1,000 target
sequence copies per mm.sup.2 to about 65,000 target sequence copies
mm.sup.2. Those of skill in the art will recognize that the surface
density of target sequence copies may have any value within this
range, e.g., about 49,600 target sequence copies per mm.sup.2.
[0151] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
buffer formulations may result in a surface density of
clonally-amplified target (or sample) oligonucleotide molecules (or
clusters) ranging from about from about 100 molecules per mm.sup.2
to about 1.times.10.sup.12 colonies per mm.sup.2. In some
instances, the surface density of clonally-amplified molecules may
be at least 100, at least 500, at least 1,000, at least 5,000, at
least 10,000, at least 15,000, at least 20,000, at least 25,000, at
least 30,000, at least 35,000, at least 40,000, at least 45,000, at
least 50,000, at least 55,000, at least 60,000, at least 65,000, at
least 70,000, at least 75,000, at least 80,000, at least 85,000, at
least 90,000, at least 95,000, at least 100,000, at least 150,000,
at least 200,000, at least 250,000, at least 300,000, at least
350,000, at least 400,000, at least 450,000, at least 500,000, at
least 550,000, at least 600,000, at least 650,000, at least
700,000, at least 750,000, at least 800,000, at least 850,000, at
least 900,000, at least 950,000, at least 1,000,000, at least
5,000,000, at least 1.times.10.sup.7, at least 5.times.10.sup.7, at
least 1.times.10.sup.8, at least 5.times.10.sup.8, at least
1.times.10.sup.9, at least 5.times.10.sup.9, at least
1.times.10.sup.10, at least 5.times.10.sup.10, at least
1.times.10.sup.11, at least 5.times.10.sup.11, or at least
1.times.10.sup.12 molecules per mm.sup.2. In some instances, the
surface density of clonally-amplified molecules may be at most
1.times.10.sup.12, at most 5.times.10.sup.11, at most
1.times.10.sup.11, at most 5.times.10.sup.10, at most
1.times.10.sup.10, at most 5.times.10.sup.9, at most
1.times.10.sup.9, at most 5.times.10.sup.8, at most
1.times.10.sup.8, at most 5.times.10.sup.7, at most
1.times.10.sup.7, at most 5,000,000, at most 1,000,000, at most
950,000, at most 900,000, at most 850,000, at most 800,000, at most
750,000, at most 700,000, at most 650,000, at most 600,000, at most
550,000, at most 500,000, at most 450,000, at most 400,000, at most
350,000, at most 300,000, at most 250,000, at most 200,000, at most
150,000, at most 100,000, at most 95,000, at most 90,000, at most
85,000, at most 80,000, at most 75,000, at most 70,000, at most
65,000, at most 60,000, at most 55,000, at most 50,000, at most
45,000, at most 40,000, at most 35,000, at most 30,000, at most
25,000, at most 20,000, at most 15,000, at most 10,000, at most
5,000, at most 1,000, at most 500, or at most 100 molecules per
mm.sup.2. Any of the lower and upper values described in this
paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of clonally-amplified molecules may range from about 5,000
molecules per mm.sup.2 to about 50,000 molecules per mm.sup.2.
Those of skill in the art will recognize that the surface density
of clonally-amplified colonies may have any value within this
range, e.g., about 48,800 molecules per mm.sup.2.
[0152] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
buffer formulations may result in a surface density of
clonally-amplified target (or sample) oligonucleotide molecules (or
clusters) ranging from about from about 100 molecules per mm.sup.2
to about 1.times.10.sup.9 colonies per mm.sup.2. In some instances,
the surface density of clonally-amplified molecules may be at least
100, at least 500, at least 1,000, at least 5,000, at least 10,000,
at least 15,000, at least 20,000, at least 25,000, at least 30,000,
at least 35,000, at least 40,000, at least 45,000, at least 50,000,
at least 55,000, at least 60,000, at least 65,000, at least 70,000,
at least 75,000, at least 80,000, at least 85,000, at least 90,000,
at least 95,000, at least 100,000, at least 150,000, at least
200,000, at least 250,000, at least 300,000, at least 350,000, at
least 400,000, at least 450,000, at least 500,000, at least
550,000, at least 600,000, at least 650,000, at least 700,000, at
least 750,000, at least 800,000, at least 850,000, at least
900,000, at least 950,000, at least 1,000,000, at least 5,000,000,
at least 1.times.10.sup.7, at least 5.times.10.sup.7, at least
1.times.10.sup.8, at least 5.times.10.sup.8, at least
1.times.10.sup.9, at least molecules per mm.sup.2. In some
instances, the surface density of clonally-amplified molecules may
be at 1.times.10.sup.9, at most 5.times.10.sup.8, at most
1.times.10.sup.8, at most 5.times.10.sup.7, at most
1.times.10.sup.7, at most 5,000,000, at most 1,000,000, at most
950,000, at most 900,000, at most 850,000, at most 800,000, at most
750,000, at most 700,000, at most 650,000, at most 600,000, at most
550,000, at most 500,000, at most 450,000, at most 400,000, at most
350,000, at most 300,000, at most 250,000, at most 200,000, at most
150,000, at most 100,000, at most 95,000, at most 90,000, at most
85,000, at most 80,000, at most 75,000, at most 70,000, at most
65,000, at most 60,000, at most 55,000, at most 50,000, at most
45,000, at most 40,000, at most 35,000, at most 30,000, at most
25,000, at most 20,000, at most 15,000, at most 10,000, at most
5,000, at most 1,000, at most 500, or at most 100 molecules per
mm.sup.2. Any of the lower and upper values described in this
paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of clonally-amplified molecules may range from about 5,000
molecules per mm.sup.2 to about 50,000 molecules per mm.sup.2.
Those of skill in the art will recognize that the surface density
of clonally-amplified colonies may have any value within this
range, e.g., about 48,800 molecules per mm.sup.2.
[0153] In some instances, the use of the disclosed low-binding
supports alone or in combination with optimized amplification
buffer formulations may result in a surface density of
clonally-amplified target (or sample) oligonucleotide colonies (or
clusters) ranging from about from about 100 colonies per mm.sup.2
to about 1.times.10.sup.9 colonies per mm.sup.2. In some instances,
the surface density of clonally-amplified colonies may be at least
100, at least 500, at least 1,000, at least 5,000, at least 10,000,
at least 15,000, at least 20,000, at least 25,000, at least 30,000,
at least 35,000, at least 40,000, at least 45,000, at least 50,000,
at least 55,000, at least 60,000, at least 65,000, at least 70,000,
at least 75,000, at least 80,000, at least 85,000, at least 90,000,
at least 95,000, at least 100,000, at least 150,000, at least
200,000, at least 250,000, at least 300,000, at least 350,000, at
least 400,000, at least 450,000, at least 500,000, at least
550,000, at least 600,000, at least 650,000, at least 700,000, at
least 750,000, at least 800,000, at least 850,000, at least
900,000, at least 950,000, at least 1,000,000, at least 5,000,000,
at least 1.times.10.sup.7, at least 5.times.10.sup.7, at least
1.times.10.sup.8, at least 5.times.10.sup.8, at least
1.times.10.sup.9, at least 5.times.10.sup.9, at least
1.times.10.sup.10, at least 5.times.10.sup.10, at least
1.times.10.sup.11, at least 5.times.10.sup.11, or at least
1.times.10.sup.12 colonies per mm.sup.2. In some instances, the
surface density of clonally-amplified colonies may be at most
1.times.10.sup.12, at most 5.times.10.sup.11, at most
1.times.10.sup.11, at most 5.times.10.sup.10, at most
1.times.10.sup.10, at most 5.times.10.sup.9, at most
1.times.10.sup.9, at most 5.times.10.sup.8, at most
1.times.10.sup.8, at most 5.times.10.sup.7, at most
1.times.10.sup.7, at most 5,000,000, at most 1,000,000, at most
950,000, at most 900,000, at most 850,000, at most 800,000, at most
750,000, at most 700,000, at most 650,000, at most 600,000, at most
550,000, at most 500,000, at most 450,000, at most 400,000, at most
350,000, at most 300,000, at most 250,000, at most 200,000, at most
150,000, at most 100,000, at most 95,000, at most 90,000, at most
85,000, at most 80,000, at most 75,000, at most 70,000, at most
65,000, at most 60,000, at most 55,000, at most 50,000, at most
45,000, at most 40,000, at most 35,000, at most 30,000, at most
25,000, at most 20,000, at most 15,000, at most 10,000, at most
5,000, at most 1,000, at most 500, or at most 100 colonies per
mm.sup.2. Any of the lower and upper values described in this
paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of clonally-amplified colonies may range from about 5,000
colonies per mm.sup.2 to about 50,000 colonies per mm.sup.2. Those
of skill in the art will recognize that the surface density of
clonally-amplified colonies may have any value within this range,
e.g., about 48,800 colonies per mm.sup.2.
[0154] In some cases the use of the disclosed low-binding supports
alone or in combination with optimized amplification reaction
formulations may yield signal from the amplified and labeled
nucleic acid populations (e.g., a fluorescence signal) that has a
coefficient of variance of no greater than 50%, such as 50%, 40%,
30%, 20%, 15%, 10%, 5%, or less than 5%.
[0155] Similarly, in some cases the use of optimized amplification
reaction formulations in combination with the disclosed low-binding
supports yield signal from the nucleic acid populations that has a
coefficient of variance of no greater than 50%, such as 50%, 40%,
30%, 20%, 10% or less than 10%.
[0156] In some cases, the support surfaces and methods as disclosed
herein allow amplification at elevated extension temperatures, such
as at 15 C, 20 C, 25 C, 30 C, 40 C, or greater, or for example at
about 21 C or 23 C.
[0157] In some cases, the use of the support surfaces and methods
as disclosed herein enable simplified amplification reactions. For
example, in some cases amplification reactions are performed using
no more than 1, 2, 3, 4, or 5 discrete reagents.
[0158] In some cases, the use of the support surfaces and methods
as disclosed herein enable the use of simplified temperature
profiles during amplification, such that reactions are executed at
temperatures ranging from a low temperature of 15 C, 20 C, 25 C, 30
C, or 40 C, to a high temperature of 40 C, 45 C, 50 C, 60 C, 65 C,
70 C, 75 C, 80 C, or greater than 80 C, for example, such as a
range of 20 C to 65 C.
[0159] Amplification reactions are also improved such that lower
amounts of template (e.g., target or sample molecules) are
sufficient to lead to discernable signals on a surface, such as 1
pM, 2 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70
pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM,
700 pM, 800 pM, 900 pM, 1,000 pM, 2,000 pM, 3,000 pM, 4,000 pM,
5,000 pM, 6,000 pM, 7,000 pM, 8,000 pM, 9,000 pM, 10,000 pM or
greater than 10,000 pM of a sample, such as 500 nM. In exemplary
embodiments, inputs of about 100 pM are sufficient to generate
signals for reliable signal determination.
[0160] Fluorescence imaging of support surfaces: The disclosed
solid-phase nucleic acid amplification reaction formulations and
low-binding supports may be used in any of a variety of nucleic
acid analysis applications, e.g., nucleic acid base discrimination,
nucleic acid base classification, nucleic acid base calling,
nucleic acid detection applications, nucleic acid sequencing
applications, and nucleic acid-based (genetic and genomic)
diagnostic applications. In many of these applications,
fluorescence imaging techniques may be used to monitor
hybridization, amplification, and/or sequencing reactions performed
on the low-binding supports.
[0161] Fluorescence imaging may be performed using any of a variety
of fluorophores, fluorescence imaging techniques, and fluorescence
imaging instruments known to those of skill in the art. Examples of
suitable fluorescence dyes that may be used (e.g., by conjugation
to nucleotides, oligonucleotides, or proteins) include, but are not
limited to, fluorescein, rhodamine, coumarin, cyanine, and
derivatives thereof, including the cyanine derivatives Cyanine
dye-3 (Cy3), Cyanine dye-5 (Cy5), Cyanine dye-7 (Cy7), etc.
Examples of fluorescence imaging techniques that may be used
include, but are not limited to, wide-field fluorescence microscopy
fluorescence microscopy imaging, fluorescence confocal imaging,
two-photon fluorescence, and the like. Examples of fluorescence
imaging instruments that may be used include, but are not limited
to, fluorescence microscopes equipped with an image sensor or
camera, wide-field fluorescence microscopy, confocal fluorescence
microscopes, two-photon fluorescence microscopes, or custom
instruments that comprise a suitable selection of light sources,
lenses, mirrors, prisms, dichroic reflectors, apertures, and image
sensors or cameras, etc. A non-limiting example of a fluorescence
microscope equipped for acquiring images of the disclosed
low-binding support surfaces and clonally-amplified colonies (or
clusters) of target nucleic acid sequences hybridized thereon is
the Olympus IX83 inverted fluorescence microscope equipped with)
20.times., 0.75 NA, a 532 nm light source, a bandpass and dichroic
mirror filter set optimized for 532 nm long-pass excitation and Cy3
fluorescence emission filter, a Semrock 532 nm dichroic reflector,
and a camera (Andor sCMOS, Zyla 4.2) where the excitation light
intensity is adjusted to avoid signal saturation. Often, the
support surface may be immersed in a buffer (e.g., 25 mM ACES, pH
7.4 buffer) while the image is acquired.
[0162] In some instances, the performance of nucleic acid
hybridization and/or amplification reactions using the disclosed
reaction formulations and low-binding supports may be assessed
using fluorescence imaging techniques, where the contrast-to-noise
ratio (CNR) of the images provides a key metric in assessing
amplification specificity and non-specific binding on the support.
CNR is commonly defined as: CNR=(Signal-Background)/Noise. The
background term is commonly taken to be the signal measured for the
interstitial regions surrounding a particular feature (diffraction
limited spot, DLS) in a specified region of interest (ROI). While
signal-to-noise ratio (SNR) is often considered to be a benchmark
of overall signal quality, it can be shown that improved CNR can
provide a significant advantage over SNR as a benchmark for signal
quality in applications that require rapid image capture (e.g.,
sequencing applications for which cycle times must be minimized),
as shown in the example below. At high CNR the imaging time
required to reach accurate discrimination (and thus accurate
base-calling in the case of sequencing applications) can be
drastically reduced even with moderate improvements in CNR.
[0163] In most ensemble-based sequencing approaches, the background
term is typically measured as the signal associated with
`interstitial` regions. In addition to "interstitial" background
(B.sub.inter), "intrastitial" background (B.sub.intra) exists
within the region occupied by an amplified DNA colony. The
combination of these two background signals dictates the achievable
CNR, and subsequently directly impacts the optical instrument
requirements, architecture costs, reagent costs, run-times,
cost/genome, and ultimately the accuracy and data quality for
cyclic array-based sequencing applications. The B.sub.inter
background signal arises from a variety of sources; a few examples
include auto-fluorescence from consumable flow cells, non-specific
adsorption of detection molecules that yield spurious fluorescence
signals that may obscure the signal from the ROI, the presence of
non-specific DNA amplification products (e.g., those arising from
primer dimers). In typical next generation sequencing (NGS)
applications, this background signal in the current field-of-view
(FOV) is averaged over time and subtracted. The signal arising from
individual DNA colonies (i.e., (S)--B.sub.inter in the FOV) yields
a discernable feature that can be classified. In some instances,
the intrastitial background (B.sub.intra) can contribute a
confounding fluorescence signal that is not specific to the target
of interest, but is present in the same ROI thus making it far more
difficult to average and subtract.
[0164] As will be demonstrated in the examples below, the
implementation of nucleic acid amplification on the low-binding
substrates of the present disclosure may decrease the B.sub.inter
background signal by reducing non-specific binding, may lead to
improvements in specific nucleic acid amplification, and may lead
to a decrease in non-specific amplification that can impact the
background signal arising from both the interstitial and
intrastitial regions. In some instances, the disclosed low-binding
support surfaces, optionally used in combination with the disclosed
hybridization and/or amplification reaction formulations, may lead
to improvements in CNR by a factor of 2, 5, 10, 100, or 1000-fold
over those achieved using conventional supports and hybridization,
amplification, and/or sequencing protocols. Although described here
in the context of using fluorescence imaging as the read-out or
detection mode, the same principles apply to the use of the
disclosed low-binding supports and nucleic acid hybridization and
amplification formulations for other detection modes as well,
including both optical and non-optical detection modes.
[0165] The disclosed low-binding supports, optionally used in
combination with the disclosed hybridization and/or amplification
protocols, yield solid-phase reactions that exhibit: (i) negligible
non-specific binding of protein and other reaction components (thus
minimizing substrate background), (ii) negligible non-specific
nucleic acid amplification product, and (iii) provide tunable
nucleic acid amplification reactions. Although described herein
primarily in the context of nucleic acid hybridization,
amplification, and sequencing assays, it will be understood by
those of skill in the art that the disclosed low-binding supports
may be used in any of a variety of other bioassay formats
including, but not limited to, sandwich immunoassays, enzyme-linked
immunosorbent assays (ELISAs), etc.
[0166] Plastic surface: Examples of materials from which the
substrate or support structure may be fabricated include, but are
not limited to, glass, fused-silica, silicon, a polymer (e.g.,
polystyrene (PS), macroporous polystyrene (MPPS),
polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene
(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic
olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene
terephthalate (PET), or any combination thereof. Various
compositions of both glass and plastic substrates are
contemplated.
[0167] Modification of a surface for the purposes disclosed herein
involves making surfaces reactive against many chemical groups
(--R), including amines. When prepared on an appropriate substrate,
these reactive surfaces can be stored long term at room temperature
for example for at least 3 months or more. Such surfaces can be
further grafted with R-PEG and R-primer oligomer for on-surface
amplification of nucleic acids, as described elsewhere herein.
Plastic surfaces, such as cyclic olefin polymer (COP), may be
modified using any of a large number of methods known in the art.
For example, they can be treated with Ti: Sapphire laser ablation,
UV-mediated ethylene glycol methacrylate photografting, plasma
treatment, or mechanical agitation (e.g., sand blasting, or
polishing, etc.) to create hydrophilic surfaces that can stay
reactive for months against many chemical groups, such as amines.
These groups may then allow conjugation of passivation polymers
such as PEG, or biomolecules such as DNA or proteins, without loss
of biochemical activity. For example, attachment of DNA primer
oligomers allows DNA amplification on a passivated plastic surface
while minimizing the non-specific adsorption of proteins,
fluorophore molecules, or other hydrophobic molecules.
[0168] Additionally, surface modification can be combined with,
e.g., laser printing or UV masking, to create patterned surfaces.
This allows patterned attachment of DNA oligomers, proteins, or
other moieties, providing for surface-based enzymatic activity,
binding, detection, or processing. For example, DNA oligomers may
be used to amplify DNA only within patterned features, or to
capture amplified long DNA concatemers in a patterned fashion. In
some embodiments, enzyme islands may be generated in the patterned
areas that are capable of reacting with solution-based substrates.
Because plastic surfaces are especially amenable to these
processing modes, in some embodiments as contemplated herein,
plastic surfaces may be recognized as being particularly
advantageous.
[0169] Furthermore, plastic can be injection molded, embossed, or
3D printed to form any shape, including microfluidic devices, much
more easily than glass substrates, and thus can be used to create
surfaces for the binding and analysis of biological samples in
multiple configurations, e.g., sample-to-result microfluidic chips
for biomarker detection or DNA sequencing.
[0170] Specific localized DNA amplification on modified plastic
surfaces can be prepared and can produce spots with an ultra-high
contrast to noise ratio and very low background when probed with
fluorescent labels.
[0171] Hydrophilized and amine reactive cyclic olefin polymer
surface with amine-primer and amine-PEG can be prepared and it
supports rolling circle amplification. When probed with fluorophore
labeled primers, or when labeled dNTPs added to the hybridized
primers by a polymerase, bright spots of DNA amplicons were
observed that exhibited signal to noise ratios greater than 100
with backgrounds that are extremely low, indicating highly specific
amplification, and ultra-low levels of protein and hydrophobic
fluorophore binding which are hallmarks of the high accuracy
detection systems such as fluorescence-based DNA sequencers.
[0172] Oligonucleotide primers and adapter sequences: In general,
at least one layer of the one or more surface modification or
polymer layers applied to the capillary or channel lumen surface
may comprise functional groups for covalently or non-covalently
attaching oligonucleotide adapter or primer sequences, or the at
least one layer may already comprise covalently or non-covalently
attached oligonucleotide adapter or primer sequences at the time
that it is grafted to or deposited on the support surface. In some
aspects, the capillary or the microfluidic channel comprises an
oligonucleotide population directed to sequence a prokaryotic
genome. In some aspects, the capillary or the microfluidic channel
comprises an oligonucleotide population directed to sequence a
transcriptome.
[0173] The central region of the flow cell devices or systems can
include a surface having at least one oligonucleotide tethered
thereto. In some embodiments, the surface can be an interior
surface of a microfluidic channel or capillary tube. In some
aspects, the surface is a locally planar surface. In some
embodiments, the oligonucleotide is directly tethered to the
surface. In some embodiments, the oligonucleotide is tethered to
the surface through an intermediate molecule.
[0174] The oligonucleotide tethered to the interior surface of the
central region can include segments that bind to different targets.
In some instance, the oligonucleotide exhibits a segment that
specifically hybridizes to a eukaryotic genomic nucleic acid
segment. In some instance, the oligonucleotide exhibits a segment
that specifically hybridizes to a prokaryotic genomic nucleic acid
segment. In some instance, the oligonucleotide exhibits a segment
that specifically hybridizes to a viral nucleic acid segment. In
some instance, the oligonucleotide exhibits a segment that
specifically hybridizes to a transcriptome nucleic acid segment.
When the central region comprises a surface having one or more
oligonucleotide tethered thereto, the interior volume of the
central region can be adjusted based on the types of sequencing
performed. In some embodiments, the central region comprises an
interior volume suitable for sequencing a eukaryotic genome. In
some embodiments, the central region comprises an interior volume
suitable for sequencing a prokaryotic genome. In some embodiments,
the central region comprises an interior volume suitable for
sequencing a transcriptome. For example, in some embodiments, the
interior volume of the central region may comprise a volume of less
than 0.05 .mu.l, between 0.05 .mu.l and 0.1 .mu.l, between 0.05
.mu.l and 0.2 .mu.l, between 0.05 .mu.l and 0.5 .mu.l, between 0.05
.mu.l and 0.8 .mu.l, between 0.05 .mu.l and 1 .mu.l, between 0.05
.mu.l and 1.2 .mu.l, between 0.05 .mu.l and 1.5 .mu.l, between 0.1
.mu.l and 1.5 .mu.l, between 0.2 .mu.l and 1.5 .mu.l, between 0.5
.mu.l and 1.5 between 0.8 .mu.l and 1.5 .mu.l, between 1 .mu.l and
1.5 .mu.l, between 1.2 .mu.l and 1.5 .mu.l, or greater than 1.5
.mu.l, or a range defined by any two of the foregoing. In some
embodiments, the interior volume of the central region may comprise
a volume of less than 0.5 .mu.l, between 0.5 .mu.l and 1 .mu.l,
between 0.5 .mu.l and 2 .mu.l, between 0.5 .mu.l and 5 .mu.l,
between 0.5 .mu.l and 8 .mu.l, between 0.5 .mu.l and 10 .mu.l,
between 0.5 .mu.l and 12 .mu.l, between 0.5 .mu.l and 15 .mu.l,
between 1 .mu.l and 15 .mu.l, between 2 .mu.l and 15 .mu.l, between
5 .mu.l and 15 .mu.l, between 8 .mu.l and 15 .mu.l, between 10
.mu.l and 15 .mu.l, between 12 .mu.l and 15 .mu.l, or greater than
15 .mu.l, or a range defined by any two of the foregoing. In some
embodiments, the interior volume of the central region may comprise
a volume of less than 5 .mu.l, between 5 .mu.l and 10 .mu.l,
between 5 .mu.l and between 5 .mu.l and 500 .mu.l, between 5 .mu.l
and 80 .mu.l, between 5 .mu.l and 100 .mu.l, between 5 .mu.l and
120 .mu.l, between 5 .mu.l and 150 .mu.l, between 10 .mu.l and 150
.mu.l, between 20 .mu.l and 150 .mu.l, between 50 .mu.l and 150
.mu.l, between 80 .mu.l and 150 .mu.l, between 100 .mu.l and 150
.mu.l, between 120 .mu.l and 150 .mu.l, or greater than 150 .mu.l,
or a range defined by any two of the foregoing. In some
embodiments, the interior volume of the central region may comprise
a volume of less than 50 .mu.l, between 50 .mu.l and 100 .mu.l,
between 50 .mu.l and 200 .mu.l, between 50 .mu.l and 500 .mu.l,
between 50 .mu.l and 800 .mu.l, between 50 .mu.l and 1000 .mu.l,
between 50 .mu.l and 1200 .mu.l, between 50 .mu.l and 1500 .mu.l,
between 100 .mu.l and 1500 .mu.l, between 200 .mu.l and 1500 .mu.l,
between 500 .mu.l and 1500 .mu.l, between 800 .mu.l and 1500 .mu.l,
between 1000 .mu.l and 1500 .mu.l, between 1200 .mu.l and 1500
.mu.l, or greater than 1500 .mu.l, or a range defined by any two of
the foregoing. In some embodiments, the interior volume of the
central region may comprise a volume of less than 500 .mu.l,
between 500 .mu.l and 1000 .mu.l, between 500 .mu.l and 2000 .mu.l,
between 500 .mu.l and 5 ml, between 500 .mu.l and 8 ml, between 500
.mu.l and 10 ml, between 500 .mu.l and 12 ml, between 500 .mu.l and
15 ml, between 1 ml and 15 ml, between 2 ml and 15 ml, between 5 ml
and 15 ml, between 8 ml and 15 ml, between 10 ml and 15 ml, between
12 ml and 15 ml, or greater than 15 ml, or a range defined by any
two of the foregoing. In some embodiments, the interior volume of
the central region may comprise a volume of less than 5 ml, between
5 ml and 10 ml, between 5 ml and 20 ml, between 5 ml and 50 ml,
between 5 ml and 80 ml, between 5 ml and 100 ml, between 5 ml and
120 ml, between 5 ml and 150 ml, between 10 ml and 150 ml, between
20 ml and 150 ml, between 50 ml and 150 ml, between 80 ml and 150
ml, between 100 ml and 150 ml, between 120 ml and 150 ml, or
greater than 150 ml, or a range defined by any two of the
foregoing. In some embodiments, the methods and systems described
herein comprise an array or collection of flow cell devices or
systems comprising multiple discrete capillaries, microfluidic
channels, fluidic channels, chambers, or lumenal regions, wherein
the combined interior volume is, comprises, or includes one or more
of the values within a range disclosed herein.
[0175] One or more types of oligonucleotide primer may be attached
or tethered to the support surface. In some instances, the one or
more types of oligonucleotide adapters or primers may comprise
spacer sequences, adapter sequences for hybridization to
adapter-ligated template library nucleic acid sequences, forward
amplification primers, reverse amplification primers, sequencing
primers, and/or molecular barcoding sequences, or any combination
thereof.
[0176] The tethered oligonucleotide adapter and/or primer sequences
may range in length from about 10 nucleotides to about 100
nucleotides. In some instances, the tethered oligonucleotide
adapter and/or primer sequences may be no more than 10, at least
10, at least 20, at least 30, at least 40, at least 50, at least
60, at least 70, at least 80, at least 90, or at least 100
nucleotides in length. In some instances, the tethered
oligonucleotide adapter and/or primer sequences may be at most 100,
at most 90, at most 80, at most 70, at most 60, at most 50, at most
40, at most 30, at most 20, or at most 10 nucleotides in length.
Any of the lower and upper values described in this paragraph may
be combined to form a range included within the present disclosure,
for example, in some instances the length of the tethered
oligonucleotide adapter and/or primer sequences may range from
about 20 nucleotides to about 80 nucleotides. Those of skill in the
art will recognize that the length of the tethered oligonucleotide
adapter and/or primer sequences may have any value within this
range, e.g., about 24 nucleotides.
[0177] The number of coating layers and/or the material composition
of each layer is chosen so as to adjust the resultant surface
density of oligonucleotide primers (or other attached molecules) on
the coated capillary lumen surface. In some instances, the surface
density of oligonucleotide primers may range from about 1,000
primer molecules per .mu.m.sup.2 to about 1,000,000 primer
molecules per .mu.m.sup.2. In some instances, the surface density
of oligonucleotide primers may be at least 1,000, at least 10,000,
at least 100,000, or at least 1,000,000 molecules per .mu.m2. In
some instances, the surface density of oligonucleotide primers may
be at most 1,000,000, at most 100,000, at most 10,000, or at most
1,000 molecules per .mu.m.sup.2. Any of the lower and upper values
described in this paragraph may be combined to form a range
included within the present disclosure, for example, in some
instances the surface density of primers may range from about
10,000 molecules per .mu.m.sup.2 to about 100,000 molecules per
.mu.m.sup.2. Those of skill in the art will recognize that the
surface density of primer molecules may have any value within this
range, e.g., about 455,000 molecules per .mu.m.sup.2. In some
instances, the surface properties of the capillary or channel lumen
coating, including the surface density of tethered oligonucleotide
primers, may be adjusted so as to optimize, e.g., solid-phase
nucleic acid hybridization specificity and efficiency, and/or
solid-phase nucleic acid amplification rate, specificity, and
efficiency.
[0178] Capillary flow cell cartridges: Also disclosed herein are
capillary flow cell cartridges that may comprise one, two, or more
capillaries to create independent flow channels. FIG. 2 provides a
non-limiting example of capillary flow cell cartridge that
comprises two glass capillaries, fluidic adaptors (two per
capillary in this example), and a cartridge chassis that mates with
the capillaries and/or fluidic adapters such that the capillaries
are held in a fixed orientation relative to the cartridge. In some
instances, the fluidic adaptors may be integrated with the
cartridge chassis. In some instances, the cartridge may comprise
additional adapters that mate with the capillaries and/or capillary
fluidic adapters. In some instances, the capillaries are
permanently mounted in the cartridge. In some instances, the
cartridge chassis is designed to allow one or more capillaries of
the flow cell cartridge to be interchangeable removed and replaced.
For example, in some instances, the cartridge chassis may comprise
a hinged "clamshell" configuration which allows it to be opened so
that one or more capillaries may be removed and replaces. In some
instances, the cartridge chassis is configured to mount on, for
example, the stage of a microscope system or within a cartridge
holder of an instrument system.
[0179] The capillary flow cell cartridges of the present disclosure
may comprise a single capillary. In some instances, the capillary
flow cell cartridges of the present disclosure may comprise 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or
more than 20 capillaries. The one or more capillaries of the flow
cell cartridge may have any of the geometries, dimensions, material
compositions, and/or coatings as described above for the single
capillary flow cell devices. Similarly, the fluidic adapters for
the individual capillaries in the cartridge (typically two fluidic
adapters per capillary) may have any of the geometries, dimensions,
and material compositions as described above for the single
capillary flow cell devices, except that in some instances the
fluidic adapters may be integrated directly with the cartridge
chassis as illustrated in FIG. 2. In some instances, the cartridge
may comprise additional adapters (i.e., in addition to the fluidic
adapters) that mate with the capillaries and/or fluidic adapters
and help to position the capillaries within the cartridge. These
adapters may be constructed using the same fabrication techniques
and materials as those outlined above for the fluidic adapters.
[0180] In some embodiments, one or more devices according to the
present disclosure may comprise a first surface in an orientation
generally facing the interior of the flow channel, wherein said
surface may further comprise a polymer coating as disclosed
elsewhere herein, and wherein said surface may further comprise one
or more oligonucleotides such as a capture oligonucleotide, an
adapter oligonucleotide, or any other oligonucleotide as disclosed
herein. In some embodiments, said devices may further comprise a
second surface in an orientation generally facing the interior of
the flow channel and further generally facing or parallel to the
first surface, wherein said surface may further comprise a polymer
coating as disclosed elsewhere herein, and wherein said surface may
further comprise one or more oligonucleotides such as a capture
oligonucleotide, an adapter oligonucleotide, or any other
oligonucleotide as disclosed herein. In some embodiments, a device
of the present disclosure may comprise a first surface in an
orientation generally facing the interior of the flow channel, a
second surface in an orientation generally facing the interior of
the flow channel and further generally facing or parallel to the
first surface, a third surface generally facing the interior of a
second flow channel, and a fourth surface, generally facing the
interior of the second flow channel and generally opposed to or
parallel to the third surface; wherein said second and third
surfaces may be located on or attached to opposite sides of a
generally planar substrate which may be a reflective, transparent,
or translucent substrate. In some embodiments, an imaging surface
or imaging surfaces within a flowcell may be located within the
center of a flowcell or within or as part of a division between two
subunits or subdivisions of a flowcell, wherein said flowcell may
comprise a top surface and a bottom surface, one or both of which
may be transparent to such detection mode as may be utilized; and
wherein a surface comprising oligonucleotides or polynucleotides
and/or one or more polymer coatings, may be placed or interposed
within the lumen of the flowcell. In some embodiments, the top
and/or bottom surfaces do not include attached oligonucleotides or
polynucleotides. In some embodiments, said top and/or bottom
surfaces do comprise attached oligonucleotides and/or
polynucleotides. In some embodiments, either said top or said
bottom surface may comprise attached oligonucleotides and/or
polynucleotides. A surface or surfaces placed or interposed within
the lumen of a flowcell may be located on or attached one side, an
opposite side, or both sides of a generally planar substrate which
may be a reflective, transparent, or translucent substrate. In some
embodiments, an optical apparatus as provided elsewhere herein or
as otherwise known in the art is utilized to provide images of a
first surface, a second surface, a third surface, a fourth surface,
a surface interposed within the lumen of a flowcell, or any other
surface provided herein which may contain one or more
oligonucleotides or polynucleotides attached thereto.
[0181] Microfluidic chip flow cell cartridges: Also disclosed
herein are microfluidic channel flow cell cartridges that may a
plurality of independent flow channels. A non-limiting example of
microfluidic chip flow cell cartridge that comprises a chip having
two or more parallel glass channels formed on the chip, fluidic
adaptors coupled to the chip, and a cartridge chassis that mates
with the chip and/or fluidic adapters such that the chip is posited
in a fixed orientation relative to the cartridge. In some
instances, the fluidic adaptors may be integrated with the
cartridge chassis. In some instances, the cartridge may comprise
additional adapters that mate with the chip and/or fluidic
adapters. In some instances, the chip is permanently mounted in the
cartridge. In some instances, the cartridge chassis is designed to
allow one or more chips of the flow cell cartridge to be
interchangeable removed and replaced. For example, in some
instances, the cartridge chassis may comprise a hinged "clamshell"
configuration which allows it to be opened so that one or more
capillaries may be removed and replaces. In some instances, the
cartridge chassis is configured to mount on, for example, the stage
of a microscope system or within a cartridge holder of an
instrument system. Even through only one chip is described in the
non-limiting example, it is understood that more than one chip can
be used in the microfluidic channel flow cell cartridge.
[0182] The flow cell cartridges of the present disclosure may
comprise a single microfluidic chip or a plurality of microfluidic
chips. In some instances, the flow cell cartridges of the present
disclosure may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, or more than 20 microfluidic chips. In some
instances, the microfluidic chip can have one channel. In some
instances, the microfluidic chip can have 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20
channels. The one or more chips of the flow cell cartridge may have
any of the geometries, dimensions, material compositions, and/or
coatings as described above for the single microfluidic chip flow
cell devices. Similarly, the fluidic adapters for the individual
chip in the cartridge (typically two fluidic adapters per
capillary) may have any of the geometries, dimensions, and material
compositions as described above for the single microfluidic chip
flow cell devices, except that in some instances the fluidic
adapters may be integrated directly with the cartridge chassis. In
some instances, the cartridge may comprise additional adapters
(i.e., in addition to the fluidic adapters) that mate with the chip
and/or fluidic adapters and help to position the chip within the
cartridge. These adapters may be constructed using the same
fabrication techniques and materials as those outlined above for
the fluidic adapters.
[0183] The cartridge chassis (or "housing") may be fabricated from
metal and/or polymer materials such as aluminum, anodized aluminum,
polycarbonate (PC), acrylic (PMMA), or Ultem (PEI), while ohter
materials are also consistent with the disclosure. A housing may be
fabricated using CNC machining and/or molding techniques, and
designed so that one, two, or more than two capillaries are
constrained by the chassis in a fixed orientation to create
independent flow channels. The capillaries may be mounted in the
chassis using, e.g., a compression fit design, or by mating with
compressible adapters made of silicone or a fluoroelastomer. In
some instance, two or more components of the cartridge chassis
(e.g., an upper half and a lower half) are assembled using, e.g.,
screws, clips, clamps, or other fasteners so that the two halves
are separable. In some instances, two or more components of the
cartridge chassis are assembled using, e.g., adhesives, solvent
bonding, or laser welding so that the two or more components are
permanently attached.
[0184] Some flow cell cartridges of the present disclosure further
comprise additional components that are integrated with the
cartridge to provide enhanced performance for specific
applications. Examples of additional components that may be
integrated into the cartridge include, but are not limited to,
fluid flow control components (e.g., miniature valves, miniature
pumps, mixing manifolds, etc.), temperature control components
(e.g., resistive heating elements, metal plates that serve as heat
sources or sinks, piezoelectric (Peltier) devices for heating or
cooling, temperature sensors), or optical components (e.g., optical
lenses, windows, filters, mirrors, prisms, fiber optics, and/or
light-emitting diodes (LEDs) or other miniature light sources that
may collectively be used to facilitate spectroscopic measurements
and/or imaging of one or more capillary flow channels).
[0185] Systems and system components: The flow cell devices and
flow cell cartridges disclosed herein may be used as components of
systems designed for a variety of chemical analysis, biochemical
analysis, nucleic acid analysis, cell analysis, or tissue analysis
application. In general, such systems may comprise one or more
fluid flow control modules, temperature control modules,
spectroscopic measurement and/or imaging modules, and processors or
computers, as well as one or more of the single capillary flow cell
devices and capillary flow cell cartridges or the microfluidic chip
flow cell devices and flow cell cartridges described herein.
[0186] The systems disclosed herein may comprise 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more than 10 single capillary flow cell devices or
capillary flow cell cartridges. In some instances the single
capillary flow cell devices or capillary flow cell cartridges may
be removable, exchangeable components of the disclosed systems. In
some instances, the single capillary flow cell devices or capillary
flow cell cartridges may be disposable or consumable components of
the disclosed systems. The systems disclosed herein may comprise 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 single microfluidic
channel flow cell devices or microfluidic channel flow cell
cartridges. In some instances the single microfluidic channel flow
cell devices or microfluidic channel flow cell cartridges may be
removable, exchangeable components of the disclosed systems. In
some instances, the flow cell devices or flow cell cartridges may
be disposable or consumable components of the disclosed
systems.
[0187] FIG. 3 illustrates one embodiment of a simple system
comprising a single capillary flow cell connected to various fluid
flow control components, where the single capillary is optically
accessible and compatible with mounting on a microscope stage or in
a custom imaging instrument for use in various imaging
applications. A plurality of reagent reservoirs are
fluidically-coupled with the inlet end of the single capillary flow
cell device, where the reagent flowing through the capillary at any
given point in time is controlled by means of a programmable rotary
valve that allows the user to control the timing and duration of
reagent flow. In this non-limiting example, fluid flow is
controlled by means of a programmable syringe pump that provides
precise control and timing of volumetric fluid flow and fluid flow
velocity.
[0188] FIG. 4 illustrates one embodiment of a system that comprises
a capillary flow cell cartridge having integrated diaphragm valves
to minimize dead volume and conserve certain key reagents. The
integration of miniature diaphragm valves into the cartridge allows
the valve to be positioned in close proximity to the inlet of the
capillary, thereby minimizing dead volume within the device and
reducing the consumption of costly reagents. The integration of
valves and other fluid control components within the capillary flow
cell cartridge also allows greater fluid flow control functionality
to be incorporated into the cartridge design.
[0189] FIG. 5 shows an example of a capillary flow cell
cartridge-based fluidics system used in combination with a
microscope setup, where the cartridge incorporates or mates with a
temperature control component such as a metal plate that makes
contact with the capillaries within the cartridge and serves as a
heat source/sink. The microscope setup consists of an illumination
system (e.g., including a laser, LED, or halogen lamp, etc., as a
light source), an objective lens, an imaging system (e.g., a CMOS
or CCD camera), and a translation stage to move the cartridge
relative to the optical system, which allows, e.g., fluorescence
and/or bright field images to be acquired for different regions of
the capillary flow cells as the stage is moved.
[0190] FIG. 6 illustrates one non-limiting example for temperature
control of the flow cells (e.g., capillary or microfluidic channel
flow cells) through the use of a metal plate that is placed in
contact with the flow cell cartridge. In some instances, the metal
plate may be integrated with the cartridge chassis. In some
instances, the metal plate may be temperature controlled using a
Peltier or resistive heater.
[0191] FIG. 7 illustrates one non-limiting approach for temperature
control of the flow cells (e.g., capillary or microfluidic channel
flow cells) that comprises a non-contact thermal control mechanism.
In this approach, a stream of temperature-controlled air is
directed through the flow cell cartridge (e.g., towards a single
capillary flow cell device or a microfluidic channel flow cell
device) using an air temperature control system. The air
temperature control system comprises a heat exchanger, e.g., a
resistive heater coil, fins attached to a Peltier device, etc.,
that is capable of heating and/or cooling the air and holding it at
a constant, user-specified temperature. The air temperature control
system also comprises an air delivery device, such as a fan, that
directs the stream of heated or cooled air to the capillary flow
cell cartridge. In some instances, the air temperature control
system may be set to a constant temperature T.sub.1 so that the air
stream, and consequently the flow cell or cartridge (e.g.,
capillary flow cell or microfluidic channel flow cell) is kept at a
constant temperature T.sub.2, which in some cases may differ from
the set temperature T.sub.1 depending on the environment
temperature, air flow rate, etc. In some instances, two or more
such air temperature control systems may be installed around the
capillary flow cell device or flow cell cartridge so that the
capillary or cartridge may be rapidly cycled between several
different temperatures by controlling which one of the air
temperature control systems is active at a given time. In another
approach, the temperature setting of the air temperature control
system may be varied so the temperature of the capillary flow cell
or cartridge may be changed accordingly.
[0192] Fluid flow control module: In general, the disclosed
instrument systems will provide fluid flow control capability for
delivering samples or reagents to the one or more flow cell devices
or flow cell cartridges (e.g., single capillary flow cell device or
microfluidic channel flow cell device) connected to the system.
Reagents and buffers may be stored in bottles, reagent and buffer
cartridges, or other suitable containers that are connected to the
flow cell inlets by means of tubing and valve manifolds. The
disclosed systems may also include processed sample and waste
reservoirs in the form of bottles, cartridges, or other suitable
containers for collecting fluids downstream of the capillary flow
cell devices or capillary flow cell cartridges. In some
embodiments, the fluid flow control (or "fluidics") module may
provide programmable switching of flow between different sources,
e.g. sample or reagent reservoirs or bottles located in the
instrument, and the central region (e.g., capillary or microfluidic
channel) inlet(s). In some embodiments, the fluid flow control
module may provide programmable switching of flow between the
central region (e.g., capillary or microfluidic channel) outlet(s)
and different collection points, e.g., processed sample reservoirs,
waste reservoirs, etc., connected to the system. In some instances,
samples, reagents, and/or buffers may be stored within reservoirs
that are integrated into the flow cell cartridge itself. In some
instances, processed samples, spent reagents, and/or used buffers
may be stored within reservoirs that are integrated into the flow
cell cartridge itself.
[0193] Control of fluid flow through the disclosed systems will
typically be performed through the use of pumps (or other fluid
actuation mechanisms) and valves (e.g., programmable pumps and
valves). Examples of suitable pumps include, but are not limited
to, syringe pumps, programmable syringe pumps, peristaltic pumps,
diaphragm pumps, and the like. Examples of suitable valves include,
but are not limited to, check valves, electromechanical two-way or
three-way valves, pneumatic two-way and three-way valves, and the
like. In some embodiments, fluid flow through the system may be
controlled by means of applying positive pneumatic pressure to one
or more inlets of the reagent and buffer containers, or to inlets
incorporated into flow cell cartridge(s) (e.g., capillary or
microfluidic channel flow cell cartridges). In some embodiments,
fluid flow through the system may be controlled by means of drawing
a vacuum at one or more outlets of waste reservoir(s), or at one or
more outlets incorporated into flow cell cartridge(s) (e.g.,
capillary or microfluidic channel flow cell cartridges).
[0194] In some instances, different modes of fluid flow control are
utilized at different points in an assay or analysis procedure,
e.g. forward flow (relative to the inlet and outlet for a given
capillary flow cell device), reverse flow, oscillating or pulsatile
flow, or combinations thereof. In some applications, oscillating or
pulsatile flow may be applied, for example, during assay wash/rinse
steps to facilitate complete and efficient exchange of fluids
within the one or more flow cell devices or flow cell cartridges
(e.g., single capillary flow cell devices or cartridges and
microfluidic chip flow cell devices or cartridges).
[0195] Similarly, in some cases different fluid flow rates may be
utilized at different points in the assay or analysis process
workflow, for example, in some instances, the volumetric flow rate
may vary from -100 ml/sec to +100 ml/sec. In some embodiment, the
absolute value of the volumetric flow rate may be at least 0.001
ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, at least 1
ml/sec, at least 10 ml/sec, or at least 100 ml/sec. In some
embodiments, the absolute value of the volumetric flow rate may be
at most 100 ml/sec, at most 10 ml/sec, at most 1 ml/sec, at most
0.1 ml/sec, at most 0.01 ml/sec, or at most 0.001 ml/sec. The
volumetric flow rate at a given point in time may have any value
within this range, e.g. a forward flow rate of 2.5 ml/sec, a
reverse flow rate of -0.05 ml/sec, or a value of 0 ml/sec (i.e.,
stopped flow).
[0196] Temperature control module: As noted above, in some
instances the disclosed systems will include temperature control
functionality for the purpose of facilitating the accuracy and
reproducibility of assay or analysis results. Examples of
temperature control components that may be incorporated into the
instrument system (or capillary flow cell cartridge) design
include, but are not limited to, resistive heating elements,
infrared light sources, Peltier heating or cooling devices, heat
sinks, thermistors, thermocouples, and the like. In some instances,
the temperature control module (or "temperature controller") may
provide for a programmable temperature change at a specified,
adjustable time prior to performing specific assay or analysis
steps. In some instances, the temperature controller may provide
for programmable changes in temperature over specified time
intervals. In some embodiments, the temperature controller may
further provide for cycling of temperatures between two or more set
temperatures with specified frequency and ramp rates so that
thermal cycling for amplification reactions may be performed.
[0197] Spectroscopy or imaging modules: As indicated above, in some
instances the disclosed systems will include optical imaging or
other spectroscopic measurement capabilities. For example, any of a
variety of imaging modes known to those of skill in the art may be
implemented including, but not limited to, bright-field,
dark-field, fluorescence, luminescence, or phosphorescence imaging.
In some embodiments, the central region comprises a window that
allows at least a part of the central region to be illuminated and
imaged. In some embodiments, the capillary tube comprises a window
that allows at least a part of the capillary tube to be illuminated
and imaged. In some embodiments, the microfluidic chip comprises a
window that allows at least a part of the chip channel to be
illuminated and imaged.
[0198] In some embodiments, single wavelength excitation and
emission fluorescence imaging may be performed. In some
embodiments, dual wavelength excitation and emission (or
multi-wavelength excitation or emission) fluorescence imaging may
be performed. In some instances, the imaging module is configured
to acquire video images. The choice of imaging mode may impact the
design of the flow cells devices or flow cell cartridges in that
all or a portion of the capillaries or cartridge will necessarily
need to be optically transparent over the spectral range of
interest. In some instances, a plurality of capillaries within a
capillary flow cell cartridge may be imaged in their entirety
within a single image. In some embodiments, only a single capillary
or a subset of capillaries within a capillary flow cell cartridge,
or portions thereof, may be imaged within a single image. In some
embodiments, a series of images may be "tiled" to create a single
high resolution image of one, two, several, or the entire plurality
of capillaries within a cartridge. In some instances, a plurality
of channels within a microfluidic chip may be imaged in their
entirety within a single image. In some embodiments, only a single
channel or a subset of channels within a microfluidic chip, or
portions thereof, may be imaged within a single image. In some
embodiments, a series of images may be "tiled" to create a single
high resolution image of one, two, several, or the entire plurality
of capillaries or microfluidic channels within a cartridge.
[0199] A spectroscopy or imaging module may comprise, e.g., a
microscope equipped with a CMOS of CCD camera. In some instances,
the spectroscopy or imaging module may comprise, e.g., a custom
instrument configured to perform a specific spectroscopic or
imaging technique of interest. In general, the hardware associated
with the imaging module may include light sources, detectors, and
other optical components, as well as processors or computers.
[0200] Light sources: Any of a variety of light sources may be used
to provide the imaging or excitation light, including but not
limited to, tungsten lamps, tungsten-halogen lamps, arc lamps,
lasers, light emitting diodes (LEDs), or laser diodes. In some
instances, a combination of one or more light sources, and
additional optical components, e.g. lenses, filters, apertures,
diaphragms, mirrors, and the like, may be configured as an
illumination system (or sub-system).
[0201] Detectors: Any of a variety of image sensors may be used for
imaging purposes, including but not limited to, photodiode arrays,
charge-coupled device (CCD) cameras, or complementary
metal-oxide-semiconductor (CMOS) image sensors. As used herein,
"imaging sensors" may be one-dimensional (linear) or
two-dimensional array sensors. In many instances, a combination of
one or more image sensors, and additional optical components, e.g.
lenses, filters, apertures, diaphragms, mirrors, and the like, may
be configured as an imaging system (or sub-system). In some
instances, e.g., where spectroscopic measurements are performed by
the system rather than imaging, suitable detectors may include, but
are not limited to, photodiodes, avalanche photodiodes, and
photomultipliers.
[0202] Other optical components: The hardware components of the
spectroscopic measurement or imaging module may also include a
variety of optical components for steering, shaping, filtering, or
focusing light beams through the system. Examples of suitable
optical components include, but are not limited to, lenses,
mirrors, prisms, apertures, diffraction gratings, colored glass
filters, long-pass filters, short-pass filters, bandpass filters,
narrowband interference filters, broadband interference filters,
dichroic reflectors, optical fibers, optical waveguides, and the
like. In some instances, the spectroscopic measurement or imaging
module may further comprise one or more translation stages or other
motion control mechanisms for the purpose of moving capillary flow
cell devices and cartridges relative to the illumination and/or
detection/imaging sub-systems, or vice versa.
[0203] Total internal reflection: In some instances, the optical
module or sub-system may be designed to use all or a portion of an
optically transparent wall of the capillaries or microfluidic
channels in flow cell devices and cartridges as a waveguide for
delivering excitation light to the capillary or channel lumen(s)
via total internal reflection. When incident excitation light
strikes the surface of the capillary or channel lumen at an angle
with respect to a normal to the surface that is larger than the
critical angle (determined by the relative refractive indices of
the capillary or channel wall material and the aqueous buffer
within the capillary or channel), total internal reflection occurs
at the surface and the light propagates through the capillary or
channel wall along the length of the capillary or channel Total
internal reflection generates an evanescent wave at the lumen
surface which penetrates the lumen interior for extremely short
distances, and which may be used to selectively excite fluorophores
at the surface, e.g., labeled nucleotides that have been
incorporated by a polymerase into a growing oligonucleotide through
a solid-phase primer extension reaction.
[0204] Imaging processing software: In some instances, the system
may further comprise a computer (or processor) and
computer-readable medium that includes code for providing image
processing and analysis capability. Examples of image processing
and analysis capability that may be provided by the software
include, but are not limited to, manual, semi-automated, or
fully-automated image exposure adjustment (e.g. white balance,
contrast adjustment, signal-averaging and other noise reduction
capability, etc.), automated edge detection and object
identification (e.g., for identifying clonally-amplified clusters
of fluorescently-labeled oligonucleotides on the lumen surface of
capillary flow cell devices), automated statistical analysis (e.g.,
for determining the number of clonally-amplified clusters of
oligonucleotides identified per unit area of the capillary lumen
surface, or for automated nucleotide base-calling in nucleic acid
sequencing applications), and manual measurement capabilities (e.g.
for measuring distances between clusters or other objects, etc.).
Optionally, instrument control and image processing/analysis
software may be written as separate software modules. In some
embodiments, instrument control and image processing/analysis
software may be incorporated into an integrated package.
[0205] System control software: In some instances, the system may
comprise a computer (or processor) and a computer-readable medium
that includes code for providing a user interface as well as
manual, semi-automated, or fully-automated control of all system
functions, e.g., control of the fluidics module, the temperature
control module, and/or the spectroscopy or imaging module, as well
as other data analysis and display options. The system computer or
processor may be an integrated component of the system (e.g. a
microprocessor or mother board embedded within the instrument) or
may be a stand-alone module, for example, a main frame computer, a
personal computer, or a laptop computer. Examples of fluid control
functions provided by the system control software include, but are
not limited to, volumetric fluid flow rates, fluid flow velocities,
the timing and duration for sample and reagent addition, buffer
addition, and rinse steps. Examples of temperature control
functions provided by the system control software include, but are
not limited to, specifying temperature set point(s) and control of
the timing, duration, and ramp rates for temperature changes.
Examples of spectroscopic measurement or imaging control functions
provided by the system control software include, but are not
limited to, autofocus capability, control of illumination or
excitation light exposure times and intensities, control of image
acquisition rate, exposure time, and data storage options.
[0206] Processors and computers: In some instances, the disclosed
systems may comprise one or more processors or computers. The
processor may be a hardware processor such as a central processing
unit (CPU), a graphic processing unit (GPU), a general-purpose
processing unit, or a computing platform. The processor may be
comprised of any of a variety of suitable integrated circuits,
microprocessors, logic devices, field-programmable gate arrays
(FPGAs) and the like. In some instances, the processor may be a
single core or multi core processor, or a plurality of processors
may be configured for parallel processing. Although the disclosure
is described with reference to a processor, other types of
integrated circuits and logic devices are also applicable. The
processor may have any suitable data operation capability. For
example, the processor may perform 512 bit, 256 bit, 128 bit, 64
bit, 32 bit, or 16 bit data operations.
[0207] The processor or CPU can execute a sequence of
machine-readable instructions, which can be embodied in a program
or software. The instructions may be stored in a memory location.
The instructions can be directed to the CPU, which can subsequently
program or otherwise configure the CPU to implement, e.g., the
system control methods of the present disclosure. Examples of
operations performed by the CPU can include fetch, decode, execute,
and write back.
[0208] Some processors are a processing unit of a computer system.
The computer system may enable cloud-based data storage and/or
computing. In some instances, the computer system may be
operatively coupled to a computer network ("network") with the aid
of a communication interface. The network may be the internet, an
intranet and/or extranet, an intranet and/or extranet that is in
communication with the internet, or a local area network (LAN). The
network in some cases is a telecommunication and/or data network.
The network may include one or more computer servers, which may
enable distributed computing, such as cloud-based computing.
[0209] The computer system may also include computer memory or
memory locations (e.g., random-access memory, read-only memory,
flash memory), electronic storage units (e.g., hard disk),
communication interfaces (e.g., network adapters) for communicating
with one or more other systems, and peripheral devices, such as
cache, other memory units, data storage units and/or electronic
display adapters. In some instances, the communication interface
may allow the computer to be in communication with one or more
additional devices. The computer may be able to receive input data
from the coupled devices for analysis. Memory units, storage units,
communication interfaces, and peripheral devices may be in
communication with the processor or CPU through a communication bus
(solid lines), such as may be incorporated into a motherboard. A
memory or storage unit may be a data storage unit (or data
repository) for storing data. The memory or storage units may store
files, such as drivers, libraries and saved programs. The memory or
storage units may store user data, e.g., user preferences and user
programs.
[0210] The system control, image processing, and/or data analysis
methods as described herein can be implemented by way of
machine-executable code stored in an electronic storage location of
the computer system, such as, for example, in the memory or
electronic storage unit. The machine-executable or machine-readable
code can be provided in the form of software. During use, the code
can be executed by the processor. In some cases, the code can be
retrieved from the storage unit and stored in memory for ready
access by the processor. In some situations, the electronic storage
unit can be precluded, and machine-executable instructions are
stored in memory.
[0211] In some instances, the code may be pre-compiled and
configured for use with a machine having a processer adapted to
execute the code. In some instances, the code may be compiled
during runtime. The code can be supplied in a programming language
that can be selected to enable the code to execute in a
pre-compiled or as-compiled fashion.
[0212] Some aspects of the systems and methods provided herein can
be embodied in software. Various aspects of the technology may be
thought of as "products" or "articles of manufacture" typically in
the form of machine (or processor) executable code and/or
associated data that is carried on or embodied in a type of
machine-readable medium. Machine-executable code can be stored on
an electronic storage unit, such as memory (e.g., read-only memory,
random-access memory, flash memory) or a hard disk. "Storage" type
media can include any or all of the tangible memory of the
computers, processors or the like, or associated modules thereof,
such as various semiconductor memories, tape drives, disk drives
and the like, which may provide non-transitory storage at any time
for the software programming. All or portions of the software may
at times be communicated through the Internet or various other
telecommunication networks. Such communications, for example, may
enable loading of the software from one computer or processor into
another, for example, from a management server or host computer
into the computer platform of an application server. Thus, another
type of media that may bear the software elements includes optical,
electrical and electromagnetic waves, such as used across physical
interfaces between local devices, through wired and optical
landline networks and over various air-links. The physical elements
that carry such waves, such as wired or wireless links, optical
links or the like, also may be considered as media bearing the
software. As used herein, unless restricted to non-transitory,
tangible "storage" media, terms such as computer or machine
"readable medium" refer to any medium that participates in
providing instructions to a processor for execution.
[0213] In some instances, the system control, image processing,
and/or data analysis methods of the present disclosure may be
implemented by way of one or more algorithms. An algorithm may be
implemented by way of software upon execution by the central
processing unit.
[0214] Nucleic acid sequencing applications: Nucleic acid
sequencing provides one non-limiting example of an application for
the disclosed flow cell devices and cartridges (e.g., capillary
flow cell or microfluidic chip flow cell devices and cartridges).
Many "second generation" and "third generation" sequencing
technologies utilize a massively parallel, cyclic array approach to
sequencing-by-synthesis (SBS), in which accurate decoding of a
single-stranded template oligonucleotide sequence tethered to a
solid support relies on successfully classifying signals that arise
from the stepwise addition of A, G, C, and T nucleotides by a
polymerase to a complementary oligonucleotide strand. These methods
typically require the oligonucleotide template to be modified with
a known adapter sequence of fixed length, affixed to a solid
support (e.g., the lumen surface(s) of the disclosed capillary or
microfluidic chip flow cell devices and cartridges) in a random or
patterned array by hybridization to surface-tethered probes of
known sequence that is complementary to that of the adapter
sequence, and then probed through a cyclic series of single base
addition primer extension reactions that use, e.g.,
fluorescently-labeled nucleotides to identify the sequence of bases
in the template oligonucleotides. These processes thus require the
use of miniaturized fluidics systems that offer precise,
reproducible control of the timing of reagent introduction to the
flow cell in which the sequencing reactions are performed, and
small volumes to minimize the consumption of costly reagents.
[0215] Existing commercially-available NGS flow cells are
constructed from layers of glass that have been etched, lapped,
and/or processed by other methods to meet the tight dimensional
tolerances required for imaging, cooling, and/or other
requirements. When flow cells are used as consumables, the costly
manufacturing processes required for their fabrication result in
costs per sequencing run that are too high to make sequencing
routinely accessible to scientists and medical professionals in the
research and clinical spaces.
[0216] This disclosure provides a low-cost flow cell architecture
that includes low cost glass or polymer capillaries or microfluidic
channels, fluidics adapters, and cartridge chassis. Utilizing glass
or polymer capillaries that are extruded in their final
cross-sectional geometry eliminates the need for multiple
high-precision and costly glass manufacturing processes. Robustly
constraining the orientation of the capillaries or channels and
providing convenient fluidic connections using molded plastic
and/or elastomeric components further reduces cost. Laser bonding
the components of the polymer cartridge chassis provides a fast and
efficient means of sealing the capillary or the microfluidic
channels and structurally-stabilizing the capillaries or channels
and flow cell cartridge without requiring the use of fasteners or
adhesives.
[0217] Applications of flow cell devices and systems: The flow cell
devices and systems described herein can be used in a variety of
applications such as sequencing analysis to improve the efficient
use of the costly reagents. For examples, a method of sequencing a
nucleic acid sample and a second nucleic acid sample can include
delivering a plurality of oligonucleotides to an interior surface
of an at least partially transparent chamber; delivering a first
nucleic acid sample to the interior surface; delivering a plurality
of nonspecific reagents through a first channel to the interior
surface; delivering a specific reagent through a second channel to
the interior surface, wherein the second channel has a lower volume
than the first channel; visualizing a sequencing reaction on the
interior surface of the at least partially transparent chamber; and
replacing the at least partially transparent chamber prior to a
second sequencing reaction. In some aspects, flowing an air current
past an exterior surface of the at least partially transparent
surface. In some aspects, the described method can include
selecting the plurality of oligonucleotides to sequence a
eukaryotic genome. In some aspects, the described method can
include selecting a prefabricated tube as the at least partially
transparent chamber. In some aspects, the described method can
include selecting the plurality of oligonucleotides to sequence a
prokaryotic genome. In some aspects, the described method can
include selecting the plurality of oligonucleotides to sequence a
transcriptome. In some aspects, the described method can include
selecting a capillary tube as the at least partially transparent
chamber. In some aspects, the described method can include
selecting a microfluidic chip as the at least partially transparent
chamber.
[0218] The described devices and systems can also be used in a
method of reducing a reagent used in a sequencing reaction,
comprising providing a first reagent in a first reservoir;
providing a second reagent in a first second reservoir, wherein
each of the first reservoir and the second reservoir are fluidicaly
coupled to a central region, and wherein the central region
comprises a surface for the sequencing reaction; and sequentially
introducing the first reagent and the second reagent into a central
region of the flow cell device, wherein the volume of the first
reagent flowing from the first reservoir to the inlet of the
central region is less than the volume of the second reagent
flowing from the second reservoir to the central region.
[0219] An additional use of the described devices and systems is a
method of increasing the efficient use of a regent in a sequencing
reaction, comprising: providing a first reagent in a first
reservoir; providing a second reagent in a first second reservoir,
wherein each of the first reservoir and the second reservoir are
fluidicaly coupled to a central region, and wherein the central
region comprises a surface for the sequencing reaction; and
maintaining the volume of the first reagent flowing from the first
reservoir to the inlet of the central region to be less than the
volume of the second reagent flowing from the second reservoir to
the central region.
[0220] In general, the first reagent is more expensive than the
second agent. In some aspects, the first reagent is selected from
the group consisting of a polymerase, a nucleotide, and a
nucleotide analog.
[0221] Method of fabricating the microfluidic chip: The
microfluidic chip can be manufactured by a combination of
microfabrication process. The method of manufacturing the
microfluidic chip described herein includes providing a surface;
and forming at least one channel on the surface. The method of
manufacturing can also include providing a first substrate which
has at least a first planar surface, wherein the first surface has
a plurality of channels; providing a second substrate having at
least a second planar surface; and binding the first planar surface
of the first substrate to the second planar surface of the second
substrate. In some instances, the channels on the first surface
have an open top side and closed bottom side, and the second
surface is bond to the first surface through the bottom side of the
channels and therefore leaving the open top side of the channels
unaffected. In some instances, the method described herein further
includes providing a third substrate having a third planar surface,
and bonding the third surface to the first surface through the open
top side of the channels. The bonding conditions can include, e.g.,
heating the substrates, or applying an adhesive to one of the
planar surfaces of the first or second substrate.
[0222] Typically, because the devices are microfabricated,
substrate materials will be selected based upon their compatibility
with known microfabrication techniques, e.g., photolithography, wet
chemical etching, laser ablation, laser irradiation, air abrasion
techniques, injection molding, embossing, and other techniques. The
substrate materials are also generally selected for their
compatibility with the full range of conditions to which the
microfluidic devices may be exposed, including extremes of pH,
temperature, salt concentration, and application of illumination or
electric fields. Accordingly, in some preferred aspects, the
substrate material may include silica based substrates, such as
borosilicate glass, quartz, as well as other substrate
materials.
[0223] In additional preferred aspects, the substrate materials
will comprise polymeric materials, e.g., plastics, such as
polymethylmethacrylate (PMMA), polycarbonate,
polytetrafluoroethylene (TEFLON.TM.), polyvinylchloride (PVC),
polydimethylsiloxane (PDMS), polysulfone, and the like. Such
polymeric substrates are readily manufactured using available
microfabrication techniques, as described above, or from
microfabricated masters, using well known molding techniques, such
as injection molding, embossing or stamping, or by polymerizing the
polymeric precursor material within the mold (See U.S. Pat. No.
5,512,131). Such polymeric substrate materials are preferred for
their ease of manufacture, low cost and disposability, as well as
their general inertness to most extreme reaction conditions. Again,
these polymeric materials may include treated surfaces, e.g.,
derivatized or coated surfaces, to enhance their utility in the
microfluidic system, e.g., provide enhanced fluid direction.
[0224] The channels and/or chambers of the microfluidic devices are
typically fabricated into the upper surface of the first substrate,
as microscale channels (e.g., grooves, indentations) using the
above described microfabrication techniques. The first substrate
comprises a top side having a first planar surface and a bottom
side. In the microfluidic devices prepared in accordance with the
methods described herein, the plurality of channels (e.g., grooves
and/or indentations) are formed on the first planar surface. In
some instances, the channels (e.g., grooves and/or indentations)
formed in the first planar surface (prior to adding a second
substrate) has bottom and side walls with the top remaining open.
In some instances, the channels (e.g., grooves and/or indentations)
in the first planar surface (prior to adding a second substrate)
has bottom and side walls and the top remaining closed. In some
instances, the channels (e.g., grooves and/or indentations) in the
first planar surfaces (prior to adding a second substrate) has only
side walls and no top or bottom surface.
[0225] When the first planar surface of the first substrate is
placed into contact with, and bonded to the planar surface of the
second substrate, the second substrate can cover and/or seal the
grooves and/or indentations in the surface of the first substrate,
to form the channels and/or chambers (e.g., the interior portion)
of the device at the interface of these two components.
[0226] After the first substrate is bonded to a second substrate,
the structure can further placed into contact with and bonded to a
third substrate. The third substrate can be placed into contact
with the side of the first substrate that is not in contact with
the second substrate. In some embodiments, the first substrate is
placed between the second substrate and the third substrate. In
some embodiments, the second substrate and the third substrate can
cover and/or seal the grooves, indentations, or apertures on the
first substrate to form the channels and/or chambers (e.g., the
interior portion) of the device at the interface of these
components.
[0227] The device can have openings that are oriented such that
they are in communication with at least one of the channels and/or
chambers formed in the interior portion of the device from the
grooves or indentations. In some embodiments, the openings are
formed on the first substrate. In some embodiments, the openings
are formed on the first and the second substrate. In some
embodiments, the openings are formed on the first, the second, and
the third substrate. In some embodiments, the openings are
positioned at the top side of the device. In some embodiments, the
openings are positioned at the bottom side of the device. In some
embodiments, the openings are positioned at the first and/or the
second ends of the device, and the channels run along the direction
from the first end to the second end.
[0228] Conditions under which substrates may be bonded together are
generally widely understood, and such bonding of substrates is
generally carried out by any of a number of methods, which may vary
depending upon the nature of the substrate materials used. For
example, thermal bonding of substrates may be applied to a number
of substrate materials, including, e.g., glass or silica based
substrates, as well as polymer based substrates. Such thermal
bonding typically comprises mating together the substrates that are
to be bonded, under conditions of elevated temperature and, in some
cases, application of external pressure. The precise temperatures
and pressures will generally vary depending upon the nature of the
substrate materials used.
[0229] For example, for silica-based substrate materials, i.e.,
glass (borosilicate glass, Pyrex.TM., soda lime glass, etc.),
quartz, and the like, thermal bonding of substrates is typically
carried out at temperatures ranging from about 500.degree. C. to
about 1400.degree. C., and preferably, from about 500.degree. C. to
about 1200.degree. C. For example, soda lime glass is typically
bonded at temperatures around 550.degree. C., whereas borosilicate
glass typically is thermally bonded at or near 800.degree. C.
Quartz substrates, on the other hand, are typically thermally
bonded at temperatures at or near 1200.degree. C. These bonding
temperatures are typically achieved by placing the substrates to be
bonded into high temperature annealing ovens.
[0230] Polymeric substrates that are thermally bonded, on the other
hand, will typically utilize lower temperatures and/or pressures
than silica-based substrates, in order to prevent excessive melting
of the substrates and/or distortion, e.g., flattening of the
interior portion of the device, i.e., channels or chambers.
Generally, such elevated temperatures for bonding polymeric
substrates will vary from about 80.degree. C. to about 200.degree.
C., depending upon the polymeric material used, and will preferably
be between about 90.degree. C. and 150.degree. C. Because of the
significantly reduced temperatures required for bonding polymeric
substrates, such bonding may typically be carried out without the
need for high temperature ovens, as used in the bonding of
silica-based substrates. This allows incorporation of a heat source
within a single integrated bonding system, as described in greater
detail below.
[0231] Adhesives may also be used to bond substrates together
according to well known methods, which typically comprise applying
a layer of adhesive between the substrates that are to be bonded
and pressing them together until the adhesive sets. A variety of
adhesives may be used in accordance with these methods, including,
e.g., UV curable adhesives, that are commercially available.
Alternative methods may also be used to bond substrates together in
accordance with the present invention, including e.g., acoustic or
ultrasonic welding and/or solvent welding of polymeric parts.
[0232] Typically, a number of the described microfluidic chips or
devices will be manufactured at a time. For example, polymeric
substrates may be stamped or molded in large separable sheets which
can be mated and bonded together. Individual devices or bonded
substrates may then be separated from the larger sheet. Similarly,
for silica-based substrates, individual devices can be fabricated
from larger substrate wafers or plates, allowing higher throughput
of the manufacturing process. Specifically, a number of channel
structures can be manufactured into a first substrate wafer or
plate which is then overlaid with a second substrate wafer or
plate, and optionally further overlaid with a third substrate wafer
or plate. The resulting multiple devices are then segmented from
the larger substrates using known methods, such as sawing, scribing
and breaking, and the like.
[0233] As noted above, the top or second substrate is overlaid upon
the bottom or first substrate to seal the various channels and
chambers. In carrying out the bonding process according to the
methods of the present invention, the bonding of the first and
second substrates is carried out using vacuum to maintain the two
substrate surfaces in optimal contact. In particular, the bottom
substrate may be maintained in optimal contact with the top
substrate by mating the planar surface of the bottom substrate with
the planar surface of the top substrate, and applying a vacuum
through the holes that are disposed through the top substrate.
Typically, application of a vacuum to the holes in the top
substrate is carried out by placing the top substrate on a vacuum
chuck, which typically comprises a mounting table or surface,
having an integrated vacuum source. In the case of silica-based
substrates, the bonded substrates are subjected to elevated
temperatures in order to create an initial bond, so that the bonded
substrates may then be transferred to the annealing oven, without
any shifting relative to each other.
[0234] Alternate bonding systems for incorporation with the
apparatus described herein include, e.g., adhesive dispensing
systems, for applying adhesive layers between the two planar
surfaces of the substrates. This may be done by applying the
adhesive layer prior to mating the substrates, or by placing an
amount of the adhesive at one edge of the adjoining substrates, and
allowing the wicking action of the two mated substrates to draw the
adhesive across the space between the two substrates.
[0235] In certain embodiments, the overall bonding system can
include automatable systems for placing the top and bottom
substrates on the mounting surface and aligning them for subsequent
bonding Typically, such systems include translation systems for
moving either the mounting surface or one or more of the top and
bottom substrates relative to each other. For example, robotic
systems may be used to lift, translate and place each of the top
and bottom substrates upon the mounting table, and within the
alignment structures, in turn. Following the bonding process, such
systems also can remove the finished product from the mounting
surface and transfer these mated substrates to a subsequent
operation, e.g., separation operation, annealing oven for
silica-based substrates, etc., prior to placing additional
substrates thereon for bonding.
[0236] In some instances, the manufacturing of the microfluidic
chip includes the layering or laminating of two or more layers of
substrates, in order to produce the chip. For example, in
microfluidic devices, the microfluidic elements of the device are
typically produced by laser irradiation, etching or otherwise
fabricating features into the surface of a first substrate. A
second substrate is then laminated or bonded to the surface of the
first to seal these features and provide the fluidic elements of
the device, e.g., the fluid channels.
EXAMPLES
[0237] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1
[0238] Nucleic acid clusters were established within a capillary
and subjected to fluorescence imaging. A flow device having a
capillary tube was used for the test. The resulting cluster images
were presented in FIG. 8. The figure demonstrated that clusters
within the lumen of a capillary system as disclosed herein can be
reliably amplified and visualized.
Example 2
[0239] Flow cell device can be constructed from one, two, or three
layer of glasses using one of the steps as shown in FIG. 9. In FIG.
9, the flow cell devices can be made form one, two, or three layers
of glasses. The glasses can be either quarts or borosilicate glass.
FIGS. 9A-9C show the methods to make such devices at wafer level
with technologies such as focused femtosecond laser radiation (1
piece) and/or laser glass bonding (2 or 3 piece construction).
[0240] In FIG. 9A, the first layer of wafer is processed with a
laser (e.g., femtosecond laser radiation) to ablate the wafer
material and provide a patterned surface. The patterned surface can
be a plurality of channels on the surface such as 12 channels per
wafer. The wafer has a diameter of 210 mm. The processed wafer can
be then placed on a support plate to form channels that can be used
to direct fluid flow through a particular direction.
[0241] In FIG. 9B, the first layer of wafer having a patterned
surface can be placed in contact with and bonded to a second layer
of wafer. The bonding can be performed using a laser glass bonding
technology. The second layer can cover and/or seal the grooves,
indentations, or apertures on the wafer having the patterned
surface to form the channels and/or chambers (e.g., the interior
portion) of the device at the interface of these components. The
bonded structure with two layers of wafer can then be placed on a
support plate. The patterned surface can be a plurality of channels
on the surface such as 12 channels per wafer. The wafer can have a
diameter of 210 mm.
[0242] In FIG. 9C, the first layer of wafer having a patterned
surface can be placed in contact with and bonded to a second layer
of wafer on one side, and a third layer of wafer can be bonded to
the first wafer layer on the other side so that the first player of
wafer is positioned between the second and the third layers of
wafer. The bonding can be performed using a laser glass bonding
technology. The second layer and the third layer of wafers can
cover and/or seal the grooves, indentations, or apertures on the
wafer having the patterned surface to form the channels and/or
chambers (e.g., the interior portion) of the device. The bonded
structure with three layers of wafer can then be placed on a
support plate. The patterned surface can be a plurality of channels
on the surface such as 12 channels per wafer. The wafer can have a
diameter of 210 mm.
Example 3
[0243] FIG. 10A shows a one-piece glass flow cell design. In this
design, flow channels and inlet outlet holes can be fabricated
using focused femtosecond laser radiation method. There are two
channels/lanes on the flow cell, and each channel has 2 rows with
26 frames in each row. The channel can have a depth of about 100
.mu.m. Chanel 1 has an inlet hole A1 and an outlet hole A2, and
channel 2 has an inlet hole B1 and an outlet hole B2. The flow cell
can also have a 1D linear and human readable code, and optionally a
2D matrix code.
[0244] FIG. 10B shows a two-piece glass flow cell. In this design,
flow channels and inlet and outlet holes can be fabricated using
focused femtosecond laser radiation or chemical etching technology.
The 2 pieces can be bonded together with laser glass bonding
technology. The inlet and outlet holes can be positioned on the top
layer of the structure and oriented in a way such that they are in
communication with at least one of the channels and/or chambers
formed in the interior portion of the device. There are two
channels on the cell, and each channel has 2 rows with 26 frames in
each row. The channel can have a depth of about 100 .mu.m. Chanel 1
has an inlet hole A1 and an outlet hole A2, and channel 2 has an
inlet hole B1 and an outlet hole B2. The flow cell can also have a
1D linear and human readable code, and optionally a 2D matrix
code.
[0245] FIG. 10C shows a three-piece glass flow cell. In this
design, flow channels and inlet and outlet holes can be fabricated
using focused femtosecond laser radiation or chemical etching
technology. The 3 pieces can be bonded together with laser glass
bonding technology. The first layer of wafer having a patterned
surface can be bonded to a second layer of wafer on one side, and a
third layer of wafer can be bonded to the first wafer layer on the
other side so that the first player of wafer is positioned between
the second and the third layers of wafer. The inlet and outlet
holes can be positioned on the top layer of the structure and
oriented in a way such that they are in communication with at least
one of the channels and/or chambers formed in the interior portion
of the device. There are two channels on the cell, and each channel
has 2 rows with 26 frames in each row. The channel can have a depth
of about 100 .mu.m. Chanel 1 has an inlet hole A1 and an outlet
hole A2, and channel 2 has an inlet hole B1 and an outlet hole B2.
The flow cell can also have a 1D linear and human readable code,
and optionally a 2D matrix code.
Example 4
[0246] Flow cells were coated by washing prepared glass channels
with KOH followed by rinsing with ethanol and silanization for 30
minutes at 65.degree. C. Channel surfaces were activated with
EDC-NHS for 30 min. followed by grafting of primers by incubation
with 5 .mu.m primer for 20 min., and then passivation with 30 .mu.m
PEG-NH2.
[0247] Multilayer surfaces are made following the approach of
Example 4, where following PEG passivation, a multi-armed PEG-NHS
is flowed through the channels following addition of the PEG-NH2,
optionally followed by another incubation with PEG-NHS, and
optionally another incubation with multi-armed PEG-NH2. For these
surfaces, primer may be grafted at any step, especially following
the last addition of multi-armed PEG-NH2.
[0248] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
any combination in practicing the invention. It is intended that
the following claims define the scope of the invention and that
methods and structures within the scope of these claims and their
equivalents be covered thereby.
* * * * *