Note: Descriptions are shown in the official language in which they were submitted.
CA 02316045 2000-08-16
Devices and Methods for Producing Microarrays of Biological
Samples
FIELD OF THE INVENTION
The invention relates to systems and methods for producing microarrays for
biological investigation. In particular, this invention is directed to systems
and methods
for producing microarrays using improved slide platforms and vacuum manifolds,
and
variable pin contact velocity.
BACKGROUND OF THE INVENTION
Information about genes is critical to understanding the biological processes
which underlie life: cellular communication, growth, movement, reproduction,
and
control. Once obtained, the structural and functional features of the genetic
sequences
(polypeptides and polynucleotides) enable better diagnostics and treatment of
disease
and defects, whether genetic or external in origin.
Traditional gene expression research has involved manual pipetting of samples
onto gels, membranes.or filters, or into multiwell plates. These methodologies
are
extremely time consuming, laborious, low throughput and expensive (on a per
gene
basis).
Modern microarray technologies get around a number of the above problems by
automating the spotting process using robotics which permits high density
spotting of
slides, also known as microarray slides, which allows thousands of gene
fragments to be
analyzed in a single experiment (Schena M, Shalon D, Davis RW, Brown PO.
"Quantitative monitoring of gene expression patterns with a complementary DNA
microarray", Science 270, 467-470 (1995); Southern, E, Mir K, and Shchepinov,
M,
Molecular Interactions on microarrays, Nature Genet. 21, 5 - 9 (1999)). Each
spot
leaves a sample of volume in the nanolitre range, the centres of adjacent
spots
separated by micrometers.
Array types include oligonucleotide arrays, cDNA arrays and genomic DNA
arrays. For example, one application is to identify the genes, the expression
or
repression of which results in the difference between a normal human cell and
a mutant
CA 02316045 2000-08-16
human cell. Cells contain thousands of genes, a few thousand in lower
organisms such
as yeast and over 100,000 in humans; a cDNA is made for each gene and spotted
onto
gene chips as part of the microarray. Another application concerns the
construction of
olionucleotide arrays. It is also possible for genomic DNA arrays (chromosomal
DNA) to
be fabricated using modern microarray technology. Arrays are also useful for
DNA
sequencing.
Typically, a microan-ayer has a number of components including: (1 ) a robotic
mechanism; (2) a dispenser assembly; (3) means for replenishing the spotting
dispensers with the biological sample; (4) a platform to support the
microarray slides
during spotting; (5) means for cleaning the spotters; and (6) software to
operate the
robot mechanism and provide an interface with a user. Samples are typically
stored in
source plates. Source plate plastics may be polystyrene, polypropylene or
polycarbonate. In general, plates may have 1536, 384 or 96 wells. DNA may be
attached to the substrate through any suitable technique (e.g. covalent or
ionic bonding).
Dispensers for spotting, also known as spotting members, include pins, which
in turn
include solid pins, split or quill pins, pin and ring systems, capillaries, or
inkjet systems.
Pins include the Telechem Chipmaker 2 and the Telechem Chipmaker 3 (Stealth
Pins).
Recent advances in robotics such as that disclosed by the invention in US
patent
6,048,373, have made possible a number of features desired in an ideal
microarrayer:
(1) high resolution; (2) repeatability; and (3) precision. Resolution of a
system is the
ability to distinguish two points as being separate; it is also the minimum
distance that
can be measured by an encoder. Repeatability refers to the ability of the
robot to return
to the same place. The difference between the position that the robot desires
to occupy
and the actual position occupied is the precision of the system. The density
of the array
is a function of the spatial resolution of the robot.
The mechanism for dispensing the biological sample typically uses pins as the
part of the print head that performs the actual spotting. The preferred
approach is a set
of pins, either in the solid or in the split form of the pin (with a slot),
typically arranged in
a rectangular matrix. The biological samples are loaded into/onto the pins
from the
source plates. It is critical that deposition of probe biological sample, such
as cDNA,
yield regularly spaced spots of uniform morphology. Not all deposition pins
designed to
the same specifications behave in a similar manner. Each will load an amount
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characteristic of the pin. Consequently, the size of the first spots produced
from a set of
pins can be significantly variable. The greatest concern is that deposition of
excessive
material on the microarrays may yield overlapping spots. This will result in
contamination of the material spotted on the arrays as well as the material in
the probe
plates. As spotting proceeds, the excess material is removed and the spots
become
uniform until the exhaustion of the material on the pin. There is thus a need
to
effectively remove the excess material prior to spotting onto the microarray
slides.
It is also important to have a well-designed platform (also known as a platen)
onto which the microarray slides are placed for array printing. Existing art
uses a
platform that had "rails" cut into it. These rails would serve to hold the
slides in place in
the X-axis and/or Y-axis, and allow for just enough space into which to place
the slides;
however in certain cases, the lack of a uniform standard on slide sizes means
that
certain commercially available slides would not fit into the tracks of a
particular
microarrayer. The rails have to be machined with great precision to hold the
slides
without allowing for movement. Other solutions use a set of spring loaded
plastic pins,
which hold the slides in place. These pins offer some compliance to allow for
subtle
variations in slide size (such as the difference between metric and imperial
measure
slides). Other units utilize a combination of machined holders for the slides
with a
vadnur~ manifold. The vacuum manifold helps hold the slides down firmly which
allows
the depression into which the slide sits to not be an exact fit. Again in this
case slight
size variations are possible allowing imperial and metric slides to be used.
These solutions do not provide for a great deal of flexibility. In addition,
these
slide platforms/holders are difficult to load, which causes the operation to
be time
consuming and increases the risk of damage to the slides. Some of these units
require
such a platform/holder design because the slide platform sits on top of one of
the robotic
actuators. In such a case the slide platform moves during the printing process
and thus
the slides need to be held firmly to prevent them from shifting in place.
Where the robot
mechanism uses an overhead gantry system; the print head travels over the
slide
platform in all three axes, and the slide platform remains stationary. In such
a situation,
there is little movement, which will cause the slides to shift. There is thus
a need for a
platform/slide holder, which allows for much greater flexibility, and much
greater ease of
use.
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In order to clean the spotting pins, a vacuum or forced air removes liquid
from the
pins, usually present on the pins (and in the slot of the pins) as a result of
dipping into a
water bath for cleaning after spotting. Known vacuum manifolds are comprised
of a
chamber containing a series of holes (as many as there are pins for the print
head) into
which the pins fit. The tips of pins are generally placed into the holes,
either at the
opening of the hole, or completely into the vacuum chamber. These solutions
are not
sufficient for optimal cleaning of the pins. There is a need for a vacuum
manifold and
associated method to optimally clean the spotting pins.
o SUMMARY OF THE INVENTION
The invention relates to a vacuum manifold for use in removing liquid from
microarray spotting members, including:
~ a plate defining a plurality of fluid flow channel members formed through
the plate,
each channel member defining an inlet and an outlet in fluid communication;
and
~ structure for coupling the plate to communicate with a source of vacuum to
draw fluid
from the microarray spotting members through the fluid flow channel members.
The invention also includes a microarrayer including the vacuum manifold.
Another embodiment of the invention relates to a method of removing liquid
from
a plurality of microarray spotting members, including applying a source of
vacuum to the
manifold of claim 1 and reciprocating the microarray spotting members
proximate to the
inlets of a manifold of the invention to create air turbulence between the
spotting
members and the inlets.
The invention also relates to a support device for holding microarray
substrates
in place during microarrayer operation, including:
~ a flat platform on which substrates may be placed, the flat platform
including an array
surface including first, second, third and fourth peripheral edges;
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~ a first bar on the first peripheral edge;
~ a second bar on the second peripheral edge, the second bar perpendicular to
the
first bar;
~ a third bar on the third peripheral edge, the third bar perpendicular to the
second bar
and opposed to the first bar, the third bar capable of applying force to the
substrates
to hold them in place during microarray operation;
~ a plurality of end bars perpendicular to the first and third bars and
opposed to the
second bar, the end bars capable of being located on the fourth peripheral
edge or
on the array surface spaced apart from the fourth peripheral edge, the end
bars
capable of applying force to the substrates to hold them in place during
microarray
operation.
The invention also includes a microarrayer including the support device.
The invention relates to a blotting device for blotting liquid from the
exterior of
microarray spotting members, including:
~ a blotting surface for drawing liquid from the microarray spotting members
when the
microarray spotting members contact the blotting surface;
~ structure for contacting the microarray spotting members with the blotting
surface.
The invention includes a microan-ayer including the blotting system.
The invention also includes a method of delivering liquid from a spotting
member
onto a microarray substrate for a microarray operation, including:
~ advancing the spotting member from a first position to a second position,
the spotting
member spaced apart from the substrate in the first position and the spotting
member engaging the substrate in the second position for delivering liquid,
the
spotting member advancing from the first position to the second position at
pre-
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determined, variable velocity, the spotting member velocity reduced when the
spotting member approaches the second position from the first position.
permitting the spotting member to engage the substrate for a pre-determined
period of time to allow the liquid to form a spot on the substrate suitable
for
microarray operation.
The invention also relates to a method of drawing liquid from a well into a
spotting member for a microarray operation, including:
advancing the spotting member from a first position to a second position, the
spotting member spaced apart from the well in the first position and the
spotting
member proximate to the bottom of the well for drawing liquid in the second
position, the spotting member advancing from the first position to the second
position at pre-determined, variable velocity, the spotting member velocity
reduced when the spotting member approaches the second position from the first
position.
permitting the spotting member to remain proximate to the bottom of the well
for
a pre-determined period of time to draw the liquid into the spotting member.
The invention also includes a substrate with a surface comprising 3000 or more
groups of DNA molecules attached to the surface in discrete known regions, the
3000 or
more groups of DNA molecules occupying a total area of less than 1 cm2 on the
substrate.
In one embodiment, the invention relates to a manifold assembly for removing
liquid from a plurality of microarray spotting members, the spotting members
each
having a spotting member body and a first open end portion for printing a spot
on a
microarray slide, comprising:
a plate, the plate defining a plurality of fluid flow apertures extending
through the
plate, the apertures having an axis and a first diameter, the spotting member
bodies having a second diameter wherein the second diameter is greater than
the first diameter, and wherein the first open end portion of the spotting
member
is adapted to extend into the aperture; and
turbulence means for creating turbulence between the spotting members and the
apertures for removing liquid from the first open end portions of the spotting
members through the apertures.
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The turbulence means preferably comprises i) aligning means for aligning the
spotting members proximate to the apertures with each spotting member body
extending
generally axially away from the corresponding aperture, ii) reciprocating
means for
reciprocating the spotting members generally axially toward and away from the
apertures and iii) a vacuum means for drawing air around the spotting members
to flow
through the apertures. The vacuum means preferably comprises a source of
vacuum
and a structure for coupling the plate to communicate with the source of
vacuum to draw
liquid from the microarray spotting members through the apertures.
In the assembly, the apertures preferably comprise channels, each channel
defining an inlet and an outlet in fluid communication. The apertures are
preferably
arranged in parallel rows. The spotting members preferably comprise pins
selected from
the group consisting of solid pins and split pins. The manifold optionally
comprises 48
apertures capable of simultaneously removing liquid from 48 spotting members
or 32
apertures capable of simultaneously removing liquid from 32 spotting members.
In one variation, the assembly further comprises a cover secured parallel to
the
plate over the inlets, the cover defining a plurality of cover apertures
therethrough, each
cover aperture concentric with an inlet of the plate, and the diameter of each
cover
aperture being less than the diameter of its concentric inlet.
Optionally, the first open end portion is tapered. Optionally, approximately
half of
the tapered first open end portion is adapted to extend into the aperture. In
one
embodiment, the first open end portion comprises a tip and the spotting member
is
reciprocable by the reciprocating means between first and second positions,
the tip
located outside the aperture in the first position and the tip located inside
the aperture in
the second position.
The source of vacuum optionally provides a pressure of 50 to 90 psi, 60 psi or
at
least 90 psi. The invention also includes a microarrayer comprising the
manifold
assembly of the invention.
The invention also includes a method of removing liquid from a plurality of
microarray spotting members, comprising applying a source of vacuum to the
assembly
of the invention and reciprocating the microarray spotting members proximate
to the
apertures of the manifold to create air flow and turbulence between the
spotting
members and the apertures. The spotting members are preferably concentric with
the
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apertures during reciprocation. The spotting members are preferably about 100
micrometers away from the inlet prior to reciprocation. The spotting members
are
optionally reciprocated about a distance of 1 mm. In one embodiment, each
spotting
member includes a tapered first open end portion adapted to extend into the
aperture,
the tapered first open end portion received in the aperture during at least
part of the
reciprocation. In a variation, the tapered first open end portion reciprocates
in and out of
the aperture. In another embodiment, each spotting member includes a tapered
first
open end portion adapted to extend into the aperture, the tapered first open
end portion
spaced apart from the aperture during reciprocation.
The invention also includes a method of removing liquid from a plurality of
microarray spotting members, the spotting members each having a spotting
member
body having a second diameter and a first open end portion for printing a spot
on a
microarray slide, the liquid removed through a manifold having a plurality of
apertures
extending therethrough, the apertures having an axis and a first diameter,
wherein the
second diameter is greater than the first diameter, the method comprising:
first generally axially aligning the spotting members proximate to the
manifold
apertures with each spotting member body extending generally axially away from
the apertures;
applying a vacuum for drawing air around the spotting members through the
apertures; and
reciprocating the spotting members generally toward and away from the
corresponding apertures,
wherein turbulence is created between the spotting members and the apertures
for removing liquid from the first open ends of the spotting members through
the
apertures.
In the methods of the invention, the spotting member is preferably
reciprocated
between first and second positions, the tip outside the aperture in the first
position and
the tip inside the aperture in the second position. In the methods of the
invention, the
removed liquid optionally comprises a cleaning solution for cleaning the
spotting
members. The invention also includes the use of the manifold assembly or
manifold of
the invention for cleaning the spotting members, wherein the removed liquid
comprises a
cleaning solution.
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The invention also includes a manifold for use with a microarray spotting
apparatus for removing liquid from microarray spotting members, the microarray
spotting
members each having a spotting member body having a second diameter and a
first
open end portion for printing a spot on a microarray slide, the manifold
comprising: a
plate, the plate defining a plurality of fluid flow apertures extending
through the plate, the
apertures having a first diameter, wherein the second diameter is greater than
the first
diameter, and wherein the first open end potion of the spotting member is
adapted to
extend into the corresponding aperture for removal of liquid from the first
open end
portion through the corresponding aperture by a turbulence means for creating
turbulence between the spotting member and the aperture. The apertures
optionally
comprise channels, each channel defining an inlet and an outlet in fluid
communication.
The apertures are preferably arranged in parallel rows.
The manifold optionally includes 48 apertures capable of simultaneously
removing liquid from 48 spotting members or 32 apertures capable of
simultaneously
removing liquid from 32 spotting members.
The invention also includes a vacuum manifold for removing liquid from
microarray spotting members, comprising:
a plate defining a plurality of fluid flow channel members formed through the
plate, each channel member defining an inlet and an outlet in fluid
communication, wherein the diameter of the inlet is less than the spotting
member diameter, and the spotting member includes a first tapered open end, a
portion of the first tapered open end capable of extending into the inlet and
the
fluid flow channel member; and
structure for coupling the plate to communicate with a source of vacuum to
draw
fluid from the microarray spotting members through the fluid flow channel
members.
The channel members are preferably arranged in parallel rows. The spotting
members optionally comprise pins. The manifold optionally comprises 48 fluid
flow
channel members capable of simultaneously removing liquid from 48 spotting
members.
The source of vacuum optionally provides a pressure of 50 to 90 psi 60psi or
at least 90
psi. The invention also includes a microarrayer comprising the vacuum
manifold.
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The invention also includes a method of removing liquid from a plurality of
microarray spotting members, comprising
applying a source of vacuum to a vacuum manifold for removing liquid from
microarray spotting members, the vacuum manifold comprising a) a plate
defining a plurality of fluid flow channel members formed through the plate,
each
channel member defining an inlet and an outlet in fluid communication, wherein
the diameter of the inlet is less than the spotting member diameter, and the
spotting member includes a first tapered open end, a portion of the first
tapered
open end capable of extending into the inlet and the fluid flow channel
member;
and b) structure for coupling the plate to communicate with a source of vacuum
to draw fluid from the microarray spotting members through the fluid flow
channel
members; and
reciprocating the microarray spotting members proximate to the inlets of the
manifold to create air turbulence between the spotting members and the inlets.
In the method, preferably each microarray spotting member is concentric with
the
inlet during reciprocation. The spotting members are optionally about 100
micrometers
away from the inlet prior to reciprocation. The spotting members are
optionally
reciprocated about a distance of 1 mm. Each spotting member optionally
includes a first
tapered open end having a portion capable of extending into the inlet, the
first tapered
open end portion received in the inlet during reciprocation. The first tapered
open end
portion preferably reciprocates in and out of the inlet.
In another embodiment, the invention relates to a manifold assembly for
removing liquid from a plurality of microarray spotting members, the spotting
members
each having a spotting member body and a first open end portion for printing a
spot on a
microarray slide, comprising:
a plate, the plate defining a plurality of fluid flow apertures extending
through the
plate, each aperture located to cooperate with a corresponding spotting
member,
each aperture having an axis, a first diameter, an upstream edge forming an
inlet
and a downstream edge forming an outlet, the edges defining the aperture, the
corresponding spotting member body having a second diameter wherein the
second diameter is greater than the first diameter so that the spotting member
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CA 02316045 2003-12-08
may not entirely pass through the aperture, and wherein the first open end
portion of the spotting member is adapted to extend into the corresponding
aperture to a position where there is space between the spotting member and
the
aperture; and
turbulence means for creating turbulence in air flowing from the inlet to the
outlet
in the space between the spotting member and the aperture for removing liquid
from the first open end portions of the spotting members.
The turbulence means optionally comprises i) aligning means for aligning the
spotting members proximate to the apertures with each spotting member body
extending
generally axially away from the corresponding aperture, ii) reciprocating
means for
repeatedly reciprocating the spotting members generally axially toward and
away from
the apertures while maintaining the spotting members axially aligned with the
corresponding apertures and limiting axially inward travel to provide
clearance with the
apertures in the limiting position and iii) vacuum means for drawing air
around the
spotting members to flow from the inlet to the outlet.
Another aspect of the invention relates to a microarrayer, said microarrayer
comprising:
a) a manifold assembly for removing liquid from a plurality of microarray
spotting
members;
b) a plurality of spotting members each having a spotting member body and a
first open end portion for printing a spot on a microarray slide;
c) a plate, the plate defining a plurality of fluid flow apertures extending
through
the plate, each aperture located to cooperate with a corresponding spotting
member, each aperture having an axis, a first diameter, an upstream edge
forming an inlet and a downstream edge forming an outlet, the edges defining
the
aperture, a corresponding spotting member body having a second diameter
wherein the second diameter is greater than the first diameter so that the
spotting
member may not entirely pass through the aperture, and wherein the first open
end portion of the spotting member is adapted to extend into the corresponding
aperture to a position where there is space between the spotting member and
the
aperture; and
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d) turbulence means for creating turbulence in air flowing from the inlet to
the
outlet in the space between the spotting member and the aperture for removing
liquid from the first open end portions of the spotting members..
Another aspect of the invention relates to a method of removing liquid from a
plurality of microarray spotting members, comprising creating turbulence in
air flowing
from the inlet to the outlet of the assembly or the microarrayer in the space
between the
spotting member and the aperture for removing liquid from the first open end
portions of
the spotting members. The turbulence is optionally created in air by:
applying a source of vacuum to the spotting members of the assembly to draw
air
around the spotting members to flow from the inlet to the outlet,
aligning the spotting members proximate to the apertures with each spotting
member body extending generally axially away from the corresponding aperture,
repeatedly reciprocating the microarray spotting members generally axially
toward and away from the apertures and limiting axially inward travel to
provide
clearance with the aperture in the limiting position to create turbulence in
air
flowing from the inlet to the outlet in the space between the spotting member
and
the aperture for removing liquid from the first open end portions of the
spotting
members.
The invention also includes a method of removing liquid from a plurality of
microarray spotting members, the spotting members each having a spotting
member
body and a first open end portion for printing a spot on a microarray slide,
the liquid
removed through a plate, the plate defining a plurality of fluid flow
apertures apertures
extending through the plate, each aperture located to cooperate with a
corresponding
spotting member, each aperture having an axis, a first diameter, an upstream
edge
forming an inlet and a downstream edge forming an outlet, the edges defining
the
aperture, a corresponding spotting member body having a second diameter,
wherein the
second diameter is greater than the first diameter so that the spotting member
may not
entirely pass through the aperture, and wherein the first open end portion of
the spotting
member is adapted to extend into the corresponding aperture to a position
where there
is space between the spotting member and the aperture the method comprising:
first aligning the spotting members proximate to the apertures with each
spotting
member body extending generally axially away from the corresponding aperture;
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drawing air around the spotting members to flow from the inlet to the outlet;
and
repeatedly reciprocating the spotting members generally toward and away from
the corresponding apertures while maintaining the spotting members axially
aligned with the corresponding apertures and limiting axially inward travel to
provide clearance with the apertures in the limiting position,
wherein turbulence is created in air flowing from the inlet to the outlet in
the
space between the spotting member and the aperture wall for removing liquid
from the first open ends of the spotting members through the apertures.
The spotting member is optionally reciprocated between first and limiting
positions, the tip outside the aperture in the first position and the tip
inside the aperture
In the limiting position.
BRIEF DESCRIPTIQN 4F THE DRAWINGS
Embodiments of the invention will be described by way of example and with
reference to the drawings in which:
Figure 1: A top plan view of an embodiment of the vacuum manifold that
Includes
placing a piece of material, such as adhesive aluminium foil, over the
manifold. Smaller
holes (micro apertures) may be created, for example, by using either an off
spec
spotting pin or a 28 gauge needle. The holes preferably have an approximate
diameter
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CA 02316045 2002-09-23
of 1 /32 of an inch for Telechem Chipmaker 2 or 3 pin but are variable
according to the
size of the pins.
Figure 2: A perspective view showing the side, top, and front of the
embodiment shown
in Figure 1.
Figure 3(A): A top plan view of a second embodiment of the invention which
includes a
manifold into which the holes that were machined had approximately 1/32 of an
inch
diameter. This avoids the need to use the adhesive foil, which is prone to
wear over
time.
Figure 3(B): A perspective view showing the side, top, and front of the
embodiment
shown in Figure 3(A).
Figure 4(A): Side view of a vacuum manifold with the pins set such that the
tips were at
the opening of the hole. In this way increased air flow is able to pass over
the pin
helping to draw off any liquid.
Figure 4(B): Side view of an alternative vacuum manifold which allows the tips
of the
pins to pass completely into the chamber. The pins almost seal the chamber
allowing
the vacuum to pull the liquid off of the pin.
Figure 5: Side view of a manifold with micro apertures. One preferably sets
the robot
up such that the pin tip just passes the opening of the hole at the down
position (A), and
is reciprocated up to a position a few hundred microns above the opening (B).
Reciprocation up and down is used to increase air turbulence, increasing the
cleaning
efficiency.
Figure 6: A manifold including a gasketed chamber, with a series of 32 holes
at the
same spacing as the pins. When not all of the holes were in use, a piece of
adhesive foil
could be used to cover the unused holes thus increasing airflow.
Figure 7(A): The slide platform is preferably ultra-flat machined aluminum
platform.
This platform is level so that there is less than 200 micrometres variance
from one
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CA 02316045 2000-08-16
corner to another. Two guidebars are used to push the slides up against thus
keeping
them in place. A steel bar proximate to the aluminum platform is used to act
as a
magnetic surface for the end bar magnetic slide holders. An additional
moveable heavy
steel bar holds the slides in the other axis. (B) A full complement of slides
was to use all
of the magnetic holders and the heavy bar to keep the slides in place. (C)
When only a
partial complement of slides is used, only some of the end bars are required,
and the
heavy steel bar can be moved to hold the slides in place.
Figure 8: A perspective view of the slide holder with a full complement of
slides. All of
the end bars and the heavy steel bar are used to hold the slides securely in
place.
Figure 9: A photograph of a complete microarray device.
~5 DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to systems and methods for producing
microarrays using an improved slide platform and vacuum manifold, and variable
pin
contact velocity. Figure 9 shows a view of a preferred microarrayer. The
invention may
be used with any suitable microan-ayer, such as the SDDC-2 microarrayer (The
Microarray Centre; Princess Margaret Hospital/Ontario Cancer Institute,
Toronto,
Canada; www.oci.utoronto.ca/serviceslmicroarrav).
Vacuum Manifold
One embodiment of the present invention (see Figures 4(A), 5, and 6) relates
to
a vacuum manifold 4 for removing liquid from microarray spotting members 2
after
spotting, preferably comprising a plate with channels or holes 3 drilled in
the plate, the
holes leading to a chamber 5 which is connected to a source of vacuum. Each
spotting
member 2 is placed in or near a hole on the plate; liquid on the spotting
members,
typically a cleaning solution, is drawn away by the pressure created by the
vacuum. As
3o a result the spotting member 2 is cleaned after spotting. Preferably, the
channels or
holes 3 are arranged in parallel rows, in the same fashion the spotting
members are
arranged on the printing head.
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CA 02316045 2002-09-23
The spotting members 2 are preferably pins, of either the solid or split
types.
Some commercially available pins include Telechem Chipmaker 2 pins, Telechem
Chipmaker 3 pins. A combination of the two may also be used as the spotting
members
of a microarrayer.
Significantly improved cleaning is achieved by using this vacuum manifold 4.
First, the inlets (holes/apertures) of the vacuum manifold are reduced in
cross-sectional
area. Alternatively, the inlets may be effectively reduced by placing a piece
of sticky
aluminum foil over the manifold and making new holes with a 28 gauge needle,
or with
an off-spec spotting pin (Figure 2). Alternatively, the manifold could be
constructed with
smaller apertures 3 (Figure 2). The magnitude of reduction of the cross-
sectional area is
preferably such that only a portion (for example, approximately half) of the
pin tip 1 could
fit through the aperture 3 and the pin body 2 cannot enter the aperture.
Secondly, the
pins 2 are preferably set to be about 100 micrometres above the manifold. From
this
point the pins 2 are reciprocated up and down to create further air
turbulence, which
result in excellent cleaning (3 to 5 percent carryover at maximum).
Slide Platform
One embodiment of the present invention relates to a support device for
holding
microarray substrates in place during microarrayer operation, comprising a
flat platform
8 on which the substrates are placed, the flat platform including an array
surface
including a first bar on one peripheral edge; a second bar on a second
peripheral edge
perpendicular to the first bar; a third bar on a third peripheral edge
perpendicular to the
second bar and opposed to the first bar, the third bar capable of applying
force to the
substrates to hold them in place during microarray operation; and a plurality
of end bars
perpendicular to the first and third bars and opposed to the second bar, the
end bars
capable of being located on the fourth peripheral edge or on the array surface
spaced
apart from the fourth peripheral edge, the end bars capable of applying force
to the
substrates to hold them in place during microarray operation.
The flat slide platform 8 is preferably less than 200 micrometre variation
from one
corner to the other. This design allows for the use of any size of slide 9,
thus providing
extreme flexibility. (See Figures 7 and 8 for details of one variation.) There
is no
limitation to the use of either imperial or metric 1 x 3 inch slides 9, but
can, if required,
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CA 02316045 2002-O1-28
use larger slides or smaller slides 9 (1 x 1 inch, 2 x 3 inch or any other
size, both custom
and standard stock). Loading and unloading of the platform 8 is also extremely
easy to
perform on a flat platform 8 because we simply place the slides 9 on the
surface and
slide them into position. This allows extremely quick loading of the arrayer,
and results
in overall efficiency.
A heavy bar 10 is preferably used to push up against the sides in one axis to
hold
the slides 9 in place. This helps to ensure that vibration from the arrayer
does not cause
the slides 9 to move in the x-y plane. In addition it helps to make sure the
slides 9 are
not bumped out of place when a technician is accessing the arrayer. End bars
11, which
are preferably small bars around the length of a standard microscope slide 9
are
preferably used to hold the slides 9 in place in the second axis. In this
case, several of
these °unit-sized" bars 11 could be used depending on the number of
slides 9 on the
platform 8. Magnets are preferably used for these small bars 11, as this would
provide a
downward force to hold these bars 11 in place. The platform 8 itself is
preferably
constructed of aluminium. Aluminum is preferred due to the precision with
which it can
be machined and the lightness of the material. To allow for the magnets to
work, a
ferromagnetic bar, plate or sheet, typically steel in construction, is
preferably added to
the bottom or edge of the platform 8.
2o Blotting
One embodiment of this invention concerns a blotting device for blotting
liquid
from the exterior of microarray spotting members, comprising a blotting
surface for
drawing liquid from the microarray spotting members when the microarray
spotting
members contact the blotting surface; and structure for contacting the
microarray
spotting members with the blotting surface. One variation involves a glass
surface.
Another aspect of the embodiment includes a processing system for directing
the
microarray spotting members to make more than one contact with the blotting
surface in
a predetermined pattern so that no portion of the blotting surface is
contacted by more
than one microarray spotting member. The processing system preferably includes
a
software-driven system.
CA 02316045 2002-09-23
During a typical run both blot slides and plates containing probe material,
are
preferably removed and replaced simultaneously. It is advantageous to replace
blot
slides concomitantly with probe plates. Plates containing probes elements
typically
possess 384 wells. If each sample is blotted 10 times, the software and
blotting slides
must accommodate 3840 spots with each spot separated by sufficient distance.
Two objectives are fulfilled during the blotting process. First, a sufficient
number
of spots are produced on the blots to remove excess material from the pins
that may
yield large spots on the array; and secondly, successive rounds of blotting
(with material
loaded onto pins at the beginning of each spotting cycle) must not overlap
previous
spots.
These two criteria translate into two basic motions that occur during the
process
of printing on the blot slides. In one direction (x), a series of spots is
produced to
remove excess material from the pins. Prior to the printing of each new set of
probes
the pins preferably move in a perpendicular direction (y) before printing the
next series of
blotting spots. The number of iterations required in each direction is
determined
empirically. This is accomplished by examining the size and morphology of
spots
produced by a set of pins. A centre to centre distance of approximately 0.5 mm
for
adjacent spots is sufficient. Blotting protocols can be modified to allow for
greater
distances.
For blotting, such as for a centre to centre distance of 0.5 mm, appropriate
and
sufficient blotting can be fulfilled if the following parameters are adopted.
In both the x
and y directions, the distances (in millimeters) the pins move between blots
is preferably
equal to the half number of pins in the direction of motion. For a single pin
(one pin in x
and one pin in y), the distance traversed between spots (x direction) and in
the y
direction (after each cycle of printing) is preferably 1 divided by 2 mm. For
4 pins in the
y direction and 1 pin the x direction (4 pins total). The required motions for
sufficient
spacing of blot spots is approximately 2 mm and 0.5 mm respectively. Twelve
print
cycles are preferably required when using 32 pins (8 pins in the x and 4 pins
in the y
direction), to spot all probe samples in a single 384 plate. The required
distances in the
x and y direction over the blot slides are preferably 2 mm in the y direction
and 4 mm in
the x direction. As demonstrated above the basic principles are sufficiently
flexible to
accommodate a variety of pin configurations.
11
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CA 02316045 2002-09-23
Adhering to these conditions will help ensure the synthesis of well-ordered
and
well-configured microarrays.
Variable Pin Contact Velocity
Robotic fabrication of microarrays requires several changes of joint velocity
in
order to provide the best quality of printing. There are three critical points
at which
velocity should to be changed: during printing onto the slides 9, during pin 2
blotting, and
during the loading of the pins 2 with the biological probe sample.
Velocity change during printing onto the slides
During printing onto the slides 9 it is necessary to both approach and depart
from
the slides 9 at a relatively slow speed in order to promote optimal spot
quality. If the pins
2 approach the slide 9 too quickly they will create "micro splashes" which
will disrupt
spot morphology. Similarly, if the pins 2 are pulled away from the slide 9 too
quickly,
then the spots can be pulled in such a way that morphology is disrupted. The
following
exemplary aspects were determined:
1) Due to the floating architecture of certain preferred pins, the pins may be
moved past the point of the initial touch with the substrate, typically glass,
without damaging the tips: they simply lift out of the print head slightly.
The
"down position", otherwise known as the second position, for the pins on the
slides should be set such that these are moving approximately 200
micrometres, as an example, past the point at which contact is made
between the tip and the glass. Setting the down position in this way does two
things. The first is that it provides a more gentle touch onto the slide, and
the
second is that it in effect allows for a dwell time in which the pins remain
in
contact with the slides. In other words, the pins come in contact with the
slide, and remain in contact while the print head continues downward for
about 200 micrometres, and as it returns up away from the slide.
2) The "up position", otherwise known as the first position, for the pins over
the
slides should be approximately 2 millimetres, as an example, above the value
selected for the "down position". This ensures that there is proper clearance
12
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CA 02316045 2002-09-23
above the platform such that during lateral movement the pins will not hit
anything causing them damage.
3) The robot is set with an overall "safe position" which is significantly
high
above the platform to allow any gross movements without collision. This
distance is often about 50 mm or more above the platform.
4) During printing the robot will travel from this "safe position" to the "up
position"
at the user-selected velocity for all gross movements.
5) As a spot is made, the robot performs the movement from the "up position"
to
the "down position" (hereafter referred to as the printing movement) via a
timed, "velocity independent" movement. Regardless of the user-selected
velocity for gross movements, the printing movement is done at a timed
speed. Studies have found that a rate of about 1 mm per 50 to 100
milliseconds was appropriate. As such, the approximately 2 mm printing
movement is set to take about 100 to 200 milliseconds to complete. Similarly
the departure movement as the pins move away from the slide back to the
"up position", also takes about 100 to 200 milliseconds to complete.
As an example of how the robot may be programmed for printing onto the chips
using timed Z-axis movements, the first instruction of a piece of code
commands the
robot to move in the z-axis only to a calibrated point from its current
position. The robot
is preferably instructed to perform this movement in about 100 milliseconds.
The next
two instructions output the robot's current position as determined by the
encoders as
well as the desired position of the robot in the x-axis (for monitoring
precision). The
fourth instruction moves the robot back up in the z-axis to another calibrated
point,
preferably also in 100 milliseconds. At this point the robot again displays
the actual and
desired y-axis positions of the robot for monitoring precision.
This procedure leads to ideal spot morphology with one particular set up (3X
SSC spotting solution, DNA concentration of 0.1 to 0.2 micrograms/microlitre),
however
differing spotting solutions and DNA concentrations require different timings
due to
changes in viscosity. To a person in the art, it is clear that variations to
the mentioned
parameters may also be used for proper performance, for example the extent
that the
13
CA 02316045 2002-09-23
pins are allowed to drop past touching the slide (approximately 200
micrometres in this
case), the height of the pins in the "up position" (about 2 mm above the "down
position"),
and the duration the pin rests on the slide. These numbers are offered as
examples.
Lower viscosity solutions are likely to splash more easily but will make
larger spots. As
a result it is generally preferred that (1) the approach speed be reduced; (2)
the distance
past touching be reduced; and (3) the departure speed be reduced. Typically
higher
viscosity solutions will have (1) the distance past touching increased to
increase dwell
time; or (2) an additional step to provide a dwell time of defined duration
after touching.
With higher viscosity solutions approach and departure speeds can be increased
which
will compensate for the required dwell time.
Velocity Changes During Blotting
It is important that deposition of probe DNA yield regularly spaced spots of
uniform morphology. Not all deposition or spotting pins designed to the same
specifications behave in a similar manner. Each will load an amount
characteristic of the
pin. Consequently, the size of the first spots produced from a set of pins
will be
significantly variable. The greatest concern is that deposition of excessive
material on
the microarrays may yield overlapping spots. The results of which will effect
contamination of the material spotted on the arrays as well as the material in
the probe
plates. As spotting proceeds the excess material is removed and the size of
spots
become uniform. The purpose of the blot slide or blot pad is to remove the
excess
material from the pins prior to the spotting on to the microarray slides. This
has helped
ensure the production of well order arrays with uniform spot configuration.
The Blot slides are preferably composed of polished glass or similar material.
High-quality microscope slides work very well. To maximize the space allocated
to the
printing of arrays, it is important to determine the minimum size required for
the Blot
Slide to perform adequately. Sufficient distances are allowed between spotted
material
to preclude the possibility of overlapping of spots. A centre to centre
distance of about
0.5 mm (millimeter) is optimal. Sufficient numbers of spots are printed to
ensure
uniformity of spots to be subsequently printed on to the arrays.
Typically, all the parameters for blotting a pin on a blot slide are the same
as for
printing on a slide, although slightly faster velocities can be used, such as
at about 75-
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CA 02316045 2000-08-16
100 milliseconds, to travel the typically 2 mm distance from the pin up
position (the first
position) to the down position (the second position).
A robot blotting routine using timed Z-axis movements preferably involve a
first
step loading a counter with the number of blots to be performed. The second
step sets
up a loop which is to be executed the number of times by the counter.. The
first step of
the loop moves the robot down in the z-axis to a calibrated position from its
current
position in preferably 100 milliseconds. The next step moves the robot back up
in the z-
axis to another calibrated point in preferably 100 milliseconds. After this
movement, the
robot is instructed to move laterally (for example, parallel to the x-axis) a
set distance
between two blots. This movement is set to take about 200 milliseconds to
avoid abrupt
movement of the robot. In a subsequent step the robot finishes its lateral
movement
before proceeding on to the next step. The robot then moves back up to a safe
position.
To a person in the art, it is clear that variations to the above mentioned
parameters may also be used for proper performance of the microarray
microarrayer.
The above parametric values are offered as examples. Again, lower viscosity
solutions
are likely to splash more easily but will make larger spots. As a result it is
generally
preferred that (1) the approach speed be reduced; (2) the distance past
touching be
reduced; and (3) the departure speed be reduced. Typically higher viscosity
solutions
will have either (1) the distance past touching increased to increase dwell
time; or (2) an
additional step to provide a dwell time of defined duration after touching.
With higher
viscosity solutions approach and departure speeds can be increased which will
compensate for the required dwell time.
Velocity Changes During Loading of the Pins
Loading of the pins 2 involves dipping the pins into the solution, which is to
be
printed. This solution is contained within the °well" of a multi-well
source plate. Each pin
2 dips into a discrete well. When using multi-well plates that have relatively
large
diameter wells, such as those found in a 96-well plate (diameter is 6 mm),
loading of the
pins 2 is relatively simple. The pins 2 can be dipped into the solution within
the well and
withdrawn with relatively quick movements. However optimal loading appears to
be a
function of velocity for these plates. When using source plates with a smaller
diameter
well opening, like in a 384-well plate (diameter is typically 3 mm), loading
becomes even
CA 02316045 2000-08-16
more velocity dependent. When using plates such as these, the pins 2 must be
more
carefully positioned in the well. In addition, the solution in the well is
more prone to stick
to the sides of the plates. Quick movements of the pin 2 in and out of the
well will cause
the solution to be °splashed" or "dragged" up the sides of the well,
which can in fact
overload the pins 2. Pin overloading (i.e. solution is present on the outside
of the pin
where it should not be) will lead to sample wastage as well as decreased spot
uniformity.
The following are examples of ways to use changes in velocity to ensure
optimal
pin 2 loading:
1 ) The pins should be lowered into the source plate slowly in a timed
movement. This lowering is done at a rate independent of the user-selected
velocity for gross movements of the robot.
2) The pins should also be withdrawn from the source plate using a slow timed
movement such that the solution is allowed to drain off of the pins rather
than
being dragged up on the pin.
3) Normally the plates are calibrated to have two vertical (z-axis) positions,
the
first being the up position outside of the well and the second being the down
position inside of the well. The addition of an extra calibration points)
leads
to added ability to change velocity during loading.
4) Although the following uses 3 z-axis calibration points, but this
methodology
could be extended to include any additional calibration points:
30
a. The first calibration point is the "safe position" of the robot. This
position is the same for all points of the robot and is a position high
enough over the base of the robot such that the pins cannot collide
with any objects.
b. The second calibration point is the "up position" (also known as the
first position) which is set such that the tips of the pins are just outside
of the wells of the source plate.
16
CA 02316045 2002-09-23
c. The third calibration point is the "down position" (the down position).
This position is selected to be the point at which the pins are just
touching the bottom of the wells (or perhaps a little past the point of
touching).
5) The velocities are preferably controlled in the following way:
a. Movement from the safe position to the up position (the first position)
is performed at the user-selected velocity. This is a gross movement
and does not need to be slowed down. Slowing down such a large
movement would only serve to waste valuable time.
b. Movement from the up position to the down position is timed.
Depending on the depth of the well, this time may change. With the
384-well source plates, this distance is covered in approximately one
second (the overall rate then is about 12 -15 millimetres per second).
c. Movement out of the well from the down position to the up position is
also timed and with one typical set up takes 1 second (again a rate of
about 12 - 15 mm per second)]. The up position is typically about 12
to 15 mm away from the down position and the movement is set to
take approximately 1 second.
d. The final movement from the up position back to the safe position is
performed again at the user-selected velocity to maximize efficiency.
These timings are again variable according due to the set up and the viscosity
of
the solution. Again we are using 3X SSC and 0.1 to 0.2 micrograms per
microlitre DNA
concentrations. To a person in the art, it is clear that variations to the
mentioned
parameters may also be used for proper performance of the microarrayer. These
numbers are offered as examples. Lower viscosity solutions are likely to
splash more
easily but will make larger spots. As a result it is generally required that
(1) the
approach speed be reduced; (2) the distance past touching be reduced; and (3)
the
departure speed be reduced. Higher viscosity solutions will require that
either (1) the
distance past touching be increased to increase dwell time, or (2) an
additional
17
t . CA 02316045 2002-O1-28
command be added to provide a dwell time of defined duration after touching.
With
higher viscosity solutions approach and departure speeds can be increased
which will
compensate for the required dwell time.
The robot may be programmed for loading of the pins 2 with biological material
which first directs the robot to move to a calibrated position (the up
position) which is just
above the source plate. This movement is carried out at the user-selected or
default
velocity. The following step instructs the robot to wait until movement is
finished before
proceeding onto the next command. The third step involves the robot moving
down in
the z-axis to the calibrated position (the down position), which is a point at
which the pins
touch the bottom of the well. This movement is set to take, as an example,
about 1000
milliseconds (1 second}. Again the robot is instructed to wait until movement
is finished
before proceeding. At this point the robot is instructed to move back up to
the up
position coordinate at which point the pins are out of the source plate. Again
this
movement is to take, as an example, 1000 milliseconds. After the movement is
finished,
the robot moves back up to the safe position using the user-selected or
default velocity.
It will be appreciated that the above description relates to the preferred
embodiments by way of example only. Many variations on the apparatus for
delivering
the invention will be obvious to those knowledgeable in the field, and such
obvious
variations are within the scope of the invention as described and claimed,
whether or not
expressly described.
18
