focal point
HARVARD-MIT DIVISION
BY KATRIN KNEIPP AND HARALD KNEIPP
WELLMAN CENTER FOR PHOTOMEDICINE
OF HEALTH SCIENCES AND TECHNOLOGY
HARVARD UNIVERSITY, MEDICAL SCHOOL
BOSTON, MASSACHUSETTS 02114
Single Molecule Raman
Scattering
INTRODUCTION
D
etecting and identifying a
single molecule represents
the ultimate sensitivity limit
in chemical analysis. Tracking and
counting single molecules, characterizing their chemical structures, and
monitoring their structural changes
offer far-reaching opportunities in
basic and applied research. The first
spectroscopic detection of a single
molecule was achieved using laserinduced fluorescence.1–3 However,
the amount of molecular structural
information that can be obtained
from a fluorescence signal is limited,
particularly under ambient conditions. Ideally, one would wish for a
spectroscopic tool that detects a single molecule and simultaneously
identifies its chemical structure.
Therefore, vibrational spectroscopy,
such as Raman spectroscopy, would
be a preferred method for single
molecule studies due to the wealth of
chemical information it generates.
However, Raman scattering is a very
weak effect. Extremely small crosssections, typically ⬃10⫺30 to 10⫺25
cm 2, with the larger values occurring
only under favorable resonance Raman conditions, require a large number of molecules to achieve adequate
conversion rates from excitation laser photons to Raman photons,
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thereby precluding the use of Raman
spectroscopy as a method for single
molecule detection. For example, the
number of Stokes photons can be estimated as the product of the Raman
cross-section and the excitation intensity: Assuming a Raman line with
a scattering cross-section of 10⫺29
cm 2 and 100 mW excitation light focused to 1 ␮m 2, a single molecule
scatters ⬃10⫺4 photons per second,
which means that one must wait
more than an hour for a Stokes photon from a single molecule. Such
simple estimates made single molecule Raman spectroscopy science
fiction.
This situation is dramatically improved if surface-enhanced Raman
scattering (SERS) is used. The exciting phenomenon of a strongly increased Raman signal from molecules attached to a rough silver surface was discovered in 1977.4,5 Within the next few years, strongly
enhanced Raman signals were verified for many different molecules
that had been attached to various
metallic nanostructures. 6 For an
overview of SERS, see Refs. 7–15.
Estimated enhancement factors of
the Raman signal started with modest factors of 103 to 105 in the first
reports on SERS. Later, many authors claimed enhancement factors
of about 1010 to 1011 for dye mole-
cules in surface-enhanced resonance
Raman experiments (SERRS).16–22
At that time, it was already stated
that single molecule detection using
SERS should be possible and that
this technique would provide some
advantages over fluorescence, 21
which at that point was becoming a
single molecule detection tool.
About 20 years after the discovery
of SERS, new methods for determining effective cross-sections resulted in
unexpectedly large cross-sections on
the order of at least 10⫺16 cm2 per molecule, corresponding to enhancement
factors of about fourteen orders of
magnitude as compared with ‘‘normal’’ nonresonant Raman scattering.23
These enhancement factors exceeded
all former estimates by at least two
orders of magnitude. Interestingly,
these effective cross-sections were inferred for near-infrared (NIR) excitation that was not in resonance with
electronic transitions in the target molecule. The experiments demonstrated
that effective nonresonant SERS
cross-sections can reach the size of
fluorescence cross-sections of good laser dyes or may even exceed these
numbers. Effective cross-sections of
such size enable the detection of Raman scattering signals of a single molecule using NIR excitation.24–26 Single
molecule Raman spectroscopy can
also be achieved using surface-
molecules, or we will probe molecules exhibiting absorption in the ultraviolet range. These nonresonant
single molecule Raman experiments
can be understood in terms of an extremely high SERS enhancement
factor that overcomes a gap of fourteen orders of magnitude between a
nonresonant Raman cross-section
and cross-sections on the order of
⬃10⫺16 cm 2 typically required for
single molecule spectroscopy.
In the following section, we discuss the physics behind SERS at
such an extremely high enhancement
level, which is mainly based on
strongly enhanced and spatially
highly confined local optical fields in
the vicinity of gold and silver nanostructures. The third section describes single molecule SERS experiments in two different configurations; one deals with single molecule
Raman spectroscopy in solution using gold and silver nanoclusters as
SERS active substrates, and the second describes single molecule detection on a ‘‘dry’’ fractal silver surface. The next section covers single
molecule SERS spectroscopy at the
anti-Stokes side of the excitation laser. Due to the extremely high effective SERS cross-section, spontaneous anti-Stokes Raman scattering becomes effectively a two-photon process. The last section describes
potential applications of single molecule Raman spectroscopy. We briefly compare SERS and fluorescence
as single molecule tools and conclude with a short review regarding
the perspectives and limitations of
single molecule Raman scattering.
FIG. 1. (a) Schematic of surface-enhanced Raman scattering.11 (b) Typical silver (left)
and gold (right) nanoaggregates in different sizes.
PHYSICS BEHIND SINGLE
MOLECULE RAMAN
SCATTERING:
SURFACE-ENHANCED
RAMAN SCATTERING AT
EXTREMELY HIGH
ENHANCEMENT LEVELS
enhanced resonance Raman scattering
(SERRS).27–29
This Focal Point article will address single molecule Raman scattering. We will focus on single molecule SERS performed without ad-
Figure 1a shows the schematic of
a SERS experiment, where molecules are in the close vicinity of a
metallic nanostructure.11 In the schematic, this nanostructure is a small
aggregate built from spherical silver
or gold nanoparticles; for compari-
ditional contributions of intrinsic
molecular resonance Raman enhancement of the target molecules,
i.e., we will use near-infrared excitation light, which is nonresonant to
the electronic transitions of the target
APPLIED SPECTROSCOPY
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focal point
son, see the electron micrograph of
colloidal silver and gold particles in
Fig. 1b. Two effects might be operative in the SERS system shown in
Fig. 1a. (1) Electromagnetic enhancement: The excitation takes
place in the enhanced local optical
fields of the metal nanostructures.
Additionally, the Raman emission is
enhanced due to the nanostructure
working as a resonant nanoantenna.
(2) Chemical or electronic enhancement, first layer effect: A molecule
in contact with a metal (nanostructure) exhibits a ‘‘new Raman process’’ at a larger cross-section than
the Raman cross-section of a free
molecule. For more explanation of
the origin of ‘‘electromagnetic’’ and
‘‘chemical’’ SERS enhancement, see
Refs. 7–15.
The formula shown in Fig. 1a estimates the scattering signal in a
SERS experiment. The total Stokes
SERS signal PSERS (␯S ) is proportional to the Raman cross-section ␴RS
ads ,
the excitation laser intensity I(␯L),
and the number of molecules N⬘ involved in the SERS process. A(␯L)
and A(␯S ) express enhancement factors for the laser and for the Raman
scattered field, respectively. ␴RS
ads describes an enhanced cross-section of
the adsorbed molecule due to ‘‘electronic interaction’’ with the metal
compared to the cross-section ␴RS
free in
a ‘‘normal’’ Raman experiment.
From Fig. 1a we can define an effective SERS cross-section as
2
2 (1)
␴SERS
⫽ ␴RS
S
ads 円A(␯L)円 円 A(␯S )円
with
PSERS ⫽ N⬘␴SERS
nL
S
(2)
where nL is the number of photons
per cm 2 and the second corresponding I(␯L).
The total SERS enhancement factor is determined by the ratio of the
effective SERS cross-section and the
‘‘normal’’ Raman cross-section ␴RS
free:
GSERS ⫽
␴ RS
ads
円A(␯L )円 2 円A(␯S )円 2
␴ RS
free
(3)
With A(␯L) ⬃ A(␯S ), Eqs. 1–3 show
that the effective SERS cross-section
and the electromagnetic SERS enhancement factor depend on the field
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FIG. 2. Stokes and anti-Stokes Raman
transitions in a molecular level scheme
and the rate equation for population
and depopulation of the first excited vibrational state by Raman scattering.
enhancement A(␯) to the power of
four.
In general, GSERS can be experimentally inferred from a comparison
between surface-enhanced Raman
signals and normal Raman scattering
or fluorescence, taking into account
the different numbers of molecules
contributing to surface-enhanced and
‘‘normal’’ effects.11 However, SERS
enhancement factors were underestimated in all these experiments for
many years because of the incorrect
assumption that all molecules in a
SERS experiment contribute to the
observed SERS signal. In order to
avoid this assumption, a different approach was invented in which both
surface-enhanced Stokes and antiStokes Raman scattering were used
to extract information on the effective SERS cross-section. 23 The idea
behind this experiment was that a
very strong Raman process should
measurably populate the first excited
vibrational level in addition to the
thermal population. Figure 2 illustrates Stokes and anti-Stokes scattering in a molecular level scheme.
Population and depopulation of the
first excited vibrational state by Raman Stokes and anti-Stokes transitions can be described by the rate
equation shown in Fig. 2.11,23 In antiStokes Raman scattering, photons
are scattered by molecules in the first
excited vibrational state. Therefore,
vibrational population pumping to
this state by the strong Raman process should result in an unexpectedly
high anti-Stokes signal. For extremely high SERS enhancement factors,
this higher anti-Stokes signal can indeed be observed and the effective
Raman cross-section operative in the
vibrational pumping process can be
estimated from the deviation of the
anti-Stokes to Stokes signal ratio
from the expected Boltzmann population. Anti-Stokes SERS under the
condition of vibrational pumping is
described in more detail in another
section below. At this point, using
this approach, we inferred effective
SERS cross-sections on the order of
at least 10⫺16 cm 2. In order to make
the large cross-section consistent
with the observed Stokes signal, we
must draw the conclusion that the
number of molecules involved in the
scattering process must be extremely
small. 23
It should be noted that the extremely high level of SERS enhancement has been obtained at near-infrared excitation energies that are not in
resonance with electronic transitions
in the target molecule. Under these
conditions, effective cross-sections
on the order of 10⫺16 cm 2 imply
SERS enhancement factors on the
order of 1014. However, under resonant conditions, the requirements for
the surface enhancement effect can
be greatly lessened. For example,
‘‘normal’’ resonance Raman scattering (RRS) that brings RRS crosssections to a level of about 10⫺26 cm 2
lessens the requirement for SERS
enhancement in single molecule experiments to ⬃1010. The resonance
contribution in single molecule Raman spectroscopy is particularly pronounced for single wall carbon nanotubes (SWNTs), which exhibit an extremely strong resonance Raman effect based on resonances of the
excitation and/or scattered photons
with the van Hove singularities in
the electronic energy levels of these
one-dimensional structures.30 The
high intrinsic resonance Raman
cross-sections of SWNTs allow the
detection of strong SERS signals
from a single SWNT, exploiting relatively low electromagnetic SERS
enhancement factors of ⬃103, as can
be obtained in so-called tip-enhanced SERS.31
Here we want to focus on single
molecule SERS without the contribution of molecular resonance and
we discuss how the extremely high
SERS enhancement factors required
for these experiments can be
achieved. Vibrational pumping experiments give evidence for the existence of such high SERS enhancement factors, but they do not provide
information about the origin of the
high enhancement level. In principle,
electromagnetic and chemical effects
can account for this effect.
Interestingly, to our best knowledge, extremely high SERS enhancement and single molecule sensitivity
has never been observed for isolated
metal nanoparticles. The fact that extremely high SERS enhancement
factors are always associated with
composites formed by gold or silver
nanoparticles provides strong indication that single molecule Raman
spectroscopy is primarily a phenomenon associated with extremely high
local optical fields in the vicinity of
specific silver or gold nanostructures. 24
Mainly two kinds of composites
of metal nanoparticles can give rise
to giant electromagnetic SERS enhancement along with a high confinement of the local field, so-called
‘‘hot spots’’. Extremely high field
enhancement can be obtained for dimers and small aggregates formed
from silver and gold nanoparticles.32,33 Strong field enhancement
exists particularly at the intersection
between two nanoparticles. The other class of SERS active structures
that provides an extremely high local
optical field are fractal types of nanostructures.34–40 Electromagnetic effects in self-similar structures can result in SERS enhancement factors up
to 1012 and even higher, leaving a
factor of 10–100 that should be ascribed to a chemical enhancement
effect. Other studies mainly use the
chemical mechanisms involving ballistic electrons in order to explain extremely large SERS enhancement
factors in single molecule SERS. 29,41
A strong chemical contribution in
single molecule Raman experiments
might also be supported by a recent
report claiming that small silver
clusters consisting of a few silver atoms can result in strongly enhanced
Stokes and anti-Stokes Raman signals.42 On the other hand, recent
studies show that combining plasmon resonances and photonic resonances can give rise to electromagnetic enhancement factors sufficient
for single molecule Raman detection
without chemical enhancement.43 In
general, the extent of chemical/electronic enhancement to single molecule SERS remains a subject of discussion. The key role of the electromagnetic enhancement mechanism is
also strongly supported by experimental results obtained for nonlinear
Raman effects such as the scaling
of the enhancement factors for surface-enhanced Raman (SERS) and
surface-enhanced hyper Raman
(SEHRS) scattering, as they can be
predicted for electromagnetic field
enhancement.44
So far, aggregates formed by silver or gold nanoparticles provide the
highest enhancement level, where
nanoparticles of both metals show
totally different SERS enhancement
factors depending on whether they
are used as isolated particles or as
aggregates. A gap of ten orders of
magnitude exists between SERS enhancement factors for isolated gold
nanoparticles measured at 514 nm
excitation and gold nanoclusters
measured at NIR excitation.37 For
silver nanoclusters, the SERS enhancement factor measured at 830
nm excitation has been found to be
seven orders of magnitude higher
than enhancement factors for isolated silver colloidal particles, even
when excited at their plasmon resonance at 407 nm. 26
To study the dependence of the
enhancement factor on the size of the
aggregates, Fig. 3 compares Stokes
and anti-Stokes signals measured
from clusters from ⬃1 ␮m in size
and from silver clusters ⬃200 nm in
size. 26 Comparing only the Stokes
(or anti-Stokes) signals in both experiments shows that the 1 ␮m cluster exhibits a higher signal level
compared to smaller clusters. This
could lead to the erroneous conclusion that when the number of particles in the aggregate increases, the
relative SERRS activity also increases.45 However, as Fig. 3 demonstrates, the ratios between antiStokes and Stokes SERS signals are
constant within the accuracy of our
measurement. That means that the
vibrational pumping rate, or, in other
words, the SERS enhancement factor
in the hot spots, must be the same
for both cluster sizes. The fact that
SERS enhancement factors are independent of the size and shape of
the clusters has important consequences for single molecule spectroscopy as it results in almost the
same scattering signal observed from
a single molecule. This has useful
consequences for the capabilities of
SERS as a single molecule tool, such
as the potential for counting of single molecules.
An interesting question involves
the spatial dimensions of the hot
spots on silver or gold clusters or aggregates. Since SERS takes place in
the local fields of metallic nanostructures, the probed volume is determined by the confinement of these
local fields. The spectroscopic selection of single SWNTs within a bundle shows that the SERS signal must
have been collected from dimensions
smaller than 5 nm in order to select
a single or very few SWNTs from
the neighboring tubes in the bundle.46,47 The strongly confined hot
spots provide the opportunity to
probe volumes that can be two orders of magnitude smaller than the
diffraction limit. Figure 4 illustrates
that SERS can spectroscopically select single nano-objects or molecules
within a larger population.14 Despite
the density of rhodamine 6G molecules on the fractal silver surface,
which results in ⬃100 molecules in
the spot size of the laser, when scanning over the surface, there are a few
APPLIED SPECTROSCOPY
325A
focal point
FIG. 3. Stokes and anti-Stokes SERS spectra of 10 ⫺8 M CV adsorbed on silver clusters of different sizes measured at 830 nm excitation.
FIG. 4. (Top) Many molecule and (bottom) single molecule SERS spectra of rhodamine
6G measured at different places on a fractal silver surface with approximately 100
molecules in the 1 ␮m laser spot.
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points where the collected spectra
are associated with a single molecule
compared to the inhomogeneously
broadened spectra usually measured
from this sample.
Enhanced and strongly confined
fields are always associated with
high field gradients. These large field
gradients may also influence the Raman spectrum, leading to relaxed selection rules or changed depolarization ratios for single molecule spectra that are always measured in such
high field gradients.48,49 Of particular
interest is the prediction that high local optical fields may also induce
and increase the effect of Raman optical activity.50 This was confirmed
in recent experiments showing surface-enhanced Raman optical activity (SEROA).51
Interesting and of direct importance for single molecule Raman detection is the observation that high
field gradients may direct molecules
to the hot spot and hold them there
for detection.14 This ensures that
most of the molecules that are attached to a nanostructure will ‘‘find’’
a spot and will be detected in a
SERS experiment.
SINGLE MOLECULE
SURFACE-ENHANCED
RAMAN SCATTERING
EXPERIMENTS
As mentioned above, we focus on
single molecule experiments where
no resonance Raman effect for the
target molecule is required. In the
previous discussion we have shown
that for colloidal silver and gold
clusters strong SERS enhancement
factors exist in the NIR wavelength
range. Fortunately, high-intensity
NIR diode lasers are easily available,
making this region also attractive for
compact, low-cost Raman instrumentation. Further, the development
of low noise, high quantum efficiency multichannel detectors (chargecoupled device (CCD) arrays), combined with high-throughput singlestage spectrographs used in combination with holographic laser
rejection filters, has led to high sensitivity Raman spectrometers. In
general, a state-of-the-art Raman
system has components that allow
the performance of single molecule
SERS experiments.
Single Molecule Surface-Enhanced Raman Scattering on Silver and Gold Nanoclusters in Solution. For single molecule detection
in solution, small silver and gold colloidal clusters in sizes between about
100 and 500 nm are very useful
SERS active substrates. At very low
analyte concentrations of the target
molecule (approximately 10⫺11 M
and lower), when the concentration
of target molecules becomes comparable to or smaller than the concentration of the colloidal clusters,
no analyte-induced cluster–cluster
aggregation occurs, and the SERS
spectra show a very good reproducibility. The smallest colloidal clusters we used in single molecule
SERS experiments are ⬃150 nm in
size, formed by only 5–10 individual
nanoparticles. These nanoclusters
provide at least one hot spot suitable
for single molecule measurements.14
No ‘‘cold’’ clusters were obtained as
reported for dimers and trimers.
Figure 5 shows a schematic of a
typical single molecule SERS experiment performed in silver or gold
colloidal solution. 24–26 Spectra are
excited by 830 nm laser light. A microscope attachment is used for laser
excitation and collection of the Raman scattered light. It is important,
particularly for ensuring the ‘‘free’’
Brownian motion of the nanoparticles into and out of the probed volume, to keep the NIR excitation laser
at relatively low intensities (⬍106 W
cm⫺2) in order to avoid any trapping
effect for the nanoclusters. The effect of trapping is discussed in detail
in Ref. 52. Here we avoid the effect
in order to perform a correct statistical analysis of the scattering signals.
Analyte concentrations on the order of 10⫺12 to 10⫺14 M and probed
volumes whose sizes are on the order
of femtoliters to picoliters result in
average numbers of one or fewer target molecules in the focus volume.
The Brownian motion of single-analyte-molecule-loaded silver or gold
clusters into and out of the probed
volume results in strong statistical
changes in the Raman signals measured from such a sample in time sequence. Figure 5b shows typical unprocessed SERS spectra measured
from a sample with an average of 0.6
crystal violet molecules in the
probed 30 pL volume. Figure 5c displays the peak heights of one characteristic Raman line of the target
molecule measured time sequence.
The signals appear at different power
intervals, which can be assigned
‘‘0’’, ‘‘1’’, ‘‘2’’, and ‘‘3’’-molecule
events. The statistical distribution of
the ‘‘0.6 molecule SERS signal’’ exhibits four relative maxima that are
fit by the superposition of four
Gaussian curves. The gradation of
the areas of the four statistical peaks
are consistent with a Poisson distribution for an average number of 0.5
molecules. This reflects the probability of finding 0, 1, 2, or 3 molecules in the scattering volume during
the actual measurement. Comparing
the measured Poisson distribution
with the 0.6 molecule concentration/
volume estimate shows that about
80% of molecules are detected by
SERS. In the meantime, these SERS
experiments in solutions using small
silver colloidal aggregates as SERS
active substrates have been repeated
by several groups, and the Poisson
distribution of single molecule SERS
signals has been corroborated in several studies.53,54
In general, single molecule experiments should not suffer from the
problem that one does not know how
many molecules contribute to the
SERS signal. Due to the Poisson statistics the measured signal comes
from 1, 2, or 3 molecules and therefore these experiments should allow
one to infer SERS enhancement factors in the correct order of magnitude
in a straight forward way by comparing SERS signals of single molecules with normal Raman signals of
a known large number of molecules.
Figure 6 shows Raman spectra measured from an aqueous solution of
silver nanoaggregates containing
10⫺14 M pseudoicocyanine (PIC) and
5 M methanol, which yields one PIC
molecule and ⬃1014 methanol molecules in the probed volume. The experiment shows that the SERS signals of a single PIC molecule appear
at the same signal level as the nonenhanced Raman signal of the 1014
methanol molecules, confirming
SERS enhancement factors on the
order of fourteen orders of magnitude, in agreement with the results
obtained from anti-Stokes to Stokes
signal ratios (see also the next section). As we discussed regarding Fig.
5, and also Fig. 6, Raman lines assigned to PIC appear at varying signal levels due to Brownian motion of
the silver colloidal cluster carrying
single PIC molecules into and out of
the probed volume. Data analysis of
the PIC SERS signals results in a
Poisson distribution, whereas the Raman signal of the 1014 methanol molecules remains statistically constant
within a Gauss distribution (data not
shown here, see Ref. 25).
In addition to changes in single
molecule SERS signal strengths that
APPLIED SPECTROSCOPY
327A
focal point
FIG. 5. Single molecule SERS spectroscopy. (A) Schematic of the experimental setup. (B) Typical SERS spectrum of a single molecule
of crystal violet attached to a silver nanocluster; the 1170 cm ⫺1 Raman line used for the statistical analysis is marked. (C) Peak
heights of the 1174 cm ⫺1 crystal violet Raman line for the 100 SERS spectra collected in time sequence, 1 second each. (D) Statistical analysis of the SERS signals shown in (C).
follow the expected Poisson statistics, SERS spectra collected at the
single molecule level in time sequence can also show fluctuations
and changes in the spectrum, such as
the appearance of new Raman lines.
Often the new spectral features do
not correlate with the spectral signal
strengths of the target molecule.
These ‘‘spectral fluctuations’’, or
‘‘blinking’’, can have different reasons depending on the experimental
situation. We will come back to the
temporal changes in SERS spectra in
the next section. In SERS experiments performed on silver or gold
nanoaggregates in solution as described above, the ‘‘new’’ Raman
lines can be ascribed to signals of
impurities on the surface of the col328A
Volume 60, Number 12, 2006
loidal particles, which are probably
introduced during the chemical preparation process. The impurity spectra
change due to different colloidal
clusters loaded with different impurities, which move into the focal volume. In SERS experiments using
larger probed volumes and extremely low concentrations (in order to
achieve one or less target molecules
in the probed volume), SERS signals
of the target molecule can vanish in
a background of impurities. This observation was discussed in the first
studies of SERS spectroscopy at the
single molecule level. 21,55 Figure 7
demonstrates this effect for the
SERS spectrum of rhodamine 6G in
silver colloidal solution.
Single Molecule Surface-En-
hanced Raman Scattering on
Fixed Fractal Silver and Gold
Cluster Structures. Particularly
high local optical fields can also exist on extended silver and gold fractal structures such as larger aggregates of colloidal particles or evaporated metal island films.35,56 Here
we demonstrate the capability of
fractal silver surfaces for single molecule detection using the small protein enkephalin as a target molecule.57 Enkephalin is a mixture of
two pentapeptides, (Leu)enkephalin,
and (Met)enkephalin. It was brought
to the silver surface in a concentration resulting in an average of one
molecule in the ⬃1 ␮m laser spot.
The molecule can be detected based
on the strongly enhanced ring-
FIG. 6. Raman spectra measured from a probed volume that contains an average of
one pseudoicocyanine molecule attached to a silver colloidal cluster and ⬃10 14 methanol molecules, ⬃100 mW cw ex-citation at 830 nm, 1 second collection time per spectrum. 25
FIG. 7. SERS spectra of rhodamine 6G in silver colloidal solution in concentrations
between 10 ⫺11 and 10 ⫺16 M along with SERS spectra of impurities.
breathing mode of phenylalanine
(⬃1000 cm⫺1), which is a building
block of both pentapeptides. Measuring only one typical SERS line of
the target molecule and using this
Raman line as an intrinsic spectroscopic signature for the specific molecule is a useful tool for detecting a
known molecule without the use of
labels.
The top panel of Fig. 8 shows selected single molecule SERS spectra
measured in a time interval in a
spectral window, which displays the
1000 cm⫺1 SERS line of phenylalanine and a line at about 750 cm⫺1,
which can be ascribed to impurities
on the silver colloids. The bottom
panel of Fig. 8 displays the Raman
signal measured at 1000 cm⫺1 shift
from the 830 nm excitation from the
same ⬃1 ␮m spot in a time sequence. Over the first 95 seconds, no
SERS signal was measured. Obviously, the laser spot was in an area
where statistically no target molecule
is present. Then, a SERS signal appears relatively abruptly, stays for
about 20 seconds at the same level,
and vanishes again. Such behavior,
namely, the appearance of the 1000
cm⫺1 SERS signal within a 10–30
second time window, has been observed in many measurements on
single enkephalin molecules. If the
enkephalin signal appears, it always
appears at the same level. Spectra B
through E in the top panel of Fig. 8
were measured in the time window
between 95 and 115 seconds, while
spectrum A shows a situation when
the 1000 cm⫺1 SERS signal does not
exceed the noise level. A possible
explanation for these changes in
scattering power is that a single enkephalin molecule is diffusing on the
colloidal silver cluster and can only
be ‘‘seen’’ when it enters a hot spot.
Entering and leaving a spot can be
explained by the discontinuous illumination: the laser was illuminating
the sample during the one-second
collection time and was closed by a
shutter for about one second between
the measurements.
Fluctuations in scattering power
and/or sudden spectral shifts and
changes that appear as ‘‘blinking’’ of
APPLIED SPECTROSCOPY
329A
focal point
the SERS signals has been reported
by several authors. 27,28,58 Different effects can account for these spectral
changes, such as thermally and nonthermally activated diffusion of molecules,59–61 as well as real transformations, such as protonation and deprotonation.62,63 Although blinking
had been claimed as a hallmark of
single molecule detection, it has become evident that this behavior is
not necessarily connected to single
molecule SERS. The effect has also
been observed in lower concentration ‘‘many-molecule’’ SERS spectra.64
PUMPED ANTI-STOKES
SURFACE-ENHANCED
RAMAN SCATTERING:
A TWO-PHOTON EXCITED
RAMAN EFFECT
As described before, SERS can result in unexpectedly high anti-Stokes
to Stokes signal ratios associated
with the population of the excited vibrational state due to vibrational
pumping. 23 It should also be noted
that anomalies in anti-Stokes to
Stokes SERS signal ratios could be
explained by molecular or charge
transfer resonance effects, which can
be selectively efficient for Stokes
and anti-Stokes scattering.65,66 Similar ‘‘asymmetry effects’’ can occur
for a situation when Stokes or antiStokes have different electromagnetic enhancement conditions due to
different resonances with surface
←
FIG. 8. (Top) Selected single molecule
SERS spectra of enkephalin from one
fixed spot on a fractal silver surface
showing the 1000 cm ⫺1 SERS line of
phenylalanine and a line at 750 cm ⫺1,
which can be ascribed to impurities on
the silver colloids. (Bottom) Time series of
the 1000 cm ⫺1 phenylalanine signal
measured from one fixed spot on a sample with an average of one enkephalin
molecule in the laser spot. Spectra were
observed over a time interval of 120
seconds, 1-second collection time each.
The signal level of 100 cps represents
approximately the background level.
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Volume 60, Number 12, 2006
plasmons resulting in different effective cross-sections for Stokes and
anti-Stokes SERS. 23 All these abnormal anti-Stokes to Stokes signal ratios are independent of the excitation
intensity. However, this situation is
different for vibrational pumping.
Simple equations for the antiStokes (Eq. 4) and Stokes signals
(Eq. 5) can be derived from Fig. 2
assuming steady state and weak saturation (exp(⫺h␯M /kT) ⱕ ␴SERS
␶1nL
S
K 1). Under steady-state and weak
saturation conditions a continuous
wave (cw) laser-excited Raman process populates the first excited vibrational state comparable to or higher
than the Boltzmann population, but
still far away from approaching equilibrium population between N0 and
N1:
PSERS
⫽ (N0 e⫺h␯M /kT ⫹ N0 ␴ SERS ␶1 nL )
aS
⫻ ␴ SERS nL
(4)
PSERS
⫽ N0 ␴ SERS n L
S
(5)
where ␶1 is the lifetime of the first
excited vibrational state, T is the
sample temperature, and h and k are
the Planck and Boltzmann constants,
respectively. The first term in Eq. 4
describes the anti-Stokes signal due
to thermal population of the first excited vibrational state. The second
term occurs due to vibrational pumping by the strong Stokes process. In
normal Raman scattering this term
can be neglected compared to the
thermal population since for normal
Raman scattering the product
␴RS (␯m)·␶1(␯m) is on the order of 10⫺40
cm 2 s. That means that about 1038 laser photons cm⫺2 s⫺1 (approximately
10 20 W cm⫺2 excitation intensity) are
required to make the Raman pumping comparable to the thermal population of the first vibrational level.
In order to account for the experimental observations in anti-Stokes
and Stokes SERS, the product of
cross-section and vibrational lifetime
in Eq. 4 must be on the order of
10⫺27 cm 2 s. Assuming vibrational
lifetimes on the order of 10 ps, the
surface-enhanced Raman cross-section is then estimated to be at least
⬃10⫺16 cm 2, corresponding to SERS
FIG. 9. Anti-Stokes and Stokes signal versus excitation intensity measured from crystal violet attached to fixed silver nanoclusters.
enhancement factors on the order of
1014.23
As the second term in Eq. 4
shows, anti-Stokes Raman scattering
starting from a pumped vibrational
level is a two-photon Raman process: one photon populates the excited vibrational state, while a second photon generates the anti-Stokes
scattering. The anti-Stokes signal PaS
depends quadratically on the excitation laser intensity, whereas the
Stokes signal PS remains linearly dependent (Eq. 5). Figure 9 shows quadratic and linear fits to the experimental data of SERS anti-Stokes and
Stokes signal powers of crystal violet versus excitation intensity, displaying the predicted quadratic and
linear dependence.
In general, a nonlinear dependence of the anti-Stokes signal on
the excitation intensity could also be
caused by an increase of temperature
due to laser heating. 23,46 In order to
exclude this effect and to provide evidence that the quadratic dependence
is due to vibrational pumping, SERS
studies have been performed on single wall carbon nanotubes (SWNTs)
on silver aggregate clusters.46,67
SWNTs show a strong dependence
of their Raman frequencies on temperature. This allows for the monitoring of temperature changes during
the SERS measurement. The observed very small shifts in frequencies along with a quadratic dependence of the anti-Stokes signal show
that temperature changes in the sample are too small to explain the observed nonlinear dependence on the
laser excitation intensity.
Of course, vibrational pumping
also exists in cases of surface-enhanced resonance Raman scattering.
Under resonance conditions, possible heating (and photodecomposition) of the target molecule can result in various regimes of interferences between heating and pumping.68,69
Considering only the second term,
we can write Eq. 4 as:
SERS 2
PSERS
aS,nl ⫽ N0 ␴aS,nl nL
(6)
using an effective two-photon crosssections ␴SERS
aS,nl with
SERS ␴SERS ␶
␴SERS
aS,nl ⫽ ␴S
aS
1
(7)
Assuming a SERS cross-section of
approximately 10⫺16 cm 2 and a viAPPLIED SPECTROSCOPY
331A
focal point
FIG. 10.
Potential application of single molecule SERS for DNA sequencing (see text).
brational lifetime on the order of 10
picoseconds, effective two-photon
cross-sections can be inferred to be
about 10⫺43 cm4 s. This cross-section
can be understood in terms of the
‘‘real’’ intermediate state in this twophoton process formed by the excited vibrational state.70
APPLICATIONS OF
SINGLE MOLECULE
SURFACE-ENHANCED
RAMAN SCATTERING
The observation of Raman signals
at the single molecule level is of basic scientific interest as it provides
insight into the intrinsic properties of
a molecule or a nanostructure as well
as structural changes without ensemble averaging. But detection and differentiation of single molecules using their specific SERS signatures is
also of practical interest. Applications range from the use of Raman
332A
Volume 60, Number 12, 2006
spectroscopic characterization of
specific DNA fragments down to
structurally sensitive detection of
single bases without the use of fluorescent or radioactive tags. One of
the most spectacular potential applications of single molecule SERS
might be in the field of rapid DNA
sequencing at the single molecule
level. The idea is described in Fig.
10. The nucleotide bases show welldistinguished surface-enhanced Raman spectra, also shown in Fig. 10.
Thus, after cleaving single native nucleotides from the DNA or RNA
strand into a medium containing colloidal silver or gold clusters, direct
detection and identification of single
native nucleotides should be possible
using the unique SERS spectra of
their bases. SERS active silver or
gold nanoclusters can be provided in
a flowing stream of colloidal solution or onto a moving surface with
silver or gold cluster structures, enabling the detection of the bases in
order when the nucleotide-loaded
nanoclusters move through the laser
beam. 26 Effective SERS cross-sections on the order of 10⫺16 cm 2 can
be inferred for adenosine monophosphate (AMP) and for adenine on colloidal silver clusters. NIR SERS
spectra for both compounds are identical, indicating that sugar and phosphate bonds do not interfere with the
strong SERS effect of adenine.
It is interesting to estimate the
detection rate of single nucleotides
in such an experiment. Single molecule adenine spectra can be measured at good signal-to-noise ratios
of 10 in 1-second collection time at
3 ⫻ 105 W cm⫺2 excitation. Assuming a SERS cross-section on the order of 10⫺17 to 10⫺16 cm 2 and a vibrational lifetime on the order of 10
picoseconds, saturation of SERS will
be achieved at 108 to 109 W cm⫺2
excitation intensity. Extrapolation to
saturation conditions shows that single molecule SERS spectra over the
complete fingerprint region (approximately 700–1700 cm⫺1) should be
measurable in one millisecond or at
a detection rate of one kHz.
PERSPECTIVES AND
LIMITATIONS OF SINGLE
MOLECULE RAMAN
SPECTROSCOPY
Numerous experiments conducted
during the past decade have demonstrated that SERS enables the measurement of Raman spectra of single
molecules, one at a time. When the
target molecule is attached to a silver
or gold nanostructure, the nonresonant Raman signal can be enhanced
up to 14 orders of magnitude, and
effective Raman cross-sections can
reach and even exceed fluorescence
cross-sections of dyes used as fluorescence labels. In principle, electromagnetic and/or chemical effects can
account for this enhancement level.
There is evidence that single molecule SERS is primarily a phenomenon associated with strongly enhanced and geometrically confined
local optical fields.
As a single molecule tool, SERS
opens up exciting capabilities as
compared with fluorescence, another
widely used single molecule technique. Currently, because of the high
structural information content of a
Raman spectrum, SERS is the only
way to detect a single molecule and
simultaneously identify its chemical
structure. Additionally, measuring
only one SERS line, which is characteristic for the target molecule, and
using this Raman line as an intrinsic
marker for the specific molecule is
of interest for detecting and tracking
single molecules without the use of
fluorescence or radioactive labels.
Raman scattering is a very general
property of a molecule, and almost
every molecule has Raman active
molecular vibrations that can be
‘‘seen’’ by the Raman effect at a
cross-section of at least 10⫺30 cm 2,
whereas far fewer molecules show
fluorescence. For the detection of a
molecule by SERS, it has to be attached to a SERS active substrate in
order to increase its effective Raman
cross-section by 12 to 14 orders of
magnitude. Due to the primarily
electromagnetic origin of this enhancement, it should be possible to
achieve an equally strong SERS effect for each molecule, thus making
SERS a single molecule tool for a
broad range of molecules.
Another interesting aspect of
SERS for single molecule detection
involves the total number of photons
that can be emitted by a molecule.
This is determined by the maximum
number of excitation–emission cycles a molecule survives. In fluorescence experiments, this number is
limited by the rate of photobleaching
of the molecule. As a process that is
electronically nonresonant to the target molecule, SERS avoids photodecomposition. Also in SERRS experiments, photodecomposition of
the molecules adsorbed on the metal
is strongly reduced as compared to
molecules in solution. 27
Another number that is of particular interest for the rapid detection
and screening of single molecules is
the maximum number of photons
that can be emitted by a molecule
per time interval. Under saturation
conditions, this number is inversely
proportional to the lifetime of the excited molecular states involved in the
optical detection process. Due to the
shorter vibrational relaxation times
as compared to the electronic relaxation times, a molecule can go
through more Raman cycles than
fluorescence cycles per time interval.
Therefore, the number of Raman
photons per time interval that can be
emitted by a molecule under saturation conditions can be higher than
the number of fluorescence photons
by a factor of 10 2 to 103.21
Limitations of SERS spectroscopy
are attributed to the fact that target
molecules must be attached to silver
or gold nanostructures. However,
chemically inert nanometer gold particles might be the favorite choice in
several applications compared to
fluorescent dyes or radioactive labels, particularly for biomedical ap-
plications. Moreover, in some applications, this attachment of the target
molecule to a ‘‘bigger’’ nanoparticle
can be an advantage for single molecule detection, as increased diffusion times minimize the possibility
of the target molecule escaping from
the focal volume of the laser too rapidly.
ACKNOWLEDGMENTS
This work is supported in part by DOD
grant #AFOSR FA9550-04-1-0079 and by the
generous gift of Dr. and Mrs. J. S. Chen to
the optical diagnostics program of the Massachusetts General Hospital, Wellman Center
for Photomedicine.
1. M. Eigen and R. Rigler, Proc. Natl. Acad.
Sci. U.S.A. 91, 5740 (1994).
2. P. M. Goodwin, W. P. Ambrose, and R. A.
Keller, Acc. Chem. Res. 29, 607 (1996).
3. S. M. Nie, D. T. Chiu, and R. N. Zare,
Science (Washington, D.C.) 266, 1018
(1994).
4. D. L. Jeanmaire and R. P. V. Duyne, J.
Electroanal. Chem. 84, 1 (1977).
5. M. G. Albrecht and J. A. Creighton, J.
Am. Chem. Soc. 99, 5215 (1977).
6. H. J. Seki, J. Electron Spectrosc. Relat.
Phenom. 39, 239 (1986).
7. A. Otto, in Light Scattering in Solids IV.
Electronic Scattering, Spin Effects, SERS
and Morphic Effects, M. Cardona and G.
Guntherodt, Eds. (Springer-Verlag, Berlin, Germany, 1984), pp. 289–418.
8. M. Moskovits, Rev. Mod. Phys. 57, 783
(1985).
9. K. Kneipp, Exp. Techniques Phys. 38, 3
(1990).
10. A. Campion and P. Kambhampati, Chem.
Soc. Rev. 27, 241 (1998).
11. K. Kneipp, H. Kneipp, I. Itzkan, R. R.
Dasari, and M. S. Feld, Chem. Rev. 99,
2957 (1999).
12. M. Moskovits, J. Raman Spectrosc. 36,
485 (2005).
13. C. L. Haynes, C. R. Yonzon, X. Y. Zhang,
and R. P. Van Duyne, J. Raman Spectrosc.
36, 471 (2005).
14. K. Kneipp, H. Kneipp, and J. Kneipp,
Acc. Chem. Res. 39, 443 (2006).
15. K. Kneipp, M. Moskovits, and H. Kneipp,
Eds., Surface-Enhanced Raman Scattering (Springer, Heidelberg, 2006).
16. A. Bachackashvilli, S. Efrima, B. Katz,
and Z. Priel, Chem. Phys. Lett. 94, 571
(1983).
17. K. Kneipp, G. Hinzmann, and D. Fassler,
Chem. Phys. Lett. 99, 503 (1983).
18. P. Hildebrandt and M. Stockburger, J.
Phys. Chem. 88, 5935 (1984).
19. K. Kneipp, H. Kneipp, and M. Rentsch,
J. Mol. Struct. 156, 331 (1987).
20. B. Pettinger and K. Krischer, J. Electron
Spectrosc. Relat. Phenom. 45, 133 (1987).
21. K. Kneipp, Experimentelle Technik der
Physik 36, 161 (1988).
APPLIED SPECTROSCOPY
333A
focal point
22. B. Pettinger, K. Krischer, and G. Ertl,
Chem. Phys. Lett. 151, 151 (1988).
23. K. Kneipp, Y. Wang, H. Kneipp, I. Itzkan,
R. R. Dasari, and M. S. Feld, Phys. Rev.
Lett. 76, 2444 (1996).
24. K. Kneipp, Y. Wang, H. Kneipp, L. T.
Perelman, I. Itzkan, R. R. Dasari, and M.
S. Feld, Phys. Rev. Lett. 78, 1667 (1997).
25. K. Kneipp, H. Kneipp, G. Deinum, I. Itzkan, R. R. Dasari, and M. S. Feld, Appl.
Spectrosc. 52, 175 (1998).
26. K. Kneipp, H. Kneipp, V. B. Kartha, R.
Manoharan, G. Deinum, I. Itzkan, R. R.
Dasari, and M. S. Feld, Phys. Rev. E 57,
R6281 (1998).
27. S. Nie and S. R. Emory, Science (Washington, D.C.) 275, 1102 (1997).
28. H. X. Xu, E. J. Bjerneld, M. Kall, and L.
Borjesson, Phys. Rev. Lett. 83, 4357
(1999).
29. A. M. Michaels, M. Nirmal, and L. E.
Brus, J. Am. Chem. Soc. 121, 9932
(1999).
30. M. S. Dresselhaus, G. Dresselhaus, A. Jorio, A. G. Souza, G. G. Samsonidze, and
R. Saito, J. Nanosci. Nanotechnol. 3, 19
(2003).
31. A. Hartschuh, E. J. Sanchez, X. S. Xie,
and L. Novotny, Phys. Rev. Lett. 90,
095503 (2003).
32. M. Inoue and K. Ohtaka, J. Phys. Soc.
Jpn. 52, 3853 (1983).
33. H. X. Xu, J. Aizpurua, M. Kall, and P.
Apell, Phys. Rev. E 62, 4318 (2000).
34. K. Li, M. I. Stockman, and D. J. Bergman, Phys. Rev. Lett. 91, 227402 (2003).
35. M. I. Stockman, V. M. Shalaev, M. Moskovits, R. Botet, and T. F. George, Phys.
Rev. B 46, 2821 (1992).
36. E. Y. Poliakov, V. M. Shalaev, V. A. Markel, and R. Botet, Opt. Lett. 21, 1628
(1996).
37. K. Kneipp, H. Kneipp, R. Manoharan, E.
334A
Volume 60, Number 12, 2006
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
B. Hanlon, I. Itzkan, R. R. Dasari, and M.
S. Feld, Appl. Spectrosc. 52, 1493 (1998).
Y. Yamaguchi, M. K. Weldon, and M. D.
Morris, Appl. Spectrosc. 53, 127 (1999).
Z. J. Wang, S. L. Pan, T. D. Krauss, H.
Du, and L. J. Rothberg, Proc. Natl. Acad.
Sci. U.S.A. 100, 8638 (2003).
M. Stockman, in Surface Enhanced Raman Scattering, K. Kneipp, M. Moskovits, and H. Kneipp, Eds. (Springer, Heidelberg, 2006), pp. 47–66.
A. Otto, J. Raman Spectrosc. 36, 497
(2005).
L. P. Capadona, J. Zheng, J. I. Gonzalez,
T. H. Lee, S. A. Patel, and R. M. Dickson,
Phys. Rev. Lett. 94, 058301 (2005).
S. L. Zou and G. C. Schatz, Chem. Phys.
Lett. 403, 62 (2005).
K. Kneipp, H. Kneipp, I. Itzkan, R. R.
Dasari, and M. S. Feld, Chem. Phys. 247,
155 (1999).
I. Khan, D. Cunningham, R. E. Littleford,
D. Graham, W. E. Smith, and D. W. McComb, Anal. Chem. 78, 224 (2006).
K. Kneipp, H. Kneipp, P. Corio, S. D. M.
Brown, K. Shafer, J. Motz, L. T. Perelman, E. B. Hanlon, A. Marucci, G. Dresselhaus, and M. S. Dresselhaus, Phys.
Rev. Lett. 84, 3470 (2000).
K. Kneipp, H. Kneipp, M. S. Dresselhaus,
and S. Lefrant, Philos. Trans. R. Soc.
London, Ser. A 362, 2361 (2004).
E. J. Ayars, H. D. Hallen, and C. L. Jahncke, Phys. Rev. Lett. 85, 4180 (2000).
E. J. Ayars, C. L. Jahncke, M. A. Paesler,
and H. D. Hallen, J. Microsc.-Oxf. 202,
142 (2001).
S. Efrima, Chem. Phys. Lett. 102, 79
(1983).
H. Kneipp, J. Kneipp, and K. Kneipp,
Anal. Chem. 78, 1363 (2006).
F. Svedberg and M. Kall, Faraday Discuss. 132, 35 (2006).
53. A. R. Bizzarri and S. Cannistraro, Appl.
Spectrosc. 56, 1531 (2002).
54. P. Etchegoin, R. C. Maher, L. F. Cohen,
H. Hartigan, R. J. C. Brown, M. J. T. Milton, and J. C. Gallop, Chem. Phys. Lett.
375, 84 (2003).
55. K. Kneipp, Y. Wang, R. R. Dasari, and
M. S. Feld, Appl. Spectrosc. 49, 780
(1995).
56. V. A. Podolskiy and V. M. Shalaev, Laser
Phys. 11, 26 (2001).
57. K. Kneipp, H. Kneipp, S. Abdali, R. W.
Berg, and H. Bohr, Spectr.-Int. J. 18, 433
(2004).
58. M. Futamata, Y. Maruyama, and M. Ishikawa, J. Mol. Struct. 735–736, 75
(2005).
59. D. B. Lukatsky, G. Haran, and S. A. Safran, Phys. Rev. E 67, 062402 (2003).
60. Z. J. Wang and L. J. Rothberg, J. Phys.
Chem. B 109, 3387 (2005).
61. S. R. Emory, R. A. Jensen, T. Wenda, M.
Y. Han, and S. M. Nie, Faraday Discuss.
132, 249 (2006).
62. A. Weiss and G. Haran, J. Phys. Chem. B
105, 12348 (2001).
63. A. R. Bizzarri and S. Cannistraro, J. Phys.
Chem. B 109, 16571 (2005).
64. C. J. L. Constantino, T. Lemma, P. A. Antunes, P. Goulet, and R. Aroca, Appl.
Spectrosc. 57, 649 (2003).
65. T. L. Haslett, L. Tay, and M. Moskovits,
Chem. Phys. 113, 1641 (2000).
66. A. G. Brolo, A. C. Sanderson, and A. P.
Smith, Phys. Rev. B 69, 045424 (2004).
67. P. V. Teredesai, A. K. Sood, A. Govindaraj, and C. N. R. Rao, Appl. Surf. Sci.
182, 196 (2001).
68. E. C. Le Ru and P. G. Etchegoin, Faraday
Discuss. 132, 63 (2006).
69. R. C. Maher, L. F. Cohen, E. C. Le Ru,
and P. G. Etchegoin, Faraday Discuss.
132, 77 (2006).
70. K. Kneipp, H. Kneipp, I. Itzkan, R. R.
Dasari, and M. S. Feld, J. Phys.: Condens.
Matter 14, R597 (2002).