U.S. patent application number 12/059736 was filed with the patent office on 2008-08-21 for optical sensor with layered plasmon structure for enhanced detection of chemical groups by sers.
Invention is credited to Vladimir Poponin.
Application Number | 20080198376 12/059736 |
Document ID | / |
Family ID | 35429016 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080198376 |
Kind Code |
A1 |
Poponin; Vladimir |
August 21, 2008 |
OPTICAL SENSOR WITH LAYERED PLASMON STRUCTURE FOR ENHANCED
DETECTION OF CHEMICAL GROUPS BY SERS
Abstract
An optical sensor and method for use with a visible-light laser
excitation beam and a Raman spectroscopy detector, for detecting
the presence chemical groups in an analyte applied to the sensor
are disclosed. The sensor includes a substrate, a plasmon resonance
mirror formed on a sensor surface of the substrate, a plasmon
resonance particle layer disposed over the mirror, and an optically
transparent dielectric layer about 2-40 nm thick separating the
mirror and particle layer. The particle layer is composed of a
periodic array of plasmon resonance particles having (i) a coating
effective to binding analyte molecules, (ii) substantially uniform
particle sizes and shapes in a selected size range between 50-200
nm (ii) a regular periodic particle-to-particle spacing less than
the wavelength of the laser excitation beam. The device is capable
of detecting analyte with an amplification factor of up to
10.sup.12-10.sup.14, allowing detection of single analyte
molecules.
Inventors: |
Poponin; Vladimir; (San
Francisco, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 1208
SEATTLE
WA
98111-1208
US
|
Family ID: |
35429016 |
Appl. No.: |
12/059736 |
Filed: |
March 31, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11133632 |
May 19, 2005 |
7351588 |
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12059736 |
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60572959 |
May 19, 2004 |
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Current U.S.
Class: |
356/301 |
Current CPC
Class: |
G01N 21/658 20130101;
G02B 5/204 20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
356/301 |
International
Class: |
G01J 3/44 20060101
G01J003/44 |
Goverment Interests
[0002] This work was supported in part by U.S. Government Agency
and U.S. Department of Defense Air Force Contract No. AFOSR
F49620-03-C-0058. The United States Government may have certain
rights in this invention.
Claims
1. An optical sensor for use with a visible-light laser excitation
beam, and a Raman spectroscopy detector, for detecting the presence
chemical groups in an analyte applied to the sensor, comprising (a)
a substrate; (b) a plasmon resonance mirror formed on a sensor
surface of the substrate; (c) disposed over said mirror, a plasmon
resonance layer composed of a periodic array of plasmon resonance
particles having (i) a coating effective to binding analyte
molecules, (ii) substantially uniform particle sizes and shapes in
a selected size range between 50-200 nm (ii) a regular periodic
particle-to-particle spacing that is less than 700 nm, and (d) an
optically transparent dielectric layer having a selected thickness
in the thickness range between 2-40 nm separating said mirror from
said particle layer; wherein irradiation of analyte bound to said
particle layer with said laser excitation beam is effective to
produce in said detector, a Raman spectrum of said analyte that
with an amplification factor of at least 10.sup.10.
2. The sensor of claim 1, wherein said mirror is a silver, gold or
aluminum mirror having mirror thickness between about 30-500
nm.
3. The sensor of claim 1, wherein of said particles have a selected
maximum dimension in the size range 50-150 nm.
4. The sensor of claim 3, wherein said particles are formed of
silver, gold, or aluminum solid or coated particles.
5. The sensor of claim 4, wherein said mirror and particles are
both gold or both silver.
6. The sensor of claim 5, wherein said particles are substantially
spherical.
7. The sensor of claim 5, wherein said particles are cylinders or
strips.
8. The sensor of claim 1, wherein said particle layer is formed of
holes in an expanse of a plasmon metal layer.
9. The sensor of claim 6, wherein said particle layer is formed of
a regular array of closed packed plasmon resonance particles having
a particle-to-particle spacing of particle dimension plus 0 and 20
nm.
10. The sensor of claim 6, wherein said particle layer includes a
periodic array of at least 50 particles in at least one
direction.
11. The sensor of claim 6, wherein said particle layer includes a
periodic array of at least 50 particles in each of two planar
directions.
12. The sensor of claim 1, which includes one or more additional
particle layers, each separated from the immediately underlying
particle layer by an optical dielectric layer having a thickness of
between 2-40 nm.
13. The sensor of claim 1, wherein said substrate is a particle
bead having a curved sensor surface.
14. A method of detecting chemical groups in an analyte with an
amplification factor of at least 10.sup.10, comprising (a) binding
the analyte to the surface of plasmon resonance particles in a an
optical device composed of (a) a substrate; (b) a plasmon resonance
mirror formed on a sensor surface of the substrate; (c) disposed
over said mirror, a plasmon resonance particle layer composed of a
periodic array of such plasmon resonance particles having (i) a
coating effective to binding analyte molecules, (ii) substantially
uniform particle sizes and shapes in a selected size range between
50-200 nm (ii) a regular periodic particle-to-particle spacing that
is less than the wavelength of the laser excitation beam, and (d)
an optically transparent dielectric layer having a selected
thickness in the thickness range between 2-40 nm separating said
mirror from said particle layer; (b) irradiating analyte molecules
bound to said particles with a visible-light laser excitation beam,
and (c) recording the Raman spectrum produced by said
irradiating.
15. The method of claim 14, which is effective to produce an
amplification factor of at least 10.sup.12, and is capable of
detecting chemical groups in one or a small number of analyte
molecules.
16. The method of claim 15, wherein said irradiating is carried out
at a beam power level of between 0.1 and 1 mW.
17. The optical sensor of claim 1, which is produced by forming a
dielectric layer on a substrate having a plasmon resonance mirror
formed on its surface, and depositing a suspension of plasmon
resonance particles on the dielectric layer, under conditions in
which the particles in the suspension self-assemble to form said
plasmon resonance layer.
18. The optical sensor of claim 1, which is produced by forming a
dielectric layer on a substrate having a plasmon resonance mirror
formed on its surface, forming a self-assembled, close-packed
monolayer of plasmon resonance particles on the surface of a
liquid, and contacting said monolayer with the dielectric layer on
the substrate, to transfer the particle monolayer to the substrate
to form said plasmon resonance layer.
Description
[0001] This patent application is a continuation of copending U.S.
patent application Ser. No. 11/133,632 filed May 19, 2005 which
claims priority to U.S. Provisional Patent Application No.
60/572,959 filed May 19, 2004, now abandoned, both of which are
incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0003] The present invention in general relates to a novel optical
sensor composed of SERS-active plasmon particles over a plasmon
mirror for enhanced localized optical phenomena, and the use of
this effect for ultrasensitive chemical and biological sensing with
high structural specificity and with high detection
sensitivity.
REFERENCES
[0004] The references below are cited as part of the background of
the invention and/or as providing methodologies that may be applied
to certain aspects of the present invention. These references are
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nanocluster technology-from optical coding and high quality
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[0024] A. Leitner et al., "Optical properties of a metal island
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BACKGROUND OF THE INVENTION
[0040] A variety of methods for confinement of light and for
localization and enhancement of electromagnetic field in
nanostructures, for the purpose of enhancing various localized
linear and nonlinear optical phenomena are known in the prior art
(See, for example, A. Wokaun, 1984: M. Moskovits, 1985). Most
attention in the prior art has been related to the phenomena of
Surface Enhanced Raman Scattering (SERS), based on localization and
confinement of light near the surfaces of substrates with nanoscale
structure. SERS has proven to be a powerful analytical tool for
ultra sensitive chemical and biochemical analysis (K. Kneipp et
al., 1999).
[0041] One SERS-based structure that has been proposed employs an
optical structure composed of a metal island film (MIF) over a
smooth metal surface (H.-G. Binger et al., 1995, G. Bauer et al.,
2003). A metal island film consists of a random two-dimensional
array of metal particles, each of several (typically, 2-10) nm in
largest size dimension. The shapes of the metal particles are also
variable, so it is difficult to characterization the particles
structurally. (The particles form a stochastic array of particles
resembling oblate spheroids with all minor axis oriented normal to
substrate surface, e.g., glass, quartz, or silicon.) For a variety
of reasons that will become clear below.
[0042] The metal island film MIF is separated from a smooth metal
layer by an intermediate spacer layer made from optically
transparent dielectrical material, the thickness of which controls
the strength of the interaction between the plasmons localized on
the islands and the surface plasmons of smooth metal layer. The
metal particles (islands) can be thought of as nanoscopic antennas,
collecting the incident radiation and then transferring the energy
into the nearby gap modes, that may be trapped into guided modes
propagating in all directions in plane of surface (omnidirectional
coupling). The ability of structure to absorb light at specific
wavelength depends on the existence of an optimal spacer layer
thickness that will maximize absorption in structure for specific
wavelength close to that of excitation light (Leitner et al., Appl
Opt 1993; W. R. Holland et al., 1983, T. Kune et al., 1995). For a
variety of reasons that will become clear below, the maximum
enhancement achievable with such MIF structures is limited to
between about 10.sup.6-10.sup.8.
[0043] The phenomenon of interaction of localized plasmons (LP)
with surface plasmon polaritons (SPP) in plasmon materials has been
discovered and new method of excitation of SPP in plasmon resonant
smooth films mediated by nanoparticles has been proposed (S.
Hayashi et al., 1996). An interesting phenomenon associated with
SPP excitation is the generation of a strong electromagnetic field
near the metallic surface. It is a generally accepted mechanism
that a strong electromagnetic field leads to enhancement of various
linear and nonlinear optical processes near the surface via a
mechanism of surface-enhanced spectroscopy (M. Moskovits, 1985; G.
C. Schatz and R. P. Van Duyne, 2002). According to this mechanism,
the enhancement of SERS signal is proportional to E.sup.4, where E
is electromagnetic field near metal surface.
[0044] One typical application of this phenomenon is the surface
enhanced Raman scattering of molecules adsorbed on metallic
surfaces that support plasmon resonances at both the excitation and
scattering wavelengths. Typical enhancement achieved by using
electrolysis roughened silver or by using substrate prepared by
nanosphere lithography (J. C. Hulteen et al., 1999) is in the range
10.sup.6-10.sup.8. In general, the degree of enhancement seen is
not uniform across the sensor nor reproducible.
[0045] The inability to control parameters of MIF metal surface and
intrinsic limitations in size of metal particles to less than 5 nm
(V. Matyushin, A et al., 2004) precludes their use for SERS (H.-G.
Binger et al., 1995) limits the sensitivity of such a system since
MIF-metal substrate structures do not have strong enhancement of
Raman signal. Therefore MIF-metal substrate have been reduced to
practice only for enhancement of fluorescence in so called
"resonant nanocluster biochip" technology (G. Bauer et al., 2003;
T. Schalkhammer et al., 2003).
SUMMARY OF THE INVENTION
[0046] The invention includes, in one aspect, an optical sensor for
use with a visible or near infrared (NIR) laser excitation beam and
a Raman spectroscopy detector, for detecting the presence of
chemical groups in an analyte applied to the sensor. The sensor
includes a substrate, a plasmon resonance mirror formed on a sensor
surface of the substrate, a plasmon resonance particle layer
disposed over the mirror, and an optically transparent dielectric
layer about 2-40 nm thick separating the mirror and particle layer.
The particle layer is composed of a periodic array of plasmon
resonance particles having (i) a coating effective to binding
analyte molecules, (ii) substantially uniform particle sizes and
shapes in a selected size range between 50-200 nm (ii) a regular
periodic particle-to-particle spacing less than the wavelength of
the laser excitation beam. The particles may have high symmetry or
reduced symmetry shape, and more generally, as will be considered
below, may be spherical, spheroid, rod like, cylindrical, nanowire,
tubes, toroid, or other shapes that, when uniform, can be arranged
with regular periodicity. A particle layer, as defined herein, is
also intended to encompass a regular array of holes in a planar
plasmon layer, where the holes have the dimensions set out above
for the particles. The device is capable of detecting analyte with
an amplification factor of up to 10.sup.12-10.sup.14, allowing
detection of single analyte molecules.
[0047] The mirror may be a silver, gold or aluminum layer having a
layer thickness between about 30-500 nm. The particle have a
preferred dimension in a selected size range of between 50-150 nm,
and may be formed from silver, gold, or aluminum solid or particles
having a shell formed of such metals. In an exemplary embodiment,
the mirror and particles are either both gold or both silver, and
the particles are substantially spherical.
[0048] The particle layer may be formed of a regular array of
closed packed plasmon resonance particles having a
particle-to-particle spacing of about 20 nm of less, including
direct particle-to-particle contact. The particle layer may include
a periodic array of at least 50 particles in at least one
direction, preferably at least 50 particles in each of two planar
directions, e.g., orthogonal directions or directions diagonal
directions dictated by close packing. The sensor may include one or
more additional particle layers, each separated from the
immediately underlying particle layer by an optical dielectric
layer having a thickness of between 2-40 nm. The substrate may have
a planar or curved shape, e.g., when formed on spherical beads or
inside pores in a porous filter.
[0049] In another aspect, the invention includes a method of
detecting chemical groups in an analyte with an amplification
factor of at least 10.sup.10. In practicing the method, molecules
of analyte are bound to plasmon resonant particles in the particle
layer of an optical sensor of the type described above, the sensor
surface is irradiated with a visible or NIR laser beam, and the
Raman spectrum produced by the irradiating is recorded. The method
may be effective to produce an amplification factor of at least
10.sup.12, and therefore capable of detecting chemical groups in
one or a small number of analyte molecules. The method allows Raman
spectrum analysis at an irradiating beam power as low as 1-100
.mu.W (micro W).
[0050] These and other objects and features of the invention will
be more fully understood when the following detailed description of
the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 shows the arrangement of components of a basic planar
structure for confinement, localization, and enhancement of EM
field according to one embodiment of the invention, and illustrates
how it is used for measurement of SERS spectra.
[0052] FIGS. 2A and 2B show schematically the structure of GMs and
SPPs in the same embodiment and illustrates in general how key
principal mechanism of invention works.
[0053] FIGS. 3A and 3B shows an embodiment of the invention in
which the periodic structure is a 2-dimensional array of nanosize
holes in metallic film.
[0054] FIGS. 4A and 4B shows an embodiment of the invention in
which the periodic structure is a 2-dimensional array of nanosize
tubes imbedded in metallic film.
[0055] FIGS. 5A through 5D show an embodiment of the invention in
which the periodic structure is a metallic grating consisting of a
one-dimensional array of metallic strips or cylinders.
[0056] FIG. 6 is an AFM topographic image of a 2 micron by 2 micron
area of surface of a planar SERS-active substrate fabricated
according to Example 2. The image demonstrates the uniformity and
high density of packing of nanoparticle placement on the
surface.
[0057] FIGS. 7A-7C show various aspects of an experimental set up
with a Raman microscope and fluidic cell used for measurement of
SERS spectra from liquid samples.
[0058] FIG. 8 shows a SERS spectra for Rhodamine 6G (R6G) molecules
obtained in a fluidic cell using a Raman microscope
Horiba-Jobin-Yvon Lab Ram HR 800 and Argon laser.
[0059] FIG. 9. shows a SERS spectra of Rhodamine 6G molecules
obtained in fluidic cell using Raman microscope.
[0060] FIG. 10A is a Raman image of 20.times.20.mu. area for main
intensity peak (1280 and 1400 cm-1) of Rhodamine 6 G molecules with
a baseline correction in %.
[0061] FIG. 10B is a SERS spectra of Rhodamine 6 G molecules at
maximum and minimum intensity with baseline correction.
[0062] FIG. 11A is a Raman imaging map of Rhodamine 6G (R6G)
molecules on SERS slide of area 20.times.20 micron. Dotted lines
present spots from which Raman spectra have been collected.
[0063] FIG. 11B is a SERS spectra of Rhodamine 6G molecule along
Line 1 out of 21, from the top of map demonstrating uniformity of
"hot spots" across the surface of substrate;
[0064] FIGS. 12A through 12D illustrate the use of SERS-active
structure of the present invention integrated into a filter based
optical SERS sensor with a planar (12A and B) and nonplanar (12C
and D) SERS-active surface. The filter is made of an optically
transparent porous silica. Part of the internal surface of the
porous material is covered by the resonant SERS-active structure of
the present invention.
[0065] FIGS. 13A and 13B show a diagram of a fiber-optic coupled
optical sensor for remote detection and identification of
environmental contaminants and hazardous materials;
[0066] FIGS. 14A and 14B show a diagram of a microbead-based
optical SERS sensor with a nonplanar spherical SERS-active
surfaces;
[0067] FIG. 15 Illustrates the use of a bead aerosol to detect
distantly biological and chemical warfare agents and explosives
with a Raman standoff system such as LIDAR;
[0068] FIG. 16 Illustrates an embodiment of a planar microfluidic
optical SERS sensor, in accordance with another embodiment of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0069] The terms below have the following meaning, unless otherwise
indicated.
[0070] "Plasmon resonant metal" includes any metal, such as gold,
silver, or aluminum which can support surface electromagnetic
modes--surface plasmon polaritons (SPP), which are coupled modes of
photons and plasmons.
[0071] "Chemical group" in a sample may include subunits in a
polymer, or subunit moieties, such as nucleic acid bases, or
chemical constituent groups, such as hydroxyl, amine, alkyl, acid,
or aldehyde groups. Such chemical groups are characterized by a
unique enhanced Raman spectral signatures or features.
[0072] "Gap modes" or "GMs" refer to electromagnetic normal modes
or electromagnetic eigenmodes that are excited by external
electromagnetic field in a space between two or more plasmon
resonance particles and when plasmon resonance particles are placed
near (less than 40 nm) a metal surface, preferably a plasmon
resonant metal surface.
[0073] "Plasmon resonance particles" (PRPs) are particles are
particles formed of a plasmon-resonance metal, such as gold,
silver, or aluminum, or particles having a shell of such metal. In
the present invention, PRP have their largest dimension typically
in the 50 nm to 200 nm size range.
[0074] "Gap-mode enhanced Raman spectrum" of a sample refers to
spectral features in a Raman spectrum of the sample that are
enhanced by the presence of gap modes at the sample.
[0075] "Photonic crystals" refers to 1-, 2-, 3-dimensional
structures with periodic distribution of refraction index that
results in a band-gap structure, with the result that photons with
energies corresponding to this band gap cannot propagate on
photonic crystal and may exist only in localized state.
[0076] "Photonic band gap" refers to a range of energy of photons
in which they cannot propagate in photonic crystal structures.
[0077] "Visible light" refers to the portion of the electromagnetic
spectrum that is visible to the human eye, generally in the
wavelength range between 400 nm to 700 nm range.
[0078] "Near infrared" refers to the portion of the electromagnetic
spectrum with a wavelength longer than visible light, but shorter
than microwave radiation, generally in the wavelength range between
700 nm and 1 mm.
B. General Description of the Invention
[0079] The present invention provides a plasmon resonance
nanostructure that allows precise control and tunability of its
optical response through plasmon resonance effects. This is
achieved by one or more periodic plasmon layers operating as 2-D or
3-D photonic crystals with appropriate photon band gap structure
enhanced by coupling to a plasmon mirror through an optically
transparent dielectric layer having a selected thickness of less
than about 40 nm.
[0080] The general design of structure according to concept of the
invention, which will be referred to "periodic plasmon
nanostructure over plasmon mirror" consists of a continuous plasmon
resonant material referred to as a "plasmon mirror" and at least
one particle layer consisting of a 1-D or 2-D periodic array of
plasmon resonance particles (or other regular nanostructures, as
discussed below) in which localized plasmons (LPs) may be excited.
Plasmon resonance coupling between the particle layer and mirror is
through a selected-thickness, optically transparent dielectric
layer having a selected "tuned" thickness between about 2-40 nm,
preferably 2-20 nm.
[0081] The particles forming the particle layer are substantially
uniform in size and shape, in a selected size range between about
50-200 nm, preferably 80-150 nm, depending on the excitation
wavelength. The particles may have high symmetry or reduced
symmetry shape, and more generally, as will be considered below,
may be spherical, spheroid, rod like, cylindrical, nanowire, tubes,
toroid, or other shapes that, when uniform, can be arranged with
regular periodicity. They may be homogeneous consisting from one
material-silver, or gold, or from composite such as nanoshells (J.
West et al., "Metal Nanoshells for Biosensing Applications", U.S.
Pat. No. 6,699,724, Mar. 2, 2004.). The periodicity of the particle
layer(s), i.e., the spacing between adjacent particles in any
direction, may vary from a close-packed arrangement, in which the
particles are separated from one another by a spacing of between
particle size plus 0-20 nm, or with a periodic spacing up to the
wavelength of incident light, with optimal coupling and enhancement
of signal being observed in the close-packed arrangement,
preferably with spherical particles. A particle layer, as defined
herein, is also intended to encompass a regular array of holes in a
planar plasmon layer, where the holes have the dimensions set out
above for the particles. The particles in the particle layer are
separated by or embedded in a dielectric material which may be air
or a solid, optically transparent dielectric material, such as like
that forming the dielectric layer.
[0082] The plasmon resonance response of the nanostructure is
tunable and may be controlled by adjustment of the parameters of
the nanostructure including the spacing between layers, the size
and shape of the nanoparticles, the spacing between nanoparticles,
the periodicity of the particles forming the particle layer, and,
and the dielectric constant and thickness of the dielectric layer.
Maximum localization and enhancement of EM field is achieved when
the frequency of the excitation light is the same as or close to
the frequency of plasmon resonance of the nanostructure as a whole,
or more precisely, the plasmon resonance frequency should be
between the frequency of incident light and that of scattered
light. Plasmon resonance frequency and shape of plasmon resonance
response in such complex metal-dielectric nanostructure depends on
many parameters (size, material, shape of nanoparticles, and their
arrangement with respect to each other and with respect to plasmon
mirror surface). However, strongest plasmon responses are obtained
on dipole plasmon resonance excitations of LPs on isolated
nanoparticles. Maximum confinement and localization and enhancement
of the EM field in the structure is achieved through a mechanism of
excitation of gap electromagnetic modes (GMs) or eigenmodes of the
particle layer, and surface plasmon polariton modes (SPPs) excited
on the smooth surface of the mirror. This mechanism operates
through coupling and interactions between these modes and between
the electromagnetic field of the excitation light.
[0083] An additional advantage of regular array of LP oscillators
over a continuum of SPP under condition of coupling between them
through GMs (close proximity between two layers separated by
dielectric) is the mechanism of synchronization of LPs through SPPs
that result in the narrowing of plasmon resonance and additional
dramatic enhancement of local field and corresponding Raman signal.
However, this effect exists in relatively narrow range of spectra.
Typically the narrow collective plasmon resonances are in a range
of 450-800 nm, but best enhancement achieved in range 500-600 nm
for silver NP and 600-750 nm for gold NP.
[0084] A general advantage of periodic regular array in the
particle layer is that it now has both high plasmon resonant
response and properties of a photonic crystal that result in
additional effect of focusing and confinement of incident light
beam due to confinement in photon band gap structure. This is in
contrast to a random array of LPs over SPP continuum, where both
effects synchronization between LP and focusing of incident light
beam (by mechanism of Anderson localization disclosed in D.
Wiersma, "Localization of light in a disordered medium", Nature,
390, 671-673 (1997)) are present, but overall the effect of EM
field enhancement is significantly less, since the density of "hot
spots" is relatively small. According to the generally accepted
paradigm of SERS (M. Moskovits, 1985: G. Schats et al., 2002),
enhancement of Raman signal happens through local field enhancement
due to plasmon excitation in so called "hot spots." The general
structure of "hot spots" in different array structures is explained
and illustrated on FIGS. 2-5 below. From a practical stand point,
this enhanced interaction from a periodic particle layer and
plasmon mirror allows for a highly reproducible high quality Raman
spectra with extremely low excitation power (typically 10-100
microwatt, and less than 1 microwatt for some samples).
[0085] The plasmon resonance nanostructure of the invention may be
used in a variety of applications in analytical instrumentation,
analytical chemistry and spectroscopy. As an example it may be used
as a substrate in mass spectrometry devices for improvement of
Laser Desorption Ionization, such as MALDI-TOF, SELDI-TOF). Another
major field of use is enhancement of a variety of localized linear
and nonlinear optical phenomena such as Generation of Harmonics,
Coherent Anti-Stokes Raman scattering (CARS) and in particular as
SERS-active substrate.
[0086] In particle, the nanostructure of the invention may be used
for enhancement of Raman signal in various optical devices and
optical sensor devices. In particular, one important practical
application of the invention is its use as a SERS-active sensor for
real-time all optical ultrasensitive detection and identification
of chemical groups in chemical and biological analytes in samples
in solid, liquid and gaseous environment. Four major embodiments of
optical devices and optical sensors using these fundamental
interactions are discussed below in Section D1-D4, and include:
[0087] 1. Optical devices and sensors with planar SERS-active
surfaces according to present invention (SERS-based), implemented,
for example, in microfluidic chip platform;
[0088] 2. Filter based optical devices and sensors with nonplanar
SERS-active surfaces (SERS based) from optically transparent porous
and mesoporous membranes and materials with all or part of internal
surfaces covered by the resonant structure of present invention.
This sensor is especially useful for continuous monitoring of
environmental contaminants in liquid and gaseous phase;
[0089] 3. Fiber optic coupled optical devices and sensor with both
planar and nonplanar SERS-active surfaces (SERS based) for distant
sensing (detection and identification) of environmental
contaminants and hazardous materials; and
[0090] 4. Mirobead based optical devices and sensors with nonplanar
(spherical or spheroid shape) SERS-active surfaces (SERS
based)--possible use are in microfluidic flow as well as in aerosol
samples.
C. Basic Optical Sensor of the Invention
[0091] The structural requirement in the optical sensor of the
invention can be understood from the following basic description of
the physical interactions responsible for the giant EM enhancement
it provides. Under plasmon resonance conditions, corresponding to
plasmon oscillation of individual NPs, the EM field excites LP
oscillation on each particle. For silver NPs in the range of 50-150
nm, plasmon resonance frequency is in a range 460-520 nm. This
geometry of excitation is also optimal for excitation of two types
of Gap electromagnetic modes (GMs). The first type is the GM
between adjacent NPs in the layer array, and the second type,
between NPs and the plasmon mirror surface. For efficient
excitation of GMs, the spacing between adjacent particles (the
periodicity of the layer) should be regular and less than
wavelength of the EM field in the dielectric media (typically is
250-700 nm, since dielectric constant of transparent matrix and
spacer layer is in a range 1.5-2.5), but best results are for a
close-packed arrangement having a periodicity close to the diameter
of the NPs plus up to 20 nm.
[0092] If the NP array (particle layer) is in close proximity to
the plasmon surface (a distance less than about 40 nm), then SPPs
in the mirror are excited and propagate in all directions in the
surface plane. Due to coupling with the LPs of nanoparticles, SPPs
creates a new mechanism (in addition to EM wave) of long range
interaction between LP oscillations. Long range interactions
produce synchronization of phases of LP oscillations in the NP
array and results in narrowing bandwidth of plasmon resonance, so
called collective plasmon oscillation. Optimal parameters for such
synchronization to occur are: NP sizes in range 50-200 nm
preferably 80-150 nm, and a regular periodicity
(particle-to-particle spacing), preferably in both directions in
the particle layer, of less than the wavelength of the excitation
light, and preferably a close packed arrangement having a
periodicity of the NP size plus up to 20 nm. Best amplification is
achieved for a perfect periodic array with a number of NPs along
one dimension of more than 50. Any deviation from perfect
periodicity and from uniformity in NP size will reduce the
enhancement effect since it results in disruption of
synchronization and broadening of plasmon resonance shape. This
explains why random arrays and fractal structures from NP are less
efficient than the periodic nanostructure over plasmon mirror
disclosed in the invention.
[0093] As an example of an exemplary nanostructure constructed in
accordance with the invention, reference will be made to the
optical sensor shown in FIGS. 1 and 2. The structure consists from
substrate 10 providing an upper sensing surface. The substrate may
be any dielectric support, such as glass, ceramic, or silicon waver
slide or waver. Formed on the sensing surface of the substrate is a
plasmon resonance mirror 20 which is formed from a material, such
as silver, gold, or aluminum, capable of supporting surface plasmon
polaritons (SPPs). This layer can be created by standard vacuum
deposition technique (e.g., V. Matyushin, A et al., 2004). The
thickness of the layer could be in a range 20-500 nm or more as
long as it can function as a mirror surface in the optical range of
spectra.
[0094] Spacer layer 30 formed over the mirror is composed from
optically transparent dielectric material, for example, LiF formed
by vacuum deposition, or dried polymer films, as described below.
The thickness of layer is in a range less than 50 nm, preferably
less than 40 nm, and more preferably 3-20 nm, e.g., 5-25 nm. If a
self-assembling method is used for making the layer of
nanoparticles on the dielectric layer, the layer is preferably
formed of a polyamine or the like capable of forming covalent
chemical bonds to the particles (and with the mirror layer). The
dielectric spacer layer can be produced with a controlled thickness
by using, for example, a micromachined piezo driving system. In
this case, the optical plasmonic properties of the substrate can be
dynamically controlled to allow optimizing absorption maxima.
[0095] A nanoparticle layer 40 may be formed, for example, by a
method of self assembling (B. E. Baker et al., 1996), which allow
plasmon particles of any size (e.g., 80-100 nm sizes), employing
particles with high uniformity in shape and size. Composite
(Gold-Silver or Silica-Silver Shell) nanoparticles may also be
used, as may low symmetry nanoparticles such as `nanobowls` (Y. Lu
et al., 2004). By using template directed self assembling
techniques (Y. Xia et al., 2003) perfectly ordered (cubic or
hexagonal or other symmetry) arrays of particles can be created
with controlled surface density and interparticle distances.
Plasmon particles can be covered by a protective layer
individually.
[0096] A protective coating layer 50 may be, for example, formed
from SiO or other dielectric optically transparent material. In an
embodiment in which the particles have individual protective
coating, a protective layer is not necessary. The thickness of the
protective layer is less than 5 nm, preferably less than 2 nm. The
protective layer or the coating on the individual particles may be
derivatized with analyte binding molecules, such as antibodies,
ligands, DNA fragments, and the like, or analyte binding to the
coating or protective surface may be by non-specific absorption. In
some embodiments, individual particles may be coated by a molecular
imprinted polymer (MIP) to bind specific target analyte (K. Haupt,
"Imprinted polymers-Tailor-made mimics of antibodies and
receptors", Chem. Comm., 2003, 171-178) or by monoclonal antibodies
for specific analytes. In either case, the surface of the sensor is
exposed to analyte under conditions in which analyte molecules bind
to the coating surface, typically placing the analyte within 0-5 nm
from a PRE in the particle surface. However in some cases when
analyte molecules may penetrate and bind directly to particle
surface enhancement may be even larger. The figures show analyte
molecules 80 placed on surface of coating 50.
[0097] In its optical sensing mode, the sensor surface is
irradiated with a visible or NIR laser beam 60 through a focusing
lens 70. As shown in FIGS. 2A and 2B, the incident light, indicated
at 110, excites Gap Modes 130 (GMs) localized presumably within the
particle layer and between the particles and the plasmon mirror,
and gap modes 140 between nanoparticles (NPs) 100 forming the
particle layer. Although not shown, localized plasmons (LPs) are
formed about each particle. Surface plasmon polaritons (SPPs)
formed on the surface of the metal film are shown at 150. The
sinusoidal wave representation of the SPPs is intended to indicate
that the SPPs are propagating, and not stationary. As seen, the GMs
produce extremely high local electric field in close proximity to
the particle surfaces. An end enhanced EM field results in
enhancement of a Raman cross section that scales as E.sup.4 (M.
Moskovits, 1985: G. C. Schatz, and R. P. Van Duyne, 2002). Enhanced
Raman signal light, indicated at 120, is generated by analyte
molecules is collected in backscattering arrangement and is send to
dispersive element of Raman spectrometer detector (not shown),
where spectra of substance are analyzed and information about
chemical groups is identified.
[0098] FIGS. 3-5 demonstrate other embodiments of "plasmon lattice
over plasmon mirror" structure operating according to same general
principle of work as described above. For example, in FIGS. 3A and
3B, the 2-D periodic plasmon structure is a metallic film 20a with
a periodic array of nanoholes 102 with diameters in the range
20-200 nm and spacing between holes in a range less than wavelength
of incident light. Between the plate with nanoholes 20b and a
plasmon mirror 20a, there is dielectric layer 30 with thickness in
the range 2-40 nm. Incident electromagnetic wave 110 excites LPs on
the surface of each nanohole and SPPs 150 on the surface of the
metal film. Due to resonance effects of anomalous transmission of
light through array of subwavelenth nanoholes (T. Ebessen et al.,
Nature, 391, 667, 1998) electromagnetic field penetrates into the
volume between the plasmon mirror and array of nanoholes and
excites GMs 132 and two types of SPPs (shown at 150) in the surface
of the plasmon mirror and on both surfaces of metal films with the
array of nanoholes 20b. The SPPs and GMs interact with each other
through the dielectric layer 30 of less than 40 nm thickness. Due
to close proximity this additional long range interaction between
SPPs and LPs stimulate synchronization of phases of LP oscillations
in array and as a result plasmon resonance gets narrowed and local
field on surfaces of NP substantially enhanced.
[0099] The nanohole lattice structure shown in FIGS. 3A and 3B may
be formed, for example, by using photolithographic etch techniques
to form a silver or gold layer containing an array of holes, each
hole having a selected diameter in the 50-200 nm range, a firm
thickness in the range 20-200 nm, and a hole-to-hole spacing in the
range of up to the excitation wavelength and preferably in the
range of hole diameter up to 20 nm. This film, once formed, can
then be transferred to a structure containing the substrate mirror
layer and dielectric layer to form the optical sensor nanostructure
of the invention.
[0100] Analyte molecules 82 on the lattice layer may be adsorbed on
the surface inside or near nanohole 102 and became exposed to
strongly enhanced local field of NPs. Due to the SERS effect
described above, Raman scattered signal 130 is enhanced, and this
signal is detected by an optical system and subjected to spectral
analysis in Raman spectrometer device.
[0101] FIGS. 4A and 4B illustrate similar embodiments to that
presented on FIGS. 3A and 3B, except that the geometric parameters
of the plasmon array lattice consist of metallic film 20b with
sub-wavelength size holes and nanotubes 104 attached to each hole.
The lattice period in this case has same range as that discussed,
namely less than wavelength of the excitation light. The geometric
structure of GMs between the lattice layer and mirror (shown at
134) will be slightly different with this configuration; however,
the fundamental mechanism of interaction through excitation of LPs,
GMs and SPPs and the effect of synchronization in an array of
nanostructures is basically the same.
[0102] The nanotube lattice structure shown in FIGS. 4A and 4B may
be formed, for example, by using self-assembly techniques to form
an assembled array of sliver or gold nanotubes which are then
transferred to a structure containing the substrate mirror layer
and dielectric layer, to form the optical sensor nanostructure of
the invention. Alternatively, the nanotube layer that is
transferred to the mirror structure can be formed by
photolithographic techniques in which both the tubes and the tube
interiors are produced by etching of photoactivated regions of the
array. In this embodiment, each tube has a selected ID in the
50-200 nm range, a film thickness (tube length) in the range 20-200
nm, and a tube-to-tube spacing in the range of up to the excitation
wavelength and preferably in the range of tube diameter up to 20
nm.
[0103] It is worth noting that due to the symmetry of the lattice
in FIGS. 2-4, the excitation of SPPs is omnidirectional and
therefore the efficiency of excitation does not depend on
polarization of incident light under perpendicular incidence
geometry.
[0104] An alternative embodiment of the "plasmon lattice over
plasmon mirror" structure is presented on FIGS. 5A-5D, which
illustrates a structure in which the plasmon lattice is a 1-D array
of nanocylinders 106 in FIGS. 5A and 5B or nanostrips 106 in FIGS.
5C and 5D which form, in effect, a plasmon metal grating as a
lattice. (Other structures common to those shown in FIGS. 2-5 are
identified with the same numerals in all of these figures). The
range of geometric parameters is the same as in previous examples.
All geometrical dimensions of the structure, including the diameter
of the cylinders or strips, and the periodicity of the surface
structures are less than wavelength of light. Specifically, each
cylinder or strip has a selected OD or width in the 50-200 nm
range, and the spacing between cylinders or widths of the strips is
such as to give a periodicity of up to the wavelength of the
excitation light and preferably the range cylinder OD (or strip
width) plus up to 20 nm.
[0105] The nanocylinder lattice structure shown in FIGS. 5A and 5B
may be formed, for example, by using self-assembly techniques to
form an assembled array of sliver or gold nanocylinders which are
then transferred to a structure containing the substrate mirror
layer and dielectric layer, to form the optical sensor
nanostructure of the invention. Alternatively, the nanocylinder
layer that is transferred to the mirror structure is formed by
photolithographic techniques.
[0106] The mechanism of operation through cylinder-to-cylinder GMs,
indicated at 146, and cylinder-to-mirror GMs, indicated at 134, and
SPPs, indicated at 150, are substantially the same as above.
However, due to the reduced symmetry in 1-D, the excitation
efficiency now depends on the orientation of polarization vector in
lateral plane. More efficient excitation of GMs is achieved if the
direction of the electric field in EM wave is perpendicular to the
direction of cylinders and strips in lateral plane.
[0107] The principles of operation of SERS-active structures in
optical sensor devices for analyte detection are the same as
described in case of FIG. 2, and can be easily understood by
analogy.
D. Description of Specific Embodiments
[0108] This section describes four applications of the optical
nanostructures described above. In these embodiments, which are
illustrated in FIGS. 12-16, the structure represented by numeral
150 in FIGS. 12 and 13 is the optical sensor nanostructure
described above. For all embodiments, the range of optical
nanostructures is intended to encompass the general structures
described above.
D1. Planar Microfluidic Optical SERS Sensor
[0109] In its basic embodiment, the optical structures is used as
an optical sensor for detection of analytes to which the sensor is
exposed, e.g., in a planar microfluidic SERS chip platform that may
be used for analysis of liquid samples with application to disease
or environmental monitoring. The general schematic diagram of use
of a planar microfluidic optical SERS sensor with a table top Raman
microscope is illustrated on FIG. 16. The SERS active structure of
present invention according to embodiments as illustrated in FIGS.
1-5 above may be integrated into each channel of a microfluidic
chip 370 which is placed on a motorized translation table 360 and
controlled by an electronic device 350 through a computer 180.
Sample analyte flow through channels and analyte molecules are
adsorbed into SERS-active surface and analyzed in Raman microscope.
Light from a light source 300 through a beam splitter 312 and
focusing optics 70 and microscope objective 72 is directed to a
sample on the surface of SERS substrate. Raman signal generated in
backscattering geometry through optical system is sent to
dispersive element 330 and spectra are detected by CCD detector 340
and analyzed in computer 180. In another embodiment of sensor
portable version of Raman spectrometer may be used. This sensor has
broad range of use including, but not limited to: Environmental
monitoring, Genomics and Proteomics research, DNA analysis,
Pharmaceutical and Drug Industry, Agriculture and Food analysis,
Biomedical diagnostics, Biodefence, Industrial monitoring, Forensic
Analysis etc.
D2. Filter-Based Optical SERS Sensor
[0110] That embodiment is illustrated the use of SERS-active
structure of the present invention integrated into a filter based
optical SERS sensor with a planar (12A and B) or nonplanar (12C and
D) SERS-active surface. The SERS-active structure, indicated at
150, is integrated into porous filters made of optically
transparent material such as porous silica in planar architecture
as illustrated by FIGS. 12A and 12B. Filters from optically
transparent porous silica may be the best for this sensor. Diameter
of pores 190 may be in a range of 1-100 microns, depending on the
purpose of the filter. The nanostructure 150 may be integrated into
porous silica by coating pores by silver layer using electroless
deposition method and subsequent functionalization of silver
surface by nanoparticle as described in Example 1 and 2. A
non-planar arrangement of pores covered with an SERS-active surface
150 is shown in FIGS. 12C and D. With an analyte solution flowing
through the filter, a laser system with spectrometer can be used
for continuous monitoring of contaminants in solution or water 200
flowing through the pores of the filter 190. That is, the intended
application is for continuous monitoring of contaminants and
hazardous materials in a fluid system, such as a water supply
system.
D3. Fiber Based Optical SERS Sensor
[0111] An application of the invention to a fiber optic sensor is
illustrated by FIGS. 13A and B. Here the SERS-active structure 150
is integrated into a sensor probe 240 which is connected by an
imaging fiber 232 (that contains between 1000 and 1000,000 of
individual fibers fused together into single bundle) with a
multichannel Raman analysis system 170. Excitation light from a
light source 220 through fiber 232 is delivered to the SERS-active
surface. Water with target analyte flows through a channel having
an inflow 200a flow in and outflow 200b. Contaminants in the
flow-through water are adsorbed to surface 150 and detected by
enhanced Raman scattering. This type of sensor is particularly
useful for applications involving monitoring the quality of an
aqueous environment.
D3. Bead-Based Optical SERS Sensor
[0112] In still another embodiment, the invention contemplates
microbeads covered by the SERS-active coating of present invention,
as illustrated in FIGS. 14A and B. Here, spherical beads 210 formed
of polyester or a similar material and having diameters in the
range 3-10 micron are covered by silver layer 20 by method of
vacuum deposition, and this layer in turn is covered by a
dielectric layer 30 having thickness in a range 2-40 nm. The coated
bead is then covered by NPs 100 which have diameter in a range
50-150 nm. As shown, the NPs are also covered by dielectric coating
30. SERS-active beads can be used as a suspension in a microfluidic
optical sensor device or in application requiring aerosols.
[0113] The use of SERS-active beads in a microfluidic optical
sensor is illustrated by FIG. 14B. Sample analyte in solution is
injected through a channel 202 and suspension of SERS-active beads,
through a channel 200. In mixing chamber 250 analyte is mixed with
beads, and analyte molecules are adsorbed onto the surface of
beads. In detection area 260 analyte is detected by SERS.
[0114] SERS-active beads in form of aerosol may be used for distant
detection of warfare biological and chemical agents and explosives
as illustrated by FIG. 15. Here an aerosol of SERS-active beads 290
is injected from an injector 280 into a cloud 270 of gas to be
analyzed. Analyte and beads are mixed in the cloud and analyte is
adsorbed onto surface of SERS-active beads. Following this, the
beads are collected, e.g., by a gas filter, or may be analyzed in
situ by a Raman system 300 for example Raman LIDAR.
[0115] From the foregoing, it can be appreciated how various
objects and features of the invention have been met. Model SERS
plates constructed in accordance with the invention were prepared
and tested with different Raman systems using adenosine molecule as
analyte. A comparison of the results with that for a commercially
available SERS plates and with an Intel porous silica covered by
silver SERS plates demonstrates an amplification better at least 6
orders of magnitude over these prior art structures. The results
are robust and reproducible, in that the same results were obtained
on multiple different set nanostructures over a period of several
months. The nanostructure plates are stable, since they sustain
SERS activity for at least 3 month.
[0116] In accordance with the invention, and for the first time,
substantial SERS signal in a range up to 7000 counts per second was
obtained with new SERS plates at illumination power as low as 5
microW at sample and in some cases even 0.4 microwatt with R6G.
This level of signal is comparable or better than that achieved in
sensors based on luminescent detection; however required
illumination power is at least 3 orders of magnitude less. Assuming
an amplification factor of Intel substrates in a range
10.sup.6-10.sup.8, one can estimate an amplification factor for
SERS plates of present invention 10.sup.12-10.sup.14.
[0117] The following examples illustrate various methods of forming
and using the nanostructures of the invention, but are in no way
intended to limit the scope of the invention.
EXAMPLE 1
Preparation of a Silver-Silver Particle Nanostructure by
Self-Assembly of Ag Nanoparticles
[0118] For each of a number of slides, a silver mirror was
deposited on a clean glass microscope slide by thermal evaporation
of the silver (99.995%) using vacuum deposition system (E302,
Edwards). The slides were immersed in a 1% aqueous polylysine
solution for one hour, forming a polylysine dielectric layer over
the silver film. Following rinsing in copious amount of water, the
slides were exposed overnight to a silver nanoparticle suspension
of optical density 5 at extinction maximum of 450 nm. The
self-assembly of the silver particles on the surface resulted in
the yellow hue (appearance) of the mirrors. The slides were then
rinsed with water and exposed to different analytes for various
time periods. After the adsorption of analyte molecules slides were
interrogated with Raman spectrometer yielding SERS spectra.
[0119] In the second example, silver nanoparticles were adsorbed on
the surface of the mirror using poly(vinylpyridine) as the surface
modifier (forming the dielectric layer). Poly(vinylpyridine) was
adsorbed on the mirrored silver and gold surfaces from 1% ethanolic
solutions for duration of several hours.
EXAMPLE 2
Preparation of Samples with Self Assembled Silver Particle
Nanostructure by Microcontact Printing
[0120] In this example, a method of microcontact printing as
disclosed for example in reference (H. S. Shin, et. al. "Direct
patterning of silver colloids by microcontact printing: possibility
as SERS substrate array", Vibrational Spectroscopy, v. 29, p.
79-82, 2002, H. Fan et al., "Self-Assembly of Ordered, Robust,
Three-Dimensional Gold Nanocrystal/Silica Arrays", Science, 304,
567-571 (2004), was used to form a close-packed array of silver
nanoparticles on a silver mirror.
[0121] Silver nanoparticles were prepared by method disclosed in
Lee P. C., Meisel, D. J., J. Phys. Chem., 86, p. 3391 (1982),
Poly(vinyl pyrrolidone) was used as the capping agent. First,
silver nitrate (0.2 g, Aldrich, 99+%) was dissolved into 3 mL
ethylene glycohol (Aldrich, 99.8%). 1 g polyvinyl pyrrolidone
(Aldrich, MW.apprxeq.40 000) was added into 15 mL ethylene glycohol
and the mixture was stirred and heated to 197.degree. C. The silver
nitrate in ethylene glycohol solution was subsequently injected
into heated poly(vinyl pyrrolidone). This reaction mixture was then
heated at 197.degree. C. for 1 hour. The silver nanoparticles were
precipitated by centrifugation. Specifically, the reaction mixture
was cooled to room temperature, diluted with acetone (about 10
times by volume), and centrifuged at 4000 rpm for 20 min, with the
liquid phase being removed using a pipette. The nanoparticles are
rinsed with water, and washed with acetone and water for 2-3 times,
to remove extra surfactants/polyvinyl pyrrolidone.
[0122] Glass slides used for silver deposition were first cleaned
by soaking in NaOH (Aldrich, 99%) solution (0.1 M NaOH in 75%
ethanol aqueous solution). After 2 hours, glass slides are washed
with ultrapure water and air-dried. A sliver thin film
(thickness=100 nm) was deposited on the cleaned glass slides by
Edwards EB3 e-beam evaporator in 432A. The obtained glass slides
were soaked into 1 wt % poly(vinyl pyridine) (Sigma, Mw.apprxeq.37
500) solution. After 4 hours, the slides were rinsed with ultrapure
water and air-dried. The slides were subsequently placed on a hot
plate and baked at 50.degree. C. for 15 minutes.
[0123] Silver nanoparticles in hexane solution were carefully
dropped onto water surface, where the hexane spreads on the water
surface to form a thin oil film. As hexane evaporates, the film
surface shrinks until all the hexane is gone and silver
nanoparticles are self-assembled into a close-packed monolayer.
[0124] These silver monolayers were transferred to the slice
surface by bringing the slide parallel to the water surface and
lightly touching the substrate to the nanoparticle film. Multiple
layers of silver nanoparticle could be achieved by repeating this
process. (shown as followed figure)
[0125] The slides were baked on a hot plate at 50.degree. C. for 15
minutes.
[0126] The method used in slide preparation is similar to method
disclosed in H. S. Shin, et. al. "Direct patterning of silver
colloids by microcontact printing: possibility as SERS substrate
array", Vibrational Spectroscopy, v. 29, p. 79-82, 2002, H. Fan et
al., "Self-Assembly of Ordered, Robust, Three-Dimensional Gold
Nanocrystal/Silica Arrays", Science, 304, 567-571, 2004.
[0127] An AFM topographic image of typical SERS substrate prepared
by this protocol is presented in FIG. 6, showing a high density
array of NP is close to periodic structure.
EXAMPLE 3
Experimental Measurements on the Analyte Rhodamine 6 G (R6G)
[0128] The experimental system set up used in present experiments
is shown on FIG. 7A-7C. Measurements were carried using
Horiba-Jobin Yvon Raman microscope LabRam HR 800.
[0129] Measurement of SERS spectra from liquid samples was carried
out using a fluidic cell made from borosilicate glass. A schematic
diagram of a fluidic cell is presented on FIG. 7A (top view) and in
FIG. 7B (cross sectional view). A glass fluidic cell contains
fluidic a channel 84 formed on a glass slide 14 to a depth of about
1.2 to 2.0 mm. The thickness' of the SERS-active structure 150 was
0.8 mm. During the experiment, the optimal value of parameters such
as depth of fluidic channel was determined, e.g., the best
conditions for focusing of the laser light beam through the
confocal objective in Raman microscope. Use of the glass cover slip
16 was critical in order to maintain the same thickness of analyte
layer during all sets of measurements. As a result an optimum depth
of fluidic channel of about 1.5 mm was determined.
[0130] Use of the fluidic cell also allows for determining an
accurate detection limit for analytes in solution, in terms of
concentration of analyte molecules in solution measured in units of
mole/liter. For that purposes a Langmuir adsorption isotherm was
determined for each analyte.
[0131] Aqueous solutions of Rhodamine 6 G (R6G) were prepared in a
range of concentrations from lowest 10.sup.-10 moles/l up to
10.sup.-3 moles/l. As a first step, measurements of Raman spectra
were taken from solutions with the lowest concentration of analyte
and subsequent measurement were done with the same SERS-active
plate but with increasing concentration of analyte. At each step of
the procedure, analyte solution was injected into the fluidic cell
using a pipette 160a, then covered by glass cover slip. After
measurement of the Raman spectra, analyte solution was replaced by
a new one at higher concentration, and the measurement was repeated
under conditions of focusing the illumination beam. The focus of
the Raman microscope was adjusted to obtain optimal illumination
condition, and these settings were used for all subsequent
measurements. During each next step, solution in fluidic cell was
replaced by solution with increasing concentration of analyte,
using pipette 160b to remove analyte solution.
[0132] FIG. 7C shows the experimental setup employed in the
measurements. In this figure, optical sensor nanostructure 150 is
irradiated by an optical beam 70 which is focused by a lens
assembly 72. Scattered light from the sample is focused in assembly
72, and directed by a beam-splitter 160 to a multichannel Raman
analysis system 170, with spectral analysis carried out on computer
180. This method allows for the use of the same SERS-active
substrate in multiple measurements. It also allowed for testing the
robustness of the substrate.
[0133] The results of representative experimental data obtained
with R6G using fluidic cell 1.5 mm deep and Raman microscope
Horiba-Jobin Yvon LabRam HR 800 are shown on FIGS. 8 to 11. From
these data, the quantitative limit of detection (LOD) was
determined for R6G to be 100 nano M/l. The LOD was define as the
first concentration at which distinctive spectral features of R6G
first appeared in Raman spectra.
[0134] FIG. 8 show SERS spectra of R6G at concentration 500
nanoM/L, with a laser power at sample of 4.1 .mu.W, integration
time 10 sec, wavelength of excitation light beam 514 nm, objective
50.times./0.45, with the light beam focused on the surface of the
substrate, and a diameter of focal spot at sample of 2 micron. This
spectra was obtained without subtraction of background. It can be
seen that that even at very low illumination power, Raman signal is
very strong, yielding 7000 counts per second for strongest
lines.
[0135] FIG. 9 shows SERS spectra of R6G at the same conditions and
set up as for the FIG. 8 experiment, except that the excitation
power at the sample was extremely small, as low as 0.4 .mu.W.
Although the Raman signal is less in this case (about 200 counts
per second), the signal to noise ratio that characterizes a quality
SERS spectra is still is very high, more than 100.
[0136] FIGS. 10A and 10B show Raman spectra images obtained by
mapping a 20 micron by 20 micron area of SERS substrate. Excitation
power in this experiment was 32 .mu.W, collection time for each
individual spectra was 1 second, mapped area was 20.times.20
micron, and the measurement of map was done with a 1 micron step
and a total number of spectra was 400 points. The whole map was
done for 7 min using automated motorized table system of
Horiba-Jobin Yvon LabRam HR 800 Raman Microscope.
[0137] FIG. 10A shows a Raman image for intensity of main peak of
R6G, integrated over the interval 1280-1400 cm.sup.-1 with baseline
correction, where intensity is given in %. The results show high
uniformity across the surface of enhancement properties of
SERS-substrate according to present invention. Maximum variation of
intensity of major spectral feature is less than 25% as illustrated
by FIG. 10B, where spectra with maximum and minimum intensity are
presented for comparison.
[0138] FIGS. 11A and 11B show the same data, but where the set of
SERS spectra are along one line consisting of 20 points, presented
as a 3-D plot in FIG. 11B.
[0139] The data demonstrate high uniformity across the surface of
the enhancement properties of SERS-substrate in the present
invention, meaning that a high density of "hot spot" that is
critical for practical use of SERS-substrate is achieved, and shows
the superiority of this SERS-substrate over others available in
prior art.
[0140] In particular, it has been discovered that substrates
prepared by the present invention have unusually strong enhancement
of Raman signal compared with other SERS substrates. Most
impressive is the fact that a strong Raman signal is achieved even
at 0.4 microWatt of illumination power (See data presented on FIG.
9). Experimental data show that SERS plates of the present
invention exceed the amplification of Raman signals achievable in
the currently existing SERS plates developed by Intel Precision
Biology Group (S. Chan et al., "Surface Enhanced Raman Scattering
of Small Molecules from Silver-coated silicon nanopore", Advanced
Materials, 15, 1595-1598, 2003, at least 5 to 6 orders of
magnitude. This means that the substrate of present invention can
provide a reproducible and stable amplification factor up to
10.sup.12 to 10.sup.14, where allowing for single molecule
sensitivity.
[0141] While the invention has been described with respect to
certain embodiments and applications, it will be appreciated how
various modifications and changes, and additional applications can
be made without departing from the invention.
* * * * *