Note: Descriptions are shown in the official language in which they were submitted.
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AUTOMATED INFORMATION PROCESSING IN RANDOMLY ORDERED ARRAYS
FIELD OF THE INVENTION
The invention relates to the use of a computer system to compare images
generated from a randomly
ordered array. This system preserves the relative position of each site within
the array so that the
same site can be compared in different images.
BACKGROUND OF THE INVENTION
There are a number of assays and sensors for the detection of the presence
andlor 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 ligandlantiligands)
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, etc.
Of particular use in these sensors are detection mechanisms utilizing
luminescence or fluorescence.
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.
1
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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,
FL, 1991, 2nd
Volume.
When using an optical fiber in an in vitro~n vivo sensor, one or more light
absorbing dyes are located
near its distal end. Typically, light from an appropriate source is used to
illuminate the dyes through
the fiber's proximal end. The light propagates along the length of the optical
fiber; and a portion of this
propagated light exits the distal end and is absorbed by the dyes. The light
absorbing dye may or may
not be immobilized; may or may not be directly attaches!-.tc the optical fiber
itself; may or may not be
suspended in a fluid sample containing one or more analytes of interest; and
may or may not be
retainable for subsequent use in a second optical determination.
Once the light has been absorbed by the dye, some light of varying wavelength
and intensity returns,
conveyed through either the same fiber or collection fibers) to a detection
system where it is observed
and measured. The interactions between the light conveyed by the optical fiber
and the properties of
the light absorbing dye provide an optical basis for both qualitative and
quantitative determinations.
Of the many different classes of light absorbing dyes which conventionally are
employed with bundles
of fiber strands and optical fibers for different analytical purposes are
those more common
compositions that emit light after absorption termed "fluorophores" and those
which absorb light and
internally convert the absorbed light to heat, rather than emit it as light,
termed "chromophores."
Fluorescence is a physical phenomenon based upon the ability of some molecules
to absorb light
(photons) at specified wavelengths and then emit light of a longer wavelength
and at a lower energy.
Substances able to fluoresce share a number of common characteristics: the
ability to absorb light
energy at one wavelength dab; reach an excited energy state; and subsequently
emit light at another
light wavelength, Vim. The absorption and fluorescence emission spectra are
individual for each
fluorophore and are often graphically represented as two separate curves that
are slightly overlapping.
The same fluorescence emission spectrum is generally observed irrespective of
the wavelength of the
exciting light and, accordingly, the wavelength and energy of the exciting
light may be varied within
limits; but the light emitted by the fluorophore will always provide the same
emission spectrum. Finally,
the strength of the fluorescence signal may be measured as the quantum yield
of light emitted. The
3 0 fluorescence quantum yield is the ratio of the number of photons emitted
in comparison to the number
of photons initially absorbed by the fluorophore. For more detailed
information regarding each of these
characteristics, the following references are recommended: Lakowicz, J. R.,
Principles of
Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D.,
Physical Biochemistry,
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second edition, W. H. Freeman and Company, New York, 1982; "Molecular
Luminescence
Spectroscopy Methods and Applications: Part I" (S.G. Schulman, editor) in
Chemical Analysis, vot. 77,
Wiley & Sons, Inc., 1985; The Theory of Luminescence, Stepanov and
Gribkovskii, iliffe Books, Ltd.,
London, 1968.
In comparison, substances which absorb tight and do not fluoresce usually
convert the light into heat
or kinetic energy. The ability to internally convert the absorbed light
identifies the dye as a
"chromophore." Dyes which absorb light energy as chromophores do so at
individual wavelengths of
energy and are characterized by a distinctive molar absorption coefficient at
that wavelength.
Chemical analysis employing fiber optic strands and absorption spectroscopy
using visible and
ultraviolet 1'~ght wavelengths in combination with the absorption coefficient
allow for the determination
of concentration for speck analyses of interest by spectral measurement. The
most common use of
absorbance measurement via optical fibers is to determine concentration which
is calculated in
accordance with Beers' law; accordingly, at a single absorbance waveiength_the
greater the quantity
of the composition which absorbs light energy at a given wavelength, the
greater the optical dens'dy for
the sample. In this way, the total quantity of light absorbed directly
correlates with the quantity of the
composition in the sample.
Many of the recent improvements employing optical fiber sensors in both
qualitative and quantitative
analytical determinations concern the desirability of depositing andlor
immobilizing various light
absorbing dyes at the distal end of the optical fiber. In this manner, a
variety of different optical fiber
chemical sensors and methods have been reported for specific analytical
determinations and
applications such as pH measurement, oxygen detection, and carbon dioxide
analyses. These
developments are exemplified by the following publications: Freeman, et al.,
Anal Chem. 53:98 (1983);
Lippitsch et al., Anal_ Chem. Acta. 205;1, (1988); Wolfbeis et al., Anal.
Chem. 60:2028 (1988); Jordan,
et al., Anal. Chem. 59:437 (i 987); Lubbers et al., Sens. Actuate 1983;
Munkholm et al., Talanta
35:109 (1988); Munkholm et al., Anal Chem.-58:1427 (1986); Seitz, W. R., Anal.
Chem. 56:16A-34A
(1984); Peterson, et al., Anal. Chem. 52:864 (1980): Saari, et al., Anal.
Chem. 54:821 (1982); Saari, et
al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal. Chem. Acta. 160:47
(1984); Schwab, et af., Anal.
Chem. 56:2199 (1984); Wolfbeis, O.S., "Fiber Optic Chemical Sensors", Ed CRC
Press, Boca Raton,
FL, 1991, 2nd Volume; and Pantano, P., Walt, D.R., Anal. Chem., 481A-487A,
Vol_ 67, (1995).
3 0 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
Wak, et al. disclose
systems for affixing multiple, different dyes on the distal end of the
bundle..
The disclosed configurations enable
separate optical fibers of the bundle to optically access individual dyes.
This avoids the problem of
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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.
United States Patents 6,023,540 and 6,327,410
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.
The use of fiducials for the registration of
sequential images has been used in screen printing
(U. S. Patent No. 5,129,155) and in implants in the human
body (U. S. Patent No. 4,991,579) and in various image
processing (see U.S. Patent Nos. 5,245,676 and 5,129,014).
Accordingly, it is an object of the present
invention to provide biosensors comprising random arrays,
generally comprising beads distributed at discrete sites on
the surface of a substrate, that utilize computer systems
and fiducials to allow comparison of sequential data images
of the arrays.
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CA 02359352 2004-O1-23
SUMMARY OF THE INVENTION
According to one aspect of the invention, there is
provided an array composition comprising: a) a substrate
with a surface comprising discrete sites; b) a population of
microspheres comprising at least a first and a second
subpopulation, wherein each subpopulation comprises a
bioactive agent and at least one subpopulation further
comprises an identifier binding ligand, wherein said
microspheres are distributed on said surface; and c) at
least one fiducial.
The invention provides, in a further aspect, an
array composition comprising: a) a fiber optic bundle with
a surface comprising discrete sites; b) a population of
microspheres comprising at least a first and a second
subpopulation, wherein each subpopulation comprises a
bioactive agent and at least one subpopulation further
comprises an identifier binding ligand, wherein said
microspheres are distributed on said surface; and c) at
least one fiducial.
The invention also provides a composition
comprising a computer readable memory to direct a computer
to function in a specified manner, said computer readable
memory comprising: a) an acquisition module for receiving a
data image of a random array comprising: i) a substrate
with a surface comprising a plurality of discrete sites;
ii) a population of microspheres comprising at least a first
and a second subpopulation wherein each subpopulation
comprises a bioactive agent; wherein said microspheres are
distributed on said surface; b) a registration module for
registering a data image; and c) a comparison module for
comparing registered data images.
4a
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There is also provided, a method of making an
array composition comprising: a) forming a surface
comprising discrete sites on a substrate; b) distributing
microspheres of said surface such that said discrete sites
contain microspheres, wherein said microspheres comprise at
least a first and a second subpopulations each comprising a
bioactive agent and at least one subpopulation further
comprises an identifier binding ligand; and c) incorporating
at least one fiducial onto said surface, according to
another aspect of the invention.
In accordance with a still further aspect of the
invention, there is provided a method for comparing separate
data images of a random array comprising: a) providing a
random array composition comprising: i) a substrate with a
surface comprising discrete sites; and ii) a population of
microspheres comprising at least a first and a second
subpopulation, wherein each subpopulation comprises a
bioactive agent and at least one subpopulation further
comprises an identifier binding ligand; wherein said
microspheres are distributed on said surface; b) using a
fiducial to generate a first data image; c) using a computer
system to register said first data image of said random
array to produce a registered first data image; d) using
said computer system to register a second data image of said
random array to produce a registered second data image; and
e) comparing said first and said second registered data
image to determine any differences between them.
According to another aspect of the invention,
there is provided a method of decoding a random array
composition comprising: a) providing a random array
composition comprising: i) a substrate with a surface
comprising discrete sites; and ii) a population of
microspheres comprising at least a first and a second
4b
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CA 02359352 2004-O1-23
subpopulation, wherein each subpopulation comprises a
bioactive agent; wherein said microspheres are distributed
on said surface; b) adding a first plurality of decoding
binding ligands to said array composition and creating a
first data image; c) using a fiducial to generate a first
registered data image; d) adding a second plurality of
decoding binding ligands to said array composition and
creating a second data image; and e) using said fiducial to
generate a second registered data image; and f) using a
computer system to compare said first and said second
registered data image to identify the location of at least
two bioactive agents.
The invention provides, in a further aspect, a
method of determining the presence of a target analyte in a
sample comprising: a) acquiring a first data image of a
random array composition comprising: i) a substrate with a
surface comprising discrete sites; ii) a population of
microspheres comprising at least a first and a second
subpopulation each comprising a bioactive agent and at least
one subpopulation further comprises an identifier binding
ligand; and iii) a fiducial; wherein said microspheres are
randomly distributed on said surface such that said discrete
sites contain microspheres; b) using a fiducial to register
said first data image to create a registered first data
image; c) contacting said random array composition with said
sample; d) acquiring a second data image from said array
with said sample; e) using said fiducial to register said
second data image to create a registered second data image;
and f) comparing said first and said second registered data
images to determine the presence or absence of said target
analyte.
In another aspect, the present invention provides
array compositions comprising a substrate with a surface
4c
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comprising discrete sites, at least one fiducial, and a
population of microspheres comprising at least a first and a
second subpopulation. Each subpopulation comprises a
bioactive agent, and the microspheres are distributed on
said surface. Each subpopulation may optionally comprise a
unique optical signature, an identifier binding ligand that
will bind a decoder binding ligand such that the
identification of the bioactive agent can be elucidated, or
both.
In an additional aspect, the invention provides
compositions comprising a computer readable memory to direct
a computer to function in a specified manner. The computer
readable memory comprises an acquisition module for
receiving a data image of a random array comprising a
plurality of discrete sites, a registration module for
registering a data image, and a comparison module for
comparing registered data images. Each module comprises
computer code for carrying out its function. The
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registration module may utilize any number of fiducials, including a fiducial
fiber when the substrate
comprises a fiber optic bundle, a fiducial microsphere, or a fiducial template
generated from the
random array.
In a further aspect, the invention provides methods of making the array
compositions of the invention
comprising forming a surface comprising individual sites on a substrate,
distributing microspheres on
the surface such that the individual sites contain microspheres, and
incorporating at least one fiducial
onto the surface. When the array has complete rotational freedom, at least two
fiducials are preferred
in the array to allow for correction of rotation.
In an additional aspect, the invention provides methods for comparing separate
data images of a
random array. The methods comprise using a computer system to register a first
data image of the
random array to produce a registered first data image, using the computer
system to register a second
data image of the random array to produce a registered second data image, and
comparing the first
and the second registered data images to determine any differences between
them.
In a further aspect, the invention provides methods of decoding a random array
composition
comprising providing a random array composition as outlined herein. A first
plurality of decoding
binding ligands is added to the array composition and a first data image is
created. A fiducial is used
to generate a first registered data image. A second plurality of decoding
binding ligands is added to
the array composition and a second data image is created. The fiducial is used
to generate a second
registered data image. A computer system is used to compare the frst and the
second registered
2 0 data image to identify the location of at least two bioactive agents.
In an additional aspect, the invention provides methods of determining the
presence of a target analyte
in a sample. The methods comprise acquiring a first data image of a random
array composition, and
registering the first data..image to create a registered first data image. The
sample is then added to
the random array and a second data image is acquired from the array. The
second data image is
registered to create a registered second data image. Then the first and the
second registered data
images are compared to determine the presence or absence of the target
analyte. Optionally, the data
acquisition may be at different wavelengths.
FIGURES
Figure 1 illustrates a fiber optic bundle with fiducial fibers.
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Figure 2 illustrates the components of a multi-multi fiber including fiducial
markers, optical fiber
bundles (multl fiber) and the components of a single optical fiber (mono
fiber).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the use of randomly ordered arrays
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, i.e. each
bead goes down arbitrarily or indescriminately on to a site. 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, or on a
different substrate and
then put onto the beads, and then the beads are randomly distributed on a
patterned surface.
However, the random placement of the beads means that all or part of the array
must be 'decoded'
after synthesis; that is, 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 ode. This encoding/decoding can be done in a number of ways,
as is generally described in United States Patents 6,023,540;
6,327,410 and 6,406,845, for exan~le. These methods include:
(1) "encoding" the beads with unique optical signatures,
generally fluorescent dyes, that can be used to identify the chemical
functionality on any particular
bead; (2) using 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;
(3) the use of 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; (4) the use of selective decoding, wherein only those beads that bind
to a target are decoded;
or (5) comfainations of any of these. In some cases, as is more fu8y 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.
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 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
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done prior to, during or after the assay as well. The target analytes will
bind to the bioactive agents as
is more fully outlined below, and results in a change in an optical signal of
a particular bead.
The present invention is directed to compositions and methods that allow
comparisons of sequential
data images taken during decoding and assay analysis. That is, in the broadest
sense, the invention
provides computer systems comprising processors and computer readable memory
that allow the
storage and analysis of multiple captured images of the same array, whether to
compare a decoding
image and an experimental image, several experimental images or several
decoding images. That is,
a first data image is taken of a random array, and using either a fiducial
template or an external
fiducial, the data image is registered. A second data image is then taken and
registered, and the twa_
registered data images can now be compared, as is more fully outlined below.
In a preferred embodiment, the present invention provides a variety of
"registration" techniques that
allow the comparison of a variety of these images in a uniform and reliable
way. That is, in order to
compare multiple data images from an array comprising a plurality of unique
sites, it is important that
the correct individual sites be compared during analysis. In a highly complex
and small system,
methods are needed to ensure that a first site in a first data image is
correctly matched to the first site
in a second data image. Accordingly, the present invention provides the
incorporation of one or more
reference features, also referred to herein as "markers" or "fiducials" or
"registration points", that allow
this registration from image to image. It is generally preferred to have a
number of spatially separated
fiducials so that small amounts of skew and reduction/enlargement can be
determined and taken into
2 0 account.
As is further described below, these fiducials can take a number of forms. For
example, when the
random array comprises beads, the fiducial may be a bead with a unique optical
signature or other
characteristic (Figure 1). When the random array comprises a fiber optic
bundle, the fiducial may be a
fiber element with a unique shape or optical properties. Alternatively, the
substrate 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 edges) or comprise the entire edge (Figure
2). Alternatively, the
fiducials may 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".
Accordingly, the present invention provides random array compositions
comprising at least a first
3 0 substrate with a surface comprising individual sites. By "random" array
herein is meant an array that
is manufactured under conditions that results in the identification of the
agent in at least some, if not
all, of the sites of the array being initially unknown; that is, each agent is
put down arbitrarily on a site
of the array in a generally non-reproducible manner. What is important in
random arrays, and what
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makes the present invention so useful, is that random arrays generally require
at least one, and
generally several "decoding" steps that produce data images that must be
compared. In addition,
while the techniques of the invention can be used on a variety of random
arrays, the discussion below
is directed to the use of arrays comprising microspheres that are laid down
randomly on a surface
comprising discrete sites. However, as will be appreciated by those in the
art, other types of random
arrays, i.e. those not containing beads, may also utilize the methods of the
invention.
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 (i.e. different beads) to many millions can be made, with
very large fiber opti ~ 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 (all numbers are per cm2) are from about
10,000,000 to about
2,000,000,000, 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
particularly 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 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.
2 5- 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 Nm
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 or more (in some instances, 1 million) different fibers
and beads in a 1 mm2
fiber optic bundle, with densities of greater than 25,000,000 individual beads
and fibers (again, in
3 0 some instances as many as 100 million) per 0.5 cm2 obtainable.
By "substrate" or "solid support" or other grammatical equivalents herein is
meant any material that
can be modified to contain discrete individual sites appropriate for the
attachment or association of
beads and is amenable to at least one detection method. As will be appreciated
by those in the art,
the number of possible substrates is very large. Possible substrates include,
but are not limited to,
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glass and modified or functionalized glass, plastics (including acrylics,
polystyrene and copolymers of
styrene and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon ~1, etc.),
polysaccharides, nylon or nitrocellulose, resins, silica or silica-based
materials including silicon and
modified silicon, carbon, metals, inorganic glasses, plastics, optical finer
bundles, and a variety of
other polymers. In general, the substrates allow optical, detection and do not
themselves appreciably
fluoresce.
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 confiocal 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 addition, as is more fully outlined below, the substrate may include a
coating, edging or sheath of
material, generally detectable, that defines a substrate edge that may serve
as one or more fiducials.
In a preferred embodiment, the substrate is an optical fiber
bundle or array, as is generally described in United States
Patent 6,200,737, PCT US98/05025, and PCT US98/09163, for example.
Preferred embodin~nts utilize preformed unitary fiber
optic arrays. 8y "preformed unitary fiber optic array" herein is meant an
array of discrete individual
2 0 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
manipulatable without intentionally
treating the preformed unitary array with agents that separate them, for
example treating a preformed
array susceptible to acid with an acid such that the interstitial material is
etched and thus the individual
cores can be separated. However, absent these intentional treatments, one
strand generally cannot
be physically separated at any point along its length from another fiber
strand.
At least one surtace of the substrate is modified to contain discrete,
individual sites for later
association of microspheres. These sites may also be referred to in some
embodiments as "features".
These sites may comprise physically al#ered sites, i.e. physical
configurations such as wells or small
3 0 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,
hydrophobicatlyl hydrophilically
functionalized sites, spots of adhesive, etc.
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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. However, it should be noted that these
sites 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, hut they
end up at discrete
sites.
In a preferred embodiment, the surface of the substrate is modified to contain
wells, Le. depressions in
the surface of the substrate. This may be done as is generally known in the
art using a variety of
techniques, including, but not limited to, photolithography, stamping
techniques, molding 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.
In a preferred embodiment, physical alterations are made in a surface of the
substrate to produce the
sites. In a preferred embodiment, the substrate is a fiber optic bundle and
the surface of the substrate,
is a terminal end of the fiber bundle, as is generally
described in United States Patents 6,023,540 and 6,327,410,
for example. 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 ane 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.
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.
In a preferred embodiment, the surface of the substrate is mod~ed to contain
chemically modified
sites, that can be used to attach, either covalently or non-covalently, the
microspheres of the invention
3 0 to the discrete sites or locations on the substrate. 'Chemically modified
sites' in this context includes,
but is not limited to, the addifron 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
CA 02359352 2001-07-17
WO 00/47996 PCT/US00/03375
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. 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.
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
2 0 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
graphited, 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
IN is a helpful
guide.
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
tag attachment. The bead sizes range from nanometers, i.e. 100 nm, to
millimeters, i.e. 1 mm, with
3 0 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 larger or
smaller beads may be
used.
11
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WO 00/47996 PCT/US00/03375
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.
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 the on the
synthetic methods. By "candidate bioactive agent" or "bioactive agent" or
"chemical functionality" or
"binding ligand" herein is meant 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.
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, preferably at least two of the functional chemical groups. The
bioactive agents often
2 0 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
proteins.
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
3 0 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.
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WO 00/47996 PCT/US00/03375
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.
In one preferred embodiment, the bioactive.agents are naturally occurring
proteins or fragments of
naturally occuring 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.
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
2 0 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
2 5 library of randomized bioactive proteinaceous agents.
In a preferred embodiment, a library of bioactive agents are 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
3 0 difficult to gauge the required absolute size of an interaction library,
nature provides a hint with the
immune response: a diversity of 10'-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 10' to 108 is
sufficient to find structures with
affinity for the target. Thus, in a preferred embodiment, at least 106,
preferably at least 10', more
13
CA 02359352 2004-O1-23
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preferably at least 10° and most preferably at least 10s different
bioactive agents are simultaneously
analyzed in the subject methods. Preferred methods maximize library size and
diversity.
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, tyrosine or histidines for phosphorylation sites, etc.,
or to purines, etc.
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 (east two nucleotides covalently linked together. A nucleic
acid of the present
invention will generally contain phosphodiester bonds, although in some cases,
as outlined below,
nuGeic 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. Or4. Chem., 35:3800 (1970); Sprinzl, et al., Eur. J. Biochem., 81:579
(1977); Letsinger, et al., Nucl.
Acids Res., 14:3487 (1986); Sawai, ef al., Chem. Lett., 805 (1984), Letsinger,
et aG, J. Am. Chem.
Soc.; 110:4470 (1988); and Pauwels, et al., Chemica Scriota, 26:141 (1986)),
phosphorothioate (Mag,
et aG, Nucleic Acids Res., 19:1437 (1991); and U.S. Patent 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. En4l., 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et
al., Nature, 380:207
(1996))). Other analog nucleic acids include those with
positive backbones (Denpcy, ef al., Proc. Natl. Acad. Sci. USA, 92:6097
(1995)); non-ionic backbones
(U.S. Patent Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;
Kiedrowshi, et al.,
Anctew. Chem. Intl. Ed. En4lish, 30:423 (1991); Letsinger, et aG, J. Am. Chem.
Soc., 110:4470 (1988);
Letsinger, et aL, Nucleosides 8 Nucleotides, 13:1597 (1994); Chapters 2 and 3,
ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S.
8anghui and P. Dan Cook;
Mesmaeker, et aL, Biooroanic 8 Medicinal Chem. Lett., 4:395 (1994); Jeffs, et
al., J. Biomolecular
NMf~, 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
3 5 nucleic acids (see Jenkins, et aL, Chem. Soc. Rev., (1995) pp. 169-176).
Several nucleic acid
14
CA 02359352 2004-O1-23
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analogs are described in Ravels, C 8 E News, June 2, 1987, page 35.
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 dsoxyribo- 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 described above generally for proteins, nucleic acid bioactive agents may
be naturally occuring
nucleic acids, random nucleic acids, or "biased" random nucleic acids. For
example, digests of
procaryotic or eukaryotic genomes may be used as is outlined above for
proteins.
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
2 0 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
2 5 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 at.
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
3 0 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'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
3 5 sequence at equilibrium (as the target sequences are present in excess, at
Tm, 50% of the probes are
CA 02359352 2001-07-17
WO 00/47996 PCT/US00/03375
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°C for short probes
(e.g. 10 to 50 nucleotides) and at
least about 60°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.
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.
In a preferred embodiment, the bioactive agents are organic chemical moieties,
a wide variety of which
are available in the literature.
In a preferred embodiment, each bead comprises a single type of bioactive
agent, although a plurality
2 0 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.
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 both
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, such as
3 0 peptides, organic moieties, and nucleic acids. It is a relatively
straightforward matter to adjust the
current synthetic techniques to use beads.
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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 surtace 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.
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 sulfhydryl 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-tinkers, pages 155-
200) 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 surtace 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
Torchitin et al., Critical Rev. Therapeutic Drua Carrier Systems. 7(41:275-308
(1991)).
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., Bioconi.
C a . 2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Biooonj.
Cherr~3:323-327 (1992);
King et al., Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconiuaate
Chem. 5:220-235
(1994)). It should be understood that the
3 0 candidate agents may be attached in a variety of ways, inGuding those
listed above. What is
important is that 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.
Specific techniques for immobilizing enzymes on microspheres are known in the
prior art. tn one case,
NHz surface chemistry microspheres are used. Surface activation is achieved
with a 2.5%
17
CA 02359352 2004-O1-23
61051-3220
gtutaraldehyde in phosphate buffered saline (10 mM) providing a pH of 6.9.
(138 mM NaCI, 2.7 mM,
KCI). 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 % finreen 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 0.45Nm amicon micropure filter.
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
L 0 a binding partner pair. By "specifically bind' herein 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, inGuding 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''-10a M'', with less
than about 105 to 10'~ M'' being preferred and less than about 10'' -
10'° M'' being particularly
preferred.
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-
figands, complementary
nucleic acids, and carbohydrates and their binding partners are also suitable
binding pairs. Nucleic
acid - nucleic acid binding proteins pairs are also useful. Similarly, as is
generally described in U.S.
Patents 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459,
5,683,867,5,705,337, and related
patents, wucleic acid'aptomers" can be developed for binding to
virtually any target; such a aptomer-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.
3 0 In a prefierred embodiment, the IBL is a molecule whose color or
luminescence properties change in
the presence of a selectively-binding DBL. For example, the IBL may be a
fluorescent pH indicator
whose emission intensity changes with pH. Similarly, the IBL may be a
fluorescent ion indicator,
whose emission properties change with ion concentration.
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Alternatively, the IBL is a molecule whose color or luminescence properties
change in the presence of
various solvents. For_example, the IBL may be a fluorescent molecule such as
an ethidium salt whose
fluorescence intensity increases in hydrophobic environments. Similarly, the
IBL may be a derivative
of fluorescein whose color changes between aqueous and nonpolar solvents.
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.
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.
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.
In a preferred embodiment, the microspheres comprise an optical
signature that can be used to identify the attached bioactive agent,
as is generally outlined in United States Patents 6,023,540 and
6,327,410, for example. That is, each subpopulation of mi.crospheres
comprise a unique optical signature or optical tag that can be used to
identify the unique bioactive
agent of that subpopulation of microspheres; a bead comprising the unique
optical signature may be
distinguished from beads at other locations with different optical signatures.
As is outlined herein,
each bioacfjve agent will have an associated unique optical signature such
that any microspheres
comprising that bioactive agent will be identlftable on the basis of the
signature: As is more fully
outlined below, it is possible to reuse or duplicate optical signatures within
an array, for example, when
another level of identification is used, for example when beads of different
sizes are used, or when the
array is loaded sequentially with different batches of beads.
In a preferred embodiment, the optical signature is generally a mixture of
reporter dyes, preferably
fluorescent. By varying both the composition of the mixture (i.e. the ratio of
one dye to another) and
3 0 the concentration of the dye (leading to differences in signal intensity),
matrices of unique tags 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
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but are preferably fluorescent dyes, which due to their strong signals provide
a good signal-to-noise
ratio for decoding. Suitable dyes for use in the invention 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 BIueTM, Texas Red, and others described in
the 1989-1991
Molecular Probes Handbook by Richard P. Haugland.
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.
iwo different fluors) or, alternatively, one label at two different
concentrations or intensity.
In a preferred embodiment, the dyes are covalentfy 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.
In a preferred embodiment, the dyes are non-covalently associated with the
beads, generally by
entrapping the dyes in the bead matrix or pores of the beads. Fluorescent dyes
are generally
preferred because the strength of the fluorescent signal provides a better
signal-to-noise ratio when.
decoding. 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.
In one embodiment, the dyes are added to the bioactive agent, rather than the
beads, although this is
generally not preferred.
In one embodiment, the microspheres do not contain an optical signature.
In a preferred embodiment, the present invention does not rely solely on the
use of optical properties
to decode the arrays. 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 of optical signatures one some beads. Thus, for example, using one "set"
of decoding molecules,
3 0 the use of two populations of beads, one with an optical signature and one
without, allows the effective
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doubling of the array size. The use of multiple optical signatures similarly
increases the possible size
of the array.
In a preferred embodiment, each subpopulation of beads comprises a plurality
of different identifier
binding ligands ("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 2~, 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 2'° possible variants
Hovivever, 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 3". 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.
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.
2 0 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.
The system is similar for proteins but requires a larger number of different
IBLs to identify each
3 0 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
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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.
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.
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 1X concentration of IBL, and
a second bead
comprising a second agent has a 10X concentration of IBL, using saturating
concentrations of the
corresponding labelled DBL allows the user to distinguish between the two
beads.
In a preferred embodiment, the compositions of the invention further comprise
at least one fiducial. By
"fiducial" or "marker" or "registration point" herein is meant a physical
reference feature or
characteristic that allows precise comparisons of sequential data images of an
array. The use of
fiducials is useful for a variety of reasons. In general, the assays involve
monitoring of objects, i.e.
bioactive agents, located at spatially distinct locations (features) over the
course of several data image
frames taken over time. Any shifting that occurs from frame to frame
complicates the analysis of the
agents. By incorporating permanent fiducials into the assay structure, each
data image can be
aligned, either manually or automatically, to allow accurate comparison of the
images, and control for
translation (i.e. a shift in an X-Y direction) and/or rotation as well as
reduction or enlargement of the
image. In addition, when fluorescence based assays are used (either for
decoding or analyte
assaying or both), in any given image, a particular region or feature may or
may not emit fluorescence,
2 0 depending on the label characteristics and the wavelength being
interrogated, or the presence or
absence of an analyte or DBL, etc. The presence of fluorescence is detected as
a positive change in
feature intensity with respect to the background intensity, which is then used
to draw a software
"segment" over the core. In situations where the core is dark, i.e. no
fluorescence is detected at that
particular feature, it is difficult to accurately draw the segment over the
core.
2 5 Accordingly, in a preferred embodiment, at least one fiducial is
incorporated into the array. In a
preferred embodiment, a plurality of fiducials are used, with the ideal number
depending on the size of
the array (i.e. features per fiducial), the density of the array, the shape of
the array, the irregularity of
the array, etc. In general, at least three non-linear fiducials are used; that
is, three fiducials that define
a plane (i.e. are not in a line) are used. In addition, it is preferred to
have at least one of the fiducials
3 0 be either on or close to the periphery of the array.
In a preferred embodiment, the substrate is a fiber optic bundle and the
fiducial is a fiducial fiber. As
will be appreciated by those in the art, the characteristics of a fiducial
fiber may vary widely. For
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example, in a preferred embodiment, the fiducial fibers may have unique or
special optical properties;
for example, fiducial fibers made of stock glass that exhibits broad
fluorescence across the visible
range of the spectrum; glasses are available in a wide range of compositions,
and often possess
intrinsic fluorescence due to the presence of impurities or dopants in the
glass material. In a preferred
embodiment, the fiducial fibers may have a different shape or size, or both,
from the other fibers of the
array. In addition, as is true for all the fiducial techniques herein, it is
often preferred to have different
fiducials have different characteristics, i.e. asymmetry among the fiducials,
to allow for an extra level of
registration. For example, in an square array format with fiducials at the
corners, one of the four
fiducials could be of a different shape or size than the other three or
positionally offset.
The fiducial fibers may each be labeled with the same or with different
labels. In a preferred
embodiment a fiber is coated with a single label. In addition, multiple fibers
to be incorporated into an
array are labeled with the same label. In an alternative embodiment, each of a
plurality of fiducial
fibers is labeled with a different or discrete label. In a preferred
embodiment a fiducial fiber comprises
a detectable label, such as a dye, fluorescent organic dye or fluorescent
inorganic particles such as
quantum dots.
Arrays comprising fiducial fibers are generally made in a variety of ways.
In one embodiment, the fiber is doped with fluorescent organic dyes or
fluorescent inorganic particles
such as quantum dots at the melting stage, prior to the machining of the glass
into rods for drawing.
In a preferred embodiment, the glass rod material is dipped into or covered
with a solution of a label
such as a fluorescent dopant material. Preferred embodiments utilize inorganic
nanoparticles
(quantum dots) as they are small in size, exhibit high fluoresence quantum
efficiencies, and are
extremely photostable over long period of time (i.e. resistant to
photobleaching). The fluorescent
characteristics of quantum dots are known to be directly related to the size
of the particles. In a
perferred embodiment, a polydisperse collection of particles is employed to
give rise to broad
2 5 absorbtive and emissive properties.
In another embodiment predoped fibers such as terbium-doped fibers are used as
a foundation for the
fiducial, and a label such as a fluorescent particle is added to the exterior
of these core glasses to give
rise to a fiber that exhibits fluorescent properties of both the internal and
external dopants.
Following the coating of the outside of either of the above-described core
bars with a label, the bar is
3 0 then inserted into a cladding tube of lower refractive index and drawn. By
cladding the coated fiber,
excitation light can be made to propagate down the core, exciting any
fluorescent material present
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either in the core itself or, in this case, at the interface between the core
and the clad. The
fluorescence is then coupled into the core and light-guided back up the core
to the proximal face of the
fiber where it is detected, for example by a CCD camera.
In an alternative embodiment each of a plurality of fibers are incorporated
into an array and each is
coated with a different label. By increasing the number of labels incorporated
into fiducial fibers, the
number and complexity of labels and/or registers increases.
In a preferred embodiment, the fiducial is a fiducial bead of the random
array. Similar to the fiducial
fibers, fiducial beads may be added to the random array in any position. Thus,
for example, a few
fiducial beads may be added to the array prior to or simultaneously with the
addition of the beads
comprising bioactive agents. In this case the fiducial beads may go down
randomly on the array.
Alternatively, when wells are used, targeted addition of fiducial beads may be
done; for example, by
creating larger wells in defined locations (for example by using a few larger
fibers and etching
techniques), large fiducial beads may be laid down in certain sites. As above
for fiducial fibers, the
detectable properties of the fiducial beads may be different than the
properties of the beads
comprising agents. In addition, when randomly laid down fiducial beads are
used, it should be noted
that an advantage of the resulting array is that the fiducial pattern is
essentially a "signature" of the
individual array. That is, since the likelihood of two arrays containing the
same spatial arrangement of
fiducial beads is very low, individual arrays can be visually distinguished,
serving as a sort of internal
"label" for the array.
In another embodiment, the marker bead comprises no label, while the remaining
beads or
microspheres are labeled. Thus, the absence of a label serves to identify the
marker bead on the
array.
In a preferred embodiment an array comprises at least one marker bead,
although more than one
marker bead is particularly preferred.
2 5 The marker beads) can be added to the substrate or array at any time prior
to, simultaneously with or
following the addition of other microspheres.
In a preferred embodiment, the fiducial is a defined edge or edges of the
substrate. As will be
appreciated by those in the art, this may be done in a variety of ways. In one
embodiment, a coating
or sheath of fiducial material, such as highly fluorescent glass, is
incorporated into the array
3 0 composition. The fiducial material can have any number of physical
characteristics to allow
registration; for example, a "stripe" of fiducial material may have notches,
dark impurities, or other
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identifying features along its length. Alternatively, the fiducial material is
placed in discrete spots or
discrete shapes; basically, any orientation that allows translation, rotation,
enlargement or reduction of
images to be detected can be used.
In a preferred embodiment, an exogeneous fiducial is not used; rather,
inherent characteristics of the
array are used. That is, rather than incorporate a special feature into the
array to serve as a fiducial,
the inherent variability of the features of an array is used to create a sort
of "fiducial template". In this
embodiment, an image of the array is taken under conditions in which all the
features are illuminated
evenly and can be differentiated from one another. For example, the surface of
the substrate can be
illuminated w. ith white light in such a way that all the features are
illuminated evenly. This finds
particular use when the substrate is a fiber optic bundle with etched wells,
in that the illumination angle
and intensity is chosen such that the light reflecting off the beads differs
in intensity from the light
reflecting off the cladding and spacer material. Preferred embodiments utilize
polarized light or light
impinging at various angles. Alternatively, the surface of the array may be
contacted with a
fluorescent solution, allowing fluorescence to be collected equally by all the
features.
In a preferred embodiment, the invention provides the use of an image produced
by a randomly
ordered array to identify and/or label the array. When forming a random array,
many, but not all, of the
microwells on an array are filled with microspheres. The filled versus
unfilled sites on the array are
randomized; thus, an image or a composite of images of an array that details
the filled from unfilled
locations on the array serves as a unique identifier of the array. Thus, the
image of a particular array
2 0 is statistically different and distinct from an image or a composite of
images of another array even
though the different arrays have functional equivalence. By "statistically
different" is meant that
although there is a theoretical probability that two arrays may be similar,
the probability is so small as
to be insignificant or unimportant.
In one embodiment, the arrays have at least one subpopulation of microspheres.
The pattern on the
array created by the random assembly of microspheres on the array serves to
identify the particular
array. The image of the array registers the location of each bead such that
composite images taken
from the array can be compared directly. For example, an image produced by an
array after exposure
to a first substance can be directly compared with the same array exposed to a
second substance.
Alternatively, a single population of microspheres can be analyzed by multiple
wavelengths and
3 0 directly compared.
In another embodiment, these arrays have two or more subpopulations
represented in each array.
Because the arrays are assembled randomly, the individual locations of beads
representing each
subpopulation are randomized. Thus, an image or a composite of images that
registers the location of
CA 02359352 2001-07-17
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each bead in a particular subpopulation will be statistically different from
an image or composite of
images of another array even though the different arrays have
functional.equivalence. Likewise, the
image or composite of images for another subpopulation within the same array
will be statistically
different from an image or composite of images of another array.
In addition, the number of beads of each subpopulation that actually populate
a given array can vary.
The specific number of beads within a subpopulation on an array is defined by
a Poisson distribution.
The variation in number of beads representing a subpopulation adds another
dimension to identifying
individual arrays.
The recognition that functionally equivalent arrays result in different images
affords one the possibility
of using that difference to "fingerprint" each array. Essentially, each random
array has a built in
method for identifying and tracking that array.
Thus, the invention facilitates the use of innate features within a random
array to identify and track a
specific array. The ability to identify and track a specific array has
important functionality ain quality
control monitoring, inventory monitoring, performance monitoring and use
monitoring. For example,
the ability to identify an array will allow one to determine when it is used
and whether it is reused.
In a preferred embodiment, the template image is used to define a "grid" which
is placed upon the data
images. The use of a template image to define the location of features is
optional, although currently
preferred. Using standard image processing software such as Image Pro (Media
Cybernetics) a
template is built based on this grid. This type of software allows the user to
create simultaneous
software segments to calculate the mean feature intensity over a region of
interest using a simple, one
step segmentation function. This software-based fiducial template can then be
mapped onto each data
image in the assay protocol to allow data collection for each region for each
data image. See for
example U.S. Patent No. 5,768,412. This allows the location of each array
feature to be defined.
Once the microspheres comprising the candidate agents and the unique tags are
generated, they are
added to the substrate to form an array. 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
attachment of the beads. This may be done in a variety of buffers, including
aqueous and organic
3 0 solvents, and mixtures. The solvent can evaporate, and excess beads
removed.
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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.
It should additionally be noted that in some cases, empty sites can serve as
fiducials. That is,
consistently "dark" sites can also be used as fiducials. This finds particular
use when the filling
efficiencies of the array are high; that is, when most sites contain a bead.
In addition, the dark sites
also can be used to "fingerprint" the array as described above. That is, the
image of light and dark
sites serves to define or identify a particular array. This image also serves
to register the array for
comparison purposes.
In some embodiments, for example when chemical attachment is done, it is
possible to attach 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.
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.
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.
In general, both
decoding and the experimental assay to determine the presence or absence of a
target analyte, both
of which are described below, requires the comparison of sequential data
images to determine the
differences between two data images. In general, this is done by taking a
first or initial data image,
using the fiducial to create a registered first data image, subjecting the
array to decoding conditions
and taking a second data image. The same fiducial is used to create a
registered second data image,
and then the two registered images can be compared. In this context, a "data
image" includes a
primary data image or a reduction of the image; for example, the image may be
reduced to a set of X-
Y coordinates with corresponding intensity values.
In a preferred embodiment, this is done using a computer system comprising a
processor and a
computer readable memory. The computer readable memory comprises an
acquisition module that
3 0 comprises computer code that can receive a data image from a random array
and a registration
module comprising computer code that can register the data image using at
least one fiducial,
including a fiducial template, to generate a registered data image. This
registered data image can
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then be stored in a storage module as needed. This same computer code, or
different code, if
required, can be used to receive additional data images and generate
additional registered data
images, which also can be stored. The computer readable memory further
comprises a comparison
module comprising computer code that can compare the registered data images to
determine the
differences between them, to allow both decoding of the array and target
analyte detection. That is,
when decoding is done, the comparison of at least two registered data images
allows the identification
of the location of at least two unique bioactive agents on the array.
Thus, using the systems described herein, a random array is decoded. 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 mass spectral tags.
In a preferred embodiment, atomic force microscopy (AFM) is used to decode the
array. In this
embodiment, an AFM tip, comprising a DBL, is positioned at the site to be
decoded, that comprises an
IBL. The force of interaction between the IBUDBL is measured using AFM. IN
addition, since AFM
has atomic resolution, a variety of other physical characteristics, including
physical size and shape can
be used for decoding. For example, different "shaped" molecules could be used
as IBLs; in this
embodiment, the AFM tip can comprise a DBL or just a moiety that can detect
different surfaces. In
addition, AFM could be used as "nanotweezers" to deliver or recover beads to
and from specific
locations on the array.
As will be appreciated by those in the art, this may also be done in systems
where the array is not
3 0 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
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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 the postives are fluorescent, and
sorting can proceed. The
characterization of the attached bioactive agent may be done directly, for
example using mass
spectroscopy.
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.
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 distinquish 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).
In an alternate preferred embodiment, the sites of attachment 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.
3 0 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.
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In a preferred embodiment, as outlined above, the DBL binds to the IBL.
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.
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.
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
2 0 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
2 5 to in an assay, during the assay, or after the assay.
In one embodiment, a single decoding step is done. In this embodiment, each
DBL is labeled with a
unique label, such that the the number of unique tags 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
3 0 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 tag, 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
CA 02359352 2001-07-17
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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.
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 prob~5 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.
The decoder probes are
pooled so that each pool 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. 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
X n sequential
hybridizations can ideally distinguish 4" sequences, although in some cases
more steps may be
required. After the hybridization of each pool, the hybrids are denatured and
the decoder probes
2 0 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).
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
3 0 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.
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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.
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, 'ode sharing" can
occur as well.
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 fluoroscent. 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 intensihr),
matrices of unique optical signatures may be generated. This may be done by
cx»ralently 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, which
due to their strong
signals provide a good signal-to-noise ratio for decoding. Suitable dyes for
use in the invention
include, but are not limited to, fluorescent lanthanide complexes, including
those of Europium and
Terbium, fluorescein, rhodamine, tetramethykhodamine, eosin, erythrosin,
coumarin, methyl-
coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BIueT'",
Texas Red, and others
described in the 6th Edition of the Molecular Probes Handbook by Richard P.
Haugland,
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.
3 0 two different floors) or, aftematively, one label at two different
concentrations or intensity.
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
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surface of the beads. As will be appreciated by those in the art, these
attachments are done to
minimize the effect on the dye.
In a preferred embodiment, the dyes are non-covalently associated with the
beads, generally by
entrapping the dyes in the pores of the beads.
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.
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 tags
(i.e. telephone
numbers) of other subarrays, that are separated by virtue of the location of
the subarray. Thus, for
example, the same unique tags can be reused from bundle to bundle. Thus, the
use of 50 unique tags
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, i.e. beads
containing unique tags for each
subarray.
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
2 0 microspheres. Optical fiber arrays can be fabricated containing features
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.
3 0 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
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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,
In a preferred embodiment, codes can be "shared" in several ways. In a first
embodiment, a single
code (i.e. IBUDBL 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.
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
2 0 presence of a particular gene, then the probes are functionally
equivalent, even though they may differ
in sequence. Similarly, if classes 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.
Once made, the compositions of the invention find use in a number of
applications. In a preferred
3 0 embodiment, the compositions are used to probe a sample solution for the
presence or absence of a
target analyte, including the quantification 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
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analyte can be used which binds a bioactive agent or for which a binding
partner (i.e. drug candidate)
is sought.
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.
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.
In a preferred embodiment, the target analyte is a nucleic acid. These assays
find use in a wide
variety of applications.
In a preferred embodiment, the probes are used in genetic diagnosis. For
example, probes can be
2 0 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.
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
3 0 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
CA 02359352 2001-07-17
WO 00/47996 PCT/US00/03375
detected in this way. Bacterial infections such as tuberculosis, chlamydia and
other sexually
transmitted diseases, may also be detected.
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.
In a further embodiment, the probes are used for forensic "DNA fingerprinting"
to match crime-scene
DNA against samples taken from victims and suspects.
In an additional embodiment, the probes in an array are used for sequencing by
hybridization.
The present invention also finds use as a methodology 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
2 0 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 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
3 0 bioactive agent results in the recruitment of the label to the bead, with
subsequent detection.
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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" herein 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'°-10~ M-', with less than about 10-5 to 10-9 M-' being preferred and
less, than about 10-' -10-9 M-'
being particularly preferred.
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.
In a preferred embodiment,.the change in optical signal occurs as a result of
the binding of a target
2 5 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 fluorophor, for example
during PCR amplification as is
known in the art. Alternatively, upon binding of the target sequences, a
hybridization indicator may be
3 0 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
3 5 signal generated at that site, which then may be detected.
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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.
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.
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.
Again, as outlined above for decoding, the assay for the presence or absence
of a target analyte
utilizes sequential processing of data images using a computer system. Thus,
in a preferred
embodiment, a first data image of a random array is acquired using an
acquisition module of the
computer system. This initial data image may be decoded, i.e. the location of
some or all of the
bioactive agents may be known, or decoding may occur either during or after
the assay. A registration
module of the computer system is used to create a registered first data image,
using either an
exogeneous fiducial or a fiducial template generated by acquiring a template
data image as outlined
herein, for example by evening illuminating the array. The sample is then
added to the array, and a
second data image is acquired using the acquisition module. The fiducial and
registration module are
then used to create a registered second data image. A comparision module of
the computer system is
then used to compare the registered data images to determine the presence or
absence of said target
2 0 analyte.
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.
In a preferred embodiment, two-color competitive hybridization assays are run.
These assays can be
3 0 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
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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 optimzed
for every probe
simultaneously. Therefore, a competitive assay system can be used to help
compensate for non-
optimal conditions for mismatch discrimination.
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 or other chain terminating nucleotides. 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
2 0 interactions, this provides increased stringency which is particularly
helpful for the analysis of complex
samples.
In addition, primer extension is possible; extension of a primer bound to
template in liquid phase is
followed by capture of the extended primer on the array.
In addition, when the target analyte and the DBL both bind to the agent, it is
also possible to do
detection of non-labelled target analytes via competition of decoding.
In a preferred embodiment, the methods of the invention are useful in array
quality control. Prior to
this invention, no methods have been described that provide a positive test of
the performance of
every probe on every array. Decoding of the array not only provides this test,
it also does so by
making use of the data generated during the decoding process itself.
Therefore, no additional
3 0 experimental work is required. The invention requires only a set of data
analysis algorithms that can
be encoded in software.
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The quality control procedure can identify a wide variety of systematic and
random problems in an
array. For example, random specks of dust or other contaminants might cause
some sensors to give
an incorrect signal-this can be detected during decoding. The omission of one
or more agents from
multiple arrays can also be detected. An advantage of this quality control
procedure is that it can be
implemented immediated prior to the assay itself, and is a true functional
test of each individual
sensor. Therefore any problems that might occur between array assembly and
actual use can be
detected. In applications where a very high level of confidence is required,
and/or there is a significant
chance of sensor failure during the experimental procedure, decoding and
quality control can be
conducted both before and after the actual sample analysis.
In a preferred embodiment, the arrays can be used to do reagent quality
control. In many instances,
biological macromolecules are used as reagents and must be quality controlled.
For example, large
sets of oligonucleotide probes may be provided as reagents. It is typically
difficult to perform quality
control on large numbers of different biological macromolecules. The approach
described here can be
used to do this by treating the reagents (formulated as the DBLs) as variable
instead of the arrays.
In a preferred embodiment, the methods outlined herein are used in array
calibration. For many
applications, such as mRNA quantitation, it is desirable to have a signal that
is a linear response to the
concentration of the target analyte, or, alternatively, if non-linear, to
determine a relationship between
concentration and signal, so that the concentration of the target analyte can
be estimated.
2 0 Accordingly, the present invention provides methods of creating
calibration curves in parallel for
multiple beads in an array. The calibration curves can be created under
conditions that simulate the
complexity of the sample to be analyzed. Each curve can be constructed
independently of the others
(e.g. for a different range of concentrations), but at the same time as all
the other curves for the array.
Thus, in this embodiment, the sequential decoding scheme is implemented with
different
2 5 concentrations being used as the code "labels", rather than different
fluorophores. In this way, signal
as a response to concentration can be measured for each bead. This calibration
can be carried out
just prior to array use, so that every probe on every array is individually
calibrated as needed.
In a preferred embodiment, the methods of the invention can be used in assay
development as well.
Thus, for example, the methods allow the identification of good and bad
probes; as is understood by
3 0 those in the art, some probes do not function well because they do not
hybridize well, or because they
cross-hybridize with more than one sequence. These problems are easily
detected during decoding.
The ability to rapidly assess probe performance has the potential to greatly
reduce the time and
expense of assay development.
CA 02359352 2004-O1-23
61051-3220
Similarly, in a preferred embodiment, the methods of the invention are useful
in quantitation in assay
development. Major challenges of many assays is the ability to detect
differences in analyte
concentrations between samples, the ability to quantitate these differences,
and to measure absolute
concentrations of analytes, ail in the presence of a complex mixture of
related analytes. An e~mple
of this problem is the quantitation of a specific mRNA in the presence of
total cellular mRNA One
approach that has been developed as a basis of mRNA quantitation makes use of
a multiple match
and mismatch probe pairs (Lockhart et al., 1996).
While this approach is simple, it requires relatively large numbers of probes.
In this approach, a
quantitative response to concentration is obtained by averaging the signals
from a set of different
probes to the gene or sequence of interest. This is necessary because only
some porbes respond
quantitatively, and it is not possible to predict these probes with certainty.
In the absence of prior
knowledge, only the average response of an appropriately chosen collection of
probes is quantitative.
However, in the present invention, that can be applied generally to nucleic
acid based assays as well
as other assays. In essence, the approach is to identify the probes that
respond quantitatively in a
particular assay, rather than average them with other probes. This is done
using the array calibration
scheme outlined above, in which concentration-based codes are used. Advantages
of this approach
include: fewer probes areweeded; the accuracy of the measurement is less
dependent on the number
of probes used; and that the response of the sensors is known with a high
level of certainty, since
each and every sequence can be tested in an efficient manner. it is important
to note that probes that
perfomr well are selected empiriically, which avoids the difficulties and
uncertainties of predicting
probe pertormance, particularly in complex sequence mixtures. In contrast, in
experiments described
to date with ordered arrays, relatively small numbers of sequences are checked
by perfomring
quantitative spiking experiments, in which a known mRNA is added to a mixture.
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