U.S. patent application number 11/378735 was filed with the patent office on 2006-08-17 for high-throughput screening with multi-through hole testing plate.
Invention is credited to Amy Deming Liu, Volker Schellenberger.
Application Number | 20060183171 11/378735 |
Document ID | / |
Family ID | 26955315 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060183171 |
Kind Code |
A1 |
Schellenberger; Volker ; et
al. |
August 17, 2006 |
High-throughput screening with multi-through hole testing plate
Abstract
A high throughput screening method. At least one liquid sample
is provided that includes an analyte that directly or indirectly
produces a detectable characteristic in one or more first assays. A
plurality of through holes is filled with portions of the liquid
sample in a testing assembly that has a pair of opposing surfaces,
each through hole extending from one of the opposing surfaces to
the other of the opposing surfaces, with the through holes arranged
in group. Surface tension holds the respective portions in the
respective plurality of through holes. A detection is made of which
of the through holes contains a liquid sample portion that includes
said analyte. The detectable characteristic may be produced in
various assays such as an absorbance transcription assay, a
fluorescent transcription assay, a fluorescent secreted enzyme
assay, or a microorganism screening assay.
Inventors: |
Schellenberger; Volker;
(Palo Alto, CA) ; Liu; Amy Deming; (Mountain View,
CA) |
Correspondence
Address: |
BROMBERG & SUNSTEIN LLP
125 SUMMER STREET
BOSTON
MA
02110-1618
US
|
Family ID: |
26955315 |
Appl. No.: |
11/378735 |
Filed: |
March 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10223893 |
Aug 20, 2002 |
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11378735 |
Mar 17, 2006 |
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09970578 |
Oct 4, 2001 |
6436632 |
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10223893 |
Aug 20, 2002 |
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|
09528085 |
Mar 17, 2000 |
6306578 |
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09970578 |
Oct 4, 2001 |
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09471852 |
Dec 23, 1999 |
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09528085 |
Mar 17, 2000 |
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09272122 |
Mar 19, 1999 |
6027873 |
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09471852 |
Dec 23, 1999 |
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Current U.S.
Class: |
435/7.72 |
Current CPC
Class: |
B01J 2219/00286
20130101; B01L 2300/0809 20130101; B01J 2219/00662 20130101; B01J
2219/0052 20130101; B01J 2219/00524 20130101; G01N 35/1065
20130101; B01J 19/0046 20130101; B01L 3/50857 20130101; B01J
2219/00596 20130101; G01N 2035/00237 20130101; B01J 2219/00319
20130101; B01L 2300/0654 20130101; B01J 2219/00533 20130101; B01L
3/5025 20130101; B01J 2219/00317 20130101; B01L 3/5085 20130101;
B01J 2219/00659 20130101; C40B 60/14 20130101; B01J 2219/00644
20130101; B01L 2300/161 20130101; B01J 2219/00707 20130101 |
Class at
Publication: |
435/007.72 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A high throughput screening method comprising: a. providing at
least one liquid sample that includes an analyte that directly or
indirectly produces a detectable characteristic in one or more
first assays; b. providing a testing assembly having a pair of
opposing surfaces and a plurality of through holes, each of said
through holes extending from one of the opposing surfaces to the
other of the opposing surfaces, the through holes arranged in
groups, each of the groups comprising at least two rows and two
columns of through holes; and c. at least partially filling a
plurality of said holes of said assembly with at least portions of
said at least one liquid sample, where surface tension holds the
respective portions in the respective plurality of through holes;
and detecting which of said plurality of through holes contains a
liquid sample portion that includes said analyte.
2. The high throughput screening method of claim 1, wherein said
detectable characteristic is produced in an absorbance
transcription assay.
3. The high throughput screening method of claim 1, wherein said
detectable characteristic is produced in a fluorescent
transcription assay.
4. The high throughput screening method of claim 1, wherein said
detectable characteristic is produced in a fluorescent secreted
enzyme assay.
5. The high throughput screening method of claim 1, wherein said
detectable characteristic is produced in a microorganism screening
assay.
6. The high throughput screening method of claim 1, further
comprising subjecting the respective portions in the respective
plurality of holes to reaction conditions to cause a reaction prior
to detecting which of said plurality of holes contains a liquid
sample portion that includes said analyte.
7. A method comprising: providing a testing assembly having a pair
of opposing surfaces and a plurality of through holes, each of said
through holes extending from one of the opposing surfaces to the
other of the opposing surfaces, the through holes arranged in
groups, each of the groups comprising at least two rows and two
columns of through holes; at least partially filling a plurality of
said through holes with respective liquid sample portions, wherein
surface tension holds the respective liquid sample portions in the
respective plurality of through holes; and subjecting the
respective liquid sample portions to one or more first assays in
the respective plurality of through holes.
8. The method of claim 7, wherein said at least partially filling
comprises immersing the testing assembly into a liquid sample.
9. The method of claim 7, wherein the said one or more first assays
comprises one or more of an absorbance transcription assay, a
fluorescent transcription assay, a fluorescent secreted enzyme
assay, and a microorganism screening assay.
10. The method of claim 7, further comprising analyzing at least
one of said liquid sample portions in at least one of said through
holes.
11. The method of claim 7, wherein at least one of said respective
liquid sample portions includes an analyte that directly or
indirectly produces a detectable characteristic in one or more
first assays; and wherein said method further comprises: detecting
which of said plurality of through holes contains a liquid sample
portion that includes said analyte.
12. The method of claim 7, further comprising stacking the testing
assembly on top of another testing assembly.
13. The method of claim 7, wherein each of said respective through
holes has a constant diameter from one of the opposing surfaces to
the other of the opposing surfaces.
14. The method of claim 13, wherein the constant diameter is about
1 millimeter.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of copending
Ser. No. 10/223,893, filed Aug. 20, 2002, which is a continuation
of U.S. Ser. No. 09/970,578 (issued as U.S. Pat. No. 6,436,632),
which is a continuation of U.S. Ser. No. 09/528,085 (issued as U.S.
Pat. No. 6,306,578), which is a continuation-in-part of U.S. Ser.
No. 09/471,852 (abandoned), which is a continuation of U.S. Ser.
No. 09/272,122 (issued as U.S. Pat. No. 6,027,873). Each of the
patent applications described in this paragraph is hereby
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention is related generally to a screening method
and, more particularly, to a method employing multi-through hole
testing plate for high throughout screening.
BACKGROUND OF THE INVENTION
[0003] Prior testing apparatuses have consisted of a testing plate
with a pair of opposing surfaces and plurality of wells. The wells
extend in from one of the opposing surfaces, but do not extend
through to the other opposing surfaces. The wells are used to hold
samples of solution to be analyzed.
[0004] Although these testing apparatuses work there are some
problems. For example, the wells in these testing apparatuses are
difficult to fill. Special delivery systems, such as large pipette
systems, are needed to fill each of the wells with samples of
solution. These special delivery systems are often expensive and
difficult to operate. As a result, the overall cost of the testing
procedure is increased.
[0005] Another problem with these prior testing apparatuses is with
their construction. The bottom of the wells in these testing plates
need to be transparent so that light can be transmitted through the
samples during testing. However, the rest of the testing plate
needs to be constructed of a non-transparent material. The
construction of a testing apparatus with these characteristics is
difficult and expensive.
[0006] Yet another problem with these prior testing apparatuses is
with the operator locating a particular well in the testing
apparatus. Typically, these testing apparatuses each include large
numbers of wells which are equidistantly spaced apart. As a result,
locating a particular well within the large number of wells is
difficult.
[0007] Accordingly, there is a need for an improved testing
apparatus for high throughput screening.
SUMMARY OF THE INVENTION
[0008] A method for holding samples in accordance with one
embodiment of the present invention includes several steps. First,
a testing plate with a pair of opposing surfaces and a plurality of
holes is provided. Each of the holes extends from one of the
opposing surfaces to the other one of the opposing surfaces. Next,
at least one of the opposing surfaces of the testing plate is
immersed in a solution to be analyzed. A portion of the solution
enters openings for each of the holes in the immersed opposing
surface and any gases in the holes escape though openings for each
of the holes in the other opposing surface. Next, the testing plate
is removed from the solution. Surface tension holds some of the
solution in each of the holes. The opposing surfaces of the testing
plate are then held above a supporting surface and the solution
held in at least one of the holes is analyzed.
[0009] A method for identifying the location at least one sample of
a solution in accordance with another embodiment of the present
invention includes several steps. First, a testing plate with a
pair of opposing surfaces and a plurality of holes is provided.
Each of the holes in the testing plate extend from one of the
opposing surfaces to the other one of the opposing surfaces. The
holes in the plate are arranged in groups. Each of the groups
comprises at least two rows and two columns of holes. Once a
testing plate has been provided, solution is loaded into the holes
and is then analyzed. Based on this analysis, the solution in at
least one hole is identified for further study. The location of the
identified hole is marked based upon the group in which the hole is
found.
[0010] A method for screening a sample in accordance with another
embodiment of the present invention includes several steps. First,
a solution of the sample is prepared for screening. Next, a testing
plate with a pair of opposing surfaces and a plurality of holes is
provided. Each of the holes extends from one of the opposing
surfaces to the other one of the opposing surfaces in the testing
plate. Next, at least one of the opposing surfaces of the testing
plate is immersed in a solution. A portion of the solution enters
openings for each of the holes in the immersed opposing surface of
the testing plate. Once the solution has enter into the holes, the
testing plate is removed from the solution and the surface tension
holds at least some of the solution in the holes. Next, the
solution in one or more of the holes is analyzed.
[0011] An apparatus for holding samples of a solution with cells
for analysis in accordance with another embodiment of the present
invention includes a testing plate with a pair of opposing surfaces
and a plurality of through holes. Each of the holes extends from an
opening in one of the opposing surfaces in the testing plate to an
opening in the other one of the opposing surfaces and is sized to
hold a plurality of the cells. A portion of at least one of the
opposing surfaces of the testing plate where the holes are located
is recessed so that the openings in the testing plate are spaced in
from the opposing surface.
[0012] An apparatus for holding samples for analysis in accordance
with yet another embodiment of the present invention also includes
a testing plate with a pair of opposing surfaces and a plurality of
holes. Each of the holes extends from one of the opposing surfaces
to the other one of the opposing surfaces. The holes are arranged
in groups on the testing plate, where each of the groups comprises
at least two rows and two columns of holes.
[0013] The method and apparatus for holding samples for analysis in
accordance with the present invention provides a number of
advantages. For example, the present invention simplifies testing
procedures. The samples of solution to be analyzed can be loaded
into the testing plate by simply dipping or flooding one of the
surfaces of the testing plate into the solution. As a result, the
present invention does not require the use of a separate delivery
systems for loading solution into the wells on the testing
plate.
[0014] The present invention also simplifies the construction of
the testing apparatus. The testing apparatus merely needs one of
the opposing surfaces of the testing apparatus to be spaced away by
additional spacers or machined to create a recessed portion and
then a plurality of holes need to be drilled through the plate in
the recessed portion. Unlike prior testing apparatuses, the present
invention does not require any special construction techniques to
male the bottom of the wells transparent because the holes extend
all of the way through the plate.
[0015] The present invention also permits an operator to more
easily identify a particular hole filled with a sample for further
analysis. Instead of spacing the holes equidistantly over the
testing plate, the present invention arranges the holes in groups
of at least two columns and two rows of holes and arranges the
groups in sets of at least two or more. The groups are spaces
further apart then the holes within each group and sets of groups
are spaced further apart then the groups are spaced apart. As a
result, an operator can more easily identify a particular hole
based upon which set, group, row, and column the hole is located in
on the testing plate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a top view of a multi-through hole testing plate
in accordance with one embodiment of the present invention;
[0017] FIG. 2 is a cross-sectional view of the multi-through hole
testing plate shown in FIG. 1 taken along lines 2-2;
[0018] FIG. 3 is a perspective, exploded view of another
multi-through hole testing plate in accordance with the present
invention between a pair of evaporation plates;
[0019] FIG. 4 is a block diagram of a testing apparatus with a
multi-through hole testing plate in accordance with another
embodiment of the present invention;
[0020] FIG. 5 is a top view of the multi-through hole testing plate
in accordance with another embodiment of the present invention;
[0021] FIG. 6 is a cross-sectional view of the multi-through hole
testing plate shown in FIG. 5 taking along the lines 6-6;
[0022] FIG. 7 is a top view of a multi-through hole testing plate
in accordance with yet another embodiment of the present
invention;
[0023] FIG. 8 is a top view of a testing plate assembly according
to an embodiment of the present invention; and
[0024] FIG. 9 is a perspective view of the assembly of FIG. 8 shown
in partial cut-away.
DETAILED DESCRIPTION
[0025] A testing apparatus 10 in accordance with one embodiment of
the present invention is illustrated in FIG. 1. The testing
apparatus 10 includes a testing plate 12 with a pair of opposing
surfaces 14 and 16 (surface 16 is shown in FIG. 2) and a plurality
of through holes 18. The through holes 18 are located in recessed
portions 20 and 22 on each side of the testing plate 12. The
through holes 18 are also arranged in groups 24 of at least two
columns and two rows of holes 18 and in sets 26 of two or more
groups of holes 18. The testing apparatus 10 provides a number of
advantages including simplifying the procedure for loading samples
of solution S into the holes 18 in the testing apparatus 10,
simplifying the construction of the testing apparatus 10, and
making the identification of a particular hole 18 filled easier for
an operator.
[0026] Referring to FIGS. 1 and 2, the testing apparatus 10
includes the testing plate 12 which in this particular embodiment
is made of a non-transparent material, such as aluminum and
polypropylene, although other types of materials, such as teflon,
polystyrene, stainless steel, polythylene, any metal or plastic,
can be used. The testing plate 12 could also be made of transparent
materials, such as glass of transparent plastic, when non-optical
means are used for analysis, such as analyzing the materials
blotted on membranes.
[0027] The testing plate 12 includes the pair of opposing surfaces
14 and 16. In this particular embodiment, the opposing surfaces 14
and 16 are substantially planar, except where the recessed portions
20 and 22 are located, although the surfaces 14 and 16 could have
other relationships with respect to each other. Each of the
opposing surfaces 14 and 16 includes one of the recessed portions
20 and 22 which are machined into the testing plate 12, although
other techniques for forming the recessed portions 20 and 22, such
as by molding or adding spaces, can be used. When either of the
opposing surfaces 14 and 16 of the testing plate 12 rests on a
supporting surface 28, the recessed portion 14 or 16 along with the
plurality of holes 18 located in the recessed portion 14 or 16 are
spaced away from the supporting surface 28. If openings 30 and 32
to the holes 18 contacted the supporting surface 28, then any
solutions in the holes 18 would drain out of the holes 18. In this
particular embodiment, a ridge 34 if formed in each of the opposing
surfaces 14 and 16 by the recessed portions 20 and 22 which extends
around the outer circumference of the testing plate 12. Although
the holes 18 are spaced from the support surface 28 by a recessed
portion 20 or 22 formed in the testing plate 12, the holes 18 can
be spaced from the supporting surface 28 with other types of
supporting structures, such as a bracket attached to the testing
plate which supports the testing plate 12 and holes 18 above the
supporting surface 28.
[0028] Referring to FIGS. 5 and 6, another testing apparatus 50 in
accordance with one embodiment of the present invention is
illustrated. The testing apparatus 50 is identical to the test
apparatus 10 shown in FIGS. 1 and 2 except that the testing
apparatus 50 does not include a pair of recessed portions. Instead,
the testing apparatus 50 has a recessed portion 52 and a protruding
portion 54. When the testing plate 51 is placed on a supporting
surface, the recessed portion 52 must be facing the supporting
surface so that the holes are spaced from the supporting surface.
Although one example of the testing apparatus 50 is shown, the
opposing surfaces of the testing plate 51 could have other
configurations. For example, protruding portion 54 could be made
flush with the upper surface of testing plate 51.
[0029] Referring to FIGS. 1-3, the testing plate 12 also includes
an optional handle 36 and an opening 38 on one side of the testing
plate 12 to receive one end of the handle 36, although other
techniques for connecting the handle 36 to the testing plate 12 can
be used, such as connecting the handle 36 with bolts. The handle 36
extends out from the side of the testing plate 12 and is used to
maneuver the testing plate 12 during loading and testing.
[0030] A plurality of through holes 18 are located in the testing
plate 12. The holes 18 extend from openings 30 in the recessed
portion 20 of one of the opposing surfaces 14 to openings 32 in the
recessed portion 22 of the other opposing surface 16. In this
particular embodiment, the holes 18 have a substantially
cylindrical shape, although the holes 18 could have other shapes,
such as a hexagonal cross-sectional shape or a cone shape. In this
particular embodiment, each of the holes 18 has a diameter of about
one millimeter and can hold about hold about 5.5 microliters of
solutions S and cells C, although the diameter, volume and number
of cells C each hole 18 can hold can vary as needed or desired. The
solution S along with cells C in the solution S are held in the
holes 18 by surface tension as shown in FIG. 4. More specifically,
the size of the holes 18 may need to change depending upon the
solution S to be analyzed and that solution's surface tension
properties. For example as understood by one of ordinary skill in
the art, a buffer solution might have different surface tension
properties than a culture media containing salt. There must be
sufficient surface tension to keep the samples of solution S in the
holes 18.
[0031] One of the advantages of the present invention is that the
testing plate 12 is easy to manufacture. A plate having opposing
surfaces can have an appropriate number of holes drilled there
through. The plate can include one or more recessed portions 20,
22, and the through holes can pass through the recessed portion of
the plate 12. Since the holes 18 extend all of the way through,
there is no need for a transparent bottom in each hole 18. Light
transmitted into the holes 18 will pass through during testing.
With prior wells, the testing apparatus also needed to be
non-transparent, but since the wells did not extend through the
apparatus, the bottom of the wells needed to be made of a
transparent material to permit light to pass through the sample for
optical analysis. Constructing these prior testing apparatuses was
difficult and expensive.
[0032] Referring to FIG. 1, the testing plate 12 has about
two-thousand holes 18 which extend through from one opposing
surface 14 to the other opposing surface 16, although the number of
holes 18 can vary as needed or desired. To assist an operator in
identifying a particular hole 18 in this particular embodiment the
holes 18 are arranged in groups and sets of holes 18. Each group 24
contains at least two rows and two columns of holes 18 and each set
26 includes at least two rows and two columns of groups 24. In this
particular embodiment, each group 24 of holes 18 and five rows and
five columns of holes 18 and there are eighty groups 24 of
twenty-five holes 18 in this example, although the number can vary
as needed or desired. The holes 18 in this example are spaced about
1.5 mm apart between rows of holes 18 and between columns of holes
18 within each group 24, although this distance can vary and the
spacing between rows of holes 18 and columns of holes 18 within
each group 24 can be different as needed or desired. In this
particular embodiment, each set of groups 24 includes two rows of
groups 24 and ten rows of groups 24 and there are four sets 26
which contain twenty groups 24 of holes 18 each in this example,
although the number can vary as needed or desired. The groups 24
within a set 26 in this example are spaced about 2.0 mm apart and
the sets 26 of groups 24 of holes 18 in this example are spaced
about 2.5 mm apart, although these distances can vary as needed or
desired.
[0033] By arranging the holes 18 in sets 26 and groups 24, it is
much easier for an operator to identify a particular hole 18 in the
testing plate 12 and retrieve a particular sample. The sets 26 of
holes 18 help the operator identify the general area of the hole 18
and then the groups 24 help the operator to begin to narrow down
the location of the hole 18. The column and row of the hole 18 in
each group 24 provides the precise location of the hole 18. The
spacing between sets 26, groups 24, and rows and columns are
different to make it visually easier for an operator to identify a
particular hole 18. When the holes 18 are all spaced equidistantly
apart, then it is more difficult to identify a particular hole 18
and it is easier for an operator to lose his/her place and select a
sample from the wrong hole 18.
[0034] Although the holes 18 are arranged in groups 24 and sets 26
in testing apparatuses 10 and 50 to aid human operators, other
arrangements for the holes 18 may also be used. For example, when
the testing apparatuses are used by robotics, instead of human
operators, the holes 18 can also be spaced equidistantly apart as
shown in the embodiment of the testing apparatus 60 illustrated in
FIG. 7. The testing apparatus 60 is identical to the testing
apparatuses 10 and 50 described and illustrated earlier except for
the that the holes 18 are equidistantly spaced apart.
[0035] Referring to FIG. 3, the testing apparatus 10 may also
include a pair of optional evaporation plates 40 and 42. The
evaporation plates 40 and 42 are each secured to the one of the
opposing surfaces 14 and 16 of the testing plate 10. The
evaporation plates 40 and 42 are secured to the testing plate 12 by
bolts, clamps, or other mechanical means. When the evaporation
plates 40 and 42 are secured to the testing plate 12 over the
recessed portions 20 and 22, the recessed portions 20 and 22 in the
opposing surfaces 14 and 16 of the testing plate 12 still space the
openings 30 and 32 of the through holes 18 away from the
evaporation plates 40 and 42. The evaporation plates 40 and 42 help
to preserve the samples of solution S in the holes 18 in the
testing plate 12 from evaporation and contamination.
[0036] Instead of a recessed portion in the plate 12, an assembly
comprising the plate and evaporation plates can be provided with
spacers between the testing plate and the evaporation plates to
space the openings of the through holes away from the evaporation
plates. The evaporation plates could be provided with recesses
portions in addition to, or instead of, spacers between the testing
plate and the evaporation plates. Any combination of recessed
portions in the testing plate, recessed portions in the evaporation
plates, or spacers can be used to provide the spacing between the
openings of the through holes and the evaporation plates.
[0037] According to an embodiment of the present invention,
stackable testing plates are provided which may or may not have
evaporation plates in-between testing plates. The stackable testing
plates may be provided with recessed portions or evaporation plates
with recessed portions can be provided between a stacked testing
plate. Any combination of recessed portions in the testing plates,
recessed portions in the evaporation plates, or spacers can be used
to provide a stack of testing plates wherein each testing plate is
spaced from the surface of an adjacent testing plate, evaporation
plate, or both.
[0038] One example of one application of the present invention will
be discussed with reference to testing apparatus 10 shown in FIGS.
1-4. In this particular example, cells C are mutagenized using
ultraviolet, chemical mutagenesis, or other mutagenesis technology.
The cells C are grown to allow for segregation. Once the cells C
have grown, the cells C are diluted to one cell C per ten
microliters in a medium containing a fluorgenic or chromogenic
substrate. For purposes of this example, the medium with the cells
C is referred to as the solution S. As a result, the cells will be
randomly distributed in the holes 18 and many of the holes 18 will
contain one or more cells C.
[0039] Although one example of preparing the solution S and cells C
is disclosed, other methods and techniques for preparing samples to
be used with the testing apparatus 10 can be used as is readily
understood by one of ordinary skill in the art.
[0040] Next, a testing plate 12 with a pair of opposing surfaces 14
and 16 and a plurality of holes 18 which extend from one of the
opposing surfaces 14 to the other one of the opposing surfaces 16
is provided. At least one of the opposing surfaces 14 of the
testing plate is immersed in the prepared solution S. The solution
S enters openings 30 and 32 for each of the holes 18 in testing
plate 12 and any gases in the holes 18 may escape through openings
30 and 32 at the opposite end of the holes 18. Alternatively, the
testing plate 12 may be flooded with solution S so that the
solution S enters through the top opening 30 to each hole 18.
[0041] One of the advantages of the present invention is the ease
with which solution S can be loaded into each of the holes 18. As
illustrated in the description above, all of the holes 18 in the
testing plate 12 can be loaded with samples of solution S in a
relatively short period of time and without any type of specialized
solution delivery system. Prior testing apparatuses with wells
required specialized solution delivery system, such as large
pipette devices, to be able to load solution into each of the
wells. These specialized solution delivery systems are difficult to
use and are expensive.
[0042] Once the solution S has been drawn into the holes 18, the
testing plate 12 is removed from the solution S. Surface tension
holds the solution S in each of the holes 18. In this particular
embodiment, each hole 18 has a diameter of about one millimeter and
holds about 5.5 microliters of solution S and cells C as shown in
FIG. 4, although the diameter and volume of each hole 18 can vary
as needed or desired for the particular application. The handle 36
can be used to manipulate the position of the testing plate 12
during the above-described operations.
[0043] Once the testing plate 12 is removed from the solution S,
the testing plate 12 can be placed on a supporting surface 28.
Since the holes 18 are located in a recessed portion 22 of the
testing plate 12, the openings 22 to the holes 18 are spaced from
the supporting surface 28 so that any solution S being held by
surface tension remains in the holes 18. A pair of evaporation
plates 40 and 42 may be attached to the opposing surfaces 14 and 16
of the testing plate 12 to prevent the samples of solution S in the
testing plate 12 from evaporating or becoming contaminated.
[0044] In this particular example, the testing plate 12 is then
optionally incubated at a controlled temperature of about
37.degree. C. and a humidity of about 70%, although the temperature
and humidity will vary based upon the particular application.
During the incubation, the cells multiply and produce a protein of
interest (the cells could produce an enzyme, an antibody, or a
metabolite which could be of interest). The ability of the protein,
such as an enzyme, to hydrolyze a substrate is analyzed, such as by
measurement of fluorogenic or chromogenic groups liberated by the
hydrolysis.
[0045] Although one example of processing the samples of solution S
in the testing plate 12 is disclosed, other methods and techniques
for processing and analysis the samples can also be used and are
know to those of ordinary skill in the art.
[0046] Next, in this particular example the samples of solution S
with cells C in the holes 18 (as shown in FIG. 4) are tested using
an image analyzer with a light source 44 and a detector 46 in this
particular example. Light is transmitted from the light source 44
towards the openings 30 for the holes 18 in the testing plate 12
and through the solution S in the holes 18 of the testing plate 12.
The detector 46 is positioned on the opposing side of the testing
plate 12 and detects the light which has been transmitted through
the solution S in the holes 18. Based upon the changes in the
detected light from the transmitted light, information about the
characteristics of the particular samples of solution S can be
determined in a manner well known to those of ordinary skill in the
art. In this particular example, the image analyzer is able to
determine which holes 18 contain solution S with the highest
concentration of converted substrate and consequently the highest
amount of enzyme. The target in this case is to retrieve the cells
C which produced the largest amount of enzyme. In a similar way,
cells C which produced the largest amount of a protein or a
chemical of interest could be identified.
[0047] Although one example of analyzing the samples of solution S
in the testing plate 12 using optics is disclosed, other methods
and techniques for analyzing the samples, such as non-optical
methods, can also be used. For example, a plate containing samples
of solution S with cells C could be blotted onto a membrane and
used for performing Western blot analysis or alternatively, the
samples S with cells C could be blotted onto substrate containing
material whereby modification of the substrate is measured
visually. As a result, when non-optical means are used to analyze
the samples of solution in the testing plate 12, the testing plate
12 can be made of a transparent material.
[0048] Next, in this particular example the operator retrieves the
samples of solution S which contain the highest concentration of
converted substrate. The holes 18 with the solution S with the
highest concentration of converted substrate can be identified and
located based upon which set 26 of groups 24, which group 24, and
which row and column within each group 24 each identified hole 18
is located. One of the advantages of the present invention is the
arrangement of the holes in groups 24 and sets 26 which enables an
operator to easily identify a particular hole 18 on the testing
plate 12. Once the desired samples are retrieved, the operator can
conduct further analysis on those samples in manners well known to
those of ordinary skill in the art.
[0049] Although one example of retrieving one or more of the
samples of solution S in the testing plate 12 is disclosed, other
methods and techniques for retrieving samples can also be used. For
example, if robotics are used to located and retrieve a particular
sample, a different testing apparatus, such as testing apparatus 60
shown in FIG. 7, could be used. The robotics would not need the
holes 18 to be arranged in groups 24 and sets 26 of holes 18,
although such an arrangement may even aid the robotics in
identifying and retrieving the desired sample.
[0050] According to some embodiments of the present invention, the
testing plate is in the form of an assembly or substrate. For
example, the plate can comprise a plurality of individual
components which together make up an assembly having opposing
surfaces and a plurality of through holes extending from one
surface to the other. An example of the present invention wherein
the testing plate comprises such an assembly is a plate made of a
bundle of capillary tubes as shown in FIGS. 8 and 9.
[0051] As shown in FIGS. 8 and 9, a plate, substrate or assembly 70
comprises a bundle of capillary tubes 72 bound together by a band
74. The through holes of the assembly according to this embodiment
are the longitudinally-extending holes through the center of each
capillary tube. The band 74 may have opposing surfaces 76 and 78,
each of which is substantially planar and substantially parallel to
the other. The band can be made of metal, plastic, glass, rubber,
elastomeric compound, or any other suitable material. Each
capillary tube 72 has a first end 80 and a second end 82. The first
ends 80 of the capillary tubes make up an opposing surface 84 of
the substrate or assembly 70 and the second ends 82 of the
capillary tubes 72 made up an opposing surface 86 of the substrate
or assembly.
[0052] As can be seen in FIGS. 8 and 9, each capillary tube 72 of
the bundle which makes up substrate or assembly 70 has a length
between its first end 80 and its second end 82 which is at least
two times greater than the average diameter of each tube.
Preferably, the length of each tube is more than four times greater
than the average diameter of each tube and is preferably many times
greater than the average diameter. Each capillary tube may be, for
example, in the form of a microcapillary tube or a hollow
fiberoptic fiber.
[0053] The capillary tubes may be hollow cylindrical in shape or
may have other rounded, oval, or polygonal cross-sections. The
average diameter of each capillary tube preferably ranges from
about 0.001 millimeter to about 1 millimeter, and the length of
each tube preferably ranges from about 1 mm to about 1 cm. The
dimensions of the capillary tubes are preferably such that each
tube has the capacity to hold from about 0.0001 microliter to about
10 microliters of liquid sample, for example, about 5.5
microliters, although the diameters, lengths, and holding
capacities of the capillary tubes may vary as needed or desired.
According to some embodiments of the present invention, it is not
necessary to have a band for holding the capillary tubes together
in a bundle as the tubes may instead be fused or otherwise bonded,
adhered, or maintained together in a bundle.
[0054] The number of capillary tubes of the embodiment in FIGS. 8
and 9 is preferably from about 100 to over 1,000 capillary tubes,
for example, from about 500 to about 1,500. Preferably, the tubes
are arranged in rows and preferably the rows are arranged in
columns. Although in the embodiment shown in FIGS. 8 and 9 the
bundle of capillary tubes 72 has a circular cross-section and the
band 74 is ring shaped, other shapes of the bundle and band are
also within the scope of the present invention. For example, a
rectangular or square array of capillary tubes can be provided and
surrounded by a band, and the band would also preferably be of
rectangular or square shape. With rectangular or square-shaped
arrays of capillary tubes, distinct columns and rows of capillary
tubes can be easily identified, facilitating the identification of
a single capillary tube within the array.
[0055] In embodiments such as the one shown in FIGS. 8 and 9, the
band 74 surrounding the bundle of capillary tubes has a length
between opposing surfaces 76 and 78 that is greater than the length
between the opposing ends 80 and 82 of the capillary tubes. As a
result, the banded assembly can be placed on a surface of, for
example, an analytical device, without the ends of the capillary
tubes touching the surface. In addition, the assemblies can be
stacked without disturbing the capillary holding forces in the
through holes.
[0056] The assembly shown in FIGS. 8 and 9, as with the plates of
FIGS. 1-7, can be loaded or filled with a starting liquid sample to
provide a plurality of samples, each constituting a portion of the
starting liquid sample. Alternatively, the assembly can be loaded
with more than one starting liquid sample, with each starting
liquid sample filling at least one of the through holes. Herein, by
"loaded" or "filled", what is meant is at least partially filled,
but not necessarily fully filled. The through-holes can be loaded
or filled, for example, by immersing the assembly or plate in a
liquid sample, contacting at least one of the opposing surfaces of
the assembly or plate with a liquid sample, or contacting the inner
walls of the respective through holes with a liquid sample or with
respective liquid samples.
[0057] Contact between a liquid sample and an opposing surface can
be made by flooding, immersing, pipetting, dropping, pouring, or
otherwise loading or at least partially filling a plurality of the
capillary tubes or through holes such that capillary action pulls
portions of the liquid sample into the respective capillary tubes
or through holes. Upon removal or discontinued contact of the
liquid sample with the assembly or plate, the opposing surfaces of
the assembly or plate are preferably made free of liquid sample
such that the portions of the sample that remain held within the
respective capillary tubes are isolated from one another.
[0058] Automated filling devices can be used and are preferred if
it is important that the respective liquid samples or liquid sample
portions are to only contact the inner walls of the through holes
and avoid contacting the opposing surfaces of the assembly.
[0059] According to embodiments of the present invention, a high
throughput screening method is provided. The method can screen for
at least one liquid sample that includes a target component or
substance to be analyzed. Herein, the target component or substance
to be analyzed may be referred to as an "analyte". The analyte may
be, but is not necessarily, a biological sample. The analyte
exhibits a detectable property or produces a detectable
characteristic in the presence of or upon reaction with a marker
compound or the like. For example, the analyte may itself exhibit a
fluorescent property. After the liquid sample is at least partially
filled into a plurality of the through holes, the portions of the
liquid sample that contain the analyte can be detected by
determining which of the through holes contains a sample portion
that exhibits the fluorescent property.
[0060] In another example, the analyte itself does not exhibit a
detectable property but may instead cause a marker component to
exhibit a detectable property upon reaction with the marker
component. According to such an embodiment, the through holes of
the testing assembly can be pre-loaded or post-loaded with one or
more marker components such that after loading the liquid sample
into the plurality of through holes, the sample portions containing
an analyte can react with the marker compound and thus enable the
marker compound to exhibit a detectable property. In such a case,
it is not the analyte itself that exhibits the detectable property,
but rather the analyte is detected indirectly as the presence of
the analyte causes the detectable property of the marker component
which in turn is directly detected. In so doing, the methods of the
present invention provide a way to partition and isolate analytes
from an original liquid sample.
[0061] According to the high throughput screening method, portions
of the liquid sample are loaded into a testing assembly having a
pair of opposing surfaces and a plurality of through holes, with
each of the through holes extending from one of the opposing
surfaces to the other of the opposing surfaces. Loading preferably
results in at least partially filling a plurality of the through
holes with at least portions of the liquid sample, and surface
tension holds the respective portions in the respective plurality
of through holes. Multiple liquid samples can instead be loaded
into respective through holes or into respective pluralities of
through holes. The method then involves detecting which of the
plurality of sample portions in the through holes exhibit the
detectable property.
[0062] According to embodiments of the present invention, the high
throughput screening assembly preferably comprises at least about
100 through holes, more preferably at least about 500 through
holes, and according to some embodiments of the present invention,
up to about 1,000,000 through holes. High throughput screening
methods can be used in conjunction with these devices to test over
100,000,000 samples or sample portions per assembly per day. The
analyte to be screened may be, for example, a biological cell, a
mixture of biological cells, a mutant cell, a secretable protein,
an enzyme, a microorganism, a mixture of microorganisms, a
contaminant, or combinations thereof. The analyte can be a
population of random mutants of one or more organisms. If the
analyte is a mixture of biological cells it could be a random
sample isolated from a natural environment. The detectable property
may be, for example, a fluorescence or adsorption property. Prior
to filling the high throughput assembly, the liquid sample may be
diluted with a suitable diluent to obtain a concentration of the
analyte in the liquid sample such that when the sample is filled
into the plurality of through holes, at least one of the analytes
is introduced into from about one-quarter to about one-half of the
plurality of through holes.
[0063] In some cases, it is possible to identify an organism with
desirable properties even if the organism is introduced into a
plurality of through holes as a mixture with other organisms. Under
such conditions, the mixture of other organisms, e.g., mixture of
biological cells, may be diluted prior to filling such that several
organisms or cells will be introduced into each through hole. Using
such a dilution technique, it is possible to detect the presence of
an analyte. For example, it is possible to detect one particular
mutant from a collection of many biological cells and mutants
thereof despite having many cells from the mixture present in each
through hole. Thus, for example, if a sample contains 1,000,000
cells and only one of them is a target mutant cell, referred to as
the "analyte", and a testing plate having 10,000 through holes is
employed, the sample can be diluted such that the 1,000,000 cells
fill the through holes with sample portions wherein each portion
contains about 100 cells. In cases where the detectable
characteristic of the analyte is detectable despite the presence of
many other cells within the same through hole, it is possible to
isolate the analyte from 99.99% of the sample in a single
assay.
[0064] The testing plates used in accordance with the present
invention, including the plates of FIGS. 1-7 and the assemblies of
FIGS. 8 and 9, can comprise hydrophilic materials or coatings,
hydrophobic materials or coatings, or a combination thereof to
facilitate loading of liquid sample portions into the through
holes. For example, the opposing surfaces of the assembly can be
made of, or treated with, a hydrophobic material such that liquid
samples tend to be repelled from the surface except in areas
immediately adjacent the through hole openings on the opposing
surface. According to such an embodiment, liquid sample portions
can be drawn into the through holes by capillary action without
wetting-out onto the opposing surfaces of the plate. As a result,
once the plate is loaded with and separated from a liquid sample no
fluid communications are provided between individual through holes
and contamination of the partitioned sample portions is minimized.
According to some embodiments of the present invention, the through
holes can include inner walls made of, or coated with, a
hydrophilic material that can be easily wetted by an aqueous sample
or medium. The entire inner walls of each through hole can be made
of or treated with a hydrophilic material or only portions of the
inner wall can be so made or treated. Plates having hydrophilic
inner walls for the through holes and hydrophobic opposing surfaces
provide excellent means to restrain, isolate, or limit the position
of liquid samples in the through holes of the testing plate while
keeping adjacent surface regions of the opposing surfaces
substantially free of liquid sample.
[0065] According to some embodiments of the present invention, to
facilitate the capillary reaction, it may be desirable to provide a
hydrophilic material immediately adjacent the opening to each
through hole on an opposing surface while maintaining or providing
the remaining area of the opposing surface hydrophobic or
non-hydrophilic. Either or both opposing surfaces of the testing
plate can be made of or treated with hydrophobic, hydrophilic, or
both materials as discussed above although if the through holes are
to be loaded by an immersion technique, it is preferred that the
opposing surface which will come in contact with the liquid sample
is treated with or formed of a hydrophobic material except in areas
immediately adjacent and preferably surrounding the through hole
openings in the opposing surface.
[0066] Exemplary high throughput screening methods that can be used
with the assemblies and other plates of the present invention
include absorbance transcription assays, fluorescent transcription
assays, fluorescent secreted enzyme assays, and microorganism
screening assays. These and other suitable assays that can benefit
from the plates and methods of the present invention are described,
for example, in: Arndt et al., A rapid genetic screening system for
identifying gene-specific suppression constructs for use in human
cells, Nucleic Acids Res., 28(6): E15 (2000); Rolls et al., A
visual screen of a GFP-fusion library identifies a new type of
nuclear envelope membrane protein, J. Cell Biol, 146(1): 29-44
(1999); Sieweke, Detection of transcription factor partners with a
yeast one hybrid screen, Methods Mol. Biol., 130: 59-77 (2000); and
WO 97/37036, all of which are herein incorporated in their
entireties by reference.
[0067] Having thus described the basic concept of the invention, it
will be rather apparent to those skilled in the art that the
foregoing detailed disclosure is intended to be presented by way of
example only, and is not limiting. Various alternations,
improvements, and modifications will occur and are intended to
those skilled in the art, though not expressly stated herein. These
alterations, improvements, and modifications are intended to be
suggested hereby, and are within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims and equivalents thereto.
* * * * *