U.S. patent application number 10/990722 was filed with the patent office on 2006-01-19 for bead dispensing system.
This patent application is currently assigned to Applera Corporation. Invention is credited to Dennis Lehto, Charles S. Vann.
Application Number | 20060012130 10/990722 |
Document ID | / |
Family ID | 22948791 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060012130 |
Kind Code |
A1 |
Vann; Charles S. ; et
al. |
January 19, 2006 |
Bead dispensing system
Abstract
A bead dispensing system is provided for delivering small
amounts of substances onto substrates. The system can include, for
example, a movable support structure having an array of
spaced-apart projections depending from its lower side. An
attraction source, such as a vacuum, magnetic, and/or electrostatic
force, is operable at each projection end region to attract and
retain one bead. The projection array can be aligned with an array
of bead-receiving regions of a substrate, e.g., an array of
spaced-apart wells of a micro-plate or card. In one embodiment, a
plurality of reagent-carrying beads are picked up, retained at
respective projection end regions, and moved to a location over a
multi-well plate. The beads are then released in a fashion
permitting each bead to land in a respective well. The system of
the invention is particularly useful for fabricating arrays of
reagents.
Inventors: |
Vann; Charles S.;
(Burlingame, CA) ; Lehto; Dennis; (Santa Clara,
CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.;APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
22948791 |
Appl. No.: |
10/990722 |
Filed: |
November 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10990714 |
Nov 16, 2004 |
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10990722 |
Nov 16, 2004 |
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10603049 |
Jun 24, 2003 |
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10990714 |
Nov 16, 2004 |
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10211131 |
Aug 2, 2002 |
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10603049 |
Jun 24, 2003 |
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09506870 |
Feb 15, 2000 |
6887431 |
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10211131 |
Aug 2, 2002 |
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09250697 |
Feb 16, 1999 |
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09506870 |
Feb 15, 2000 |
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Current U.S.
Class: |
277/595 |
Current CPC
Class: |
B01J 2219/00369
20130101; B01J 2219/00722 20130101; C40B 40/06 20130101; B01J
2219/00702 20130101; C40B 70/00 20130101; B01J 2219/00689 20130101;
B01L 2400/043 20130101; Y10T 436/255 20150115; B01J 2219/00315
20130101; B01J 2219/00545 20130101; G01N 35/10 20130101; G01N
35/1065 20130101; B82Y 30/00 20130101; B01J 2219/00468 20130101;
B01L 3/0289 20130101; B01J 2219/00549 20130101; B01J 2219/00659
20130101; B01L 3/021 20130101; Y10T 436/11 20150115; B01J
2219/00691 20130101; B01J 2219/00585 20130101; B01J 2219/00693
20130101; B01L 3/563 20130101; B01L 2200/0657 20130101; B01L
2400/0487 20130101; B01L 2200/143 20130101; B01J 2219/00317
20130101; B01J 2219/0065 20130101; B01L 2200/0647 20130101; B01L
2200/021 20130101; B01J 2219/005 20130101; B01J 2219/00283
20130101; B01L 2400/049 20130101; Y10T 436/2575 20150115; B01J
2219/00596 20130101; B01L 2300/0864 20130101; C40B 60/14 20130101;
B01J 2219/00677 20130101; B01J 19/0046 20130101; B01L 2300/0654
20130101; B01L 2400/0415 20130101; G01N 2035/00574 20130101; B01J
2219/00648 20130101; B01J 2219/00308 20130101 |
Class at
Publication: |
277/595 |
International
Class: |
F02F 11/00 20060101
F02F011/00 |
Claims
1. A bead-like particle for real time fluorescence-based
measurements of nucleic acid amplification products, comprising:
(i) a reagent core comprising (a) first and second oligonucleotide
primers having sequences effective to hybridize to opposite end
regions of complementary strands of a selected polynucleotide
analyte segment, for amplifying the segment by primer-initiated
polymerase chain reaction, and (b) a fluorescer-quencher
oligonucleotide capable of hybridizing to the analyte segment in a
region downstream of one of the primers, for producing a detectable
fluorescent signal when the analyte is present in the sample; and
(ii) a coating covering said reagent core; wherein said coating is
configured to permit access to the reagent core under controllable
conditions.
2. The particle of claim 1, wherein said coating comprises a
gelatin.
3. The particle of claim 1, wherein said coating is configured to
permit access to the reagent core upon exposure to a particular
solvent.
4. The particle of claim 3, wherein said coating is
dissolvable.
5. The particle of claim 3, wherein said coating is swellable.
6. The particle of claim 1, having a diameter no greater than 3.90
millimeters.
7. The particle of claim 6, having a diameter no greater than one
millimeter.
8. A system for real time fluorescence-based measurements of
nucleic acid amplification products, comprising: a container; and
one or more bead-like particles disposed in said container, said
one or more bead-like particles comprising (i) a reagent core
comprising (a) first and second oligonucleotide primers having
sequences effective to hybridize to opposite end regions of
complementary strands of a selected polynucleotide analyte segment,
for amplifying the segment by primer-initiated polymerase chain
reaction, and (b) a fluorescer-quencher oligonucleotide capable of
hybridizing to the analyte segment in a region downstream of one of
the primers, for producing a detectable fluorescent signal when the
analyte is present in the sample, and (ii) a coating covering said
reagent core; wherein said coating is configured to permit access
to the reagent core under controllable conditions.
9. The system of claim 8, further comprising a tracking number on
said container.
10. The system of claim 9, wherein said tracking number comprises a
machine-readable format.
11. The system of claim 10, wherein said tracking number comprises
a bar code.
12. The system of claim 8, wherein said coating comprises a
gelatin.
13. The system of claim 8, wherein said coating permits access to
the reagent core upon exposure to a particular solvent.
14. The system of claim 13, wherein said coating is
dissolvable.
15. The system of claim 13, wherein said coating is swellable.
16. The system of claim 8, where each of said one or more bead-like
particles has a diameter no greater than 3.90 millimeters.
17. The system of claim 16, where each of said one or more
bead-like particles has a diameter no greater than one
millimeter.
18. A system for real time fluorescence-based measurements of
nucleic acid amplification products, comprising: a microcard
defining a plurality of wells; one or more bead-like particles
disposed in each of said wells, said one or more bead-like
particles comprising (i) a reagent core comprising (a) first and
second oligonucleotide primers having sequences effective to
hybridize to opposite end regions of complementary strands of a
selected polynucleotide analyte segment, for amplifying the segment
by primer-initiated polymerase chain reaction, and (b) a
fluorescer-quencher oligonucleotide capable of hybridizing to the
analyte segment in a region downstream of one of the primers, for
producing a detectable fluorescent signal when the analyte is
present in the sample, and (ii) a coating covering said reagent
core; wherein said coating is configured to permit access to the
reagent core under controllable conditions.
19. The system of claim 18, wherein said coating comprises a
gelatin.
20. The system of claim 18, wherein said coating permits access to
the reagent core upon exposure to a particular solvent.
21. The system of claim 20, wherein said coating is
dissolvable.
22. The system of claim 20, wherein said coating is swellable.
23. The system of claim 18, where each of said one or more
bead-like particles has a diameter no greater than 3.90
millimeters.
24. The system of claim 23, where each of said one or more
bead-like particles has a diameter no greater than one
millimeter.
25. The system of claim 18, wherein each well of said microcard has
a diameter of no greater than 1 millimeter.
26. The system of claim 18, wherein said plurality of wells define
an array.
27. The system of claim 26, wherein said plurality of wells
comprises at least 384 wells.
28. The system of claim 18, wherein said microcard comprises an
upper surface and a lower surface; and further comprising a vacuum
source disposed in fluid communication with the lower surface of
the card.
29. The system of claim 26, wherein said plurality of wells
comprises at least 1,024 wells.
30. A reagent-holding device, comprising: a micro-plate comprising
at least 1,024 wells; wherein each well comprises an upper-end
diameter of no greater than about 1 mm; an analyte-specific reagent
disposed in each of said wells, wherein said reagent comprises
components for real time fluorescence-based measurements of nucleic
acid amplification products; and an optically-clear cover disposed
over, and sealing, said wells.
31. The device of claim 30, further comprising a registration
feature formed in said micro-plate.
32. The device of claim 31, wherein said registration feature
comprises a bore, a slot, or a combination thereof.
33. The device of claim 30, further comprising a bead in each of
said wells, with said reagent being held by said bead.
34. The device of claim 30, wherein said micro-plate comprises a
length of no greater than about 127.8 mm and a width of no greater
than about 85.5 mm.
35. The device of claim 30, further comprising a bar code disposed
on said micro-plate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Application No.
______, filed Nov. 16, 2004 (Case no. 4424C2), which is a
continuation of U.S. application Ser. No. 10/603,049, filed Jun.
24, 2003, which is a continuation of U.S. application Ser. No.
10/211,131, filed Aug. 2, 2002, which is a divisional of U.S.
patent application Ser. No. 09/506,870, filed Feb. 15, 2000, which
is a continuation-in-part of U.S. patent application Ser. No.
09/250,697 filed Feb. 16, 1999, now abandoned, which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the manipulation of small
beads capable of carrying substances such as reagents or samples,
and the like. More particularly, the invention provides a method
and apparatus for fabricating an array of such beads on a
micro-card or plate.
BACKGROUND OF THE INVENTION
[0003] Multi-well arrays have long been popular for separately
performing numerous chemical and/or biological reactions at
substantially the same time. Perhaps the most popular multi-well
format in recent years has been the 96-well microplate. Typically,
several microliters of reagents are placed in each of the 96
reaction wells, per assay. In an effort to decrease reagent costs,
as well as to increase throughput, many laboratory directors are
now moving toward the use of even higher-density plates having very
small wells, such as 384- and 1536-well formats with wells about 1
millimeter in diameter, or smaller. With the higher density well
formats, comes the need for distributing even smaller amounts of
substances (e.g. <1 nL) into extremely compact arrays.
[0004] Most conventional automated micro-volume deposition systems
dispense substances in fluid form, using robotic delivery
assemblies. In a typical system, a robot aspirates fluid into one
or more ejectors, moves a loaded ejector to a well in a micro-card
or plate, and delivers an aliquot of fluid. Commonly used ejectors
include "non-contact" devices, such as ink jet nozzles, and
"contact" devices, such as a pens or quills. Ink jets, pens, and
quills are well-known devices used in a variety of applications.
Unfortunately, for the purpose of depositing numerous substances
into the wells of a micro-card or plate, each of these devices is
associated with certain disadvantages. For example, ink jets
generally work fine when the fluid of interest has been carefully
optimized for the nozzle. However, when depositing many different
fluids through the same nozzle, optimization for each separate
fluid is often impractical. As a result, the nozzles can become
clogged. With regard to pens and quills, these devices can collide
with the well walls, and are generally too slow for cost-effective
operations.
[0005] The task of delivering micro-volumes of fluidic substances
can be especially challenging when the substance deposited at each
location is unique to one or only a few positions in the array.
Further complications can arise when multiple fluidic substances
are serially deposited into each well. For example, liquids can
drip and splatter, contaminating reagents in neighboring wells. As
another disadvantage, all devices contacting a fluid reagent must
be cleaned, or disposed of, before being used with a different
fluidic reagent. This is necessary to prevent mixing (i.e.,
contamination) of one reagent with another. It should be
appreciated that multiple rounds of cleaning and aspiration can be
time consuming and expensive, as well. This is especially true for
applications requiring a large number of different substances. As a
further disadvantage, it is often difficult to control the volume
of fluid dispensed with a high degree of accuracy. Also, small
amounts of dispensed liquid can be difficult to detect with
standard imaging systems. Accordingly, dispensing errors can go
undetected and, thus, uncorrected.
[0006] The need is apparent for an apparatus and process capable of
fabricating an array of substances on a micro-card or plate in a
relatively fast, efficient and accurate manner.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention provides a system for
picking up a plurality of relatively small beads from a supply and
transferring them to a desired location.
[0008] According to one embodiment, the system includes a plurality
of projections, e.g., in the nature of tubes or rods, depending
from a support at spaced-apart locations defining an array. Each
projection has a lower end region, distal from the support, with a
cavity formed therein. Each such cavity is characterized by a lower
opening, an upper ceiling region, and a sidewall extending between
the lower opening and upper ceiling region. An attraction source,
such as a vacuum, electrostatic and/or magnetic force, is operable
at each projection end region in a manner effective to draw beads
from the supply into the cavities and to releasably retain the
beads therein.
[0009] In one embodiment, the sidewall about each cavity is formed,
at least in part, is of a resiliency flexible material, such as
tetrafluoroethylene (TFE) tubing, or the like. The resiliency
flexible sidewall can have, for example, a generally cylindrical or
tubular shape, with both an inner diameter and a longitudinal depth
of less than about 1.5 mm. In an exemplary arrangement, intended
for use with submillimeter beads, the inner diameter and
longitudinal depth are between about 100-1,250 micrometers. In a
particularly preferred construction, especially useful in
connection with substantially spherical microbeads having a
diameter of between about 275-325 micrometers, the inner diameter
and longitudinal depth are between about 350-425 micrometers.
[0010] In an exemplary arrangement, the sidewall about each cavity
has a substantially constant inner diameter along a region
extending between its lower opening and its upper ceiling region,
such that lines extending longitudinally along confronting inner
surfaces of each sidewall are substantially parallel to one
another.
[0011] According to one embodiment, the lower opening of each
cavity has a diameter of between about 100-1,250 micrometers. The
longitudinal length of the sidewall, measured from the lower
opening to the upper ceiling, is preferably between about 0.50-1.25
times the diameter of the lower opening.
[0012] In another embodiment, the lower opening of each cavity has
a diameter of between about 250-750 micrometers, and preferably
between about 350-425 micrometers. Further in this embodiment, the
longitudinal length of the measured from the lower opening to the
upper ceiling, is between about 0.75-1.10 times the diameter of the
lower opening. In one particularly preferred embodiment, the
diameter of the lower opening and the longitudinal length of the
sidewall are approximately equal.
[0013] In one embodiment, the cavity at the lower end region of
each projection is configured to receive between about 0.50-1.25
beads, and preferably between about 0.75-1.10 beads. In a
particularly preferred embodiment, each cavity can receive one
entire bead. The cavities, in this preferred construction, are
sized to preclude entry of a substantial portion (e.g., >20%) of
a second bead once a first bead has become situated therein.
[0014] The system of the present invention can further include a
plurality of ampules for containing the bead supply. In a preferred
arrangement, a plurality of plastic ampules are disposed in an
array alignable with the projection array. Each ampule can be
provided with a cover member configured to extend over an upper
opening thereof. The covers can be, for example, plastic domes or
caps, and/or frangible polymeric membranes (films).
[0015] The support from which the projections depend can include,
or be attached to, a frame adapted to pivot about a generally
vertical axis, rendering the projection array movable along a
generally arcuate or circular pathway. The frame can further be
adapted for reciprocal linear motion along a generally vertical
pathway. By this construction, the projections can be aligned over
the ampule array, and each projection can be lowered into a
respective one of the ampules.
[0016] The ampules can hold any desired reagents. In one
embodiment, for example, each ampule holds beads carrying two
primers, two probes and buffer for use in real-time PCR. It should
be appreciated that the reagents in the various ampules can be the
same or different. In one embodiment, one of the ampules holds a
plurality of submillimeter beads that carry a first set of
analyte-specific reagents, and another of the ampules holds a
plurality of submillimeter beads that carry a second set of
analyte-specific reagents. The first and second reagent sets can
differ from one another, for example, by at least one
analyte-specific component.
[0017] The cavities at the lower end regions of the projections can
be formed, for example, by fitting a resiliency flexible, tubular
sleeve, or sheath, over their free ends such that an overhang
region is left extending below the terminal end of each projection.
In this construction, the overhand region can define the sidewall
laterals bounding each cavity and the terminal end of each
projection, facing the cavity, can define the upper ceiling
region.
[0018] According to one embodiment, each of the projections is a
capillary tube having an axial lumen extending therethrough. Each
lumen, in this embodiment, is provided with a first end that opens
into a respective one of the cavities through its ceiling region,
and a second end disposed in fluid communication with a
pressure-control assembly. The pressure-control assembly can
include, for example, a vacuum pump operable to establish a reduced
pressure within each of the lumens, and/or a pump operable to
establish an increased pressure within each of the lumens.
Regarding the latter, such an increased pressure can be utilized to
displace (blow out) any beads retained in the cavities.
[0019] In one preferred construction, each lumen is formed with an
inner diameter at its first end that is smaller than the diameter
of a respective cavity into which it opens. For example, each
cavity can have an inner diameter, at a location directly adjacent
its ceiling region, of greater than 275 micrometers (e.g., between
about 300-400 micrometers), and each of the lumens can have an
inner diameter at its first end of between about 100-275
micrometers.
[0020] A detection system can be used to sense the presence or
absence of a bead retained in the various cavities. One embodiment,
for example, provides a detection system having a field of view
extending along each of the projection end regions. In one
particular arrangement, the detection system includes a plurality
of elongated light-conductive (optical) fibers. One end of each
fiber, in this arrangement, extends along one of the projections
and faces a respective cavity. The other end of each fiber can be
disposed in communication with a camera device, such as a CCD
camera.
[0021] The system of the present invention can further include a
conduit assembly having a plurality of conduits for separately
guiding or funneling a plurality of beads released from the various
cavities to desired locations on a substrate (e.g., into wells of a
micro-plate or card). In one embodiment, the conduits have (i)
large openings at their upper ends disposed in an array having a
center-to-center pitch substantially like that of the projection
array such that the large openings are generally alignable
thereunder, and (ii) small openings at their lower ends.
[0022] The small openings can be disposed in an array having a
center-to-center pitch substantially like that of the large-opening
array, or the two arrays can differ in pitch. In one embodiment,
for example, the small openings are disposed in an array having a
center-to-center pitch substantially smaller than that of the
large-opening array. In one particular arrangement, the
center-to-center pitch of the small-opening array is reduced by a
factor of at least about 2, and preferably at least about 3, as
compared to that of the large-opening array.
[0023] In one embodiment, the substrate onto which the beads are
deposited is a micro-plate or card having a plurality of wells
disposed in an array alignable under the small-opening array of the
conduit assembly. For example, the substrate can be a plastic
96-well plate. The plate can be of standard dimensions, or custom
dimensions. For example, the plate can have an 8.times.12 regular
rectangular array of wells, with each well having a diameter, at
its upper end, of about 1 mm.
[0024] The system can further include a detection system operable
to sense the presence or absence of a bead in each well of a
micro-plate or card. In one embodiment, for example, a detection
system is utilized having a field of view extending through each of
the conduits and down onto the substrate.
[0025] Another aspect of the present invention provides a system
for channeling a plurality of beads to desired locations on a
substrate, such as a micro-plate or card.
[0026] According to one embodiment, the system includes an array of
bead supports, such as the previously described projections, with
each support being adapted to releasably hold, from above, no more
than one bead. A plurality of conduits are disposed under the
micro-bead support array. The conduits are provided with (i) large
openings at their upper ends disposed in an array having a
center-to-center pitch substantially like that of the support array
such that the large openings are generally alignable thereunder,
and (ii) small openings at their lower ends.
[0027] In one embodiment, each of the large openings of the conduit
assembly has a diameter of greater than about 1 mm (e.g., between
1-6 mm), and each of the small openings has a diameter of less than
about 1 mm (e.g., between 0.15-1 mm).
[0028] The small openings can be disposed in an array having a
center-to-center pitch substantially like that of the large-opening
array, or the two arrays can differ. In one embodiment, the small
openings of the conduit assembly are disposed in an array having a
center-to-center pitch substantially smaller than that of the
large-opening array. In an exemplary arrangement, the
center-to-center pitch of the small-opening array is reduced by a
factor of at least about 2, and preferably at least about 3, as
compared to that of the large-opening array. In another
arrangement, the center-to-center pitch of the large-opening array
is greater than about 3 mm (e.g., between about 3-9 mm), and the
center-to-center pitch of the small-opening array is less than
about 3 mm (e.g., about 1-3 mm).
[0029] A parallelogram linkage assembly can be used to support the
conduit assembly for reciprocal arcuate movement between a raised
position, above and vertically offset from a substrate, and a
lowered position, directly over and in close proximity to a
substrate. In one such embodiment, a parallelogram linkage assembly
is supported on a carousel surface for pivotal movement radially of
the carousel's rotational axis. A substrate holding area is also
provided on the carousel surface, radially outward of and adjacent
to the parallelogram linkage assembly. Further in this embodiment,
a stationary rail extends along an inner region of the carousel,
having a bearing surface in mechanical communication with the
parallelogram linkage arrangement. The construction is arranged to
permit the parallelogram linkage assembly to ride along the rail as
rotational movement of the carousel advances it. In a preferred
arrangement, the bearing surface includes a first arcuate region
disposed a first distance from the central axis at a first vertical
height, and a second arcuate region disposed a second distance from
the central axis at a second vertical height. In this arrangement,
the second distance is shorter than the first distance, and the
second vertical height is higher than the first vertical height.
When the parallelogram linkage arrangement rides along the first
arcuate region, the conduit assembly assumes the lowered position
over the substrate holding area. When the parallelogram linkage
arrangement rides along the second arcuate region, on the other
hand, the conduit array assumes the raised position. The bearing
surface can also include transition regions bridging the first and
second arcuate regions.
[0030] According to one embodiment, the substrate is a micro-plate
or card having a plurality of wells disposed in an array alignable
under the small-opening array of the conduit assembly. For example,
the substrate can be a plate having 96 wells, or more. The
micro-plate or card can include a pair of spaced-apart indexing
bores and/or slots (holes), with each being configured for
alignment with a respective indexing pin depending from a lower
side of the conduit assembly. Upon inserting the indexing pins into
the indexing bores or slots, the conduit assembly's small-opening
array becomes substantially aligned with the substrate's array of
wells.
[0031] In one embodiment, a detection system is provided having a
field of view extending into each of the conduits of the conduit
assembly. The detection system, in this embodiment, is adapted to
sense the presence or absence of a bead on the micro-plate or card
under each of the small openings. In an exemplary arrangement, the
detection system includes a radiation source, such as a laser,
adapted to illuminate the micro-plate or card at locations, e.g.,
wells, below each of the small openings. The system can further
include a plurality of elongated light-conductive (optical) fibers,
with each fiber having one end facing, or extending into, a
respective one of the large openings to receive light traveling up
through a respective conduit and a second end communicating with a
camera device, such as a CCD camera.
[0032] In another of its aspects, the present invention provides a
system for covering an array of wells formed in a micro-plate or
card.
[0033] According to one embodiment, the system includes a web of an
optically clear cover material mounted for movement from a supply
reel to a take-up reel. Shearing blades are mounted for reciprocal
linear motion along a direction substantially normal to the web for
cutting out a portion of the cover material at a region between the
supply and take-up reels. A resiliency compliant, generally planar
surface is provided between the blades for pressing the web against
the upper surface of the micro-card, over the wells.
[0034] In one embodiment, a pair of indexing pins extends below the
compliant surface. Further in this embodiment, the micro-card is
provided with a pair of spaced-apart indexing bores or slots, with
each being alignable with one of the indexing pins. Registration of
the indexing pins with the bores or slots serves to orient the
shearing blade over the micro-card for effecting a cut in the web.
In a related embodiment, the shearing blade has a cutting edge
defining a quadrilateral, such as a square or rectangle. Upon
registering the indexing pins with the indexing bores, a cut can be
made in the web having (i) two sides substantially parallel to the
side edges of the web and (ii) two sides substantially normal to
the side edges of the web. Preferably, the web has a side-to-side
width that is longer than the cut along the direction normal to the
side edges of the web, so that the web is not severed in two upon
making the cut.
[0035] A further aspect of the present invention provides an
apparatus for delivering a substance (e.g., a liquid solvent or
reagent) onto a substrate.
[0036] In accordance with one embodiment, the apparatus includes a
plurality of elongated conduits disposed in fixed, spaced relation
in a common support structure. The support structure can be, for
example, a block, tray, plate, frame or the like. In one
embodiment, the support structure is made substantially of glass.
Each of the conduits is provided with a large opening at one end
and a small opening at its other end. Between its two ends, each
conduit can have a tapered or funnel-shaped region. The large
openings are disposed in an array along one side of the support
structure, and the small openings are disposed in an array along an
opposite side of the support structure. The large- and
small-opening arrays can be arranged with a similar pitch
(center-to-center spacing), or they can differ. In one embodiment,
the large-opening array is arranged with a center-to-center pitch
substantially larger (e.g., greater than about 2:1, and preferably
greater than about 3:1) than that of the small-opening array.
[0037] In one preferred arrangement, a region of each conduit
extending from a respective one of the small openings is of
capillary size, such that a liquid placed in contact with the
small-opening array can be drawn at least partially into each
conduit. For example, the capillary-size region of each conduit can
have an inner diameter of less than about 1 mm. In one embodiment,
the capillary-size region of each conduit is provided with an inner
sidewall that is hydrophilic.
[0038] In yet another of its aspects, the present invention
provides a method for simultaneously transferring a plurality of
beads from one location to another location. According to one
embodiment, the method includes the steps of: (!) picking up, in a
substantially simultaneous fashion, a plurality of beads from a
supply and retaining the beads at respective, spaced-apart
locations defining an array; [0039] (ii) releasing the beads, in a
substantially simultaneous fashion, over a substrate having an
array of separate bead-holding regions; and [0040] (iii)
individually channeling or guiding each of the released beads, in a
substantially simultaneous fashion, to a respective one of the
bead-holding regions.
[0041] The step of picking up beads can be effected, for example,
by establishing a [0042] reduced pressure (vacuum) at each of the
locations, and the step of releasing the beads can be effected by
establishing an increased pressure (positive gas flow) at each of
the locations.
[0043] In one embodiment, the array of bead-holding regions (e.g.,
wells of a micro-plate or card) has a center-to-center pitch
substantially smaller than that of the retained-bead array. In an
exemplary arrangement, the center-to-center pitch of the array of
bead-holding regions is reduced by a factor of at least about 2,
and preferably at least about 3, as compared to that of the
retained-bead array.
[0044] According to one embodiment the bead supply is provided in
an array of plastic capsules or ampules. A cover can be provided
over an upper opening of each ampule. The covers can be removed, or
punctured, to provide access to the beads. Suitable covers can
include, for example, polymeric film-like membranes, or plastic
caps/domes.
[0045] One embodiment provides, between step (i) and step (ii), a
step of inspecting each location of the retained-bead array for the
presence of a bead; and picking up and retaining a bead for
locations lacking a bead. After step (iii), each of the
bead-holding regions can be inspected for beads deposited
therein.
[0046] In one embodiment, each of the bead-holding regions can be
covered with an optically clear film, or the like. The film can be
sealed over the bead-holding regions using adhesives and/or
heat-sealing techniques.
[0047] These and other features and advantages of the present
invention will become clear from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] 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:
[0049] FIG. 1 is a partially schematic side elevational view, with
portions shown in phantom, of a system for fabricating an array of
reagent-carrying beads on a substrate, constructed in accordance
with one embodiment of the present invention.
[0050] FIG. 2 is a side elevational view, with portions shown in
phantom, of an ampule containing a plurality of reagent-carrying
beads, and having a dome-like cover member over an upper opening,
according to an embodiment of the present invention.
[0051] FIG. 3(A) is a side elevational view, with portions shown in
phantom, of an ampule containing a plurality of reagent-carrying
beads, and having a film-like cover member over an upper opening,
according to a further embodiment of the present invention.
[0052] FIG. 3(B) is a side elevational view of the ampule of FIG.
3(A). further showing an elongated projection, in the nature of a
tube, that has punctured the cover member to gain access to the
beads contained therein, as taught by an embodiment of the present
invention.
[0053] FIG. 4(A) is a side elevational view, with a portion broken
away and others shown in phantom, of an elongated projection, in
the nature of a tube, for use in attracting and retaining
reagent-carrying beads, according to an embodiment of the present
invention.
[0054] FIG. 4(B) is a side elevational view of the projection shown
in FIG. 4(A), further illustrating a bead disposed in a cavity at
the lower end region of the projection.
[0055] FIG. 5 is a side cross-sectional view of one preferred
construction for a projection useful in attracting and retaining
reagent-carrying beads, as taught by an embodiment of the present
invention.
[0056] FIG. 6 is a partial view of the system depicted in FIG. 1,
illustrating the simultaneous removal of a plurality of dome-like
cover members from a like number of ampules containing a bead
supply, according to an embodiment of the present invention.
[0057] FIG. 7 is a side elevational view, with portions shown in
phantom, of a projection, in the nature of an elongated tube,
having a resiliency flexible lower end region that can bend when
pressed against the bottom region of an ampule, in accordance with
an embodiment of the present invention.
[0058] FIG. 8A is a side cross-sectional view of an array of
projections, like that of FIG. 1, with each projection having an
optical fiber extending therethrough for determining the presence
of a bead in a respective cavity at its lower end region, according
to an embodiment of the present invention.
[0059] FIG. 8B a top view of a horizontal crossbar and bead
extractors of the system shown in FIG. 1, with portions of the bead
extractors broken away in the phantom-depicted positions to
illustrate cameras employed to capture an image of the lower end of
a projection array for determining the presence or absence of a
target object, according to an embodiment of the invention.
[0060] FIG. 8C is a schematic view of a display device, which may
be used in connection with the cameras shown in FIG. 8B, for
displaying an image of the lower end of a projection array.
[0061] FIG. 9 is a partially schematic side elevational view, with
portions shown in phantom, of a system for fabricating a
micro-array of reagent-carrying beads on a substrate, constructed
in accordance with an embodiment of the present invention.
[0062] FIG. 10(A) is a side cross-sectional view of a conduit
assembly having an array of conduits configured for separately
directing a plurality of beads into the wells of a micro-plate or
card, in accordance with an embodiment of the present
invention.
[0063] FIG. 10(B) is a top plan view of the conduit assembly of
FIG. 10(A), showing an array of large openings of the conduits.
[0064] FIG. 10(C) is a plan view from beneath the conduit assembly
of FIG. 10(A), showing an array of small openings of the conduits,
as well as a pair of indexing pins on either side of the
small-opening array.
[0065] FIG. 11 is a partially schematic side elevational view, with
portions depicted in phantom, of a parallelogram linkage assembly
for reciprocally moving a conduit assembly between a raised
position, shown in dashed lines, and a lowered position over a
bead-receiving substrate, shown in solid lines, in accordance with
an embodiment of the present invention.
[0066] FIG. 12(A) is a top plan view of a multi-well micro-card
having an indexing bore formed along one side and an indexing slot
formed along an opposing side, according to the teachings of the
present invention.
[0067] FIG. 12(B) shows, in side-sectional view, the multi-well
card of FIG. 12(A) resting on a substrate-holding area, and a pair
of indexing pins extending from a conduit assembly passed through
the bore and slot of the card, in accordance with an embodiment of
the present invention.
[0068] FIG. 13 is a perspective view of a high-throughput system
for fabricating an array of beads on a micro-plate or card,
according to one preferred embodiment of the present invention.
[0069] FIG. 14 is a partially schematic side-sectional view of a
detection system for determining the presence of beads at
bead-receiving locations of a substrate, in accordance with an
embodiment of the present invention.
[0070] FIG. 15 is a partially schematic side-sectional view of a
detection system, similar to that of FIG. 14, further including an
array of optical fibers configured to extend down into the conduits
of a conduit assembly positioned over a bead-receiving substrate,
according to a further embodiment of the present invention.
[0071] FIG. 16(A) is a partially schematic side-sectional view of a
system for covering an array of wells formed in a substrate, such
as a micro-plate or card, with a film-like cover material,
according to an embodiment of the present invention.
[0072] FIG. 16(B) is a top plan view of a length of cover material
on a take-up reel of the device of FIG. 16(A), showing areas that
have been cut out for use in covering the wells of substrates, in
accordance with the teachings of the present invention.
[0073] FIG. 17(A) is a side elevational view, with portions shown
in phantom, of a hand-operable system for fabricating an array of
reagent-carrying beads on a multi-well plate, constructed in
accordance with an embodiment of the present invention.
[0074] FIG. 17(B) is a top plan view, with portions shown in
phantom, of the hand-operable system of FIG. 17(A).
[0075] FIG. 18 is a side cross-sectional view of a fluid
distributor having an array of conduits for transferring a liquid
from a vessel to an array of wells of a micro-plate or card,
according to an embodiment of the present invention.
[0076] FIG. 19(A) is a perspective view showing an array of small
openings, surrounded by a channel, formed in one side of the fluid
distributor of FIG. 18.
[0077] FIG. 19(6) is a perspective view showing an array of large
openings, surrounded by an upstanding peripheral wall, formed on
one side of the fluid distributor of FIG. 18.
[0078] FIG. 20 is a side cross-sectional view of the fluid
distributor of FIGS. 18-19 disposed over a vessel containing a
liquid, and showing portions of the liquid drawn partially into the
conduits of the fluid distributor by capillary action, in
accordance with the teachings of the present invention.
[0079] FIG. 21 (A) is a side cross-sectional view of the fluid
distributor of FIGS. 18-20 inverted over a multi-well plate,
showing aliquots of liquid that have traveled downward through
respective conduits to form drops at each of the large openings,
over respective bead-containing wells of the plate.
[0080] FIG. 21 (B) is a perspective view of the multi-well plate
shown under the fluid distributor in FIG. 21 (A).
[0081] FIG. 22 is a side cross-sectional view of the inverted fluid
distributor of FIG. 21 (A), with the drops having been pulled into
the wells of the multi-well plate due to adhesive forces with the
beads.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0082] 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.
[0083] One aspect of the present invention provides a system for
picking up a plurality of small, reagent-carrying beads from a
supply or source area and transferring them onto a substrate, e.g.,
wells in a micro-card or plate. Generally, the system includes a
plurality of projections depending from a movable support structure
at fixed, spaced-apart locations. A cavity is provided at a lower
end region of each of the projections, defined by a (i) lower
opening, (ii) an upper ceiling, and (iii) a sidewall extending
between the lower opening and upper ceiling. An attraction source
is operable at each of the projection end regions in a manner
effective to draw individual beads from the supply into respective
cavities and to releasably retain them therein. While retained in
the cavities, the beads can be moved from one place to another with
movement of the support structure. Once the beads have been
positioned at a desired location, they can be released from the
cavities. A plurality of conduits can be used to separately guide
or channel the beads, once released from the cavities, to desired
locations on the substrate.
[0084] More particularly, and with initial reference to the
exemplary embodiment of FIG. 1, a reagent supply is indicated at
12, having a plurality of spaced-apart reagent-supply locations,
arranged in an array. Each reagent-supply location is defined by a
welt, such as 16a-16f, of a reagent plate 20. While only six such
locations, arranged side-by-side in a linear fashion, are visible
in the view of FIG. 1, it should be understood that any reasonable
number of supply locations can be disposed in any desired spatial
configuration. For example, a reagent plate, like plate 20, can
include 24, 48, 96, 384, 1024, 1536 wells, or more, with each well
being configured to support a plurality of reagent-carrying beads.
In such arrangements, the wells will typically be arranged in a
regular array, e.g., an 8.times.12, 16.times.24, 32.times.32, or a
32.times.48 rectangular array, though other layouts are possible.
As indicated above, each reagent-supply location 16a-16f can hold a
plurality of beads, such as 24a-24f, respectively. Each bead, in
turn, can carry a desired reagent.
[0085] A wide variety of reagent-carrying beads can be used with
the present invention. Generally, the beads should resist
substantial physical deformations when exposed for a relatively
short time to moderately stressful conditions, e.g., being pulled
upon by an attractive force such as a vacuum, or a magnetic or
electrostatic field, as discussed more fully below. Certain
embodiments, for example, contemplate the use of beads having a
substantially rigid outer shell, or a soft gelatinous coating.
Several exemplary types of beads are described next.
[0086] In one embodiment, the beads are formed by applying a
coating material, such as a gelatin, to a reagent core. The coating
cures to form a substantially solid shell about the reagent. The
coating can be dissolvable or swellable to permit access to the
reagent under controllable conditions (e.g., upon exposure to a
particular solvent). Guidance for preparing coated beads, or
micro-particles, is provided, for example, in: [1] R. Pommersheim,
H. Lowe, V. Hessel, W. Ehrfeld (1998), "Immobilation of living
cells and enzymes by encapsulation," Institut fur Mikrotechnik
Mainz GmbH, IBC Global Conferences Limited; [2] F. Lim A. Sun
(1980), Science 210, 908; [3] R. Pommersheim, J Schrezenmeir, W.
Vogt (1994), "Immobilization of enzymes and living cells by
multilayer microcapsules" Macromol Chem. Phys 195, 1557-1567; and
[4] W. Ehrfeld, V. Hessel, H. Lehr, "Microreactors for Chemical
Synthesis and Biotechtechnology-Current Developments and Future
Applications" in: Topics in Current Chemistry 194, A. Manz, H.
Becker, Microsystem Technology in Chemistry and Life Science,
Springer Verlag, Berlin Heidelberg (1998), 233-252; each expressly
incorporated herein by reference.
[0087] In another embodiment, a plurality of bead-like particles
act as solid supports for the reagents. For example, reagents can
be synthesized on the beads, or absorbed thereto. In still a
further embodiment, a slurry or dispersion comprised of a reagent
and binding material is used to form a plurality of bead-like
particles, with each individual bead having a substantially
homogenous consistency. Methods for preparing such beads are well
known to those skilled in the art.
[0088] A plurality of different reagents can be formed into
respective collections or groups of reagent beads, referred to
herein as "lots." For example, 10,000 different reagents can be
formed into 10,000 different bead lots, with each lot comprised of
a plurality of substantially like beads carrying a respective
reagent. To assist in distinguishing beads from different lots, and
to provide a means for quickly determining the type of reagent
carried by any one particular bead, beads from each lot can be
formed to display a particular, pre-assigned color. For example,
yellow beads can carry reagent "A," blue beads can carry reagent
"B," and red beads can carry reagent "C." Beads from each lot can
be placed at respective reagent-supply locations.
[0089] In one embodiment, a plurality of bead lots are formed,
wherein each bead includes a reagent core covered with a coating
material, such as a gelatin, having well-defined physical and
chemical properties. Preferably in this embodiment, all beads in
all lots bear substantially the same outer coating (i.e., a
"generic" coating), with the coatings for each lot differing only
in color, as discussed above. It should be appreciated that this
arrangement reduces the risk of equipment contamination due to
contact with the reagents themselves. If any residues are left
behind as the reagents move through the system, such residues will
all be of the same, known coating material. Preferably, the coating
material is chosen so that any residues are innocuous to the
system. It should further be appreciated that a higher speed for
depositing substances can be achieved using such beads, as compared
to conventional liquid deposition systems, because the hardware
delivering the beads will not require frequent cleaning, nor is
time spent aspirating fluids.
[0090] While beads of substantially any shape can be used with the
present invention, beads having a generally spherical geometry are
particularly well suited for use herein. Also, the system of the
invention can be used with beads of various sizes. For example, one
embodiment contemplates the use of spherical beads having a
diameter of less than about 1 mm. In one such arrangement, each
bead is formed with a diameter of between about 275-325
micrometers, and most preferably about 300 micrometers. In another
embodiment, the beads are larger, such that each bead substantially
fills one well of the reagent plate. For example, each bead can
have a diameter of between about 1.0-4.0 mm, and preferably about
3.7 mm. Each well of the reagent plate, in turn, can be configured
with an inner diameter slightly larger than the diameter of a bead.
The lower end of each well, in this embodiment, can be shaped to
complement the contour of the bead's outer surface. The beads can
carry any desired reagent. As used herein, the term "reagent" can
refer to a single substance, or a grouping of substances. According
to one preferred embodiment, the reagent carried by each bead
includes components useful for real time fluorescence-based
measurements of nucleic acid amplification products (such as PCR)
as described, for example, in PCT Publication WO 95/30139 and U.S.
patent application Ser. No. 08/235,411, each of which is expressly
incorporated herein by reference.
[0091] In an exemplary arrangement, each bead carries an
analyte-specific reagent effective to react with a selected analyte
that may be present in a sample. For example, for polynucleotide
analytes, the analyte-specific reagent can include first and second
oligonucleotide primers having sequences effective to hybridize to
opposite end regions of complementary strands of a selected
polynucleotide analyte segment, for amplifying the segment by
primer-initiated polymerase chain reaction. The analyte-specific
detection reagent can further include a fluorescer-quencher
oligonucleotide capable of hybridizing to the analyte segment in a
region downstream of one of the primers, for producing a detectable
fluorescent signal when the analyte is present in the sample.
[0092] A plurality of beads from the same or substantially
identical lots can be packed into an ampule or capsule. A plurality
of such ampules (e.g., tens, hundreds, thousands, or more) can be
dispensed into respective, designated wells of a multi-well plate,
with the various ampules containing the same or different reagents,
as desired. As depicted in FIG. 1, for example, a plurality of
plastic ampules 26a-26f, each holding beads carrying a different
reagent, are removably seated in respective wells, 16a-f, of
reagent plate 20. Ampules of any desired size and shape can be
used. One arrangement, for example, contemplates the use of
generally bullet-shaped ampules having an open top and a rounded,
closed bottom. Exemplary dimensions for each ampule are as follows:
(i) about 10 mm in height, (ii) an outer diameter of about 3.7 mm,
and (iii) an inner diameter of about 3.0 mm. About 1,000
substantially spherical reagent beads, each having a diameter of
about 300 micrometers, can be loaded into each such ampule. An
exemplary reagent plate, useful for holding the just-described
ampules, is configured with an array of wells (e.g., an 8.times.12,
16.times.24, or 32.times.32 array), with each well having an inner
diameter of about 4.0 mm and a depth of between about 6-9 mm.
[0093] The ampules can be placed in the wells of the reagent plate
in any suitable manner. In one embodiment, an operator manually
places the ampules in the wells. In another embodiment, the ampules
are serially dispensed from respective containers supported in an
array of holding cells disposed above the reagent plate, as taught
in U.S. application Ser. No. 09/251,232 filed Feb. 16, 1999 and
expressly incorporated herein by reference.
[0094] Each ampule can be provided with a cover member over an
upper opening thereof. The cover member can be, for example, a
removable cap or dome, such as 30a of FIG. 2, having an open end
32a configured to fit snugly about an opening defined by an upper
rim, or lip, 28a of an ampule 26a. Or, a sheet-like film or
membrane, such as 34a of FIG. 3(A), can be applied to an upper rim
28a about the opening of an ampule 26a. For example, a polymeric
film, such as a polystyrene, polyester, polypropylene or
polyethylene film, between about 0.05-0.40 millimeters thick, can
cover the upper opening of each ampule. In one embodiment, the
cover is a thin polyvinylidene chloride (PVDC) film, such as that
sold under the trade name SARAN WRAP by Dow Chemical Co., (Midland,
Mich.).
[0095] As described more fully below, access to the beads can be
gained, for example, by displacing the covers using the projection
array.
[0096] In one embodiment, the cover member over each ampule forms a
substantially airtight seal, sequestering the contents of the
ampule from the external atmosphere. The seal can be effected or
enhanced, for example, using conventional adhesives, elastomers
and/or by heating-sealing techniques. In an exemplary arrangement,
frictional engagement between a plastic dome-shaped cap and the
upper region of a plastic ampule is sufficient to provide an
airtight seal. The sealed ampules can further contain an inert gas,
such as nitrogen or the like, surrounding the beads.
[0097] In another embodiment, each cover member is primarily
designed to prevent against spillage, or other loss, of a
respective ampule's contents. In this embodiment, the interface
between each cover member and its respective ampule is not
necessarily airtight.
[0098] To prevent against inadvertent dislodgment of the seated
ampules, means can be provided for holding the ampules in place at
each reagent-supply location. For example, FIG. 1 shows a vacuum
manifold, indicated generally at 40, formed in plate 20 under wells
16a-16f. Manifold 40 includes a plurality of generally vertical
passageways that communicate a central chamber of the manifold with
the bottom regions of respective wells 16a-16f. It should be noted
that the uppermost end of each vertical passageway, which opens
into a respective well, has a smaller diameter than the outer
diameter of a seated ampule. A pressure-control source such as
vacuum pump 44, is disposed in fluid communication with a lower
region of manifold 40 by way of a connector line, denoted as 46.
Upon activating pressure-control source 44, a reduced pressure can
be established at the bottom region of each well 16a-16f. The
reduced pressure is effective to draw upon the bottom of each
seated ampule 26a-26f, thereby preventing disiodgment from the
wells. Upon deactivating pressure-control source 44, the ampules
can be readily removed from the wells, if desired.
[0099] Instead of loading the beads into an ampule or capsule,
which is then placed at a reagent-supply location, one embodiment
contemplates placement of the beads directly into the wells, or
other holding areas, of a reagent plate or the like. The wells of
the reagent plate, in this embodiment, can be formed with fully
closed bottoms.
[0100] With continuing reference to the embodiment of FIG. 1, a
pair of bead extractors, denoted generally by the reference
numerals 50a and 50b, are adapted for positioning at respective
locations that are elevated with respect to reagent plate 20. Each
extractor 50a, 50b includes a plurality of projections, such as
54a-54f and 54g-541, depending from a respective support structure,
58a and 58b, at fixed, spaced-apart locations. Each projection can
be shaped, for example, as an elongated nub, tube, rod, or the
like, extending from the support. Preferably, the longitudinal axes
of projections 54a-54f, 54g-541 are disposed such that they are
generally parallel to one another.
[0101] Projections 54a-54f, 54g-541 can be formed integrally with
their respective support structures 58a, 58b, or they can be formed
separately and attached by any suitable means. According to one
embodiment, for example, a plurality of separately-formed
projections bear threads at one end for mating engagement with
respective threaded bores extending into the lower side of a
respective support. Together, each group of projections 54a-54f,
54g-541 defines a respective projection array, denoted as 56a and
56b. Each projection array 56a, 56b is configured with
substantially the same center-to-center spacing as the array of
reagent-supply locations (wells) 16a-16f in reagent plate 20.
Preferably, each projection array 56a, 56b includes as many
projections as the reagent plate has reagent-supply locations, or a
substantial fraction thereof. By this construction, each projection
array 56a, 56b is alignable with locations of the supply.
[0102] A substantially T-shaped frame, denoted generally as 60,
supports both extractors 50a, 50b. More particularly, frame 60
includes an upper, horizontal crossbar, having co-linear arm
portions 60a, 60b. The upper side of each support structure 58a,
58b is rigidly attached to an outer end region of a respective arm
60a, 60b. A rotation motor, depicted schematically at 64, under the
direction of a control computer (not shown), is adapted to rotate a
central vertical shaft 60c of frame 60 about its longitudinal axis,
thereby rendering movable the projection arrays 56a, 56b along
arcuate or circular pathways. In addition, extractors 50a, 50b are
adapted for reciprocal linear motion along respective vertical
pathways. Such movement can be effected, for example, by way of a
linear motor, as at 66, operatively arranged to move shaft 60c up
and down along its longitudinal axis. As with rotation motor 64,
linear motor 66 is preferably adapted for operation under the
direction of a control computer.
[0103] The range of motion, just described, permits a number of
operations, such as (i) aligning a projection array with the ampule
array, (ii) lowering the projections into respective ampules to
attract and retain reagent beads, (iii) lifting retained beads
above the ampule array, and (iv) transferring the retained beads to
a desired location. Such operations are set out in greater detail
below.
[0104] It should be appreciated that any reasonable number of
extractors can be employed. For example, instead of using two
extractors, as described above, one embodiment of the invention
includes only a single extractor. Further embodiments, on the other
hand, contemplate the use of several (e.g., 3, 4, 5, 6, or more)
extractors. The number of crossbar or arm assemblies for the
supportive frame, in these embodiments, will be determined by the
number of extractors included in the system.
[0105] With reference to the enlarged view of projection 54a shown
in FIG. 4(A), a cavity, denoted as 70, is provided at the
projection's lower end region. In this embodiment, cavity 70 is
defined by (i) a lower opening formed by a terminal rim or lip 72,
(ii) an upper ceiling region 74, and (iii) a sidewall 76 extending
between the lower opening and upper ceiling. The composite of these
elements is generally that of an inverted cup-like structure.
[0106] Cavity 70 can be constructed, for example, by forming an
axial bore into one end of an elongated rod or tube. The rod or
tube can be of any suitable material, such as plastic, glass,
aluminum, and the like. In one embodiment, a bore is formed in a
polyurethane tube. The bore can be formed in any manner, e.g.,
drilling with a bit having a diameter sufficiently smaller than the
outer diameter of the tube or rod.
[0107] Where a tube already having a longitudinally extending bore
or lumen, such as at 86a in FIG. 4(A), is used, cavity 70 can be
formed by drilling a counterbore into an end of the tube, with the
counterbore having a diameter greater than the inner diameter of
the lumen 86a, yet smaller than the outer diameter of the tube.
[0108] The cavity at the end region of each projection is
preferably configured to receive no more than about one entire
bead. FIG. 4(B), for example, shows a single bead 24a contained
within cavity 70. Notably, in this embodiment, cavity 70 is
substantially filled by bead 24a, leaving insufficient room to
accommodate a second entire bead, or even a substantial portion of
a second bead. It should be appreciated that the particular size of
the cavity will generally be determined by the size of the beads
used with the system. Thus, for substantially spherical beads a
little less than 1 mm in diameter (e.g., 0.75-0.95 mm), a suitably
sized cavity can have an inner diameter, from one sidewall region
to a directly opposing sidewall region, of about 1 mm. The
longitudinal depth, from the ceiling region to the lowermost
opening, will generally be from between about 50%-125% of the size
of the inner diameter of the cavity. Accordingly, in this example,
the longitudinal depth is between about 0.50-1.25 mm. Preferably,
the longitudinal depth is between about 75%-100% of the cavity's
inner diameter; and most preferably the longitudinal depth and
inner diameter are approximately equal. In a particularly preferred
embodiment, wherein substantially spherical beads having a diameter
of between about 275-325 micrometers are utilized, each cavity is
configured with both an inner diameter and longitudinal depth of
between about 330-500 micrometers, and most preferably about 375
micrometers. The cavity, in this embodiment, is formed at the end
of a polyurethane rod or tube having an outer diameter of about 0.5
mm.
[0109] FIG. 5 shows one preferred construction for a projection,
denoted generally as 54a', for use in a projection array, such as
56a and 56b of FIG. 1. In this embodiment, each cavity 70' is
formed, for example, by fitting a hollow sheath, or sleeve, 80 over
the end of an elongated tube or rod 82, and leaving an overhang
region of the sheath, as at 80a, extending below a terminal end 82a
of the tube. In this arrangement, the overhang region 80a of sheath
80 defines sidewalls 76', and the terminal end 82a of the tube 82,
facing cavity 70', defines a ceiling region 74'. Frictional
engagement of sheath 80 about the end of tube 82 can maintain the
relative positioning of these elements. Optionally, conventional
adhesives, abrasives, and/or shrink-fitting techniques can be used
to hold sheath 80 in place on tube 82. Tube 82 can be constructed,
for example, of a substantially rigid material, such as glass,
plastic, metal, and the like. Sheath 80 can be formed, for example,
from a material that is resiliently expandable and flexible, such
as tetrafluoroethylene (TFE), or the like. Suitable tubing, for use
in constructing sheath 80, is available commercially, for example,
from McMaster-Carr Supply Co. (Chicago, Ill.) under the product
name "Thinwall Teflon TFE Spaghetti Tubing."
[0110] The ceiling region 74 can have any suitable shape or
contour. In the exemplary arrangement of FIG. 4(A), ceiling region
74 is generally conical or funnel-shaped, with an inwardly and
upwardly sloped surface (along the direction away from the lower
end of the tube). In a similar arrangement (not shown), the ceiling
region is generally hom-shaped. In still a further embodiment, the
surface of the ceiling region is substantially planar, as shown,
for example, at 74' in FIG. 5.
[0111] As previously indicated, an attraction source is operable at
each of the projection end regions in a manner effective to draw
individual beads from the supply into respective cavities and to
releasably retain them therein. In a typical operation, the
attraction source will be engaged after an array of projections,
such as 56a or 56b (FIG. 1), has been aligned with a corresponding
array of reagent-supply locations, such as wells 16a-16f, and the
projections have been lowered to respective positions proximate a
plurality of reagent beads, such as 24a-24f, held therein. The
attraction source can be, for example, a reduced pressure (vacuum),
an electrostatic force, and/or a magnetic force. In one preferred
embodiment, the attraction source is a vacuum. In the embodiment of
FIG. 1, for example, each of the projections, 54a-54f and 54g-541,
is a capillary tube having an axial lumen, respectively denoted as
86a-86f and 86g-861, extending therethrough. As best seen in the
detailed view of FIGS. 4(A)-4(B), lumen 86a provides a passageway
for a vacuum to extend longitudinally through the projection 54a.
Lumen 86a has a lower end that opens into a respective cavity 70
through a central area of ceiling 74. At its lower end, the inner
diameter of lumen 86a is smaller than the diameter of cavity 70, at
a location adjacent the ceiling region 74. In an exemplary
arrangement, each of the cavities has a diameter of at least 275
micrometers (e.g., between about 300-350 micrometers, and
preferably about 325 micrometers), and each of the lumens has an
inner diameter at its lower end of less than 275 micrometers (e.g.,
between about 230-270 micrometers, and preferably about 250
micrometers).
[0112] In an alternative embodiment, a plurality of beads are
attracted to, and retained at, respective projection end regions
using electrostatic means. Techniques for attracting and retaining
micro-beads using an electrostatic force are disclosed, for
example, in U.S. Pat. Nos. 5,788,814 and 5,846,595; each of which
is expressly incorporated herein by reference.
[0113] Referring again to FIG. 1, the upper end of each lumen
86a-86f, 86g-861 leads to a respective manifold, such as 88a and
88b, formed in support structures 58a, 58b. Each manifold 88a, 88b,
in turn, is disposed in fluid communication with a respective
pressure-control source. For example, manifold 88a can communicate
with vacuum pump 94 via flow line 92a, and manifold 88b can
communicate with vacuum pump 96 via flow line 92b. Each vacuum pump
94, 96 is operable, e.g., under the direction of a control computer
(not shown), to establish a reduced pressure in a respective line
92a, 92b and, consequently, within a respective array of lumens
86a-86f, 86g-861.
[0114] The manifolds can be of a generally monolithic construction,
e.g., molded of plastic or metal; or they can be assembled from
sub-component parts. Regarding the latter, one embodiment (not
shown) contemplates a manifold comprised of several sub-component
layers, stacked one on top of the other. One such multi-layered
arrangement includes a rectangular frame member, having a central
opening, sandwiched between upper and lower rectangular plate
members. Together, the layers form a box-like structure having a
central open region, or chamber. Particularly, the upper surface of
the lower plate member defines a floor region; the inner edge of
the rectangular frame member provides lateral sidewalls; and the
lower surface of the upper plate member defines a ceiling region. A
rectangular gasket can be interposed between confronting regions of
the frame member and each plate member to promote airtight
interfaces. The lower plate can be formed with an array of bores
extending fully between its two broad surfaces. Each bore can be
suitably threaded to receive, from below, a threaded end of a
respective tubular projection, and to communicate a lumen extending
longitudinally through such projection with the region (chamber)
above the plate. Conventional washers and/or gaskets can be used to
promote an airtight interface between each projection and the lower
plate. The upper plate, which can be attached to a rotatable frame,
such as 60, can have one or more conduits formed therethrough for
communicating the main chamber with a like number of remotely
positioned pressure-control sources.
[0115] In an exemplary operation, wells 16a-16f of reagent plate 20
are loaded with respective ampules 26a-26f, each of which contains
a plurality of beads 24a-24f carrying a particular reagent. A
projection array, such as 56a, is then aligned with the array of
wells 16a-16f in plate 20. This can be accomplished, for example,
by placing reagent plate 20 at a location underlying the arcuate or
circular pathway along which the array 56a moves when the central
vertical shaft 60c of frame 60 is rotated about its longitudinal
axis. Rotational motor 64 can then rotate frame 60 until array 56a
assumes a position directly over, and in alignment with, the
ampules 26a-26f of plate 20. Next, linear motor 66 can lower the
projections 54a-54f toward respective ampules 26a-26f.
[0116] If the ampules are provided with cover members, such as dome
30a (FIG. 2) or film 34a (FIG. 3(A)), then the cover members must
be displaced from the opening region of each ampule, at least
partially, in order to access the beads therein. With regard to
film-type covers, e.g., a thin polymeric membrane, as the
projections 54a-54f are lowered, each can engage and rupture the
cover, as exemplified in FIG. 3(B). In this way, all of the covers
in the array can be displaced at substantially the same time.
[0117] An exemplary process for removing cap or dome-type covers is
depicted in FIG. 6. This process is particularly useful for
simultaneously removing a plurality of covers that are frictionally
fitted about the upper rim or lip region of respective ampules; the
frictional engagement being such that each cover and ampule can be
pulled apart using only a moderate amount of force. Projections
54a-54f are lowered until each engages the top of a respective
dome-type cover 30a-30f. Pressure-control source 44 is actuated to
generate a reduced pressure in flow line 46 and, thus, in manifold
40 and at the lower regions of wells 16a-16f. Pressure-control
source 94 is actuated to generate a reduced pressure in flow line
92a and, thus, in manifold 88a and at the lower regions of
projections 54a-54f. Responsive to the vacuum forces, the bottom of
each ampule is drawn downward against the lower region of its
respective well, and the top of each cover member is drawn up
against a lower rim or lip of a respective projection.
[0118] Maintaining a vacuum hold on the domes, projections 54a-54f
can be lifted, as exemplified in FIG. 6, thereby pulling domes
30a-30f off of, and away from, ampules 26a-26f and exposing beads
24a-24f. Once the domes have vertically cleared the ampules,
rotational motor 64 can rotate frame 60 until the domes are
positioned over a depository area (not shown). At this point, the
vacuum can be discontinued, permitting the covers to fall, for
example, into a collection container (not shown). Rotational motor
64 can then rotate the projection array back into alignment with
the ampule array.
[0119] Referring again to FIG. 1, once the covers have been
removed, or in the event that no covers are utilized, the
projections 54a-54f can be lowered to respective positions
proximate the beads 24a-24f in the various ampules 26a-26f.
Pressure-control source 94 can be actuated to generate a vacuum
reaching through each of lumens 86a-86f to a respective cavity,
such as cavity 70 of FIGS. 4(A)-4(B), at the lower end region of
each projection. The vacuum force that is established at the lower
end region of each projection is sufficiently strong to attract a
reagent bead from a respective ampule 26a-26f, and to retain the
bead in a cavity of the projection. In one embodiment, for example,
a pressure of about 15 psi is established at each projection end
region to attract and retain spherical beads, each about 300
micrometers in diameter, in a cavity measuring about 325
micrometers in diameter and longitudinal depth.
[0120] FIG. 4(B), for example, shows a spherical bead 24a that has
been drawn into cavity 70 by a vacuum force pulling in a direction
extending up through lumen 86a. The relatively large diameter of
bead 24a, compared to the diameter of lumen 86a, as well as the
bead's resistance to significant physical deformation, prevents the
bead from being sucked up into lumen 86a. As also exemplified in
FIG. 4(B), owing to the conical or funnel shape of ceiling region
74, bead 24a becomes situated at an upper, central area of cavity
70.
[0121] In one embodiment (not shown), as the projections are being
lowered into the ampules, with a reduced pressure established at
each projection end region, a stream of gas is directed upwardly
from a bottom region of each ampule in a fashion effective to blow
beads up towards the projections. For example, a small hole can be
formed through the bottom of each ampule. A gas-permeable membrane
can cover each hole. Although permeable to gases (e.g., air), the
membrane is configured to prevent beads from falling out through
the holes. Flow lines can communicate a positive-pressure pump with
the various holes. A partial cover can extend over an upper region
of each ampule that permits passage of a projection, but prevents
beads from being blown past the projections and out through the top
of the ampules.
[0122] As previously noted, one embodiment of the invention
contemplates a resiliency flexible lower portion for each
projection of the projection array. With reference to projection
54a of FIG. 7, for example, sidewall 76 is formed of a resiliency
flexible material, while the rest of the projection, above sidewall
76, is made of a substantially rigid material, such as glass,
plastic or metal. This construction permits the lower end region of
the projection to bend as it encounters the interior contours of an
ampule, such as the curved or rounded bottom region of ampule 26a.
This feature is especially useful when only one or a few beads
remain in an ampule, since the opening at the projection's end
region can bend to face beads gathered in a lower central region of
the ampule.
[0123] Referring again to FIG. 1, once projections 54a-54f have
attracted and retained respective beads 24a-24f from supply 12,
linear motor 66 can vertically raise the projections, along with
the retained beads, clear of plate 20. Rotational motor 64 can then
rotate vertical shaft 60c of frame 60 about its central axis so
that the beads, retained on the projection array, are moved to a
deposit location. At this point, the vacuum retaining force can be
discontinued. For some applications, the force of gravity, alone,
will be sufficient to cause each bead to fall from a respective
cavity down to a substrate at the deposit location. In other
applications, it may be desirable to further urge the beads out of
the cavities. In this regard, one embodiment contemplates the
establishment of an increased pressure in the manifold above each
projection array. The pressure increase is sufficient to cause gas
to flow down through the lumen of each projection, thereby
"blowing" the beads out of the cavities. A pressure-control source
in communication with the manifold above each projection array can
effect the pressure increase. As shown in FIG. 1, for example, pump
102 can communicate with manifold 88a via flow line 98a; and pump
104 can communicate with manifold 88b via flow line 98b. Upon
activating one of the pumps and generating an increased pressure in
a respective manifold, gas will flow downward through the lumens of
a respective projection array in a manner tending to blow any
retained objects away from the projection end regions.
[0124] Detection instrumentation can be included in the system of
the invention for monitoring the various operations. In one
embodiment, for example, means are provided for determining whether
or not a target object, such as a cover member or bead, is present
at the lower end region of each projection. In an exemplary
arrangement, cessation of gas flow and/or attainment of an expected
(predetermined) low-pressure value in the vacuum flow lines can be
used as an indicator that each projection has properly engaged and
attracted a target object, such that a substantially airtight seal
is formed across the lower opening of each projection. If gas
continues to flow through a vacuum line, and/or a higher than
expected pressure is measured in a vacuum line, then it is possible
that at least one of the projections failed to attract and retain a
target object. A conventional gas-flow and/or pressure sensor (not
shown) can be located along the flow lines for this purpose. The
sensor(s) can be read by an operator, and/or communicate with a
control computer. In the latter case, the computer can alert an
operator to a potential error, and/or automatically initiate
corrective actions.
[0125] In another exemplary arrangement, the means for determining
the presence of a target object at the lower end region of each
projection includes a visual detection assembly. As will become
apparent, this arrangement is particularly useful in connection
with target objects that are substantially opaque or just slightly
translucent. Referring now to FIG. 8A, for example, a bundle of
light-conductive fibers, denoted generally as 112, can extend
through a flow line 92a leading to a projection array 56a. In
manifold 88a, above the projection array, the individual fibers
112a-112f of bundle 112 diverge and extend towards respective
projections 54a-54f. Each individual fiber is arranged with a
first, or "receiving," end extending at least partially into the
lumen of a respective projection, and with its terminal face at
this end generally directed toward a respective cavity at a lower
end region thereof. A radiation source (not shown) can provide
light, direct or reflected, that can pass up into each projection,
toward such receiving ends, when the projection end region is empty
and clear. In one preferred embodiment, a diffuse light source,
e.g., a fiber-optic bundle, illuminates substantially the entire
upper surface of the reagent plate. Diffusely reflected light, in
turn, irradiates each projection end region, from below. The
second, or "transmitting," end of each fiber is disposed in
communication with a camera device (not shown). In this regard, the
fibers can be bundled and supported at their second end such that
their terminal faces generally define a plane that is proximate to,
and generally parallel with, a planar array of photo-detectors of
the camera device. One or a few of the photo-detectors can be
assigned to each fiber in the transmitting-end bundle. In an
exemplary arrangement, the detector array is part of a CCD having a
range of view that is generally confined to a respective grouping
of fiber terminal-end faces.
[0126] A properly attracted and" retained object will extend across
a section of the projection's lower end region, thereby blocking
light from reaching the receiving end of a respective fiber.
Consequently, the photo-detector(s) assigned to such fiber will not
receive light (above background). In the absence of an object
blocking a section along the projection's terminal end region, on
the other hand, light will reach a respective fiber's receiving end
and will travel to its transmitting end. From the transmitting end,
the light will impinge upon one or more assigned photo-detectors.
The photo-detector(s) can then produce an output signal that can be
visualized on a CRT screen, or the like, for inspection by an
operator.
[0127] The output signals can produce bright spots at pre-assigned
locations of an otherwise dark CRT screen. Thus, a bright spot on
the screen can be used to alert an operator to the absence of a
target object at the end region of a particular projection.
Alternatively, or in addition, the output signals can be
transmitted through an appropriate interface to a control computer.
The computer, then, can alert an operator to any projections that
have apparently failed to pick up a target object, and/or
automatically initiate corrective actions.
[0128] In one embodiment, a standard optical fiber having a
diameter of between about 30-70 micrometers, and preferably about
50 micrometers, extends into the longitudinal lumen of each
projection. Suitable optical fibers are commercially available, for
example, from Edmund Scientific Co., (Barrington, N.J.). Each
lumen, in this embodiment, can have a diameter of between about
230-270 micrometers, and preferably about 250 micrometers. The
transmitting end of each fiber, in turn, can be disposed for
communication with one or several photo-detectors of a CCD camera.
Any of numerous commercially available CCD cameras can be used in
the present invention, and suitable cameras can readily be chosen
by those skilled in the art. One particular CCO camera,
contemplated for use herein, is available commercially under the
trade name PANASONIC GP-KR222.
[0129] Rather than running only a single fiber into the lumen of
each projection, as shown in FIG. 8A, it should be appreciated that
any reasonable number (e.g., 2, 3, 4, 5, or more) of fibers can be
employed. Various factors that can be considered in determining an
appropriate number of fibers to use include (i) the outer diameter
of each fiber, (ii) the inner diameter of the respective lumens,
(Hi) the inner diameter of the flow lines passing through the
supportive frame, and (iv) the number, size and spatial
configuration of the photo-detectors in the camera device.
Importantly, those embodiments relying upon a vacuum extending
through each projection to attract and retain the beads, the fibers
should not substantially hinder gas flow through the various flow
lines, manifolds and projection lumens. Preferably, any reduction
in flow rate due to the presence of the fibers is less than about
50%.
[0130] With further regard to the optical fibers, instead of
running each fiber through a flow line passing through the frame
structure and into the lumens of the various projections, as
illustrated in FIG. 8A, one embodiment (not shown) provides a small
bore formed through an upper region of each projection through
which an end region of a fiber can be passed. In an exemplary
construction, each projection includes a plastic top portion with a
metal tube extending therefrom. A small bore is drilled through the
metal part of each projection, dimensioned to receive one end of an
optical fiber. In this embodiment, each fiber forms a substantially
airtight seal with its respective bore so as not to interfere with
the establishment of a desired pressure (e.g., a vacuum) in the
system.
[0131] Rather than directing light up through each projection, the
detection system can be arranged to operate in a "reverse" manner
from that described above. That is, the fiber ends distal from the
projections can act as "receiving" ends communicating with a light
source. The fiber ends in the projections, on the other hand, can
act as "transmitting" ends adapted to direct light down and out of
each projection end region. In operation, prior to picking up
objects with the projections, a camera can image the projection end
regions, from below. For example, the central frame can be rotated
about an angle sufficient to pass each projection over a linear
photo-detector array. Those projections that have light passing out
of their lower end regions are determined to be empty and
clear--i.e., available for picking up an object. After a pick-up
operation, the camera can again image the projection end regions. A
properly picked up and retained object will block light from
reaching the camera's photo-detector array. A pick-up failure is
indicated for any projections that have light passing out of their
lower end regions. Corrective actions can be taken, e.g., a re-load
attempt, in the case of a pick-up failure.
[0132] In another exemplary arrangement, illustrated in FIGS. 8B
and 8C, the means for determining the presence or absence of a
target object, such as a bead, at the lower end region of each
projection includes one or more cameras or other suitable image
capture devices positioned below, but in substantially vertical
alignment with, the arcuate or circular path in which extractors
50a and 50b and corresponding projection arrays 56a and 56b travel
between the "picking up" and "releasing" steps.
[0133] In the illustrated embodiment, where two extractors are
employed, two cameras 500a and 500b can be used. In this
embodiment, after extractor 50a and its projection array 56a
executes a bead "pick up" operation in the position denoted by the
letter A and after extractor 50b and its projection array 56b
executes a bead "release" operation in position C, the rotation
motor 64 is controlled to rotate extractors 50a and 50b to
positions B and D, respectively, where the extractors are
temporarily held while the individual projections of corresponding
projection arrays 56a and 56b are checked for the presence or
absence of beads. When in position B, the lower end of projection
array 56a is positioned above, and in the field of view of, a lens
502a of camera 500a which captures an image 506 of the lower end,
depicting the ends of all of the individual projections, preferably
as seen along the axis of the projections. As shown in FIG. 8C, the
image is transmitted to a suitable display device 504 such as CRT
or LCD display where the image is displayed to enable an operator
to visually determine if any projection failed to pick up a bead,
or if any projection picked up more than one bead. Similarly, when
in position D, the lower end of projection array 56b is in the
field of view of a lens 502b of camera 500b. In this case, the
image captured by camera 500b and displayed on the monitor is used
to determine if any projection of projection array 56b failed to
release a bead. By looking at the exemplary image 506 on the
display device in FIG. 8C it can easily be seen that that each of
the projections, except the one in row 2, column 2 contain a bead.
It should be noted that the display device may be part of the image
capture device, or may be a separate unit in communication
therewith.
[0134] If the check step performed with cameras 500a and 500b
reveals that any projection of projection array 56a failed to pick
up a bead in position A, or that any projection of projection array
56b failed to release a bead in position C, rotation motor 64 is
controlled to rotate extractors 50a and 50b back to positions A and
C, respectively, where the corresponding pick up and/or release
step is again executed, as required. Afterward, projection arrays
56a and 56b are again rotated to positions B and D, respectively,
where they are once again checked for the presence or absence of
beads by cameras 500a and 500b before continuing to positions C and
A, respectively.
[0135] Although, in the illustrated embodiment, each camera is
positioned about 90.degree. between the pick up and release
positions, this is not necessary. It should be appreciated that the
cameras may be positioned at other locations along the arcuate
travel path. For example, one camera may be offset 45.degree. in
the counterclockwise direction from position A while the other
camera is offset the same degree in the same direction relative to
position C.
[0136] It should also be appreciated that more or less than two
cameras may be employed, depending on the number of extractors
used. For example, in another embodiment of the invention where
only a single extractor is used, only a single camera, located at
either position B or D, need be used. In this case, the extractor
will move back and forth along an arcuate path defined by either
ABC or ADC, depending on where the camera is located. Thus, the
single camera will perform both the check after pick up and the
check after release. In other embodiments, where three or more
extractors are used, two or more cameras may be employed.
[0137] As previously mentioned, the attracted and retained beads
can be moved to a deposit position whereat the beads can be
released. In one embodiment, for example, the deposit location is
over a substrate, such as a micro-plate or card, having a plurality
of bead-receiving locations. FIG. 1 shows, for example, a
substrate, denoted as 122, having a plurality of spaced-apart
wells, such as 124a-124f, for receiving and holding beads.
[0138] The receiving wells of substrate 122 can be configured for
alignment with the projections 54a-54f, 54g-541 of projection
arrays 56a, 56b. In this regard, wells 124a-124f can be formed with
the same center-to-center spacing as projections 54a-54f, 54g-541.
In one particular embodiment, a bead-receiving substrate is
provided as an injection-molded plate, or tray, made of any
suitable material, such as acrylic, polycarbonate, polypropylene,
polysulfone, or the like. Preferably, the length and width of the
tray conform to the commonly used standard of 5.03''.times.3.37''
(127.8 mm and 85.5 mm), although other outer dimensions can be
used. A regular array of depressions, or wells, are provided in the
tray for separately receiving and holding reagent beads dropped
from above. For example, the instant embodiment contemplates a
16.times.24 array of wells integrally formed with the tray, with
adjacent wells spaced apart about 4.50 mm center-to-center. Each
well, in this embodiment, has an interior region, or lumen, that is
substantially square in horizontal cross-section, and a bottom or
floor region that is generally flat. It should be appreciated,
however, that wells of any desired geometrical configuration (e.g.,
oval, square, rectangular, triangular, etc.) can be used.
Similarly, the wells may be of any desired shape when viewed along
their longitudinal axes, e.g., straight, tapered or other shape.
For the square wells of the present embodiment, the four sidewalls
of each well are provided with a slight inward taper (i.e., the
distance between opposing sidewalls continuously decreases) along
the direction extending from the well's upper, receiving end toward
the floor region. Preferred dimensions for each well in this
embodiment are as follows: (i) about 1 mm in depth (top to bottom);
(ii) about 1 mm.times.1 mm across an uppermost opening; and (iii)
about 0.50 mm.times.0.50 mm across a flat, bottom floor region.
[0139] The depending projections of an extractor, intended for use
with the just-described tray, can similarly be arranged in a
16.times.24 array, with adjacent projections spaced apart about
4.50 mm center-to-center. By this construction, the extractor can
be positioned over the 16.times.24 array of wells in the tray, with
the two arrays in alignment. In use, a plurality of beads retained
by the extractor can be deposited directly into the wells of the
tray. For example, a plurality of retained beads on such a
projection array can be lowered into close proximity with
respective openings of the well array. From this position, the
beads can be released from the projections, in a substantially
simultaneous fashion, so that each bead falls into a respective
well.
[0140] In another embodiment, a generally square micro-card, about
1''.times.1'', is provided with a 32.times.32 array of wells. As
with the previous embodiment, each well of the array is formed with
a substantially square horizontal cross-section, and a generally
flat bottom or floor region. Again, it should be noted that other
well configurations can be used. Preferred dimensions for each
well, according to this embodiment, are as follows: (!) about 1 mm
in depth (top to bottom); (ii) about 0.60 mm.times.0.60 mm across
an uppermost opening; and (iii) about 0.35 mm.times.0.35 mm across
a bottom floor region. Downwardly convergent (tapered) sidewalls
extend between the top opening and floor of each well. Adjacent
wells in the array are spaced about 1-2 mm apart
(center-to-center), and preferably about 1.50 mm. So constructed,
each well can hold, for example, up to three substantially
spherical reagent beads, each having a diameter of between about
275-325 micrometers, and preferably about 300 micrometers.
[0141] An extractor, suitable for use with the just-described
micro-card, can be provided with an array of projections disposed
in an array that is complementary to the array of wells in the
card, permitting direct alignment of each of the projection arrays
with the array of wells.
[0142] Instead of depositing the beads directly onto a substrate,
as described above, one embodiment provides a means for guiding or
channeling each bead, once released from a projection end region,
to a respective receiving location on the substrate. Such means can
include, for example, a conduit or channel assembly adapted for
positioning between the projection array and substrate. Referring
to the embodiment of FIG. 1, for example, a conduit assembly,
indicated generally at 126, includes a plurality of conduits
128a-128f in a support structure 130. Support structure 130
maintains the conduits in fixed, spaced relation to one another. In
one embodiment, the support structure takes the form of a frame, or
rack, into which individual conduits can be secured (e.g., snap
fit). In another embodiment, the conduits are integrally formed
with the support structure. For example, the conduit assembly can
be constructed of plastic using an injection molding process; or
each conduit can be formed by boring through a block of material,
such as glass, plastic, metal, or the like.
[0143] The top of conduit assembly 126 is provided with an array of
openings for receiving beads that have been released from a
projection array, such as 56a or 56b. In a preferred embodiment,
the conduit upper-opening array is configured for alignment with
each of the projection arrays. In this regard, the upper openings
of conduit assembly 126 and the projections 54a-54f, 54g-541 of
each projection array 56a, 56b can be arranged with substantially
the same pitch (center-to-center spacing). The bottom of conduit
assembly 126 provides an array of openings through which beads can
egress. The conduit lower-opening array can be configured for
alignment with the array of wells of the substrate. For example,
the openings at the bottom of conduit assembly 126 and the wells
124a-124f of substrate 122 can be formed with substantially the
same pitch.
[0144] In order to facilitate passing of a released bead from a
projection end region down into a conduit positioned thereunder
(i.e., ease the tolerance of bead delivery by the extractor), the
upper end of each conduit 128a-128f can be formed with an expanded,
or enlarged-diameter, opening. In one embodiment, for example, each
conduit upper opening is at least 150%, and preferably greater than
250%, the size of an opening defined by the lower rim or lip of a
respective projection end region. To facilitate passing of a bead
from a conduit down into a respective well of a bead-receiving
substrate, the lower opening of each conduit can be formed with a
diameter that is generally no larger than the upper opening of a
respective receiving well. In a preferred embodiment, the lower
opening of each conduit 128a-128f is smaller than the upper opening
of a respective receiving well 124a-124f. In an exemplary
construction, each conduit lower opening is between about 40-95%,
and preferably about 70%, the size of the opening of a respective
receiving well. For example, a generally circular lower opening of
a conduit, having a diameter of about 400 micrometers, can be
positioned over a substantially square receiving well having an
upper opening measuring about 600.times.600 micrometers.
[0145] From the above discussion, it should be appreciated that it
will often be advantageous to utilize a conduit assembly wherein
each conduit has a large upper opening and a small lower opening
(relative to one another). In one embodiment, for example, each
conduit upper opening has a diameter of between about 1 mm-10 mm,
and each lower opening has a diameter of less than 1 mm. In one
particular construction, each large opening has a diameter of
between about 1-6 mm; and preferably about 2 mm; and each small
opening has a diameter of between about 0.25-0.75 mm, and
preferably about 0.40 mm.
[0146] With continuing reference to FIG. 1, between its upper and
lower ends, each conduit 128a-128f of conduit assembly 126 can be
shaped to promote ready passage of a bead therethrough and down
onto a substrate, such as 122, thereunder. One embodiment, for
example, provides downwardly convergent (tapered) sidewalls between
the upper and lower openings of each conduit. In the exemplary
arrangement of FIG. 1, the sidewalls of each conduit are generally
straight along the longitudinal direction, defining generally
cone-shaped conduits. In another exemplary arrangement, the
sidewalls are curved along the longitudinal direction, defining
generally horn-shaped conduits.
[0147] As previously indicated, it is often advantageous to utilize
a conduit assembly having an upper-opening array configured with
substantially the same pitch as the projection array, and further
having a lower-opening array configured with substantially the same
pitch as the receiving-well array of a substrate. Thus, in systems
where the pitch of both the projection and receiving-well arrays
are substantially the same, the pitch of the conduit upper- and
lower-opening arrays will be about equal. For example, FIG. 1 shows
an embodiment wherein each of (i) the projection arrays, (ii) the
receiving-well array, and (iii) the conduit upper- and
lower-opening arrays all have substantially the same
center-to-center spacing.
[0148] In systems where the pitch of the projection and
receiving-well arrays differ, on the other hand, it will often be
advantageous to utilize a conduit assembly having upper- and
lower-opening arrays that differ in pitch, as well. In one
exemplary system of this type, the conduit lower-opening array is
provided with a center-to-center pitch that is smaller than that of
the conduit upper-opening array. FIG. 9 shows, for example, an
embodiment much like that of FIG. 1, except that receiving wells
124a'-124f of substrate 122' are arranged in an array having a
pitch that is substantially smaller than that of the projection
arrays 56a, 56b. Consequently, the upper- and lower-opening arrays
of conduit assembly 126', in this embodiment, differ in pitch in a
similar fashion. For example, the center-to-center pitch of the
lower-opening array can be between about 1/2 to 1/4 that of the
upper-opening array. In one particular embodiment, the
center-to-center pitch of the lower-opening array is about 1/3 that
of the upper-opening array.
[0149] With further regard to shape, it should be noted that each
of conduits 128a'-128f of FIG. 9 is curved along its longitudinal
direction such that it roughly defines an "S" shape. In another
embodiment, one or more of the conduits are substantially straight.
In this regard, attention is drawn to conduit assembly 126'' of
FIGS. 10(A)-10(C). In this arrangement, a 10.times.10 array of
conduits, such as at 128'', are formed in a support structure, such
as block 130''. Block 130'', in turn, is situated over a micro-card
122'' having a 10.times.10 array of receiving wells that are
aligned with the conduit assembly's lower-opening array. The
conduit assembly's upper-opening array, as depicted in FIG. 10(B),
can have, for example, 4 mm diameter openings disposed at a 6 mm
pitch, and the lower-opening array, shown in FIG. 10(C) can have
0.4 mm diameter openings disposed at a 1.5 mm pitch.
[0150] The conduit assembly can be manually placed over the
bead-receiving substrate, or it can be placed over the substrate in
an automated fashion. In either case, it will often be helpful to
include in the system a means for registering the bead-receiving
locations of the substrate with the lower-opening array of the
conduit assembly. In one embodiment, indexing pins, such as at 132,
134 in FIG. 1, depending from the lower side of conduit assembly
126, can assist in registering the wells of micro-plate 122 with
the lower-opening array of conduit assembly 126.
[0151] Particularly, each indexing pin 132, 134 is alignable with a
respective indexing bore, such as 136, 138, formed through a
corresponding region of substrate 122. Insertion of the indexing
pins in the indexing bores substantially aligns the lower-opening
array of the conduit assembly with the array of wells of the
micro-card.
[0152] With further regard to placement of the conduit assembly
over a substrate, one embodiment contemplates the inclusion of a
conventional motor or pneumatic lifter in the system, such as at
140 and 140' in FIGS. 1 and 9, respectively. Lifter 140 is adapted
to raise and lower the conduit array along a generally vertical
pathway. Those skilled in the art can readily select a suitable
lifter from the devices that are available from commercial sources.
Preferably, lifter 140 is adapted for operation under the direction
of a control computer (not shown).
[0153] In another embodiment, a parallelogram linkage assembly
supports a conduit assembly for reciprocal movement between a
raised position and a lowered position. FIG. 11, for example, shows
an exemplary parallelogram linkage assembly, indicated generally as
144, supporting conduit assembly 126'' for such movement. In the
illustrated arrangement, first and second side links, denoted
respectively as 146 and 148, are provided along one side of linkage
assembly 144. The lower ends of side links 146, 148 are pivotally
attached at respective, spaced-apart locations, or pivot points,
150, 152 proximate a supportive surface 154, and pivotally attached
at their upper ends, in a similarly spaced-apart fashion, at pivot
points 156, 158 along a horizontal link 160. By this construction,
horizontal link 160 can be moved up and down along a generally
arcuate pathway between a raised position, as depicted in broken
lines, and a lowered position, as shown in solid lines. Although
not visible in FIG. 11, an additional pair of side links, like side
links 146 and 148, are provided on the opposite side of linkage
assembly 144. A substrate holding area, indicated generally as 164,
is provided adjacent to parallelogram linkage assembly 144 for
receiving and holding a bead-receiving substrate, such as 122''.
Conduit assembly 126'' becomes positioned over substrate 122'' when
linkage assembly 144 is at its lowered position (solid lines).
[0154] Indexing pins 132'', 134'' are adapted to mate with
respective indexing bores 136'', 138'' in substrate 122'' to assist
in aligning the lower-opening array of conduit assembly 126'' with
the well array of substrate 122''. Details of a similar indexing
arrangement are shown in FIGS. 12(A)-12(B). Here, one indexing pin,
such as 132''', can be aligned with a bore 136''' through
micro-card 122''', and a second pin 134''' can be aligned with slot
138''' formed in micro-card 122'''. Returning to the view of FIG.
11, a passageway 166 can open into a central region of the
substrate holding area 164. By connecting conduit 166 to a remote
pressure-control source, a vacuum can be established reaching to,
and drawing upon, the lOWerSUrface Of 3 bead-receiving substrate
122'', thereby maintaining the substrate in a seated position.
[0155] It should be noted that there are other ways of positioning
the conduit assembly relative to the micro-card. For example, a
linear arrangement of two motors may be used. In this arrangement,
one motor can be used to move the conduit assembly into place over
the micro-card. The other motor can then be used to push the
micro-card into the conduit assembly to connect them after the
conduit assembly has been moved into place.
[0156] With additional reference to the perspective view of FIG.
13, a plurality of parallelogram linkage assemblies, such as 144,
each carrying a respective conduit assembly 126', can be seen in
combination with a carousel arrangement, denoted generally as 168.
Rotational motion of carousel 168 causes the various linkage
assemblies to revolve about the carousel's central axis "A".
Preferably, such motion of the carousel is carried out under the
direction of a control computer (not shown). Each conduit assembly
is disposed along a region of a respective horizontal link 160
lying radially outward of axis "A". In one embodiment, for example,
each horizontal link is rigidly attached to, or integrally formed
with, a frame structure having a central opening (not visible in
FIG. 13) configured to receive and support a respective conduit
assembly. The other end of each horizontal link 160 rigidly
attaches to, or is integrally formed with, an elongated arm 172
that extends in the direction of the carousel's rotational axis
"A," reaching to and engaging a rail 174 running along the inner
region of the carousel's supportive surface. As best seen in FIG.
11, rail 174 provides a bearing surface 178, further described
below, along which each linkage assembly 144 can ride as it is
advanced by carousel 168. In this regard, elongated arm 172
includes a downwardly angled, terminal bend 180 adapted to slide
along bearing surface 178. A bearing material can be attached to
bend 180 along a region confronting bearing surface 178.
Preferably, the bearing material is selected to provide a contact
interface with low sliding friction. For example, FIG. 11
illustrates a boss 182 formed of a low-friction material, such as
polytetrafluroethylene (PTFE) or the like, bonded to bend 180 at a
region adjacent bearing surface 178.
[0157] As mentioned above, and with particular reference to the
perspective view of FIG. 13, it can be seen that rail 174 runs
along an inner region of the carousel's supportive surface 170.
More particularly, the bearing surface 178 of rail 174 includes (i)
a first arcuate section disposed a first distance R1 from
rotational axis "A" at a first vertical height H1 above the
carousel's supportive surface; and (ii) a second arcuate section
disposed a second distance R2 from axis "A," shorter than distance
R1, at a second vertical height H2, higher than vertical height H1.
The configuration of each such arcuate section is nearly that of a
semicircle, measuring between about 60-85 degrees. Transition
sections, as at 183 and 184, bridge the first and second arcuate
sections. Together, the first and second arcuate sections, and the
transition sections, provide a continuous, bearing surface,
appearing roughly oblong in top plan view (not shown).
[0158] In operation, as each parallelogram linkage assembly 144 is
advanced along the first arcuate section of rail 174, a respective
conduit assembly 126* will be located at the lowered position,
directly over a substrate 122'. As each parallelogram linkage
assembly is moved along the second arcuate section, its respective
conduit assembly will locate at the raised position, above and
offset from the substrate.
[0159] Detection instrumentation can be included in the system of
the invention for determining the presence of a bead at target
locations of a bead-receiving substrate, such as in the wells of a
micro-card. In one embodiment, all beads carrying a particular
reagent are formed to display a unique, pre-assigned color. The
detection instrumentation, in this embodiment, is adapted to
inspect each target well for a bead of such color. In the exemplary
arrangement of FIG. 14, an illumination source, such as laser 186,
directs an expanding radiation beam 188 towards a lens 190. Lens
190 focuses the beam to irradiate the upper-opening array of
conduit assembly 126'' so that a fraction of the beam passes down
through each conduit to the wells of plate 122''. Upon striking
each well, and its contents (if any), a retro-beam 192 of reflected
light travels back up through each conduit, towards lens 190. To
facilitate passage of the retro-beam through the conduits, each
conduit can have a reflective inner surface, e.g., a highly
polished metallic surface. Lens 190 focuses the retro-beam 192 to
pass through an aperture 193 and fall upon a collimating lens 194
which, in turn, directs the beam to a color filter 196. Color
filter 196 only permits light of a designated wavelength (or range)
to pass to a sensor array of an adjacent camera device, such as CCD
camera 198. Each well of micro-card 122'' is assigned to one or a
few photo-detectors of the CCD camera 198. Target wells that have
been successfully loaded with a bead of the designated color will
provide a retro-beam capable of passing through the color filter
and striking respective photo-detectors of the CCD camera. The
photo-detectors, in turn, can communicate an output signal for
visualization on a video screen and/or transmission via an
appropriate interface to a computer. In either case, a positive
signal is indicative of successful loading. Any target well(s)
failing to hold a bead of the designated color, on the other hand,
will not be able to generate a retro-beam capable of reaching the
CCD camera. Accordingly, the photo-detector(s) assigned to such
well(s) will not produce a signal and a loading failure is
indicated. A new loading attempt can then be made, or the card can
be rejected.
[0160] Rather than relying only upon reflected light to provide a
retro-beam from each well, the coating on each bead can be of a
type that fluoresces upon being illuminated with light of a certain
wavelength. In this way, each bead can generate fluorescent
emissions of a particular, pre-assigned color indicative of the
reagent that it carries.
[0161] In another embodiment, similar to that of FIG. 14 (described
above), an array of optical fibers communicates an illuminating
beam with a plurality of target locations of a bead-receiving
substrate, and further communicates a retro-beam leaving each
target location with a light-sensitive camera device. As shown in
FIG. 15, for example, an expanding beam 188' emanating from laser a
186' is directed through a lens 190' onto the terminal ends of an
array of optical fibers, including fibers 202. The other terminal
end of each fiber is disposed above, or in, a respective conduit of
conduit assembly 126''. In one particular arrangement, each fiber
extends down through a respective conduit to a location slightly
above a respective bead-receiving well. A retro-beam, leaving each
well, can travel up through its respective fiber towards lens 190'.
Lens 190' focuses the retro-beam 192' to pass through an aperture
193'and fall upon a collimating lens 194' that, in turn, directs
the beam to a color filter 196'. As with the previous embodiment,
color filter 196' only permits light of a designated wavelength (or
range) to pass to a sensor array of an adjacent camera device, such
as CCD camera 198'. Additional details are substantially like those
of the previous embodiment.
[0162] In certain applications, it may be desirable to detect beads
of different colors, e.g., blue beads, red beads and green beads.
To this end, either of the above-described detection assemblies
(FIGS. 14 and 15) can be provided with a plurality of color
filters, with each filter being independently movable in and out of
the retro-beam path prior to the camera device. By selecting an
appropriate filter, and moving it into position for interception of
the retro-beam, beads of a particular color can be detected.
Another embodiment contemplates the use of multiple, separate
detection assemblies, each being configured to detect beads of a
particular color. The detection assemblies can be substantially
alike, except that each includes a uniquely colored filter. For
example, one detection assembly can include a filter adapted for
the detection of blue beads, another can include a filter for
detecting red beads, and a third can have a filter for detecting
green beads. The different detection assemblies can be arranged in
series along a conveyor apparatus carrying bead-receiving
substrates.
[0163] The broken-line rectangle 204 surrounding certain of the
detection assembly components in FIGS. 14 and 15 schematically
represents a housing or case within which the detection assembly
components can be mounted. In one preferred embodiment, the housing
is adapted for reciprocal linear movement along a generally
vertical pathway over a conduit assembly. With particular reference
to the embodiment of FIG. 15, for example, laser 186', lenses 190*
and 194', aperture 193', color filter 196' and CCD camera 198' can
all be mounted in housing 204. The upper region of each optical
fiber 202 is also mounted in the housing, with the terminal end of
each fiber facing lens 190'. The lower ends of the optical fibers
project out through the bottom wall of housing 204. The projecting
fiber ends are arranged in an array complementary to the conduit
upper-opening array. By this construction, the detection assembly
can be lowered over a conduit assembly and micro-card, such as
126'' and 122'', in a fashion permitting each fiber end to enter,
and extend down into, a respective conduit. Once the detection has
been completed, the detection assembly can be raised and another
conduit assembly/micro-card can be moved into position under the
detection assembly. Instead of inspecting for the presence of beads
in the wells of a substrate while a conduit assembly is lowered
thereover, as described above, one embodiment contemplates removal
(lifting) of the conduit assembly prior to detection. In this
embodiment, the detection assembly irradiates the wells directly,
and detects for the presence of beads on the basis of color.
[0164] Another feature of the present invention provides a system
for covering an array of wells formed in a substrate, such as a
micro-plate or card. According to one embodiment, depicted in the
side-sectional view of FIG. 16(A), the system includes a continuous
web of a cover material, denoted as 212, mounted for movement from
a supply position, such as idler reel 214, to a take-up position,
such as driven reel 216. Shearing blades 218 are mounted on the
lower face of a movable piston 222 for reciprocal linear motion
along a direction substantially normal to the web for cutting out a
portion of the cover material 212 at a region between the supply
position 214 and take-up position 216. A resiliency compliant,
generally planar surface, indicated at 220, is provided on the
lower face of piston 222 along a region between blades 218 for
pressing the cover material against the upper surface of a
substrate, such as micro-card 122''.
[0165] Indexing pins 232, 234, depending from the lower face of
piston 222, can register with indexing bores 136'', 138'' of the
substrate 122'' to orient the shearing blade 218 thereover for
effecting a desired cut in web 212. Although not visible in the
sectional view of FIG. 16(A), the shearing blades collectively
provide a cutting edge defining a quadrilateral, such as a square
or rectangle. In this regard, FIG. 16(B) illustrates sequential
cuts 224, 226 made in web 212 by shearing blades 218, with each cut
having (i) two sides substantially parallel to the side edges of
the web and (ii) two sides substantially normal to the side edges
of the web.
[0166] Any suitable cover material can be utilized. Preferred
materials are substantially chemically inert with the reagents
placed in the wells. One embodiment contemplates the use of a cover
material that is capable of forming a substantially fluid-tight
seal with the upper surface of a multi-well tray, or appropriate
regions thereof (e.g., an upstanding rim or lip about the opening
of each well). Such a seal can be effected, for example, using
conventional adhesives and/or heat sealing techniques. Suitable
heat-sealable materials include, for example, polymeric films, such
as polystyrene, polyester, polypropylene and/or polyethylene films.
Such materials are available commercially, for example, from
Polyfiltronics, Inc. (Rockland, Mass.) and Advanced Biotechnologies
(Epsom, Surrey England UK). One embodiment contemplates the use of
a substantially clear polymeric film, e.g., between about 0.05-0.50
millimeters thick, that permits optical measurement of reactions
taking place in the covered wells. In this regard, it will be
recalled that the present invention contemplates real time
fluorescence-based measurements of nucleic acid amplification
products (such as PCR). Generally, in such a technique, an
excitation beam is directed through a sealing cover sheet into each
of a plurality of fluorescent mixtures separately contained in an
array of reaction wells, wherein the beam has appropriate energy to
excite the fluorescent centers in each mixture. Measurement of the
fluorescence intensity indicates, in real time, the progress of
each reaction. For purposes of permitting such real time
monitoring, each sheet in this embodiment is formed of a
heat-sealable material that is transparent, or at least transparent
at the excitation and measurement wavelength(s). One suitable
heat-sealable sheet, in this regard, is a co-laminate of
polypropylene and polyethylene. A heatable platen (not shown) can
be used to engage the sheet, once cut and placed over an array of
wells, and to apply heat so that the sheet bonds to the
substrate.
[0167] At this point, attention is directed to the perspective view
of FIG. 13, wherein a number of the above-described features of the
invention can be seen embodied in a high-throughput system for
fabricating an array of beads on a micro-plate or card. Generally,
a linear conveyor 252 transports, in serial fashion, empty
micro-cards 122' from a supply area 254 to a position adjacent a
first robot 260. As the robot 260 picks up an empty card 122' from
the conveyor 252, a rotatable carousel 168 advances a platform 154
and an associated, movable conduit assembly 126' to a location also
adjacent the robot 260. Notably, the conduit assembly 126' is
disposed at a raised position, above and vertically offset from a
substrate-holding area 164 of the platform 154. The robot 260
places the card 122' at the unoccupied substrate-holding area 164,
and the carousel 168 rotates to advance the card 122' to a position
adjacent a scanning assembly 264. With the conduit assembly 126'
still raised, the scanning assembly 264, e.g., a bar-code reader,
scans a label on the side of the card 122'. This operation can
serve, for example, to ensure that the card has been properly
placed at the substrate-holding area and to read identifying
information into a control computer (not shown). Next, a
parallelogram linkage assembly 144 lowers the conduit assembly 126'
to a position directly over the card 122', with a lower-opening
array of the conduit assembly 126' aligned with a complementary
array of wells in the card 122'. The card 122' is then advanced to
a loading position adjacent a bead dispensing arrangement, as at 8,
which can be constructed substantially as shown in FIG. 9. The
dispensing arrangement 8 is operable to pick up a plurality of
beads from a reagent plate 20 and, upon rotating about 180.degree.,
to deposit the beads into the micro-card 122' via the conduit
assembly 126', as previously described. Further regarding the
dispensing arrangement 8, it should be appreciated that while one
bead extractor 50a deposits a set of beads into the wells of a
micro-card, the other extractor 50b can simultaneously pick up
another set of beads from the reagent plate 20 for placement in the
next card advanced to the loading position. This operation can
continue until all of the empty cards have been filled and/or the
supply of reagent beads has been exhausted.
[0168] Having received beads from the dispensing arrangement 8, the
card 122' is then advanced to a position below a detection assembly
204, e.g., like that of FIG. 15, that inspects each well of the
card 122' for the presence of a bead. As the card 122' leaves the
detection assembly 204, the conduit assembly 126' is returned to
the raised position, and the card 122' is presented to a sealer 268
that places a cover, such as an optically clear membrane, over the
wells. The sealer can be constructed, for example, substantially as
shown in FIG. 16(A). A second camera, as at 272, then inspects the
card 122' to ensure proper placement of the cover. Finally, the
card 122' is advanced to a second robot 274, located between the
carousel 168 and conveyor 252, which lifts the card 122' from the
substrate-holding area 164. If the inspection operations indicate
that the card 122' has been properly loaded with beads and
effectively sealed, the card 122' is then placed back on the linear
conveyor 252 and transported to a storage location 280. If a
failure in either of these regards has been indicated, on the other
hand, the robot 274 can instead deposit the card 122' in a reject
bin, as at 284.
[0169] A control computer (not shown) can integrate the operation
of the various assemblies, for example through a program written in
an event driven language such as LABVIEW.RTM. or LABWINDOWS.RTM.
(National Instruments Corp., Austin, Tex.). In particular, the
LABVIEW software provides a high level graphical programming
environment for controlling instruments. U.S. Pat. Nos. 4,901,221;
4,914,568; 5,291,587; 5,301,301; 5,301,336; and 5,481,741 (each
expressly incorporated herein by reference) disclose various
aspects of the LABVIEW graphical programming and development
system. The graphical programming environment disclosed in these
patents allows a user to define programs or routines by block
diagrams, or "virtual instruments." As this is done, machine
language instructions are automatically constructed which
characterize an execution procedure corresponding to the displayed
procedure. Interface cards for communicating the computer with the
motor controllers are also available commercially, e.g., from
National Instruments Corp.
[0170] A further aspect of the invention provides a hand-held unit
for simultaneously picking up a plurality of reagent-carrying beads
and depositing the beads at a desired location. An exemplary
arrangement of one such device, denoted generally by the reference
numeral 350, is depicted in FIGS. 17(A)-17(B). Here, a rectangular
frame member 360, having substantially planar upper and lower broad
surfaces, is provided with a handle 363 rigidly attached thereto.
Handle 363 is roughly U- or arch-shaped and oriented over frame 360
so as to provide a gap 365 between its central region and the upper
surface of frame 360. Preferably, gap 365 is dimensioned to
accommodate the fingers of an operator's hand upon grasping handle
363.
[0171] A generally rectangular support 358, also having
substantially planar upper and lower broad surfaces, is attached to
an underside of frame 360. Frame 360 and support 358 are disposed
in fixed, spaced relation, with their confronting surfaces
generally parallel to one another. A plurality of projections, such
as 354, depend from the lower surface of support 358. Preferably,
the projections are arranged in an array, such as an 8.times.12,
16.times.24, or 32.times.32 array, although other layouts are
possible. Each of the projections has a cavity at a lower end
region thereof, adapted to receive a bead, as previously
described.
[0172] Elongated legs 367, upon which the unit can rest, extend
downwardly from respective corner regions of frame 360. Preferably,
the legs are of a type permitting adjustments to the height of the
unit. For example, each leg can have threads formed about its outer
circumference adapted for mating engagement with internal threads
of respective bores formed through the frame's corner regions. A
knob, such as 369, can rigidly attach to the upper end of each leg
367 to facilitate manual height adjustments, e.g., by rotating the
legs.
[0173] Upon grasping handle 363, an operator can pick up the unit
350 and place it over a reagent supply, such as plate 20 of FIG. 1.
The reagent-supply locations (wells) of the plate are preferably
arranged in an array having a center-to-center spacing
substantially like the hand-held unit's projection array 356 so
that the two arrays can be aligned. By this construction, each
projection 354 of the projection array 356 can be lowered into a
respective bead-holding well to attract a reagent bead.
[0174] An attraction source is operable at each of the projection
end regions in a manner effective to draw individual beads from the
supply into respective cavities and to releasably retain them
therein. In one embodiment, the attraction source includes a
pressure-control assembly capable of generating a reduced pressure
(vacuum) at each projection end region. With continued reference to
FIGS. 17(A)-17(B), for example, a chamber or manifold 388 can
extend through support 358, over the projection array 356. Each
projection of the projection array can have an axially extending
lumen (not shown) providing fluidic communication between the
cavity at its lower end region and the chamber 388 in support 358.
In this regard, the projections can be formed, for example,
substantially as shown in FIGS. 4(A)-4(B) or FIG. 5. Chamber 388 is
further disposed in fluid communication with a pressure-control
source. Any suitable pressure control source can be utilized. For
example, the pressure control source can be a pump, or a
syringe-type device, operable in one mode to evacuate chamber, and
in a second mode to pressurize chamber. In one preferred
arrangement, shown in FIGS. 17(A)-17(B), the pressure-control
source is a resiliency deformable squeeze-type bottle 391 that is
mounted to an upper side of handle 363. A connector line 393
permits fluid communication between chamber 388 and bottle 391.
Upon deforming or crushing bottle 391 by, for example, a human
hand, fluid (e.g., a gas, such as air) can be forced out, thereby
effecting a pressure increase in chamber 388 and at each projection
end region. When released, bottle 391 substantially returns to its
original shape, creating a suction or vacuum force tending to draw
air out of chamber 388.
[0175] The evacuated chamber 388, in turn, generates a reduced
pressure at each projection end region. To provide control over the
flow of fluid between bottle 391 and chamber 388, a valve assembly,
such as at 395, can be situated along flow line 393. Conveniently,
a thumb-depressible, spring-biased button 397 located near one end
of handle 363, permits manual control (e.g., "on/off") over the
flow of fluid through line 393.
[0176] Once beads have been attracted to, and retained in,
respective cavities of the projection array 356, the beads can be
moved to a deposit location and released. For example, the
hand-held unit 350 can be placed over a bead-receiving substrate,
such as micro-plate 322 of FIG. 17(A), having a plurality of wells
(e.g., 96, 384, 1,024, or more) formed therein. In a preferred
embodiment, the wells are arranged with a center-to-center spacing
substantially like the projection array 356 so that the two arrays
can be aligned. Release of the beads can be accomplished, for
example, by discontinuing the retaining force. For some
applications, the force of gravity, alone, will be sufficient to
cause each bead to fall from a respective cavity down into a
respective well. In other applications, it may be desirable to
further urge the beads out of the cavities. In this regard, one
embodiment contemplates the establishment of an increased pressure
in the chamber above the projection array. This can be effected,
for example, by pressing upon bottle 391 to further deform (crush)
it, thereby forcing some of the remaining air from bottle 391 into
chamber 388 and, consequently, down through the lumen of each
projection. In this way, the beads can be "blown" out of the
cavities down towards the wells.
[0177] In one embodiment, the beads are deposited directly into the
wells of micro-plate 322. In another embodiment, a conduit
assembly, such as 326, is utilized to guide or channel each bead
into a respective well. In the exemplary arrangement of FIG. 17(A),
conduit assembly 326 includes a plurality of large openings formed
along one side, arranged in an array alignable with the projection
array 356. A plurality of small openings are formed in the other
side of the conduit assembly 326, alignable with the array of wells
in micro-plate 322. A generally cone- or funnel-shaped conduit,
such as at 328, extends between each large opening and a respective
one of the small openings.
[0178] Indexing pins, such as 332 and 334, that depend from the
lower side of conduit assembly 326, can assist in registering the
wells of micro-plate 322 with the lower-opening array of the
conduit assembly. Particularly, each indexing pin is alignable with
a respective indexing bore formed through a corresponding region of
micro-plate 322. Insertion of the indexing pins into the indexing
bores substantially aligns the lower-opening array of the conduit
assembly with the array of wells of micro-plate.
[0179] At this point, it can be well appreciated that the bead
dispensing system of the present invention offers a number of
advantages over typical liquid dispensing arrangements. For
example, the volume of each bead can be accurately determined by
measuring it off-line. Also, because beads are solid, they do not
drip or splatter, thereby reducing the likelihood of
cross-contamination. Further, the bead dispensers of the present
invention can be used to dispense multiple reagents without
frequent cleaning, and without a substantial risk of clogging. And,
because of their high-contrast edges, the presence or absence of a
bead at a selected location can be readily determined.
[0180] It is also noteworthy that the process of bead dispensing,
as taught here, is less energetic than typical fluid ejection
systems. For example, ink-jets generally eject fluid at a rate of
kilohertz and at a velocity of several meters per second. The
present invention, on the other hand, can be used to drop beads
into wells at a rate of only one or a few per second. Accordingly,
problems associated with very energetic spot deposition, such as
splattering, breakage, and misdirected ejection (satellites) can
generally be avoided. Notwithstanding the relatively slow rate, as
compared to other deposition methods, the system of the present
invention is comparatively quite fast owing, among other reasons,
to its highly parallel approach.
[0181] In another of its aspects, the present invention provides a
fluid distributor useful, for example, for depositing a liquid in
an array of wells of a micro-plate or card. According to one
embodiment, the fluid distributor includes a plurality of elongated
conduits disposed in fixed, spaced relation in a common support
structure. Each conduit has a large opening at one end and a small
opening at its other end. The large openings are disposed in an
array along one side of the support structure, and the small
openings are disposed in an array along an opposite side of the
support structure. A region of each conduit extending from a
respective one of the small openings is of capillary size, such
that a liquid placed in contact with the small-opening array can be
drawn at least partially into the conduits by capillary action. Any
inner diameter which effects the desired capillary action can be
utilized within the scope of this invention. For example, the
capillary-size regions can be formed with an inner diameter of less
than about 1 mm. To further encourage the desired capillary action,
the capillary-size region of each conduit can be provided with
inner sidewalls that are hydrophilic.
[0182] With reference to FIGS. 18 and 19(A)-19(B), an exemplary
fluid distributor 402 is shown. An array of small openings, as at
404, are provided on one side of distributor 402 and an array of
large openings, as at 406, are provided on an opposite side. The
two arrays of openings can be arranged with a like pitch, or they
can differ from one another. In the illustrated arrangement,
small-opening array 404 is arranged with a substantially reduced
pitch as compared to large-opening array 406. A conduit extends
between each large opening and a respective one of the small
openings. FIG. 18 shows conduits 408a-408f, each having sidewalls
that taper (i.e., decrease in diameter) along the direction from a
respective large opening to a small opening. As shown in FIGS. 18
and 19(A), a groove or channel 412 extends around the small-opening
array 404. And as shown in FIGS. 18 and 19(B), an upstanding
peripheral wall 414 extends around the large-opening array 406 on
the opposite side of the distributor 402.
[0183] With additional reference to FIG. 20, a vessel 418 is
provided for holding a selected fluid, as at 427. Vessel 418 is
provided with a bottom, sidewalls, and an open top. The sidewalls
of vessel 418 are configured to register with channel 412 about the
small-opening array 404 so that each small opening can be placed in
contact with the fluid 427. The small openings, and a region of
their respective conduits, are sized such that capillary action can
draw some of the fluid into each conduit, as at 433 in FIG. 20. The
amount of fluid drawn into each conduit will depend upon the nature
of the fluid, the material composition of the conduit, and the
diameter of each opening and section of conduit thereabove. This
amount can be determined experimentally and/or calculated using
principles well known to those skilled in the art. Upon lifting the
fluid distributor from the vessel, the drawn-in fluid will remain
inside the conduits. The fluid distributor 402 can then be turned
over and placed over a substrate, such as multi-well plate 422
shown in FIGS. 21(A)-21(B) and FIG. 22. It should be noted that the
wells 423 of plate 422 are disposed in an array alignable with the
large-opening array 406 of the fluid distributor 402. Fluid 427 can
then be permitted to flow from the small-opening end to the
large-opening end of each conduit, eventually forming a meniscus at
each end of the inverted large-opening array, as illustrated in
FIG. 21 (A). Upon contacting an object adjacent each large opening,
such as a reagent bead 435, adhesive forces between the liquid and
the object can pull the fluid out of the tubes and into the wells,
as illustrated in FIG. 22.
[0184] It should be noted that vessel 418 can hold any desired
fluid. For example, the fluid deposited in the bead-containing
wells can be a solvent capable of dissolving and/or swelling a
coating material enveloping each bead 435, thereby providing access
to a reagent core.
[0185] Also, it should be appreciated that certain disadvantages
often associated with the deposition of a liquid into a well
already holding a liquid, such as cross-contamination due to
splashing, can be avoided using the fluid distributor of the
present invention, wherein a liquid is touched to a bead in each
well.
[0186] 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. 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.
* * * * *