U.S. patent application number 10/172428 was filed with the patent office on 2003-11-13 for nanoparticle probs with raman spectrocopic fingerprints for analyte detection.
This patent application is currently assigned to Northwestern University. Invention is credited to Cao, Yunwei, Jin, Rongchao, Mirkin, Chad A..
Application Number | 20030211488 10/172428 |
Document ID | / |
Family ID | 29407691 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211488 |
Kind Code |
A1 |
Mirkin, Chad A. ; et
al. |
November 13, 2003 |
Nanoparticle probs with Raman spectrocopic fingerprints for analyte
detection
Abstract
The invention encompasses reagents comprising particles with at
least one Raman dye and a specific binding members bound thereto
and methods of using such reagents. The invention also encompases
reagents of a specific binding member and two or more different
Raman dyes and methods for using such reagents. New types of
particle probes having a specific binding member bound thereto are
described. These reagents are used in a novel detection strategy
that utilizes the catalytic properties of the Au nanoparticles to
generate a silver coating that can behave as a surface-enhanced
Raman scattering (SERS) promoter for the dye-labeled particles that
have been captured by target and an underlying chip in microarray
format. The strategy provides the high sensitivity and high
selectivity attributes of grey-scale scanometric detection but
provides a route to multiplexing and ratioing capabilities since a
very large number of probes can be designed based upon the concept
of using a Raman tag as a spectroscopic fingerprint in detection.
These spectra are used as fingerprints to differentiate
oligonucleotide or other targets in one solution. This method has
been used to distinguish six dissimilar DNA targets with six Raman
labeled nanoparticle probes, and also two RNA targets with single
nucleotide polymorphisms (SNPs).
Inventors: |
Mirkin, Chad A.; (Wilmette,
IL) ; Cao, Yunwei; (Evanston, IL) ; Jin,
Rongchao; (Evanston, IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Northwestern University
633 Clark Street
Evanston
IL
60208
|
Family ID: |
29407691 |
Appl. No.: |
10/172428 |
Filed: |
June 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60378538 |
May 7, 2002 |
|
|
|
60383630 |
May 28, 2002 |
|
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|
Current U.S.
Class: |
435/6.12 ;
436/525 |
Current CPC
Class: |
Y02A 50/30 20180101;
G01N 33/583 20130101; G01N 33/587 20130101; G01N 33/54373 20130101;
C12Q 1/6816 20130101; C12Q 1/6816 20130101; C12Q 2565/632 20130101;
C12Q 2565/519 20130101; C12Q 2537/125 20130101 |
Class at
Publication: |
435/6 ;
436/525 |
International
Class: |
C12Q 001/68; G01N
033/553 |
Claims
What is claimed:
1. A reagent comprising a particle having bound there to at least
one Raman label and a specific binding member wherein the Raman
label can be activated to provide a SERS effect.
2. The reagent of claim 1 wherein the particle has two or more
different Raman labels.
3. The reagent of claim 1 wherein the specific binding member is a
DNA, RNA, polypeptide, antibody, antigen, carbohydrate or small
molecule.
4. The reagent of claim 1 wherein the particle is a gold, Ag, Cu,
Pt, Ag/Au, Pt/Au, Cu/Au coreshell and alloy particles.
5. The reagent of claim 1 where the Raman label is activated by a
silver, gold or copper stain.
6. A reagent comprising a specific binding member having two or
more different known labels bound thereto.
7. A reagent of claim 6 wherein the specific binding member is a
DNA, RNA, antibody, antigen, polypeptide or carbohydrate.
8. A method for detecting an analyte comprising: (a) forming a
complex of the reagent of claim 1 and the analyte; (b) binding the
complex to a substrate; (c) staining the complex on the substrate
to activate the SERS effect in the Raman label; and (d) measuring
the SERS effect.
9. The method of claim 8 wherein the complex is bound to the
substrate through one or more specific binding substances.
10. A method for detecting an analyte comprising: (a) binding the
analyte to a substrate; (b) complexing the reagent of claim 1 with
the analyte on the substrate (c) staining the complex on the
substrate to activate the SERS effect in the Raman label; (d)
measuring the SRS effect.
11. The method of claim 10 wherein the reagent is indirectly bound
to the analyte on the substrate through one or more specific
binding substances.
12. The method of claim 8 or 10 wherein the target analyte
comprises an antibody, an antigen, a hapten, a receptor, a ligand,
a protein, a peptide, a polypeptide, a nucleic acid, a membrane or
membrane fraction, a lipid, a membrane-protein complex, a
carbohydrate, a virus, a cell or macromolecule or molecular
complex.
13. The method of claim 8 or 10 wherein the specific binding member
comprises an antibody, an antigen, a receptor, a ligand, a protein,
a polypeptide, small molecule or a nucleic acid.
14. The method of claim 8 or 10 wherein the substrate has a
plurality of different first specific binding member attached
thereto in an array to allow for the detection of multiple types of
target analytes.
15. The method of claim 8 or 10 wherein the substrate comprises a
glass slide, microplate well, beads or optical fiber.
16. A method for detecting for the presence or absence of one or
more target analytes in a sample, the target analytes having at
least two binding sites, said method comprising: providing a
substrate having bound thereto one or more types of a first
specific binding member for immobilizing the target analyte onto
said substrate; providing one or more types of particles having
bound thereto (a) one or more Raman labels; and (b) a second
specific binding member for binding to a specific target analyte,
wherein (i) the Raman active label bound to each type of particle
is different and serves as an identifier for a specific target
analyte; (ii) the second specific binding member bound to each type
of particle is different and is targeted to a specific target
analyte; and (iii) the Raman label comprises at least one Raman
label. contacting the particles, the sample and the substrate under
conditions effective for specific binding interactions between the
target analyte and first and second specific binding member so as
to form a test substrate having particles complexed thereto in the
presence of one or more target analytes in the sample; contacting
the test substrate with a staining material to produce a detection
substrate having a surface capable of causing surface-enhanced
Raman scattering (SERS); and determining for the presence of said
particle complexes on said detection substrate as an indication of
the presence of one or more target analytes in the sample by
obtaining and analyzing a SERS spectrum.
17. A method for detecting for the presence or absence of one or
more target nucleic acids in a sample, the sequence of the nucleic
acid having at least two portions, said method comprising:
providing a substrate having oligonucleotides bound thereto, the
oligonucleotides bound to the substrate having a sequence that is
complementary to a first portion of the nucleic acid; providing one
or more types of particles comprising oligonucleotides bound
thereto and a Raman active label bound to a portion of the
oligonucleotides, wherein (i) at least some of the oligonucleotides
attached to each type of particle have a sequence that is
complementary to a second portion of the sequence of a specific
target nucleic acid; and (ii) the Raman label bound to each type of
particles is different and serves as an identifier for a specific
target nucleic acid, said Raman label comprising at least one Raman
label. contacting the particles, the substrate, and the sample
under conditions effective for hybridization of the
oligonucleotides bound to the substrate with the first portion of
the nucleic acid and for hybridization of the oligonucleotides
attached to the particle with the second portion of the nucleic
acid so as to form a test substrate having one or more particle
complexes bound thereto when one or more target nucleic acids are
present in said sample; contacting the test substrate with a
staining material to produce a detection substrate having a surface
capable of causing surface-enhanced Raman scattering (SERS); and
determining for the presence of said particle complexes on said
detection substrate as an indication of the presence of one or more
target nucleic acids in the sample by obtaining and analyzing a
SERS spectrum.
18. A method for detecting for the presence or absence of a target
nucleic acid in a sample, the sequence of the nucleic acid having
at least two portions, said method comprising: providing a
substrate having oligonucleotides bound thereto, the
oligonucleotides bound to the substrate having a sequence that is
complementary to a first portion of the nucleic acid; providing a
particle comprising oligonucleotides bound thereto and a Raman
label bound to a portion of the oligonucleotides, wherein (i) at
least some of the oligonucleotides attached to the particle have a
sequence that is complementary to a second portion of the nucleic
acid; and (ii) the Raman label bound to particles serves as an
identifier for the target nucleic acid, said Raman label comprising
at least one Raman label providing a detectable or measurable Raman
scattering signal when illuminated by radiation capable of inducing
a Raman scattering; contacting the particles, the substrate, and
the sample under conditions effective for hybridization of the
oligonucleotides bound to the substrate with the first portion of
the nucleic acid and for hybridization of the oligonucleotides
attached to the particle with the second portion of the nucleic
acid so as to form a test substrate having a particle complex bound
thereto when said target nucleic acid is present in said sample;
contacting the test substrate with a staining material to produce a
detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and determining for the
presence of said particle complex on said detection substrate as an
indication of the presence of the target nucleic acid in the sample
by obtaining and analyzing a SERS spectrum.
19. A method for screening one or more molecules to determine
whether the molecule is a ligand to one or more specific receptors,
the molecules are present in a sample, said method comprising:
providing a substrate having bound thereto one or more specific
receptors; providing conjugates comprising particles,
oligonucleotides bound to the particles, a Raman active label bound
to a portion of the oligonucleotides, and the molecule from said
sample bound to a portion of the oligonucleotides, wherein said
Raman active label comprising at least one Raman active molecule
providing a detectable or measurable Raman scattering signal when
illuminated by radiation capable of inducing a Raman scattering;
contacting the particles, sample and substrate under conditions
effective for specific binding interactions between the molecule
bound to the particles with the specific receptor bound to the
substrate so as to form a test substrate having particles complexed
thereto when the molecule is a ligand to a specific receptor;
contacting the test substrate with a staining material to produce a
detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and determining for the
presence of said particle complexes on said detection substrate as
a confirmation of a ligand to a specific receptor by obtaining and
analyzing a SERS spectrum.
20. A test kit comprising the reagent of claim 1 in one container
and a Raman enhancer stain in another container.
21. A test kit comprising the reagent of claim 1 in one container
and a silver, gold or copper stain Raman enhancer in another
container.
22. A fiber optic detection device comprising a bundle of optical
fibers terminating with ends of the optical fiber wherein a
plurality of the optical fibers have a reagent of claim 1 located
at the ends of the optical fiber.
23. The fiber optic detection device of claim 20 wherein two or
more of the reagents of claims 1 at the ends of the optical fiber
have different specific binding member and different Raman labels.
Description
CROSS-REFERENCE
[0001] This application claims priority based on U.S. provisional
applications Nos. 60/378,538, filed May 7, 2002 (case no. 02-338);
and 60/383,630, filed May 28, 2002 (case no. 02-338-A) which are
hereby incorporated by reference in their entirety. The work
described in this application has been supported in part from
grants from the Air Force Office of Scientific Research, DARPA, and
the NSF. Accordingly, the United States government may have some
rights to the invention.
BACKGROUND OF THE INVENTION
[0002] The development of high-sensitivity, high-selectivity
detection formats for chemical and biological molecules is of
paramount importance for realizing the full potential of genomics
and proteomics advances made over the past decade..sup.1-4 High
density gene chips have made it possible to monitor the levels of
expression of thousands of genes simultaneously. Lower density
chips have shown promise for both laboratory and clinical
identification of many potential biohazards in one sample. Although
the core accepted and utilized labeling technology is currently
based upon molecular fluorophore markers, recent advances in
nanoparticle technology have pointed toward systems with
significantly higher sensitivities and selectivities and
potentially more straightforward and versatile readout hardware
than conventional fluorescence-based approaches..sup.5-17 A strong
argument is being made for nanoparticles as the next generation
labeling technology for biodiagnostic research.
[0003] One of the most sensitive and selective detection formats
for DNA relies on oligonucleotide-functionalized nanoparticles as
probes, a particle-initiated silver developing technique for signal
enhancement, and a flatbed scanner for optical readout..sup.8 The
current demonstrated detection limit for this "scanometric DNA
detection" format is 100 .mu.M, and the utility of the system has
been demonstrated with short synthetic strands, PCR products, and
genomic DNA targets..sup.17,18 A limitation of this approach is
that it is inherently a one color system based upon grey scale. The
flexibility and applicability of all DNA detection systems benefit
from access to multiple types of labels with addressable and
individually discernable labeling information. In the case of
fluorescence, others have demonstrated that one can use multiple
fluorophores, including quantum dots, to prepare encoded structures
with optical signatures that depend upon the types of fluorophores
used and their signal ratio within the probes..sup.11,19 These
approaches typically use micron size probes so that they can obtain
encoded structures with the appropriate signal intensities and
uniformities. Moreover, in the case of molecular fluorophores, due
to overlapping spectral features and non-uniform fluorophore
photobleaching rates,.sup.1,11 this approach has several potential
complications.
[0004] The art describes the use of Surface Enhanced Raman
Spectroscopy (SERS) to detect various analytes. For example, U.S.
Pat. No. 5,306,403 describes a method and apparatus for DNA
sequencing using SERS. U.S. Pat. No. 5,266,498 describes the use of
SERS to detect analytes in general. U.S. Pat. No. 5,445,972
describes the use of a Raman label bound to a specific binding
molecule. U.S. Pat. No. 5,376,556 describes the use of SERS in
immunoassays. U.S. Pat. No. 6,127,120 describes the use of SERS,
the detection of nucleic acid and nucleic acid subunits. U.S. Pat.
Nos. 6,242,264 and 6,025,202 describe the use of silver to form a
SERS active substrate to enhance Raman scattering of adsorbed
molecules.
[0005] In the present invention, a reagent comprising a particle
having a Raman labeled and specific binding member bound to the
particle is used for assays of analytes. This reagent is
particularly advantageous in that it can be bound to a binding
partner analyte to form a complex and directly or indirectly bound
to a support. The Raman label in the label complex on the support
can then be SERS activated by staining, for example, silver, gold
or copper enhancement to achieve a SERS effect when irradiated with
a laser. Generally this complex is captured on a solid support and
treated with silver to provide a SERS effect. Alternatively, the
complex can be directly or indirectly reacted with an analyte which
has already been bound to a solid support substrate. In the present
invention, the SERS effect is produced near the time it is
measured. This reagent can advantageously include multiple
different Raman dyes bound to be particle carriers as a way
distinguishing particular carriers with particular specific binding
members as a way of indexing a vast number of reagent for multiplex
application.
[0006] Another advantageous reagent of this invention is a
conjugate of several different Raman dyes bound to a specific
binding substance such as DNA, RNA, polypeptide, antibody, antigen,
small molecules, etc. This also serves as a reagent indexing
tool.
[0007] The invention is particularly distinguished from the prior
art method in that the SERS technology is used in conjunction with
nanoparticle assay techniques to provide extraordinary sensitivity
and specificity of detection of analytes which is particularly
amenable to multiplexed determination of analtyes.
DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 illustrates a chip-based DNA detection method using
nanoparticles functionalized with oligonucleotides and Raman
labels.
[0009] FIG. 2 illustrates a flatbed scanner image of microarrays
after hybridized with nanoparticles functionalized with Cy3 labels,
before (A) and after (B) silver staining. (C) A typical Raman
Spectrum acquired from one of the silver stained spots. (D) A
profile of Raman intensity at 1192 cm.sup.-1 as a function of
position on the chip; the laser beam from the Raman instrument is
moved over the chip from left to right as defined by the line in
"B".
[0010] FIG. 3 illustrates the unoptimized detection limit of DNA
using the Raman scanning method. (A) A microarray-based sandwich
detection format; (B) A flatbed scanner image of microarrays for 20
fM target concentration after hybridized with nanoparticles
functionalized with Cy3.5 labels; (C) A typical Raman spectrum
acquired from one of the silver-stained spots; (D) A profile of
Raman intensity at 1199 cm.sup.-1 as a function of position on the
chip; the laser beam from the Raman instrument is moved over the
chip from left to right as defined by the line in "B".
[0011] FIG. 4 illustrate Left: The Raman spectra of six dyes. Each
dye correlates with a different color in our labeling scheme (see
rectangular boxes). Right: six DNA target analysis systems. The
information of target strand sequences were obtained from the web
site of the National Center for Biological Information
(http://www2.ncbi.nlm.nih.gov/Genbank/index.ht- ml).
[0012] FIG. 5 illustrates (A) Flatbed scanner images of
silver-stained microarrays and (B) corresponding Raman spectra. The
colored boxes correlate with the color coded Raman spectra in FIG.
4.
[0013] FIG. 6 illustrates the differentiation of two RNA targets
(Target 1: perfect; Target 2: with one-base difference).
[0014] FIG. 7 illustrates hybridization of pure RNA target 1 or 2,
or mixture of target 1 and 2, to microarrays (A) before stringency
wash, (B) after stringency wash.
[0015] FIG. 8 illustrates (A) Typical flatbed scanner images of
microarrays hybridized with nanoparticles, (1) before and (2) after
stringency wash but prior to silver enhancing, and (3) after silver
enhancing. Flatbed scanner image of microarrays hybridized with
nanoparticles (4) before stringency wash but after silver
enhancement. (B) A typical Raman spectrum (purple line) of the
silver enhanced spots in (4), compared with the spectrum (black
line) for mixed probes (1:1, probe 1:probe 2, after silver
enhacement). (C) Raman spectrum of the mixed probes (probe 1:probe
2, 1:1, after silver enhacement) compared with the spectra for
probe 1 (with only TMR, blue line) or probe 2 (with only Cy3, red
line).
[0016] FIG. 9 illustrates (A) typical flatbed scanner images of
nanoparticle-functionalized microarrays, (1) before and (2) after
stringency wash but prior to silver staining, and (3) after silver
staining. (B) Raman spectra (1550.about.1750 cm.sup.-1) from the
stained spots at different ratios of target 1 and target 2: (a)
1:0; (b) 5:1; (c) 3:1; (d) 1:1; (e) 1:2; (f) 1:3; (g) 1:5; (h) 0:1.
The full Raman spectra from 400 to 1800 cm.sup.-1 are shown in the
supporting information. The inset is a profile of Raman intensity
ratio (I.sub.b/I.sub.1) verse target ratio (T.sub.2/T.sub.1), where
I.sub.1 is the Raman Intensity at 1650 cm.sup.-1 (from probe 1: TMR
labeled gold oligonucleotide conjugate); I.sub.2 is the Raman
Intensity at 1588 cm.sup.-1 (from probe 2: Cy3 labeled gold
oligonucleotide conjugate).
[0017] FIG. 10 illustrates Raman spectra (400.about.1800 cm.sup.-1)
from the silver enhanced spots at different target 1 to target 2
ratios: (a) 1:0; (b) 5:1; (c) 3:1; (d) 1:1; (e) 1:3; (f) 1:5; and
(g) 0:1.
[0018] FIG. 11 illustrates (A) Scheme for screening protein-small
molecule interactions. (B) Flatbed scanner images of silver-stained
microarrays and (C) corresponding Raman spectra according to the
color coded scheme in FIG. 4. Biotin was labeled with Cy3, DIG with
Cy3.5 and DNP with Cy5. See supporting information for probe
preparation details.
[0019] FIG. 12 illustrates the Raman-based detection format for
proteins.
[0020] FIG. 13 illustrates (A1-4) Flatbed scanner images of
silver-stained microarrays associated with the protein-protein
screening experiments. (B) Color code for the Raman identification
of the probes in the silver stained spots; no cross reactivity is
observed. Anti-Mouse IgG was labeled with Cy3
modified-alkylthiol-capped poly adenine (A.sub.10), anti-ubiquitin
by Cy3.5 modified-alkylthiol-capped Poly adenine (A.sub.10), and
anti-human protein C by Cy5 modified-alkylthiol-capped Poly adenine
(A.sub.10). The A.sub.10 oligonucleotide spacer was used to enhance
the stability of the particle probes..sup.33
[0021] FIG. 14 illustrates the examples for creating Raman-labeled
nanoparticle probes with mulplexing capabilities. R1, R2, R3, are
different Raman dyes.
[0022] FIG. 15 illustrates the creation of massive nanoparticle
probes with multiple Raman labels.
[0023] FIG. 16 illustrates Left: Raman spectrum of a probe with two
Raman labels (Cy3:TMR=1:1, black line) after Ag staining in
microarray form compared with the spectra for probes with only TMR
(blue line) or Cy3 (red line). Right: Raman spectra of two-dye
functionalized nanoparticle probes as a function of Cy3 to TMR
ratio.
[0024] FIG. 17 illustrates Left and Right: two Raman spectra of
three-dye composite labels (black line) compared with the spectra
of TMR (blue line), Cy3 (red line) and Cy3.5 (green line).
[0025] FIG. 18 illustrates the microbead-based detection format
using the scanning Raman method.
[0026] FIG. 19 illustrates (A) and (B): The eight DNA target
analysis systems. Each of the probe strands was marked by a
single-dye or two-dye labels (see rectangular boxes and circles,
corresponding Raman spectra. The colored boxes and circles
correlate with the color coded Raman spectra in FIG. 20.
[0027] FIG. 20 illustrates the Raman spectra of six single dyes and
two mixed dyes, each spectra correlates with a different color in
our labeling scheme (see rectangular boxes and circles).
[0028] FIG. 21 illustrates microscopy image of silver-stained
microspheres. The colored circles correlate with the color coded
Raman spectra in FIG. 20.
[0029] FIG. 22 illustrates optical microscope image of aligned
silver-stained microspheres. The colored boxes correlate with the
color coded Raman spectra in FIG. 20.
[0030] FIG. 23 illustrates the fiberoptic-based detection format
using microbeads.
[0031] FIG. 24 illustrates the synthesis of Raman labeled
oligonucleotides.
SUMMARY OF THE INVENTION
[0032] The invention relates to reagents comprising particles
having specific binding members and Raman labels bound to the
particle wherein the particle can be treated with an enhancing
stain such as silver, gold or copper to provide a SERS effect when
irradiated.
[0033] This reagent may be complexed with analyte which binds to
the specific binding member and the resulting complex can be
directly or indirectly captured on a substrate. The Raman label in
the complex on the substrate is treated with a staining agent such
as silver, gold or copper to activate the SERS effect when
irradiated with a laser. Alternatively the analyte may be captured
on the solid support substrate and reacted directly or indirectly
with the reagent prior to staining and SERS measurement.
[0034] The invention also encompasses a reagent of a specific
binding substance having two or more different Raman labels bound
thereto.
[0035] The use of two or more different Raman labels on a reagent
particle or a specific binding substance provides a way of indexing
vast numbers of different particles and reagents for multiplexing
applications.
[0036] The invention includes methods of detecting analytes using
these reagents and test kits containing reagents and other
materials for carrying methods of the invention.
[0037] More particularly the present invention relates to particles
or carriers or Raman dye carriers functionalized with specific
binding members and Raman labels, coupled with surface-enhanced
Raman scattering (SERS) spectroscopy, to perform multiplexed
detection of analytes. This is exemplified for DNA and RNA targets
in FIG. 1. Although oligonucleotides can be directly detected by
SERS on aggregated particles,.sup.26 the structural similarities of
oligonucleotides with different sequences results in spectra that
are difficult to distinguish. Therefore, one must use different
Raman dyes to label different oligonucleotides to distinguish
oligonucleotide sequences..sup.20,21 Previously a SERS-based
detection methodology that allows for single or multiplexed
sandwich hybridization assay formats had not been demonstrated. In
part, this conspicuous technological absence is due to the
difficulty in reproducibly generating and functionalizing stable
SERS-active substrates.sup.23 as well as a lack of an appropriate
probe design strategy to enable multiplexed detection. To get the
benefits of high sensitivity and high selectivity detection coupled
with multiple labeling capabilities, a new type of particle probe
has been designed that can be used, for example, for DNA (or RNA)
detection (FIG. 1), but is equally applicable to other specific
binding substances. These probes consist of 13-nm gold particles
functionalized with Raman-dye labeled oligonucleotides. Particles
of various size, shape and materials may be used. The Raman
spectroscopic fingerprint, which can be designated through choice
of Raman label can be read out after silver enhancing via scanning
Raman spectroscopy (FIG. 1). Because the SERS-active substrate in
this strategy is generated prior to the detection event, a large
and reproducible Raman scattering response can be obtained.
[0038] Accordingly, in one embodiment of the invention, a method
for detecting for the presence or absence of one or more target
analytes in a sample, the target analytes having at least two
binding sites, is provided. The method comprises:
[0039] providing a substrate having bound thereto one or more types
of a first specific binding complements for immobilizing the target
analyte onto said substrate;
[0040] providing one or more types of particles having bound
thereto (a) one or more Raman active labels; and (b) a second
specific binding complement for binding to a specific target
analyte, wherein (i) the Raman active label bound to each type of
particle is different and serves as an identifier for a specific
target analyte; (ii) the second specific binding complement bound
to each type of particle is different and is targeted to a specific
target analyte; and (iii) the Raman active label comprises at least
one Raman active molecule providing a detectable or measurable
Raman scattering signal when illuminated by radiation capable of
inducing a Raman scattering;
[0041] contacting the particles, the sample and the substrate under
conditions effective for specific binding interactions between the
target analyte and first and second specific binding complements so
as to form a test substrate having particles complexed thereto in
the presence of one or more target analytes in the sample;
[0042] contacting the test substrate with a staining material to
produce a detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and
[0043] determining for the presence of said particle complexes on
said detection substrate as an indication of the presence of one or
more target analytes in the sample by obtaining and analyzing a
SERS spectrum.
[0044] In another embodiment of the invention, a method for
detecting for the presence or absence of one or more target nucleic
acids in a sample, the sequence of the nucleic acid having at least
two portions, is provided. The method comprises:
[0045] providing a substrate having a oligonucleotides bound
thereto, the oligonucleotides bound to the substrate having a
sequence that is complementary to a first portion of the nucleic
acid;
[0046] providing one or more types of particles comprising
oligonucleotides bound thereto and a Raman active label bound to a
portion of the oligonucleotides, wherein (i) at least some of the
oligonucleotides attached to each type of particle have a sequence
that is complementary to a second portion of the sequence of a
specific target nucleic acid; and (ii) the Raman active label bound
to each type of particles is different and serves as an identifier
for a specific target nucleic acid, said Raman active label
comprising at least one Raman active molecule providing a
detectable or measurable Raman scattering signal when illuminated
by radiation capable of inducing Raman scattering;
[0047] contacting the particles, the substrate, and the sample
under conditions effective for hybridization of the
oligonucleotides bound to the substrate with the first portion of
the nucleic acid and for hybridization of the oligonucleotides
attached to the particle with the second portion of the nucleic
acid so as to form a test substrate having one or more particle
complexes bound thereto when one or more target nucleic acids are
present in said sample;
[0048] contacting the test substrate with a staining material to
produce a detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and
[0049] determining for the presence of said particle complexes on
said detection substrate as an indication of the presence of one or
more target nucleic acids in the sample by obtaining and analyzing
a SERS spectrum.
[0050] In yet another embodiment of the invention, a method for
detecting for the presence or absence of a target nucleic acid in a
sample, the sequence of the nucleic acid having at least two
portions, is provided. The method comprises:
[0051] providing a substrate having oligonucleotides bound thereto,
the oligonucleotides bound to the substrate having a sequence that
is complementary to a first portion of the nucleic acid;
[0052] providing a particle comprising oligonucleotides bound
thereto and a Raman label bound to a portion of the
oligonucleotides, wherein (i) at least some of the oligonucleotides
attached to the particle have a sequence that is complementary to a
second portion of the nucleic acid; and (ii) the Raman active label
bound to particles serves as an identifier for the target nucleic
acid, said Raman active label comprising at least one Raman active
molecule providing a detectable or measurable Raman scattering
signal when illuminated by radiation capable of inducing a Raman
scattering;
[0053] contacting the particles, the substrate, and the sample
under conditions effective for hybridization of the
oligonucleotides bound to the substrate with the first portion of
the nucleic acid and for hybridization of the oligonucleotides
attached to the particle with the second portion of the nucleic
acid so as to form a test substrate having a particle complex bound
thereto when said target nucleic acid is present in said
sample;
[0054] contacting the test substrate with a staining material to
produce a detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and
[0055] determining for the presence of said particle complex on
said detection substrate as an indication of the presence of the
target nucleic acid in the sample by obtaining and analyzing a SERS
spectrum.
[0056] In yet another embodiment of the invention, a method for
detecting for the presence or absence of a single nucleotide
polymorphism in a nucleic acid in a sample, the sequence of the
nucleic acid having at least two portions, is provided. The method
comprises:
[0057] providing a substrate having a oligonucleotides bound
thereto, the oligonucleotides bound to the substrate having a
sequence that is complementary to a first portion of the nucleic
acid;
[0058] providing one or more types of particles comprising
oligonucleotides bound thereto and a Raman active label bound to a
portion of the oligonucleotides, wherein (i) at least some of the
oligonucleotides attached to each type of particle have a sequence
that is believed to be complementary to a second portion of the
sequence of the nucleic acid, said second portion of the sequence
of the nucleic acid is suspected of having a single nucleotide
substitution when compared to a wild type sequence of the nucleic
acid; and (ii) the Raman active label bound to each type of
particles is different and serves as an identifier for a specific
sequence having a single nucleotide substitution, said Raman active
label comprising at least one Raman active molecule providing a
detectable or measurable Raman scattering signal when illuminated
by radiation capable of inducing a Raman scattering;
[0059] contacting the particles, the substrate, and the sample
under conditions effective for hybridization of the
oligonucleotides bound to the substrate with the first portion of
the nucleic acid and for hybridization of the oligonucleotides
attached to the particle with the second portion of the nucleic
acid so as to form a test substrate having one or more particle
complexes bound thereto;
[0060] applying a stringency wash to the substrate to substantially
remove any non-specifically bound particles and any particle
complexes having oligonucleotides that are not complementary to the
second portion of the nucleic acid sequence;
[0061] contacting the test substrate with a staining material to
produce a detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and
[0062] determining for the presence of any particle complexes on
said detection substrate as an indication of the existence of a
single nucleotide morphism in said nucleic acid in the sample by
obtaining and analyzing a SERS spectrum.
[0063] In the foregoing methods, the nucleic acid is first
contacted with the substrate so that the first portion of the
nucleic acid sequence hybridizes with complementary
oligonucleotides bound to the substrate and then the nucleic acid
bound to the substrate is contacted with the particles having
oligonucleotides bound thereto so that at least some of the
oligonucleotides bound to the particles hybridize with the second
portion of the sequence of the nucleic acid bound to the
substrate.
[0064] In another aspect of the invention, the nucleic acid is
first contacted with the particles having oligonucleotides bound
thereto so that at least some of the oligonucleotides bound to the
particles hybridize with a second portion of the sequence of the
nucleic acid; and then contacting the nucleic acid bound to the
particles with the substrate so that the first portion of the
sequence of the nucleic acid bound to the particles hybridizes with
complementary oligonucleotides bound to the substrate. In another
embodiment, the substrate has a plurality of types of
oligonucleotides attached thereto in an array to allow for the
detection of multiple portions of a single type of nucleic acid,
the detection of multiple types of nucleic acids, or both.
[0065] In another aspect of the invention, at least two or more
different Raman active labels are used. The ratio of the two or
more types of Raman labels may be the same or different.
[0066] In yet another embodiment of the invention, a reagent is
provided. The reagent comprises having at least one type of Raman
active label bound thereto and a specific binding complement for
binding to a specific target analyte, wherein (i) the Raman active
label serves as an identifier for a specific target analyte; and
(ii) the Raman active label comprises at least one Raman active
molecule providing a detectable or measurable Raman scattering
signal when illuminated by radiation capable of inducing a Raman
scattering.
[0067] In yet another embodiment, a reagent is provided. The
reagent comprises a particle, oligonucleotides bound to the
particle and at least one type of Raman label bound to a portion of
the oligonucleotides, wherein at least some of the oligonucleotides
bound to the particle have a sequence that is complementary to at
least a portion of a target nucleic acid.
[0068] In another aspect of the invention, the reagent comprises a
particle, oligonucleotides bound to the particle, an
oligonucleotide connector having first and second portions, an
oligonucleotide having at least one type of Raman label bound
thereto, wherein at least some of the oligonucleotides bound to the
particles have a sequence that is complementary to the first
portion of the oligonucleotide connector, the oligonucleotide
having the Raman active label bound thereto has a sequence that is
complementary to the second portion of the oligonucleotide
connector, and at least a portion of the oligonucleotides bound to
the particles have a sequence that is complementary to a target
nucleic acid.
[0069] In yet another aspect of the invention, the reagent
comprises a particle, oligonucleotides bound to the particle, an
oligonucleotide connector having first and second portions, an
oligonucleotide having at least one type of Raman label bound
thereto, and an oligonucleotide having a specific binding
complement to a target analyte, wherein at least some of the
oligonucleotides bound to the particles have a sequence that is
complementary to the first portion of the oligonucleotide
connector, the oligonucleotide having the Raman active label bound
thereto has a sequence that is complementary to the second portion
of the oligonucleotide connector, and the oligonucleotide having
the specific binding complement bound thereto has a sequence that
is complementary to the second portion of the oligonucleotide
connector.
[0070] In another embodiment of the invention, a kit is provided
for the detection of one or more target analytes in a sample. The
kit has in one container a reagent comprising a particle having a
specific binding member and at least one Raman label bound to the
particle; a staining reagent; and a substrate having a capture
reagent. A representative kit comprises:
[0071] one or more types of conjugates comprising particles,
oligonucleotides bound to the particles, a Raman label bound to a
portion of the oligonucleotides, wherein (i) at least some of the
oligonucleotides attached to each type of particle have a sequence
that is complementary to a second portion of the sequence of a
specific target nucleic acid; and (ii) the Raman active label bound
to each type of particles is different and serves as an identifier
for a specific target nucleic acid, said Raman active label
comprising at least one Raman active molecule providing a
detectable or measurable Raman scattering signal when illuminated
by radiation capable of inducing a Raman scattering;
[0072] an optional substrate having oligonucleotides bound there,
the oligonucleotides bound to the substrate have a sequence that is
complementary to a first portion of a sequence of the target
nucleic acid; and
[0073] optional stain reagents for creating a substrate surface
capable of causing surface-enhanced Raman scattering (SERS).
[0074] In another embodiment of the invention, a kit is provided
for the detection of one or more target analytes in a sample, the
sequence of the nucleic acid having at least two portions. The kit
comprises:
[0075] particles comprising oligonucleotides bound thereto, a Raman
label bound to a portion of the oligonucleotides, wherein (i) at
least some of the oligonucleotides attached to the particle have a
sequence that is complementary to a second portion of the sequence
of the target nucleic acid; and (ii) the Raman active label bound
to the particles serves as an identifier for the target nucleic
acid, said Raman active label comprising at least one Raman active
molecule providing a detectable or measurable Raman scattering
signal when illuminated by radiation capable of inducing a Raman
scattering; and
[0076] an optional substrate having oligonucleotides bound there,
the oligonucleotides bound to the substrate have a sequence that is
complementary to a first portion of a sequence of the target
nucleic acid; and
[0077] In another embodiment of the invention, a kit is provided
for the detection of one or more target nucleic acids in a sample,
the sequence of the nucleic acid having at least two portions. The
kit comprises:
[0078] a first container including oligonucleotides having Raman
active labels attached thereto, wherein the oligonucleotides the
Raman active label comprising at least one Raman active molecule
providing a detectable or measurable Raman scattering signal when
illuminated by radiation capable of inducing a Raman
scattering;
[0079] a second container including conjugates comprising particles
and oligonucleotides bound to the particles, wherein at least some
of the oligonucleotides attached to each type of particle have a
sequence that is complementary to at least a portion of the
sequence of the oligonucleotides having Raman active labels;
and
[0080] an optional substrate having oligonucleotides bound there,
the oligonucleotides bound to the substrate have a sequence that is
complementary to a first portion of a sequence of the target
nucleic acid; and
[0081] optional stain reagents for creating a substrate surface
capable of causing surface-enhanced Raman scattering (SERS).
[0082] In another embodiment of the invention, a kit is provided
for the detection of one or more target nucleic acids in a sample,
the sequence of the nucleic acid having at least two portions. The
kit comprises:
[0083] one or more containers including oligonucleotides having one
or more types of Raman active labels attached thereto, wherein the
Raman active label comprising at least one Raman active molecule
providing a detectable or measurable Raman scattering signal when
illuminated by radiation capable of inducing a Raman
scattering;
[0084] a second container including conjugates comprising particles
and oligonucleotides bound to the particles, wherein at least some
of the oligonucleotides attached to each type of particle have a
sequence that is complementary to at least a portion of the
sequence of the oligonucleotides having Raman active labels;
and
[0085] an optional substrate having oligonucleotides bound there,
the oligonucleotides bound to the substrate have a sequence that is
complementary to a first portion of a sequence of the target
nucleic acid and optional staining material reagents.
[0086] In another embodiment of the invention, a method for
screening one or more molecules to determine whether the molecule
is a ligand to one or more specific receptors, the molecules are
present in a sample, is provided. The method comprises:
[0087] providing a substrate having bound thereto one or more
specific receptors;
[0088] providing reagents comprising particles, specific binding
substance bound to the particles, a Raman active label bound to a
portion of the specific binding substance, and the molecule from
said sample bound to a portion of the specific binding substance,
wherein said Raman active label comprising at least one Raman
active molecule providing a detectable or measurable Raman
scattering signal when illuminated by radiation capable of inducing
a Raman scattering;
[0089] contacting the particles, sample and substrate under
conditions effective for specific binding interactions between the
molecule bound to the particles with the specific receptor bound to
the substrate so as to form a test substrate having particles
complexed thereto when the molecule is a ligand to a specific
receptor;
[0090] contacting the test substrate with a staining material to
produce a detection substrate having a surface capable of causing
surface-enhanced Raman scattering (SERS); and
[0091] determining for the presence of said particle complexes on
said detection substrate as a confirmation of a ligand to a
specific receptor by obtaining and analyzing a SERS spectrum.
[0092] The invention also includes in another aspect a fiber optic
analyte detection device in which a particle reagent with specific
binding substance and Raman labels is associated with the ends of
optical fibers in an optical cable.
[0093] These and other embodiments of the invention will be
apparent in light of the detailed description below.
DETAILED DESCRIPTION OF THE INVENTION
[0094] (A) Definitions
[0095] "Analyte," as used herein, is the substance to be detected
in the test sample using the present invention. The analyte can be
any substance for which there exists a naturally occurring specific
binding member (e.g., an antibody, polypeptide, DNA, RNA, cell,
virus, etc.) or for which a specific binding member can be
prepared, and the analyte can bind to one or more specific binding
members in an assay. "Analyte" also includes any antigenic
substances, haptens, antibodies, and combinations thereof. The
analyte can include a protein, a peptide, an amino acid, a
carbohydrate, a hormone, asteroid, a vitamin, a drug including
those administered for therapeutic purposes as well as those
administered for illicit purposes, a bacterium, a virus, and
metabolites of or antibodies to any of the above substances.
[0096] "Analyte-analog", as used herein, refers to a substance
which cross reacts with an analyte specific binding member although
it may do so to a greater or lesser extent than does the analyte
itself. The analyte-analog can include a modified analyte as well
as a fragmented or synthetic portion of the analyte molecule so
long as the analyte analog has at least one epitopic site in common
with the analyte of interest.
[0097] "Analyte epitope," as used herein, denotes that part of the
analyte which contacts one member of the specific ligand binding
pair during the specific binding event. That part of the specific
binding pair member which contacts the epitope of the analyte
during the specific binding event is termed the "paratope."
[0098] "Analyte-mediated ligand binding event," as used herein,
means a specific binding event between two members of a specific
ligand binding pair, the extent of the binding is influenced by the
presence, and the amount present, of the analyte. This influence
usually occurs because the analyte contains a structure, or
epitope, similar to or identical to the structure or epitode
contained by one member of the specific ligand binding pair, the
recognition of which by the other member of the specific ligand
binding pair results in the specific binding event. As a result,
the analyte specifically binds to one member of the specific ligand
binding pair, thereby preventing it from binding to the other
member of the specific ligand binding pair.
[0099] "Ancillary Specific binding member," as used herein, is a
specific binding member used in addition to the specific binding
members of the captured reagent and the indicator reagent and
becomes a part of the final binding complex. One or more ancillary
specific binding members can be used in an assay. For example, an
ancillary specific binding member can be used in an assay where the
indicator reagent is capable of binding the ancillary specific
binding member which in turn is capable of binding the analyte.
[0100] "Associated," as used herein, is the state of two or more
molecules and/or particulates being held in close proximity to one
another.
[0101] "Capture reagent," as used herein, is a specific binding
member, capable of binding the analyte or indicator reagent, which
can be directly or indirectly attached to a substantially solid
material. The solid phase capture reagent complex can be used to
separate the bound and unbound components of the assay.
[0102] "Conjugate," as used herein, is a substance formed by the
chemical coupling of one moiety to another. An example of such
species include the reaction product of bovine serum albumin with
chemically activated theophylline molecules and the reaction
product of chemically activated Raman-active labels with a protein
molecule, such as an antibody, or with a ligand, such as
biotin.
[0103] "Enhancer," a stain such as a silver or gold stain that
provides for activating Raman labels on particles to produce a SERS
effect.
[0104] "Indicator reagent," as used herein comprises a detectable
label directly or indirectly attached to a specific binding member
or metal surface.
[0105] "Intervening molecule," as used herein, is any substance to
which both a specific binding pair member and a Raman-active label
are attached.
[0106] "Particles," as used herein, is any substance which can be
dispersed in a liquid and which will support the phenomenon of a
surface-enhanced Raman light scattering (SERS) or surface-enhanced
resonance Raman light scattering (SERRS). Examples of particles
include, but are not limited to: Colloids of gold or silver, Pt,
Cu, Ag/Au, Pt/Au, Cu/Au, coreshell or alloy particles; particles or
flakes of gold, silver, copper, or other substances displaying
conductance band electrons. As the particle surface participates in
the SERS and SERRS effect, flakes or particles of substances not
displaying conductance band electrons, which have been coated with
a substance which does, also become suitable particulates.
[0107] "Radiation," as used herein, is an energy in the form of
electromagnetic radiation which, when applied to a test mixture,
causes a Raman spectrum to be produced by the Raman-active label
therein.
[0108] "Raman label," as used herein, is any substance which
produces a detectable Raman spectrum, which is distinguishable from
the Raman spectra of other components present, when illuminated
with a radiation of the proper wavelength. Other terms for a
Raman-active label include dye and reporter molecule. Such labels
are shown on pp 25.
[0109] "SERRS (Surface Enhanced Resonance Raman Scattering)"
results when the adsorbate at a SERS active surface is in resonance
with the laser excitation wavelength. The resultant enhancement is
the product of the resonance and surface enhancement.
[0110] "SERS (Surface-Enhanced Raman Scattering)" means the
increase in Raman scattering exhibited by certain molecules in
proximity to certain metal surfaces.
[0111] "Specific binding member," as used herein, is a member of a
specific binding pair, i.e., two different molecules where one of
the molecules, through chemical or physical means, specifically
binds to the second molecule. In addition to antigen and
antibody-specific binding pairs, other specific binding pairs
include biotin and avidin, carbohydrates and lectins, complementary
nucleotide sequences (including probe and captured nucleic acid
sequences used in DNA hybridization assays to detect a target
nucleic acid sequence), complementary peptide sequences, effector
and receptor molecules, enzyme cofactors and enzymes, enzyme
inhibitors a and enzymes, cells, viruses and the like. Furthermore,
specific binding pairs can include members that are analogs of the
original specific binding member. For example a derivative or
fragment of the analyte, i.e., an analyte-analog, can be used so
long as it has at least one epitope in common with the analyte.
Immunoreactive specific binding members include antigens, haptens,
antibodies, and complexes thereof including those formed by
recombinant DNA methods or peptide synthesis.
[0112] "Test mixture," as used herein, means a mixture of the test
sample and other substances used to apply the present invention for
the detection of analyte in the test sample. Examples of these
substances include: Specific binding members, ancillary binding
members, analyte-analogs, Raman-active labels, buffers, diluents,
and particulates with a surface capable of causing a
surface-enhanced Raman spectroscopy, and others.
[0113] "Test sample," as used herein, means the sample containing
the analyte to be detected and assayed using the present invention.
The test sample can contain other components besides the analyte,
can have the physical attributes of a liquid, or a solid, and can
be of any size or volume, including for example, a moving stream of
liquid. The test sample can contain any substances other than the
analyte as long as the other substances do no interfere with the
specific binding of the specific binding member or with the analyte
or the analyte-analog. Examples of test samples include, but are
not limited to: Serum, plasma, sputum, seminal fluid, urine, other
body fluids, and environmental samples such as ground water or
waste water, soil extracts, air and pesticide residues.
[0114] (B) Reagents
[0115] The present invention contemplates the use of any suitable
particle having Raman labels and specific binding substances
attached thereto that are suitable for use in detection assays. In
practicing this invention, however, nanoparticles are preferred.
The size, shape and chemical composition of the particles will
contribute to the properties of the resulting probe including the
DNA barcode. These properties include optical properties,
optoelectronic properties, electrochemical properties, electronic
properties, stability in various solutions, pore and channel size
variation, ability to separate bioactive molecules while acting as
a filter, etc. The use of mixtures of particles having different
sizes, shapes and/or chemical compositions, as well as the use of
nanoparticles having uniform sizes, shapes and chemical
composition, are contemplated. Examples of suitable particles
include, without limitation, nano- and microsized core particles,
aggregate particles, isotropic (such as spherical particles) and
anisotropic particles (such as non-spherical rods, tetrahedral,
prisms) and core-shell particles such as the ones described in U.S.
patent application Ser. No. 10/034,451, filed Dec. 28, 2002 and
International application no. PCT/US01/50825, filed Dec. 28, 2002,
which are incorporated by reference in their entirety.
[0116] Nanoparticles usefuil in the practice of the invention
include metal (e.g. gold, silver, copper and platinum),
semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS)
and magnetic (e.g., ferromagnetite) colloidal materials. Other
nanoparticles useful in the practice of the invention include ZnS,
ZnO, TiO.sub.2, AgI, AgBr, HgI.sub.2, PbS, PbSe, ZnTe, CdTe,
In.sub.2S.sub.3, In.sub.2Se.sub.3, Cd.sub.3P.sub.2,
Cd.sub.3As.sub.2, InAs, and GaAs. The size of the nanoparticles is
preferably from about 1.4 nm to about 150 nm (mean diameter), more
preferably from about 5 to about 50 nm, most preferably from about
10 to about 30 nm. The nanoparticles may also be rods, prisms,
cubes, tetrahedra, or core shell particles.
[0117] Methods of making metal, semiconductor and magnetic
nanoparticles are well-known in the art. See, e.g., Schmid, G.
(ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A.
(ed.) Colloidal Gold: Principles, Methods, and Applications
(Academic Press, San Diego, 1991); Massart, R., IEEE Taransactions
On Magnetics, 17, 1247 (1981); Ahmadi, T. S. et al., Science, 272,
1924 (1996); Henglein, A. et al., J. Phys. Chem., 99, 14129 (1995);
Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27, 1530
(1988).
[0118] Methods of making ZnS, ZnO, TiO.sub.2, AgI, AgBr, HgI.sub.2,
PbS, PbSe, ZnTe, CdTe, In.sub.2S.sub.3, In.sub.2Se.sub.3,
Cd.sub.3P.sub.2, Cd.sub.3As.sub.2, InAs, and GaAs nanoparticles are
also known in the art. See, e.g., Weller, Angew. Chem. Int. Ed.
Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988);
Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53,
465 (1991); Bahncmann, in Photochemical Conversion and Storage of
Solar Energy (eds. Pelizetti and Schiavello 1991), page 251; Wang
and Herron, J. Phys. Chem., 95, 525 (1991); Olshavsky et al., J.
Am. Chem. Soc., 112, 9438 (1990); Ushida et al., J. Phys. Chem.,
95, 5382 (1992).
[0119] Suitable nanoparticles are also commercially available from,
e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold) and
Nanoprobes, Inc. (gold).
[0120] Presently preferred for use in detecting analytes are gold
nanoparticles. Gold colloidal particles have high extinction
coefficients for the bands that give rise to their beautiful
colors. These intense colors change with particle size,
concentration, interparticle distance, and extent of aggregation
and shape (geometry) of the aggregates, making these materials
particularly attractive for calorimetric assays. For instance,
hybridization of oligonucleotides attached to gold nanoparticles
with oligonucleotides and nucleic acids results in an immediate
color change visible to the naked eye.
[0121] (C) Attachment of Specific Binding Members
[0122] The particles, the specific binding member or both are
functionalized in order to attach to the particles. Such methods
are well known in the art. For instance, oligonucleotides
functionalized with alkanethiols at their 3'-termini or 5'-termini
readily attach to gold nanoparticles. See Whitesides, Proceedings
of the Robert A. Welch Foundation 39th Conference On Chemical
Research Nanophase Chemistry, Houston, Tex., pages 109-121 (1995).
See also, Mucic et al. Chem. Commun. 555-557 (1996) (describes a
method of attaching 3' thiol DNA to flat gold surfaces; this method
can be used to attach oligonucleotides to nanoparticles). The
alkanethiol method can also be used to attach oligonucleotides to
other metal, semiconductor and magnetic colloids and to the other
nanoparticles listed above. Other functional groups for attaching
oligonucleotides to solid surfaces include phosphorothioate groups
(see, e.g., U.S. Pat. No. 5,472,881 for the binding of
oligonucleotide-phosphorothioates to gold surfaces), substituted
alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4, 370-377
(1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103,
3185-3191 (1981) for binding of oligonucleotides to silica and
glass surfaces, and Grabar et al., Anal. Chem., 67, 735-743 for
binding of aminoalkylsiloxanes and for similar binding of
mercaptoaklylsiloxanes). Oligonucleotides terminated with a 5'
thionucleoside or a 3' thionucleoside may also be used for
attaching oligonucleotides to solid surfaces. The following
references describe other methods which may be employed to attached
oligonucleotides to nanoparticles: Nuzzo et al., J. Am. Chem. Soc.,
109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir,
1, 45 (1985) (carboxylic acids on aluminum); Allara and Tompkins,
J. Colloid Interface Sci., 49, 410-421 (1974) (carboxylic acids on
copper); Iler, The Chemistry Of Silica, Chapter 6, (Wiley 1979)
(carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem.,
69, 984-990 (1965) (carboxylic acids on platinum); Soriaga and
Hubbard, J. Am. Chem. Soc., 104, 3937 (1982) (aromatic ring
compounds on platinum); Hubbard, Acc. Chem. Res., 13, 177 (1980)
(sulfolanes, sulfoxides and other functionalized solvents on
platinum); Hickman et al., J. Am. Chem. Soc., 111, 7271 (1989)
(isonitriles on platinum); Maoz and Sagiv, Langmuir, 3, 1045 (1987)
(silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034 (1987)
(silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989)
(silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951 (1987)
(aromatic carboxylic acids, aldehydes, alcohols and methoxy groups
on titanium dioxide and silica); Lec et al., J. Phys. Chem., 92,
2597 (1988) (rigid phosphates on metals).
[0123] U.S. patent application Ser. Nos. 09/760,500 and 09/820,279
and international application nos. PCT/US01/01190 and
PCT/US01/10071 describe oligonucleotides functionalized with a
cyclic disulfide which are useful in practicing this invention. The
cyclic disulfides preferably have 5 or 6 atoms in their rings,
including the two sulfur atoms. Suitable cyclic disulfides are
available commercially or may be synthesized by known procedures.
The reduced form of the cyclic disulfides can also be used.
[0124] Those skilled in the art recognize a large variety of
methods by which antigen, antibodies, small molecules or
carbohydrates can be bound to particles.
[0125] (D) Substrates
[0126] Any substrate can be used which allows observation of the
detectable change. Suitable substrates include transparent solid
surfaces (e.g., glass, quartz, plastics and other polymers), opaque
solid surface (e.g., white solid surfaces, such as TLC silica
plates, filter paper, glass fiber filters, cellulose nitrate
membranes, nylon membranes), and conducting solid surfaces (e.g.,
indium-tin-oxide (ITO)). The substrate can be any shape or
thickness, but generally will be flat and thin. Preferred are
transparent substrates such as glass (e.g., glass slides or glass
beads) or plastics (e.g., wells of microtiter plates). The ends of
optical fiber in a fiber optical cable serve as a substrate in one
embodiment of the invention.
[0127] (E) Attachment of Capture Probes to a Substrate
[0128] Any suitable method for attaching an analyte to a substrate
may be used. For instance, oligonucleotides can be attached to the
substrates as described in, e.g., Chrisey et al., Nucleic Acids
Res., 24, 3031-3039 (1996); Chrisey et al., Nucleic Acids Res., 24,
3040-3047 (1996); Mucic et al., Chem. Commun., 555 (1996);
Zimmermann and Cox, Nucleic Acids Res., 22, 492 (1994); Bottomley
et al., J. Vac. Sci Technol. A, 10, 591 (1992); and Hegner et al.,
FEBS Lett., 336, 452 (1993).
[0129] When a substrate is employed, a plurality of capture probes
may be attached to the substrate in an array for detecting multiple
different target analytes. For instance, a substrate may be
provided with rows of spots, each spot containing a different type
of capture probes designed to bind a reagent analyte complex. A
sample containing one or more analytes is applied to each spot, and
the rest of the assay is performed in one of the ways described
above using appropriate reagents of the invention.
[0130] (F) Raman Labels
[0131] The Raman labels, can be any one of a number of molecules
with distinctive Raman scattering spectra. Unlike the enzymes used
in enzyme imununoassays, these label species can be stable, simple,
inexpensive molecules which can be chemically modified as
required.
[0132] The following attributes enhance the effectiveness of the
label in this application: (a) A strong absorption band in the
vicinity of the laser excitation wavelength (extinction coefficient
near 10.sup.4; (b) A functional group which will enable covalent
attachment to a specific binding member; (c) Photostability; (d)
Sufficient surface and resonance enhancement to allow detection of
analyte in the subnanogram range; (e) Minimal interference in the
binding interaction between the labeled and unlabeled specific
binding members; (f) Minimal exhibition of strong fluorescence
emission at the excitation-wavelength used; (g) A relatively simple
scattering pattern with a few intense peaks; and/or (h) Labels with
scattering patterns which do not interfere with each other so
several indicator molecules may be analyzed simultaneously.
[0133] The following is a listing of some, but not all potential
candidates for these Raman-active label:
4-(4-Aminophenylazo)phenylarsoni- c acid monosodium salt, arsenazo
I, basic fuchsin, Chicago sky blue, direct red 81, disperse orange
3, HABA (2-(4-hydroxyphenylazo)-benzoic acid), erythrosin B, trypan
blue, ponceau S, ponceau SS, 1,5-difluoro-2,4-dinitrobenzene,
cresyl violet and p-dimethylaminoazobenzene. The chosen labels may
be covalently attached to the specific binding members of interest
or attached or associated with.
[0134] An important aspect of the invention is that multiple Raman
or Raman labels may be bound to the particle to provide a
multicoding Rarnan labels for indexing different particles. Thus,
the invention includes a reagent which has multiple Raman dyes and
a specific binding substance, such as DNA, RNA, antibody, antigen,
small molecule bound to the particle.
[0135] The multiple Raman label also need not be bound to the
particle but may be complexed to the particle through specific
binding reactions. Thus, the invention encompasses multiple SERS
reagents bound to a specific binding ligand such as DNA, RNA,
antibody, antigen, small molecule, cell or virus. This embodiment
may be envisioned as follows:
Raman.sub.1-Raman.sub.2-Raman.sub.3-(specific binding ligand)
[0136] (G). Excitation Sources
[0137] In the preferred embodiment, a laser serves as the
excitation source. The laser may be of an inexpensive type such as
a helium-neon or diode laser. An operating lifetime of such lasers
may be in excess of 50,000 hours.
[0138] In one embodiment, a diode laser is used to excite at or at
the near IR spectrum, minimizing fluorescence interference. The
excitation sources used need not necessarily be monochromatic and
they also need not necessarily have to be of high intensity. Lamps
may also be used.
[0139] The SERS effect can be excited by direct illumination of the
surface or by evanescent waves from a waveguide beneath the
plasmon-active surface.
[0140] (H.) Raman Labeled Probes
[0141] Several different conjugates could be prepared from specific
binding members having different specificities, each type with a
different Raman active label having a distinctive scattering
pattern. Mixing these conjugates in an assay would allow the
simultaneous analysis of several different analytes in the same
sample. In another aspect of the invention, the conjugate may
include two or more different Raman labels.
[0142] It is important to note that in contrast with conventional
fluorescence-based chip detection, the ratio of Raman intensities
can be extracted from a single Raman spectrum using single laser
excitation. Moreover, the number of available Raman dyes is much
larger than the number of available and discernable fluorescent
dyes..sup.20,21,26 A Raman dye can be either fluorescent or
non-fluorescent. A minor chemical modification of a dye molecule
can lead to a new dye with different Raman spectra even though the
two dyes exhibit virtually indistinguishable fluorescence
spectra..sup.26 Therefore, this Raman fingerprinting method offers
potentially greater flexibility, a larger pool of available and
non-overlapping probes, and higher multiplexing capabilities than
conventional fluorescence-based detection approaches. This approach
has been extended to random array, bead based format where high
multiplexing capabilities are essential are underway.
[0143] (I). SERS Enhancement (Enhancer)
[0144] Initially, the Raman-labeled probes have little or no
detectable SERS activity. Staining material such as silver stains
provide strong SERS enhancment. When a substrate is employed, a
detectable change can be produced or further enhanced by silver
staining. Silver staining can be employed with any type of
nanoparticles that catalyze the reduction of silver. Preferred are
nanoparticles made of noble metals (e.g., gold and silver). See
Bassell, et al., J. Cell Biol., 126, 863-876 (1994); Braun-Howland
et al., Biotechniques, 13, 928-931 (1992). If the nanoparticles
being employed for the detection of a nucleic acid do not catalyze
the reduction of silver, then silver ions can be complexed to the
nucleic acid to catalyze the reduction. See Braun et al., Nature,
391, 775 (1998). Also, silver stains are known which can react with
the phosphate groups on nucleic acids.
[0145] Silver, gold or copper staining can be used to produce or
enhance a detectable change in any assay performed on a substrate,
including those described above. In particular, silver staining has
been found to provide a huge increase in sensitivity for assays
employing a single type of nanoparticle so that the use of layers
of nanoparticles can often be eliminated.
[0146] (J). Detection of Raman Scattering
[0147] Several methods are available for detecting Raman
scattering. These generally can be used with different types of
spectrometers. In SERS, the primary measurement is one of light
scattering intensity at particular wavelengths. SERS requires
measuring wavelength-shifted scattering intensity in the presence
of an intense background from the excitation beam. The use of a
Raman-active substance having a large Stokes shift simplifies this
measurement.
[0148] Several concepts for further simplifying the readout
instrument have been proposed. These include the use of wavelength
selective mirrors, filters or holographic optical elements for
scattered light collection.
[0149] Neither the angle of the incident light beam to the surface
nor the position of the detector is critical using SERS. With flat
surfaces positioning the surface of the laser beam at 60 degrees to
the normal is commonly done and detection at either 90 degrees or
180 degrees to the beam are standard. SERS excitation can be
performed in the near infrared range which would suppress intrinsic
sample fluorescence. It may also be possible to perform SERS-based
ligand binding assays using evanescent waves produced by optical
waveguides.
[0150] No signal development time is required as readout begins
immediately upon illumination and data can be collected for as long
as desired without decay of signal unless the excitation light is
extremely intense and chemical changes occur. The signal cannot
overdevelop as in systems dependent on optical absorbance. Unlike
fluorescent readout systems. SERS reporter groups will not
self-quench so the signal can be enhanced by increasing the number
of Raman reporter groups on the probe molecule. Fluorescent
molecules near the SERS-active surface will also be
surface-quenched.
[0151] (K.) Instrumentation
[0152] The present invention is adaptable for use as an automatic
analyzer. Since the instrument would monitor discrete Stokes
shifted spectral lines, the need for an elaborate monochromator
system is not necessary. Recent advances in state-of-the-art optics
technology, such as holographic optical elements, allow the design
of a suitable spectrometer with cost and complexity below that of
the laboratory grade device.
[0153] Optical readout energies as a result of SERS are above that
which require ultra-sensitive photon counting devices. In fact,
some SERRS spectrometers now in use incorporate silicon photodiode
detectors. The optical efficiency of a typical monochromator used
in a laboratory grade spectrometer is less than 10%. The advances
in optical materials and components mentioned above should make
possible two to three-fold increases in optical efficiency for a
simple spectrometer dedicated to only a few specific spectral
lines. This also addresses one of the previously major concerns,
blocking of the Rayleigh scattering line. With blocking
capabilities of newer filters on the order of 10.sup.-9,
substitution of filters for one or more stages of the typical
monochrometer system should be possible with significant cost
savings.
EXAMPLES
Example 1
[0154] Microarray Fabrication.
[0155] Oligonucleotide capture strands were immobilized onto the
SMPB-(succinimidyl 4-(malemidophenyl)-butyrate) functionalized
glass slide by spotting 5'-hexyl-thiol-capped oligoucloetides (1 mM
in a 0.15 M NaCl, pH 6.5 phosphate buffer solution (PBS, 10 mM
phosphate)) with a commercial arrayer (GMS 417 arrayer, Genotic
MicroSystems, Inc). After spotting the chip with the capture
oligonucleotides (.about.200 .mu.m spots), the chip was kept in a
humidity chamber for 12 hours to effect the coupling reaction
between SMPB and the hexylthiol-capped oligonucleotides. Then the
chip was washed copiously with Nanopure water. Passivation of the
areas of the chip surrounding the oligonucleotide spots was carried
out by immersing the chip in a solution of hexylthiol-capped
poly-adenine (A.sub.15) (0.1 mM) for 4 h and then in a solution of
3-mercapto-propane sulfonic acid, sodium salt (0.2 M) for 30
minutes to cap off the remaining SMPB sites. Finally, the chip was
washed with Nanopure water and dried by a microarray centrifuge
(2000 g).
Example 2
[0156] Synthesis and Purification of
Cy3-labeled-(propylthiol)-capped Oligonucleotides.
[0157] This Example describes the synthesis of an oligonucleotide
having a Raman label attached thereto: (3'HS-Cy3-A.sub.10-AAT CTC
AAC GTA CCT, (SEQ ID NO 1. in FIG. 19a)
3'HS-Cy3-A.sub.10-CTC-CCT-AAT-AAC-AAT) (SEQ ID NO. 25 in FIG.
1)
[0158] The Cy3-modified, (propylthiol)-capped oligonucleotides were
synthesized on a 1 .mu.mol scale using standard phosphoramidite
chemistry 5 with a Thiol-Modifier C3 S--S CPG (controlled-pore
glass) solid support on a commercial synthesizer (Expedite). The
Cy3-CE phospboramidite (Indodicarbocyanine 3,
1'-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-
-diisopropyl)-phosphoramidite (Glen Research) was used to
incorporate the Cy3 unit in the oligonucleotides. To aid
purification, the final dimethoxytrityl (DMT) protecting group was
not removed. After synthesis, the CPG-supported oligonucleotides
were placed in 1 mL of concentrated ammonium hydroxide for 8 h at
55 C.sup.o to cleave the oligonucleotide from the solid support and
remove the protecting groups from the bases. In each case, cleavage
from the solid support via the succinyl ester linkage produced a
mixed disulfide composed of the (mercaptopropyl) oligonucleotide
and a mercaptopropanol linker. After evaporation of anmmonia, the
crude oligonucleotides were purified by preparative reverse-phase
HPLC using an HP ODS Hypersil column (300 .DELTA., 250.times.10 mm,
retention time=32 min) with 0.03 M triethylammonium acetate (TEAA),
pH 7 and a 1%/min gradient of 95% CH.sub.3CN/5% 0.03 M TEAA at a
flow rate of 3 mL/min, while monitoring the UV signal of DNA at 254
nm and 550 nm. The DMT was cleaved by dissolving the purified
oligonucleotides in an 80% acetic acid solution for 30 min,
followed by evaporation; the oligonucleotides were redispersed in
500 .mu.L of water, and the solutions were extracted with ethyl
acetate (3.times.300 .mu.L). After evaporation of the solvent, the
oligonucleotides were redispersed in 400 .mu.L of a 0.1 M
dithiothreotol (DTT), 0.17 M phosphate buffer (pH 8) solution at
room temperature for 2 h to cleave the 3' disulfide. Aliquots of
this solution (<10 ODs) were purified through a desalting NAP-5
column (Amersham Pharrnacia Biotech AB).
Example 3
[0159] Synthesis and Purification of TMR-, Cy3.5- and
Cy5-Labeled-(propylthiol)-Capped Oligonucleotides
[0160] This Example describes the syntheses of three
oligonucleotides having Raman labels bound thereto: 3'
HS-TMR-A.sub.10-AAC CGA AAG TCA ATA [SEQ ID NO. 2 in FIG. 19a]; 3'
HS-Cy3.5-A.sub.10-CCT CAT TTA CAA CCT [SEQ ID NO. 3 in FIG. 19a];
and 3'HS-Cy5-A.sub.10-CTC CCT AAT AAC AAT [SEQ ID NO. 4 in 19b].
Because the dyes are sensitive to standard cleavage reagent
(ammonia), ultramild base monomers (from Glen Research) were used
here to allow the deprotection reaction under ultramild conditions:
phenoxyacetyl (Pac) protected dA, 4-isopropyl-phenoxyacetyl
(iPr-Pac) protected dG, and acetyl (Ac) protected dC. TAMRA-dT
(TMR-dT,
5'-Dimethoxytrityloxy-5-[N-((tetramethylrhodaminyl)-aminohexyl)-3-acrylim-
ido]-2'-deoxy
Uridine-3'-[2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-
),Cy3.5-CE phosphoramidite (Indodicarbocyanine 3.5,
1'-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphora-
midite), and Cy5-CE phosphoramidite (Indodicarbocyanine 5,
1'-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphora-
midite) were used to label the oligonucleotides, respectively.
After synthesis of the oligonucleotides, the synthesis column
contents were transferred to a 2 mL reaction vial and treated with
1 mL of 0.05M potassium carbonate in anhydrous methanol for 4 h at
room temperature. Then the supernatant was pipetted from the
support and neutralized with 1.5 mL of 2M triethyammonium acetate.
Further purification was carried out as described above for the
synthesis of the Cy3-labeled-oligonucleoti- des. HPLC retention
times are 28, 32, 30 min for TMR-, Cy3.5- and Cy5-labeled,
propylthiol-capped oligonucleotides, respectively.
Example 4
[0161] Synthesis and Purification of Rhodamine 6G-, and Texas
Red-Labeled-(propylthiol)-Capped Oligonucleotides
[0162] This Example describes the synthesis of two olignucleotides
have Raman labels attached thereto: 3' HS-Rd-A.sub.10-TCA ACA TTG
CCT TCT [SEQ ID NO. 5 in FIG. 19b] and 3' HS-TR-A.sub.10-TCT TCT
ATA AAC CTT ATT [SEQ ID NO. 6 in FIG. 19a]. See FIG. 24. Both of
these oligonucleotides were prepared via two-step syntheses. In the
first step, amino-modified oligonucleotides
(3'-S--S-(NH.sub.2)-A.sub.10-TCA ACA TTG CCA TCT and
3'-S--S-(NH.sub.2)-A.sub.10-TCT TCT ATA AAC CTT ATT)were
synthesized via literature procedures..sup.5 The amino-modifier C6
dT(5'-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2'-d-
eoxyUridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)
was placed in position 5 in the synthesizer (Expedite), and
amino-modified oligonucleotides were obtained by conventional
automated syntheses. The cleavage, deprotection, and purification
of the oligonucleotides were carried out by the procedures
described for the synthesis of the Cy3-modified oligonucleotide
(above), retention time=26 min. In the second step, succinimide
ester modified Rhod 6G (5-carboxyl-rhodamine 6G, succinimidyl
ester) and Texas Red (Texas Red-X-succinimidyl ester) were coupled
to the amino-modified oligonucleotides, respectively. In a typical
experiment, an amino-modified, alkylthiol-capped-oligonucleotide
(0.15 .mu.mol) was dissolved in a sodium borate buffer (0.1M,
pH=8.5, 0.5 ml), and a DMSO solution (150 .mu.l) containing 2.5 mg
of the succiuimide ester modified Rhod 6G (or Texas Red) was added
to the oligonucleotide buffer solution, FIG. 24. The solution was
stirred at room temperature for 12 hr. Then, the Rhod 6G-(or Texas
red-) labeled oligonucleotide was purified by ethanol precipitation
(3 times) and further by HPLC in the conditions as described
above.
Example 5
[0163] DNA Detection Assay
[0164] In a typical experiment for DNA detection, a three-component
sandwich assay is used in microarray format (FIG. 1). Gold
nanoparticles (13.+-.2 nm in diameter) modified with Cy3-labeled,
alkylthiol-capped oligonucleotide strands (Supporting Information)
were used as probes to monitor the presence of specific target DNA
strands. On average, there are 110 oligonucleotide strands on each
13-nm gold nanoparticle. The Cy3 group was chosen as a Raman label
due to its large Raman cross section..sup.23 A chip spotted with
the appropriate 15 mer capture strands was coated with a 0.6 M NaCl
PBS buffer solution (10 mM of phosphate, pH 7) containing a 30 mer
target sequence (100 pM) in a humidity chamber at room temperature.
After 4 h, the chip was washed four times with 0.6 M NaCl PBS
buffer solution to remove nonspecifically bound target. Then, the
chip was treated with a 0.6 M NaCl PBS solution of nanoparticle
probes (2 nM) for 1.5 hour to effect hybridization with the
overhanging region of the target sequence (FIG. 1). The chip was
then washed with 0.6 M NaNO.sub.3 PBS buffer solution to remove
chloride ions and nonspecifically bound nanoparticle probes. The
chip was immediately treated with a silver enhancement solution
(Ted Pella, Inc) for 8 minutes, subsequently rinsed with Nanopure
water, and dried with a microarray centrifuge (2000 g). The chip,
which exhibits grey spots visible to the naked eye, could be imaged
with a flatbed scanner (Expression 1600, Epson) via literature
procedures, FIG. 2A and B..sup.8 The spots also were imaged by
Raman spectroscopy in a 0.3 M NaCl PBS buffer solution (Solution
Raman 633 spectrometer from Detection Limit Inc., 30 mW He--Ne
laser), FIG. 2C. The chip was scanned with a fiber-optic probe with
a 0.65 N.A. adapter (25 .mu.m laser spot), and each spot shows a
consistent and strong Raman response at 1192 cm.sup.-1 (FIG.
2D).
[0165] Prior to silver enhancing, the nanoparticle probes were
invisible to the naked eye, and no Raman scattering signal was
detectable (FIG. 2A). This is due to a lack of electromagnetic
field enhancement for the undeveloped nanoparticles (13 nm in
diameter) in this state..sup.24-26 Others have shown that closely
spaced gold nanoparticles in such sizes can give surface-enhanced
Raman scattering enhancement,.sup.27-30 but for DNA detection at
technologically relevant target concentrations (<1 nM),
nanoparticle spacings are too large to yield such effects. After
silver enhancing, the Ag particles can grow around the Cy3-labeled
nanoparticle probes leading to large Raman scattering enhancements.
Typically, the obtained spectra include both sharp (15 to 30
cm.sup.-1) Raman lines and a concomitant broad underlying continuum
as noted by Brus et. al. in their studies of Rhodamine 6G molecules
on Ag particles..sup.30-31 Importantly, the Raman scattering
signals arise almost exclusively from the Cy3 dye molecules
immobilized on the particles; no signals were observed from other
species such as the oligonucleotides, solvent molecules, and the
succinimidyl 4-(maleimidophenyl)-butyrate (SMPB) on the glass
surface. Moreover, the Raman scattering frequency for each Raman
line remains constant from experiment to experiment, deviating by
less than 2 cm.sup.-1. Since consistent SERS signals from the
Cy3-labeled nanoparticle probes were obtained, the Raman spectrum
of Cy3 can be used as a spectroscopic fingerprint to monitor the
presence of a specific target oligonucleotide strand.
Example 6
[0166] Detection of DNA at Low Target Concentration (Example: 20
fM)
[0167] In a typical experiment, a chip spotted with the appropriate
capture strands (FIG. 3A) was coated with a 0.75M NaCl PBS buffer
solution (10 mM of phosphate, pH 7) containing a 30-mer target
sequence (20 fM) in a humidity chamber at room temperature. After 8
h, the chip was washed with 0.75M NaCl PBS buffer solution to
remove nonspecifically bound target. Then, the chip was treated
with a 0.75 M NaCl PBS solution of nanoparticle probes (500 pM) for
3 h to effect hybridization with the overhanging region of the
target sequence (FIG. 3A). The chip was washed with 0.75 M
NaNO.sub.3 PBS buffer solution to remove chloride ions and
nonspecifically bound nanoparticle probes. The chip was immediately
treated with silver enhancement solution (from Ted Pella, Inc) for
15 min, subsequently rinsed with Nanopure water, and dried with a
microarray centrifuge (2000 g). The spots can be imaged in the dry
state with a flatbed scanner (FIG. 3B) or by Raman spectroscopy in
the wet state (0.3 M NaCl, pH7, PBS buffer solution), FIG. 3C and
D. The current unoptimized detection limit with this technique is
10 fM.
Example 7
[0168] Detection of Multiple Oligonucleotide Targets
[0169] This Example describes detection of multiple
oligonucleotides using a plurality of Raman labeled probes. One can
utilize the approach described in Example 5 and nanoparticle probes
functionalized with dyes other than Cy3 to create a large number of
probes with distinct and measurable SERS signals. This allows
multiplexed detection of a large number of oligonucleotide targets
simultaneously. To demonstrate this point, six commercially
available dyes were selected with distinct Raman spectra that can
be incorporated into oligonucleotides through standard automated
DNA-syntheses. Six types of Raman labeled and
oligonucleotide-modified gold nanoparticle probes were prepared
with sequences that were respectively complementary to
statistically unique 30-36 mer sequences for: (A) Hepatitis A virus
Vall7 polyprotein gene (HVA), (B) Hepatitis B virus surface antigen
gene (HVB), (C) HIV, (D) Ebola Virus (EV), (E) Variola virus
(smallpox, VV), and (F) Bacillus anthracis (BA) protective antigen
gene (FIG. 4)..sup.32 With these probes, the multiplexing
capabilities of the novel scanning Raman technique for the six
target analytes can be demonstrated.
[0170] Eight separate tests were carried out to evaluate the
selectivity of the system and our ability to determine the number
and types of strands in solutions containing mixtures of the
different targets (FIG. 4 and 5). The concentrations of the target
strands were kept constant for all of these experiments (100 pM
each), and the hybridization conditions were as described above. In
the first test (FIG. 5, row 1), all spots show the same intense
grey color associated with silver deposition. However, they can be
differentiated simply by using the Raman scanning method, and once
the spectroscopic fingerprint of the Ag-containing spot has been
determined the correct Raman label and, therefore, target sequence
can be identified. To simplify the analysis, a color (rectangular
box) to each Raman labeled probe (FIG. 4 and FIG. 5B) was assigned.
In the first test (FIG. 5A), all six targets were present, and all
show strong grey scale values when measured via the flatbed scanner
and the expected Raman fingerprints. In the next seven tests, one
or more of the targets to evaluate the suitability of this method
for multiplexing were systematically removed. Note that with the
single color grey scale method one cannot determine if any cross
hybridization has occurred. However, with this "multiple color"
scanning Raman method, one can carefully study the SERS spectra of
each spot to determine which labels make up each spot. For the
experiments described in FIG. 5, where the sequences are very
dissimilar, it was found that other than the expected spectroscopic
probe signature for each target, there are virtually no other
detectable Raman lines, which means that there is no
cross-hybridization between different targets and probes.
[0171] It should be mentioned that the obtained SERS signal only
comes from areas of the substrate where the Raman dye-labeled gold
particles have initiated Ag formation. Therefore, this "multiple
color" scanning Raman detection method does not record background
signal due to silver deposition where Au particles do not exist.
This is not the case for the previous grey-scale scanometric
approach, especially at ultra-low target concentrations (<50
fM)..sup.8
Example 8
[0172] Discrimination and Ratioing of Single Nucleotide
Polymorphisms (SNPs) in Oligo-Ribonucleic Acid (RNA) Targets.
[0173] This Example describes the use of oligonucleotides having
Raman labels in detection systems to differentiate single
nucleotide polymorphisms (SNPs), and in the case of gene expression
studies, one would like access to RNA detection with single spot
signal ratioing capabilities. It is well known that nanoparticle
probes heavily functionalized with oligonucleotides exhibit
extraordinarily sharp thermally-induced denaturation transitions
that lead to substantially higher selectivity than conventional
molecular fluorophore probes in DNA detection..sup.5,8,9 However,
nothing is known about the behavior of these probes in the context
of RNA detection. To further test the selectivity of this Raman
based system and its ability to identify SNP targets, two RNA
targets were chosen that can bind to the same capture strand DNA
but have a single-base mutation in the probe binding regions
(target 1:T.sub.1, normal; target 2:T.sub.2, single-base
difference, FIG. 6). Therefore, two DNA-functionalized probes
(probe 1: P.sub.1, probe 2: P.sub.2), which differ in sequence and
Raman label, are required to differentiate these two RNA target
strands (FIG. 6). Seven separate tests were performed to
demonstrate not only how the two targets (T.sub.1 and T.sub.2) can
be differentiated but also how mixtures of the two targets can be
analyzed in semi-quantitative fashion.
[0174] In a typical experiment, the appropriate capture strands
(FIG. 6) were spotted in quadruplicate on SMPB functionalized glass
slides. These slides were coated with 0.3M NaCl PBS buffer
solutions (10 mM of phosphate, pH 7) containing pure RNA target 1
or target 2, or mixtures of 1 and 2 (1 nM total oligonucleotide
concentration) in a humidity chamber at room temperature. After 2
h, the chip was washed four times with 0.3M NaCl PBS buffer
solution to remove nonspecifically bound target. Then, the chip was
treated with a 0.3 M NaCl PBS solution of nanoparticle probes (2
nM, probel: probe2=1:1) for 1.5 h to effect hybridization with the
overhanging region of the target sequences (FIG. 7). The chip was
washed with 0.3 M NaNO.sub.3 PBS buffer solution to remove chloride
ions and nonspecifically bound nanoparticle probes. If the chips
were developed by silver enhancing, the Raman measurements on the
grey spots at different target ratios yield similar spectra (FIG.
8), which are nearly identical to the spectrum for the sample
containing probe I and probe 2 in equal amounts. This result
indicates that there are equal amounts of probe 1 and probe 2 on
the chip. This is because the stabilities of the perfectly matched
and single-mismatched oligonucleotide duplexes are close in
magnitude, and therefore, nanoparticle probes (1 and 2) bound to
the spots on the chips in nearly equally amounts at all of the
target ratios. Under these conditions the two targets cannot be
differentiated.
[0175] In each of these tests, a slide was treated with a 0.3 M
NaCl PBS buffer solution containing T.sub.1 and T.sub.2 in
different ratios (total concentration=1 nM) in a humidity chamber.
After 2 h, the chip was washed with a 0.3 M NaCl PBS buffer to
remove nonspecifically bound target. Then, the chip was treated
with nanoparticle probes (P.sub.1 and P.sub.2 at 1:1 ratio, 2nM
total concentration) for 1.5 h to effect hybridization with the
overhanging region of the target sequences (FIG. 6). The chip was
washed with 0.3 M NaNO.sub.3 PBS buffer solution to remove chloride
ions and nonspecifically bound nanoparticle probes. Note that there
are four possible hybridization modes, namely, T.sub.1:P.sub.1,
T.sub.2:P.sub.2, T.sub.1:P.sub.2, and T.sub.2:P.sub.1 (FIG. 6). If
the chip was developed by silver enhancing without prior stringency
wash, the Raman measurements on the grey spots which correspond to
different solution target ratios yield nearly identical spectra in
all seven experiments; these spectra also are almost identical to
those obtained for a sample containing a 1:1 ratio of probe 1 and
probe 2 (see Supporting Information). These data show that probe 1
and probe 2 are bound to the spots on the chip in equal amounts,
regardless of the target composition on the spot.
[0176] Therefore, in order to identify the target composition on
the spots, a salt or temperature-based stringency wash must be
applied. Accordingly, a salt stringency wash (8 mM NaCl PBS buffer)
was employed to selectively denature the imperfect duplexes
(T.sub.1:P.sub.2 and/or T.sub.2:P.sub.1, FIG. 6C and 6D) but not
the duplexes formed from the perfectly complementary
oligonucleotides (T.sub.1:P.sub.1 and/or T.sub.2:P.sub.2, FIG. 6A
and 6B)..sup.9 After stringency wash and subsequent silver
staining, the Raman measurements on the grey spots can be used to
readily identify the target composition on the spots by the
obtained spectra. In tests where only pure RNA target 1 or 2 are
present, only signals for probe 1 or 2, respectively are observed
(compare FIG. 9B "a" and "g"). In the case of mixtures, signals for
both probes (I.sub.1: 1650 cm.sup.-1 from probe 1 and I.sub.2: 1588
cm.sup.-1 from probe 2) are detected, and the intensity ratios are
proportional to the ratios of the two targets in each experiment
(inset of FIG. 9B).
Example 9
[0177] Screening of Protein: Small Molecule Interaction
[0178] This Raman detection format also can be used in protein
microarray applications for screening protein-small molecule and
protein-protein interactions. For the detection of protein-small
molecule interactions, we selected three unrelated small molecules
for which the specific protein receptors are commercially
available: biotin and its mouse monoclonal antibody; DIG (steroid
digoxigenin) and its mouse monclonal antibody; DNP (dinitrophenyl)
and its mouse monoclonal antibody. The three small molecules were
labeled with Raman dye-functionalized gold particles: the gold
particles (13 nm in diameter) were modified with a small-molecule
capped, Raman dye and alkylthiol-functionalized
poly-adenine(A.sub.20) (FIG. 11A). In a typical detection
experiment, the proteins from all three pairs were immobilized in
triplet onto aldehyde-functionalized glass slides by spotting the
protein solution (200 .mu.g/ml, 5% glycerol) with a commercial
arrayer (FIG. 11A)..sup.33,34 After 4-hour incubation in a humidity
chamber, the protein chip was washed with PBS buffer (0.173 M NaCl,
0.027 M KCl, 4.3 mM Na.sub.2BPO.sub.4, 1.4 mM KH2PO.sub.4, pH=7.4)
containing 0.5% bovine serum albumin (BSA), and immersed into such
solution for 4 hour to passivate the unreacted aldehydes on the
protein chip. After being washed with a PBS solution (0.173 M NaCl,
0.027 M KCl, 4.3 mM Na.sub.2HPO.sub.4, 1.4 mM KH.sub.2PO.sub.4,
pH=7.4), the protein chip was treated with Raman labeled small
molecule probes (for 2 hours at 4.degree. C. After washed with a
buffer solution (0.2 M NaNO.sub.3, 5 mM phosphate, pH=7.4), the
gold particle functionallized protein chip was treated with the
silver enhancement solution for 8 minutes and washed with Nanopure
water. Before Raman measurements, the silver stained chip was
immersed in a 2.times.PBS solution for 10 minutes.
[0179] In the first test, the protein chip was exposed to all a
solution containing all three Raman-labeled small molecule probes.
After silver enhancement, the triplet dot array is clearly visible,
even to the naked eye (FIG. 11B-1). When measuring the Raman
spectra of the dots, we obtained the correct probe spectra with no
evidence of cross reactivity (i.e. less than 1%, Cy3 for biotin,
Cy3.5 for DIG, and Cy5 for DNP). Next we studied the same type chip
but in the presence of the DIG and DNP probes, and gain obtained
the expected results, FIG. 11B-2 and C2). We did this for all other
possible two probe combinations and again obtained the expected
results, demonstrating the high selectivity of the system (FIG.
11B-3, C-3 and 11B-4, C-4). In the two probe experiments, one probe
for the array is absent, serving as a control for screening the
other interaction pairs.
Example 10
[0180] Screening Protein-Protein Interactions
[0181] For screening protein-protein interactions, we chose three
pairs of proteins to study: mouse immunoglobulin G (IgG) and its
antibody; ubiquitin and its antibody; human protein C and its
antibody. Mouse IgG, ubiqutin, and human protein C were spotted in
quadruplicate on aldehyde slides, respectively. Gold nanoparticles
were first functionalized with antibodies and then with Raman-dye
labeled oligonucleotides. The labeling procedure is shown in FIG.
12: an antibody (10 .mu.g, pH=9.2) was put into a solution of gold
particles (13 nm, 10 nM, 1 mL, pH=9.2) for 20 minutes, and then the
Raman dye capped-alkylthiol-functionalized poly-adenine (A.sub.10,
0.2 OD at 260 mn) was added to the solution. After 12 hours, 10%
BSA solution (0.3 mL) was added to the solution to further
passivate the surface of the gold particles. The solution was
allowed to stand for 10 minutes. The Raman-dye capped gold
particle-antibody conjugates were purified by centrifugation
(14,000 rpm), which precipittaes the particles. The supernatant
containing excess oligonucleotide, BSA, and antibodies can be
decanted from the particles. The particle probes are then be
redispersed in PBS buffer. The probes (2 nM for gold nanoparticles,
about 2 .mu.g/ml for the antibodies) were then used to develop the
protein chips. The protocol for screening the protein-protein
interactions is similar to that for protein-small molecule
interactions (described above).
[0182] The chip in FIG. 13 A-4 was probed with all the three Raman
labeled antibodies simultaneously. After silver enhancement, all
three two-by-two dots array are clearly visible after silver
develioping. Raman analysis shows no detectable cross reactivity
and all of the correct dyes are in the correct spots (FIG. 13).
[0183] Just like fluorophore-based methods, this new scanometric
detection format provides a general approach for genomic and
porteomic detection but with a higher sensitivity and a higher
multiple labeling capability. The number of available Raman dyes is
much larger than the number of available and discernable
fluorescent dyes..sup.20,21 A Raman dye can be a fluorescent dye
and also a non-fluorescent dye. A small modification of a dye can
lead to a new dye with different Raman spectra and even the dyes
which show undistinguishable fluorescent spectra can be
distinguished by Raman spectroscopy..sup.16 In the conventional
multicolor fluorescent dyes labeling format, the data readout
requires multi-lasers and multiple scans..sup.1 By contrast, only a
single laser and individual scan are required in this Raman
scanometric detection format, suggesting a potential for a high
throughput reading process. Although quantum-dot-labeled
fluorescence detection requires only single laser, multicolors are
usually generated from different size and shape quantum-dot
nanoparticles..sup.6,7 Different sized and shaped nanoparticles
associated biological labels will have different thermodynamic and
kinetic properties, which are problematic for parallel microarray
biological detection. In the Raman scanometric detection format, in
contrast, only one-sized gold nanoparticle (13 nm, here) carriers
are required, and labeling information from different Raman dyes.
Therefore, most of the labels described here have similar
thermodynamic and kinetic target binding properties, which are
essential for faster, more-accurate, high-throughput microarray
based mapping and screening of biomolecules..sup.1
Example 11
[0184] Multiple Raman-Dye Labeled Nanoparticle Probes
[0185] All the Raman labels described above are single-dye systems:
one carrier and Raman dye. One can load two or multiple Raman dyes
onto a nano-sized nanoparticle carrier. Massively encoded Raman
labels can be generated by tailoring the ratio between the
components (FIG. 14 and 15). In a two-dye system, two alkylthiol
capped-oligonucleotide strands with same base sequences but
different the Raman labels (Cy3 and TMR) were used to modify 13-nm
gold nanoparticles simultaneously, and therefore a composite Raman
label was generated. This two-dye labeled nanopaticle probe has
similar thermodynamic and kinetic properties as the single-dye
labeled nanoparticle probe (i.e. same hybridization kinetics and
melting temperatures with identical strands). In a typical DNA
detection experiment (target concentration is 100 pM, FIG. 1), a
Raman spectrum from a silver-stained spot clearly shows
characteristic Raman lines from both of Cy3 and TMR (FIG. 16,
left). By varying the ratio of Cy3 and TMR, different composite
Raman spectra are obtained (FIG. 16, right). These Raman spectra
are distinguishable from each other by differences in relative
intensities for the main bands in the region interrogated. The
multiple reference windows increase the accuracy for identifying
different Raman labels, making this two dye Raman labeling
methodology practically usable. Beyond two-dye systems, two
examples of three-dye labels, which have different amount ratio
between Cy3, TMR and Cy3.5, are shown in FIG. 17.
[0186] One can use one-dye, two-dye, three-dye and even larger
combination-labeled systems. A significant question is: how many
labels can be achievable in this Raman labeling system? In a
two-dye system, assuming five intensity levels (0, 1, 2, 3, 4),
there are 13 labels that can be generated. Five million labels and
three billion labels can be generated with 10-dye and 14-dye
systems, respectively (FIG. 15).
Example 12
[0187] Microbead-Based Biological Detection
[0188] Large numbers of parallel labeling techniques are of
particular importance in microbead-based biological detection
strategies. Microbead technology is emerging as an important
biological analysis format for gene expression monitoring, SNP
genotyping, proteomic screening, and drug discovering..sup.1,13
Compared with the microarray technique, microbead detection shows
more flexibility in hybridization-based procedures, faster analyte
diffusion kinetics, and they are easier and cheaper to produce. The
microbead detection without the positional encoding in the
microarrys, however, must rely on some sort of barcoding strategy
for the particle probes. A major problem in the current
fluorescent-dye-based encoding approach is that the number of
distinguishable labels are limited due to the broad emission
spectra and energy transfer between organic dyes..sup.11 Raman
labeling, in contrast, can overcome these difficulties.
[0189] For a typical DNA target detection system, a three-component
sandwich assay format can be used. In our experiments, glass
microbeads (210-250 mm in diameter) were functionalized with
oligonucleotide capture strands (FIG. 18). Gold nanoparticles (13
nm in diameter) modified with pure or mixed Raman dye-labeled and
alkylthiol-capped oligonucleotides probe strands were synthesized.
Then the Raman dye and gold particle associated probes (2 nM for
gold particles) co-hybridized with the target strands onto the
surface of the capture strand oligonucleotide functionalized glass
microbeads in a 4.times.PBS buffer solution (0.6 M of NaCl, 10 mM
of phosphate buffer( pH=7) ) for 2 hours and washed with a second
buffer solution (0.6 M NaNO3, 10 mM phosphate) to remove chloride
ions, and non-specifically bound nanoparticle labels, and
immediately treated with a silver enhancement solution (from
BBInternational) for 8 minutes. Before Raman measurements, the
microbeads were immersed in a 2.times.PBS buffer for 10 min to
further enhance Raman scattering signal.
[0190] To demonstrate the multiplexing capabilities of the novel
scanning Raman technique in microbead detection format, we chose an
eight-target analyte detection experiment. The sequences of target,
capture and probe oligonucleotide strands are shown in FIG. 19a and
b. The corresponding Raman spectra (marked by colored circle and
rectangular boxes) are listed in FIG. 20. In a typical experiment,
eight capture strands were loaded onto microbeads, respectively.
Mixing all the microbeads together, a flexible "random microarray"
was built. Then the eight targets (100 pM) and Raman-labeled
nanoparticle probes (2 nM) are introduced to the random microarray
solution under hybridization conditions as described above. After
washing and silver staining, the microbeads are show up as
dark-grey spheres and exhibit the expected Raman signatures (FIG.
21). To achieve an easy readout process, we can align these
microbeads mechanically (FIG. 22 top) and read them in serial
fashion via scanning Raman spectroscopy. (FIG. 22, bottom).
Moreover, the Raman fingerprints of the micorbeads can also be read
out by fiber optics (FIG. 23).
[0191] Beside this new Raman labeling technique, two recent
strategies show the practical potential for massively parallel
labeling abilities: quantum-dot-tagged microbeads and submicrometer
metallic barcodes..sup.11,35 However, both of these strategies
achieve multiple labeling based on micron-size structures. In
contrast, Raman labeling here is a nano-size labeling methodology,
and has much more flexibility than those micro-size ones. In
particular, the footprints of the probes are smaller and the
specificity and sensitivity of systems based on the probes can be
dramatically improved over the systems based upon larger
structures. This new nanoparticle-based methodology is important
for a variety of reasons. First, in contrast with conventional
fluorescence-based chip detection, the ratio of Raman intensities
can be extracted from a single Raman spectrum using single laser
excitation. Second, the number of available Raman dyes is much
larger than the number of available and discernable fluorescent
dyes..sup.20,21,26 Indeed, a Raman dye can be either fluorescent or
non-fluorescent, but a minor chemical modification of a dye
molecule can lead to a new dye with a different Raman spectrum even
though the two dyes exhibit virtually indistinguishable
fluorescence spectra..sup.26 Therefore, this fingerprinting method
offers potentially greater flexibility, a larger pool of available
and non-overlapping probes, and higher multiplexing capabilities
than conventional fluorescence-based detection approaches. Finally,
the method incorporates all of the previous advantages of
gold-nanoparticle based detection, including several orders of
magnitude higher sensitivity and many orders of magnitude higher
selectivity than the analogous molecular fluorescence based
approach..sup.8,9
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[0228]
Sequence CWU 1
1
27 1 15 DNA Hepatitis A virus 1 tccatgcaac tctaa 15 2 15 DNA
Hepatitis B virus 2 ataactgaaa gccaa 15 3 15 DNA Ebola virus 3
tccaacattt actcc 15 4 15 DNA Bacillus anthracis 4 taacaataat ccctc
15 5 15 DNA Variola virus 5 tcttccgtta caact 15 6 18 DNA Human
immunodeficiency virus type 1 6 ttattccaaa tatcttct 18 7 12 DNA
Francisella tularensis 7 agccacctaa cc 12 8 15 DNA Hog cholera
virus 8 acatgtccaa tttcc 15 9 15 DNA Hepatitis A virus 9 agaaagagga
gttaa 15 10 15 DNA Hepatitis B virus 10 taccacatca tccat 15 11 15
DNA Ebola virus 11 ttgttgatac tgttc 15 12 15 DNA Bacillus anthracis
12 atcctttaca atatt 15 13 15 DNA Variola virus 13 ctgattacta ttgca
15 14 15 DNA Human immunodeficiency virus type 1 14 tgcatccagg
tcatg 15 15 15 DNA Francisella tularensis 15 cttttgcatc atcag 15 16
15 DNA Hog cholera virus 16 tggttcacct ttgac 15 17 30 DNA Hepatitis
A virus 17 ttagagttgc atggattaac tcctctttct 30 18 30 DNA Hepatitis
B virus 18 ttggctttca gttatatgga tgatgtggta 30 19 30 DNA Ebola
virus 19 ggagtaaatg ttggagaaca gtatcaacaa 30 20 30 DNA Bacillus
anthracis 20 gagggattat tgttaaatat tgtaaaggat 30 21 30 DNA Variola
virus 21 agttgtaacg gaagatgcaa tagtaatcag 30 22 33 DNA Human
immunodeficiency virus type 1 22 agaagatatt tggaataaca tgacctggat
gca 33 23 27 DNA Francisella tularensis 23 ggttaggtgg ctctgatgat
gcaaaag 27 24 30 DNA Hog cholera virus 24 ggcaattgga catgtgtgaa
aggtgaacca 30 25 15 DNA Artificial Sequence misc_feature (1)..(15)
Synthetic probe strand 25 taacaataat ccctc 15 26 15 DNA Artificial
Sequence misc_feature (1)..(15) Synthetic capture strand 26
atccttatca atatt 15 27 30 DNA Artificial Sequence misc_feature
(1)..(30) Synthetic target sequence 27 gagggattat tgttaaatat
tgataaggat 30
* * * * *
References