U.S. patent application number 09/846467 was filed with the patent office on 2001-08-30 for apparatus and method for transfering small volumes of substances.
This patent application is currently assigned to PE Corporation (NY). Invention is credited to Kowallis, Reid B..
Application Number | 20010018216 09/846467 |
Document ID | / |
Family ID | 23129993 |
Filed Date | 2001-08-30 |
United States Patent
Application |
20010018216 |
Kind Code |
A1 |
Kowallis, Reid B. |
August 30, 2001 |
Apparatus and method for transfering small volumes of
substances
Abstract
The present invention provides a method and apparatus for
dispensing small volumes of selected substances, such as biological
reagents or samples, onto substrates. According to one general
embodiment, a plurality of spaced, tandemly-arranged substrates are
advanced, e.g., by way of a conveyor, along a transport pathway
extending over a reagent-supply location, such as a reservoir
supported at a fixed position in a base. From a position over the
reagent-supply location and the pathway, a reagent-transfer
instrument, or tip, is extended along an axis through an
intervening region, e.g., an opening defined by a surface of the
conveyor, separating an adjacent pair of advancing substrates to
contact reagent held at the reagent-supply location. The
reagent-transfer instrument is then withdrawn, along with a portion
of such reagent, through the intervening region to a position above
the transport pathway. Once the conveyor has advanced a selected
substrate, upstream of the intervening region, to a position
aligned with the axis of the reagent-transfer instrument, a
selected amount of reagent is transferred from the instrument onto
a selected site of the substrate. Advantageously, the apparatus and
method are readily adaptable for the production of micro-arrays
having a great number of closely spaced spots.
Inventors: |
Kowallis, Reid B.;
(Burlingame, CA) |
Correspondence
Address: |
PATTI SELAN, PATENT ADMINISTRATOR
APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
PE Corporation (NY)
|
Family ID: |
23129993 |
Appl. No.: |
09/846467 |
Filed: |
April 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09846467 |
Apr 30, 2001 |
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09293659 |
Apr 16, 1999 |
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6245297 |
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Current U.S.
Class: |
436/44 ;
436/180 |
Current CPC
Class: |
Y10T 436/25625 20150115;
Y10T 436/2575 20150115; B01J 2219/00459 20130101; B01J 2219/0059
20130101; Y10T 436/25 20150115; Y10T 436/110833 20150115; C40B
60/14 20130101; B01J 2219/00367 20130101; B01J 2219/00605 20130101;
Y10T 436/114998 20150115; B01L 3/0262 20130101; Y10T 436/112499
20150115; G01N 35/00009 20130101; Y10T 436/11 20150115; B01J
2219/00585 20130101; B01L 2300/0838 20130101; B01J 2219/00648
20130101; B01J 2219/00659 20130101; G01N 35/10 20130101; B01L
3/0244 20130101; B01J 2219/00527 20130101 |
Class at
Publication: |
436/44 ;
436/180 |
International
Class: |
G01N 021/00 |
Claims
It is claimed:
1. An apparatus for spotting a reagent on one or more substrates,
comprising in operative condition: a base adapted to hold such a
reagent; a conveyor having a surface with a plurality of spaced,
tandemly-arranged substrate-support regions, each adapted to
support such a substrate, said conveyor being operable to advance
such substrates along a transport pathway over said base; said
conveyor surface defining an opening between each adjacent pair of
substrate-support regions; a transfer head having a spotting tip,
said tip being mounted for movement along an axis, toward and away
from a raised position at which the tip is disposed above the
conveyor surface; an actuator operatively connected to said tip for
moving the same along said axis; and a control unit operatively
connected to the conveyor and actuator, operative, for one or more
selected substrates on the conveyor surface, to (i) shift said tip
away from its raised position through a selected opening in said
conveyor surface to contact reagent in said base, (ii) withdraw
said tip from the reagent and through the opening by shifting the
tip toward its raised position, and (iii) shift said tip away from
its raised position toward a selected substrate upstream of the
selected opening, to transfer a selected amount of reagent from
said tip to a selected region of the selected substrate.
2. The apparatus of claim 1, which further includes one or more
additional transfer heads and associated actuators disposed at
spaced positions along said transport pathway, and structure in
said base adapted to hold one or more reagents at each of said
spaced positions.
3. The apparatus of claim 2, wherein at least one of said transfer
heads is laterally offset from the other transfer heads.
4. The apparatus of claim 2, wherein a plurality of transfer heads
are disposed in a row extending laterally or obliquely across the
conveyor surface at one or more of said spaced positions along said
transport pathway.
5. The apparatus of claim 1, further comprising a channel extending
through at least a portion of said base.
6. The apparatus of claim 5, further comprising a flow line having
an outlet disposed to direct a selected fluid into said
channel.
7. The apparatus of claim 6, wherein said base is adapted to hold
one or more reagent reservoirs such that a lower region of each
reservoir extends at least partially into said channel.
8. The apparatus of claim 6, wherein said transfer tip, when
shifted away from its raised position with said axis unobstructed,
can enter at least partially into said channel.
9. The apparatus of claim 1, wherein said tip is a pin having a
distal end less than about 500 .mu.m in diameter.
10. The apparatus of claim 1, wherein said tip includes a channel
of capillary size adapted to draw in reagent, when shifted into
contact therewith, by way of capillary action.
11. The apparatus of claim 1, wherein said tip is a
micropipette.
12. The apparatus of claim 1, wherein said transfer head includes a
plurality of spotting tips mounted side-by-side, in spaced
relation; each tip being adapted for movement along a respective
axis, toward and away from a raised position at which the tip is
disposed above the conveyor surface.
13. The apparatus of claim 1, wherein an elongate web defines said
conveyor surface, with each of said substrate-support portions
defining a substrate suitable for spotting.
14. The apparatus of claim 13, wherein said web material is a
flexible membrane material.
15. The apparatus of claim 1, wherein an elongate flexible belt
defines said conveyor surface.
16. An apparatus for spotting a reagent onto one or more
substrates, comprising: a conveyor belt comprising a plurality of
substrate-support regions separated from one another by intervening
open regions therebetween; a base located beneath the conveyor belt
for supporting one or more reagent reservoirs; a transfer head,
disposed above said base and said conveyor belt, which has a
reagent spotting tip mounted for movement between (1) a raised
position above the conveyor belt, (2) a reagent dispensing position
for depositing reagent on a substrate carried by one of said
substrate-support regions, and (3) an extended position below the
conveyor belt which is achieved by passing the tip through one of
said open regions; means for moving the conveyor belt along a
transport pathway such that the substrate-support regions pass
between said base and said transfer head; means for shifting the
spotting tip between said extended, reagent dispensing, and raised
positions; and one or more controllers operatively connected to the
moving means and shifting means effective to (i) shift the spotting
tip from its raised position to its extended position by traversing
a selected open region in said conveyor belt, for withdrawing
reagent from a reservoir supported by the base, (ii) raise the
spotting tip after step (i) to a position above the conveyor belt,
(iii) move the conveyor belt so that a selected substrate is
positioned below the raised spotting tip, (iv) move the spotting
tip to a reagent dispensing position so that reagent is deposited
onto a selected region of the selected substrate, (v) after reagent
deposition, raise the spotting tip to its raised position, (vi)
move the conveyor belt so that the spotting tip is positioned above
another open region, and (vii) repeat steps (i) through (vi) a
selected number of times.
17. An apparatus for spotting a reagent on one or more substrates,
comprising in operative condition: a base adapted to hold such a
reagent; a conveyor having a surface defining (i) a plurality of
spaced, tandemly-arranged substrate regions, and (ii) an opening
between each adjacent pair of substrate regions; said conveyor
being operable to advance such regions along a transport pathway
over said base; a transfer head having a spotting tip, said tip
being mounted for movement along an axis, toward and away from a
raised position at which the tip is disposed above the conveyor
surface; an actuator operatively connected to said tip for moving
the same along said axis; and a control unit operatively connected
to the conveyor and actuator, operative, for one or more selected
substrate regions defined by the conveyor surface, to (i) shift
said tip away from its raised position through a selected opening
in said conveyor surface to contact reagent in said base, (ii)
withdraw said tip from the reagent and through the opening by
shifting the tip toward its raised position, and (iii) shift said
tip away from its raised position toward a selected substrate
region upstream of the selected opening, to transfer a selected
amount of reagent from said tip to a selected site of the selected
substrate region.
18. The apparatus of claim 17, wherein said conveyor surface is a
flexible web material.
19. A method for spotting a reagent on one or more substrates,
comprising the steps of: (i) advancing a plurality of spaced,
tandemly-arranged substrates along a transport pathway extending
over a reagent-supply location; (ii) from a position over said
reagent-supply location and said pathway, (a) extending a
reagent-transfer instrument through an intervening region
separating an adjacent pair of advancing substrates to contact
reagent held at said reagent-supply location, (b) withdrawing the
reagent-transfer instrument, along with a portion of such reagent,
through said intervening region to a position above said transport
pathway, and (c) transferring a selected amount of reagent from
said reagent-transfer instrument onto a selected region of a
selected substrate upstream of said intervening region.
20. The method of claim 19, wherein said substrates are integrally
formed as spaced-apart expansive portions provided along an
elongate web material, and each of said intervening regions is an
opening formed through said web material between adjacent substrate
portions.
21. The method of claim 19, wherein said substrates are advanced
using a conveyor having a movable belt with a plurality of
tandemly-arranged substrate-support regions; with each substrate
being placed at a defined location on a respective one of said
substrate-support regions.
22. The method of claim 19, wherein said transport pathway extends
over a plurality of reagent-supply locations, disposed at spaced
positions along said pathway; and wherein step (ii) is performed at
two or more of said spaced positions in a fashion effective to
produce a plurality of reagent spots on the selected substrate.
23. The method of claim 22, wherein at least one of said reagent
spots on the selected substrate is laterally offset from the other
reagent spots.
24. The method of claim 22, wherein step (ii) is performed at least
twice, in a substantially parallel fashion, using separate
reagent-transfer instruments at one or more of said spaced
positions.
25. The method of claim 19, further comprising the steps of:
removing any reagent(s) being held at said reagent-supply location;
extending at least a portion of said reagent-transfer instrument
into said reagent-supply location; flowing a cleaning fluid along
said reagent-supply location so that it contacts and cleans said
reagent-transfer instrument.
26. The method of claim 25, further comprising the step of: with at
least a portion of said reagent-transfer instrument extended into
said reagent-supply location, flowing a gas along said
reagent-supply location so that it contacts and dries the cleaned
reagent-transfer instrument.
27. The method of claim 19, further comprising the steps of:
placing a vessel containing a selected liquid reagent at said
reagent-supply location; and flowing a cooling fluid along said
reagent-supply location so that it contacts said vessel, thereby
reducing evaporative loss of the selected liquid reagent.
28. The method of claim 19, further comprising the steps of: prior
to step (i), retrieving a vessel containing a selected reagent from
a storage location, and placing the vessel at said reagent-supply
location; and subsequent to step (ii), retrieving the vessel from
said reagent-supply location, and returning the vessel to its
storage location.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
09/293,659, filed Apr. 16, 1999, which is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the dispensing of
substances, such as biological reagents and samples. More
particularly, the invention provides an apparatus and method for
transferring small volumes of substances onto one or more
substrates.
BACKGROUND OF THE INVENTION
[0003] As the sensitivity of analytical techniques continues to
improve, it is increasingly desirable to carry out chemical and
biochemical assays using very small volumes of samples/reagents.
This is especially true in situations involving expensive
substances. Accordingly, it is now popular to utilize very small
volumes of such substances laid down as "spots" on the surface of a
substrate, such as a slide, micro-card, chip or membrane.
[0004] Not only is it often desirable to provide ultra-small
volumes of individual samples and/or reagents in the form of spots,
it is becoming increasingly popular to arrange numerous such spots
in close proximity to one another in the form of an array on a
substrate. For example, a lab technician might need to evaluate a
specimen for the presence of a wide assortment of target biological
and/or chemical compounds, or to determine the reaction of many
different specimens against one or more reagents, such as labeled
probes. High-density array formats, or "microarrays," permit many
reactions to be carried out in a highly parallel fashion, saving
space, time and money.
[0005] A variety of methods are currently available for making
microarrays. Microarrays can be made, for example, by a robotic arm
device having a spotting tip that moves successively between a
sample-pickup well in a sample array, e.g., a microtitre plate, and
a selected array position. Although high-density arrays of selected
substances can be constructed by this approach, the production time
and efficiency is limited by the fact that the regions of the
microarray (or microarrays, if several are being constructed at
once) are deposited one-by-one in a serial fashion. Additional time
and effort is required where a plurality of different substances
are laid down in the array, as the spotting tip must be cleaned and
dried prior to being used with each new substance.
[0006] Multi-channel micropipette devices are available for laying
down several reagent spots at once. Devices of this type typically
have 8 or 12 micropipettes, fixed side-by-side in a linear array.
Generally, these devices are unsuitable for quickly producing very
dense arrays, as the size of each micropipette and any associated
service connections (e.g., supply tubing, electrical connections,
etc.) limits the minimal center-to-center spacing (pitch) that can
be achieved for adjacent spots. Also, since only a few spots
(usually 8 or 12) can be laid down at a time with such devices, the
production of very dense arrays, e.g., having hundreds or thousands
of spots with a submillimeter pitch, tends to be a very tedious and
time-consuming process.
[0007] Another technique employs an array of pins arranged to
simultaneously dip into an array of reservoirs, e.g., the 96 wells
of a microtitre plate, to pick up one or more selected substances
for transfer to a substrate, such as a membrane. Similar to the
multi-channel pipette devices, the pitch spacing is limited by the
size of each pin. Also, the pins of such arrays are typically
arranged to match the pitch of a conventional supply-well array,
typically 21/4, 41/2, or 9 mm center-to-center. Thus, similar to
the multi-channel pipetters, the production of very dense arrays
can only be accomplished by sequentially laying down a number of
sub-arrays, e.g., in a staggered or interleaved fashion--a very
cumbersome and inefficient endeavor.
[0008] As an additional disadvantage, most of the known spotting
techniques require the handling or transfer of substances between
multiple receptacles (e.g., pipettes, flasks, vials, etc.) and/or
flow lines (e.g., channels, hoses, tubing). Such transfers
frequently result in a loss or contamination of the substance,
thereby reducing the overall efficiency and sensitivity of the
assay. Particularly with regard to expensive substances, it is
generally desirable to keep such losses to a minimum.
[0009] In view of the above, the need is apparent for a device and
method useful for delivering a micro-volume of a substance onto a
substrate in a quick and efficient manner. Preferably, the device
should be relatively easy to use, cost effective and readily
adaptable for the production of micro-arrays having a great number
of individual spots.
SUMMARY OF THE INVENTION
[0010] In one of its aspects, the present invention provides an
apparatus for spotting a selected substance (e.g., a liquid sample
or reagent, or micro-particles such as beads) onto one or more
substrates.
[0011] In one general embodiment, the apparatus of the invention
includes a base, adapted to hold one or more reagents, and a
conveyor. The conveyor includes a movable surface defining (i) a
plurality of spaced, tandemly-arranged substrate-support regions,
each of which is adapted to support a substrate, and (ii) an
opening between each adjacent pair of substrate-support regions.
The conveyor is operable to advance the substrate-support regions
along a transport pathway extending over the base. Further included
is a transfer instrument or head having a spotting tip mounted for
movement along an axis, toward and away from a raised position at
which the tip is disposed above the conveyor surface. Shifting
means, e.g., an actuator (such as a solenoid, or the like), are
operatively connected to the tip for moving the same along its
axis. A control unit is operatively connected to the conveyor and
the actuator. At the direction of the control unit, a selected
opening of the conveyor surface can be advanced to a position
generally aligned with the axis of the transfer tip, at which point
the control unit can signal the shifting means to shift the tip
away from its raised position through such opening to contact
reagent in the base. The shifting means can then withdraw the tip
from the reagent and through the opening by shifting the tip toward
its raised position. A selected site of a substrate-support region
upstream of the selected opening can then be advanced to a position
generally aligned with the axis of the transfer tip, at which point
the control unit can signal the shifting means to shift the tip
away from its raised position toward such site to transfer a
selected amount of reagent from the tip to a selected region of a
substrate at the substrate-support region.
[0012] According to one embodiment, one or more additional transfer
heads and associated shifting means are disposed at spaced
positions along the transport pathway, and structure is provided in
the base for holding one or more reagents at each of the spaced
positions. Such structure can include, for example, one or more
tube holders (e.g., apertures or bores formed along a top surface
of the base). The various transfer heads can be positioned along a
line running parallel with the transport pathway, or one or more of
the transfer heads can be laterally offset from the other transfer
heads.
[0013] In one embodiment, a plurality of transfer heads are
disposed in a row extending laterally or obliquely across the
conveyor surface at one or more of the spaced positions along the
transport pathway.
[0014] One embodiment contemplates a channel or cavity extending
through at least a portion of the base. For example, an elongate
channel can extend longitudinally through a central region the
base. Optionally, a flow line can communicate a remote fluid source
with the channel. In one such arrangement, a fluid flow line is
connected to a fitting at one end of the channel. An outlet of the
flow line is arranged so as to direct a selected fluid, passed
through the line, into and along the channel. The channel can
further include an egression port, e.g., at a distal end, through
which any fluid(s) directed into the channel can exit.
[0015] In one embodiment, the base of the apparatus is adapted to
hold one or more reagent reservoirs (e.g., tubes, vials, or the
like) such that a lower region of each reservoir extends at least
partially into a channel of the base, such as the channel just
described. For example, apertures can be formed along the top of
the base into which respective reagent-holding tubes can be
inserted. Each aperture, in this exemplary construction,
communicates the interior of the channel with a region between the
base and the transport pathway. With the tubes in place along the
base, a cooling fluid (e.g., a gas, or water) passed through the
channel can impinge upon the accessible external surfaces of the
tubes, thereby cooling the tubes so as to discourage evaporation of
the reagent(s) held therein.
[0016] According to one exemplary design, the transfer tip, when
shifted away from its raised position, with its axis of motion
unobstructed (i.e., through an opening defined by the conveyor
surface), is adapted to enter at least partially into a channel
extending through the base. At this position, a cleaning fluid
(e.g., a liquid solvent) passed through the channel can clean the
tip. Optionally, a dry, warm gas subsequently passed through the
channel can be used to dry the cleaned tip.
[0017] Any suitable transfer instrument or head can be used,
including contact and/or non-contact type devices. For example, the
apparatus can employ a transfer head having an elongated tip in the
nature of a-pin or rod. In a typical construction, a relatively
narrow rod is employed, e.g., one having a distal end less than
about 500 .mu.m in diameter, and preferably less than about 250
.mu.m in diameter. In another exemplary arrangement the tip
includes a channel of capillary size (e.g., less than about 1 mm in
diameter) adapted to draw in a liquid reagent, when shifted into
contact therewith, by way of capillary action. Still further
embodiments contemplate the use of a micropipette, syringe device,
jetting apparatus, or other "sip and spit" assembly, as the
transfer tip.
[0018] Preferably, the transfer tips of the transfer head or
instrument are of an independent construction. The tips are not
permanently fixed spatially with respect to one another. Each
individual transfer tip can be attached to and detached from the
head, without affecting or otherwise disturbing any other transfer
tip(s) of the apparatus.
[0019] One embodiment of the invention teaches a transfer head
having a plurality of spotting tips mounted side-by-side, in spaced
relation. Each tip, in this embodiment, is adapted for movement
along a respective axis, toward and away from a raised position at
which the tip is disposed above the conveyor surface.
[0020] The conveyor of the transfer apparatus can be, for example,
a linear-type conveyor or a carousel-type arrangement, among
others. In one embodiment, the conveyor surface takes the form of
an elongate web. Each of the substrate-support portions of the web,
in this embodiment, defines a substrate portion or region that can
be spotted. That is, the web and substrates are of an integral
construction. In one particular arrangement, the web material is a
flexible, membrane material. In another embodiment, the conveyor
surface takes the form of an elongate flexible belt, e.g., a rubber
or metallic endless belt, upon which separately formed substrates
(e.g., 1".times.3" micro-cards) can be removably placed. In one
particular arrangement, the belt includes a pocket at each of its
substrate-support regions for receiving respective micro-cards and
maintaining the position of each at a known location as it is
advanced along the transport pathway and spotted.
[0021] Another general embodiment of the spotting apparatus, as
taught herein, includes a conveyor belt comprising a plurality of
substrate-support regions separated from one another by intervening
open regions therebetween. A base is located beneath the conveyor
belt for supporting one or more reagent reservoirs (e.g., tubes,
vials, or the like). A transfer instrument or head is disposed
above the base and the conveyor belt, having a spotting tip mounted
for movement between (1) a raised position above the conveyor belt,
(2) a reagent dispensing position for depositing reagent on a
substrate carried by one of the substrate-support regions, and (3)
an extended position below the conveyor belt which is achieved by
passing the tip through one of the open regions. Further included
are means for moving the conveyor belt (including, for example, a
motor, drive train, and driven roller) along a transport pathway
such that the substrate-support regions pass generally along a
plane extending between the base and the transfer head. Shifting
means (e.g., an actuator, such as a z-motion actuator) are
operatively connected to the spotting tip for shifting it between
the extended, reagent dispensing, and raised positions. One or more
controllers are operatively connected to the moving means and
shifting means, the controllers being operable to (i) shift the
spotting tip from its raised position to its extended position by
traversing a selected open region in the conveyor belt, for
withdrawing reagent from a reservoir supported by the base, (ii)
raise the spotting tip after step (i) to a position above the
conveyor belt, (iii) move the conveyor belt so that a selected
substrate is positioned below the raised spotting tip, (iv) move
the spotting tip to a reagent dispensing position so that reagent
is deposited onto a selected region of the selected substrate, (v)
after reagent deposition, raise the spotting tip to its raised
position, (vi) move the conveyor belt so that the spotting tip is
positioned above another open region. If desired, the controller
can repeat steps (i)-(vi) a selected number of times.
[0022] A further general embodiment of a spotting apparatus, as
taught herein, includes a base, adapted to hold a reagent, and a
conveyor. The conveyor includes a surface defining (i) a plurality
of spaced, tandemly-arranged substrate regions, and (ii) an opening
between adjacent substrate regions. The conveyor is operable to
advance such regions along a transport pathway extending over the
base. A transfer instrument or head is provided, having a spotting
tip mounted for movement along an axis, toward and away from a
raised position at which the tip is disposed above the conveyor
surface. Shifting means, e.g., an actuator, are operatively
connected to the tip for moving the same along its axis. A control
unit is operatively connected to the conveyor and the shifting
means. At the direction of the control unit, a selected opening of
the conveyor surface can be advanced to a position generally
aligned with the axis of the transfer tip, at which point the
control unit can signal the shifting means to shift the tip away
from its raised position through such opening to contact reagent in
the base. The shifting means can then withdraw the tip from the
reagent and through the opening by shifting the tip toward its
raised position. A selected site of a substrate region, upstream of
the selected opening, can then be advanced to a position generally
aligned with the axis of the transfer tip, at which point the
control unit can signal the shifting means to shift the tip away
from its raised position toward such site to transfer a selected
amount of reagent from the tip thereto.
[0023] In one particular arrangement of the spotting apparatus, the
conveyor surface is a flexible web material, such as a membrane or
the like.
[0024] In another of its aspects, the present invention provides a
method for spotting a selected substance (or substances) onto one
or more substrates.
[0025] According to one general embodiment, the method includes the
steps of:
[0026] (i) advancing a plurality of spaced, tandemly-arranged
substrates along a transport pathway extending over a
reagent-supply location;
[0027] (ii) from a position over the reagent-supply location and
the pathway,
[0028] (a) extending a reagent-transfer instrument, or tip, through
an intervening region separating an adjacent pair of advancing
substrates to contact reagent held at the reagent-supply
location,
[0029] (b) withdrawing the reagent-transfer instrument, along with
a portion of such reagent, through the intervening region to a
position above the transport pathway, and
[0030] (c) transferring a selected amount of reagent from the
reagent-transfer instrument onto a selected region of a selected
substrate upstream of the intervening region.
[0031] In one embodiment, the substrates are integrally formed as
spaced-apart expansive portions provided along an elongate web of
material (e.g., a membrane material), and each of the intervening
regions is an opening formed through the web of material (e.g., a
cut-out region) between adjacent substrate portions.
[0032] In another embodiment, the substrates are advanced using a
conveyor having a belt (e.g., a flexible endless belt) with a
plurality of tandemly-arranged substrate-support regions. Each of
the substrates, in this embodiment, is placed at a respective one
of the substrate-support regions.
[0033] According to one embodiment, the transport pathway extends
over a plurality of reagent-supply locations, disposed at spaced
positions along the pathway. Step (ii), in this embodiment, is
performed at two or more of the spaced positions in a fashion
effective to produce a plurality of reagent spots on the selected
substrate. The reagent spots can be placed along a line extending
substantially parallel to the transport pathway, and/or one or more
of the reagent spots can be placed at positions that are laterally
offset from the other reagent spots.
[0034] In one embodiment, step (ii) is performed at least twice, in
a substantially parallel fashion, using separate reagent-transfer
instruments at one or more of the spaced positions.
[0035] Another embodiment contemplates the additional steps of:
removing any reagent(s) being held at the reagent-supply
location(s); extending at least a portion of each reagent-transfer
instrument into a respective reagent-supply location; and flowing a
cleaning fluid (e.g., a liquid solvent) along the reagent-supply
location so that it contacts and cleans each reagent-transfer
instrument or tip. Optionally, a drying fluid (e.g., a warm, dry
gas) can be passed along the reagent-supply location, subsequent to
such cleaning step, such that it contacts and dries each transfer
instrument.
[0036] In one embodiment, one or more reagent reservoirs, or
vessels, are placed at respective reagent-supply locations; and a
cooling fluid is passed along the reagent-supply location so that
it contacts the vessel, thereby reducing evaporative loss of any
liquid reagent held therein.
[0037] A further embodiment contemplates, prior to step (i), the
additional step of retrieving a vessel containing a selected
reagent from a storage location, and placing the vessel at a
reagent-supply location; and, subsequent to step (ii), the step of
retrieving the vessel from the reagent-supply location, and
returning the vessel to its storage location. In this way, the use
of intermediate vessels, and consequent loss of reagent, is
avoided.
[0038] Still a further aspect of the invention provides a
substrate, bearing one or more reagent spots (e.g., a micro-array),
produced in accordance with the method taught herein.
[0039] These and other features and advantages of the present
invention will become clear from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The structure and manner of operation of the invention,
together with the further objects and advantages thereof, may best
be understood by reference to the following description taken in
conjunction with the accompanying drawings, in which:
[0041] FIG. 1 is a partially schematic, perspective view of a
reagent-transfer apparatus, along with several exemplary
substrates, according to the teachings of the present
invention;
[0042] FIG. 2 is a partial exploded view of the apparatus and
substrates shown in FIG. 1;
[0043] FIG. 3 is a perspective view of another embodiment of a
reagent-transfer apparatus, according to the present invention;
[0044] FIG. 4 is a cross-sectional view showing a base portion of
the reagent-transfer apparatus of the present invention, as
contemplated by one embodiment, with a reagent reservoir seated in
an aperture in the upper wall of the base; and
[0045] FIGS. 5A-5G depict an exemplary operation wherein a reagent
spot is formed on a selected substrate, in accordance with the
teachings of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The following discussion of the preferred embodiments of the
present invention is merely exemplary in nature. Accordingly, this
discussion is in no way intended to limit the scope of the
invention.
[0047] One aspect of the invention provides an apparatus for
transferring a selected substance or substances, such as biological
reagents and/or samples, onto one or more substrates. In one
general embodiment, the apparatus includes a base adapted to hold a
supply of reagent, e.g., in a reservoir. Means are provided for
moving a plurality of tandemly-arranged substrates, separated from
one another by intervening open regions, along a transport pathway
extending over the base. A reagent-transfer instrument is mounted
for movement toward and away from a raised position at which a
transfer tip, along one end of the instrument, is disposed above
the transport pathway. Shifting means are provided for moving the
transfer tip along an axis, toward and away from its raised
position. A control unit is operative to (i) shift the transfer tip
away from its raised position through a selected open region to
contact reagent held at the reagent-supply location, (ii) withdraw
the tip, along with a portion of such reagent, through the open
region to a position above the transport pathway, and (iii) to
shift the tip away from its raised position toward a selected
substrate upstream of the selected open region, to transfer a
selected amount of reagent from the tip to a selected region of the
selected substrate.
[0048] An exemplary arrangement of a reagent-transfer apparatus, as
provided by the present invention, is indicated generally by the
reference numeral 8 in FIG. 1. In this embodiment, a conveyor,
denoted generally as 12, is adapted to move a plurality of
tandemly-arranged substrates, such as 14a-14d, separated from one
another by intervening open regions, as at 28a-28c, along a
transport pathway, "P," that extends over a base 50. Conveyor 12
includes a driven roller 16 at one of its ends, and an idler roller
18 at its other end. A flexible belt, denoted as 22, extends over
the driven and idler rollers. Optionally, conventional support
rollers (not shown) can be provided between the drive and idler
rollers, and any known slack adjusting mechanism (not shown) can by
used to maintain a desired tension in the belt.
[0049] Any suitable flexible belt can be used for supporting and
transporting the substrates. In one embodiment, the conveyor belt
is formed of a flexible polymer material, e.g., rubber or the like.
In another embodiment, a flexible metal belt, such as a steel band,
is utilized. While the particular material composition of the belt
is not critical, it is important that the belt includes at least
one region configured to support a substrate, and an opening
downstream of the substrate-support region. For example, in the
present embodiment, belt 22 includes (i) a plurality of
substrate-support regions, such as 26a-26d (FIG. 2), and (ii) an
opening, as at 28a-28c, between adjacent substrate-support
regions.
[0050] Although not visible in the figures, the conveyor belt can
further include features for locating and maintaining each
substrate at a desired position and orientation thereon. For
example, a sunken region or pocket can be formed at each
substrate-support region of the conveyor belt, dimensioned to
closely fit the outer dimensions of a given type of substrate,
e.g., a 1".times.3" micro-card.
[0051] A motor, as at 32 in FIG. 1, is provided for advancing
conveyor 12. Motor 32 can be of any suitable, known type.
Preferably motor 32 is a stepper motor, though other motors can be
used, e.g., servos, etc. Motor 32 is operatively connected to a
drive train, denoted generally as 34, for driving the conveyor in
accordance with the commands of a control unit, as indicated
schematically at 36. Any suitable drive train can be employed. In
the illustrated arrangement, an endless chain or belt 38 couples a
drive sprocket 40 affixed to an output shaft of motor 32 with a
driven sprocket 42 of roller 16. As will be discussed, the drive
may be intermittent or continuous.
[0052] Instead of placing a plurality of separately formed
substrates on the conveyor belt, as shown in FIGS. 1 and 2, the
belt itself can provide a plurality of substrates. That is, the
belt and substrates can be of an integral construction-- e.g., with
each substrate-support region, itself, defining a substrate. In the
exemplary arrangement of FIG. 3, a web 22' of a flexible material
(e.g., a membrane) extends from a supply roll 18', over base 50, to
a driven take-up roll 16'. Web 22', in this embodiment, includes a
plurality of tandemly arranged substrate portions, such as at
14a'-14d', each defined by an expansive region of the belt. Each
adjacent pair of substrate portions of the web, in this embodiment,
are separated from one another by intervening open regions in the
nature of apertures or holes, as at 28a'-28c', defined by web 22'.
Upon driving take-up roll 16' in a clockwise direction, the
substrate portions are moved along transport pathway P, over base
50. From the take-up roll, the substrate portions can be subjected
to further processing (e.g., severing the various substrate
portions into separate, individual sheets), if desired.
[0053] Returning now to FIG. 1, a reagent-transfer instrument or
head, as at 44, is mounted for movement over the transport pathway
P. In the illustrated arrangement, transfer head 44 includes a
transfer tip 46 movable along an axis, denoted as "A."
Particularly, tip 46 is adapted for movement toward and away from a
raised position at which it is disposed above the transport pathway
P.
[0054] The type of transfer tip utilized is not critical, provided
only that it is capable of picking up a selected reagent from a
reservoir and transferring the reagent to a selected substrate. The
particular type of transfer tip used will often be determined, at
least in part, by the nature of the reagent employed and the
desired spot size (e.g., volume) to be formed on each substrate.
Exemplary tips useful for the transfer of liquid reagents include
pins, rods, quills, syringes, pipettes, jetting devices (e.g., "sip
and spit" devices), among others. Exemplary tips useful for
transferring solid or semi-solid reagents, such as micro-beads,
include electrostatic and/or magnetic pins or rods, as well as
vacuum capillary tubes, and the like. While only one transfer tip
is shown on the head in FIG. 1, other embodiments contemplate
multiple tips (2 or more) associated with each head.
[0055] Shifting means 48 are operatively connected to transfer tip
46 for moving it along axis A, toward and away from its raised
position. The shifting means can be, for example, an actuator, such
as a z-motion actuator adapted to move the transfer tip in a linear
or vertical fashion. In one exemplary arrangement, a solenoid
assembly includes a solenoid piston movable between two positions.
The lower end of the piston, in this embodiment, is connected to
the upper end of the transfer tip. Upon activation, the piston is
drawn downwardly (z direction), thereby shifting the transfer tip
to its lowered position. Upon release, the piston returns to its
normal, raised position, e.g., under spring bias, thereby shifting
the transfer tip to its raised position. Many solenoids are
available from commercial sources, and suitable models can be
readily chosen by those skilled in the art. In one embodiment, the
solenoid is operable to shift the transfer tip up and down over a
stroke of from about 2 to about 3 cm, and preferably about 2.5
cm.
[0056] Other devices, useful as shifting means, include, for
example, pneumatic, hydraulic, magnetostrictive, and piezoelectric
actuators, as well as motor assemblies (e.g., steppers) operable to
generate a downward motive force followed by reciprocation. Several
particular assemblies which can be adapted for use herein as the
shifting means are disclosed, for example, in U.S. Pat. Nos.
3,164,304; 3,329,964; 3,334,354; 5,443,791; 5,525,515; 5,551,487;
5,601,980; and 5,807,522; each of which is expressly incorporated
herein by reference.
[0057] Positioning means can be utilized to move the transfer tip
linearly or in an x-y plane to locate the transfer head at a
selected deposition position over the transport pathway. In one
exemplary arrangement of the positioning means, the transfer device
is carried on a movable arm or support that can be moved to a
desired position and then releasably clamped or locked down. Such
positioning can be accomplished in a manual or automated fashion,
as desired. Both manual and automated positioning arrangements are
well known, and suitable arrangements can be readily chosen by
those skilled in the art.
[0058] Exemplary automated devices useful for positioning include,
for example, robots with electronically controlled linked or
crossed movable arms, such as a SCARA, gantry and Cartesian robots.
In one embodiment, an x-y positioning assembly is employed,
comprising a motorized x-y carriage or rail arrangement. In another
embodiment, the transfer head is threadedly mounted on a worm screw
that can be driven (rotated) in a desired direction by a stepper
motor, as directed by the control unit. It is understood, of
course, that any other robotic mechanism could be used in
accordance with the present invention so long as it can accomplish
substantially the same purposes and secure substantially the same
result. Several exemplary x-y positioning assemblies which can be
readily adapted for use herein as the positioning means are
disclosed, for example, in U.S. Pat. Nos. 5,443,791; 5,551,487; and
5,587,522; each of which is expressly incorporated herein by
reference.
[0059] In an exemplary embodiment of a manually operable
positioning assembly, the transfer head attaches to a support
adapted to ride along a rail extending laterally or obliquely over
the transport pathway. The transfer head can be positioned at a
desired location by manually sliding the support along the rail,
and then locked down by turning a securing bolt threadedly received
in a bore extending through the support, so that the bolt's
terminal end pressingly engages the rail. In another embodiment, a
manually adjustable x- and/or y-axis lead screw arrangement is
employed.
[0060] As previously noted, a base, indicated generally as 50, is
provided under transport pathway P. Base 50 includes at least one
reagent-holding region whereat one or more selected reagents can be
placed (such as the region of base 50 below transfer head 44 in
FIG. 1). In a typical operation, each reagent-holding region
remains stationary (at a fixed position along the base below an
associated transfer tip) while one or more substrates are moved
thereover along the transport pathway, as by way of conveyor
12.
[0061] Further regarding the reagent holding region(s), in one
embodiment, one or more reservoirs are provided in the base itself,
each suitable for directly receiving and holding a selected
reagent. For example, a reservoir in the nature of a well or cup
can be integrally formed in, or attached to, a surface of the base
confronting the transport pathway. In another embodiment, the base
is provided with one or more locating features for removably
receiving a reagent-containing reservoir, such as a tube or vial,
at a desired position. In the exemplary arrangement of FIGS. 1
through 4, base 50 takes the form of an elongated structure having
upper and lower walls, denoted as 52 and 54 (FIG. 2), respectively,
joined by opposed, lateral sidewalls 56, 58. The long dimension of
base 50 is disposed substantially parallel to the direction of
substrate movement along transport pathway P. The four walls (52,
54, 56, 58) of base 50 define an elongated internal cavity or
channel, indicated at 63, having a generally rectangular
cross-sectional profile, as visible in FIG. 4.
[0062] A plurality of apertures, such as 62a-62c shown in FIG. 2,
extend through upper wall 52 of base 50 at spaced locations
therealong. According to one preferred embodiment, at least one of
the apertures in the base is laterally offset from the other
apertures. Instead of providing only one aperture at each of the
spaced locations along the base, other embodiments provide two or
more apertures disposed laterally or obliquely across the base at
each location.
[0063] Each aperture is configured to receive a reagent-holding
reservoir, such as one of tubes 70a-70d. The reservoirs can be held
in any suitable manner. For example, as best seen with regard to
tube 70d of FIG. 4, each tube can include a circumferential rim 71
about its upper opening. The diameter of tube 70d, measured across
rim 71, is greater than the diameter across any one of the
apertures in base 50. The region of tube 70d below its rim 71, on
the other hand, is configured with a diameter smaller than the
diameter of the apertures. By this construction, tube 70d can be
inserted, bottom first, into a selected one of the apertures, until
the lower side of its rim abuts the region of base 50
circumscribing the aperture. It will be understood that the tube
depicted represents but one variety of many types of tubes, or
other reservoirs, that can be used, and that other tube
configurations can be accommodated in the base with equal
effectiveness. For example, a tube of another form may be longer
and thereby rest against the bottom wall of base, instead of being
supported at its rim. Similarly, the apertures may be sized to
accommodate tubes of larger or smaller diameters than the tube
shown.
[0064] It should be appreciated that any desired type or form of
reagent can be held in the various reagent reservoirs (e.g.,
liquids, slurries, micro-beads, etc.). The reagents in the various
reservoirs can all be the same, or they can differ. For example,
1,000 different tubes along the transport pathway can contain,
respectively, 1,000 different primer sets for spotting onto one or
more substrates for use in primer-initiated polymerase chain
reaction (PCR).
[0065] The upper wall of the base can be of unitary construction,
e.g., an elongated strip of plastic, metal and/or wood; or,
alternatively, it can be of a modular design. In a general
embodiment of the latter, the upper wall is comprised of a
plurality of individual subunits, in the nature of panels or tiles,
laid down end-to-end. Each subunit, in this embodiment, includes
one or more tube-receiving apertures formed therethrough. One
particular embodiment provides numerous sets of panels, with the
members of each set being characterized by a particular aperture
configuration/pattern. In use, panels suitable for the task at hand
are chosen from the various sets and laid down (and, optionally,
locked in place) to provide an upper wall having a custom
arrangement of apertures therealong. Selected reagent tubes can
then be inserted into the various apertures for use in a spotting
operation (described below). Between spotting operations, the
panels can be dissembled and reassembled, as desired.
[0066] In one embodiment, the abutting edges of walls 52, 54, 56,
58 are adjoined in a substantially fluid-tight fashion, permitting
the flow of one or more selected fluids (gas and/or liquid) through
channel 63. In this regard, one or more flow lines, such as 64 and
65 in FIG. 2, can be connected at one end of base 50, via
respective fittings 67, 68. Each flow line, in the illustrated
arrangement, is adapted to communicate a selected remote fluid
source (not shown) with the channel 63 of base 50. One or more exit
ports (not shown) can be provided at the other end of base to allow
egression of any fluid(s) passed through the base. As previously
described, with the reagent tubes in their seated positions, a
substantial portion of each tube (e.g., the region below its upper
lip) extends down into channel 63. Accordingly, with the tubes in
place, fluid(s) passed through the channel (e.g., a coolant) can
contact the accessible exterior surfaces of such reservoirs. With
the reservoirs removed, and the transfer tips shifted through the
(empty) apertures to their lowered positions, fluid(s) passed
through the channel (e.g., a cleaning fluid) can contact the
accessible surfaces of such tips. Exemplary operations utilizing
such features are described more fully below.
[0067] For those embodiments employing a contact-type transfer tip,
it may be desirable to utilize means for preventing significant
deflection of the belt or web as a result of such contact. For
example, one or more shutter mechanisms (not shown) can be provided
along the base. In one embodiment, a shutter mechanism occupies a
narrow, substantially planar region between the lower surface of
the belt or web and the upper regions of the reservoirs, at each of
the spaced locations along the base. Such shutter mechanisms are
operable, at the direction of the control unit, to intermittently
form substantially rigid surfaces for supporting regions of the
belt during spotting operations. Particularly, each shutter is open
when the transfer tip is shifted through an opening to its lowered
position to pick up reagent from a reagent reservoir, and closed
when the transfer tip is shifted from its raised position into
contact with a substrate for forming a reagent spot thereon.
Alternatively, or in addition, the belt or web can be subjected to
an increased tension during contact spotting, using conventional
belt tensioning assemblies.
[0068] The positions of the substrates along the conveyor belt or
web can be monitored by any suitable means. In certain embodiments,
for example, the position of each substrate is monitored in terms
of conveyor travel length increments. In this regard, one preferred
embodiment of the invention contemplates the use of a stepper motor
mechanically engaged with the conveyor belt or web such that each
rotational step of the motor induces movement of the conveyor belt
or web a given travel length increment. The control unit is
programmed to track the steps of the motor, in accordance with
conventional principles, and thereby determine the position of each
substrate along the belt. Optionally, the stepper motor control
system can include a home switch associated with the motor that
will allow the control unit to determine a starting or reference
"home" position.
[0069] Another embodiment contemplates the use of a servo motor
mechanically engaged with the conveyor belt or web such that
rotation of the motor's drive shaft through a given angle induces
movement of the belt or web a known travel length increment. Here,
an encoder monitors the rotation of the motor's shaft and generates
a pulse for each chosen increment of shaft rotation. The encoder is
electrically connected to the control unit which counts or
otherwise tracks the encoder pulses. By monitoring the increments
of shaft rotation in this way, corresponding increments of linear
travel of the conveyor belt can be readily determined.
[0070] Still a further embodiment of position-monitoring means
includes a conventional position detector mounted adjacent the
conveyor so as to have a field of view directed at the transport
pathway. The detector can be, for example, an optic detector, or
the like, operable to generate an output signal when a substrate or
substrate-support region is positioned underneath the transfer
head. The output of the sensor is fed to the input of the control
unit.
[0071] The control unit of the reagent-transfer apparatus serves to
actuate the conveyor motor and shifting means in a sequence
designed for automated operation of the apparatus in forming at
least one reagent region on one or more substrates. In this regard,
the control unit is constructed, according to conventional
microprocessor control principles, to provide appropriate signals
to the shifting means and conveyor motor, in a given timed sequence
and for an appropriate signaling time. The construction of the
unit, and the settings that are selected by the user to achieve a
desired reagent-spot pattern, will be understood from the following
description of a typical apparatus operation.
[0072] In one general embodiment of a spotting method according to
the present invention, a plurality of spaced, tandemly-arranged
substrates are advanced, e.g., by way of a conveyor, along a
transport pathway extending over one or more reagent-supply
locations, such as reservoirs held in a base. From a position over
the reagent-supply location(s) and the pathway, a reagent-transfer
instrument, or tip, is extended along an axis through an
intervening region separating an adjacent pair of advancing
substrates to contact reagent held at the reagent-supply location.
The reagent-transfer instrument is then withdrawn, along with a
portion of such reagent, through the intervening region to a
position above the transport pathway. Next, a selected amount of
reagent is transferred from the reagent-transfer instrument onto a
selected region or site of a selected substrate or substrate region
upstream of the intervening region.
[0073] With primary reference to the embodiment of FIGS. 5A through
5G, a typical operation will now be described wherein a reagent
spot is placed on a substrate--in this case substrate 14b. The
operation is described in connection with an apparatus essentially
as depicted in FIGS. 1, 2 and 4. With transfer tip 46 disposed at
its raised position, the conveyor motor is signaled to advance belt
22, and any substrates thereon, along the transport pathway (toward
the right in the figures) until an open region downstream of the
selected substrate 14b, such as opening 28b, becomes positioned
under transfer tip 46 (i.e., generally aligned with axis A), at
which point the belt is stopped (FIG. 5A). Shifting means 48 is
then signaled to shift the transfer tip away from its raised
position and through opening 28b to contact a reagent 72 held in
base 50 (FIG. 5B). Shifting means 48 then withdraws the tip, along
with a portion of such reagent, through the opening to a position
above the transport pathway (FIG. 5C). The control unit then
signals the conveyor motor to advance belt 22 until a selected
region or site of substrate 14b intersects the transfer head's axis
A, at which point the belt is again stopped (FIG. 5D). Next,
shifting means 48 is signaled to shift the transfer tip away from
its raised position toward the selected region of substrate 14b, to
transfer a selected amount of reagent, e.g., in the form of a spot
74, from the tip to such region of the substrate (FIGS. 5E and 5F).
If desired, the spotting tip can again be shifted, one or more
times, to transfer additional reagent to the substrate. Such
additional reagent can be placed at the already-laid spots, or,
upon incrementally advancing the substrate under the spotting tip,
at previously unspotted regions of the substrate. The just-spotted
substrate can then be transported downstream (FIG. 5G) for
additional spotting at one or more downstream spotting heads, as
desired; and the next selected, upstream substrate can be advanced
for spotting.
[0074] In some cases, it is desired to spot out the reagents in a
humid environment so that the droplets do not dry until the
arraying operation is complete. For similar reasons, low-volatility
solvents are also preferable in such cases.
[0075] The just-described operation contemplates an indexed mode of
operation, wherein the belt stops and starts repeatedly. It should
be appreciated, however, that a continuous mode could be employed
instead. If used in the continuous mode, the control unit controls
the speed of the conveyor motor, and thus the speed at which
substrates are moved along the transport pathway. The control unit
also monitors the positions of the various substrates, and signals
shifting of the various transfer tips in a fashion permitting
reagent retrieval and deposition-- without pausing the movement of
the belt/substrates.
[0076] It should be noted that while only one transfer head is
shown in FIG. 1, a device like 44 can be provided at each of the
spaced locations along the transport pathway having a reagent
reservoir. Each substrate, by this arrangement, can be spotted at
one or more of the spaced locations, as desired, during its
movement along the transport pathway. Also, in this arrangement, a
plurality of spotting operations, such as described above with
regard to substrate 14a, can be carried out substantially
simultaneously. By providing a spotting head at each of the five
reagent-supply locations shown in FIG. 1, for example, a reagent
spot can be placed on each of five tandemly-arranged substrates at
substantially the same time. The illustrated arrangement can be
extended to any desired number of spotting heads. One exemplary
embodiment contemplates the use of 1,000 spotting heads disposed
sequentially at spaced locations along the transport pathway, at
various pre-selected laterally offset positions. By this
arrangement, 1,000 spots can be laid per second, or so, with one
such spot being placed on each of 1,000 substrates along the
transport pathway. In another embodiment, four spotting heads are
disposed laterally or obliquely across the transport pathway (along
with respective reagent reservoirs thereunder) at each of 250
spaced locations--again, in a selected laterally offset pattern.
The various spotting heads at each of the spaced locations can be
operated individually, or in mass. Preferably, each transfer tip is
independent of all other transfer tips and can therefor be adjusted
to accommodate a wide range of spot spacing. This feature reduces
tolerance problems associated with conventional transfer devices
having fixed spatial relationships. By the above arrangements and
methods, a very compact interleaving of reagent spots can be formed
on each substrate's surface--the practical density being limited
only by the volume of liquid transferred and the wetability or
surface feature size of the substrate. Fully arrayed substrates can
be pulled off of the conveyor at the end of the transport pathway
at a relatively rapid rate (e.g., 1 substrate about every 1 to 2
seconds).
[0077] The independent construction of each transfer device, as
provided herein, also allows for the service/replacement of each
transfer device on an individual basis. As mentioned above, each
individual transfer tip can be attached to and detached from the
head, without affecting or otherwise disturbing any other transfer
tip(s) of the apparatus. Conventional transfer schemes that utilize
permanently fixed arrays of transfer devices, on the other hand,
are inherently deficient if one member of the array malfunctions.
In such conventional assemblies, if one of the devices should
require service, repair, or replacement, then the entire array of
transfer devices must be disassembled and/or replaced. Moreover, in
the latter case, any reagents that cannot be retrieved from such
conventional devices must also be discarded and replaced.
[0078] Preferably, the shifting motion (stroke) of each transfer
tip, as taught herein, is kept to a minimum throughout the transfer
operation. That is, at its raised position, the transfer tip clears
the uppermost region of a substrate by only a small distance, e.g.,
less than about 2 mm, and preferably less than about 1 mm.
Similarly, at its lowered position, the transfer tip only enters
the reagent tube to the extent necessary to pick up a desired
amount of reagent. Regarding the latter, reagent levels in each
tube can be monitored (e.g., optically detected) or calculated so
that the control unit can avoid overextending the various transfer
tips. This limited motion saves time and, particularly for liquid
reagents, avoids concentration variability associated with
evaporation.
[0079] With a set of reagent tubes in place along the base, any
suitable cooling fluid (e.g., a gas, or water) can be passed
through the channel of the base to contact the accessible exterior
regions of the reagent reservoirs. This can be useful to discourage
evaporative loss of the reagents.
[0080] After one or more reagent transfer operations have been
carried out, it will sometimes be desired to clean the
reagent-transfer tips so that a new, different set of reagents can
be utilized without substantial risk of cross-contamination. In one
embodiment, the reagent reservoirs (tubes) are removed from the
apertures along the base, and the transfer tips are then shifted to
their lowered positions-- with the reagent-contacting portion of
each tip passed through a respective aperture to a location inside
the base's channel. A suitable cleaning solvent is then flowed
through the channel, thereby cleaning the various transfer tips in
a substantially simultaneous fashion. Optionally, after such a
cleaning operation, a dry, warm gas can be flowed through the
channel to dry the transfer tips.
[0081] Preferably, the tubes, or other reagent reservoirs, that are
supported in the base for supplying the transfer tips with reagents
are also the same containers in which the reagents are stored. For
example, prior to a spotting operation, an operator or robot can
retrieve one or more vessels containing selected reagents from a
storage location (e.g., a file cabinet), then open each vessel and
place it at a respective supply location along the base. Once the
spotting operation has been completed, the vessel can simply be
resealed and returned to its storage location. Thus, each reagent
remains in its own vessel throughout storage and use. This is
contrasted to most conventional schemes where the reagent vessel
must be retrieved and then a portion of its contents transferred to
another vessel or reservoir--with still further manipulations at
the actual point of use. Not only does the present invention
provide for reduced handling of each reagent, saving time, it also
offers reduced reagent loss as compared to most conventional
deposition systems. As just described, reagents that are deposited
onto a substrate are preferably transferred from a storage tube, or
other reservoir, directly onto the surface of a substrate without
the use intermediate containers or lines. After use, the reagent
tube is simply resealed prior to storage. It should be appreciated
that intermediate containers typically waste fluid because of
residues and films that are unavoidably left behind. For
applications requiring expensive reagents, intermediate containers
can waste an unacceptable amount of fluid.
[0082] Certain embodiments of the present invention contemplate the
use of an automated shuttle means (e.g., including robots,
conveyors, etc.) for retrieving selected reagents from storage and
placing each at an appropriate location along the base.
Additionally, the shuttle means can be used to return reagents to
their storage locations after use. For example, while one set of
reagents is in use, the next set of selected reagents can be
retrieved by the shuttle and brought to respective locations along
the transport pathway adjacent their points of use. At an
appropriate time, the shuttle means can remove the just-used set,
place the new set in the base, and return the used set to storage.
These steps can be repeated (cycled), preferably under the
direction of a programmed computing device, as many times as
desired.
[0083] It should be appreciated that any desired substrate(s) can
be used with the present invention, including slides, cards,
plates, trays, chips, membranes, and the like. In one general
embodiment, the substrate surface is relatively hydrophilic, i.e.,
wettable. For example, the surface can have native, bound or
covalently attached charged groups. One such surface is a glass
surface having an absorbed layer of a polycationic polymer, such as
poly-l-lysine. In one embodiment, for example, an aqueous or
predominantly aqueous reagent solution or biological sample is
spotted onto a slide having a hydrophilic surface. In another
embodiment, the substrate surface has or is formed to have a
relatively hydrophobic character, i.e., one that causes aqueous
medium deposited on the surface to bead. A variety of known
hydrophobic polymers, such as polystyrene, polypropylene, or
polyethylene have desired hydrophobic properties, as do a variety
of lubricant or other hydrophobic films that may be applied to the
substrate surface.
[0084] Those skilled in the art can now appreciate from the
foregoing description that the broad teachings of the present
invention can be implemented in a variety of forms. For example,
rather than utilizing a single linear belt or web for advancing
substrates, as illustrated in the drawings, a plurality of
conveyors can be arranged to pass off substrates from one conveyor
to the next. In one such arrangement, two or more conveyors are
arranged end-to-end, with the various conveyors collectively
forming a transport pathway of greater length than any one of them.
Also, a non-linear conveyor can be utilized, e.g., a carousel-type
arrangement, instead of a linear arrangement as depicted in the
drawings. Further, the transport pathway can change direction one
or more times during or between spotting operations, e.g., two
steps forward, two steps back, four steps forward, two steps back,
etc. Therefore, while this invention has been described in
connection with particular embodiments and examples thereof, the
true scope of the invention should not be so limited. Various
changes and modification may be made without departing from the
scope of the invention, as defined by the appended claims.
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