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Description  |
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FIELD OF THE INVENTION
The present invention relates to DNA gene probes, biosensors and methods
for gene identification, particularly non-radioactive gene probes,
biosensors and methods for oligonucleotide identification and more
particularly to non-radioactive gene probes, biosensors and methods based
on surface enhanced Raman scattering (SERS) label detection.
BACKGROUND OF THE INVENTION
There is currently strong interest in the development of nonradioactive DNA
probes for use in a wide variety of applications, such as gene
identification, gene mapping, DNA sequencing, medical diagnostics, and
biotechnology. Among the various methods for gene identification,
technologies using radioactive labels are currently the most widely used.
Radioactive label techniques suffer from several disadvantages however.
The principal isotope used, Phosphorus-32, has a limited shelflife because
it has a 14-day half-life. Secondly, because there is one principal label
for gene probes, DNA can only be probed for one sequence at a time. Due to
material limitations, probing immobilized DNA with different .sup.32
P-labeled sequences can only be performed a few (3-4) times. Therefore,
the researcher must have idea about the sequence prior to probing. In
addition to these inconveniences, the potential safety hazard associated
with use of radioactive materials makes the technology undesirable.
Shipping, handling and waste disposal of radioactive materials are
strictly regulated by federal and state guidelines.
Recently, luminescence labels such as fluorescent or chemiluminescent
labels have been developed for gene detection. Although sensitivities
achieved by luminescence techniques are adequate, alternative techniques
with improved spectral selectivities must be developed to overcome the
need for radioactive labels and the poor spectral specificity of
luminescent labels.
Spectroscopy is an analytical technique concerned with the measurement of
the interaction of radiant energy with matter and with the interpretation
of the interaction both at the fundamental level and for practical
analysis. Interpretation of the spectra produced by various spectroscopic
instrumentation has been used to provide fundamental information on atomic
and molecular energy levels, the distribution of species within those
levels, the nature of processes involving change from one level to
another, molecular geometries, chemical bonding, and interaction of
molecules in solution. Comparisons of spectra have provided a basis for
the determination of qualitative chemical composition and chemical
structure, and for quantitative chemical analysis.
Vibrational spectroscopy is a useful technique for characterizing molecules
and for determining their chemical structure. The vibrational spectrum of
a molecule, based on the molecular structure of that molecule, is a series
of sharp lines which constitutes a unique fingerprint of that specific
molecular structure. If the vibrational spectrum is to be measured by an
optical absorption process, optical fibers must be used so that optical
energy from a source is delivered to a sample via one fiber, and after
passage through the sample, an optical signal generated by the exciting
optical energy is collected by the same or, more preferably, another
fiber. This collected light is directed to a monochrometer/or a
photodetector for analyzing its wavelength and /or intensity.
One particular spectroscopic technique, known as Raman spectroscopy,
utilizes the Raman effect, which is a phenomenon observed in the
scattering of light as it passes through a material medium, whereby the
light suffers a change in frequency and a random alteration in phase. When
exciting optical energy of a single wavelength interacts with a molecule,
the optical energy scattered by the molecule contains small amounts of
optical energy having wavelengths different from that of the incident
exciting optical energy. The wavelengths present in the scattered optical
energy are characteristic of the structure of the molecule, and the
intensity of this optical energy is dependent on the concentration of
these molecules.
Raman spectroscopy is a spectrochemical technique that is complementary to
fluorescence, and has been an important analytical tool due to its
excellent specificity for chemical group identification. Raman
spectroscopy provides a means for obtaining similar molecular vibrational
spectra over optical fibers using visible or near infrared light that is
transmitted by the optical fibers without significant absorption losses.
In Raman spectroscopy, monochromatic light is directed to a sample and the
spectrum of the light scattered from the sample is determined. One of the
major limitations of Raman spectroscopy is its low sensitivity. Recently,
the Raman technique has been rejuvenated following the discovery of
enormous Raman enhancement of up to 10.sup.6 for molecules adsorbed on
microstructures of metal surfaces.
Raman spectroscopy is a useful tool for chemical analysis due to its
excellent capability of chemical group identification. One limitation of
conventional Raman spectroscopy is its low sensitivity, often requiring
the use of powerful and costly laser sources for excitation. However, a
renewed interest has recently developed among Raman spectroscopists as a
result of observation that Raman scattering efficiency can be enhanced by
factors of up to 10.sup.8 when a compound is adsorbed on or near special
metal surfaces. Spectacular enhancement factors due to the microstructured
metal surface scattering process is responsible for increasing the
intrinsically weak normal Raman scattering (NRS). The technique associated
with this phenomenon is known as surface-enhanced Raman scattering (SERS)
spectroscopy. The Raman enhancement process is believed to result from a
combination of several electromagnetic and chemical effects between the
molecule and the metal surface.
Deoxyribonucleic acid (DNA) is the main carrier of genetic information in
most living organisms. DNA is essentially a complex molecule built up of
deoxyribonucleotide repeating units. Each unit comprises a sugar,
phosphate, and a purine or pyrimidine base. The deoxyribonucleotide units
are linked together by the phosphate groups, joining the 3' position of
one sugar to the 5' position of the next. The alternate sugar and
phosphate residues form the backbone of the molecule, and the purine and
pyrimidine bases are attached to the backbone via the 1' position of the
deoxyribose. This sugar-phosphate backbone is the same in all DNA
molecules. What gives each DNA its individuality is the sequence of the
purine and pyrimidine bases.
OBJECTS OF THE INVENTION
It is an object of the invention to provide a nonradioactive gene probe
biosensor based upon surface enhanced Raman scattering (SERS) label
detection for identifying target oligonucleotide strands such as
Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA) and Peptide nucleic
acid (PNA) in a variety of samples such as environmental samples or
biological samples. It is another object of the invention to provide a
SERS gene probe biosensor for the identification of bacterial and viral
gene sequences.
It is a further object of the invention to provide a SERS gene probe
detection system for the detection and identification of biotargets such
as DNA, RNA and PNA in bacteria and viruses. It is yet another object of
the invention to provide methods for using a SERS gene probe biosensor for
hybridization, detection and identification of hybridized target
oligonucleotides such as DNA, RNA and PNA in bacteria and viruses in a
variety of samples such as environmental or biological samples. Further
and other objects of the present invention will become apparent from the
description contained herein.
SUMMARY
The subject invention is a new type of gene probe biosensor based on
surface enhanced Raman scattering label detection. The surface enhanced
Raman (SER) gene probes do not require the use of radioactive labels and
have great potential to provide both sensitivity and selectivity. The SER
gene probe is used to detect DNA biotargets such as gene sequences,
bacteria and viral oligonucleotide strands via hybridization to
oligonucleotide strands complementary to the SER gene probe.
In accordance with one object of the invention, a SER gene probe biosensor
comprises a support means, a SER gene probe having at least one
oligonucleotide strand labeled with at least one SERS label, and a SERS
active substrate disposed on the support means and having at least one of
the SERS gene probe adsorbed thereon.
In accordance with another object of the invention, a SER gene probe
detection system comprises a SERS active substrate having at least one SER
gene probe adsorbed thereon wherein the SER gene probe has at least one
oligonucleotide strand labeled with at least one SERS label. The system
further comprises an energy source, a means for transmitting optical
energy from an optical energy source to the SERS active substrate in order
to generate a Raman signal, a means for collecting the Raman signal and
transmitting the Raman signal for detection, and an analyzing means for
detecting and processing the Raman signal.
In accordance with yet another object of the invention, a method for using
a SER gene probe for hybridization and detection to identify hybridized
target oligonucleotide strands comprising the steps of: a) preparing a
sampling medium with immobilized oligonucleotide strands of known sequence
adsorbed thereon wherein the immobilized oligonucleotide strands are
complementary to the target oligonucleotide strands; b) synthesizing SER
gene probes wherein a SER gene probe comprises at least one
oligonucleotide strand of unknown sequence having at least one SERS active
label; c) preparing a SER gene probe solution comprising at least one SER
gene probe wherein the SERS label is unique to the oligonucleotide strand
of a particular sequence; d) incubating the sample medium in an amount of
SER gene probe solution sufficient enough to hybridize the immobilized
oligonucleotide strands on the sample medium with the target
oligonucleotide strands that are complementary to the immobilized
oligonucleotide strands, incubating for a time period sufficient enough as
for the SER gene probes to contact the immobilized oligonucleotide strands
and sufficient enough as for hybridization to occur, thereby producing
hybridized oligonucleotide material; e) removing the oligonucleotide
strands that did not hybridize to the immobilized oligonucleotide strands;
f) recovering the hybridized oligonucleotide material from the sampling
medium; g) transferring to a SERS active substrate a small amount of the
recovered hybridized oligonucleotide material in an amount sufficient
enough as to provide a detectable quantity of hybridized oligonucleotide
material; and h) analyzing the SERS active substrate containing the
hybridized oligonucleotide material.
In accordance with still another object of the invention, a method for
using a SER gene probe for hybridization and direct detection to identify
hybridized target oligonucleotide strands comprising the steps of: a)
preparing a SERS active substrate having adsorbed thereon at least one SER
gene probe complementary to the target oligonucleotide strand wherein the
SER gene probe comprises at least one oligonucleotide strand of known
sequence labeled with a SERS label unique for the target oligonucleotide
strands of a particular sequence; b) introducing the SERS active substrate
into a sample suspected of containing target oligonucleotide strands and
contacting the SER gene probe with the target oligonucleotide strands for
a time sufficient enough as for the contact to occur and hybridization to
occur between the target oligonucleotide strand and the complementary SER
gene probe, thereby producing hybridized oligonucleotide material; c)
removing from the SERS active substrate, remaining sample containing
nonhybridized oligonucleotide strands; and d) analyzing the SERS active
substrate containing the hybridized oligonucleotide material.
In accordance with still yet another object of the invention, a method for
using a SER gene probe for detection and identification of target DNA
strands that have been amplified through Polymerase Chain Reaction
comprising the steps of: a) preparing a SERS active substrate having
adsorbed thereon two unlabeled DNA strands of known sequence, being
complementary to a target region of a target DNA strand, said target DNA
strand comprising double strands of DNA complementary to one another, and
said SERS active substrate being disposed on a support means;
b) synthesizing two SER gene probes as primers wherein each of said SER
gene probes comprises an oligonucleotide strand complementary to sites on
the opposite DNA strands of said target DNA strand wherein each primer has
a sequence which is identical to the 5' end of one DNA strand of said
target DNA strand, each of said SER gene probes further comprises a SERS
label attached to said oligonucleotide strand; c) heating said target DNA
strand to a temperature sufficient for denaturization of said double
strands of said target DNA to occur to form single-stranded DNA templates;
d) annealing said two primers to said DNA templates at a temperature
ranging from 40.degree.-60.degree. C. wherein each primer binds to said
complementary sequence at the 3' end of said opposite DNA strand of said
target DNA strand; e) adding DNA polymerase to extend the DNA molecule
through said target region of said target DNA strand yielding amplified
products, said amplified products being SERS labeled amplified DNA
segments; f) immersing said SERS active substrate in a sample containing
said amplified products; g) incubating said SERS active substrate in said
sample for a time sufficient enough as for hybridization between said SERS
labeled amplified DNA segments and said unlabeled DNA strands on said
substrate to occur to completion and a SERS signal is detected; and h)
analyzing said SERS signal.
BRIEF DESCRIPTION OF THE DRAWINGS
For a better understanding of the present invention, together with other
and further objects, advantages and capabilities thereof, reference is
made to the following disclosure and appended claims when read in
connection with the appended drawings, wherein:
FIG. 1 illustrates a SER Gene Probe Detection System.
FIG. 2 shows a diagram of the method of using a SER Gene Probe for
hybridization and detection with the SER Gene Probe attached to the
nucleotide strands to be identified.
FIG. 3a is a SERS Spectrum of only cresyl fast violet label.
FIG. 3b is a SERS Spectrum of cresyl fast violet label attached to 18
deoxyribonucleotide oligomers, p(dT).sub.18.
FIG. 4a is a spectrum showing detection of the SER Gene Probe that has
hybridized to a DNA fragment complementary to the probe.
FIG. 4b is a spectrum showing no SERS detection.
FIG. 5a shows the absorption spectrum of cresyl fast violet.
FIG. 5b shows the fluorescence spectrum of cresyl fast violet.
FIG. 6a is a diagram illustrating the method of direct detection with the
SER Gene Probe being attached to the SERS Active Substrate.
FIG. 6b illustrates the same direct detection method as FIG. 6a, except no
hybridization occurs due to absence of target oligonucleotides.
FIG. 7 is a schematic diagram of a SER Gene Probe Fiberoptic Biosensor.
FIG. 8 shows a close-up view of the dotted area of the SER Gene Probe
Fiberoptic Biosensor in FIG. 7 with SER Gene Probes being attached to the
SERS active substrate on the fiberoptic probe tip.
FIG. 9 shows an alternate embodiment to the SER Gene Probe Fiberoptic
Biosensor of FIG. 8.
FIG. 10 is a schematic diagram of an array of SER Gene Probe Fiberoptic
Biosensors each having a SERS active substrate with immobilized SER Gene
Probes used for conducting simultaneous multiple assays.
FIG. 11 illustrates SER Gene Probes attached to a SERS active substrate on
the surface of a Waveguide Biosensor.
FIG. 12 is a diagram of SER Gene Probes attached on a SERS active substrate
on the surface of waveguide microsensor arrays with charge-coupled devices
or photodiode arrays.
FIG. 13 illustrates a method for using the SER gene probe in conjunction
with Polymerase Chain Reaction to detect target oligonucleotide strands.
DETAILED DESCRIPTION OF THE INVENTION
The possibility of using Raman and/or SERS for in situ monitoring has been
reported in the past few years as well as development of efficient SERS
active substrates for trace organic analysis in environmental and
biological applications. The SERS technique has also been applied to trace
detection of pesticides, dyes, food products, and metabolites of chemical
exposure. The subject invention herein discloses the use of the SERS
technique as a tool for detecting specific nucleic acid sequences. An
example of a hybridization experiment using the SER gene probe illustrates
the usefulness of this technology. Hybridization of a nucleic acid probe
to DNA biotargets such as gene sequences, bacteria and viral DNA permits a
very high degree of accuracy for identifying DNA sequences complementary
to that probe.
Applicant's SERS gene probe technology can rapidly detect microorganisms
from multiple environmental samples. Examples include detection of
Salmonella bacteria, the causative agent for food poisoning, during food
processing; detection of Legionaella bacteria, the causative agent for
pneumonia, from water samples; detection of Giardlia lamblia, causative
agent for diarrhea, from water samples; and detection of Hepatitis virus
from shellfish. Applicant's SERS gene probe biosensor can also have a
global impact on biosensor technology in cancer detection. Applicant's
biosensor is able to detect both DNA and RNA viruses and retroviruses in
particular which can play a part in transforming healthy cells into cancer
cells. Examples of this global impact include the detection of DNA viruses
such as Papovavirus and its many strains which play a part in causing
benign warts and carcinoma of uterine cervix worldwide. The detection of
Hepadnavirus (Hepatitis B), which plays a part in causing liver cancer
mainly in southeast Asia and tropical Africa, is now possible. Also, the
detection of Herpesvirus, which plays a role in causing lymphocyte cancer
and nasopharyngeal carcinoma mainly in west Africa, southern China and
Greenland. Applicant's probe can also make a global impact on the
detection of the HIV-1 virus, the causative agent for Kaposi's carcinoma
and AIDS, worldwide. Another example is the detection of human T-cells and
HTLV-1 virus which play a part in adult T-cell mainly in Japan (Kyushu)
and detection of leukemia/lymphoma, mainly in the West Indes. Applicant's
probe is a sensitive DNA biosensor that can detect viral diseases at the
early stage of the infection. Yet other viruses and bacteria that can be
detected are included in the causative agents that play a role in AIDS,
Lyme Disease, Rocky Mountain Spotted Fever, Tuberculosis, Toxoplasmosis
and Cancer. These bacteria and viruses can dwell in numerous different
mediums which can be analyzed by Applicant's SERS gene probe biosensor.
These different mediums include bodily fluids, blood, sputum, cat feces,
raw meat and other tissues.
Applicant's invention is a Surface Enhanced Raman (SER) gene probe
biosensor used for the detection and identification of hybridized
oligonucleotide strands labeled with a SERS label wherein identification
of a target oligonucleotide is based on the detection of the SERS label.
The SER gene probe biosensor comprises a support means, a SER gene probe
and a SERS active substrate disposed on the support means. The SERS active
substrate has at least one SERS gene probe adsorbed onto the substrate.
The SER gene probe has at least one oligonucleotide strand labeled with at
least one SERS label. Oligonucleotides include DNA, RNA and PNA. The
oligonucleotide of the SER gene probe either has the SERS label attached
to the strand or if two oligonucleotide strands are used, the SERS label
can be intercalated between the two oligonucleotide strands, enveloped by
the oligonucleotide strands holding the label in place. If the SERS label
is attached to the oligonucleotide strand, the label can be attached
either at the end of the strand or at any site between the strand ends.
More than one SERS label can be used to label as long as it does not
interfere with hybridization. Many different SERS labels can be used.
Examples of the different SERS labels that can be used include cresyl fast
violet, cresyl blue violet, para-aminobenzoic acid, erythrosin and
aminoacridine. Other SERS labels that can be used that are inert to
hybridization are chemical elements or structures that exhibit a
characteristic Raman or SERS emission. These chemical elements or
structures include cyanide, a methyl group, a thiol group, a chlorine,
bromine, phosphorus and sulfur.
In one embodiment, Applicant's invention requires the oligonucleotide
strand to be labeled with a SERS label for detection and identification.
In another embodiment, a SERS label can be entrapped or intercalated into
a double strand of oligonucleotide. The label is a specific chemical group
that can be detected using the SERS spectrographic technique. Raman
spectroscopy is a spectrochemical technique that is complementary to
fluorescence, and is an important analytical tool due to its excellent
specificity for chemical group identification. Recently, however, there
has been enormous Raman enhancement of up to 10.sup.8 for molecules
adsorbed on SERS active substrates, microstructures of metal surfaces.
See, for example, D. J. Jeanmaire and R. P. Van Duyne J. Electronal.
Chem., 84, (1977).
The SERS active substrate includes a support base having a roughened metal
surface having a degree of roughness sufficient to induce the SERS effect
described above. The roughened surface is preferably formed by applying a
microparticle or microstructure layer to the surface of the support base
and then depositing a metal layer onto the microstructure layer. The
roughened surface may be formed using conventional techniques, such as
described in U.S. Pat. No. 4,674,878, incorporated herein by reference.
For the SERS active substrate to be effective for detecting and identifying
a target oligonucleotide strand, the target oligonucleotide strand must be
in the vicinity of the roughened surface. An overcoat of silica, metal
oxides, self-assembled organic monolayer layer or organic polymer can be
applied to the metallic microstructure layer. The coating is applied to
the roughened surface to sorb the oligonucleotide material which are not
easily adsorbed by the roughened surface and which are capable of either
penetrating into the coating or being attached onto the coating. The
oligonucleotide material thereby is adsorbed and becomes positioned in the
vicinity of the roughened surface and exhibit the SERS effect. Thus, in
essence, the coating serves to "alter" the adsorptivity of the roughened
surface. The oligonucleotide material immobilized on the roughened surface
of the SERS active substrate comprises either the labeled SER gene probe
immobilized on the SERS active substrate which later hybridizes with the
target oligonucleotide strand before analysis or it comprises an
oligonucleotide strand of known sequence immobilized on the SERS active
substrate which later hybridizes to the SERS labeled target
oligonucleotide strand of unknown sequence. Therefore, the SER gene probe
can be immobilized on the SERS active substrate before hybridization with
a target oligonucleofide strand FIG. 6 or the SER gene probe is attached
to an known oligonucleotide sequence complementary to a target
oligonucleotide strand and is hybridized to the immobilized
oligonucleotide strand on the substrate.
The coating may be an organic or inorganic sorbent material such as silica
or self-assembled organic monolayer, or is an organic sorbent polymer
coating, such as polymethyl-methacrylate. Selection of the polymer is
based on the sorbtivity of the polymer for the oligonucleotide material to
be identified. Selection criteria for coatings may be based upon the
desired physical (e.g., size selectivity, permeability), chemical (e.g.
polarity, chemical selectivity), electrical, magnetic, nuclear
radiation-hardening and biological properties of the coating materials.
Examples of other coating materials include carnauba wax, ethyl cellulose,
ethylene maleic anhydride copolymer, methyl vinyl ether, octadecyl vinyl
ether, phenoxy resin, poly 2-ethylhexyl methacrylate, poly (caprolactone),
poly (caprolactone) triol, poly-1-butadiene, poly-n-butyl acrylate,
poly-p-vinyl phenol, polybutadiene oxide, polybutadiene hydroxy
terminated, polybutadiene-methylacrylated, polycutadiene acrylonitrile,
polydecyl acetate, polyethyl acrylate, polyethylene, polyethylene glycol
methyl ether, polyhexyl methacrylate, poly 1 butene, polymethacrylate,
polystyrene, polyvinyl butyryl, polyvinyl carbazone, polyvinyl chloride,
polyvinyl isobutyl ether, polyvinyl methyl ether, polyvinyl stearate, and
vinyl alcohol/vinyl/actate copolymer.
In most cases, oligonucleotides such as DNA have to be attached onto SERS
active substrates which can have as its support a glass microscope slide,
a surface of a fiberoptic probe biosensor, a fiberoptic probe biosensor
array, a waveguide or waveguide microsensor arrays. Since most of SERS
coatings are based on silver or gold, the binding of oligonucleotides on
the metal surface can be based on thiol chemistry or other standard
chemical binding methods. The thiols are known to strongly chemisorb to
silver and gold surfaces to form monolayers that possess supramolecular
properties, as found in G. Whitesides and P. Laibnis, Langmuir, 6, 87-95,
1990, incorporated herein by reference.
If the overcoat is silica, the gene probe is bound to the silica coating.
The silica surface is derivatized with silane by incubation in a 2%
3-aminopropyl triethoxysilane (APTS) for 24 hr. at room temperature,
washed in acetone and dried in vacuum. The silanyl groups are activated by
incubation in 1% glutaraldehyde in water for one hour at room temperature.
Excess glutaraldehyde is removed by washing in water and rinsing with
phosphate buffered saline (PBS). Oligonucleotide probe molecules
containing amino linkers are attached to the silica surface by incubating
for 24 hr at 4.degree. C. with a probe solution (e.g., concentration 10
mg/ml). Unbound probe is washed away with PBS. A. Pal et al, Analytical
Chemistry, 67, 3154, 1995, is incorporated herein by reference.
The oligonucleotide strand of the SER gene probe is complementary to a
target oligonucleotide strand when the SER gene probe is immobilized on
the SERS active substrate. The SERS label is unique for a particular
target oligonucleotide of a particular sequence that is characteristic of
a particular bacteria or virus. So, if there are more than one SER gene
probe utilized to assay for more than one particular oligonucleotide
sequence characteristic of a particular bacteria or virus, then each SER
gene probe that is unique for a particular target oligonucleotide strand
will have a different, separate unique SERS label. Target oligonucleotide
strands in a multiple assay having the same sequence are designated for
the same SERS label.
FIG. 1 is a schematic diagram of a SERS gene probe detection system 1. The
system comprises an energy source 5, a bandpass filter 10, a mirror 15, a
SERS active substrate 20, the SERS active substrate 20 having SER gene
probes 25, a collection of optics 30, a Raman holographic filter 35,
optical fiber 40, coupling optics 45, a detector 50 being a signal
analyzer and a data processor 55. The bandpass filter 10 and the mirror 15
provide a means for transmitting optical energy from the energy source 5
to the SERS active substrate 20 to generate a Raman optical signal from
the SER gene probe 25 being labeled with a SERS label. The collection of
optics 30, the Raman holographic filter 35, the optical fiber 40 and the
coupling optics 45 provide a means for collecting the Raman optical signal
and transmitting the signal for detection by a signal analyzer 50. The
signal analyzer 50 and the data processor 55 provide an analyzing means
for detecting and identifying hybridized target oligonucleotide strands.
Instrumentation for the experimental was as follows. Raman measurements
were conducted with a SPEX Model 1403 double-grating spectrometer (SPEX
Inc.) equipped with a thermoelectrically cooled gallium arsenide
photomultipler tube (RCA, Model C31034), operated in the
single-photon-counting mode. Data storage and processing were handled
using a personal computer (PC) with SPEX Datamate software. The
monochromator bandpass was 2 cm.sup.-1. Laser excitation was the 620-nm
line extracted from the emission band of a rhodamine 6G loaded dye laser
(Coherent, CR-599-21) pumped by an argon ion laser (Coherent, Innova-70).
Tuning of a birefringent filter plus the use of a bandpass filter
permitted a narrow excitation bandpass centered at 620 nm. Laser power was
25 mW for all measurements. A right-angle geometry of the laser excitation
source and the scattered radiation was employed. SERS measurements were
performed using two experimental systems. The SPEX-based system was used
to generate the basic SERS spectra. An ICCD-based system was used to
generate spectra in hybridized experiments. In this system, the 632.8-nm
line from a helium-neon laser was used with an excitation power of
approximately 5 mW. A bandpass filter was used to spectrally isolate the
632.8-nm line before focusing onto the sample. Scattered radiation was
collected with a two-lens system which efficiently coupled the collected
radiation to a 600 .mu.m diameter silica fiber (NA-0.26, General Fiber
Optics). Signal collection was performed at 180.degree. with respect to
the incident laser beam. A Raman holographic filter was used to reject the
Rayleigh scattered radia | | |
