U.S. patent application number 14/779517 was filed with the patent office on 2016-03-03 for method for treating a semiconductor device.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to James A. BALL, Brian REED.
Application Number | 20160061773 14/779517 |
Document ID | / |
Family ID | 50686202 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160061773 |
Kind Code |
A1 |
BALL; James A. ; et
al. |
March 3, 2016 |
METHOD FOR TREATING A SEMICONDUCTOR DEVICE
Abstract
A sensor array includes a plurality of sensors. A sensor of the
plurality of sensors has a sensor pad exposed at a surface of the
sensor array. A method of treating the sensor array includes
exposing at least the sensor pad to a wash solution including
sulfonic acid and an organic solvent and rinsing the wash solution
from the sensor pad.
Inventors: |
BALL; James A.; (Ledyard,
CT) ; REED; Brian; (Guilford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
50686202 |
Appl. No.: |
14/779517 |
Filed: |
March 28, 2014 |
PCT Filed: |
March 28, 2014 |
PCT NO: |
PCT/US14/32214 |
371 Date: |
September 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61806603 |
Mar 29, 2013 |
|
|
|
61817805 |
Apr 30, 2013 |
|
|
|
Current U.S.
Class: |
134/27 ;
134/28 |
Current CPC
Class: |
C11D 7/34 20130101; C11D
7/5022 20130101; C11D 3/162 20130101; C11D 3/3418 20130101; C11D
7/5027 20130101; B08B 3/08 20130101; G01N 27/4145 20130101; B08B
9/08 20130101; C11D 3/43 20130101; C11D 7/5013 20130101 |
International
Class: |
G01N 27/414 20060101
G01N027/414; C11D 7/50 20060101 C11D007/50; C11D 7/34 20060101
C11D007/34; B08B 3/08 20060101 B08B003/08 |
Claims
1.-53. (canceled)
54. A method of treating a sensor array, the sensor array including
a plurality of sensors, a sensor of the plurality of sensors having
a sensor pad exposed at a surface of the sensor array, the method
comprising: exposing at least the sensor pad to a wash solution
including acid and an organic solvent; and rinsing the wash
solution from the sensor pad.
55. The method of claim 54, wherein the acid includes sulfonic
acid.
56. The method of claim 55, wherein the sulfonic acid includes
alkyl sulfonic acid, alkyl aryl sulfonic acid, or a combination
thereof.
57. The method of claim 56, wherein the alkyl aryl sulfonic acid
includes an alkyl group having between 1 and 20 carbons.
58. The method of claim 55, wherein the sulfonic acid includes
dodecyl benzene sulfonic acid.
59. The method of claim 54, wherein the wash solution includes
between 10 mM and 500 mM of the acid.
60. The method of claim 54, wherein the wash solution includes
between 0.5 wt % and 25 wt % of the acid.
61. The method of claim 54, wherein the organic solvent is
non-polar.
62. The method of claim 54, wherein the organic solvent has a
normal boiling point in a range of 36.degree. C. to 345.degree.
C.
63. The method of claim 54, wherein the organic solvent is an
alkane having between 6 and 24 carbons.
64. The method of claim 54, wherein the organic solvent is a polar
aprotic solvent.
65. The method of claim 64, wherein the polar aprotic solvent
includes tetrahydrofuran, ethylacetate, acetone, dimethylformamide,
acetonitrile, dimethyl sulfoxide, N-methyl pyrrolidone, or
combinations thereof.
66. The method of claim 54, further comprising heating the sensor
pad and the wash solution while exposing the sensor pad to the wash
solution at a temperature in a range of 35.degree. C. to 70.degree.
C.
67. The method of claim 54, wherein exposing includes exposing for
a period in a range of 30 seconds to 30 minutes.
68. The method of claim 54, further comprising exposing at least
the sensor pad to a basic solution following exposing the sensor
pad to the wash solution.
69. The method of claim 68, wherein the basic solution includes
between 0.005M and 1.5M sodium hydroxide.
70. The method of claim 54, wherein rinsing includes rinsing with a
rinse organic solvent miscible with water and the organic
solvent.
71. The method of claim 70, wherein the low boiling organic solvent
includes alcohol.
72. A method of treating a sensor array, the sensor array including
a plurality of sensors, a sensor of the plurality of the sensors
including a sensor pad, a well structure defining a well array
corresponding with the sensor array, a well of the well array
exposing the sensor pad, a cap attached over the sensor array and
the well structure and including an fluid port, a space defined
between the cap and the well structure, the method comprising:
applying a wash solution through the fluid port into the space and
waiting for a first period between 30 seconds and 30 minutes, the
wash solution including acid and an organic solvent; applying a
basic solution through the fluid port into the space and waiting
for a second period between 20 seconds and 15 minutes; and applying
a rinse solution through the fluid port.
73. The method of claim 72, further comprising repeating applying
the wash solution, applying the basic solution, and applying the
rinse solution.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims benefit of U.S. Provisional
Application No. 61/806,603, filed Mar. 29, 2013, which is
incorporated herein by reference in its entirety.
[0002] This application claims benefit of U.S. Provisional
Application No. 61/817,805, filed Apr. 30, 2013, which is
incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0003] This disclosure, in general, relates to methods for treating
sensor arrays, sensor arrays formed by such methods, and solutions
for use in such methods.
BACKGROUND
[0004] Arrays of sensors formed in semiconductor substrates are
increasingly being used in fields such as analytical chemistry and
molecular biology. For example, when analytes are captured on or
near sensor pads of a sensor array, the analytes or byproducts of
reactions associated with the analytes can be detected and used to
elucidate information regarding the analyte. In particular, such
sensor arrays have found use in genetic analysis, such as genetic
sequencing or quantitative amplification.
[0005] During manufacture, various semiconductor processing
techniques can alter the nature of the surface of sensor array and
the surface of well structures around the sensor array. Such
processing can also leave residues on the surface. Altered surface
chemistry or residues can prevent or limit the capture of analytes
proximate to the sensors. As such, the effectiveness of such sensor
arrays is reduced and signals resulting from such sensor arrays may
include erroneous data or no data.
SUMMARY
[0006] In an aspect, a sensor device including a sensor array and
optionally a well array corresponding to the sensor array or a cap
attached over the sensor array can be treated with a wash solution.
The wash solution can include an organic solvent and an acid, such
as a sulfonic acid, e.g., alkyl benzene sulfonic acid. The sensor
device can be further treated with a basic solution, such as a NaOH
solution, or can be rinsed with a low boiling organic solvent or
water. Optionally, the sensor device can be dried.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The present disclosure may be better understood, and its
numerous features and advantages made apparent to those skilled in
the art by referencing the accompanying drawings.
[0008] FIG. 1 includes an illustration of an exemplary measurement
system.
[0009] FIG. 2 includes an illustration of an exemplary measurement
component.
[0010] FIG. 3 includes an illustration of exemplary array of
measurement components.
[0011] FIG. 4 includes an illustration of exemplary well
configurations.
[0012] FIG. 5 includes an illustration of exemplary well and sensor
configurations.
[0013] FIG. 6 and FIG. 7 include illustrations of exemplary sensor
devices.
[0014] FIG. 8, FIG. 9, and FIG. 10 include flow diagrams
illustrating exemplary methods.
[0015] FIG. 11 includes an illustration of exemplary methods for
preparing a sequencing device.
[0016] The use of the same reference symbols in different drawings
indicates similar or identical items.
DETAILED DESCRIPTION
[0017] In an exemplary embodiment, a method of treating a sensor
array includes applying a wash solution to the sensor array and
rinsing the wash solution from the sensor array. In particular, the
wash solution includes an organic solvent and an acid, such as a
sulfonic acid. The sulfonic acid can include an alkyl or alkyl aryl
sulfonic acid. In an example, the alkyl or alkyl aryl sulfonic acid
can have an alkyl group having between 9 and 18 carbons. For
example, the sulfonic acid can include dodecyl benzene sulfonic.
The organic solvent can be a non-polar solvent or an aprotic polar
solvent. In an example, the organic solvent can have a normal
boiling point a range of 65.degree. C. to 275.degree. C. In an
example, the organic solvent includes heptane. In another example,
the organic solvent includes undecane. In an additional example,
the organic solvent includes dimethylformamide, acetonitrile,
dimethyl sulfoxide, or combinations thereof. Following treatment
with the wash solution, the sensor array can be rinsed with a rinse
solution. In an example, the rinse solution includes a low boiling
organic solvent such as alcohol, for example, ethanol or
isopropanol. In another example, the rinse solution can include
water. The method may further include applying a basic solution or
a weak acid solution following application of the wash solution and
before rinsing. The basic solution can have a pH of at least 7,
such as at least 8, and can include a strong base, such as sodium
hydroxide. In an example, the wash solution and the basic solution
can be applied repeatedly, such as 2 or more times, 3 or more times
or even 4 or more times, but generally not greater than 10
times.
[0018] In a particular embodiment, a wash solution is applied to at
least a sensor pad of the sensor array. The sensor array can
include a plurality of sensors. A sensor of the sensor array can
include a sensor pad. Optionally, a well structure can be defined
over the sensor array and include a plurality of wells correspond
with sensor pads of the sensor array. A well of the well array can
expose a sensor pad of a sensor. Optionally, a cap including at
least one fluid port can be disposed or attached over the sensor
array and the well structure. A space for fluid can be defined
between the cap and the well structure or sensor array and can be
in fluid communication with the fluid port of the cap. The wash
solution can be applied through the fluid port and into the space
between the cap and the well structure. Optionally, a basic
solution can be applied through the fluid port to the space between
the cap and the well structure following application of the wash
solution. Application of the wash solution followed by the basic
solution can be repeated, such as at least twice or even at least 3
times. A rinse solution can be applied through the fluid port, for
example, including alcohol or water. Optionally, the system can be
dried.
[0019] In another exemplary embodiment, a sensor array includes a
plurality of sensors. A sensor of the plurality of sensors includes
a sensor pad. A well structure is disposed over the sensor array
and includes a well array that operatively corresponds with the
sensor array. A well of the well array exposes a sensor pad of a
sensor. The wash solution including an acid, such as a sulfonic
acid, and an organic solvent can be applied to the sensor array for
a period between 30 seconds and 30 min. The sensor array can be
rinsed with a rinse solution. In an example, the rinse solution can
include a low boiling organic solvent such as an alcohol. The
sensor array can be rinsed one or more times with the low boiling
organic solvent. In another example or in addition, the sensor
array can be rinsed with water, such as deionized water. The sensor
array can be dried and a cap can be attached over the well
structure and the sensor array. The cap can include at least one
fluid port. A space is defined between the cap and the sensor array
or the well structure and is in fluid communication with the fluid
port.
[0020] In a particular embodiment, a sensor system includes a flow
cell in which a sensory array is disposed, includes communication
circuitry in electronic communication with the sensory array, and
includes containers and fluid controls in fluidic communication
with the flow cell. In an example, FIG. 1 illustrates an expanded
and cross-sectional view of a flow cell 100 and illustrates a
portion of a flow chamber 106. A reagent flow 108 flows across a
surface of a well array 102, in which the reagent flow 108 flows
over the open ends of wells of the well array 102. The well array
102 and a sensor array 105 together can form an integrated unit
forming a lower wall (or floor) of flow cell 100. A reference
electrode 104 can be fluidically coupled to flow chamber 106.
Further, a flow cell cover 130 encapsulates flow chamber 106 to
contain reagent flow 108 within a confined region.
[0021] FIG. 2 illustrates an expanded view of a well 201 and a
sensor 214, as illustrated at 110 of FIG. 1. The volume, shape,
aspect ratio (such as base width-to-well depth ratio), and other
dimensional characteristics of the wells can be selected based on
the nature of the reaction taking place, as well as the reagents,
byproducts, or labeling techniques (if any) that are employed. The
sensor 214 can be a chemical field-effect transistor (chemFET),
more specifically an ion-sensitive FET (ISFET), with a floating
gate 218 having a sensor plate 220 optionally separated from the
well interior by a material layer 216. In addition, a conductive
layer (not illustrated) can be disposed over the sensor plate 220.
In an example, the material layer 216 includes an ion sensitive
material layer. The material layer 216 can be a ceramic layer, such
as an oxide of zirconium, hafnium, tantalum, aluminum, or titanium,
among others, or a nitride of titanium. In an example, the material
layer 216 can have a thickness in a range of 5 nm to 100 nm, such
as a range of 10 nm to 70 nm, a range of 15 nm to 65 nm, or even a
range of 20 nm to 50 nm. Together, the sensor plate 220 and the
material layer 216 form a sensor pad.
[0022] While the material layer 216 is illustrated as extending
beyond the bounds of the illustrated FET component, the material
layer 216 can extend along the bottom of the well 201 and
optionally along the walls of the well 201. The sensor 214 can be
responsive to (and generate an output signal related to) the amount
of a charge 224 present on material layer 216 opposite the sensor
plate 220. Changes in the charge 224 can cause changes in a current
between a source 221 and a drain 222 of the chemFET. In turn, the
chemFET can be used directly to provide a current-based output
signal or indirectly with additional circuitry to provide a
voltage-based output signal. Reactants, wash solutions, and other
reagents can move in and out of the wells by a diffusion mechanism
240.
[0023] In an embodiment, reactions carried out in the well 201 can
be analytical reactions to identify or determine characteristics or
properties of an analyte of interest. Such reactions can generate
directly or indirectly byproducts that affect the amount of charge
adjacent to the sensor plate 220. If such byproducts are produced
in small amounts or rapidly decay or react with other constituents,
multiple copies of the same analyte can be analyzed in the well 201
at the same time in order to increase the output signal generated.
In an embodiment, multiple copies of an analyte can be attached to
a solid phase support 212, either before or after deposition into
the well 201. In an example, the solid phase support 212 can be a
particle, such as a polymeric particle or an inorganic particle. In
another example, the solid phase support 212 can be a polymer
matrix, such as a hydrophilic polymer matrix, for example, a
hydrogel matrix or the like.
[0024] The well 201 can be defined by a wall structure, which can
be formed of one or more layers of material. In an example, the
wall structure can have a thickness extending from the lower
surface to the upper surface of the well in a range of 0.01
micrometers to 10 micrometers, such as a range of 0.05 micrometers
to 10 micrometers, a range of 0.1 micrometers to 10 micrometers, a
range of 0.3 micrometers to 10 micrometers, or a range of 0.5
micrometers to 6 micrometers. In particular, the thickness can be
in a range of 0.01 micrometers to 1 micrometer, such as a range of
0.05 micrometers to 0.5 micrometers, or a range of 0.05 micrometers
to 0.3 micrometers. The wells 201 can have a characteristic
diameter, defined as the square root of 4 times the cross-sectional
area (A) divided by Pi (e.g., sqrt(4*A/.pi.), of not greater than 5
micrometers, such as not greater than 3.5 micrometers, not greater
than 2.0 micrometers, not greater than 1.6 micrometers, not greater
than 1.0 micrometers, not greater than 0.8 micrometers or even not
greater than 0.6 micrometers. In an example, the wells 201 can have
a characteristic diameter of at least 0.01 micrometers.
[0025] While FIG. 2 illustrates a single-layer wall structure and a
single-layer material layer 216, the system can include one or more
wall structure layers, one or more conductive layers or one or more
material layers. For example, the wall structure can be formed of
one or more layers, including an oxide of silicon or TEOS or
including a nitride of silicon.
[0026] In a particular example illustrated in FIG. 3, a system 300
includes a well wall structure 302 defining an array of wells 304
disposed over or operatively coupled to sensor pads of a sensor
array. The well wall structure 302 defines an upper surface 306. A
lower surface 308 associated with the well is disposed over a
sensor pad of the sensor array. The well wall structure 302 defines
a sidewall 310 between the upper surface 306 and the lower surface
308. As described above, a material layer in contact with sensor
pads of the sensor array can extend along the lower surface 308 of
a well of the array of wells 304 or along at least a portion of the
wall 310 defined by the well wall structure 302. The upper surface
306 can be free of the material layer. In particular, a polymer
matrix can be disposed in the wells of the array of wells 304. The
upper surface 306 can be substantially free of the polymer matrix.
For example, the upper surface 306 can include an area that is free
of the polymer matrix, such as at least 70% of the total area, at
least 80% of the total area, at least 90% of the total area or
approximately 100% of the total area.
[0027] While the wall surface of FIG. 2 is illustrated as extending
substantially vertically and outwardly, the wall surface can extend
in various directions and have various shapes. Substantially
vertically denotes extending in a direction having a component that
is normal to the surface defined by the sensor pad. For example, as
illustrated in FIG. 4, a well wall 402 can extend vertically, being
parallel to a normal component 412 of a surface defined by a sensor
pad. In another example, the wall surface 404 extends substantially
vertically, in an outward direction away from the sensor pad,
providing a larger opening to the well than the area of the lower
surface of the well. As illustrated in FIG. 4, the wall surface 404
extends in a direction having a vertical component parallel to the
normal component 412 of the surface 414. In an alternative example,
a wall surface 406 extends substantially vertically in an inward
direction, providing an opening area that is smaller than an area
of the lower surface of the well. The wall surface 406 extends in a
direction having a component parallel to the normal component 412
of the surface 414.
[0028] While the surfaces 402, 404, or 406 are illustrated by
straight lines, some semiconductor or CMOS manufacturing processes
can result in structures having nonlinear shapes. In particular,
wall surfaces, such as wall surface 408 and upper surfaces, such as
upper surface 410, can be arcuate in shape or take various
nonlinear forms. While the structures and devices illustrated
herewith are depicted as having linear layers, surfaces, or shapes,
actual layers, surfaces, or shapes resulting from semiconductor
processing can differ to some degree, possibly including nonlinear
and arcuate variations of the illustrated embodiment.
[0029] FIG. 5 includes an illustration of exemplary wells including
ion sensitive material layers. For example, a well structure 502
can define an array of wells, such as exemplary wells 504, 506, or
508. The wells (504, 506, or 508) can be operatively coupled to an
underlying sensor (not illustrated) or linked to such an underlying
sensor. Exemplary well 504 includes an ion sensitive material layer
510 defining the bottom of the well 504 and extending into the
structure 502. While not illustrated in FIG. 5, a conductive layer,
such as a gate, for example, a floating gate of ion sensitive field
effect transistor can reside below the ion sensitive material layer
510.
[0030] In another example, as illustrated by well 506, an ion
sensitive material layer 512 can define the bottom of the well 506
without extending into the structure 502. In a further example, a
well 508 can include an ion sensitive layer 514 that extends along
at least a portion of a sidewall 516 of the well 508 defined by the
structure 502. As above, the ion sensitive material layers 512 or
514 can reside over conductive layers or gates of underlying
electronic devices.
[0031] As illustrated in FIG. 6, the sensor device can include a
sensor array 602 formed within a semiconductor substrate. A well
structure is defined over the sensor array. A cap 604 is attached
to the well structure or the sensor array 602. For example, the cap
604 can be adhered to the sensor array or well structure 602 using
an adhesive. The cap 604 includes fluid ports 606 or 608, which are
in fluid communication with a flow cell 610 defined between the cap
604 and the sensor array and well array 602. Fluid applied to one
of the fluid ports 606 or 608 can flow through the flow cell 610
and optionally out the other port 608 or 606. In another example
illustrated in FIG. 7, a cap 702 can include fluid ports 704 and
706 and can define a larger flow space. The cap can be adhered over
a well array or sensor array, for example, using an adhesive.
[0032] As illustrated in the method 800 of FIG. 8, a sensor array
can be exposed to a wash solution including an acid, such as
sulfonic acid, phosphonic acid, or a combination thereof, as
illustrated 802. The wash solution can further include an organic
solvent.
[0033] An exemplary sulfonic acid includes an alkyl sulfonic acid,
an alkyl aryl sulfonic acid, or a combination thereof. An exemplary
alkyl sulfonic acid includes an alkyl group having 1 to 18 carbons,
such as 1 to 14 carbons, 1 to 10 carbons, or 1 to 5 carbons. In
another example, the alkyl group of the alkyl sulfonic acid has 10
to 14 carbons. For example, an alkyl sulfonic acid can include
methanesulfonic acid, ethanesulfonic acid, propane sulfonic acid,
butane sulfonic acid, or combinations thereof. In another example,
the alkyl group can be functionalized, for example, with a terminal
functional group opposite the sulfonic acid functional group. An
exemplary functionalized alkyl sulfonic acid includes an alkyl
sulfonic acid functionalized with a terminal amine group, such as
taurine. In a further example, the alkyl groups of the sulfonic
acid can be halogenated, such as fluorinated.
[0034] In a further example, the sulfonic acid includes an alkyl
aryl sulfonic acid. The alkyl aryl sulfonic acid, for example,
alkyl benzene sulfonic acid, can include an alkyl group having
between 1 and 20 carbons. For example, the alkyl group can have
between 9 and 18 carbons, such as between 10 and 14 carbons. In a
particular example, the alkyl aryl sulfonic acid includes dodecyl
benzene sulfonic acid. The dodecyl benzene sulfonic acid can be a
purified form of dodecyl benzene sulfonic acid having at least 90%,
such as at least 95% of alkyl aryl sulfonic acid having an alkyl
group with 12 carbons. Alternatively, the dodecyl benzene sulfonic
acid can include a blend of alkyl benzene sulfonic acid having
alkyl groups with an average of 12 carbons. The alkyl aryl sulfonic
acid can be alkylated at a blend of positions along the alkyl
chain. In another example, the alkyl group can have between 1 and 6
carbons. For example, the alkyl aryl sulfonic acid can include
toluene sulfonic acid.
[0035] The wash solution can have a concentration between 10 mM and
500 mM acid, such as sulfonic acid. For example, the wash solution
can have a concentration between 50 mM and 250 mM acid. In another
example, the wash solution includes between 0.5 wt % and 25 wt % of
the acid, such as sulfonic acid. For example, the wash solution can
include between 1 wt % and 10 wt % of the acid, such as sulfonic
acid, such as between 2.5 wt % and 5 wt % of the acid, such as
sulfonic acid.
[0036] The organic solvent within the wash solution is a
non-aqueous solvent providing solubility for the acid (e.g.,
sulfonic acid) to at least the concentrations above. In an example,
the organic solvent can be aprotic. The organic solvent can be a
non-polar organic solvent. In another example, the organic solvent
can be a polar aprotic solvent. In an example, the organic solvent
can have a normal boiling point in a range of 36.degree. C. to
345.degree. C. For example, the normal boiling point can be in a
range of 65.degree. C. to 275.degree. C. In another example, the
normal boiling point can be in a range of 65.degree. C. to
150.degree. C. Alternatively, the normal boiling point is in a
range of 150.degree. C. to 220.degree. C.
[0037] In a particular example, the non-polar organic solvent
includes an alkane solvent, an aromatic solvent, or a combination
thereof. An alkane solvent can have between 6 and 20 carbons. For
example, the alkane can have between 6 and 14 carbons, such as
between 6 and 9 carbons. Alternatively, the alkane can have between
10 and 14 carbons. In a particular example, the alkane is a linear
alkane. For example, the alkane solvent can include pentane,
hexane, heptanes, octane, decane, undecane, dodecane, or
combinations thereof. In another example, the alkane is
halogenated. An exemplary branched alkane can include hydrogenated
dimers of C11 or C12 alpha olefins.
[0038] In a further example, the organic solvent can include a
polar aprotic solvent. For example, the polar aprotic solvent can
include tetrahydrofuran, ethylacetate, acetone, dimethylformamide,
acetonitrile, dimethyl sulfoxide, N-methyl pyrrolidone (NMP), or
combinations thereof. In another example, the organic solvent may
be free of ether solvents and, for example, may include
dimethylformamide, acetonitrile, dimethyl sulfoxide, or
combinations thereof.
[0039] As illustrated at 804, the wash solution and sensor device
can be heated while the sensor pad is exposed to wash solution. In
an example, the wash solution and the sensor device can be heated
at a temperature in a range of 35.degree. C. to 60.degree. C. For
example, the temperature can be in a range of 40.degree. C. to
50.degree. C.
[0040] As illustrated 806, the sensor array can be exposed to the
wash solution and optionally heated for a period in a range of 30
seconds to 30 min. For example, the period can be in a range of 30
seconds to 10 min., such as a range of 30 seconds to 5 min., or
even a range of 1 min. to 2 min.
[0041] An organic solvent or water can be used to rinse the sensor
array, as illustrated 808, following exposure to the wash solution.
The organic solvent can be a low boiling organic solvent. An
exemplary low boiling organic solvent can have a normal boiling
point range of 25.degree. C. to 100.degree. C., such as a normal
boiling point in a range of 50.degree. C. to 100.degree. C. In an
example, the low boiling organic solvent includes an alcohol, such
as ethanol or propanol. Alternatively, the organic solvent can be
N-methyl pyrrolidone (NMP). Alternatively or in addition, the
sensor array can be rinsed with water, such as deionized water. In
a particular example, the organic solvent is at least partially
miscible with water and the organic solvent of the acid/solvent
mixture.
[0042] As illustrated at 810, the sensor array can be dried. In an
example, the sensor array can be dried under an inert atmosphere,
such as using dry nitrogen or helium. In another example, the
sensor can be heated under a dry atmosphere. In a further example,
the sensor array can be dried under vacuum.
[0043] In a further example illustrated in FIG. 9, a method 900
includes exposing a sensor array to a wash solution including an
acid as described above, for example, sulfonic acid, as illustrated
902. A sensor device can include the sensor array and optionally a
well structure defining a well array operatively corresponding to
the sensor array. A well of the well array can expose a sensor pad
of a sensor of the sensor array. The wash solution can have the
composition described above. Optionally, the sensor array and the
wash solution can be heated as illustrated 904. For example, the
sensor array and the wash solution can be heated to temperatures
described above.
[0044] In a further example, the sensor array can be exposed to the
wash solution and optionally heated for a period in a range of 30
seconds to 30 min., as illustrated 906. Alternatively, the sensor
array can be exposed to a wash solution and optionally heated for
the time periods described above.
[0045] Following exposure to the wash solution, the sensor array
can be rinsed with a solvent, e.g., a low boiling solvent, as
illustrated 908. Exemplary low boiling solvents are described
above. Alternatively, the solvent can be N-methyl pyrrolidone
(NMP). The sensor array can be rinsed with the low boiling solvent
one time or more than one time.
[0046] Following rinsing with the low boiling organic solvent, the
sensor array can be rinsed with water, such as deionized water, as
illustrated at 910. Following rinsing with water, the sensor array
can be dried as illustrated 912. For example, the sensor array can
be exposed to dry nitrogen gas.
[0047] Once dried, a cap can be applied over the sensor array, as
illustrated 914. For example, the cap can be attached to the sensor
array or an intervening well structure using an adhesive. In
particular, the cap includes fluid ports in fluid communication
with a space defined between the cap and a well structure over the
sensor array. Optionally, the method can be applied to a wafer
prior to separating the wafer into arrays and capping individual
arrays.
[0048] In a further exemplary embodiment illustrated in FIG. 10, a
method 1000 includes exposing a sensor array to a wash solution
that includes acid as described above, for example, sulfonic acid,
as illustrated at 1002. The wash solution can further include an
organic solvent. In particular, the sensor array can include a well
structure defining an array of wells operatively corresponding to
the array of sensors. A sensor pad of a sensor is exposed by a well
of the well array. A cap can be attached over the sensor array or
the well array. The cap includes fluid ports in communication with
a space defined between the cap and the well array or sensor array.
The wash solution can be applied through the fluid port into the
space. The composition of a wash solution can be as described
above.
[0049] Optionally, as illustrated at 1004, the wash solution and
the sensor array can be heated. For example, the wash solution and
the sensor array can be heated at the temperatures described
above.
[0050] The sensor array can be exposed to the wash solution and
optionally heated for a first period, as illustrated at 1006. The
first period can have a range of 30 seconds to 30 min., such as the
ranges of times periods described above.
[0051] In addition, a basic solution can be applied to the sensor
array for a second period, as illustrated at 1008. For example, the
basic solution can be applied through a fluid port of a cap and
into the space defined between the cap and the well structure or
sensor array. The basic solution has a pH greater than 7, such as a
pH greater than 8, or even a pH greater than 9. The basic solution
includes a strong base, such as sodium or potassium hydroxide or a
tetraalkyl ammonium hydroxide, such as tetramethyl ammonium
hydroxide or tetraethyl ammonium hydroxide. The base is in a
concentration between 0.05M and 1.5M. For example, the base can be
in a concentration between 0.05M and 1.0M, such as between 0.05M
and 0.5M. The sensor array can be exposed to the basic solution for
a period of between 20 seconds and 15 min., such as a period of
between 20 seconds and 5 min. or a period of between 30 seconds and
2 min. Alternatively, a weak acid solution can be substituted for
the basic solution.
[0052] The sensor array can be exposed to the wash solution
followed by exposure to the basic solution r or the weak acid
repeatedly, such as k times. For example, k can be 1, 2, or 3, but
generally is not greater than 10.
[0053] Following exposure to the wash solution and the basic
solution, the sensor array can be rinsed with the organic solvent
or water, as illustrated at 1010. Exemplary low boiling organic
solvents include those described above. Alternatively, the organic
solvent can be N-methyl pyrrolidone (NMP). The sensor array can be
rinsed with low boiling organic solvents one or more times followed
by a rinse with water. Optionally, the sensor array can be dried,
such as with exposure to dry nitrogen, as illustrated at 1012.
[0054] The method of FIG. 10 can alternatively be applied to a
wafer including multiple arrays. The wafer can be separated into
individual arrays and caps applied to the individual arrays
following drying the wafer.
[0055] Such treatment of sensor arrays has been shown to improve
loading of polymeric beads that incorporating an analyte and secure
the analyte in proximity to sensor pads of the sensor array. In
particular, such treatment improves loading of polymeric beads that
include polynucleotide analytes amplified thereon. For example,
such treatment methods improve the performance of sequence
sequencing devices. Such treatment of sensor arrays has been shown
to improve sensor signal and signal-to-noise ratio (SNR),
particularly in FET-based sensor arrays.
[0056] For example, FIG. 11 illustrates and exemplary sequencing
system enhanced by the above described treatment methods. In a
particular example illustrated in FIG. 11, polymeric particles can
be used as a support for polynucleotides during sequencing
techniques. For example, such hydrophilic particles can immobilize
a polynucleotide for sequencing using fluorescent sequencing
techniques. In another example, the hydrophilic particles can
immobilize a plurality of copies of a polynucleotide for sequencing
using ion-sensing techniques. Alternatively, the above described
treatments can improve polymer matrix bonding to a surface of a
sensor array. The polymer matrices can capture analytes, such as
polynucleotides for sequencing.
[0057] In general, the polymeric particle can be treated to include
a biomolecule, including nucleosides, nucleotides, nucleic acids
(oligonucleotides and polynucleotides), polypeptides, saccharides,
polysaccharides, lipids, or derivatives or analogs thereof. For
example, a polymeric particle can bind or attach to a biomolecule.
A terminal end or any internal portion of a biomolecule can bind or
attach to a polymeric particle. A polymeric particle can bind or
attach to a biomolecule using linking chemistries. A linking
chemistry includes covalent or non-covalent bonds, including an
ionic bond, hydrogen bond, affinity bond, dipole-dipole bond, van
der Waals bond, and hydrophobic bond. A linking chemistry includes
affinity between binding partners, for example between: an avidin
moiety and a biotin moiety; an antigenic epitope and an antibody or
immunologically reactive fragment thereof; an antibody and a
hapten; a digoxigen moiety and an anti-digoxigen antibody; a
fluorescein moiety and an anti-fluorescein antibody; an operator
and a repressor; a nuclease and a nucleotide; a lectin and a
polysaccharide; a steroid and a steroid-binding protein; an active
compound and an active compound receptor; a hormone and a hormone
receptor; an enzyme and a substrate; an immunoglobulin and protein
A; or an oligonucleotide or polynucleotide and its corresponding
complement.
[0058] As illustrated in FIG. 11, a plurality of polymeric
particles 1104 can be placed in a solution along with a plurality
of polynucleotides 1102. The plurality of particles 1104 can be
activated or otherwise prepared to bind with the polynucleotides
1102. For example, the particles 1104 can include an
oligonucleotide complementary to a portion of a polynucleotide of
the plurality of polynucleotides 1102. In another example, the
polymeric particles 1104 can be modified with target
polynucleotides 1104 using techniques such as biotin-streptavidin
binding.
[0059] In a particular embodiment, the hydrophilic particles and
polynucleotides are subjected to polymerase chain reaction (PCR)
amplification or recombinase polymerase amplification (RPA). For
example, dispersed phase droplets 1106 or 1108 are formed as part
of an emulsion and can include a hydrophilic particle or a
polynucleotide. In an example, the polynucleotides 1102 and the
hydrophilic particles 1104 are provided in low concentrations and
ratios relative to each other such that a single polynucleotide
1102 is likely to reside within the same dispersed phase droplets
as a single hydrophilic particle 1104. Other droplets, such as a
droplet 1108, can include a single hydrophilic particle and no
polynucleotide. Each droplet 1106 or 1108 can include enzymes,
nucleotides, salts or other components sufficient to facilitate
duplication of the polynucleotide.
[0060] In a particular embodiment, an enzyme such as a polymerase
is present, bound to, or is in close proximity to the hydrophilic
particle or hydrogel particle of the dispersed phase droplet. In an
example, a polymerase is present in the dispersed phase droplet to
facilitate duplication of the polynucleotide. A variety of nucleic
acid polymerase may be used in the methods described herein. In an
exemplary embodiment, the polymerase can include an enzyme,
fragment or subunit thereof, which can catalyze duplication of the
polynucleotide. In another embodiment, the polymerase can be a
naturally-occurring polymerase, recombinant polymerase, mutant
polymerase, variant polymerase, fusion or otherwise engineered
polymerase, chemically modified polymerase, synthetic molecules, or
analog, derivative or fragment thereof.
[0061] Following PCR or RPA, particles are formed, such as particle
1110, which can include the hydrophilic particle 1112 and a
plurality of copies 1114 of the polynucleotide. While the
polynucleotides 1114 are illustrated as being on a surface of the
particle 1110, the polynucleotides can extend within the particle
1110. Hydrogel and hydrophilic particles having a low concentration
of polymer relative to water can include polynucleotide segments on
the interior of and throughout the particle 1110 or polynucleotides
can reside in pores and other openings. In particular, the particle
1110 can permit diffusion of enzymes, nucleotides, primers and
reaction products used to monitor the reaction. A high number of
polynucleotides per particle produces a better signal.
[0062] In embodiments, polymeric particles from an
emulsion-breaking procedure can be collected and washed in
preparation for sequencing. Collection can be conducted by
contacting biotin moieties (e.g., linked to amplified
polynucleotide templates which are attached to the polymeric
particles) with avidin moieties, and separation away from polymeric
particles lacking biotinylated templates. Collected polymeric
particles that carry double-stranded template polynucleotides can
be denatured to yield single-stranded template polynucleotides for
sequencing. Denaturation steps can include treatment with base
(e.g., NaOH), formamide, or pyrrolidone.
[0063] In an exemplary embodiment, the particle 1110 can be
utilized in a sequencing device. For example, a sequencing device
1116 can include an array of wells 1118. The sequencing device 1116
can be treated with a wash solution including sulfonic acid, as
described above. A particle 1110 can be placed within a well
1118.
[0064] In an example, a primer can be added to the wells 1118 or
the particle 1110 can be pre-exposed to the primer prior to
placement in the well 1118. In particular, the particle 1110 can
include bound primer. The primer and polynucleotide form a nucleic
acid duplex including the polynucleotide (e.g., a template nucleic
acid) hybridized to the primer. The nucleic acid duplex is an at
least partially double-stranded polynucleotide. Enzymes and
nucleotides can be provided to the well 1118 to facilitate
detectible reactions, such as nucleotide incorporation.
[0065] Sequencing can be performed by detecting nucleotide
addition. Nucleotide addition can be detected using methods such as
fluorescent emission methods or ion detection methods. For example,
a set of fluorescently labeled nucleotides can be provided to the
system 1116 and can migrate to the well 1118. Excitation energy can
be also provided to the well 1118. When a nucleotide is captured by
a polymerase and added to the end of an extending primer, a label
of the nucleotide can fluoresce, indicating which type of
nucleotide is added.
[0066] In an alternative example, solutions including a single type
of nucleotide can be fed sequentially. In response to nucleotide
addition, the pH within the local environment of the well 1118 can
change. Such a change in pH can be detected by ion sensitive field
effect transistors (ISFET). As such, a change in pH can be used to
generate a signal indicating the order of nucleotides complementary
to the polynucleotide of the particle 1110.
[0067] In particular, a sequencing system can include a well, or a
plurality of wells, disposed over a sensor pad of an ionic sensor,
such as a field effect transistor (FET). In embodiments, a system
includes one or more polymeric particles loaded into a well which
is disposed over a sensor pad of an ionic sensor (e.g., FET), or
one or more polymeric particles loaded into a plurality of wells
which are disposed over sensor pads of ionic sensors (e.g., FET).
In embodiments, an FET can be a chemFET or an ISFET. A "chemFET" or
chemical field-effect transistor, includes a type of field effect
transistor that acts as a chemical sensor. The chemFET has the
structural analog of a MOSFET transistor, where the charge on the
gate electrode is applied by a chemical process. An "ISFET" or
ion-sensitive field-effect transistor, can be used for measuring
ion concentrations in solution; when the ion concentration (such as
H+) changes, the current through the transistor changes
accordingly.
[0068] In embodiments, the FET may be a FET array. As used herein,
an "array" is a planar arrangement of elements such as sensors or
wells. The array may be one or two dimensional. A one dimensional
array can be an array having one column (or row) of elements in the
first dimension and a plurality of columns (or rows) in the second
dimension. The number of columns (or rows) in the first and second
dimensions may or may not be the same. The FET or array can
comprise 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7
or more FETs.
[0069] In embodiments, one or more microfluidic structures can be
fabricated above the FET sensor array to provide for containment or
confinement of a biological or chemical reaction. For example, in
one implementation, the microfluidic structure(s) can be configured
as one or more wells (or microwells, or reaction chambers, or
reaction wells, as the terms are used interchangeably herein)
disposed above one or more sensors of the array, such that the one
or more sensors over which a given well is disposed detect and
measure analyte presence, level, or concentration in the given
well. In embodiments, there can be a 1:1 correspondence of FET
sensors and reaction wells.
[0070] Returning to FIG. 11, in another example, a well 1118 of the
array of wells can be operatively connected to measuring devices.
For example, for fluorescent emission methods, a well 1118 can be
operatively coupled to a light detection device. In the case of
ionic detection, the lower surface of the well 1118 may be disposed
over a sensor pad of an ionic sensor, such as a field effect
transistor.
[0071] One exemplary system involving sequencing via detection of
ionic byproducts of nucleotide incorporation is the Ion Torrent
PGM.TM. or Proton.TM. sequencer (Life Technologies), which is an
ion-based sequencing system that sequences nucleic acid templates
by detecting hydrogen ions produced as a byproduct of nucleotide
incorporation. Typically, hydrogen ions are released as byproducts
of nucleotide incorporations occurring during template-dependent
nucleic acid synthesis by a polymerase. The Ion Torrent PGM.TM. or
Proton.TM. sequencer detects the nucleotide incorporations by
detecting the hydrogen ion byproducts of the nucleotide
incorporations. The Ion Torrent PGM.TM. or Proton.TM. sequencer can
include a plurality of template polynucleotides to be sequenced,
each template disposed within a respective sequencing reaction well
in an array. The wells of the array can each be coupled to at least
one ion sensor that can detect the release of H+ ions or changes in
solution pH produced as a byproduct of nucleotide incorporation.
The ion sensor comprises a field effect transistor (FET) coupled to
an ion-sensitive detection layer that can sense the presence of H+
ions or changes in solution pH. The ion sensor can provide output
signals indicative of nucleotide incorporation which can be
represented as voltage changes whose magnitude correlates with the
H+ ion concentration in a respective well or reaction chamber.
Different nucleotide types can be flowed serially into the reaction
chamber, and can be incorporated by the polymerase into an
extending primer (or polymerization site) in an order determined by
the sequence of the template. Each nucleotide incorporation can be
accompanied by the release of H+ ions in the reaction well, along
with a concomitant change in the localized pH. The release of H+
ions can be registered by the FET of the sensor, which produces
signals indicating the occurrence of the nucleotide incorporation.
Nucleotides that are not incorporated during a particular
nucleotide flow may not produce signals. The amplitude of the
signals from the FET can also be correlated with the number of
nucleotides of a particular type incorporated into the extending
nucleic acid molecule thereby permitting homopolymer regions to be
resolved. Thus, during a run of the sequencer multiple nucleotide
flows into the reaction chamber along with incorporation monitoring
across a multiplicity of wells or reaction chambers can permit the
instrument to resolve the sequence of many nucleic acid templates
simultaneously.
[0072] In a first aspect, a sensor array includes a plurality of
sensors. A sensor of the plurality of sensors has a sensor pad
exposed at a surface of the sensor array. A method of treating the
sensor array includes exposing at least the sensor pad to a wash
solution including acid and an organic solvent and rinsing the wash
solution from the sensor pad.
[0073] In a second aspect, a sensor array includes a plurality of
sensors. A sensor of the plurality of the sensors includes a sensor
pad. A well structure defines a well array corresponding with the
sensor array. A well of the well array exposes the sensor pad. A
cap is attached over the sensor array and the well structure and
includes a fluid port. A space is defined between the cap and the
well structure. A method of treating the sensor array includes
applying a wash solution through the fluid port into the space and
waiting for a first period between 30 seconds and 30 minutes. The
wash solution includes acid and an organic solvent. The method
further includes applying a basic solution through the fluid port
into the space and waiting for a second period between 20 seconds
and 15 minutes and applying a rinse solution through the fluid
port. In an example of the second aspect, the method further
includes repeating applying the wash solution and applying the
basic solution.
[0074] In a third aspect, a sensor array includes a plurality of
sensors. A sensor of the plurality of sensors includes a sensor
pad. A well structure defines a well array corresponding with the
sensor array. A well of the well array exposes the sensor pad. A
method of treating the sensor array includes applying a wash
solution to at least the sensor pad and waiting for a first period
between 30 seconds and 30 minutes. The wash solution includes acid
and an organic solvent. The method further includes rinsing at
least the sensor pad with a low boiling organic solvent and drying
the sensor array. In an example of the third aspect, the method
further includes rinsing with water following rinsing with the low
boiling organic solvent and prior to drying. In another example of
the third aspect and the above examples, the method further
includes attaching a cap over the sensor array and the well array,
the cap including a fluid port, a space defined between the cap and
the well array in fluid communication with the fluid port.
[0075] In an example of the first, second, and third aspects and
the above examples, the acid includes a sulfonic acid. The sulfonic
acid can include an alkyl sulfonic acid, alkyl aryl sulfonic acid,
or a combination thereof. In an example, the alkyl aryl sulfonic
acid includes an alkyl group having between 1 and 20 carbons. For
example, the alkyl group has between 9 and 18 carbons, such as
between 10 and 14 carbons. In another example, the alkyl group has
between 1 and 6 carbons.
[0076] In another example of the first, second, and third aspects
and the above examples, the sulfonic acid includes dodecyl benzene
sulfonic acid. In a further example of the first, second, and third
aspects and the above examples, the sulfonic acid includes
methanesulfonic acid, ethanesulfonic acid, propane sulfonic acid,
butane sulfonic acid, or combinations thereof. In an additional
example of the first, second, and third aspects and the above
examples, the sulfonic acid includes para toluene sulfonic
acid.
[0077] In a further example of the first, second, and third aspects
and the above examples, the wash solution includes between 10 mM
and 500 mM of the acid. For example, the wash solution includes
between 50 mM and 250 mM of the acid.
[0078] In an additional example of the first, second, and third
aspects and the above examples, the wash solution includes between
0.5 wt % and 25 wt % of the acid. For example, the wash solution
includes between 1 wt % and 10 wt % of the acid, such as between
2.5 wt % and 5 wt % of the acid.
[0079] In another example of the first, second, and third aspects
and the above examples, the organic solvent is non-polar.
[0080] In a further example of the first, second, and third aspects
and the above examples, the organic solvent has a normal boiling
point in a range of 36.degree. C. to 345.degree. C. For example,
the normal boiling point is in a range of 65.degree. C. to
275.degree. C. In an example, the normal boiling point is in a
range of 65.degree. C. to 150.degree. C. In an alternative example,
the normal boiling point is in a range of 150.degree. C. to
220.degree. C.
[0081] In an additional example of the first, second, and third
aspects and the above examples, the organic solvent is an alkane
having between 6 and 24 carbons. For example, the alkane has
between 6 and 14 carbons, such as between 6 and 9 carbons or
alternatively, between 10 and 14 carbons.
[0082] In a further example of the first, second, and third aspects
and the above examples, the organic solvent is an aprotic polar
solvent. For example, the polar aprotic solvent includes
tetrahydrofuran, ethylacetate, acetone, dimethylformamide,
acetonitrile, dimethyl sulfoxide, N-methyl pyrrolidone (NMP), or
combinations thereof.
[0083] In another example of the first, second, and third aspects
and the above examples, the method further includes heating the
sensor pad and the wash solution while exposing the sensor pad to
the wash solution. For example, heating includes heating at a
temperature in a range of 35.degree. C. to 70.degree. C. For
example, the temperature is in a range of 40.degree. C. to
55.degree. C.
[0084] In a further example of the first, second, and third aspects
and the above examples, exposing includes exposing for a period in
a range of 30 seconds to 30 minutes. For example, the period is in
a range of 30 seconds to 10 minutes, such as in a range of 30
seconds to 5 minutes or in a range of 1 minute to 3 minutes.
[0085] In an additional example of the first, second, and third
aspects and the above examples, the method further includes
exposing at least the sensor pad to a basic solution. For example,
the basic solution includes between 0.005M and 1.5M sodium
hydroxide, such as between 0.01M and 1.0M sodium hydroxide or
between 0.01M and 0.5M sodium hydroxide. In an additional example,
exposing at least the sensor pad to the basic solution occurs
following exposing the sensor pad to the wash solution. The method
can further includes repeating exposing at least the sensor pad to
the wash solution and exposing at least the sensor pad to the basic
solution.
[0086] In another example of the first, second, and third aspects
and the above examples, rinsing includes rinsing with a low boiling
organic solvent having a normal boiling point in a range of
25.degree. C. to 100.degree. C. For example, the low boiling
organic solvent has a normal boiling point in a range of 50.degree.
C. to 100.degree. C. In a particular example, the low boiling
organic solvent includes alcohol. For example, the alcohol includes
ethanol or isopropanol.
[0087] In a further example of the first, second, and third aspects
and the above examples, rinsing includes rinsing with water.
[0088] In a fourth aspect, a system includes a sensor device
treated by the method of any one of the above aspects and
examples.
[0089] In a fifth aspect, a solution includes 2.5 wt % to 5 wt %
sulfonic acid and a non-polar linear alkane having between 6 and 18
carbons.
[0090] In a sixth aspect, a kit includes a nucleotide solution
including at least one nucleotide type, polymeric particles, and a
wash solution including 0.5 wt % to 20 wt % sulfonic acid and an
organic solvent having a normal boiling point in a range of
65.degree. C. to 275.degree. C.
[0091] Note that not all of the activities described above in the
general description or the examples are required, that a portion of
a specific activity may not be required, and that one or more
further activities may be performed in addition to those described.
Still further, the order in which activities are listed are not
necessarily the order in which they are performed.
[0092] Particular salts of above described acids or bases can have
activity, and may be useful in the above described methods. Salts
include alkali or alkali earth metal salts, or tetraalkylammonium
salts of the above acids or bases.
[0093] In the foregoing specification, the concepts have been
described with reference to specific embodiments. However, one of
ordinary skill in the art appreciates that various modifications
and changes can be made without departing from the scope of the
invention as set forth in the claims below. Accordingly, the
specification and figures are to be regarded in an illustrative
rather than a restrictive sense, and all such modifications are
intended to be included within the scope of invention.
[0094] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are intended to cover a non-exclusive inclusion. For
example, a process, method, article, or apparatus that comprises a
list of features is not necessarily limited only to those features
but may include other features not expressly listed or inherent to
such process, method, article, or apparatus. Further, unless
expressly stated to the contrary, "or" refers to an inclusive-or
and not to an exclusive-or. For example, a condition A or B is
satisfied by any one of the following: A is true (or present) and B
is false (or not present), A is false (or not present) and B is
true (or present), and both A and B are true (or present).
[0095] Also, the use of "a" or "an" are employed to describe
elements and components described herein. This is done merely for
convenience and to give a general sense of the scope of the
invention. This description should be read to include one or at
least one and the singular also includes the plural unless it is
obvious that it is meant otherwise.
[0096] Benefits, other advantages, and solutions to problems have
been described above with regard to specific embodiments. However,
the benefits, advantages, solutions to problems, and any feature(s)
that may cause any benefit, advantage, or solution to occur or
become more pronounced are not to be construed as a critical,
required, or essential feature of any or all the claims.
[0097] After reading the specification, skilled artisans will
appreciate that certain features are, for clarity, described herein
in the context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features
that are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any
subcombination. Further, references to values stated in ranges
include each and every value within that range.
* * * * *