Note: Descriptions are shown in the official language in which they were submitted.
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ASSAY DEVICES FOR COMBINATORIAL LIBRARIES
Field
[0001] This disclosure provides for devices and methods for conducting
assays for large
scale combinatorial libraries. In particular, the devices and methods
disclosed herein allow
for conducting simultaneous assays on libraries of up to ten million
compounds.
State of the Art
[0002] Combinatorial libraries are well known in the literature and often
utilize beads.
Each of these beads contain multiple copies of a single compound bound by a
linker to the
bead. In addition, the bead typically contains a reporting element such as DNA
that allows
for assessing the structure of the single compound on the bead. Many of these
libraries are
limited by the fact that the compound being tested remains on the bead during
the assay. As
such, the biological data generated by the assay is potentially compromised by
the possibility
that the bound compound is not able to effectively bind to the target of
choice. This could be
due to physical interference from the bead as well as possible steric
interference due to the
attachment of a linker connecting the compound to a bead. As to the latter,
this linkage could
inhibit the ability of an otherwise potent compound from binding properly to
the target,
resulting in assay results that evidence less than the actual potency of the
compound.
[0003] One option for addressing this problem includes the use of cleavable
linkers that
cleave under proper stimulation (e.g., light) thereby freeing the compound
from the bead.
Once the compound is in solution, such as in a test well, it is free to orient
itself in a manner
that provides maximum potency in the assay. Still further, release of these
compounds can be
conducted in a manner such that the amount of compound released is controlled
so as to
provide meaningful dose dependent data. See, e.g., US Patent Application Pub.
No.
2019/0358629.
Summary
[0004] While the use of cleavable linkers can help avoid the steric
hindrance problem
posed by beads and/or linkers, the scale-up of the number of individual wells
on an assay
device to accommodate larger libraries raises yet another problem. If adjacent
wells are too
proximate to each other, then a portion of the test solution in one well may
spill-over and
contaminate the test solution in an adjacent well. Any such spill-over can
alter the results by
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providing for either a false positive or dilute the reported activity of an
active compound.
The former can occur when the test compound in solution is active and a
portion of that
solution "spills-over" to a test well with an inactive compound. The spill-
over results in the
well with the inactive compound now having active compound which then
erroneously
reports that there is activity in that well. The latter can occur when spill-
over from a well
with an inactive compound contaminates a well with an active compound and
reduces the
concentration of the active compound such that the reported activity is less
than the actual
activity when reported in a dose-dependent manner.
[0005] The spill-over problem is particularly relevant when the assay
device contains a
large number of wells in close proximity to each other. In order to maintain a
workable size
for the device, well density is increased to the point that aqueous solutions
in one well can
spill over and contaminate an adjacent well. At such a density, the assay
results become less
reliable with individual well reliability decreasing with increasing well
density. This creates
a conundrum for the technician ¨ either use an assay device that separates the
well by such a
distance that it no longer can accommodate a desired well density, or allow
for spill-over that
reduces the reliability of the data generated during the assay.
[0006] Still further, each well in an assay device comprises a target which
is the intended
binding site of the test compound. The target location is preferably at or
near the center of
the well. However, when the target is a viable cell, after deposition, the
cell can translocate
into the corner of the well where visualization of these cells becomes more
difficult. As the
assay results are often measured by visualizing the cell, the failure to
properly visualize is a
significant drawback on the ability of the assay to convey reliable
information regarding the
activity of cells.
[0007] In view of the above, it would be beneficial to provide for an assay
device that
inhibits spill-over and, when appropriate, impedes translocation of the target
when placed
into the well.
[0008] In one embodiment, this disclosure provides for an assay device
containing a high
density of wells that is configured to inhibit spill-over of a portion of an
aqueous solution
from a first well into a second well. In one embodiment, this disclosure
provides for an assay
device that impedes translocation of a target, such as a viable cell,
positioned in a well. For
example, impeding translocation of a target can reduce the risk of the target
translocating to a
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site within the well that is difficult to reliably detect the resulting
biological consequences of
the soluble compound being absorbed into the cell.
[0009] Accordingly, in one of the device embodiments, there is provided an
assay device
(1) comprising a high density of wells (2) aligned thereon wherein each of
said wells (2)
comprises:
a) a floor wall (8) and side walls (7) that are configured to retain one or
more beads (6) and one or more targets (16) in an aqueous solution (17); and
b) partitions (3) separating adjacent wells (2) from each other provided
that each of said partitions is at least about 10 microns in length from the
nearest edge
of a first well (2) to the nearest edge of a second well (2') wherein said
second well
(2') is the nearest neighbor from the first well (2); wherein at least a
surface portion of
said partitions (3) comprises a hydrophobic water repellant layer (4) that is
incorporated therein and encompasses the surface thereof or extends from the
surface
thereof.
[0010] In embodiments, a well of the device (2) contains one or more beads
(6) each of
which contains multiple copies of a single compound which are releasably bound
to said
bead(s) (6) in a dose dependent manner. In embodiments, said floor wall (8)
comprises a
target capturing element (5) that captures said target (16) and which is
capable of impeding
target movement within the well (2) after placement of the target (16)
therein.
[0011] In one embodiment, one or more of said beads further comprises a
mRNA
capturing component.
[0012] In another of the device embodiments, there is provided an assay
device (1)
comprising a high density of wells (2) aligned thereon wherein each of said
wells (2)
comprises:
a) a floor wall (8) and side walls (7) that comprises one or more beads (6)
and one or more targets (16) in an aqueous solution (17) wherein the bead or
beads (6)
in an individual well (2) contains multiple copies of a single compound which
are
releasably bound to said bead(s) (6) in a dose dependent manner and further
wherein
each of said beads (6) comprises a mRNA capturing component;
b) partitions (3) separating adjacent wells (2) from each other provided
that each of said partitions (3) is at least about 10 microns in length from
the nearest
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edge of a first well (2) to the nearest edge of a second well (2') wherein
said second
well (2') is the nearest neighbor from the first well (2);
wherein said floor wall (8) comprises a target capturing element (5) that
captures said target (16) and impedes target movement within the well (2)
after
placement of the target (16) therein; and
further wherein at least a surface portion of said partitions (3) comprises a
hydrophobic water repellant layer (4) that is incorporated therein or extends
upward
therefrom and is substantially free of said aqueous solution.
[0013] In still another of the device embodiments, there is provided an
assay device (1)
comprising a multiplicity of wells (2) aligned thereon wherein each of said
wells (2)
comprises
a) a floor wall (8) and side walls (7) that comprises one or more beads (6)
and one or more targets (16) in an aqueous solution (17) wherein the bead or
beads (6)
in an individual well (2) contains multiple copies of a single compound which
are
releasably bound to said bead(s) (6) in a dose dependent manner and further
wherein
each of said beads (6) comprises a RNA capturing component;
b) partitions (3) separating adjacent wells (2) from each other provided
that each of said partitions (3) is at least about 10 microns in length from
the nearest
edge of a first well (2) to the nearest edge of a second well (2') wherein
said second
well (2') is the nearest neighbor from the first well (2);
wherein said floor wall (8) comprises a cell capturing element (5) that
captures
a mammalian cell and impedes cell movement within the well (2) after placement
of
the cell therein;
further wherein at least a surface portion of said partitions (3) comprises a
hydrophobic water repellant layer (4) that is incorporated therein or extends
upward
therefrom and is substantially free of said aqueous solution; and
still further wherein the top surface of the aqueous solution (17) in each of
the
wells (2) is covered with a hydrophobic fluid (18).
[0014] In one preferred embodiment, the device comprises a well density of
at least 10
wells per square millimeter and, preferably, at least about 1,000 to
10,000,000 wells per
device. For example, a device may comprise at least 1,000 wells, or at least
about 10,0000
wells, or at least about 100,000 wells, or at least about 1,000,000 wells.
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[0015] In another preferred embodiment, each of said partitions (3) is
about 20 microns in
length from the nearest edge of a first well (2) to the nearest edge of a
second well (2')
wherein said second well (2') is the nearest neighbor from the first well (2).
In embodiments,
a preferred range of partition (3) lengths is from at least about 10 microns
to about 30
microns and preferably from about 15 microns to about 25 microns.
[0016] In one embodiment, a single well (2) contains a target or multiple
copies of that
target (16) optionally in the presence of an aqueous solution (17). In one
embodiment, the
target (16) is a mammalian cell and the aqueous solution (17) is a growth
medium for that
cell so as to maintain the viability of the cell in solution. In one
embodiment, the mammalian
cell is a human cell.
[0017] In one embodiment, the target (16) is a mammalian cell and the
target capturing
element (5) comprises a compound (including polymers) that binds to or
complexes with the
cell so as to impede cell movement within the well.
[0018] In one embodiment, there is provide a method to inhibit spill-over
in an assay
device having a high density of wells each of which comprise an aqueous
solution which
method comprises:
a) providing for a density of wells on said device of at least 10 wells per
mm2 wherein said wells are aligned on the device such that the edge of each of
said
wells is placed at least about 10 microns from the closest edge of its nearest
neighboring well thereby providing for a partition (3) between said wells (2);
b) applying to at least a portion of said partitions (3) a biocompatible,
hydrophobic water repellent film or layer (4) that overlays the material
otherwise
comprising the device (1) thereby creating an impediment to transfer of a
portion of
the aqueous solution in one well (2) to an adjacent well (2).
[0019] In one embodiment, there is provided a method to impede
translocation of a target
(16) placed proximate to the middle of the bottom surface of well (2) said
method comprises
applying a target capturing element (5) in sufficient amounts so that target
(16) translocation
is impeded.
[0020] An apparatus suitable for conducting an assay for a combinatorial
library is
provided. The apparatus may comprise an assay device. The assay device may
comprise at
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least 10,000 wells on a top surface of the assay device. Each of the at least
10,000 wells may
comprise a floor and side walls configured to retain one or more beads and one
or more
targets in an aqueous solution. The assay device may comprise surface
partitions separating a
first well of the at least 10,000 wells from a second well of the at least
10,000 wells.
[0021] A distance along the top surface of the assay device from a nearest
edge of the
first well to a nearest edge of the second well may be from about 10 microns
([tm) to about
50 pm.
[0022] The second well may be a nearest neighboring well to the first well.
[0023] At least a portion of each of the surface partitions may comprise a
hydrophobic
layer.
[0024] The hydrophobic layer may be configured to restrict spill-over of
the aqueous
solution from the first well to the second well.
[0025] The assay device may have a top surface area. A density of the at
least 10,000
wells on the top surface area may be at least 10 wells per square millimeter
(mm2).
[0026] Each of the wells may have a well diameter from about 30 [tm to
about 250 [tm.
Each of the wells may have a well depth from about 30 [tm to about 400 [tm.
[0027] The density may be from at least 10 wells per mm2 to about 400 wells
per mm2.
[0028] The density may be from about 40 wells per mm2 to about 150 wells
per mm2.
[0029] The distance along the top surface from the nearest edge of the
first well to the
nearest edge of the second well may be from about 10 [tm to about 30 [tm.
[0030] The apparatus may further comprise a mammalian cell maintained in an
aqueous
growth medium for the mammalian cell. The aqueous growth medium may be
configured to
maintain the viability of the mammalian cell in solution.
[0031] The aqueous growth medium may be maintained in at least one of the
at least
10,000 wells.
[0032] The mammalian cell may be a human cell.
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[0033] The target capturing element may comprise poly-D-lysine.
[0034] The distance along the top surface from the nearest edge of the
first well to the
nearest edge of the second well may be from about 151.tm to about 251.tm.
[0035] The assay device may have at least about 100,000 wells on the top
surface.
[0036] The hydrophobic layer may comprise a biologically compatible,
hydrophobic
material selected from polyethylene, polypropylene, block copolymers of
ethylene and
propylene, polytetrafluoroethylene, (trichloro)octadecyltsilane (OTS),
amorphous
fluoropolymers, and polydimethylsiloxane (PDMS).
[0037] The floor of at least one of the at least 10,000 wells may further
comprise a target
capturing element that captures the mammalian cell.
[0038] At least a portion of the floor of at least one of the at least
10,000 wells may be
hydrophilic.
Brief Description of the Drawings
[0039] Provided herein are figures that illustrate certain aspects of assay
devices of this
invention. These devices comprise required components as well as optional
components.
Each of these components in these figures are numbered for ease of reference
and common
components found in multiple figures have the same numbers. It is understood
that the
components described herein are non-limiting and are provided for illustrative
purposes only.
Equivalents of individual components are included within the scope of this
invention.
[0040] Figure 1A and Figure 1B illustrate a cross-sectional overview of a
portion of one
embodiment of a device (1) of this invention. Figure 1A is a top view. Figure
1B is a side
view.
[0041] Figures 2A and 2B illustrate a cross-section of a portion of the
device (1)
described in Figures 1A and 1B wherein the device (1) comprises wells (2), a
bead (6) in said
well (2), a target capturing element (5) in the well (2), and a hydrophobic
water repellent
layer (4) forming part of the surface that partitions one well from another.
Figure 2A shows
the leftmost well (2) with a bead (6) disposed therein, while the other two
wells (2) middle
and rightmost are empty (for clarity). Figure 2B shows the device of Figure 2A
in which the
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rightmost well is filled with bead (6), target (16) and solution (17). As in
Figure 2A, other
well (2) content is omitted solely for clarity.
[0042] Figure 2C illustrates a cross-section of another embodiment of a
portion of a
device (1) described herein wherein the device (1) comprises wells (2), a bead
(6) in the well
(2), a target capturing element (5) in the well (2), and a hydrophobic water
repellant layer (4)
extending upward from at least a portion of the partition (3).
[0043] Figure 3 illustrates an optional aspect of this invention where a
hydrophobic liquid
(18) such as silicon oil is applied to the top of device (1) so as to provide
an oil layer over the
device thereby further inhibiting spill-over from one well to an adjacent
well. Figure 3 also
shows optional walls (28) extending upward to contain hydrophobic liquid (18).
[0044] Figure 4 illustrates one process for forming the hydrophobic, water
repellent layer
(4) on the partitions (3) of the assay devices described herein.
Detailed Description
[00451 Disclosed are devices and methods for conducting assays for large
scale
combinatorial libraries. However, prior to describing this invention in more
detail, the
following terms will first be defined. If not defined, terniti used herein
have their generally
accepted scientific meaning.
[0046] The terminology used herein is for the purpose of describing
particular
embodiments only and is not intended to be limiting of the invention. As used
herein, the
singular forms "a", "an" and "the" are intended to include the plural forms as
well, unless the
context clearly indicates otherwise.
[0047] "Optional" or "optionally" means that the subsequently described
event or
circumstance can or cannot occur, and that the description includes instances
where the event
or circumstance occurs and instances where it does not.
[0048] The term "about" when used before a numerical designation, e.g.,
temperature,
time, amount, concentration, and such other, including a range, indicates
approximations
which may vary by ( + ) or ( -) 10%, 5%, 1%, or any subrange or subvalue there
between.
Preferably, the term "about" means that the dose may vary by +/- 10%.
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[0049] "Comprising" or "comprises" is intended to mean that the
compositions and
methods include the recited elements, but not excluding others.
[0050] "Consisting essentially of' when used to define compositions and
methods, shall
mean excluding other elements of any essential significance to the combination
for the stated
purpose. Thus, a composition consisting essentially of the elements as defined
herein would
not exclude other materials or steps that do not materially affect the basic
and novel
characteristic(s) of the claimed invention.
[0051] "Consisting of' shall mean excluding more than trace elements of
other
ingredients and substantial method steps. Embodiments defined by each of these
transition
terms are within the scope of this invention.
[0052] The term "assay device" refers to a device that is capable of
simultaneously
assaying multiple test compounds against a target. Such devices contain a
multiplicity of
wells where each individual well preferably contains multiple copies of
substantially the
same compound. The device comprises a material that transmits light
therethrough. For
example, the light may be exposed onto the device or the light may be
generated from within
the device. In one embodiment, the light transmitted therethrough is at a
wavelength and an
intensity that at least a portion of the cleavable bonds attaching each of the
multiple copies of
substantially the same compound to a bead is cleaved from the bead so as to
generate a
solution having a concentration of that compound in the well. In one
embodiment, the light
transmitted therethrough is fluorescence that is generated from molecules in a
given well
where these molecules are preferably not bound to the bead. As the
fluorescence is
transmitted through the device, the so generated fluorescence is capable of
being detected
outside of the device.
[0053] In one embodiment, the assay device comprises upwards of 1,000,000
wells and
preferably up to about 10,000,000 wells. In one embodiment, the assay device
comprises
from about 10,000 to about 10,000,000 wells and preferably from about 50,000
to about
2,000,000 wells. In one preferred embodiment, the size of the device is up to
about 10,000
square millimeters.
[0054] The term "target" means a material such as a biological material
that one wishes
to assess the binding affinity of a test compound to that target and/or the
biological
consequences of such binding. Exemplary targets include monoclonal or
polyclonal
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antibodies, fragments of monoclonal or polyclonal antibodies, mammalian cells,
DNA, RNA,
siRNA, proteins (e.g., fusion proteins, enzymes, cytokines, chemokines and the
like), viruses,
and the like. In one preferred embodiment, the target is a mammalian cell,
such as a human
cell.
[0055] The term "target capturing element" means a biocompatible layer or
film of a
compound or mixture of compounds. In one embodiment, the layer or film binds
to or
complexes with the target on the bottom surface of the well with sufficient
strength so as to
impede target movement within the well. In another embodiment, the target
capturing
element is a biocompatible layer or film that does not interfere with the
integrity of a target in
suspension or solution. In another embodiment, the complex between the target
and the
target capturing element is defined by a dissociation constant (Ka) of less
than 1 x 10-3
[tmol/ .L. In one embodiment when multiple cells are employed in a single
well, then the
target capturing element further inhibits cell clumping.
[0056] The term "releasably bound" means that a compound bound to the bead
can be
released by application of a stimulus that breaks the bond. Such bonds are
sometimes
referred to as "cleavable" bonds. The appropriate stimulus to release the
compounds depends
on the bond used. The art is replete with examples of such bonds and the
appropriate
stimulus that breaks the bond. Non-limiting examples of cleavable bonds
include those that
are released by pH changes, enzymatic activity, oxidative changes, redox, UV
light, infrared
light, ultrasound, changes in magnetic field, to name a few. A comprehensive
summary of
such cleavable bonds and the corresponding stimuli required to cleave these
bonds is
provided by Taresco, et al., Self-Responsive Prodrug Chemistries for Drug
Delivery, Wiley
Online Library, 2018, onlinelibrary.wiley.com/doi/ful1/10.1002/adtp.201800030.
[0057] The term "compound," which is interchangeable with "test compound,"
means a
compound that is being evaluated for its binding affinity to a target and/or
the biological
consequences of such binding. Such compounds are typically part of a structure-
activity
relationship (SAR) analysis as it relates to a specific target. The analysis
of what compounds
bind or do not bind to the target provides meaningful data to the skilled
artisan as to the
consequences of changes in the structure of the compound. Likewise, assessing
the
biological consequences (or activity) of such binding provides still further
information to skill
artisan as to what structural differences alter these biological consequences.
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[0058] The term "substantially the same," used in reference to compounds,
means that a
majority of the compounds on a bead are the same. In one embodiment, at least
80% of the
compounds are the same and preferably at least 90% and more preferably at
least 95%. The
compounds that are not the same are typically the result of incomplete
reactions on the bead
such that these compounds are either starting materials or intermediates to
the final product.
Such compounds are anticipated as lacking sufficient structure to meaningfully
interact with
the target.
[0059] The term "fluid" means a liquid or a flowable powder.
[0060] The term "releasably bound to said bead(s) (6) in a dose dependent
manner"
means that the compounds are bound to the bead via a cleavable linker, where
cleavage is
titratable so that the amount of compound released can be controlled. In one
embodiment,
the amount of compound released by the cleavable linker is assessed by linkage
of multiple
copies of a companion marker such as a fluorescent compound bound to the same
or different
beads by the identical cleavable linker. When bound to the bead, a non-
cleavable quencher
molecule is attached proximate thereto to reduce or eliminate fluorescence of
that fluorescent
compound. A standardized plot of fluorescent intensity versus the amount of
fluorescent
compound cleaved from the bead by the cleaving agent (e.g., UV light of a
defined
wavelength and defined intensity) is generated over set periods of time. UV
light is then
applied equally to the test bead(s) having cleavable test compounds and to the
beads having
cleavable fluorescent compounds. The extent of cleavage of the fluorescent
compounds as
evidenced by the standardized plot of fluorescent intensity is then correlated
to the amount of
test compound released. In such a manner, once can control the amount of test
compound
released and correlate that to the amount the concentration of the test
compound in solution,
as the amount of solution per test well is known.
[0061] The term "biocompatible" refers to materials that are compatible
with each of
components used in the devices including without limitation the beads, the
targets, the target
capturing elements, the compounds, the mRNA, the aqueous solutions employed,
and the
like. In the case where the target is a viable cell, the biocompatible
materials must maintain
the viability of the cells during use. Likewise, for proteins, polypeptides,
antibodies, DNA,
mRNA, the biocompatible materials must retain the functional properties of
these
components.
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Device
[0062] The ability to assay a very large combinatorial library of compounds
is limited by
the size constraints of the overall device and the density of wells on the
device. As the size of
the wells decrease, the ability to place more wells on a per square millimeter
basis increases.
However, there is a limit to such increases as the well integrity requires
that there be a
minimal distance between adjacent wells. For example, if wells are too close
together, a
portion of the aqueous solution in one well may spill over to another well
rendering the
evaluation of both wells suspect. Generally, the minimal distance between
wells is at least
about 50 microns which ensures that spill over from one well to another is
substantially
reduced/prevented. However, such a separation distance is contrary to a high
density of
wells.
[0063] In the device described herein, the design of the wells allows for
the minimal
distance between wells to be reduced to about 10 microns and as low as about 5
microns
while maintaining well integrity, as the hydrophobic water repellent surface
or protrusion
between the wells inhibits spill-over. This allows for significantly more
wells per millimeter
square. Thus, in embodiments, well separation may be less than 50 microns, or
less than 40
microns, or less than 30 microns, or less than 20 microns, each with a minimum
distance of
separation of about 5 microns, or about 10 microns, or about 15 microns,
including any
values or ranges in between the recited values, including fractions thereof.
[0064] The diameter of each of the wells also controls the density of wells
on the device.
For example, a device having wells with a diameter of about 40 microns, can
allow for a
significantly greater density of wells than a device where the wells are about
150 microns in
diameter. In practical terms, the devices described herein have a high density
of wells, such
as those having at least 10 wells per millimeter square of the device surface
that comprises
wells.
[0065] Finally, the device of this invention should be sized for easy use
by a skilled
technician. For example, a conventional 96 well plate is about 128 mm by 85 mm
(or about
7.4 inches by 3.3 inches). These plates provide a well density of about
0.00885 wells per
mm2. Whereas the devices described herein are contemplated as having a well
density of up
to about 400 wells per mm2 and, preferably, at least 10 wells per mm2 and,
more preferably,
from about 40 wells per mm2 to about 150 wells per mm2 In embodiments, the
wells have a
well diameter of from about 60 to 150 microns. In perspective, a well density
of about 200
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wells per mm2 provides for over 2,100,000 wells when sized to be compatible
with a
conventional 96 well plate. However, many different device sizes are feasible
with a
preferred maximum size of from no more than about 12 inches (300 mm ¨ X axis)
to no more
than about 12 inches (300 mm ¨ Y axis). The high well density devices
described herein
allow for exceptionally high throughput of a combinatorial library.
[0066] Turning now to Figures 1A and 1B, there is provided an overview
illustrating an
exemplary portion of the surface of device 1 having a thickness (100) of about
1 mm and
where each of the illustrated wells (2) have a maximum diameter (105)
(measured along its
longest axis) of about 150 microns, a well (2) depth (110) of about 150
microns, and a
distance of at least 20 microns from the nearest edge of one well to the
nearest edge of a
second well that is its nearest neighbor.
[0067] In more general terms, device 1 of Figures 1A and 1B has a top to
bottom
thickness (100) of at least about 0.1 mm and contains a multiplicity of wells
(2) on the
surface thereof. Each well (2) has a diameter (105) of from about 30 to about
250 microns
and preferably from about 50 to about 150 microns. Each well (2) has a depth
(110) of from
about 30 to about 400 microns and preferably about 150 microns. This provides
for a volume
within the well of 2.65 x 106 cubic microns or 0.00265 microliters when the
well diameter is
about 150 microns and a depth of about 150 microns.
[0068] The devices described herein can comprise any of a number
biocompatible,
materials including but not limited to polymers such as Cyclo Olefin Polymer
(COP) which is
commercial available from Zeon Specialty Materials, Inc. (San Jose,
California, USA), cyclic
olefin copolymers (COC) which are commercially available from a number of
sources such
as Polyplastics USA, Inc. (Farmington Hills, Michigan, USA), polyimides which
are
commercially available from a number of sources such as Putnam Plastics
(Dayville,
Connecticut, USA), polycarbonates which are commercially available from a
number of
sources such as Foster Corporation (Putnam, Connecticut, USA),
polydimethylsiloxane
which are commercially available from Edge Embossing (Medford, Massachusetts,
USA) and
polymethylmethacryate which is commercially available from Parchem Fine &
Specialty
Chemicals (New Rochelle, New York, USA).
[0069] The devices of this invention can be readily prepared by hot
embossing methods
which are well known in the art and comprise stamping a pattern into a polymer
softened by
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heating the polymer to a temperature just above its glass transition
temperature. Subsequent
cooling of the polymer provides for a high density of wells in the devices
described herein.
Alternatively, mold injection techniques can be used and are well known in the
art. Still
further, a solid block of a biocompatible polymer can be laser etched to
introduce the desired
number of wells having the appropriate size, volume and shape as well as with
the desired
well density.
[0070] Figures 1A and 1B illustrate a portion of partially formed device
(1) which
includes a multiplicity of wells (2) and partitions (3) that separate wells
(2) from each other
(For an expanded view of partitions (3) see Figures 2A-C). In one embodiment,
each
partition (3) is at least about 10 microns in length distant from a first well
(2) to its nearest
neighboring well (2'). This minimal distance between wells (2) ensures well
integrity such
that a homogenous aqueous solution (no spill-over) is included in each well
(2) and that each
well (2) contain one or more beads where the bead(s) contain multiple copies
of the same test
compound bound thereto. In a preferred embodiment, the partitions (3) have a
length as
measured from the nearest neighbor well of about 5, 10 or 20 microns and, more
preferably
from about 20 microns to less than about 50 microns in length.
[0071] When generating wells (2) by a hot embossing method having
partitions (3) that
are about 10 microns in length as per above, the sheet of thermoplastic
polymer is heated to a
temperature slightly higher than its glass transition temperature as described
above. A stamp
is selected that comprises a number of circular prongs that are preferably
uniformly placed on
its surface at a desired density. Each prong is sized to have diameter and a
depth correlating
to the size of the wells (2) described above. The distance between any two
adjacent prongs is
at least about 10 microns (i.e., partition (3) is at least about 10 microns
thick). The stamp is
sized so that the portion comprising the prongs fits within the top surface of
the sheet.
Sufficient force is applied to the stamp so as to ensure that the full length
of the prongs sink
into the sheet. The force required is dependent on the degree of softness of
the sheet and is
readily ascertainable by the skilled artisan. As the sheet cools, the prongs
are removed so as
to provide for a sheet now containing wells (2) and partitions (3) as per
Figures 1A and 1B.
[0072] Alternatively, the partially formed device (1) of Figures 1A and 1B
can be
prepared by conventional injection molding using two mold halves ¨ one with
protrusions
corresponding to those of the stamp (male mold half) and the other forming the
base of the
device (female mold half). The mold halves are juxtaposed to each other so as
to form a
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cavity in the shape of the device (1) illustrated in Figures 1A and 1B.
Injection of a monomer
or reactive oligomer composition into this cavity followed by polymerization
provides for a
device (1) now containing wells (2) and partitions (3) as per Figures 1A and
1B.
[0073] In one embodiment, after heat embossment or mold formation, a
silicon dioxide
coating may be applied to the top surface of device (1) including a bottom
surface (i.e., floor
wall of well (2); see Figure 2A) (8) of wells (2) by conventional sputtering
technology.
Preferably, the thickness of the silicon dioxide layer is from about 0.5 to
about 100
nanometers and more preferably about 10 to 50 nanometers. The silicon dioxide
coating
provides a reactive layer that binds both a water repelling, biocompatible
layer (4) as well as
the target capturing element (5) that are to be formed.
[0074] Figures 2A, 2B, and 2C illustrate different aspects of device (1)
during different
stages of construction. For example, Figure 2A illustrates device (1) having
wells (2) with a
side surface (7) and a bottom surface (8) as well as a biologically
compatible, hydrophobic,
water repellant layer (4) defining the top surface of partitions (3). In a
first well (2), a target
capturing layer (5) and bead (6) is illustrated.
[0075] Figure 2B further includes target (16) in an aqueous solution (17)
in well (2). And
Figure 2C illustrates an alternative form for the biologically compatible,
hydrophobic, water
repellant layer (4) from that disclosed in Figure 2A. In Figure 2C, water
repelling layer (4) is
formed only over a portion of the partitions (3) and such can be formed by
laser etching the
water repelling layer (4) after formation to reduce the length of said
partition (4).
[0076] As to the specifics of construction of device (1), after application
of the silicon
dioxide coating on the top surfaces of device (1) including the bottom surface
(8) of wells (2),
each partition (3) is then modified to include a biologically compatible,
hydrophobic, water
repellant layer (4) that inhibits spill-over of aqueous solution (17) from one
well to another as
illustrated in Figure 4. The water repelling layer (4) comprises a
biologically compatible,
hydrophobic, water repellant material such as polyethylene, polypropylene,
block copolymers
of ethylene and propylene, polytetrafluoroethylene,
(trichloro)octadecyltsilane (OTS),
amorphous fluoropolymers (such as CYTOP ), and polydimethylsiloxane (PDMS),
and the
like.
[0077] The biocompatible water repellent layer (4) is generated by
conventional coating
techniques. For example, as illustrated in Step 1 of the process of Figure 4,
one such
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technique involves applying a solution of a biocompatible water repellent
material dissolved
in a suitable solvent compatible with the device (e.g., ethanol) onto a disc
(24). Disc (24) is
then spun (not shown) so as to create a thin solution film (23) of about 1-5
microns. The
spinning is stopped and then top surface of device (1) is placed onto/into the
thin film (23) as
shown in Step 2 of Figure 4. Device (1) is disengaged from the disc (24)
within about 1 to 5
minutes as shown in Step 3 and then dried to form water repellent layer (4)
which is about 1
to 5 microns in thickness.
[0078] In an alternative embodiment, formation of the water repellent
biocompatible
layer (4) is then conducted by injection molding to a desired thickness. As
the addition of the
water repelling biocompatible layer (4) adds to the depth of each of the
wells, it is understood
that the total depth of the wells described above refers to that depth after
formation of the
water repelling layer (4).
[0079] Application of the target capturing (layer) element (5) onto the
bottom of wells (2)
is achieved as per Figure 4, Steps 4-5. In Step 4, the target capturing
element (5) is poly-D-
lysine (PDL) which is used for illustrative purposes only. Sufficient PDL is
dissolved into an
aqueous solution so as to achieve a concentration of, e.g., about 0.1 mg/mL.
PDL is
commercially available from numerous sources. One preferred source of PDL is
from
ThermoFisher Scientific, 10010 Mesa Rim Road, San Diego, California USA as
catalog no.
A389040. Other examples of target capturing element (5) include: fibronectin
(ThermoFisher
Scientific, catalog no. 33016015), vitronectin (Sigma Aldrich, catalog no.
5051), and the like.
[0080] Partially formed device (1), without the PDL target capturing
element (5), is
immersed into the container comprising the PDL solution as shown in Figure 4,
Step 4. The
immersion continues for about 1 hour. Device (1) is then removed and then
dried as shown
in Figure 4, Step E. The hydrophobic coating on the top surface of device (1)
inhibits
deposition of PDL on that surface thereby providing the target capturing
element on the
bottom surface (8) of wells (2) and perhaps on the side walls (7) of well (2).
[0081] Target capturing element (5) is biologically compatible with the
bottom surface
(8) of well (2) and either adheres to the target (17) at the site of
deposition so as to impede
target translocation once deposited or is biologically compatible with the
target (1) when
target (1) is in solution or is a suspension. Preferably, the overall
character of target
capturing element (5) is hydrophilic although areas of hydrophobicity are
permitted. In one
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embodiment, target capturing element (5) is selected to adhere to the bottom
surface (8) of
well (2) and to the target (17) deposited thereon. Target capturing element
(5) includes
materials such as poly(amino acids), DNA, RNA, siRNA, antibodies, antibody
fragments,
proteins, polypeptides, and the like. The particular target capturing element
(5) is selected
relative to the target (16) employed and such a selection is well known to the
skilled artisan.
In one embodiment, the target (16) is a mammalian cell, such as a human Hela
cell, and the
target capturing element (5) is a polymer of D-lysine (PDL). Polymers of D-
Lysine having
from about 1 x 109 to about 1 x 1014 lysine residues are preferred.
[0082] When the water repelling biocompatible layer (4) is used in
combination with a
target capturing element (5), the devices (1) described herein allow for very
high densities of
wells per square millimeter as well as maintaining reproducible detection of a
cell deposited
in well (2) using electromagnetic energy detection means (e.g., light). The
presence of a
water repelling biocompatible layer (4) described herein inhibits or
eliminates spill-over of
the aqueous solution from adjacent wells.
[0083] The presence of the target capturing element (5) assists in
obviating a problem
associated with translocation of the target deposited proximate to or at the
middle of the
bottom of well 2 to its corners. When so translocated, application and reading
of
electromagnetic energy applied to and retrieved from the target 5 becomes less
reliable.
[0084] Preferably, the target capturing element (5) binds to target (1)
that deposits on
surface (8) by non-covalent interactions including electrostatic, hydrophilic
(e.g., hydrogen
bonds), hydrophobic, and Van der Waal forces. Such binding can be measured by
an
equilibrium disassociation constant (Kd ¨ sometime referred to as KD) where
lower values
correlate to stronger binding interactions. In one embodiment, the target
capturing element
(5) binds to target (1) with a sufficient disassociation constant so as to
impede translocation
of target (1) within well (2). Preferably, the binding of the target to the
target capturing
element provides for a Kd of no more than about 1 x 10-3 and more preferably
no more than
about 1 x 10-5 i.tmo1/11.L.
[0085] The above process provides for a method for forming an assay device
(1) wherein
said device contains a multiplicity of wells (2). This method comprises:
a) heating a biocompatible thermoplastic material to just above the
glass
transition temperature so as to soften the material;
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b) applying a stamp to the surface of said heated material wherein said
stamp contains a number of prongs wherein each prong is sized to have diameter
and
a depth correlating to the size of the wells (2) to be formed, wherein the
distance
between any two adjacent prongs is at least about 10 microns;
c) applying sufficient pressure to the stamp so as to ensure that the full
length of the prongs sink into the sheet and then subsequently removed to
provide for
wells (2) having partitions (3) separating each well from adjacent wells (2),
having a
bottom surface (8) and side surface (7);
d) optionally applying a layer of silicon dioxide to the exposed surfaces
of the partitions (3) and wells (2);
e) applying a layer of a biocompatible, water repellent, hydrophobic
material (4) to the partitions (3); and
applying a layer of a target capturing element (5) to the bottom surface
of wells (2)
thereby providing for device (1) that is capable of inhibiting spill-over of
an
aqueous solution (17) from one well (2) to an adjacent well (2) while impeding
a
target deposited in well (2) from translocating within said well (2).
[0086] In another embodiment illustrated in Figure 3, the outside edges
(28) of device (1)
are extended slightly upward to allow for the addition of a layer of
hydrophobic fluid (18)
which is less dense than water. This layer (18) provides for additional
protection against
spill-over as well as preventing contamination of the wells (2) by
contaminants such as dust,
pollen, etc. that can affect the test results. Hydrophobic fluid 18 is
biocompatible and has a
density of less than 0.99 grams per cubic centimeter at 25 C so that the fluid
forms a layer
over the aqueous solution. One preferred hydrophobic fluid 18 is silicon oil
which is
available from many commercial vendors such as SigmaAldrich, Inc., St. Louis,
Missouri,
USA. Hydrophobic fluid 18 can be applied in any manner including by a
dispenser that sits
over device (1) and applies a mist of the fluid in a manner that does not
cause any spill-over
of aqueous solution (17) from one well (2) to another well (2). One means to
provide the
hydrophobic fluid layer (18) is provided in US Application No. 16/774,875
entitled "Caps for
Assay Devices" (Attorney Docket No. 057698-503F01U5). In accordance with
Figure 3,
there is provided a method of preventing spill-over and evaporation comprising
providing the
device with optional walls (28) and placing hydrophobic liquid (18) over
filled wells (2).
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[0087] The following example is provided for illustrative purposes only and
does not
constitute any limitation for the claimed invention. All temperatures are
Centrigrade unless
stated otherwise and all conditions are at atmospheric pressure unless stated
otherwise. In
this example, the following abbreviations have the following meanings:
mL = milliliter
!MU = millimeter
MM2 millimeters squared
OTS = trichloro(octadecyl)silane
PMMA = polymethyl methacrylic acid
rpm = rotations per minute
IAL= microliters
microns
Example 1 - Formation of Device (1)
[0088] A sheet of thermoplastic PMMA (available from Lucite International
Cassel
Works, Billingham UK) measuring 76 mm (X-axis) by 50 mm (Y-axis) by 1 mm (Z-
axis) is
heated to a temperature slightly higher than its glass transition temperature
(Tg) of about
125 C in order to soften the plastic. A stamp is selected that comprises a
number of circular
prongs uniformly placed into 4 rows on its surface at a density of about 40
prongs per mm2 in
each row. Each row of prongs is approximately 50 mm long and 7 mm wide.
[0089] Each prong has a diameter of about 150 i_tm and a depth from the
base to the end
of the prong of about 150 Jim. The distance between any two adjacent prongs is
about 20
The stamp is sized so that each of the rows of prongs fits within the top
surface of the
sheet. Sufficient force is applied to the stamp so as to ensure that the full
length of the prongs
sink into the top surface of the sheet. The force required is dependent on the
degree of
softness of the sheet and is readily ascertainable by the skilled artisan. As
the sheet cools,
the prongs are removed so as to provide for a partially formed device (1)
having wells (2) and
partitions (3) as depicted in Figure 1.
[0090] Device (1) having wells (2) and partitions (3) is then coated with a
thin layer of
silicon dioxide (5i02) by conventional sputtering technology well known in the
art. The
sputtering process is continued until a silicon dioxide film of about 30
nanometers in
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thickness is formed. The purpose of this film is used to enhance the adhesion
of both the
water repelling hydrophobic layer (4) and the target capturing element (5) to
device (1).
[0091] The next steps in preparing device (1) are illustrated in Figure 4.
[0092] Figure 4 illustrates the formation of a water repellent element (3)
on the top
surface of the partially formed device (1) with the silicon dioxide layer in
place. Specifically,
a rotatable disc (24) is placed on a spinner and a solution of OTS in ethanol
at a concentration
of about 25 micromolar is applied thereto. The spinner is initiated and
rotated at a rate of
about 1000 rpm. Spinning is continued until the solution (23) is uniformly
deposited on the
disc. Typically, spinning is continued for about less than 1 minute and then
stopped and the
thickness of the solution (23) is about 0.1 microns to about 2 microns.
[0093] In Figure 4, Step 2, top surface of partially formed device (1) is
placed into the
solution (23) on the now stationary disc (24) and maintained there for up to
about 5 minutes.
In Figure 4, Step 3, partially formed device (1) is removed from the disc and
then dried to
form water repellent layer (4) which is about 1 to 2 microns in thickness.
[0094] Figure 4, Step 4 illustrates the formation of the target capturing
element (6) on the
bottom surface of wells (2). In Figure 4, Step 4, a container (25) is filled
with a solution (26)
of poly-D-lysine obtained from ThermoFisher Scientific, 10010 Mesa Rim Road,
San Diego,
California USA as catalog no. A389040. Sufficient PDL is dissolved into an
aqueous
solution so as to achieve a concentration of, e.g., about 0.1 mg/mL. Partially
formed device
(1), without the PDL target capturing element (5), is immersed into the
container comprising
the PDL solution (26). The immersion continues for about 1 hour. Device (1) is
then
removed and then dried as per Figure 4, Step 5. The hydrophobic coating on the
top surface
of device (1) inhibits deposition of PDL on that surface thereby providing the
target capturing
element (4) on the bottom surface (8) of wells (2) and perhaps on the side
walls (7) of well
(2).
[0095] The above example is provided for illustrative purposes only and is
non-limiting.
Other techniques may be used to form device (1).
