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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to a practical substrate, preferably
flexible, for surface-enhanced Raman spectroscopy, and to an apparatus for
using the substrate in trace analysis, particularly of organic compounds,
in either a continuous or a static monitoring mode.
The U.S. government has rights in this invention pursuant to a contract
awarded by the U.S. Department of Energy.
A number of optical spectroscopic techniques have been developed to
characterize solid-gas (vacuum), solid-liquid (electrolyte) and
solid-solid interfaces. In particular, the chemical identity of
surface-adsorbed molecular species can be determined with specificity
using surface analysis spectroscopy (SAS), such as infrared transmission
spectroscopy and electron energy loss spectroscopy, instead of surface
electronic absorption spectroscopy or photoacoustic spectroscopy. For
example, SAS techniques can be used in the analysis of molecules sorbed at
the surface of an electrode within a working electrochemical cell.
Among the SAS methods, surfce-enhanced Raman spectrometry (SERS) has
recently received considerable attention. Enhancements by factors of
10.sup.3 to 10.sup.6 can be realized in the Raman scattering intensity for
adsorbates on or near special rough metal surfaces. This phenomenon has
been verified for adsorbates at silver, copper, and gold metal surfaces
under both solution and vacuum conditions. See, e.g., Albrecht &
Creighton, 99 J. AM. CHEM. SOC. 5215 (1977). These spectacular enhancement
factors help overcome the normally low sensitivity of Raman spectroscopy
which had often necessitated the use of powerful, costly laser sources for
excitation.
In spite of the current interest in the SERS phenomenon, there has been no
report on a generalized application of this effect for trace analysis.
Most of the basic studies reported in the literature deal with samples of
concentrations between 10.sup.-1 and 10.sup.-3 M, well above the
concentration range of interest to analytical spectroscopists. Also,
previous SERS studies have involved only rigid substrates and specific
surfaces, such as glass plates covered with silver particles or the like
and microscopically roughened electrodes, and have dealt mainly with
highly polarizable, small monocyclic molecules, such as pyridine and its
derivatives, and with a few ionic species, such as the cyanide radical
CN.sup.- and the anion of dithiozone. See A. Otto in 6 APPLICATIONS OF
SURFACE SCIENCE 309-55 (North-Holland Publ. Co. 1980); Pemberton & Buck,
53 ANAL. CHEM. 2284 (1981) Vo-Dinh et al, 56 ANAL. CHEM. 1667 (1984), and
references cited therein. As a consequence, no information on the
reproducibility and general applicability of the SERS technique is
available.
Furthermore, one of the greatest barriers to the analytical applications of
SERS, especially for continuous monitors, is the lack of practical
substrate materials that can be easily prepared and that can provide data
with sufficient reproducibility and accuracy for analytical purposes.
Heretofore, rigid surfaces were prepared for SERS via a variety of
techniques, such as electrochemical roughening of electrode surfaces,
lithographic etching, and the "prolade post" method. In the prolade post
method, a SiO.sub.2 support was first coated with a thin (4 to 5 nm) layer
of etch-resistant metal, such as silver or aluminum, and the resulting
metal layer was then disrupted by heating to form metal "islands" on the
SiO.sub.2 surface. Thereafter, the substrate was exposed to an SiO.sub.2
-etching plasma, so that surface areas between the metal islands were
etched to produce metal-capped "posts." After the metal caps were removed
by a acid wash, a SERS-active metal was deposited, e.g., by thermal
evaporation, onto the ends of the posts to produce the SERS substrate.
This approach is elaborate and time-consuming.
SUMMARY OF THE INVENTION
it is therefore an object of the present invention to provide a practical
and efficient SERS-active substrate that can be easily prepared, is
inexpensive, and gives reproducible results.
It is another object of the present invention to provide a flexible
substrate material that permits the extension of SERS analytical
capabilities to include trace organic analysis.
It is yet another object of the present invention to provide SERS apparatus
utilizing the aforesaid flexible substrate for rapid, accurate detection
and continuous measurement of organic compounds present in trace
(subnanogram) quantities.
In accomplishing the foregoing objects, there has been provided, in
accordance with one aspect of the present invention, a substrate for
surface-enhanced Raman spectroscopy, comprising a flexible support having
at least one SERS-active surface which carries (a) a coating comprised of
microbodies, the coating being immediately adjacent to the surface; and
(b) a metallized outer layer. In a preferred embodiment, the aforesaid
substrate is the product of a process comprising the steps of coating the
surface of the flexible substrate with an aqueous suspension of the
above-mentioned microbodies, then spinning the substrate to effect a
substantially even distribution of the microbodies across the surface;
after the spinning step, drying the substrate surface; and thereafter
depositing a metallic layer onto the surface coated with the microscopic
bodies.
In accordance with another aspect of the present invention, there has been
provided spectroscopy apparatus comprising (a) a laser excitation source;
(b) a substrate as described above; (c) means for exposing a predetermined
portion of the SERS-active surface to the source; (d) means for
positioning the portion of the SERS-active surface in a predetermined
relation to the source; and (e) spectrometric means for detecting a
surface Raman signal from the portion of the SERS-active surface. In a
preferred embodiment, the substrate comprises a tape which is advanced
through the apparatus.
Other objects, features, and advantages of the present invention will
become apparent from the following detailed description. It should be
understood, however, that the detailed description and specific examples,
while indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications within the
spirit and scope of the invention will become apparent to those skilled in
the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 and 2 are schematic representations of apparatus within the present
invention.
FIG. 3 is a schematic representation of a portion of apparatus modified
from FIG. 2 to accommodate Raman spectrometric analysis of gaseous
samples.
FIGS. 4, 5 and 6 are scanning electron microscope (SEM) photographs showing
substrates before (FIG. 4) and after (FIGS. 5 and 6) the application of a
metallized overcoating in accordance with the present invention.
FIG. 7 is a graph showing the Raman emission spectrum of a 3.6 ng sample of
benzoic acid applied to a substrate of the present invention.
FIG. 8 is a graph showing the results of trace analyses of 1-aminopyrene,
obtained using substrate and apparatus within the present invention.
FIG. 9 is a graph showing results of continuous, sequential
Raman-spectrometric analyses conducted on four samples comprising
differing concentrations of 1-nitropyrene.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
For the purposes of this description, a surface is characterized as
"SERS-active" if it has the degree of roughness required to induce the
SERS effect described above. In the present invention, the requisite
surface roughness can be achieved by providing a support material, which
is preferably flexible, e.g., cellulosic material like filter paper, with
a coating of latex microspheres manufactured by Duke Scientific
Corporation (Palo Alto, CA). The commercially available microspheres are
made from polystyrene, polyvinyltoluene and polybutadiene, respectively,
and are generally uniform in size and shape. However, beads in the
submicron size range made from other materials, such as
polytetrafluoroethylene (TEFLON.RTM.), can be used in this context.
Suitable TEFLON.RTM. beads are manufactured by E. I. duPont de Nemours &
Co. (Wilmington, DE). Also, nonspherical particles, such as platinum
particles produced in accordance with Brugger et al, 103 J. AM. CHEM. SOC.
2923 (1981), and zirconium phosphate-base particles produced in accordance
with Maya & Danis, 190 J. CHROMATOG. 145 (1980), as well as spherical
bodies are suitable, so long as sufficient roughness is imparted thereby
to the support surface. The term "microbodies" will be employed herein to
refer to the class of roughness-imparting microspheres, submicron-sized
beads, nonspherical particles and the like which are suitable for use in
the present invention.
Preferably, microbodies applied to a substrate in accordance with the
present invention are uniform in shape, and fall within the size range
characteristic of the above-mentioned microspheres, i.e., between about
0.038 and 0.497 microns. As indicated above, the texture and degree of
roughness of the substrate surface thus treated can be regulated, and the
SERS effective optimized, by modifying the size of the microbodies used.
The support itself can be made from any material that can support a coating
of microbodies and, in addition, a metallized overcoating, described in
more detail below. Flexible support materials are preferred, especially
for continuous monitors; for example, one or more optical fibers, plastic
sheets, thin layer chromatographic (silica gel) sheets and thin metal
sheets are suitable supports. Cellulosic support materials exemplified by
filter paper (Whatman 50 or Millipore, e.g., 0.45 micron pore size) are
especially preferred because of their relatively low cost and ready
availability. Also, the surface protrusions and fibrous structure
characteristic of cellulosic support material provide the advantages of
added SERS effect-inducing roughness and increased surface area for
sorption, respectively.
The microbodies can be applied in an aqueous suspension onto the support
surface. To effect a uniform distribution of microbodies across the
surface, as is preferred, the coated substrate can be spun in a
conventional spinning device and then dried, preferably at room
temperature. After drying, a metallized overcoating should be applied to
the support, covering the microbodies previously provided. This can be
accomplished by mounting the microbody coated substrate in the vacuum
chamber of a conventional vacuum evaporator device and then depositing a
layer of metal on the substrate. Alternatively, the overcoating metal can
be sputtered onto the microbody-coated support surface, using known
techniques.
The thickness of the metallized overcoating should be sufficient to cover
the microbodies, so that a thicker overcoating is required when larger
microbodies are used. Too thick a deposit can smoothen the substrate
surface, decreasing the SERS effect; conversely, too thin a deposit may
leave interstitial regions of the substrate uncoated with metal. In
practice, the thickness of the metallized overcoating can be adjusted on a
case-by-case basis to maximize Raman emissions from the SERS-active
surface ("surface Raman signals"). Layer thicknesses of between about 100
and 2000 angstroms have been used.
A variety of metals can be used in the present invention, including gold,
copper, tungsten and, to a lesser extent, platinum. Silver is preferred,
however, for forming the metallized overcoating.
To use a substrate of the present invention in trace analysis, e.g., of
organic molecules, the substrate must be exposed to a laser source such
that the surface Raman signals can be detected, and the resulting Raman
spectra employed, to characterize molecules previously sorbed at or (in
cases where the sample is applied to the substrate in multiple layers)
near the substrate surface. If the substrate is in the form of a plate or
disk, e.g., of a suitable plastic material, a test sample can be applied
to the SERS-active surface thereof by hand and the substrate positioned in
a frame, as shown in FIG. 1, to permit precise irradiation of the
substrate surface thus exposed to an excitation laser beam. The resulting
surface Raman signals can be directed via a conventional optical system to
a Raman spectrometer (see FIG. 1), where the spectra are then quantified
and analyzed.
In a preferred embodiment of the present invention, a tape is supplied with
an extended SERS-active surface and then drawn, preferably continuously,
through apparatus as shown in FIG. 2. To produce such a tape, a ribbon of
suitable support material is coated with microbodies (there being no
spinning step) and then mounted on a cassette, such that only a portion of
the ribbon is exposed at any given time. The cassette is sized to fit
conveniently into the vacuum chamber of a conventional vacuum evaporator
or sputtering device. Once mounted in a vacuum chamber, the cassette is
advanced in such a way that successive portions of the microbody-coated
surface are exposed and provided with the metallized overcoating, until
the entire surface is rendered SERS-active. Alternatively, a plurality of
substrates, each with a SERS-active surface, can be mounted in sequence on
a tape which is drawn through the apparatus of FIG. 2.
With reference to FIG. 2, a tape-like carrier as described above,
preferably comprising a cellulosic support material such as filter paper,
is drawn from a roll 10 through a housing 11 by a stepping motor-driven
roller 12 pressurized against a spring-loaded idler 13. The tape, with
SERS-active surface outwardly exposed, follows a path, defined by a series
of stationary guides 14, within the housing. A sample delivery unit 15 (an
automated pipette or similar device) dispenses measured liquid test
samples through a port in the top of the housing onto the SERS-active
surface of the moving tape. A sample drying unit 16 provides heat from a
heating lamp (or similar device) through a port in the housing to dry each
passing sample on the tape. (For certain samples, e.g., those that are
thermally sensitive, no heating step may be required.) Each sample is then
moved past an excitation detection port 17, where the sample is exposed to
a laser excitation source 18. The resulting surface Raman signals are
collected and directed by a conventional optical system 19 to a Raman
spectrometer 20. Sample delivery system 15 and a stepping motor 21 can be
interfaced with a microcomputer 22 programmed to control the speed of the
tape and the sample delivery interval, respectively.
In another embodiment, shown in FIG. 3, apparatus of the present invention
is equipped with a conduit to deliver a gaseous sample to the SERS-active
surface of a tape-like substrate as described above. Molecular species
contained in the carrier gas of the sample are sorbed onto the active
surface and exposed to the laser source, generating characteristic Raman
spectra.
EXAMPLE 1
Preparation of a substrate based on filter paper support
An aqueous dispersion of 0.038 micron latex microspheres (10 wt.-% solids)
manufactured by Duke Scientific Corp. (Palo Alto, CA) was diluted with
distilled water in a ratio of approximately 1:10. About 100 .mu.l of the
diluted microsphere suspension was applied to a Whatman 50 filter paper
support. The coated support was spun at 800-2000 rpm for approximately 20
seconds in a conventional rotating spinning device, and then was allowed
to dry in air at room temperature. A scanning electron microscope (SEM)
photograph of the resulting microsphere-bearing surface is shown in FIG.
4.
After drying, the support was mounted on a holder inside a vacuum chamber
(Thermionics Laboratory, Inc., Boston, MA) where silver was allowed to
thermally evaporate onto the microsphere-bearing surface. The time for
evaporation, the evaporation rate, and the silver coating thickness were
precisely controlled via the thermal evaporation unit. The thickness of
the silver deposit was measured with a Model QM311 quartz crystal
thickness monitor (Kronos, Inc., Torrance, CA) to be approximately 1500
angstroms. A SEM photograph of the final substrate is shown in FIG. 5.
(Note the distance scale at the lower right corner.) Because the ultimate
resolution of the SEM is approximately 0.030 .mu.m, the microspheres are
too small to be seen. However, the fibrous structure of the paper support
is plainly visible.
FIG. 6 shows an SEM photograph of Millipore paper coated with 0.497 micron
microspheres and a 2000 angstrom overcoating of silver, following the same
general procedure as described above but omitting the spinning step. The
fibrous structure of the paper substrate increased the effective surface
area available for the microspheres and for the sorbate molecules.
Although the orientations of the paper fibers was not uniform, the
diameter of the excitation laser beam can compensate for this
nonuniformity, since the beam diameter is much larger than the surface
roughness. (Typically, the laser beam diameter is over 20 times the width
of FIG. 2.) As a consequence, the total number of microspheres illuminated
by the laser is large enough to give a reproducible surface Raman signal.
EXAMPLE 2
Use of filter-base substrate for surface-enhanced Raman spectroscopy
Surface-enhanced Raman measurements were successfully conducted for several
organic compounds, including benzoic acid, pyrene, acridine, carbazole,
1-nitropyrene and 1-aminopyrene. Benzoic acid was selected as the model
compound for detailed investigation because the Raman spectrum of this
compound has been previously investigated. The substrates used for these
measurements were prepared by the procedure described above, using Whatman
50 filter paper as the support, 0.091 micron microspheres, and a 2000
angstrom-thick overcoating of silver. The substrates were each mounted in
a sample holder of an apparatus as shown in FIG. 1.
Samples of the test compounds were added to the substrates by spotting a 3
.mu.l aliquot of an ethanolic solution (10.sup.-6 M) of the compound onto
the substrate. FIG. 7 shows an example of the detection of 3.6 ng benzoic
acid sorbed onto the SERS-active surface of the substrate. The laser used
had a power of 50 mW and an excitation wavelength of 514.5 nm. The optical
limit of detection for benzoic acid was 0.3 ng. The signal accumulation
time per data point was only 100 milliseconds. The detection limits per
sample spot for most of the other compounds investigated were in the
nanogram and subnanogram levels: carbazole (0.2 ng) at 1061 cm.sup.-1 and
1-aminopyrene (1.4 ng) at 1185 cm.sup.-1. It should be emphasized that
these limits of detection are given per sample spot and, hence, do not
account for the laser/sample illumination ratio. Since the sample area
actually illuminated by the laser beam was only 1/100 of the total sample
spot, the actual limits of detection are only 36 pg, 2 pg and 14 pg for
p-aminobenzoic acid, carbazole and 1-aminopyrene, respectively.
A typical analytical curve of 1-aminopyrene obtained in accordance with the
above-described protocol is illustrated in FIG. 8. The data were obtained
with the 1185 cm.sup.-1 Raman band, using a 633 nm laser excitation. The
slope of the log-log analytical curve is close to unity over two orders of
magnitude. Measurements performed with benzoic acid and carbazole showed
that the calibration curve is linear from 10.sup.-3 M to 10.sup.-6 M and
10.sup.-7 M, respectively. Saturation effects apparently occurred above
10.sup.-3 M as the surface Raman signal-concentration curves tended to be
nonlinear beyond this concentration. Results of multiple measurements
conducted on samples identically prepared gave a relative standard
deviation of 15-20%. This reproducibility would be quite satisfactory for
most analytical studies.
EXAMPLE 3
Performance of continuous apparatus using fiber-base substrate
A series of samples was analyzed in sequence wih a continuous monitor
device as shown in FIG. 2 in order to demonstrate the feasability of
continuous SERS monitoring, as well as the speed and detection accuracy of
apparatus within the present invention. The samples were solutions having
a volume of 4 .mu.l each and containing, respectively, concentrations of
1-nitropyrene of 10.sup.-4 M, 5.times.10.sup.-4 M, 10.sup.-3 M and
5.times.10.sup.-3 M. Each sample was applied to a separate substrate
comprised of Whatman 50 filter paper, a coating thereon of 0.038 .mu.m
polystyrene latex microspheres, and a silver metal overcoating. Each
substrate was mounted on a paper carrier tape, as described above, and run
in sequence through apparatus as shown in FIG. 2. The results of these
tests are shown in FIG. 9.
The substrate of the present invention will find wide application in
SERS-based analyses for trace amounts of organic compounds. Apparatus
within the present invention can be adapted for use as continuous field
monitors of energy-related pollutants, toxic chemicals, indoor and outdoor
air pollutants, and other hazardous substances encountered in the
environment.
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