U.S. patent application number 16/457667 was filed with the patent office on 2020-01-09 for interposer with first and second adhesive layers.
This patent application is currently assigned to ILLUMINA, Inc.. The applicant listed for this patent is ILLUMINA, Inc.. Invention is credited to M. Shane Bowen, Gerald Kreindl, Steven H. Modiano, Arthur J. Pitera, LiangLiang Qiang, Randall Smith, Hai Quang Tran, Dajun Yuan, Maxwell Zimmerley.
Application Number | 20200009556 16/457667 |
Document ID | / |
Family ID | 63294406 |
Filed Date | 2020-01-09 |
View All Diagrams
United States Patent
Application |
20200009556 |
Kind Code |
A1 |
Zimmerley; Maxwell ; et
al. |
January 9, 2020 |
INTERPOSER WITH FIRST AND SECOND ADHESIVE LAYERS
Abstract
An interposer for a flow cell comprises a base layer having a
first surface and a second surface opposite the first surface. The
base layer comprises black polyethylene terephthalate (PET). A
first adhesive layer is disposed on the first surface of the base
layer. The first adhesive layer comprises methyl acrylic adhesive.
A second adhesive layer is disposed on the second surface of the
base layer. The second adhesive layer comprises methyl acrylic
adhesive. A plurality of microfluidic channels extends through each
of the base layer, the first adhesive layer, and the second
adhesive layer.
Inventors: |
Zimmerley; Maxwell; (San
Diego, CA) ; Qiang; LiangLiang; (San Diego, CA)
; Bowen; M. Shane; (Encinitas, CA) ; Modiano;
Steven H.; (San Diego, CA) ; Yuan; Dajun; (San
Diego, CA) ; Smith; Randall; (San Marcos, CA)
; Pitera; Arthur J.; (Encinitas, CA) ; Tran; Hai
Quang; (San Diego, CA) ; Kreindl; Gerald;
(Schaerding, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ILLUMINA, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
ILLUMINA, Inc.
San Deigo
CA
|
Family ID: |
63294406 |
Appl. No.: |
16/457667 |
Filed: |
June 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62693762 |
Jul 3, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502707 20130101;
B01L 2200/0642 20130101; B01L 3/56 20130101; B01L 3/502715
20130101; B01L 2300/12 20130101; B01L 3/502746 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2018 |
NL |
2021377 |
Claims
1. An interposer comprising: a base layer having a first surface
and a second surface opposite the first surface, the base layer
comprising black polyethylene terephthalate (PET); a first adhesive
layer disposed on the first surface of the base layer, the first
adhesive layer comprising acrylic adhesive; a second adhesive layer
disposed on the second surface of the base layer, the second
adhesive layer comprising acrylic adhesive; and a plurality of
microfluidic channels extending through each of the base layer, the
first adhesive layer, and the second adhesive layer.
2. The interposer of claim 1, wherein a total thickness of the base
layer, first adhesive layer, and second adhesive layer is in a
range of about 1 to about 200 microns.
3. The interposer of claim 1, wherein the base layer has a
thickness in a range of about 10 to about 100 microns, and each of
the first adhesive layer and the second adhesive layer has a
thickness in a range of about 5 to about 50 microns.
4. The interposer of claim 1, wherein the each of the first and
second adhesive layers has an auto-fluorescence in response to a
532 nm excitation wavelength of less than about 0.25 a.u. relative
to a 532 nm fluorescence standard.
5. The interposer of claim 4, wherein the each of the first and
second adhesive layers has an auto-fluorescence in response to a
635 nm excitation wavelength of less than about 0.15 a.u. relative
to a 635 nm fluorescence standard.
6. The interposer of claim 1, wherein the base layer comprises at
least about 50% black PET.
7. The interposer of claim 1, wherein the base layer consists
essentially of black PET.
8. The interposer of claim 1, wherein each of the first and second
adhesive layers is comprises at least about 5% acrylic
adhesive.
9. The interposer of claim 1, wherein each of the first and second
adhesive layers consists essentially of acrylic adhesive.
10. A flow cell comprising: a first substrate; a second substrate;
and the interposer of claim 1 disposed between the first substrate
and the second substrate, wherein the first adhesive layer bonds
the first surface of the base layer to a surface of the first
substrate, and the second adhesive layer bonds the second surface
of the base layer to a surface of the second substrate.
11. The flow cell of claim 10, wherein each of the first and second
substrates comprises glass, and wherein a bond between each of the
first and second adhesive layers and the respective surfaces of the
first and second substrates is adapted to withstand a shear stress
of greater than about 50 N/cm.sup.2 and a peel force of greater
than about 1 N/cm.
12. The flow cell of claim 10, wherein each of the first and second
substrates comprises a resin layer that is less than about one
micron thick and includes the surface that is bonded to the
respective first and second adhesive layers, and wherein a bond
between each of the resin layers and the respective first and
second adhesive layers is adapted to withstand a shear stress of
greater than about 50 N/cm.sup.2 and a peel force of greater than
about 1 N/cm.
13. The flow cell of claim 12, wherein: a plurality of wells is
imprinted in the resin layer of at least one of the first substrate
or the second substrate, a biological probe is disposed in each of
the wells, and the microfluidic channels of the interposer are
configured to deliver a fluid to the plurality of wells.
14. An interposer comprising: a base layer having a first surface
and a second surface opposite the first surface; a first adhesive
layer disposed on the first surface of the base layer; a first
release liner disposed on the first adhesive layer; a second
adhesive layer disposed on the second surface of the base layer; a
second release liner disposed on the second adhesive layer; and a
plurality of microfluidic channels extending through each of the
base layer, the first adhesive layer, and the second adhesive
layer, and the second release liner, but not through the first
release liner.
15. The interposer of claim 14, wherein: the first release liner
has a thickness in a range of about 50 to about 300 microns; and
the second release liner has a thickness in a range of about 25 to
about 50 microns.
16. The interposer of claim 14, wherein: the base layer comprises
black polyethylene terephthalate (PET); and each of the first and
second adhesive layers comprises acrylic adhesive.
17. The interposer of claim 14, wherein the first release liner is
at least substantially optically opaque and the second release
liner is at least substantially optically transparent.
18. A method comprising: forming an interposer comprising: a base
layer having a first surface and a second surface opposite the
first surface, the base layer comprising black polyethylene
terephthalate (PET), a first adhesive layer disposed on the first
surface of the base layer, the first adhesive layer comprising
acrylic adhesive, a second adhesive layer disposed on the second
surface of the base layer, the second adhesive layer comprising
acrylic adhesive; and forming microfluidic channels through at
least the base layer, the first adhesive layer, and the second
adhesive layer.
19. The method of claim 18, wherein the forming microfluidic
channels involves using a CO.sub.2 laser.
20. The method of claim 19, wherein: the interposer further
comprises: a first release liner disposed on the first adhesive
layer, and a second release liner disposed on the second adhesive
layer; and in the step of forming the microfluidic channels, the
microfluidic channels are further formed through the second release
liner using the CO.sub.2 laser, but are not formed through the
first release liner.
21. The method of claim 20, wherein the CO.sub.2 laser has a
wavelength in a range of about 5,000 nm to about 15,000 nm, and a
beam size in a range of about 50 to about 150 .mu.m.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of US Provisional
App. No. 62/693,762, filed Jul. 3, 2018, and claims priority to
Netherland Patent App. No. NL 2021377, filed July 23, 2018, the
entire disclosures of which are incorporated herein by
reference.
BACKGROUND
[0002] Various protocols in biological or chemical research involve
performing a large number of controlled reactions on local support
surfaces or within predefined reaction chambers. The desired
reactions may then be observed or detected, and subsequent analysis
may help identify or reveal properties of chemicals involved in the
reaction. For example, in some multiplex assays, an unknown analyte
having an identifiable label (e.g., fluorescent label) may be
exposed to thousands of known probes under controlled conditions.
Each known probe may be deposited into a corresponding well of a
microplate. Observing any chemical reactions that occur between the
known probes and the unknown analyte within the wells may help
identify or reveal properties of the analyte. Other examples of
such protocols include DNA sequencing processes, such as
sequencing-by-synthesis or cyclic-array sequencing. In cyclic-array
sequencing, a dense array of DNA features (e.g., template nucleic
acids) are sequenced through iterative cycles of enzymatic
manipulation. After each cycle, an image may be captured and
subsequently analyzed with other images to determine a sequence of
the DNA features.
[0003] Advances in microfluidic technology has enabled development
of flow cells that can perform rapid gene sequencing or chemical
analysis using nano-liter or even smaller volumes of a sample. Such
microfluidic devices desirably may withstand numerous high and low
pressure cycles, exposure to corrosive chemicals, variations in
temperature and humidity, and provide a high signal-to-noise ratio
(SNR).
SUMMARY
[0004] Some implementations provided in the present disclosure
relate generally to microfluidic devices. An example of a
microfluidic device is a flow cell. Some implementations described
herein relate generally to microfluidic devices including an
interposer, and in particular, to a flow cell that includes an
interposer formed from black polyethylene terephthalate (PET) and
double-sided acrylic adhesive, and having microfluidic channels
defined therethrough. The interposer may be configured to have low
auto-fluorescence, high peel and shear strength, and can withstand
corrosive chemicals, pressure and temperature cycling.
[0005] In a first set of implementations, an interposer comprises a
base layer having a first surface and a second surface opposite the
first surface. The base layer comprises black polyethylene
terephthalate (PET). A first adhesive layer is disposed on the
first surface of the base layer. The first adhesive layer comprises
acrylic adhesive. A second adhesive layer is disposed on the second
surface of the base layer. The second adhesive layer comprises
acrylic adhesive. A plurality of microfluidic channels extends
through each of the base layer, the first adhesive layer, and the
second adhesive layer.
[0006] In some implementations of the interposer, a total thickness
of the base layer, first adhesive layer, and second adhesive layer
is in a range of about 50 to about 200 microns.
[0007] In some implementations of the interposer, the base layer
has a thickness in a range of about 30 to about 100 microns, and
each of the first adhesive layer and the second adhesive layer has
a thickness in a range of about 10 to about 50 microns.
[0008] In some implementations of the interposer, each of the first
and the second adhesive layers has an auto-fluorescence in response
to a 532 nm excitation wavelength of less than about 0.25 a.u.
relative to a 532 nm fluorescence standard.
[0009] In some implementations of the interposer, each of the first
and second adhesive layers has an auto-fluorescence in response to
a 635 nm excitation wavelength of less than about 0.15 a.u.
relative to a 635 nm fluorescence standard.
[0010] In some implementations of the interposer, the base layer
comprises at least about 50% black PET. In some implementations,
the base layer consists essentially of black PET.
[0011] In some implementations of the interposer, each of the first
and second adhesive layers is made of at least about 10% acrylic
adhesive.
[0012] In some implementations of the interposer, each of the first
and second adhesive layers consists essentially of acrylic
adhesive.
[0013] In some implementations, a flow cell comprises a first
substrate, a second substrate, and any one of the interposers
described above.
[0014] In some implementations of the flow cell, each of the first
and second substrates comprises glass such that a bond between each
of the first and second adhesive layers and the respective surfaces
of the first and second substrates is adapted to withstand a shear
stress of greater than about 50 N/cm.sup.2 and a 180 degree peel
force of greater than about 1 N/cm.
[0015] In some implementations of the flow cell, each of the first
and second substrates comprises a resin layer that is less than one
micron thick and includes the surface that is bonded to the
respective first and second adhesive layers such that a bond
between each of the resin layers and the respective first and
second adhesive layers is adapted to withstand a shear stress of
greater than about 50 N/cm.sup.2 and a peel force of greater than
about 1 N/cm.
[0016] In some implementations of the flow cell, a plurality of
wells is imprinted in the resin layer of at least one of the first
substrate or the second substrate. A biological probe is disposed
in each of the wells, and the microfluidic channels of the
interposer are configured to deliver a fluid to the plurality of
wells.
[0017] In another set of implementations, an interposer comprises a
base layer having a first surface and a second surface opposite the
first surface. A first adhesive layer is disposed on the first
surface of the base layer. A first release liner is disposed on the
first adhesive layer. A second adhesive layer is disposed on the
second surface of the base layer. A second release liner is
disposed on the second adhesive layer. A plurality of microfluidic
channels extends through each of the base layer, the first adhesive
layer, and the second adhesive layer, and the second release liner,
but not through the first release liner.
[0018] In some implementations of the interposer, the first release
liner has a thickness in a range of about 50 to about 300 microns,
and the second release liner has a thickness in a range of about 25
to about 50 microns.
[0019] In some implementations of the interposer, the base layer
comprises black polyethylene terephthalate (PET); and each of the
first and second adhesive layers comprises acrylic adhesive.
[0020] In some implementations of the interposer, the first release
liner is at least substantially optically opaque and the second
release liner is at least substantially optically transparent.
[0021] The interposers and flow cells described above and herein
may be implemented in any combination to achieve the benefits as
described later in this disclosure.
[0022] In yet another set of implementations, a method of
patterning microfluidic channels, comprises forming an interposer
comprising a base layer having a first surface and a second surface
opposite the first surface. The base layer comprises black
polyethylene terephthalate (PET). A first adhesive layer is
disposed on the first surface of the base layer, the first adhesive
layer comprising acrylic adhesive, and a second adhesive layer is
disposed on the second surface of the base layer, the second
adhesive layer comprising acrylic adhesive. Microfluidic channels
are formed through at least the base layer, the first adhesive
layer, and the second adhesive layer.
[0023] In some implementations of the method, the forming
microfluidic channels involves using a CO.sub.2 laser.
[0024] In some implementations, the interposer further comprises a
first release liner disposed on the first adhesive layer, and a
second release liner disposed on the second adhesive layer. In some
implementations, in the step of forming the microfluidic channels,
the microfluidic channels are further formed through the second
release liner using the CO.sub.2 laser, but are not formed through
the first release liner.
[0025] In some implementations of the method, the CO.sub.2laser has
a wavelength in a range of about 5,000 nm to about 15,000 nm, and a
beam size in a range of about 50 to about 150 p.m.
[0026] The methods described above and herein may be implemented in
any combination to achieve the benefits as described later in this
disclosure.
[0027] All of the implementations described above, including the
interposers, flow cells, and methods, can be combined in any
configuration to achieve the benefits as described later in this
disclosure. Further the foregoing implementations and additional
implementations discussed in greater detail below (provided such
concepts are not mutually inconsistent) are contemplated as being
part of the subject matter disclosed herein, and can be combined in
any configuration.
[0028] While this specification contains many specific
implementation details, these should not be construed as
limitations on the scope of any inventions or of what may be
claimed, but rather as descriptions of features specific to
particular implementations of particular inventions. Certain
features described in this specification in the context of separate
implementations can also be implemented in combination in a single
implementation. Conversely, various features described in the
context of a single implementation can also be implemented in
multiple implementations separately or in any suitable
subcombination. Moreover, although features may be described above
as acting in certain combinations and even initially claimed as
such, one or more features from a claimed combination can in some
cases be excised from the combination, and the claimed combination
may be directed to a subcombination or variation of a
subcombination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The foregoing and other features of the present disclosure
will become more fully apparent from the following description and
appended claims, taken in conjunction with the accompanying
drawings. Understanding that these drawings depict only several
implementations in accordance with the disclosure and are
therefore, not to be considered limiting of its scope, the
disclosure will be described with additional specificity and detail
through use of the accompanying drawings.
[0030] FIG. 1 is a schematic illustration of an example flow cell,
according to an implementation.
[0031] FIG. 2 is a schematic illustration of an example interposer
for use in a flow cell, according to an implementation.
[0032] FIG. 3 is a schematic illustration of an example flow cell,
according to another implementation.
[0033] FIG. 4A is a top, perspective view of an example wafer
assembly including a plurality of flow cells, according to an
implementation; FIG. 4B is a side cross-section of the wafer
assembly of FIG. 4A taken along the line A-A shown in FIG. 4.
[0034] FIG. 5 is a flow diagram of an example method of forming an
interposer for a flow cell, according to an implementation.
[0035] FIG. 6A is a schematic illustration of a cross-section of an
example bonded and patterned flow cell and FIG. 6B is a schematic
illustration of a cross-section of an example bonded un-patterned
flow cell used to test performance of various base layers and
adhesives.
[0036] FIG. 7 is a bar chart of fluorescence intensity in the red
channel of various adhesives and flow cell materials.
[0037] FIG. 8 is a bar chart of fluorescence intensity in the green
channel of the various adhesives and flow cell materials of FIG.
7.
[0038] FIGS. 9A and 9B show schematic illustrations of an example
lap shear test and an example peel test setup, respectively, for
determining lap sheer strength and peel strength of various
adhesives disposed on a glass base layer.
[0039] FIG. 10 is an example Fourier Transform Infrared (FTIR)
spectra of an acrylic adhesive and Scotch tape.
[0040] FIG. 11 is an example gas chromatography (GC) spectrum of
acrylic adhesive and Black Kapton.
[0041] FIG. 12 is an example mass spectroscopy (MS) spectrum of an
outgas compound released from the acrylic adhesive and the outgas
compounds possible chemical structure.
[0042] Reference is made to the accompanying drawings throughout
the following detailed description. In the drawings, similar
symbols typically identify similar components, unless context
dictates otherwise. The illustrative implementations described in
the detailed description, drawings, and claims are not meant to be
limiting. Other implementations may be utilized, and other changes
may be made, without departing from the spirit or scope of the
subject matter presented here. It will be readily understood that
the aspects of the present disclosure, as generally described
herein, and illustrated in the figures, can be arranged,
substituted, combined, and designed in a wide variety of different
configurations, all of which are explicitly contemplated and made
part of this disclosure.
DETAILED DESCRIPTION
[0043] Provided herein are examples of microfluidic devices.
Implementations described herein relate generally to microfluidic
devices including an interposer, an in particular, to a flow cell
that includes an interposer formed from black polyethylene
terephthalate (PET) and double-sided acrylic adhesive, and having
microfluidic channels defined therethrough. The interposer is
configured to have relatively low auto-fluorescence, relatively
high peel and relatively high shear strength, and can withstand
corrosive chemicals, pressure and temperature cycling.
[0044] Advances in microfluidic technology has enabled development
of flow cells that can perform rapid genetic sequencing or chemical
analysis using nano-liter or even smaller volumes of a sample. Such
microfluidic devices should be capable of withstanding numerous
high and low pressure cycles, exposure to corrosive chemicals,
variations in temperature and humidity, and provide a high
signal-to-noise ratio (SNR). For example, flow cells may comprise
various layers that are bonded together via adhesives. It is
desirable to structure the various layers so that they may be
fabricated and bonded together to form the flow cell in a high
throughput fabrication process. Furthermore, various layers should
be able to withstand temperature and pressure cycling, corrosive
chemicals, and not contribute significantly to noise.
[0045] Implementations of the flow cells described herein that
include an interposer having a double-sided adhesive and defines
microfluidic channels therethrough provide benefits including, for
example: (1) allowing wafer scale assembly of a plurality of flow
cells, thus enabling high throughput fabrication; (2) providing low
auto-fluorescence, high lap shear strength, peel strength and
corrosion resistance, that can last through 300 or more thermal
cycles at high pH while providing test data with high SNR; (3)
enabling fabrication of flat optically interrogateable microfluidic
devices by using a flat interposer having the microfluidic channels
defined therein; (4) allowing bonding of two resin coated
substrates via the double-sided adhesive interposer; and (5)
enabling bonding of a microfluidic device including one or more
opaque surfaces.
[0046] FIG. 1 is a schematic illustration of flow cell [100],
according to an implementation. The flow cell [100], may be used
for any suitable biological, biochemical or chemical analysis
application. For example, the flow cell [100] may include a genetic
sequencing (e.g., DNA or RNA) or epigenetic microarrays, or may be
configured for high throughput drug screening, DNA or protein
fingerprinting, proteomic analysis, chemical detection, any other
suitable application or a combination thereof.
[0047] The flow cell [100] includes a first substrate [110], a
second substrate [120] and an interposer [130] disposed between the
first substrate [110] and the second substrate [120]. The first and
second substrates [110] and [120] may comprise any suitable
material, for example, silicon dioxide, glass, quartz, Pyrex, fused
silica, plastics (e.g., polyethylene terephthalate (PET), high
density polyethylene (HDPE), low density polyethylene (LDPE),
polyvinyl chloride (PVC), polypropylene (PP), polyvinylidene
fluoride (PVDF), etc.), polymers, TEFLON.RTM., Kapton (i.e.,
polyimide), paper based materials (e.g., cellulose, cardboard,
etc.), ceramics (e.g., silicon carbide, alumina, aluminum nitride,
etc.), complementary metal-oxide semiconductor (CMOS) materials
(e.g., silicon, germanium, etc.), or any other suitable material.
In some implementation, the first and/or the second substrate [110]
and [120] may be optically transparent. In other implementations,
the first and/or the second substrate [110] and [120] may be
optically opaque. While not shown, the first and/or and the second
substrate [110] and [120] may define fluidic inlets or outlets for
pumping a fluid to and/or from microfluidic channels
[0048] defined in the interposer [130]. As described herein, the
term "microfluidic channel" implies that at least one dimension of
a fluidic channel (e.g., length, width, height, radius or
cross-section) is less than 1,000 microns.
[0049] In various implementations, a plurality of biological probes
may be disposed on a surface [111] of the first substrate [110]
and/or a surface [121] of the second substrate [120] positioned
proximate to the interposer [130]. The biological probes may be
disposed in any suitable array on the surface [111] and/or [121]
and may include, for example, DNA probes, RNA probes, antibodies,
antigens, enzymes or cells. In some implementations, chemical or
biochemical analytes may be disposed on the surface [111] and/or
[121]. The biological probes may be covalently bonded to, or
immobilized in a gel (e.g., a hydrogel) on the surface [111] and/or
[121] of the first and second substrate [110] and [120],
respectively. The biological probes may be tagged with fluorescent
molecules (e.g., green fluorescent protein (GFP), Eosin Yellow,
luminol, fluoresceins, fluorescent red and orange labels, rhodamine
derivatives, metal complexes, or any other fluorescent molecule) or
bond with target biologics that are fluorescently tagged, such that
optical fluorescence may be used to detect (e.g., determine
presence or absence of) or sense (e.g., measure a quantity of) the
biologics, for example, for DNA sequencing.
[0050] The interposer [130] includes a base layer [132] having a
first surface [133] facing the first substrate [110], and a second
surface [135] opposite the first surface [133] and facing the
second substrate [120]. The base layer [132] includes black PET. In
some implementations, the base layer [132] may include at least
about 50% black PET, or at least about 80% black PET, with the
remaining being transparent PET or any other plastic or polymer. In
other implementations, the base layer [132] may consist essentially
of black PET. In still other implementations, the base layer [132]
may consist of black PET. Black PET may have low auto-fluorescence
so as to reduce noise as well as provide high contrast, therefore
allowing fluorescent imaging of the flow cell with high SNR.
[0051] A first adhesive layer [134] is disposed on the first
surface [133] of the base layer [132]. The first adhesive layer
[134] includes an acrylic adhesive (e.g., a methacrylic or a
methacrylate adhesive). Furthermore, a second adhesive layer [136]
is disposed on the second surface [135] of the base layer [132].
The second adhesive layer [136] also includes acrylic adhesive
(e.g., a methacrylic or a methacrylate adhesive). For example, each
of the first adhesive layer [134] and the second adhesive layer
[136] may be include at least about 10% acrylic adhesive, or at
least about 50% acrylic adhesive, or at least about 80% acrylic
adhesive. In some implementations, the first and second adhesive
layers [134] and [136] may consist essentially of acrylic adhesive.
In some implementations, the first and second adhesive layers
[0052] and [136] may consist of acrylic adhesive. In particular
implementations, the acrylic adhesive may include the adhesive
available under the tradename MA-61ATM available from ADHESIVES
RESEARCH.RTM.. The acrylic adhesive included in the first and
second adhesive layers [134] and [136] may be pressure sensitive so
as to allow bonding of the base layer [132] of the interposer [130]
to the substrates [110] and [120] through application of a suitable
pressure. In other implementations, the first and second adhesive
layers [134] and [136] may be formulated to be activated via heat,
ultra violet (UV) light or any other activations stimuli. In still
other implementations, the first adhesive layer [134] and/or the
second adhesive layer [136] may include butyl-rubber.
[0053] In some implementations, each of the first and second
adhesive layers [134] and [136] has an auto-fluorescence in
response to a 532 nm excitation wavelength (e.g., a red excitation
laser) of less than about 0.25 arbitrary units (a.u.) relative to a
532 nm fluorescence standard. Furthermore, each of the first and
second adhesive layers [134] and [136] may have an
auto-fluorescence in response to a 635 nm excitation wavelength
(e.g., a green excitation laser) of less than about 0.15 a.u.
relative to a 635 nm fluorescence standard. Thus, the first and
second adhesive layer [134] and [136] also have low
auto-fluorescence such that the combination of the black PET base
layer [132] and the first and second adhesive layers [134] and
[136] including acrylic adhesive contribute negligibly to the
fluorescent signal generated at the biological probe interaction
sites and therefore provide high SNR.
[0054] A plurality of microfluidic channels [138] extends through
each of the first adhesive layer [134], the base layer [132] and
the second adhesive layer [136]. The microfluidic channels [138]
may be formed using any suitable process, for example, laser
cutting (e.g., using a UV nanosecond pulsed laser, a UV picosecond
pulsed laser, a UV femtosecond pulsed laser, a CO.sub.2laser or any
other suitable laser), stamping, die cutting, water jet cutting,
physical or chemical etching or any other suitable process.
[0055] In some implementations, the microfluidic channels [138] may
be defined using a process which does not significantly increase
auto-fluorescence of the first and second adhesive layers [134] and
[136], and the base layer [132], while providing a suitable surface
finish. For example, a UV nano, femto or picosecond pulsed laser
may be able to provide rapid cutting, smooth edges and corners,
therefore providing superior surface finish which is desirable, but
may also modify the surface chemistry of the acrylic adhesive
layers [134] and [136] and/or the black PET base layer [132] which
may cause auto-fluorescence in these layers.
[0056] In contrast, a CO.sub.2 laser may provide a surface finish,
which while in some instances may be considered inferior to the UV
lasers but remains within design parameters, but does not alter the
surface chemistry of the adhesive layers [134] and [136] and/or the
base layer [132] so that there is no substantial increase in
auto-fluorescence of these layers. In particular implementations, a
CO.sub.2 laser having a wavelength in a range of about 5,000 nm to
about 15,000 nm (e.g., about 5,000, about 6,000, about 7,000, about
8,000, about 9,000, about 10,000, about 11,000, about 12,000, about
13,000, about 14,000 or about 15,000 nm inclusive of all ranges and
values therebetween), and a beam size in a range of about 50 .mu.m
to about 150 .mu.m (e.g., about 50, about 60, about 70, about 80,
about 90, about 100, 1 about 10, about 120, about 130, about 140 or
about 150 .mu.m, inclusive of all ranges and values therebetween)
may be used to define the microfluidic channels [138] through the
first adhesive layer [134], the base layer [132] and the second
adhesive layer [136].
[0057] As shown in FIG. 1 the first adhesive layer [134] bonds the
first surface [133] of the base layer [132] to a surface [111] of
the first substrate [110]. Moreover, the second adhesive layer
[136] bonds the second surface [135] of the base layer [132] to a
surface [121] of the second substrate [120]. In various
implementations, the first and second substrates [110] and [120]
may comprise glass. A bond between each of the first and second
adhesive layers [134] and [136] and the respective surfaces [111]
and [121] of the first and second substrates [110] and [120] may be
adapted to withstand a shear stress of greater than about 50
N/cm.sup.2 and a 180.degree. peel force of greater than about 1
N/cm. In various implementations, the bond may be able withstand
pressures in the microfluidic channels [138] of up to about 15 psi
(about 103,500 Pascal).
[0058] For example, the shear strength and peel strength of the
adhesive layers [134] and [136] may be a function of their chemical
formulations and their thicknesses relative to the base layer
[132]. The acrylic adhesive included in the first and second
adhesive layers [134] and [136] provides strong adhesion to the
first and second surface [133] and [135] of the base layer [132]
and the surface [111] and [121] of the first and second substrates
[110] and [120], respectively. Furthermore, to obtain a strong bond
between the substrates [110] and [120] and the base layer [132], a
thickness of the adhesive layers [134] and [136] relative to the
base layer [132] may be chosen so as to transfer a large portion of
the peel and/or shear stress applied on the substrates [110] and
[120] to the base layer [132].
[0059] If the adhesive layers [134] and [136] are too thin, they
may not provide sufficient peel and shear strength to withstand the
numerous pressure cycles that the flow cell [100] may be subjected
to due to flow of pressurized fluid through the microfluidic
channels [138]. On the other hand, adhesive layers [134] and [136]
that are too thick may result in void or bubble formation in the
adhesive layers [134] and [136] which weakens the adhesive strength
thereof. Furthermore, a large portion of the stress and shear
stress may act on the adhesive layers [134] and [136] and is not
transferred to the base layer [132]. This may result in failure of
the flow cell due to the rupture of the adhesive layers [134]
and/or [136].
[0060] In various arrangements, the base layer [132] may have a
thickness in a range of about 25 to about 100 microns, and each of
the first adhesive layer [134] and the second adhesive layer [136]
may have a thickness in a range of about 5 to about 50 microns
(e.g., about 5, about 10, about 20, about 30, about 40 or about 50
microns, inclusive of all ranges and values therebetween). Such
arrangements, may provide sufficient peel and shear strength, for
example, capability of withstanding a shear stress of greater than
about 50 N/cm.sup.2 and a peel force of greater than about 1 N/cm
sufficient to withstand numerous pressure cycles, for example, 100
pressure cycles, 200 pressure cycles, 300 pressure cycles or even
more. In particular arrangements, a total thickness of the base
layer [132], first adhesive layer [134], and second adhesive layer
[136] may be in a range of about 50 to about 200 microns (e.g.,
about 50, about 100, about 150 or about 200 microns inclusive of
all ranges and values therebetween).
[0061] In various implementations, adhesion promoters may also be
included in the first and second adhesive layers [134] and [136]
and/or may be coated on the surfaces [111] and [121] of the
substrates [110] and [120], for example, to promote adhesion
between the adhesive layers [134] and [136] and the corresponding
surfaces [111] and [121]. Suitable adhesion promoters may include,
for example, silanes, titanates, isocyanates, any other suitable
adhesion promoter or a combination thereof.
[0062] The first and second adhesive layers [134] and [136] may be
formulated to withstand numerous pressure cycles and have low
auto-fluorescence, as previously described herein. During
operation, the flow cell may also be exposed to thermal cycling
(e.g., from about -80 degrees to about 100 degrees Celsius), high
pH (e.g., a pH of up to about 11), vacuum and corrosive reagents
(e.g., formamide, buffers and salts). In various implementations,
the first and second adhesive layers [134] and [136] may be
formulated to withstand thermal cycling in the range of about -80
to about 100 degrees Celsius, resists void formation even in
vacuum, and resists corrosion when exposed to a pH of up to about
11 or corrosive reagents such as formamide.
[0063] FIG. 2 is a schematic illustration of an interposer [230],
according to an implementation. The interposer [230] may be used in
the flow cell [100] or any other flow cell described herein. The
interposer [230] includes the base layer [132], the first adhesive
layer [134] and the second adhesive layer [136] which were
described in detail with respect to the interposer [130] included
in the flow cell [100]. The first adhesive layer [134] is disposed
on the first surface [133] of the base layer [132] and the second
adhesive layer [136] is disposed on the second surface [135] of the
base layer [132] opposite the first surface [133]. The base layer
[132] may include black PET, and each of the first and second
adhesive layers [134] and [136] may include an acrylic adhesive, as
previously described herein. Furthermore, the base layer [132] may
have a thickness B in a range of about 30 to about 100 microns
(about 30, about 50, about 70, about 90 or about 100 microns
inclusive of all ranges and values therebetween), and each of the
first and second adhesive layers [134] and [136] may have a
thickness A in a range of about 5 to about 50 microns (e.g., about
5, about 10, about 20, about 30, about 40 or about 50 microns
inclusive of all ranges and values therebetween).
[0064] A first release liner [237] may be disposed on the first
adhesive layer [134]. Furthermore, a second release liner [239] may
be disposed on the second adhesive layer [136]. The first release
line [237] and the second release liner [239] may serve as
protective layers for the first and second release liners [237] and
[239], respectively and may be configured to be selectively peeled
off, or otherwise mechanically removed, to expose the first and
second adhesive layers [134] and [136], for example, for bonding
the base layer [132] to the first and second substrates [110] and
[120], respectively.
[0065] The first and second release liners [237] and [239] may be
formed from paper (e.g., super calendared Kraft (SCK) paper, SCK
paper with polyvinyl alcohol coating, clay coated Kraft paper,
machine finished Kraft paper, machine glazed paper, polyolefin
coated Kraft papers, etc.), plastic (e.g., biaxially oriented PET
film, biaxially oriented polypropylene film, polyolefins, high
density polyethylene, low density polyethylene, polypropylene
plastic resins, etc.), fabrics (e.g., polyester), nylon, Teflon or
any other suitable material. In some implementations, the release
liners [237] and [239] may be formed from a low surface energy
material (e.g., any of the materials described herein) to
facilitate peeling of the release liners [237] and [239] from their
respective adhesive layers [134] and [136]. In other
implementations, a low surface energy material (e.g., a silicone,
wax, polyolefin, etc.) may be coated at least on a surface of the
release liners [237] and [239] which is disposed on the respective
adhesive layers [134] and [136] to facilitate peeling of the
release liners [237] and [239] therefrom.
[0066] A plurality of microfluidic channels [238] extends through
each of the base layer [132], the first adhesive layer [134], the
second adhesive layer [136], and the second release liner [239],
but not through the first release liner [237]. For example, the
second release liner [239] may be a top release liner of the
interposer [230] and defining the microfluidic channels [238]
through the second release liner [239], but not in the first
release liner [237], may indicate an orientation of the interposer
[230] to a user, thereby facilitating the user during fabrication
of a flow cell (e.g., the flow cell [100]). Furthermore, a
fabrication process of a flow cell (e.g., the flow cell [100]) may
be adapted so that the second release liner [239] is initially
peeled off from the second adhesive layer [136] for bonding to a
substrate (e.g., the second substrate [220]). Subsequently, the
first release liner [237] may be removed and the first adhesive
layer [134] bonded to another substrate (e.g., the substrate
[110]).
[0067] The first and second release liners [237] and [239] may have
the same or different thicknesses. In some implementations, the
first release liner [237] may be substantially thicker than the
second release liner [239] (e.g., about 2X, about 4X, about 6X,
about 8X, or about 10X, thicker, inclusive), for example, to
provide structural rigidity to the interposer [230] and may serve
as a handling layer to facilitate handling of the interposer [230]
by a user. In particular implementations, the first release liner
[237] may have a first thickness L1 in a range of about 50 to about
300 microns (e.g., about 50, about 100, about 150, about 200, about
250 or about 300 microns inclusive of all ranges and values
therebetween), and the second release liner [239] may have a second
thickness L2 in a range of about 25 to about 50 microns (e.g.,
about 25, about 30, about 35, about 40, about 45 or about 50
microns inclusive of all ranges and values therebetween).
[0068] The first and second release liners [237] and [239] may be
optically opaque, transparent or translucent and may have any
suitable color. In some implementations, the first release liner
[237] may be at least substantially optically opaque (including
completely opaque) and the second release liner [239] may be at
least substantially optically transparent (including completely
transparent). As previously described herein, the second release
liner [239] may be removed first from the second adhesive layer
[136] for bonding to a corresponding substrate (e.g., the second
substrate [120]). Providing optical transparency to the second
release liner [239] may allow easy identification of the second
release liner [239] from the opaque first release liner [237].
Furthermore, the substantially optically opaque second release
liner [239] may provide a suitable contrast to facilitate optical
alignment of a substrate (e.g., the second substrate [120]) with
the microfluidic channels [238] defined in the interposer [230].
Moreover, having the second release liner [239] being thinner than
the first release liner [237] may allow preferential peeling of the
second release liner [239] relative to the first release liner
[237], therefore preventing unintentional peeling of the first
release liner [237] while peeling the second release liner [239]
off the second adhesive layer [136].
[0069] In some implementations, one or more substrates of a flow
cell may include a plurality of wells defined thereon, each well
having a biological probe (e.g., an array of the same biological
probe or distinct biological probes) disposed therein. In some
implementations, the plurality of wells may be etched in the one or
more substrates. For example, the substrate (e.g., the substrate
[110] or [120]) may include glass and an array of wells are etched
in the substrate using a wet etch (e.g., a buffered hydrofluoric
acid etch) or a dry etch (e.g., using reactive ion etching (RIE) or
deep RIE).
[0070] In other implementations, the plurality of wells may be
formed in a resin layer disposed on a surface of the substrate. For
example, FIG. 3 is a schematic illustration of a flow cell [300],
according to an implementation. The flow cell [300] includes the
interposer [130] including the base layer [132], the first adhesive
layer [134] and the second adhesive layer [136] and having a
plurality of microfluidic channels [138] defined therethrough, as
previously described in detail herein.
[0071] The flow cell [300] also includes a first substrate [310]
and a second substrate [320] with the interposer [132] disposed
therebetween. The first and second substrates [310] and [320] may
be formed from any suitable material, for example, silicon dioxide,
glass, quartz, Pyrex, plastics (e.g., polyethylene terephthalate
(PET), high density polyethylene (HDPE), low density polyethylene
(LDPE), polyvinyl chloride (PVC), polypropylene (PP), etc.),
polymers, TEFLON.RTM., Kapton or any other suitable material. In
some implementation, the first and/or the second substrate [310]
and [320] may be transparent. In other implementations, the first
and/or the second substrate [310] and [320] may be opaque. As shown
in FIG. 3, the second substrate [320] (e.g., a top substrate)
defines a fluidic inlet [323] for communicating to the microfluidic
channels [138], and a fluidic outlet [325] for allowing the fluid
to be expelled from the microfluidic channels [138]. While shown as
including a single fluid inlet [323] and a single fluidic outlet
[325], in various implementations, a plurality of fluidic inlets
and/or fluidic outlets may be defined in the second substrate
[320]. Furthermore, fluidic inlets and/or outlets may also be
provided in the first substrate [310] (e.g., a bottom substrate).
In particular implementations, the first substrate [310] may be
significantly thicker than the second substrate [320]. For example
the first substrate [310] may have a thickness in a range of about
350 to about 500 microns (e.g., about 350, about 400, about 450 or
about 500 microns inclusive of all ranges and values therebetween),
and the second substrate [320] may have a thickness in a range of
about 50 to about 200 microns (e.g., about 50, about 100, about 150
or about 200 microns inclusive of all ranges and values
therebetween).
[0072] The first substrate [310] includes a first resin layer [312]
disposed on a surface [311] thereof facing the interposer [130].
Furthermore, a second resin layer [322] is disposed on a surface
[321] of the second substrate [320] facing the interposer [130].
The first and second resin layers [312] and [322] may include, for
example, polymethyl methacrylate (PMMA), polystyrene, glycerol
1,3-diglycerolate diacrylate (GDD), Ingacure 907, rhodamine 6G
tetrafluoroborate, a UV curable resin (e.g., a novolac epoxy resin,
PAK-01, etc.) any other suitable resin or a combination thereof. In
particular implementations, the resin layers [312] and [322] may
include a nanoimprint lithography (NIL) resin (e.g., PMMA).
[0073] In various implementations, the resin layers [312] and [322]
may be less than about 1 micron thick and are bonded to the
respective first and second adhesive layers [134] and [136]. The
first and second adhesive layers [134] and [136] are formulated
such that a bond between each of the resin layers [312] and [322]
and the respective first and second adhesive layers [134] and [136]
is adapted to withstand a shear stress of greater than about 50
N/cm.sup.2 and a peel force of greater than about 1 N/cm. Thus, the
adhesive layers [134] and [136] form a sufficiently strong bond
directly with the respective substrate [310] and [320] or the
corresponding resin layers [312] and [322] disposed thereon.
[0074] A plurality of wells [314] is formed in the first resin
layer [312] by NIL. A plurality of wells [324] may also be formed
in the second resin layer [322] by NIL. In other implementations,
the plurality of wells [314] may be formed in the first resin layer
[312], the second resin layer [322], or both. The plurality of
wells may have diameter or cross-section of about 50 microns or
less. A biological probe (not shown) may be disposed in each of the
plurality of wells [314] and [324]. The biological probe may
include, for example, DNA probes, RNA probes, antibodies, antigens,
enzymes or cells. In some implementations, chemical or biochemical
analytes may be additionally or alternatively disposed in the
plurality of wells [314] and [324].
[0075] In some implementations, the first and/or second resin
layers [312] and [322] may include a first region and a second
region. The first region may include a first polymer layer having a
first plurality of functional groups providing reactive sites for
covalent bonding of a functionalized molecule (e.g., a biological
probe such as an oligonucleotide). The first and/or second resin
layers [312] and [322] also may have a second region that includes
the first polymer layer and a second polymer layer, the second
polymer layer being on top of, directly adjacent to, or adjacent to
the first polymer layer. The second polymer layer may completely
cover the underlying first polymer layer, and may optionally
provide a second plurality of functional groups. It should also be
realized that the second polymer layer may cover only a portion of
the first polymer layer in some implementations. In some
implementations the second polymer layer covers a substantial
portion of the first polymer layer, wherein the substantial portion
includes greater than about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
or about 99% coverage of the first polymer layer, or a range
defined by any of the two preceding values. In some
implementations, the first and the second polymer layers do not
comprise silicon or silicon oxide.
[0076] In some implementations, the first region is patterned. In
some implementations, the first region may include micro-scale or
nano-scale patterns. In some such implementations, the micro-scale
or nano-scale patterns first and/or second resin layers [312] and
[322] channels, trenches, posts, wells, or combinations thereof.
For example, the pattern may include a plurality of wells or other
features that form an array. High density arrays are characterized
as having features separated by less than about 15 .mu.m. Medium
density arrays have features separated by about 15 to about 30
.mu.m, while low density arrays have sites separated by greater
than about 30 .mu.m. An array useful herein can have, for example,
features that are separated by less than about 100 .mu.m, about 50
.mu.m, about 10 .mu.m, about 5 .mu.m, about 1 .mu.m, or about 0.5
.mu.m, or a range defined by any of the two preceding values.
[0077] In particular implementations, features defined in the first
and/or second resin layer [312] and [322] can each have an area
that is larger than about 100 nm.sup.2, about 250 nm.sup.2, about
500 nm.sup.2, about 1 .mu.m.sup.2, about 2.5 .mu.m.sup.2, about 5
.mu.m.sup.2, about 10 .mu.m.sup.2, about 100 .mu.m.sup.2, or about
500 .mu.m.sup.2, or a range defined by any of the two preceding
values. Alternatively or additionally, features can each have an
area that is smaller than about 1 mm.sup.2, about 500 .mu.m.sup.2,
about 100 .mu.m.sup.2, about 25 .mu.m.sup.2, about 10 .mu.m.sup.2,
about 5 .mu.m.sup.2, about 1 .mu.m.sup.2, about 500 nm.sup.2, or
about 100 nm.sup.2, or a range defined by any of the two preceding
values.
[0078] As shown in FIG. 3, the first and/or second resin layers
[312] and [322] include a plurality of wells [314] and [324] but
may also include other features or patterns that include at least
about 10, about 100, about 1.times.10.sup.3, about
1.times.10.sup.4, about 1.times.10.sup.5, about 1.times.10.sup.6,
about 1.times.10.sup.7, about 1.times.10.sup.8, about
1.times.10.sup.9 or more features, or a range defined by any of the
two preceding values. Alternatively or additionally, first and/or
second resin layers [312] and [322] can include at most about
1.times.10.sup.9, about 1.times.10.sup.8, about 1.times.10.sup.7,
about 1.times.10.sup.6, about 1.times.10.sup.5, about
1.times.10.sup.4, about 1.times.10.sup.3, about 100, about 10 or
fewer features, or a range defined by any of the two preceding
values. In some implementations an average pitch of the patterns
defined in the first and/or second resin layers [312] and [322] can
be, for example, at least about 10 nm, about 0.1 .mu.m, about 0.5
.mu.m, about 1 .mu.m, about 5 .mu.m, about 10 .mu.m, about 100
.mu.m or more, or a range defined by any of the two preceding
values. Alternatively or additionally, the average pitch can be,
for example, at most about 100 .mu.m, about 10 .mu.m, about 5
.mu.m, about 1 .mu.m, about 0.5 .mu.m, about 0.1 .mu.m or less, or
a range defined by any of the two preceding values.
[0079] In some implementations, the first region is hydrophilic. In
some other implementations, the first region is hydrophobic. The
second region can, in turn be hydrophilic or hydrophobic. In
particular cases, the first and second regions have opposite
character with regard to hydrophobicity and hydrophilicity. In some
implementations, the first plurality of functional groups of the
first polymer layer is selected from C.sub.8-14 cycloalkenes, 8 to
14 membered heterocycloalkenes, C.sub.8-14 cycloalkynes, 8 to 14
membered heterocycloalkynes, alkynyl, vinyl, halo, azido, amino,
amido, epoxy, glycidyl, carboxyl, hydrazonyl, hydrazinyl, hydroxy,
tetrazolyl, tetrazinyl, nitrile oxide, nitrene, nitrone, or thiol,
or optionally substituted variants and combinations thereof. In
some such implementations, the first plurality of functional groups
is selected from halo, azido, alkynyl, carboxyl, epoxy, glycidyl,
norbornene, or amino, or optionally substituted variants and
combinations thereof.
[0080] In some implementations, the first and/or second resin
layers [312] and [322] may include a photocurable polymer
composition containing a silsesquioxane cage (also known as a
"POSS"). An example of POSS can be that described in Kehagias et
al., Microelectronic Engineering 86 (2009), pp. 776-778, which is
incorporated by reference herein in its entirety. In some cases, a
silane may be used to promote adhesion between the substrates [310]
and [320] and their respective resin layers [312] and [322]. The
ratio of monomers within the final polymer (p:q:n:m) may depend on
the stoichiometry of the monomers in the initial polymer
formulation mix. The silane molecule contains an epoxy unit which
can be incorporated covalently into the first and lower polymer
layer contacting the substrates [310] or [320]. The second and
upper polymer layer included in the first and/or second resin
layers [312] and [322] may be deposited on a semi-cured first
polymer layer which may provide sufficient adhesion without the use
of a silane. The first polymer layer will naturally propagate
polymerization into the monomeric units of the second polymer layer
covalently linking them together.
[0081] The alkylene bromide groups in the well [314] and [324]
walls may act as anchor points for further spatially selective
functionalization. For example, the alkylene bromide groups may be
reacted with sodium azide to create an azide coated well [314] and
[324] surface. This azide surface could then be used directly to
capture alkyne terminated oligos, for example, using copper
catalyzed click chemistry, or bicyclo[6.1.0] non-4-yne (BCN)
terminated oligos using strain promoted catalyst-free click
chemistry. Alternatively, sodium azide can be replaced with a
norbornene functionalized amine or similar ring-strained alkene or
alkyne, such as dibenzocyclooctynes (DIBCO) functionalized amine to
provide strained ring moiety to the polymer, which can subsequently
undergoing catalyst-free ring strain promoted click reaction with a
tetrazine functionalized oligos to graft the primers to
surface.
[0082] Addition of glycidol to the second photocurable polymer
composition may yield a polymer surface with numerous hydroxyl
groups. In other implementations, the alkylene bromide groups may
be used to produce a primary bromide functionalized surface, which
can subsequently be reacted with 5-norbornene-2-methanamine, to
create a norbornene coated well surface. The azide containing
polymer, for example,
poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM),
may then be coupled selectively to this norbornene surface
localized in the wells [314] and [324], and further be grafted with
alkyne terminated oligos. Ring-strained alkynes such as BCN or
DIBCO terminated oligos may also be used in lieu of the alkyne
terminated oligos via a catalyst-free strain promote cycloaddition
reaction. With an inert second polymer layer covering the
interstitial regions of the substrate, the PAZAM coupling and
grafting is localized to the wells [314] and [324]. Alternatively,
tetrazine terminated oligos may be grafted directly to the polymer
by reacting with the norbornene moiety, thereby eliminating the
PAZAM coupling step.
[0083] In some implementations, the first photocurable polymer
included in the first and/or second resin layers [312] and [322]
may include an additive. Various non-limiting examples of additives
that may be used in the photocurable polymer composition included
in the first and/or second resin layer [312] and [322] include
epibromohydrin, glycidol, glycidyl propargyl ether,
methyl-5-norbornene-2,3-dicarboxylic anhydride, 3-azido-1-propanol,
tert-butyl N-(2-oxiranylmethyl)carbamate, propiolic acid,
11-azido-3,6,9-trioxaundecan-1-amine, cis-epoxysucclmc acid,
5-norbornene-2-methylamine, 4-(2-oxiranylmethyl)morpholine,
glycidyltrimethylammonium chloride, phosphomycin disodium salt,
poly glycidyl methacrylate, poly(propylene glycol) diglycidyl
ether, poly(ethylene glycol) diglycidyl ether,
poly[dimethylsiloxane-co-(2-(3,4-epoxycyclohexyl)ethyl)methylsiloxane],
poly[(propylmethacryl-heptaisobutyl-PS S)-co-hydroxyethyl
methacrylate], poly[(propylmethacryl-heptaisobutyl-PSS)-co-(t-butyl
methacrylate)],
[(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane,
trans-cyclohexanediolisobutyl POSS, aminopropyl isobutyl POSS, octa
tetramethylammonium POSS, poly ethylene glycol POSS, octa
dimethylsilane POSS, octa ammonium POSS, octa maleamic acid POSS,
trisnorbornenylisobutyl POSS, fumed silica, surfactants, or
combinations and derivatives thereof.
[0084] Referring to the interposer [130] of FIG. 3, the
microfluidic channels [138] of the interposer [130] are configured
to deliver a fluid to the plurality of wells [314] and [324]. For
example, the interposer [130] may be bonded to the substrates [310]
and [320] such that the microfluidic channels [138] are aligned
with the corresponding wells [314] and [324]. In some
implementations, the microfluidic channels [138] may be structured
to deliver the fluid (e.g., blood, plasma, plant extract, cell
lysate, saliva, urine, etc.), reactive chemicals, buffers,
solvents, fluorescent labels, or any other solution to each of the
plurality of wells [314] and [324] sequentially or in parallel.
[0085] The flow cells described herein may be particularly amenable
to batch fabrication. For example, FIG. 4A is a top perspective
view of a wafer assembly [40] including a plurality of flow cells
[400]. FIG. 4B shows a side cross-section view of the wafer
assembly [40] taken along the line A-A in FIG. 4A. The wafer
assembly [40] includes a first substrate wafer [41], a second
substrate wafer [42], and an interposer wafer [43] interposed
between the first and second substrate wafers [41], [42]. As shown
in FIG. 4B the wafer assembly [40] includes a plurality of flow
cells [400]. The interposer wafer [43] includes a base layer [432]
(e.g., the base layer [132]), a first adhesive layer [434] (e.g.,
the first adhesive layer [134]) bonding the base layer [432] to a
surface of the first substrate wafer [41], and a second adhesive
layer [436] e.g., the second adhesive layer [136]) bonding the base
layer [432] to a surface of the second substrate wafer [42].
[0086] A plurality of microfluidic channels [438] is defined
through each of the base layer [432] and the first and second
adhesive layers [434] and [436]. A plurality of wells [414] and
[424] may be defined on each of the first substrate wafer [41] and
the second substrate wafer [42] (e.g., etched in the substrate
wafers [41] and [42], or defined in a resin layer disposed on the
surfaces of the substrate wafers [41] and [42] facing the
interposer wafer [43]. A biological probe may be disposed in each
the plurality of wells [414] and [424]. The plurality of wells
[414] and [424] is fluidly coupled with corresponding microfluidic
channels [438] of the interposer wafer [43]. The wafer assembly
[40] may then be diced to separate the plurality of flow cells
[400] from the wafer assembly [40]. In various implementations, the
wafer assembly [40] may provide a flow cell yield of greater than
about 90%.
[0087] FIG. 5 is flow diagram of a method [500] for fabricating
microfluidic channels in an interposer (e.g., the interposer [130],
[230]) of a flow cell (e.g., the flow cell [100], [300], [400]),
according to an implementation. The method [500] includes forming
an interposer, at [502]. The interposer (e.g., the interposer
[130], [230]) includes a base layer (e.g., the baser layer [132])
having a first surface and a second surface opposite the first
surface. The base layer includes black PET (e.g., at least about
50% black PET, consisting essentially of black PET, or consisting
of black PET). A first adhesive layer (e.g., the first adhesive
layer [134]) is disposed on the first surface of the base layer,
and a second adhesive layer (e.g., the second adhesive layer [136])
is disposed on the second surface of the base layer. The first and
second adhesive layer include an acrylic adhesive (e.g., at least
about 10% acrylic adhesive, at least about 50% acrylic adhesive,
consisting essentially of acrylic adhesive, or consisting of
acrylic adhesive). In some implementations, the adhesive may
include butyl-rubber. The base layer may have a thickness of about
30 to about 100 microns, and each of the first and second adhesive
layer may have a thickness of about 10 to about 50 microns such
that the interposer (e.g., the interposer [130]) may have a
thickness in a range of about 50 to about 200 microns.
[0088] A first release line (e.g., the first release liner [237])
may be disposed on the first adhesive layer, and a second release
liner (e.g. the second release liner [239]) may be disposed on the
second adhesive layer. The first and second release liners may be
formed from paper (e.g., super calendared Kraft (SCK) paper, SCK
paper with polyvinyl alcohol coating, clay coated Kraft paper,
machine finished Kraft paper, machine glazed paper, polyolefin
coated Kraft papers, etc.), plastic (e.g., biaxially oriented PET
film, biaxally oriented polypropylene film, polyolefins, high
density polyethylene, low density polyethylene, polypropylene
plastic resins, etc.), fabrics (e.g., polyester), nylon, Teflon or
any other suitable material. In some implementations, the release
liners may be formed from a low surface energy material (e.g., any
of the materials described herein) to facilitate peeling of the
release liners from their respective adhesive layers. In other
implementations, a low surface energy materials (e.g., a silicone,
wax, polyolefin, etc.) may be coated at least on a surface of the
release liners disposed on the corresponding adhesive layers [134]
and [136] to facilitate peeling of the release liners [237] and
[239] therefrom. The first release liner may have a thickness in a
range of about 50 to about 300 microns (e.g., about 50, about 100,
about 150, about 200, about 250, or about 300 microns, inclusive)
and in some implementations, may be substantially optically opaque.
Furthermore, the second release liner may have a thickness in a
range of about 25 to about 50 microns (e.g., about 25, about 30,
about 35, about 40, about 45, or about 50 microns, inclusive) and
may be substantially transparent.
[0089] At [504], microfluidic channels are formed through at least
the base layer, the first adhesive layer, and the second adhesive
layer. In some implementations in the step of forming the
microfluidic channels, the microfluidic channels are formed using a
CO.sub.2 laser. In some implementations, the microfluidic channels
are further formed through the second release liner using the
CO.sub.2 laser, but are not formed through the first release liner
(though in other implementations, the microfluidic channels can
extend partially into the first release liner). The CO.sub.2 laser
may have a wavelength in a range of about 5,000 nm to about 15,000
nm, and a beam size in a range of about 50 to about 150 .mu.m. For
example, the CO.sub.2 laser may have a wavelength in a range of
about 3,000 to about 6,000 nm, about 4,000 to about 10,000 nm,
about 5,000 to about 12,000 nm, about 6,000 to about 14,000 nm,
about 8,000 to about 16,000 nm or about 10,000 to about 18,000 nm.
In particular implementations, the CO.sub.2 laser may have a
wavelength of about 5,000, about 6,000, about 7,000, about 8,000,
about 9,000, about 10,000, about 11,000, about 12,000, about
13,000, about 14,000 or about 15,000 nm inclusive of all ranges and
values therebetween. In some implementations, the CO.sub.2 laser
may have a beam size of about 40 to about60 .mu.m, about 60 to
about 80 .mu.m, about 80 to about 100 .mu.m, about 100 to about 120
.mu.m, about 120 to about 140 .mu.m or about 140 to about 160
.mu.m, inclusive. In particular implementations, may have a beam
size of about 50, about 60, about 70, about 80, about 90, about
100, about 110, about 120, about 130, about 140 or about 150 .mu.m
inclusive of all ranges and values therebetween.
[0090] As previously described herein, various lasers may be used
to form the microfluidic channels in the interposer. Important
parameters include cutting speed which defines total fabrication
time, edge smoothness which is a function of the beam size and
wavelength of the laser and chemical changes caused by the laser to
the various layers included in the interposer which is a function
of the type of the laser. UV pulsed lasers may provide a smaller
beam size, therefore providing smoother edges. However, UV lasers
may cause changes in the edge chemistry of the adhesive layers, the
base layer or debris from the second release liner that may cause
auto-fluorescence. The auto-fluorescence may contribute
significantly to the fluorescence background signal during
fluorescent imaging of a flow cell which includes the interposer
described herein, thereby significantly reducing SNR. In contrast,
a CO.sub.2 laser may provide a suitable edge smoothness, while
being chemically inert, therefore not causing any chemical changes
in the adhesive layers, the base layer or any debris generated by
the second release liner. Thus, forming the microfluidic channels
in the interposer using the CO.sub.2 laser does not contribute
significantly to auto-fluorescence and yields higher SNR.
[0091] Non-Limiting Experimental Examples
[0092] This section describes various experiments demonstrating the
low auto-fluorescence and superior adhesiveness of adhesiveness of
an acrylic adhesive. The experimental examples described herein are
only illustrations and should not be construed as limiting the
disclosure in any way.
[0093] Material Properties: Properties of various materials to bond
a flow cell and produce high quality sequencing data with low cost
were investigated. Following properties are of particular
importance: 1) No or low auto-fluorescence: gene sequencing is
based on fluorescence tags attached to nucleotides and the signal
from these tags are relative weak than normal. No light emitted or
scattered from the edge of bonding materials is desirable to
improve the signal to noise ratio from the DNA cluster with
fluorophores; (2) Bonding strength: Flow cells are often exposed to
high pressure (e.g., 13 psi or even higher). High bonding strength
including peel and shear stress is desirable for flow cell bonding;
(3) Bonding quality: High bonding quality without voids and leakage
is the desirable for high quality flow cell bonding; (4) Bonding
strength after stress: Gene sequencing involves a lot of buffers
(high pH solutions, high salt and elevated temperature) and may
also include organic solvents. Holding the flow cells substrates
(e.g., a top and bottom substrate) together under such stress is
desirable for a successful sequencing run; (5) Chemical stability:
It is desirable that the adhesive layers and the base layer are
chemically stable and do not release (e.g., out gas) any chemical
into the solutions because the enzymes and high purity nucleotides
used in gene sequencing are very sensitive to any impurity in the
buffer.
[0094] Flow Cell Configurations: Pressure sensitive adhesives (PSA)
were applied to two different flow cell configurations as shown in
FIGS. 6A and 6B. FIG. 6A is a schematic illustration of a
cross-section of a bonded and patterned flow cell, i.e., a flow
cell including wells patterned in a NIL resin disposed on a surface
of glass substrates having an interposer bonded therebetween, and
FIG. 6B is a schematic illustration of a cross-section of a bonded
un-patterned flow cell having an interposer bonded directly to the
glass substrate (i.e., does not have a resin on the substrates).
FIG. 6A demonstrates the configuration on patterned flow cell with
100 micron thickness adhesive tape formed from about 25 micron
thick pressure sensitive adhesives (PSAs) on about 50 micron thick
black PET base layer. The patterned surface containing low surface
energy materials which showed low bonding strength for some of the
PSAs.
[0095] Material Screening Process: There were 48 different
screening experiments for the full materials screening process. In
order to screen the adhesive and carrier materials in high
throughput, the screening processes were divided into five
different priorities as summarized in Table I. Many adhesives
failed after stage 1 tests. The early failures enabled screening of
a significant number of materials (>20) in a few weeks.
TABLE-US-00001 TABLE I Material screening process. Priority # Test
Type Surface Type Method 1 1 Optical Fluorescence(532 nm) /
Typhoon, 450PMT BPG1 filter 1 2 Optical Fluorescence(635 nm) /
Typhoon, 475PMT LPR filter 1 3 Adhesion Lap shear(N/cm.sup.2) Glass
Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi Lamination, 3 day cure 1
4 Adhesion Peel(N/cm) Glass Kapton, 5 .times. 10 mm, 40 mm/min, 20
psi lamination, 3 day cure 1 5 Adhesion Easy to bond Glass Visual
check for voids after bond 1 6 FTIR FTIR Glass 4000-500 cm-1, FTIR-
ATR 1 7 Buffer Stress Lap shear(N/cm.sup.2) Glass 3 day, pH 10.5,
1M NaCl, 0.05% tween 20, 60 degrees Celsius. Kapton, 5 .times. 10
mm, 40 mm/min, 20 psi lamination 1 8 Buffer Stress Peel(N/cm) Glass
3 day, pH 10.5, 1M NaCl, 0.05% tween 20, 60 degrees Celsius.
Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi lamination 1 9
Dimensions Thickness (um) / Adhesive, liner and carrier thickness
by micrometer 2 10 Adhesion Lap shear(N/cm.sup.2) NIL Kapton, 5
.times. 10 mm, 40 mm/min, 20 psi lamination 2 11 Adhesion
Peel(N/cm) NIL Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi
lamination 2 12 Buffer Stress Lap shear(N/cm.sup.2) NIL 3 day, pH
10.5, 1M NaCl, 0.05% tween 20, 60 degrees Celsius Kapton, 5 .times.
10 mm, 5 mm/min, 20 psi lamination 2 13 Buffer Stress Peel(N/cm)
NIL pH 10.5, 1M NaCl, 0.05% tween 20, 60 degrees Celsius Kapton, 5
.times. 10 mm, 5 mm/min, 20 psi lamination 2 14 Formamide Lap
shear(N/cm.sup.2) Glass 24 hr, 60 degrees Celsius, stress
formamide. Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi lamination 2
15 Formamide Peel(N/cm) Glass 24 hr, 60 degrees Celsius, stress
formamide. Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi lamination 2
16 Vacuum Voids Glass 24 hr, 60 degrees Celsius, Vacuum, 5 .times.
20 mm adhesive bonded glass on both sides, Nikon imaging system 3
17 Formamide Lap shear(N/cm.sup.2) NIL 24 hr, 60 degrees Celsius,
stress formamide. Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi
lamination 3 18 Formamide Peel(N/cm) NIL 24 hr, 60 degrees Celsius,
stress formamide. Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi
lamination 3 19 Vacuum Voids NIL 24 hr, 60 degrees Celsius, Vacuum,
5 .times. 20 mm adhesive bonded glass on both sides, Nikon imaging
system 3 20 Overflow, Overflow, Laser cut Glass 10x Microscope
image Laser cut 3 21 Overflow, Overflow, Plot cut Glass 10x
Microscope image Plot cut 3 22 Swell in Thermogravimetric / 24 hr
buffer soaking at 60 Buffer analysis (TGA) degrees Celsius, TGA 32-
200 C., 55 Celsius/min, calculate weight loss 3 23 Swell in TGA /
24 hr formamide soaking Formamide at 60 degrees Celsius, TGA 32-200
Celsius, 5 C./min, calculate weight loss 3 24 Solvent TGA / TGA
32-200 Celsius and Outgas FTIR 3 25 4 degrees Lap shear(N/cm.sup.2)
Glass 24 hr 4 Celsius. Kapton, Celsius 5 .times. 10 mm, 40 mm/min,
stress 20 psi lamination, 3 day cure 3 26 4 degrees Peel(N/cm)
Glass 24 hr 4 degrees Celsius, Celsius Kapton, 5 .times. 10 mm,
stress 40 mm/min, 20 psi lamination, 3 day cure 3 27 -20 degrees
Lap shear(N/cm.sup.2) Glass 24 hr -20 degrees Celsius, Celsius
Kapton, 5 .times. 10 mm, stress 40 mm/min, 20 psi lamination, 3 day
cure 3 28 -20 degrees Peel(N/cm) Glass 24 hr -20 degrees Celsius,
Celsius Kapton, 5 .times. 10 mm, stress 40 mm/min, 20 psi
lamination, 3 day cure 4 29 Vacuum Lap shear(N/cm.sup.2) Glass 24
hr, 60 degrees Celsius, vacuum, Kapton, 5 .times. 10 mm, 40 mm/min,
20 psi lamination, 3 day cure 4 30 Vacuum Peel(N/cm) Glass 24 hr,
60 degrees Celsius, vacuum, Kapton, 5 .times. 10 mm, 40 mm/min, 20
psi lamination, 3 day cure 4 31 Vacuum Lap shear(N/cm.sup.2) NIL 24
hr, 60 degrees Celsius, vacuum, Kapton, 5 .times. 10 mm, 40 mm/min,
20 psi lamination, 3 day cure 4 32 Vacuum peel(N/cm) NIL 24 hr, 60
degrees Celsius, vacuum, Kapton, 5 .times. 10 mm, 40 mm/min, 20 psi
lamination, 3 day cure 5 33 Curing Time Lap shear(N/cm.sup.2) Glass
1 day 5 34 Curing Time Lap shear(N/cm.sup.2) Glass 2 day 5 35
Curing Time Lap shear(N/cm.sup.2) Glass 3 day 5 36 Curing Time
Peel(N/cm) Glass 1 day 5 37 Curing Time Peel(N/cm) Glass 2 day 5 38
Curing Time Peel(N/cm) Glass 3 day 5 39 Curing Time Lap
shear(N/cm.sup.2) NIL 1 day 5 40 Curing Time Lap shear(N/cm.sup.2)
NIL 2 day 5 41 Curing Time Lap shear(N/cm.sup.2) NIL 3 day 5 42
Curing Time Peel(N/cm) NIL 1 day 5 43 Curing Time Peel(N/cm) NIL 2
day 5 44 Curing Time Peel(N/cm) NIL 3 day 5 45 Outgas GC-MS / 60
degrees Celsius 1 hr and GC-MS 5 46 Chemical DNA sequencing Glass
PR2, 60 degrees Celsius, leaching 24 hr baking, pumping between
each cycles 5 47 Sequencing DNA sequencing Glass PR2, 60 degrees
Celsius, by synthesis 24 hr baking, pumping compatibility between
each cycles 5 48 Thermal Peel(N/cm) Glass -20 C. to 100 degrees
Cycle Celsius
[0096] Auto-fluorescence properties: The auto-fluorescence
properties were measured by confocal fluorescence scanner (Typhoon)
with green (532 nm) and red (635 nm) laser as excitation light
source. A 570 nm bandpass filter was used for green laser and a 665
long pass filter was used for red laser. The excitation and
emission set up was similar to that used in an exemplary gene
sequencing experiment. FIG. 7 is a bar chart of fluorescence
intensity in the red channel of various adhesives and flow cell
materials. FIG. 8 is a bar chart of fluorescence intensity in the
green channel of the various adhesives and flow cell materials of
FIG. 7. Table II summarizes the auto-fluorescence from each of the
materials.
TABLE-US-00002 TABLE II Auto-fluorescence measurements summary.
Name Fluorescence (532 nm) Fluorescence (635 nm) Tape Sample 1 102
72 Tape Sample 2 176 648 Tape Sample 2-Base 82 514 layer only Tape
Sample 3 238 168 Tape Sample 4-Base 83 81 layer only ND-C 130 77
Acrylic adhesive 68 70 PET-3 71 70 PET-1 76 77 PET-2 69 70 Tape
Sample-5 114 219 Tape Sample-6 / / Kapton 1 252 354 Kapton 2 92 113
Kapton 3 837 482 Black Kapton 100 100 Polyether ketone 3074 2126
(PEEK) Glass 61 62 Adhesive tape 100 100 Reference 834 327 Ref 777
325 BJK 100 100 Acrylic adhesive- 76.3 161.4 Batch 2 Acrylic
adhesive-75 75.2 76.4 microns thick Acrylic adhesive-65 75.6 76.8
microns thick Tape Sample 7 74.2 73.2 Tape Sample 8 99.7 78.3
[0097] Tape Samples 1-4 and 7-8 were adhesives including thermoset
epoxies, the Tape Sample-5 adhesive include a butyl rubber
adhesive, and Tape Sample-6 includes an acrylic/silicone base film.
As observed from FIGS. 7, 8 and Table II, the Black Kapton
(polyimide) and Glass were employed as negative control. In order
to meet the low fluorescence requirement in this experiment, any
qualified material should emit less light than Black Kapton. Only a
few adhesives or carriers pass this screening process including
methyl acrylic adhesive, PET-1, PET-2, PET-3, Tape Sample 7 and
Tape Sample 8. Most of the carrier materials such as Kapton 1, PEEK
and Kapton 2 failed due to high fluorescence background. The
acrylic adhesive has an auto-fluorescence in response to a 532 nm
excitation wavelength of less than about 0.25 a.u. relative to a
532 nm fluorescence standard (FIG. 7), and has an auto-fluorescence
in response to a 635 nm excitation wavelength of less than about
0.15 a.u. relative to a 635 nm fluorescence standard (FIG. 8),
which is sufficiently low to be used in flow cells.
[0098] Adhesion with and without stress: The bonding quality,
especially adhesion strength, should be evaluated for flow cell
bonding. The lap shear stress and 180 degree peel test were
employed to quantify the adhesion strength. FIGS. 9A and 9B show
the lap shear and peel test setups used to test the lap shear and
peel stress of the various adhesives. As show in FIGS. 9A and 9B,
the adhesive stacks were assembly in sandwich structure. The bottom
surface is glass or NIL surface which is similar to a flow cell
surface. On the top of adhesive is thick Kapton film which
transfers the force from instrument to adhesive during shear or
peel test. Table III summarizes results from the shear and peel
tests.
TABLE-US-00003 TABLE III Shear and Peel Test Results Unit N/cm
N/cm.sup.2 Peel Lap Lap on Shear Lap Shear Peel NIL Easy Lap after
Shear NIL after Peel on after to Name Shear Stress NIL Stress Peel
Stress NIL Stress Bond Sample 1 113 .+-. 1.3 51 .+-. 1.1 66.7 77
9.2 .+-. 3.4 0.25 .+-. 0.11 0.73 .+-. 0.28 2.1 .+-. 0.38 + ND-C 131
.+-. 4.7 122 .+-. 1.4 / / 5.1 .+-. 0.2 2.5 .+-. 0.2 / / ++ Acrylic
111.7 .+-. 1.8 74.8 .+-. 0.4 65.2 .+-. 1.8 49.2 .+-. 7.0 3.6 .+-.
0.4 3.8 .+-. 0.6 3.35 .+-. 0.52 2.6 .+-. 0.16 +++ Adhesive PET-3
106.2 .+-. 0.6 117.5 .+-. 4.5 / / 0.6 .+-. 1.8 4.6 .+-. 1.4 / / -
PET-1 90.9 .+-. 8.3 96.4 .+-. 4.0 / / 0.4 .+-. 0.2 1.9 .+-. 0.2 / /
- PET-5 100.5 .+-. 2.9 98.1 .+-. 1.2 / / 0.9 .+-. 0.4 6.3 .+-. 0.8
/ / - Tape 49.8 .+-. 3.3 24.8 .+-. 2.1 / / 1.8 .+-. 0.1 0.53 .+-.
0.08 / / - Sample-5 Tape 89.8 .+-. 4.4 24.1 .+-. 0.6 56.4 .+-. 1.4
13.5 1.6 .+-. 0.1 0.71 .+-. 0.29 0.75 .+-. 0.17 Fell + Sample 6
apart Adhesive 500 .+-. 111 tape
[0099] The initial adhesion of the adhesives test is shown in Table
III. Most of the adhesives meet the minimum requirements (i.e.,
demonstrate >50 N/cm.sup.2 shear stress and >1 N/cm peel
force) on glass surface except PET-1, PET-2 and PET-3 which failed
in peel test and also have voids after bonding. The Tape Sample 1
adhesive has relatively weak peel strength on NIL surface and
failed in the test. The adhesives were also exposed to high salt
and high pH buffer (1M NaCl, pH 10.6 carbonate buffer and 0.05%
tween 20) at about 60 degrees Celsius for 3 days as a stress test.
Tape Sample 5 and Tape Sample 1 lost more than about 50% of lap
shear stress and peel strength. After the auto-fluorescence and
bonding strength screening, the acrylic adhesive was the leading
adhesive demonstrating all the desirable characteristics. ND-C was
the next best material and showed about 30% higher background in
red fluorescence channel relative to the acrylic adhesive.
[0100] Formamide, high temperature and low temperature stress: To
further evaluate the performance of the adhesive in the application
of flow cell bonding, more experiments were conducted on the
acrylic, Tape Sample 5 and Tape Sample 1 adhesives. These included
soaking in formamide at about 60 degrees Celsius for about 24
hours, cold storage at about -20 degrees Celsius and about 4
degrees Celsius for about 24 hour and vacuum baking at about 60
degrees Celsius for about 24 hour. All of the results are
summarized in Table IV.
TABLE-US-00004 TABLE IV Summary of formamide, high temperature and
low temperature stress tests. Acrylic Tape Tape Name Adhesive
Sample 5 Sample 1 Peel test, formamide exposure, 1.41 .+-. 0.2 1.47
.+-. 0.12 60 degrees Celsius for 24 hours Peel test, -20 degrees
for 24 3.36 .+-. 0.5 1.9 .+-. 0.1 hours Peel test, 4 degrees
Celsius for 4.1 .+-. 0.7 2.12 .+-. 0.14 24 hours Peel test, vacuum
bake, 60 3.5 .+-. 0.4 1.3 .+-. 0.3 2.36 degrees Celsius and NIL
resin on substrate Lap shear, formamide exposure, 77.8 .+-. 1.2
61.6 .+-. 4.4 60 degrees Celsius for 24 hours Lap shear, vacuum
bake, 60 68.6 .+-. 2.4 35.7 .+-. 3.6 92.8 degrees Celsius and NIL
resin on substrate Lap shear, -20 degrees Celsius 76.4 .+-. 4.2
63.3 .+-. 1.1 for 24 hours Lap shear, 4 deg. Celsius 24 hr 72.3
.+-. 3.4 69.4 .+-. 5.7
[0101] Both adhesives pass most of the tests. However, Tape Sample
5 adhesive showed a lot of voids developed after vacuum baking and
lost more than 40% of shear stress and didn't meet the minimum
requirement. The acrylic adhesive also lost significant part of
peel strength after formamide stress but still meets the minimum
requirement.
[0102] Solvent outgas and overflow: Many reagents used in gene
sequencing are very sensitive to impurities in the buffers or
solutions which may affect the sequencing matrix. In order to
identify any potential hazard materials released from the
adhesives, thermogravimetric analysis (TGA), Fourier transform
infrared (FTIR) and gas chromatography-mass spectroscopy (GC-MS)
were used to characterize the basic chemical structures of adhesive
and out gas from adhesive. According to TGA measurement, the dry
acrylic, ND-C and Tape Sample 5 adhesives show very little weight
loss (0.5%). Tape Sample 1 showed more than 1% weight loss which
may indicate higher risk of release harmful material during
sequencing run.
[0103] The adhesive weight loss was also characterized after
formamide and buffer stress. Acrylic adhesive showed about 1.29%
weight loss which indicate this adhesive is more suspected to
formamide and aligned with previous stress test in formamide. Tape
Sample 5 showed more weight loss after buffer stress (about 2.6%)
which also explained the poor lap shear stress after buffer stress.
The base polymer of the acrylic adhesive and ND-C were classified
as acrylic by FTIR. Biocompatibility of acrylic polymer is well
known and reduces the possibility of harmful materials being
released during a sequencing run. FIG. 10 is a FTIR spectrum of the
acrylic adhesive and scotch tape. Table V summarize the results of
TGA and FTIR measurements.
TABLE-US-00005 TABLE V Summary of TGA and FTIR measurements.
Acrylic Name adhesive ND-C Fralock-1 3M-EAS2388C TGA(32 to 200
0.41% 0.43% 0.48% 1.06% degrees Celsius TGA after buffer 0.41% /
2.60% / stress TGA after 1.29% / 0.84% / formamide FTIR Acrylic
Acrylic Butyl Rubber Acrylic- Silicone
[0104] To further investigate the outgas from the acrylic adhesive,
acrylic adhesive and Black Kapton were analyzed by GC-MS. Both
samples were incubated at about 60 degrees Celsius for one hour and
outgas from these materials was collected by cold trap and analyzed
by GC-MS. As show in FIG. 11, there is no detectable out gas from
Black Kapton and about 137 ng/mg of total volatiles was detected
from acrylic adhesive after one hour baking at 60 degrees Celsius.
The amount of out gas compounds is very limited and only about
0.014% of the total weight of the acrylic adhesive. All of the out
gas compounds were analyzed by GC-MS, there are all very similar to
each other and originated from acrylic adhesives including
acrylate/methacrylate monomer and aliphatic side chains etc. FIG.
12 demonstrated the typical MS spectra of these out gas compounds
with inset showing the possible chemical structure of the out
gassed compound. Since acrylic and methacrylic adhesives are
generally known to be biocompatible, the small of amount of
acrylate/methacrylate out gas is not expected to have any negative
impact on the gene sequencing reagents.
[0105] The following implementations are encompassed by the present
disclosure:
[0106] 1. An interposer, comprising: a base layer having a first
surface and a second surface opposite the first surface; a first
adhesive layer disposed on the first surface of the base layer; a
second adhesive layer disposed on the second surface of the base
layer; and a plurality of microfluidic channels extending through
each of the base layer, the first adhesive layer, and the second
adhesive layer.
[0107] 2. The interposer of clause 1, wherein: the base layer
comprises black polyethylene terephthalate (PET); the first
adhesive layer comprises acrylic adhesive; the second adhesive
layer comprises acrylic adhesive.
[0108] 3. The interposer of clause 2, wherein a total thickness of
the base layer, first adhesive layer, and second adhesive layer is
in a range of about 1 to about 200 microns.
[0109] 4. The interposer of clause 2 or 3, wherein the base layer
has a thickness in a range of about 10 to about 100 microns, and
each of the first adhesive layer and the second adhesive layer has
a thickness in a range of about 5 to about 50 microns.
[0110] 5. The interposer of any of clauses 1-4, wherein the each of
the first and second adhesive layers has an auto-fluorescence in
response to a 532 nm excitation wavelength of less than about 0.25
a.u. relative to a 532 nm fluorescence standard.
[0111] 6. The interposer of any of the preceding clauses, wherein
the each of the first and second adhesive layers has an
auto-fluorescence in response to a 635 nm excitation wavelength of
less than about 0.15 a.u. relative to a 635 nm fluorescence
standard.
[0112] 7. The interposer of any of clauses 2-6, wherein the base
layer comprises at least about 50% black PET.
[0113] 8. The interposer of clause 7, wherein the base layer
consists essentially of black PET.
[0114] 9. The interposer of any of clauses 2-8, wherein each of the
first and second adhesive layers is comprises at least about 5%
acrylic adhesive.
[0115] 10. The interposer of clause 9, wherein each of the first
and second adhesive layers consists essentially of acrylic
adhesive.
[0116] 11. The interposer of any of the preceding clauses, further
comprising: a first release liner disposed on the first adhesive
layer; a second release liner disposed on the second adhesive
layer; wherein the plurality of microfluidic channels extends
through each of the base layer, the first adhesive layer, and the
second adhesive layer, and the second release liner, but not
through the first release liner.
[0117] 12. The interposer of clause 11, wherein: the first release
liner has a thickness in a range of about 50 to about 300 microns;
and the second release liner has a thickness in a range of about 25
to about 50 microns.
[0118] 13. The interposer of clause 11 or 12, wherein: the base
layer comprises black polyethylene terephthalate (PET); and each of
the first and second adhesive layers comprises acrylic
adhesive.
[0119] 14. The interposer of any of clauses 11-13, wherein the
first release liner is at least substantially opaque and the second
release liner is at least substantially transparent. 15. A flow
cell comprising: a first substrate; a second substrate; and the
interposer of any of clauses 2-10 disposed between the first
substrate and the second substrate, wherein the first adhesive
layer bonds the first surface of the base layer to a surface of the
first substrate, and the second adhesive layer bonds the second
surface of the base layer to a surface of the second substrate.
[0120] 16. The flow cell of clause 15, wherein each of the first
and second substrates comprises glass, and wherein a bond between
each of the first and second adhesive layers and the respective
surfaces of the first and second substrates is adapted to withstand
a shear stress of greater than about 50 N/cm.sup.2 and a peel force
of greater than about 1 N/cm.
[0121] 17. The flow cell of clause 15, wherein each of the first
and second substrates comprises a resin layer that is less than
about one micron thick and includes the surface that is bonded to
the respective first and second adhesive layers, and wherein a bond
between each of the resin layers and the respective first and
second adhesive layers is adapted to withstand a shear stress of
greater than about 50 N/cm.sup.2 and a peel force of greater than
about 1 N/cm.
[0122] 18. The flow cell of clause 17, wherein: a plurality of
wells is imprinted in the resin layer of at least one of the first
substrate or the second substrate, a biological probe is disposed
in each of the wells, and the microfluidic channels of the
interposer are configured to deliver a fluid to the plurality of
wells.
[0123] 19. A method of patterning microfluidic channels,
comprising: forming an interposer comprising: a base layer having a
first surface and a second surface opposite the first surface, the
base layer comprising black polyethylene terephthalate (PET), a
first adhesive layer disposed on the first surface of the base
layer, the first adhesive layer comprising acrylic adhesive, a
second adhesive layer disposed on the second surface of the base
layer, the second adhesive layer comprising acrylic adhesive; and
forming microfluidic channels through at least the base layer, the
first adhesive layer, and the second adhesive layer.
[0124] 20. The method of clause 19, wherein the forming
microfluidic channels involves using a CO.sub.2 laser.
[0125] 21. The method of clause 20, wherein: the interposer further
comprises: a first release liner disposed on the first adhesive
layer, and a second release liner disposed on the second adhesive
layer; and in the step of forming the microfluidic channels, the
microfluidic channels are further formed through the second release
liner using the CO.sub.2 laser, but are not formed through the
first release liner.
[0126] 22. The method of clause 21, wherein the CO.sub.2 laser has
a wavelength in a range of about 5,000 nm to about 15,000 nm, and a
beam size in a range of about 50 to about 150 p.m.
[0127] It should be appreciated that all combinations of the
foregoing concepts and additional concepts discussed in greater
detail below (provided such concepts are not mutually inconsistent)
are contemplated as being part of the inventive subject matter
disclosed herein. In particular, all combinations of claimed
subject matter appearing at the end of this disclosure are
contemplated as being part of the inventive subject matter
disclosed herein
[0128] As used herein, the singular forms "a", "an" and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, the term "a member" is intended to
mean a single member or a combination of members, "a material" is
intended to mean one or more materials, or a combination
thereof.
[0129] As used herein, the terms "about" and "approximately"
generally mean plus or minus 10% of the stated value. For example,
about 0.5 would include 0.45 and 0.55, about 10 would include 9 to
11, about 1000 would include 900 to 1100.
[0130] As utilized herein, the terms "substantially" and similar
terms are intended to have a broad meaning in harmony with the
common and accepted usage by those of ordinary skill in the art to
which the subject matter of this disclosure pertains. It should be
understood by those of skill in the art who review this disclosure
that these terms are intended to allow a description of certain
features described and claimed without restricting the scope of
these features to the precise arrangements and/or numerical ranges
provided. Accordingly, these terms should be interpreted as
indicating that insubstantial or inconsequential modifications or
alterations of the subject matter described and claimed are
considered to be within the scope of the inventions as recited in
the appended claims.
[0131] It should be noted that the term "example" as used herein to
describe various implementations is intended to indicate that such
implementations are possible examples, representations, and/or
illustrations of possible implementations (and such term is not
intended to connote that such implementations are necessarily
extraordinary or superlative examples).
[0132] The terms "coupled" and the like as used herein mean the
joining of two members directly or indirectly to one another. Such
joining may be stationary (e.g., permanent) or moveable (e.g.,
removable or releasable). Such joining may be achieved with the two
members or the two members and any additional intermediate members
being integrally formed as a single unitary body with one another
or with the two members or the two members and any additional
intermediate members being attached to one another.
[0133] It is important to note that the construction and
arrangement of the various exemplary implementations are
illustrative only. Although only a few implementations have been
described in detail in this disclosure, those skilled in the art
who review this disclosure will readily appreciate that many
modifications are possible (e.g., variations in sizes, dimensions,
structures, shapes and proportions of the various elements, values
of parameters, mounting arrangements, use of materials, colors,
orientations, etc.) without materially departing from the novel
teachings and advantages of the subject matter described herein.
Other substitutions, modifications, changes and omissions may also
be made in the design, operating conditions and arrangement of the
various exemplary implementations without departing from the scope
of the present invention.
* * * * *