Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND DEVICE FOR CREATING MICRO-ARRAYS
BACKGROUND OF THE INVENTION
Field of the Invention
The invention relates to a method and device capable of
simultaneously creating a series of identical micro-arrays,
each micro-array comprising hundreds or thousands of analyte-
assay regions on a solid support, each analyte-specifi.c reagent
useful, for example, in detecting labeled cDNA in hybridization
assays.
Description of the Related Art
Micro-arrays of hundreds or thousands of biological
analyte-assay regions are widely used for biological analysis.
Tiny droplets, each containing a different known reagent,
usually distinct polynucleotide or polypeptide biopolymers such
as known DNA fragments, are deposited and immobilized in a
regular array on a solid substrate such as a glass microscope
slide. The array of dried droplets is exposed to a solution
containing an unknown, for example complementary DNA (cDNA)
fragments pre-labeled with fluorescent or radioactive chemical
tags. Binding reactions or hybridizations occur wherever there
is a match between the complementary sequence polynucleotides
in the array and the cDNA. Subsequent optical or
radiosensitive scanning determines which spots contain tags,
thereby identifying the complementary compounds present in the
solution.
While micro-arrays provide a useful tool for rapid
biological analysis, the processes by which the micro-arrays
are produced remain time consuming and expensive.
For example, it is known from US Patent 5,807,522 (Shalon
et al.) to use capillary pens of various geometries to print or
spot droplets onto substrates, one substrate at a time.
Although multiple (typically 8 or 16) pens may be used
simultaneously, often under robotic control, each pen or group
of pens is loaded with only one reagent per pen. The pen(s)
are then touched to one substrate after another, depositing
nearly identical droplets on each. After each of the set of
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substrates to be prepared (typically a few dozen to several
hundred) has been spotted with a first set of reagent droplets,
the set of pens is washed and dried, reloaded with the next set
of reagents and the next set of droplets are printed onto the
same substrates at adjacent locations. This procedure is time
consuming and requires expensive and elaborate equipment to
achieve precision and speed.
Another known method involves long flexible capillary
tubes to carry fluid from sets of storage wells to the tips of
the tubes, which tips are applied to one substrate after
another in a manner similar to Shalon et al. This method
suffers all of the same disadvantages as Shalon et al. and also
requires a significant volume of expensive reagent to be stored
in the capillary tubes.
Still another known method is disclosed in US Patent
5,800,992 (Fodor et al.) and involves combinatorial chemistry
to synthesize oligonucleotides on the substrate with a series
of chemical reactions (Affymetrix). This method is limited to
oligonucleotides and is not suitable for long stranded cDNA's.
In addition, the sequence of the oligonucleotides must be known
in advance. This method also suffers the disadvantages of
requiring cumbersome expensive equipment and involving time
consuming reaction steps.
In order to amplify the fluorescence or radioactivity
signal indicative of a binding reaction, US Patent 5,843,767
(Beattie) teaches the provision of a multiplicity of discrete
channels running through the substrate and arranged in groups,
and with binding reagent immobilized on the walls of the
channels. The channels increase the amount of surface area in
the substrate available for the binding, thus theoretically
improving detection sensitivity and efficiency. However, in
practice, it has been found that the improvements were not as
great as expected, since the detection optics will still be
limited to direct reception only from the projected area.
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SUMMARY OF THE INVENTION
In view of the foregoing disadvantages inherent in
known methods of manufacture and types of micro-arrays, it
is an object of the present invention to provide an improved
method and device for simultaneously creating a plurality of
identical micro-arrays of biological samples.
The invention utilizes a plurality of substrates,
each of which having a top side, a bottom side, and a
pattern of through-holes. Each through-hole has a wider
upper cross-section, a narrower lower cross-section, and
preferably a step or plateau parallel to the top side of the
substrate formed in the transition area. When a number of
substrates are stacked, the corresponding through-holes are
in registry and form tunnels extending through the stack of
substrates. Reagents of interest are caused to flow through
the tunnels and deposit on the step or plateau area.
Thereby all substrates in the stack are "spotted"
simultaneously, at precise locations and with a precise
amount of reagent. A barrier layer may be provided between
substrates to prevent leak-through between neighboring
holes.
After the desired reagents have been deposited,
the substrates are separated. In this manner a series of
micro-arrays, each capable of containing hundreds or
thousands of biological samples such as cDNA fragments, is
formed simultaneously.
Thus, according to one aspect of the present
invention, there is provided a substrate for use in making
micro-arrays, said substrate having a top side, a bottom
side, and multiple through-holes extending between said
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substrate top side and bottom side, each through-hole having
an area of wider cross-section, an area of narrower cross-
section, and a plateau formed in a transition area, wherein
each substrate through-hole receives a deposit of an
immobilized reagent.
According to another aspect of the present
invention, there is provided a stack of substrates for use
in making micro-arrays, said stack comprising at least two
substrates, each substrate having a top side, a bottom side,
and multiple through-holes extending between said substrate
top side and bottom side, each through-hole having an area
of wider cross-section, an area of narrower cross-section,
and a plateau formed in a transition area wherein each
substrate through-hole receives a deposit of an immobilized
reagent, wherein in said stack of substrates through-holes
in corresponding positions are in registry such that they
form continuous tunnels extending through said stack of
substrates.
According to still another aspect of the present
invention, there is provided a device for simultaneously
forming a set of identical micro-arrays, said device
comprising: a releasable retaining means; a stack of
substrates held by said releasable retaining means, said
stack comprising at least two substrates, each substrate
having a top side, a bottom side, and multiple through-holes
extending between said substrate top side and bottom side,
each through-hole having an area of wider cross-section, an
area of narrower cross-section, and a plateau formed in the
transition area, wherein in said stack of substrates said
through-holes are in registry and form continuous tunnels
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extending through said stack of substrates; and a means for
introducing reagent to discrete through-holes at the upper
or lower surface of said stack such that the reagent is
immobilized upon each substrate of the stack.
According to yet another aspect of the present
invention, there is provided a method for simultaneously
creating a series of identical micro-arrays, said method
comprising: forming a stack of substrates, said stack
comprising at least two substrates, each substrate having a
top side, a bottom side, and multiple through-holes
extending between said substrate top side and bottom side,
each through-hole having an area of wider cross-section, an
area of narrower cross-section, and a plateau former in the
transition area, wherein in said stack of substrates said
through-holes are in registry and form continuous tunnels
extending through said stack of substrates; introducing at
least a first reagent into a first tunnel such that the
first reagent is immobilized upon each substrate of the
stack along the first tunnel and a second reagent into a
second tunnel such that the second reagent is immobilized
upon each substrate of the stack along the second tunnel;
and separating said substrates.
According to a further aspect of the present
invention, there is provided a method for simultaneously
creating a series of identical micro-arrays, said method
comprising: (a) forming a stack of alternating spacers and
substrates, said stack comprising at least two spacers and
two substrates, each substrate having a top side, a bottom
side, and multiple through-holes extending between said
substrate top side and bottom side, each spacer having a top
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side, a bottom side, and multiple through-holes extending
between said spacer top side and bottom side, wherein said
spacer through-holes are of greater diameter than said
substrate through-holes, and wherein said spacer through-
holes are in registry with said substrate through-holes,
such that aligned spacer and substrate through-holes form
columns with a continuous flow path through said stack of
alternating spacers and substrates, and such that said
spacers form barriers separating through-holes of adjacent
columns; (b) passing reagent through said continuous flow
path to cause deposition of and immobilization of reagent
upon said substrates; and (c) separating said substrates to
reveal a micro-array of reagents on each of said at least
two substrates.
The foregoing has outlined rather broadly the more
pertinent and important features of the present invention in
order that the detailed description of the invention that
follows may be better understood and so that the present
contribution to the art can be more fully appreciated.
Additional features of the invention will be described
hereinafter which form the subject of the claims of the
invention. It should be appreciated by those skilled in the
art that the conception and the specific embodiments
disclosed may be readily utilized as a basis for modifying
or designing other methods and devices for producing micro-
arrays and for carrying out the same purposes of the present
invention. It should also be realized by those skilled in
the art that such
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equivalent structures do not depart from the spirit and scope
of the invention as set forth in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
For a fuller understanding of the nature and objects of
the present invention reference should be made by the following
detailed description in conjunction with the accompanying
drawings in which:
Fig. 1 is an isometric drawing of stack of substrates,
showing the matching holes and pockets.
Fig. 2 is a cross-sectional view through part of one row
of holes in a stack of substrates.
Fig. 3 is a cross-sectional view of a stack of substrates
combined with a means for filling them using a pipette. This
figure also shows a clamping means.
Fig. 4 is a cross-sectional view of a stack of substrates
combined with a means for filling them using vacuum suction and
tubes to a micro-titre tray.
Fig. 5 is a cross section through a stack of substrates as
in Fig. 2 but in an alternative arrangement.
Fig. 6a is an exploded view of a stacking arrangement of
alternating layers of wide aperture gasket and narrow aperture
substrate, the layers not-to-scale, as used in an alternative
embodiment of the invention.
Fig. 6b is a cross-sectional view through two gasket and
two substrate layers according to Fig. 6a.
Fig. 6c is a view of a section of a micro-array prepared
using the assembly of Figs. 6a and 6b, showing six of the
hundreds or thousands of analyte-assay regions formed on a
solid support.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is concerned with a method of
forming a micro-array of analyte-assay regions on a solid
support, where each region in the array has a known amount of a
selected, analyte-specific reagent. More generally, there is
provided a substrate for use in detecting binding of labeled
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polynucleotides to one or more of a plurality different-
sequence, immobilized polynucleotides.
Micro-arrays, and reagents used in the formation thereof,
are well known in the art and thus need not be described herein
in any great detail. The reagents are preferably distinct
polynucleotide or polypeptide biopolymers fixed to the
substrate.
Methods of using -the micro-arrays, such as by contacting
fluorescent reporter-labeled cDNAs with a micro-array of
polynucleotides representing a plurality of known-DNA fragments
under conditions that result in hybridization of the labeled
cDNAs to complementary-sequence polynucleotides in the array
followed by examination. by fluorescence under fluorescence
excitation conditions, are also well known in the art and thus
need not be described herein in greater detail. For a
discussion of techniques reference may be made to US Patent
5,800,992 (Fodor et al.) and US Patent 5,807,522 (Brown et
al. ),
The present invention is specifically concerned with the
method and device with which a plurality of identical micro-
arrays of biological samples can be easily and quickly
produced.
A significant and distinguishing feature of the present
invention resides in the utilization of a plurality of
substrates, each of which having a top side, a bottom side, and
a pattern of through-holes. Each through-hole has a wider
upper cross-section, a narrower lower cross-section, and
preferably a step or plateau parallel to the top side of the
substrate formed in the transition area. When a number of
substrates are stacked, the corresponding through-holes are in
registry and form tunnels extending through the stack 'of
substrates. Reagents of interest are caused to flow through
the tunnels and deposit on the step or plateau area. Thereby
all substrates in the stack are " spotted " simultaneously, at
'' the precise location and with a precise amount of reagent. A
barrier layer may be provided between substrates to prevent
leak-through between neighboring holes.
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After the desired reagents have been deposited, the
substrates are separated. In this manner a series of micro-
arrays, each capable of containing hundreds or thousands of
biological samples such as cDNA fragments, is formed
simultaneously.
Thus, in comparison to the prior art planar array of
substrates which are addressed one at a time by devices
designed to deposit reagent onto only the surface of one
substrate after another, the present invention comprises a
stack of preferably identical substrates each having the same
pattern of through-holes, with one hole in each substrate
corresponding to each spot of analyte-specific reagent intended
in the final array. For example, if it were desired to create
100 identical arrays with one array per substrate and with each
array having 10,000 different spots, then 100 identical
substrates will be used, each manufactured with 10,000 through-
holes, the through-holes are in registry when the substrates
are stacked.
The term " registry " as used herein simply means that
through-holes of adjacent substrates in the stack are in
communication. In a preferred embodiment of the invention, the
stack is formed such that horizontal step or plateau areas of
each column of through-holes appear to be superimposed. These
horizontal step or plateau area can be referred to for brevity
as a discrete assay region or a" spot zone ". In order for
micro-array bearing substrates to be used interchangeably, it
is preferred that the spot zones on each substrate are
identical, such that assay spotting can be carried out by
robotic means programmed to spot at specific x,y coordinates.
The areas of narrower cross-section are preferably provided on
one side of the spot zone, i.e., at one of the edges of the
spot zone. More preferably, the area of narrower cross section
of the through-hole of even numbered slides in a stack are
provided on one side of the spot zone (e.g., right side), and
the area of narrower cross section of the through-hole of odd
numbered slides in the same stack are provided on the opposite
side of the spot zone (e.g., left side), such that reagent
flowing through the tunnel is caused to " slalom " back and
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forth, washing over each of the spot zones with reagent, and
ensuring that no bubbles are trapped in the tunnel.
In a preferred embodiment of the invention, the " spot
zones " are patterned on a substrate in a pattern which has 180
symmetry, i.e., when a first substrate is rotated about 180 and
stacked on top of a second, non-rotated substrate, through-
holes remain in registry and tunnels are formed. This makes it
possible to form all substrates using a single manufacturing
technique, and to provide the areas of narrow diameter on one
side of odd numbered slides, and to provide the areas of narrow
diameter on the opposite side of even numbered slides, by
simply rotating alternate numbered slides about 180 while
stacking.
There is no theoretical limit on the size of the
substrates, but they may typically be 0.5 to 5 cm in lateral
dimensions, and 0.05 to 3 mm thick, and may be the size of a
conventional microscope slide. Different sizes would be
appropriate for different applications. The number, size and
spacing of spots, and the number of substrates will depend on
the number and the amounts of reagent to be used in the array.
When the identical substrates are arranged in a stack such
that superimposed through-holes are in registry and form
tunnels extending through the stack of substrates, they are
preferably provided with a barrier seal between the slides such
that no lateral leakage can occur from one tunnel to any other
tunnel. Further, each hole in each substrate may be associated
with a counterbore, countersink, or other (possibly eccentric)
pocket in the substrate. These pockets create tiny.volumes to
the side of the line of holes through the substrates, when seen
in cross-section. In addition, these pockets provide small
areas of substrate surface area roughly parallel to the overall
surface of each substrate. Preferably, each through-hole has a
wider upper cross-section, a narrower lower cross-section, and
preferably a step or plateau formed in the transition area
parallel to the top side of the substrate.
The sealing means may be a hydrophobic viscous substance
such as grease, wax, a weak adhesive, or any other bonding or
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sealing agent compatible with (inert to) the particular
chemistry being used for the arrays. For other applications, a
very thin elastomer layer (gasket) will suffice. It has also
been found that the extremely smooth surfaces characteristic of
the micro-machining processes proposed for manufacture of the
substrates makes the sealing relatively simple. Indeed, for
some applications it may be possible to rely entirely on the
super-smooth surfaces of the substrates, such as glass slides,
wherein adjacent slides are in continuous contact with each
other with the exception of the through-holes.
The respective reagents used to create the array are
injected at one end of each tunnel, generally the upper end of
the tunnel, with each tunnel receiving (in general) a different
reagent. The injections may be done one at a time or in groups
or, preferably, simultaneously to all tunnels. The injection
may be done with syringes, tubes, or other means; manually or
automatically; with the aid of pumps of various sorts, with
capillary action or with vacuum.
As the reagents flow through the tunnels extending through
the stack substrates, including the side pockets formed by the
areas of the through-holes with greater diameter, they will
react with, and bond to, the exposed surfaces of said tunnels
with side pockets, dependent on the chemistry of the particular
reagents and surface in use, in a manner analogous to that
which occurs in the prior art when droplets are physically
deposited on flat surfaces. Drying can occur after deposition,
also in a manner analogous to that which occurs in the
conventional techniques. Thus, all of the same chemistries and
combinations now in use in the state of the art may be used to
advantage for particular applications with the method and
device of the present invention.
After whatever reactions are desired in the entire stack
have been completed and the stack has dried, the stack may be
separated into its individual substrates by simply releasing
the sealing means, if any. At this point multiple identical
individual substrates are available for hybridization, etc. as
with the conventional techniques. However, instead of having
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spotted each of dozens or hundreds of slides, the spotting
process was only carried out once.
The identical patterns of holes (and associated side-
pockets or areas of greater diameter) are preferably
manufactured using silicon or glass micro-lithography and
micro-machining techniques. This technology is ideally suited
for inexpensive production of multiple identical patterns in
the small sizes desired. However, other techniques, including
but not limited to laser machining, plasma etching, and
conventional machining or abrading may be used, as well as a
technique involving the arrangement of dissimilar glass
materials, one of which is acid etchable (channel glass) and in
the form of fibers corresponding to the through-holes to be
formed, the other of which is inert, followed by chemical
etching to remove the etchable glass.
Protrusions (bosses) on one side of each substrate, for
example on the top side of the substrate, and corresponding
depressions on the opposite side, for example the bottom side,
may be used to align the substrates to create a stack with all
holes in registry. Alternately, pins can be placed through
alignment holes provided in all of the substrates to achieve
the same end. Further yet, if the machining is sufficiently
accurate, the substrates can be aligned with reference to their
edges by providing guides against which to rest all of the
layers. Other methods for aligning will be obvious to anyone
skilled in the art.
It is important to note that the position of the spots is
determined during this initial manufacturing step and not by
robotic sample deposition. Thus, tremendous precision can be
achieved at relatively low cost at a central high volume
substrate manufacturing location. No expensive devices are
required for the subsequent injection steps which might be
carried out at a large number of different laboratories with
different reagents. The central manufacturing step for
manufacturing the substrates does not involve or determine the
various reagents or site-selection or arrangement of spots,
which can be chosen by the individual user laboratories.
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A variety of schemes can be used to connect a reagent
injection means to the stack of substrates. Simple arrays of
passive micro-funnels or channels can mediate the transition
from a relatively coarse injection means to a relatively fine
array spacing. Alternatively or additionally, simple but
precise x-y positioning devices can be used to move the stack
of arrays with respect to the injection means. Since hundreds
or more substrates are being injected simultaneously,
reasonable production rates are possible without the expense of
fast robots as used in the prior art.
In the following the invention will be described in
greater detail by reference to an illustrative embodiment shown
in the figures. In Fig. 1 substrates 1 are stacked with
intermediate adhesive layers 4. The adhesive layers have
through-holes corresponding to the holes in the substrates to
permit reagent to flow through the tunnel, and serve as
horizontal barriers to prevent leakage of reagent between
tunnels. Each substrate layer has an array of through-holes 3
in registry with the through holes of adjacent substrates. The
adhesive is coated onto one or both of the top and bottom
planar surfaces of the substrate in a manner such that the
adhesive is interrupted at the location of the holes.
Alternatively, the adhesive may be provided on the substrate
prior to the step of forming the holes, in which case adhesive
is removed at the same time and in the same areas in which the
holes are formed.
As shown in Fig. 1, each through-hole has a wider upper
cross-section, a narrower lower cross-section, and preferably a
step or plateau 2 parallel to the top side of the substrate
formed in the transition area. This step or plateau 2
ultimately forms the presentation area of the analyte-assay
regions. Locating pins 14 and/or bosses 15 may be used for
initial alignment or to maintain alignment of the stack.
As can be seen in Fig. 2, the areas of the holes having
the narrower cross-section 3 are in registry and connect to
form a tunnel which extends completely through the stack from
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top to bottom, including extending through the adhesive layers
4. Side pockets are formed in the area of the step or plateau
2. Adhesive may or may not be on the substrate in the area of
the plateau.
Fig. 3 shows a device which can be used for injecting in
conjunction with a stack of the substrates 1 of the present
invention. The stack of substrateg is clamped between an
adapter plate 6 and a vacuum manifold 9. Clamping pressure is
provided by clamp 10 and frame 8. The adapter plate forms an
injection mask with tapered holes 7 having a larger upper
diameter for easy access for the injecting means such as a
pipette tip 5 and narrow lower diameter in registry with the
through-holes. In the process of introducing reagent, an
aliquot of reagent is introduced. into the adapter plate and
permitted to flow downwards by gravity or capillary action, or
the flow is assisted by a pressure differential such as created
by application of a slight vacuum to the lower end of each
tunnel. Due to the cost of reagent, care is taken to ensure
that sufficient reagent is drawn into the tunnel to completely
fill the tunnel, but that no or only little excess is expelled
from the bottom of_ the tunnel. This can usually be
accomplished by adjusting the vacuum to be great enough to
assist in drawing reagent into the tunnel, but not sufficiently
large to overcome capillary forces which tend to keep the
reagent inside the tunnel.
Fig. 4 represents an alternative embodiment of the
invention, and shows a stack of substrates 1 clamped between an
upper vacuum manifold 9 and a lower tubing adapter plate 13.
Tubes 11 spread out to adapt to the spacing of wells in a
micro-titre tray 12, and are in communication with the reagent
provided in the wells of the micro-titre tray 12.
Fig. 5 is a cross section through a stack of substrates as
in Fig. 2 but in an alternative arrangement wherein the pattern
of through-holes 3 is staggered or alternating, defining a
slalom path for the reagent. As long as there is a continuous
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path through each column of through-holes and pockets, the
exact position of each need not be repeated exactly in adjacent
or alternating layers. This alternating path may have the
advantage in some situations of mixing the filling flow for
better coverage of the bottoms of the pockets. It may be
simplest to alternate between two different patterns, but it is
also possible to have repeating patterns every three slides or
random patterns. It is also simple to have the pockets all in
line for the greatest simplicity in later observation and
automated data acquisition, but it is not absolutely necessary
for the proper operation of the method. Many possible
variations will be obvious to one skilled in the art.
The invention may also be carried out using the embodiment
shown in Figs. 6a (exploded) and 6b (assembled) , with only two
of potentially dozens of stacked alternating substrates and
spacers being shown. In this embodiment, the substrates 17a,
17b have only narrow through-holes 18a, 18b machined through
them, representing a simplification in the manufacturing
process as compared to the stepped or countersunk substrates
shown in Figs. 1, 2 and 5. The individual planar deposition
areas on the substrates are actually defined by holes 19a, 19b
in the spacers 16a, 16b.
The spacers may be separable from the substrates, or
alternatively a spacer layer may coated onto a substrate
followed by etching of through-holes, or a spacer layer may be
silk screen printed, offset printed, or otherwise printed onto
the substrate, or a solid elastomeric or other film with pre-
formed through-holes may be laminated onto a substrate layer.
In order to minimize wastage of reagent, it is preferred that
the spacer is comprised of a material which does not absorb
reagent, and more preferably resists deposition of reagent,
such as plastic (preferably an elastomeric polymer), rubber,
wax, glass, and metal. Any of the materials discussed above
with respect to the first embodiment of the invention can be
used in the second embodiment of the invention.
The spacers are interposed between the substrate layers,
with the holes in the separable spacers being larger than the
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through holes 18a, 18b in the substrate 17a, 17b. The spacer
layer is of finite thickness, usually thinner than the
substrate, thus creating a pocket between adjacent substrates,
the pocket allowing reagent to contact and be deposited on the
substrate as it flows through. The staggered substrate
through-hole option as discussed above with respect to Fig. 5
may optionally used in the embodiment of Figs. Ga and 6b.
Fig. 6b is a cross-sectional view through two spacer and
two substrate layers, showing the two sets of layers according
to Fig. 6a in the assembled condition. It is apparent that
reagent can flow continuously through spacer apertures 19a, 19b
in the spacer 16a, 16b and the through holes 18a, 18b in the
substrate 17a, 17b.
Fig. 6c is a view of a section of a micro-array prepared
using the assembly of Figs. 6a and 6b, showing six of the
hundreds or thousands of analyte-assay regions remaining on the
solid support after removal of the spacer 16a, 16b. Obviously,
in the case that the spacer is coated or laminated onto the
substrate, one spacer layer would remain adhered to each
substrate, either on the top or on the bottom of the substrate.
Actually, since the reagent will coat both sides of the
substrate, either side of the substrate can be considered the
top or useable side.
It is preferred that the spacer be kept as thin as
possible in order to minimize the amount of reagent required
and to minimize reagent deposition on the spacer. The
selection and thickness of the spacer and substrate materials
is a matter of preference and can be readily determined by
those working in this art.
With respect to the above description then, it is to be
realized that the optimum dimensional relationships for the
parts of the invention, to include variations in size,
materials, shape, form, function and manner of operation,
assembly and use, are deemed readily apparent and obvious to
one skilled in the art, and all equivalent relationships to
those illustrated in the drawings and described in the
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specification are intended to be encompassed by the present
invention.
Therefore, the foregoing is considered as illustrative
only of the principles of the invention. Further, since
numerous modifications and changes will readily occur to those
skilled in the art, it is not desired to limit the invention to
the exact construction and operation shown and described, and
accordingly, all suitable modifications and equivalents may be
resorted to, falling within the scope of the invention.
Now that the invention has been described,
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