U.S. patent application number 10/852447 was filed with the patent office on 2004-11-04 for method for making a multiwell plate having transparent well bottoms.
Invention is credited to Martin, Gregory R., Tanner, Allison J., Wang, Hongming.
Application Number | 20040216835 10/852447 |
Document ID | / |
Family ID | 25452017 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040216835 |
Kind Code |
A1 |
Tanner, Allison J. ; et
al. |
November 4, 2004 |
Method for making a multiwell plate having transparent well
bottoms
Abstract
A method is described herein for making a multiwell plate that
is used for assaying samples and is made from a plastic upper plate
which forms the sidewalls of one or more wells and a glass lower
plate which forms the bottom walls of the wells. The plastic upper
plate and glass lower plate are attached and bound to one another
by an enhanced adhesive. The enhanced adhesive includes an adhesive
mixed with an additive that interacts with the adhesive, the
plastic upper plate and the glass bottom plate in a manner that
strengthens a bond between the plastic upper plate and the glass
lower plate.
Inventors: |
Tanner, Allison J.;
(Portsmouth, NH) ; Martin, Gregory R.; (Acton,
MA) ; Wang, Hongming; (Lee, NH) |
Correspondence
Address: |
CORNING INCORPORATED
SP-TI-3-1
CORNING
NY
14831
|
Family ID: |
25452017 |
Appl. No.: |
10/852447 |
Filed: |
May 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10852447 |
May 24, 2004 |
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09925638 |
Aug 9, 2001 |
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6767607 |
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Current U.S.
Class: |
156/272.2 ;
156/285 |
Current CPC
Class: |
C09J 2400/143 20130101;
Y10T 428/24331 20150115; B32B 17/10 20130101; Y10T 428/24273
20150115; B32B 3/10 20130101; C09J 2425/006 20130101; C09J 5/00
20130101 |
Class at
Publication: |
156/272.2 ;
156/285 |
International
Class: |
B32B 031/00 |
Claims
What is claimed is:
1. A method for making a multiwell plate, said method comprising
the steps of: providing an upper plate that forms sidewalls of at
least one well, said upper plate made from a polymeric material;
providing a lower plate that forms a bottom wall of the at least
one well, said lower plate made from an inorganic material; joining
said upper plate to said lower plate using an adhesive that
contains a silane monomer which interacts with said adhesive, said
upper plate and said lower plate to strengthen a bond between said
upper plate and said lower plate; wherein said polymeric material
was subjected to a process to create reactive groups that interact
with said silane monomer in said adhesive to further strengthen the
bond between said adhesive and said upper plate.
2. The method of claim 1, wherein said polymeric material was
subjected to a plasma treatment process.
3. The method of claim 1, wherein said polymeric material is
polystyrene.
4. The method of claim 1, wherein said inorganic material was
subjected to a process to free silanol groups that interact with
said silane monomer in said adhesive to further strengthen the bond
between said adhesive and said lower plate.
5. The method of claim 4, wherein said inorganic material was
subjected to a pyrolysis process.
6. The method of claim 1, wherein said inorganic material is
glass.
7. The method of claim 1, wherein said step of joining further
includes the steps of: applying a substantially thin film of said
adhesive/silane mononer onto one of said plates; placing said other
plate onto the said adhesive/silane monomer; and applying a vacuum
to bring said upper plate and said lower plate into close proximity
while said silane monomer polymerizes to form a compatible network
with said adhesive and also interacts with said upper plate and
said lower plate to strengthen the bond between said upper plate
and said lower plate.
8. The method of claim 1, wherein said adhesive is a non-cytotoxic
adhesive.
9. The method of claim 1, wherein said silane monomer is one of the
following: 3-(trimethoxysilyl)propyl methacrylate;
3-(mercaptopropyl)trimethoxy silane; or tris2-(methoxyethoxy)vinyl
silane.
10. A method for making a multiwell plate, said method comprising
the steps of: providing an upper plate that forms sidewalls of at
least one well, said upper plate made from a polymeric material;
providing a lower plate that forms a bottom wall of the at least
one well, said lower plate made from an inorganic material; joining
said upper plate to said lower plate using an adhesive that
contains a silane monomer which interacts with said adhesive, said
upper plate and said lower plate to strengthen a bond between said
upper plate and said lower plate; and wherein said inorganic
material was subjected to a process to free silanol groups that
interact with said silane monomer in said adhesive to further
strengthen the bond between said adhesive and said lower plate.
11. The method of claim 10, wherein said inorganic material was
subjected to a pyrolysis process.
12. The method of claim 10, wherein said inorganic material is
glass.
13. The method of claim 10, wherein said polymeric material was
subjected to a process to create reactive groups that interact with
said silane monomer in said adhesive to further strengthen the bond
between said adhesive and said upper plate.
14. The method of claim 13, wherein said polymeric material was
subjected to a plasma treatment process.
15. The method of claim 10, wherein said polymeric material is
polystyrene.
16. The method of claim 10, wherein said step of joining further
includes the steps of: applying a substantially thin film of said
adhesive/silane mononer onto one of said plates; placing said other
plate onto the said adhesive/silane monomer; and applying a vacuum
to bring said upper plate and said lower plate into close proximity
while said silane monomer polymerizes to form a compatible network
with said adhesive and also interacts with said upper plate and
said lower plate to strengthen the bond between said upper plate
and said lower plate.
17. The method of claim 10, wherein said adhesive is a
non-cytotoxic adhesive.
18. The method of claim 10, wherein said silane monomer is one of
the following: 3-(trimethoxysilyl)propyl methacrylate;
3-(mercaptopropyl)trimethoxy silane; or tris2-(methoxyethoxy)vinyl
silane.
19. A method for making a multiwell plate, said method comprising
the steps of: providing a frame that forms sidewalls of at least
one well, said frame made from a polymeric material; providing a
layer that forms a bottom wall of the at least one well, said layer
made from an inorganic material; joining said frame to said layer
using an adhesive that contains a silane monomer which interacts
with said adhesive, said frame and said layer to strengthen a bond
between said frame and said layer; wherein said polymeric material
was subjected to a process to create reactive groups that interact
with said silane monomer in said adhesive to further strengthen the
bond between said adhesive and said frame; and wherein said
inorganic material was subjected to a process to free silanol
groups that interact with said silane monomer in said adhesive to
further strengthen the bond between said adhesive and said
layer.
20. The method of claim 19, wherein said polymeric material is
polystyrene that was subjected to a plasma treatment process.
21. The method of claim 19, wherein said inorganic material is
glass that was subjected to a pyrolysis process.
22. The method of claim 19, wherein said step of joining further
includes the steps of: applying a substantially thin film of said
adhesive/silane mononer onto said frame or said layer; placing said
layer or said frame onto the said adhesive/silane monomer; and
applying a vacuum to bring said frame and said layer into close
proximity while said silane monomer polymerizes to form a
compatible network with said adhesive and also interacts with said
frame and said layer to strengthen the bond between said frame and
said layer.
23. The method of claim 19, wherein said adhesive is a
non-cytotoxic adhesive.
24. The method of claim 19, wherein said silane monomer is one of
the following: 3-(trimethoxysilyl)propyl methacrylate;
3-(mercaptopropyl)trimethoxy silane; or tris2-(methoxyethoxy)vinyl
silane.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 09/925,638, filed Aug. 9, 2001, now
pending.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates in general to the
biotechnology field and, in particular, to a multiwell plate made
from a plastic upper plate and a glass bottom plate that are joined
to one another using an enhanced adhesive.
[0004] 2. Description of Related Art
[0005] The recent growth in many areas of biotechnology has
increased the demand to perform a variety of studies, commonly
referred to as assays, of biochemical systems. These assays include
for example, biochemical reaction shifts, DNA and protein
concentration measurements, excitation/emission of fluorescent
probes, enzyme activities, enzyme co-factor assays, homogeneous
assays, drug metabolite assays, drug concentration assays,
dispensing confirmation, volume confirmation, solvent
concentration, and salvation concentration. Also, there are a
number of assays which use intact living cells that require visual
examination.
[0006] Assays of biochemical systems are carried out on a large
scale in both industry and academia, so it is desirable to have an
apparatus that allows these assays to be performed in a convenient
and inexpensive fashion. Because they are relatively easy to
handle, are low in cost, and generally disposable after a single
use, multiwell plates are often used for such studies. Multiwell
plates are typically formed from a polymeric material and consist
of an ordered array of individual wells. Each well includes
sidewalls and a bottom so that an aliquot of sample can be placed
within each well. The wells may be arranged in a matrix of mutually
perpendicular rows and columns. Common sizes for multiwell plates
include matrices having dimensions of 8.times.12 (96 wells),
16.times.24 (384 wells), and 32.times.48 (1536 wells).
[0007] The materials used to construct a multiwell plate are
selected based on the samples to be assayed and the analytical
techniques to be used. For example, the materials of which the
multiwell plate is made should be chemically inert to the
components of the sample or any biological or chemical coating that
has been applied to the multiwell plate. Further, the materials
should be impervious to radiation or heating conditions to which
the multiwell plate is exposed during the course of an experiment
and should possess a sufficient rigidity for the application at
hand.
[0008] In many applications, a transparent window in the bottom of
each well is needed. Transparent bottoms are primarily used in
assay techniques that rely on emission of light from a sample
within the well and subsequent spectroscopic measurements. Examples
of such techniques include liquid scintillation counting and
techniques which measure light emitted by luminescent labels, such
as bioluminescent or chemiluminescent labels, fluorescent labels,
or absorbance labels. Optically transparent bottom wells also
enable microscopic viewing of specimens and living cells within the
well. Currently, optically transparent and ultraviolet transparent
bottomed multiwell plates exist in the market and are used for the
aforementioned purposes. These multiwell plates are typically made
from a hybrid of different polymeric materials, one material making
up the sidewalls of the wells and another material making up the
bottom walls of the wells.
[0009] Preferably, multiwell plates that are used for spectroscopic
and microscopic measurements would have well bottoms made from
glass. Glass has the advantage of being chemically inert, has
superior optical properties in the visible range, is rigid, and is
highly resistant to any deformation process caused by heating, due
to its high melting temperature. Further and unlike most polymers,
glass can be formulated and processed to provide a surface which
produces very little background signal (barring absorbance) and
which may be manufactured to extreme smoothness. While it is simple
to make glass in sheets, it is not possible to injection mold
articles made from glass, and it is extremely difficult to press a
molten gob of glass into an industry standard multiwell plate
format. A solution to the problem, is to join a plastic upper plate
that forms the sidewalls of the wells of a microplate to a
substantially flat transparent glass lower plate that forms the
bottom walls of the wells. One commonly employed method of joining
a plastic upper plate and a glass lower plate to one another is to
use an adhesive. Unfortunately, the multiwell plate that uses a
traditional adhesive to bond together the plastic upper plate and
glass lower plate does not perform well under normal cell culture
conditions. In particular, the adhesive bond that holds together
the plastic upper plate and glass lower plate is known to degrade
such that the two plates can easily separate or the contents in one
well can leak into other wells. Accordingly, there is a need for a
multiwell plate that has a strong adhesive bond between the plastic
upper plate and the glass lower plate. This need and other needs
are satisfied by the multiwell plate and the method of the present
invention.
BRIEF DESCRIPTION OF THE INVENTION
[0010] The present invention includes a multiwell plate that is
used for assaying samples and is made from a plastic upper plate
which forms the sidewalls of one or more wells and a glass lower
plate which forms the bottom walls of the wells. The plastic upper
plate and glass lower plate are attached and bound to one another
by an enhanced adhesive. The enhanced adhesive includes an adhesive
mixed with an additive that interacts with the adhesive, the
plastic upper plate and the glass bottom plate in a manner that
strengthens a bond between the plastic upper plate and the glass
lower plate. The present invention also includes a method for
making such multiwell plates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] A more complete understanding of the present invention may
be had by reference to the following detailed description when
taken in conjunction with the accompanying drawings wherein:
[0012] FIG. 1 is a perspective view of a multiwell plate in
accordance with the present invention;
[0013] FIG. 2 is a cut-away partial perspective view of the
multiwell plate shown in FIG. 1;
[0014] FIG. 3 is a cross-sectional side view of the multiwell plate
shown in FIG. 1;
[0015] FIG. 4 is a flowchart illustrating the steps of a preferred
method for making the multiwell in accordance with the present
invention; and
[0016] FIG. 5 is a flowchart illustrating in greater detail the
joining operation of step 406 of the preferred method 400 shown in
FIG. 4.
DETAILED DESCRIPTION OF THE DRAWINGS
[0017] Referring to FIG. 1, there is illustrated a perspective view
of an exemplary multiwell plate 10 of the present invention. The
multiwell plate 10 (e.g., microplate) includes a peripheral skirt
12 and a top surface 14 having an array of wells 16 each of which
is capable of receiving an aliquot of sample to be assayed.
Preferably, the multiwell plate 10 conforms to industry standards
for multiwell plates; that is to say, the multiwell plate 10 is
bordered by a peripheral skirt 12, laid out with ninety-six wells
16 in an 8.times.12 matrix (mutually perpendicular 8 and 12 well
rows). In addition, the height, length, and width of the multiwell
plate 10 preferably conform to industry standards. However, the
present invention can be implemented in a multiwell plate that has
any number of wells and is not limited to any specific dimensions
and configurations.
[0018] Referring to FIGS. 2 and 3, there are illustrated two cross
sectional views of the multiwell plate 10 shown in FIG. 1. The
multiwell plate 10 is of two-part construction including an upper
plate 20 and a lower plate 22. The upper plate 20 forms the
peripheral skirt 12, the top surface 14 and the sidewalls 24 of the
wells 16. The lower plate 22 forms the bottom walls 26 of the wells
16. During the manufacturing process, the upper plate 20 and lower
plate 22 are joined together at an interface by an enhanced
adhesive 28. For clarity, a more detailed discussion about the
manufacturing process and the enhanced adhesive 28 is provided
below after a brief discussion about the exemplary structures of
the multiwell plate 10.
[0019] The upper plate 20 includes a frame that forms the sidewalls
24 of an array of open-ended sample wells 16 in addition to the
peripheral skirt 12, and the top surface 14. The upper plate 20 is
preferably molded from a polymeric material (e.g., polystyrene)
that becomes intertwined upon heating and bonds together in a
non-covalent mechanism upon cooling, thereby forming an
interpenetrating polymer network. Further, the upper plate 20 need
not be molded, instead the upper plate 20 can be laminated so that
each layer has desired properties. For example, a top most layer
may be anti-reflective, a middle layer may form the sidewalls of
the wells and can be hydrophobic for meniscus control, and the
bottommost layer may be a polymeric material.
[0020] The lower plate 22 is preferably made from a layer of glass
material that can be purchased from a variety of manufacturers
(e.g. Erie Scientific, Corning, Inc.) as a sheet. This sheet can
then be altered to fit the dimensions of the desired size multiwell
plate 10. The glass material forms a transparent bottom wall 26 for
each sample well 16 and permits viewing therethrough. The
transparent lower plate 22 also allows for light emissions to be
measured through the bottom walls 26 of the wells 16. As shown, the
lower plate 22 is substantially flat and is sized to form the
bottom walls 26 for all of the wells 16 of the upper plate 20. It
should be noted that one or more chemically active coatings (not
shown) can be added to a top surface of the bottom walls 26 of the
wells 16.
[0021] Although the lower plate 22 as a whole is substantially
flat, it may have relief features formed upon its surface such as
ridges, curves, lens, raised sections, diffraction gratings,
dimples, concentric circles, depressed regions, etc. Such features
may be located on the lower plate 22 such that they shape or
otherwise become features of the bottom walls 26 themselves, and
may in turn enhance the performance of an assay, enhance or enable
detection (as in the case with lenses and gratings), or serve to
mechanically facilitate bonding with the upper plate 20. These
relief features may be formed by any number of known methods
including vacuum thermoforming, pressing, or chemical etching,
laser machining, abrasive machining, embossing, or precision
rolling.
[0022] Each well 16 includes sidewalls 24 and a bottom wall 26. To
prevent light transmission between adjacent wells 16, the sidewalls
24 are preferably formed from an opaque organic polymeric material
or filled with an inorganic TiO.sub.2 material. For assaying
techniques which require the detection of very small amounts of
light, as in liquid scintillation counting, the pigmentation used
to render the plastic upper plate 20 opaque is preferably light in
color (e.g. white) so as to be highly reflective and non-absorptive
to ensure high counting efficiency with respect to radioactive
samples. The white coloration is typically achieved with TiO.sub.2.
However the sidewalls 24 may be optically transparent. In some
types of luminescence and fluorescence assays, it is preferred that
the sidewalls 24 of the wells 16 be non-reflective and absorptive,
in which case the sidewalls 24 are formed from a black pigmented
polymer. As is commonly known and practiced, the black coloration
of the polymer may be achieved by the addition of a pigment
material such as carbon black to the polymer blend at
concentrations readily known and practiced in the art.
[0023] As described above, the bottom wall 26 of a well 16 is
formed from a transparent material. Preferably, the transparent
material is an inorganic such as glass, but may be pure silica,
mica, or even metallic coated films. More preferably, the glass is
of a high optical quality and flatness such as boroaluminosilicate
glass (Corning Inc. Code 1737). Optical flatness of the bottom
walls 26 of the wells 16 is important particularly when the
multiwell plate 10 is used for microscopic viewing of specimens and
living cells within the wells 16. This flatness is also important
in providing even cell distribution and limiting optical variation.
For example, if the bottom wall 26 of a well 16 is domed, the cells
will tend to pool in a ring around the outer portion of the bottom
26. Conversely, if the bottom wall 26 of a well 16 is bowed
downwards, the cells will pool at the lowest point. Glass
microscope slides are typically flat within microns to ensure an
even distribution. Preferably, the bottom walls 26 of the wells 16
are formed from a glass sheet having a thickness similar to
microscope slide cover slips, which are manufactured to match the
optics of a particular microscope lens. Although the bottom walls
26 may be of any thickness, for microscopic viewing it is preferred
that the bottom wall 26 thickness is less than or equal to 500
microns and their flatness is in the range of 0-10 microns across
the diameter of the outer bottommost surface of an individual well
16.
[0024] Moreover, the wells 16 can be any volume or depth, but in
accordance with the 96 well industry standard, the wells 16
preferably have a volume of approximately 300 ul and a depth of
approximately 12 mm. Spacing between wells 16 is approximately 9 mm
between center lines of rows in the x and y directions. The overall
height, width, and length dimensions of the multiwell plate 10 are
preferably standardized at 14 mm, 85 mm and 128 mm, respectively.
Wells 16 can be made in any cross sectional shape (in plan view)
including, square sidewalls 24 with flat or round bottoms, conical
sidewalls 24 with flat or round bottoms, and combinations
thereof.
[0025] The preferred process of manufacturing the multiwell plate
10 of the present invention includes employing an enhanced adhesive
28 to join the upper plate 20 and the lower plate 22. The use of
the enhanced adhesive 28 to bond together the upper plate 20 and
the lower plate 22 of the multiwell plate 10 is a marked
improvement over the traditional multiwell plate in that the
multiwell plate 10 of the present invention performs well under
normal cell culture conditions. In contrast, the traditional
multiwell plate does not perform well under normal cell culture
conditions because the adhesive bond that holds together the
plastic upper plate and glass lower plate is known to degrade such
that the two plates can easily separate or the contents in one well
can leak into other wells.
[0026] In the preferred embodiment, the enhanced adhesive 28
includes a non-cytotoxic adhesive (e.g., NOA-63 manufactured by
Norland Products Inc.) mixed with approximately 2.5% or greater
volume of an additive such as a silane monomer (e.g., Dow Corning
Product Z-6030). The silane monomer polymerizes to form a
compatible network with the non-cytotoxic adhesive and also
interacts with the plastic upper plate 20 and the glass lower plate
22 to strengthen a bond between the plastic upper plate 20 and the
glass lower plate 22. Details on how the silane monomer strengthens
the bond between the plastic upper plate 20 and the glass lower
plate 22 are provided below after a brief discussion about the
various properties of the NOA-63 adhesive and the silane
monomer.
[0027] The NOA-63 adhesive is a clear, colorless, liquid
photopolymer that cures when exposed to ultraviolet light. Below
are listed some of the physical properties of the NOA-63 adhesive
sold under the brand name of Norland Optical Adhesive 63:
[0028] Physical state: Liquid
[0029] Boiling point: NA
[0030] ph: NA
[0031] Percent volatile by volume: <0.1
[0032] Freezing point: NAv
[0033] Evaporation rate: <<Butyl Acetate
[0034] Vapor Density: >1 (Air=1)
[0035] Vapor Pressure (mm Hg): <0.1 @ 20.degree. C.
[0036] Specific Gravity: 1.2 (H.sub.2O=1)
[0037] Odor: Slightly sulfurous odor
[0038] Odor Threshold (ppm): NAv
[0039] Viscosity at 25.degree. C.: 2500 cps
[0040] Refractive index of cured polymer: 1.56
[0041] Elongation at failure: approx. 6%
[0042] Modulus of Elasticity: 240,000 psi
[0043] Tensile strength: 5000 psi
[0044] Hardness-Shore D: 90
[0045] In this application, the NOA-63 adhesive can withstand
temperatures of -15.degree. C. to 60.degree. C. and upto 90.degree.
C. if the adhesive is spread in a thin film. The NOA-63 adhesive
also contains a mercapto-ester and has a slight sulfurous odor. In
addition, the NOA-63 adhesive cures well in thick sections and has
low shrinkage and a slight resiliency to minimize strain.
Typically, the NOA-63 adhesive is cured by ultraviolet light and
has a maximum absorption in the range of 350 to 380 nanometers. For
instance, the energy required to perform a full cure is
approximately 4.5 Joules/cm.sup.3 of long wavelength ultraviolet
light. Some of the light sources that can be used to cure the
NOA-63 adhesive are sunlight, mercury lamps and fluorescent black
lights. It should be noted that a variety of adhesives now known or
subsequently developed that have similar properties to the NOA-63
adhesive can be used in the present invention.
[0046] Referring now to the additive, the silane monomer
effectively interacts with the NOA-63 adhesive, the plastic upper
plate 20 and the glass lower plate 22 to strengthen the bond
between the plastic upper plate 20 and the glass lower plate 22.
Below are listed some of the physical properties of the silane
monomer sold under the brand name of Dow Corning Z-6030:
[0047] Product name: 3-(trimethoxysilyl)propyl methacrylate
[0048] Synonyms*: [[3-(methacryloyloxy)propyl]
trimethoxysilane]
[0049] Molecular formula(s):
H.sub.2C=C(CH.sub.3)CO(CH.sub.2).sub.3Si(OCH.- sub.3).sub.3 or
C.sub.10H.sub.20O.sub.5Si
[0050] Molecular weight: 248.35
[0051] Density: 1.045 g/ml Assay: 98%
[0052] Boiling point: 190/760.degree. C.
[0053] Refractive index: 1.4310
[0054] Flash point: 92.2.degree. C.
[0055] Infrared spectrum: conforms to structure
[0056] Explosion limits in air: 0.9%-5.4%
[0057] Physical state: liquid
[0058] Vapor Pressure: 10 MM 130.degree. C.
[0059] Specific Gravity: 1.045 (H.sub.2O=1) * Other synonyms
include: dynasylan memo; KBM 503; KH 570; M 8550; methacrylic acid,
3-(trimethoxysilyl)propyl ester;
gamma-methacryloxypropyltrimethoxysilane- ; MOPS-M; NUCA 174;
2-propenoic acid, 2-methyl-, 3-(trimethoxysilyl)propyl ester;
silan, (3-hydroxypropyl) trimethoxy-, methacrylate; silicone A-174;
3-(trimethoxysilyl)-1-propanol methacrylate;
trimethoxysilyl-3-propylester kyseliny methakrylove (Czech); and
Union Carbide A-174.
[0060] It should be noted that a variety of additives now known or
subsequently developed that have similar properties to the silane
monomer can be used in the present invention. Examples of suitable
additives include 3-(mercaptopropyl)trimethoxy silane
(C.sub.6H.sub.16O.sub.3Si/A18- 9) and tris 2-(methoxyethoxy)vinyl
silane (C.sub.11H.sub.24O.sub.6Si/A172)- .
[0061] As mentioned before, the enhanced adhesive 28 including the
additive (e.g., silane monomer) mixed with an adhesive (e.g.,
NOA-63) strengthens the bond between the plastic upper plate 20 and
the glass lower plate 22. In particular, the silane monomer
polymerizes and forms a compatible network with the NOA-63 adhesive
which increases the bond strength of the enhanced adhesive 28
between the plastic upper plate 20 and the glass lower plate 22.
Polymerization of NOA-63 adhesive in this case is stimulated by a
photoinitiator after receiving energy from ultraviolet light. The
silane monomer becomes incorporated into the growing polymer
network. The silane functional groups are then free to form bonds
with the silanol groups on the glass lower plate 22 as well as with
the plasma treated polymer upper surface 20. Of course, the silane
monomer must be reactive enough to polymerize within the NOA-63
adhesive, but not polymerize so rapidly that the silane fuctional
groups in the silane monomer become unavailable for interacting
with the silanol groups on the glass lower plate 22. As such, since
there is increased interaction between the enhanced adhesive 28 and
the glass lower plate 22, the bond between the glass lower plate 22
and the plastic upper plate 20 is more effective than a bond
without the silane monomer.
[0062] Pyrolysis at 350.degree. C. for 3 hours, of the glass lower
plate 22 cleans the surface of bound contaminants freeing silanol
groups for interaction with the enhanced adhesive. Cleansing the
glass of contaminants also facilitates adhesion of cells in
culture. As for the plastic upper plate 20, the plastic can be
polystyrene which has been treated with a plasma to create reactive
groups that interact with the silane monomer as it polymerizes in a
way similar to the manner in which the silane monomer binds to the
reactive (silanol) groups on the glass lower plate 22. It should be
noted that the silane monomer increases the strength of the bond
between the plastic upper plate 20 and the glass lower plate 22
without affecting the non-cytotoxic status of the adhesive.
[0063] A series of tests have been performed in which it was
determined that the multiwell plate 10 built using the enhanced
adhesive performs well under normal cell culture conditions.
Whereas, the same tests showed that the traditional multiwell plate
built using the adhesive (e.g., NOA-63 adhesive) alone did not
perform well under normal cell culture conditions. To conduct the
test, cells were seeded in the wells within an aqueous nutrient
solution (media) supplemented with animal serum (usually fetal
bovine). The multiwell plates are then incubated at 37.degree.
C./85% relative humidity/5% carbon dioxide for at least 3 days. The
fluid in the wells is aspirated off and a dye solution is then
added to only the middle column of wells. Thereafter, the multiwell
plates are incubated at room temperature for 5 minutes and then the
dye solution is removed. If leaking occurs, the dye can be detected
in wells other than those in which the dye was placed. A second
test requires turning the multiwell plates over so that the glass
is now the upper portion of the multiwell plates. A rounded object
is then used to push up through the well under the glass to see if
the bond between the glass lower plates and the plastic upper
plates releases or if the round object pushes through the glass in
that well. The multiwell plates 10 assembled with the silane
monomer mixed with the NOA-63 adhesive passed these tests. In
contrast, the traditional multiwell plates assembled using only the
NOA-63 adhesive failed these tests.
[0064] In addition, a number of tests have been conducted to assess
different characteristics between the traditional multiwell plate
and multiwell plate 10. For instance, autofluorescence has been
examined at 350, 450 and 550 nm and there was very little
difference in autofluorescence property between the traditional
multiwell plate and multiwell plate 10.
[0065] Referring to FIG. 4, there is a flowchart illustrating the
steps of the preferred method 400 for making the multiwell plate
100. Although the multiwell plate 10 is described herein as having
ninety-six functional wells arranged in a grid having a plurality
of rows and columns, again it should be understood that the present
invention is not limited to any specific number of wells.
Accordingly, the multiwell plate 10 and preferred method 400 should
not be construed in such a limited manner.
[0066] The multiwell plate 10 can be manufactured by providing
(step 402) an upper plate 20 and also providing (step 404) a lower
plate 22. The upper plate 20 has a frame that forms the sidewalls
24 of one or more wells 16 and is preferably made from a polymeric
material such as polystyrene. And, the lower plate 22 has a layer
that forms the bottom walls 26 of the wells 16 and is preferably
made from an inorganic material such as glass.
[0067] The next step in manufacturing the multiwell plate 10
includes joining (step 406) the upper plate 20 to the lower plate
22 using an enhanced adhesive 28. Again, the enhanced adhesive 28
includes an adhesive mixed with an additive that functions to
strengthen a bond between the upper plate 20 and the lower plate
22. In the preferred embodiment, the enhanced adhesive 28 includes
the non-cytotoxic NOA-63 adhesive mixed with approximately 2.5% or
greater volume of a silane monomer.
[0068] As described above, the silane monomer interacts with the
NOA-63 adhesive to strengthen the bond between the plastic upper
plate 20 and the glass lower plate 22. In particular, the silane
monomer polymerizes and forms a compatible network with the NOA-63
adhesive which increases the bond strength of the enhanced adhesive
28 between the plastic upper plate 20 and the glass lower plate 22.
Polymerization of NOA-63 adhesive in this case is stimulated by a
photoinitiator after receiving energy from ultraviolet light. The
silane monomer becomes incorporated into the growing polymer
network. The silane functional groups are then free to form bonds
with the silanol groups on the glass lower plate 22 as well as with
the plasma treated polymer upper surface 20. As such, since there
is increased interaction between the enhanced adhesive 28 and the
glass lower plate 22, the bond between the glass lower plate 22 and
the plastic upper plate 20 is more effective than without the
silane monomer. As for the plastic upper plate 20, the plastic can
be polystyrene which has been treated with a plasma to create
reactive groups that interact with the silane monomer as it
polymerizes in a way similar to the manner in which the silane
monomer binds to the reactive (silanol) groups on the glass lower
plate 22.
[0069] Referring to FIG. 5, there is a flowchart illustrating in
greater detail an exemplary joining operation of step 406 of the
preferred method 400. It should be understood that the present
invention is not limited to the steps described below that make-up
the joining step 406. Instead, it should be understood that there
are numerous other ways of joining the upper plate 20 to the lower
plate 22 of the multiwell plate 10.
[0070] In joining the plastic upper plate 20 to the glass lower
plate 22, the proper proportions of the adhesive (e.g., NOA-63
adhesive) and additive (e.g., silane monomer) can be mixed (step
502) in a container before being applied (step 504) to either the
plastic upper plate 20 or the glass lower plate 22. Alternatively,
the adhesive and additive can be mixed in proper proportions as
they are being dispensed just before they are applied (step 504) to
the plastic upper plate 20 or the glass lower plate 22. In the
preferred embodiment, a Byrd bar is used to apply (step 504) a thin
film of the mixed adhesive and additive (enhanced adhesive 28) to a
carrier belt that transfers the enhanced adhesive 28 onto the
plastic upper plate 20 and then the glass lower plate 22 is placed
(step 506) on top of the mixed adhesive and additive. Thereafter, a
vacuum is applied (step 508) to bring the plastic upper plate 20
into close proximity of the glass lower plate 22. After exposure to
ultraviolet light, the additive polymerizes to form a compatible
network with the NOA-63 adhesive and also interacts with the
plastic upper plate 20 and the glass lower plate 22 to strengthen a
bond between the plastic upper plate 20 and the glass lower plate
22.
[0071] Although one embodiment of the present invention has been
illustrated in the accompanying Drawings and described in the
foregoing Detailed Description, it should be understood that the
invention is not limited to the embodiment disclosed, but is
capable of numerous rearrangements, modifications and substitutions
without departing from the spirit of the invention as set forth and
defined by the following claims.
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