U.S. patent application number 11/396098 was filed with the patent office on 2008-07-10 for novel gold nanoparticle aggregates and their applications.
Invention is credited to Christian D. Grant, Claire Gu, Thaddeus Norman, Tammy Y. Oshiro, Adam Schwartzberg, Leo Seballos, Rebecca Sutphen, Jin Zhang, Yi Zhang.
Application Number | 20080166706 11/396098 |
Document ID | / |
Family ID | 39594616 |
Filed Date | 2008-07-10 |
United States Patent
Application |
20080166706 |
Kind Code |
A1 |
Zhang; Jin ; et al. |
July 10, 2008 |
Novel gold nanoparticle aggregates and their applications
Abstract
The invention is drawn to novel gold nanoparticles that are used
in a dual optical method for sensitive and selective detection of
antigens. The gold nanoparticle aggregates are synthesized from
gold hydrochloride and sulfur salts in an aqueous solution. The
aggregates can be selectively sized using a spectral notch filter
that results in an improved product with versatile uses. The gold
nanoparticles can also be used in improved optical communications
devices.
Inventors: |
Zhang; Jin; (Santa Cruz,
CA) ; Schwartzberg; Adam; (Santa Cruz, CA) ;
Norman; Thaddeus; (San Jose, CA) ; Oshiro; Tammy
Y.; (Santa Cruz, CA) ; Grant; Christian D.;
(San Jose, CA) ; Sutphen; Rebecca; (Tampa, FL)
; Seballos; Leo; (Santa Cruz, CA) ; Zhang; Yi;
(Santa Cruz, CA) ; Gu; Claire; (Santa Cruz,
CA) |
Correspondence
Address: |
BELL & ASSOCIATES
201 WARREN DRIVE
SAN FRANCISCO
CA
94131
US
|
Family ID: |
39594616 |
Appl. No.: |
11/396098 |
Filed: |
March 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60667151 |
Mar 30, 2005 |
|
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|
60711808 |
Aug 26, 2005 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 436/524; 977/902; 977/924 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 21/658 20130101; G01N 33/587 20130101; G01N 33/553 20130101;
G01N 33/54346 20130101; G01N 33/585 20130101; G01N 33/54313
20130101 |
Class at
Publication: |
435/6 ; 436/524;
435/287.2; 977/902; 977/924 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; G01N 33/551 20060101
G01N033/551 |
Claims
1. A chemical sensor comprising a plurality of particles, each
particle comprising: a core, a shell having at least one surface
and having contact with the core and wherein the shell comprises a
sulfur-oxygen molecular species, and wherein the particle has been
selectively sized using a notch filter and electromagnetic
radiation, the electromagnetic radiation having a spectral
wavelength of between about 350 nm and about 1075 nm.
2. The chemical sensor of claim 1 wherein the core comprises a
metal selected from the group consisting of gold, silver, platinum,
copper, aluminum, palladium, cadmium, iridium, and rhodium.
3. The chemical sensor of claim 1 wherein the core comprises
gold.
4. The chemical sensor of claim 1 wherein the shell that further
comprises a linker molecule, the linker molecule selected from the
group consisting of a thiol group, a sulphide group, a phosphate
group, a sulphate group, a cyano group, a piperidine group, an Fmoc
group, and a Boc group.
5. The chemical sensor of claim 1 wherein the electromagnetic
radiation has a spectral wavelength of between about 350 nm and
about 650 nm and between about 950 nm and about 1075 nm.
6. The chemical sensor of claim 1 wherein the electromagnetic
radiation has a spectral wavelength of between about 350 nm and
about 775 nm and between about 875 nm and about 1075 mn.
7. The chemical sensor of claim 1 wherein the particle has a size
in the range of about 60 and 200 nm.
8. The chemical sensor of claim 1 further comprising a support.
9. The chemical sensor of claim 8 wherein the support comprises a
medium that is permeable to an analyte of interest.
10. The chemical sensor of claim 1 wherein the surface can induce
surface enhanced Raman scattering.
11. The chemical sensor of claim 1 further comprising a detecting
molecule, wherein the detecting molecule is bound to the
surface.
12. The chemical sensor of claim 11 wherein the detecting molecule
is selected from the group consisting of proteins, peptides,
antibodies, antigens, nucleic acids, peptide nucleic acids, sugars,
lipids, glycophosphoinositols, and lipopolysaccharides.
13. The chemical sensor of claim 11 wherein the detecting molecule
is an antibody.
14. The chemical sensor of claim 11 wherein the detecting molecule
is an antigen.
15. The chemical sensor of claim 1 further comprising a
semiconductor quantum dot.
16. The chemical sensor of claim 15 wherein the semiconductor
quantum dot further comprises a linker molecule, the linker
molecule selected from the group consisting of a thiol group, a
sulphide group, a phosphate group, a sulphate group, a cyano group,
a piperidine group, an Fmoc group, and a Boc group.
17. The chemical sensor of claim 15 wherein the semiconductor
quantum dot further comprises a detecting molecule, wherein the
detecting molecule is bound to the semiconductor quantum dot.
18. The chemical sensor of claim 15 wherein the detecting molecule
is selected from the group consisting of proteins, peptides,
antibodies, antigens, nucleic acids, peptide nucleic acids, sugars,
lipids, glycophosphoinositols, and lipopolysaccharides.
19. The chemical sensor of claim 15 wherein the detecting molecule
is an antibody.
20. The chemical sensor of claim 15 wherein the detecting molecule
is an antigen.
21. The chemical sensor of claim 20 wherein the detecting molecule
is an antigen that binds to an ovarian cancer marker antibody with
an affinity (K.sub.a) of at least 10.sup.6 l/mole.
22. The chemical sensor of claim 21 wherein the K.sub.a is at least
10.sup.8 l/mole.
23. A method for detecting an analyte in a sample using a chemical
sensor, the method comprising the steps of: i) providing a sample;
ii) providing a semiconductor quantum dot comprising a linker
molecule (LM-SQD) iii) conjugating the analyte in the sample with
the LM-SQD thereby producing an analyte-LM-SQD conjugate; iv)
providing the chemical sensor of claim 11; v) incubating the
analyte-LM-SQD conjugate with the chemical sensor for a
predetermined time period; and vi) measuring the extent of binding
between the analyte-LM-SQD conjugate and the chemical sensor;
thereby detecting the analyte in the sample.
24. The method of claim 23 wherein the analyte is an ovarian cancer
marker antibody.
25. The method of claim 23 wherein the detecting molecule in the
chemical sensor is an antigen that binds to an ovarian cancer
marker antibody with an affinity (K.sub.a) of at least 10.sup.6
l/mole.
26. The method of claim 25 wherein the K.sub.a is at least 10.sup.8
l/mole.
27. An optical communications device comprising a fiber and the
chemical sensor of claim 1.
28. The optical communications device of claim 27 wherein the fiber
is selected from the group consisting of ceramics, glasses, and
polymers.
29. The optical communications device of claim 27 wherein the fiber
cross-section is D-shaped.
30. The optical communications device of claim 27 wherein the
chemical sensor is disposed upon a surface of the fiber.
Description
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 60/667,151 entitled "Novel Gold
Nanoparticle Aggregates and Their Applications", filed Mar. 30,
2005, and U.S. Provisional Patent Application Ser. No. 60/711,808
entitled "Novel Gold Nanoparticle Aggregates and Their
Applications", filed Aug. 26, 2005, which are herein incorporated
by reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to particles comprising a
metallic core and sulfur species on their surface with useful
properties. The invention further relates to methods of using the
particles for detecting chemical and biological analytes, and in
use in optical communications.
BACKGROUND
[0003] During the 1980s Raman Scattering in fibers was demonstrated
by Lin, Stolen, and other co-workers of AT&T Bell Laboratories
in Holmdel, N.J. using Raman lasers operating between 0.3 to 2.0
.mu.m. In the early years of the Raman fiber before extensive work
had begun, no one perceived that a Raman fiber could be pumped by a
practical semiconductor laser-based source or that an efficient
CW-pumped Raman Fiber Laser was possible. However, with the
development of Cladding-pumped Fiber Lasers and Fiber Bragg
Gratings, diode-laser-based CW Raman Fiber Lasers have been made
efficient, emitting at various wavelengths throughout the infrared
spectrum a reality. (See van Gisbergen et al. (1996) Chem. Phys.
Lett. 259: 599-604.)
[0004] Raman spectroscopy is a powerful optical technique for
detecting and analyzing molecules. Its principle is based on
detecting light scattered off a molecule that is shifted in energy
with respect to the incident light. The shift, called Raman shift,
is characteristic of individual molecules, reflecting their
vibrational frequencies that are like fingerprints of molecules. As
a result, the key advantage of Raman spectroscopy is its molecular
specificity while its main limitation is the small signal due to
low quantum yield of Raman scattering. One way to enhance the Raman
signal is to tune the excitation wavelength to be on resonance with
an electronic transition, so called resonance Raman scattering.
This can usually produce an enhancement on the order of
10.sup.2-10.sup.3.
[0005] Another technique to enhance Raman scattering is surface
enhancement by roughened metal surfaces, notably silver and gold,
that provides an enhancement factor on the order of
10.sup.6-10.sup.8. This is termed surface enhanced Raman
spectroscopy (SERS). Similar or somewhat larger enhancement factors
(.about.10.sup.8-10.sup.10) have been observed for metal, mostly
silver or gold, nanoparticles.
[0006] In the last few years, it has been shown that an even larger
enhancement (.about.10.sup.10-10.sup.15) is possible for aggregates
of metal nanoparticles (MNPs), silver and gold. The largest
enhancement factor of 10.sup.14-10.sup.15 has been reported for
rhodamine 6G (R6G) on single silver nanoparticle aggregates. This
huge enhancement is thought to be mainly due to significant
enhancement of the local electromagnetic field of the nanoparticle
aggregate that strongly absorbs the incident excitation light for
the Raman scattering process. With such large enhancement, many
important molecules that are difficult to detect with Raman
normally can now be easily detected. This opens many interesting
and new opportunities for detecting and analyzing molecules using
SERS with extremely high sensitivity and molecular specificity.
[0007] SERS can also be developed into a molecular imaging
technique for biomedical and other applications. Existing Raman
imaging equipment should be usable for SERS imaging. SERS will
provide a much-enhanced signal and thereby significantly shortened
data acquisition time, making the technique practically useful for
medical or other commercial and industrial applications including
chip inspection or chemical monitoring. SERS is also useful for
detecting other cancer biomarkers that can interact or bind to the
MNP surface. For example, Sutphen et al. have recently shown that
lysophospholipids (LPL) are potential biomarkers of ovarian cancer
(Sutphen et al. (2004) Cancer Epidemiol. Biomarker Prev. 13:
1185-1191).
[0008] For many practical applications, for example SERS and
optical filters, it is highly desirable to narrow the distribution
of size/shape of nanoparticle aggregates. For SERS in particular,
the incident light has to be on resonance with the substrate
absorption. Only those nanoparticle aggregates that have resonance
absorption of the incident light are expected to be SERS active. It
is thus extremely beneficial to have a narrow size/shape
distribution and thereby narrow optical absorption.
[0009] Fluorescent nanoparticles (quantum dots (QDs) such as
semiconductor quantum dots, SQDs) have been used recently as
fluorescent biological markers and have been found to be extremely
effective. They offer advantages including higher stability,
stronger fluorescence, tunability of color, and possibility of
optical encoding based on different sized or colored SQDs.
[0010] A method of synthesis for gold nanoparticle aggregates
(GNAs) has been described in the prior art (see Norman et al.
(2002) J. Phys. Chem. B, 106: 7005-7012). Norman used Na.sub.2S and
HAuCl.sub.4 (chloroauric acid). Norman suggested that the product
of the reaction is elemental sulfur, elemental gold, free protons,
and free chlorine ions. This contrasts with the alternative dogma
that the aggregates comprise an Au.sub.2S core enveloped by an Au
shell. Therefore Norman concluded that the reaction produces
aggregates of gold nanoparticles having amorphous sulfur on their
surface.
[0011] Metal nanoparticles have been recognized for their unique
optical properties that could be exploited in optoelectronic
devices. Nanoparticle systems composed of gold, for example, have
distinct optical properties that make them amenable to study by
Raman scattering. The Raman spectrum of the adsorbed species is
significantly enhanced by 10 to 15 orders of magnitude when the
metal nanoparticles have aggregated, leading to enhanced
electromagnetic field effects near the surface that increases the
Raman scattering intensity. The greater sensitivity found in the
surface enhanced Raman spectroscopy (SERS) of metal nanoparticle
aggregates facilitates the detection and analysis of a whole host
of molecules that were previously difficult to study.
[0012] Wang et al. disclose a method of using SQDs (dye-conjugated
CdTe nanoparticles, CT-NPs) to detect interactive binding between
Ag-CT-NPs and Ab-CT-NPs (Wang et al. (2002) NanoLett. 2: 817-822).
The interactions were determined by differential quenching or
enhancement fluorescence activity of two different sized SQDs (red
or green) measured during the analysis.
[0013] The chemical methods used historically for the production of
gold nanoparticle aggregates (GNAs) results in a wide distribution
of aggregate size. This distribution leads to a broadened
absorption spectrum. Accordingly, researchers have attempted to
narrow the lineshape of the spectral peak due to the aggregates by
homogenizing the size of the GNAs after they have been produced. By
eliminating certain ranges of aggregate size, absorption spectrum
peaks should narrow appreciably and concomitantly increase in
intensity, resulting in more sensitive detection. Previous attempts
to select for a narrow size range of aggregates have employed
mechanical techniques such as passing a solution of aggregates
through a filter. For example, Emory & Nie have employed
size-selective fractionation using membrane filters to select for
optically active silver nanoparticles (Emory and Nie, (1997) J.
Phys. Chem. B, 102: 493-497).
[0014] The use of SERS for analyte detection of biomolecules has
been previously studied. U.S. Pat. No. 6,699,724 to West et al.
describes a chemical sensing device and method (nanoshell-modified
ELISA technique) based on the enzyme-linked immunoadsorbant assay
(ELISA). The chemical sensing device can comprise a core comprising
gold sulfide and a surface capable of inducing surface enhanced
Raman scattering (SERS). In much of the patent disclosure, the
nanoparticle is disclosed as having a silica core and a gold shell.
The patent discloses that an enhancement of 600,000-fold
(6.times.10.sup.5) in the Raman signal using conjugated
mercaptoaniline was observed.
[0015] In the nanoshel-modified ELISA technique, antibodies are
directly bound to the metal nanoshells. Raman spectra are taken of
the antibody-nanoshell conjugates before and after the addition of
a sample containing a possible antigen, and binding of antigen to
antibody is expected to cause a detectable shift in the
spectra.
[0016] The conjugation of quantum dots to antibodies used for
ultrasensitive nonisotopic detection for use in biological assays
has also been studied. U.S. Pat. No. 6,468,808 B1 to Nie et al.
disclosed an antibody is conjugated to a water-soluble quantum dot.
The binding of the quantum dot-antibody conjugate to a targeted
protein will result in agglutination, which can be detected using
an epi-fluorescence microscope. In addition, Nie et al. described a
system in which a quantum dot is attached to one end of an
oligonucleotide and a quenching moiety is attached to the other.
The preferred quenching moiety in the Nie patent is a
nonfluorescent organic chromophore such as
4-[4'-dimethylaminophenylazo]benzoic acid (DABCYL).
[0017] Raman amplifiers are also expected to be used globally as a
key device in next-generation optical communications, for example,
in wavelength-division-multiplexing (WDM) transmission systems.
Raman scattering occurs when an atom absorbs a photon and another
photon of a different energy is released. The energy difference
excites the atom and causes it to release a photon with low energy;
therefore, more light energy is transferred to the photons in the
light path.
[0018] FIG. 6 shown how the Raman amplifier operates. The Raman
amplification process begins as a seed beam (incoming light) passes
through the optical fiber. While it is traveling, a stronger pump
beam is released from another light source and is deflected using a
refractive material, such as a mirror. The pump beam and seed beam
then come in contact with each other and the seed beam depletes the
energy of the pump beam; therefore the intensity of the light
increases and the signal is amplified. Now the signal is capable of
traveling long distances, for example, more than 70 km, without
losing a signal. (See, for example, U.S. Pat. No. 6,292,288; Vinson
and Webb (2001) Light Amplification: The Future Of Optical
Communications, Optical Engineering UCSC, Summer Ventures of
Science and Math, 2001, 7 pp.)
[0019] There is therefore a need in the art for use in the
biomedical analytical industries and the optical communications
industries to provide more sensitive compositions and devices that
are inexpensive to manufacture and easy to use.
BRIEF DESCRIPTION OF THE INVENTION
[0020] The present invention provides a chemical sensor comprising
a plurality of particles, each particle comprising: a core, a shell
having at least one surface and having contact with the core and
wherein the shell comprises a sulfur-oxygen molecular species, and
wherein the particle has been selectively sized using a notch
filter and electromagnetic radiation, the electromagnetic radiation
having a spectral wavelength of between about 350 nm and about 1075
nm. In one embodiment the particle has a size in the range of about
60 and 200 nm. In another embodiment the particle has a size
selected from the range of between about 60 and 150 nm, between
about 60 and 100 nm, between about 60 and 80 nm, between about 80
and 200 nm, between about 80 and 150 nm, between about 80 and 100
nm, between about 100 and 200 nm, between about 100 and 150 nm, and
between about 150 and 200 nm.
[0021] In a preferred embodiment the core comprises a metal
selected from the group consisting of gold, silver, platinum,
copper, aluminum, palladium, cadmium, iridium, and rhodium. In a
more preferred embodiment the core comprises gold.
[0022] In another preferred embodiment the electromagnetic
radiation has a spectral wavelength of between about 350 nm and
about 650 nm and between about 950 nm and about 1075 nm. In yet a
more preferred embodiment the electromagnetic radiation has a
spectral wavelength of between about 350 nm and about 775 nm and
between about 875 nm and about 1075 nm.
[0023] In another embodiment, the chemical sensor comprises a shell
that further comprises a linker molecule, the linker molecule
selected from the group consisting of a thiol group, a sulfide
group, a phosphate group, a sulfate group, a cyano group, a
piperidine group, an Fmoc group, and a Boc group.
[0024] In yet a further embodiment, the chemical sensor comprises a
support. In a preferred embodiment, the support comprises a medium
that is permeable to an analyte of interest.
[0025] In another preferred embodiment, the chemical sensor has a
surface wherein the surface can induce surface enhanced Raman
scattering (SERS).
[0026] In still another preferred embodiment, the chemical sensor
further comprises at least one detecting molecule, wherein the
detecting molecule is bound to the surface. In a more preferred
embodiment the detecting molecule is selected from the group
consisting of proteins, peptides, antibodies, antigens, nucleic
acids, peptide nucleic acids, sugars, lipids,
glycophosphoinositols, and lipopolysaccharides.
[0027] In a yet more preferred embodiment the detecting molecule is
an antibody. In another preferred embodiment, the detecting
molecule is an antigen.
[0028] In another embodiment, the invention provides a chemical
sensor further comprising at least one semiconductor quantum dot.
In a preferred embodiment the semiconductor quantum dot further
comprises a linker molecule, the linker molecule selected from the
group consisting of a thiol group, a sulfide group, a phosphate
group, a sulfate group, a cyano group, a piperidine group, an Fmoc
group, and a Boc group.
[0029] In a still further embodiment, the invention provides a
chemical sensor comprising at least one semiconductor quantum dot
wherein the semiconductor quantum dot further comprises a detecting
molecule, wherein the detecting molecule is bound to the
semiconductor quantum dot. In a more preferred embodiment, the
detecting molecule is selected from the group consisting of
proteins, peptides, antibodies, antigens, nucleic acids, peptide
nucleic acids, sugars, lipids, glycophosphoinositols, and
lipopolysaccharides.
[0030] In a more preferred embodiment, the detecting molecule is an
antibody. In the alternative, a more preferred embodiment comprises
a chemical sensing device wherein the detecting molecule is an
antigen.
[0031] Another embodiment of the invention provides a method for
detecting an analyte in a sample using a chemical sensor, the
method comprising the steps of: i) providing a sample; ii)
providing a semiconductor quantum dot comprising a linker molecule
(LM-SQD); iii) conjugating the analyte in the sample with the
LM-SQD thereby producing an analyte-LM-SQD conjugate; iv) providing
a chemical sensor comprising a plurality of particles, each
particle comprising: a core, a shell having at least one surface
and having contact with the core and wherein the shell comprises a
sulfur-oxygen molecular species, and wherein the particle has been
selectively sized using a notch filter and electromagnetic
radiation, the electromagnetic radiation having a spectral
wavelength of between about 350 nm and about 1075 nm, the shell
surface further comprising a detecting molecule; v) incubating the
analyte-LM-SQD conjugate with the chemical sensor for a
predetermined time period; and vi) measuring the extent of binding
between the analyte-LM-SQD conjugate and the chemical sensor;
thereby detecting the analyte in the sample.
[0032] In a preferred embodiment the invention provides a method
for detecting an analyte that is an ovarian cancer marker antibody.
In one embodiment of the invention the detecting molecule in the
chemical sensing device is an antigen that binds to an ovarian
cancer marker antibody with an affinity (K.sub.a) of at least
10.sup.6 l/mole. In a more preferred embodiment the K.sub.a is at
least 10.sup.8 l/mole. In another preferred embodiment the analyte
is a phospholipid. In a most preferred embodiment the phospholipid
is lysophosphatidic acid (LPA).
[0033] In another embodiment, the invention provides an optical
fiber, the fiber being shaped and adapted to provide a substrate
surface for the chemical sensor. The fiber has a proximal end and a
distal end. In one embodiment, the fiber is shaped having a D-shape
cross-section; in another embodiment the distal end of the fiber is
tapered to provide a large substrate surface. In a more preferred
embodiment the fiber has at least two substrate surfaces.
[0034] In a yet additional embodiment, the invention provides an
optical communications device comprising a fiber, a plurality of
particles, each particle comprising: a core, a shell having at
least one surface and having contact with the core and wherein the
shell comprises a sulfur-oxygen molecular species, and wherein the
particle has been selectively sized using a notch filter and
electromagnetic radiation, the electromagnetic radiation having a
spectral wavelength of between about 350 nm and about 1075 nm.
[0035] In a more preferred embodiment the optical communications
device comprises a fiber, wherein the fiber is selected from the
group consisting of ceramics, glasses, polymers, and metal-polymer
composites. In one preferred embodiment the fiber cross-section is
D-shaped. In another preferred embodiment the chemical sensor is
disposed upon a surface of the fiber.
[0036] The invention also provides a method for synthesizing a
chemical sensor comprising gold nanoparticle aggregates, the method
comprising the steps of (i) providing one volume (1V) of a solution
of 0.1 M HAuCl.sub.4; (ii) diluting the solution of HAuCl.sub.4
with Milli-Q water to a final concentration of between
4.times.10.sup.-4-6.times.10.sup.-4 M HAuCl.sub.4; (iii) combining
the diluted solution of HAuCl.sub.4 with a 0.1 M solution of
Na.sub.2S to a final concentration of HAuCl.sub.4 of between
4.times.10.sup.-5-6.times.10.sup.-5M HAuCl.sub.4; (iv) incubating
the combined solution for about between 60-120 minutes; and (v)
measuring the extended plasmon band of the combined solution until
the near-infra-red (NIR) absorption is at a wavelength longer than
600 nm, thereby synthesizing a chemical sensing particle comprising
gold nanoparticle aggregates. In one alternative, the 0.1 M
solution of Na.sub.2S of step (iii) is substituted with an equal
volume of 0.1M Na.sub.2S.sub.2O.sub.3.
[0037] The invention also provides a method for coating gold
nanoparticle aggregates onto a substrate, the method comprising the
steps of (i) providing the gold nanoparticle aggregates as
disclosed above; (ii) submerging the gold nanoparticle aggregates
in a 5 mM aqueous solution of a tethering molecule, the tethering
molecule selected from the group consisting of
trimethoxy[3-(methylamino)propyl]silane (APS) and
(3-mercaptopropyl)trimethoxy silane (MPS), a compound having a
silane terminus and a thiol or amine terminus, and the like; (iii)
incubating the gold nanoparticle aggregates with the tethering
molecule to allow the gold nanoparticle aggregates to adsorb the
tethering molecules; (iv) providing a substrate, wherein the
substrate is selected from the group consisting of silicon dioxide,
silicon, and the like; (v) sonicating the substrate in contact with
a 2% solution of surfactant, the surfactant selected from the group
consisting of HELLMANEX, ALCONOX, a small molecule alkaline
surfactant, and the like; (vi) sonicating the substrate with 18
m.OMEGA. water; (vii) drying the substrate under nitrogen gas;
(viii) depositing a volume of the adsorbed gold nanoparticle
aggregates and tethering molecules solution onto the surface of the
substrate; (ix) incubating the substrate with the solution for five
seconds; (x) blowing the substrate dry with nitrogen gas; wherein
the incubation time for step (iii) is selected from the group
consisting of 30 minutes, 60 minutes, 90 minutes, and 120 minutes,
the method resulting in gold nanoparticle aggregates coated onto a
substrate.
[0038] The invention also provides a method for creating a chemical
sensor with improved sensitivity, the method comprising the steps
of: (i) providing the chemical sensing particle comprising gold
nanoparticle aggregates as disclosed above; (ii) illuminating the
gold nanoparticle aggregates with an amplified femtosecond beam at
a flux of approximately 0.1 mJ/cm.sup.2 for 1 hour; (iii)
illuminating the gold nanoparticle aggregates using a tunable
picosecond laser, thereby creating a chemical sensor with improved
sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows representative electronic absorption spectra of
gold nanoparticle aggregates in water during the growth and
aggregation process.
[0040] FIG. 2 shows results from a persistent spectral hole burning
experiment using .about.800 nm laser light at 200 .mu.J/pulse.
[0041] FIG. 3 shows an illustration of how narrowing aggregate
size/shape distribution using light is achieved.
[0042] FIG. 4 shows a schematic illustration of how
photoluminescence (PL) of an SQD-Ab conjugate is quenched by an
MNP-Ag conjugate.
[0043] FIG. 5 shows the results of two SDQ quenching experiments:
two SERS spectra of an SQD-Ab (polyclonal donkey anti-goat Ab)
conjugate having been quenched by an MNP-Ag conjugate (pink and
dark red) and one control SERS spectrum of an SQD without a
conjugated Ab (blue).
[0044] FIG. 6 is an illustration of how a Raman amplifier functions
to intensify a photon beam.
[0045] FIG. 7A is an illustration of an exemplary optical fiber
having a substrate surface parallel to the axis of the fiber.
[0046] FIG. 8A illustrates an alternative exemplary optical fiber
having a single distal substrate surface at an angle to the axis of
the fiber.
[0047] FIG. 9 illustrates the path of a photon within the fiber and
shown interacting with a nanoparticle aggregate conjugated compound
on the substrate surface of the fiber and the resulting SERS
photon.
[0048] FIG. 10A illustrates another alternative exemplary optical
fiber having two distal substrate surfaces.
[0049] FIG. 11 illustrates the path of photons within the fiber and
shown interacting with nanoparticle aggregate conjugated compound
on the substrate surfaces of the fiber and the resulting SERS
photons.
[0050] FIG. 12 illustrates the distal end of a fiber positioned in
proximity to the ends of two additional fibers that transmit the
SERS photon to a detector.
[0051] FIG. 13 illustrates the angle of the distal substrate
surface to the longitudinal plane of the fiber.
[0052] FIG. 14 shows a representative UV-visible absorption
spectrum of silver nanoparticles. The absorption towards the 780 nm
region is believed to be sufficient for SERS to occur.
[0053] FIG. 15 shows a Raman spectrum of bulk lysophosphatidic acid
crystals (780 nm excitation, 3 mW power) for 16:0 LPA.
[0054] FIG. 16 shows a Raman spectrum of bulk lysophosphatidic acid
crystals (780 nm excitation, 3 mW power) for 18:0 LPA.
[0055] FIG. 17 shows SERS spectra of one hundred samples of
10.sup.-6 M solutions of 16:0 LPA and 18:0 LPA dried on silver
nanoparticles (780 nm excitation, 3 mW power).
[0056] FIG. 18 shows the SERS region between 1050 cm.sup.-1-1150
cm.sup.-1 for 100.times.10.sup.-6 M solutions of 16:0 LPA and 18:0
LPA to show the distinguishable mode at 1097 cm.sup.-1 and 1101
cm.sup.-1 (780 nm excitation, 3 mW power).
[0057] FIG. 19 illustrates a schematic of the Raman probe with a
D-shaped (or side-polished) fiber coated with SERS substrate on the
flat surface. Only the end segment of the fiber (about 1 cm) that
is polished is shown. The rest of the unpolished fiber is about 0.5
m.
[0058] FIG. 20 illustrates the intensity distribution as light
propagates through the D-shaped fiber covered with a silver film.
Area shown is a cross-section parallel to the polished surface and
through the center of the fiber core (top view). Inset: contour
plot of the intensity distribution in a cross-section perpendicular
to the fiber axis (end view). The semi-circle area is the fiber
core with the cladding outside the half-circle. The thick black
line above the semi-circle represents a 0.1 mm metal particle thin
film and above the film (the top blue rectangle) is the air.
[0059] FIG. 21 illustrates a representative SERS spectra of
rhodamine 6G on silver nanoparticles dried onto a D-shaped fiber
collected with excitation laser incident to fiber surface (A) and
coupled into the fiber (B).
DETAILED DESCRIPTION OF THE INVENTION
[0060] The embodiments disclosed in this document are illustrative
and exemplary and are not meant to limit the invention. Other
embodiments can be utilized and structural changes can be made
without departing from the scope of the claims of the present
invention.
[0061] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a particle" includes a plurality of such particles, and a
reference to "a surface" is a reference to one or more surfaces and
equivalents thereof, and so forth.
1: Unique Properties of Novel Gold Nanoparticle Aggregates
(GNA)
[0062] We have recently discovered that the reaction of chloroauric
acid (HAuCl.sub.4) with sodium sulfide (Na.sub.2S) results in
generation of novel gold nanoparticle aggregates (GNAs). These GNAs
have unique optical and surface properties that are useful for
applications, for example, surface enhanced Raman scattering
(SERS), as a chemical sensor. SERS is a known and powerful
technique for detecting molecules with high sensitivity and
specificity. First, the GNAs we discovered are in aqueous solution
and thereby naturally compatible with biological samples in water.
Second, the GNAs have strong near IR absorption (650-1200 nm) that
is ideal for biological applications due to better tissue
penetration in this spectral region. Third, these GNAs have unique
surface properties due to sulfur species on their surface that not
only causes the aggregation in the first place but also provides a
strong driving force for binding with chemical and biological
molecules with high affinity for sulfur. Some of the potential
applications will be outlined separately in the following
sections.
[0063] The surface properties are undoubtedly due to the component
sulfur and oxygen atoms (such as, but not limited to molecules of
negatively charged S.sub.xO.sub.y, wherein x=1 or 2 and y=1, 2, 3,
or 4).
[0064] The particles can have a size range of about between 60 and
200 nm.
[0065] FIG. 1 shows some representative electronic absorption
spectra of the GNAs in water during the growth process. The
spectrometer used limits the measurements to about 850 nm in the
near IR. The strong near IR band is clearly visible in the
spectra.
2. Optical Control and Manipulation of Distribution of Size/Shape
of Gold Nanoparticle Aggregates and its Application for Optical
Filters
[0066] We have also discovered based on transient absorption and
hole burning studies that the broad near IR absorption of the GNAs
is inhomogeneously broadened due to a distribution of sizes and/or
shapes (see FIG. 2). Since the hole burned is permanent, this
immediately led us to propose the idea of using light (such as, but
not limited to, high power lasers) to narrow the distributions of
size/shape distribution. The idea is outlined in FIG. 3 using a
combination of "white light" and notch filters to convert GNAs
absorbing outside the notch filter covered region into GNAs that
absorb only in the notch filter covered region. A significant
increase in the number of GNAs in the notch filter covered region
through this process is achieved.
[0067] This is useful since for many practical applications, for
example, SERS and optical filters, it is highly desirable to narrow
the distribution of size/shape of nanoparticle aggregates. For SERS
in particular, the incident light has to be on resonance with the
substrate absorption. Only those GNAs that have resonance
absorption of the incident light are expected to be SERS active. It
is thus extremely beneficial to have a narrow size/shape
distribution and thereby narrow optical absorption.
[0068] A white light source with a particular wavelength blocked by
an optical notch filter is used to irradiate a sample of GNA having
a broad distribution of aggregate sizes and shapes. All the
aggregates can be either destroyed and/or converted into the
aggregates that absorb at the blocked wavelength. This can
significantly reduce the size and shape distribution of the
aggregates. Only aggregates that absorb (on resonance) with the
blocked wavelength will remain and such aggregate samples are
stable. This principle can work for many other metal nanoparticle
aggregates such as silver, Pt, and Pd, etc. The size/shape-narrowed
aggregates are useful for many applications such as SERS or can be
used as an optical filter with a fairly narrow bandwidth. The
aggregates can further be patterned onto a solid substrate and used
for any application that can benefit from narrow distributed metal
nanoparticle aggregates.
[0069] FIG. 2 shows the results of a persistent spectral hole
burning experiment using .about.800 nm laser light at 200
.mu.J/pulse. Trace a) is prior to laser irradiation. Trace b) is
after 2.5 hours of hole burning. A hole is clearly seen in trace b)
as well as a growth and shift of the maximum absorbance to bluer
wavelengths. The SERS enhancement has been observed as being at
about one-billion-fold (10.sup.9) that is approximately three to
four orders of magnitude greater than that disclosed in the prior
art. Such an increase in enhancement therefore results in a reagent
with greater sensitivity than other systems.
[0070] One potential application using this optical narrowing
effect is proposed here. By exposing the aggregate solution to
varying wavelengths of light it may be possible to burn away all
but a narrow absorption band anywhere from 650 nm to 950 nm. This
is shown schematically in the left portion of FIG. 3. As shown in
the right portion of FIG. 3, a composition comprising particles
having a narrow range of absorbance is created. By suspending these
particles in glass, a low cost notch filter is produced. Current
notch filters are highly expensive and difficult to produce. With
this technology it is possible to produce good quality notch
filters at very low costs. While these filters might not be as high
quality as current high cost filters, there is a large market for
low cost, low-end filters where high precision is not required. The
market mainly comprises fiber-optical communications devices that
are used to transmit information using photons instead of
electrons. The devices comprise light amplifiers that retain the
intensity of light as it travels through fiber optic networks.
Light amplifiers are used to increase the intensity of weak light
signals as they travel through long distances of fiber optic
networks. The most commonly used form of light amplification is the
Raman amplifier because it has proven to be the most efficient.
This technique increases the frequency of transmitted light signals
within a fiber optic network to prevent a loss of transferred data.
This market has not currently been tapped in notch filters.
3. SERS Detection Applications for Sensing and Imaging
[0071] Raman spectroscopy is a powerful optical technique for
detecting and analyzing molecules. Its principle is based on
detecting light scattered off a molecule that is shifted in energy
with respect to the incident light. The shift, called Raman shift,
is characteristic of individual molecules, reflecting their
vibrational frequencies that are like figure prints of molecules.
As a result, the key advantage of Raman spectroscopy is its
molecular specificity while its main limitation is the small signal
due to low quantum yield of Raman scattering. One way to enhance
the Raman signal is to tune the excitation wavelength to be on
resonance with an electronic transition, so called resonance Raman
scattering. This can usually produce an enhancement on the order of
10.sup.2-10.sup.3. Another technique to enhance Raman scattering is
surface enhancement by roughened metal surfaces, notably silver and
gold, that provides an enhancement factor on the order of
10.sup.6-10.sup.8. Similar or somewhat larger enhancement factors
(.about.10.sup.8-10.sup.10) have been observed for metal, mostly
silver, nanoparticles.
[0072] In the last few years, it has been shown that an even larger
enhancement (.about.10.sup.10-10.sup.15) is possible for aggregates
of metal nanoparticles, for example, comprising silver and/or gold.
The largest enhancement factor of 10.sup.14-10.sup.15 has been
reported for rhodamine 6G (R6G) on single silver nanoparticle
aggregates. This huge enhancement is thought to be mainly due to
significant enhancement of the local electromagnetic fields of the
nanoparticle aggregates that absorb strongly the incident
excitation light for the Raman scattering process. With such large
enhancement, many important molecules that are difficult to detect
with Raman normally can now be easily detected. This provides many
interesting and new opportunities for detecting and analyzing
molecules using SERS with extremely high sensitivity and molecular
specificity.
[0073] Of emphasis are the unique surface properties of the
aggregates. It is these surface properties that make the gold
nanoparticles aggregate in the first place and make the aggregates
useful for SERS as a substrate with desirable properties.
[0074] Given the particular surface and optical features of the
GNAs we have found, they can be suitable for SERS detection and
analysis of a large number of molecules, including, but not limited
to, proteins, DNA, explosives, chemical and biological warfare
agents, toxins, and even virus and biological cells. We have
demonstrated that the GNAs are SERS active for amino acids and DNA
bases as well as antibodies for cancer detection. As discussed in
Section 2 above, the possibility of narrowing the optical
absorption of the GNAs for SERS is an important added
advantage.
[0075] SERS can also be developed into a molecular imaging
technique for biomedical and other applications. Exciting Raman
imaging equipment may be usable for SERS imaging. SERS can provide
an enhanced signal and thereby significantly shortened data
acquisition time, making the technique practically useful for
medical or other commercial and industrial applications including,
but not limited to, chip inspection or chemical monitoring.
4. Antigen/Antibody Detection with Metal and Semiconducting
Nanoparticles
[0076] Fluorescent nanoparticles (semiconductor quantum dots, SQDs)
have been used recently as fluorescent biological markers and have
been found to be extremely effective. They offer advantages
including higher stability, stronger fluorescence, tunability of
color, and possibility of optical encoding based on different sized
or colored SQDs.
[0077] GNAs of the invention can be used to detect an analyte. Such
an analyte can be, for example, but not limited to, an antigen, an
antibody, a biochemical metabolite, an organic compound, a compound
or element having biological activity, or the like.
[0078] For example, the GNAs of the invention can be used in a
novel dual optical scheme for sensitive and selective detection of
antigens and is illustrated in FIG. 4. The technique is based on
detection of photoluminescence from SQDs with antibody (Ab)
attached and SERS (surface enhanced Raman scattering) spectra of
antigen (Ag) attached to the GNAs that comprise metal nanoparticles
(MNP). SERS spectrum is a measure of the Raman spectrum of the Ag
that can be significantly enhanced by a metal nanoparticle. The
Raman spectrum, similar to an infra-red (IR) spectrum, is
characteristic of specific molecules due to the unique set of
vibrational frequencies of each molecule. Before the Ag and Ab
interact or bind to one another, we expect strong photoluminescence
(PL) from the SQD and a well-defined SERS spectrum. Upon binding of
Ab with the complementary Ag, two important consequences can occur.
First, the PL from the SQD will be significantly, if not
completely, quenched by the GNA-Ab complex. Second, the SERS
spectrum of the Ag will change with small but noticeable frequency
shift and/or relative spectral intensity changes due to Ag-Ab
interaction. It is well known that PL from fluorophores can be
significantly quenched when brought near a metal surface (bulk or
nanostructured). The distance at which quenching occurs is a known
parameter and is of the order of between about 1-2 nm for effective
quenching. The distance will be dependent on the sizes of the SQD,
GNA, Ag, and Ab, the length of a linker molecule (LM) between the
SQD and the Ag (or alternatively, between the SQD and the Ab,
between the GNA and Ab, or between the GNA and Ag), as well as on
their relative binding configuration. We can estimate at this point
that a certain favorable configuration and size will allow
quenching. This is supported by a recent report from Wang et al.
that PL quenching of a small, green-emitting QD with antibody
attached by large, red-emitting QD with antigen attached occurs
when the Ag and Ab interact (Wang et al. (2002) NanoLett., 2:
817-822). This experiment demonstrates that the distance can be
close enough for effective PL quenching due to resonance energy
transfer from the large QD to the smaller QD.
[0079] As for the SERS aspect, we have very recently demonstrated
that we can detect the SERS spectrum of a polyclonal Ab attached to
gold nanoparticles through electrostatic interaction. FIG. 5 shows
two representative SERS spectra where the reproducible peaks can be
attributed to the Ab. To our best knowledge, this is the first
demonstration of SERS detection of Ab. Since Raman or SERS spectrum
is extremely sensitive to the structure, configuration and
environment of a molecule, we anticipate that the SERS spectrum of
an Ab can change upon interaction with its Ag.
[0080] The proposed scheme offers a novel technique for detecting
antigen with high sensitivity (offered by both PL and SERS) and
specificity (offered by both SERS and Ag-Ab interaction). In a
typical experiment, we choose a laser wavelength that is on
resonance with absorption of the QD (usually in the near UV and
visible region) for measuring PL from the SQD with Ab attached,
with and without binding to Ag attached to GNA. We then choose a
laser wavelength that is off-resonance for the SQD absorption and
produces no PL for SERS measurement (usually near IR) of the Ag
attached to GNA, with and without binding with Ab attached to SQD.
Comparing the two situations with and without Ag-Ab binding, we can
see PL quenching of the SQD and SERS spectral change upon binding.
Since the PL quenching of the SQD by a GNA is expected to be much
more effective than a large SQD, the PL can be completely quenched
in the situation with Ag-Ab binding. This is thus a zero-background
experiment and can be much more sensitive than conventional PL
detection that is typically not zero-background. For SERS, since
the enhancement can be as high as 10.sup.9, it is almost as
sensitive as fluorescence but has the extremely important advantage
of direct molecular specificity between the Ag and the Ab.
[0081] In another example, the Ag can be replaced by a second Ab,
the second Ab being specific for binding the first Ab. The second
antibody can be from the same animal species as the first Ab, or
can be from another animal species. Such first and second Abs are
well known to those in the art and can be raised in and isolated
from an animal such as, but not limited to, a rabbit, a human, a
mouse, a rat, a monkey, an ape, a goat, a sheep, a cow, a pig, a
donkey, a horse, a guinea pig, a whale, a wombat, a platypus, or
the like.
[0082] SERS is also useful for detecting other cancer biomarkers
that can interact or bind to the GNA surface. For example, Sutphen
et al. have recently shown that lysophospholipids (LPL) are
potential biomarkers of ovarian cancer (Sutphen et al. (2004)
Cancer Epidemiol Biomarker Prev., 13: 1185-1191). Based on the
molecular structure of LPL molecules, a favorable interaction
between LPL molecules with GNA through electrostatic interaction
can occur at the appropriate pH. In the case of the SERS experiment
using a polyclonal Ab shown in FIG. 5, the strongest interaction
with GNA occurs at the isoelectrostatic pH, i.e. pH at which the
GNA has equal number of positive and negative charges. The pH is
varied to adjust the charge on the GNA to determine the optimal pH
or charge for strong interaction with LPL.
[0083] By conjugating fluorescent nanoparticle QDs to antigens and
mixing the Ag-QD conjugate with a GNA-Ab composition, quenching of
fluorescence upon binding of the antigen/antibody pair can be
observed. The Ag and/or the Ab can be conjugated to the QD or GNA
using a linker molecule (LM). A decrease in fluorescence can
indicate the presence of the antibody for that particular antigen
to which the fluorescing QDs have been attached. Depending on which
antigen is utilized a wide array of antibodies can be detected.
This can allow for the rapid detection of cancers or diseases that
currently can take days or weeks to diagnose. Likewise, the scheme
can work as well if antibody is attached to a fluorescent QD and
the respective antigen to a metal nanoparticle. Metal particles
have no florescence with visible excitation. The fluorescence
quenching by metal nanoparticles can be more effective than
quenching by larger QDs. This approach is sensitive and specific.
The distance between the metal nanoparticle and QD is important for
this to work (for example, the distance can be less than 2 nm). The
interaction between the two components can be adjusted to achieve
the maximum quenching effect.
5. Detection of Tumor Markers
[0084] Surface-enhanced Raman scattering using silver nanoparticles
was applied to detect various forms of lysophosphatidic acid (LPA)
to examine its potential application as an alternative to current
detection methods of LPA as biomarkers of ovarian cancer.
Enhancement of the Raman modes of the molecule, especially those
related to the acyl chain within the 800-1300 cm.sup.-1 region, was
observed. In particular, the C--C vibration mode of the
gauche-bonded chain around 1100 cm.sup.-1 was enhanced to allow the
discrimination of two similar LPA molecules. Given the molecular
selectivity of this technique, the detection of LPA using SERS may
eliminate the need for partial purification of samples prior to
analysis in cancer screening.
[0085] Lysophosphatidic acid (LPA), originally known for its role
as an intermediate in intracellular lipid metabolism, has now been
recognized as an important multifunctional biological mediator that
can elicit cellular responses including mitogenic and antimitogenic
effects on the cell cycle, actin skeleton regulation, and cellular
motility (see Tigyi et al. (1994) Proc. Nat. Acad. Sci. 91:
1908-1912; van Corven et al. (1989) Cell 59: 45-54; Ridley and Hall
(1992) Cell 70: 389-399; and Zhou et al (1995) J. Biol. Chem. 270:
25549-25556). The involvement of LPA in inducing cell
proliferation, migration and survival implicates it in the
initiation and progression of malignant disease, and has been
proposed as a sensitive biomarker for ovarian cancer (see Xu et al
(1998) JAMA 280: 719-723; Mills and Moolenaar (2003) Nature Reviews
3: 582-591; Fang et al (2004) J. Biol. Chem. 279: 9653-9661; and
Sutphen et al (2004) Cancer Epidemiol. Biomark. Prev. 13:
1185-1191).
[0086] Typically, the detection of LPA has been conducted using
chromatography and mass spectroscopy assays that require a partial
purification of the samples using thin layer chromatography (TLC)
prior to analysis. Although this method is effective, an
underestimation of LPA concentration can result during the recovery
process due in part to the varying mobility of the LPA salts (free
acid, sodium and calcium salts) when subjected to chromatography by
TLC. The low stability of LPA also calls for fast and sensitive
detection techniques.
[0087] A powerful optical detection technique based on
surface-enhanced Raman scattering (SERS) offers a unique
combination of high sensitivity and molecular specificity. With
SERS, the Raman signal of a molecule is increased by many orders of
magnitude as a result of strong enhancement of the excitation light
through the resonance of the metal's surface electrons called the
surface plasmon (see Moskovitz (1985) Rev. Modern Physics 57:
783-828; Otto et al. (1992) J. Phys. Condense Matter 4: 1143-1212;
and Campion and Kambhampati (1998) Chem. Soc. Rev. 27: 241-250).
SERS has been successfully used in the detection and analysis of a
large number of chemicals and biological molecules (see Albrecht
and Creighton (1977) J. Am. Chem. Soc. 99: 5215-5217; Nie and Emory
(1997) Science 275: 1102-1106; Keating et al. (1998) J. Phys. Chem.
B 102: 9414-9425; Kneipp et al (1998) Phys. Rev. E 57: R6281-R6284;
and Schwartzberg et al. (2004) J. Phys. Chem. B 108:
19191-19197).
6. SERS Application for Detection and Analysis of Semiconductor
Nanoparticles
[0088] Another application of SERS based on the gold nanoparticle
system is for measuring Raman spectrum of semiconductor
nanoparticles (QDs). Similar to molecules, normal Raman signals are
very small and thus Raman spectrum is challenging to measure. SERS
as an enhanced Raman technique for measuring Raman for
semiconductor nanoparticles have not been reported before. The
surface chemistry of the metal nanoparticles and the semiconductor
QDs must be compatible for this to work. The sulfur species on the
surface of the GNAs are ideal for II-VI SQDs to bind, enabling SERS
detection of the SQDs. This provides a powerful method for
detecting and analyzing semiconductor nanoparticles.
7. SERS for Raman Amplifier in Optical Communications
[0089] Raman amplifiers have been used to amplify signal in optical
communications (see, for example, FIG. 6). SERS can provide more
amplification than normal Raman amplifiers. By doping MNPs, for
example GNAs, into glass or polymer fibers, Raman scattering from
the glass or polymer matrix can be used to amplify optical signal
with the proper wavelength.
8. Synthesis of Biological Molecules
Chemical Synthesis of Peptides
[0090] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker molecule. A linker
molecule such as methylamine-derivatized polyethylene glycol is
attached to poly(styrene-co-divinylbenzene) to form the support
resin. The amino acid residues are N-.alpha.-protected by acid
labile Boc (t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin.
[0091] Trifluoroacetic acid or piperidine are used to remove the
protecting group in the case of Boc or Fmoc, respectively. Each
additional amino acid is added to the anchored residue using a
coupling agent or pre-activated amino acid derivative, and the
resin is washed. The full-length peptide is synthesized by
sequential deprotection, coupling of derivatized amino acids, and
washing with dichloromethane and/or N,N-dimethylformamide. The
peptide is cleaved between the peptide carboxy terminus and the
linker group to yield a peptide acid or amide. These processes are
described in the Novabiochem 1997/98 Catalog and Peptide Synthesis
Handbook (San Diego Calif. pp. S1-S20). Automated synthesis may
also be carried out on machines such as the ABI 431A peptide
synthesizer (ABI). A protein or portion thereof may be purified by
preparative high performance liquid chromatography and its
composition confirmed by amino acid analysis or by sequencing
(Creighton (1984) Proteins, Structures and Molecular Properties, W
H Freeman, New York N.Y.).
[0092] In particular, a purified antigen may be used to produce
antibodies or to screen libraries of pharmaceutical agents to
identify those that specifically bind an antigen. Antibodies to an
antigen may also be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies (i.e., those which inhibit dimer formation)
are especially preferred for therapeutic use.
[0093] For the production of polyclonal antibodies, various hosts
including goats, rabbits, rats, mice, humans, and others may be
immunized by injection with an antigen or with any fragment or
oligopeptide thereof that has immunogenic properties. Rats and mice
are preferred hosts for downstream applications involving
monoclonal antibody production. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface-active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable. (For review of
methods for antibody production and analysis, see, for example,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.)
[0094] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to an antigen have an amino
acid sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 14 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of antigen amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
Antibodies
[0095] Monoclonal antibodies to an antigen may be prepared using
any technique that provides for the production of antibody
molecules by continuous cell lines in culture. These include, but
are not limited to, the hybridoma technique, the human B-cell
hybridoma technique, and the EBV-hybridoma technique. (See, for
example, Kohler et al. (1975) Nature 256: 495-497; Kozbor et al.
(1985) J. Immunol. Methods 81: 31-42; Cote et al. (1983) Proc.
Natl. Acad. Sci. 80: 2026-2030; and Cole et al. (1984) Mol. Cell
Biol. 62: 109-120.)
[0096] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See, for
example, Morrison et al. (1984) Proc. Natl. Acad. Sci. 81:
6851-6855; Neuberger et al. (1984) Nature 312: 604-608; and Takeda
et al. (1985) Nature 314: 452-454.) Alternatively, techniques
described for the production of single chain antibodies may be
adapted, using methods known in the art, to produce
antigen-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, for example, Burton (1991) Proc.
Natl. Acad. Sci. 88: 10134-10137.)
[0097] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, for example, Orlandi
et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter et
al. (1991) Nature 349: 293-299.)
[0098] Antibody fragments that contain specific binding sites for
an antigen may also be generated. For example, such fragments
include, but are not limited to, F(ab')2 fragments produced by
pepsin digestion of the antibody molecule and Fab fragments
generated by reducing the disulfide bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity. (See, for example, Huse
et al. (1989) Science 246: 1275-1281.)
[0099] Various immunoassays may be used for screening to identify
antibodies having the desired specificity and minimal
cross-reactivity. Numerous protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal
antibodies with established specificities are well known in the
art. Such immunoassays typically involve the measurement of complex
formation between an antigen and its specific antibody. A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering antigen epitopes is preferred, but
a competitive binding assay may also be employed. (Maddox,
supra.)
[0100] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for an antigen. Affinity is expressed as an
association constant, K.sub.a, which is defined as the molar
concentration of antigen-antibody complex divided by the molar
concentrations of free antigen and free antibody under equilibrium
conditions. The K.sub.a determined for a preparation of polyclonal
antibodies, which are heterogeneous in their affinities for
multiple antigen epitopes, represents the average affinity, or
avidity, of the antibodies for an antigen. The K.sub.a determined
for a preparation of monoclonal antibodies, which are monospecific
for a particular antigen epitope, represents a true measure of
affinity. High-affinity antibody preparations with K.sub.a ranging
from about 10.sup.9 to 10.sup.12 l/mole are preferred for use in
immunoassays in which the antigen-antibody complex must withstand
rigorous manipulations. Low-affinity antibody preparations with
K.sub.a ranging from about 10.sup.6 to 10.sup.7 l/mole are
preferred for use in immunopurification and similar procedures
which ultimately require dissociation of antigen, preferably in
active form, from the antibody. (See Catty (1988) Antibodies,
Volume I: A Practical Approach, IRL Press, Washington, D. C.; and
Liddell and Cryer (1991) A Practical Guide to Monoclonal
Antibodies, John Wiley & Sons, New York, N.Y.)
[0101] The titre and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody.ml.sup.-1, preferably 5-10 mg specific
antibody.ml.sup.-1, is preferred for use in procedures requiring
precipitation of antigen-antibody complexes. Procedures for
evaluating antibody specificity, titer, and avidity, and guidelines
for antibody quality and usage in various applications, are
generally available. (See, for example, Catty, supra, and Coligan
et al. supra.)
Preparation and Screening of Antibodies
[0102] Various hosts including, but not limited to, goats, rabbits,
rats, mice, and human cell lines may be immunized by injection with
antigen or any portion thereof. Adjuvants such as Freund's, mineral
gels, and surface-active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and
dinitrophenol may be used to increase immunological response. The
oligopeptide, peptide, or portion of protein used to induce
antibodies should consist of at least about five amino acids, more
preferably ten amino acids, which are identical to a portion of the
natural protein. Oligopeptides may be fused with proteins such as
KLH in order to produce antibodies to the chimeric molecule.
[0103] Monoclonal antibodies may be prepared using any technique
that provides for the production of antibodies by continuous cell
lines in culture. These include, but are not limited to, the
hybridoma technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique. (See, for example, Kohler et al. (1975)
Nature 256:495-497; Kozbor et al. (1985) J. Immunol. Methods
81:31-42; Cote et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030;
and Cole et al. (1984) Mol. Cell. Biol. 62: 109-120.)
[0104] Alternatively, techniques described for antibody production
may be adapted, using methods known in the art, to produce
epitope-specific, single chain antibodies. Antibody fragments that
contain specific binding sites for epitopes of the protein may also
be generated. For example, such fragments include, but are not
limited to, F(ab')2 fragments produced by pepsin digestion of the
antibody molecule and Fab fragments generated by reducing the
disulfide bridges of the F(ab)2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (See, for example, Huse et al. (1989) Science 246:
1275-1281.)
[0105] The antigen, or a portion thereof, may be used in screening
assays of phagemid or B-lymphocyte immunoglobulin libraries to
identify antibodies having the desired specificity. Numerous
protocols for competitive binding or immunoassays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between the protein and its
specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering
epitopes is preferred, but a competitive binding assay may also be
employed (Pound (1998) Immunochemical Protocols, Humana Press,
Totowa N.J.).
[0106] In the alternative, an antibody can be substituted by, for
example, a chimeric protein that comprises a portion or fragment of
a T-cell receptor (TCR). TCRs have an immunoglobulin domain that
binds a cell-surface antigen comprising a host or a non-host
molecule. Such molecules can be of viral origin or can be a
particular cancer marker protein. The chimeric protein can also
comprise a soluble protein (i.e. present in a bodily fluid or the
cell cytoplasm) or a cell membrane-associated protein (such as a
ligand receptor, an ion channel, or a molecule involved in signal
transduction.
[0107] Metal nanoparticles are currently studied for a wide variety
of biomedical applications including contrast imaging, ultrasonic
imaging, thermal destruction of specific cancer cells, and laser
tissue welding. All applications of this type rely on the optical
and physical properties associated with metal nanoparticles,
nominally of gold. Much of this work has focused on gold nanoshells
due to their near IR optical absorption where tissue transmission
is at its peak, making in-vivo applications feasible. This gold
nanoparticle aggregate system possesses these same optical features
with multiple advantages. While nanoshells can be tuned to absorb
in a particular region, their absorption is inhomogenously broad
and cannot be narrowed without significant purification. Therefore
a significant percentage of nanoshells will be functionally useless
at a given wavelength. Gold aggregates on the other hand can be
tuned to have a very narrow absorption through the optical hole
burning technique. With the absorption tuned to a given wavelength
all aggregates will be utilized making them significantly more
efficient for any of the above applications.
[0108] One of the most exciting of these applications is thermal
destruction of cancer cells. The nanoparticle aggregates are
selectively attached to cancer cells in a tumor by a passive
mechanism that has been termed an "enhanced permeability and
retention effect". The tumor mass is then illuminated with near IR
laser light which passes harmlessly through the tissue, but is
absorbed strongly by the aggregates, causing them to heat
drastically, killing only the cancerous cells. (See O'Neal et al.
(2004) Cancer Lett. 209: 171-176, herein incorporated by reference
in its entirety.) This technology has been utilized with
gold-silica nanoshells further comprising "stealthing" polymers,
such as poly(ethyleneglycol) and derives thereof, or liposomes;
however this can be done better with gold nanoparticle aggregates
of the present invention.
9. Detection of Specific Compounds Using Optical Fibers
[0109] The nanoparticle aggregates can be used to detect specific
compounds that may be at very low levels in a sample. Such a sample
can be blood, urine, saliva, lung lavage, gastric fluid, lymphatic
fluid, any other body fluid, or the like. In addition, the sample
can be a sample of water or other aqueous medium, such as water
from a spring, a stream, a river, a pond, a lake, a sea, or an
ocean. The sample can be a geological sample such as from a
geothermal spring, a lava evaporate or exudate, a hydrocarbon, or
from an abyssal trench; a plant sample such as from the xylem or
phloem of a stalk or trunk; a sample from a fluid in a man-made
structure such as concrete, cement, aggregate, or the like; a
sample of fluid from a piece of machinery such as an engine, motor,
compressor, or the like.
[0110] The nanoparticle aggregates can be conjugated with antibody,
the antibody having been synthesized to bind a specific compound.
Such a specific compound can be a protein, a fatty acid, a
carbohydrate, an organic compound based upon a benzene ring
structure, an organic compound based upon a short chain
hydrocarbon, a medium chain hydrocarbon or a long chain
hydrocarbon. The specific compound can be modified with a reactive
group. Such reactive groups are well known to those of skill in the
art and can include phosphate groups, methyl groups, hydroxyl
groups, sulphate groups, acetyl groups, or the like.
[0111] The nanoparticle aggregate-antibody conjugate complex has an
altered SERS profile when the specific compound binds to the
complex. The complex can be applied and incorporated onto a
substrate surface of an optical fiber as disclosed herein. The
exterior surface of the fiber comprises a compound that reflects
photons. Such compounds are well known to those in the fiberoptic
arts. The optical fiber has a proximal end or proximal section and
a distal end or distal section. The distal end of a conventional
fiber has a circular cross-section. A modified substrate surface
can be created to create a substrate surface having an area larger
than that of the distal end. The substrate surface of the fiber is
created by removing a distal portion of the fiber section thereby
creating a fiber with a distal end cross-section that is different
from the distal end of the original fiber. The cross-section can be
D-shaped, diamond-shaped, triangular, oval, or another
non-symmetrical shape. Removing a portion of the fiber results in
substrate surface with a larger surface area than the surface area
of the original end of the fiber.
[0112] The resulting substrate surface can have a surface area that
is up to at least about 8,000-fold larger than the distal end
surface of the original fiber. The diameter of the fiber can be
from between about 0.01 .mu.m to about 10 .mu.m. In one
alternative, the diameter is from between about 0.1 .mu.m to about
1 .mu.m. In another alternative, the diameter is between about 0.2
.mu.m to about 8 .mu.m.
[0113] The complex is applied and incorporated onto the substrate
surface and light is directed longitudinally through the fiber. The
light can be coherent and/or non-coherent. The light interacts with
the nanoparticle aggregate-antibody conjugate complex and a
resulting SERS profile can be compared with a SERS profile from the
nanoparticle aggregate-antibody conjugate complex that is bound
with a known amount of specific compound. The SERS radiation is
detected using a photon detector suitably disposed to detect the
SERS radiation. The detector can be disposed at or near the
substrate surface of the fiber at the distal end or distal section
of the fiber, at or near the proximal end or proximal section of
the fiber, or at another position as disclosed herein.
[0114] The fiber can have one or more such substrate surfaces. In
the case of two substrate surfaces, the second substrate surface
can reflect the SERS signal from the first substrate surface to the
detector longitudinally along the length of the fiber, resulting in
a markedly improved amplification of the SERS signal. Similarly,
the first substrate surface can reflect a SERS signal from the
second substrate surface to the detector.
[0115] In another alternative, at least one additional optical
fiber can be positioned in proximity to the distal end or distal
section of the fiber. The end of the additional fiber can have the
same shape as the shape of the distal end or distal section of the
fiber, such that SERS radiation emitted from the fiber is conducted
through the additional fiber to a detector. Two additional fibers
can be used in parallel where there are two new substrate surface s
on the fiber.
[0116] The optical fiber can additionally have a non-uniform
diameter, for example, the distal end having a cross-section
perpendicular to the longitudinal plane that is larger in magnitude
than a cross-section of the proximal end. Such a shape can further
increase the amount of SERS radiation produced by a photon
source.
[0117] The optical fiber can be made using glass, ceramics, or the
like; or a polymeric compound such as cyclic olefin polymer (COP),
polysulfone (for example, UDEL and RADEL resins), fluorinated
terpolymers (such as those synthesized from tertafluoroethylene,
hexafluoropropylene, and vinylidene fluoride), polycarbonate,
polyacrylate, polystryrene, or the like.
[0118] As illustrated in FIG. 13, the surface of the substrate
surface (2) of the fiber (1) is at an angle .theta. (7) to the
longitudinal plane (8) of the fiber. The angle .theta. is between
15.degree. and 75.degree.; preferably between 15.degree. and
65.degree.; more preferably between 15.degree. and 45.degree.; and
most preferably between 15.degree. and 35.degree.. In one example,
the angle .theta. is 22.5.degree.. The optimal preferable angle can
be determined empirically to find the optimal angle for
back-scattering of the SERS radiation.
[0119] One of skill in the art can readily determine which angle
.theta. is optimal using data collected from experimentation as
described above and knowledge of the composition of the fiber. It
is well known in the art that different compositions have different
refractive indices and one of skill in that art would know that
different compositions will have a particular optimal refractive
index. One of skill in the art would also know that it is not
always necessary to create a fiber having a distal end section
having a substrate surface at the optimal angle .theta. to the
plane of the fiber since the nanoparticle aggregate-antibody
conjugate complex can have different effects upon the refractivity
of a fiber compound. It would require relatively little
experimentation by one of skill in the art to determine the optimal
preferable angle .theta..
[0120] The SERS radiation can be further enhanced approximately
4-5-fold if an electrical field of a few Volts per centimeter
(V/cm) is applied across the fiber, approximately perpendicular to
the substrate surface. The potential difference can be maintained
through an electrically conducting solution. The electrically
conducting solution can be aqueous or non-aqueous but should not
quench SERS radiation to the extent that the SERS enhancement due
to the electrical field is quenched by the electrically conducting
solution.
[0121] Examples of different optical fiber ends are illustrated in
FIGS. 7 through 12. FIG. 7A illustrates a cylindrical fiber (1)
having an exterior surface (3) and having a longitudinal portion
removed from the distal section of the fiber thereby creating a
substrate surface (2). FIG. 7B shows a cross-section of the distal
section.
[0122] FIG. 8A illustrates an alternative distal section whereby a
longitudinal portion is removed from the fiber at an included plane
to the fiber resulting in a tapered distal end of the fiber. FIGS.
8B, 8C, and 8D illustrate three cross-sections of the distal
section at different positions along the length of the fiber. FIG.
9 illustrates the path of a photon (4) from a photon source and the
path of a SERS photon (5) from the substrate surface of the fiber
to a detector (6).
[0123] FIG. 10A illustrates another alternative distal section
whereby two longitudinal portions are removed from the fiber
resulting in a tapered distal end of the fiber. FIGS. 10B, 10C, and
10D illustrate three cross-sections of the distal section at
different positions along the length of the fiber. FIG. 11
illustrates the path of a photon (4) from the photon source and the
path of a SERS photon (5) from the substrate surface (2) of the
fiber to a detector (6).
[0124] FIG. 12 illustrates the distal end of a fiber positioned in
proximity to the ends of two additional fibers that transmit the
SERS photon to a detector. FIG. 13 illustrates the angle between
the substrate surface (2) and the longitudinal plane (8) or axis of
the fiber.
[0125] The nanoparticle aggregates can be formed and shaped into a
desired shape, such as a sphere, a cylinder, a rod, a cone, a
pyramid, or other shape, not limited to regular shapes, and
deposited upon a substrate at a desired density using means well
known t to hose of skill in the art. (See, for example, Fan et al.
(2005) J. Vac. Sci. Technol. 8: 947-953; Chaney et al. (2005) Appl.
Phys. Lett. 87: pub. no. 031908.)
EXAMPLES
[0126] The invention will be more readily understood by reference
to the following examples, which are included merely for purposes
of illustration of certain aspects and embodiments of the present
invention and not as limitations.
Example I
Synthesis of Gold Nanoparticle Aggregates
[0127] The synthesis of the gold nanoparticle aggregates (GNAs) was
performed as follows: 400-600 ul of a 0.02 M HAuCl.sub.4 stock
solution was diluted to 4.times.10.sup.-4-6.times.10.sup.-4 M with
Milli-Q water in glassware cleaned in aquaregia and rinsed with
Milli-Q water to avoid contamination. To this, 40-60 .mu.l of a 0.1
M solution of Na.sub.2S that has been aged for 2-3 months was
added. After approximately 60-120 minutes, the color changed from a
straw yellow to deep purple with the extended plasmon band (EPB)
growing in between 600-1000 nm, indicating reaction completion. The
aggregate formation was signified by strong near-infra-red (NIR)
absorption at wavelengths longer than 600 nm. This reaction is also
performed with sodium thiosulfate (Na.sub.2S.sub.2O.sub.3) by
replacing 1:1 the sodium sulfide solution, however, while these
particles are optically identical to those that are sodium sulfide
generated, they work poorly for SERS and should only be used for
non detection applications. HAuCl.sub.4, Na.sub.2S,
Na.sub.2S.sub.2O.sub.3 and were obtained from Sigma-Aldrich (St.
Louis Mo.) at the highest level of purity available.
Example II
Coating Gold Nanoparticle Aggregates onto Substrates/Fiber
[0128] The coating of gold nanoparticle aggregates onto a
substrate/fiber was performed in two ways. First, by spin coating
or drop casting a dilute solution of the aggregates onto the
substrate. This yields a relatively thick film, however, it is not
as stable as is necessary for many applications. The second method
utilizes a tethering molecule, in this case
trimethoxy[3-(methylamino)propyl]silane (APS). The substrate was
cleaned prior to the silanization step by sonication in 2% solution
of HELLMANEX or other surfactant, followed by 18 m.OMEGA.
water.
[0129] The gold nanoparticle aggregates were then submerged in a 5
mM aqueous solution of APS to deposit the tethering molecules.
After 1-2 minutes the substrate was rinsed with water, dried under
nitrogen, and 40 .mu.l of the aggregate solution was placed on the
surface. After several seconds exposed to the solution, it was
blown dry with nitrogen. This provided a significantly thinner film
than those made by spin or drop casting, however, it was extremely
stable and robust under use. This film was made to near monolayer
coverage by increasing the substrate exposure time to the APS and
aggregate solutions to approximately 1 hour. APS was obtained from
Sigma-Aldrich.
Example III
Band Narrowing in Gold Nanoparticle Aggregates
[0130] The extended plasmon band of the gold nanoparticle
aggregates were tuned and narrowed via a regeneratively amplified,
mode locked femtosecond Ti-sapphire laser system. This was done by
illuminating the sample with the amplified femtosecond beam at a
flux of approximately 0.1 mJ/cm.sup.2. After approximately 1 hour,
a deep, broad hole was burned in the absorption spectrum at near
800 nm while absorption to the blue drastically increased.
[0131] In order to tune the band to one particular wavelength a
tunable picosecond laser is required. Due to the broad spectral
linewidth of the femtosecond pulses it is impossible to completely
narrow the extended plasmon band (EPB). By using the spectrally
narrow picosecond pulses it is possible to selectively destroy the
EPB to the blue and red of the desired excitation wavelength,
enhancing the absorption more than two fold.
Example IV
Single Particle SERS/Luminescence and Bulk SERS
[0132] Samples for single particle experiments were prepared by
immobilizing the GNAs or semiconductor quantum dots (SQD) on glass
coverslips with trimethoxy[3-(methylamino)propyl]silane (APS).
Coverslips were cleaned prior to the silanization step by
sonication in 2% solution of Hellmanex, followed by 18 M.OMEGA.
water. They were then submerged in 5 mM aqueous solution of APS to
deposit the tethering molecules. After 1-2 minutes the coverslips
were rinsed with water, dried under nitrogen, and 40 .mu.l of the
aggregate or SQD solution was placed on one surface. After several
seconds exposed to the solution, it was blown dry with
nitrogen.
[0133] Single particle experiments were performed on a custom
designed confocal microscope built onto an inverted fluorescence
microscope (Axiovert 100, Carl Zeiss, Inc., Thornwood N.Y.). A
helium-neon or Argon ion laser depending on desired excitation
wavelength was coupled into the back port of the microscope and
directed into a high numerical aperture objective (Apochromat
100.times., 1.4 NA) that focused the light onto the sample surface.
The sample was then raster scanned across the focused laser to
generate an image using a commercially available piezoelectric
scanner (Physik Instrumente, Auburn Mass.) and control electronics
(Digital Instruments (Veeco Instruments Inc.) Woodbury N.Y.). The
Raman scattered light or fluorescence was collected with the same
objective used for excitation and focused onto a confocal aperture.
The Rayleigh scattered light was then removed using a holographic
notch filter (Kaiser Optical Systems, Inc., Ann Arbor Mich.) and
the remaining scattered light was focused onto an avalanche
photodiode (EG&G (ClTY STATE)). Once a nanoparticle aggregate
or QD was located, it was centered on the focused laser and the
Raman scattering was directed into a spectrograph (Acton
Instruments, Acton Mass.) that dispersed the light onto a liquid
nitrogen cooled CCD camera (Princeton Instruments, Trenton N.J.).
Typically six spectra (30 seconds each) were averaged. Bulk SERS
experiments were performed on a Renishaw MICRORAMAN instrument
(Renishaw Plc, Wotton-under-Edge, GL12 8JR, United Kingdom) with a
783 nm diode excitation laser. A drop of sample was placed on a
quartz substrate and the laser was focused into the solution.
Typically 4 spectra (30 seconds) were averaged.
Example V
Surface-Enhanced Raman Scattering Detection of Lysophosphatidic
Acid
[0134] Lysophosphatidic acid (LPA), originally known for its role
as an intermediate in intracellular lipid metabolism, has now been
recognized as an important multifunctional biological mediator that
can elicit cellular responses including mitogenic and antimitogenic
effects on the cell cycle, actin skeleton regulation, and cellular
motility. The involvement of LPA in inducing cell proliferation,
migration and survival implicates it in the initiation and
progression of malignant disease, and has been proposed as a
sensitive biomarker for ovarian cancer.
[0135] Typically, the detection of LPA has been conducted using
chromatography and mass spectroscopy assays that require a partial
purification of the samples using thin layer chromatography (TLC)
prior to analysis. Although this method is effective, an
underestimation of LPA levels can result during the recovery
process due in part to the varying mobility of the LPA salts (free
acid, sodium and calcium salts) when subjected to chromatography by
TLC. The low stability of LPA also calls for fast and sensitive
detection techniques.
[0136] A powerful optical detection technique based on
surface-enhanced Raman scattering (SERS) offers a unique
combination of high sensitivity and molecular specificity. With
SERS, the Raman signal of a molecule is increased by many orders of
magnitude as a result of strong enhancement of the excitation light
through the resonance of the metal's surface electrons called the
surface plasmon. SERS has been successfully used in the detection
and analysis of a large number of chemicals and biological
molecules.
[0137] Here we report for the first time to our knowledge, the
application of SERS using silver nanoparticles as a potential
alternative technique for detecting LPA with high sensitivity and
molecular specificity. Experimental results obtained for 16:0 LPA
and 18:0 LPA successfully demonstrated not only that SERS of LPA
can be measured but also that the SERS spectra of the two very
similar LPA molecules were shifted enough in the 1100 cm.sup.-1
region to uniquely identify them. The results suggested the strong
potential for practical LPA detection using SERS-based techniques
that are fast, sensitive, and molecular specific.
[0138] Powder samples of 16:0 LPA
(1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphate(sodium salt)) and
18:0 LPA (1-Stearoyl-2-hydroxy-sn-glycero-3-phosphate(sodium salt))
were purchased from Avanti Polar Lipids, Inc. Silver nitrate and
sodium citrate were purchased from Sigma Aldrich (St Louis Mo.).
Raman spectra were obtained using a Renishaw micro-Raman setup with
a 50.times. objective lens and 780 nm excitation laser at 3 mW.
[0139] Silver nanoparticles were prepared using a synthesis from
Lee and Meisel using silver nitrate as the metal precursor and a
sodium citrate reducing agent (Lee and Meisel (1982) J. Phys. Chem.
86: 3391-3395). Formation of the silver nanoparticles was monitored
using UV-visible spectroscopy using a HP 8452A spectrometer with 2
nm resolution. This nanoparticle solution was then concentrated by
a factor of 10 via centrifugation prior to application. For the
SERS experiment, 2 .mu.l drops of the concentrated silver
nanoparticles were placed on a glass slide and allowed to dry. Its
Raman signal was obtained with one accumulation of a 30 second
scan. After the silver had dried, 4 .mu.l of a 100 .mu.M solution
of either the 16:0 LPA or 18:0 LPA (dissolved in Milli-Q water) was
added on top of the silver to dry. The 4 .mu.l volume ensured the
complete coverage of the silver that had dried on the glass. The
Raman signal was then collected using the same scan parameters. For
comparison, Raman of the crystalline LPA samples was collected with
five accumulations of a two-minute scan.
[0140] The primary goal of this work was to demonstrate the ability
of SERS to be selective, reproducible, and sensitive in detecting
16:0 LPA versus 18:0 LPA and show its potential as a viable
alternative to current detection methods. FIG. 14 presents the
UV-vis absorption spectrum of the silver nanoparticles used in this
experiment. The surface plasmon band of these particles peaks near
400 nm, and contains very weak absorption around 780 nm, possibly
due to some nanoparticle aggregation. For SERS to work effectively,
resonance absorption of the metal nanoparticle substrate with the
incident wavelength is essential (Schwartzberg et al. (2004)
supra). The fact that there is only weak absorption near 780 nm
while SERS has been demonstrated to work effectively suggests that
either the weak absorption at 780 nm is enough for SERS occur, or
the nanoparticles have further aggregated upon concentrating and
drying for the SERS experiment that may have caused a red-shift of
the absorption band and increased absorption at 780 nm similar to
what has been observed for gold nanoparticles (Norman et al. (2002)
J. Phys. Chem. B 106: 7005-7012).
[0141] The Raman spectra and molecular structures of the bulk
crystal of 16:0 LPA and 18:0 LPA are presented in FIG. 15 and FIG.
16, respectively. With the only difference between the two LPAs
being the length of their acyl chains, the ability to apply SERS
for detection applications depends on its capacity to detect the
acyl peaks. Hence, experimental measurements were performed between
800-1400 cm.sup.-1 where many of the acyl peaks occur. The
vibrational modes were assigned based on characteristic Raman
frequencies (Lin-Vien et al. (1991) In: Infrared and Raman
Characteristic Frequencies of Organic Molecules. Academic Press,
Inc, San Diego Calif.). The band at 889 cm.sup.-1 is representative
of methylene rocking, and the 1294 cm.sup.-1 band is typical of
methylene twisting. The C--C skeletal stretching vibrations
appeared between 1060 cm.sup.-1-1130 cm.sup.-1. In this region,
information about the conformation of the carbon chain can also be
obtained. The bands observed around 1060 cm.sup.-1 and 1130
cm.sup.-1 are characteristic of trans bonded carbon and the band
observed around 1100 cm.sup.-1 denotes the vibration of a gauche
bonded chain. The analysis of the spectra obtained for these LPA
samples compared well to Raman spectra of various lipids that have
been previously analyzed (Dai et al (2005) Colloids and Surfaces B
42: 21-28; Krafft et al. (2005) Spectrochim. Acta (61): 1529-1535;
Saint-Pierre Chazelet et al. (1994) Thin Solid Films 244: 852-856;
and Suh (1992) Chem. Phys. Lett. 193: 327-330). Very strong
similarities between the spectra of these two LPA molecules were
noted, in particular the 889 cm.sup.-1, 1294 cm.sup.-1, 1060
cm.sup.-1, and 1128 cm.sup.-1 bands that they commonly share.
Fortunately, the 16:0 LPA was distinguishable from the 18:0 LPA by
the shift of the C--C vibration of the gauche-bonded chain from
1097 cm.sup.-1 for 16:0 LPA to 1101 cm.sup.-1 for 18:0 LPA.
[0142] FIG. 17 presents the SERS spectra of the two LPA samples
that were dried on the silver nanoparticles after subtracting the
background signal of the dried nanoparticle solution. Attempts to
obtain the Raman spectra of dried samples of LPA solutions without
any silver present resulted in no signal, indicating that the
presence of the silver enhanced the LPA Raman signal and made the
detection of low concentrations possible. As expected, the bands of
the acyl chain were enhanced using this technique with little to no
shift from their bulk Raman positions. The SERS signal of the 16:0
LPA maintained its peaks of 889 cm.sup.-1, 1097 cm.sup.-1, 1128
cm.sup.-1, and 1294 cm.sup.-1, while 18:0 LPA maintained its peaks
of 889 cm.sup.-1, 1101 cm.sup.-1, 1128 cm.sup.-, and 1294
cm.sup.-1. FIG. 18 shows the region between 1050 cm.sup.-1-1130
cm.sup.-1, demonstrating the ability of this procedure to detect
the gauche peak that allows the acyl chains of the LPA samples to
be distinguished from each other is clearly observed.
[0143] In SERS, the relative enhancement of a given mode implies
the preferred orientation of the adsorbate to the surface of the
metal. Typically, enhancement of a given mode is best when it is
closest to the surface of the metal. Comparing the band intensities
of the SERS spectrum of either LPA sample to its respective bulk
spectrum shows that the two are quite similar (see Table 1). In
other words, the intensity distribution of the SERS modes of 16:0
LPA exhibited a similar pattern of its bulk spectrum. The same
pattern was observed for the 18:0 samples. This implied that no
strong attraction was occurring between the nanoparticle surface
and functional groups on the molecule to promote a specific
orientation of the adsorbate. Also, with any strong surface
interaction between the adsorbate and metal present, one would
expect a shift in some bands of the SERS spectrum compared to the
bulk Raman spectrum due to vibrational hindrance that would result
from the adsorbate-metal surface interaction. This phenomenon was
not observed in the LPA SERS spectra. The conclusion that no strong
interaction was present between the metal surface and adsorbate
could also be made from the fact that no immediate SERS was
observed for mixed solutions of silver and LPA. The interaction
between LPA and the nanoparticle surface was only strong enough for
SERS to be observed when the molecule was dried on top of the
silver.
TABLE-US-00001 TABLE 1 Comparison of the intensity of the assigned
vibrational modes of the Raman spectrum of the bulk samples of LPA
with its SER signal (W = weak, M = medium, S = strong, VS = very
strong) 16:0 18:0 18:0 Mode (cm.sup.-1) Assignment LPA 16:0 SERS
LPA SERS 889 CH.sub.2 rock M M M M 1060 C--C vib (trans) S -- S --
1097 or 1101 C--C vib (gauche) M M M M 1128 C--C vib (trans) S S VS
VS 1294 CH.sub.2 (twist) S W VS M
[0144] Experiments to improve the sensitivity of this technique in
terms of its ability to detect lower concentrations of various LPA
in mixed samples along with actual samples of plasma/blood where
other lysophospholipids besides LPA are present are performed. Some
preliminary experiments using a prepared sample of mixed 16:0 and
18:0 LPA solutions had shown that this technique was able to
distinguish the two different LPA molecules from each other in
millimolar concentrations. However, in order to apply SERS for
practical LPA detection, this technique should detect in micromolar
quantities. As the surface interaction between the molecule and the
nanoparticles play an important role in the effective enhancement
of this technique, experiments are conducted with other metal
nanoparticles capped with various surface agents that may induce
stronger interactions between the acyl chain of the adsorbate and
metal. We also can apply SERS detection of LPA using different
shaped metal nanoparticles, as non-spherical particles with sharp
edges or corners show stronger SERS activities than spherical
particles (Schatz (1984) Acc. Chem. Res. 17: 370-376; Gersten
(1980) J. Chem. Phys. 72: 5779-5780).
Example VI
Detection of Ab-GNP Binding Interaction Using a Secondary Ab
[0145] The effect of binding an antigen to its antibody is observed
by taking the Raman spectrum of the antibody before and after
exposure to the antigen through the use of SERS. To study the
applicability of this method, a primary antibody (SC2020, Santa
Cruz Biotechnology Santa Cruz Calif.) and a secondary antibody
(SC1616, Santa Cruz Biotechnology Santa Cruz Calif.) were used.
SC2020 was obtained at a concentration of 400 .mu.g/ml and diluted
by a factor of two with 20 mM HEPES buffer (pH 7.4). This solution
was mixed equal volume with a GNP solution that was also diluted by
a factor of two with 20 mM HEPES buffer. After twenty minutes of
interaction, a SERS spectrum was obtained. An equal amount of
SC1616 was added to the system and the SERS spectrum was obtained
again. The binding of the secondary antibody (SC1616) to the
primary antibody (SC2020) caused the SERS intensity of the
secondary antibody to increase by 20-50%. This method provides an
indirect means of detecting antigens in a system.
Example VII
Detection of Tumour-Antigens in Bodily Fluids
[0146] A murine monoclonal antibody raised against the CA125
ovarian cancer marker (OC125; Bast et al. (1981) J. Clin. Invest.
68: 1331-1337; Cat. No. AB19551, AbCam Ltd., Cambridge, UK) is
incubated at a final concentration of 100 .mu.g/ml in HEPES buffer
(pH 7.4) with GNA as prepared above at a final concentration of 1
mg/ml for twenty minutes at ambient temperature. The mixture is
then washed four times with excess sample buffer, then stored at
4.degree. C. until use. A fraction is subjected to SERS to obtain
baseline values.
[0147] Fluid samples from individuals with diagnosed ovarian cancer
are incubated with SQD in the presence of a conjugating agent and
linker molecule for 20 minutes at ambient temperature. The mixture
is washed four times and resuspended in HEPES buffer (pH 7.4) to
produce SQD-Ag conjugate. A fraction is subjected to SQD
luminescence to obtain baseline values.
[0148] The SQD-Ag conjugate is added to OC125-GNA mixture in HEPES
incubation medium (pH 7.4) at ambient temperature for 8 hours.
Control samples are from individuals without diagnosed disease or
disorders. The samples are then washed four times with incubation
medium, resuspended in sample buffer, and then divided into two
fractions. One fraction is subjected to SQD luminescence. The other
fraction is subjected to SERS. Baseline values obtained earlier are
then compared with the values obtained under experimental
conditions.
Example VIII
Production of Antigen Specific Antibodies
[0149] Antigen substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, for example, Harrington (1990) Methods
Enzymol. 182: 488-495) or other purification techniques is used to
immunize rabbits and to produce antibodies using standard
protocols. The antigen amino acid sequence is analyzed using
DNASTAR software (DNASTAR Inc., Madison Wis.) to determine regions
of high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, for example, Ausubel et al. supra,
chapter 11.)
[0150] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an Applied Biosystems Peptide Synthesizer
Model 431 A using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich,
St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase
immunogenicity. (See, for example, Ausubel et al. supra.) Rabbits
are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity, for example, by binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG. In the alternative, a
non-peptide antigen is used and is conjugated to KLH.
Example IX
Purification of Naturally Occurring Antigen Using Specific
Antibodies
[0151] Naturally occurring or recombinant antigen is substantially
purified by immunoaffinity chromatography using antibodies specific
for the antigen. An immunoaffinity column is constructed by
covalently coupling anti-antigen antibody to an activated
chromatographic resin, such as CNBr-activated Sepharose (Pharmacia
& Upjohn, Kalamazoo Mich.). After the coupling, the resin is
blocked and washed according to the manufacturer's
instructions.
[0152] Media containing antigen are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of antigen (for example, high ionic
strength buffers in the presence of detergent). The column is
eluted under conditions that disrupt antibody/antigen binding (for
example, a buffer of pH 2 to pH 3, or a high concentration of a
chaotrope, such as urea or thiocyanate ion), and antigen is
collected.
Example X
Identification of Molecules That Interact with Antigen
[0153] Antigen, or biologically active fragments thereof, are
labeled with [.sup.125I] Bolton-Hunter reagent. (See, for example,
Bolton and Hunter (1973) Biochem. J. 133: 529-539.) Candidate
molecules previously arrayed in the wells of a multi-well plate are
incubated with the labeled antigen, washed, and any wells with
labeled antigen complex are assayed. Data obtained using different
concentrations of antigen are used to calculate values for the
number, affinity, and association of antigen with the candidate
molecules.
Example XI
SERS Sensor Based on D-Shaped Fiber
[0154] Fiber surface enhanced Raman scattering (SERS) sensors show
great potential for in vivo and in vitro detection, however,
current probes based on end polished fibers suffer from small
signal due to their small active region. To overcome this, we
propose and demonstrate a D-shaped fiber configuration to increase
the detection area. Initial modeling has shown that most of the
light can be absorbed by the SERS active layer coated on the
polished fiber surface. Several orders of magnitude increase in
surface area leads to substantially more detectable Raman scattered
photons than those in end tip configurations. The SERS sensor based
on D-shaped fibers has been demonstrated, for the first time, with
excellent results using rhodamine 6G.
[0155] The majority of previous work in this area has utilized
colloidal solutions or single use films to great effect, however,
for practical applications the substrate must be portable,
reusable, and robust (see Schwarzberg et al. (2004) supra; Tao et
al. (2003) Nano Letters 3: 1229; van Duyne (2002) Abstracts of
Papers of the American Chemical Society 223: 3; Sagmulleret al.
(2003) J. Mol. Struct. 661: 279; Michaels et al. (2000) J. Phys.
Chem. B 104: 11965; and Jana (2003) Analyst 128: 954). Recently,
significant advancements have been made to this end. End tip fiber
optic SERS probes have been shown to produce excellent results with
high stability and portability, where a fiber with a flat, angled,
or tapered tip was modified with silver island films, roughened
silver films, or silver film over nanospheres to produce the SERS
substrate (see Stokes et al. (2004) Applied Spectroscopy 58: 292;
Stokes and Vo-Dinh (2000) Sensors and Actuators B-Chemical 69: 28;
Mullen and Carron (1991) Anal. Chem. 63: 2196; Gessner et al.
(2004) Analyst 129: 1193; Viets and Hill (1998) Sensors and
Actuators B-Chemical 51: 92; Viets and Hill (2000) J. Raman
Spectrosc. 31: 625; and Viets and Hill (2001) J. Mol. Struct. 565:
515). This configuration is highly advantageous, being
reproducible, facile to fabricate, and inexpensive. The main
limitation is the small number of SERS particles on the active
fiber region, requiring high laser intensity and long integration
times to attain reasonable SERS spectrum.
[0156] To overcome this hurdle, we chose to use a D-shaped fiber
(DSF) configuration, so named because of the cross sectional
D-shape formed by side polishing the fiber (see FIG. 19). Light can
be coupled out of the polished fiber into silver or gold
nanostructured films coated onto the polished surface, which can
potentially increase the active surface area by several orders of
magnitude. DSF has been used in a variety of applications including
humidity, temperature, strain sensing, communication, optical
switching, attenuators, and polarizers, but never for SERS
detection, to our knowledge (see McCallion and M. Shimazu (1998)
Laser Focus World 34: S19; Sohn et al. (2002) Sensors and Actuators
A-Physical 101: 137; Alvarez-Herrero et al. (2000) IEEE Photonics
Technol. Lett. 12: 1043; and Gu et al. (2003a) J. Optics A-Pure
Appl. Optics 5: S420; Gu et al. (2003b), Optical Materials 23:
219).
[0157] Procedures for polishing the DSF have been described
previously (Xu (2003) In-line fiber optical components for
telecommunication in electrical engineering, University of
California, Santa Cruz, Santa Cruz, Calif.). The surface of the
flat polished plane was purposely left rough to facilitate
nanoparticle binding and increase active surface area. Silver
nanoparticles were synthesized by the method of Lee and Meisel (Lee
and Meisel (1982) J. Phys. Chem. 86: 3391).
[0158] The SERS fibers were created by mixing 20 .mu.l of silver
nanoparticles with 5 .mu.l of 0.1 mM rhodamine 6G (R6G) and drying
a drop of this solution on the fiber under ambient conditions. SERS
experiments were then performed on these devices in two
configurations. A 780 nm diode laser is either coupled into the
fiber (FIG. 19) or into a Renishaw micro-Raman spectrometer with a
20.times. objective. In both configurations the micro-Raman
spectrometer collects the scattered light from the DSF. Samples
were nominally integrated for 40 seconds for one scan with a laser
intensity of .about.3 mW.
[0159] On a highly polished DSF, little or no light would be
coupled out of the fiber core. When the surface is roughened and
coated with a layer of metal such as silver nanoparticles, light
can be coupled into the coating layer to induce SERS. The amount of
light coupled into the film is determined by several parameters
including polishing depth, film thickness and density, and nature
of the metal. The proper polishing depth should be determined by
calculation, however, drop-casting a silver nanoparticle film onto
the DSF ensures a thick coating which will couple the maximum
amount of light from the fiber core into the SERS substrate.
[0160] To determine how the light will be coupled out of the fiber
into the SERS substrate, we simulated light propagation inside a
D-shaped fiber covered with a solid silver film as the colloidal
film, using FIMMWAVE software. Various film thickness, polishing
depth, refractive index, and absorption constants were used in our
simulation. We found that the amount of light being coupled into
the metal layer strongly depends on the refractive index and the
absorption constant of the metal. As an example, FIG. 20 shows the
simulation result of a Corning 28e fiber covered with a silver
nanoparticle aggregate layer. The plot in FIG. 20 shows the
intensity distribution across the polished surface (top view
illustrated). The fiber parameters were: core diameter 48 .mu.m,
n.sub.core=1.450, cladding diameter 125 .mu.m, n.sub.clad=1.4447,
and the polished surface cuts through the center of the fiber core.
The metal film parameters were: thickness=0.1 .mu.m,
n.sub.Ag=0.147, .alpha..sub.Ag=82000/cm. Here a large absorption
constant was chosen because of the resonant absorption of the Ag
nanoparticle aggregates at 780 nm wavelength. With this choice of
parameters, as light propagated through the side-polished section
(from left to right in FIG. 20), the total intensity decreased due
to coupling into the metal film and metal absorption, with only 30%
remaining in the core at the end of the 1 cm side-polished fiber
segment. The absorbed light activated SERS scattering over the
entire 1 cm.times.8 .mu.m=80,000 .mu.m.sup.2 surface region above
the fiber core, as opposed to the 50 .mu.m.sup.2 of an end polished
fiber of the same kind. As shown in the inset of FIG. 20, within a
cross-section perpendicular to the fiber axis, light was mostly
confined in the core region. Utilizing the DSF would increase the
SERS active area, and consequently Raman scattered light, by as
much as three orders of magnitude since light can be coupled into
the metal layer over a substantial distance.
[0161] Having confirmed the viability of the concept, SERS
experiments were conducted using rhodamine 6G as a reference
molecule resulting in excellent SERS data. With the laser coupled
through the microscope and aligned perpendicular to the DSF
surface, large enhancements are observed in the Raman modes of
rhodamine 6G (FIG. 21, B). As expected, all peaks observed in the
SERS spectrum were consistent with previous SERS results of
rhodamine 6G (see Hildebrandt and Stockburger (1984) J. Phys. Chem.
88: 5935). More interestingly, instead of coupling light through
the Raman optical microscope, light coupled into the fiber from the
end of the fiber (see FIG. 19) produced similarly intense signals
emitted and detected from the DSF surface (FIG. 21, A). In this
case, light coupled from the core of the fiber into the silver
nanoparticle film on the surface activates the plasmon mode and
induced SERS of R6G on the film. It is important to note that after
background subtraction the SERS spectra measured from both
configurations were completely consistent. While peak intensities
vary slightly, peak positions remained nearly constant. This
indicated that the scattering mechanism was the same, independent
of the excitation configuration.
[0162] One should be careful when comparing these spectra since
their excitation intensities are different. The intensity of
illumination through the microscope objective was high and confined
to a small area due to the strong focusing of the excitation beam.
The light coupled through the fiber had lower intensity over a
larger area. Excitation intensity and surface area affect the
scattering strength. The variations of peak intensities in the two
spectra collected using two different illumination configurations
could be partly due to the difference in excitation intensity.
[0163] In conclusion, we proposed and demonstrated a SERS sensor
based on D-shaped fibers. Initial modeling had shown that as much
as 70% of light coupled into the fiber may be absorbed into the
SERS active layer across much of the 1 cm.times.8 .mu.m surface,
yielding an 80,000 .mu.m.sup.2 active region for a D-shaped fiber
as compared to .about.50 .mu.m.sup.2 of an end polished fiber of
the same kind. This leads to as much as three orders of magnitude
increase in Raman scattered photons compared to end tip fiber
probes. The device was tested with rhodamine 6G with light directly
illuminating the fiber surface and coupled through the fiber. Both
configurations yield excellent and consistent SERS spectra of R6G.
The experimental results, in conjunction with the theoretical
modeling, demonstrated successfully that D-shaped fibers can serve
as a convenient platform for SERS sensors that can potentially
provide extremely high sensitivity and molecular specificity.
[0164] Those skilled in the art will appreciate that various
adaptations and modifications of the just-described embodiments can
be configured without departing from the scope and spirit of the
invention. Other suitable techniques and methods known in the art
can be applied in numerous specific modalities by one skilled in
the art and in light of the description of the present invention
described herein. Therefore, it is to be understood that the
invention can be practiced other than as specifically described
herein. The above description is intended to be illustrative, and
not restrictive. Many other embodiments will be apparent to those
of skill in the art upon reviewing the above description. The scope
of the invention should, therefore, be determined with reference to
the appended claims, along with the full scope of equivalents to
which such claims are entitled.
* * * * *