U.S. patent application number 12/359503 was filed with the patent office on 2009-12-10 for detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers).
Invention is credited to Xing Su, Lei Sun.
Application Number | 20090303461 12/359503 |
Document ID | / |
Family ID | 36228828 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090303461 |
Kind Code |
A1 |
Sun; Lei ; et al. |
December 10, 2009 |
DETECTION OF ENHANCED MULTIPLEX SIGNALS BY SURFACE ENHANCED RAMAN
SPECTROSCOPY (SERS)
Abstract
Various methods of using Raman-active or SERS-active probe
constructs to detect analytes in biological samples, such as the
nucleic acid and/or protein-containing analytes in a body fluid are
provided.
Inventors: |
Sun; Lei; (Santa Clara,
CA) ; Su; Xing; (Cupertino, CA) |
Correspondence
Address: |
Pillsbury Winthrop Shaw Pittman LLP
P.O. Box 10500
McLean
VA
22102
US
|
Family ID: |
36228828 |
Appl. No.: |
12/359503 |
Filed: |
January 26, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11016613 |
Dec 17, 2004 |
7485471 |
|
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12359503 |
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Current U.S.
Class: |
356/36 ; 356/301;
356/72; 530/300; 530/350; 530/391.1; 536/1.11; 536/23.1;
977/773 |
Current CPC
Class: |
G01N 21/658 20130101;
G01N 33/54306 20130101 |
Class at
Publication: |
356/36 ; 356/72;
536/23.1; 530/350; 530/300; 530/391.1; 536/1.11; 356/301;
977/773 |
International
Class: |
G01N 1/44 20060101
G01N001/44; G01N 21/65 20060101 G01N021/65; C07H 21/02 20060101
C07H021/02; C07H 21/04 20060101 C07H021/04; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00; C07H 1/00 20060101
C07H001/00 |
Claims
1-26. (canceled)
27. A biological target complex comprising: a target analyte bound
to a first specific binding member; a second specific binding
member that binds to the first specific binding member forming a
target complex, wherein the second specific binding member
comprises a seed particle suitable for catalyzing the formation of
a surface enhanced Raman scattering (SERS) substrate, wherein the
SERS substrate is suitable to be activated to provide a SERS
effect; a capture reagent bound to a solid substrate, wherein the
capture reagent comprises a Raman label, wherein the target analyte
binds to the capture reagent forming a biological target complex;
and a layer of roughened metal over the substrate or the biological
target complex.
28. The biological target complex of claim 27, wherein the layer of
roughened metal comprises roughness features on the order of tens
of nanometers.
29. The biological target complex of claim 28, wherein plasmon
excitation due to electromagnetic irradiation of the biological
target complex is confined to the roughness features.
30. The biological target complex of claim 29, wherein the layer of
roughened metal comprises a thickness of approximately one-half the
wavelength of the electromagnetic irradiation.
31. The biological target complex of claim 27, wherein the layer is
transparent.
32. The biological target complex of claim 27, wherein the layer of
roughened metal comprises gold, silver, copper, or aluminum.
33. The biological target complex of claim 27, wherein the target
analyte is a DNA, RNA, polypeptide, antibody, antigen, carbohydrate
or small molecule.
34. The biological target complex of claim 27, wherein the capture
reagent is a DNA, RNA, polypeptide, antibody, antigen, carbohydrate
or small molecule.
35. The biological target complex of claim 27, wherein the first or
second specific binding member is a DNA, RNA, antibody, antigen,
polypeptide or carbohydrate.
36. The biological target complex of claim 27, wherein the target
analyte further comprises an ancillary specific binding member.
37. A method comprising: a) providing a target analyte bound to a
first specific binding member; b) providing a capture reagent bound
to a solid substrate, wherein the capture reagent comprises a Raman
label; c) contacting the target analyte of a) with the capture
reagent of (b) under conditions suitable for forming a target
analyte-capture reagent complex; d) contacting, prior to,
concurrently with, or subsequent to c) the first specific binding
partner with a second specific binding member functionally
associated with a seed particle suitable for associating with a
SERS substrate, wherein the first specific binding member binds to
the second specific binding member; and e) coating either the
substrate or the target analyte-capture reagent complex with a
layer of roughened metal; f) contacting the target analyte-capture
reagent complex with electromagnetic radiation suitable for
detecting a specific property associated with the analyte-capture
reagent complex by Raman spectroscopy.
38. The method of claim 37, wherein the layer of roughened metal
comprises roughness features on the order of tens of
nanometers.
39. The method of claim 37, wherein plasmon excitation due to the
electromagnetic irradiation is confined to the roughness
features.
40. The method of claim 37, wherein the layer comprises a thickness
of approximately one-half the wavelength of the electromagnetic
irradiation.
41. The method of claim 37, wherein the layer of roughened meta is
transparent.
42. The method of claim 37, wherein the layer of roughened metal
comprises gold, silver, copper, or aluminum.
43. The method of claim 37, comprising forming the layer of
roughened metal by vapor deposition of metal particles or
application of metal colloids.
44. The method of claim 43, wherein application of metal colloids
comprises subjecting a colloidal solution of metal cations to
reducing conditions to form metal nanoparticles in situ.
45. The method of claim 43, wherein application of metal colloids
comprises using seed particle to precipitate nanoparticles from a
metal colloid solution.
46. The method of claim 45, wherein the seed particle is selected
from the group consisting of gold, Ag, Cu, Pt, Ag/Au, Pt/Au, Cu/Au
coreshell and alloy particles.
47. The method of claim 37, further comprising detecting a single
analyte molecule.
48. A system comprising: biological target complex comprising a
target analyte bound to a first specific binding member, a second
specific binding member that binds to the first specific binding
member forming a target complex, the second specific binding member
comprising a seed particle suitable for catalyzing the formation of
a surface enhanced Raman scattering (SERS) substrate, a capture
reagent bound to a solid substrate, the capture reagent comprising
a Raman label, wherein the target analyte binds to the capture
reagent forming the biological target complex and a layer of
roughened metal over the substrate or the biological target
complex; and an electromagnetic radiation source.
49. The system of claim 48, further comprising a Raman detection
unit.
50. The system of claim 48, wherein the electromagnetic radiation
source comprises a frequency doubled Nd:YAG laser, a frequency
doubled Ti:sapphire laser, a nitrogen laser, a helium-cadmium laser
a light emitting diode, an Nd:YLF laser, ion lasers, or dye
lasers.
51. The system of claim 48, wherein the radiation source is either
pulsed or continuous.
52. The system of claim 48, further comprising confocal optics and
a microscope objective.
53. The system of claim 48, further comprising a flow through
cell.
54. The system of claim 48, further comprising a monochromator.
55. The system of claim 52, wherein the confocal optics comprises
one or more of dichroic filters, barrier filters, holographic notch
filters, confocal pinholes, lenses, and mirrors.
56. The system of claim 49, wherein the Raman detection unit
comprises an avalanche photodiode interfaced with a computer.
57. The system of claim 56, wherein the Raman detection unit is
configured to count and digitize a signal.
58. The system of claim 49, wherein the Raman detection unit
comprises a double-grating spectrophotometer with a
gallium-arsenide photomultiplier tube, Fourier-transform
spectrographs, charged injection devices, photodiode arrays, InGaAs
detectors, electronmultiplied CCD, intensified CCD or
phototransistor arrays.
59. The system of claim 49, wherein the Raman detection unit
comprises a red-enhanced intensified charge-coupled device
(RE-ICCD) detection system.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to methods and
devices useful to identify the presence of an analyte in a
sample.
[0003] 2. Background Information
[0004] Surface-enhanced Raman scattering (SERS) is a sensitive
spectroscopic method for detection of an analyte. Raman
Spectroscopy probes vibrationally excitable levels of an analyte.
Once a vibrational level is excited by a photon, the energy of the
photon shifts by an amount equal to that of the level (Raman
scattering). A Raman spectrum, similar to an infrared spectrum,
consists of a wavelength distribution of bands corresponding to
molecular vibrations specific to the sample being analyzed (the
analyte). In the practice of Raman spectroscopy, the beam from a
radiation source is focused upon the sample to thereby generate
inelastically scattered radiation, which is optically collected and
directed into a wavelength-dispersive spectrometer in which a
detector converts the energy of impinging photons to electrical
signal intensity. In SERS, analyte molecules are adsorbed on noble
metal nanoparticles. These nanoparticles, once excited by light,
set up plasmon modes, which, in turn, create near fields around
each particle. These fields can couple to analyte molecules in the
near field regions. As a result, concentration of the incident
light occurs at close vicinity of the nanoparticles enhancing the
Raman scattering from the analyte molecules. This method can
enhance the detection of biological systems by as much as a factor
of 10.sup.14.
[0005] Multiplexing is a demanding approach for high throughput
assays in various areas such as biological research, clinical
diagnosis and drug screening because of its great potentials in
increasing efficiencies of chemical and biochemical analyses. In a
multiplex assay, multiple probes are used that have specificities
to corresponding analytes in a sample mixture. One of the critical
challenges in establishing a multiplex platform is to develop a
probe identification system that has distinguishable components for
each individual probe in a large probe set.
[0006] Previous methods have utilized SERS in combination with
multiplex analysis. Such methods utilize target-coated gold
particles and DNA probes co-modified with both a Raman dye and
thiol group (Cao et al, Science 297:1536). However, such reagents
are generally expensive to manufacture and labor intensive to use.
In addition, coupling the Raman dye and thiol group to the analyte
does not provide flexibility in application and/or removal of the
dye or SERS substrate. Thus, there exists a need for compositions
and methods that provide lower costs and increased flexibility in
labeling analytes and capture reagents during multiplex
analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1A is a schematic flow chart illustrating an improved
method for detection of an analyte. Capture reagents bound to a
solid support (e.g., antibodies) include a Raman label. The binding
of a target analyte is subsequently detected by Raman
detection.
[0008] FIG. 1B is a schematic flow chart illustrating previous
methods for detection of an analyte absent interchangeable binding
members and Raman labeled capture reagents.
[0009] FIG. 2 is a schematic diagram illustrating an improved
method for detection of an analyte. The exemplary diagram
illustrates protein targets binding to antibodies immobilized to a
solid surface and labeled with Raman labels.
[0010] FIG. 3A is a schematic flow chart illustrating an improved
method for detection of a nucleic acid sequence (e.g., SNP).
Capture nucleic acids bound to a solid support include hybridize a
target nucleic acid associated with a first binding member.
Hybridization of the target nucleic acid is subsequently detected
by Raman detection.
[0011] FIG. 3B is a schematic flow chart illustrating previous
methods for detection of a nucleic acid sequence absent
interchangeable binding members.
[0012] FIG. 4 is a schematic diagram illustrating an improved
method for detection of a nucleic acid sequence. The exemplary
diagram illustrates target nucleic acids hybridizing to immobilized
capture nucleic acid molecules.
[0013] The following detailed description contains numerous
specific details in order to provide a more thorough understanding
of the disclosed embodiments of the invention. However, it will be
apparent to those skilled in the art that the embodiments can be
practiced without these specific details. In other instances,
devices, methods, procedures, and individual components that are
well known in the art have not been described in detail herein.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The various embodiments of the invention relate to signal
amplification methods for multiplex biological assays. In general,
biological target complexes are tagged by a seed substance that can
catalyze the formation of a surface-enhanced Raman scattering
(SERS) substrate. The target complexes can then bind to capture
reagents which include a Raman label. The SERS substrate is then
generated on the seed substance through reduction of metal cations.
The target signals are detected by SERS measurement of the Raman
labels. More specifically, embodiments of the invention provide
target analytes functionalized with specific binding members
comprising seed particles. Embodiments of the invention further
relate to target complexes formed between such target analytes and
a capture reagent bound to a solid substrate. The capture reagent
optionally includes a Raman label. Other embodiments of the
invention relate to methods of detecting binding of a target
analyte to a capture reagent coupled with surface-enhanced Raman
scattering (SERS) spectroscopy, to perform multiplexed detection of
analytes. This is exemplified for polypeptide targets in FIGS. 1A,
1B and 2, and for nucleic acid targets in FIGS. 3A, 3B and 4.
[0015] Accordingly, in one embodiment, a biological target complex
including a target analyte associated with a first specific binding
member is provided. The target complex further includes a second
specific binding member that binds to the first specific binding
member forming a target complex. The second specific binding member
includes a seed particle suitable for catalyzing the formation of a
surface enhanced Raman scattering (SERS) substrate. Subsequently,
the SERS substrate can be activated to provide a SERS effect. The
complex further includes a capture reagent bound to a solid
substrate. The capture reagent can include a Raman label.
[0016] "Target 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 capture reagent (e.g., an antibody, polypeptide, DNA,
RNA, cell, virus, etc.) or for which a capture reagent can be
prepared, and the target analyte can bind to one or more capture
reagents in an assay. "Target analyte" also includes any antigenic
substances, haptens, antibodies, and combinations thereof. The
target 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
[0017] "Target analyte-analog", as used herein, refers to a
substance which cross reacts with an analyte capture reagent
although it may do so to a greater or lesser extent than does the
target analyte itself. The target analyte-analog can include a
modified target analyte as well as a fragmented or synthetic
portion of the target analyte molecule so long as the target
analyte analog has at least one epitopic site in common with the
target analyte of interest.
[0018] "Specific binding member," as used herein, is a member of a
specific binding pair, i.e., two different molecules where one of
the molecules (e.g., a first specific binding member), through
chemical or physical means, specifically binds to the second
molecule (e.g., a second specific binding member). 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.
[0019] "Ancillary Specific binding member," as used herein, is a
specific binding member used in addition to the specific binding
members of the target analyte and the capture reagent and becomes a
part of the final complex. One or more ancillary specific binding
members can be used in an assay. "Binding," as used herein, is any
process resulting in the formation of coupled moieties. The process
of "binding" refers to the direct or indirect attachment of one
moiety to another through the formation of at least one bond, which
can include covalent, ionic, coordinative, hydrogen, or Van der
Waals bonds, or non-chemical interactions, for example, hydrophobic
interactions. It is understood that two moieties can be coupled to
each other by numerous ways. Such coupling can include, but is not
limited to, specific non-covalent affinity interactions, for
example streptavidin: or avidin:biotin interactions and
hapten:antibody interactions; hydrophobic interactions; magnetic
interactions; polar interactions, for example, "wetting"
associations between two polar surfaces or between
oligonucleotide/polyethylene glycol; formation of a covalent bond,
for example, an amide bond, a disulfide bond, a thioether bond, an
ether bond, a carbon-carbon bond; or via other crosslinking agents;
or via an acid-labile linker. Exemplary coupled moieties include,
but are not limited to, antibody-epitope complexes, receptor-ligand
complexes or complementary nucleic acid complexes. Exemplary target
analyte-capture reagent complexes include a target nucleic acid
sequence (i.e., a target analyte) hybridizing to a complementary
nucleic acid sequence (i.e. a capture reagent). Other exemplary
target analyte-capture reagent complexes include a target
polypeptide (i.e., a target analyte) binding to a receptor or
antibody (i.e. a capture reagent), thus forming a ligand binding
pair. The extent of the binding is influenced by the presence, and
the amount present, of the target analyte. "Associated," as used
herein, is the state of two or more molecules and/or particulates
being held in close proximity to one another.
[0020] "Capture reagent," as used herein, is a molecule or compound
capable of binding the target analyte or target reagent, which can
be directly or indirectly attached to a substantially solid
material. The capture agent can be any substance for which there
exists a naturally occurring target analyte (e.g., an antibody,
polypeptide, DNA, RNA, cell, virus, etc.) or for which a target
analyte can be prepared, and the capture reagent can bind to one or
more target analytes in an assay.
[0021] "Seed particle," as used herein, is any substance that can
precipitate formation of a nanoparticle from a metal colloid
solution and support the phenomenon of a surface-enhanced Raman
light scattering (SERS) or surface-enhanced resonance Raman light
scattering (SERRS). Examples of seed 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.
[0022] "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
label include "Raman-active label," "Raman dye" and "Raman reporter
molecule." A Raman label includes any organic or inorganic
molecule, atom, complex or structure, including but not limited to
synthetic molecules, dyes, naturally occurring pigments such as
phycoerythrin, organic nanostructures such as C60, buckyballs and
carbon nanotubes, metal nanostructures such as gold or silver
nanoparticles or nanoprisms and nano-scale semiconductors such as
quantum dots. Numerous examples of Raman labels are disclosed
below. Exemplary Raman labels are provided in Table 1 below. The
skilled artisan will realize that such examples are not limiting,
and that "Raman label" encompasses any organic or inorganic atom,
molecule, compound or structure known in the art that can be
detected by Raman spectroscopy. A particular type of "Raman label"
includes "Raman dyes." Examples of Raman dyes include chemical
labels such as cresyl fast violet (CFV, Fluka), brilliant cresyl
blue (BCB, Allied Chemical and Dye) and p-aminobenzoic acid (PABA,
Aldrich). Additional dyes include Cy3, Cy3.5, Cy5, TAMRA (TMR),
Texas-Red (TR) and Rhodamine 6G (RD).
[0023] Raman labels offer the advantage of producing sharp spectral
peaks, allowing a greater number of distinguishable labels to be
attached to probes. Additional non-limiting examples of
Raman-active labels of use include TRIT (tetramethyl rhodamine
isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye,
phthalic acid, terephthalic acid, isophthalic acid, cresyl fast
violet, cresyl blue violet, brilliant cresyl blue,
para-aminobenzoic acid, erythrosine, biotin, digoxigenin,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein, TET
(6-carboxy-2',4,7,7'-tetrachlorofluorescein), HEX
(6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein), Joe
(6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein)
5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxy rhodamine, Tamra (tetramethylrhodamine),
6-carboxyrhodamine, Rox (carboxy-X-rhodamine), R6G (Rhodamine 6G),
phthalocyanines, azomethines, cyanines (e.g. Cy3, Cy3.5, Cy5),
xanthines, succinylfluoresceins,
N,N-diethyl-4-(5'-azobenzotriazolyl)-phenylamine and aminoacridine.
These and other Raman-active tags can be obtained from commercial
sources (e.g., Molecular Probes, Eugene, Oreg.).
[0024] It is contemplated that the Raman-active label may comprise
one or more double bonds, for example carbon to nitrogen double
bonds. It is also contemplated that the Raman-active labels may
comprise a ring structure with side groups attached to the ring
structure, such as polycyclic aromatic compounds in general.
Compounds with side groups that increase Raman intensity include
compounds with conjugated ring structures, such as purines,
acridines, Rhodamine dyes and Cyanine dyes. The overall polarity of
a polymeric active molecular Raman code is contemplated to be
hydrophilic, but hydrophobic side groups can be included. Other
tags that can be of use include cyanide, thiol, chlorine, bromine,
methyl, phosphorus and sulfur.
[0025] Included herein are guidelines useful for designing and
manufacturing Raman labels. Briefly, exemplary Raman labels
provided herein encompass those that comprise any combination of
the following attributes: 1) a conjugated aromatic system
(generally two or more rings); 2) one or more nitrogen or sulphur
atoms with a lone pair of electrons (for Ag binding), preferably
two of such atoms on the same side of the molecule so that they can
chelate a metal atom; 3) as few oxygen atoms as possible; 4) few or
no free OH groups in proximity to the Ag binding site; and 5) few
competing Ag binding modes.
[0026] Accordingly, molecules useful for providing metal surface
adsorption generally include at least one N or S atoms with a lone
pair of electrons (for Ag binding). In some aspects, it is useful
to have two of such atoms on the same side of the molecule so that
they can chelate metal atoms. Additionally, in other aspects, a
positive charge in the molecule, such as N.sup.+, S.sup.+, or
C.sup.+, is included.
[0027] Generally, silver colloids are stable with a relatively
large surface potential (-60 mV or lower); when organic compound
molecules are adsorbed on to silver colloid surfaces, the potential
(zeta potential) is reduced and thus cause colloid agglutination
with the organic compounds as the "glue". The present study has
identified compounds with a conjugated aromatic system that can
more efficiently induce aggregation of Ag particles. N or S atoms
are suitable for stable binding to Ag surface, and a single binding
mode anchored by two chelating electron-donor atoms aids in
generating strong Raman signals with simple signature. In general,
O atoms, especially those from free hydroxyl groups, compete with N
and S for Ag surface binding.
[0028] Molecules suitable for providing a strong Raman signal
generally include those possessing strong absorption of UV-Visible
light (conjugated double bonds and aromatic system). Molecules with
strong absorption near the Raman excitation wavelength are included
because of their resonance effect. Also included are those
molecules with vibration modes such as C--N bond stretching, C--C
bond stretching, and 6-member ring breath modes in an aromatic
system. Also included are those molecules with few O atoms because,
generally, C--O, O--H, and C.dbd.O bonds do not provide strong
Raman signals.
[0029] Also provided are chemical structures that impart a unique
Raman signature for a Raman label. The Raman shift of a particular
mode can be "moved" to either longer wavelength or shorter
wavelength based on its chemical structure environment. For
example, neighboring electron-withdrawing group (conjugated
aromatic ring, CN, etc.) may move the Raman peak to higher wave
number, and electron-donating groups (Amine, thiol, etc.) do the
opposite. In addition, unique Raman signatures can be imparted to a
molecule by avoiding same vibration modes occurring at different
parts of the molecule unless they are symmetrical. Such structure
gives double peaks or broadened single peak, which generally
complicate the Raman signature.
[0030] In certain embodiments, the Raman-active labels used in the
invention methods and complexes can be independently selected from
the group consisting of nucleic acids, nucleotides, nucleotide
analogs, base analogs, fluorescent dyes, peptides, amino acids,
modified amino acids, organic moieties, quantum dots, carbon
nanotubes, fullerenes, metal nanoparticles, electron dense
particles and crystalline particles, or a combination of any two or
more thereof.
[0031] 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.
[0032] Nanoparticles useful 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 mm, most preferably from about
10 to about 30 nm. The nanoparticles may also be rods, prisms,
cubes, tetrahedra, or core shell particles.
[0033] "SERS (Surface-Enhanced Raman Scattering)" means the
increase in Raman scattering exhibited by certain molecules in
proximity to certain metal surfaces. "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.
[0034] "SERS substrate," as used herein, includes a stain such as a
silver or gold stain that provides for activating Raman labels on
particles to produce a SERS effect. "Stain," as used herein,
includes material, e.g., gold, silver, etc., that can be used to
produce or enhance a detectable change in any assay described
herein. For example, silver staining can be employed with any type
of nanoparticles that catalyze the reduction of silver. Thus, gold
colloid exposed to a staining solution containing AgNO.sub.3 can
serve as nucleation sites for the deposition of Ag.
[0035] "Intermediary molecule," as used herein, is any substance
that includes a specific binding member and binds to a target
analyte. Exemplary intermediary molecules include antibodies
attached to a specific binding member. As shown in FIG. 2, an
exemplary intermediary molecule includes second antibody comprising
nanogold seeds. Such molecules generally bind to the target analyte
following binding of the target analyte to the immobilized capture
reagent.
[0036] "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.
[0037] The complexes and methods provided herein are
distinguishable from previous reports combining SERS detection with
Raman label applications. For example, gold nanoparticles combined
with surface-enhanced Raman scattering spectroscopy for detection
and identification of single dye molecules has been described by
Cao et al (Science 297:1536). Cao et al designed a probe that is
built around a 13 nm gold nanoparticle. The nanoparticles are
coated with hydrophilic oligonucleotides containing a Raman dye at
one end and terminally capped with a small molecule recognition
element (e.g. biotin). This molecule is catalytically active and
will be coated with silver in the solution of Ag(I) and
hydroquinone. After the probe is attached to a small molecule or an
antigen it is designed to detect, the substrate is exposed to
silver and hydroquinone solution. The silver-plating occurs in
close proximity to the Raman dye, which allows for dye signature
detection with a standard Raman microscope. In contrast, the
complexes disclosed herein are comprised of a Raman dye that is
separate from, for example, a gold nanoparticle suitable for
supporting a SERS substrate. The biological target complexes
comprise a seed particle capable of catalyzing the formation of a
SERS substrate. The seed particle is associated with a second
binding member which binds to a first binding member associated
with a target analyte. Once the target analyte binds to a capture
reagent associated with a Raman label the SERS substrate is
generated through reduction of metal cations. Alternatively, when a
nucleic acid is a the target analyte, the Raman label is added to
the SERS substrate subsequent to formation (see e.g., FIGS. 3A, 3B
and 4).
[0038] Accordingly, in another embodiment, a method for detecting
an analyte-capture reagent complex by Raman spectroscopy is
provided. The method includes providing a target analyte associated
with a first specific binding member and providing a capture
reagent bound to a solid substrate. The capture reagent includes a
Raman label. The method further includes contacting the target
analyte with the capture reagent of under conditions suitable for
forming a target analyte-capture reagent complex. The first
specific binding partner is contacted with a second specific
binding member functionally associated with a seed particle
suitable for associating with a SERS substrate. The target
analyte-capture reagent complex is then contacted with
electromagnetic radiation suitable for detecting a specific
property associated with the analyte-capture reagent complex by
Raman spectroscopy.
[0039] In still another embodiment, the invention provides methods
for multiplex detection of analytes in a sample by contacting
target analytes in a sample under conditions suitable to form
complexes with a set of capture reagents. In the case of a target
analyte that is a non-nucleic acid, the capture reagent is
conjugated with an Raman label. Each capture reagent can be
conjugated to a unique Raman label, such as a Raman dye, associated
with a unique optical signature. Following complex formation and
SERS activation of the SERS substrate, the unique optical
signatures are detected in a multiplex manner with a suitable
detection device. Since each specifically binding target analyte is
bound to a specific capture reagent conjugated to a known Raman dye
that emits a distinguishable optical signature, such as a SERS
signal, individual optical signatures detected from the constructs
are thus associated with the identity of a target analyte in the
sample.
[0040] "Test sample," as used herein, means the sample containing
the target analyte to be detected and assayed using the present
invention. The test sample can contain other components besides the
target 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 target analyte as long as the other substances do no
interfere with the binding of the target analyte with the capture
reagent or the specific binding of the first binding member to the
second binding member. 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.
[0041] The optical detection procedure or combination of optical
detection procedures to be used will depend on the nature of the
analytes, the separation device or matrix, as well as the structure
and properties of the capture reagents. The separated complexes can
be detected by one or a combination of optical techniques selected
from adsorption, reflection, polarization, refraction,
fluorescence, Raman spectra, SERS, resonance light scattering,
grating-coupled surface plasmon resonance, using techniques
described herein and as known in the art.
[0042] The Raman active labels are selected from a Raman-active
dye, amino acid, nucleotide, or a combination thereof. Examples of
Raman active amino acids suitable for incorporation into the
Raman-active tag include arginine, methionine, cysteine, and
combinations thereof. Examples of Raman-active nucleotides suitable
for incorporation into the Raman-active tag include adenine,
guanine and derivatives thereof. The Raman-active labels are small
molecules that are highly active in producing a Raman signal and
typically have a molecular weight of less than 1 kDa. Raman-active
tags that meet these requirements include dyes (e.g., R6G, Tamra,
Rox), amino acids (e.g., arginine, methionine, cysteine), nucleic
acid bases (e.g., adenine, or guanine), or any combination thereof.
Naturally occurring or synthetic compounds having the
above-described characteristics, such as suitable molecular weight
and Raman characteristics, can also be used. The Raman-active tags
can be placed in any position along the molecular backbone and a
single backbone can have more than one such tag. Raman signatures
of the members of the set can be adjusted by changing the type,
number and relative positions of the Raman-active tags along the
backbone during synthesis of the molecular Raman codes.
[0043] The bound complexes including the Raman-active label is
covered with a thin layer of metal, as described herein, to enhance
Raman signals from the complex. The metal layer, being in close
proximity to the analyte, will produce SERS signals and the
complete solid support can be irradiated with a single light source
while SERS signals are collected from the bound complexes, for
example by SERS scanning. One or more SERS spectra obtained from a
discrete site associates the capture reagent with the presence of a
particular target analyte in the sample or identifies the capture
reagent as having affinity (e.g., heretofore unknown) for a
molecule or complex in the sample.
[0044] Antibodies and receptors are non-limiting examples of the
capture reagents attached to the discrete locations on the solid
support. Nucleic acids, phage-displayed peptides, nucleic acids,
aptamers, ligands, lectins, and combinations thereof can also be
used as capture reagents in the invention methods. The sample is
not necessarily a body fluid, although it may be, but can comprise
any mixed pool of analytes, including proteins, gluco-proteins,
lipid proteins, nucleic acids, virus particles, polysaccharides,
steroids, and combinations thereof. In one aspect, the sample
comprises a pool of body fluid of patients known or suspected of
having a particular disease.
[0045] The term "nucleic acid" is used broadly herein to mean a
sequence of deoxyribonucleotides or ribonucleotides that are linked
together by a phosphodiester bond. For convenience, the term
"oligonucleotide" is used herein to refer to a polynucleotide that
is used as a primer or a probe. Generally, an oligonucleotide
useful as a probe or primer that selectively hybridizes to a
selected nucleotide sequence is at least about 10 nucleotides in
length, usually at least about 15 nucleotides in length, for
example between about 15 and about 50 nucleotides in length.
[0046] A nucleic acid can be RNA or can be DNA, which can be a gene
or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid
sequence, or the like, and can be single stranded or double
stranded, as well as a DNA/RNA hybrid. In various embodiments, a
polynucleotide, including an oligonucleotide (e.g., a probe or a
primer) can contain nucleoside or nucleotide analogs, or a backbone
bond other than a phosphodiester bond. In general, the nucleotides
comprising a polynucleotide are naturally occurring
deoxyribonucleotides, such as adenine, cytosine, guanine or thymine
linked to 2'-deoxyribose, or ribonucleotides such as adenine,
cytosine, guanine or uracil linked to ribose. However, a
polynucleotide or oligonucleotide also can contain nucleotide
analogs, including non-naturally occurring synthetic nucleotides or
modified naturally occurring nucleotides. Such nucleotide analogs
are well known in the art and commercially available, as are
polynucleotides containing such nucleotide analogs (Lin et al.,
Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry
34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73
(1997).
[0047] The covalent bond linking the nucleotides of a nucleic acid
generally is a phosphodiester bond. However, the covalent bond also
can be any of numerous other bonds, including a thiodiester bond, a
phosphorothioate bond, a peptide-like bond or any other bond known
to those in the art as useful for linking nucleotides to produce
synthetic polynucleotides (see, for example, Tam et al., Nucl.
Acids Res. 22:977-986 (1994); Ecker and Crooke, BioTechnology
13:351360 (1995)). The incorporation of non-naturally occurring
nucleotide analogs or bonds linking the nucleotides or analogs can
be particularly useful where the polynucleotide is to be exposed to
an environment that can contain a nucleolytic activity, including,
for example, a tissue culture medium or upon administration to a
living subject, since the modified polynucleotides can be less
susceptible to degradation.
[0048] As used herein, the term "selective hybridization" or
"selectively hybridize," refers to hybridization under moderately
stringent or highly stringent conditions such that a nucleotide
sequence preferentially associates with a selected nucleotide
sequence over unrelated nucleotide sequences to a large enough
extent to be useful in identifying the selected nucleotide
sequence. It will be recognized that some amount of non-specific
hybridization is unavoidable, but is acceptable provided that
hybridization to a target nucleotide sequence is sufficiently
selective such that it can be distinguished over the non-specific
cross-hybridization, for example, at least about 2-fold more
selective, generally at least about 3-fold more selective, usually
at least about 5-fold more selective, and particularly at least
about 10-fold more selective, as determined, for example, by an
amount of labeled oligonucleotide that binds to target nucleic acid
molecule as compared to a nucleic acid molecule other than the
target molecule, particularly a substantially similar (i.e.,
homologous) nucleic acid molecule other than the target nucleic
acid molecule. Conditions that allow for selective hybridization
can be determined empirically, or can be estimated based, for
example, on the relative GC:AT content of the hybridizing
oligonucleotide and the sequence to which it is to hybridize, the
length of the hybridizing oligonucleotide, and the number, if any,
of mismatches between the oligonucleotide and sequence to which it
is to hybridize (see, for example, Sambrook et al., "Molecular
Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press
1989)).
[0049] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42EC (moderate stringency conditions); and 0.1.times.SSC at
about 68EC (high stringency conditions). Washing can be carried out
using only one of these conditions, e.g., high stringency
conditions, or each of the conditions can be used, e.g., for 10-15
minutes each, in the order listed above, repeating any or all of
the steps listed. However, as mentioned above, optimal conditions
will vary, depending on the particular hybridization reaction
involved, and can be determined empirically.
[0050] As used herein, the term "antibody" is used in its broadest
sense to include polyclonal and monoclonal antibodies, as well as
antigen binding fragments of such antibodies. An antibody useful in
a method of the invention, or an antigen binding fragment thereof,
is characterized, for example, by having specific binding activity
for an epitope of an analyte. Alternatively, as explained below,
the analyte can be the probe antibody, particularly in embodiments
of the invention methods wherein antibodies used as probes (e.g.
active agents) are exposed to body fluids to screen a set of
antibodies for utility as drug candidates.
[0051] The antibody, for example, includes naturally occurring
antibodies as well as non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric,
bifunctional and humanized antibodies, as well as antigen-binding
fragments thereof. Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, can be produced
recombinantly or can be obtained, for example, by screening
combinatorial libraries consisting of variable heavy chains and
variable light chains (see Huse et al., Science 246:1275-1281
(1989)). These and other methods of making, for example, chimeric,
humanized, CDR-grafted, single chain, and bifunctional antibodies
are well known to those skilled in the art (Winter and Harris,
Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546,
1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring
Harbor Laboratory Press, 1988); Hilyard et al., Protein
Engineering: A practical approach (IRL Press 1992); Borrabeck,
Antibody Engineering, 2d ed. (Oxford University Press 1995)).
Monoclonal antibodies suitable for use as probes may also be
obtained from a number of commercial sources. Such commercial
antibodies are available against a wide variety of targets.
Antibody probes can be conjugated to molecular backbones using
standard chemistries, as discussed below.
[0052] The term "binds specifically" or "specific binding
activity," when used in reference to an antibody means that an
interaction of the antibody and a particular epitope has a
dissociation constant of at least about 1.times.10.sup.-6,
generally at least about 1.times.10.sup.-7, usually at least about
1.times.10.sup.-8, and particularly at least about
1.times.10.sup.-9 or 1.times.10.sup.-10 or less. As such, Fab,
F(ab').sub.2, Fd and Fv fragments of an antibody that retain
specific binding activity for an epitope of an antigen, are
included within the definition of an antibody.
[0053] In the context of the invention, the term "ligand" denotes a
naturally occurring specific binding partner of a receptor, a
synthetic specific-binding partner of a receptor, or an appropriate
derivative of the natural or synthetic ligands. The determination
and isolation of ligands is well known in the art (Lerner, Trends
Neurosci. 17:142-146, 1994). As one of skill in the art will
recognize, a molecule (or macromolecular complex) can be both a
receptor and a ligand. In general, the binding partner having a
smaller molecular weight is referred to as the ligand and the
binding partner having a greater molecular weight is referred to as
a receptor.
[0054] In certain aspects, the invention pertains to methods for
detecting an analyte in a sample. By "analyte" is meant any
molecule or compound for which a probe can be found. An analyte can
be in the solid, liquid, gaseous or vapor phase. By "gaseous or
vapor phase analyte" is meant a molecule or compound that is
present, for example, in the headspace of a liquid, in ambient air,
in a breath sample, in a gas, or as a contaminant in any of the
foregoing. It will be recognized that the physical state of the gas
or vapor phase can be changed by pressure, temperature as well as
by affecting surface tension of a liquid by the presence of or
addition of salts etc.
[0055] The analyte can be a molecule found directly in a sample
such as a body fluid from a host. The sample can be examined
directly or can be pretreated to render the analyte more readily
detectable. Furthermore, the analyte of interest can be determined
by detecting an agent probative of the analyte of interest such as
a specific binding pair member complementary to the analyte of
interest, whose presence will be detected only when the analyte of
interest is present in a sample. Thus, the agent probative of the
analyte becomes the analyte that is detected in an assay. The body
fluid can be, for example, urine, blood, plasma, serum, saliva,
semen, stool, sputum, cerebral spinal fluid, tears, mucus, and the
like.
[0056] As used herein, the term "colloid" refers to metal ions
suspended in a liquid, usually water. Typical metals contemplated
for use in invention metal colloids and to from nanoparticles
include the transparent metals, for example, silver, gold,
platinum, aluminum, and the like.
[0057] To enhance the Raman spectra produced by Raman-active
substrates, a thin layer of a transparent metal, wherein the layer
has a roughened surface, is deposited over the upper layer of the
substrate and/or the bound complexes thereon. The roughness
features are on the order of tens of nanometers; small, compared to
the wavelength of the incident excitation radiation. The small size
of the particles allows the excitation of the metal particle's
surface plasmon to be localized on the particle. Metal roughness
features at the metal surface can be developed in a number of ways;
for example; vapor deposition of metal particles or application of
metal colloids onto the upper layer of the biosensor. Since the
surface electrons of the metal are confined to the particle, whose
size is small, the plasmon excitation is also confined to the
roughness feature. The resulting electromagnetic field of the
plasmon is very intense, greatly enhancing the SERS signal as
compared to a Raman signal.
[0058] It has been estimated that only 1 in 10 analyte molecules
inelastically scatter in Raman Spectroscopy. However, in
embodiments of the invention methods wherein the intensity of Raman
signal from a scattering molecule is greatly enhanced under SERS
conditions, low concentrations of a Raman-active analyte can be
detected at concentrations as low as pico- and femto-molar. In some
circumstances, the invention methods can be used to detect the
presence of a single analyte molecule in a complex biological
sample, such as blood serum, by depositing a thin layer of a
transparent metal so as to be in contact with the bound complexes
containing a Raman label. Gold, silver, copper and aluminum are the
transparent metals most useful for this technique.
[0059] A roughened metal surface can be produced using one of
several methods. The term "a thin metal layer" as used herein means
a metal layer deposited by chemical vapor deposition over the bound
complexes containing a Raman label. Alternatively, a thin metal
layer means a layer of nanoparticles formed by subjecting a
colloidal solution of metal cations to reducing conditions to form
metal nanoparticles in situ. In some embodiments, the nanoparticles
will contain the bound complexes. Alternatively, seed particles,
for example attached to the Raman codes, can precipitate formation
of the nanoparticles from a metal colloid solution. In this
context, "thin" means having a thickness of about one-half the
wavelength of the irradiating light source (usually a laser) to
achieve the benefit of SERS, for example from about 15 nm to about
500 nm, such as about 100 nm to about 200 nm.
[0060] The "analytes", as the term is used herein, includes nucleic
acids, proteins, peptides, lipids, carbohydrates, glycolipids,
glycoproteins or any other potential target for which a specific
probe can be prepared. As discussed above, antibody or aptamer
probes can be incorporated into the invention active molecular
Raman codes and used to identify any target for which an aptamer or
antibody can be prepared. The presence of multiple analytes in a
sample can be assayed simultaneously, since each member of a set
can be distinguishably labeled and detected. Quantification of the
analyte can be performed by standard techniques, well known in
spectroscopic analysis. For example, the amount of analyte bound to
an invention Raman probe construct can be determined by measuring
the signal intensity produced and comparison to a calibration curve
prepared from known amounts of similar Raman probe construct
standards. Such quantification methods are well within the routine
skill in the art.
[0061] A "substrate" as the term is used herein, includes such well
known devices as chips or microtiter plates, may comprise a
patterned surface containing individual discrete sites that can be
treated as described herein bind to individual analytes or types of
analytes. Alternatively, in embodiments wherein the probe Raman
construct is attached to the substrate, a correlation between the
location of an individual site on the array with the Raman code or
probe located at that particular site can be made.
[0062] Array compositions may include at least a surface with a
plurality of discrete substrate sites. The size of the array will
depend on the end use of the array. Arrays containing from about 2
to many millions of different discrete substrate sites can be made.
Generally, the array will comprise from two to as many as a billion
or more such sites, depending on the size of the surface. Thus,
very high density, high density, moderate density, low density or
very low density arrays can be made. Some ranges for very
high-density arrays are from about 10,000,000 to about
2,000,000,000 sites per array. High-density arrays range from about
100,000 to about 10,000,000 sites. Moderate density arrays range
from about 10,000 to about 50,000 sites. Low-density arrays are
generally less than 10,000 sites. Very low-density arrays are less
than 1,000 sites.
[0063] The sites comprise a pattern, i.e. a regular design or
configuration, or can be randomly distributed. A regular pattern of
sites can be used such that the sites can be addressed in an X-Y
coordinate plane. The surface of the substrate can be modified to
allow attachment of analytes at individual sites. Thus, the surface
of the substrate can be modified such that discrete sites are
formed. In one embodiment, the surface of the substrate can be
modified to contain wells, i.e. depressions in the surface of the
substrate. This can be done using a variety of known techniques,
including, but not limited to, photolithography, stamping
techniques, molding techniques and microetching techniques. As will
be appreciated by those in the art, the technique used will depend
on the composition and shape of the substrate. Alternatively, the
surface of the substrate can be modified to contain chemically
derived sites that can be used to attach analytes or probes to
discrete locations on the substrate. The addition of a pattern of
chemical functional groups, such as amino groups, carboxy groups,
oxo groups and thiol groups can be used to covalently attach
molecules containing corresponding reactive functional groups or
linker molecules.
[0064] Biological "analytes" may comprise naturally occurring
proteins or fragments of naturally occurring proteins. Thus, for
example, cellular extracts containing proteins, or random or
directed digests of proteinaceous cellular extracts, can be used.
In this way libraries of procaryotic and eukaryotic proteins can be
made for screening the systems described herein. For example
libraries of bacterial, fungal, viral, and mammalian proteins can
be generated for screening purposes.
[0065] Methods for oligonucleotide synthesis are well known in the
art and any such known method can be used. For example,
oligonucleotides can be prepared using commercially available
oligonucleotide synthesizers (e.g., Applied Biosystems, Foster
City, Calif.). Nucleotide precursors attached to a variety of tags
can be commercially obtained (e.g. Molecular Probes, Eugene, Oreg.)
and incorporated into oligonucleotides or polynucleotides.
Alternatively, nucleotide precursors can be purchased containing
various reactive groups, such as biotin, diogoxigenin, sulfhydryl,
amino or carboxyl groups. After oligonucleotide synthesis, tags can
be attached using standard chemistries. Oligonucleotides of any
desired sequence, with or without reactive groups for tag
attachment, may also be purchased from a wide variety of sources
(e.g., Midland Certified Reagents, Midland, Tex.).
[0066] The metal required for achieving a suitable SERS signal is
inherent in the COIN, and a wide variety of Raman-active organic
compounds can be incorporated into the particle. Indeed, a large
number of unique Raman signatures can be created by employing
nanoparticles containing Raman-active organic compounds of
different structures, mixtures, and ratios. Thus, the methods
described herein employing COINs are useful for the simultaneous
detection of many analytes in a sample, resulting in rapid
qualitative analysis of the contents of "profile" of a body fluid.
In addition, since many COINs can be incorporated into a single
nanoparticle, the SERS signal from a single COIN particle is strong
relative to SERS signals obtained from Raman-active materials that
do not contain the nanoparticles described herein as COINs. This
situation results in increased sensitivity compared to
Raman-techniques that do not utilize COINs.
[0067] COINs are readily prepared for use in the invention methods
using standard metal colloid chemistry. The preparation of COINs
also takes advantage of the ability of metals to adsorb organic
compounds. Indeed, since Raman-active organic compounds are
adsorbed onto the metal during formation of the metallic colloids,
many Raman-active organic compounds can be incorporated into the
COIN without requiring special attachment chemistry.
[0068] In general, the COINs used in the invention methods are
prepared as follows. An aqueous solution is prepared containing
suitable metal cations, a reducing agent, and at least one suitable
Raman-active organic compound. The components of the solution are
then subject to conditions that reduce the metallic cations to form
neutral, colloidal metal particles. Since the formation of the
metallic colloids occurs in the presence of a suitable Raman-active
organic compound, the Raman-active organic compound is readily
adsorbed onto the metal during colloid formation. This simple type
of COIN is referred to as type I COIN. Type I COINs can typically
be isolated by membrane filtration. In addition, COINs of different
sizes can be enriched by centrifugation.
[0069] In alternative embodiments, the COINs can include a second
metal different from the first metal, wherein the second metal
forms a layer overlying the surface of the nanoparticle. To prepare
this type of SERS-active nanoparticle, type I COINs are placed in
an aqueous solution containing suitable second metal cations and a
reducing agent. The components of the solution are then subject to
conditions that reduce the second metallic cations so as to form a
metallic layer overlying the surface of the nanoparticle. In
certain embodiments, the second metal layer includes metals, such
as, for example, silver, gold, platinum, aluminum, and the like.
This type of COIN is referred to as type II COINs. Type II COINs
can be isolated and or enriched in the same manner as type I COINs.
Typically, type I and type II COINs are substantially spherical and
range in size from about 20 nm to 60 nm. The size of the
nanoparticle is selected to be about one-half the wavelength of
light used to irradiate the COINs during detection.
[0070] Typically, organic compounds are attached to a layer of a
second metal in type II COINs by covalently attaching organic
compounds to the surface of the metal layer Covalent attachment of
an organic layer to the metallic layer can be achieved in a variety
ways well known to those skilled in the art, such as for example,
through thiol-metal bonds. In alternative embodiments, the organic
molecules attached to the metal layer can be crosslinked to form a
molecular network.
[0071] The COIN(s) used in the invention methods can include cores
containing magnetic materials, such as, for example, iron oxides,
and the like. Magnetic COINs can be handled without centrifugation
using commonly available magnetic particle handling systems.
Indeed, magnetism can be used as a mechanism for separating
biological targets attached to magnetic COIN particles tagged with
particular biological probes.
[0072] As used herein, "Raman-active organic compound" refers to an
organic molecule that produces a unique SERS signature in response
to excitation by a laser. A variety of Raman-active organic
compounds are contemplated for use as components in COINs. In
certain embodiments, Raman-active organic compounds are polycyclic
aromatic or heteroaromatic compounds. Typically the Raman-active
organic compound has a molecular weight less than about 300
Daltons.
[0073] Additional, non-limiting examples of Raman-active organic
compounds useful in COINs include TRIT (tetramethyl rhodamine
isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye,
phthalic acid, terephthalic acid, isophthalic acid, cresyl fast
violet, cresyl blue violet, brilliant cresyl blue,
para-aminobenzoic acid, erythrosine, biotin, digoxigenin,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino
phthalocyanines, azomethines, cyanines, xanthines,
succinylfluoresceins, aminoacridine, and the like. These and other
Raman-active organic compounds can be obtained from commercial
sources (e.g., Molecular Probes, Eugene, Oreg.).
[0074] In certain embodiments, the Raman-active compound is
adenine, adenine, 4-amino-pyrazolo(3,4-d)pyrimidine,
2-fluoroadenine, N6-benzolyadenine, kinetin,
dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine,
8-azaguanine, 6-mercaptopurine,
4-amino-6-mercaptopyrazolo(3,4-d)pyrimidine, 8-mercaptoadenine, or
9-amino-acridine 4-amino-pyrazolo(3,4-d)pyrimidine, or
2-fluoroadenine. In one embodiment, the Raman-active compound is
adenine.
[0075] When "fluorescent compounds" are incorporated into COINs,
the fluorescent compounds can include, but are not limited to,
dyes, intrinsically fluorescent proteins, lanthanide phosphors, and
the like. Dyes useful for incorporation into COINs include, for
example, rhodamine and derivatives, such as Texas Red, ROX
(6-carboxy-X-rhodamine), rhodamine-NHS, and TAMRA
(5/6-carboxytetramethyl rhodamine NHS); fluorescein and
derivatives, such as 5-bromomethyl fluorescein and FAM
(5'-carboxyfluorescein NHS), Lucifer Yellow, IAEDANS, 7-Me.sub.2,
N-coumarin-4-acetate, 7-OH-4-CH.sub.3-coumarin-3-acetate,
7-NH.sub.2-4CH.sub.3-coumarin-3-acetate (AMCA), monobromobimane,
pyrene trisulfonates, such as Cascade Blue, and
monobromotrimethyl-ammoniobimane.
[0076] Exemplary uses for the methods described herein is to detect
a target nucleic acid. Such a method is useful, for example, for
detection of a single nucleotide polymorphism (SNP), for detection
of infectious agents within a clinical sample, detection of an
amplification product derived from genomic DNA or RNA or message
RNA, or detection of a gene (cDNA) insert within a clone. For
certain methods aimed at detection of a target polynucleotide, an
oligonucleotide probe is synthesized using methods known in the
art.
[0077] In the practice of the present invention, the Raman
spectrometer can be part of a detection unit designed to detect and
quantify Raman signals of the present invention by Raman
spectroscopy. Methods for detection of Raman labeled analytes, for
example nucleotides, using Raman spectroscopy are known in the art.
(See, e.g., U.S. Pat. Nos. 5,306,403; 6,002,471; and 6,174,677).
Variations on surface enhanced Raman spectroscopy (SERS), surface
enhanced resonance Raman spectroscopy (SERRS) and coherent
anti-Stokes Raman spectroscopy (CARS) have been disclosed.
[0078] A non-limiting example of a Raman detection unit is
disclosed in U.S. Pat. No. 6,002,471. An excitation beam is
generated by either a frequency doubled Nd:YAG laser at 532 nm
wavelength or a frequency doubled Ti:sapphire laser at 365 nm
wavelength. Pulsed laser beams or continuous laser beams can be
used. The excitation beam passes through confocal optics and a
microscope objective, and is focused onto the flow path and/or the
flow-through cell. The Raman emission light is collected by the
microscope objective and the confocal optics and is coupled to a
monochromator for spectral dissociation. The confocal optics
includes a combination of dichroic filters, barrier filters,
confocal pinholes, lenses, and mirrors for reducing the background
signal. Standard full field optics can be used as well as confocal
optics. The Raman emission signal is detected by a Raman detector,
that includes an avalanche photodiode interfaced with a computer
for counting and digitization of the signal.
[0079] Another example of a Raman detection unit is disclosed in
U.S. Pat. No. 5,306,403, including a Spex Model 1403 double-grating
spectrophotometer with a gallium-arsenide photomultiplier tube (RCA
Model C31034 or Burle Industries Model C3103402) operated in the
single-photon counting mode. The excitation source includes a 514.5
nm line argon-ion laser from SpectraPhysics, Model 166, and a 647.1
nm line of a krypton-ion laser (Innova 70, Coherent).
[0080] Alternative excitation sources include a nitrogen laser
(Laser Science Inc.) at 337 nm and a helium-cadmium laser (Liconox)
at 325 nm (U.S. Pat. No. 6,174,677), a light emitting diode, an
Nd:YLF laser, and/or various ions lasers and/or dye lasers. The
excitation beam can be spectrally purified with a bandpass filter
(Corion) and can be focused on the flow path and/or flow-through
cell using a 6.times. objective lens (Newport, Model L6X). The
objective lens can be used to both excite the Raman-active probe
constructs and to collect the Raman signal, by using a holographic
beam splitter (Kaiser Optical Systems, Inc., Model HB 647-26N18) to
produce a right-angle geometry for the excitation beam and the
emitted Raman signal. A holographic notch filter (Kaiser Optical
Systems, Inc.) can be used to reduce Rayleigh scattered radiation.
Alternative Raman detectors include an ISA HR-320 spectrograph
equipped with a red-enhanced intensified charge-coupled device
(RE-ICCD) detection system (Princeton Instruments). Other types of
detectors can be used, such as Fourier-transform spectrographs
(based on Michaelson interferometers), charged injection devices,
photodiode arrays, InGaAs detectors, electronmultiplied CCD,
intensified CCD and/or phototransistor arrays.
[0081] Any suitable form or configuration of Raman spectroscopy or
related techniques known in the art can be used for detection of
the target complex of the present invention, including but not
limited to normal Raman scattering, resonance Raman scattering,
surface enhanced Raman scattering, surface enhanced resonance Raman
scattering, coherent anti-Stokes Raman spectroscopy (CARS),
stimulated Raman scattering, inverse Raman spectroscopy, stimulated
gain Raman spectroscopy, hyper-Raman scattering, molecular optical
laser examiner (MOLE) or Raman microprobe or Raman microscopy or
confocal Raman microspectrometry, three-dimensional or scanning
Raman, Raman saturation spectroscopy, time resolved resonance
Raman, Raman decoupling spectroscopy or UV-Raman microscopy.
[0082] In certain aspects of the invention, a system for detecting
the target complex of the present invention includes an information
processing system. An exemplary information processing system may
incorporate a computer that includes a bus for communicating
information and a processor for processing information. In one
embodiment of the invention, the processor is selected from the
Pentium.RTM. family of processors, including without limitation the
Pentium.RTM. II family, the Pentium.RTM. III family and the
Pentium.RTM. 4 family of processors available from Intel Corp.
(Santa Clara, Calif.). In alternative embodiments of the invention,
the processor can be a Celeron.RTM., an Itanium.RTM., or a Pentium
Xeon.RTM. processor (Intel Corp., Santa Clara, Calif.). In various
other embodiments of the invention, the processor can be based on
Intel.RTM. architecture, such as Intel.RTM. IA-32 or Intel.RTM.
IA-64 architecture. Alternatively, other processors can be used.
The information processing and control system may further comprise
any peripheral devices known in the art, such as memory, display,
keyboard and/or other devices.
[0083] In particular examples, the detection unit can be operably
coupled to the information processing system. Data from the
detection unit can be processed by the processor and data stored in
memory. Data on emission profiles for various Raman labels may also
be stored in memory. The processor may compare the emission spectra
from the target complexes in the flow path and/or flow-through cell
to identify the Raman-active moiety in complexed with the target
analyte or the capture reagent. The information processing system
may also perform standard procedures such as subtraction of
background signals or comparison of signals from different
samples.
[0084] While certain methods of the present invention can be
performed under the control of a programmed processor, in
alternative embodiments of the invention, the methods can be fully
or partially implemented by any programmable or hardcoded logic,
such as Field Programmable Gate Arrays (FPGAs), TTL logic, or
Application Specific Integrated Circuits (ASICs). Additionally, the
disclosed methods can be performed by any combination of programmed
general purpose computer components and/or custom hardware
components.
[0085] Following the data gathering operation, the data will
typically be reported to a data analysis operation. To facilitate
the analysis operation, the data obtained by the detection unit
will typically be analyzed using a digital computer such as that
described above. Typically, the computer will be appropriately
programmed for receipt and storage of the data from the detection
unit as well as for analysis and reporting of the data
gathered.
[0086] In certain embodiments of the invention, custom designed
software packages can be used to analyze the data obtained from the
detection unit. In alternative embodiments of the invention, data
analysis can be performed using an information processing system
and publicly available software packages.
[0087] Exemplary Raman labels are provided below in Table 1.
TABLE-US-00001 TABLE 1 Raman labels selection Abbreviation Name
Structure AAD (AA) 8-Aza-Adenine ##STR00001## BZA (BA)
N-Benzoyladenine ##STR00002## APP 4-Amino-pyrazolo
[3,4-d]pyrimidine ##STR00003## ZEN Zeatin ##STR00004## MBL
Methylene Blue ##STR00005## AMA (AM) 9-Amino-acridine ##STR00006##
EBR Ethidium Bromide ##STR00007## BMB Bismarck Brown Y ##STR00008##
THN Thionin acetate ##STR00009## DAH 3,6-Diaminoacridine
##STR00010## AIC 4-Amino-5-imidazole- carboxamide hydrochloride
##STR00011## DII 1,3-Diiminoisoindoline ##STR00012## R6G Rhodamine
6G ##STR00013## CRV Crystal Violet ##STR00014## BFU Basic Fuchsin
##STR00015## NBA N-Benzyl-aminopurine ##STR00016## MBI
2-Mercapto-benzimidazole (MBI) ##STR00017## CYP 6-Cyanopurine
##STR00018## ANB Aniline Blue diammonium salt ##STR00019## ACA
N-[(3-(Anilinomethylene)- 2-chloro-1-cyclohexen-1-yl)
methylene]aniline mono- hydrochloride ##STR00020## ATT
O-(7-Azabenzotriazol-1-yl)- N,N,N',N'- tetramethyluronium
hexafluorophosphate ##STR00021## AMF 9-Aminofluorene hydrochloride
##STR00022## BBL Basic Blue ##STR00023## DDA 1,8-Diamino-4,5-
dihydroxyanthraquinone ##STR00024## PFV Proflavine hemisulfate salt
hydrate ##STR00025## VRA Variamine Blue RT Salt ##STR00026## ABZ
2-Amino-benzothiazole ##STR00027## MEL Melamine ##STR00028## PPN
3-(3-Pyridylmethyl amino)propionitrile ##STR00029## SSD Silver(I)
sulfadiazine ##STR00030## AMPT 4-Amino6-Mercaptopyrazolo
[3,4-d]pyrimidine ##STR00031## APU 2-Am-Purine ##STR00032## ATH
Adenine Thiol ##STR00033## FAD F-Adenine ##STR00034## MCP
6-Mercaptopurine ##STR00035## AMP 4-Amino-6-mercaptopyrazolo
[3,4-d]pyrimidine ##STR00036## R110 Rhodamine 110 ##STR00037## DAB
4-([4-(Dimethylamino) phenyl]azo)benzoic acid ##STR00038##
[0088] Although the invention has been described with reference to
the above example, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *