U.S. patent application number 10/856039 was filed with the patent office on 2004-11-04 for methods of detecting targets on an arrary.
Invention is credited to Carlson, Edward, Coblentz, Kenneth D., Dickinson, Todd.
Application Number | 20040219590 10/856039 |
Document ID | / |
Family ID | 25460499 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040219590 |
Kind Code |
A1 |
Dickinson, Todd ; et
al. |
November 4, 2004 |
Methods of detecting targets on an arrary
Abstract
The invention relates to methods of detecting a target analyte
in a biological sample using composite microsphere arrays having
first and second assay locations. Preferred target analytes include
nucleic acid, and more specifically, nucleic acid having one or
more single nucleotide polymorphisms (SNPs).
Inventors: |
Dickinson, Todd; (San Diego,
CA) ; Coblentz, Kenneth D.; (Del Mar, CA) ;
Carlson, Edward; (Oceanside, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
25460499 |
Appl. No.: |
10/856039 |
Filed: |
May 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10856039 |
May 27, 2004 |
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09931271 |
Aug 16, 2001 |
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6770441 |
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09931271 |
Aug 16, 2001 |
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09782588 |
Feb 12, 2001 |
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60181631 |
Feb 10, 2000 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
B01J 19/0046 20130101;
G01N 21/6452 20130101; B01J 2219/00511 20130101; B01J 2219/00619
20130101; B01J 2219/0072 20130101; C40B 60/14 20130101; B01J
2219/00659 20130101; B01L 2300/0654 20130101; B01J 2219/00317
20130101; B01J 2219/00662 20130101; G01N 21/6428 20130101; B01J
2219/0061 20130101; B01J 2219/00644 20130101; B01J 2219/00596
20130101; B01J 2219/005 20130101; B01J 2219/00637 20130101; B01J
2219/00274 20130101; B01J 2219/00677 20130101; B01L 2300/0636
20130101; B01J 2219/00648 20130101; B01J 2219/00605 20130101; B01J
2219/00702 20130101; B01J 2219/00585 20130101; B01L 3/5085
20130101; B01J 2219/00626 20130101; B01J 2219/00612 20130101; B01J
2219/00621 20130101; B82Y 30/00 20130101; B01J 2219/0063 20130101;
B01J 2219/00628 20130101; B01L 2300/0822 20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. A method of detecting a target analyte in a biological sample,
comprising: a) providing a composite array comprising: a substrate
having a surface; a first and a second assay location on said
surface, wherein said assay locations comprise a population of
microspheres, and wherein said microspheres comprise bioactive
agents; a physical partition separating said first assay location
from said second assay location; b) adding said biological sample
to said first assay location under conditions sufficient to allow
said target analyte to bind to said bioactive agents; and c)
detecting the binding of said bioactive agents to said target
analyte.
2. The method of claim 1, wherein the binding of said bioactive
agents to said target analyte is detected by a change in an optical
signature of the microspheres.
3. The method of claim 1, wherein said target analyte comprises a
nucleic acid.
4. The method of claim 3, wherein said bioactive agents comprise a
nucleic acid.
5. The method of claim 4, wherein said microspheres further
comprise labeled single-stranded nucleic acid sequences hybridized
to said bioactive agents.
6. The method of claim 1, wherein said microspheres are randomly
distributed on said surface at said assay locations.
7. The method of claim 1, wherein said microspheres are separated
by a distance of less than 25 .mu.m.
8. The method of claim 1, wherein said microspheres are separated
by a distance of less than 15 .mu.m.
9. The method of claim 1, further comprising detecting a target
analyte in a second biological sample by adding said second
biological sample to said second assay location and thereafter
detecting the binding of said bioactive agents to said target
analyte.
10. The method of claim 1, wherein said substrate comprises a
microscope slide.
11. The method of claim 1, wherein said first and second assay
locations are separately sealed to form hybridization chambers.
12. The method of claim 11, wherein said hybridization chambers
comprise flexible membranes.
13. The method of claim 1, wherein said physical partition is a
non-permanent sealant.
14. The method of claim 13, wherein said non-permanent sealant is
selected from the group consisting of: rubber, silicon, petroleum
jelly, wax and parafilm.
15. The method of claim 1, wherein said physical partition is a
ridge or rim of sufficient width and height to prevent said
bioactive agents from moving from said first assay location to said
second assay location.
16. The method of claim 1, wherein said physical partition is a
trough of sufficient width and depth to prevent said bioactive
agents from moving from said first assay location to said second
assay location.
17. The method of claim 1, wherein said physical partition is a
gasket.
18. The method of claim 17, wherein said gasket is adapted to fit
within an indentation or channel on the substrate.
19. The method of claim 1, wherein said target analyte comprises a
detectable optical label.
20. The method of claim 19, wherein said optical label is a
fluorophore.
21. The method of claim 1, wherein said biological sample comprises
blood.
22. The method of claim 1, wherein said bioactive agents comprise a
detectable optical label.
23. The method of claim 1, wherein said detectable label is a
fluorophore.
24. A method of detecting a target nucleic acid in a biological
sample, wherein said target nucleic acid comprises one or more
single nucleotide polymorphisms (SNPs) at one or more predetermined
positions, comprising: a) providing a composite array comprising: a
substrate having a surface; a population of microspheres, wherein
said microspheres are linked to capture probes configured to bind
to said target nucleic acid at said one or more predetermined
positions; a first and second assay location on said surface,
wherein said assay locations comprise said population of
microspheres; a physical partition separating said first assay
location from said second assay location; b) adding said biological
sample to said first assay location under conditions sufficient to
allow said target nucleic acid to bind to said capture probes; and
c) detecting the binding of said target nucleic acid to said
capture probes.
25. The method of claim 24, wherein the binding of said capture
probes to said target nucleic acid is detected by a change in an
optical signature of the microspheres.
26. The method of claim 24, wherein said microspheres are randomly
distributed on said surface at said assay locations.
27. The method of claim 24, wherein said microspheres are separated
by a distance of less than 25 .mu.m.
28. The method of claim 24, wherein said microspheres are separated
by a distance of less than 15 .mu.m.
29. The method of claim 24, further comprising detecting a target
analyte in a second biological sample by adding said second
biological sample to said second assay location and thereafter
detecting the binding of said capture probes to said target
analyte.
30. The method of claim 24, wherein said substrate comprises a
microscope slide.
31. The method of claim 24, further comprising adding a SNP
allele-specific probe, configured to hybridize to said target
nucleic acid, wherein said SNP allele-specific probe is labeled
with a fluorophore.
32. The method of claim 24, wherein said physical partition is a
non-permanent sealant.
33. The method of claim 32, wherein said non-permanent sealant is
selected from the group consisting of rubber, silicon, petroleum
jelly, wax and parafilm.
34. The method of claim 24, wherein said physical partition is a
ridge or rim of sufficient width and height to prevent said capture
probes from moving from said first assay location to said second
assay location.
35. The method of claim 24, wherein said physical partition is a
trough of sufficient width and depth to prevent said capture probes
from moving from said first assay location to said second assay
location.
36. The method of claim 24, wherein said physical partition is a
gasket.
37. The method of claim 36, wherein said gasket is adapted to fit
within an indentation or channel on the substrate.
38. The method of claim 24, wherein said biological sample
comprises blood.
39. The method of claim 24, wherein said target nucleic acid having
one or more SNPs is causatively linked to a human disease.
40. The method of claim 39, wherein said disease is Alzheimer's
disease.
41. The method of claim 24, wherein said target nucleic acid
comprises a detectable optical label.
42. The method of claim 41, wherein said optical label is a
fluorophore.
43. The method of claim 24, wherein said microspheres further
comprise labeled single-stranded nucleic acid sequences hybridized
to said capture probes.
44. The method of claim 24, wherein said capture probes comprise a
detectable optical label.
45. The method of claim 44, wherein said optical label is a
fluorophore.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/931,271, filed Aug. 16, 2001, which is a
continuation-in-part of U.S. application Ser. No. 09/782,588, filed
Feb. 12, 2001, which claims priority to Provisional Application No.
60/181,631, filed Feb. 10, 2000, all of which are incorporated by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to methods of detecting a target
analyte in a biological sample using composite microsphere arrays
having first and second assay locations. Preferred target analytes
include nucleic acid, and more specifically, nucleic acid having
one or more single nucleotide polymorphisms (SNPs).
[0004] 2. Description of the Related Art
[0005] There are a number of assays and sensors for the detection
of the presence and/or concentration of specific substances in
fluids and gases. Many of these rely on specific ligand/antiligand
reactions as the mechanism of detection. That is, pairs of
substances (i.e. the binding pairs or ligand/antiligands) are known
to bind to each other, while binding little or not at all to other
substances. This has been the focus of a number of techniques that
utilize these binding pairs for the detection of the complexes.
These generally are done by labeling one component of the complex
in some way, so as to make the entire complex detectable, using,
for example, radioisotopes, fluorescent and other optically active
molecules, enzymes, quantum dots etc.
[0006] Of particular use in these sensors are detection mechanisms
utilizing luminescence. Recently, the use of optical fibers and
optical fiber strands in combination with light absorbing dyes for
chemical analytical determinations has undergone rapid development,
particularly within the last decade. The use of optical fibers for
such purposes and techniques is described by Milanovich et al.,
"Novel Optical Fiber Techniques For Medical Application",
Proceedings of the SPIE 28th Annual International Technical
Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz,
W.R., "Chemical Sensors Based On Immobilized Indicators and Fiber
Optics" in C.R.C. Critical Reviews In Analytical Chemistry, Vol.
19, 1988, pp. 135-173; Wolfbeis, O. S., "Fiber Optical
Fluorosensors In Analytical Chemistry" in Molecular Luminescence
Spectroscopy, Methods and Applications (S. G. Schulman, editor),
Wiley & Sons, New York (1988); Angel, S. M., Spectroscopy 2
(4):38 (1987); Walt, et al., "Chemical Sensors and
Microinstrumentation", ACS Symposium Series, Vol. 403, 1989, p.
252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors, Ed. CRC
Press, Boca Raton, Fla., 1991, 2nd Volume.
[0007] More recently, fiber optic sensors have been constructed
that permit the use of multiple dyes with a single, discrete fiber
optic bundle. U.S. Pat. Nos. 5,244,636 and 5,250,264 to Walt, et
al. disclose systems for affixing multiple, different dyes on the
distal end of the bundle, the teachings of each of these patents
being incorporated herein by this reference. The disclosed
configurations enable separate optical fibers of the bundle to
optically access individual dyes. This avoids the problem of
deconvolving the separate signals in the returning light from each
dye, which arises when the signals from two or more dyes are
combined, each dye being sensitive to a different analyte, and
there is significant overlap in the dyes' emission spectra.
[0008] U.S. Ser. Nos. 08/818,199 and 09/151,877 describe array
compositions that utilize microspheres or beads on a surface of a
substrate, for example on a terminal end of a fiber optic bundle,
with each individual fiber comprising a bead containing an optical
signature. Since the beads go down randomly, a unique optical
signature is needed to "decode" the array; i.e. after the array is
made, a correlation of the location of an individual site on the
array with the bead or bioactive agent at that particular site can
be made. This means that the beads may be randomly distributed on
the array, a fast and inexpensive process as compared to either the
in situ synthesis or spotting techniques of the prior art. Once the
array is loaded with the beads, the array can be decoded, or can be
used, with full or partial decoding occurring after testing, as is
more fully outlined below.
[0009] In addition, compositions comprising silicon wafers
comprising a plurality of probe arrays in microtiter plates have
been described in U.S. Pat. No. 5,545,531.
SUMMARY OF THE INVENTION
[0010] Embodiments of the invention relate to methods of detecting
a target analyte in a biological sample, comprising providing a
composite array comprising a substrate having a surface; a first
and a second assay location on said surface, wherein said assay
locations comprise a population of microspheres, and wherein said
microspheres comprise bioactive agents; a physical partition
separating said first assay location from said second assay
location; adding said biological sample to said first assay
location under conditions sufficient to allow said target analyte
to bind to said bioactive agents; and detecting the binding of said
bioactive agents to said target analyte.
[0011] In more specific embodiments, the binding of the bioactive
agents to the target analyte can be detected by a change in an
optical signature of the microspheres. Target analytes and
bioactive agents can include a nucleic acid, for example.
[0012] In other embodiments, the microspheres can include labeled
single-stranded nucleic acid sequences hybridized to said bioactive
agents. In other embodiments, microspheres can be randomly
distributed on said surface at said assay locations. In further
aspects, microspheres can be separated by a distance of less than
25 .mu.m. In more specific embodiments, the microspheres can be
separated by a distance of less than 15 .mu.m.
[0013] Methods herein, can further comprise detecting a target
analyte in a second biological sample by adding said second
biological sample to said second assay location and thereafter
detecting the binding of said bioactive agents to said target
analyte. In certain aspects the substrate can include a microscope
slide.
[0014] In further embodiments, the first and second assay locations
can be separately sealed to form hybridization chambers. In an
advantageous embodiment, the hybridization chambers include
flexible membranes.
[0015] The physical partitions described herein can be a
non-permanent sealant, such as rubber, silicon, petroleum jelly,
wax and parafilm. In other embodiments, the physical partition is a
ridge or rim of sufficient width and height to prevent the
bioactive agents from moving from the first assay location to the
second assay location. In still further aspects, the physical
partition can be a trough of sufficient width and depth to prevent
the bioactive agents from moving from the first assay location to
the second assay location.
[0016] In advantageous embodiments, the physical partition is a
gasket. More particularly, the gasket can be adapted to fit within
an indentation or channel on the substrate.
[0017] In other embodiments, the target analyte includes a
detectable optical label, such as a fluorophore, for example.
[0018] Advantageous biological samples can include blood, for
example. Additionally, bioactive agents can include a detectable
optical label, such as a fluorophore, for example.
[0019] Further methods include detecting a target nucleic acid in a
biological sample, wherein said target nucleic acid includes one or
more single nucleotide polymorphisms (SNPs) at one or more
predetermined positions, comprising providing a composite array
comprising a substrate having a surface; a population of
microspheres, wherein said microspheres are linked to capture
probes configured to bind to said target nucleic acid at said one
or more predetermined positions; a first and second assay location
on said surface, wherein said assay locations comprise said
population of microspheres; a physical partition separating said
first assay location from said second assay location; adding said
biological sample to said first assay location under conditions
sufficient to allow said target nucleic acid to bind to said
capture probes; and detecting the binding of said target nucleic
acid to said capture probes.
[0020] In more specific embodiments, the binding of said capture
probes to said target nucleic acid can be detected by a change in
an optical signature of the microspheres. Advantageously, the
microspheres can be randomly distributed on said surface at said
assay locations.
[0021] In other embodiments, the microspheres can be separated by a
distance of less than 25 .mu.m, or by a distance of less than 15
.mu.m.
[0022] Methods herein, can further comprise detecting a target
analyte in a second biological sample by adding the second
biological sample to the second assay location and thereafter
detecting the binding of the capture probes to the target
analyte.
[0023] In particular embodiments, the substrate can include a
microscope slide.
[0024] Further embodiments, include adding a SNP allele-specific
probe, configured to hybridize to said target nucleic acid, wherein
the SNP allele-specific probe is labeled with a fluorophore.
[0025] Additional aspects of the teachings herein include using a
target nucleic acid having one or more SNPs causatively linked to a
human disease, such as Alzheimer's disease.
[0026] Further methods include using microspheres having labeled
single-stranded nucleic acid sequences hybridized to the capture
probes. In particular aspects, the capture probes can include a
detectable optical label, such as a fluorophore, for example.
[0027] The invention also provides array compositions comprising a
rigid support; a molded layer with at least a first assay location
comprising discrete sites, where the molded layer is adhered to the
rigid support; a layer of bonding agent adhering the rigid support
to the molded layer; and a population of microspheres comprising at
least a first and a second subpopulation, where the first
subpopulation comprises a first bioactive agent and the second
subpopulation comprises a second bioactive agent where the
microspheres are randomly distributed on the sites.
[0028] The invention also provides a method for making an array
composition containing at least a first assay location having
discrete sites comprising the steps of contacting a surface of a
template structure, the surface comprising one or more sets of
projections, with a moldable material; removing the moldable
material from the surface of the template structure, whereby the
removed moldable material forms a molded layer with at least a
first assay location comprising discrete sites; adhering the molded
layer to a rigid support; and randomly distributing microspheres on
the molded layer such that individual discrete sites comprise
microspheres, where the microspheres comprise at least a first and
a second subpopulation, where the first subpopulation comprises a
first bioactive agent and the second subpopulation comprises a
second bioactive agent.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A, 1B, 1C, 1D and 1E depict several different "two
component" system embodiments of the invention. In FIG. 1A, a bead
array is depicted. The first substrate 10 has array locations 20
with wells 25 and beads 30. The second substrate 40 has assay
locations 45. An optional lens or filter 60 is also shown; as will
be appreciated by those in the art, this may be internal to the
substrate as well. FIG. 1B is similar except that beads are not
used; rather, array locations 20 have discrete sites 21, 22, 23,
etc. that may be formed using spotting, printing, photolithographic
techniques, etc. FIGS. 1C-F depict the use of a plurality of first
substrates. FIG. 1C depicts a "bead of beads" that may have
additional use for mixing functions. FIG. 1D depicts a plurality of
bead arrays and FIG. 1E depicts a plurality of non-bead arrays.
FIG. 1F depicts the use of binding functionalities to "target"
first substrates 10 to locations on the second substrate 40; as
will be appreciated by those in the art, this may be done on flat
second substrates or on compartmentalized second substrates. FIG.
1F utilizes binding ligand pairs 70/70', 71/71', 72/72', etc. These
may be either chemical functionalities or biological ones, such as
are described for IBL/DBL pairs, such as oligonucleotides, etc.
[0030] FIGS. 2A and 2B depict two different "one component"
systems. FIG. 2A depicts a bead array, with the substrate 50 having
assay locations 45 with wells 25 comprising beads 30. FIG. 2B
depicts a non-bead array; each assay location 45 has discrete sites
21, 22, 23, etc.
[0031] FIG. 3 depicts clustering in hyperspectral alpha space
(a1=I1/SII, a2=I2/SIi, a3=I3/SIi, etc.). A set of 128 different
bead types present on a fiber bundle were decoded with by
hybridizing set of complementary oligonucleotides labeled with four
dyes: Bodipy-493, Bodipy-R6G, Bodipy-TXR, and Bod-564 (only one dye
per oligonucleotide). Shown is the second stage of a four stage
decode in which 4013 beads were decoded. Ovals are drawn around
zones of hue clusters.
[0032] FIG. 4 Illustrates a two color decoding process wherein
either FAM-labeled or Cy3-labeled oligo complements are use to
"paint" (label) the different bead types on the array.
[0033] FIG. 5 depicts the decoding 128 different bead types with
four colors and four decode stages. (inset shows a single decode
stage using four different dyes to decode 16 bead types.)
[0034] FIG. 6 depicts grey scale decoding of 16 different bead
types. (A) Combinatorial pooling scheme for complementary decoding
oligos. A (B) Two independent normalizing images were acquired, and
the resulting bead intensities compared. (C) The alpha values
(ratio of bead intensity in indicated decode stage to intensity in
normalization image) are plotted for three decodes stage described
in (A).
[0035] FIG. 7 schematically depicts the lid and base plate. A.
Depicts the lid 80 and base plate 90 of the hybridization chamber.
Ports 82 in the lid allow for fiber optic bundles 84 to be inserted
through the lid and contact the sample in the wells of the
microtiter plate 86 in the base cavity 88 of the base plate 90. B.
Depicts the base cavity 88 of the base plate 90.
[0036] FIG. 8 schematically depicts the hybridization chamber
including the lid 80 and base plate 90. Also shown are the
peripheral seal 94, the clamp 96 and clamp receptacle 98, fiber
optic bundles 84 inserted through the lid and into the well of the
microtiter plate 86.
[0037] FIG. 9 depicts a base plate with holes 102. A Depicts the
holes 102 in the base plate. B Depicts channels 100 connecting the
holes 102.
[0038] FIG. 10 depicts variable solution volume and localization on
the membrane caused by pressure and/or vacuum. A. +P indicates
pressure; -P indicates vacuum. Upward bending of the membrane in
response to pressure in all chambers and holes. B. Fluid is moved
to the left side of the membrane when vacuum is applied to the left
chambers and pressure is applied to the middle and right chambers.
C. When vacuum is first applied to the left section, fluid fills
the wells. When vacuum is subsequently applied to the middle and
right chambers, empty wells are formed. D. Fluid moves to the
center of the membrane when vacuum is applied to the center and
pressure is applied to left and right chambers. E. Fluid fills in
wells formed by high vacuum in the center. Empty wells form on the
left and right when low vacuum is applied. F. Fluid moves to the
right when vacuum is applied to the right chamber and pressure is
applied to the left and middle chambers.
[0039] FIG. 11 depicts a flow chart of a representative assay
scheme that finds use with the hybridization chamber.
[0040] FIG. 12 depicts an array of arrays in a microscope slide
format.
[0041] FIG. 13 depicts a mold for making arrays.
[0042] FIG. 14 depicts formation of a submaster structure and use
of the submaster structure to form a molded layer adhered to a
rigid support in accordance with an embodiment of the method of the
invention. In FIG. 14A a master structure 300 is used to form a
submaster structure 310 which can be coated with a bonding agent
320 and adhered to a rigid support 330. In FIG. 14B a submaster
structure 310 acts as a template structure to form a molded layer
340 which can be coated with a bonding agent 320 and adhered to a
rigid support 330.
[0043] FIG. 15 depicts a series of configurations in which the
template structure can be either an even or odd generation
submaster structure.
[0044] FIGS. 16A, 16B and 16C depict three different embodiments
for attaching a target sequence to an array. The solid support 105
has microsphere 110 with capture probe 120 linked via a linker 115.
FIG. 16A depicts direct attachment; the capture probe 120
hybridizes to a first portion of the target sequence 125. FIG. 16B
depicts the use of a capture extender probe 130 that has a first
portion that hybridizes to the capture probe 120 and a second
portion that hybridizes to a first domain of the target sequence
125. FIG. 16C shows the use of an adapter sequence 135, that has
been added to the target sequence, for example during an
amplification reaction as outlined herein.
DETAILED DESCRIPTION OF THE INVENTION
[0045] Overview
[0046] The present invention is directed to the formation of very
high density arrays that can allow simultaneous analysis, i.e.
parallel rather than serial processing, on a number of samples.
This is done by forming an "array of arrays", i.e. a composite
array comprising a plurality of individual arrays, that is
configured to allow processing of multiple samples. For example,
each individual array can be present within each well of a
microtiter plate. Thus, depending on the size of the microtiter
plate and the size of the individual array, very high numbers of
assays can be run simultaneously; for example, using individual
arrays of 2,000 distinct species (with high levels of redundancy
built in) and a 96 well microtiter plate, 192,000 experiments can
be done at once; the same arrays in a 384 microtiter plate yields
768,000 simultaneous experiments, and a 1536 microtiter plate gives
3,072,000 experiments.
[0047] Composite arrays of the present invention are not limited to
the configuration of a microtiter plate, and can be formed to have
any of a variety of dimensions and designs, as described below. The
composite arrays of the present invention are produced using a
process designed for production-scale manufacture of surfaces with
fine features (i.e., features as small as sub-micron scale).
Briefly, this process, which is also contemplated herein as an
embodiment of the present invention, includes contacting a template
structure with a moldable material, where the template structure
has one or more sets of projections on its surface. The moldable
material is then removed from the template structure as a molded
layer, which is then adhered to a rigid support. The resultant
composite array will contain surface features of the molded layer
that are complementary to that of the template structure and
contain at least one array of discrete sites, and will also have
sufficient rigidity to permit accurate optical interrogation of the
discrete sites.
[0048] Generally, the array compositions of the invention can be
configured in several ways. In a preferred embodiment, as is more
fully outlined below, a "one component" system is used. That is, a
first substrate comprising a plurality of assay locations
(sometimes also referred to herein as "assay wells"), such as a
microtiter plate, is configured such that each assay location
contains an individual array. That is, the assay location and the
array location are the same. For example, the plastic material of
the microtiter plate can be formed to contain a plurality of "bead
wells" in the bottom of each of the assay wells. Beads containing
bioactive agents can then be loaded into the bead wells in each
assay location as is more fully described below. It should be noted
that while the disclosure herein emphasizes the use of beads, beads
need not be used in any of the embodiments of the invention; the
bioactive agents can be directly coupled to the array locations.
For example, other types of arrays are well known and can be used
in this format; spotted, printed or photolithographic arrays are
well known; see for example WO 95/25116; WO 95/35505; PCT
US98/09163; U.S. Pat. Nos. 5,700,637; 5,807,522 and 5,445,934; and
U.S. Ser .Nos. 08/851,203 09/187,289; and references cited within,
all of which are expressly incorporated by reference. In one
component systems, if beads are not used, preferred embodiments
utilize non-silicon wafer substrates.
[0049] The present invention is generally based on previous work
comprising a bead-based analytic chemistry system in which beads,
also termed microspheres, carrying different chemical
functionalities are distributed on a substrate comprising a
patterned surface of discrete sites that can bind the individual
microspheres. The beads are generally put onto the substrate
randomly, and thus several different methodologies can be used to
"decode" the arrays. In one embodiment, unique optical signatures
are incorporated into the beads, generally using fluorescent dyes,
that can be used to identify the chemical functionality on any
particular bead. This allows the synthesis of the candidate agents
(i.e. compounds such as nucleic acids and antibodies) to be
divorced from their placement on an array; i.e., the candidate
agents may be synthesized on the beads, and then the beads are
randomly distributed on a patterned surface. Since the beads are
first coded with an optical signature, this means that the array
can later be "decoded", i.e. after the array is made, a correlation
of the location of an individual site on the array with the bead or
candidate agent at that particular site can be made. This means
that the beads may be randomly distributed on the array, a fast and
inexpensive process as compared to either the in situ synthesis or
spotting techniques of the prior art. These methods are generally
outlined in PCT US98/05025; PCT US98/21193; PCT US99/20914; PCT
US99/14387; and U.S. Ser. Nos. 08/818,199; 09/315,584; and
09/151,877, all of which are expressly incorporated herein by
reference. In addition, while the discussion herein is generally
directed to the use of beads, the same configurations can be
applied to cells and other particles; see for example PCT
US99/04473.
[0050] In these systems, the placement of the bioactive agents is
generally random, and thus a coding/decoding system is required to
identify the bioactive agent at each location in the array. This
may be done in a variety of ways, as is more fully outlined below,
and generally includes: a) the use of a decoding binding ligand
(DBL), generally directly labeled, that binds to either the
bioactive agent or to identifier binding ligands (IBLs) attached to
the beads; b) positional decoding, for example by either targeting
the placement of beads (for example by using photoactivatible or
photocleavable moieties to allow the selective addition of beads to
particular locations), or by using either sub-bundles or selective
loading of the sites, as are more fully outlined below; c)
selective decoding, wherein only those beads that bind to a target
are decoded; or d) combinations of any of these. In some cases, as
is more fully outlined below, this decoding may occur for all the
beads, or only for those that bind a particular target analyte.
Similarly, this may occur either prior to or after addition of a
target analyte.
[0051] Once the identity (i.e. the actual agent) and location of
each microsphere in the array has been fixed, the array is exposed
to samples containing the target analytes; although as outlined
below, this can be done prior to or during the analysis as well.
The target analytes will bind to the bioactive agents as is more
fully outlined below, resulting in a change in the optical signal
of a particular bead.
[0052] Array
[0053] Accordingly, the present invention provides array
compositions comprising a molded layer with a surface comprising
one or more assay locations. By "array" herein is meant a plurality
of candidate agents in an array format; the size of the array will
depend on the composition and end use of the array. Arrays
containing from about 2 different bioactive agents (e.g., different
beads) to many millions can be made, with very large fiber optic
arrays being possible. Generally, the array will comprise from two
to as many as a billion or more, depending on the size of the beads
and the substrate, as well as the end use of the array; thus very
high density, high density, moderate density, low density and very
low density arrays may be made. Preferred ranges for very high
density arrays are from about 10,000,000 to about 2,000,000,000,
(with all numbers being per square centimeter) with from about
100,000,000 to about 1,000,000,000 being preferred. High density
arrays range about 100,000 to about 10,000,000, with from about
1,000,000 to about 5,000,000 being particularly preferred. Moderate
density arrays range from about 10,000 to about 100,000 being
preferred, and from about 20,000 to about 50,000 being especially
preferred. Low density arrays are generally less than 10,000, with
from about 1,000 to about 5,000 being preferred. Very low density
arrays are less than about 1,000, with from about 10 to about 1000
being preferred, and from about 100 to about 500 being particularly
preferred. In some embodiments, the compositions of the invention
may not be in array format; that is, for some embodiments,
compositions comprising a single bioactive agent may be made as
well. In addition, in some arrays, multiple substrates may be used,
either of different or identical compositions. Thus for example,
large arrays may comprise a plurality of smaller substrates.
[0054] In addition, one advantage of the present compositions is
that particularly through the use of fiber optic technology,
extremely high density arrays can be made. Thus for example,
because beads of 200 .mu.m or less (with beads of 200 nm possible)
can be used, and very small fibers are known, it is possible to
have as many as 40,000-50,000 or more (in some instances, 1
million) different fibers and beads in a 1 mm.sup.2 fiber optic
bundle, with densities of greater than 15,000,000 individual beads
and fibers (again, in some instances as many as 25-50 million) per
0.5 cm.sup.2 obtainable.
[0055] By "composite array" or "combination array" or grammatical
equivalents herein is meant a plurality of individual arrays, as
outlined above. The number of arrays in a composite array is not
necessarily limited to any particular number, and will be
determined according to any of a variety of factors considered by
one skilled in the art, including corresponding equipment, number
of samples to be tested, convenience of experimental design, and
the like. For example, the number of individual arrays can be set
by the size of the microtiter plate used; thus, 96 well, 384 well
and 1536 well microtiter plates utilize composite arrays comprising
96, 384 and 1536 individual arrays, although as will be appreciated
by those in the art, not each microtiter well need contain an
individual array. It should be noted that the composite arrays can
comprise individual arrays that are identical, similar or
different. That is, in some embodiments, it may be desirable to do
the same 2,000 assays on 96 different samples; alternatively, doing
192,000 experiments on the same sample (i.e. the same sample in
each of the 96 wells) may be desirable. Alternatively, each row or
column of the composite array could be the same, for
redundancy/quality control. As will be appreciated by those in the
art, there are a variety of ways to configure the system. In
addition, the random nature of the arrays may mean that the same
population of beads may be added to two different surfaces,
resulting in substantially similar but perhaps not identical
arrays.
[0056] As will be appreciated by one skilled in the art, a
configuration of a composite array is not limited to the dimensions
of a microtiter plate. A composite array configuration may be a
single array, or may be a one-dimensional composite of arrays, i.e.
a composite array having only one array in a first dimension and a
plurality of arrays in a second dimension. Furthermore, a composite
array can be a square, e.g., 2.times.2, 3.times.3, etc., or any
other configuration, including, but not limited to, concentric
circles, spiral, rectangular, triangular, and the like. Preferably,
the composite of arrays contain regularly spaced arrays in lattice
configuration. In a preferred embodiment, the composite of arrays
forms a square or rectangular lattice.
[0057] By "substrate" or other grammatical equivalents herein is
meant a rigid structure containing at least one assay location
having discrete sites. In a preferred embodiment, a substrate of
the invention array composition contains a combination of a rigid
support and a molded layer adhered thereto, where the molded layer
contains at least one assay location having discrete sites. In a
preferred embodiment the substrate is in the form or dimensions of
a standard microscope slide.
[0058] As used herein, a "molded layer" refers to a structural
component of the substrate that contains discrete individual sites
appropriate for the attachment or association of beads, and which
is amenable to at least one detection method. As outlined below,
typically the discrete sites of the molded layer are formed using a
template structure, such as a template structure having one or more
sets of projections, which impresses the discrete sites into the
moldable material. The molded layer can generally be any thickness
which permits any structural features such as discrete sites and
fluid barriers to be maintained and functional. In one embodiment,
the thickness of the layer will be determined according to the
desired optical properties of the molded layer, as discussed below.
The molded layer will usually be at least about 10 .mu.m in
thickness and can be 1 .mu.m or more in thickness. Typically, the
molded layer will range from about 50 .mu.m to about 500 .mu.m in
thickness.
[0059] As will be appreciated by those in the art, the possible
compositions that can be used to form a molded layer are quite
varied. In a particularly preferred embodiment, a molded layer
comprises a material that can be molded to form discrete individual
sites and can be subsequently hardened to maintain the form of the
discrete individual sites. A molded layer will typically be
selected to have one or more of the following properties: resistant
to microcracking; resistant to moisture uptake; having a low
thermal expansion coefficient; having low fluorescence; robust and
non-deformable; capable of being rigid; capable of being robust and
non-deformable; capable of being planar; capable of permanently
adhering to an underlying rigid support without use of a bonding
agent; capable of permanently adhering to an underlying rigid
support with use of a bonding agent; capable of retaining
microspheres. Possible molded layer compositions include, but are
not limited to resins, including polyimides, epoxies, and the like;
plastics, including polycarbonate, acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyurethanes, TeflonJ, and the like;
polysaccharides; nylon; nitrocellulose; or a variety of other
polymers. Exemplary molded layer compositions are disclosed in U.S.
Ser. No. 4,582,885, which is herein incorporated by reference. In a
preferred embodiment the molded layer is a resin, most preferably
an epoxy. In another preferred embodiment the molded layer is made
of a thermosetting or thermoplastic polymer. In yet another
embodiment of the invention the molded layer is a plastic, most
preferably polycarbonate.
[0060] In another embodiment, in addition to the above properties
of a molded layer, a molded layer is further suitable for
large-scale production, having characteristics such as being
inexpensive, easily formed in large quantities, capable of setting
rapidly; capable of being rapidly adhered to a rigid support;
capable of being stored for long periods of time, and the like. In
one embodiment, the molded layer is a polymer with well-
characterized molding properties. Examples of such polymers include
resins such as epoxies and polycarbonate.
[0061] In one preferred embodiment, the molded layer is
sufficiently rigid such that it need not be adhered to a rigid
support in order to retain flatness or planarity. Thus, the
invention also provides a rigid molded layer. In this embodiment,
the array composition of the invention can comprise a molded layer,
and need not also comprise a rigid support nor the corresponding
bonding agent for bonding the molded layer to the rigid support.
Such a rigid molded layer will, in addition to having properties of
a molded layer, have one or more properties of a rigid support as
discussed herein. Preferably, a rigid molded layer will be capable
of being substantially flat or planar. In addition, a rigid molded
layer is preferably flat or planar such that substantially all
discrete sites can be accurately and simultaneously optically
interrogated. Rigid molded layers can be formed from a variety of
materials including polymers such as thermosetting plastics and
polycarbonates; a preferred material is polycarbonate.
[0062] In another embodiment, the molded layer will be selected
according to the desired optical properties of the molded layer.
The desired optical properties will be determined according to one
or more of a variety of factors, including but not limited to, the
type of radiation to be detected, the type and position of the
detector, the manner of carrying out excitation (if desired), the
strength of the detected signal, the proximity of the discrete
sites, and the like. For example, a molded layer can be selected to
have particular properties with respect to excitory or detectable
radiation such as: having low autofluorescence; being transparent;
being absorbent; being selectively absorbent, for example, to one
or more wavelengths of light; being selectively transparent, for
example of a narrow range of wavelengths, such as a filter; being
opaque; or being reflective. In general, the molded layer allows
optical detection and does not itself appreciably fluoresce.
[0063] In another embodiment, the molded layer can comprise a layer
with desired optical properties, such as a reflective layer, which
can be employed to enhance signal collection from the arrays. Such
a reflective layer can be formed from, for example, a metal layer.
Examples of reflective layers are generally described in U.S. Ser.
Nos. 5,080,465 and 5,999,318, which are herein incorporated by
reference. Such a molded layer comprising more than one layers is
discussed below.
[0064] In a preferred embodiment, the molded layer comprises a
compound that prolongs the desired optical properties of the molded
layer. For example, the molded layer can comprise a compound that
prolongs the transparency of the molded layer that has been exposed
to ultraviolet light or other radiation. A molded layer can also
comprise a compound that, for example reduces oxidation-mediated
degradation of the optical properties of the molded layer.
[0065] In a preferred embodiment, the molded layer is selected
according to the hydrophobicity properties of the sample-containing
solvent used in methods of the invention for detecting a target
analyte. For example, if an aqueous solvent is used, the molded
layer will be sufficiently hydrophilic to permit the sample to
contact the microspheres in the discrete sites. Similarly, if a
hydrophobic or apolar solvent, such as hexanes, toluene, ether, and
the like, is used, the molded layer will be sufficiently apolar to
permit the sample to contact the microspheres in the discrete
sites.
[0066] In a preferred embodiment, the molded layer is selected
according to a desired level of wetability of the discrete sites.
As used to herein, wetability refers to the ability of the entire
area of a surface to come into contact with an aqueous or polar
solvent which has been placed thereon. The wetability can be
achieved in any of a number of ways, including selecting a
composition for the entire molded layer which has the desired
wetability properties, selecting a molded layer comprising a layer
with the desired wetability properties; and modifying the surface
of the discrete sites to improve wetability of the discrete sites.
Methods of modifying the surface of discrete sites to improve
wetability include, but are not limited to acid etching, ion
bombardment, and acid/tin etching such as that disclosed in U.S.
Ser. No. 3,666,527.
[0067] In another embodiment, the molded layer can be selected to
increase the ability for a nonpolar or hydrophobic solvent to come
into contact with the discrete sites on the surface of the molded
layer. This can be achieved in any of a number of ways, including
selecting a composition for the entire molded layer which has the
desired hydrophobic contacting properties, selecting a molded layer
comprising a layer with the desired hydrophobic contacting
properties; and modifying the surface of the discrete sites to
improve hydrophobic contacting properties of the discrete
sites.
[0068] In one embodiment, in addition to a molded layer, a
substrate also contains a rigid support. As used herein, a "rigid
support" refers to a structural component of a substrate that
causes a substrate to be inflexible or to retain structural memory.
That is, a rigid support will either substantially prevent the
substrate from deforming or will, upon deformation of the
substrate, cause a substrate to substantially return to the
substrate's intended shape. As will be recognized by one skilled in
the art, "substantially prevent", "substantially return" and like
terms refer to structural properties where the shape of the
structure is maintained or reinstated in such a way as to permit
the desired optical interrogation of the invention arrays, in
accordance with the methods taught and cited herein. In a preferred
embodiment, a rigid support is flat or planar such that
substantially all discrete sites of an array can be accurately and
simultaneously detected. In another preferred embodiment, a rigid
support is inflexible, such as, for example, a glass slide. In
addition, the rigid support can serve to keep the molded layer in a
planar or flat configuration. As is known in the art, many
detection techniques, for example, fluorescence detection, rely on
very shallow depth of field detection methods using, for example,
CCD cameras and confocal microscopes. Since many molded materials
cannot be made sufficiently flat or planar for such detection
methods, a rigid support is used to maintain the molded layer in a
flat or planar configuration.
[0069] A rigid support can be formed from any of a variety of
materials and will be selected according to the desired properties
of the rigid support, including, but not limited to the
above-discussed structural properties and other structural
properties such as flatness, strength, stiffness, low thermal
expansion coefficient, optical properties and chemical properties
such as molded layer compatibility. For example, a rigid structure
can be selected to have optical properties that include, but are
not limited to having low autofluorescence, or being transparent,
selectively transparent, absorptive, selectively absorptive, opaque
or reflective. In addition, a metal or metal-coated rigid structure
can be employed to enhance signal collection from the arrays.
[0070] Compositions for a rigid support which demonstrate the above
properties include metals, such as aluminum, iron, steel, various
alloys, and the like; ceramics; composites such as fiberglass;
silicon or other semiconductor materials; glass; rigid plastics or
polymers; and the like.
[0071] In a preferred embodiment, a substrate containing discrete
sites is formed according to the method of the invention, as
described in detail below. Briefly, this method includes the steps
of: contacting a surface of a template structure, which surface has
one or more sets of projections, with a moldable material; then
removing the moldable material from the surface of the template
structure, such that the removed moldable material forms a molded
layer with at least a first assay location having discrete sites;
and then adhering the molded layer to a rigid support.
[0072] In a preferred embodiment of the method of the invention the
invention further includes the step of randomly distributing
microspheres on the molded layer such that individual discrete
sites comprise microspheres, where the microspheres comprise at
least a first and a second subpopulation, where the first
subpopulation comprises a first bioactive agent and the second
subpopulation comprises a second bioactive agent.
[0073] In addition, the arrays can be prepared by microfabrication
techniques as are known in the art. Such techniques include, but
are not limited to, injection molding, hot-embossing, UV
lithography, surface micromachining, photopolymerization, etching,
microstereolithography and electroplating.
[0074] In addition, the shape of the sites or wells on the molded
layer can be modified to alter the signal production. That is, the
wells can be square, round or polygonal in shape. These surface
modifications and additional surface modifications to improve
signal output and /or detection are described in more detail in
U.S. Ser. No. 09/651,181, filed Aug. 30, 2000 and PCT/US00/23830,
filed Aug. 30, 2000, both of which are expressly incorporated
herein by reference.
[0075] Generally the substrate is flat (planar), although as will
be appreciated by those in the art, other configurations of
substrates may be used as well; for example, three dimensional
configurations can be used, for example by embedding the beads in a
porous block of plastic that allows sample access to the beads and
using a confocal microscope for detection. Similarly, the beads may
be placed on the inside surface of a tube, for flow-through sample
analysis to minimize sample volume. Preferred substrates include
optical fiber bundles as discussed below, and flat planar
substrates such as glass, polystyrene and other plastics and
acrylics. In some embodiments, silicon wafer substrates are not
preferred. In one embodiment the substrate is in the shape of or is
a microscope slide (see FIG. 12).
[0076] That is, in a preferred embodiment, the substrate is a
microscope slide or a substrate of substantially the same
dimensions as a standard microscope slide. As one of ordinary skill
in the art appreciates, a microscope slide is approximately about
3" or about 7.5 cm, by about 1" or 2.5 cm by a thickness of about
0.04" or about 1 mm, although different dimensions could be used.
Thus, in a preferred embodiment, the substrate of the present
invention is approximately 7.5 cm by 2.5 cm by approximately 1 mm
(FIG. 12). An advantage of using substrates of this size is that
existing instrumentation, i.e. detectors can be used to analyze
signals on the substrate. That is, existing scanning-based
instrumentation including, but not limited to, that sold by General
Scanning, Molecular Dynamics, Gene Machine, Genetic Microsystems,
Vysis, Axon and Hewlett-Packard can be used to analyze arrays of
the present invention.
[0077] The substrate will comprise one or more assay locations,
i.e. the location where the assay for the detection of a target
analyte will occur. A substrate that contains a first assay
location can also contain a set of discrete sites into which
microspheres can be located, forming an array location. A substrate
that contains a first and a second assay locations can contain two
sets of discrete sites which can contain microspheres and thus form
two array locations. In one embodiment, the first and second assay
locations are separated by a fluid barrier. When used in reference
to separating assay locations, a "fluid barrier" refers to a
physical or physico-chemical structure or coating which prohibits
or prevents flow of a liquid between a first and second assay
location. In one embodiment, the assay locations are physically
separated from each other; for example, a first and second assay
location can have a wall physically blocking a liquid sample from
flowing between first assay and second assay locations.
[0078] In some configurations, the assay locations can be separated
by physico-chemical barrier coating, which acts to prohibit a
sample from passing across the coating due to incompatibility of
the barrier coating with the liquid. For example, a
physico-chemical barrier can comprise a hydrophobic or apolar
material which prohibits aqueous or polar liquids from flowing over
the hydrophobic or apolar material. In another example, a
physico-chemical barrier can comprise a hydrophilic or polar
material which prohibits hydrophobic or apolar liquids from flowing
over the hydrophilic or polar material. Typically, this
physico-chemical barrier will have a low propensity to interact
with the flowing liquid relative to the propensity of the assay
location to interact with the flowing liquid, which assay location
contains the discrete sites.
[0079] As discussed above, the assay locations, need not be limited
to the configuration of a microtiter plate. That is, any separable
location on a substrate serves as an assay location. By separable
location is meant a location on a substrate that is physically
separated from other regions on the substrate. The physical
separation can be any border between assay locations. The
separation can be a partition, structure or coating, or
alternatively, the separation can simply be spacing between assay
locations sufficient at least to distinguish one from the other.
When it is desired to maintain separate solutions in each assay
location, there need only be sufficient separation such that
reagents delivered to one assay location will not cross contaminate
another assay location. However, in some embodiments, such a
physical barrier is not necessary, or in some instances, not even
desired. As such, the assay locations need only be separated enough
to distinguish one from the other. When partitions or borders are
used between assay locations, preferred borders include but are not
limited to hydrophobic regions surrounding an assay location;
ridges or rims of sufficient width and height to prevent sample
migration between assay locations; or troughs of sufficient width
and depth to prevent sample migration between assay locations. In
some embodiments, the borders are made of gaskets including, but
not limited to rubber or silicon. That is, In a preferred
embodiment, the border comprises a sealing mechanism to prevent
leakage of the sample or reagents between wells of the substrate.
As will be appreciated by those in the art, this may take on a
variety of different forms. In one embodiment, there is a gasket on
the substrate comprising the array, comprising sheets, tubes or
strips. Alternatively, there may be a rubber or silicone strip or
tube used. In one embodiment the substrate contains an indentation
or channel into which the gasket fits. Furthermore, adhesives can
be used to attach the gasket to the substrate. When hydrophobic
regions are used to surround an assay location, the hydrophobic
regions effectively contain or force the solutions to localize over
the sites contained within the region surrounded by the hydrophobic
region. In some embodiments the borders or partitions are made of
printable materials including, but not limited to gels.
[0080] Also, in some embodiments, it is desirable to provide
channels for fluid flow between wells. In this embodiment, the
channels can be etched into the substrate as described herein. In
an alternative embodiment, printing techniques are used for the
creation of desired fluid guiding pathways; that is, patterns of
printed material can permit directional fluid transport. Thus, the
build-up of "ink" can serve to define a flow channel. In addition,
the use of different "inks" or "pastes" can allow different
portions of the pathways having different flow properties.
Multi-material fluid guiding pathways can be used when it is
desirable to modify retention times of reagents in fluid guiding
pathways. Furthermore, printed fluid guiding pathways can also
provide regions containing reagent substances, by including the
reagents in the "inks" or by a subsequent printing step. See for
example U.S. Pat. No. 5,795,453, herein incorporated by reference
in its entirety.
[0081] In a particularly preferred embodiment fluid channels,
barriers, or combinations of both are formed in the molded layer
during process of forming the discrete sites in the molded layer,
in accordance with the method of the invention. In this method, the
channels, barriers, or combinations of both comprise the same
material as the material of the molded layer.
[0082] In an alternative embodiment, the fluid barriers can be
added in a step subsequent to formation of the molded layer. In
this method, the fluid barrier can comprise the same material as
the molded layer or any other material useful for forming a fluid
barrier, and can particularly be useful when the fluid barrier
comprises a physico-chemical fluid barrier.
[0083] In one embodiment, the assay locations are depressed regions
in the substrate. As described herein the depressed regions, or
assay locations contain discrete sites or wells.
[0084] In a preferred embodiment assay locations on the substrate
are fiber optic bundles. That is, the fiber optic bundles are
attached to or inserted through the substrate, as described in more
detail below, to form discrete assay locations. Although not
required in all embodiments, in some embodiments the fiber optic
bundles are physically separated from one another by partitions
that include but are not limited to those described above, e.g.
hydrophobic regions, ridges or troughs. Alternatively, each fiber
bundle is separated by sufficient distance to distinguish one from
the other.
[0085] In a preferred embodiment, the substrate is an optical fiber
bundle or array, as is generally described in U.S. Ser. Nos.
08/944,850 and 08/519,062, PCT US98/05025, and PCT US98/09163, all
of which are expressly incorporated herein by reference. Preferred
embodiments utilize preformed unitary fiber optic arrays. By
"preformed unitary fiber optic array" herein is meant an array of
discrete individual fiber optic strands that are co-axially
disposed and joined along their lengths. The fiber strands are
generally individually clad. However, one thing that distinguished
a preformed unitary array from other fiber optic formats is that
the fibers are not individually physically manipulable; that is,
one strand generally cannot be physically separated at any point
along its length from another fiber strand.
[0086] In a preferred embodiment, the assay locations comprise a
plurality of discrete sites. Thus, the assay location is the same
as the array location, as described herein. In this embodiment, at
least one portion of the molded layer is modified to contain
discrete, individual sites for later association of microspheres
(or, when microspheres are not used, for the attachment of the
bioactive agents). These discrete sites may comprise physically
altered sites, i.e. physical configurations such as wells or small
depressions in the substrate that can retain the beads, such that a
microsphere can rest in the well, or the use of other forces
(magnetic or compressive), or chemically altered or active sites,
such as chemically functionalized sites, electrostatically altered
sites, hydrophobically/ hydrophilically functionalized sites, spots
of adhesive, etc.
[0087] The sites may be a pattern, i.e. a regular design or
configuration, or randomly distributed. A preferred embodiment
utilizes a regular pattern of sites such that the sites may be
addressed in the X-Y coordinate plane. "Pattern" in this sense
includes a repeating unit cell, preferably one that allows a high
density of beads on the substrate.
[0088] However, it should be noted that these sites on an invention
substrate may not be discrete sites. That is, it is possible to use
a uniform surface of adhesive or chemical functionalities, for
example, that allows the attachment of beads at any position. That
is, the surface of the substrate is modified to allow attachment of
the microspheres at individual sites, whether or not those sites
are contiguous or non-contiguous with other sites. Thus, the
surface of the substrate may be modified such that discrete sites
are formed that can only have a single associated bead, or
alternatively, the surface of the substrate is modified and beads
may go down anywhere, but they end up at discrete sites.
[0089] In a preferred embodiment, the surface of the substrate is
modified to contain wells, i.e. depressions in the surface of the
substrate. In a preferred embodiment, the depression in the surface
of the substrate comprise depressions in the molded layer of an
invention substrate that comprises a molded layer and a rigid
support. In such an embodiment, the depressions in the molded layer
will comprise the discrete sites of an assay location, and the
depressions and discrete sites will be formed simultaneously in the
method of the invention which comprises use of a template structure
to form the surface of the molded layer. Also contemplated herein,
depressions in the substrate may be formed using a variety of
techniques generally known in the art, including, but not limited
to, photolithography, stamping techniques and microetching
techniques. As will be appreciated by those in the art, the
technique used will depend on the composition and shape of the
substrate.
[0090] In a preferred embodiment, the sites or wells are separated
with spaces between each other. As is appreciated by those skilled
in the relevant art, bead spacing is determined by calculating the
distance between centers. Varying the spacing between sites results
in the formation of arrays of high density, medium density or lower
density. High density arrays are characterized as having sites
separated by less than about 5 to 15 .mu.m. Medium density arrays
have sites separated by about 15 to 30 .mu.m, while low density
arrays have sites separated by greater than 30 .mu.m. Generally,
the sites are separated by less than 100 .mu.m; preferably less
than 50 .mu.m and most preferably less than 15-20 .mu.m. A
particular advantage of spacing wells apart is that commercial
scanners can be used to analyze the arrays. That is, the resolution
of scanners varies and arrays can be formed that allow for
detection on high or low resolution scanners. For high density
arrays, high resolution scanners (<5 .mu.m) can be employed.
These scanners effectively analyze arrays with close spacing
(<15 .mu.m) between features, i.e. beads. For lower resolution
scanners (>5 .mu.m), increased bead spacing, i.e. >10 .mu.m
can be utilized, with from 15 to 20 .mu.m being preferred. In both
cases, various software packages are used, such as but not limited
to, GENEPIX software package by AXON instruments or others that are
provided with conventional fluorescent microscope scanning
equipment. In a preferred embodiment, the software employs
contrast-based or other image processing algorithms to resolve the
beads and extract signal intensity information (see also U.S. Ser.
No. 09/651,181, filed Aug. 30, 2000 and PCT/US00/23830, filed Aug.
30, 2000, both of which are expressly incorporated herein by
reference).
[0091] While in the above described embodiment the spacing between
features is accomplished by physically altering the spacing of the
sites on the substrate, in an alternative embodiment, when beads in
bead wells form the array, density is modulated by adding to a
population of beads comprising bioactive agents, a population of
beads that do not comprise a bioactive agent. That is, a population
of beads with no bioactive agent, and in some embodiments no
detectable signal or label, is added to at least one population of
beads that does comprise a bioactive agent. The beads lacking a
bioactive agent, i.e. "blank beads", dilute the concentration of
beads with a bioactive agent. When applied to or distributed on a
substrate, this results in increased spacing between beads with
bioactive agents. That is, in the absence of blank beads, beads
with bioactive agents will fill substantially all of the wells on a
substrate at an average density of not more than one bead per well.
When the spacing of wells is close, only high resolution scanners
effectively analyze the array. However, upon the addition of a
population of blank beads, blank beads will be distributed with the
beads that have bioactive agents thereby increasing the distance
between beads with bioactive agents. Thus, in a preferred
embodiment, the distance between centers of beads with bioactive
agents is at least 5 .mu.m; more preferably between 10 to 50 .mu.m;
and most preferably between 15 to 25 .mu.m.
[0092] In one embodiment, the ratio between beads with bioactive
agents and blank beads is adjusted to achieve proper density of
beads with bioactive agents on the array. The ratio depends on the
desired spacing between beads. That is, when it is desired to have
beads with bioactive agents n beads apart, the ratio beads with
bioactive agents to blank beads is n.sup.2 That is, if it is
desired to have beads with bioactive agents separated by six blank
beads, the ratio of beads with bioactive agents to blank beads is
1:6.sup.2 or 1:36. While in some embodiments it may only be
necessary to include a small number of blank beads, i.e. the ratio
is about 10:1 or greater, in preferred embodiments, the ratio is at
least 1:36 or more, with 1:100 being particularly preferred.
[0093] In an alternative embodiment, the array comprises a first
population of beads with a first bioactive agent and a second
population of beads with a second population of bioactive agents.
When modulating the spacing of beads on an array so that
conventional scanners can be used, it is useful in this embodiment
for each population of beads to be labeled or tagged with different
tags. The tags are preferably detectable in distinct channels. As
such, in this embodiment, only one population of beads is analyzed
at a time. Accordingly, the beads that are not being analyzed serve
as spacer beads although they do contain a bioactive agent and can
be analyzed in a different channel. As such, the spacing of the
beads from each population will be adequately spaced for analysis,
while the number of beads to be analyzed is increased relative the
above-described assay that uses blank beads. That is, when
analyzing the first population of beads in a first channel, which
does not detect the second population of beads, the second
population of beads serve as spacing beads or blank beads. The
second population of beads serves to increase the spacing between
the first population of beads. In turn, when analyzing the second
population of beads in a second channel, the first population of
beads serves as spacing or "blank" beads that separate the second
population of beads.
[0094] Accordingly, the present invention also includes an array as
described above and a detector. In a preferred embodiment the array
is in the detector. In a particularly preferred embodiment the
substrate is formatted to the shape of a microscope slide and
includes a molded layer containing the assay locations and array
locations and at least one array location is in the detector.
[0095] In a preferred embodiment, physical alterations are made in
a surface of the substrate to produce the sites. In a preferred
embodiment, for example, when a first substrate contains more than
one assay locations and a second substrate is a fiber optic bundle
containing more than one array locations, the surface of the second
substrate is a terminal end of the fiber bundle, as is generally
described in Ser. Nos. 08/818,199 and 09/151,877, both of which are
hereby expressly incorporated by reference. In this embodiment,
wells are made in a terminal or distal end of a fiber optic bundle
comprising individual fibers. In this embodiment, the cores of the
individual fibers are etched, with respect to the cladding, such
that small wells or depressions are formed at one end of the
fibers. The required depth of the wells will depend on the size of
the beads to be added to the wells.
[0096] Generally in this embodiment, the microspheres are
non-covalently associated in the wells, although the wells may
additionally be chemically functionalized as is generally described
below, cross-linking agents may be used, or a physical barrier may
be used, i.e. a film or membrane over the beads.
[0097] The invention also provides a method for making a substrate
containing one or more assay locations having discrete sites. This
method is carried out by contacting a surface of a template
structure with a moldable material, where the surface comprising
one or more sets of projections. The moldable material is removed
from the surface of the template structure, such that the removed
moldable material forms a molded layer with at least a first assay
location comprising discrete sites. This molded layer is adhered to
a rigid support. FIG. 14B shows an example of how a template
structure 310 can be used to form the molded layer 340 which can be
adhered to the rigid support 330.
[0098] The method of the present invention draws from the
methodology used in the replication process for forming ruled
diffraction gratings. The general teachings of the formation of
ruled diffraction gratings can be found, for example, in the
Diffraction Grating Handbook, 4th ed. by Thermo RGL (formerly known
as Richardson Grating Laboratory) available directly from Thermo
RGL. Although these references will serve as general resource, it
will be appreciated that the considerations for the method of
forming molded layers formed in accordance with the present
invention will differ from those for a method of forming ruled
gratings since the principle purpose of the invention molded layers
is not high precision dispersion of polychromatic light into its
monochromatic components. For example, the requirement for low
tolerance for flaws forming diffraction gratings (for example, to
improve wavelength resolving power and reduce scattering and
ghosts) will not be so stringent in the method of the invention.
The important factors in forming a molded layer in accordance with
the method of the invention include: forming discrete sites
separated by as little as 5 .mu.m, preferably in a pattern, where
the discrete sites are each preferably depressions capable of
accommodating a microsphere; forming discrete sites that are
non-deformable and highly uniform; and forming the discrete sites
in a manner that will permit a suitable detection system to
optically interrogate discrete sites individually. Accordingly, one
skilled in the art will recognize that a flaw in one or a few
discrete sites will not appreciably change the effectiveness of an
array of the invention.
[0099] The method of the invention includes a step of contacting a
surface of a template structure with a moldable material. As used
herein a "template structure" refers to a model form whose surface
is used as a mold to create an inverse replica of the template
structure surface. Preferably, the template structure contains one
or more sets of projections on its surface. The sets of projections
of the template structure will create sets of impressions in the
moldable material upon contact of the template structure and the
moldable material. These sets of impressions ultimately correspond
to discrete sites present in a molded layer. Accordingly, a
template structure that contains one or more sets of projections
will be used to form a molded layer containing one or more sets of
discrete sites; similarly, a template structure that contains two
or more sets of projections will be used to form a molded layer
containing two or more sets of discrete sites. The template
structure can further contain additional projections which can
create impressions in the moldable material corresponding in the
molded layer to channels for the flow of fluids, including a fluid
sample, along the surface of the molded layer. Further, the
impressions can correspond to any other desired depressed feature
on the substrate surface as discussed above. The template structure
can also contain depressions on its surface. The depressions of the
template structure will create projections in the moldable material
upon contact of the template structure and the moldable material.
These projections can ultimately correspond to, for example,
physical fluid barriers present in a molded layer. Further, the
projections can correspond to any other desired raised feature on
the substrate surface as discussed above.
[0100] The moldable material which contacts the template structure
in the method of the invention is any material that adopts and is
capable of maintaining the shape of the molded layer after removing
the moldable material from the surface of the template structure.
The moldable material will comprise materials discussed above in
describing the molded layer, including resins such as expoxides,
polyimides, and the like.
[0101] In the step of contacting a surface of a template structure
with a moldable material carried out in the method of the
invention, the contacting can be of any form, duration and extent
which results in formation of one or more sets of discrete sites in
the resultant molded layer. For example, a moldable material can
contact a surface of a template structure by spraying the moldable
material onto the template structure, where the moldable material
is a liquid in a stream, spray or mist form. Alternatively, the
surface of the template structure can be dipped into a tank
containing liquid moldable material, or can be pressed into a
partially solidified moldable material which can be shaped to
complement the surface of the template structure contacting the
moldable material. In one embodiment, the template structure has a
cylindrical shape, and the template structure is contacted with a
moldable material, the moldable material is permitted to solidify
until the shape of the moldable material is capable of remaining
unchanged, and then the moldable material is unrolled from the
cylinder as a molded layer. In a preferred embodiment, when the
template structure is cylindrical, the method of contacting the
template structure is a continuous process in which at a first
portion of the cylinder, the cylinder is contacted with a moldable
material and at a second portion of the cylinder, solidified
moldable material is removed from the cylinder as a molded
layer.
[0102] A template structure can be either a master structure or a
submaster structure. A "master structure", as used herein, refers
to the original mold formed by a process other than the molding
method of the invention. One skilled in the art will recognize that
a variety of methods can be used for forming the master structure,
including, but not limited to physical etching (e.g., diamond
etching), photoresist etching, or any other method capable of
forming a detailed design on a substance useful as a template
structure in the method of the invention. In addition to use of a
master structure in the method of the invention as a template
structure, a submaster structure can also be used as a template
structure in the method of the invention. As used herein, a
"submaster structure" refers to a template structure that has been
formed according to the method of the invention or in a similar
molding process such that the design of the template structure
originates from a separate, master structure. A submaster structure
can be formed directly using a master structure as the template
structure in a method such as the method of the invention.
Additionally, a second submaster structure can be formed from using
a first submaster structure as the template structure in a method
such as the method of the invention. A submaster structure formed
from a master structure is referred to as a first generation
submaster structure. A submaster structure formed from a first
generation submaster structure is referred to as a second
generation submaster structure.
[0103] It is within the scope of the present invention that a
template structure can be a submaster structure from any of a large
number of generations. One skilled in the art will appreciate that
the number of submaster generations available for use as template
structures in the method of the invention will correspond to the
degree to which the original design of the master structure is
maintained from generation to generation. While some elements of
the original design of the master structure can be lost, damaged or
distorted between generations, it will be recognized that only
those elements that are important to the desired use of the
invention substrate array composition will be considered in
determining the value of a submaster generation or a particular
submaster structure as a template structure in the method of the
invention. Such elements include, but are not limited to: formation
of discrete sites separated by as little as 5 .mu.m, preferably in
a pattern, where the discrete sites are each preferably depressions
capable of accommodating a microsphere having a diameter that
varies from about 0.5 .mu.m to about 50 .mu.m, preferably from
about 1 .mu.m to about 5 .mu.m; and forming the discrete sites in a
manner that will permit a suitable detection system to optically
interrogate discrete sites individually. Although the number of
generations for which submasters are useful can be very large, such
as 50, typically submaster structures used as template structures
in the method of the invention will be 20 generation submaster
structures or less, preferably 10 generations or less, more
preferably 6 generations or less.
[0104] One skilled in the art will appreciate that by using a
submaster to create multiple submasters in the next generation, a
large number of multi-generation submasters can be formed. For
example, if a submaster of each generation is used to form 10
submasters of the next generation and 6 generations of submasters
are formed, more than 1 million submasters will result.
Accordingly, formation of the template structure used in the method
of the invention will typically occur by forming a
multiple-generation submaster structure, according to the number of
desired template structures and the capability of forming
multi-generation submaster structures. One skilled in the art will
also appreciate that each odd-numbered submaster generation (first
generation, third generation, fifth generation, etc.) will have the
inverse form of the original design of the master structure, and
each even-numbered submaster generation (second generation, fourth
generation, etc.) will have a replica of the original design of the
master structure. When the master structure represents the desired
design of the molded layer formed using the method of the
invention, one skilled in the art will know that odd-numbered
submaster generations are to be used as the template structure in
the method of the invention (see, for example FIG. 14). Similarly,
when the master structure represents the inverse design of the
desired design of the molded layer formed using the method of the
invention, even-numbered submaster generations are to be used as
the template structure in the method of the invention. When the
when either the actual design or the inverse design can be the
desired design of the molded layer, for example, when the design of
the master structure is substantially equivalent to the inverse
design of the master structure (for example, an egg-crate design or
equivalent, see FIG. 15), one skilled in the art will recognize
that all generations of submaster structure can be used as template
structures in the method of the invention.
[0105] In a preferred embodiment, prior to the step of contacting
the surface of the template structure with a moldable material, a
releasing agent is applied to the surface of the template
structure. As used herein a "releasing agent" is any compound,
composition, or other substance, which aids in the separation of
the moldable material from the surface of the template structure in
forming a molded layer. Useful releasing agents include silicone
oil, polyvinyl alcohol, fluorinated silane or other known releasing
agent. Selection of the type and amount of a releasing agent will
depend on several easily determinable factors such as the strength
of the template structure and of the moldable material, the
environmental conditions of the contacting and molding process, the
degree of tolerance for distortions or imperfections in the molded
layer, and the like.
[0106] In another preferred embodiment, a molded layer actually
contains two or more layers. This embodiment will find particular
utility when it is desirable for a molded layer to possess
particular chemical or physical properties. In this instance, the
molded layer contains at least one layer that provides and
maintains the shape of the molded layer (the "shape layer") and at
least one layer that provides the desired properties on the surface
of the molded layer (the "active layer"). The shape layer will
comprise materials discussed above, and include resins such as
expoxides, polyimides, and the like. The active layer can provide
chemical properties such as permitting a bioactive agent or other
molecule to be chemically bound thereto, or physical properties
such as affinity for particular fluids, desired optical properties,
and the like. The active layer can also be selected to have
properties for favorable interaction with sample-containing
solvents or for favorable wetability properties, as described
above. The active layer can also serve as a protective layer to
prevent the underlying shape layer from degradation as a result of,
for example, irradiation, fluid contact, oxidation, and the like.
In another embodiment, the active layer can be selected according
to desired optical properties, such as a reflective layer, which
can be employed to enhance signal collection from the arrays. Such
a reflective layer can be formed from, for example, a metal layer
such as a layer of silver, aluminum or chromium. Exemplary active
layers include, but are not limited to, glass, quartz, fused
silica, magnesium fluoride, and the like.
[0107] The active layer can, but need not, contribute to
maintaining the shape of the molded layer; accordingly, the active
layer can quite thin (i.e., <1 .mu.m), though typically the
active layer will be about 0.1 .mu.m. The shape layer is typically
thicker than the active layer, having any thickness sufficient to
insure that the shape layer maintains the shape of the molded
layer. This thickness of the shape layer will usually be at least
about 10 .mu.m in thickness and can be 1 .mu.m or more in
thickness. Typically, the shape layer will range from about 50
.mu.m to about 500 .mu.m in thickness.
[0108] In another embodiment, the active layer can itself comprise
two layers, three layers, or more. For example, an active layer can
comprise a layer having desired optical properties such as
reflectance, and an overlying layer which serves as a protective
layer to prevent the underlying optical active layer from
degradation. Any of a number of multiple active layer combinations
can be used in the present invention, as will be apparent to one
skilled in the art. For example, the active layer can comprise a
reflective layer such as a silver, aluminum or chromium layer, and
further comprise an overlying layer that prevents the underlying
optical layer from degradation.
[0109] The method of the invention also includes a step of removing
the moldable material from the surface of the template structure to
form a molded layer, where the molded layer has at least one assay
location having discrete sites. At some point which can occur
before or after removing the moldable material from the surface of
the template structure, the moldable material will transition to a
state in which the shape of the moldable material is substantially
unchanged; transition to this state represents the formation of the
moldable layer. A moldable material having a shape that is
substantially unchanged refers to a state of the moldable material
where its shape does not change in such a way as to alter the
intended properties of the resultant molded layer; for example, the
size or shape of the discrete sites is not changed in such a way
that the changes intended properties of accommodating a
microsphere, or transmitting or reflecting luminescence, or the
like. Generally, the step of removing the moldable material from
the surface of the template structure will be carried out in such a
way as to permit the moldable material to maintain a shape that is
fully complementary to the template structure, and thus, the
moldable material is removed from the surface of the template
structure in the form of the molded layer.
[0110] In another embodiment, the molded material will undergo a
change of shape after removal from the template structure. This can
be, for example, a rounding or smoothing of features formed in the
moldable material that are complementary to the projections of the
template structure, which can result in improved microsphere
interaction properties, improved optical properties, or other
desired property.
[0111] Once the features (i.e., discrete sites, fluid barriers,
fluid channels, and the like) formed in the molded material are
substantially unchanged, the moldable material is then termed the
molded layer. Although the molded layer is characterized as having
substantially unchanged features, it will be understood that the
molded layer is not required to be rigid or retain structural
memory beyond that of maintaining the shape of the features. For
example, in one embodiment of the invention, a molded layer can be
a flexible film containing discrete sites. Such a flexible film can
be stored in a compact form, such as in rolled form on a spool, as
stacked sheets, or any other configuration for convenient storage.
A substrate containing one or more assay locations having discrete
sites is formed in accordance with the invention by adhering this
flexible molded layer to a rigid structure, as will be further
discussed below.
[0112] The moldable material or a molded layer in flexible form,
can comprise a substance that can be subjected to a hardening step
to decrease the deformability of the moldable material or molded
layer. The hardening step can be carried out using any of a variety
of substances known to be useful for hardening including: heat
treatment; radiation treatment using radiation such as UV
radiation, particle beam radiation; chemical treatment using an
oxidative substance or other chemically reactive substance; and the
like. The hardening step can be carried out to any extent desired
to convey to the moldable material or molded layer the desired
structural properties, including carried out to completion where
the resultant molded layer is inflexible. In one embodiment of the
invention, the hardening step is carried out prior to the step of
removing the moldable material from the surface of the template
structure. In another embodiment, the hardening step is carried out
after adhering the molded layer to the rigid support.
[0113] The molded layer formed in accordance with the method of the
invention is adhered to a rigid support. As discussed above, a
rigid support is a structural component of a substrate that causes
a substrate to be inflexible or to retain structural memory. By
adhering the molded layer to the rigid support, the molded layer
can retain a shape that is optically interrogatable. The step of
adhering the molded layer to the rigid support is carried out by
contacting the molded layer directly to the rigid support or by
coating either the molded layer or the rigid support with a bonding
agent and then contacting the molded layer/bonding agent to the
rigid support or rigid support/bonding agent to the molded layer.
The result of the adhering step will be to cause the molded layer
to be attached to the rigid support, preferably such that it does
not rotate, bubble, warp, curl, tear, or otherwise deform in a
manner which adversely influences the ability to optically
interrogate the discrete sites or which adversely influences the
flow of fluids over the molded layer and selected assay locations
therein. In a preferred embodiment, the moldable layer is adhered
to the rigid support by directly contacting the molded layer to the
rigid support.
[0114] A bonding agent useful in the adhering step of the method of
the invention can be any substance that is capable of securing the
attachment of the molded layer to the rigid support without
adversely influencing the ability to optically interrogate the
discrete sites and which does not cause the adverse degradation of
the rigid support or the molded layer. When a bonding agent is
used, it will preferably form a thin, even coating (about 1 .mu.m
in thickness) between the molded layer and the rigid support.
Alternatively, only portions of either the rigid support or the
molded layer are coated with the bonding agent. For example, the
bonding agent can be placed only in portions of either the rigid
support or the molded layer, or both, such that the bonding agent
does not lie in a position that will cause excitory radiation or a
detectable signal to pass through the bonding agent during the
optical interrogation of the discrete sites. Such an example
includes placement of the bonding agent in locations on the rigid
support or moldable layer, or both, that do not lie directly
beneath the discrete sites of the molded layer. In such an
instance, a bonding agent need not be limited to a substance that
does not influence the optical interrogation of the discrete sites.
In another example, the bonding agent can be placed in a variety of
partially-coating configurations, such as serpentine, concentric
circles or ovals, spiral, and the like.
[0115] As will be appreciated by one skilled in the art, when the
molded layer is coated with the bonding agent, the back surface of
the molded layer will be coated; that is, the surface coated with
the bonding agent is the surface of the molded layer not containing
the formed features such as discrete sites, fluid barriers, fluid
channels, and the like. Suitable bonding agents include, but are
not limited to, liquid epoxies; glues or adhesives that are added
to either the molded layer or the rigid support; sheets of epoxies;
rolls of epoxy tape, glues or adhesives in gel form or attached to
a solid support; solvents which dissolve a small portion of the
surface of the rigid support or molded layer and result in
adherence of the molded layer to the rigid support upon evacuation
of the solvent; compounds which become permanently adhesive upon
heat setting, photo-setting, UV setting, and the like; and any
other adhesive substance known in the art.
[0116] In another embodiment, the surface of the substrate is
modified to contain modified sites, particularly chemically
modified sites, that can be used to attach, either covalently or
non-covalently, the microspheres of the invention to the discrete
sites or locations on the substrate. "Chemically modified sites" in
this context includes, but is not limited to, the addition of a
pattern of chemical functional groups including amino groups,
carboxy groups, oxo groups and thiol groups, that can be used to
covalently attach microspheres, which generally also contain
corresponding reactive functional groups; the addition of a pattern
of adhesive that can be used to bind the microspheres (either by
prior chemical functionalization for the addition of the adhesive
or direct addition of the adhesive); the addition of a pattern of
charged groups (similar to the chemical functionalities) for the
electrostatic attachment of the microspheres, i.e. when the
microspheres comprise charged groups opposite to the sites; the
addition of a pattern of chemical functional groups that renders
the sites differentially hydrophobic or hydrophilic, such that the
addition of similarly hydrophobic or hydrophilic microspheres under
suitable experimental conditions will result in association of the
microspheres to the sites on the basis of hydroaffinity. For
example, the use of hydrophobic sites with hydrophobic beads, in an
aqueous system, drives the association of the beads preferentially
onto the sites.
[0117] In addition, biologically modified sites may be used to
attach beads to the substrate. For example, binding ligand pairs as
are generally described herein may be used; one partner is on the
bead and the other is on the substrate. Particularly preferred in
this embodiment are complementary nucleic acid strands and
antigen/antibody pairs.
[0118] Furthermore, the use of biological moieties in this manner
allows the creation of composite arrays as well. This is analogous
to the system depicted in FIG. 1F, except that the substrate 10 is
missing. In this embodiment, populations of beads comprise a single
binding partner, and subpopulations of this population have
different bioactive agents. By using different populations with
different binding partners, and a substrate comprising different
assay or array locations with spatially separated binding partners,
a composite array can be generated. This embodiment also a reuse of
codes, as generally described below, as each separate array of the
composite array may use the same codes.
[0119] As outlined above, "pattern" in this sense includes the use
of a uniform treatment of the surface to allow attachment of the
beads at discrete sites, as well as treatment of the surface
resulting in discrete sites. As will be appreciated by those in the
art, this may be accomplished in a variety of ways.
[0120] As noted above, arrays of the present invention and in
particular, arrays of the one-component system are formed by
altering the surface of a substrate so that it contains discrete
sites or wells. Preferably the wells are formed to contain not more
than one microsphere as described herein. As noted herein, in a
one-component system, in a preferred embodiment the assay locations
(which also form the array locations in the one-component system)
are formed in the molded layer. As such, in an alternative
embodiment, the invention provides improved methods for making
arrays comprising a molded layer formed using a template
structure.
[0121] In another preferred embodiment the assay locations are
fiber optic bundles. As such, in a related alternative embodiment,
the invention provides improved methods for making arrays
comprising a segment of a fiber optic bundle on at least one
discrete site on an array.
[0122] In general in this embodiment, at least one, but generally a
plurality of fiber optic bundles is attached to or inserted through
the planar substrate to form the array of arrays on a planar
substrate. When the fiber optic bundle is attached to the
substrate, the method includes providing a fiber optic bundle of
length L, wherein L can in theory be any length, and cutting the
bundle, by methods as are known in the art such as, but not limited
to the use of a diamond saw or water jets, to form a plurality of
small fiber optic bundle segments. The segments are then attached
to the array by methods that include, but are not limited to
placing the segments in a pre-formed well sized to accommodate the
bundle segments attaching with adhesives, or melting the substrate
such that the fiber optic bundle is embedded into the
substrate.
[0123] In an alternative embodiment, the method includes inserting
at least one fiber optic bundle through a block of substrate
material such as plastic or ceramic and then cutting the substrate
including the bundle, to the desired thickness (see FIG. 13). As
such, a cross-sectional portion of the fiber bundle is framed by
the substrate material. Generally, at least a first and a second
bundle will be inserted into the substrate material. Generally, the
bundles will be in an array format as described herein. As one of
skill in the art appreciates, this method markedly facilitates the
production of a large number of uniform array of arrays. That is,
in theory, there is no upper limit to the length of the fiber
bundle or thickness. As such, very long bundles can be inserted
into very thick substrate blocks, i.e. up to or even thicker than I
meter. For example, when one considers that the typical thickness
of the substrate of an array is less than about 0.5 cm, a 1 m block
(into which a potentially 1 m bundle is inserted) results in the
formation of at least about 200 uniform array of arrays.
[0124] In this method, the fiber bundle(s) is inserted into the
substrate substantially perpendicularly to the plane of the surface
of the block. Generally, the substrate is drilled or machined to
form an orifice into which the bundle is inserted. In some
embodiments, the orifice is lined with a sealant or gasket which
surrounds the bundle. Alternatively, a sealant or epoxy is applied
to the substrate surrounding the bundles after the block is cut to
form the plurality of substrates. In some embodiments this is
advantageous as the sealant may not only prevent leaks of any assay
solutions or beads through the substrate around the bundle, but
also the sealant forms a barrier around the bundle that isolates a
bundle from the other bundles. That is, once the arrays are formed,
a substance is applied to the surface of the substrate surrounding
at least a first fiber optic bundle; the substance not only anchors
the fiber bundle in place, but forms a partition separating the
different fiber bundles. In some embodiments the substance, i.e.
epoxy, does not form a partition, but rather holds the fiber bundle
in place.
[0125] In an alternative embodiment a fiber optic bundle is placed
into a liquid or molten solution of a substance that will be the
substrate. That is, in this embodiment, the substrate is made from
a meltable material such as, but not limited to plastic or wax. In
a preferred embodiment the substrate is made from thermosetting or
thermoplastic polymers. In one embodiment, at least one fiber optic
bundle is retained by one end by a holder and immersed into the
molten substrate solution.
[0126] The use of a molten substrate finds a number of uses in
forming the substrate of the array. A particular advantage is the
ability to add substances to the molten solution that will be
incorporated into the substrate once hardened. That is, substances
can be added to modify properties of the substrate such as
rendering it reflective or opaque. In a particularly preferred
embodiment carbon black is added to the liquid substrate substance.
The addition of carbon black causes the resulting substrate to be
opaque.
[0127] It is understood that container may take any shape, and that
a heating mechanism may be implemented in a variety of fashions.
However it is the function of container and heating mechanism to
create and retain a bath of molten substrate into which at least
those portions of the array of bundles projecting from temporary
holder may be immersed or submerged. In a preferred embodiment the
container is of the same dimensions as a microscope slide such that
the resulting substrate is the same size as a microscope slide.
[0128] The molten substrate will fill the space between individual
bundles. The heat source is turned off and the substrate is allowed
to harden, and the temporary holder is removed. Bundles are
embedded within the substrate, for at least a fraction of the
length L of the bundles. The substrate is then cut as described
above to form a plurality of substrates containing fiber
bundles.
[0129] In some embodiments, the exposed ends of the bundles are
machined or processed, typically by lapping and polishing to
planarize the surface of the ends. Concave well regions may be
formed in the surface of exposed ends of each strand, and a bead
deposited in each well, or into a substantial number of the wells,
as described herein.
[0130] Note that the individual bundles are retained in tight
registration with each other by the solidified substrate. The
longitudinal axis of each bundle will remain substantially parallel
to each other, and substantially perpendicular to the plane of the
top surface of substrate.
[0131] In one embodiment an advantage of the substrate is that
individual bundles may be removed and replaced, if necessary. For
example, one or more bundles might become damaged. Rather than
discard the entire array, when a meltable substrate like wax is
used, the damaged bundles may be removed by heating the substrate
surrounding the bundles in question. For example, a thin walled
hollow tube, whose inner diameter exceeds the outer diameter D of a
the bundle to be removed, can be heated and pushed into and through
the wax probe, to surround the bundle in question. This localized
heating enables the damaged bundle to be removed and replaced with
a new or different bundle, around which molten wax can then be
deposited to retain the replacement bundle within the
substrate.
[0132] In a preferred embodiment, the invention also includes a
substrate holder. The substrate holder is a device into which the
substrate fits. The holder allows for easy handling of the array.
In addition , the holder provides rigidity to prevent warping of
the substrate/array. While the holder can be formed from any rigid
substance, in a preferred embodiment the holder is metal.
[0133] In a preferred embodiment the holder comprises a metal frame
that surrounds each edge of the substrate, i.e. the microscope
slide array. In some embodiments the holder comprises a lid that
optionally includes hinges to allow for opening and closing of the
lid. A hinged lid as such facilitates insertion and removal of the
array from the holder. While the lid can be made of any material,
it is preferably a translucent material that allows for detection
of signals from the array.
[0134] In an alternative embodiment the holder further comprises a
base to which the frame is attached. The base may be any rigid
material, but in a preferred embodiment is translucent.
Alternatively the base is metal. What is important is that the
holder remain rigid and prevent the substrate from warping.
[0135] As will be appreciated by those in the art, there are a
number of possible configurations of the system, as generally
depicted in the Figures. In addition to the standard formats
described herein, a variety of other formats may be used. For
example, as shown in FIGS. 1C-1F, "pieces" of substrates may be
used, that are not connected to one another. Again, these may be
the same arrays or different arrays. These pieces may be made
individually, or they may be made as a large unit on a single
substrate and then the substrate is cut or separated into different
individual substrates. Thus, for example, FIGS. 1 C and 1 D depict
a plurality of bead arrays that are added to the wells of the
second substrate; FIG. 1 C is a "bead of beads" that is configured
to maximize mixing. FIG. 1D utilizes a plurality of planar first
substrates; as will be appreciated by those in the art, these may
or may not be attached to the second substrate. In one embodiment,
no particular attachment means are used; alternatively, a variety
of attachment techniques are used. For example, as outlined for
attachment of beads to substrates, covalent or non-covalent forces
may be used, including the use of adhesives, chemistry,
hydrophobic/hydrophilic interactions, etc. In addition, the
substrate may be magnetic and held in place (and optionally mixed)
magnetically as well. Thus, for example, as depicted in FIG. 1F,
binding moieties can be used; these can be covalent linkages or
non-covalent linkages. They may be used simply for attachment, or
for targeting the first substrate arrays to particular locations in
or on the second substrate. Thus, for example, different
oligonucleotides may be used to target and attach the first
substrate to the second.
[0136] In a preferred embodiment, there are optical properties
built into the substrate used for imaging. Thus, for example,
"lensing" capabilities may be built into the substrate, either in a
one component or two component system. For example, in a one
component system, the portion below of one or more of the assay
locations, either within the molded layer or within the rigid
support, may have unique or special optical components, such as
lenses, filters, etc.
[0137] In addition, preferred embodiments utilize configurations
that facilitate mixing of the assay reaction. For example,
preferred embodiments utilize two component systems that allow
mixing. That is, in some embodiments, the arrays project from the
block and can be used as a "stick" that stirs the reaction to
facilitate good mixing of the assay components, increase the
kinetics of the reaction, etc. As will be appreciated by those in
the art, this may be accomplished in a variety of ways. In a
preferred embodiment, the first and second substrates are
configured such that they can be moved relative to one another,
either in the X-Y coordinate plane, the X-Z coordinate plane, the
Y-Z coordinate plane, or in three dimensions (X-Y-Z). Preferred
embodiments utilize a block jig that allows the block to move
freely in either the plane of the plate or orthogonal to it. This
is particularly useful when the reaction volumes are small, since
standard mixing conditions frequently do not work well in these
situations.
[0138] In addition to this, or in place of it, there may be
additional mixing components as part of the system. For example,
there may be exogeneous mixing particles added; one embodiment for
example utilizes magnetic particles, with a magnet that is moved to
force mixing; for example small magnetic mixing bars and magnetic
stir plates may be used.
[0139] Alternatively, mixing in either one or two component systems
can be accomplished by sealing the system and shaking it using
standard techniques, optionally using mixing particles.
[0140] In a preferred embodiment, the system is configured to
reduce evaporation and facilitate small sample size and handling.
That is, the system is closed or sealed by enclosing a defined
space to maintain control over the small sample volumes. In this
regard the invention provides a hybridization chamber that
encompasses or encloses the array and/or sample. As is more fully
outlined below, preferred embodiments utilize the hybridization
chambers comprising a base plate and alignment moieties that find
particular use in the two-component system, although they also find
use in the one-component system.
[0141] One advantage of the enclosed system is that it reduces or
dampens vibration. That is, because of the small sample volume, the
arrays may be susceptible to disturbances caused by vibration, for
example, by platform shaking, motor vibration, or air circulation.
By enclosing the array, and placing the array on the base plate,
the samples and arrays are less susceptible to disturbances caused
by vibration as the base plate dampens the vibration.
[0142] An additional advantage of this aspect of the invention is
that the enclosed array allows for the use of increasingly small
volumes. In an open array format, small sample volumes may
evaporate resulting in a variety of problems including sample
variation, alteration and inconsistent concentration of solutes in
the solution. For example, when small sample volumes are present in
different assay locations, differential evaporation of the solution
may result in dramatically altered solute concentration. Such
differences may alter hybridization kinetics, for example, and make
it difficult to interpret and compare results obtained from such
open arrays. However, by enclosing the array, for example in the
hybridization chamber outlined herein, such sample variance is
minimized thereby rendering the data obtained from the enclosed
array more reliable. Accordingly, any of the methods described
herein, find use with the hybridization chamber.
[0143] Also, the enclosed array allows for prolonged
assay/incubation times relative to incubation times in an open
array. Again, the sealed or enclosed array prevents sample
evaporation, allowing for prolonged incubation periods.
[0144] In addition, the enclosed array facilitates mixing of the
sample, when necessary. In general, when using small sample
volumes, adequate mixing of the sample may be difficult to achieve.
However, as is more fully outlined below, in one embodiment the
hybridization chamber facilitates mixing when flexible membranes
are used with a pneumatic device that provides vacuum and/or
pressure.
[0145] When a "two-component" system is used, a hybridization
chamber may be used. That is, both of the components i.e. the
substrate comprising a plurality of assay locations and the
substrate comprising a plurality of array locations, are enclosed
within the hybridization chamber. In a preferred embodiment, these
components include but are not limited to a fiber optic array and a
multi-well microtiter plate that are enclosed in the hybridization
chamber.
[0146] In a preferred embodiment the hybridization chamber contains
a base plate upon which or into which one of the components is
placed. By base plate is meant any platform or holder onto which
one of the array components is placed. The base plate may be made
of any material including plastic, glass or metal or any materials
outlined herein for substrates; when the base plate is metal, it is
preferably made of aluminum. Aluminum provides for a light weight
yet durable base plate. In addition, aluminum allows for efficient
and/or rapid heat transfer to the chamber. However, when the base
plate is made of plastic or glass, the component is directly
contacted with the base plate. Alternatively, metal sheets or
templates may be inserted into or placed on the base plate. The
metal sheets or templates can be designed to hold any of a variety
of shapes to accommodate a variety of component sizes and/or
shapes. As previously described, metal offers the advantage of
being a rapid and efficient heat conductor.
[0147] In one embodiment the base plate contains at least one
depression or base cavity into which the array component is placed.
That is, when a microtiter plate is the component, for example, the
depression or base cavity is formed such that the microtiter plate
is placed directly into it and preferably fits tightly to avoid
extra vibration and allow efficient heat transfer. The depression
may be molded into the base plate. In addition, the base plate may
contain multiple depressions or cavities such that multiple
separate array components are placed on a single base plate.
Alternatively, the base plate may be flat, and preferably comprise
hooks or other attachment moieties to keep the arrays in place.
[0148] In addition preferred embodiments utilize a lid with the
hybridization chamber. The lid can be made of any material (again,
as listed for substrates herein), but glass, plastics or metal is
preferred. The lid is preferably matched to the base plate such
that when the lid is placed on the base plate, a closed chamber is
formed.
[0149] In another embodiment the lid comprises at least one
component placement port. By component placement port is meant a
site in the lid to which a component is immobilized. That is, the
placement port allows for attachment of one of the components to
the lid. In a preferred embodiment, the port is a hole in the lid
through which the component is inserted. For example, when a fiber
optic bundle is the component, the bundle is inserted through the
port. In this embodiment, the port additionally comprises a sealant
surrounding the attachment site, such that an airtight seal is
formed between the component, i.e. the distal end of the fiber
optic bundle, and the lid. This sealant may be any material
including silicon, rubber, plastic, etc., as outlined below.
Alternatively, the seal may be a gel-based substance such as
petroleum jelly, or a film based substance such as PARAFILM.
[0150] In an additional embodiment, the lid comprises a plurality
of ports in the lid. That is, when multiple components are to be
used, it is necessary to have a separate port for each component.
For example, when multiple fiber optic bundles are used, each fiber
optic bundle is placed in a separate port. However, although it is
possible for each fiber optic bundle to be inserted into one port
at a time, it is also possible for the same fiber optic bundle to
be inserted into different ports successively. That is, there is
nothing to limit the number of ports into which a component is
inserted successively. For example, as shown in FIG. 7A the lid 80
contains multiple ports 82 into which fiber optic bundles 84 are
placed. The lid is then placed onto a microtiter plate 86 in the
base cavity 88 of the base plate 90. A base plate 90 is depicted in
FIG. 7B and shows the base plate 90 and base cavity 88.
[0151] In a preferred embodiment, the port seal reduces or prevents
solution cross contamination. That is, the seal surrounding the
individual port/component forms a seal against the base plate or
array component such that the solution from the sample
corresponding to a particular port/component is separated or sealed
from the other components.
[0152] In an alternative embodiment, not all ports are filled with
components at all times. When it is appropriate or desired to have
less than maximal filling of the ports, plugs can be inserted into
the ports that do not contain components. In this manner, the lid
still forms an airtight seal with the base plate, despite the
presence of ports without components. The plugs can be in the form
of a rubber stopper, a gasket, a film, a gel and the like.
[0153] In a preferred embodiment around the periphery of the
chamber between the lid and base plate resides a sealant. The
sealant may be of any material that results in an airtight seal
being formed between the lid and base plate. In a preferred
embodiment, the sealant is formed of rubber, such as a rubber or
silicon gasket or O-ring 94 (see FIG. 8). The sealant may be fixed
to either the lid or baseplate. To this end, the sealant may be
permanently affixed to the lid or baseplate. Alternatively, the
sealant may fit into a groove in either the lid or base plate. As
such, the sealant is immobilized to the lid or base plate, but the
immobilization is not necessarily permanent. Alternatively, the
sealant may be formed from a liquid sealant such as petroleum jelly
or from a pliable film material such as PARAFILM or other
waxes.
[0154] In a preferred embodiment, when a two-component system is
used, the hybridization chamber further comprises alignment
moieties. By alignment moieties is meant a feature of the chamber
that facilitates alignment of the lid with the base plate. The
importance of the alignment moieties resides not only in the
alignment of a single lid and base plate, but also reproducible
alignment of multiple lids and base plates. That is, the alignment
moieties facilitate the physical alignment between any array
components and any multiple well microtiter plate configuration.
When fiber optic bundles in the lid are to be aligned with a
microtiter plate on the base plate, the alignment moieties allow
for alignment of the vertical center axis of the fiber bundle with
their corresponding well center axis. In a preferred embodiment,
alignment is such that all fiber bundles clear, i.e. do not touch,
the inner walls of the wells. This alignment may be important for
sequential imaging.
[0155] In another embodiment, the alignment moieties facilitate the
physical alignment between any array components and a detection
instrument, such as a microscope.
[0156] In one embodiment the alignment moiety is a complementary
male/female fitting. The male fitting may be affixed to the lid or
base plate, although it need not be permanently affixed. When a
male fitting is used as an alignment moiety in either the base
plate or lid of the chamber, it is preferable that the opposite
chamber piece contain a slot or hole (female fitting) into which
the male fitting is inserted. One of ordinary skill in the art
appreciates the variations of this male/female fitting that find
use with the invention. In this regard, the features may be indexer
pins or bumps on one chamber piece and holes or complementary
grooves on the other piece.
[0157] In a preferred embodiment, fiducials are used; see U.S. Ser.
Nos. 60/119, 323, and 09/500, 555 and PCT/US00/03375, hereby
incorporated by reference in their entirety.
[0158] In an alternative embodiment, the chamber may also contain
clamp features to maintain secure contact between the lid and base
plate. The advantage of clamping is to distribute uniform loading
throughout the chamber to accomplish uniform seal compression. By
"clamp features" or "clamps" is meant any feature that allows for
the application and maintenance of increased pressure or a seal
between the lid and base plate. In one embodiment, the clamp
feature includes a rotating stud/receptacle mechanism. That is, a
stud 96 is inserted into a receptacle 98 and rotated to depress the
lid and base plate together (see FIG. 8). Alternatively, the
mechanism may include a hook and latch mechanism. One of ordinary
skill in the art appreciates the number of clamping mechanisms that
find use with the invention. In addition, one of ordinary skill in
the art appreciates that the method of clamping is not limited to
manual clamping. As such, it may also be automated.
[0159] In an alternative embodiment, the chamber includes features
around the periphery for handling the chamber. In a preferred
embodiment the features are slots that are wide enough to permit a
users fingers to manually handle the chamber/array. In an
alternative embodiment, the features are slots, grooves, handles
and the like and may find particular use in automatic or robotic
movement of the chambers. These additional features may also be
distributed asymmetrically to facilitate robotic handling.
[0160] As described above, an advantage of the hybridization
chamber is that small sample volumes can be used without the loss
of sample solution. In a further embodiment, the chamber may
contain one or more reservoirs to hold additional solutions. As
such, the hybridization chamber also functions as a humidity
chamber. The inclusion of additional solution in the reservoir
further prevents evaporation of sample.
[0161] In an alternative embodiment, for example when no microtiter
plate is used, the sample may be applied to a membrane that is on
the surface of a base plate. Advantages of using the membrane
include ease of cleaning or even disposing of the membrane after
each use and the flexible membrane will not damage pipette tips or
fiber optic tips due to contacting the tips with the bottom of the
sample well.
[0162] In this embodiment, the base plate contains a series of
small openings 102, for example in microplate format (FIG. 9A).
Thus, the membrane is depressed into the openings forming separate
assay locations. A variety of membranes are useful with the
invention. What is important is that the membrane is flexible. In
some embodiments it may be desired to have a chemically inert
membrane, while in some embodiments it may be desirable to have a
membrane to which assay components will interact, for example
nylon, nitrocellulose membranes and the like.
[0163] In a preferred embodiment, channels connect each of the
openings (FIG. 9B). The channels 100 may connect to a pneumatic
device that produces vacuum and/or pressure. Thus, when vacuum is
applied, the membrane deforms into the openings 102 to form small
pockets or wells. The sample can then be applied to the pockets. By
applying different amounts of vacuum to the membrane through the
openings, the volume of the well formed by the deformed membrane
and fluid height can be changed. Furthermore, applying intermittent
vacuum to the membrane through the channel can also agitate or mix
the liquid in the wells. Such a mixing method is advantageous
because the entire system does not have to be vibrated and stir
bars or tumblers are not required. Furthermore, when subsets of
openings are connected to different channels, different subsets can
be mixed independently in the same base plate.
[0164] When positive pressure is applied, the membrane deforms up
or stays flat depending on the magnitude of the pressure, whether
there is a load on top of the membrane and the size and shape of
the opening. This has significant advantages particularly in
washing or cleaning of the chamber.
[0165] When pressure and vacuum are applied to different ports in
certain sequences, small amounts of solutions can be made to
migrate to different portions of the membrane. That is, as shown in
FIG. 10A-F, differential application of pressure and vacuum results
in a membrane that is elevated in some places and depressed in
other places. Thus, a solution that is applied to the membrane will
migrate to the lower sections of the membrane. This has the
advantage of allowing incubations of a sample on the membrane to
proceed for precise times. That is following the particular time,
vacuum can be released and if necessary pressure applied to remove
the solution. This will allow the incubation in small sections to
achieve uniform incubation time between the first and last wells
across an array.
[0166] Advantages of regulating sample volume through the
application of vacuum or pressure, include reducing consumption
volume of reagents, such as hybridization solutions; increasing the
ease of mixing small sample volumes and increasing the ease of
cleaning the membrane.
[0167] In a preferred embodiment the channels connect to common
fluid handling devices to pump in or suck out sample solutions such
as hybridization mixtures or wash fluids. Again, in one embodiment
all openings are connected to a single channel. As such, all wells
are treated with the same solution. Alternatively, subpopulations
of openings are connected to different channels allowing for
differential application of solutions to the subpopulations.
[0168] When the channels are connected to fluid handling devices,
it will be necessary to include a feature for the application and
removal of the liquid from the sample. That is, for liquid to be
added and removed through the opening in the base plate, the
membrane must be penetrated to allow the fluid to be moved. In this
regard, a needle, for example, is useful for puncturing the
membrane to apply and remove the fluid. When needles are used, it
may be necessary to use a resealable membrane, or apply a sealant
to the puncture location to prevent undesired leakage of the
solution.
[0169] In some embodiments the chamber includes heat transfer
features. That is, when elevated temperatures are required or
desired, the chamber is designed to maintain elevated temperatures.
In one embodiment, this includes the application of an insulating
material to the chamber. Then, when pre-warmed solution is
introduced into the chamber, the elevated temperature is
maintained. That is, the solution can be easily heated outside of
the chamber prior to being pumped into the chamber. The simple
chamber geometry will facilitate the maintenance of equal
temperatures between liquid in different wells.
[0170] In an alternative embodiment, the chamber includes a heating
mechanism to maintain the elevated temperature in the chamber. In
one embodiment, the chamber is heated uniformly by the heating
apparatus. In an alternative embodiment, the heating apparatus
heats different sections of the chamber independently.
[0171] As described above, the use of metal such as aluminum on the
base plate facilitates heat transfer because the metal is a fast
and efficient conductor of heat.
[0172] When a "one-component" system is used, a lid and a sealing
mechanism can be used. That is, as described above, the lid forms
an airtight seal with the base plate. Thus, like the lid above, the
lid of the "one-component" system also includes a sealant between
the lid and base plate. In one embodiment, the lid and base plate
also include alignment moieties as described above for the
"two-component" system. Alternatively, in one embodiment the
chamber of the one-component system does not include alignment
moieties. In this respect, the necessity for stringent alignment of
the lid and base plate in the one-component system is lower than
that for the two-component system. That is, because the
one-component system does not have array components in the lid to
be aligned with array locations on the base plate, alignment is not
as stringent. However, alignment may still be important for
imaging.
[0173] Furthermore, as described above, the lid of the chamber in
the one-component system can be made of glass, plastic or metal.
Again, the use of metal facilitates the maintenance of temperature
as the metal is a fast and efficient heat conductor.
[0174] In addition, the system may comprise additional elements as
well. These include a holder or holders for the probes or fiber
optic bundles. Such holders are more fully described in U.S. Ser.
Nos. 60/135,089, filed May 20, 1999, and 09/574,962 filed May 19,
2000, and PCT US00/13772 filed May 19, 2000. In addition, the
system may include cells as described in U.S. Ser. Nos. 09/033,462
and 09/260,963 and PCT/US99/04473. In addition, the system may
include fiducials as described in U.S. Ser. Nos. 60/119,323, and
09/500,555 and PCT/US00/03375, all of which are expressly
incorporated herein by reference.
[0175] In a preferred embodiment, the methods and compositions of
the invention comprise a robotic system. Systems can be directed to
the use of 96 (or more) well microtiter plates, but as will be
appreciated by those in the art, any number of different plates or
configurations of arrays may be used. In addition, any or all of
the steps outlined herein may be automated; thus, for example, the
systems may be completely or partially automated.
[0176] As will be appreciated by those in the art, there are a wide
variety of components which can be used, including, but not limited
to, one or more robotic arms; plate handlers for the positioning of
the plates containing the one or more arrays; automated lid
handlers to remove and replace lids for wells on non-cross
contamination plates; tip assemblies for sample distribution with
disposable tips; washable tip assemblies for sample distribution;
96 well or other configuration loading blocks; cooled reagent
racks; plate pipette positions (optionally cooled); stacking towers
for plates and tips; and computer systems.
[0177] Fully robotic systems include automated liquid- and
particle-handing, including high throughput pipetting to perform
all steps of screening applications. This includes liquid, and
particle manipulations such as aspiration, dispensing, mixing,
diluting, washing, accurate volumetric transfers; retrieving, and
discarding of pipet tips; and repetitive pipetting of identical
volumes for multiple deliveries from a single sample aspiration.
These manipulations are cross-contamination-free liquid and
particle transfers.
[0178] In a preferred embodiment, chemically derivatized particles,
plates, tubes, magnetic particle, or other solid phase matrix with
specificity to the ligand or variant proteins are used. The binding
surfaces of plates, tubes or any solid phase matrices include
non-polar surfaces, highly polar surfaces, modified dextran coating
to promote covalent binding, antibody coating, affinity media to
bind fusion proteins or peptides, surface-fixed proteins such as
recombinant protein A or G, nucleotide resins or coatings, and
other affinity matrix are useful in this invention.
[0179] In a preferred embodiment, platforms for multi-assay
location plates, multi-tubes, minitubes, deep-well plates,
microfuge tubes, cryovials, square well plates, filters, chips,
optic fibers, beads, and other solid-phase matrices or platform
with various volumes are accommodated on an upgradable modular
platform for additional capacity. This modular platform includes a
variable speed orbital shaker, and multi-position work decks for
source samples, sample and reagent dilution, assay plates, sample
and reagent reservoirs, pipette tips, and an active wash
station.
[0180] In a preferred embodiment, thermocycler and thermoregulating
systems are used for stabilizing the temperature of the heat
exchangers such as controlled blocks or platforms to provide
accurate temperature control of incubating samples from 4.degree.
C. to 100.degree. C.
[0181] In a preferred embodiment, Interchangeable pipet heads
(single or multi-channel ) with single or multiple magnetic probes,
affinity probes, or pipetters robotically manipulate the liquid and
particles. Multi-well or multi-tube magnetic separators or
platforms manipulate liquid and particles in single or multiple
sample formats.
[0182] In some preferred embodiments, the instrumentation will
include CCD cameras to capture and transform data and images into
quantifiable formats; and a computer workstation. These will permit
data analysis. CCD cameras used in the method of the invention will
permit simultaneous detection of one or more subpopulations of
beads. The CCD cameras used in detection can be coupled with
additional optics devices including lenses, fiber optics, image
intensifiers, and the like, for amplifying, focusing, reducing or
expanding the images produced in the detection methods of the
invention. One skilled in the art will be readily able to determine
the detector and optics required according to the desired screening
methods and the dimensions of the invention composite of
arrays.
[0183] In other embodiments, the instrumentation will include
confocal microscopes. Confocal microscopes used in the method of
the invention will permit simultaneous imaging of one or more
subpopulations of beads. The confocal microscope will typically be
configured for receiving a composite array of the present invention
which is formatted to the dimensions of a microscope slide.
[0184] Exemplary detection instruments useful with the composite
array of the invention are well known in the art and include, for
example, the GenePix 4000B Array Scanner from Axon Instruments,
Inc., GSI Scanarray 3000 from GSI Luminomics, array WoRx Microarray
Scanner from Applied Precision, and the like.
[0185] In one embodiment of the invention, the array composition
will be attached via a liquid and/or vapor tight seal to a fluid
flow device. Such a fluid flow device will permit sample, solvent,
probes, rinsing solutions, reacting solutions, and the like, to
flow over the discrete sites and the microspheres positioned
thereon. Thus, a fluid flow device will be capable of carrying out
functions such as those disclosed above with regard to the
pneumatic device or robotic device, or other such devices known in
the art. Fluid flow devices known in the art include flow cells
such as those provided by manufacturers such as Mindrum Precision,
Inc., Rancho Cucamonga, Calif.; and Upchurch Scientific, Oak
Harbor, Wash.
[0186] In a preferred embodiment of the invention, the array
composition of the invention is attached to a fluid flow device and
positioned with respect to a detection instrument such as a
confocal microscope or a CCD camera in such a way as to permit
numerous iterations of sample loading, detection, rinsing, and the
like, without requiring the array composition to be moved or
manipulated in any manner beyond fluid flow through the fluid flow
device.
[0187] The hardware used in the apparatus and detection methods of
the invention preferably orients the invention composite arrays
with respect to the detector optics or to the CCD camera or other
optical detection device such that substantially all discrete sites
(i.e., 80% or more, preferably 90% or more, more preferably 95% or
more) of an array, and, in a preferred embodiment, all arrays
(i.e., 80% or more, preferably 90% or more, more preferably 95% or
more), can be accurately and simultaneously detected. The present
invention provides a composite array, typically containing a rigid
support, which composite array is sufficiently rigid to permit the
various discrete sites on the composite array to be positioned for
accurate, simultaneous measurement by, for example, all discrete
sites being oriented at the same distance from the optics or
detector.
[0188] Accordingly, detection methods which include the use of
instruments such as CCD cameras and confocal microscopes will find
increased efficacy when the substrate, such as an invention
composite array, is planar and the detector is positioned
substantially parallel to the substrate such that a plurality of
signals from the discrete sites can be measured. Thus, the array
composition of the present invention will be substantially planar
and flat to within about 20 .mu.m. As referred to herein,
substantially planar means that about 80% of the discrete sites lie
in the same plane, within a non-planarity tolerance of about 20
.mu.m. Preferably, about 90% of the discrete sites will lie in the
same plane, within the above tolerance; more preferably, about 95%
of the discrete sites lie in the same plane; most preferably about
98% of the discrete sites lie in the same plane.
[0189] The above non-planarity tolerance will typically have a
flatness or planarity to within about 20 .mu.m. The flatness or
planarity of the substrate can correspond to the flatness of the
discrete sites within the active area of the detection device, or
can alternatively correspond to the flatness or planarity of the
discrete sites throughout the entire substrate. Flatness or
planarity within a particular distance refers to the characteristic
of a substrate in which discrete sites differ by no more than the
specified distance along the dimension perpendicular to the plane
of the substrate. For example, discrete sites that are flat or
planar to within about 20 .mu.m are the discrete sites which are
sufficiently close to the plane of the substrate such that each
discrete site is no more than 20 .mu.m "higher" than another
discrete site (where height is measured as a distance along the
dimension perpendicular to the plane of the substrate). In one
embodiment the discrete sites have a flatness or planarity such
that they preferably lie within the depth of field of the detection
device. Preferably, the non-planarity tolerance will be within
about 10 .mu.m. More preferably, the non-planarity tolerance will
be within about 5 .mu.m. Most preferably the non-planarity
tolerance will be within about 1 .mu.m.
[0190] In a preferred embodiment, the composite arrays of the
invention utilize fiducials to allow comparison of sequential data
images of the arrays. Such a fiducial can be a bead with a unique
optical signature or other characteristic. Alternatively, the
substrate or composite array may have other types of physical
fiducials, such one or more defined edges that have characteristic
optical properties that can be either spaced along the edge(s) or
comprise the entire edge. Alternatively, the fiducials can be an
inherent characteristic of the array; for example, small
irregularities in the sites (features) of the array can be
exploited to serve as fiducials, generating a "fiducial template".
Other such fiducials and methods for using arrays comprising
fiducials are taught in U.S. Ser. No. 09/500,555, which is herein
incorporated by reference.
[0191] The flexible hardware and software allow instrument
adaptability for multiple applications. The software program
modules allow creation, modification, and running of methods. The
system diagnostic modules allow instrument alignment, correct
connections, and motor operations. The customized tools, labware,
and liquid and particle transfer patterns allow different
applications to be performed. The database allows method and
parameter storage. Robotic and computer interfaces allow
communication between instruments.
[0192] In a preferred embodiment, the robotic workstation includes
one or more heating or cooling components. Depending on the
reactions and reagents, either cooling or heating may be required,
which can be done using any number of known heating and cooling
systems, including Peltier systems.
[0193] In a preferred embodiment, the robotic apparatus includes a
central processing unit which communicates with a memory and a set
of input/output devices (e.g., keyboard, mouse, monitor, printer,
etc.) through a bus. The general interaction between a central
processing unit, a memory, input/output devices, and a bus is known
in the art. Thus, a variety of different procedures, depending on
the experiments to be run, are stored in the CPU memory.
[0194] In a preferred embodiment, the compositions of the invention
further comprise a population of microspheres. By "population"
herein is meant a plurality of beads as outlined above for arrays.
Within the population are separate subpopulations, which can be a
single microsphere or multiple identical microspheres. That is, in
some embodiments, as is more fully outlined below, the array may
contain only a single bead for each bioactive agent; preferred
embodiments utilize a plurality of beads of each type. By
"microspheres" or "beads" or "particles" or grammatical equivalents
herein is meant small discrete particles. The composition of the
beads will vary, depending on the class of bioactive agent and the
method of synthesis. Suitable bead compositions include those used
in peptide, nucleic acid and organic moiety synthesis, including,
but not limited to, plastics, ceramics, glass, polystyrene,
methylstyrene, acrylic polymers, paramagnetic materials, thoria
sol, carbon graphite, titanium dioxide, latex or cross-linked
dextrans such as Sepharose, cellulose, nylon, cross-linked micelles
and Teflon may all be used. "Microsphere Detection Guide" from
Bangs Laboratories, Fishers, Ind. is a helpful guide.
[0195] The beads need not be spherical; irregular particles may be
used. In addition, the beads may be porous, thus increasing the
surface area of the bead available for either bioactive agent
attachment or IBL attachment. The bead sizes range from nanometers,
i.e. 100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2
micron to about 200 microns being preferred, and from about 0.5 to
about 5 micron being particularly preferred, although in some
embodiments smaller beads may be used.
[0196] It should be noted that a key component of the invention is
the use of a substrate/bead pairing that allows the association or
attachment of the beads at discrete sites on the surface of the
substrate, such that the beads do not move during the course of the
assay.
[0197] In some embodiments, each microsphere comprises a bioactive
agent, although as will be appreciated by those in the art, there
may be some microspheres which do not contain a bioactive agent,
depending on the synthetic methods. Alternatively, as described
herein, in some embodiments it is desirable that a population of
microspheres does not contain a bioactive agent. By "candidate
bioactive agent" or "bioactive agent" or "chemical functionality"
or "binding ligand" as used herein describes any molecule, e.g.,
protein, oligopeptide, small organic molecule, coordination
complex, polysaccharide, polynucleotide, etc. which can be attached
to the microspheres of the invention. It should be understood that
the compositions of the invention have two primary uses. In a
preferred embodiment, as is more fully outlined below, the
compositions are used to detect the presence of a particular target
analyte; for example, the presence or absence of a particular
nucleotide sequence or a particular protein, such as an enzyme, an
antibody or an antigen. In an alternate preferred embodiment, the
compositions are used to screen bioactive agents, i.e. drug
candidates, for binding to a particular target analyte.
[0198] Bioactive agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 Daltons. Bioactive agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, and preferably at least two
of the functional chemical groups. The bioactive agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Bioactive agents are also found among
biomolecules including peptides, nucleic acids, saccharides, fatty
acids, steroids, purines, pyrimidines, derivatives, structural
analogs or combinations thereof. Particularly preferred are nucleic
acids and peptides.
[0199] Bioactive agents can be obtained from a wide variety of
sources including libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides. Alternatively,
libraries of natural compounds in the form of bacterial, fungal,
plant and animal extracts are available or readily produced.
Additionally, natural or synthetically produced libraries and
compounds are readily modified through conventional chemical,
physical and biochemical means. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification and/or amidification to
produce structural analogs.
[0200] In a preferred embodiment, the bioactive agents are
proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and norleucine
are considered amino acids for the purposes of the invention. The
side chains may be in either the (R) or the (S) configuration. In
the preferred embodiment, the amino acids are in the (S) or
L-configuration. If non-naturally occurring side chains are used,
non-amino acid substituents may be used, for example to prevent or
retard in vivo degradations.
[0201] In one preferred embodiment, the bioactive agents are
naturally occurring proteins or fragments of naturally occurring
proteins. Thus, for example, cellular extracts containing proteins,
or random or directed digests of proteinaceous cellular extracts,
may be used. In this way libraries of procaryotic and eukaryotic
proteins may be made for screening in the systems described herein.
Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, and mammalian proteins, with the latter
being preferred, and human proteins being especially preferred.
[0202] In a preferred embodiment, the bioactive agents are peptides
of from about 5 to about 30 amino acids, with from about 5 to about
20 amino acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or
"biased" random peptides. By "randomized" or grammatical
equivalents herein is meant that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized bioactive proteinaceous agents.
[0203] In a preferred embodiment, a library of bioactive agents is
used. The library should provide a sufficiently structurally
diverse population of bioactive agents to effect a
probabilistically sufficient range of binding to target analytes.
Accordingly, an interaction library must be large enough so that at
least one of its members will have a structure that gives it
affinity for the target analyte. Although it is difficult to gauge
the required absolute size of an interaction library, nature
provides a hint with the immune response: a diversity of 107-108
different antibodies provides at least one combination with
sufficient affinity to interact with most potential antigens faced
by an organism. Published in vitro selection techniques have also
shown that a library size of 107 to 108 is sufficient to find
structures with affinity for the target. Thus, in a preferred
embodiment, at least 106, preferably at least 107, more preferably
at least 108 and most preferably at least 109 different bioactive
agents are simultaneously analyzed in the subject methods.
Preferred methods maximize library size and diversity.
[0204] In a preferred embodiment, the library is fully randomized,
with no sequence preferences or constants at any position. In a
preferred embodiment, the library is biased. That is, some
positions within the sequence are either held constant, or are
selected from a limited number of possibilities. For example, in a
preferred embodiment, the nucleotides or amino acid residues are
randomized within a defined class, for example, of hydrophobic
amino acids, hydrophilic residues, sterically biased (either small
or large) residues, towards the creation of cysteines, for
cross-linking, prolines for SH-3 domains, serines, threonines,
tyrosines or histidines for phosphorylation sites, etc., or to
purines, etc.
[0205] In a preferred embodiment, the bioactive agents are nucleic
acids (generally called "probe nucleic acids" or "candidate probes"
herein). By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage, et
al., Tetrahedron, 49(10):1925 (1993) and references therein;
Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl, et al., Eur. J.
Biochem., 81:579 (1977); Letsinger, et al., Nucl. Acids Res.,
14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,
et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al.,
Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag, et al.,
Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No. 5,644,048),
phosphorodithioate (Briu, et al., J. Am. Chem. Soc., 111:2321
(1989)), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc., 114:1895 (1992); Meier, et al.,
Chem. Int. Ed. Engl., 31:1008 (1992); Nielsen, Nature, 365:566
(1993); Carlsson, et al., Nature, 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include
those with positive backbones (Denpcy, et al., Proc. Natl. Acad.
Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S. Pat. Nos.
5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;
Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);
Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger,
et al., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2
and 3, ASC Symposium Series 580, "Carbohydrate Modifications in
Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker,
et al., Bioorganic & Medicinal Chem. Lett., 4:395 (1994);
Jeffs, et al., J. Biomolecular NMR, 34:17 (1994); Tetrahedron
Lett., 37:743 (1996)) and non-ribose backbones, including those
described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters
6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in
Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic
acids containing one or more carbocyclic sugars are also included
within the definition of nucleic acids (see Jenkins, et al., Chem.
Soc. Rev., (1995) pp. 169-176). Several nucleic acid analogs are
described in Rawls, C & E News, Jun. 2, 1997, page 35. All of
these references are hereby expressly incorporated by reference.
These modifications of the ribose-phosphate backbone may be done to
facilitate the addition of additional moieties such as labels, or
to increase the stability and half-life of such molecules in
physiological environments; for example, PNA is particularly
preferred. In addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xanthanine,
hypoxanthanine, isocytosine, isoguanine, and base analogs such as
nitropyrrole and nitroindole, etc. As used herein, the term
"nucleoside" includes nucleotides as well as nucleoside and
nucleotide analogs, and modified nucleosides such as amino modified
nucleosides. In addition, "nucleoside" includes non-naturally
occurring analog structures. Thus for example the individual units
of a peptide nucleic acid, each containing a base, are referred to
herein as a nucleoside.
[0206] In a preferred embodiment, the bioactive agents are
libraries of clonal nucleic acids, including DNA and RNA. In this
embodiment, individual nucleic acids are prepared, generally using
conventional methods (including, but not limited to, propagation in
plasmid or phage vectors, amplification techniques including PCR,
etc.). The nucleic acids are preferably arrayed in some format,
such as a microtiter plate format, and beads added for attachment
of the libraries.
[0207] Attachment of the clonal libraries (or any of the nucleic
acids outlined herein) may be done in a variety of ways, as will be
appreciated by those in the art, including, but not limited to,
chemical or affinity capture (for example, including the
incorporation of derivatized nucleotides such as AminoLink or
biotinylated nucleotides that can then be used to attach the
nucleic acid to a surface, as well as affinity capture by
hybridization), cross-linking, and electrostatic attachment,
etc.
[0208] In a preferred embodiment, affinity capture is used to
attach the clonal nucleic acids to the beads. For example, cloned
nucleic acids can be derivatized, for example with one member of a
binding pair, and the beads derivatized with the other member of a
binding pair. Suitable binding pairs are as described herein for
IBL/DBL pairs. For example, the cloned nucleic acids may be
biotinylated (for example using enzymatic incorporate of
biotinylated nucleotides, for by photoactivated cross-linking of
biotin). Biotinylated nucleic acids can then be captured on
streptavidin-coated beads, as is known in the art. Similarly, other
hapten-receptor combinations can be used, such as digoxigenin and
anti-digoxigenin antibodies. Alternatively, chemical groups can be
added in the form of derivatized nucleotides, that can then be used
to add the nucleic acid to the surface.
[0209] Preferred attachments are covalent, although even relatively
weak interactions (i.e. non-covalent) can be sufficient to attach a
nucleic acid to a surface, if there are multiple sites of
attachment per nucleic acid. Thus, for example, electrostatic
interactions can be used for attachment, for example by having
beads carrying the opposite charge to the bioactive agent.
[0210] Similarly, affinity capture utilizing hybridization can be
used to attach cloned nucleic acids to beads. For example, as is
known in the art, polyA+RNA is routinely captured by hybridization
to oligo-dT beads; this may include oligo-dT capture followed by a
cross-linking step, such as psoralen crosslinking. If the nucleic
acids of interest do not contain a polyA tract, one can be attached
by polymerization with terminal transferase, or via ligation of an
oligoA linker, as is known in the art.
[0211] Alternatively, chemical crosslinking may be done, for
example by photoactivated crosslinking of thymidine to reactive
groups, as is known in the art.
[0212] In general, special methods are required to decode clonal
arrays, as is more fully outlined below.
[0213] As described above generally for proteins, nucleic acid
bioactive agents may be naturally occurring nucleic acids, random
nucleic acids, or "biased" random nucleic acids. For example,
digests of prokaryotic or eukaryotic genomes may be used as is
outlined above for proteins.
[0214] In general, probes of the present invention are designed to
be complementary to a target sequence (either the target analyte
sequence of the sample or to other probe sequences, as is described
herein), such that hybridization of the target and the probes of
the present invention occurs. This complementarity need not be
perfect; there may be any number of base pair mismatches that will
interfere with hybridization between the target sequence and the
single stranded nucleic acids of the present invention. However, if
the number of mutations is so great that no hybridization can occur
under even the least stringent of hybridization conditions, the
sequence is not a complementary target sequence. Thus, by
"substantially complementary" herein is meant that the probes are
sufficiently complementary to the target sequences to hybridize
under the selected reaction conditions. High stringency conditions
are known in the art; see for example Maniatis et al., Molecular
Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols
in Molecular Biology, ed. Ausubel, et al., both of which are hereby
incorporated by reference. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, "Overview of
principles of hybridization and the strategy of nucleic acid
assays" (1993). Generally, stringent conditions are selected to be
about 5-10.degree. C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions will be those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g. greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. In another
embodiment, less stringent hybridization conditions are used; for
example, moderate or low stringency conditions may be used, as are
known in the art; see Maniatis and Ausubel, supra, and Tijssen,
supra.
[0215] The term "target sequence" or grammatical equivalents herein
means a nucleic acid sequence on a single strand of nucleic acid.
The target sequence may be a portion of a gene, a regulatory
sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or
others. It may be any length, with the understanding that longer
sequences are more specific. As will be appreciated by those in the
art, the complementary target sequence may take many forms. For
example, it may be contained within a larger nucleic acid sequence,
i.e. all or part of a gene or mRNA, a restriction fragment of a
plasmid or genomic DNA, among others. As is outlined more fully
below, probes are made to hybridize to target sequences to
determine the presence or absence of the target sequence in a
sample. Generally speaking, this term will be understood by those
skilled in the art.
[0216] The target sequence may also be comprised of different
target domains; for example, in "sandwich" type assays as outlined
below, a first target domain of the sample target sequence may
hybridize to a capture probe or a portion of capture extender
probe, a second target domain may hybridize to a portion of an
amplifier probe, a label probe, or a different capture or capture
extender probe, etc. In addition, the target domains may be
adjacent (i.e. contiguous) or separated. For example, when OLA
techniques are used, a first primer may hybridize to a first target
domain and a second primer may hybridize to a second target domain;
either the domains are adjacent, or they may be separated by one or
more nucleotides, coupled with the use of a polymerase and dNTPs,
as is more fully outlined below. The terms "first" and "second" are
not meant to confer an orientation of the sequences with respect to
the 5'-3' orientation of the target sequence. For example, assuming
a 5'-3' orientation of the complementary target sequence, the first
target domain may be located either 5' to the second domain, or 3'
to the second domain. In addition, as will be appreciated by those
in the art, the probes on the surface of the array (e.g. attached
to the microspheres) may be attached in either orientation, either
such that they have a free 3' end or a free 5' end; in some
embodiments, the probes can be attached at one ore more internal
positions, or at both ends.
[0217] As outlined herein, the invention provides a number of
different primers and probes. By "primer nucleic acid" herein is
meant a probe nucleic acid that will hybridize to some portion,
i.e. a domain, of the target sequence. The size of the primer
nucleic acid may vary, as will be appreciated by those in the art,
in general varying from 5 to 500 nucleotides in length, with
primers of between 10 and 100 being preferred, between 15 and 50
being particularly preferred, and from 10 to 35 being especially
preferred, depending on the use and amplification technique. In
addition, the different amplification techniques may have further
requirements of the primers, as is more fully described below.
[0218] In general, either direct or indirect detection of the
target products can be done. "Direct" detection as used in this
context, as for the other reactions outlined herein, requires the
incorporation of a label, in this case a detectable label,
preferably an optical label such as a fluorophore, into the target
sequence, with detection proceeding as outlined below. In this
embodiment, the label(s) may be incorporated in a variety of ways:
(1) the primers comprise the label(s), for example attached to the
base, a ribose, a phosphate, or to analogous structures in a
nucleic acid analog; (2) modified nucleosides are used that are
modified at either the base or the ribose (or to analogous
structures in a nucleic acid analog) with the label(s); these
label-modified nucleosides are then converted to the triphosphate
form and are incorporated into a newly synthesized strand by a
polymerase; (3) modified nucleotides are used that comprise a
functional group that can be used to add a detectable label; (4)
modified primers are used that comprise a functional group that can
be used to add a detectable label or (5) a label probe that is
directly labeled and hybridizes to a portion of the target sequence
can be used. Any of these methods result in a newly synthesized
strand or reaction product that comprises labels, that can be
directly detected as outlined below.
[0219] In a preferred embodiment, a secondary detectable label is
used. A secondary label is one that is indirectly detected; for
example, a secondary label can bind or react with a primary label
for detection, can act on an additional product to generate a
primary label (e.g enzymes), or may allow the separation of the
compound comprising the secondary label from unlabeled materials,
etc. Secondary labels find particular use in systems requiring
separation of labeled and unlabeled probes, such as SBE, OLA,
invasive cleavage reactions, etc; in addition, these techniques may
be used with many of the other techniques described herein.
Secondary labels include, but are not limited to, one of a binding
partner pair; chemically modifiable moieties; nuclease inhibitors,
enzymes such as horseradish peroxidase, alkaline phosphatases,
lucifierases, etc.
[0220] In a preferred embodiment, the secondary label is a binding
partner pair. For example, the label may be a hapten or antigen,
which will bind its binding partner. In a preferred embodiment, the
binding partner can be attached to a solid support to allow
separation of extended and non-extended primers. For example,
suitable binding partner pairs include, but are not limited to:
antigens (such as proteins (including peptides)) and antibodies
(including fragments thereof (FAbs, etc.)); proteins and small
molecules, including biotin/streptavidin; enzymes and substrates or
inhibitors; other protein-protein interacting pairs;
receptor-ligands; and carbohydrates and their binding partners.
Nucleic acid - nucleic acid binding proteins pairs are also useful.
In general, the smaller of the pair is attached to the NTP for
incorporation into the primer. Preferred binding partner pairs
include, but are not limited to, biotin (or imino-biotin) and
streptavidin, digeoxinin and Abs, and Prolinx.TM. reagents.
[0221] In a preferred embodiment, the binding partner pair
comprises biotin or imino-biotin and streptavidin. Imino-biotin is
particularly preferred as imino-biotin disassociates from
streptavidin in pH 4.0 buffer while biotin requires harsh
denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at
95.degree. C.).
[0222] In a preferred embodiment, the secondary label is a
chemically modifiable moiety. In this embodiment, labels comprising
reactive functional groups are incorporated into the nucleic acid.
The functional group can then be subsequently labeled with a
primary label. Suitable functional groups include, but are not
limited to, amino groups, carboxy groups, maleimide groups, oxo
groups and thiol groups, with amino groups and thiol groups being
particularly preferred. For example, primary labels containing
amino groups can be attached to secondary labels comprising amino
groups, for example using linkers as are known in the art; for
example, homo-or hetero-bifunctional linkers as are well known (see
1994 Pierce Chemical Company catalog, technical section on
cross-linkers, pages 155-200, incorporated herein by
reference).
[0223] In a preferred embodiment, the bioactive agents are organic
chemical moieties, a wide variety of which are available in the
literature.
[0224] In a preferred embodiment, each bead comprises a single type
of bioactive agent, although a plurality of individual bioactive
agents are preferably attached to each bead. Similarly, preferred
embodiments utilize more than one microsphere containing a unique
bioactive agent; that is, there is redundancy built into the system
by the use of subpopulations of microspheres, each microsphere in
the subpopulation containing the same bioactive agent.
[0225] As will be appreciated by those in the art, the bioactive
agents may either be synthesized directly on the beads, or they may
be made and then attached after synthesis. In a preferred
embodiment, linkers are used to attach the bioactive agents to the
beads, to allow good attachment, sufficient flexibility to allow
good interaction with the target molecule, and to avoid undesirable
binding reactions. In a preferred embodiment, the bioactive agents
are synthesized directly on the beads. As is known in the art, many
classes of chemical compounds are currently synthesized on solid
supports, including beads, such as peptides, organic moieties, and
nucleic acids.
[0226] In a preferred embodiment, the bioactive agents are
synthesized first, and then covalently attached to the beads. As
will be appreciated by those in the art, this will be done
depending on the composition of the bioactive agents and the beads.
The functionalization of solid support surfaces such as certain
polymers with chemically reactive groups such as thiols, amines,
carboxyls, etc. is generally known in the art. Accordingly, "blank"
microspheres may be used that have surface chemistries that
facilitate the attachment of the desired functionality by the user.
Some examples of these surface chemistries for blank microspheres
include, but are not limited to, amino groups including aliphatic
and aromatic amines, carboxylic acids, aldehydes, amides,
chloromethyl groups, hydrazide, hydroxyl groups, sulfonates and
sulfates.
[0227] These functional groups can be used to add any number of
different candidate agents to the beads, generally using known
chemistries. For example, candidate agents containing carbohydrates
may be attached to an amino-functionalized support; the aldehyde of
the carbohydrate is made using standard techniques, and then the
aldehyde is reacted with an amino group on the surface. In an
alternative embodiment, a sulthydryl linker may be used. There are
a number of sulfhydryl reactive linkers known in the art such as
SPDP, maleimides, a- haloacetyls, and pyridyl disulfides (see for
example the 1994 Pierce Chemical Company catalog, technical section
on cross-linkers, pages 155-200, incorporated herein by reference)
which can be used to attach cysteine containing proteinaceous
agents to the support. Alternatively, an amino group on the
candidate agent may be used for attachment to an amino group on the
surface. For example, a large number of stable bifunctional groups
are well known in the art, including homobifunctional and
heterobifunctional linkers (see Pierce Catalog and Handbook, pages
155-200). In an additional embodiment, carboxyl groups (either from
the surface or from the candidate agent) may be derivatized using
well known linkers (see the Pierce catalog). For example,
carbodiimides activate carboxyl groups for attack by good
nucleophiles such as amines (see Torchilin et al., Critical Rev.
Therapeutic Drug Carrier Systems, 7(4):275-308 (1991), expressly
incorporated herein). Proteinaceous candidate agents may also be
attached using other techniques known in the art, for example for
the attachment of antibodies to polymers; see Slinkin et al.,
Bioconj. Chem. 2:342-348 (1991); Torchilin et al., supra;
Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al.,
Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate
Chem. 5:220-235 (1994), all of which are hereby expressly
incorporated by reference). It should be understood that the
candidate agents may be attached in a variety of ways, including
those listed above. Preferably, the manner of attachment does not
significantly alter the functionality of the candidate agent; that
is, the candidate agent should be attached in such a flexible
manner as to allow its interaction with a target. In addition,
these types of chemical or biological functionalities may be used
to attach arrays to assay locations, as is depicted in FIG. 1F, or
individual sets of beads.
[0228] Specific techniques for immobilizing enzymes on microspheres
are known in the prior art. In one case, NH.sub.2 surface chemistry
microspheres are used. Surface activation is achieved with a 2.5%
glutaraldehyde in phosphate buffered saline (10 mM) providing a pH
of 6.9. (138 mM NaCl, 2.7 mM, Kcl). This is stirred on a stir bed
for approximately 2 hours at room temperature. The microspheres are
then rinsed with ultrapure water plus 0.01% tween 20 (surfactant)
-0.02%, and rinsed again with a pH 7.7 PBS plus 0.01% tween 20.
Finally, the enzyme is added to the solution, preferably after
being prefiltered using a 0.45 mm Amicon micropure filter.
[0229] In some embodiments, the microspheres may additionally
comprise identifier binding ligands for use in certain decoding
systems. By "identifier binding ligands" or "IBLs" herein is meant
a compound that will specifically bind a corresponding decoder
binding ligand (DBL) to facilitate the elucidation of the identity
of the bioactive agent attached to the bead. That is, the IBL and
the corresponding DBL form a binding partner pair. By "specifically
bind" as used herein with respect to IBL/DBL binding is meant that
the IBL binds its DBL with specificity sufficient to differentiate
between the corresponding DBL and other DBLs (that is, DBLs for
other IBLs), or other components or contaminants of the system. The
binding should be sufficient to remain bound under the conditions
of the decoding step, including wash steps to remove non-specific
binding. In some embodiments, for example when the IBLs and
corresponding DBLs are proteins or nucleic acids, the dissociation
constants of the IBL to its DBL will be less than about
10.sup.-4-10.sup.-6 M.sup.-1, with less than about 10.sup.-5 to
10.sup.-9 M.sup.-1 being preferred and less than about 10.sup.-7
-10.sup.-9 M.sup.-1 being particularly preferred.
[0230] IBL-DBL binding pairs are known or can be readily found
using known techniques. For example, when the IBL is a protein, the
DBLs include proteins (particularly including antibodies or
fragments thereof (FAbs, etc.) or small molecules, or vice versa
(the IBL is an antibody and the DBL is a protein). Metal ion--metal
ion ligands or chelators pairs are also useful. Antigen-antibody
pairs, enzymes and substrates or inhibitors, other protein-protein
interacting pairs, receptor-ligands, complementary nucleic acids
(including nucleic acid molecules that form triple helices), and
carbohydrates and their binding partners are also suitable binding
pairs. Nucleic acid--nucleic acid binding proteins pairs are also
useful, including single-stranded or double-stranded nucleic acid
binding proteins, and small molecule nucleic acid binding agents.
Similarly, as is generally described in U.S. Pat. Nos. 5,270,163,
5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337,
and related patents, hereby incorporated by reference, nucleic acid
"aptamers" can be developed for binding to virtually any target;
such an aptamer-target pair can be used as the IBL-DBL pair.
Similarly, there is a wide body of literature relating to the
development of binding pairs based on combinatorial chemistry
methods.
[0231] In a preferred embodiment, the IBL is a molecule whose color
or luminescence properties change in the presence of a
selectively-binding DBL.
[0232] In one embodiment, the DBL may be attached to a bead, i.e. a
"decoder bead", that may carry a label such as a fluorophore.
[0233] In a preferred embodiment, the IBL-DBL pair comprise
substantially complementary single-stranded nucleic acids. In this
embodiment, the binding ligands can be referred to as "identifier
probes" and "decoder probes". Generally, the identifier and decoder
probes range from about 4 basepairs in length to about 1000, with
from about 6 to about 100 being preferred, and from about 8 to
about 40 being particularly preferred. What is important is that
the probes are long enough to be specific, i.e. to distinguish
between different IBL-DBL pairs, yet short enough to allow both a)
dissociation, if necessary, under suitable experimental conditions,
and b) efficient hybridization.
[0234] In a preferred embodiment, as is more fully outlined below,
the IBLs do not bind to DBLs. Rather, the IBLs are used as
identifier moieties ("IMs") that are identified directly, for
example through the use of mass spectroscopy.
[0235] Alternatively, in a preferred embodiment, the IBL and the
bioactive agent are the same moiety; thus, for example, as outlined
herein, particularly when no optical signatures are used, the
bioactive agent can serve as both the identifier and the agent. For
example, in the case of nucleic acids, the bead-bound probe (which
serves as the bioactive agent) can also bind decoder probes, to
identify the sequence of the probe on the bead. Thus, in this
embodiment, the DBLs bind to the bioactive agents. This is
particularly useful as this embodiment can give information about
the array or the assay in addition to decoding. For example, as is
more fully described below, the use of the DBLs allows array
calibration and assay development. This may be done even if the
DBLs are not used as such; for example in non-random arrays, the
use of these probe sets can allow array calibration and assay
development even if decoding is not required.
[0236] In a preferred embodiment, the microspheres do not contain
an optical signature. That is, as outlined in U.S. Ser. Nos.
08/818,199 and 09/151,877, previous work had each subpopulation of
microspheres comprising a unique optical signature or optical tag
that is used to identify the unique bioactive agent of that
subpopulation of microspheres; that is, decoding utilizes optical
properties of the beads such that a bead comprising the unique
optical signature may be distinguished from beads at other
locations with different optical signatures. Thus the previous work
assigned each bioactive agent a unique optical signature such that
any microspheres comprising that bioactive agent are identifiable
on the basis of the signature. These optical signatures comprised
dyes, usually chromophores or fluorophores, that were entrapped or
attached to the beads themselves. Diversity of optical signatures
utilized different fluorochromes, different ratios of mixtures of
fluorochromes, and different concentrations (intensities) of
fluorochromes.
[0237] In the present invention, "decoding" refers to identifying
one or more bioactive ligands located in an invention array.
Decoding can be carried out using any of a variety of methods,
including using optical signatures, decoding binding ligands that
are added during a decoding step, or a combination of these
methods. The decoding binding ligands will bind either to a
distinct identifier binding ligand partner that is placed on the
beads, or to the bioactive agent itself, for example when the beads
comprise single-stranded nucleic acids as the bioactive agents. The
decoding binding ligands are either directly or indirectly labeled,
and thus decoding can occur by detecting the presence of the label.
By using pools of decoding binding ligands in a sequential fashion,
it is possible to greatly minimize the number of required decoding
steps.
[0238] Thus, the present invention need not rely solely on the use
of optical properties to decode the arrays, although in some
instances it may. However, as will be appreciated by those in the
art, it is possible in some embodiments to utilize optical
signatures as an additional coding method, in conjunction with the
present system. Thus, for example, as is more fully outlined below,
the size of the array may be effectively increased while using a
single set of decoding moieties in several ways, one of which is
the use in combination with optical signatures one beads. Thus, for
example, using one "set" of decoding molecules, the use of two
populations of beads, one with an optical signature and one
without, allows the effective doubling of the array size. The use
of multiple optical signatures similarly increases the possible
size of the array.
[0239] In a preferred embodiment, each subpopulation of beads
comprises a plurality of different IBLs. By using a plurality of
different IBLs to encode each bioactive agent, the number of
possible unique codes is substantially increased. That is, by using
one unique IBL per bioactive agent, the size of the array will be
the number of unique IBLs (assuming no "reuse" occurs, as outlined
below). However, by using a plurality of different IBLs per bead,
n, the size of the array can be increased to 2n, when the presence
or absence of each IBL is used as the indicator. For example, the
assignment of 10 IBLs per bead generates a 10 bit binary code,
where each bit can be designated as "1" (IBL is present) or "0"
(IBL is absent). A 10 bit binary code has 210 possible variants
However, as is more fully discussed below, the size of the array
may be further increased if another parameter is included such as
concentration or intensity; thus for example, if two different
concentrations of the IBL are used, then the array size increases
as 3n. Thus, in this embodiment, each individual bioactive agent in
the array is assigned a combination of IBLs, which can be added to
the beads prior to the addition of the bioactive agent, after, or
during the synthesis of the bioactive agent, i.e. simultaneous
addition of IBLs and bioactive agent components.
[0240] Alternatively, when the bioactive agent is a polymer of
different residues, i.e. when the bioactive agent is a protein or
nucleic acid, the combination of different IBLs can be used to
elucidate the sequence of the protein or nucleic acid.
[0241] Thus, for example, using two different IBLs (IBL1 and IBL2),
the first position of a nucleic acid can be elucidated: for
example, adenosine can be represented by the presence of both IBL1
and IBL2; thymidine can be represented by the presence of IBL1 but
not IBL2, cytosine can be represented by the presence of IBL2 but
not IBL1, and guanosine can be represented by the absence of both.
The second position of the nucleic acid can be done in a similar
manner using IBL3 and IBL4; thus, the presence of IBL1, IBL2, IBL3
and IBL4 gives a sequence of AA; IBL1, IBL2, and IBL3 shows the
sequence AT; IBL1, IBL3 and IBL4 gives the sequence TA, etc. The
third position utilizes IBL5 and IBL6, etc. In this way, the use of
20 different identifiers can yield a unique code for every possible
10-mer.
[0242] The system is similar for proteins but requires a larger
number of different IBLs to identify each position, depending on
the allowed diversity at each position. Thus for example, if every
amino acid is allowed at every position, five different IBLs are
required for each position. However, as outlined above, for example
when using random peptides as the bioactive agents, there may be
bias built into the system; not all amino acids may be present at
all positions, and some positions may be preset; accordingly, it
may be possible to utilize four different IBLs for each amino
acid.
[0243] In this way, a sort of "bar code" for each sequence can be
constructed; the presence or absence of each distinct IBL will
allow the identification of each bioactive agent.
[0244] In addition, the use of different concentrations or
densities of IBLs allows a "reuse" of sorts. If, for example, the
bead comprising a first agent has a 1.times. concentration of IBL,
and a second bead comprising a second agent has a 10.times.
concentration of IBL, using saturating concentrations of the
corresponding labeled DBL allows the user to distinguish between
the two beads.
[0245] Once the microspheres comprising the candidate agents and
the unique IBLs are generated, they are added to the substrate to
form an array. It should be noted that while most of the methods
described herein add the beads to the substrate prior to the assay,
the order of making, using and decoding the array can vary. For
example, the array can be made, decoded, and then the assay done.
Alternatively, the array can be made, used in an assay, and then
decoded; this may find particular use when only a few beads need be
decoded. Alternatively, the beads can be added to the assay
mixture, i.e. the sample containing the target analytes, prior to
the addition of the beads to the substrate; after addition and
assay, the array may be decoded. This is particularly preferred
when the sample comprising the beads is agitated or mixed; this can
increase the amount of target analyte bound to the beads per unit
time, and thus (in the case of nucleic acid assays) increase the
hybridization kinetics. This may find particular use in cases where
the concentration of target analyte in the sample is low;
generally, for low concentrations, long binding times must be
used.
[0246] In addition, adding the beads to the assay mixture can allow
sorting or selection. For example, a large library of beads may be
added to a sample, and only those beads that bind the sample may be
added to the substrate. For example, if the target analyte is
fluorescently labeled (either directly (for example by the
incorporation of labels into nucleic acid amplification reactions)
or indirectly (for example via the use of sandwich assays)), beads
that exhibit fluorescence as a result of target analyte binding can
be sorted via Fluorescence Activated Cell Sorting (FACS) and only
these beads added to an array and subsequently decoded. Similarly,
the sorting may be accomplished through affinity techniques;
affinity columns comprising the target analytes can be made, and
only those beads which bind are used on the array. Similarly, two
bead systems can be used; for example, magnetic beads comprising
the target analytes can be used to "pull out" those beads that will
bind to the targets, followed by subsequent release of the magnetic
beads (for example via temperature elevation) and addition to an
array.
[0247] In general, the methods of making the arrays and of decoding
the arrays is done to maximize the number of different candidate
agents that can be uniquely encoded. The compositions of the
invention may be made in a variety of ways. In general, the arrays
are made by adding a solution or slurry comprising the beads to a
surface containing the sites for association of the beads. This may
be done in a variety of buffers, including aqueous and organic
solvents, and mixtures. The solvent can evaporate, and excess beads
removed.
[0248] In a preferred embodiment, when non-covalent methods are
used to associate the beads to the array, a novel method of loading
the beads onto the array is used. This method comprises exposing
the array to a solution of particles (including microspheres and
cells) and then applying energy, e.g agitating or vibrating the
mixture. This results in an array comprising more tightly
associated particles, as the agitation is done with sufficient
energy to cause weakly-associated beads to fall off (or out, in the
case of wells). These sites are then available to bind a different
bead. In this way, beads that exhibit a high affinity for the sites
are selected. Arrays made in this way have two main advantages as
compared to a more static loading: first of all, a higher
percentage of the sites can be filled easily, and secondly, the
arrays thus loaded show a substantial decrease in bead loss during
assays. Thus, in a preferred embodiment, these methods are used to
generate arrays that have at least about 50% of the sites filled,
with at least about 75% being preferred, and at least about 90%
being particularly preferred. Similarly, arrays generated in this
manner preferably lose less than about 20% of the beads during an
assay, with less than about 10% being preferred and less than about
5% being particularly preferred.
[0249] In this embodiment, the substrate comprising the surface
with the discrete sites is immersed into a solution comprising the
particles (beads, cells, etc.). The surface may comprise wells, as
is described herein, or other types of sites on a patterned surface
such that there is a differential affinity for the sites. This
differential affinity results in a competitive process, such that
particles that will associate more tightly are selected.
Preferably, the entire surface to be "loaded" with beads is in
fluid contact with the solution. This solution is generally a
slurry ranging from about 10,000:1 beads:solution (vol:vol) to 1:1.
Generally, the solution can comprise any number of reagents,
including aqueous buffers, organic solvents, salts, other reagent
components, etc. In addition, the solution preferably comprises an
excess of beads; that is, there are more beads than sites on the
array. Preferred embodiments utilize two-fold to billion-fold
excess of beads.
[0250] The immersion can mimic the assay conditions; for example,
if the array is to be "dipped" from above into a microtiter plate
comprising samples, this configuration can be repeated for the
loading, thus minimizing the beads that are likely to fall out due
to gravity.
[0251] Once the surface has been immersed, the substrate, the
solution, or both are subjected to a competitive process, whereby
the particles with lower affinity can be disassociated from the
substrate and replaced by particles exhibiting a higher affinity to
the site. This competitive process is done by the introduction of
energy, in the form of heat, sonication, stirring or mixing,
vibrating or agitating the solution or substrate, or both.
[0252] A preferred embodiment utilizes agitation or vibration. In
general, the amount of manipulation of the substrate is minimized
to prevent damage to the array; thus, preferred embodiments utilize
the agitation of the solution rather than the array, although
either will work. As will be appreciated by those in the art, this
agitation can take on any number of forms, with a preferred
embodiment utilizing microtiter plates comprising bead solutions
being agitated using microtiter plate shakers.
[0253] The agitation proceeds for a period of time sufficient to
load the array to a desired fill. Depending on the size and
concentration of the beads and the size of the array, this time may
range from about 1 second to days, with from about 1 minute to
about 24 hours being preferred.
[0254] It should be noted that not all sites of an array may
comprise a bead; that is, there may be some sites on the substrate
surface which are empty. In addition, there may be some sites that
contain more than one bead, although this is not preferred.
[0255] In some embodiments, for example when chemical attachment is
done, it is possible to associate the beads in a non-random or
ordered way. For example, using photoactivatible attachment linkers
or photoactivatible adhesives or masks, selected sites on the array
may be sequentially rendered suitable for attachment, such that
defined populations of beads are laid down.
[0256] The arrays of the present invention are constructed such
that information about the identity of the candidate agent is built
into the array, such that the random deposition of the beads in the
fiber wells can be "decoded" to allow identification of the
candidate agent at all positions. This may be done in a variety of
ways, and either before, during or after the use of the array to
detect target molecules.
[0257] Thus, after the array is made, it is "decoded" in order to
identify the location of one or more of the bioactive agents, i.e.
each subpopulation of beads, on the substrate surface. FIG. 11
depicts a flow chart exemplifying, but not limiting, the assays
that can be performed with the arrays and hybridization chamber of
the invention.
[0258] In a preferred embodiment, a selective decoding system is
used. In this case, only those microspheres exhibiting a change in
the optical signal as a result of the binding of a target analyte
are decoded. This is commonly done when the number of "hits", i.e.
the number of sites to decode, is generally low. That is, the array
is first scanned under experimental conditions in the absence of
the target analytes. The sample containing the target analytes is
added, and only those locations exhibiting a change in the optical
signal are decoded. For example, the beads at either the positive
or negative signal locations may be either selectively tagged or
released from the array (for example through the use of
photocleavable linkers), and subsequently sorted or enriched in a
fluorescence-activated cell sorter (FACS). That is, either all the
negative beads are released, and then the positive beads are either
released or analyzed in situ, or alternatively all the positives
are released and analyzed. Alternatively, the labels may comprise
halogenated aromatic compounds, and detection of the label is done
using for example gas chromatography, chemical tags, isotopic tags,
or mass spectral tags.
[0259] As will be appreciated by those in the art, this may also be
done in systems where the array is not decoded; i.e. there need not
ever be a correlation of bead composition with location. In this
embodiment, the beads are loaded on the array, and the assay is
run. The "positives", i.e. those beads displaying a change in the
optical signal as is more fully outlined below, are then "marked"
to distinguish or separate them from the "negative" beads. This can
be done in several ways, preferably using fiber optic arrays. In a
preferred embodiment, each bead contains a fluorescent dye. After
the assay and the identification of the "positives" or "active
beads", light is shown down either only the positive fibers or only
the negative fibers, generally in the presence of a light-activated
reagent (typically dissolved oxygen). In the former case, all the
active beads are photobleached. Thus, upon non-selective release of
all the beads with subsequent sorting, for example using a
fluorescence activated cell sorter (FACS) machine, the
non-fluorescent active beads can be sorted from the fluorescent
negative beads. Alternatively, when light is shown down the
negative fibers, all the negatives are non-fluorescent and the
positives are fluorescent, and sorting can proceed. The
characterization of the attached bioactive agent may be done
directly, for example using mass spectroscopy.
[0260] Alternatively, the identification may occur through the use
of identifier moieties ("IMs"), which are similar to IBLs but need
not necessarily bind to DBLs. That is, rather than elucidate the
structure of the bioactive agent directly, the composition of the
IMs may serve as the identifier. Thus, for example, a specific
combination of IMs can serve to code the bead, and be used to
identify the agent on the bead upon release from the bead followed
by subsequent analysis, for example using a gas chromatograph or
mass spectroscope.
[0261] Alternatively, rather than having each bead contain a
fluorescent dye, each bead comprises a non-fluorescent precursor to
a fluorescent dye. For example, using photocleavable protecting
groups, such as certain ortho-nitrobenzyl groups, on a fluorescent
molecule, photoactivation of the fluorochrome can be done. After
the assay, light is shown down again either the "positive" or the
"negative" fibers, to distinguish these populations. The
illuminated precursors are then chemically converted to a
fluorescent dye. All the beads are then released from the array,
with sorting, to form populations of fluorescent and
non-fluorescent beads (either the positives and the negatives or
vice versa).
[0262] In an alternate preferred embodiment, the sites of
association of the beads (for example the wells) include a
photopolymerizable reagent, or the photopolymerizable agent is
added to the assembled array. After the test assay is run, light is
shown down again either the "positive" or the "negative" fibers, to
distinguish these populations. As a result of the irradiation,
either all the positives or all the negatives are polymerized and
trapped or bound to the sites, while the other population of beads
can be released from the array.
[0263] In a preferred embodiment, the location of every bioactive
agent is determined using decoder binding ligands (DBLs). As
outlined above, DBLs are binding ligands that will either bind to
identifier binding ligands, if present, or to the bioactive agents
themselves, preferably when the bioactive agent is a nucleic acid
or protein.
[0264] In a preferred embodiment, as outlined above, the DBL binds
to the IBL.
[0265] In a preferred embodiment, the bioactive agents are
single-stranded nucleic acids and the DBL is a substantially
complementary single-stranded nucleic acid that binds (hybridizes)
to the bioactive agent, termed a decoder probe herein. A decoder
probe that is substantially complementary to each candidate probe
is made and used to decode the array. In this embodiment, the
candidate probes and the decoder probes should be of sufficient
length (and the decoding step run under suitable conditions) to
allow specificity; i.e. each candidate probe binds to its
corresponding decoder probe with sufficient specificity to allow
the distinction of each candidate probe.
[0266] In a preferred embodiment, the DBLs are either directly or
indirectly labeled. By "labeled" herein is meant that a compound
has at least one element, isotope or chemical compound attached to
enable the detection of the compound. In general, labels fall into
three classes: a) isotopic labels, which may be radioactive or
heavy isotopes; b) magnetic, electrical, thermal; and c) colored or
luminescent dyes; although labels include enzymes and particles
such as magnetic particles as well. Preferred labels include
luminescent labels. In a preferred embodiment, the DBL is directly
labeled, that is, the DBL comprises a label. In an alternate
embodiment, the DBL is indirectly labeled; that is, a labeling
binding ligand (LBL) that will bind to the DBL is used. In this
embodiment, the labeling binding ligand-DBL pair can be as
described above for IBL-DBL pairs. Suitable labels include, but are
not limited to, fluorescent lanthanide complexes, including those
of Europium and Terbium, fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade Blue.TM., Texas Red, FITC, PE, cy3, cy5 and others
described in the 6th Edition of the Molecular Probes Handbook by
Richard P. Haugland, hereby expressly incorporated by
reference.
[0267] In one embodiment, the label is a molecule whose color or
luminescence properties change in the presence of the IBL, due to a
change in the local environment. For example, the label may be: (1)
a fluorescent pH indicator whose emission intensity changes with
pH; (2) a fluorescent ion indicator, whose emission properties
change with ion concentration; or (3) a fluorescent molecule such
as an ethidium salt whose fluorescence intensity increases in
hydrophobic environments.
[0268] A label can also include a semiconductor nanocrystal, also
known as a quantum dot. Semiconductor nanocrystal compositions and
applications are known in the art, as is demonstrated in the
teachings of U.S. Pat. No. 5,571,018; U.S. Pat. No. 5,505,928; U.S.
Pat. No. 5,262,357; U.S. Pat. No. 5,990,047; U.S. Pat. No.
6.207,392; and EP 0 990 903, each of which is incorporated by
reference in their entirety. Quantum dot labels will find
particularly preferred use for applications such as large-scale
multiplexing, continued use of the label, broad wavelength
irradiation, narrow wavelength detection, particle or high energy
beam irradiation, distance-sensitive assays, and the like.
[0269] Accordingly, the identification of the location of the
individual beads (or subpopulations of beads) is done using one or
more decoding steps comprising a binding between the labeled DBL
and either the IBL or the bioactive agent (i.e. a hybridization
between the candidate probe and the decoder probe when the
bioactive agent is a nucleic acid). After decoding, the DBLs can be
removed and the array can be used; however, in some circumstances,
for example when the DBL binds to an IBL and not to the bioactive
agent, the removal of the DBL is not required (although it may be
desirable in some circumstances). In addition, as outlined herein,
decoding may be done either before the array is used in an assay,
during the assay, or after the assay.
[0270] In one embodiment, a single decoding step is done. In this
embodiment, each DBL is labeled with a unique label, such that the
number of unique labels is equal to or greater than the number of
bioactive agents (although in some cases, "reuse" of the unique
labels can be done, as described herein; similarly, minor variants
of candidate probes can share the same decoder, if the variants are
encoded in another dimension, i.e. in the bead size or label). For
each bioactive agent or IBL, a DBL is made that will specifically
bind to it and contains a unique label, for example one or more
fluorochromes. Thus, the identity of each DBL, both its composition
(i.e. its sequence when it is a nucleic acid) and its label, is
known. Then, by adding the DBLs to the array containing the
bioactive agents under conditions which allow the formation of
complexes (termed hybridization complexes when the components are
nucleic acids) between the DBLs and either the bioactive agents or
the IBLs, the location of each DBL can be elucidated. This allows
the identification of the location of each bioactive agent; the
random array has been decoded. The DBLs can then be removed, if
necessary, and the target sample applied.
[0271] In a preferred embodiment, the number of unique labels is
less than the number of unique bioactive agents, and thus a
sequential series of decoding steps are used. To facilitate the
discussion, this embodiment is explained for nucleic acids,
although other types of bioactive agents and DBLs are useful as
well. In this embodiment, decoder probes are divided into n sets
for decoding. The number of sets corresponds to the number of
unique tags. Each decoder probe is labeled in n separate reactions
with n distinct tags. All the decoder probes share the same n tags.
Each pool of decoders contains only one of the n tag versions of
each decoder, and no two decoder probes have the same sequence of
tags across all the pools. The number of pools required for this to
be true is determined by the number of decoder probes and the n.
Hybridization of each pool to the array generates a signal at every
address comprising an IBL. The sequential hybridization of each
pool in turn will generate a unique, sequence-specific code for
each candidate probe. This identifies the candidate probe at each
address in the array. For example, if four tags are used, then
4.times.n sequential hybridizations can ideally distinguish 4n
sequences, although in some cases more steps may be required. After
the hybridization of each pool, the hybrids are denatured and the
decoder probes removed, so that the probes are rendered
single-stranded for the next hybridization (although it is also
possible to hybridize limiting amounts of target so that the
available probe is not saturated. Sequential hybridizations can be
carried out and analyzed by subtracting pre-existing signal from
the previous hybridization).
[0272] As will be appreciated by one of ordinary skill in the art,
hybridization or incubation times vary. Generally, hybridization or
incubation times last from seconds to minutes or up to hours or
days or more. When the hybridization chamber as described herein is
utilized, hybridization or incubation times can be increased
relative to incubation times without the hybridization chamber.
[0273] An example is illustrative. Assuming an array of 16 probe
nucleic acids (numbers 1-16), and four unique tags (four different
fluors, for example; labels A-D). Decoder probes 1-16 are made that
correspond to the probes on the beads. The first step is to label
decoder probes 1-4 with tag A, decoder probes 5-8 with tag B,
decoder probes 9-12 with tag C, and decoder probes 13-16 with tag
D. The probes are mixed and the pool is contacted with the array
containing the beads with the attached candidate probes. The
location of each tag (and thus each decoder and candidate probe
pair) is then determined. The first set of decoder probes are then
removed. A second set is added, but this time, decoder probes 1, 5,
9 and 13 are labeled with tag A, decoder probes 2, 6, 10 and 14 are
labeled with tag B, decoder probes 3, 7, 11 and 15 are labeled with
tag C, and decoder probes 4, 8, 12 and 16 are labeled with tag D.
Thus, those beads that contained tag A in both decoding steps
contain candidate probe 1; tag A in the first decoding step and tag
B in the second decoding step contain candidate probe 2; tag A in
the first decoding step and tag C in the second step contain
candidate probe 3; etc. As will be appreciated by those in the art,
the decoder probes can be made in any order and added in any
order.
[0274] In one embodiment, the decoder probes are labeled in situ;
that is, they need not be labeled prior to the decoding reaction.
In this embodiment, the incoming decoder probe is shorter than the
candidate probe, creating a 5' "overhang" on the decoding probe.
The addition of labeled ddNTPs (each labeled with a unique tag) and
a polymerase will allow the addition of the tags in a sequence
specific manner, thus creating a sequence-specific pattern of
signals. Similarly, other modifications can be done, including
ligation, etc.
[0275] In addition, since the size of the array will be set by the
number of unique decoding binding ligands, it is possible to
"reuse" a set of unique DBLs to allow for a greater number of test
sites. This may be done in several ways; for example, by using some
subpopulations that comprise optical signatures. Similarly, the use
of a positional coding scheme within an array; different
sub-bundles may reuse the set of DBLs. Similarly, one embodiment
utilizes bead size as a coding modality, thus allowing the reuse of
the set of unique DBLs for each bead size. Alternatively,
sequential partial loading of arrays with beads can also allow the
reuse of DBLs. Furthermore, "code sharing" can occur as well.
[0276] In a preferred embodiment, the DBLs may be reused by having
some subpopulations of beads comprise optical signatures. In a
preferred embodiment, the optical signature is generally a mixture
of reporter dyes, preferably fluorescent or quantum dot. By varying
both the composition of the mixture (i.e. the ratio of one dye to
another) and the concentration of the dye (leading to differences
in signal intensity), matrices of unique optical signatures may be
generated. This may be done by covalently attaching the dyes to the
surface of the beads, or alternatively, by entrapping the dye
within the bead. The dyes may be chromophores or phosphors but are
preferably fluorescent dyes or quantum dots, which due to their
strong signals provide a good signal-to-noise ratio for decoding.
Suitable dyes for use in the invention include those listed for
labeling DBLs, above.
[0277] In a preferred embodiment, the encoding can be accomplished
in a ratio of at least two dyes, although more encoding dimensions
may be added in the size of the beads, for example. In addition,
the labels are distinguishable from one another; thus two different
labels may comprise different molecules (i.e. two different fluors)
or, alternatively, one label at two different concentrations or
intensity.
[0278] In a preferred embodiment, the dyes are covalently attached
to the surface of the beads. This may be done as is generally
outlined for the attachment of the bioactive agents, using
functional groups on the surface of the beads. As will be
appreciated by those in the art, these attachments are done to
minimize the effect on the dye.
[0279] In a preferred embodiment, the dyes are non-covalently
associated with the beads, generally by entrapping the dyes in the
pores of the beads.
[0280] Additionally, encoding in the ratios of the two or more
dyes, rather than single dye concentrations, is preferred since it
provides insensitivity to the intensity of light used to
interrogate the reporter dye's signature and detector
sensitivity.
[0281] In a preferred embodiment, a spatial or positional coding
system is done. In this embodiment, there are sub-bundles or
subarrays (i.e. portions of the total array) that are utilized. By
analogy with the telephone system, each subarray is an "area code",
that can have the same labels (i.e. telephone numbers) of other
subarrays, that are separated by virtue of the location of the
subarray. Thus, for example, the same unique labels can be reused
from bundle to bundle. Thus, the use of 50 unique labels in
combination with 100 different subarrays can form an array of 5000
different bioactive agents. In this embodiment, it becomes
important to be able to identify one bundle from another; in
general, this is done either manually or through the use of marker
beads; these can be beads containing unique tags for each subarray,
or the use of the same marker bead in differing amounts, or the use
of two or more marker beads in different ratios.
[0282] In alternative embodiments, additional encoding parameters
can be added, such as microsphere size. For example, the use of
different size beads may also allow the reuse of sets of DBLs; that
is, it is possible to use microspheres of different sizes to expand
the encoding dimensions of the microspheres. Optical fiber arrays
can be fabricated containing pixels with different fiber diameters
or cross-sections; alternatively, two or more fiber optic bundles,
each with different cross-sections of the individual fibers, can be
added together to form a larger bundle; or, fiber optic bundles
with fiber of the same size cross-sections can be used, but just
with different sized beads. With different diameters, the largest
wells can be filled with the largest microspheres and then moving
onto progressively smaller microspheres in the smaller wells until
all size wells are then filled. In this manner, the same dye ratio
could be used to encode microspheres of different sizes thereby
expanding the number of different oligonucleotide sequences or
chemical functionalities present in the array. Although outlined
for fiber optic substrates, this as well as the other methods
outlined herein can be used with other substrates and with other
attachment modalities as well.
[0283] In a preferred embodiment, the coding and decoding is
accomplished by sequential loading of the microspheres into the
array. As outlined above for spatial coding, in this embodiment,
the optical signatures can be "reused". In this embodiment, the
library of microspheres each comprising a different bioactive agent
(or the subpopulations each comprise a different bioactive agent),
is divided into a plurality of sublibraries; for example, depending
on the size of the desired array and the number of unique tags, 10
sublibraries each comprising roughly 10% of the total library may
be made, with each sublibrary comprising roughly the same unique
tags. Then, the first sublibrary is added to the fiber optic bundle
comprising the wells, and the location of each bioactive agent is
determined, generally through the use of DBLs. The second
sublibrary is then added, and the location of each bioactive agent
is again determined. The signal in this case will comprise the
signal from the "first" DBL and the "second" DBL; by comparing the
two matrices the location of each bead in each sublibrary can be
determined. Similarly, adding the third, fourth, etc. sublibraries
sequentially will allow the array to be filled.
[0284] In a preferred embodiment, codes can be "shared" in several
ways. In a first embodiment, a single code (i.e. IBL/DBL pair) can
be assigned to two or more agents if the target analytes different
sufficiently in their binding strengths. For example, two nucleic
acid probes used in an mRNA quantitation assay can share the same
code if the ranges of their hybridization signal intensities do not
overlap. This can occur, for example, when one of the target
sequences is always present at a much higher concentration than the
other. Alternatively, the two target sequences might always be
present at a similar concentration, but differ in hybridization
efficiency.
[0285] Alternatively, a single code can be assigned to multiple
agents if the agents are functionally equivalent. For example, if a
set of oligonucleotide probes are designed with the common purpose
of detecting the presence of a particular gene, then the probes are
functionally equivalent, even though they may differ in sequence.
Similarly, if classes or "families" of analytes are desired, all
probes for different members of a class such as kinases or
G-protein coupled receptors could share a code. Similarly, an array
of this type could be used to detect homologs of known genes. In
this embodiment, each gene is represented by a heterologous set of
probes, hybridizing to different regions of the gene (and therefore
differing in sequence). The set of probes share a common code. If a
homolog is present, it might hybridize to some but not all of the
probes. The level of homology might be indicated by the fraction of
probes hybridizing, as well as the average hybridization intensity.
Similarly, multiple antibodies to the same protein could all share
the same code.
[0286] In a preferred embodiment, decoding of self-assembled random
arrays is done on the bases of pH titration. In this embodiment, in
addition to bioactive agents, the beads comprise optical
signatures, wherein the optical signatures are generated by the use
of pH-responsive dyes (sometimes referred to herein as "pH dyes")
such as fluorophores. This embodiment is similar to that outlined
in PCT US98/05025 and U.S. Ser. No. 09/151,877, both of which are
expressly incorporated by reference, except that the dyes used in
the present invention exhibits changes in fluorescence intensity
(or other properties) when the solution pH is adjusted from below
the pKa to above the pKa (or vice versa). In a preferred
embodiment, a set of pH dyes is used, each with a different pKa,
preferably separated by at least 0.5 pH units. Preferred
embodiments utilize a pH dye set of pKa's of 2.0, 2.5, 3.0, 3.5,
4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,
10.5, 11, and 11.5. Each bead can contain any subset of the pH
dyes, and in this way a unique code for the bioactive agent is
generated. Thus, the decoding of an array is achieved by titrating
the array from pH 1 to pH 13, and measuring the fluorescence signal
from each bead as a function of solution pH.
[0287] In a preferred embodiment, there are additional ways to
increase the number of unique or distinct tags. That is, the use of
distinct attributes on each bead can be used to increase the number
of codes. In addition, sequential decoding allows a reuse of codes
in new ways. These attributes are independent of each other, thus
allowing the number of codes to grow exponentially as a function of
the number of decoding steps and the number of attributes (e.g.
distinct codes). However, by increasing the amount of decoding
information obtained in a single decoding step, the number of
decoding steps is markedly reduced. Alternatively, the number of
distinct codes is markedly increased. By increasing the number of
attributes per decoding step, fewer decoding steps are required for
a given number of codes. Thus, in a preferred embodiment, a variety
of methods are used to generate a number of codes for use in the
process of decoding the arrays, while minimizing the necessary
decoding steps. For example, a variety of different coding
strategies can be combined: thus, different "colors", combinations
of colors ("hues"), different intensities of colors or hues or
both, etc. can all be combined.
[0288] In a preferred embodiment DBLs rely on attaching or
embedding a quantitative or discrete set of physical attributes to
the bead, i.e. labeling the bead. Preferred physical attributes of
a bead include but are not limited to: surface "smoothness" or
"roughness", color (Fluorescent and otherwise), color intensity,
size, detectable chemical moieties, chemical reactivity,
magnetization, pH sensitivity, energy transfer efficiency between
dyes present, hydrophobicity, hydrophilicity, absorptivity, charge,
pH sensitivity, etc.
[0289] A bead decoding scheme includes assigning/imbuing a single
quantifiable attribute to each bead type wherein each bead type
differs in the quantifiable value of that attribute. For instance,
one can attach a given number of fluorophores to a bead and
quantitate the number of attached fluorophores in the decoding
process; however, in practice, attaching a "given amount" of an
attribute to a bead and accurately measuring the attribute may be
problematic. In general, the goal is to reduce the coefficient of
variation (CV). By coefficient of variation is meant the
variability in labeling a bead in successive labelings. This CV can
be determined by labeling beads with a defined given number of
label (fluorophore, for example) in multiple tests and measuring
the resulting signal emitted by the bead. A large CV limits the
number of useable and resolvable "levels" for any given
attribute.
[0290] A more robust decoding scheme employs ratiometric rather
than absolute measurements for segmenting a quantitative attribute
into codes. By ratiometric decoding is meant labeling a bead with a
ratio of labels (i.e. 1:10, 1:1, and 10:1). In theory any number of
ratios can be used so long as the difference in signals between the
ratios is detectable. This process produced smaller CVs and
allowing more attribute segmentation within a given dynamic range.
Thus, in a preferred embodiment, the use of ratiometric decoding
reduces the coefficient of variability.
[0291] In addition, as will be appreciated by those in the art,
ratiometric decoding can be accomplished in a different way. In
this embodiment, rather than add a given number of DBLs with a
first dye (or dye combination) intensity in the first decoding
reaction and a second number with a second dye intensity in the
sequential second decoding reaction, this ratiometric analysis may
be done by using a ratio of labeled: unlabelled DBLs. That is,
given a set saturating concentration of decoding beads, for example
100,000 DBLs/reaction, the first intensity decoding step may be
done by adding 100,000 labeled DBLs and the second step can be done
by adding 10,000 labeled DBLs and 90,000 unlabeled DBLs.
Equilibrium dictates that the second step will give one tenth the
signal intensity.
[0292] Because of the spread in values of a quantitatively measured
attribute value, the number of distinct codes is practically
limited to less than a dozen or so codes. However, by serially
"painting" (i.e. temporarily attaching an attribute level to a
bead) and "stripping" (removing the attribute level) a bead with
different attribute values, the number of possible codes grows
exponentially with the number of serial stages in the decoding
process.
[0293] An example is illustrative. For instance, nine different
bead types and three distinguishable attribute distributions (Table
1). "Painting" (labeling) the beads with different attribute values
in a combinatorially distinct pattern in the two different stages,
generates a unique code for each bead type, i.e. nine distinct
codes are generated. Thus, in a preferred embodiment beads are
labeled with different attributes in a combinatorially distinct
pattern in a plurality of stages. This generates unique codes for
each bead type. Examples of different attributes are described
above. Labeling of beads with different attributes is performed by
methods known in the art.
1TABLE 1 Serial decode generates unique codes using a small number
of attribute levels stage 2 stage 1 attribute attribute Bead Type
value value Code 1 L L (L, L) 2 L M (L, M) 3 L H (L, H) 4 M L (M,
L) Number of unique codes = 5 M M (M, Number of attributes * M)
Number of stages 6 M H (M, H) 7 H L (H, L) 8 H M (H, M) 9 H H (H,
H)
[0294] Fluorescent colors are a particularly convenient attribute
to use in a decoding scheme. Fluorescent colors can be attached to
any agent that recognizes an IBL to form a labeled DBL. The
discussion is directed to oligonucleotides (including nucleic acid
analogs) as the DBLs. A fluorescently labeled oligonucleotide is a
particularly useful DBL since it can specifically and reversibly
"paint" (label) any desired subset of beads with a particular color
simply by the process of hybridization and dehybridization (i.e. to
the DBL with a complementary sequence). Moreover, fluorescence is
easily imaged and quantitated using standard optical hardware and
software. In order to "paint" a given bead type with a particular
color, the bead type must be labeled with a unique hybridizable DNA
sequence (IBL) and the decoding solution must contain the
color-labeled complement of that sequence.
[0295] One consideration in implementing a decoding scheme is to
minimize the number of images collected. In a color-based scheme,
the number of images collected is the product of the number of
colors and the number of stages. The number of images can be
reduced by "painting" a bead with multiple colors for each given
stage. By assigning multiple colors to a bead, the number of
effective codes is increased. As an example, in a 24 bit three
color scheme (e.g. red, green, blue) coloring process used by
computers, a total of 256*256*256=16.7 million different "hues" can
be generated from just three colors (red, green, blue).
[0296] Thus, in a preferred embodiment DBts are labeled with a
combination of colored fluorophores. As such, this method finds use
in increasing the number of available codes for labeling DBLs using
only a handful of different dyes (colors). Increasing the number of
codes available at each decoding step will greatly decrease the
number of decoding steps required in a given decoding process.
[0297] In one embodiment a population of oligonucleotides encoding
a single DBL is labeled with a defined ratio of colors such that
each bead to which the DBL binds is identified based on a
characteristic "hue" formulated from the combination of the colored
fluorophores. In a preferred embodiment two distinct colors are
used. In a preferred embodiment, three or more distinct dyes
(colors) are available for use. In this instance the number of
differentiable codes generated by labeling a population of
oligonucleotides encoding a single DBL with any given color is
three. However by allowing combinations of colors and color levels
in the labeling, many more codes are generated.
[0298] For decoding by hybridization, a preferred number of
distinguishable color shades is from 2 to 2000; a more preferred
number of distinguishable color shades is from 2 to 200 and a most
preferred number of distinguishable color shades is from 2 to 20.
Utilizing three different color shades (intensities) and three
colors, the number of different hues will be 34=81. Combining a hue
with sequential decoding allows a virtually limitless number of
codes to be generated.
[0299] As previously described, the DBL can be any agent that binds
to the IBL. In a preferred embodiment, a single DBL is labeled with
a pre-determined ratio of colors. This ratio is varied for each DBL
thus allowing for a unique "hue" for each DBL labeled as such.
Following treatment of the beads with the DBL, the bead is analyzed
to determine the "hue" associated with each bead, thereby
identifying the bead with its associated bioactive agent.
[0300] For instance, with four primary colors and two intensity
levels (color is present or absent), fifteen different hues/stage
are possible. If four dyes and three different intensity levels are
used (absent, half-present, fully present), then 73 different
hues/stage are possible. In this case, acquisition of only 4 color
images is sufficient to obtain information on 73 different coding
hues.
[0301] In a preferred embodiment, the present invention provides
array compositions comprising a first substrate with a surface
comprising discrete sites. Preferred embodiments utilize a
population of microspheres distributed on the sites, and the
population comprises at least a first and a second subpopulation.
Each subpopulation comprises a bioactive agent, and, in addition,
at least one optical dye with a given pKa. The pKas of the
different optical dyes are different.
[0302] In a preferred embodiment, when for example the array
comprises cloned nucleic acids, there are several methods that can
be used to decode the arrays. In a preferred embodiment, when some
sequence information about the cloned nucleic acids is known,
specific decoding probes can be made as is generally outlined
herein.
[0303] In a preferred embodiment, "random" decoding probes can be
made. By sequential hybridizations or the use of multiple labels,
as is outlined above, a unique hybridization pattern can be
generated for each sensor element. This allows all the beads
representing a given clone to be identified as belonging to the
same group. In general, this is done by using random or partially
degenerate decoding probes, that bind in a sequence-dependent but
not highly sequence-specific manner. The process can be repeated a
number of times, each time using a different labeling entity, to
generate a different pattern of signals based on quasi-specific
interactions. In this way, a unique optical signature is eventually
built up for each sensor element. By applying pattern recognition
or clustering algorithms to the optical signatures, the beads can
be grouped into sets that share the same signature (i.e. carry the
same probes).
[0304] In order to identify the actual sequence of the clone
itself, additional procedures are required; for example, direct
sequencing can be done. By using an ordered array containing the
clones, such as a spotted cDNA array, a "key" can be generated that
links a hybridization pattern to a specific clone whose position in
the set is known. In this way the clone can be recovered and
further characterized.
[0305] Alternatively, clone arrays can be decoded using binary
decoding with vector tags. For example, partially randomized oligos
are cloned into a nucleic acid vector (e.g. plasmid, phage, etc.).
Each oligonucleotide sequence consists of a subset of a limited set
of sequences. For example, if the limits set comprises 10
sequences, each oligonucleotide may have some subset (or all of the
10) sequences. Thus each of the 10 sequences can be present or
absent in the oligonucleotide. Therefore, there are 210 or 1,024
possible combinations. The sequences may overlap, and minor
variants can also be represented (e.g. A, C, T and G substitutions)
to increase the number of possible combinations. A nucleic acid
library is cloned into a vector containing the random code
sequences. Alternatively, other methods such as PCR can be used to
add the tags. In this way it is possible to use a small number of
oligo decoding probes to decode an array of clones.
[0306] In a preferred embodiment, discriminant analysis and cluster
algorithms and computer apparatus are used to analyze the decoding
data from the arrays of the invention. The potentially large number
of codes utilized in the invention, coupled with the use of
different intensities and "hues" of fluorophores in multi-step
decoding processes requires good classification of the data. The
data, particularly intensity data, is acquired in a multi-step
process during which beads are reversibly labeled (for example by
hybridizing dye-labeled complementary decoding oligonucleotides to
the IBL probes on the beads, or the formation of binding ligand
pairs for non-nucleic acid IBL-DBL pairs) with different colors or
mixtures of colors ("hues") at each stage. The challenge is to
accurately classify a bead as to which color with which it was
painted at each step. The more closely related the labels are to
one another (as determined by the optical imaging system), the more
difficult the classification.
[0307] The proximity of the dyes as seen by the imaging system is
determined by the spectral properties of the decoding dyes and the
spectral channel separation of the imaging system. Better color
separation is achieved by employing fluorescent dyes with narrow
emission spectra, and by employing an optical system with narrow
band pass excitation and emission filters which are designed to
excite the dye "on peak" and measure its emission "on peak". The
process of optically imaging the dyes on the beads is similar to
the human vision process in which our brain sees color by measuring
the ratio of excitation in the three different cone types within
our eye. However, with an optical imaging system, the number of
practical color channels is much greater than the three present in
the human eye. CCD based imaging systems can "see" color from 350
nm up to 850 nm whereas the cones in the eye are tuned to the
visible spectrum from 500-600 nm.
[0308] The problem of decoding bead arrays is essentially a
discriminant analysis classification problem. Thus, in a preferred
embodiment, an analysis of variance in hyperspectral alpha space is
performed on a known set of bead colors or hues. The center of the
bead clusters in alpha space are termed the centroids of the
clusters, and the scatter of the points within a cluster determines
the spread of the cluster. A robust classification scheme requires
that the distance between the centroids of the different bead
classes (hues) is much greater than the spread of any cluster
class. Moreover, the location of the centroids should remain
invariant from fiber to fiber and from experiment to
experiment.
[0309] Thus, in a preferred embodiment, a hue "zone" is defined as
a region in alpha space surrounding the hue centroid and extending
out to the spread radius of the cluster. Given a reference set of
hue centroids and spread radii, as determined empirically, the
classification of a new set of data can be accomplished by asking
whether a given bead point falls closest to or within the "zone" of
a hue cluster. This is accomplished by calculating the Mahalanobis
distance (in this case, it is simply a Euclidean distance metric)
of the bead point from the centroids of the different hue classes.
For the data shown in FIG. 3, the location of the centroids and
their distances from one another are indicated in Table 2.
2 TABLE 2 Centroid position Distance between centroids dye/channel
Blue Green Yellow Red Bod-493 Bod-R6G Bod-564 Bod-TXR Bod-493 0.63
0.22 0.11 0.03 0.00 Bod-R6G 0.03 0.51 0.37 0.09 0.72 0.00 Bod-564
0.06 0.04 0.57 0.32 0.81 0.55 0.00 Bod-TXR 0.09 0.05 0.04 0.82 0.99
0.93 0.73 0.00
[0310] For classifying the different beads into a particular hue
class, a Euclidean distance cutoff of 0.3 was chosen. The closest
two centroids, the Bod-R6G and Bod-564 (dist=0.55), have a slight
overlap in their decoding zones when using a Euclidean or
Mahalanobis distance of 0.3. An improvement in classification can
be achieved by decreasing this distance, and by weighting the
different coordinate axes appropriately.
[0311] Accordingly, the present invention provides computer methods
for analyzing and classifying the color of a bead. The
classification of the color of the bead is done by viewing the bead
in hyperspectral "alpha" space (a1=I1/SII, a2=I2/SIi, a3=I3/SIi,,
etc.) in which each coordinate axis represents the fraction of the
bead intensity within a given imaging channel. For instance, if
four imaging channels are used to image the beads, the color or hue
of a bead can be represented by a point in 3-D alpha space (the
fourth dimension is not necessary since Sai=1). Given a set of
different primary dyes by which to label the beads, the number of
hues that can be generated from these dyes is unlimited since the
dyes can be combined in varying ratios and in varying combinatorial
patterns. The number of practical hues is experimentally determined
by the separation of the different hue clusters in hyperspectral
alpha space.
[0312] FIG. 3 shows a hyperspectral alpha plot of beads labeled
with four different hues imaged in four separate imaging channels.
Note that the beads form four distinct clusters. The fact that
these four clusters are well separated allows a robust decode
classification scheme to be implemented.
[0313] In a preferred embodiment, a quality control analysis of the
decoding process is done. This is achieved by performing a cluster
analysis of alpha space for each decoding stage. The number of
clusters determined will be fixed by the expected number of hues.
The positions of the cluster centroids will be monitored and any
deviations from the expected position will be noted.
[0314] Thus the invention provides an apparatus for decoding the
arrays of the invention. In addition to the compositions outlined
herein, the apparatus includes a central processing unit which
communicates with a memory and a set of input/output devices (e.g.,
keyboard, mouse, monitor, printer, etc.) through a bus. The general
interaction between a central processing unit, a memory,
input/output devices, and a bus is known in the art. One aspect of
the present invention is directed toward the hyperspectral "alpha"
space classification system stored in the memory.
[0315] The classification system program includes a data
acquisition module that receives data from the optical reader or
confocal microscope (or other imaging system). In general, the
classification program also includes an analysis module, that can
analyze the variance in hyperspectral alpha space, calculate the
centroids of the clusters, calculate the scatter of the cluster
(the spread) and define the hue zone and distance cutoff. In
general, the analysis module will further determine whether a data
point falls within the hue zone by calculating the Mahalanobis
distance.
[0316] Finally, the analysis module will analyze the different
sequential decoding information to finally assign a bioactive agent
to a bead location.
[0317] In this way, sequential decoding steps are run, with each
step utilizing the discriminant analysis calculations to assign
each bead in the array to a hue cluster at each step. The buildup
of the sequential decoding information allows the correlation of
the location of a bead and the chemistry contained on it.
[0318] Once made, the compositions of the invention find use in a
number of applications. In a preferred embodiment, the compositions
are used to probe a sample solution for the presence or absence of
a target analyte, including the quantitation of the amount of
target analyte present. By "target analyte" or "analyte" or
grammatical equivalents herein is meant any atom, molecule, ion,
molecular ion, compound or particle to be either detected or
evaluated for binding partners. As will be appreciated by those in
the art, a large number of analytes may be used in the present
invention; basically, any target analyte can be used which binds a
bioactive agent or for which a binding partner (e.g a drug
candidate) is sought.
[0319] Suitable analytes include organic and inorganic molecules,
including biomolecules. When detection of a target analyte is done,
suitable target analytes include, but are not limited to, an
environmental pollutant (including pesticides, insecticides,
toxins, etc.); a chemical (including solvents, polymers, organic
materials, etc.); therapeutic molecules (including therapeutic and
abused drugs, antibiotics, etc.); biomolecules (including hormones,
cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular
membrane antigens and receptors (neural, hormonal, nutrient, and
cell surface receptors) or their ligands, etc); whole cells
(including procaryotic (such as pathogenic bacteria) and eukaryotic
cells, including mammalian tumor cells); viruses (including
retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and
spores; etc. Particularly preferred analytes are nucleic acids and
proteins.
[0320] In a preferred embodiment, the target analyte is a protein.
As will be appreciated by those in the art, there are a large
number of possible proteinaceous target analytes that may be
detected or evaluated for binding partners using the present
invention. Suitable protein target analytes include, but are not
limited to, (1) immunoglobulins; (2) enzymes (and other proteins);
(3) hormones and cytokines (many of which serve as ligands for
cellular receptors); and (4) other proteins.
[0321] Suitable protein target analytes include, but are not
limited to, (1) immunoglobulins, particularly IgEs, IgGs and IgMs,
and particularly therapeutically or diagnostically relevant
antibodies, including but not limited to, for example, antibodies
to human albumin, apolipoproteins (including apolipoprotein E),
human chorionic gonadotropin, cortisol, a-fetoprotein, thyroxin,
thyroid stimulating hormone (TSH), antithrombin, antibodies to
pharmaceuticals (including antieptileptic drugs (phenytoin,
primidone, carbariezepin, ethosuximide, valproic acid, and
phenobarbitol), cardio active drugs (digoxin, lidocaine,
procainamide, and disopyramide), bronchodilators (theophylline),
antibiotics (chloramphenicol, sulfonamides), antidepressants,
immunosuppresants, abused drugs (amphetamine, methamphetamine,
cannabinoids, cocaine and opiates) and antibodies to any number of
viruses (including orthomyxoviruses, (e.g. influenza virus),
paramyxoviruses (e.g respiratory syncytial virus, mumps virus,
measles virus), adenoviruses, rhinoviruses, coronaviruses,
reoviruses, togaviruses (e.g. rubella virus), parvoviruses,
poxviruses (e.g. variola virus, vaccinia virus), enteroviruses
(e.g. poliovirus, coxsackievirus), hepatitis viruses (including A,
B and C), herpesviruses (e.g. Herpes simplex virus,
varicella-zoster virus, cytomegalovirus, Epstein-Barr virus),
rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus
(e.g. rabies virus), retroviruses (including HIV, HTLV-I and -II),
papovaviruses (e.g. papillomavirus), polyomaviruses, and
picomaviruses, and the like), and bacteria (including a wide
variety of pathogenic and non-pathogenic prokaryotes of interest
including Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g.
Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella,
e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae;
Clostridium, e.g. C. botulinum, C. tetani, C. difficile,
C.perfringens; Comyebacterium, e.g C. diphtheriae; Streptococcus,
S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus;
Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis,
N. gonorrhoeae; Yersinia, e.g. G. lambliaY. pestis, Pseudomonas,
e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis;
Bordetella, e.g. B. pertussis; Treponema, e.g T. palladium; and the
like); (2) enzymes (and other proteins), including but not limited
to, enzymes used as indicators of or treatment for heart disease,
including creatine kinase, lactate dehydrogenase, aspartate amino
transferase, troponin T, myoglobin, fibrinogen, cholesterol,
triglycerides, thrombin, tissue plasminogen activator (tPA);
pancreatic disease indicators including amylase, lipase,
chymotrypsin and trypsin; liver function enzymes and proteins
including cholinesterase, bilirubin, and alkaline phosphotase;
aldolase, prostatic acid phosphatase, terminal deoxynucleotidyl
transferase, and bacterial and viral enzymes such as HIV protease;
(3) hormones and cytokines (many of which serve as ligands for
cellular receptors) such as erythropoietin (EPO), thrombopoietin
(TPO), the interleukins (including IL-1 through IL-17), insulin,
insulin-like growth factors (including IGF-1 and -2), epidermal
growth factor (EGF), transforming growth factors (including TGF-a
and TGF-b), human growth hormone, transferrin, epidermal growth
factor (EGF), low density lipoprotein, high density lipoprotein,
leptin, VEGF, PDGF, ciliary neurotrophic factor, prolactin,
adrenocorticotropic hormone (ACTH), calcitonin, human chorionic
gonadotropin, cotrisol, estradiol, follicle stimulating hormone
(FSH), thyroid-stimulating hormone (TSH), leutinzing hormone (LH),
progeterone, testosterone; and (4) other proteins (including a-
fetoprotein, carcinoembryonic antigen CEA.
[0322] In addition, any of the biomolecules for which antibodies
may be detected may be detected directly as well; that is,
detection of virus or bacterial cells, therapeutic and abused
drugs, etc., may be done directly.
[0323] In a preferred embodiment, the target analyte is a nucleic
acid. These assays find use in a wide variety of applications, as
is generally outlined in U.S. Ser. Nos. 60/160,027; 60/161,148;
09/425,633; 60/160,917 and 60/244,119, all of which are expressly
incorporated herein by reference.
[0324] In a preferred embodiment, the probes are used in genetic
diagnosis. For example, probes can be made using the techniques
disclosed herein to detect target sequences such as the gene for
nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which
is a gene associated with a variety of cancers, the Apo E4 gene
that indicates a greater risk of Alzheimer's disease, allowing for
easy presymptomatic screening of patients, mutations in the cystic
fibrosis gene, cytochrome p450s or any of the others well known in
the art.
[0325] In an additional embodiment, viral and bacterial detection
is done using the complexes of the invention. In this embodiment,
probes are designed to detect target sequences from a variety of
bacteria and viruses. For example, current blood-screening
techniques rely on the detection of anti-HIV antibodies. The
methods disclosed herein allow for direct screening of clinical
samples to detect HIV nucleic acid sequences, particularly highly
conserved HIV sequences. In addition, this allows direct monitoring
of circulating virus within a patient as an improved method of
assessing the efficacy of anti-viral therapies. Similarly, viruses
associated with leukemia, HTLV-I and HTLV-II, may be detected in
this way. Bacterial infections such as tuberculosis, chlamydia and
other sexually transmitted diseases, may also be detected.
[0326] In a preferred embodiment, the nucleic acids of the
invention find use as probes for toxic bacteria in the screening of
water and food samples. For example, samples may be treated to lyse
the bacteria to release its nucleic acid, and then probes designed
to recognize bacterial strains, including, but not limited to, such
pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae,
Leishmania, enterotoxic strains of E. coli, and Legionnaire's
disease bacteria. Similarly, bioremediation strategies may be
evaluated using the compositions of the invention.
[0327] In a preferred embodiment, the compositions of the invention
are used to screen bioactive agents to find an agent that will
bind, and preferably modify the function of, a target molecule. As
above, a wide variety of different assay formats may be run, as
will be appreciated by those in the art. Generally, the target
analyte for which a binding partner is desired is labeled; binding
of the target analyte by the bioactive agent results in the
recruitment of the label to the bead, with subsequent
detection.
[0328] In a preferred embodiment, the binding of the bioactive
agent and the target analyte is specific; that is, the bioactive
agent specifically binds to the target analyte. By "specifically
bind" as used herein with respect to bioactive agent/ligand binding
is meant that the agent binds the analyte, with specificity
sufficient to differentiate between the analyte and other
components or contaminants of the test sample. However, as will be
appreciated by those in the art, it will be possible to detect
analytes using binding which is not highly specific; for example,
the systems may use different binding ligands, for example an array
of different ligands, and detection of any particular analyte is
via its "signature" of binding to a panel of binding ligands,
similar to the manner in which "electronic noses" work. This finds
particular utility in the detection of chemical analytes. The
binding should be sufficient to remain bound under the conditions
of the assay, including wash steps to remove non-specific binding,
although in some embodiments, wash steps are not desired; i.e. for
detecting low affinity binding partners. In some embodiments, for
example in the detection of certain biomolecules, the dissociation
constants of the analyte to the binding ligand will be less than
about 10.sup.-4 to 10.sup.-6 M.sup.-1, with less than about
10.sup.-5 to 10.sup.-9 M.sup.-1 being preferred and less than about
10.sup.-7 to 10.sup.-9 M.sup.-1 being particularly preferred.
[0329] Generally, a sample containing a target analyte (whether for
detection of the target analyte or screening for binding partners
of the target analyte) is added to the array, under conditions
suitable for binding of the target analyte to at least one of the
bioactive agents, i.e. generally physiological conditions. The
presence or absence of the target analyte is then detected. As will
be appreciated by those in the art, this may be done in a variety
of ways, generally through the use of a change in an optical
signal. This change can occur via many different mechanisms. A few
examples include the binding of a dye-tagged analyte to the bead,
the production of a dye species on or near the beads, the
destruction of an existing dye species, a change in the optical
signature upon analyte interaction with dye on bead, or any other
optical interrogatable event.
[0330] In a preferred embodiment, the change in optical signal
occurs as a result of the binding of a target analyte that is
labeled, either directly or indirectly, with a detectable label,
preferably an optical label such as a fluorochrome. Thus, for
example, when a proteinaceous target analyte is used, it may be
either directly labeled with a fluor, or indirectly, for example
through the use of a labeled antibody. Similarly, nucleic acids are
easily labeled with fluorochromes, for example during PCR
amplification as is known in the art. Alternatively, upon binding
of the target sequences, a hybridization indicator may be used as
the label. Hybridization indicators preferentially associate with
double stranded nucleic acid, usually reversibly. Hybridization
indicators include intercalators and minor and/or major groove
binding moieties. In a preferred embodiment, intercalators may be
used; since intercalation generally only occurs in the presence of
double stranded nucleic acid, only in the presence of target
hybridization will the label light up. Thus, upon binding of the
target analyte to a bioactive agent, there is a new optical signal
generated at that site, which then may be detected.
[0331] Alternatively, in some cases, as discussed above, the target
analyte such as an enzyme generates a species that is either
directly or indirectly optical detectable.
[0332] Furthermore, in some embodiments, a change in the optical
signature may be the basis of the optical signal. For example, the
interaction of some chemical target analytes with some fluorescent
dyes on the beads may alter the optical signature, thus generating
a different optical signal.
[0333] As will be appreciated by those in the art, in some
embodiments, the presence or absence of the target analyte may be
done using changes in other optical or non-optical signals,
including, but not limited to, surface enhanced Raman spectroscopy,
surface plasmon resonance, radioactivity, etc.
[0334] The assays may be run under a variety of experimental
conditions, as will be appreciated by those in the art. A variety
of other reagents may be included in the screening assays. These
include reagents like salts, neutral proteins, e.g. albumin,
detergents, etc., which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite binding.
Various blocking and washing steps may be utilized as is known in
the art.
[0335] Once made, the compositions of the invention find use in a
number of applications. In a preferred embodiment, the compositions
are used to probe a sample solution for the presence or absence of
a target sequence, including the quantitation of the amount of
target sequence present.
[0336] The compositions of the invention are useful for any of a
variety of nucleic acid assays including SNP identification,
sequencing, amplification, genotyping, nucleic acid quantitation,
and the like; methods for carrying out such assays are taught in
U.S. Ser. No. 60/244,119, which is herein incorporated by
reference.
[0337] In more specific embodiments, the teachings herein are
useful for the detection of mutations or mismatches in target
nucleic acid sequences. For example, recent focus has been on the
analysis of the relationship between genetic variation and
phenotype by making use of polymorphic DNA markers. Previous work
utilized short tandem repeats (STRs) as polymorphic positional
markers; however, recent focus is on the use of single nucleotide
polymorphisms (SNPs), which occur at an average frequency of more
than 1 per kilobase in human genomic DNA. Some SNPs, particularly
those in and around coding sequences, are likely to be the direct
cause of therapeutically relevant phenotypic variants. There are a
number of well known polymorphisms that cause clinically important
phenotypes; for example, the apoE2/3/4 variants are associated with
different relative risk of Alzheimer's and other diseases (see
Cordor et al., Science 261(1993). Multiplex PCR amplification of
SNP loci with subsequent hybridization to oligonucleotide arrays
has been shown to be an accurate and reliable method of
simultaneously genotyping at least hundreds of SNPs; see Wang et
al., Science, 280:1077 (1998); see also Schafer et al., Nature
Biotechnology 16:33-39 (1998). The compositions of the present
invention may easily be substituted for the arrays of the prior
art; in particular, single base extension (SBE) and pyrosequencing
techniques are particularly useful with the compositions of the
invention.
[0338] In a preferred embodiment, two-color competitive
hybridization assays are run. These assays can be based on
traditional sandwich assays. The beads contain a capture sequence
located on one side (upstream or downstream) of the SNP, to capture
the target sequence. Two SNP allele-specific probes, each labeled
with a different fluorophor, are hybridized to the target sequence.
The genotype can be obtained from a ratio of the two signals, with
the correct sequence generally exhibiting better binding. This has
an advantage in that the target sequence itself need not be
labeled. In addition, since the probes are competing, this means
that the conditions for binding need not be optimized. Under
conditions where a mismatched probe would be stably bound, a
matched probe can still displace it. Therefore the competitive
assay can provide better discrimination under those conditions.
Because many assays are carried out in parallel, conditions cannot
be optimized for every probe simultaneously. Therefore, a
competitive assay system can be used to help compensate for
non-optimal conditions for mismatch discrimination.
[0339] In a preferred embodiment, dideoxynucleotide
chain-termination sequencing is done using the compositions of the
invention. In this embodiment, a DNA polymerase is used to extend a
primer using fluorescently labeled ddNTPs. The 3' end of the primer
is located adjacent to the SNP site. In this way, the single base
extension is complementary to the sequence at the SNP site. By
using four different fluorophors, one for each base, the sequence
of the SNP can be deduced by comparing the four base-specific
signals. This may be done in several ways. In a first embodiment,
the capture probe can be extended; in this approach, the probe must
either be synthesized 5'-3' on the bead, or attached at the 5' end,
to provide a free 3' end for polymerase extension. Alternatively, a
sandwich type assay can be used; in this embodiment, the target is
captured on the bead by a probe, then a primer is annealed and
extended. Again, in the latter case, the target sequence need not
be labeled. In addition, since sandwich assays require two specific
interactions, this provides increased stringency which is
particularly helpful for the analysis of complex samples.
[0340] Accordingly, in one embodiment the invention provides a
method of preparing a universal array as described above. In
addition, the invention provides a method of linking a variety of
molecules to a universal bead. That is, the method includes
attaching an adapter sequence to a target analyte to form an
adapter/target analyte complex. This complex is then immobilized to
a bead to which an adapter has been attached. In a preferred
embodiment the adapter is a hairpin adapter. A particular advantage
of the present invention is that universal adapter beads can be
prepared in bulk. That is, there is no need to prepare different
adapter beads because the adapters are the same. In addition,
because the association of the bead adapters and adapter/target
analyte complexes is based on affinity, the immobilized complexes
are more resistant to the presence of impurities.
[0341] Accordingly, the present invention includes a method of
affinity purifying a target analyte. The method includes attaching
an adapter to the target analyte to form an adapter/target analyte
complex and isolating the complex by hybridization to a bead
adapter. In a preferred embodiment the adapter is a hairpin
adapter.
[0342] In an additional embodiment the invention provides a method
of regulating the packing density of target analytes linked to the
bead surface. The method includes adding adapters that are free
from a target analyte (dummy adapter) but hybridize with the
bead-based adapter. This dummy adapter will hybridize with a
fraction of the bead-based adapters, depending on the
concentration. As such, the dummy adapters serve as spacers.
[0343] In an additional embodiment the invention provides a method
of selecting full length sequences. That is the invention provides
a method of isolating target sequences from their 5' terminus. As
is known in the art, obtaining the 5' end of a target sequence can
at times be challenging. However, the present invention allows for
the selection of full length sequences. When the adapter is
designed such that the 3' terminus is recessed for ligation, the
hybridization and ligation reaction can be used to select for
full-length products from a 3' to 5' chemical synthesis of an
oligonucleotide. Only full-length, 5' phosphorylated products will
be able to link covalently to the adapter. If the reaction is
performed under conditions where non-covalently linked molecules
are transiently stable, full-length molecules are selected from the
mixture of other sequences. That is, following hybridization and
ligation of the full-length target sequence, the non-covalently
bound products are removed. The remaining sequences are full-length
sequences. In effect, a solid-phase affinity-based purification of
full-length products is performed.
[0344] In one embodiment the invention provides for specific array
formats. That is, arrays are designed with an eye to monitoring a
particular factor in a target sample. Such factors include the
presence of microorganisms in a sample, the presence of recombinant
molecules in a sample, the presence of contamination in a sample,
and the like. In particular, the invention provides an array for
the detection of genetically modified foods. That is, nucleic acid
arrays are constructed to detect a subset or all known genetically
modified variants of crop or livestock species including
commercially important species such as, corn or cattle. The arrays
would contain elements or combinations of elements that would
uniquely identify each GM (genetically modified) strain of the
organism in question. Such elements might identify any of the
following signatures for a given strain: the presence of
transgenes, the absence of deleted genes, the boundaries created by
such deletions or insertions, and the like. DNA is extracted from
food products or other samples of the tested organisms, and
hybridized to the array. The combination of hybridizing signals or
absence of hybridizing signals identifies the GMOs (genetically
modified organisms) present in the sample.
[0345] A GMO array allows food manufacturers to examine the quality
control of each lot of incoming ingredients or outgoing products to
ensure that only the intended strains are included. Likewise,
industrial farms would be able to use such arrays to check their
seed lots, and manufacturers of GMO-free products would use such
arrays to validate that their product is truly GMO free.
[0346] In an additional embodiment the array can be designed to
detect reporter genes in a target species. As is known in the art,
reporter genes are genes whose products generate easily detected
signals such as luminescence, fluorescence, or measurable
biochemical products. They are used for a wide range of
applications in biotechnology, including monitoring the acquisition
of transgenic DNA, measuring promoter activity, and many others.
Because they are useful for so many purposes, a small number of
reporter genes have been inserted into a variety of species,
including those that are widely studied, such as bacteria, fungi,
plants, invertebrates and vertebrates, including cultured cells of
humans.
[0347] Developing a microarray for a new organism or even a new
subset of genes from the same organism presents the need to rapidly
develop reliable protocols for sample preparation and array
validation. Organisms and even different tissues in an organism
differ in their biochemical composition and rates of degradation of
RNA, all of which make necessary developing and validating new
procedures for sample preparation and storage for each new
biological system under investigation. Accordingly, the invention
provides a method of calibrating sensor arrays.
[0348] In one embodiment the invention provides a method of
generating standardized array elements for the detection of
reporter genes. The method includes developing a collection of
sensors, each specifically detecting a particular reporter gene or
reporter gene product. The sensors are characterized to determine
the sensitivity and dynamic range with which they detect their
targets. Concurrent with or prior to the development of an array
for a new organism or a new tissue type, the standard sensors is
used to establish sample preparation procedures. The samples would
then be hybridized to the array and the quality of the specimen
preparations could be monitored by the detection of the reporter
gene transcript.
[0349] In one embodiment when a new array is made, a replicate
batch of the reporter gene sensors is cosynthesized with the
sensors in the new array. In testing the array, both the newly
synthesized sensors and the well-characterized standard sensors
used in the previous pilot experiments are loaded onto the array
with the organism-specific sensors. If a hybridization failure
occurs with the new array, the behavior of the two groups of
sensors would facilitate the tracing of the problem. If all the
sensors fail, it suggests a problem in the sample. If the
cosynthesized sensors fail but the original sensors work, it
suggests a problem in the synthesis of the new array. Because
reporter gene activity is by definition easily monitored, one
obtains an independent measurement of the reporter gene's activity
in the source tissue; in this way it is possible to eliminate
variation in expression levels as a reason for failing to obtain or
detect hybridization signals in the array.
[0350] All references cited herein are incorporated by reference in
their entireties.
* * * * *