U.S. patent number 7,572,643 [Application Number 11/284,155] was granted by the patent office on 2009-08-11 for nanoparticle composite-coated glass microspheres for use in bioassays.
This patent grant is currently assigned to E. I. du Pont de Nemours and Company. Invention is credited to Kevin Michael Croker, Michael B. Damore, Kostantinos Kourtakis, Michael P. Perry, James M. Prober, Paul Douglas Stull.
United States Patent |
7,572,643 |
Croker , et al. |
August 11, 2009 |
Nanoparticle composite-coated glass microspheres for use in
bioassays
Abstract
A microsphere for use in a bioassay comprising a glass core
coated with a nanoparticle composite comprising a bioactive probe
is provided. The nanoparticle composite coating enhances the
density of bioprobe loading on the surface of the microspheres,
resulting in enhanced dynamic range and sensitivity in bioassays.
The particle may be used in detection systems where resonant light
scattering properties of the particle are useful.
Inventors: |
Croker; Kevin Michael
(Hockessin, DE), Damore; Michael B. (Wilmington, DE),
Kourtakis; Kostantinos (Media, PA), Perry; Michael P.
(Landenberg, PA), Prober; James M. (Wilmington, DE),
Stull; Paul Douglas (Wilmington, DE) |
Assignee: |
E. I. du Pont de Nemours and
Company (Wilmington, DE)
|
Family
ID: |
38054038 |
Appl.
No.: |
11/284,155 |
Filed: |
November 21, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070117224 A1 |
May 24, 2007 |
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Current U.S.
Class: |
436/524; 435/5;
435/6.12; 435/7.2; 435/7.21; 435/7.32; 435/7.5; 436/525;
436/527 |
Current CPC
Class: |
B82Y
30/00 (20130101); G01N 33/54346 (20130101) |
Current International
Class: |
G01N
33/551 (20060101); G01N 33/552 (20060101); G01N
33/553 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 246 757 |
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Nov 1987 |
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EP |
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02097581 |
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Apr 1990 |
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JP |
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10-0467770 |
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Jan 2005 |
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KR |
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WO 01/46471 |
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Jun 2001 |
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WO |
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Other References
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by other .
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Inc., New York, NY, 1935 (Book Not Supplied). cited by other .
Bradley et al., Metal Alkoxides, Academic Press, New York, NY, 1978
(Book Not Supplied). cited by other .
Emanuele Ostuni et al., A Survey of Structure-Proeprty
Relationships of Surfaces That Resist the Adsorption of Protein,
Langmuir, vol. 17:5605-5620, 2001. cited by other .
Danhua Wang et al., Novel Benzo-15-Crown-5 Sol-Gel Coating for
Solid-Phase, Journal of Chromatography A, vol. 1005:1-12, 2003.
cited by other .
Zhihong Wang et al., Dense PZT Thick Films Derived From Sol-Gel
Based Nanocomposite Process, Materials Science and Engineering,
vol. B99:56-62, 2003. cited by other .
Y. Castro et al., Coatings Produced by Electrophoretic Deposition
From Nano-Particulate Silica Sol-Gel Suspensions, Surface and
Coatings Technology, vol. 182:199-203, 2004. cited by other .
Christopher J. Larson et al., A High-Capacity Column for Affinity
Purification of Sequence-Specific DNA-Binding Proteins, Nucleic
Acids Research, vol. 20(13):3525, 1992. cited by other .
James T. Kadonaga, Purification of Sequence-Specific Binding
Proteins by DNA Affinity Chromatography, Methods in Enzymology,
vol. 208:10-23, 1991. cited by other .
Rolf Lohrmann et al., New Solid Supports for DNA Synthesis, Fourth
Annual Congress for Recombinant DNA Research, p. 122. cited by
other .
H. Schmidt et al., The Sol-Gel Process as a Basic Technology for
Nanoparticle-Dispersed Inorganic-Organic Composites, Journal of
Sol-Gel Science and Technology, vol. 19:39-51, 2000. cited by other
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Kulwinder Flora et al., Comparison of Formats for the Development
of Fiber-Optic Biosensors Utilizing Sol-Gel Derived Materials
Entrapping Fluorescently-Labelled Protein, Analyst, vol.
124:1455-1462, 1999. cited by other .
Sarita V. Malik et al., Immobilization of Porcine Pancreas Lipase
on Zirconia Coated Alkylamine Glass Using Glutaraldehyde, Indian
Journal of Chemical Technology, vol. 7:64-67, 2000. cited by other
.
G. Carturan, Preparation of Supports for Catalysis by the "Gel
Route", Journal of Non-Crystalline Solids, vol. 63:273-281, 1984.
cited by other .
A. Peter Jardine, Synthesis and Characterization of PZT Coatings on
Glass Microspheres, Mat. Res. Soc. Symp. Proc., vol. 372:151-154,
1995. cited by other .
M. Haraguchi et al., Fabrication and Optical Characterization of a
TiO2 Thin Film on a Silica Microsphere, Surface Science, vol.
548:59-66, 2004. cited by other .
Joanne D. Andreadis et al., Use of Immobilized PCR Primers to
Generate Covalently Immobilized DNAs for In Vitro
Transcription/Translation Reactions, Nucleic Acids Research, vol.
28(2):ii-vii, 2000. cited by other .
Toribio & Ovejero, J. Mat. Eng. Perform. 9:272-79 (2000). cited
by other.
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Primary Examiner: Chin; Christopher L
Claims
What is claimed is:
1. A microsphere for use in a bioassay comprising a glass core
coated with a nanoparticle composite coating comprising a bioactive
probe, wherein the nanoparticle composite coating comprises: a) a
colloidal oxide of an element selected from the group consisting of
silicon, zirconium, aluminum, titanium, cerium, antimony, and
mixtures thereof, wherein the colloidal oxide has a particle size
of about 2 nanometers to about 100 nanometers; and b) a
non-collodial oxide or oxyhydroxide of an element selected from the
group consisting of silicon, zirconium, aluminum, titanium,
tantalum, niobium, and mixtures thereof.
2. A microsphere according to claim 1 wherein the bioactive probe
is selected from the group consisting of proteins, polypeptides,
polynucleotides, antibodies, antibody fragments, biological cells,
microorganisms, cellular organelles, cell membrane fragments,
bacteriophages, bacteriophage fragments, whole viruses, and viral
fragments.
3. A microsphere according to claim 1 wherein the bioactive probe
is synthesized on the surface of the coated microsphere.
4. A microsphere according to claim 1 wherein the bioactive probe
is isolated from natural sources or synthesized separately prior to
being attached to the surface of the coated microsphere.
5. A microsphere according to claim 4 wherein the bioactive probe
is attached to the coated microsphere using linker chemistries or
crosslinking chemistries.
6. A microsphere according to claim 5 wherein the linker
chemistries employ linker groups selected from the group consisting
of hydroxyl, amino, carboxyl, aldehyde, amide, sulfonate, and
sulfate.
7. A microsphere according to claim 5 wherein the crosslinking
chemistries employ reactive groups selected from the group
consisting of s-triazines, epoxides, maleamides, haloacetyl
derivatives, isothiocyanates, succinimidyl esters, sulfonyl
halides, and carbodiimides.
8. A microsphere according to claim 1 wherein the bioactive probe
is one member of a binding pair.
9. A microsphere according to claim 8 wherein the one member of a
binding pair is selected from binding pair combinations consisting
of: antigen/antibody, antigen/antibody fragment, Protein
A/antibody, Protein G/antibody, hapten/anti-hapten, biotin/avidin,
biotin/streptavidin, folic acid/folate binding protein;
hormone/hormone receptor, lectin/carbohydrate, enzyme/cofactor,
enzyme/substrate, enzyme/inhibitor, peptide nucleic
acid/complimentary nucleic acid, polynucleotide/polynucleotide
binding protein, vitamin B12/intrinsic factor; complementary
nucleic acid segments; pairs comprising sulfhydryl reactive groups,
pairs comprising carbodiimide reactive groups, and pairs comprising
amine reactive groups.
10. A microsphere according to claim 1 wherein the glass core
comprises a composition selected from the group consisting of: A)
[Ba.sub.1-xTi.sub.ySi.sub.y'B.sub.y''Ca.sub.y'''O.sub.(1-x+2y+2y'+3/2y''+-
y''')].sub.1-a(AO.sub.z).sub.a, wherein 0.6>y>0.1;
0.6>y'>0.05; 0.6>y''.gtoreq.0; 0.4>y'''.gtoreq.0;
x=y+y'+y''+y'''; A is any of, or a combination of Na, Fe, Sr, and
Zr; 0.01>a .gtoreq.0, and 2.gtoreq.z.gtoreq.0.5; B)
[Ba.sub.1-xLa.sub.ySi.sub.y'Ti.sub.y''B.sub.y'''Ca.sub.y''''O.sub.(1-x+3/-
2y+2y'+2y''+3/2y'''+2y'''')].sub.1-a(AO.sub.z).sub.a; wherein
0.5>y>0.1; 0.6>y'>0.05; 0.6>y''>0.04;
0.4>y'''.gtoreq.0; 0.3.gtoreq.y''''.gtoreq.0;
x=y+y'+y''+y'''+y''''; and wherein A is any of, or a combination of
Cr, Fe, W, Na and Zr; 0.01>a .gtoreq.0; and
3.gtoreq.z.gtoreq.0.5; and C) a composition comprised of calcium,
titanium, silicon and oxygen, wherein the calcium, titanium, and
silicon content is given by: Ca.sub.1-x-yTi.sub.xSi.sub.y wherein x
and y are independently equal to 0.2 to 0.5.
11. A microsphere according to claim 1 wherein the glass core
comprises a silicon content of at least about 50 atom % and a
composition selected from the group consisting of: A)
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y'', provided that x is less
than or equal to 0.5, 0.5>y.gtoreq.0.1;0.1.gtoreq.y'.gtoreq.0,
0.1.gtoreq.y''.gtoreq.0; A is any of, or a combination of Fe, Ca,
and K; 0.1>a.gtoreq.0; and 1.5.gtoreq.z.gtoreq.0.5; and B)
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y''; provided that x is less
than or equal to 0.5; 0.5>y.gtoreq.0.1; 0.1.gtoreq.y'.gtoreq.0,
0.1.gtoreq.y''.gtoreq.0; A is any of, or a combination of Fe, Ca,
and K; 0.1>a.gtoreq.0; and 1.5.gtoreq.z.gtoreq.0.5.
12. A microsphere according to claim 11 wherein the glass core
comprises a composition of:
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y''; y=0.213; y'=0.0258;
y''=0.035; a=0.002; A is any of, or a combination of Fe, Ca, and K;
and 1.5.gtoreq.z.gtoreq.0.5.
13. A microsphere according to claim 1 wherein glass core has a
diameter of about 10 micrometers to about 100 micrometers.
14. A microsphere according to claim 1, wherein the oxide or
oxyhydroxide of (b) is aluminum tri-sec butoxide,
tetraethylorthoxisilicate, niobium ethoxide, tantalum ethoxide,
zirconium n-propoxide, or titanium ethoxide.
Description
FIELD OF THE INVENTION
The invention relates to glass microspheres for use in bioassays.
Specifically, glass microspheres are provided having a specific
nanoparticle composite coating composition that enhances the
density of bioprobe loading on the surface of the microspheres.
BACKGROUND OF THE INVENTION
There is a need for highly sensitive diagnostic tools for the
detection of biological analytes in the pharmaceutical,
diagnostics, agriculture, veterinary and health care industries.
The use of resonant light scattering as an analytical method in
these areas is an emerging technology that is ripe for further
development. A key component of such methods is particles or
microspheres having unique resonant light scattering
properties.
The use of resonant light scattering as an analytical method for
determining a particle's identity and the presence and optionally,
the concentration of one or more target analytes has been described
(Prober et al., copending and commonly owned U.S. patent
application Ser. No. 10/702,320 and U.S. Patent Application
Publication No. 2005/0019842). In that method, a microparticle is
irradiated with light of a given wavelength and the resonant light
scattering from the microparticle is detected. As the incident
wavelength is scanned (i.e., varied over an analytical wavelength
range) a scattering pattern or scattering spectrum as a function of
wavelength results. Each particle has a distinct resonance light
scattering spectrum that can be used to identify the particle. The
presence and optionally the concentration of a target analyte can
be determined from the shift in the resonance light scattering
spectrum that occurs when the analyte binds to a capture probe
attached to the surface of the particle. The magnitude of the shift
is related to the concentration of the analyte in the solution.
A key aspect of the above described method is the nature of the
particle, and the ability of the particle to both bind a
bio-analyte while at the same time retaining light scattering
properties. The dynamic range and the sensitivity of the method are
limited by the amount of bioprobe that can be attached to the
surface of the particle. Modifications of the particle that could
enhance the dynamic range and the sensitivity of the resonant light
scattering measurements would be an advance in the art. One tool
for such a modification encompasses the use of nanoparticle
composite coatings for the surface modification of these
particles.
The use of sol-gel materials to modify particle or nanoparticle
surfaces is known (see review by Schmidt et al., Journal of Sol-Gel
Science and Technology (2000), 19(1/2/3), 39-51). This technology
has been applied in processes for the analysis of bio-analytes. For
example, Flora et al., (Analyst (Cambridge, United Kingdom) (1999),
124(10), 1455-1462) teach the use of sol-gel particles for the
adsorption of proteins for fiber-optic analysis. Similarly, glass
beads comprising coatings of zirconia (Malik et al., (Indian
Journal of Chemical Technology (2000), 7(2), 64-67) or alkoxysilane
(Kuramoto et al, JP02097581) have been prepared and used to
immobilize enzymes. Additionally, colloidal sol-gel composites have
been coated on the surface of SiO.sub.2 particulate substrates to
modify light transmission properties of the substrate (Garvey et
al., EP246757), and non-colloidal sol-gel coatings have been
applied to glass beads to modify the refractive indices of the
beads (Jun et al., KR2002017667), by using the so-called sol-gel
reaction. Additional modification of glass surfaces with sol-gels
have been reported, see for example Carturan et al., (Journal of
Non-Crystalline Solids (1984), 63(1-2), 273-81) describing coating
glass beads with thin layer porous oxides comprised of
SiO.sub.2/Al.sub.2O.sub.3/Na.sub.2O; and Jardine, A. Peter,
(Materials Research Society Symposium Proceedings (1995), 37,
(Hollow and Solid Sphere and Microspheres: Science and Technology
Associated with Their Fabrication and Application), describing the
synthesis of Pb(Ti,Zr)O.sub.3 (PZT) coatings on glass microspheres
using sol-gel techniques; and Haraguchi et al. (Surface Science
(2004), 548(1-3), 59-66), reporting the fabrication of a uniform
TiO.sub.2 thin film on SiO.sub.2 microspheres.
Although the above described particle modifications are useful,
none of the disclosures teach a particle having enhanced binding
for bio-analytes or the compositions needed for detection by
resonant light scattering means. Applicants provide herein a new
glass particle having been modified with a nanoparticle composite
coating that provides for greater density of bioprobe loading on
the surface of the particle, resulting in enhanced dynamic range
and sensitivity in resonant light scattering assays.
SUMMARY OF THE INVENTION
The invention provides a microsphere for use in a bioassay.
Accordingly, the invention provides a microsphere for use in a
bioassay comprising a glass core coated with a nanoparticle
composite coating comprising a bioactive probe.
In one embodiment, the invention provides a microsphere for use in
a bioassay comprising a glass core coated with a nanoparticle
composite coating comprising a bioactive probe, wherein the
nanoparticle composite coating comprises:
a) a colloidal oxide of an element selected from the group
consisting of silicon, zirconium, aluminum, titanium, cerium,
antimony, and mixtures thereof, wherein the colloidal oxide has a
particle size of about 2 nanometers to about 100 nanometers;
and
b) an oxide or oxyhydroxide of an element selected from the group
consisting of: silicon, zirconium, aluminum, titanium, tantalum,
niobium, and mixtures thereof.
In another embodiment, the invention provides a method for the
detection of analyte binding to a nanoparticle composite-coated
microsphere comprising:
a) providing a light scanning source which produces light over an
analytical wavelength range;
b) providing at least one nanoparticle composite coated microsphere
comprising a glass core coated with a nanoparticle composite
comprising a bioactive probe, wherein the bioactive probe has
affinity for at least one analyte;
c) optionally scanning the nanoparticle composite coated
microsphere of (b) one or more times over the analytical wavelength
range to produce at least one first reference resonant light
scattering spectrum for the nanoparticle composite coated
microsphere of (b);
d) contacting the nanoparticle composite coated microsphere of (c)
with a sample suspected of containing at least one analyte where,
if the analyte is present, binding occurs between the at least one
bioactive probe and the at least one analyte;
e) scanning the nanoparticle composite coated microsphere of (d)
one or more times over the analytical wavelength range to produce
at least one second binding resonant light scattering spectrum for
each nanoparticle composite coated microsphere of (d); and
f) detecting binding of the at least one analyte to the at least
one bioactive probe by comparing the differences between the
resonant light scattering spectra selected from the group
consisting of: any of the at least one first reference light
scattering spectrum and any of the at least one second light
scattering spectrum.
BRIEF DESCRIPTION OF THE FIGURES
The invention can be more fully understood from the following
detailed description and figures, which form a part of this
application.
FIG. 1 is a schematic diagram of the imaging detection system used
to measure resonant light scattering from microspheres, as
described in Examples 2 and 3.
FIG. 2 is a digital image of scattered light from a group of
microparticles, at a single wavelength of incident light. Both the
incident and scattered light were polarized; the directions of the
polarization were parallel. The numbers 12, 3, 6, and 9 refer to
regions of the scattered light image for each particle as explained
in Examples 2 and 3.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a microsphere for use in a bioassay
comprising a glass core coated with a nanoparticle composite
comprising a bioactive probe. The nanoparticle composite coating
enhances the density of bioprobe loading on the surface of the
microspheres, thereby enhancing the dynamic range and the
sensitivity in resonant light scattering assays.
The nanoparticle composite-coated glass microspheres of the
invention have application in methods of specific analyte detection
and particle identification, which are based on the measurement of
resonant light scattering. The methods are capable of parallel
analysis with high multiplicity.
The following definitions and abbreviations are to be used for the
interpretation of the claims and the specification.
The terms "particle", "microparticle", "bead", "microbead",
"microsphere", and grammatical equivalents refer to small discrete
particles, substantially spherical in shape, having a diameter of
about 10 micrometers to about 100 micrometers, preferably about 10
micrometers to about 75 micrometers, more preferably about 10
micrometers to about 50 micrometers.
The term "bioactive" when referring to a capture probe refers to a
capture probe that is able to participate in biological
interactions, such as interactions between members of binding
pairs.
The terms "capture probe", "probe", "binding agent", "bioactive
agent", "binding ligand", "bioprobe", or grammatical equivalents,
refer to any chemical or biological structure or moiety, for
example protein, polypeptide, polynucleotide, antibody or antibody
fragment, biological cells, microorganisms, cellular organelles,
cell membrane fragments, bacteriophage, bacteriophage fragments,
whole viruses, viral fragments, organic ligand, organometallic
ligand, and the like that may be used to bind either
non-specifically to multiple analytes, or preferentially, to a
specific analyte or group of analytes in a sample.
The term "binding-pair" includes any of the class of immune-type
binding-pairs, such as, antigen/antibody, antigen/antibody
fragment, or hapten/anti-hapten systems; and also any of the class
of nonimmune-type binding-pairs, such as biotin/avidin,
biotin/streptavidin, folic acid/folate binding protein,
hormone/hormone receptor, lectin/specific carbohydrate,
enzyme/cofactor, enzyme/substrate, enzyme/inhibitor, or vitamin
B12/intrinsic factor. They also include complementary nucleic acid
fragments (including DNA sequences, RNA sequences, and peptide
nucleic acid sequences), as well as Protein A/antibody or Protein
G/antibody, and polynucleotide/polynucleotide binding protein.
Binding pairs may also include members that form covalent bonds,
such as, sulfhydryl reactive groups including maleimides and
haloacetyl derivatives; amine reactive groups such as
isothiocyanates, succinimidyl esters, carbodiimides, and sulfonyl
halides; and carbodiimide reactive groups such as carboxyl and
amino groups.
The terms "protein", "peptide", "polypeptide" and "oligopeptide"
are herein used interchangeably to refer to two or more covalently
linked, naturally occurring or synthetically manufactured amino
acids.
The term "atom %" as used herein, refers to the percentage of
silicon atoms in the glass compositions of the invention relative
to the total number of cationic atoms (i.e., excluding oxygen and
other anions) in the composition.
The terms "analyte" or "bio-analyte" refer to a substance to be
detected or assayed using the microspheres of the present
invention. Typical analytes may include, but are not limited to,
proteins, peptides, nucleic acids, peptide nucleic acids,
antibodies, receptors, molecules, biological cells, microorganisms,
cellular organelles, cell membrane fragments, bacteriophage,
bacteriophage fragments, whole viruses, viral fragments, and one
member of a binding pair.
The terms "target" and "target analyte" refer to the analyte
targeted by the assay. Sources of targets will typically be
isolated from organisms and pathogens such as viruses and bacteria
or from an individual or individuals, including but not limited to,
for example, skin, plasma, serum, spinal fluid, lymph fluid,
synovial fluid, urine, tears, blood cells, organs, tumors, and also
to samples of in vitro cell culture constituents (including but not
limited to conditioned medium resulting from the growth of cells in
cell culture medium, recombinant cells and cell components).
Additionally, targets may be from synthetic sources.
The term "resonant light scattering spectrum" refers to a plot of
resonant light scattering intensity as a function of wavelength
obtained by scanning the glass microspheres of the invention over
an analytical wavelength range and measuring the resulting resonant
light scattering signal.
The terms "spectral features", "optical resonance structures",
"identification features", "scattering resonances", and "resonant
light scattering signatures" are used interchangeably herein to
refer to features in the resonant light scattering spectrum that
may be used for particle identification, including, but not limited
to peak location, peak width, peak order, periods between peaks of
different orders, and polarization-dependent spectral
properties.
The phrase "richness of spectral features" when used in relation to
a resonant light scattering spectrum, refers to a spectrum that has
a multitude of spectral features that may be used for particle
identification.
The term "analytical wavelength range" refers to a wavelength
window over which the microspheres of the present invention are
scanned to produce resonant light scattering signatures. The window
typically has a span of about 1 to about 20 nanometers over the
optical wavelengths from about 275 to about 1900 nanometers,
preferably from about 600 to about 1650 nanometers. More
preferably, the analytical wavelength range spans a range of 10
nanometers from about 770 to about 780 nanometers. It is
contemplated that a number of scans of the particles of the
invention may be made during the process of identifying an analyte
or detecting analyte binding, however each of these scans will be
over an "analytical wavelength range" although that range may
differ from scan to scan depending on the specific object of the
assay.
The term "light scanning source" refers to a source of light whose
wavelength may be varied over the analytical wavelength range.
Light scanning sources include sources that produce light that may
be varied over the analytical wavelength range, such as scanning
diode lasers and tunable dye lasers, and polychromatic sources
which produce light having a range of wavelengths, such as
light-emitting diodes, lamps and the like, used in conjunction with
a wavelength-selecting means.
The term "reference resonant light scattering spectrum" refers to
the resonant light scattering spectrum that is produced by scanning
the microspheres of the present invention over the analytical
wavelength range after the capture probe has been applied to the
particles or in the case of detection of analyte dissociation from
the capture probe, after the analyte has bound to the capture
probe. The reference resonant light scattering spectrum may be used
to identify the particles and the probes attached thereto and may
serve as a baseline for the detection of analyte binding. A number
of reference resonant spectra may be obtained by scanning the
particles at different times.
The term "binding resonant light scattering spectrum" refers to the
resonant light scattering spectrum that is produced by scanning the
microspheres of the present invention over the analytical
wavelength range after the microspheres are contacted with the
analyte. A series of binding resonant light scattering spectra may
be obtained to follow the binding in real time. The determination
of binding is done by comparing either any one of the binding
resonant light scattering spectra to any one of the reference
resonant light scattering spectra or anyone of the plurality of
binding resonant light scattering spectra with a previous binding
resonant light scattering spectra in the series.
The invention provides a microsphere for use in a bioassay
comprising a glass core coated with a nanoparticle composite
comprising a bioactive probe. A key component of the invention is
the construction of a three dimensional nanocomposite structure
that surrounds the glass core. It is postulated that the three
dimensional nature and the porosity of the structure allows for a
higher volumetric density of probes that can bind to target
molecules. The higher density of probes enables a greater number of
target molecules to bind to the probe, which causes a
proportionally larger change in refractive index at the surface of
the microsphere. This results in a larger response in the resonant
light scattering spectrum by inducing a greater shift in the
wavelength of one or more resonance peaks. Additionally, the
sensitivity towards low concentrations of analyte should be
improved as a consequence of the improved volumetric density of
probes.
Glass Cores
For use in the invention, the glass core is a glass microbead that
is substantially spherical in shape, and has a diameter of about 10
micrometers to about 100 micrometers, preferably about 10
micrometers to about 75 micrometers, more preferably about 10
micrometers to about 50 micrometers. The term "substantially
spherical", as used herein, means that the shape of the glass core
does not deviate from a perfect sphere by more than about 10%. The
refractive index of the glass core is about 1.4 to about 2.1.
Suitable glass cores for use in the invention are comprised of
materials including, but not limited to, oxides of barium, bismuth,
titanium, iron, sodium, calcium, boron, niobium, tantalum,
lanthanum, silicon, strontium, chromium, and tungsten. Barium
titanium silicon oxide glasses have been found to be particularly
useful in resonant light scattering methods for identification of
the microspheres as well as for analyte detection because of the
richness of spectral features in their light scattering spectra.
Additionally, glass cores having a composition comprised of
calcium, titanium, silicon and oxygen, and glass cores having a
composition comprising a silicon content of at least about 50 atom
%, for example, borosilicate glasses, are particularly useful for
high sensitivity detection.
In one embodiment, the glass core has a composition of:
[Ba.sub.1-xTi.sub.ySi.sub.y'B.sub.y''Ca.sub.y'''O.sub.(1-x+2y+2y'+3/2y''+-
y''')].sub.1-a(AO.sub.z).sub.a, wherein 0.6>y>0.1;
0.6>y'>0.05; 0.6>y''.gtoreq.0; 0.4>y'''.gtoreq.0;
x=y+y'+y''+y'''; A is any of, or a combination of Na, Fe, Sr, and
Zr; 0.01>a.gtoreq.0, and 2.gtoreq.z.gtoreq.0.5.
In another embodiment, the glass core has a composition of:
[Ba.sub.1-xTi.sub.ySi.sub.y'B.sub.y''Ca.sub.y'''O.sub.(1-x+2y+2y'+3/2y''+-
y''')]1-a(AO.sub.z).sub.a; wherein x=y+y'+y''+y'''; y=0.394;
y'=0.113; y''=0.134; y'''=0.066; a=0.005; 2.gtoreq.z.gtoreq.0.5;
and wherein A is a combination of Fe, Sr, Na, and Zr.
In another embodiment, the glass core has a composition of:
[Ba.sub.1-xLa.sub.ySi.sub.y'Ti.sub.y''B.sub.y'''Ca.sub.y''''O.sub.(1-x+3/-
2y+2y'+2y''+3/2y'''+2y'''')].sub.1-a(AO.sub.z).sub.a; wherein
0.5>y>0.1; 0.6>y'>0.05; 0.6>y''>0.04;
0.4>y'''.gtoreq.0; 0.3>y''''.gtoreq.0; x=y+y'+y''+y'''+y'''';
where A is any of, or a combination of Cr, Fe, W, Na and Zr;
0.01>a.gtoreq.0; and 3.gtoreq.z.gtoreq.0.5.
In another embodiment, the glass core has a composition of:
[Ba.sub.1-xLa.sub.ySi.sub.y'Ti.sub.y''B.sub.y'''Ca.sub.y''''O.sub.(1-x+3/-
2y+2y'+2y''+3/2y'''+2y'''')].sub.1-a(AO.sub.z).sub.a; wherein
y=0.171, y'=0.401, y''=0.044, y'''=0.0614, y''''=0.0194,
x=y+y'+y''+y'''+y''''; a=0.0044; 3.gtoreq.z.gtoreq.0.5; and A is a
combination of Cr, Fe, W, Na, and Zr.
In another embodiment, the glass core has a composition comprised
of calcium, titanium, silicon and oxygen, wherein the calcium,
titanium, and silicon content is given by:
Ca.sub.1-x-yTi.sub.xSi.sub.y and wherein x and y are independently
equal to 0.2 to 0.5, preferably between 0.3 and 0.4. An example of
glass compositions of this type is:
Ca.sub.1-x-yTi.sub.xSi.sub.yO.sub.(1+x+y) wherein x and y are
independently equal to 0.2 to 0.5, preferably between 0.3 and
0.4.
In another embodiment, the glass core has a composition comprising
a silicon content of at least about 50 atom %, for example,
borosilicate glasses. An exemplary glass composition of this type
is:
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y'', provided that x is less
than or equal to 0.5; 0.5>y .gtoreq.0.1; 0.1.gtoreq.y'.gtoreq.0,
0.1.gtoreq.y''.gtoreq.0; A is any of, or a combination of Fe, Ca,
and K; 0.1>a.gtoreq.0; and 1.5.gtoreq.z.gtoreq.0.5.
In another embodiment, the glass core has a composition of:
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y''; y=0.213; y'=0.0258; and
y''=0.035; a=0.002; A is any of, or a combination of Fe, Ca, and K;
and 1.5.gtoreq.z.gtoreq.0.5.
In another embodiment, the glass core has a composition of:
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y''; provided that x is less
than or equal to 0.5; 0.5>y.gtoreq.0.1; 0.3.gtoreq.y'.gtoreq.0,
0.1.gtoreq.y''.gtoreq.0; A is any of, or a combination of Fe, Ca,
and K; 0.1>a.gtoreq.0; and 1.5.gtoreq.z.gtoreq.0.5.
In another embodiment, the glass core has a composition of:
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y''; y=0.175; y'=0.2; and
y''=0.007; a=0.025; A is any of, or a combination of Fe, Ca, and K;
and 1.5.gtoreq.z.gtoreq.0.5.
Suitable glass cores may be obtained from commercial suppliers such
as MO-SCI Specialty Products, LLC. (a subsidiary of MO-SCI
Corporation, Rolla, Mo.).
Nanoparticle Composite Coating
The nanoparticle composite coating comprises a nanoparticle
colloidal component in combination with a binder derived from an
alkoxide. The nanoparticle composite coating is a skeletal
framework of oxides and oxyhydroxides derived from the hydrolysis
and condensation of alkoxides and other reagents combined with the
larger structure introduced by the colloidal oxide, which creates a
porous layer surrounding the glass core. The scaffold material
comprises combinations of the colloidal oxides with oxides and
oxyhydroxides, or their mixtures, of aluminum, silicon, niobium,
tantalum, titanium, and niobium. For use in resonant light
scattering assays, the nanoparticle composite coating has a
thickness of less than 700 nanometers, preferably, from about 1
nanometer to about 600 nanometers. The thickness of the coating may
be determined using methods known in the art, such as scanning
electron microscopy.
The nanoparticle composite coating may be comprised of any
combination of colloidal particles, ranging in size from about 2
nanometers to about 100 nanometers. Suitable colloidal
nanoparticles include, but are not limited to, colloidal oxides of
elements such as silicon, zirconium, titanium, aluminum, cerium,
antimony, and any combination thereof. The porosity of the
nanoparticle composite coating may be tuned by appropriate choice
of size or size distribution of the colloidal oxides.
Suitable colloidal oxides are commercially available, for example,
from Nalco Chemical Company (Napperville, Ill.) or Nyacol Nano
Technologies, Inc. (Ashland, Mass.). Additionally, colloidal oxides
may be prepared using methods known in the art (see for example,
Inorganic Colloid Chemistry, volumes 1, 2 and 3, J. Wiley and Son,
Inc., New York, N.Y., 1935). Colloid formation involves either
nucleation and growth or subdivision or dispersion processes. For
example, hydrous titanium dioxide colloids may be prepared by
adding ammonium hydroxide to a solution of tetravalent titanium
salt, followed by peptization (re-dispersion) by dilute alkalis.
Zirconium oxide colloids may be prepared by dialysis of sodium
oxychlorides. Cerium oxide sol may be prepared by dialysis of a
solution of ceric ammonium nitrate.
The inorganic metal alkoxides used as starting material in
combination with the colloidal nanoparticles for preparing the
microspheres of the invention may include any alkoxide which
contains from 1 to 20 carbon atoms, and preferably from 1 to 5
carbon atoms in the alkoxide group. Suitable inorganic metal oxides
include, but not limited to, aluminum tri-sec butoxide,
tetraethylorthoxisilicate, niobium ethoxide, tantalum ethoxide,
zirconium n-propoxide, and titanium ethoxide. Alkoxides having one
to four carbon atoms, such as titanium ethoxide, and aluminum
trisecondary butoxide, are preferred. Additionally, inorganic metal
alkoxides that are soluble in the liquid reaction medium are
preferred. Suitable inorganic metal alkoxides are available
commercially. For example, commercially available alkoxides, such
as tetraethylorthosilicate and Tyzor.TM. organic titanate esters
may be used. Additionally, inorganic alkoxides may be prepared by
various routes, such as for example, direct reaction of zero valent
metals with alcohols in the presence of a suitable catalyst; and
the reaction of metal halides with alcohols. Alkoxy derivatives may
be synthesized by the reaction of the alkoxide with alcohol in a
ligand interchange reaction. Direct reactions of metal dialkamides
with alcohol also form alkoxide derivatives. Additional examples
are disclosed by Bradley et al. (Metal Alkoxides, Academic Press,
New York, N.Y., 1978).
The process to form the nanoparticle composite coating on the lass
core is carried out in a liquid medium. Generally, the liquid
medium utilized in the process should be a solvent for the
inorganic alkoxide or alkoxides used. Alkoxides that are reactive
to water at room temperature need to be dispersed in a non-aqueous
solvent, while alkoxides that are less reactive to water. (e.g.,
tetraethylorthosilicate) may be dispersed in water. The addition of
acidic or basic reagents to the liquid medium can have an effect on
the kinetics of the hydrolysis and condensation reactions, and the
microstructure of the oxide/hydroxide binder derived from the
alkoxide precursor. Generally, a pH range of 1-12 may be used,
while a pH range of 1-6 preferred.
The glass core may be optionally treated with water, acid or base
to initiate hydrolysis and condensation reactions of the alkoxides.
In some cases, the glass core may be reacted with the alkoxides
without added water, since the surface of the glass core contains
hydroxyl groups and some nascent adsorbed water for the formation
of the binder phase.
The glass core, the colloidal nanoparticles, and the inorganic
metal alkoxide are added to the liquid medium and the mixture is
refluxed under nitrogen for about 15 minutes to about 8 hours. The
resulting nanoparticle composite-coated microspheres may be
recovered using any means known in the art, such as filtration,
centrifugation, or decantation. The microspheres may be rinsed with
a nonaqueous solvent and subsequently rinsed with water.
In general, the thickness and porosity of a coating layer may be
controlled by the relative proportions of the colloidal components
and the alkoxide reagent precursors. For the silicon oxide system
(e.g., colloidal silicon oxide combined with
tetraethyoxyorthosilane), a molar ratio (colloidal
SiO.sub.2/alkoxoide derived SiO.sub.2) of about 1 yields a coating
thickness of about 400 to 600 nanometers. Lowering this ratio to
0.1 or below will result in a coating layer thickness of less than
200 nanometers. A preferred range of the molar ratio for use in the
invention is 0.1 to about 3, more preferably, about 0.5 to about 2.
Additionally, the thickness of the coating may be controlled by
repeated sequential depositions of the coating layers, provided
that the underlying layer is not disturbed.
Additionally, the microspheres comprising the nanoparticle
composite coating may be optionally heat-treated at elevated
temperatures to further modify the properties of the nanoparticle
composite coating and to help further anchor the coating to the
surface of the glass core. Typically, a temperature of about
100.degree. C. to about 600.degree. C. may be used for the heat
treatment step, depending on the melting point of the glass core.
Specifically, a temperature that would melt the glass core is not
desired. The atmosphere used for the heat treatment may be
non-oxidizing (for example nitrogen or argon) or the treatment may
be done in air.
Microspheres Comprising a Bioactive Probe
The microsphere of the invention is prepared by applying a capture
probe that is bioactive to the surface of the nanoparticle
composite-coated glass core. The capture probe may be any chemical
or biological structure or moiety, including, but not limited to,
protein, polypeptide, polynucleotide, antibody or antibody
fragment, biological cells, microorganisms, cellular organelles,
cell membrane fragments, bacteriophage, bacteriophage fragments,
whole viruses, viral fragments, organic ligand, organometallic
ligand, and the like that may be used to bind either
non-specifically to multiple analytes, or preferentially, to a
specific analyte or group of analytes in a sample.
The probe may be applied to the surface of the nanoparticle
composite-coated microspheres by either directly synthesizing the
probe on the surface or by attaching a probe that is naturally
occurring or has been synthesized, produced, or isolated separately
to the surface using methods know in the art, as described by
Prober et al. in copending and commonly owned U.S. Patent
Application Publication No. 2005/0019842, which is incorporated
herein by reference. The utility of the invention is enhanced by
using a set of microspheres, each of which has one or more unique
capture probes exposed on its surface. Such a set may be generally
referred to as a "library" of microspheres or probes.
Microspheres comprising a bioactive probe may be prepared by
derivatizing the surface of the nanoparticle composite-coated
microspheres such that the appropriate capture probes may be
attached using linker chemistries or crosslinking chemistries,
which are well known in the art. Examples of linking groups
include, but are not limited to, hydroxyl groups, amino groups,
carboxyl groups, aldehydes, amides, and sulfur-containing groups
such as sulfonates and sulfates. Examples of crosslinking
chemistries include, but are not limited to, hydroxy reactive
groups such as s-triazines and bis-epoxides, sulfhydryl reactive
groups such as maleimides and haloacetyl derivatives, amine
reactive groups such as isothiocyanates, succinimidyl esters and
sulfonyl halides and carboxyl reactive groups such as
carbodiimides.
One class of capture probes comprises proteins. By "protein" is
meant two or more covalently linked amino acids; thus the terms
"peptide", "polypeptide", "oligopeptide", and terms of similar
usage in the art are all to be interpreted synonymously in this
disclosure. Libraries of protein capture probes may be prepared,
for example, from plant or animal cellular extracts, using the
linker chemistries described above to attach the protein to the
surface of the nanoparticle composite-coated microspheres.
Particularly useful and thus preferred are libraries of human
proteins, for example human antibodies.
Another class of capture probes comprise nucleic acids or nucleic
acid mimics, such as peptide nucleic acids (PNA), which may also be
known as "DNA fragments", "RNA fragments", "polynucleotides",
"oligonucleotides", "gene probes", "DNA probes" and similar terms
used in the art, which are all to be considered synonymous in the
present disclosure. Methods for preparing nucleic acid probes or
pseudo-nucleic acid probes, such as PNA, are well known in the art.
For example, the nucleic acid probes may be prepared using standard
.beta.-cyanoethyl phosphoramidite coupling chemistry on controlled
pore glass supports using commercially available DNA
oligonucleotide synthesizers, such as that available from Applied
Biosystems (Foster City, Calif.). The synthesized nucleic acid
probes may then be coupled to the nanoparticle composite-coated
microspheres using covalent or non-covalent coupling, as is well
known in the art. Surface preparation of the nanoparticle
composite-coated glass microspheres useful for this invention may
include, for example, linker chemistry, affinity capture by
hybridization or by biotin/avidin affinity, combinatorial
chemistry, and others known in the art.
In another approach, the capture probe may be directly synthesized
on the surface of the nanoparticle composite-coated microspheres of
the invention. Probes that may be directly synthesized on the
nanoparticle composite-coated microspheres include, but are not
limited to, nucleic acids (DNA or RNA), peptide nucleic acids,
polypeptides and molecular hybrids thereof. In the direct synthesis
approach, a microsphere that is derivatized with a reactive residue
to be used to chemically or biochemically synthesize the probe
directly on the microsphere is used. The chemical linkage of the
reactive residue must not be cleavable from the microparticle
during post-synthesis deprotection and cleanup of the final
microspheres (Lohrmann et al., DNA 3, 1222 (1984); Kadonaga, J. T.,
Methods of Enzymology 208, 10-23 (1991); Larson et al., Nucleic
Acid Research 120, 3525 (1992); Andreadis et al. Nucleic Acid Res.
228, e5 (2000); and Chrisey et al. WO/0146471). This approach
allows for mass production and assembly of libraries.
In some applications, e.g., assays in complex biological fluids
such as urine, cerebrospinal fluid, serum, plasma, and the like, it
may be necessary to treat the nanoparticle composite-coated
microspheres to prevent or reduce non-specific binding of sample
matrix components. Methods to reduce non-specific binding to a
variety of solid supports in heterogeneous assays are well known in
the art and include, but are not limited to, treatment with
proteins such as bovine serum albumin (BSA), casein, and non-fat
milk. These treatments are generally done after the attachment of
the capture probe to the microspheres, but before the assay to
block potential non-specific binding sites. Additionally, surfaces
that resist non-specific binding can be formed by coating the
surface with a thin film comprising synthetic polymers, naturally
occurring polymers, or self-assembled monolayers that consist of a
single component or a mixture of components. The thin film may be
modified with adsorption-repelling moieties to further reduce
non-specific binding. For example, the thin film may be a
hydrophilic polymer such as polyethylene glycol, polyethylene
oxide, dextran, or polysaccharides, as well as self-assembled
monolayers with end functional groups that are hydrophilic, contain
hydrogen-bond acceptors but not hydrogen bond donors, and are
overall electrically neutral (Ostuni, E. et al., "A Survey of
Structure-Property Relationships of Surfaces that Resist the
Adsorption of Protein", Langmuir, 17, 5605-5620, (2001)). In this
approach, the non-specific binding resistant layer is generally
formed on the microsphere and then is chemically activated to allow
attachment of the capture probe.
Analyte Detection Using Resonant Light Scattering
Assays carried out with the microspheres of the present invention
may make use of the specific interaction of binding pairs, one
member of the pair located on the surface of the microsphere (also
referred to as the "probe", "binding partner", "receptor", or
grammatically similar terms) and the other member of the pair
located in the sample (referred to as the "target", "analyte", or
grammatically similar terms). Generally the analyte carries at
least one so-called "determinant" or "epitopic" site, which is
unique to the analyte and has enhanced binding affinity for a
complementary probe site.
The nature of assay types possible with the microspheres of the
invention varies considerably. Probe/target binding pairs may, for
example, be selected from any of the following combinations, in
which either member of the pair may be the probe and the other the
analyte: antigen and specific antibody; antigen and specific
antibody fragment; folic acid and folate binding protein; vitamin
B12 and intrinsic factor; Protein A and antibody; Protein G and
antibody; polynucleotide and complementary polynucleotide; peptide
nucleic acid and complementary polynucleotide; hormone and hormone
receptor; polynucleotide and polynucleotide binding protein; hapten
and anti-hapten; lectin and specific carbohydrate; enzyme and
cofactor; enzyme and substrate; enzyme and inhibitor; biotin and
avidin or streptavidin; and hybrids thereof, and others as known in
the art. Binding pairs may also include members that form covalent
bonds, such as, sulfhydryl reactive groups such as maleimides and
haloacetyl derivatives, and amine reactive groups such as
isothiocyanates, succinimidyl esters, sulfonyl halides, and
carbodiimide reactive groups such as carboxyl and amino groups.
Specific examples of binding assays include those for naturally
occurring targets, for example, antibodies, antigens, enzymes,
immunoglobulin (Fab) fragments, lectins, various proteins found on
the surface of cells, haptens, whole cells, cellular fragments,
organelles, bacteriophage, phage proteins, viral proteins, viral
particles and the like. These may include allergens, pollutants,
naturally occurring hormones, growth factors, naturally occurring
drugs, synthetic drugs, oligonucleotides, amino acids,
oligopeptides, chemical intermediates, and the like. Practical
applications for such assays include for example, monitoring health
status, detection of drugs of abuse, pregnancy and pre-natal
testing, donor matching for transplantation, therapeutic dosage
monitoring, detection of disease, e.g. cancer antigens, pathogens,
sensors for biodefense, medical and non-medical diagnostic tests,
and similar applications known in the art.
Assays using the microspheres of the invention may be done using
various specific resonant light scattering protocols and
instrumentation as described by Prober et al., supra. For example,
analyte binding to a microsphere may be detected and the amount of
analyte in the sample may be determined. In general, when
determining binding of an analyte by resonant light scattering
methods, at least two measurements are made, one before exposing
the particles to the analyte to establish a baseline, and one after
exposing the particles to the analyte. The determination of binding
is done by comparing the two spectra and is thus typically a
"differential" measurement. Specifically, to detect binding of an
analyte to a capture probe, at least one capture probe is applied
to the nanoparticle composite-coated microspheres of the invention.
The microspheres are optionally scanned, (i.e., irradiated with
light of varying wavelength, over an analytical wavelength range
within an optical wavelength range) one or more times over the
analytical wavelength range to produce at least one first reference
resonant scattering spectrum for each particle. The microspheres
are scanned using a light scanning source such as a scanning diode
laser or tunable dye laser. In principle, any optical wavelength
range is applicable for the measurements of this invention.
Preferably, the optical wavelength range is from about 275 to about
1900 nanometers, more preferably from about 600 to about 1650
nanometers. Preferably, the analytical wavelength range has a span
of about 1 nanometers to about 20 nanometers, more preferably about
10 nanometers in width. More preferably the analytical wavelength
range has a span of 10 nanometers from about 770 to about 780
nanometers.
The microspheres are then contacted with a sample suspected of
containing an analyte. The microspheres are then scanned over the
analytical wavelength range using the light scanning source one or
more times to produce at least one second binding resonant light
scattering spectrum for each particle. Detection of analyte binding
is done by comparing either any one of the second binding resonant
light scattering spectra to any one of the first reference resonant
light scattering spectra, preferably the one most recently
obtained, or any one of the second binding resonant light
scattering spectra with a previous second binding resonant light
scattering spectrum in the series. The amount of analyte in the
sample may be determined by comparing the differences between the
two compared resonant light scattering spectra, specifically, the
degree of shift of one or more of the spectral features in the
scattering spectrum observed upon binding. The amount of analyte in
the sample may then be determined from a calibration curve prepared
using known standards, as is well known in the art.
The microspheres of the invention may also be used for particle
identification, a combination of particle identification and
detection of binding, identification of analytes, and detection of
analyte dissociation, as described by Prober et al., supra.
EXAMPLES
The present invention is further defined in the following Examples.
It should be understood that these Examples, while indicating
preferred embodiments of the invention, are given by way of
illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various uses and conditions.
The meaning of abbreviations used is as follows: "min" means
minute(s), "h" means hour(s), ".mu.L" means microliter(s), "mL"
means milliliter(s), "L" means liter(s), "nm" means nanometer(s),
"mm" means millimeter(s), "cm" means centimeter(s), ".mu.m" means
micrometer(s), "pm" means picometer(s), "mM" means millimolar, "M"
means molar, "mmol" means millimole(s), ".mu.mole" means
micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "mg"
means milligram(s), "g" means the gravitation constant, "rpm" means
revolutions per minute.
Example 1
Microspheres having a Borosilicate Glass Core and a Nanoparticle
Composite Silicon Oxide Coating Comprising a Bioactive Probe
The purpose of this Example was to prepare microspheres having a
borosilicate glass core and a nanoparticle composite silicon oxide
coating comprising a bioactive probe. Borosilicate glass microbeads
were coated with a nanoparticle composite coating formed by
reacting colloidal silicon oxide with tetraethoxylorthosilane. The
bioactive probe biotin was then coupled to the nanocomposite
silicon oxide-coated microspheres.
Preparation of Nanocomposite Silicon Oxide-Coated Glass
Microspheres:
Borosilicate microbeads having the composition expressed by the
formula:
(Si.sub.1-xB.sub.yAl.sub.y'Na.sub.y''O.sub.(2-2x+3/2y+3/2y'+1/2y'')).sub.-
1-a(AO.sub.z).sub.a wherein x=y+y'+y''; y=0.213; y'=0.0258; and
y''=0.035; a=0.002; A is any of, or a combination of Fe, Ca, and K;
and 1.5.gtoreq.z.gtoreq.0, were obtained from MO-SCI Specialty
Products, LLC. (a subsidiary of MO-SCI Corporation, Rolla, Mo.).
The microbeads were sieved between minus 25 and plus 20 .mu.m
screens by the supplier.
The microbeads (25 g) were added to a 1-L round-bottom flask
equipped with a dropping funnel. The round-bottom flask assembly
was purged with nitrogen for one hour. Then, 170 g of
tetraethoxylorthosilane (Aldrich, Milwaukee, Wis.) and 170 grams of
colloidal silicon oxide solution (HS-30, 30 wt % in water; Nyacol
Products, Ashland, Mass.) were added to the flask. The mixture was
refluxed for 2 h, after which the product was filtered and rinsed
with alcohol and water. The product was screened on 20 to 25 mesh
screens prior to attachment of the bioactive probe.
The thickness of the nanoparticle composite silicon oxide coating
was determined to be approximately 400 to 600 nm from fracture
scanning electron microscopy images.
Preparation of Biotinylated Nanocomposite Silicon Oxide-Coated
Glass Microspheres:
A mixture of about 1 g of the nanocomposite silicon oxide-coated
microspheres, 1.0 mL of 3-aminopropyltrimethoxysilane (APS), 300
.mu.L of pyridine, and 25 mL of dry toluene in a 50-mL flask was
gently refluxed under nitrogen overnight (16-18 h). After cooling,
the liquid layer was decanted and the residual microspheres were
washed 3 times with 10 mL portions of dry toluene. The microspheres
were cured at 100.degree. C. overnight (16-18 h) under nitrogen.
The cured microspheres were stored under nitrogen at room
temperature.
The APS treated microspheres (100 mg) were combined with 200 .mu.L
of a 50 mM solution of Sulfo-NHS-SS-Biotin (Pierce Biotechnology,
Rockford, Ill., Cat #21331) in 400 .mu.L of 25% DMF/0.1 M sodium
bicarbonate, pH 8.3. The resulting suspension was mixed at room
temperature overnight on a rotating platform. Then, the
microspheres were washed multiple times with 0.1 M sodium phosphate
buffer, pH 7.4 and water, and then centrifuged at 13,000 rpm for 5
min into a Centricon.RTM. Centrifugal Filter Unit, YM-100
(Millipore Corp, Billerica, Mass.). The microspheres were stored
dry at 4.degree. C. until further use.
Examples 2 and 3
The purpose of these Examples was to demonstrate the improved
sensitivity obtained with borosilicate microbeads having a
nanocomposite silicon oxide coating compared to the uncoated
microbeads in a bioassay using resonance light scattering
detection.
The resonant light scattering of the biotinylated-nanocomposite
silicon oxide-coated microspheres was measured using the method and
instrumentation described below.
Five milliliters of PBS buffer (Sigma-Aldrich, St. Louis, Mo., Cat
#3813,) was flushed through a closed-top optical cell (034), [cell
cover shown as 036] shown in FIG. 1, which contained a
micro-machined silicon substrate containing inverted pyramidal pits
to stabilize the position of the microspheres. Approximately 1
.mu.L of the biotinylated-nanocomposite silicon oxide-coated
microspheres, prepared as described in Example 1, (dry) were placed
into 200 .mu.L of PBS buffer and gently agitated. Then, 1 .mu.L of
the microsphere suspension (035) was pipetted onto the silicon
substrate in the cell and the cell was closed. The cell was placed
on a translation stage (033) in the detection apparatus, as shown
in FIG. 1. The microspheres were manipulated by flowing PBS
solution through the cell. The microspheres that remained in place
were scanned three times using the detection system, described
below.
The microscope (026) (Model U-KMAS, Olympus Industrial) was set up
for bright field illumination using a diode laser (023) (Model
Velocity 6312, New Focus, Inc.), operating at constant current, as
the light source. The output of the laser passed through an
electro-optic power controller (024) (Model MI-10450-NIR, Brockton
Electro-Optics) which was used to flatten the gain structure of the
laser output and control the power of the laser radiation delivered
to the microscope. Upon exiting the power controller the laser beam
passed through a holographic diffuser (025) (Model
LSD5GL3-2.75/0.25, Physical Optics) spinning at 1800 rpm. This
spinning diffuser serves to eliminate the laser speckle pattern in
the illumination field, which would otherwise interfere with the
acquisition and analysis of image data. The standard beam splitter
installed by the microscope manufacturer was replaced by a
pellicle-type beam splitter (027) (National Photocolor) in order to
eliminate interference fringes in the image. To acquire scattering
spectra from a multiplicity of particles simultaneously, a set of
particles was first placed in the field of view of the microscope
and focused with the objective lens (029) (Model UMPLFL 20XW,
Olympus Industrial). Once the particles of interest were in the
field of view and focused, the laser was scanned in wavelength,
typically from 780 to 770 nm in 20 s. During this scan, the digital
camera (028) (Model KP-F120CL-S5-R2, Hitachi Instruments) acquired
a complete scattered light image of the field of view at each
wavelength. Each image was captured by an image capture board (031)
(PCI-1428, National Instruments) installed in a personal computer
(032) (Dell Precision 370 Workstation). Custom software was written
to store each image. A wavelength scan resulted in a set of linked
images, one for each wavelength in the scan. A typical Image is
shown in FIG. 2. To determine the scattering spectrum of each
particle in the field of view from a set of wavelength-linked
Images, software was written to identify a representative region or
regions of the image corresponding to each particle. For example, a
portion of the ring-shaped scattered light image (106) surrounding
the bright spot of reflected light (107) at each particle center as
seen in the image of FIG. 2. In this Example, the incident and
scattered light beams were polarized independently, with the two
axes of polarization parallel to each other. This resulted in
sectors of scattered light centered approximately at the 12:00,
3:00, 6:00, and 9:00 positions of the circle as indicated for the
center image of FIG. 2 by the numbers 12, 3, 6, and 9 respectively.
Theory predicts, and results confirm, that scattered light spectra
from the "12" and "6" regions are equivalent and scattered light
spectra from the "3" and "9" regions are equivalent. Furthermore,
spectra from the two pairs of sectors are different from one
another.
After three wavelength scans, the PBS was replaced with 2500 .mu.L
of a 2.times.SSC buffer, which was prepared by dilution of a
20.times. stock SSC Buffer (Sigma-Aldrich, St. Louis, Mo., Cat
#S-6639,) at a flow rate of 16 .mu.L/s and 3 wavelength scans were
performed as described above. The refractive index of the
2.times.SSC buffer was measured on an ARIAS 500 refractometer
(Reichert Analytical Instruments, Depew, N.Y.) and was found to be
1.33788. The 2.times.SSC buffer served as a control for the
resonant light scattering measurements. The 2.times.SSC buffer in
the optical cell was replaced with 2500 .mu.L of PBS buffer and 3
wavelength scans were taken. A 2500 .mu.L solution of streptavidin
(Pierce Biotechnology, Cat 21125) in PBS (50 .mu.g/mL) was injected
into the optical cell at a flow rate .mu.L/s. At the end of the
injection, 3 wavelength scans were taken. The microspheres were
washed with 2500 .mu.L of PBS at a flow rate of 16 .mu.L/s to
remove the unbound streptavidin and then three more wavelength
scans were obtained.
The resonance light scattering spectra of the prescan, refractive
index scan and the final scans were auto-analyzed using custom
software. The auto-annulus software performed an image intensity
thresholding of the pixels obtained in a 60-degree arc and computed
each of the values per wavelength. The values calculated are
combined to form a spectrum for each particle measured under each
experimental condition. The resultant spectral shift between the
pre and post scans was calculated using custom software. The mean
shift values obtained upon streptavidin binding for 8 microspheres
are given in Table 1.
For comparison, borosilicate microspheres having the same
composition but not having the nanocomposite silicon oxide coating
were biotinylated as described above. These microspheres were
tested using the resonance light scattering method described above.
The mean shift values obtained upon streptavidin binding for 16
microspheres are given in Table 1.
TABLE-US-00001 TABLE 1 Resonance Light Scattering Shift Values
Obtained Upon Streptavidin Binding to Biotinylated Glass
Microspheres Median Shift Mean Shift Standard Glass 2 .times. SSC
Streptavidin Deviation Example Microspheres pm pm pm 2 silicon
oxide 284 507 170 coated 3, No silicon 295 296 47 Comparative oxide
coating
As can be seen from the data in the Table, the biotinylated,
nanocomposite silicon oxide coated-microspheres gave a
significantly larger shift than the biotinylated, uncoated
microspheres upon exposure to streptavidin (i.e., a 71% shift
increase). The shifts obtained upon exposure to the control
2.times.SSC solution were comparable for both types of particles,
indicating that the particles gave essentially equivalent shifts
due to the refractive index change produced by the 2.times.SSC
solution. This result suggests that the enhanced shift obtained
with the biotinylated, nanocomposite silicon oxide
coated-microspheres was due to the silicon oxide coating, and not
to any inherent difference in the resonance light scattering
properties of the borosilicate glass microspheres used.
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