U.S. patent application number 12/690388 was filed with the patent office on 2010-05-13 for multi-well plate and method of manufacture.
Invention is credited to James G. Clements, Michael Curtis, Paul E. Gagnon, William J. Lacey, Gregory R. Martin, David M. Root, Allison J. Tanner.
Application Number | 20100119418 12/690388 |
Document ID | / |
Family ID | 26893964 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100119418 |
Kind Code |
A1 |
Clements; James G. ; et
al. |
May 13, 2010 |
MULTI-WELL PLATE AND METHOD OF MANUFACTURE
Abstract
A method of manufacture and assembly of multiwell plates
employing targeted radiation at an interface in order to achieve
bonding is disclosed. The method accommodates glass and polymer
attachment as well as polymer to polymer attachment. Resultant
plates have unique flatness and optical properties.
Inventors: |
Clements; James G.;
(Brentwood, NH) ; Curtis; Michael; (Stratham,
NH) ; Gagnon; Paul E.; (Farmington, NH) ;
Lacey; William J.; (North Andover, MA) ; Martin;
Gregory R.; (Acton, ME) ; Root; David M.;
(Westford, MA) ; Tanner; Allison J.; (Portsmouth,
NH) |
Correspondence
Address: |
CORNING INCORPORATED
SP-TI-3-1
CORNING
NY
14831
US
|
Family ID: |
26893964 |
Appl. No.: |
12/690388 |
Filed: |
January 20, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09837241 |
Apr 18, 2001 |
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12690388 |
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60198604 |
Apr 19, 2000 |
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60258913 |
Dec 29, 2000 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B29C 65/1412 20130101;
B29C 66/71 20130101; B29C 66/929 20130101; B29C 66/9241 20130101;
B29C 65/1616 20130101; B29C 66/53421 20130101; B29C 66/71 20130101;
B29C 66/71 20130101; B29C 66/727 20130101; B29K 2995/0008 20130101;
B29C 66/81267 20130101; B29C 66/001 20130101; B29C 65/1477
20130101; B29C 66/71 20130101; B29K 2709/08 20130101; B29C
2035/0822 20130101; B01L 3/5085 20130101; B29C 66/74 20130101; B29C
57/00 20130101; B29C 65/1674 20130101; B29C 66/71 20130101; B29K
2309/08 20130101; B29C 66/131 20130101; B29C 66/71 20130101; B29C
66/53423 20130101; B29C 66/73921 20130101; B29C 65/1654 20130101;
B29C 66/81455 20130101; B29C 66/71 20130101; C12M 23/12 20130101;
B29K 2083/00 20130101; B29K 2067/00 20130101; B29K 2023/14
20130101; B29K 2081/06 20130101; B29K 2027/12 20130101; B29K
2077/00 20130101; B29K 2027/06 20130101; B29K 2023/12 20130101;
B29K 2033/12 20130101; B29K 2023/18 20130101; B29K 2023/00
20130101; B29K 2023/06 20130101; B29K 2009/06 20130101; B29K
2025/06 20130101; B29K 2069/00 20130101; B29K 2025/04 20130101;
B29K 2023/38 20130101; B29C 66/53461 20130101; B29C 65/1658
20130101; B29C 66/71 20130101; B29C 66/71 20130101; B29C 66/92211
20130101; B29L 2031/601 20130101; B29C 65/1677 20130101; B29C
65/1664 20130101; B29C 66/71 20130101; B29C 66/71 20130101; B29C
66/71 20130101; B29C 66/71 20130101; B29C 66/7465 20130101; B29C
65/1635 20130101; B29C 66/71 20130101; B29C 66/71 20130101; B29C
66/71 20130101; G01N 21/03 20130101; B29K 2995/0027 20130101; B29C
66/112 20130101; B29C 66/71 20130101; B29C 65/1435 20130101; B29C
66/71 20130101; B29C 65/8253 20130101; B29C 66/8322 20130101 |
Class at
Publication: |
422/102 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Claims
1.-31. (canceled)
32. A multiwell plate for use in assaying samples compromising in
combination: an upper plate having at least one well therein
extending between open ends on opposite surfaces of said upper
plate; a lower plate of polymer film having a thickness less than 5
mils and bonded to said upper plate by means other than adhesive
attachment; and whereby said lower plate extends across the at
least one well creating a well bottom having a top surface.
33. The multiwell plate of claim 32 wherein said top surface of
said well bottom has a flatness of less than 10 microns as measured
across the diameter of said well.
34. The multiwell plate of claim 32 wherein said lower plate forms
well bottoms for a matrix of wells, said lower plate having bottom
surface having a flatness of no greater than 50 microns when
measured across said entire bottom surface on a line intersecting
the diameters of each said well bottom in said line.
35. The multiwell plate of claim 32 wherein said lower plate is
porous.
36. The multiwell plate of claim 32 wherein said top surface of
said at least one well comprises reactive coating and wherein
sidewalls of said at least one well are free of the reactive
coating.
37.-50. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/198,604, filed Apr. 19, 2000 and U.S.
Provisional Application No. 60/258,913, filed Dec. 29, 2000.
FIELD OF INVENTION
[0002] The present invention relates generally to multiwell assay
plates for use in chemical and biochemical analysis, and more
particularly multiwell plates having transparent well bottoms and
improved methods of manufacture.
BACKGROUND
[0003] 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 kinetics, DNA melting point
determinations, DNA spectral 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
solvation concentration. Also, there are a number of assays which
use intact living cells and which require visual examination.
[0004] 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 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 typically are 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 may 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).
[0005] Typically, 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 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.
[0006] In many applications, a transparent window in the bottom of
each sample well is needed. Transparent bottoms are primarily used
in assay techniques that rely on emission of light from a sample
and subsequent spectroscopic measurements. Examples of such
techniques include liquid scintillation counting, techniques which
measure light emitted by luminescent labels, such as bioluminescent
or chemoluminescent labels, fluorescent labels, or absorbance
levels. Optically transparent bottom wells also lend the advantage
of microscopic viewing of specimens and living cells within the
well.
[0007] Currently, optically transparent and UV transparent bottomed
multiwell plates exist in the market and are used for the purposes
described. These plates typically are a hybrid of different
polymeric materials, one material making up the well walls and
another making up the bottom portion of the wells.
[0008] Ideally, plates to be used for spectroscopic and microscopic
measurement 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 and which may be manufactured to extreme
smoothness. Still, its surface may be easily coated or otherwise
altered in order to promote attachment of specific targeted
molecules. For example, a silane coating may be applied to the
glass in order to extend any variety of functional groups such as
amine functionalities, for example. Such amine functionality may
can be effectively used to immobilize reactive molecules of the
types commonly used in biological assays and testing procedures,
e.g., to immobilize specific binding members (e.g., antigens,
ligands, and haptens), entire cells, proteins (e.g., binding
proteins, receptor proteins, antibodies and antibody fragments),
nucleic acids (e.g., RNA and DNA molecules), tissue and the like.
Further, the use of a polylysine coating on glass cover slips to
grow nerve cells is a standard procedure.
[0009] Unfortunately, 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 assay plate format. One solution to the problem,
offered by the present invention, is to combine an injection molded
polymeric upper plate molded to form the wells of a microplate,
with a substantially flat transparent glass lower plate to form the
well bottoms. In order to accomplish this result, the inventors
considered several known methods for combining glass and plastic.
Two commonly employed methods of joining these types of materials
are by means of adhesive bonding and by means of insert
molding.
[0010] The use of adhesives to bond together the material forming
the well bottoms and material forming the well walls is expensive
and leads to contamination of the biologically sensitive well
surface. Low molecular weight species from the polymeric material
making up the sidewalls of the wells, as well as species within the
adhesive itself, tend to migrate through the adhesive and onto the
transparent bottom surface. When this occurs, biomolecules can not
properly react with the surface as intended under particular assay
conditions. Adhesives which are UV cured or UV stabilized also have
the tendency to absorb LTV light, which may result in altering
fluorescent readings taken from a detector located above or below
the plate. The effect of the UV light is to non-specifically modify
the signal by non-specific fluorescence thus creating undesired
background readings which are highly variable from well to well and
from plate to plate.
[0011] Insert molding is another common technique for joining
together polymeric and glass parts. In this manufacturing method,
the polymer portion is molded against or around the glass portion.
Since the polymer has a much lower melting temperature than the
glass, the glass remains in solid form while the liquid polymer is
pressed against it. Once hardened, the polymer/glass interface
remains attached only by weak interactions. One way of increasing
the mechanical strength of the connection is to mold around or
encapsulate the glass with the polymer, e.g. in glass bottomed
ashtrays. Unfortunately, using this technique to combine polymeric
parts with glass sheets of microscope coverslip thickness, as
described in multiwell plate manufacture, is not practical because
the mechanical strength of glass at such thinness is extremely
low.
SUMMARY
[0012] The present invention offers an improved multiwell plate and
a method of making a multiwell plate having a transparent bottom
welled portion which allows for undistorted spectroscopic
measurement of light emissions from a sample. The method comprises
the following steps: providing an upper plate having an array of
open ended wells, the plate made from a polymeric material
containing a silane and infrared absorbing particles and/or
particles; providing a substantially flat glass sheet lower plate
which is substantially transparent to infra red radiation at
selected wavelengths; contacting the upper plate to the lower plate
to form an interface; and, heating the upper plate at the interface
above its transition temperature, through the lower plate, by means
of infra-red radiation, the molten upper plate polymer wets the
lower plate at the interface and the part is cooled such that the
upper plate and the lower plate are bonded together by covalent
attachment formed during the wetting, heating and cooling
steps.
[0013] The present method allows for the attachment of flat glass
of small thickness as the material for the transparent lower plate.
Glass has the advantage that it will not polarize or stretch, and
thereby will not distort the emission measurements obtained from
the sample wells. In addition, glass may be manufactured to extreme
optical flatness requirements and has a much higher melting
temperature than the polymeric material making up the upper plate.
As a result it is far less susceptible to any deformation from
melting and is more likely to maintain its excellent optical
properties.
[0014] The present method also allows for the attachment of like
polymers (e.g. a polystyrene black opaque upper plate with an
optically transparent polystyrene film), or attachment of unlike
polymers (e.g. a polystyrene black opaque upper plate with a PTFE
film). The resultant polymeric plates have unique characteristic
flatness both across individual wells and across the entire
plate.
BRIEF DESCRIPTION OF DRAWINGS
[0015] FIG. 1 is a three dimensional view of the multiwell plate of
the present invention.
[0016] FIG. 2 is a partial cross section of the multiwell plate of
the present invention. The upper and lower plates of the present
invention are shown, after the two have been joined.
[0017] FIG. 3 is a partial cross section of an embodiment of the
multiwell plate of the present invention, showing the bottom plate
treated with a chemically active coating.
[0018] FIG. 4 is an exploded perspective view of the multiwell
plate of the present invention.
[0019] FIG. 5 is a partial exploded cross section view of one
embodiment of the multiwell plate embodying the present invention,
showing thin ridges around the perimeter of the well bottoms.
[0020] FIG. 6 is a schematic cross sectional view of the
manufacturing apparatus used in the present invention prior to
introducing gas.
[0021] FIG. 7 is a schematic cross sectional view of the
manufacturing apparatus used in the present invention after
introduction of gas to inflate the bladder formed by the PDMS
layer.
[0022] FIG. 8 is an exploded view of an embodiment of the present
invention.
[0023] FIG. 9 is an exploded schematic cross sectional view of the
assembly of parts in the manufacturing apparatus used in the
present invention for producing a plate with a thin film
bottom.
[0024] FIG. 10 is a schematic representation of the parts of FIG. 9
as assembled and clamped within the manufacturing apparatus.
[0025] FIG. 11 is a bar graph representation comparing across plate
flatness of 96 well plates made with the present process and
similar plates made with a standard molding process.
[0026] FIG. 12 is a bar graph representation comparing across well
flatness of 96 well plates made with the present process and
similar plates made with a standard molding process.
[0027] FIG. 13 is a bar graph representation comparing across well
flatness of 384 well plates made with the present process and
similar plates made with a standard molding process.
[0028] FIG. 14 is a 300.times. scanning electron micrograph of a
cross section of the bonding zone between a polystyrene upper plate
and a polystyrene lower plate assembled according to the present
invention.
[0029] FIG. 15 is a 200.times. magnification photograph of cells
within a well of a 96 well plate created by the assembly process of
the present invention. The surface is untreated.
[0030] FIG. 16 is a 200.times. magnification photograph of cells
within a well of a 96 well plate created by the assembly process of
the present invention. The surface is treated with plasma.
[0031] FIG. 17 is a 200.times. magnification photograph of cells
within a well of a 96 well plate created by the assembly process of
the present invention. The surface is treated with plasma and
collagen.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Shown in FIG. 1 is a multiwell test plate 10 of the present
invention. The plate includes a peripheral skirt 12 and an upper
surface 13 having an array of wells 14 each of which is capable of
receiving an aliquot of sample to be assayed. Preferably, the plate
conforms to industry standards for multiwell plates; that is to
say, a plate bordered by a peripheral skirt 12, laid out with 96
wells in an 8.times.12 matrix (mutually perpendicular 8 and 12 well
rows). In addition, the height, length, and width are preferably
conform to industry standards. The present invention, however, can
be implemented in any type of multiwell plate arrangement including
384 and 1536 wells, and is not limited to any specific number of
wells or any specific dimensions.
[0033] The plate is of two-part construction. Referring to FIG. 2,
a partial cross section of multiwell plate 10, an upper plate 20
forms the well walls 24 and top surface; a lower plate 22 forms the
well bottoms. During the manufacturing process as will be described
in detail below, the two plates are integrally and chemically
joined together at an interface 28.
[0034] Each well 14 includes a top rim 16, sidewalls 24, and a
bottom 26. In order to prevent light transmission between adjacent
wells, 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 polymeric material opaque is
preferably light (e.g. white) in color so as to be highly
reflective and non-absorptive in order to ensure high counting
efficiency with respect to the radioactive samples. However the
walls may be optically transparent. In some types of luminescence
and fluorescence assays, it is preferred that the sidewalls 24 of
the sample wells 14 be non-reflective and absorptive, in which case
the well walls 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. The white coloration is typically
achieved with TiO.sub.2.
[0035] The bottom of the wells 26, in contrast to the sidewalls 24,
is formed from a transparent material. Preferably, the 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 well bottom and
plate is important particularly when the plate is used for
microscopic viewing of specimens and living cells within the wells.
This flatness is important in providing even cell distribution and
limiting optical variation. For example, if the well bottoms are
domed, the cells will tend to pool in a ring around the outer
portion of the well bottom. Conversely, if the wells are bowed
downwards, the cells will pool at the lowest point. Glass
microscope slides are typically flat within microns in order to
ensure an even distribution. Preferably, the well bottoms 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 well bottoms may be of any
thickness, for microscopic viewing it is preferred that the well
bottom thickness be in the range of 5-100 microns and have a
flatness in the range of 0-10 microns across the diameter of the
outer bottommost surface of an individual well. The inner and outer
surfaces of these wells are coplanar.
[0036] The glass material used here can be purchased from a variety
of manufacturers (e.g. Erie Scientific, Corning, Inc.) as a sheet.
These sheets can then be altered to fit the dimensions of the
desired size plate. This forms a transparent bottom wall 26 for
each sample well 14 and permits viewing therethrough. The
transparent lower plate also allows for light emissions to be
measured through the bottom of the sample wells 14.
[0037] The multiwell plate 10 is comprised of two separate parts. A
separately molded upper plate 20, comprising an array of open ended
sample wells 14, is used to form the sidewalls 24, the peripheral
skirt 12, and top surface 13. The upper plate is preferably molded
from long polymers that become intertwined with heating and bond
together in a noncovalent mechanism upon cooling, thereby forming
an interpenetrating polymer network. Further, the upper plate
preferably is chosen from a group of polymers containing a silane
functionality. Silane functional polymer can be copolymerized with
other monomers to create polymers with pendent silane groups. These
polymers will crosslink upon exposure to moisture in the
environment giving a toughened final polymer. An example of a
suitable material is poly (ethylene-co-trialkoxyvinylsilane). The
silane functionality in the polymer is important in creating a
covalent attachment with the glass lower plate that is used to form
the well bottoms 26. The two plates are brought into contact at an
interface 28 where covalent attachments between the silane
functionality of the organic polymeric material forming the upper
plate 20 and the hydroxyl functionality of the glass lower plate 22
create the covalent attachment.
[0038] It should be noted that the upper plate need not be molded.
For example, a sheet of the silane polymer may be provided into
which wells are drilled out by laser, punched out, or otherwise
extracted. Further, the upper plate may be laminated so that each
layer has desired properties. For example, a top most layer may be
anti-reflective, a middle layer forming the plate's sidewalls may
be hydrophobic for meniscus control, an the bottommost layer which
contact the glass lower plate is a silane polymer.
[0039] The wells 14 can be any volume or depth, but in accordance
with the 96 well industry standard, the wells will have a volume of
approximately 300 ul and a depth of 12 mm. Spacing between wells is
approximately 9 mm between center lines of rows in the x and y
directions. The overall height, width, and length dimensions of the
plate are preferably standardized at 14 mm, 85 mm and 128 mm,
respectively. Wells can be made in any cross sectional shape (in
plan view) including, square, sheer vertical walls with flat or
round bottoms, conical walls with flat or round bottoms, and
combinations thereof.
[0040] FIG. 3 shows a partial cross sectional view of one
embodiment of the present invention. This figure, however, also
shows a chemically active coating 30 that can be added onto the
inner surface of the lower plate 22. Depending on the assay
requirements, any number of chemically active coatings described in
more detail below, may be used to treat the glass surface of the
well bottoms, both on the inner and outer well bottom surfaces.
[0041] FIG. 4 is an exploded perspective view of the multiwell
plate of the present invention. The upper plate 20 can be seen,
containing the peripheral skirt 12, top rim 16, wells 14, and
sidewalls 24. The lower plate 22 is preferably flat and sized in
order to form well bottoms for all wells of the upper plate 20.
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 such that they shape or otherwise become
features of the well bottoms 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. 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.
[0042] Infrared Radiation Cold Welding Process:
[0043] The preferred process of manufacture for the plate of the
present invention is by employing infrared radiation absorbed at
the interface between the upper plate and lower plate that in
combination, form a multiwell plate. The upper plate is formed by
using standard injection molding techniques on the organic
polymeric material used. Infrared absorbing particles are added to
the batch mixture. In the case of a carbon black pigmented upper
plate, the carbon black itself serves as the infrared absorbent
material. If transparent well walls are required, an infrared
absorbing transparent pigment must be used (e.g. laser dyes such as
IR-792 perchlorate available from Aldrich Chemical). Concentrations
for the laser dyes are preferably greater than 5.times.10.sup.-6
g/cm.sup.2 at the interfacial region between upper and lower
plates. The organic polymeric material of choice is molded into an
upper plate 20, as shown in FIG. 4, consisting of a peripheral
skirt 12, an array of open ended, cylindrical wells 14 defined by
sidewalls 24, and having top rims 16.
[0044] Next, a glass sheet is obtained as the lower plate, the
length and width dimensions of which conform generally a size
capable of covering an entire array of wells from an industry
standard multiwell plate. The thickness is variable, but preferably
in the range previously discussed. Prior to assembly, the glass is
extensively cleaned by pyrolysis, plasma, UV/ozone, or piranah
solution. The two plates are firmly held together in contact using
an infrared assembly machine manufactured for example by Branson
Ultrasonics (Danbury, Conn.). A cross sectional schematic drawing
of the assembly is shown in FIGS. 6 and 7. A bottom metal feature
50 aligns and holds the upper plate 52. An upper feature 54 is
comprised of an infra red transparent polycarbonate sheet 56 of
sufficient thickness to withstand at least 25 psig, bolted to a
platinum catalyzed polydimethylsiloxane (PDMS) sheet 58 of
sufficient thickness also able to withstand at least 25 psig. An
appropriately lower plate 62 is placed on top of the upper plate
52. During the welding process, metal C-clamps 60 hold the upper
feature 54 and lower feature 50 together. The clamp 60 is attached
to the upper feature and lower feature by appropriate hinges,
bolts, and clamping means strong enough to withstand 25 psig and is
made stainless steel or aluminum, for example. A metal frame 51 is
placed inside the skirt of the upper plate in order to properly
align and locate the lower plate over the wells of the upper plate.
Once the clamps are securely fastened, an appropriate gas, e.g.
nitrogen, is introduced to the space between the polycarbonate
sheet 56 and the PDMS sheet 58 by means of a gas inlet 64. As shown
in FIG. 7, the PDMS sheet is forced downward by the inflating gas
pocket 66. This inflation has the effect of uniformly forcing the
two parts (upper plate and lower plate) into intimate contact. The
pressure may be adjusted, but the preferred range is 4-25 psi, more
preferably, 5-7 psig.
[0045] Energy is then supplied by an array of infra red laser
diodes transmitting at approximately 820 nm. This energy passes
through the polycarbonate sheet, the PDMS sheet, and the glass
lower plate and is targeted at the portion of the upper plate that
contacts the lower plate. The infra red absorbing molecules forming
part of the matrix polymer of the upper plate, absorb this energy,
transfer it to the polymer and thereby melt the portion of the
upper plate which interfaces the glass of the lower plate. The
organic polymeric material of the upper plate must be brought to
its melting temperature in order for it to wet the glass and then
for the covalent attachments to occur between the silane in the
polymer and the hydroxyl groups of the glass at the interface,
since the reaction of the covalent attachment is driven by the
presence of heat and moisture. Assembly should preferably take
place under clean room conditions. In fact, a carbon filter within
the unit will effectively remove smoke and residual organics
created from the welding process, as well as protect the lasers
from out-gassing, and add clean air to the system. Further, it is
helpful to sparge the unit with helium while the welding process is
initiated and carried out. This helps achieve a clean part and
limits any unwanted surface oxidation or other reactions on the
exposed surfaces.
[0046] The array of infra red diodes is focused to give a uniform
line of energy about 2 min wide on the bottom surface of the upper
plate. The clamp 60 described in FIG. 6 is translated at a constant
speed to scan the line of energy over the entire surface to be
bonded. The scan speed is variable, but preferably in the range of
0.1-1.0 inches/second. Operation power on the instrument is
typically in the range between 45 and 75%. The laser can be turned
off at the end of the scan to prevent damage to the clamp or
translation mechanism. In a preferred embodiment the metal frame 51
is used to define the area to be scanned and to hold the glass
lower plate 62 in alignment. This metal frame also takes up the
energy until the laser output is stable and to take up the energy
of any over scan. If necessary the frame can be cooled. Further,
any excess energy passing through the entire assembly of upper and
lower plates (e.g. in well areas) can be absorbed into the base of
the assembly structure which is preferably fitted with cooling
equipment. The combination of the frame and the base of the
instrument receive the laser energy not used in welding. Since
other materials are IR transparent, the heat buildup in the
polycarbonate window 56, the PDMS bladder 58, and the glass 62 is
minimal. Heating only occurs over the scan line, so after the scan
line has passed over a particular area, the lower and upper plates
in that area are bonded, while the area not yet scanned is still
held together by the clamping means previously discussed. Because
of the clamping, the registration between upper and lower plate
during the welding process remains unchanged; this is especially
important when exact alignment of features in the lower plate is
required.
[0047] The covalent grafting is achieved in a two step reaction.
First, a hydrolysis reaction occurs in which a water molecule
hydrolyzes a silicon-ethoxy bond in the silane, releasing a
molecule of ethanol and leaving a silicon hydroxide function bond.
Second, a condensation reaction occurs in which two silicon
hydroxide functionalities, one from the silane and one from the
glass surface, eliminate a water molecule, giving rise to a
siloxane bond. The resultant siloxane bonds grafting the upper and
lower plate together are extremely stable.
[0048] The infrared welding technique is considered a "cold
welding" technique because it focuses the heating only in the
contact zone interface at the bottom of the sidewalls defining the
open bottom of the upper plate. This allows the organic polymeric
material forming the remainder of the upper plate to remain rigid
and cold, just as the glass material remains so. Further, the use
of an infrared laser allows the organic polymeric material to be
heated very quickly and only a small amount of heat is added at the
interface, which is quickly radiated. This has the effect of
preventing the polymeric material of the upper plate from
expanding, and therefore remains inflexible. In contrast,
conventional methods of heating would cause the entirety of the
upper plate to stretch, and eventually become pliable, due to the
slow heating and cooling processes inherent in these methods.
[0049] FIG. 5 is a partial exploded cross section demonstrating an
alternative design of the upper plate which provides more surface
area for the interaction between the polymer and the glass lower
plate. A beaded rim 40 is molded onto the bottom of the sidewalls
42 of the sample wells making up the upper plate 44. These beads
directly contact and covalently bond to the surface of the lower
plate 46 thereby drastically increasing the contact area between
plates.
[0050] In an alternative embodiment, prior to covalently fusing the
upper and lower plate together and as shown in FIG. 3, a
biologically or chemically active coating 30 may be applied to the
surface of the lower plate 22. This way, after the lower and upper
plates are fused to form a multiwelled plate, the well bottoms will
have imparted thereupon the coating. Coatings can be introduced by
any suitable method known in the art including printing, spraying,
condensation, radiant energy, ionization techniques or dipping. The
coatings may then provide either covalent or non-covalent
attachment sites. Such sites, in or on the bottom well surface can
be used to attach moities, such as assay components (e.g., one
member of a binding pair), chemical reaction components (e.g.,
solid synthesis components for amino acid or nucleic acid
synthesis), and cell culture components (e.g., proteins that
facilitate growth or adhesion). Further, the coatings may also be
used to enhance the attachment of cells (e.g., polylysine). It can
also be conceived that an array of biomolecules (e.g., DNA
sequences) can be printed or otherwise synthesized on the surface
of the glass lower plate prior to assembly with the upper plate.
Once the upper plate is attached, each well bottom may contain a
separate such array. The manufacturing method of the present
invention is particularly useful for any embodiment involving
application of a coating or other moiety. Since only a limited
region on the polymeric upper plate and away from the area which
becomes the well bottoms is actually heated in order to join the
upper and lower plates, the coating remains substantially unaltered
and undamaged. In this embodiment, it is assumed that no materials
are added to the coatings which would absorb at the wavelength of
the particular laser diodes being employed.
[0051] Further, it may be contemplated that coatings be applied to
the outermost surface of the well bottoms. For example, coatings
which affect the optical properties of the well (e.g. color
filters, IR filters, UV filters, polarizing filters, photon sieves,
antireflective surfaces, etc.) may be added based on the specific
assay requirements.
[0052] Additionally, the lower plate may have a uniform coating on
the contacting surface from which bonding areas are ablated by
laser. This technique may be required in instances where an
extremely thick coating may affect the integrity of the bond formed
using the IR assembly instrument. Since the coating is removed from
the areas where the lower and upper plates will intersect, nothing
will be present to affect the bond strength. Alternatively, the
coating may be applied as a design print in which the bonding areas
are free of coating. This printing technique may also be employed
to create bottom wells, each having a different functionalized
surface based on different coatings. This may be accomplished by
printing the varied coatings over the contact surface of the lower
plate. The coated regions each align with a particular well from
the upper plate. Once bonded, a plate having different coatings in
the bottoms of each well is achieved.
Gasket:
[0053] In an alternative construction process to that described
above and as shown in FIG. 8, instead of having an entire upper
plate made of a silane polymer, an interfacial gasket molded from
the silane polymer material having IR absorbent characteristics, as
previously described, may be employed instead. In such an
embodiment, the gasket 70 serves as a bond facilitator for joining
the glass bottom 72 with the polymer upper plate 74. This thin
film-like gasket, which may also be formed by cutting or by
punching a film, preferably has the same footprint dimensions as
the finished plate bottom. Although the gasket thickness may vary,
it is preferred that it be between 0.5-5 mils in thickness so that
the entire gasket may be properly and entirely heated from
radiation aimed only at one side. An array of holes or punches are
formed therein to align and correspond exactly with the wells of
the upper plate. As above, a polymeric material is molded into an
upper plate consisting of a peripheral skirt, and array of wells
with sidewalls, and top rims. However, it is not necessary that
this plate contain a silane material. Preferably, the upper plate
is molded from a polymer material which is IR transparent, such as
polystyrene, polypropylene, polymethacrylate, polyvinyl chloride,
polymethyl pentene, polyethylene, polycarbonate, polysulfone,
polystyrene copolymers, cyclic olefin copolymers, polypropylene
copolymers, fluoropolymers, polyamides, fully hydrogenated styrenic
polymers, polystyrene butadiene copolymers, and polycarbonate PDMS
copolymers. Once molded, the upper plate is contacted and aligned
with the interfacial gasket, which is subsequently contacted with a
lower plate comprising a transparent glass sheet by using the infra
red assembly instrument and method previously described. The infra
red radiation is then targeted through the glass bottom plate on
the interfacial gasket between the upper and lower plate. The
radiation passes through the glass and is absorbed by the IR
absorbent particles (e.g. carbon black) in the polymer material
making up the gasket. This energy absorption heats the gasket only,
causing the polymer to reach its melting temperature. This melted
gasket material wets the glass where covalent siloxane bonding
occurs between the silane in the gasket polymer and the hydroxyl
groups of the glass by the same mechanism previously described. The
melted gasket material also simultaneously wets and melts the
interface between it and the upper plate thereby creating an
interpenetrating polymer network bond. In a preferred embodiment,
IR radiation is also targeted onto the gasket through an IR
transparent upper plate. This way, not only is the interface
between glass and gasket heated, but also the interface between
upper plate and gasket thereby enhancing bond strength at both
interfaces.
[0054] As in the previous process embodiment, this method is
advantageous in the glass sheet may be treated with a biologically
or chemically active coating prior to assembly. Since the heating
only occurs in the polymeric material of the gasket and not on the
glass itself, the coating or attached biological/chemical moiety
remains largely unaffected by the manufacturing process.
[0055] It is important to also note that the gasket material serves
as an impermeable barrier to fluid transfer between wells and as a
barrier to any optical cross-talk.
Silane Coating:
[0056] Yet another embodiment for manufacturing the glass bottomed
plates of the present invention is to apply a silane coating to the
portion of the upper plate which will interact with the glass
bottom plate during assembly. In this embodiment, a solvent
containing both the silane functionality such as an epoxy silane
(e.g., 2-7-oxabicyclo[4.1.0]hept-3-yl-ethyl]silane; Aldrich
Chemical) as well as the IR absorbent pigments or dyes in acetone,
for example, is applied to the bottom portion of a polymer (e.g. a
styrenic polymer) upper plate by means of spraying, printing,
dipping, brush coating, thermoset, or other means. Although not
necessary for practicing the invention, it is believed that the
epoxy silane material intercalates in and cross links into the
polymer material of the upper plate while the silane functionality
remains actively extended. Again, as in previous embodiments, the
coated upper plate is contacted with the glass bottom plate, by
using the infra red assembly instrument and method previously
described. The infra red radiation is then targeted through the
glass bottom plate on the silane coating at the interface between
the upper and lower plate. The radiation passes through the glass
and is absorbed by the IR absorbent particles (e.g. carbon black)
in the silane coating. This energy absorption heats the coating
only, driving the covalent bond reaction between the silane and the
glass where covalent siloxane bonding occurs between the silane and
the hydroxyl groups of the glass by the same mechanism previously
described. Although IR heating is preferred to help drive the
reaction and although not preferred, this type of assembly may be
performed without the aid of IR radiation (and therefore without
need of IR absorbent particles) simply by applying consistent
pressure for an extended period without heating.
[0057] As in the previous process embodiment, this method is
advantageous in the glass sheet may be treated with a biologically
or chemically active coating prior to assembly. Since the heating
only occurs in the IR absorbent material at the interface and not
on the glass itself, the coating or attached biological/chemical
moiety remains largely unaffected by the manufacturing process.
Magnetic Particles:
[0058] In an alternative construction process to that described
above, instead of infrared absorbing particles, magnetic particles
or ferromagnetic particles are mixed into a silane polymer blend in
molding a gasket. The magnetic particles are preferably coated with
a plasma polymerized coat to prevent the ferromagnetic material
from interacting with the well contents. As above, an array of
holes or punches are formed therein to align and correspond exactly
with the wells of the upper plate. Similarly, a polymeric material
is molded into an upper plate consisting of a peripheral skirt, and
array of wells with sidewalls, and top rims. Once molded, the upper
plate is contacted and aligned with the interfacial gasket, which
is subsequently contacted with a lower plate comprising a
transparent glass sheet, the length and width dimensions of which
are sized generally to cover all wells of the industry standard for
multiwell plates. Electromagnetic radiation is then targeted on the
interfacial gasket between the upper and lower plate. The radiation
passes through the glass and is absorbed by the magnetic particles
in the polymer material making up the gasket. This energy
absorption leads to vibration of the magnetic particles which in
turn heats the gasket only, causing the polymer to reach its
melting temperature. This melted gasket material wets the glass
where covalent siloxane bonding occurs between the silane in the
gasket polymer and the hydroxyl groups of the glass by the same
mechanism previously described. The melted gasket material also
simultaneously wets and melts the interface between it and the
upper plate thereby creating an interpenetrating polymer network
bond. As above, in a preferred embodiment, the electromagnetic
energy is also directed through the upper plate onto the gasket in
order to create a stronger bond between upper plate and gasket.
[0059] As in the previous process embodiment, this method is
advantageous in the glass sheet may be treated with a biologically
or chemically active coating prior to assembly. Since the heating
only occurs in the polymeric material of the gasket and not on the
glass itself, the coating or attached biological/chemical moiety
remains largely unaffected by the manufacturing process.
[0060] It may also be conceived that instead of a gasket, a
transfer film of ink containing magnetic particles is imparted on
the bottommost plane of the upper plate by hot-stamping, dipping,
or other known means. When this region interfaces with the lower
plate, e.g. glass sheet, it is the target of the prescribed
radiation.
Heating of the Lower Plate:
[0061] Another alternative method of production is by heating the
lower plate such that its temperature is raised to the melting
temperature of the polymeric material making up the upper plate.
Once contacted with the heated lower plate, the temperature of the
upper plate is elevated to it melting temperature. Again, as in the
embodiments previously described, the heating of the upper plate
only occurs at the interface with the lower plate. However, unlike
the other embodiments, the glass plate itself is heated, making
this embodiment less attractive for instances in which a coating or
biological/chemical moiety is attached prior to assembly. Again,
the covalent siloxane bond attachment mechanism is the same as
previously described. The process should be run in inert gas
conditions to prevent rapid oxidation.
[0062] Further, although not necessarily preferred for best
quality, it should be noted that the glass bottomed plate of the
present invention can be also made using standard insert molding
techniques where the glass lower plate is inserted into a mold and
the silane functional polymer upper plate is molded onto its
surface. The attachment between plates is still largely covalent
due to the pendent silane groups in the polymer material of the
upper plate and the hydroxyl groups on the glass surface.
Polymer-Polymer Plate Manufacture
[0063] It should also be noted that although the above embodiments
of the present invention envision the use of a glass lower plate,
the process of manufacture is equally effective for attaching a
lower plate comprised of a polymeric material or even a polymer
sheet. In that case, the attachment is not covalent as described,
but relies on a fusion bond. In such an embodiment, a polymer film
is used in place of the glass bottom plate. The film is
substantially transparent to infra red radiation. Examples of IR
transparent material that can serve as the lower plate portion
include: polystyrene, polypropylene, polymethacrylate, polyvinyl
chloride, polymethyl pentene, polyethylene, polycarbonate,
polysulfone, polyolefins, cyclic olefin copolymers, polystyrene
copolymers, polypropylene copolymers, fluoropolymers, polyesters,
polyamides, polystyrene butadiene copolymers, fully hydrogenated
styrenic polymers, and polycarbonate PDMS copolymers. The choice of
material will depend on the particular type of assay the multiwell
plate is meant to facilitate. In this embodiment, the lower plate
may take the form of a plate, film (porous or non-porous), filter,
pigment containing films, and cavity containing films. In the case
of films, the thickness can vary depending on material and assay
requirements, but the present method accommodates plate manufacture
with films as low as 1 mil in thickness. Multiwell plates made with
films having thicknesses below 5 mils are difficult if not
impossible to make using standard injection molding techniques. The
extremely thin films that may be employed as bottom wells may allow
gas exchange through the well bottom which in turn facilitates call
growth and biological activity. As in the glass lower plate
embodiment previously described, the polymer lower plate may
contain 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 such that they shape or otherwise become
features of the well bottoms 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.
[0064] An upper plate is either molded from material having IR
absorbent properties (e.g. carbon black), or has IR absorbent
characteristics at the interface at which the upper and lower
plates are to be bonded. For example, an IR transparent upper plate
may be employed onto the bottom of which an IR absorbent material
has been hot stamped, painted, thermoset, sprayed or otherwise
imparted. Examples of suitable IR absorbent materials include:
carbon black pigment, laser dye molecules, or other IR absorbent
materials commonly known to those of skill in the art. It is also
conceivable that the bottom plate have a patterned area of IR
absorbent material imparted onto its bonding surface which would
properly align with a wholly IR transparent upper plate.
[0065] Prior to assembly, the polymer lower plate, whether film or
rigid plate, should be cleaned with an ionized air stream in order
to remove any particulates as well as relieve static buildup. The
polymer may also be plasma treated to drive off any unwanted
moisture and to activate the surface with reactive functional
groups. This activation may in turn enhance the polymer-polymer
interaction between upper and lower plates as well as create a
biologically reactive surface. In fact, bonding certain unlike
polymers by the disclosed method may require plasma treatment of
the interactive surfaces. Plasma treatment may be accomplished by
using a plasma chamber (Branson 7150, Branson Ultrasonics, Danbury
Conn.). The RF frequency is preferably set at 13.56 MHz, the
pressure is 180 millitorr, using oxygen, and the time of treatment
is approximately 1.0 minute.
[0066] Again, as in previous embodiments, the upper plate is
contacted with the bottom plate, by using the infra red assembly
instrument and method previously described. The infra red radiation
is then targeted through the IR transparent bottom plate on the IR
absorbent material at the interface between the upper and lower
plate. Heating occurs only in areas that have been predetermined to
absorb IR radiation. The heating combined with pressure applied to
enhance the welding process, allows for fusion along the interface
between parts. Unlike in an injection molding operation where
conditions such as extreme heat will polarize and stretch the
polymer making up the well bottom, this selective heating method
assures that only the interface is heated, thereby preventing
deformation of the well and preserving the optical qualities of the
lower plate/film.
[0067] When using a non self-supporting thin film as a bottom
plate, it is preferred that a sheet of glass be used in the IR
assembly instrument as backing for the film in order to help create
the necessary pressure for bonding to the upper plate. FIGS. 9-10
are schematic cross-sectional representations of the clamping
mechanics within the IR assembly instrument. FIG. 9 is an exploded
view of the parts and the clamping mechanism employed in this
embodiment. An upper plate 80 taking the form of an open welled
polystyrene multiwell plate is fixed in place within the
instrument. The lower plate 82 taking the form of a polystyrene
film is located on top of the upper plate 80. A sheet of glass 84
sized to substantially cover the lower plate/film is located on top
of the lower plate 82. A polycarbonate layer 86 is held within an
aluminum frame 88. A PDMS bladder 90 is clamped to the aluminum
frame 88. In FIG. 10, all pieces are in intimate contact, gas has
been pumped into the area between the polycarbonate 86 and the
bladder 90 thereby creating uniform pressure on the glass sheet 84.
The pressure applied to the glass by the bladder may be between
4-25 PSI, but is preferably between 5-7 PSI. As previously
described, IR radiation is targeted through the polycarbonate 86,
the PDMS bladder 90, the glass 84, and the polystyrene film 82 onto
the contact surface of the polystyrene upper plate 80. The IR
radiation travels directly through the upper plate in the open
welled sections, without heating. The upper and lower plates are
thermally bonded together and the part is removed from the
instrument. The glass sheet does not bond to the lower plate and
therefore is easily removed. It is also important to note that
throughout this process, nothing contacts the portion of the film
that is to become the bottom of the wells. This is important
because it preserves the optical integrity and biological integrity
of the well bottom.
[0068] The method as described, when used with films which are
non-self supporting, creates well flatness not before seen in
plates having upper and lower plates joined together by
non-adhesive means such as insert molding, sonic welding, and heat
welding. Non-self supporting films are those which are unable to
support themselves in a horizontal plane when clamped on one end.
This may be tested by clamping a 6 cm.times.1 cm sample on one end
and held outwardly in a horizontal position. If the sample is not
sufficiently rigid to maintain the plane, it shall be considered,
for purposes of this disclosure, non-self supporting.
[0069] As with previous embodiments, the film or polymer plate
(lower plate) may be treated with a biologically or chemically
active coating prior to assembly. Since the heating occurs only at
the interface between polymer parts, the IR transparent coated
portion making up the well bottom will remain unaffected. Further,
a unique characteristic of this assembly process allows for only
the well bottoms to be coated or functionalized, and not the well
walls. This can have important advantages in many assay systems
where biomolecular attachment to the well walls can cause problems
with assay results. For example, in cell based assays, it is
important that cells adhere to the transparent bottom of the well
in a monolayer. When cells attach to the side walls of the well,
they often will later retract and settle onto the well bottom,
killing the cells of interest attached there. In a plate having
wells with non-functionalized side walls and functionalized
bottoms, one can be sure that the desired assay activity occurs on
the well bottoms. It should be noted that this advantage is equally
applicable to the glass bottomed plate embodiment.
Experiment 1
[0070] In order to demonstrate unique flatness characteristics both
across individual wells as well as across the entire bottom of a
multiwell plate, measurements were taken from plates made by the
present method as well as plates made from standard injection
molding/insert molding techniques. FIG. 11 demonstrates the
flatness of several plates as measured by a Tencor (model P20)
profilometer. Measurements were taken across the bottom portion of
several types of multiwell plates. Each plate was measured across
several different lines and results were averaged. First, several
Greiner .mu.Clear.TM. 96 well black/clear plates, made from an
insert molding technique, whereby a 5 mil thick optically clear
polystyrene film is placed in a mold and an upper plate is molded
against it, were measured. As shown in FIG. 11, column A, the
bottoms of these plates were not even flat enough across their
length to be recorded by the instrument. Next, several Corning
Costar.TM. 96 well black/clear plates, also made from an insert
molding technique whereby a 25 mil thick separately molded
optically clear polystyrene lower plate is placed in a mold and a
black polystyrene upper plate is molded against it, were measured.
The black polystyrene plate was achieved by standard practice of
adding carbon black to the batch mixture in concentration of 1 part
carbon black material (Furnace Black-Black Pearl 430, Clarion
Corp.) to 50 parts virgin polystyrene (685D, Dow Chemical Corp.),
and injection molding the plate. As shown in FIG. 11, column B, the
average flatness across the plates was in the range of 70 microns.
Next, several Corning Costar.TM. 96 well black/clear plates made
from an insert molding technique whereby a 5 mil thick optically
clear polystyrene film is placed in a mold and a black polystyrene
upper plate is molded against it, were measured. As shown in FIG.
11, column C, the average flatness across these plates were in the
range of 80 microns. Finally, several plates made by the present
method from a 5 mil optically clear polystyrene film IR radiation
bonded to a black polystyrene upper plate were measured. As shown
in FIG. 11, column D, the average flatness across these plates is
approximately 55 microns.
Experiment 2
[0071] Similarly, the plates of example 1 were measured across the
bottoms of multiple randomly selected individual wells for
flatness. FIG. 12 shows the results of the experiment. The Greiner
.mu.Clear.TM. plate, column A, has well bottoms with an average
flatness approaching 60 microns. The Corning Costar 25 mil bottom
plates, column B, have an average well flatness of approximately 35
microns. The Corning Costar 5 mil bottom plates, column C, have an
average well flatness of about 40 microns. The 5 mil bottom plates
made from the present method have an average well flatness of less
than 5 microns.
Experiment 3
[0072] Across well flatness was also measured for plates
manufactured in the same manner, with the same materials as in
Example 1, but of a 384 well format instead of 96. Again, the
flatness measurements for the plate made by the instant invention
are superior in flatness to plates made by standard insert molding
techniques for flatness across an individual well. FIG. 13 shows
the results of the experiment. The Greiner .mu.Clear.TM. 384 well
plate, column A, have well bottoms approaching an average flatness
of 16 microns. The Corning Costar.TM. 25 mil bottom plates, column
B, also have an average well flatness of approximately 16 microns.
The Corning Costar.TM. 5 mil bottom plates, column C, have an
average well flatness of about 12 microns. The 5 mil bottom 384
well plates made from the present method have an average well
flatness of less than 5 microns.
Experiment 4
[0073] Several 1 by 3 inch glass microscope slides were employed to
determine the relative strength of the silane/glass bond
interaction that plays a significant part in some embodiments of
the present invention. Two types of interactions were studied.
First, a 20 mil thick non-silane containing polymer material was
attached to a glass slide using the IR assembly instrument and
process as previously described. Next, a 20 mil thick silane
containing polymeric material was attached to a glass slide using
the IR assembly instrument and process as previously described. The
polymeric material in each instance was sized to overlap
approximately an inch beyond the glass. Once assembled, each part
was stabilized with a clamp, and the overlapping polymer portion
was clamped and pulled with a force at a right angle to the plane
established by the glass slide. The total pounds of force required
to separate the glass from the polymer on each slide was
recorded.
[0074] The non-silane polymeric material registered a result of 0
pounds of force required for separation. The silane containing
material could not be separated from the glass upon exacting the
maximum amount of force (40 lbs.) from the measuring instrument.
This experiment demonstrates the effectiveness of the bonding
interaction facilitated by the siloxane linkages.
Experiment 5
[0075] Several 96 well plates were constructed using the assembly
method of the present invention. The plates were constructed of a
high density polyethylene upper plate, and a 1 mm thick glass
bottom bonded together with a vinyl silane gasket. The plates were
then subjected to a pressure test to discern the force required to
separate the parts. An Ametec.TM. pressure gauge was used wherein
the gauge unit was inserted into various wells and pressed against
the well bottoms. No separation or distortion of the plate occurred
through 20 pounds of pressure, the outer limit of the instrument.
This test demonstrates the effectiveness of the bond created by
employing a gasket device as described herein.
Experiment 6
[0076] A black polystyrene 96-well upper plate was bonded to a 5
mil thick optically clear polystyrene film lower plate using the
infra red assembly process herein described. The plate was then cut
in cross section in order to exposed the bonding zone. FIG. 14 is
an SEM micrograph of the cross-sectional cut at a resolution that
does not differentiate the black upper portion from the optically
clear lower portion. A knit line 100 marks the boundary between
upper plate 102 and lower plate 104. The bottom 106 of the
multiwell plate is marked by the transition to air 108. This
micrograph displays that the polystyrene parts weld together so
well that the intersection between upper and lower plate cannot be
determined but for the knit line 100.
Experiment 7
[0077] In order to demonstrate the effects of the welding process
of the present invention on a pretreated surface, an experiment was
performed studying biological compatibility of plates manufacture
according to the present method. Three groups of 96 well plates
were assembled according to the present process, combining a
96-well black polystyrene upper plate with an optically clear 5 mil
thick polystyrene film lower plate. Prior to assembly, the lower
plates were treated in different ways. In a first set of plates,
the lower plates were untreated; in a second set of plates, the
lower plate surface, which was to become the well bottoms, was
treated with plasma radiation as described above; and in the third
group of plates the lower plates were treated with plasma radiation
and coated with collagen. After assembly, all fully assembled
96-well plates were tested for biological compatibility by
attempting to grow cells on the well surfaces.
[0078] In the first group of plates, as shown in the photograph of
FIG. 15, the untreated surface was not compatible with achieving a
monolayer of cell growth. Cells 110 are widely distributed and
non-confluent. Some cells are bunched around the edge of the well
112, while other areas around the well edge are completely free of
cell attachment.
[0079] In the second and third group of plates, as shown by
representative photographs FIGS. 16-17, respectively, cell growth
is robust and cells 110 are evenly distributed across the well
bottom surface. Cells have flattened out indicating good adhesion
to the surface. Further, and importantly the cell attachment and
growth occurs right up to the edge of the wells 114, 116. This
demonstrates that the cold welding assembly process does not
adversely affect these pretreated surfaces.
[0080] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose and variations can be made therein by those
skilled in the art without departing from the spirit and scope of
the invention which is defined by the following claims.
* * * * *