U.S. patent application number 12/894810 was filed with the patent office on 2011-03-24 for raman-active reagents and the use thereof.
This patent application is currently assigned to IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC.. Invention is credited to G. Brent Dawson, Robert J. Lipert, Jing Ni, Marc D. Porter.
Application Number | 20110070662 12/894810 |
Document ID | / |
Family ID | 46302705 |
Filed Date | 2011-03-24 |
United States Patent
Application |
20110070662 |
Kind Code |
A1 |
Porter; Marc D. ; et
al. |
March 24, 2011 |
RAMAN-ACTIVE REAGENTS AND THE USE THEREOF
Abstract
The present invention provides a new class of Raman-active
reagents for use in biological and other applications, as well as
methods and kits for their use and manufacture. Each reagent
includes a Raman-active reporter molecule, a binding molecule, and
a surface enhancing particle capable of causing surface enhanced
Raman scattering (SERS). The Raman-active reporter molecule and the
binding molecule are affixed to the particle to give both a strong
SERS signal and to provide biological functionality, i.e. antigen
or drug recognition. The Raman-active reagents can function as an
alternative to fluorescence-labeled reagents, with advantages in
detection including signal stability, sensitivity, and the ability
to simultaneously detect several biological materials. The
Raman-active reagents also have a wide range of applications,
especially in clinical fields (e.g., immunoassays, imaging, and
drug screening).
Inventors: |
Porter; Marc D.; (Salt Lake
City, UT) ; Ni; Jing; (Sunnyvale, CA) ;
Lipert; Robert J.; (AMES, IA) ; Dawson; G. Brent;
(Greensboro, NC) |
Assignee: |
IOWA STATE UNIVERSITY RESEARCH
FOUNDATION, INC.
AMES
IA
|
Family ID: |
46302705 |
Appl. No.: |
12/894810 |
Filed: |
September 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10931142 |
Aug 31, 2004 |
7829348 |
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12894810 |
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09961628 |
Sep 24, 2001 |
7824926 |
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10931142 |
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60234608 |
Sep 22, 2000 |
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Current U.S.
Class: |
436/501 ;
436/164 |
Current CPC
Class: |
G01N 33/54373 20130101;
G01N 21/658 20130101; G01N 33/587 20130101; G01N 33/553 20130101;
G01N 33/58 20130101; C07H 21/02 20130101; G01N 33/532 20130101 |
Class at
Publication: |
436/501 ;
436/164 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 21/00 20060101 G01N021/00 |
Claims
1. A method for determining the presence or amount of a target
analyte in a test sample, the method comprising the steps of: (a)
contacting a test sample with a Raman-active reagent, the
Raman-active reagent including a reactive group, a binding
molecule, and a surface enhancing particle capable of causing
surface enhanced Raman scattering, the Raman-active reporter
molecule being chemically linked to the surface enhancing particle
and providing a detectable or measurable Raman scattering signal
when illuminated by an excitation source capable of inducing a
Raman scattering, the reactive group operably linked to the binding
molecule, and wherein binding molecule is capable of specifically
binding to a target analyte; (b) allowing the Raman-active reagent
to bind to the target analyte to form a reagent/analyte complex;
(c) inducing a Raman scattering signal by illuminating the
reagant/analyte complex with the excitation source to induce Raman
scattering; (d) measuring the intensity of the Raman scattering
signal; and (e) determining the presence or amount of the target
analyte in the test sample.
2. The method of claim 1 wherein the binding molecule is selected
from the group of lectins, lectin fragments, lectin derivatives,
antigens, monoclonal antibodies, polyclonal antibodies,
immunoreactive fragments, immunoreactive derivatives, peptides,
haptens, aptamers, nucleic acid molecules, crown ethers,
cyclodextrins, cryptands, and calixarenes.
3. The method of claim 1 wherein the binding molecule is
anti-prostate-specific antigen (anti-PSA).
4. The method of claim 1 wherein the Raman-active reporter molecule
is selected from the group of dithiobisbenzonic acid,
4-mercaptobenzoic acid, 2-naphthalenethiol, thiophenol,
4,4'-dithiobis(succinimidylbenzoate), direct red 81, Chicago Sky
blue, p-dimethylaminoazobenzene, 4-(4-Aminophenylazo)phenylarsonic
acid monosodium salt, 1,5-difluoro-2,4-dinitrobenzene, arsenazo I,
basic fuchsin, disperse orange 3, HABA
(2-(4-hydrozyphenylazo)-benzoic acid, erythrosine B, trypan blue,
ponceau S, ponceau SS, 5,5'-dithiobis(2-nitrobenzoic acid), metal
complexes and polymeric particles.
5. The method of claim 1 wherein the reactive group is a terminal
functional group of a reactive compound that is selected from the
group consisting of succinimides, maleimides, isothiocyanates,
isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals,
epoxides, oxiranes carbodiimides, carbonates, arylating agents,
acryloyl derivatives, diazoalkanes, diazoacetyl compounds,
anhydrides, aziridines, imidoesters, or carbonyldiimidazole.
6. The method of claim 1 wherein the reactive group is a terminal
succinimide group of the reactive compound N-hydroxysuccinimide
(NHS).
7. The method of claim 1 wherein the surface enhancing particle
comprises a metallic material.
8. The method of claim 1 wherein the metallic material is either
gold, silver, copper, platinum, aluminum, gallium, indium, zinc,
cadmium, lithium, or sodium.
9. The method of claim 8 wherein the metallic material is gold.
10. The method of claim 1 wherein the surface enhancing particle is
either a silica, plastic, glass, carbon, ceramic or magnetic
material, coated with a metallic material.
11. The method of claim 10 wherein the metallic coating is either
gold, silver, copper, platinum, aluminum, gallium, indium, zinc,
cadmium, lithium or sodium.
12. The method of claim 11 wherein the metallic coating is
gold.
13. The method of claim 1 further comprising the step of contacting
the reagent/analyte complex to a substrate that binds the
reagent/analyte complex prior to inducing the Raman scattering with
the excitation source.
14. The method of claim 1 wherein the test sample is first placed
in contact with a substrate prior to being placed in contact with
the Raman-active reagent.
15. The method of claim 1 further comprising the step of exposing
the reagent/analyte complex to a magnetic force that causes the
reagent/analyte complex to be separated from the test sample.
16. The method of claim 1 wherein the Raman-active reagent permits
the separation between the reporter molecule and particle surface
to be minimized and maximizes the number of reporter molecules on
each particle.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 10/931,142, filed Aug. 31, 2004, which is a
continuation-in-part of application Ser. No. 09/961,628, filed Sep.
24, 2001, which claims the benefit of U.S. Provisional Application
60/234,608, filed Sep. 22, 2000. These applications are
incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] None.
BACKGROUND OF THE INVENTION
[0003] Many assays exist for detecting and measuring analytes of
small quantity in the presence of a large volume of other
substances. Such assays typically make use of the high binding
affinity between the analyte (the substance to be detected or
measured) and a second molecule having a high degree of specificity
for binding to that analyte. These assays are often referred to as
ligand-binding assays.
[0004] One of the most common ligand-binding assays are
immunoassays. Immunoassays typically employ an antigen and an
antibody which specifically binds to the antigen to form an
antibody/antigen complex. In order to measure the extent of the
antibody/antigen binding, one member of the complex is generally
labeled or tagged with a traceable substance. The presence of the
traceable substance, and hence the presence of the antibody or
antigen to which it is attached, may then be detected or measured
using a variety of different techniques depending upon the unique
characteristics of the label employed. These techniques may include
scintillation counting, fluorescence, absorption, electrochemistry,
chemiluminescence, Rayleigh scattering and Raman scattering. Of
these techniques, fluorescence spectroscopy has been one of the
most widely used readout methods, primarily because of its high
sensitivity.
[0005] Although fluorescence spectroscopy has seen substantial use
in scientific research and clinical diagnostics, there are
disadvantages in using fluorescence spectroscopy. For instance, the
different types of fluorescent molecules used in fluorescence
spectroscopy typically require excitation with photons of differing
wavelengths. Therefore, if the detection of multiple fluorescent
molecules is desired in a single sample, multiple light sources may
be required. Even so, the spectral overlap between the emission of
the different fluorescent molecules often limits reliable
individual and quantitative detection of multiple analytes in a
single sample.
[0006] Today, many assays require the concomitant determination of
more than one analyte in a single test sample (e.g., the screening
of cancer markers, such as a-fetoprotein and carcinoembryonic
antigen). There are two general approaches to assaying multiple
analytes in a single sample. One approach immobilizes different
binding molecules on a solid support at spatially separated
addresses. Multiple analytes can then be detected using the same
label, with identification based on address location.
Alternatively, different labels can be used to detect multiple
analytes simultaneously in the same spatial area. In this case,
each analyte obtains its own distinct label.
[0007] We have explored Raman spectroscopy as an alternative to
fluorescence spectroscopy. Raman spectroscopy measures the level of
Raman scattering induced by the application of a radiation source,
i.e. light source, on an analyte. The light incident on the analyte
is scattered due to excitation of electrons in the analyte. "Raman"
scattering occurs when the excited electron returns to an energy
level other than that from which it came, resulting in a change in
the wavelength of the scattered light and giving rise to a series
of spectral lines at both higher and lower frequencies than that of
the incident light. The series of spectral lines is generally
called the Raman spectrum.
[0008] Conventional Raman spectroscopy usually lacks sufficient
sensitivity for use as a readout method for immunoassays. Raman
spectroscopy is also unsuccessful for fluorescent materials because
the broad fluorescence emission bands tend to swamp the weaker
Raman bands.
[0009] However, a modified form of Raman spectroscopy based on
"surface enhanced" Raman scattering (SERS) has proved to be more
sensitive and thus of more general use. In the SERS form of Raman
spectroscopy, the analyte whose spectrum is being recorded is
closely associated with a roughened metal surface. This close
association leads to a large increase in detection sensitivity, the
effect being greater the closer the proximity of the analyte to the
metal surface.
[0010] The manner in which surface enhancement occurs is not yet
fully understood, but it is thought that the incident light excites
conduction electrons in roughened metal surfaces or particles,
generating an electron plasma resonance (plasmon). As a result, the
electromagnetic field in the vicinity of the metal surface is
greatly amplified, giving rise to enhanced Raman scattering for
molecules located close to the surface.
[0011] Surprisingly, there have been only a few reports on the
application of SERS for detection in immunoassays. Two of these
approaches used a sandwich-type assay, which coupled surface and
resonance enhancements. In particular, Rohr et al., Anal. Biochem.
1989, 182, 388, used labeled detection antibodies and roughened
silver films coated with a capture antibody (see also U.S. Pat. No.
5,266,498 to Tarch et al.), and Dou et al., Anal. Chem. 1997, 69,
1492, exploited the adsorption on silver colloids of an
enymatically amplified product. Another approach by White et al.,
International Application Publication No. WO 99/44065, employs an
immunoassay based on the displacement of SERS and surface enhanced
resonance Raman (SERRS) active analyte analogs which are modified
so as to have particular SERS and SERRS surface seeking properties.
Upon introduction of a sample, the analyte analogs are displaced by
the analyte of interest in the sample and exposed to a SERS or
SERRS surface, such as an etched or roughened surface, a metal sol
or an aggregatation of metal colloid particles. Raman spectroscopy
is then performed to detect the displaced analyte analog associated
with the SERS or SERRS surface to determine the presence or
quantity of the analyte in the sample.
[0012] A major barrier that prohibits using SERS for the direct
detection of biological samples is that the surface enhancement
effect diminishes rapidly with increasing distance from the
metallic surfaces. In other words, strong SERS signals are observed
only if the scattering centers are brought into close proximity
(<100 nm) to the surface. In addition, although Raman spectra of
biomolecules can be obtained on silver surfaces when coupling SERS
and resonance enhanced scattering, the spectra are usually lacking
of sufficient chemical content and/or signal amplitude to be used
for immunoassay purposes.
[0013] We have overcome these barriers by developing a novel class
of Raman-active reagents having both Raman-active reporter
molecules and binding molecules integrated with each other on the
same SERS surface. In each of the above systems, the SERS or SERRS
surface and the Raman-active molecule are not integrated with each
other, but are merely placed in close proximity to each other by
the combination of an analyte sandwiched between an antibody
immobilized on the enhancing surface and an antibody attached to a
Raman-active molecule, or the combination of the SERS or SERRS
surface with a particular SERS or SERRS surface seeking group
coupled to an analyte analog and a Raman-active molecule, after
exposure to the sample.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention is summarized as a novel class of
Raman-active reagents for use in biological and other applications,
as well as methods and kits for their use and manufacture.
[0015] The Raman-active reagents each include a Raman-active
reporter molecule, a binding molecule, and a surface enhancing
particle capable of causing surface enhanced Raman scattering. The
Raman-active reporter molecule and the binding molecule are
operably linked to the particle to give both a strong surface
enhanced Raman scattering (SERS) signal and to provide biological
functionality, e.g. antigen or drug recognition. The Raman-active
reporter molecule and the binding molecule may be either directly
linked to the surface enhancing particle or indirectly linked to
the surface enhancing particle by way of a linker molecule. In one
embodiment, the Raman-active reporter molecule and the binding
molecule are each independently linked to the surface enhancing
particle. In a second embodiment, the binding molecule is operably
linked to the Raman-active reporter molecule, which is operably
linked to the surface enhancing particle. Other variations are
possible.
[0016] The Raman-active reagents may be employed to determine the
presence or amount of a target analyte in a test sample by use of
the binding specificity of the binding molecule for the target
analyte, or a portion thereof, and the generation and measurement
of SERS signal induced by the application of a radiation onto the
reagent/analyte complex. The Raman-active reagents may be used, for
example, in clinical, forensic, and water quality testing labs for
the detection of drugs, pesticides, bacteria, viruses, microbial
toxins, hormones and biologically important proteins, industrial
chemicals, explosives, trace metals, etc.
[0017] The Raman-active reagents may be manufactured in the lab or
provided to the user in the form of a kit. The kit may include a
previously prepared Raman-active reagent, or the ingredients for
manufacturing the Raman-active reagents in the lab. The kit may
also include ingredients that minimize nonspecific binding and
ingredients that stabilize the reagent to extend its shelf life. In
addition, the kit may include a capture substrate covered with
binding molecules to immobilize analytes for subsequent detection
with the Raman-active reagent. For the simultaneous detection of
multiple analytes, the kit may also include unreactive spacer
molecules, such as molecules terminated with ethylene glycol units,
for interspersing amongst the binding molecules so as to minimize
steric interferences as well as to resist nonspecific
adsorption.
[0018] In one embodiment, the Raman-active reagents are
manufactured by coimmobilizing the Raman-active reporter molecules
and the binding molecules to metal colloid particles. In the case
of gold particles, the reporter molecules can be covalently linked
to the particle through thiol functionalities on the reporter
molecules. Binding molecules, for example, antibodies, will
spontaneously associate with unreacted areas of the particle to
form a Raman-active reagent for antigen detection. In a second
embodiment, the Raman-active reagents are manufactured by
covalently linking the Raman-active reporter molecules to the
surface enhancing particle, and by covalently linking the binding
molecules to the Raman-active reporter molecules.
[0019] It is one object of the present invention to provide a new
class of labeling reagents as an alternative to
fluorescence-labeled reagents. It is also an object of the present
invention to provide a new class of labeling reagents capable of
simultaneously detecting multiple analytes in a single test
sample.
[0020] The Raman-active reagents of the present invention serve as
an alternative to fluorescence-labeled reagents, with advantages in
detection including signal stability, sensitivity, and the ability
to simultaneously detect several analytes in a single test sample.
Because of the ability to simultaneously detect several analytes in
a single test sample, faster analysis speeds and reduced labor
costs may be obtained.
[0021] In yet another embodiment, the invention provides a novel
reagent for low-level detection in immunoadsorbent assays. The
reagent consists of gold nanoparticles modified with succinimide
ester derivatives such as, for example 5, 5'-dithiobis
(succinimidyl-2 nitrobenzoate) to integrate bioselective species
(e.g., antibodies) with molecular labels to generate SERS
responses. The reagent is constructed by coating gold nanoparticles
(30 nm) with a monolayer of an intrinsically strong Raman
scatterer. These monolayer-level labels are bifunctional bydesign
and contain disulfides for chemisorption to the nanoparticle
surface and succinimides (i.e., reactive group) for coupling to the
bioselective species.
[0022] An object of this embodiment is to provide a label design
that both minimizes the separation between label and particle
surface and maximizes the number of labels on each particle.
[0023] Another object of this embodiment is to provide a novel
approach to SERS-based labeling with the following advantages:
narrow spectral bandwidth, resistance to photobleaching and
quenching, and long-wavelength excitation of multiple labels with a
single excitation source.
[0024] Yet another aspect of this embodiment is that it enables the
detection of antigens, for example, free prostate-specific antigen
(PSA) using a sandwich assay format based on monoclonal
antibodies.
[0025] In still another aspect, this embodiment provides detection
limits of approximately 1 pg/mL in human serum and approximately 4
pg/mL in bovine serum albumin at a spectrometer readout time of 60
s.
[0026] In another aspect, this embodiment may be used in
conjunction with multianalyte assays to simultaneously determine
many complexed forms of antigens, such as for example, PSA.
[0027] Other objects, features and advantages of the present
invention will be apparent in the following Detailed Description of
the Invention when read in conjunction with the accompanying
drawings, examples and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 illustrates two possible methods for preparing
Raman-active immunogold reagents.
[0029] FIG. 2 is a graph illustrating the UV-Vis spectra for SERS
substrate characterization.
[0030] FIG. 3 is a graph illustrating the SERS spectra of
dithiobisbenzonic acid (DBA) and
4,4'-dithiobis(succinimidylbenzoate) (DSB) Raman-active reporter
molecules absorbed on gold island film substrates: (a) DBA, (b)
DSB.
[0031] FIG. 4 is a graph illustrating the dose-response curves of
the intensity of the strongest SERS band (1075 cm.sup.-1) versus
the rat IgG concentration: (a) using the colloidal detection
reagent prepared via the co-immobilized approach, (b) using the
colloidal detection reagent prepared via the covalent linking
approach.
[0032] FIG. 5 depicts an illustrative setup for SERS measurements.
P: polarization rotator; PB1 and PB2: Pellin Brocha prisms; M:
mirror; A: aperture; L1: cylindrical lens; S: sample slide; L2:
collection lens; NF: notch filter.
[0033] FIG. 6 illustrates an example of a sandwich assay employing
Raman-active reagents of the present invention.
[0034] FIG. 7 is a schematic that comparatively illustrates two
methods for preparing Raman-active immunogold reagents: (a) tracer
nanoparticles are prepared by the physisorption of antibodies on
gold colloids that had been previously coated with a partial
monolayer of RRMs (Raman reporter molecules) based on aromatic
thiols; (b) produces particles coated with a thiolate-based
monolayer that has a terminal succinimide group (i.e., a terminal
reactive functional group). This schematic (b) shows that the
terminal succinimide group can then react with the amines of a
protein to form an amide linkage.
[0035] FIG. 8 is schematic illustrating the process for preparing a
Raman reporter-labeled immunogold colloid.
[0036] FIG. 9 illustrates the experimental setup for measuring PSA
levels in human serum using surface-enhanced Raman
spectroscopy.
[0037] FIG. 10 shows an infrared reflection spectra of DSU-derived
monolayer on gold before (spectrum A) and after (spectrum B)
exposure to the anti-free PSA capture antibody.
[0038] FIG. 11 shows an infrared reflection spectra of a
DSNB-derived monolayer on gold before (spectrum A) and after
(spectrum B) exposure in the anti-PSA tracer antibody.
[0039] FIG. 12 shows a Raman spectra of the reporter compound: (A)
spectrum of DSNB powder; (B) SERS spectrum of gold nanoparticles
following reaction with DSNB.
[0040] FIG. 13 illustrates a SERS-based free PSA immunoassay: (A)
SERS spectra, offset for clarify, acquired at various PSA
concentrations; (B) dose-response curve for free PSA in human
serum. The dose-response curve was constructed by calculating the
average reading of the response for 6-8 different locations on the
surface of each sample, which typically varied by 10% as described
in the detailed description below.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention provides a novel class of Raman-active
reagents for use in determining the presence or amount of a target
analyte in a test sample. Also provided are particular methods and
kits for using the Raman-active reagents of the present invention,
as well as certain novel, preferred, methods for their
manufacture.
[0042] The Raman-active reagents according to the present invention
comprise a Raman-active reporter molecule, a binding molecule, and
a surface enhancing particle capable of causing surface enhanced
Raman scattering. The Raman-active reporter molecule and the
biological binder are operably linked, either directly or
indirectly, to the surface enhancing particle to give both a strong
surface enhanced Raman scattering (SERS) signal and specific
binding affinity to a target analyte. The Raman-active reporter
molecule and the binding molecule may be either directly linked to
the surface enhancing particle or indirectly linked to the surface
enhancing particle by way of a linker molecule. The Raman-active
reporter molecule and the binding molecule may be independently
linked to the surface enhancing particle, or the binding molecule
may be operably linked to the Raman-active reporter molecule, which
is operably linked to the surface enhancing particle.
[0043] The term "surface enhancing particle" is defined herein to
include particles capable of causing surface enhanced Raman
scattering. Particles capable of causing surface enhanced Raman
scattering are well known in the art and generally include, without
limitation, particles of metallic materials such as gold, silver,
copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium
and sodium. The particles may also include, without limitation,
other inert support structures of silica, plastic, glass, carbon,
ceramics, or other materials, including magnetic materials, coated
with a metallic material capable of causing surface enhanced Raman
scattering, such as the metallic materials listed above.
[0044] The particles used in the present invention are colloid
particles. The colloid particles are preferably of a uniform and
desired size and shape and stabilized against possible
self-aggregation. Processes for preparing unaggregated colloids are
well known in the art and typically involve, for example, the
reduction of a metal salt (e.g., silver nitrate) with a reducing
agent (e.g., citrate) to form a stable microcrystalline suspension.
Stabilization may also be realized by the use of thin films or
monolayers of various organic compounds. The colloid particles can
be of any size as long as they give rise to an SERS signal. For
example, the colloid particles may be less than 1000 nm in
diameter, and preferably less than 100 nm in diameter.
[0045] In the preferred embodiment, the surface enhancing particles
are metallic nanoparticles. The large surface enhancement observed
on metallic nanoparticles results in SERS intensities that can be
comparable to or even exceeding those for fluorescence. Such a
level of enhancement, which may lead to a high detection
sensitivity, together with the ease of handling, make metallic
nanoparticles more promising than most other types of SERS
substrates for use in ligand-binding assay applications. However,
nonmetallic particles encapsulated with an enhancing material may
also be of value in some applications. Of the various metallic
nanoparticles, gold colloids is preferred over silver colloids,
despite the fact that silver colloids provide larger enhancements
than gold. This is because the greatest Raman enhancements with
gold particles are produced with longer wavelength excitation
light. This makes it possible to minimize the generation of sample
fluorescence, which may interfere with the measurement of the Raman
scattering.
[0046] The term "Raman-active reporter molecule" is defined to
include anyone of a large number of molecules with distinctive
Raman scattering patterns. Various molecules with distinctive Raman
scattering patterns are well known in the art. Examples of such
molecules include, but is not limited to, dithiobisbenzonic acid
(DBA), 4-mercaptobenzoic acid (MBA), 2-naphthalenethiol (NT),
thiophenol (TP), direct red 81, Chicago sky blue,
4,4'-dithiobis(succinimidylbenzoate) (DSB),
p-dimethylaminoazobenzene, 1,5-difluoro-2,4-dinitrobenzene,
4-(4-aminophenylazo)phenylarsonic acid monosodium salt, arsenazo I,
basic fuchsin, disperse orange 3, HABA
(2-(4-hydroxyphenylazo)-benzoic acid, erythrosine B, trypan blue,
ponceau S, ponceau SS, 5,5'-dithiobis(2-nitrobenzoic acid), metal
complexes and polymeric particles.
[0047] For example, in a preferred embodiment of the invention the
reporter molecule 5,5'-dithiobis(2-nitrobenzoic acid) may be
converted to a corresponding succinimide ester derivative by
treatment with the reactive compound, N-hydroxy succinimide (NHS),
resulting in formation of
5,5'-dithiobis(succinimidyl-2-nitrobenzoate (DSNB). DSNB can then
be coupled to the primary amine group of a tracer antibody through
formation of an amide linkage.
[0048] The term "reactive compound" is defined to include any
molecule having a reactive group including but not limited to
succinimides, maleimides, isothiocyanates, isocyanates, acyl
azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes
carbodiimides, carbonates, arylating agents, acryloyl derivatives,
diazoalkanes, diazoacetyl compounds, anhydrides, aziridines,
imidoesters, carbonyldiimidazole, or other groups that may be amine
reactive, thiol reactive, or nucleophile reactive, such as for
example, N-hydroxysuccinimide.
[0049] The term "binding molecule" is defined to include any
molecule having a binding specificity and avidity for a molecular
component of a target analyte, or which is associated with a target
analyte. In general, binding molecules are known to those skilled
in the art and typically include, without limitation, lectins
(including fragments or derivatives thereof which retain binding
function), monoclonal and polyclonal antibodies (including
immunoreactive fragments or derivatives derived therefrom, which
fragments retain all or a portion of the binding function of the
antibody), peptides, haptens, aptamers, and nucleic acid molecules
(including single stranded RNA, single-stranded DNA, or
single-stranded nucleic acid hybrids), and any fragments and
derivatives thereof. Crown ethers, cyclodextrins, cryptands,
calixarenes, and many other types of ligands could also be
used.
[0050] The term "target analyte" is defined to include a molecule
of an organic or inorganic nature, the presence and/or quantity of
which is being tested for, which contains a molecular component
(e.g., ligand or sequence or epitope or domain or portion or
chemical group or reactive functionality or determinant) for which
a binding molecule has binding specificity. The molecule may
include, but is not limited to, a nucleic acid molecule, protein,
glycoprotein, eukaryotic cell, prokaryotic cell, lipoprotein,
peptide, carbohydrate, lipid, phospholipid, aminoglycans, chemical
messenger, biological receptor, structural component, metabolic
product, enzyme, antigen, antibody, drug, therapeutic, toxin,
inorganic chemical, organic chemical, a substrate and the like.
[0051] The term "test sample" means a sample to be tested for the
presence or amount of a target analyte. The sample may include a
target analyte or be free from the presence of the target
analyte.
[0052] The term "operably linked" is defined to mean a linkage
between two different molecules, or a molecule and a particle, of
sufficient stability for the purposes of signal enhancement and
detection according to the present invention. As known to those
skilled in the art, and as will be more apparent by the following
embodiments, there are several methods and compositions in which
two or more molecules, or a molecule and a particle, may be
operably linked utilizing reactive functionalities. Reactive
functionalities include, but are not limited to, bifunctional
reagents, linker molecules, biotin, avidin, free chemical groups
(e.g., thiol, or carboxyl, hydroxyl, amino, amine, sulfo,
phosphine, selenide, etc.), and reactive chemical groups (reactive
with free chemical groups).
[0053] The term "linker" is defined to refer to a compound or
moiety that acts as a molecular bridge to operably link two
different molecules, or a molecule and a particle, wherein one
portion of the linker is operably linked to a first molecule, and
wherein another portion of the linker is operably linked to a
second molecule or particle. The two different molecules, or the
molecule and particle, may be linked to the linker in a step-wise
manner. There is no particular size or content limitations for the
linker so long as it can fulfill its purpose as a molecular bridge.
Linkers are known to those skilled in the art to include, but are
not limited to, chemical chains, chemical compounds, carbohydrate
chains, peptides, haptens, and the like. The linkers may include,
but are not limited to, homobifunctional linkers and
heterobifunctional linkers. As an illustrative example, a linker
may comprise a carboxylic acid that has been activated by
conversion to its acid chloride to react with an amino acid (e.g.,
lysine) residue of a binding molecule comprising a monoclonal
antibody, and a thiol reactive group to link with the particle or
the Raman-active reporter molecule.
[0054] Heterobifunctional linkers are well known to those skilled
in the art and generally contain a functionality on one end that
binds to a target (e.g., a molecule or surface), and an opposite
end having a second reactive functionality to specifically link to
a different target. Heterobiofunctional photo-reactive linkers
(e.g., phenylazides containing a cleavable disulfide bond) are also
well known in the art and may be employed as linkers in accordance
with the present invention. For example, a
sulfosuccinimidyl-2-(p-azido salicylamido)
ethyl-1,3'-dithiopropionate contains a N-hydroxy-succinimidyl azide
(upon photolysis) reacts with any amino acid.
[0055] The linker may further comprise a protective group which
blocks reactivity with a functional group on the linker which is
used to react with and bind to a molecule or particle to be linked.
A deprotection reaction may involve contacting the linker to one or
more conditions and/or reagents which remove the protective group,
thereby exposing the function group to interact with the molecule
to be linked. Depending on the nature of the protective group,
deprotection can be achieved by various methods known in the art,
including, but not limited to, photolysis, acidolysis, hydrolysis,
and the like. Depending on such factors as the molecules and
particles to be linked, and the conditions in which the method of
detection is performed, the linker may vary in length and
composition for optimizing such properties as flexibility,
stability, and resistance to certain chemical and/or temperature
parameters. For example, short linkers of sufficient flexibility
include, without limitation, linkers having from 2 to 10 carbon
atoms (see, e.g., U.S. Pat. No. 5,817,795).
[0056] Any two molecules having an affinity for each other may
comprise the reagent/analyte complex according to the present
invention. Examples of ligand-binding systems include: antibodies
and antigens; hormones and their receptors; lectins and the complex
carbohydrates to which they bind; effector molecules and their
receptors; complimentary nucleotide sequences; binding molecules
designed through molecular modeling and synthesized specifically to
bind another molecule, and molecules with mutual affinity to each
other, such as avidin and biotin.
[0057] In one embodiment, the Raman-active reporter molecule and
the binding molecule are each independently linked to the
surface-enhancing particle. The Raman-active reporter molecule and
the binding molecule may be either directly linked to the surface
enhancing particle or indirectly linked to the surface enhancing
particle by way of a linker molecule.
[0058] In another embodiment, the binding molecule is operably
linked to the Raman-active reporter molecule, which is operably
linked to the surface enhancing particle. The Raman-active reporter
molecule and the binding molecule may be either directly linked to
the surface enhancing particle or indirectly linked to the surface
enhancing particle by way of a linker molecule.
[0059] The Raman-active reagents of the present invention may be
manufactured in the lab or provided to the user in the form of a
kit. The kit may include a previously prepared Raman-active
reagent, or the ingredients for manufacturing the Raman-active
reagents as described above. The kit may also include ingredients
that minimize nonspecific binding (nonspecific binding ingredient)
and ingredients that stabilize the reagent (stabilizing ingredient)
to extend its shelf life. Various nonspecific binding ingredients
and stabilizing ingredients effective in use with the present
invention are well known in the art. In addition, the kit may
include a capture substrate covered with binding molecules to
immobilize analytes for subsequent detection with the Raman-active
reagent. For the simultaneous detection of multiple analytes, the
kit may also include unreactive spacer molecules, such as molecules
terminated with ethylene glycol units, for interspersing amongst
the binding molecules so as to minimize steric interferences as
well as to resist nonspecific adsorption. For example, surfactants,
blocking agents, and buffers may be added.
[0060] Raman reporter-labeled immunogold probes can be prepared in
many different ways. For example, as depicted in FIG. 1 (a) (the
co-immobilization approach), an uncoated gold nanoparticle is
labeled with Raman-active reporter molecules through the
spontaneous adsorption of thiol-containing reporter molecules on
gold, and then integrated with antibodies. The amount of thiol is
chosen to coat only a portion of the nanoparticle surface and to
leave exposed portions of the nanoparticle surface available for
antibody immobilization. The antibodies are subsequently
immobilized on the uncoated portion of the reporter-labeled
nanoparticle through a combination of ionic and hydrophobic
interactions. Immobilization can, of course, also be achieved by
simply adsorbing an antibody directly on a coating of Raman-active
reporter molecules.
[0061] In a second example, as depicted in FIG. 1 (b) (the covalent
linker approach), an uncoated gold nanoparticle is labeled with
Raman-active reporter molecules, which are then covalently linked
to antibodies. The Raman-active reporter molecule not only carries
thiol or disulfide groups for immobilization on the gold
nanoparticle, it also contains a succinimide ester functional group
(i.e., a coupling reagent) for the covalent linking of an antibody.
The covalent linker approach enhances the Raman reporter coverage
and ultimately its sensitivity. Because the antibodies are
covalently linked to the nanoparticles, the exchange of antibodies
between nanoparticles with different Raman reporter molecules is
reduced, and hence the probe specificity in the multi-analyte
application is improved.
[0062] The Raman-active reagents of the present invention determine
the presence or amount of a target analyte, if present in a test
sample, by the binding specificity of the binding molecule for the
target analyte, or a portion thereof, and the generation and
measurement of a SERS signal induced by the application of
electromagnetic radiation onto the reagent/analyte combination. The
Raman-active reagents may be used in clinics or forensic labs for
the detection of drugs, pesticides, microbial toxins, hormones and
biologically important proteins, industrial chemicals, explosives,
pesticides, chlorophenols and other pollutants in soils, water,
air, biological materials and other matrices. Such analysis may
include in-situ testing methods (i.e., those not requiring any
separation of the analytes from the sample prior to either their
analysis or detection), as well as other in vivo, in vitro, or ex
vivo methods.
[0063] The detection or measurement of target analytes using the
Raman-active reagents according to the present invention may be
performed using anyone of a number of assaying techniques known in
the art. In general, a test sample is placed in contact with a
Raman-active reagent of the present invention under suitable
conditions to allow the binding molecule to specifically bind to
the target analyte, thus forming a reagent/analyte complex. The
sample containing the reagent/analyte complex is then exposed to an
excitation source (e.g., light source) that is suitable for
exciting the Raman-active reporter molecule to induce surface
enhanced Raman scattering. The intensity of the Raman scattering
signal can then be measured to determine the presence or amount of
the target analyte in the test sample. Absence of a Raman
scattering signal is indicative of the absence of the target
analyte in the test sample.
[0064] Techniques for detecting Raman scattering are well known in
the art. The primary measurement is one of light scattering
intensity at particular wavelengths. Neither the angle of the
incident beam nor the position of the detector is critical. With
colloidal suspensions, detection is often at an angle of 90.degree.
to the incident beam. The intensity of the Raman scattering signals
must be measured against an intense background from the excitation
source. As such, the use of Raman-active report molecules with
large Stokes shifts is preferred.
[0065] Several devices are suitable for collecting SERS signals,
including fiber-optic waveguides, wavelength selective minors, and
holographic optical elements for scattered light detection. The
choice of the detector will largely depend on the sensitivity of
detection required to carry out a particular assay. The intensity
of the signal may be measured using a silicon photodiode, a charge
coupled device (CCD), photographic film, or photomultiplier tubes
arranged either singly or in series for cascade amplification of
the signal. Photon counting electronics can also be used for
sensitive detection.
[0066] Analysis of the SERS spectrum will typically include the use
of some form of data processor such as a computer. Raman signals
consist of a series of distinct spectral lines of varying
intensity. The frequencies and relative intensities of these
spectral lines are specific to each Raman-active reporter molecule
being detected such that each Raman-active reporter has a distinct
"fingerprint". The manner in which this fingerprint is analyzed
will depend primarily on the purpose of the detection. If a SERS
analyzer is being used to selectively detect one or more analytes
out of a test sample containing multiple analytes, then an analysis
of the entire fingerprint for each reporter molecule may be
necessary to make a reliable identification. However, if the
analyzer is being used to quantify the detection of one or several
labels, each of which has a unique spectral line, then an analysis
of only the unique spectral line may be necessary.
[0067] The excitation source may be any source capable of exciting
the Raman-active reporter molecule to induce Raman scattering.
Typically, excitation will be carried out using incident light from
a laser having a frequency in the visible spectrum. However, it is
possible to envision situations in which other frequencies might be
used, for example, in the ultraviolet or near-infrared ranges. The
selection and tuning of the excitation source, with the appropriate
frequency and power, will be well within the capabilities of one
skilled in the art and will depend on the reporter molecule,
surface enhancing particle and target analyte employed. In the
preferred embodiment, a laser serves as the excitation source. The
laser may be an inexpensive type such as a helium-neon or diode
laser. Preferably, a diode laser is used at or near the IR
spectrum, minimizing fluorescence interference. Lamps may also be
used as the excitation source. Direct illumination of the surface
or by evanescent waves from a waveguide beneath the plasmon-active
surface may also employed to induce a SERS affect.
[0068] An illustration of a typical SERS measurement system is
depicted in FIG. 5.
[0069] In the preferred embodiment, the test sample may be placed
in contact with a capture substrate covered with binding molecules
that selectively immobilize analytes for subsequent detection with
the Raman-active reagent. This substrate is then treated with the
Raman-active reagent under suitable conditions to allow the
Raman-active reagent binding molecule to specifically bind to the
target analyte, forming the reagent/analyte complex. FIG. 3
illustrates one type of assay employing such a method. In this
example, a different Raman-active reporter molecule is associated
with a different antibody as different probes, with the presence of
different antigens detected by the characteristic Raman bands of
the reporters. In an alternative embodiment, the test sample may be
contacted with the Raman-active reagent under suitable conditions
to allow the Raman-active reagent binding molecule to specifically
bind to the target analyte, forming the reagent/analyte complex,
prior to its capture by the substrate.
[0070] The substrate may take the form of a generally flat surfaces
(e.g., strips, slides, gene chips, etc.) or inert support
structures of silica, carbon, plastic, glass, paper or other
materials which may be in the form of macroscopically flat or
textured pieces, slides, strips, spheroids or fibers capable of
supporting the reagent/analyte complex. Analytes and/or the
reagent/analyte complex may bind to the substrate by direct
adsorption, adsorption through a linker covalently attached to
either the particle or the reporter molecule, by covalent
attachment of the particle or reporter molecule to the substrate
directly or through a linker or by intercalation of the distal
portion of the linker into the substrate surface, by magnetic
attraction to the substrate, or by specifically binding a second
binding molecule affixed to the substrate to the target analyte or
a molecule operably linked to the particle and having a specific
affinity for the second binding molecule. For example, the
substrate may include a binding molecule identical to that found on
the Raman-active reagent that binds the target analyte, such as in
a sandwich assay. Such a system might be employed for the detection
of multiple target analytes using a limited number of different
Raman-active labels in association with multiple binding molecules.
Identification and quantification of the analytes would be
accomplished through the measurement of the distinctive spectral
fingerprints of the Raman-active labels provided for each analyte.
Alternatively, the substrate may contain address locations for the
various analytes with the specific binding molecule identified by
the address location rather than by its spectral fingerprint. This
system may also be employed for separating the target analyte from
the test sample.
[0071] In another embodiment, the method may further comprise
exposing the test sample to a magnetic force that separates the
reagent/analyte complex from the test sample. Such separation may
occur if the surface-enhancing particle comprises of a material
that is responsive to a magnetic force. In this case, the
magnetic-responsive material is likely to be coated with a metallic
material capable of emitting a SERS signal. Upon detection of the
SERS signal, the magnetic force may be applied to cause the
reagent/analyte complex to be separated from the test sample. In
addition, different Raman-active reagents having different
Raman-active reporter molecules and binding molecules may be
employed to allow for the sorting of multiple target analytes using
magnetic forces.
[0072] Assay kits for the method of the present invention are also
provided. In one preferred embodiment, the assay kit comprises a
Raman-active reagent in accordance with the present invention,
wherein the Raman-active reagent includes at least one binding
molecule having an affinity for a known target analyte. In a second
embodiment, a substrate capable of binding the analyte and/or the
reagent/analyte complex is provided. For the simultaneous detection
of multiple analytes, the kit may also include unreactive spacer
molecules, such as molecules terminated with ethylene glycol units,
for interspersing amongst the binding molecules so as to minimize
steric interferences as well as to resist nonspecific adsorption.
For example, surfactants, blocking agents, and buffers may be
added.
[0073] One aspect of the present invention is that it allows
multiple target analytes to be detected from a single test sample.
For example, simultaneous detection may be achieved by the use of
multiple binding molecules, each specific to a target analyte or a
class of target analytes and each associated with a different
Raman-active reporter molecule. Because Raman-active vibrational
modes usually yield bands one to two orders of magnitude narrower
than most fluorescence bands, it is now possible to distinguish a
much large number of different Raman labels as compared to
fluorescent labels. Alternatively, a single Raman-active reporter
molecule may also be employed with identification based on an
address location on a substrate, such as a gene chip or screening
slide, as is well known in the art.
[0074] Because Raman scattering is not affected by oxygen and other
quenchers, thus simplifying its use in many different experimental
environments, it has potential advantages as a broadly applicable
readout method in comparison to the widely used fluorescence
detection schemes. In addition, because the SERS signal is less
subject to photobleaching, lower detection limits can be obtained
by increasing the signal integration time. Raman-active vibrational
modes also usually yield bands one to two orders of magnitude
narrower than most fluorescence bands, indicating the possibility
of distinguishing a much large number of different Raman-active
labels than likely with fluorescent labels, and minimizing the need
to use spacial locations for analyte identification.
[0075] One argument favoring a fluorescence over a Raman-based
detection scheme, in the past, is the inherent detection capability
of fluorescence measurements. However, by combining the SERS effect
and the use of reporter molecules with a relatively large Raman
scattering cross section as extrinsic labels, trace amounts of
intrinsically weak Raman scatterers (e.g., antibodies) can be
indirectly detected. A detection limit of 0.1 pg/mL has been
estimated from recent experiments for the antibody detection. With
further optimization, even lower detection limits are expected.
This will include using dyes for Raman reporter molecules that
absorb the Raman excitation light. This leads to surface enhanced
resonance Raman scattering or SERRS. SERRS can be 2-6 orders of
magnitude stronger than SERS. It is important to note that in the
present invention, the reporter molecules are directly attached to
the metal nanoparticle surface, which effectively quenches
potential fluorescence from the reporter molecule that could
interfere with the Raman measurements.
[0076] The below Examples include an illustration describing an
application of one type of Raman-active gold colloidal reagent
(Raman-active reagent) used as a detection reagent in immunoassays.
It is envisioned that similar concepts can be developed for other
types of assays, target analytes and Raman-active reagents
developed in accordance with the present invention, as well as
infrared-active colloidal reagents, and in some cases, reagents for
fluorescence or electrochemical based assays.
[0077] The following examples are provided as further non-limiting
illustrations of particular embodiments of the invention.
EXAMPLES
Example 1
Synthesis of Raman-Active Reporter Molecule
4,4'-Dithiobis(Succinimidylbenzoate)
[0078] The Raman-active reporter molecule
4,4'-dithiobis(succinimidylbenzoate) (DSB) was synthesized
following a procedure similar to that used for preparing
dithiobis(succinimidylundecanoate) as described in Wagner et al.,
Biophys. J. 1996, 70, 2052, which is incorporated herein by
reference. Briefly, 0.50 g of the reporter molecule
dithiobisbenzonic acid (DBA) (1.6 mmol) (Toronto Research
Chemicals, Inc), 0.67 g of 1,3-dicyclohexylcarbodiimide (DCCD) (3.2
mmol) (obtained from Aldrich), 0.37 g of N-hydroxysuccinimide (NHS)
(3.2 mmol) (obtained from Aldrich), and 60 mL of tetrahydrofuran
were added to a 100 mL round-bottom flask equipped with a magnetic
stir bar and drying tube. The reaction mixture was stirred at room
temperature for three days. The solution was then filtered and the
solvent was removed under reduced pressure to give an orange
residue. The crude product was dissolved in hot acetone and
filtered again. Hexane was added to the filtrate until the solution
became cloudy and the product was obtained as an orange powder.
H-NMR (300 MHz, CDCl.sub.3) .delta. 8.08 (d, 4H), 7.60 (d, 4H),
2.91 (s, 8H). Infrared reflection spectroscopy: 1810 cm.sup.-1
(v.sub.(C.dbd.O) of the ester), 1772 cm.sup.-1 (v.sub.s(C.dbd.O) of
the succinimide), 1746 cm.sup.-1 (v.sub.as(C.dbd.O) of the
succinimide), 1585 cm.sup.-1 (v.sub.(C.dbd.C) of the benzene
ring).
Example 2
Preparation of Raman-Active Reagents Using Co-Immobilization
[0079] Raman-active immunogold colloidal reagents were prepared
using the coimmobilization approach depicted in FIG. 1(a). First,
25 .mu.L of ethanolic Raman reporter solution (0.5 mM DBA) was
added to 10 mL of a suspension of uncoated gold colloids (.about.30
nm diameter, 2.times.10.sup.11 particles/mL) (Ted Pella, Inc.). The
mixture was allowed to react for 5 hours at room temperature.
During this step, the reporter molecules bound via self-assembly
onto the colloid surface through the formation of sulfur-gold
linkages. We note that this amount of reporter, based on an
estimation of the colloidal surface area, will only partially cover
the colloid, leaving portions of the uncoated colloidal surface
available for protein immobilization. After separating the
reporter-labeled colloids from solution by centrifugation at 14,000
g for 4 minutes, the loosely packed, red-colored sediment was
resuspended in 10 mL of borate buffer (2 mM, pH 9).
[0080] The Raman-active colloids were next immuno-labeled by adding
230 .mu.g of goat anti-rat IgG to 10 mL of the above suspension.
The mixture was incubated at 4.degree. C. for 12 hours, during
which the IgG protein adsorbed directly onto the exposed colloidal
surface through a combination of ionic and hydrophobic
interactions. The incubation was followed by centrifugation at
14,000 g for 5 minutes, and the loose sediment of reporter-labeled
immunogold was rinsed by resuspending in 2 mM borate buffer and
collected after a second centrifugation. Finally, the labeled
colloids were suspended in 10 mM tris(hydroxymethyl)aminomethane
(Tris)-buffered saline (Tris/HCl, NaCl 10 mM, pH 7.6) giving a
concentration of approximately 2.times.10.sup.11 particles/mL.
Tween 80 (1%) (Aldrich) was also added to the suspension to
minimize nonspecific adsorption in the assays. The suspensions
usually remained uniformly dispersed for 2-3 days when stored at
4.degree. C.
Example 3
Preparation of Raman-Active Reagents Using a Covalent Linking
[0081] Raman-active immunogold colloidal reagents were prepared
using the covalent linking approach depicted in FIG. 1(b). The DSB
molecules were used as both Raman reporters and antibody linkers.
The succinimide ester group of the DSB molecule can readily react
with the primary amine group of an amino acid, such as the lysine,
present in antibodies such as IgG to form a covalent bond. As shown
in Scheme 2, the preparation of the covalently-linked colloidal
reagent follows a process very similar to that used for the
co-immobilized reagents. However, with the covalent linking
approach, the antibodies indirectly attached to the colloid through
the reporter molecules rather than directly adsorbed onto the
colloidal surface. Briefly, 25 .mu.L of a reporter-linker solution
(5 mM DSB in CHCl.sub.3) was added to 10 mL of bare gold suspension
(30 nm) under vigorous agitation. The molecules self-assemble onto
the colloid surface, with their succinimide end groups available
for protein immobilization. It is noted that this amount of the
reporter-linker is estimated to be more than enough to cover the
entire colloidal surface. The reporter-linker labeled colloids were
centrifuged, and resuspended in the aforementioned borate
buffer.
[0082] Similar to the co-immobilization approach above, 230 .mu.g
of goat anti-rat IgG were added to the 10 mL suspension of the
DSB-labeled gold colloids, followed by an incubation at 4.degree.
C. for 12 hours. This step covalently couples anti-rat IgG molecule
to the colloid surface via amide linkages that are formed by the
reactions of its amine groups with the succinimde ester groups of
DSB. Finally, the Raman-active immunogold was rinsed and
resuspended in Tris buffer, and the final concentration of the
colloids was adjusted to approximately 2.times.10.sup.11
particles/mL. The suspensions were usually stable for a few weeks
when stored at 4.degree. C.
Example 4
UV-Vis Characterization of Gold Island Films
[0083] Gold island films were used as SERS-active substrates to
examine the scattering properties of the acid-terminated DBA and
succinimide-terminated DSB reporters discussed above. Gold films
were deposited onto clean glass microscope slides by resistive
evaporation at a pressure of less than 1.3.times.10.sup.-4 Pa. Gold
island films, which were used as the SERS substrates in Raman
reporter characterization experiments, were prepared by evaporating
approximately 5 nm of gold directly onto the glass substrate. The
island films were then derivatized with reporter molecules by
immersion in 1 mM DBA (in ethanol) or 1 mM DSB (in chloroform)
solutions for 24 hours, and subsequently rinsed with the
corresponding neat solvents before SERS characterization. Smooth
gold films were prepared by first coating a glass substrate with 15
nm of chromium followed by 300 nm of gold. These substrates were
used to prepare capture antibody substrates for the immunoassay
experiment described below.
[0084] To minimize differences caused by substrate variability, the
gold island films (.about.5 nm thick) were first examined using
UV-Vis spectrometer. FIG. 4 shows the spectra of two such films
(spectra a and b) before immersion in the reporter molecule
solution. For comparison, spectrum c was collected from 5 nm
colloidal gold suspended in aqueous solution. Both island films
exhibited a plasmon resonance band with a maximum of 597 nm, while
that for colloidal gold was at 519 nm. The plasmon bands from the
island films were also wider than that observed from uniformly
dispersed 5 nm colloidal gold. The difference in the spectra of the
5 nm-thick gold island films and the 5-nm diameter gold colloid
suspension can be explained by the distribution of sizes and shapes
of the nanostructures on the two different types of samples.
Evaporated gold islands usually have a broad size distribution with
different irregular shapes. Colloidal gold, on the other hand, is
reported to be more uniform in size and have a near-spherical
shape.
[0085] It is more important to note that the spectra for the two
island films were effectively superimposable. This agreement argues
that the average sizes and shapes of the islands on the two
substrates were strongly similar. As a result, both substrates
should have had similar surface plasmon properties and therefore
produce similar magnitudes of surface enhancement for Raman
scattering.
Example 5
SERS Characterization of DBA and DSB Reporter Molecules
[0086] The DBA and DSB reporter molecules were analyzed to
determine the difference in the reporter scattering properties as a
result of altering the terminal functional groups in the reporter
molecule. The experimental setup for the SERS measurements is shown
in FIG. 2. The signal was excited with a diode laser (Hitachi
HL7851G, Thorlabs) operated at 20.degree. C. and 120 mA. These
conditions produced 50 mW of output power at the sample with a
wavelength of 785.13 nm. A polarization rotator adjusted the
polarization direction of the laser to minimize reflection losses
at the Pellin-Brocha prisms. The prisms were used to remove
background laser emission. The laser beam was then directed by a
mirror through an aperture and focused by a 50-mm focal length
cylindrical lens to a 3 mm by 0.25 mm line on the sample surface.
The laser beam irradiated the sample at an angle of approximately
60.degree. with respect to the surface normal, and the scattered
light was collected and focused onto the entrance slit of the
monochromator with a f/2 lens. A holographic notch filter
(HSPF-785.0, Kaiser Optical Systems) was used to block the Rayleigh
scattered light, while the Raman scattered light passed through the
entrance slit (200 .mu.m slit width) of a 300 f/4 spectrograph
(SpectraPro 300i, Acton Research Corp.) and illuminated onto a 1200
grooves/mm grating. The grating was blazed for 750 nm and produced
a nominal dispersion of 2.7 nm/mm. A thinned, back-illuminated,
liquid nitrogen-cooled CCD (LN/CCD-1100PB, Princeton Instruments)
was controlled by a PC for spectra acquisition. The positions of
the reporter molecule Raman bands were determined by calibration
using the known band positions of solid naphthalene.
[0087] SERS spectra (10 second integration time) of self-assembled
monolayers of DBA and DSB on the gold island films are shown in
FIG. 3. Several strong aromatic vibrational bands from the benzene
ring are present within this spectral region. The strongest band at
1075 cm.sup.-1 is from the aromatic C--H in-plane bending, and
another major band at 1585 cm.sup.-1 is from the C.dbd.C ring
stretching. To obtain maximum sensitivity, signals at 1075
cm.sup.-1 were used as readout in both the DBA and DSB-based
immunoassays. The similar intensities of the bands in the two
spectra of FIG. 3 was consistent with what was expected based on
the similarity in the molecular structures of the two types of
reporters. It was also noted that the intensity ratios of the peak
at 1075 cm.sup.-1 to the peak 1585 cm.sup.-1 in the two spectra
were slightly different, possibly reflecting the orientation
difference of the two types molecules when adsorbed on the
surface.
[0088] The existence of two additional bands was observed in the
DBA spectrum, both with very low intensities. The band at 1420
cm.sup.-1, which appears as a shoulder on the broad glass band
(i.e., Si--O stretches) around 1390 cm.sup.-1, is strongly
characteristic of a COO.sup.-.cndot.symmetric vibration, while the
1150 cm.sup.-1 band in the DBA spectra is tentatively assigned to a
COH stretching mode. In summary, replacing the carboxylate group
with succinimide group had only a minor influence on SERS signal
derived from the benzene structure in the DBA and DSB molecules.
The Raman signatures from the carboxylate group diminished in the
DSB spectrum, verifying the synthesis product.
Example 6
Preparation of Capture Antibody Substrates
[0089] Glass microscopes slides were soaked in a dilute surfactant
solution (Micro, Cole-parmer) for 12 hours, rinsed with deionized
water and ethanol, and dried in a stream of nitrogen. The slides
were then coated with 15 nm of chromium, followed by 300 nm of gold
by resistive evaporation at a pressure of less than
1.times.10.sup.-4 Pa. The gold substrates were next cut into 1
cm.times.1 cm sections and immersed in a 1 mM ethanolic solution of
thioctic acid for approximately 12 hours to form a carboxylic
acid-terminated monolayer.
[0090] The immobilization of the IgG proteins was accomplished by
first immersing the monolayer-modified substrates into 1% (w/w)
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) (Aldrich) in
anhydrous acetonitrile for 5 hours. This step activates the free
carboxyl groups of thioctic acid by forming on O-acylurea
intermediate with the EDC. The activated surface was then modified
with capture antibody by pipetting 100 .mu.L of goat anti-rat IgG
(100 .mu.g/mL, 0.1 M borate buffer, pH 9) (Pierce) onto
approximately 1-cm.sup.2 of the activated substrate. This reaction
was allowed to progress at 4.degree. C. for 12 hours. Finally, the
antibody-coated substrates were rinsed with deionized water, and
quickly dried under a stream of argon. All assays were conducted
using freshly prepared substrates.
Example 7
Dose-Response Curves
[0091] Dose-response curves were constructed based on the results
of a set of sandwich assays. Samples containing Rat IgG (Pierce) as
a model antigen were prepared at concentrations ranging from 0.01
ng/mL to 1 mg/mL in 50 mM PBS buffer
(KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, 150 mM NaCl, pH=7.6). A 100
.mu.L aliquot of each sample solution was pipetted onto the
separate capture antibody substrates described above, and allowed
to react for 1 hour at room temperature. After rinsing with copious
amounts of water, the substrates were then exposed to 100 .mu.L of
reporter-labeled immunogold solution for 3 hours. All substrates
were rinsed with deionized water and dried under argon before SERS
characterization.
[0092] The detection approach relied solely on the SERS effect by
utilizing the immunogold colloids labeled with Raman-active
reporter molecules as detection reagents. In this approach, gold
colloids were labeled with both antibodies for bio-recognition and
Raman-active reporter molecules for signal transduction. A key
feature of this concept is that the scattering center of the label
is positioned in close proximity to the colloid surface, which
strongly enhances the signal. The presence of the antigen was
therefore recognized by its detection antibody, and the SERS signal
of the co-immobilized Raman active species reported the ligation of
the antibody with the antigen.
[0093] The strongest SERS band at 1075 cm.sup.-1 was used as
readout in the immunoassay experiments. FIG. 4 plots the intensity
of this band versus the concentration of the antigen, rat IgG, with
either the co-immobilized (4a) or the covalently-linked colloids
(4b) as detection reagent. In both experiments, SERS signals show
proportional response to the antigen concentration almost over the
entire tested concentration range, representing a dynamic range of
nearly 8 orders of magnitude. The solid lines represent the curve
fitting of the immunoassay data based the four-parameter logistic
model, a common regression model used for describing sandwich type
immunoassays. The slope of the curve suggests how the readout
signals quantify samples of different concentration; the larger the
slope, the easier the distinction. Two important parameters
obtained from the curve fitting will be discussed with more details
in the later sections. One is the expected signal at zero dose,
which is also called the negative control signal, the other is the
expected signal at infinitely high or saturation dose, which is
also called the positive control signal.
[0094] When working with samples at low concentration, we found it
was more difficult to distinguish the analyte signal (S) accurately
from that at zero dose than from the spectral noise (N). For
example, even the signals from the negative control samples, were
readily distinguishable from the noise in the spectra with S/N
larger than 3 in both experiments. We therefore defined the limit
of detection (LOD) as the concentration associated with a response
three times the mean response obtained at zero dose. The LOD was
around 0.2 ng/mL when employing the co-immobilized reagent, but
lowered to 0.04 ng/mL with the use of the covalently-linked
reagent.
[0095] The difference in LOD was largely due to the different SERS
intensities observed for the two negative control samples, which
were obtained through the same assay procedure, using samples at a
concentration of zero (i.e., buffer only). Indeed, the difference
in the negative control signals, which reflect different extents of
nonspecific binding, is a major difference between the two sets of
results. The colloidal reagent prepared using the co-immobilization
approach seemed to yield a more pronounced nonspecific binding, and
therefore, a higher Raman signal (145 counts) for the negative
control sample (from curve fitting, S is 164 counts at zero
concentration). In comparison, the colloids modified via the
covalent linking approach yielded a much lower signal (22 counts)
from the negative control (32 counts based on curve fitting).
Because of the lack of a fluorescence background and lack of
photobeaching of the Raman-active reagent, LOD values could be
lowered by increasing the signal integration times.
[0096] Scanning Electron Microscopy images of these sample surfaces
showed that a higher colloid density was observed on the capture
antibody substrate when using the co-immobilized reagent,
supporting the conclusion that a higher extent of nonspecific
binding occurs with these samples. The increased nonspecific
binding of the co-immobilized colloids was attributed to the weak
interaction between the antibody and the colloid surface. This
interaction is weakened due to the partial coverage of the reporter
molecules on the colloid surface, which reduces the surface area on
a colloid that can interact with the antibody and hence weakens the
binding. This weak interaction can result in vacancies on the
reporter-labeled immunogold colloid, and lead to its nonspecific
binding with the antibodies on the capture substrate. Covalent
coupling reduces this complication, which in turn, lowers the
amount of nonspecific binding.
[0097] In addition to the negative control signal, all the spectra
obtained with the covalently-linked colloidal reagent were of lower
intensity than those obtained with the co-immobilized colloid for
samples of same concentration. Based on the characterizations on
the gold island film in FIG. 5, it is not believed that the lower
intensity observed with the covalently linked immunogold is due to
the difference in the Raman scattering intensity between the DSB
and DBA molecules. It is suspected that the lower signal in FIG.
4(b) may arise from an increased extent of antibody denaturation
due to covalent linking, which lowers the "active" detection
antibody levels on the colloid surface.
[0098] It is important to note that, although the co-immobilized
colloids yielded much higher absolute Raman signals, the relative
signals that normalized to the signal at zero dosage were always
higher when using the covalently-linked reagent for detection. The
curve fitting results show, for example, that the expected signal
at the saturation dosage for the co-immobilized reagent is almost
2.5 times higher than that obtained using the covalently-linked
reagent. However, the ratio for the signals at saturation dosage
with respect to those at zero dosage is 50% larger for the
covalently-linked reagent. The higher ratio suggests a sharper
contrast between the positive and the negative control signal and
therefore a more accurate distinction between an analyte and a
blank sample.
[0099] The colloidal suspension prepared from the covalent linking
approach was also more stable in solution and less susceptible to
aggregation. These observations explain the lower run-to-run
variation observed when using the covalently-linked reagent
(.about.10%) compared to that when using the co-immobilized reagent
(>20%). It was also noted that when starting with a new batch of
reporter-labeled immunogold reagent, the batch-to-batch variation
was even more significant and sometimes up to 100% when using the
co-immobilized reagent. It is suspected that this difference
represents the importance of the first step in the colloid
modification. It is less critical in the covalent linking approach
because DSB was always added at a level to ensure the exhaustive
coverage of the reporters on every colloid. However, it is very
critical in the co-immobilization approach since the dosage of DBA
determined the reporter coverage on each colloid and hence the
signal intensity per colloid.
Example 8
Reagents Used in Preparation of Raman-Active Molecules
[0100] Suspensions of unconjugated colloidal gold (32.2+4.4-nm
diameter, 2.times.1011 particles/ml) were purchased from Ted Pella,
Inc. The matched pair of monoclonal antibodies utilized for the
sandwich assay was obtained from research Diagnostics. The pair
consisted of mouse anti-human free PSA clone PSA-F65, which was
used as the capture antibody after immobilization on gold-coated
glass chips, and mouse anti-human PSA clone PSA-66, which was
employed as the tracer antibody after conjugation to the gold
particles as described below.
[0101] Serum PSA (10-30% free PSA) was purchased from Bios Pacific,
and buffer packs and ImmunoPure normal human serum were acquired
from Pierce Biotechnology. N-hydrosysuccinimide (NHS),
1,3-dicyclohexylcarbodiimide (DCCD), Tween 80,
5,5'-dithiobis(2-nitrobenzoid acid) (DNBA), and bovine serum
albumin (BSA) were obtained from Aldrich. Applicants note that
unless otherwise specified, all reagents were used as received or
were reconstituted according to standard methodologies. The
preparation of dithiobis (succinimide undecanoate) (DSU) followed a
modification to recent literature procedures; (Wagner et al.,
Biophys. J. 1996, 70, 2052-66.)
Example 9
Synthesis of 5,5'-Dithiobis(succinimidyl-2-nitrobenzoate)
(DSNB)
[0102] The Raman-active reporter molecule
5,5'-Dithiobis(succinimidyl-2-nitrobenzoate) (DSNB) was synthesized
following a procedure similar to that described herein above and in
Porter et al., Anal. Chem.; 2003, 1; 75(21):5936-43 (incorporated
by reference herein in its entirety). Briefly, to 50 mL of dry
tetrahydrofuran was added 0.50 g of DNBA (1.3 mmol), 0.52 g of DCCD
(2.5 mmol), and 0.29 G of NHS (2.5 mmol) in a 100-mL round-bottom
flask equipped with a drying tube. The mixture was magnetically
stirred at 25.degree. C. for 12 h, filtered, and then
rotoevaporated to remove solvent. The crude product was
recrystallized from acetone/hexane, yielding a yellow powder: 1H
NMR (CDCl.sub.3) .delta.=8.13 (d, 2H, .sup.3JH, H=8 Hz,
C.sub.6H.sub.4), 7.85 (d, 2H, .sup.3JH, H=8 Hz, C.sub.6H.sub.4),
7.97 (s, 2H, C.sub.6H.sub.4), 2.91 (s, 8H, CH.sub.2).
Example 10
Preparation of Raman Reporter-Labeled Immunogold Colloids
[0103] Raman Reporter-labeled immunogold colloids were prepared
using various derivatives of dithiobis (benzoic acid), which could
easily be converted to the corresponding succinimide ester with
NBS. Of those tested, DSNB is a particularly attractive example
because of the strong scattering cross section of its symmetric
NO.sub.2 stretch. As such, treatment of colloidal gold with this
derivative (FIG. 8) yields a coating of the thiolate of DSNB, which
can couple to the primary amines of a tracer antibody by formation
of an amide linkage. Applicants note that this design strategy
minimizes the distance between the gold surface and label
scattering center. This minimization is particularly significant
because, according to a simplified electromagnetic model,
enhancement varies inversely with the 12th power of the separation
distance between the scatterer and the metal particle center.
[0104] The particle workup consisted of two steps. In step one, 100
.mu.L of a 2.5 mM DSNB solution in acetonitrile was added to 1 mL
of the unconjugated colloidal gold suspension and the mixture
reacted for 3-5 h. The reporter-labeled colloids were then
separated from solution by centrifugation at 10000 g for 7 min. The
clear supernatant was discarded, and the loose red sediment was
resuspended in 1 mL of borate buffer (2 mM, pH 9).
[0105] In step two, mouse anti-PSA was coupled to the gold
particles via the succinimidyl terminus of the DSNB-derived
coating. As such, 35 .mu.g of detection antibody (7 .mu.L of 5
mg/mL PSA-66 solution) was added to the 1-mL suspension of the
reporter-labeled colloid. The mixture was then incubated at room
temperature for 1 h. After centrifugation at 10000 g for 7 min and
removal of the supernatant, the red sediment was resuspended in 1
mL of 2 mM Tris buffer (Tris-HCl (pH 7.6), 1% BSA). Applicants note
that the use of BSA, Tween 80, or both in all of the preparative
steps and in the assay protocol is part of a general procedure
designed to minimize complications from nonspecific adsorption.
Example 11
Preparation of Capture Antibody Substrates
[0106] Capture antibody substrates were prepared by first cleaning
glass slides in an ultrasonic bath under dilute surfactant solution
(Micro, Cole-Parmer), deionized water, and methanol, each for 30
min. The slides were then loaded into an Edwards 306A metal
evaporator and coated with 15 nm of chromium and 300 nm of gold at
0.2 nm/s at pressures less than 5.times.10-6 Torr. Next, the gold
substrates were removed from the evaporator and exposed for
.about.30 s to an octadecanethiol (ODT)-soaked
poly(dimethylsiloxane) stamp, which had a 5-mm-diameter hole cut in
its center. This step "inks" the outer portion of the gold
substrate with a monolayer of ODT. After inking, the substrates
were rinsed with ethanol, dried under a stream of nitrogen, and
immersed in a 1 mM ethanolic solution of DSU for 6-12 h. Upon
removal from solution, the substrates were rinsed again with
ethanol and dried under a stream of nitrogen. The result is a
5-mm-diameter domain of the succinimide ester-terminated monolayer
on each substrate, surrounded by a hydrophobic ODT coating.
Applicants note that the ODT coating serves as a hydrophobic
barrier that localizes aqueous protein solutions when pipetted onto
the area of the substrate defined by the DSU-derived monolayer.
[0107] Anti-free PSA antibodies (PSA-65) were immobilized by
pipetting 40 .mu.L of the protein solution (100 .mu.g/mL in 0.05 M
borate buffer (pH 9) and 1% Tween 80) onto the localized domain of
the DSU-modified monolayer. The reaction was allowed to progress
overnight at room temperature. After rinsing three times with
buffer 1 (0.01 M borate buffer (pH 9), 30 mM NaCl, 0.5% Tween 80),
40 .mu.L of blocking buffer (5% BSA in 0.05 M borate buffer (pH 9)
was pipetted onto the surface and incubated for 1 h. The substrates
were then rinsed three times with buffer 1.
Example 12
Immonoassay Protocol
[0108] PSA dose-response curves were constructed using matrixes
consisting of normal human serum, 10 mM phosphate-buffered saline
(PBS, KH2PO4/K2HPO4 (pH 7.5), 150 mM NaCl, 0.1% BSA, 0.5% Tween 80,
0.02% NaN3), and a 1:1 mixture of human serum and PBS, following
the typical procedure for a sandwich-type assay. For each matrix,
40 .mu.L aliquots of PSA solutions of various concentrations were
pipetted onto a capture antibody-coated substrate and allowed to
react for 3 h at room temperature. After rinsing three times with
buffer 2 (10 mM PBS buffer (pH 7.5), 0.5% Tween 80, 0.02% NaN3),
the sample was exposed to 40 .mu.L of the immunogold detection
reagent for 6 h. All samples were then rinsed three times with
buffer 2 and once with deionized water and dried under a stream of
nitrogen before SERS characterization. Applicants found that Tween
concentrations of 0.1-0.5% in the rinse buffer were generally
effective in minimizing nonspecifically bound protein, while
maintaining the hydrophobic integrity of the ODT domain.
Example 13
Instrumentation
(i) SERS Measurements.
[0109] A fiber-optic based Raman system, NanoRamon I, from
NanoRaman Instruments was used for all Raman data generation. The
system consists of three major subassemblies: laser light source,
spectrograph, and fiber-optic probe. FIG. 9 shows the spectroscopic
setup. The light source is a 30-mW, 632.8-nm HeNe laser, while the
spectrograph consists of an f/2.0 Czemy-Turner imaging spectrometer
(6-8-cm.sup.-1 resolution, no moving parts) and a
thermoelectrically cooled (0.degree. C.) Kodak 0401E CCD. The
fiber-optic probe (1.75.times.2.5.times.6 in) utilizes band-pass
and long-pass filters for laser light (OD 6) and fiber background
(OD 4) rejection. The probe objective provides a numerical aperture
of 0.65 while maintaining a relatively long working distance of 3
mm. The laser spot size on the sample surface is .about.22 .mu.m in
diameter. A Windows-based Visual Basic program controls the system.
All spectra were collected with a 60-s integration time. The
positions of the Raman bands were determined by comparisons to the
known positions of bands for solid naphthalene.
(ii) Infrared Spectroscopy.
[0110] Infrared reflection spectra were acquired with a Nicolet 850
FT-IR spectrometer, purged with liquid N.sub.2 boil-off, and
equipped with a liquid N.sub.2-cooled HgCdTe detector. Spectra were
obtained using p-polarized light incident at 80.degree. with
respect to the surface normal. The spectra were recorded as
-log(R/Ro), where R is the sample reflectance and Ro is the
reflectance of an octadecanethiolate-d.sub.37 monolayer-coated Au
reference. The spectra are an average of 512 sample and reference
scans, taken at 4 cm.sup.-1 resolution with Happ-Genzel
apodization.
(iii) X-ray Photoelectron Spectroscopy.
[0111] X-ray photoelectron spectra (XPS) were acquired at room
temperate with a Physical Electronics Industries 5500
multitechnique surface analysis system. This system is equipped
with a hemispherical analyzer, a toroidal monochromator, a
multichannel detector at 45.degree., and monochromatic Al Ka.
excitation radiation (1486.6 eV, 250 W). A pass energy of 29.35 eV
was used, giving a half-width of the Au (4f.sub.7/2) peak of
.about.0.8 eV.
Results and Discussion
Chip Characterization
[0112] The capture antibody substrate consisted of anti-free PSA
bound to a gold-coated glass chip via the DSU-derived coupling
agent. DSU chemisorbs to gold through cleavage of the sulfur-sulfur
bond, and the formation of the resulting gold-bond thiolate and its
subsequent coupling to anti-free PSA can be readily confirmed by
infrared reflection spectroscopy (IRS) and XPS. The IRS results are
presented in FIG. 10. The three bands around 1800 cm.sup.-1 in the
spectrum of the layer formed from DSU (FIG. 10A) are assigned to
the carbonyl stretches of the ester (1816 cm.sup.-1) and of the
succinimidyl end group (1787 (in-phase) and 1750 cm.sup.-1
(out-of-phase)). The presence of these bands, along with the
succinimidyl bands at 1219 and 1078 cm.sup.-1 and the methylene
stretches between 3000 and 2800 cm.sup.-1, verifies the formation
of the DSU-based coating.
[0113] IRS was also used to confirm the covalent binding of
anti-free PSA to the terminal group of the gold-bound coupling
layer (FIG. 10B). Since the acyl carbon of the succinimidyl ester
group is strongly susceptible to nucleophilic attack, reaction with
the sterically accessible amines in the protein should immobilize
anti-free PSA via amide linkages. As evident in FIG. 2B, treatment
of the DSU-modified substrate with anti-free PSA causes a marked
decrease in the magnitude of the bands for the succinimidyl group
(e.g., 1750, 1219, and 1078 cm.sup.-1). Moreover, three readily
identifiable bands, which are located at 3304 (N--H stretch), 1654
(amide I), and 1540 cm.sup.-1 (amide II), have appeared. The new
bands reflect the presence of amides inherent in the native
antibody as well as those formed by the reaction of the
succinimidyl groups of DSU with amines in the protein. Coupled with
earlier reports, which, in part studied the hydrolysis rate of the
succinimidyl terminal group of DSU-derived monolayer under similar
conditions, the differences in FIG. 10 support the covalent
attachment of anti-free PSA to the underlying substrate.
[0114] The XPS characterizations are in agreement with the IRS
findings. The results for both modified surfaces are given in Table
1 shown below.
TABLE-US-00001 TABLE 1 Binding Energies (eV) and Compositional
Assignments for XPS Spectra of DSU and DSNB Monolayers on Gold
before and after Antibody Derivitization Core Anti-free Anti-PSA/
level DSU/Au PSA/DSU/Au DSNB/Au DSNB/Au Au(4f.sub.7/2) 83.9 83.9
83.9 83.9 S(2p.sub.3/2) 161.8 161.8 162.2 162.2 S(2p.sub.1/2) 162.9
162.9 nd.sup.a nd C(1s) 289.0 288.5 288.4 288.1 C(1s) 284.4 284.9,
284.6 284.8, 286.4 (sh) 285.9 (sh) N(1s) 401.9 400.5 405.6, 401.2
405.2, 400.0 O(1s) 532.5, 534.9 432.1 532.2 532.2 .sup.and, not
detected.
[0115] As expected, survey spectra for the two different coatings
showed only the presence of carbon, oxygen, nitrogen, and sulfur.
For the DSU-based coating, the C (1s) region was composed of a
lower energy band (284.4 eV), attributed to the alkyl chain
structure of the coating, and a higher energy band (289.0 eV),
assigned to the different types of carbonyl carbon. Two bands were
also observed in the O(1s) and S(2p) regions. In the O(1s) region,
the band at 534.9 eV is ascribed to the oxygen of the ester linkage
and that at 532.5 eV is assigned to the remaining carbonyl oxygens.
In the S(2p) region, the positions of the features in the doublet
that arises from spin-orbit coupling (2p.sub.3/2, 161.8 eV;
2p.sub.1/2, 162.9 eV) are consistent with the presence of
gold-bound thiolates formed by the adsorption of thiols and
disulfides on gold. In contrast, there was only one band observed
in the N(1s) region. The location of this band (401.9 eV) agrees
with the presence of an electron-withdrawing group attached to
nitrogen, as is the case for the succinimidyl nitrogen.
[0116] After treatment with anti-free PSA, all XPS features undergo
a general broadening, which limited the ability to carry out an
in-depth compositional analysis. There were, however, two readily
identifiable changes that support the coupling of anti-free PSA to
the DSU-derived coating. First the N(1s) band shifts from 401.9 to
400.5 eV. This shift is ascribed to the loss of the succinimidyl
end group and the appearance of the numerous nitrogen
functionalities in the immobilized protein. Second, the intensity
of the S(2p) couplet is greatly diminished. The decrease in
intensity reflects the attenuation of the photoelectrons by the
immobilized protein.
Detection Reagent Characterization.
[0117] The new Ramen reporter molecules, which were prepared by
reacting DNBA with NHS to form the bis(succinimide ester), yield a
coating on gold that can act as a coupling agent in the same manner
as the DSU-based monolayer. FIG. 11 shows the IRS spectra for a
monolayer of DSNB spontaneously adsorbed on gold-coated glass
before and after exposure to anti-PSA. The as-formed layer has
carbonyl stretches at 1812, 1789, and 1748 cm.sup.-1 and strong
symmetric and asymmetric nitro stretches at 1343 and 1533
cm.sup.-1, respectively. As with the DSU-based monolayer, the
spectrum for the DSNB-derived monolayer undergoes a similar set of
changes following exposure to anti-PSA. Three new bands appear
(3292 (N--H stretch), 1655 (amide I), and 1530 cm.sup.-1 (amide
II)), and the three carbonyl stretches exhibit a notable decrease.
The detection of residual succinmide groups is expected because the
presence of an immobilized antibody will sterically hinder protein
binding to neighboring succinimide moieties. Thus, BSA was used in
the immunogold reagent preparation procedure to react with any
exposed residual succinmide groups in order to preclude their
participation in nonspecific binding.
[0118] The XPS characterizations (Table 1) also strongly mimic
those for the DSU-based layer. In this case, however, there are two
N(1s) bands for each sample. For the as-formed layer, bands at
401.2 and 405.5 eV are indicative of the succinimidyl nitrogen and
nitro nitrogen on the aromatic ring, respectively. After exposure
to anti-PSA, the band at 401.2 eV disappears and one at 400.0 eV
appears. This change again parallels that for the DSU-derived
coating.
SERS of Reporter-Labeled Immunogold Reagent.
[0119] Raman spectra for the Ramen reporter molecule DSNB are shown
in FIG. 12 before and after coupling to the gold nanoparticles. The
nanoparticle sample was prepared by drop casting a small amount of
the labeled colloid solution onto a gold-coated glass slide and
evaporating the water-based solvent. The powder spectrum (FIG. 12A)
is dominated by the symmetric nitro stretch at 1342 cm.sup.-1, and
we attribute the band at 851 cm.sup.-1 to the nitro scissoring
vibration. The band at 1566 cm.sup.-1 is assigned to an aromatic
ring mode (8a), and the large band at 1079 cm.sup.-1 is probably a
succinimidyl N--C--O stretch overlapping with aromatic ring
modes.
[0120] Many of the bands in the powder spectrum are present in the
spectrum after DSNB is chemisorbed onto the gold nanoparticles
(FIG. 12B), though some have undergone a small change in position.
For example, the symmetric nitro stretch has shifted from 1342 to
1338 cm.sup.-1, and the 8a mode has moved from 1566 to 1558
cm.sup.-1 These shifts are indicative of interactions between
neighboring adsorbates and between the adsorbates and the gold
surface. Additionally, since there was no detectable Raman signal
for the monolayer formed from DSNB adsorbed to a smooth gold
surface, the spectrum in FIG. 12B illustrates that the
immobilization of DSNB on the gold nanoparticles results in a
significant level of enhancement, the magnitude of which will be
examined in detail in future studies. Together, these results
confirm that the particles have been effectively modified with the
DSNB-based RRMs.
SERS Immonoassay Detection of Free PSA.
[0121] The results of our SERS-based determinations for free PSA in
normal human serum are shown in FIG. 13. Test solutions were made
by serial dilution in human serum of a 1 mg/mL PSA standard to
cover the range from 1 .mu.g/mL (30 nM) to pg/mL (30 fM). The
spectra in FIG. 13A were obtained using 60-s integrations after
completion of the immunoassay protocol outlined above. As is
evident, the features diagnostic of the DSNB-labeled nanoparticles
exhibit a strong increase as the PSA level increases. These changes
span more than 6 order of magnitude, this encompassing
concentration levels critical to prostate cancer diagnosis.
[0122] The lower limit of detection by the nonspecific adsorption
of the labeled nanoparticles, as demonstrated by the signal
observed for the blank serum sample. Blanks prepared without BSA
and Tween 80 as additives yielded signals that were several times
larger than those obtained with the use of additives. In contrast,
the packing constraints imposed by the labeled particle size should
control the upper limit of the dynamic range. Though only examined
in a preliminary manner, tests place the upper limit at .about.10
.mu.g/mL.
[0123] A more detailed treatment of applicants' findings is
presented by the dose-response curve in FIG. 13B. This curve was
constructed by plotting the scattering intensity of the symmetric
nitro stretch (1338 cm.sup.-1, full width at half-maximum of 22
cm.sup.-1). Each data point represents the average of six to eight
readings across the sample surface. Variations in signal strength
across the surface of each chip were typically .about.10%. However,
signal strengths up to twice as large as those represented in the
plot were observed .about.20% of the time. The dose-response curve
was constructed by omitting the data for these "hot spots". These
hot spots could possibly reflect the presence of domains where
there are higher localized concentrations of binding sites and,
therefore, higher particle densities are reasonably homogenous over
areas irradiating by the laser source. On the other hand, AFM
imaging revealed a small number of particle aggregates that could
account for the hot spots. A third possibility arises from the
existence of "hot particles". Recent studies have shown that
enhancement factors are strongly dependent on particle size, shape,
and excitation wavelength and that a small fraction of particles
exhibit markedly larger enhancements.
[0124] The dose-response curve shows that our detection platform
can determine free PSA at very low concentrations in human serum.
The detection limit is .about.1 pg/mL. These results compare
favorably with commercial assays based on radiometric,
chemiluminescent, and ELISA methods, which have detection limits
ranging from 3 to 1000 pg/mL free PSA. Additional studies were
performed in which PSA was added to 10 mM PBS that contained 0.1%
BSA and 0.5% Tween 80. These studies yielded a detection limit of 4
pg/mL free PSA, based on a concentration that produces a signal
three times the standard deviation of the background. Similar
results were obtained in a analyte matrix of a 1:1 mixture of
PBS/serum. Thus, the assay appears to be applicable to a range of
sample matrixes.
[0125] The ability to detect exceedingly small amounts of analyte
using our monoclonal-based assay format is underscored by a rough
estimate of the number of molecular recognition events responsible
for the response at the limit of detection. At a detection limit of
4 pg/mL, a 40 .mu.L solution of free PSA contains 160 fg
(.about.3.times.106 molecules) of free PSA. If we assume that (1)
the capture surface exhaustively binds all of the proteins, (2) the
captured antigens are uniformly distributed across the
5-mm-diameter surface of the capture substrate, and (3) the binding
stoichiometry between the nanoparticles and captured antigen is
1:1, then there are only .about.60 PSA molecules in the 22
.mu.m-diameter area irradiated by the laser source. This analysis
shows that the combination of surface enhancement with respect to
the close proximity of the scattering site to the particle surface,
the amplification due to the large number of Ramen reporter
molecules coating each particle (preliminary estimates indicate
that there are .about.10.sup.3 RRMs tethered to each nanoparticle),
and the binding affinity of monoclonal antibodies leads to an
extremely low level of detection.
[0126] Based on the estimate of the number of recognition events
detected in the above-described PSA assay, projections can be made
which strongly argue that the technique can be extended to
single-molecule detection. There are two clear avenues to reach
such a level. The first avenue uses labels that undergo both
resonance and surface enhancement. With resonance enhancement,
intensities can be 2-6 orders of magnitude greater than those based
on normal Raman scattering. The second avenue takes advantage of
recent reports that have shown that the surface enhancement for
slightly larger gold particles (e.g., 60 nm for our excitation
wavelength) is greater than that for 30-nm particles. Taken
together, the ability to detect the binding of a single antigen
appears to be well within reach and should be of immense value in
the ultra-low-level detection of a wide range of biomarkers used in
early disease diagnosis and other assay applications. Low-level
detection becomes even more important as the degree of multiplexing
increases, e.g., in instances where screening for multiple analytes
at a single location is of interest.
[0127] Therefore. this embodiment of the present invention enables
the detection of biomarkers for early cancer diagnosis in serum
samples at very low concentrations by a SERS-based readout method.
This strategy is capable of encompassing a wide range of
applications especially in view of the opportunities to multiplex
through the judicious design of more labeled nanoparticles. As
such, multiple analytes could be concurrently identified through
the position of a characteristic feature of the Raman label and
then quantified by its intensity.
[0128] It is envisioned that assays could be developed for the
high-sensitivity. simultaneous screening of a battery of cancer
markers using a single serum sample. saving time reducing assay
costs, and potentially leading to earlier diagnosis.
[0129] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding. it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications to the disclosed reagents.
methods and kits may be made thereto without departing from the
spirit or scope of the appended claims.
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