U.S. patent application number 11/503312 was filed with the patent office on 2007-02-15 for assay particles and methods of use.
Invention is credited to Wayne F. Patton.
Application Number | 20070037215 11/503312 |
Document ID | / |
Family ID | 37758262 |
Filed Date | 2007-02-15 |
United States Patent
Application |
20070037215 |
Kind Code |
A1 |
Patton; Wayne F. |
February 15, 2007 |
Assay particles and methods of use
Abstract
The invention provides assay particles useful, for example, for
detecting analytes and binding molecule interactions. One type of
assay particle includes a core portion encased by a shell portion,
wherein the shell portion comprises an inorganic phosphor that
binds selectively to a target molecule. Another type of an assay
particle includes a core portion encased by a shell portion, and a
coat portion covering the shell portion, wherein the coat portion
comprises an inorganic phosphor that binds selectively to a target
molecule. A further type of assay particle includes a core portion
encased by a shell portion, and a coat portion covering the shell
portion, wherein the coat portion comprises an inorganic phosphor
and a target selective binding moiety, and wherein the assay
particle is buoyant in aqueous media. An additional type of assay
particle includes a core portion encased by a shell portion, and a
coat portion covering the shell portion, wherein the shell portion
comprises an inorganic phosphor and the coat portion comprises a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media. Also provided are kits and related
methods.
Inventors: |
Patton; Wayne F.; (Newton,
MA) |
Correspondence
Address: |
PERKINELMER LAS, INC.;IP LEGAL DEPARTMENT
549 Albany Street
BOSTON
MA
02118
US
|
Family ID: |
37758262 |
Appl. No.: |
11/503312 |
Filed: |
August 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60707492 |
Aug 11, 2005 |
|
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|
Current U.S.
Class: |
435/7.1 ;
435/15 |
Current CPC
Class: |
C12Q 1/42 20130101; G01N
33/5432 20130101; G01N 33/542 20130101; G01N 33/573 20130101; G01N
33/54306 20130101; C12Q 1/485 20130101; G01N 33/5436 20130101; G01N
2333/9121 20130101; G01N 2333/916 20130101; G01N 33/54313
20130101 |
Class at
Publication: |
435/007.1 ;
435/015 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12Q 1/48 20060101 C12Q001/48 |
Claims
1. An assay particle, comprising a core portion encased by a shell
portion, wherein the shell portion comprises an inorganic phosphor
that binds selectively to a target molecule.
2. The assay particle of claim 1, wherein the core portion
comprises a material selected from the group of a gas, a liquid, a
solid and a mixture thereof.
3. The assay particle of claim 1, wherein the core portion
comprises a material selected from the group of air, organic
solvent and organic polymer.
4. The assay particle of claim 1, wherein the assay particle is
buoyant in aqueous media.
5. The assay particle of claim 1, wherein the target molecule is a
phosphorylated molecule.
6. The assay particle of claim 5, wherein the inorganic phosphor is
a hydrated metal oxide.
7. The assay particle of claim 1, wherein the inorganic phosphor is
selected from the group of rare-earth ion-doped yttrium oxide,
rare-earth ion-doped zirconium oxide, rare-earth ion-doped yttrium
oxysulfide and rare-earth ion-doped yttrium aluminum garnet.
8. The assay particle of claim 6, wherein the rare-earth dopant is
selected from the group of terbium (III), europium (III),
dysprosium (III), samarium (III), ruthenium (II), rhenium (I) and a
combination thereof.
9. The assay particle of claim 1, wherein the particle has a
density of less than 1 g/cm3.
10. A method for detecting a target molecule, comprising:
contacting a sample suspected of containing the target molecule
with an assay particle, wherein (i) the target molecule comprises a
moiety capable of emitting radiation, and (ii) the assay particle
comprises a shell portion comprising an inorganic phosphor capable
of binding selectively to target molecules; wherein binding of the
target molecule to the inorganic phosphor produces a light signal,
whereby the target molecule is detected.
11. A method for detecting protein kinase activity, comprising:
contacting a sample containing a protein kinase with an assay
particle, wherein the assay particle comprises a shell portion
comprising an inorganic phosphor that is capable of binding
selectively to phosphorylated molecules, and a protein kinase
substrate comprising a moiety capable of emitting radiation, under
conditions wherein the protein kinase can phosphorylatethe
substrate to produce a phosphorylated substrate, wherein binding of
the phosphorylated substrate to the inorganic phosphor produces a
light signal, and determining a protein kinase activity based on a
level of detected phosphorylated substrate.
12. A method for detecting protein phosphatase activity,
comprising: contacting a sample containing a protein phosphatase
with an assay particle, wherein the assay particle comprises a
shell portion comprising an inorganic phosphor that is capable of
binding selectively to phosphorylated molecules and wherein a
protein phosphatase substrate comprising a moiety capable of
emitting radiation is bound to the inorganic phosphor, under
conditions wherein the protein phosphatase can dephosphorylate the
substrate to produce a dephosphorylated substrate, wherein
phosphorylated substrate bound to the inorganic phosphor produces a
light signal, and determining a phosphatase activity based on a
level of detected phosphorylated substrate.
13. A method for identifying a protein kinase modulator,
comprising: contacting a sample containing a protein kinase with
(i) an assay particle comprising a shell portion comprising an
inorganic phosphor that is capable of binding selectively to
phosphorylated molecules; and (ii) a substrate comprising a moiety
capable of emitting radiation, in the presence and absence of a
candidate compound, under conditions wherein the protein kinase can
phosphorylate the substrate to produce a phosphorylated substrate,
wherein binding of the phosphorylated substrate to the inorganic
phosphor produces a light signal; and detecting light signals
produced in the presence and absence of the candidate compound,
wherein a difference in light signals produced in the presence and
absence of the compound identifies the compound as a protein kinase
modulator.
14. A method for identifying a cyclase modulator, comprising:
contacting a sample containing a cyclase with (i) an assay particle
comprising a shell portion comprising an inorganic phosphor that is
capable of binding selectively to non-cyclized nucleotides; and
(ii) radioactively labeled non-cyclized nucleotide, in the presence
and absence of a candidate compound, under conditions wherein the
cyclase can acts on the substrate to produce cyclized nucleotides,
wherein binding of the non-cyclized nucleotide to the inorganic
phosphor produces a light signal; and detecting light signals
produced in the presence and absence of the candidate compound,
wherein a difference in light signals produced in the presence and
absence of the compound identifies the compound as a cyclase
modulator.
15. A method for identifying a phosphodiesterase modulator,
comprising contacting a sample containing a phosphodiesterase with
(i) an assay particle comprising a shell portion comprising an
inorganic phosphor that is capable of binding selectively to
non-cyclized nucleotides; and (ii) radioactively labeled cyclized
nucleotide, in the presence and absence of a candidate compound,
under conditions wherein the phosphodiesterase can acts on the
substrate to produce non-cyclized nucleotides, wherein binding of
the non-cyclized nucleotide to the inorganic phosphor produces a
light signal; and detecting light signals produced in the presence
and absence of the candidate compound, wherein a difference in
light signals produced in the presence and absence of the compound
identifies the compound as a phosphodiesterase modulator.
16. A kit comprising an assay particle, comprising a core portion
encased by a shell portion, wherein the shell portion comprises an
inorganic phosphor that binds selectively to a target molecule, and
an enzyme substrate comprising a moiety capable of emitting
radiation.
17. An assay particle, comprising a core portion encased by a shell
portion, and a coat portion covering the shell portion, wherein the
coat portion comprises an inorganic phosphor that binds selectively
to a target molecule.
18. The assay particle of claim 17, wherein the core portion
comprises a material selected from the group of a gas, a liquid, a
solid and a mixture thereof.
19. The assay particle of claim 17, wherein the core portion
comprises a material selected from the group of air, organic
solvent and organic polymer.
20. The assay particle of claim 17, wherein the shell portion
comprises a material selected from glass and ceramic.
21. The assay particle of claim 17, wherein the assay particle is
buoyant in aqueous media.
22. The assay particle of claim 17, wherein the target molecule is
a phosphorylated molecule.
23. The assay particle of claim 22, wherein the inorganic phosphor
is a hydrated metal oxide.
24. The assay particle of claim 17, wherein the inorganic phosphor
is selected from the group of rare-earth ion-doped yttrium oxide,
rare-earth ion-doped zirconium oxide, rare-earth ion-doped yttrium
oxysulfide and rare-earth ion-doped yttrium aluminum garnet.
25. The assay particle of claim 24, wherein the rare-earth dopant
is selected from the group of terbium (III), europium (III),
dysprosium (III), samarium (III), ruthenium (II), rhenium (I) and a
combination thereof.
26. The assay particle of claim 17, wherein the particle has a
density of less than 1 g/cm3.
27. A method for detecting a target molecule, comprising contacting
a sample suspected of containing the target molecule with an assay
particle of claim 17, wherein (i) the target molecule comprises a
moiety capable of emitting radiation, and (ii) the assay particle
comprises a coat portion comprising an inorganic phosphor capable
of binding selectively to target molecules; wherein binding of the
target molecule to the inorganic phosphor produces a light signal,
whereby the target molecule is detected.
28. A method for detecting protein kinase activity, comprising
contacting a sample containing a protein kinase with an assay
particle of claim 17, wherein the assay particle comprises a coat
portion comprising an inorganic phosphor that is capable of binding
selectively to phosphorylated molecules, and a protein kinase
substrate comprising a moiety capable of emitting radiation, under
conditions wherein the protein kinase can phosphorylatethe
substrate to produce a phosphorylated substrate, wherein binding of
the phosphorylated substrate to the inorganic phosphor produces a
light signal, and determining a protein kinase activity based on a
level of detected phosphorylated substrate.
29. A method for detecting protein phosphatase activity, comprising
contacting a sample containing a protein phosphatase with an assay
particle of claim 17, wherein the assay particle comprises a coat
portion comprising an inorganic phosphor that is capable of binding
selectively to phosphorylated molecules and a protein phosphatase
substrate comprising a moiety that is capable of emitting radiation
is bound to the inorganic phosphor, under conditions wherein the
protein phosphatase can dephosphorylate the substrate to produce a
dephosphorylated substrate, wherein phosphorylated substrate bound
to the inorganic phosphor produces a light signal, and determining
a phosphatase activity based on a level of detected phosphorylated
substrate.
30. A method for identifying a protein kinase modulator, comprising
contacting a sample containing a protein kinase with (i) an assay
particle of claim 17 comprising a coat portion comprising an
inorganic phosphor that is capable of binding selectively to
phosphorylated molecules; and (ii) a substrate capable of emitting
radiation, in the presence and absence of a candidate compound,
under conditions wherein the protein kinase can phosphorylate the
substrate to produce a phosphorylated substrate, wherein binding of
the phosphorylated substrate to the inorganic phosphor produces a
light signal; and detecting light signals produced in the presence
and absence of the candidate compound, wherein a difference in
light signals produced in the presence and absence of the compound
identifies the compound as a protein kinase modulator.
31. A method for identifying a cyclase modulator, comprising:
contacting a sample containing a cyclase with (i) an assay particle
of claim 17 comprising a coat portion comprising an inorganic
phosphor that is capable of binding selectively to non-cyclized
nucleotides; and (ii) radioactively labeled non-cyclized
nucleotide, in the presence and absence of a candidate compound,
under conditions wherein the cyclase can acts on the substrate to
produce cyclized nucleotides, wherein binding of the non-cyclized
nucleotide to the inorganic phosphor produces a light signal; and
detecting light signals produced in the presence and absence of the
candidate compound, wherein a difference in light signals produced
in the presence and absence of the compound identifies the compound
as a cyclase modulator.
32. A method for identifying a phosphodiesterase modulator,
comprising: contacting a sample containing a phosphodiesterase with
(i) an assay particle of claim 17 comprising a coat portion
comprising an inorganic phosphor that is capable of binding
selectively to non-cyclized nucleotides; and (ii) radioactively
labeled cyclized nucleotide, in the presence and absence of a
candidate compound, under conditions wherein the phosphodiesterase
can acts on the substrate to produce non-cyclized nucleotides,
wherein binding of the non-cyclized nucleotide to the inorganic
phosphor produces a light signal; and detecting light signals
produced in the presence and absence of the candidate compound,
wherein a difference in light signals produced in the presence and
absence of the compound identifies the compound as a
phosphodiesterase modulator.
33. A kit comprising an assay particle of claim 17, comprising a
core portion encased by a shell portion, and a coat portion
covering the shell portion, wherein the coat portion comprises an
inorganic phosphor that binds selectively to a target molecule, and
an enzyme substrate capable of emitting radiation.
34. An assay particle, comprising a core portion encased by a shell
portion, and a coat portion covering the shell portion, wherein the
coat portion comprises an inorganic phosphor and a target selective
binding moiety, and wherein the assay particle is buoyant in
aqueous media.
35. The assay particle of claim 34, wherein the core portion
comprises a material selected from the group of a gas, a liquid, a
solid and a mixture thereof.
36. The assay particle of claim 34, wherein the core portion
comprises a material selected from the group of air, organic
solvent and organic polymer.
37. The assay particle of claim 34, wherein the core portion
comprises a material selected from the group of air, organic
solvent and organic polymer.
38. The assay particle of claim 34, wherein the shell portion
comprises a material selected from the group of glass, ceramic and
polymer.
39. The assay particle of claim 34, wherein the inorganic phosphor
is selected from the group of rare-earth ion-doped yttrium oxide,
rare-earth ion-doped zirconium oxide, rare-earth ion-doped yttrium
oxysulfide and rare-earth ion-doped yttrium aluminum garnet.
40. The assay particle of claim 39, wherein the rare-earth dopant
is selected from the group of terbium (III), europium (III),
dysprosium (III), samarium (III), ruthenium (II), rhenium (I) and a
combination thereof.
41. The assay particle of claim 34, wherein the target selective
binding agent is selected from the group of antibody, aptamer,
protein A, protein G, streptavidin, avidin, captavidin,
neutravidin, metal chelate, siderophore, lectin, and an
oligonucleotide.
42. The assay particle of claim 34, wherein the particle has a
density of less than 1 g/cm.sup.3.
43. A method for detecting a target molecule, comprising contacting
a sample suspected of containing the target molecule with an assay
particle of claim 34, wherein (i) the target molecule comprises a
moiety capable of emitting radiation, and (ii) the assay particle
comprises a coat portion comprising an inorganic phosphor and a
target selective binding agent, and is buoyant in aqueous media;
wherein binding of the target molecule to the target selective
binding agent activates the inorganic phosphor to produce a light
signal, whereby the target molecule is detected.
44. A method for detecting protein kinase activity, comprising
contacting a sample containing a protein kinase with (i) an assay
particle of claim 34, wherein the assay particle comprises a coat
portion comprising an inorganic phosphor and a binding agent
selective for phosphorylated molecules, and is buoyant in aqueous
media; and (ii) a protein kinase substrate comprising a moiety
capable of emitting radiation, under conditions wherein the protein
kinase can phosphorylatethe substrate to produce a phosphorylated
substrate, wherein binding of the phosphorylated substrate to the
inorganic phosphor produces a light signal, and determining a
protein kinase activity based on a level of detected phosphorylated
substrate.
45. A method for detecting protein phosphatase activity, comprising
contacting a sample containing a protein phosphatase with an assay
particle of claim 34, wherein the assay particle comprises a coat
portion comprising an inorganic phosphor and a binding agent
selective for phosphorylated molecules, and wherein a protein
phosphatase substrate comprising a moiety capable of emitting
radiation is bound to the inorganic phosphor, under conditions
wherein the protein phosphatase can dephosphorylate the substrate
to produce a dephosphorylated substrate, wherein phosphorylated
substrate bound to the inorganic phosphor produces a light signal,
and determining a phosphatase activity based on a level of detected
phosphorylated substrate.
46. A method for identifying a protein kinase modulator, comprising
contacting a sample containing a protein kinase with (i) an assay
particle of claim 34, wherein the assay particle comprises a coat
portion comprising an inorganic phosphor and a binding agent
selective for phosphorylated molecules; and (ii) a substrate
comprising a moiety capable of emitting radiation, in the presence
and absence of a candidate compound, under conditions wherein the
protein kinase can phosphorylate the substrate to produce a
phosphorylated substrate, wherein binding of the phosphorylated
substrate to the inorganic phosphor produces a light signal; and
detecting light signals produced in the presence and absence of the
candidate compound, wherein a difference in light signals produced
in the presence and absence of the compound identifies the compound
as a protein kinase modulator.
47. A method for identifying a cyclase modulator, comprising
contacting a sample containing a cyclase with (i) an assay
particle, wherein the assay particle of claim 34 comprises a coat
portion comprising an inorganic phosphor and a binding agent
selective for non-cyclized nucleotides ; and (ii) radioactively
labeled non-cyclized nucleotide, in the presence and absence of a
candidate compound, under conditions wherein the cyclase can acts
on the substrate to produce cyclized nucleotides, wherein binding
of the non-cyclized nucleotide to the inorganic phosphor produces a
light signal; and detecting light signals produced in the presence
and absence of the candidate compound, wherein a difference in
light signals produced in the presence and absence of the compound
identifies the compound as a cyclase modulator.
48. A method for identifying a phosphodiesterase modulator,
comprising contacting a sample containing a phosphodiesterase with
(i) an assay particle of claim 34, wherein the assay particle
comprises a coat portion comprising an inorganic phosphor and a
binding agent selective for non-cyclized nucleotides; and (ii)
radioactively labeled cyclized nucleotide, in the presence and
absence of a candidate compound, under conditions wherein the
phosphodiesterase can acts on the substrate to produce non-cyclized
nucleotides, wherein binding of the non-cyclized nucleotide to the
inorganic phosphor produces a light signal; and detecting light
signals produced in the presence and absence of the candidate
compound, wherein a difference in light signals produced in the
presence and absence of the compound identifies the compound as a
phosphodiesterase modulator.
49. A kit comprising an assay particle of claim 34, comprising a
core portion encased by a shell portion, and a coat portion
covering the shell portion, wherein the coat portion comprises an
inorganic phosphor and a target selective binding moiety, and
wherein the assay particle is buoyant in aqueous media, and an
enzyme substrate capable of emitting radiation.
50. An assay particle, comprising a core portion encased by a shell
portion, and a coat portion covering the shell portion, wherein the
shell portion comprises an inorganic phosphor and the coat portion
comprises a target selective binding moiety, and wherein the assay
particle is buoyant in aqueous media.
51. A method for detecting a target molecule, comprising use of the
assay particle of claim 50.
52. A kit comprising an assay particle of claim 50, comprising a
core portion encased by a shell portion, and a coat portion
covering the shell portion, wherein the shell portion comprises an
inorganic phosphor and the coat portion comprises a target
selective binding moiety, and wherein the assay particle is buoyant
in aqueous media, and an enzyme substrate capable of emitting
radiation.
Description
[0001] The present application claims the benefit of priority to
U.S. Provisional Patent Application Ser. No. 60/707,492, which was
filed on Aug. 11, 2005.
BACKGROUND
[0002] The scintillation proximity assay (SPA) is an approach for
assay development and biochemical screening that allows rapid and
sensitive measurement of molecular interactions in a homogeneous
system, obviating the need for separation and washing steps. In
theory, all that is required in the assay is mixing and
measurement. The technology has proven useful in radiometric
screening since its adoption in about 1992, with hundreds of PubMed
literature references citing SPA applications to date. SPA is
considered to be convenient, cost effective and safer than
radioactive filter binding assays, providing fewer handling steps,
no need for filters and scintillation cocktails as well as reduced
disposal costs. The signal detection for SPA can be performed using
any photomultiplier tube-based scintillation counter or CCD camera
imager. SPA has enabled advances in high throughput screening,
being both automation-friendly and requiring minimal hands-on
involvement. It has been estimated that the assay provides a
30-fold increase in productivity relative to typical filtration
assays.
[0003] Turning to the technical aspects of SPA, binding reactions
can be assayed without the washing or filtration procedures
normally used to separate bound from free fractions. Assays are
typically performed using radioactive labels that emit electrons
with only a short range (about 10 um) in water. When bound close to
a solid scintillator surface by the binding reaction the
radioactive labels are able to transfer electron energy to the
scintillator to produce photons detectable with a scintillation
counter. Electrons emitted from labeled molecules not bound close
to the surface dissipate their energy in the medium and are not
detected. The amount of light (photons) generated is proportional
to the amount of radiolabeled molecules bound to the solid
scintillant. Thus the bound fraction is detected specifically
without separation of the solution from the support.
[0004] SPA beads are microscopic beads which contain a scintillant
that can be stimulated to emit light. As is indicated above, this
stimulation event only occurs when radiolabeled molecules of
interest are bound to the surface of the bead, then blue light is
emitted that can be detected on standard scintillation counters.
Another type of SPA beads, often referred to as SPA imaging beads,
emit red light that can be detected on standard CCD cameras. Assay
plates coated with scintillant have also been used for SPA
methods.
[0005] Further applications, formats, materials and procedures for
performing SPA and SPA-like technologies are expected to contribute
further to high throughput screening capability as well as to
advances in bioanalytical and biomedical sciences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 shows exemplary assay particles encompassed within
the technology described herein. The drawings exemplify
cross-sections of assay particles having (A) a core and a shell
portion; (B) core, shell and coating portions; (C) core, shell,
coating and outer layer portions; and (D) a core portion containing
an exemplary type of encoding element.
[0007] FIG. 2 shows results from experiments in which
TiO.sub.2-coated assay particles were used to prepare a sample
enriched in phosphorylated molecules. Shown in FIG. 2A are mass
spectra of eluted and unbound samples of .alpha.-casein tryptic
digest fractionated on TiO.sub.2-coated magnetic beads. Shown in
FIG. 2B are mass spectra of eluted, and unbound and control samples
of a-casein tryptic digest fractionated on TiO.sub.2-coated glass
assay particles as well as the negative control mass spectrum of
a-casein tryptic digest eluted from uncoated glass assay
particles.
[0008] FIG. 3 shows a schematic diagram depicting an exemplary
homogenous protein kinase scintillation proximity assay using a
tritiated peptide substrate and an air-filled glass assay particle
coated with an inorganic phosphor that binds selectively to
phosphorylated molecules, according to an embodiment of the
technology described herein.
[0009] FIG. 4 is a schematic diagram of an exemplary homogenous
protein kinase time-resolved fluorescence assay using a cyanine 5
dye-labeled peptide substrate and an air-filled glass assay
particle coated with an inorganic phosphor that binds selectively
to phosphorylated molecules, according to an embodiment of the
technology described herein.
SUMMARY
[0010] The invention provides assay particles useful, for example,
for detecting analytes and molecular interactions. One type of
assay particle includes a core portion encased by a shell portion,
wherein the shell portion comprises an inorganic phosphor that
binds selectively to a target molecule. Another type of an assay
particle includes a core portion encased by a shell portion, and a
coat portion covering the shell portion, wherein the coat portion
comprises an inorganic phosphor that binds selectively to a target
molecule. A further type of assay particle includes a core portion
encased by a shell portion, and a coat portion covering the shell
portion, wherein the coat portion comprises an inorganic phosphor
and a target selective binding moiety, and wherein the assay
particle is buoyant in aqueous media. An additional type of assay
particle includes a core portion encased by a shell portion, and a
coat portion covering the shell portion, wherein the shell portion
comprises an inorganic phosphor and the coat portion comprises a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media. In an embodiment, the core portion of an
assay particle can include a material selected from the group of a
gas, a liquid, a solid and a mixture thereof, such as a material
selected from the group of air, organic solvent and organic
polymer. In an embodiment, an assay particle can be buoyant in
aqueous media. In an embodiment, the target molecule is a
phosphorylated molecule. inorganic phosphor is selected from the
group of rare-earth ion-doped yttrium oxide, rare-earth ion-doped
zirconium oxide, rare-earth ion-doped yttrium oxysulfide and
rare-earth ion-doped yttrium aluminum garnet. The rare-earth dopant
can be selected from, for example, the group of terbium (III),
europium (III), dysprosium (III), samarium (III), ruthenium (II),
rhenium (I) and a combination thereof. In an embodiment, the assay
particle has a density of less than 1 g/cm3. 1. In some
embodiments, the target selective binding agent can be, for
example, selected from the group of antibody, aptamer, protein A,
protein G, streptavidin, avidin, captavidin, neutravidin, metal
chelate, siderophore, lectin, and an oligonucleotide.
[0011] The invention provides methods for detecting a target
molecule using an assay particle described herein. In one aspect, a
method involves contacting a sample suspected of containing the
target molecule with an assay particle, wherein (i) the target
molecule comprises a moiety capable of emitting radiation, and (ii)
the assay particle comprises a shell portion comprising an
inorganic phosphor capable of binding selectively to target
molecules; wherein binding of the target molecule to the inorganic
phosphor produces a light signal, whereby the target molecule is
detected.
DETAILED DESCRIPTION
[0012] The technology described herein provides assay particles and
related methods and kits for detecting analytes and performing
protein interaction and enzyme assays, such as protease, kinase,
phosphatase, receptor binding, and molecular interaction assays, in
scintillation and luminescence proximity assay formats, as well as
fluorescence assay formats.
[0013] Assay beads have become an important tool for performing
high throughput assays in biomedical research and drug development.
Various commercial SPA methods involve using plastic beads (for
example, polystyrene, polyvinyltoluene or polyethyleneimine)
containing an organic scintillant, such as 2,4-diphenyloxazole
(PPO) or anthracene. Other SPA methods use beads made from the
inorganic scintillators yttrium silicate or yttrium oxide. Various
inositol phosphates and phosphorylated lipids, most particularly
sphingosine phosphate, have previously been detected and quantified
by SPA using commercially available solid yttrium silicate and
solid yttrium oxide particles (Brandish et al, 2003; 2004; Noremant
et al, 2002).
[0014] The technology provided herein includes assay particles that
can be used in proximity assays of various types, including SPA.
One type of assay particle provided herein has a shell portion,
which is made from an inorganic phosphor material, and which
encases a core portion. The particular inorganic phosphor material
used for this assay particle has the ability to bind to target
molecules. As an example, the inorganic phosphor material can be a
hydrated metal oxide, such as TiO.sub.2, which can bind selectively
to phosphorylated molecules. Exemplary constructions of such assay
particles include TiO.sub.2 formed into hollow microspheres (the
shell) surrounding a core of air or another gas, and TiO.sub.2
layered as a shell onto a polymer core. In either case, the
resulting assay particle can have a low density relative to the
assay solution, if desired. Another type of assay particle provided
herein also has a shell and core, but further has a coating
portion. The coating portion in this case contains an inorganic
phosphor that has the ability to bind to target molecules. Carrying
on with the TiO.sub.2 example, an exemplary construction of an
assay particle having a coating portion include a hollow
microsphere (the shell and core) coated with TiO.sub.2. A further
type of assay particle provided herein has shell, core and coat
portions, with either or both the shell and coat portions
containing an inorganic phosphor and a target selective binding
agent. This type of particle is composed of materials that allow it
to be buoyant in aqueous media. Each of the assay particles can be
relatively lightweight in comparison to typical metal oxide SPA
beads, such as those referenced above, which are solid, and can be
buoyant in aqueous media.
[0015] Therefore, the technology provides an assay particle,
comprising a core portion encased by a shell portion, wherein the
shell portion comprises an inorganic phosphor that binds
selectively to a target molecule. In another embodiment, an assay
particle of the invention includes a core portion encased by a
shell portion, and a coat portion covering the shell portion,
wherein the coat portion comprises an inorganic phosphor that binds
selectively to a target molecule. In a further embodiment, an assay
particle of the invention includes a core portion encased by a
shell portion, and a coat portion covering the shell portion,
wherein the coat portion comprises an inorganic phosphor and a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media. In yet another embodiment, an assay
particle of the invention includes a core portion encased by a
shell portion, and a coat portion covering the shell portion,
wherein the shell portion comprises and inorganic phosphor and the
coat portion comprises a target selective binding moiety, and
wherein the assay particle is buoyant in aqueous media. The
invention provides assay particles useful, for example, for
detecting analytes and binding molecule interactions. One type of
assay particle includes a core portion encased by a shell portion,
wherein the shell portion comprises an inorganic phosphor that
binds selectively to a target molecule. Another type of an assay
particle includes a core portion encased by a shell portion, and a
coat portion covering the shell portion, wherein the coat portion
comprises an inorganic phosphor that binds selectively to a target
molecule. A further type of assay particle includes a core portion
encased by a shell portion, and a coat portion covering the shell
portion, wherein the coat portion comprises an inorganic phosphor
and a target selective binding moiety, and wherein the assay
particle is buoyant in aqueous media. An additional type of assay
particle includes a core portion encased by a shell portion, and a
coat portion covering the shell portion, wherein the shell portion
comprises an inorganic phosphor and the coat portion comprises a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media. In an embodiment, the core portion of an
assay particle can include a material selected from the group of a
gas, a liquid, a solid and a mixture thereof, such as a material
selected from the group of air, organic solvent and organic
polymer. In an embodiment, an assay particle can be buoyant in
aqueous media. In an embodiment, the target molecule is a
phosphorylated molecule. The inorganic phosphor can be selected
from the group of rare-earth ion-doped yttrium oxide, rare-earth
ion-doped zirconium oxide, rare-earth ion-doped yttrium oxysulfide
and rare-earth ion-doped yttrium aluminum garnet. The rare-earth
dopant can be selected from, for example, the group of terbium
(III), europium (III), dysprosium (III), samarium (III), ruthenium
(II), rhenium (I) and a combination thereof. In an embodiment, the
assay particle has a density of less than 1 g/cm3.
[0016] As used herein, the term "core portion" when used in
reference to an assay particle of the invention means the innermost
part of the particle, which is surrounded by a shell. The core
portion can be composed of any substance containable within the
shell portion of the assay particle, including one or more of a
gas, solid, matrix, gel, colloid and liquid, and mixtures thereof.
The composition of the core portion can be selected to impart
certain physical properties to the assay particle, such as a
particular magnetic, density or buoyancy property. Exemplary gases
suitable for a core portion of an assay particle include air,
nitrogen and oxygen. Exemplary liquids suitable for a core portion
of an assay particle include oils, organic solvents, aqueous
solutions and mixtures thereof. Exemplary solids and matrices,
suitable for a core portion of an assay particle include organic
materials such as cellulose, polyethyleneimine, dextran, agarose,
polyacrylamide, polyvinyltoluene, Trisacryl, hydroxyalkyl
methacrylate, poly(vinylacetate-co-ethylene), oxirane acrylate,
polyethylene, polypropylene, poly (vinyl chloride), poly (methyl
methacrylate), phenol resin, poly (vinylidene difluoride), poly
(ethylene terephthalate), polyvinylpyrrolidone, polycarbonate and
starch, and inorganic materials such as glass, ceramic, metal,
glass, alumina, silica, zirconia, a ferromagnetic material and a
paramagnetic material.
[0017] In an embodiment, an assay particle of the invention can be
buoyant in an assay medium, typically an aqueous medium. An assay
particle can have a low density relative to an assay medium or a
density similar to an assay medium, for example, to render the
assay particle buoyant in aqueous media. Therefore, a core portion
can be selected to have a low density relative to one or both the
shell portion and the coat portion (if present) of an assay
particle, and also to have a density lower or similar to an assay
medium. A core portion can therefore have a density of less than 1
g/cm.sup.3, such as less than 0.5 g/cm.sup.3 and 0.1 g/cm.sup.3,
although a more dense core portion can be used, for example, to
obtain a buoyancy characteristic in a particular medium, such as a
viscous medium. As a specific example of a core portion of an assay
particle, described below is preparation of an assay particle
having a gas core portion (Example 1). Assay particles having lower
density than the aqueous medium, upon standing for a period of
time, will float to the surface of the media, allowing their
imaging above the assay container, which can reduce background in
comparison to imaging particles in solution. In addition, larger
particles having inorganic phosphor coatings become feasible to
employ in SPA due to reduced settling of particles associated with
the high density of inorganic particles or crystals. Larger
particles or particles are readily imaged using less sophisticated
CCD camera-based optical imaging techniques.
[0018] The term "shell portion" when used in reference to an assay
particle of the invention means a skin or thickness of material
surrounding or enveloping the core portion of the assay particle.
The composition of the shell portion generally is selected to be
compatible with the core portion of the assay particle. Thus, a
relatively non-porous material is useful for the shell when the
underlying core is a gas or liquid, whereas a non-porous or porous
material can be suitable when the underlying core is, for example,
a solid or matrix. The shell portion generally has a thickness of
about 1 nm to about 500 nm, such as from about 1 nm to about 200
nm. A thickness can be selected, for example, based on the
composition of the core, and to obtain an assay particle having a
particular physical characteristic such as weight, strength,
durability and the like. Exemplary materials for assay particle
shells include glass, ceramic, metal, metal oxides, alumina,
silica, and zirconia. Specific examples of shell materials are
hollow glass and ceramic microspheres, which typically have
relatively high strength to weight ratio. Commercially available
glass and ceramic micropheres range in density from 0.16 to 0.7
g/cm.sup.3, depending upon the specific product, with sizes
typically ranging from 15 to 200 .mu.m. Several varieties of
bubbles and microspheres are available commercially from 3M.TM.,
including Scotchlite.TM. Glass Bubbles, Scotchlite.TM. Glass
Bubbles Floated Series and Z-Light Spheres.TM. Ceramic
microspheres. These micropheres range in size from 20 to 60 .mu.m.
Procedures for preparing micropheres and assay particles based on
microspheres are described herein below.
[0019] The term "coat portion," when used in reference to an assay
particle means a layer of material that covers the surface of the
shell portion of the assay particle. A coat portion generally has a
thickness of less than 3000 .mu.m, such as less than 2000 .mu.m and
less than 1000 .mu.m, depending on the coating material. The coat
portion can be continuously or discontinuously present on the
surface of the shell potion of an assay particle. The texture of
the coat portion can vary from smooth to coarse, depending on the
materials used. For example, the coat portion can have a surface of
fine or coarse crystalline material, particulate material,
proteinacious material, gel material, organic material and the
like.
[0020] An assay particle described herein includes an "inorganic
phosphor," present in the shell portion or the coat portion. As
used herein, the term "phosphor" means a substance that emits light
when excited by radiation, such as ultraviolet light, electron
bombardment and electrical fields. A phosphor useful for an assay
particle described herein is capable of converting radiation from a
substance in a sample, such as an analyte or target molecule, into
light energy that can be detected using a photomultiplier tube, CCD
camera or the like. An inorganic phosphor useful for the assay
particles described herein is typically capable of emitting light
under conditions of a scintillation proximity assay (SPA).
[0021] An assay particle can also include a target selective
binding agent, which is a substance that interacts with a target
molecule but does not appreciably interact with other molecules in
a sample. A target selective binding agent is useful for bringing
an analyte or target molecule capable of emitting radiation into
proximity with an inorganic phosphor, such that the inorganic
phosphor absorbs radiation from the analyte or target molecule and
produces a detectable signal. A target selective binding agent can
bind with specificity to a general target species, such as a class
of antibodies, a class of post-translationally modified proteins
(for example, phosphomolecules and glycoproteins), and tags (for
example, myc, polyhistidine and FLAG tags), or bind with
specificity to a specific target species, such as a particular
polypeptide. Exemplary target selective binding agents include an
antibody, aptamer, protein A, protein G, streptavidin, avidin,
captavidin, neutravidin, metal chelate, siderophore, lectin, and an
oligonucleotide.
[0022] Examples of inorganic phosphors suitable for a shell or
coating of an assay particle described herein include yttrium
oxide, yttrium silicate, yttrium oxysulphide, yttrium aluminium
gallium oxide, yttrium aluminium garnet, sodium yttrium fluoride,
lanthanum fluoride, lanthanum oxysulphide, yttrium fluoride,
yttrium gallate, gadolinium fluoride, barium yttrium fluoride,
gadolinium oxysulphide, zinc silicate, zinc sulphide and yttrium
vanadate. An inorganic phosphor can include a rare-earth ion
dopant. Presence of a dopant in a hydrated metal oxide phosphor can
render to the material luminescent or shift the emission maximum of
the material. Non-limiting examples of metal oxides suitable for
assay particles described herein include yttrium oxide, zirconium
oxide, yttrium oxysulfide and yttrium aluminum garnet. Exemplary
rare ion dopants include terbium (III), europium (III), dysprosium
(III), samarium (III), ruthenium (II) and rhenium (I). A
combination of one or more dopants also can be used. A variety of
processes are suitable for applying an inorganic phosphor to a
selected shell material. An inorganic phosphor layer can be
deposited onto a ceramic or glass hollow microsphere, for example,
by liquid-phase deposition, chemical bath deposition, successive
ion layer adsorption and reaction (SILAR), electroless deposition,
reactive sputtering, reactive evaporation, spray pyrolysis,
track-etching, anodic oxidation, chemical vapor deposition, and
sol-gel processing. The deposited layer can be crystalline,
nanocrystalline, poorly crystallized or amorphous. In one
embodiment, an assay particle contains an inorganic phosphor
capable of selective binding to a target molecule. An example of
such an inorganic phosphor is a hydroxylated metal oxide. One
process for making a hydroxylated metal oxide layer is to form a
crystalline metal oxide layer on a particle shell or core, and
subsequently hydroxylate it. Hydroxylating can be achieved by
incubation in an aqueous-based medium until sufficient
hydroxylation has occurred to impart the phosphomolecule binding
property. The ability of the hydroxylated metal oxide layer to bind
phosphomolecules can be tested during the incubation, which
generally occurs over a period of one hour to several months,
depending on materials used. Deposition of a metal oxide can be
achieved on an ion-by-ion or particle attachment basis. When an
organic matrix or solid core is used, functionalization of an
organic material, especially with sulfonate, hydroxyl, amine or
carboxyl groups can improve depositing the inorganic phosphor.
[0023] An inorganic phosphor containing a hydrated metal oxide can
be deposited onto a core or shell by low-temperature synthesis of
thin films through direct deposition from aqueous-based solutions
(see for example Niesen and De Guire, 2001) as well as alternative
solvents, such as 2-propanol and blends of acetic acid, acetone and
water. When an organic material is used for the core or shell
portion, a solvent is selected to avoid damaging the organic
material. For example, deposition of a hydrated metal oxide onto an
organic material can occur near ambient temperature and in an
aqueous-based medium. Without wishing to be bound by theory, it
appears that heterogeneous nucleation is required for effective
coating with hydrated metal oxides. Under certain circumstances,
homogenous nucleation of supersaturated precursor metal ion
solutions predominates and precipitation results. Under other
conditions, the precursors form a metastable solution with very low
reaction rate. Heterogeneous nucleation occurs at intermediate
conditions between the metastable and supersaturated states. Often,
reaction conditions can be adjusted to maintain purely
heterogeneous nucleation and lessen homogeneous deposition, which
can result in unwanted excessive deposition on the core or shell.
Reaction conditions can be defined for a variety of suitable
starting materials by construction of a phase diagram of the
starting material concentration versus pH for a given temperature.
Once the reaction conditions for heterogeneous nucleation are
identified, the core (with or without a shell) is incubated in the
reaction solution for a period of about 0.5 to 260 hours. The
diameter of the coating gradually increases throughout the
deposition time course. Incubation time is usually adjusted to
achieve layer thicknesses that are 200 nm or less, as thicker
layers may have a tendency to peel off the substrate after
deposition. Particle sizes in the coatings typically range from a
few nanometers to a few tens of nanometers. Optionally, inclusion
of a surfactant, into the reaction solution, while suppressing the
growth of crystals somewhat, also reduces the propensity of
cracking or crazing, thus making a more durable coating. Exemplary
surfactants that can be included in a reaction mixture include
sodium dodecyl sulfate, lithium dodecyl sulfate, sodium
bis-2-ethylhexylsulphosuccinate, sodium cholate, perfluordecyl
bromide, cetyltrimethylammonium bromide, didodecylamonium bromide,
Triton X-100, polyoxyethylene 10-oleyl ether,
polyoxyethylene-10-dodecyl ether, N,N-dimethyldodecylamine-N-oxide,
Brij 35, Tween-20, Tween-80, sorbitan monooleate, lecithin,
diacylphosphatidylcholine, sucrose monolaurate and sucrose
dilaurate. Deposition of the hydrated metal oxides can occur more
readily upon hydrophilic materials, in comparison to hydrophobic
materials.
[0024] Metal oxide or hydroxide coatings also be formed through the
ligand-exchange (hydrolysis) equilibrium reaction of metal-fluoro
complex ionic species and a fluoride consumption reaction using
boric acid or aluminum metal (Niesen and De Guire, 2001). With the
technique, coatings can be formed on a variety of organic cores and
shells by immersion into the reaction solution. Multicomponent
layers containing several metals (eg europium-doped yttrium oxide
phosphor, yttrium oxysulfide phosphor, europium-doped spinel
phosphor, europium-doped gadolinium oxide phosphor, europium-doped
zirconia phosphor) also can be produced (Niesen and De Guire,
2001).
[0025] A metal oxide inorganic phosphor can also be coated onto a
ceramic core or shell by mixing of metal oxide powder with a
solution containing a binder, such as polyvinyl alcohol,
polyethylene glycol, polyethyleneimine, poly(dimethylsiloxane),
hydroxypropylcellose or polyacrylamide. Further well known
deposition methods include reactive sputtering, reactive
evaporation, spray pyrolysis, track-etching, anodic oxidation,
cold-pressed molding, chemical vapor deposition and sol-gel
processing. In general, these methods involve heating at
temperatures above 400 degrees centigrade, to obtain sufficient
crystallinity.
[0026] A metal oxide inorganic phosphor shell or coating portion of
an assay particle can have a variety of textures, for example, the
surface can be nano-porous, include ultrafine crystallites or be
poorly crystallized. When it is desired for the inorganic phosphor
to be capable of binding to phosphorylated molecules, the metal
oxide surface contains hydroxide groups. Nevertheless, processes
for preparing metal oxide coatings that result in reduced
hydroxylation of the metal oxide surface can be used (for example,
sintering), so long as the hydroxylated surface is regenerated
after process. Hydroxyl groups can be regenerated, for example, by
incubating the metal oxides in an aqueous environment.
[0027] Optimal interaction of phosphorylated molecules with
inorganic phosphor scintillants depends upon the interaction
between the hydrated oxide and the phosphate ion. In aqueous-based
media the predominant surface functional group on metal oxides is
the hydroxyl group. Without wishing to be bound by theory, it
appears that at the proper pH value the surface hydroxyl groups
are, in one embodiment, polarized and electrically charged to allow
interaction with phosphorylated molecules. The oxide surface
adsorbs and/or desorbs protons from solution, thus influencing the
surface charge. This induces electrostatic effects in the vicinity
of the charged surface, which directly impacts the capacity of the
material for sorption of different ionic species from the
aqueous-based media. At low pH values the surface charge becomes
positive, while at high pH values it becomes negative. The pH value
at which the particles possess no surface charge is referred to as
the isoelectric point or pH of zero zeta potential. The loss or
gain of protons is commonly considered as an acid-base reaction at
the metal oxide surface. A variety of different surface hydroxyl
groups can be present on a metal oxide surface. When a surface
hydroxyl group is coordinated to a single metal atom, it is
referred to as a singly coordinated or terminal hydroxyl group,
whereas if the hydroxyl group is coordinated to two, three or four
metal atoms, it is referred to as a bridging hydroxyl group. For
iron oxides, the surface hydroxyl groups may be coordinated to one,
two or even three underlying metal atoms. It is also possible for
two surface hydroxyl groups to be bound to a single metal atom. The
configurations of the different types of surface groups depend upon
the structure of the oxide and the crystal face being examined,
with different surface groups likely to display different chemical
properties. Not all of the different types of surface groups are
active in the titratable pH-range required for capture of
phosphorylated molecules. Overall, adsorption of phosphorylated
molecules is governed by a set of complex formation reactions
between the dissolved phosphorylated solute and the titratable
surface functional groups of the hydrated metal oxide surface. The
interface between metal oxide and water is surprisingly complex,
involving what has been dubbed "wet-electron" states (Onda et al,
2005). Despite this complexity, the inventor has determined that
certain base materials for inorganic phosphors, specifically
yttrium oxide and yttrium aluminum garnet, when hydrated, display
specific, high affinity for phosphorylated compounds. In the case
of titania surfaces, protonation and consequently positive charge
attributes are achieved below the material's isoelectric point of
approximately 6.0. Other metal oxide surfaces differ in the
isoelectric point that is conducive to the generation of a proper
surface for affinity capture of phosphorylated molecules. For
example, highly hydrated zirconia possesses an isoelectric point of
8.2, while hematite, yttrium oxide, and gibbsite possess
isoelectric point values of 7.5, 8.5, and 10.0, respectively.
Inclusion of certain alkali metals in the media can shift the
isoelectric point of hydrated metal oxide particles to higher pH
values, as observed with rutile particles incubated with barium,
calcium or magnesium salts. It is likely this is due to the
precipitation of hydrous metal oxides onto the surface of the
particles. Pure silica has an isoelectric point value of roughly
1.8, rendering the material unsatisfactory for the present methods
and systems. However, the coating of silica with an appropriate
hydrated metal oxide can provide a usable surface for binding
phosphorylated molecules. It should also be noted that the
isoelectric point of hydrated metal oxides depends somewhat upon
the preparation method, trace impurities and the degree of
hydration, among other factors. Overall, metal oxides of the
general formula Me.sub.2O.sub.3 tend to have isoelectric point
values of about 9.0, regardless of the metal ion incorporated in
the structure. On the other hand metal oxides of the general
formula MeO.sub.2 display isoelectric point values that increase
with the ionic radius of the metal atom and when the
electronegativity of the metal atom decreases. Collectively all
these factors can be valuable in optimizing and fine-tuning
affinity-based detection of phosphorylated molecules using SPA.
[0028] The isoelectric point of the hydrated metal oxide
effectively establishes an upper limit for the pH of binding
buffers employed in affinity-based detection of phosphorylated
molecules. When using particles comprising amorphous or weakly
crystallized hydrated metal oxide, strongly acidic (pH<3.0) and
strongly basic (pH>11.0) solutions are typically avoided due to
chemical instability of the hydrated metal oxide surface under
these conditions.
[0029] Assay particles can also be prepared using layered precursor
deposition on sacrificial organic colloidal core particles. The
organic template is used for controlled structuring of inorganic
materials by nanocasting or nanocoating (Caruso, F. (2003); Caruso,
R (2003)). The difference between the two techniques is that
casting is a filling of the porous structure of the organic
material, while coating results in a layer of the inorganic
substance on the polymer structure. One approach to generating
assay particles uses a layer-by-layer assembly process for the
creation of coated particles (core-shell colloids) which are
subsequently converted to hollow inorganic particles. Sacrificial
core template particles are coated with multiple layers of
preformed inorganic nanoparticles, or inorganic molecular
precursors, and oppositely charged polyelectrolyte, using
electrostatic attraction for construction of the layers on the
particles. Calcination of the core-shell nanocomposite particles
yields hollow inorganic particles of defined size and composition.
The wall thickness can be controlled with nanoscale precision
through the number of layers formed on the organic particles.
[0030] As is described herein, certain inorganic phosphors have the
ability to bind selectively to target molecules. Therefore, in one
embodiment, an assay particle of the invention includes an
inorganic phosphor capable of binding selectively to a target
molecule. A target specific binding agent can be used with an
inorganic phosphor capable of binding to a target molecule when
binding of additional or different target molecules to an assay
particle is desired. One or more target selective binding agents
can be present in one or both the coat portion of the assay
particle and an outer layer on the coat portion. The term "outer
layer" as used herein means a film, covering or deposition of a
material onto the surface of the coat portion of an assay particle.
The outer layer can be a continuous or discontinuous coating, film
or deposition containing the target selective binding agent.
[0031] An assay particle can have a shape and size suited for its
use. Typical assay particle shapes include spherical, cylindrical
and irregular shapes. An assay particle described herein generally
has a size of about 0.1 to about 10,000 um, such as about 1 .mu.m
to about 2000 um, about 1.5 to about 500 .mu.m in size, and about
10 .mu.m to about 100 .mu.m in size. In one aspect, the technology
described herein provides assay particles of size sufficiently
small for dispensing in micro- or nanoliter volumes. The particular
size selected will depend on equipment used for microdispensing,
for example, for dispensing into 96-, 384- and 1536-well
plates.
[0032] An assay particle described herein can also contain an
"encoding element" that contains or imparts information, such as
information about the particle, a set of particles, a sample, an
analyte, a target selective binding agent and the like. The
encoding element can be a physical element such as a radio
frequency identifier, holographic or other identifier, or a
chemical element such as one or more luminescent materials, for
example, fluorophors and mixtures thereof, quantum dots and the
like. A variety of well-recognized encoding systems are known to
those skilled in the art, and can be adapted to the assay particles
described herein. For example, Chandler et al. (U.S. Pat. No.
5,981,180) describes a particle-based system in which different
bead types are encoded by mixtures of various proportions of two or
more fluorescent dyes impregnated into polymer particles; Soini
(U.S. Pat. No. 5,028,545) describes a particle-based multiplexed
assay system that employs time-resolved fluorescence for particle
identification; Fulwyler (U.S. Pat. No. 4,499,052) describes using
beads distinguished by color and/or size; and Moon and Putnam
(2004-0179267, 2004-0132205, 2004-0130786, 2004-0130761,
2004-0126875, 2004-0125424, and 2004-0075907) describe particles
encoded by holographic barcodes. Such encoding elements can be
located in any portion of the assay particle, including the shell
portion, coating portion and outer layer. Use of encoding elements
in assay particles described herein can be useful for performed
multiplexed assays. Therefore, provided by the technology are
multiplexed assays using assay particles of the invention. In these
assays, a plurality of differently encoded assay particles is
simultaneously assayed in a method described herein.
[0033] As a specific example, one type of assay particle of the
invention is an encoded particle (i.e., a particle containing an
encoding element) that has an inorganic phosphor deposited on its
surface. A plurality of assay particles with different encoding
elements can be used for measuring different analytes. For example,
particles can be dyed with differing concentrations of two
fluorophores to generate distinct particle sets, as is performed
with Luminex beads. Each bead set is coated with a layer of
inorganic phosphor and then a target specific binding agent
specific for one particular analyte. The amount of captured analyte
is detected based upon the magnitude of the scintillation signal of
the inorganic phosphor coating, which is in direct proportion to
the amount of analyte bound. The identity of the analyte is
determined from the characteristic fluorescence properties of the
core particle itself, as determined based upon color ratios. As
another example, it is possible to classify the assay particles
based upon a variety of other optical properties, such as their
size or shape. As a further example, an organic solvent containing
different ratios of fluorophore can be entrapped in inorganic
phosphor coated assay particles to achieve similar objectives.
[0034] An assay particle as described herein can be prepared using
a variety of known methods, which will vary depending on the
particular materials selected for the core, shell and optional coat
portions of the assay particle. In some cases a core will be
prepared and encased within a shell, while in other cases the shell
and core portions will form in the same process. For example,
formation of a shell portion which is a glass microsphere will
inherently result in formation of a core portion that is typically
a gas.
[0035] In one embodiment, the shell and core of the assay particle
are the body and inside of a hollow microsphere, respectively.
Microspheres also can be synthesized by well-known techniques such
as emulsion-ion extraction techniques, sol-emulsion-gel synthesis,
or emulsion templating. The microspheres can be coated with an
inorganic phosphor, as is described in detail herein, or can be
generated directly from an inorganic phosphor. Commercial
microspheres, such as those made from glass and ceramic, also can
be used to form an assay particle of the invention. For crude
microspheres, a variety of methods, such as physical screen
separations, are suitable for further sizing the material, if
desired.
[0036] The technology provided herein includes methods for
detecting analytes and assaying enzyme activities. The methods are
applicable to SPA assays, fluorescence resonance energy transfer
assays, fluorescence polarization assays and other formats that
involve the generation of a light signal upon binding of a molecule
having a moiety capable of emitting radiation to an assay particle
having an inorganic phosphor surface.
[0037] In one embodiment, the technology described herein provides
a method for detecting a target molecule. The method involves
contacting a sample suspected of containing the target molecule
with an assay particle, wherein (i) the target molecule comprises a
moiety capable of emitting radiation, and (ii) the assay particle
comprises a shell portion comprising an inorganic phosphor capable
of binding selectively to target molecules, wherein binding of the
target molecule to the inorganic phosphor produces a light signal,
whereby the target molecule is detected.
[0038] In another embodiment, a method for detecting a target
molecule involves contacting a sample suspected of containing the
target molecule with an assay particle, wherein (i) the target
molecule comprises a moiety capable of emitting radiation, and (ii)
the assay particle comprises a coat portion comprising an inorganic
phosphor capable of binding selectively to target molecules;
wherein binding of the target molecule to the inorganic phosphor
produces a light signal, whereby the target molecule is
detected.
[0039] In a further embodiment, a method for detecting a target
molecule involves contacting a sample suspected of containing the
target molecule with an assay particle, wherein (i) the target
molecule comprises a moiety capable of emitting radiation, and (ii)
the assay particle comprises a coat portion comprising an inorganic
phosphor and a target selective binding agent, and is buoyant in
aqueous media; wherein binding of the target molecule to the target
selective binding agent activates the inorganic phosphor to produce
a light signal, whereby the target molecule is detected.
[0040] As used herein the term "moiety capable of emitted
radiation" means a chemical label or modification that emits
radiation sufficient to activate an inorganic phosphor to emit a
light signal. Non-limiting examples of such moieties include
radioactive labels and luminescent labels such as fluorescent
labels, phosphorescent labels, and chemilluminescent labels.
Exemplary radioactive labels include .sup.3H, .sup.125I, .sup.14C,
.sup.35S .sup.32P, .sup.55Fe, .sup.86Rb, .sup.109Cd and .sup.51Cr
and .sup.33P. Exemplary luminescent labels include fluorescent
labels such as cyanine-5 (PerkinElmer), cyanine-3 (PerkinElmer,
AlexaFluor 647 (Invitrogen), Quasar 670 (Bioserach Technologies),
DY 630 (Dyomics), HiLyte Fluor (Anaspec) or various lanthanide
chelates. A variety of fluorescent labels are described, for
example, in Handbook of Fluorescent Probes and Research Products,
Ninth Edition by Dr. Richard P. Haugland (Molecular Probes,
2003).
[0041] A variety of samples can be used for carrying out the
methods described herein. A sample can, for example, be a
biological sample, environmental sample, experimental sample,
diagnostic sample, or any other type of sample that contains or is
suspected to contain a target molecule of interest. In a biological
context, a sample can be prepared from or include biological
fluids, whole organisms, organs, tissues, cells, microorganisms,
culture supernatants, subcellular organelles, protein complexes,
individual proteins, recombinant proteins, fusion proteins,
viruses, viral particles, peptides and amino acids. A sample can be
processed to preserve or stabilize molecules of interest, such as
enzymes and phosphorylated molecules. Methods for preserving the
integrity of molecules in a sample are well known to those skilled
in the art. Such methods include the use of appropriate buffers
and/or inhibitors, including nuclease, protease and phosphatase
inhibitors that preserve or minimize changes in the molecules in
the sample. Such inhibitors include, for example, chelators such as
ethylenediamne tetraacetic acid (EDTA), ethylene glycol
bis(p-aminoethyl ether)N,N,N.sup.1,N.sup.1-tetraacetic acid (EGTA),
protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF),
aprotinin, leupeptin, antipain and the like, and phosphatase
inhibitors such as phosphate, sodium fluoride, vanadate and the
like. Appropriate buffers and conditions for allowing selective
interactions between molecules are well known to those skilled in
the art and can be varied depending, for example, on the type of
molecule in the sample to be characterized (see, for example,
Ausubel et al., Current Protocols in Molecular Biology (Supplement
47), John Wiley & Sons, New York (1999); Harlow and Lane, Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999);
Tietz Textbook of Clinical Chemistry, 3rd ed., Burtis and Ashwood,
eds., W. B. Saunders, Philadelphia, (1999)).
[0042] In an embodiment, the assay particles provided herein have
shell or coat portions having a surface of inorganic phosphor
capable of binding selectively to target molecules. In another
embodiment, an assay particle of the invention includes an
inorganic phosphor and a target selective binding agent. In a
particular embodiment, the shell or coat portions include a
hydrated metal oxide capable of binding to phosphorylated
molecules. A specific example of such a metal oxide is titanium
dioxide, as is described in Example 2. The ability of an inorganic
phosphor to bind to a target molecule, such as a molecule that has
been phosphorylated or otherwise modified, can be tested
empirically. Therefore, the technology described herein provides
methods for detecting phosphorylated molecules and methods for
assaying enzymes. Exemplary enzymes that can be assayed based on
levels of target molecules or substrates in a sample include
enzymes that alter phosphorylation states of their substrates, for
example, kinases and phosphatases, and enzymes that catalyze
cyclization and decyclyzation of nucleotide monophosphates, such as
cyclases and phosphodiesterases. The methods described herein are
applicable to assaying a variety of target molecules, protein
interactions, enzyme and receptor activities and the like.
[0043] An example of a protein kinase SPA assay is depicted in FIG.
3. In this example, a protein kinase present in the sample
phosphorylates a substrate peptide. The substrate peptide contains
a tritium label, which emits radiation whether or not it is
phosphorylated. A substrate peptide that becomes phosphorylated
will have an affinity for an assay particle coated with an
inorganic phosphor that binds selectively to phosphorylated
molecules, such as europium-doped yttrium oxide. A phosphorylated
substrate peptide that binds to an assay particle will activate the
particle to produce a light signal. This activation occurs because
the tritium label emits a beta particle(s) that are in turn
absorbed by the inorganic phosphor on the assay particle. As a
result of absorbing such radiation, the inorganic phosphor emits a
photon of light. In the case of europium-doped yttrium oxide, the
light is emitted at 615 nm and correlates with the amount of
phosphorylated peptide. The light signal is detectable using a
variety of devices, such as scintillation counters and CCD camera
imagers.
[0044] Another example of a protein kinase SPA assay is depicted in
FIG. 4. The depicted format involves using time-resolved
fluorescence to detect phosphorylated substrate. In the case of
homogenous time-resolved fluorescence assays, the energy donor is
the rare earth dopant in the inorganic phosphor, rather than
radioactivity. The energy acceptor is a fluorophore whose
excitation profile overlaps the emission profile of the dopant in
the inorganic phosphor. Binding events are detected as emission of
the longer wavelength fluorophore upon excitation of the shorter
wavelength emitting phosphor with mid-range ultraviolet radiation.
In this example, a protein kinase present in the sample
phosphorylates a substrate peptide. The substrate peptide contains
a cyanine 5 dye label, which can be excited by light to emit
radiation. A substrate peptide that becomes phosphorylated will
have an affinity for an assay particle coated with an inorganic
phosphor that binds selectively to phosphorylated molecules, such
as europium-doped yttrium oxide. Upon illumination with short
wavelength UV light (.about.337 nm) fluorescence resonance energy
transfer occurs between the inorganic phosphor coating and the
cyanine 5 dye label, resulting in long-lived, 665-nm photon
emission that correlates with the amount of phosphorylated peptide
product formed. Fluorescence resonance energy transfer occurs if
the cyanine 5 dye label on the substrate peptide is in close
proximity with the inorganic phosphor. Binding of the substrate
peptide to the assay particle brings it into sufficient proximity
for energy transfer to occur, whereas substrate peptides unbound to
the assay particle lack such proximity. The light signal is
detectable using a variety of devices including fluorescence
readers and CCD camera imagers.
[0045] In addition to SPA assays and fluorescence resonance energy
transfer assays, fluorescence polarization assays also can be
carried out using the methods described herein. To use a
fluorescence polarization assay format, the assay particles are
added to the kinase reaction along with a fluorescently-labeled
peptide substrate. When phosphorylated by a protein kinase, the
fluorophore-derivatized peptide substrate binds to the assay
particle. This binding event results in the rotation of the
fluorescent phosphorylated substrate being decreased, resulting in
greater fluorescence polarization of the emitted light.
[0046] The invention provides methods for detecting a target
molecule using an assay particle described herein. In one aspect, a
method involves contacting a sample suspected of containing the
target molecule with an assay particle, wherein (i) the target
molecule comprises a moiety capable of emitting radiation, and (ii)
the assay particle comprises a shell portion comprising an
inorganic phosphor capable of binding selectively to target
molecules; wherein binding of the target molecule to the inorganic
phosphor produces a light signal, whereby the target molecule is
detected. In another aspect, a method for detecting a target
molecule involves contacting a sample suspected of containing the
target molecule with an assay particle, wherein (i) the target
molecule comprises a moiety capable of emitting radiation, and (ii)
the assay particle comprises a coat portion comprising an inorganic
phosphor capable of binding selectively to target molecules;
wherein binding of the target molecule to the inorganic phosphor
produces a light signal, whereby the target molecule is detected.
In another aspect, a method for detecting a target molecule
involves contacting a sample suspected of containing the target
molecule with an assay particle, wherein (i) the target molecule
comprises a moiety capable of emitting radiation, and (ii) the
assay particle comprises a coat portion comprising an inorganic
phosphor and a target selective binding agent, and is buoyant in
aqueous media; wherein binding of the target molecule to the target
selective binding agent activates the inorganic phosphor to produce
a light signal, whereby the target molecule is detected. Similar
methods can be used for an assay particle that includes a core
portion encased by a shell portion, and a coat portion covering the
shell portion, wherein the shell portion comprises an inorganic
phosphor and the coat portion comprises a target selective binding
moiety, and wherein the assay particle is buoyant in aqueous
media.
[0047] The invention provides methods for detecting protein kinase
activity using an assay particle described herein. In one aspect, a
method involves contacting a sample containing a protein kinase
with an assay particle, wherein the assay particle comprises a
shell portion comprising an inorganic phosphor that is capable of
binding selectively to phosphorylated molecules, and a protein
kinase substrate comprising a moiety capable of emitting radiation,
under conditions wherein the protein kinase can phosphorylatethe
substrate to produce a phosphorylated substrate, wherein binding of
the phosphorylated substrate to the inorganic phosphor produces a
light signal, and determining a protein kinase activity based on a
level of detected phosphorylated substrate. In another aspect, a
method for detecting protein kinase activity involves contacting a
sample containing a protein kinase with an assay particle, wherein
the assay particle comprises a coat portion comprising an inorganic
phosphor that is capable of binding selectively to phosphorylated
molecules, and a protein kinase substrate comprising a moiety
capable of emitting radiation, under conditions wherein the protein
kinase can phosphorylatethe substrate to produce a phosphorylated
substrate, wherein binding of the phosphorylated substrate to the
inorganic phosphor produces a light signal, and determining a
protein kinase activity based on a level of detected phosphorylated
substrate. In another aspect, a method for detecting protein kinase
activity involves contacting a sample containing a protein kinase
with (i) an assay particle, wherein the assay particle comprises a
coat portion comprising an inorganic phosphor and a binding agent
selective for phosphorylated molecules, and is buoyant in aqueous
media; and (ii) a protein kinase substrate comprising a moiety
capable of emitting radiation, under conditions wherein the protein
kinase can phosphorylatethe substrate to produce a phosphorylated
substrate, wherein binding of the phosphorylated substrate to the
inorganic phosphor produces a light signal, and determining a
protein kinase activity based on a level of detected phosphorylated
substrate. Similar methods can be used for an assay particle that
includes a core portion encased by a shell portion, and a coat
portion covering the shell portion, wherein the shell portion
comprises an inorganic phosphor and the coat portion comprises a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media.
[0048] The invention provides methods for detecting protein
phosphatase activity using an assay particle described herein. In
one aspect, a method involves contacting a sample containing a
protein phosphatase with an assay particle, wherein the assay
particle comprises a shell portion comprising an inorganic phosphor
that is capable of binding selectively to phosphorylated molecules
and a protein phosphatase substrate comprising a moiety capable of
emitting radiation is bound to the inorganic phosphor, under
conditions wherein the protein phosphatase can dephosphorylate the
substrate to produce a dephosphorylated substrate, wherein
phosphorylated substrate bound to the inorganic phosphor produces a
light signal, and determining a phosphatase activity based on a
level of detected phosphorylated substrate. In another aspect, a
method for detecting protein phosphatase activity involves
contacting a sample containing a protein kinase with an assay
particle, wherein the assay particle comprises a coat portion
comprising an inorganic phosphor that is capable of binding
selectively to phosphorylated molecules and wherein a protein
phophatase substrate comprising a moiety that is capable of
emitting radiation is bound to the inorganic phosphor, under
conditions wherein the protein phosphatase can dephosphorylate the
substrate to produce a dephosphorylated substrate, wherein
phosphorylated substrate bound to the inorganic phosphor produces a
light signal, and determining a phosphatase activity based on a
level of detected phosphorylated substrate. In another aspect, a
method for detecting protein phosphatase activity involves
contacting a sample containing a protein phosphatase with an assay
particle, wherein the assay particle comprises a coat portion
comprising an inorganic phosphor and a binding agent selective for
phosphorylated molecules, and wherein a protein phosphatase
substrate comprising a moiety capable of emitting radiation is
bound to the inorganic phosphor, under conditions wherein the
protein phosphatase can dephosphorylate the substrate to produce a
dephosphorylated substrate, wherein phosphorylated substrate bound
to the inorganic phosphor produces a light signal, and determining
a phosphatase activity based on a level of detected phosphorylated
substrate. Similar methods can be used for an assay particle that
includes a core portion encased by a shell portion, and a coat
portion covering the shell portion, wherein the shell portion
comprises an inorganic phosphor and the coat portion comprises a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media.
[0049] The invention provides method for identifying a protein
kinase modulator using an assay particle described herein. In one
aspect, a method involves contacting a sample containing a protein
kinase with (i) an assay particle comprising a shell portion
comprising an inorganic phosphor that is capable of binding
selectively to phosphorylated molecules; and (ii) a substrate
comprising a moiety capable of emitting radiation, in the presence
and absence of a candidate compound, under conditions wherein the
protein kinase can phosphorylate the substrate to produce a
phosphorylated substrate, wherein binding of the phosphorylated
substrate to the inorganic phosphor produces a light signal; and
detecting light signals produced in the presence and absence of the
candidate compound, wherein a difference in light signals produced
in the presence and absence of the compound identifies the compound
as a protein kinase modulator. In another aspect, a method for
identifying a protein kinase modulator involves contacting a sample
containing a protein kinase with (i) an assay particle comprising a
coat portion comprising an inorganic phosphor that is capable of
binding selectively to phosphorylated molecules; and (ii) a
substrate capable of emitting radiation, in the presence and
absence of a candidate compound, under conditions wherein the
protein kinase can phosphorylate the substrate to produce a
phosphorylated substrate, wherein binding of the phosphorylated
substrate to the inorganic phosphor produces a light signal; and
detecting light signals produced in the presence and absence of the
candidate compound, wherein a difference in light signals produced
in the presence and absence of the compound identifies the compound
as a protein kinase modulator. In another aspect, a method for
identifying a protein kinase modulator involves contacting a sample
containing a protein kinase with (i) an assay particle, wherein the
assay particle comprises a coat portion comprising an inorganic
phosphor and a binding agent selective for phosphorylated
molecules; and (ii) a substrate comprising a moiety capable of
emitting radiation, in the presence and absence of a candidate
compound, under conditions wherein the protein kinase can
phosphorylate the substrate to produce a phosphorylated substrate,
wherein binding of the phosphorylated substrate to the inorganic
phosphor produces a light signal; and detecting light signals
produced in the presence and absence of the candidate compound,
wherein a difference in light signals produced in the presence and
absence of the compound identifies the compound as a protein kinase
modulator. Similar methods can be used for an assay particle that
includes a core portion encased by a shell portion, and a coat
portion covering the shell portion, wherein the shell portion
comprises an inorganic phosphor and the coat portion comprises a
target selective binding moiety, and wherein the assay particle is
buoyant in aqueous media. Similar methods can be used for an assay
particle that includes a core portion encased by a shell portion,
and a coat portion covering the shell portion, wherein the shell
portion comprises an inorganic phosphor and the coat portion
comprises a target selective binding moiety, and wherein the assay
particle is buoyant in aqueous media.
[0050] The invention provides methods for identifying a cyclase
modulator using an assay particle described herein. In one aspect,
a method involves contacting a sample containing a cyclase with (i)
an assay particle comprising a shell portion comprising an
inorganic phosphor that is capable of binding selectively to
non-cyclized nucleotides; and (ii) radioactively labeled
non-cyclized nucleotide, in the presence and absence of a candidate
compound, under conditions wherein the cyclase can acts on the
substrate to produce a cyclized nucleotide, wherein binding of the
non-cyclized nucleotide to the inorganic phosphor produces a light
signal; and detecting light signals produced in the presence and
absence of the candidate compound, wherein a difference in light
signals produced in the presence and absence of the compound
identifies the compound as a cyclase modulator. In another aspect a
method for identifying a cyclase modulator involves contacting a
sample containing a cyclase with (i) an assay particle comprising a
coat portion comprising an inorganic phosphor that is capable of
binding selectively to non-cyclized nucleotides; and (ii)
radioactively labeled non-cyclized nucleotide, in the presence and
absence of a candidate compound, under conditions wherein the
cyclase can acts on the substrate to produce cyclized nucleotides,
wherein binding of the non-cyclized nucleotide to the inorganic
phosphor produces a light signal; and detecting light signals
produced in the presence and absence of the candidate compound,
wherein a difference in light signals produced in the presence and
absence of the compound identifies the compound as a cyclase
modulator. In another aspect, a method for identifying a cyclase
modulator, comprising contacting a sample containing a cyclase with
(i) an assay particle, wherein the assay particle comprises a coat
portion comprising an inorganic phosphor and a binding agent
selective for non-cyclized nucleotides ; and (ii) radioactively
labeled non-cyclized nucleotide, in the presence and absence of a
candidate compound, under conditions wherein the cyclase can acts
on the substrate to produce cyclized nucleotides, wherein binding
of the non-cyclized nucleotide to the inorganic phosphor produces a
light signal; and detecting light signals produced in the presence
and absence of the candidate compound, wherein a difference in
light signals produced in the presence and absence of the compound
identifies the compound as a cyclase modulator. Similar methods can
be used for an assay particle that includes a core portion encased
by a shell portion, and a coat portion covering the shell portion,
wherein the shell portion comprises an inorganic phosphor and the
coat portion comprises a target selective binding moiety, and
wherein the assay particle is buoyant in aqueous media.
[0051] The invention provides methods for identifying a
phosphodiesterase modulator using an assay particle described
herein. In one aspect, a method involves contacting a sample
containing a phosphodiesterase with (i) an assay particle
comprising a shell portion comprising an inorganic phosphor that is
capable of binding selectively to non-cyclized nucleotides; and
(ii) radioactively labeled cyclized nucleotide, in the presence and
absence of a candidate compound, under conditions wherein the
phosphodiesterase can acts on the substrate to produce non-cyclized
nucleotides, wherein binding of the non-cyclized nucleotide to the
inorganic phosphor produces a light signal; and detecting light
signals produced in the presence and absence of the candidate
compound, wherein a difference in light signals produced in the
presence and absence of the compound identifies the compound as a
phosphodiesterase modulator. In another aspect a method for
identifying a phosphodiesterase modulator involves contacting a
sample containing a phosphodiesterase with (i) an assay particle
comprising a coat portion comprising an inorganic phosphor that is
capable of binding selectively to non-cyclized nucleotides; and
(ii) radioactively labeled cyclized nucleotide, in the presence and
absence of a candidate compound, under conditions wherein the
phosphodiesterase can acts on the substrate to produce non-cyclized
nucleotides, wherein binding of the non-cyclized nucleotide to the
inorganic phosphor produces a light signal; and detecting light
signals produced in the presence and absence of the candidate
compound, wherein a difference in light signals produced in the
presence and absence of the compound identifies the compound as a
phosphodiesterase modulator. In another aspect, a method for
identifying a phosphodiesterase modulator involves contacting a
sample containing a phosphodiesterase with (i) an assay particle,
wherein the assay particle comprises a coat portion comprising an
inorganic phosphor and a binding agent selective for non-cyclized
nucleotides; and (ii) radioactively labeled cyclized nucleotide, in
the presence and absence of a candidate compound, under conditions
wherein the phosphodiesterase can acts on the substrate to produce
non-cyclized nucleotides, wherein binding of the non-cyclized
nucleotide to the inorganic phosphor produces a light signal; and
detecting light signals produced in the presence and absence of the
candidate compound, wherein a difference in light signals produced
in the presence and absence of the compound identifies the compound
as a phosphodiesterase modulator. Similar methods can be used for
an assay particle that includes a core portion encased by a shell
portion, and a coat portion covering the shell portion, wherein the
shell portion comprises an inorganic phosphor and the coat portion
comprises a target selective binding moiety, and wherein the assay
particle is buoyant in aqueous media.
[0052] In some embodiments, the methods described herein involve
use of a substrate for a protein kinase and a substrate for a
phosphatase (that is, a phosphorylated substrate). Such substrates
are well known to those skilled in the art and are commercially
available for example, from ANASPEC, SIGMA, BIOMOL and others. In
some embodiments, the methods described herein involve use of
non-cyclized nucleotides. Procedures for tagging such molecules
with radiation emitting labels such as radioactive and fluorescent
labels are well known to those skilled in the art.
[0053] Examples of other analytes that can be assayed using the
methods described herein include antigens, antibodies, hormones,
metabolites, enzymes, proteins and drugs. Also, the assay format
can be any of a variety of generally recognized assay types,
including, for example, signal addition assays, in which a
radiolabelled donor is added to an inactive substrate bound to the
assay particle surface; signal removal assays, in which a
radiolabeled substrate, already linked to the assay particle
surface, is removed generally by the action of an enzyme, and
product capture assays, in which the radiolabeled component,
optionally in the presence of unlabelled sample component, is bound
to the target selective binding agent, by means of a specific
interaction between the component and the target selective binding
agent on the surface of the assay particle. It is understood that
an inorganic phosphor that binds selectively to a target molecule
is a type of target selective binding agent.
[0054] The methods described herein can be used for screening
candidate compounds for a modulator of an enzyme or a binding
molecule. As used herein, the term "candidate compound" refers to
any molecule that potentially acts as a modulator of a selected
enzyme or binding partner of a selected molecule. A modulator can
be determined to be, for example, an inhibitor, activator, agonist,
antagonist or ligand using the screening methods disclosed herein.
A candidate compound can be a naturally occurring macromolecule,
such as a polypeptide, nucleic acid, carbohydrate, lipid, or any
combination thereof. A candidate compound also can be a partially
or completely synthetic derivative, analog or mimetic of such a
macromolecule, or a small organic molecule prepared by
combinatorial chemistry methods. If desired in a particular assay
format, a candidate compound can be detectably labeled or attached
to a solid support.
[0055] Methods for preparing libraries of compounds, including
simple and complex organic molecules, metal-containing compounds,
carbohydrates, peptides, proteins, peptidomimetics, glycoproteins,
lipoproteins, nucleic acids, antibodies, and the like, are well
known in the art and are described, for example, in Huse, U.S. Pat.
No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428
(1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, Mol.
Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496
(1995); and the like. Libraries containing large numbers of natural
and synthetic compounds also can be obtained from commercial
sources.
[0056] The number of different candidate compounds to test will
depend on the application of the method. For example, one or a
small number of candidate compounds are often used in manual
screening procedures, or when it is desired to compare efficacy
among several predicted ligands, agonists or antagonists. However,
it is generally understood that the larger the number of candidate
compounds, the greater the likelihood of identifying a compound
having the desired activity in a screening assay. Additionally,
large numbers of compounds can be processed in high-throughput
automated screening assays. More than one compound can be screened
in a sample, if desired. Screening of compounds also can be
performed by single compound assays run in parallel.
[0057] The technology herein provides a kit containing an assay
particle described herein. The kit also can contain a substrate for
an enzyme which is labeled to be capable of emitting radiation.
Examples of such kits include kits for assaying a kinase,
phosphatase, and cyclase. A kit provided by the invention can
contain a variety of components in addition to assay particles. A
package can contain, for example, instructions for using a assay
particles, a recommendation regarding the concentration of sample
for use in a particular application, as well as guidance regarding
temperature, buffer conditions and incubation time periods. A kit
can optionally can contain other components, such as one or more of
standards, substrates, target selective binding agents, coating
materials, and assay particles to receive preparation by the user
prior to beginning an assay. Those skilled in the art will be able
to select suitable components for inclusion in a kit or other
commercial package of the invention based on such exemplary factors
as design of the assay protocol, the particular inorganic phosphor
and radiation emitting component used for performing an assay,
method of detection or measurement to be employed once the assay
has been performed, consumer price point, shipping and handling
suitability and the like.
[0058] A kit provided by the technology described herein can
include a core portion encased by a shell portion, wherein the
shell portion comprises an inorganic phosphor that binds
selectively to a target molecule, and an enzyme substrate
comprising a moiety capable of emitting radiation.
[0059] In another aspect, a kit provided by the technology
described herein can include assay particle, comprising a core
portion encased by a shell portion, and a coat portion covering the
shell portion, wherein the coat portion comprises an inorganic
phosphor that binds selectively to a target molecule, and an enzyme
substrate capable of emitting radiation.
[0060] In a further aspect a kit provided by the technology
described herein can include assay particle, comprising a core
portion encased by a shell portion, and a coat portion covering the
shell portion, wherein the coat portion comprises an inorganic
phosphor and a target selective binding moiety, and wherein the
assay particle is buoyant in aqueous media, and an enzyme substrate
capable of emitting radiation.
[0061] In yet another aspect, a kit provided by the technology
described herein can include an assay particle, comprising a core
portion encased by a shell portion, and a coat portion covering the
shell portion, wherein the shell portion comprises an inorganic
phosphor and the coat portion comprises a target selective binding
moiety, and wherein the assay particle is buoyant in aqueous media,
and an enzyme substrate capable of emitting radiation.
[0062] Although specific embodiments have been illustrated and
described herein, it will be appreciated by those skilled in the
art that any arrangement which is calculated to achieve the same
purpose may be substituted for the specific embodiments shown. This
application is intended to cover any adaptations or variations of
the present invention.
[0063] A kit can be prepared for assayed any of a variety of
anaytes, enzymes and molecular interactions. Examples of specific
kits include kits for assaying a protein kinase, which can include
assay particles that bind selectively to phosphorylated molecules
and a kinase substrate that is radioactively or fluorescently
labeled, for example, a tritium-labeled peptide substrate; kits for
assaying a protein phosphatase, which can include assay particles
that bind selectively to phosphorylated molecules and a phosphatase
substrate that is radioactively or fluorescently labeled; kits for
assaying a cyclase, which can include assay particles that bind
selectively to non-cyclized nucleotides, such as AMP and GMP, and
non-cyclized nucleotides that are radioactively labeled; and kits
for assaying a phosphodiesterase, which can include assay particles
that bind selectively to non-cyclized nucleotides, such as AMP and
GMP, and cyclized nucleotides that are radioactively labeled.
REFERENCES
[0064] Bertoglio-Matte J H Immediate ligand detection assay. U.S.
Pat. No. 4,568.649, Feb. 4, 1986.
[0065] Brandish P E, Hill L A, Zheng W, Scolnick E M. Scintillation
proximity assay of inositol phosphates in cell extracts:
high-throughput measurement of G-protein-coupled receptor
activation. Anal Biochem. 2003, 313(2):311-8.
[0066] Bryant R, McGuinness D, Turek-Etienne T, Guyer D, Yu L,
Howells L, Caravano J, Zhai Y, Lachowicz J. WGA-coated yttrium
oxide beads enable an imaging-based adenosine 2a receptor binding
scintillation proximity assay suitable for high throughput
screening. Assay Drug Dev Technol. 2004; 2(3):290-9.
[0067] Caruso, R A Nanocasting and Nanocoating. Top Curr Chem.
2003, 226:91-118.
[0068] Caruso F. Hollow inorganic capsules via colloid-templated
layer-by-layer electrostatic assembly. Top Curr Chem. 2003, 227:
145-168.
[0069] Niesen, T P, De Guire, M R Review: Deposition of ceramic
thin films at low temperatures from aqueous solutions. J.
Electroceram. 2001, 6: 169-207
[0070] Onda K, Li B, Zhao J, Jordan K D, Yang J, Petek H. Wet
electrons at the H2O/TiO2(110) surface. Science. 2005;308
(5725):1154-1158.
[0071] Larsen M R, Thingholm T E, Jensen O N, Roepstorff P,
Jorgensen T J. Highly selective enrichment of phosphorylated
peptides from peptide mixtures using titanium dioxide microcolumns.
Mol Cell Proteomics. 2005 Apr. 27; [Epub ahead of print]
[0072] Jessop, R. A. Scintillation proximity test. U.S. Pat. No.
6,524,786 B1 Feb. 25, 2003.
[0073] Thomson, J., ter Wiel, J., van Lune, H., Bosel, H M, Kremer,
G H Scintillation counting system using scintillator capsules. U.S.
Pat. No. 5,512,753 Apr. 30, 1996.
[0074] Normant E. Melendez A, Casamitjana O., and Moreau F Methods
and compsositions for screening modulators of lipid kinases. United
States Patent Aplication Publication No. US 2002/0042091 A1, Apr.
11, 2002.
[0075] Brandish P E, Hill L A Assays for inositol phosphates.
United States Patent Application Publication US 2004/0180394 A1,
Sep. 16, 2004.
[0076] Boge A., Lavis, L D, Sportsman, R., Hoekstra, M F, and
Huang, W. Molecular modification assays. United States Patent
Application Publication No. 2004/024586 A1, Dec. 9, 2004.
[0077] Potter, C., Warner, G., Oikari, T. Method for simultaneous
assay of ligands. U.S. Pat. No. 5,246,869, Sep. 21, 1993.
EXAMPLES
Example 1
[0078] This example describes preparation of assay particles based
on hollow glass micro spheres.
[0079] Two types of hollow glass microspheres (glass bubbles) (S60
10,000 and S60 HS, 3M Corporation) were used for preparing assay
particles. Both types of bubbles were received as powdery samples
and were evaluated in parallel. About 2.5 g of bubbles were
resuspended in 40 ml of water in a 50 ml conical centrifuge tube
and centrifuged at 2 KRPM for 5 min. Most of the glass bubbles
floated as expected (bubble density is about 0.6 g/cc) forming a
"creamy" thick layer on the top. A small fraction of bubbles sunk
to the bottom of the tube. About 1 ml of the bubbles floating on
the top was pipetted out and 6 ml of the 50 mM TiF4 solution was
added for coating the surfaces with TiO.sub.2. The TiF.sub.4 was
prepared as follows: weighing 0.31 g of Ti (IV) fluoride
(Sigma-Aldrich catalog # 333239) in container with cap (455 Filter
Unit Receiver); adding 20 ml of deionized water (Milli-Q) and
closing the cap right away to avoid escape of hydrofluoric acid
from the container. It takes about 30-40 min. to completely
dissolve the Titanium fluoride. Then, 25 ml of 0.1% Ammonium
Hydroxide was added, and the pH was adjusted to 1.8 by adding 1-2
drops of 1% NH4OH. The negative control of uncoated beads was
prepared in a similar way using water instead of TiF.sub.4
solution. Following the 2 hour coating at 60.degree. C., the
bubbles were centrifuged and the top layer of the floating bubbles
was collected and diluted in 40 ml of water.
[0080] This example shows successful coating of glass bubbles with
TiO.sub.2. Most of the glass bubbles remained afloat after the
TiO.sub.2-coating.
Example 2
[0081] This example describes enrichment of phosphorylated peptides
using glass assay particles coated with titanium dioxide
[0082] Using vigorous mixing, the 200 .mu.l, 100 .mu.l, and 50
.mu.l of bubble suspension, either uncoated or prepared as
described in Example 1, was dispensed into a MULTISCREEN plate
(Millipore). Two columns, 8 wells per column, were dispensed for
each volume of bead suspension and for each type of beads. The
uncoated beads were dispensed at 200 .mu./well in a FIG. 2 shows
that successful enrichment of phosphopeptides was achieved for the
.alpha.-casein tryptic digest fractionation. The S60 10,000 and S60
HS glass bubbles performed similarly in these experiments. Uncoated
beads, on the other hand yielded low levels of nonselective binding
of peptides from the trypic digest.
[0083] This example shows selective enrichment of phosphopeptides
from an .alpha.-casein tryptic digest using TiO.sub.2-coated glass
bubbles.
Example 3
[0084] This example describes a method for performing SPA-based
protein kinase detection using an assay particle of the invention.
.sup.3H-labeled Abl and glycogen synthase 1-10 peptide are
obtained. Abl peptide is a substrate for Abl tyrosine kinase and
its amino acid sequence is E-A-I-Y-A-A-P-F-A-K-K-K (MW 1336).
Glycogen synthase 1-10 peptide is a substrate for
Calcium-Calmodulin-Dependent protein Kinase II and its amino acid
sequence is P-L-S-R-T-L-S-V-S-S (MW 1045.2). The peptides are
applied individually to various wells of a 96-well microplate,
generally at a concentration of 0.01 to 10 .mu.M. The assay is
performed in any appropriate support or device, including plate,
tube, flask or vial. Multi-well plates allow multiple assays to be
performed in parallel. Next, kinase reactions are performed, for
example, in a 30 to 100 .mu.L reaction volume containing 20,000
U/mL or 1600 units enzyme (Calmodulin-Dependent protein Kinase II,
New England Biolabs, Beverly, Mass.). Alternatively, the enzyme is
obtained from a eukaryotic cell lysate comprising 0.1 to 50 .mu.g
of total proteins. The kinase reaction is performed using buffer,
CaCl.sub.2, calmodulin, and ATP supplied with the enzyme. The
supplied kinase buffer includes 50 mM Tris-HCl, 10 mM MgCl.sub.2, 2
mM dithiothreitol, 0.1 mM Na.sub.2EDTA, pH 7.5. CaCl.sub.2,
calmodulin and ATP (working concentrations range from 2 mM to 1.2
.mu.M). Typically, the amount of kinase employed in the assay
should is be sufficient to modify less than one third of the
tritiated substrate during a defined incubation period. The
reaction solution with enzyme is pipetted into the well and
incubated at 37 degrees centigrade for up to three hours,
optionally with mixing supplied by an orbital shaker. After
incubation, assay particles containing an inorganic phosphor thin
film coating are added to the reaction mixture. Europium-doped
yttrium oxide, europium-doped zirconia or europium-doped yttrium
aluminum garnet are examples of inorganic phosphors that serve both
as a target selective binding agent for phosphorylated peptides and
as a scintillant. The assay particles are optionally suspended in 0
to 70% glycerol. After further incubation for a period of 10
minutes to 24 hours, the amount or quantity of particle-bound
phosphopeptide is assessed by scintillation counting or
scintillation imaging, using either a TopCount-HTS, 12 Detector,
96/384 instrument or ViewLux.TM. ultraHTS Microplate Imager
(PerkinElmer, Boston, Mass.), respectively. Calmodulin-dependent
kinase II specifically phosphorylates the glycogen synthase 1-10
peptide, leading to detectable scintillations, while the Abl
peptide is not phosphorylated and thus does not activate the
inorganic phosphor-coated particles. Though illustrated with
descriptions relating to the detection of phosphorylated molecules
using SPA, applications of the method and materials are not
restricted to any particular analyte, class of substances, or
binding component reactant pair and in principle any binding assay
can be performed according to a method of the invention.
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