U.S. patent application number 13/135854 was filed with the patent office on 2011-11-17 for method for double-dip substrate spin optimization of coated micro array supports.
This patent application is currently assigned to SQI Diagnostics Systems Inc.. Invention is credited to Jennifer Hansen, Peter Lea, Mingfu Ling.
Application Number | 20110281028 13/135854 |
Document ID | / |
Family ID | 39432022 |
Filed Date | 2011-11-17 |
United States Patent
Application |
20110281028 |
Kind Code |
A1 |
Lea; Peter ; et al. |
November 17, 2011 |
Method for double-dip substrate spin optimization of coated micro
array supports
Abstract
Described is a method for preparing a substrate-coated support
for use in micro-array devices. The method comprises the steps of
applying a first coat of substrate to a support, making the
substrate coating ramp by subjecting the coated support to
centripetal forces, adding a second coat of substrate to the
resulting support having a ramping planar coat, and subjecting the
coated support to centripetal forces for a second time to produce a
substrate-coated membrane in which the thickness of the substrate
layer is uniform across the entire coated surface.
Inventors: |
Lea; Peter; (Toronto,
CA) ; Ling; Mingfu; (Toronto, CA) ; Hansen;
Jennifer; (Scarborough, CA) |
Assignee: |
SQI Diagnostics Systems
Inc.
Toronto
CA
|
Family ID: |
39432022 |
Appl. No.: |
13/135854 |
Filed: |
July 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11999276 |
Dec 4, 2007 |
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13135854 |
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Current U.S.
Class: |
427/240 |
Current CPC
Class: |
B01J 2219/00637
20130101; B01L 2300/16 20130101; B01J 2219/00605 20130101; B01J
2219/00527 20130101; B01L 2200/12 20130101; B01L 3/50857
20130101 |
Class at
Publication: |
427/240 |
International
Class: |
B05D 3/12 20060101
B05D003/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 4, 2006 |
CA |
2,569,971 |
Claims
1.-20. (canceled)
21. A method for preparing a substrate-coated support, the method
comprising the steps of: (a) coating at least one surface of a
support having a first edge and a second edge with a substrate
coating by dipping the support in a liquid or semi-solid substrate
coating; (b) positioning the thus coated support into a dust free
chamber having an inner diameter and an outer diameter such that
the first edge of the support is in proximity to the inner diameter
of the dust free chamber and the outer edge of the support is in
proximity to the outer diameter of the dust free chamber; (c)
centrifuging the dust free chamber at a relative centrifugal force
and for a length of time until the substrate coating on the
substrate-coated support is thinner at the first edge than at the
second edge, wherein the inner diameter is closer to an axis of
rotation about the dust free chamber than the outer diameter; (d)
removing the substrate-coated support from the dust free chamber;
and (e) applying a second coat of substrate coating to the
substrate-coated support according to steps (b) to (d) so as to
provide a double-substrate-coated support having a planar film with
uniform thickness across the substrate-coated surface.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 11/999,276, filed Dec. 4, 2007, which
application claims priority under 35 U.S.C. .sctn.119 to Canadian
Patent Application No. 2,569,971, filed Dec. 4, 2006, the entire
contents of each of which are hereby incorporated herein by this
reference.
TECHNICAL FIELD
[0002] The invention relates to the field of micro-array assay
technology. More particularly, the invention relates to methods for
the preparation of substrate-coated micro-array supports. Even more
particularly, the invention relates to the use of centripetal force
to prepare coated micro-array support surfaces evenly coated with
substrate.
BACKGROUND
[0003] Micro-array platforms are devices that comprise support
material that is coated with a substrate to localize an assay
reaction. A variety of support and coating materials have been used
and are selected based on the nature of the assay for which the
micro-array will be used. In the case of micro-arrays in which the
substrate material is coated onto the surface of a support
material, the substrate may be applied by dipping the support
material into liquid or semi-solid substrate or dripping the
substrate onto the surface of the support. Spin coating is one
method for spreading the substrate across the surface of a support
that consists of applying centripetal acceleration to the support
material after a substrate has been applied.
[0004] Prior art spin coating techniques whereby a single side of a
support is coated with a substrate require a multiplicity of
handling steps in the mounting of the support, the coating of the
substrate, the semi-curing or drying of the surface, removal of
excess substrate and remounting and repeating of the coating cycle.
Importantly, there are practical limits to the evenness of a
coating and thinness of the coating obtainable by spin coating
methods. U.S. Pat. No. 3,730,760 describes a method in which
centrifugal forces are applied to coated substrate supports. This
method creates a ramp effect, whereby the inner diameter edges of
the coated substrate are thinner in thickness than the outer
diameter edges of the coated substrate. Control of this ramp effect
is difficult but desirable as it affects the relative unit volume
density distribution of suspended particulates in the coating
substrate film.
[0005] A film formation method involving spin coating comprises
adding a coating substrate solution drop-wise to a support to be
coated and drawing the coating solution thereon by centrifugal
force, so as to form a thin film on the support. This method causes
film thickness distribution to occur. Japanese Patent 2,942,213
discloses a modification to this method wherein the drop wise
coating solution is added to a sealed cup. Japanese Patent
3,231,970 discloses a modification to the sealed cup method in
which gas is injected into the cup.
[0006] U.S. patent application 2006/073521 discloses a spin coating
method wherein the support is rotated in a state where the surface
of the support to be coated is inclined during coating. The
objective of this method is to produce a substrate film on the
surface of the support with a film thickness that is even across
the surface of the support. Each of these prior art references
describe methods in which a single coating step is used in the
preparation of a support having a film or coating of substrate
evenly distributed across its surface. However, none of these
methods adequately prevent unevenness and ramp effects in the film
thickness generated in the substrate and manifested at marginal
parts of the support.
SUMMARY OF THE INVENTION
[0007] Described is a method comprising the following steps:
[0008] (a) Preparing a substrate-coated support in which the
coating or film is sufficiently uniform in thickness to enable the
use of the coated support in a micro-array platform;
[0009] (b) Allowing controlled modification of thin and ultra-thin
coatings on a support while effectively utilizing uniform spatial
resolution of suspensions contained within the substrate; and
[0010] (c) Simultaneously modifying the substrate coating on planar
supports with substrate coated onto both planar support
surfaces.
[0011] According to one aspect of the invention there is provided a
method for preparing a substrate-coated support for use in a
micro-array comprising the steps of:
[0012] (a) providing a support having a first edge and a second
edge
[0013] (b) coating at least one surface of the support with a
substrate;
[0014] (c) providing a dust-free chamber having an inner diameter
and an outer diameter;
[0015] (d) positioning the support into the dust-free chamber such
that the first edge of the support is in proximity to the inner
diameter of the dust-free chamber and the outer edge of the support
is in proximity to the outer diameter of the chamber;
[0016] (e) centrifuging the chamber at a relative centrifugal force
and for a length of time until the substrate coating on the
substrate-coated support is thinner at the first edge than the
second edge wherein the inner diameter is closer to an axis of
rotation about the chamber than the outer diameter;
[0017] (f) removing the support from the dust-free chamber;
[0018] (g) applying a second coat of substrate to the support
according to steps (d) to (f) thereby proving a double-coated
support having a planar film with uniform thickness across the at
least one surface of the support.
[0019] The method of the invention preferably includes the steps of
first applying a single coat of a substrate onto a support having
planar surfaces by dipping, under controlled conditions, the
support in a vertical position and with its planar surfaces
exposed, into the substrate. The substrate-coated support is then
placed into a dust-free chamber and subjected to a first spin by a
centrifugal rotating means for rotating the chamber and substrate
support. The chamber is rotated about its own axis in a vertical or
horizontal position. The chamber is spun at sufficient speed such
that the centripetal acceleration force (relative g force) applied
to the support causes the substrate coating layer to form a ramped
coating layer with time-dependant increasing thickness in the
direction from the inner to the outer diameter of the centrifuge
during rotation of the substrate. Initial spin speeds generating
centripetal acceleration equivalent to 150 g may be used for glass
supports. The upper spin speed is limited by the ability of the
support to withstand the requisite, applied centripetal forces. The
time interval for spinning the coating is determined by the
physical properties of the respective substrate, including
viscosity and surface tension in order to obtain correct film and
ramp formation.
[0020] At the completion of the first spin, the support now having
a ramped substrate coating, is removed from the chamber and dipped
into the substrate a second time. The support is re-inserted into
the dust-free chamber and oriented such that the thin edge of the
ramped substrate is facing the outer diameter of the centrifuge,
which is opposite to the orientation in the first spin. The
substrate-coated support is then subjected to a second spin under
the same conditions of centripetal acceleration and time as the
first spin. At the completion of the second spin a coated support
is produced in which the thickness of the substrate coating its
planar surface is uniform.
[0021] There is also provided a method for preparing a
substrate-coated support for use in a micro-array platforms
comprising the steps of:
[0022] (a) preparing a substrate-coated support by coating at least
one surface of a support with a substrate the substrate-coated
support having a first edge and a second edge;
[0023] (b) positioning the substrate-coated support into a
dust-free chamber having an inner diameter and an outer diameter
such that the first edge of the substrate-coated support is in
proximity to the inner diameter of the dust-free chamber and the
outer edge of the substrate-coated support is in proximity to the
outer diameter of the chamber;
[0024] (c) centrifuging the chamber with the inner diameter closer
to the axis of rotation than the outer diameter and at a relative
centripetal force and for a length of time until the substrate
coating on the substrate-coated support is thinner at the inner
first edge than the second outer edge;
[0025] (d) removing the substrate-coated support from the dust-free
chamber and preparing a double-coated support by applying a second
coat of substrate to the substrate-coated support where the
double-coated support comprises a first and a second edge that
correspond to the first and second edge of the substrate-coated
support;
[0026] (e) positioning the double-coated support into a dust-free
chamber having an inner diameter and an outer diameter such that
the first edge of the double-coated support is in proximity to the
outer diameter of the dust-free chamber and the outer edge of the
double-coated support is in proximity to the inner diameter of the
chamber; and
[0027] (f) centrifuging the chamber with the inner diameter closer
to the axis of rotation than the outer diameter and at a relative
centripetal force and for a length of time until the substrate
forms a coating in which the thickness of the coating its planar
surface is uniform.
[0028] This invention will more particularly be understood by
reference to the preferred embodiments in the general
specification, and the contained general description, when read in
conjunction with the accompanying illustrations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In drawings which illustrate by way of example only a
preferred embodiment of the invention,
[0030] FIG. 1 is a graph illustrating the variation of coating
thickness across the surface of the support after the first spin.
Coating thickness is measured as a function of intensity by
measuring the intensity of a series of spot measurements across the
coating on the support.
[0031] FIG. 2 is a graph illustrating the even distribution of the
substrate coating across the surface of the support after the
second spin. Coating thickness is measured as a function of
intensity by measuring the intensity of a series of spot
measurements across the now planar coating on the carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0032] High binding capacity, planar coating uniformity and surface
density of binding sites are required for high signal intensity
distribution. The signal intensity is further highly dependent on
the interplay between the surface chemistry of the substrate, the
substrate thickness as well as the planarity of the substrate
coating and the uniform unit area density of binding sites in the
substrate.
[0033] When coating a substrate onto a support by vertical dipping
of the support into the coating substrate solution, the coating
adheres to the support and excess non-adhered coating material
drains from the surfaces. This results in a gravity imposed,
non-planar coating on the support. The non-planarity of the coating
is enhanced by the viscosity and surface tension of the coating
material as modulated by adhesive and cohesive forces.
Surprisingly, although even with the substrate coating in a
semi-solid state, irregularities in the coating are smoothed by
controlled application of centrifugal action. However, the g force
that is applied during centrifugation also causes the support to
have a thicker coating along the edges of the support furthest away
from the spin axis of the centrifuge and a thinner coating along
the edges of the support closer to the spin axis of the centrifuge
("ramping effect"). This ramping effect results in a coating on the
surfaces of the support having a thickness that is significantly
uneven. An uneven substrate thickness is unsuitable for use in a
micro array because signal intensity is also proportional to the
thickness of the substrate; therefore, signal intensity would vary
across the surface of the array.
[0034] The centripetal forces work against the adhesive force
between the substrate and support and cohesive forces, which
determine the surface tension and viscosity of the substrate to be
coated onto a support. Therefore, it is important to integrate
these forces in such a manner that the substrate flows uniformly
across the surface of the support to form a film that is contiguous
with the entire surface of the support. Should, for example, the
centripetal forces exceed adhesive forces, the substrate will not
be retained on the support surface. This phenomenon, where the
substrate flies off the spinning support during centrifugation, is
known as "skittering". When skittering is prevented, the adhesive
forces are sufficient to retain the substrate on the support
surface. Simultaneously, a ramping effect occurs in which the
cohesive forces are overcome during centrifugation resulting in a
coating that is thicker at the substrate edge furthest from the
axis of rotation of the centrifuge. The ramping effect is
demonstrated in FIG. 1 where signal intensity was measured along
the surface of a coated support after it had been dipped in the
substrate once and then subjected to a single centrifugation. The
coating thickness is proportional to the intensity of the signal
and was, therefore, estimated based on the intensity of a series of
spot measurements across the coating on the support.
[0035] Referring to FIG. 1, column 8 is a measurement of signal
intensity at the edge of the support nearer to the axis of
rotation, which is the inner diameter of the centrifuge (ID). The
substrate coating is thinner at this edge of the support. The
ramping effect is evident as measurements are taken and the
intensity of the signal increases moving progressively closer to
support edge furthest from the axis of the centrifuge, which is the
outer diameter of the centrifuge (OD) (FIG. 1, columns 7 through
1).
[0036] The invention is advantageous in that by adding a second
layer of substrate and forming a second counter elevating ramp
effect, the resulting substrate film is effectively uniform in
thickness and therefore provides for coatings on the surfaces of
the micro-array supports to become planar and have uniform
substrate coating thickness.
[0037] The method may utilize a first step in which a single coat
of a substrate is applied to a support having planar surfaces by
dipping, under controlled conditions, the support in a vertical
position and with its planar surfaces exposed, into the substrate.
The dip-coated support is then placed into a dust-free chamber and
subjected to a first spin by a centrifugal rotating means for
rotating the chamber and substrate support. The chamber is rotated
about its own axis in a vertical or horizontal position. The
chamber is spun at sufficient speed such that the relative
centripetal force applied to the support cause the substrate
coating layer to form a ramped coating layer with increasing
thickness in the direction from the inner to the outer diameter of
the centrifuge during rotation of the substrate. Initial spin
speeds in excess of 1500 rpm may be used. The upper spin speed is
limited by the ability of the support to withstand the requisite,
applied centripetal forces.
[0038] In a second step, the substrate-coated support is dip-coated
for a second time in a manner similar to the first dip-coating. The
support, having two coatings, is then subjected to a second spin
under similar conditions to the first spin. However, in the second
spin, the support is orientated such that the edge of the support
closest to the ID in the first spin is placed closest to the OD in
the second spin. Conversely, the edge of the support that was
closest to the OD in the first spin is closest to the ID in the
second spin. This relocation effectively places the area of coating
having higher thickness that resulted from the first spin closer to
the axis of rotation for the second spin. Surprisingly, when the
planarity of the substrate coating on the support is measured, the
thickness of the coating is uniform and it is planar over the
entire surface the support. After the second step of the method of
the invention, the substrate coating thickness is even and,
therefore, very suitable to support micro-array applications since
it provides a consistent background to support signal intensity
measurement as well as maintaining consistent elevation for
micro-array elements above the surface of the support in providing
a planar surface equidistant from the support surface.
[0039] FIG. 2 illustrates the same analysis conducted as described
for FIG. 1. However, in FIG. 2, the analysis was conducted on a
support coated with substrate using the method of the invention.
The intensity of the signal obtained from measurements taken at the
edge of the support nearer to the axis of rotation (column 1) when
reading closer to the edge furthest from the axis of the centrifuge
(column 2 through column 8) were all similar. Although it is
expected, as illustrated and confirmed in FIG. 2, that the variance
in signal response at different locations on the surface of the
support will be very low, surface responses measuring up to 25%
variance and coating planarity measures of up to 25% variance would
be acceptable, within the scope of the invention.
[0040] Substrates prepared according to the invention have a planar
surface finish with uniform thickness of the coated material and
are suitable for the purposes intended. For example, in micro-array
analytical analysis platforms, the substrate should be as smooth as
possible to ensure that the coating it forms on the surface of the
support is as smooth and even as possible. Substrates commonly used
in the art that may be used include nitro cellulose, neutral
hydrophilic polymers, silanized surfaces, polyethylene glycol,
amphiphilic surfactants, alkane thiols, self-assembled monolayers,
streptavidin-biotin, functionalized lipids, branched polymers, gel
surfaces, e.g., polyacrylamide, and combinations thereof.
Epoxysilane is a preferred substrate because of its environmentally
compatible chemistry, its workable viscosity and its surface
tension at room temperature.
[0041] Materials commonly used as support for micro-arrays that may
be used for the invention include glass, silicon, silica, plastic
polymers such as polystyrene as well as metal films. Additionally,
less commonly used materials that are also compatible with the
invention include various metals such as titanium, as well as glass
materials and ceramic materials, and combinations of these may be
utilized. The surfaces of the support may also be derivatized to
have suitable covalent bonds expressed on the active surfaces that
will allow cross-linking with the substrate. Examples of
derivatization surface treatments include epoxy silanization and
mercapto silanization with maleimido-succinimidyl cross linker and
thiol reactive maleimids. In the case of expoxysilane,
derivatization allows covalent links to be formed between the
silanes and the support, for detection sensitivity.
[0042] Surprisingly, this coating technique allows accurate control
of the ramp planarity as shown in Example 2. The centripetal force
applied determines substrate thickness and ramp control. Support
coating according to the invention has been carried out at
centripetal force in excess of 200 g. Thus, single speed coating is
available although multi-speed coating techniques may also be
utilizable. The basic advantage of this invention over the prior
art is that thin coatings using substrates of relatively high
solids content and high viscosity can be applied to supports by
adjusting the centrifugation speed. In particular, higher rpm may
be used if centrifugation is carried out in the vertical or
horizontal spin coating station. The second substrate coating of
the support followed by inversion of the support before applying
the second spin cycle produces substrate coating surfaces that are
consistently planar coatings and have uniform unit volume
distribution of binding sites across the coated support
surface.
[0043] The range of coating modification speeds is between the g
forces applied for coating modulation to occur by centrifugal
action without damaging the actual support. This is a function of
the viscosity of the coating material and linkage of the coating to
the surface of the support. Such parameters are easily determined
by one skilled in the art. Further, one may also wish to modify the
applied coating at a slower speed, and then "spin off" or dry the
substrate coating at a higher speed. Initial coating speeds of 2500
rpm may also be used, subject to the construction materials and
design parameters of the support.
[0044] Ramp control is achieved while using coating substrates.
Coatings of comparably consistent thickness and bonding ability are
achievable, and thinner or thicker coatings may be made when using
the double dip/double spin method of the invention.
Example 1
[0045] The uniformity of the epoxy coating was characterized using
the following method.
[0046] Following immersion in cleaning solution, the support was
centrifuged once, and then a second time in the opposite
orientation, i.e., the clean support was rotated 180 degrees. The
support was then immersed into a cleaning solution for a second
time. The support was also centrifuged a third time, and a fourth
time in the opposite orientation. The clean, dry support was then
coated with appropriate substrate.
[0047] A fluorescent-labeled protein solution was placed over the
entire epoxy surface.
[0048] After an appropriate incubation period, excess substrate
coated onto the support was washed off and the substrate-coated
support was dried. The substrate-coated support was then scanned in
a fluorescence reader to measure and map the fluorescence intensity
over the entire surface. Using computer software, the support
surface was divided into a rectangular grid containing equal sized
circles that were touching so that the centre-to-centre distance
between the circumscribed area spots was equivalent to a typical
pitch, of about 300 microns, for micro-array spot diameters. The
average fluorescence intensity of each spot was determined and
plotted to compare the amount of fluorescent-labeled protein bound
over the entire substrate of the now planar substrate on the
support.
Example 2
[0049] A clean substrate-coated support as known in the art is spun
in a dust-free holding chamber on a motorized spindle. The spindle
is preferably capable of speeds over 1000 rpm (100 g). Using
conventional glass supports, the support is coated on both sides
with an epoxysilane because of its environmentally compatible
chemistry, a workable viscosity and surface tension at room
temperature. The substrate-coated support is spun at a speed
required to yield the desired even coating thickness, as
substantiated by a plot shown for example in FIG. 2. The
comparative spot density plots, confirm the substrate coating
planarity, essentially a linearly changing thickness measure,
indicating mitigation of what is known as the ramp effect, i.e.,
thinner coating at ID thicker coating at OD. This ramp effect is
controlled by the degree to which material is brought to the
surface of the disk. Surprisingly, centrifugal force applied to the
double dip/double spin method however will result in planar
coating.
[0050] Batch to batch production uniformity of coated substrate
supports the use of this process, including applications in
clinical diagnostics. Ninety-five percent of the CVs (co-efficient
of variation) for standard proteins printed on the substrate were
measured at less than 10%, and 90% of CVs measured for
antigen/protein complexes printed on these substrates were also
measured to be less than 10% for all batches of print runs,
including epoxysilane substrate-coated supports for multiplex
micro-array assay platforms. Furthermore, assay reproducibility was
found to be better than standard ELIZAs. For printed IgG
calibration standard arrays (N=110 triplicates) over 90% of CVs
were less than 10%. For printed antigens (N=280 triplicates), 90%
of the IgG response CVs were less than 10%, as were IgM response
CVs. Assay sensitivity was measured to be in femtoMol/ml. Signal to
background ratio was very good, as was spot quality with high
signal intensity. The process was optimal when
double-dip/double-spin coating with 2.5% epoxysilane and was
demonstrated to be better than commercially available epoxysilane
on glass slide carriers. While this invention has been broadly
described, other variations are determinable to those skilled in
the art.
[0051] Various embodiments of the invention having been thus
described in detail by way of example, it will be apparent to those
skilled in the art that variations and modifications may be made
without departing from the invention. The invention includes all
such variations and modifications as fall within the scope of the
appended claims.
* * * * *