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Surface Enhanced Raman

Spectroscopy web themed issue

Guest editors: Professors Duncan Graham,

Zhongqun Tian and Richard Van Duyne

All articles in this issue will be gathered together online at www.rsc.org/sers.

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7158 Chem. Commun., 2011, 47, 7158–7160 This journal is c The Royal Society of Chemistry 2011

Cite this: Chem. Commun., 2011, 47, 7158–7160

Fluctuations of the Stokes and anti-Stokes surface-enhanced resonance

Raman scattering intensities in an electrochemical environmentwzDiego P. dos Santos,

abGustavo F. S. Andrade,

cAlexandre G. Brolo*

aand

Marcia L. A. Temperinib

Received 4th December 2010, Accepted 6th May 2011

DOI: 10.1039/c0cc05360k

Stokes and anti-Stokes SERRS intensity fluctuations were

observed from a roughened silver electrode immersed in diluted

solutions of Brilliant Green (BG), a behaviour linked to single-

molecule events. The distributions of the anti-Stokes to Stokes

ratios were obtained and their shape showed a strong dependence

on the applied potential.

Time-dependent fluctuations in surface-enhanced (resonance)

Raman scattering (SE(R)RS) intensities are generally observed

for experiments in diluted solutions.1 It is well accepted in the

field that these fluctuations are caused by a small number of

molecules probing electromagnetic hot-spots present on the

nanostructured surface.2 Statistical analysis of the SERS

intensity fluctuations in systems of two diluted analytes

provided a direct proof of the single-molecule detection

capabilities of the technique.3 Another interesting observation

that accompanies the fluctuations is unusually high anti-

Stokes to Stokes intensity ratios (IAS/IS) in some of the events.

This phenomenon was first reported by Kneipp et al., who

interpreted the unexpected ratios as a consequence of optical

pumping.4 Although the optical pumping mechanism might

play a role at cryogenic temperatures,5 other authors suggested

that the non-thermal IAS/IS could be explained by considering

the energy of the resonances of the molecular-hot-spot

system.6 Among the arguments that support this explanation

is the fact that not only the anti-Stokes intensity is enhanced

relative to the Stokes intensity, but an anomalous increase in

the Stokes intensity could also be verified. However, statistical

analysis of the IAS/IS during time-dependent single-molecule

SERS experiments has not been reported yet.

In this work, we investigated the time evolution of the anti-

Stokes to Stokes ratios under single-molecule conditions,

provided a statistical analysis and investigated the effect of

the electrochemical potential on the distributions. Brilliant

Green (BG) was chosen as the molecular probe, because of

its strong SERRS active band at B220 cm�1 (the UV-Vis

spectrum of BG in solution is presented in ESIz). The relativelylow vibrational frequency of this band allowed the measure-

ment of both the Stokes and the anti-Stokes branches simul-

taneously by our Renishaw inVia Raman microscope. The

experiments were realized using 633 nm excitation and a

63� (NA = 0.9) water immersion objective. The measure-

ments consisted of obtaining consecutive time series of 2000

spectra (1 second acquisition time each and 2 mW laser power)

to generate the histograms. The metallic substrate was an

electrochemically roughened silver electrode. The conditions

for the surface preparation and details of the data analysis,

including the procedure to eliminate the spurious spectrum

due to photodecomposition, have been published elsewhere.7

The electrode potential was controlled in all experiments using

a potentiostat–galvanostat EG&G model PAR-273A. A Pt

wire was used as a counter electrode and a Ag wire coated with

AgCl was the reference electrode. The electrolyte was 0.1 M

KBr in all measurements. The Br� electrolyte ensures that the

region around 220 cm�1 is free of spectral interferences due to

the metal-halide stretching.

Under non-resonant conditions, the IAS/IS ratio is given by

eqn (1), below:8

IAS

IS¼ sAS

sS

ðoexc þ ovibÞ3

ðoexc � ovibÞ3

" #exp � �hovib

kBT

� �1þ exp � �hovib

kBT

� �� � ð1Þ

sAS and sS are the Raman cross sections for the anti-Stokes

and the Stokes scattering, respectively. All other terms have

their usual meaning. For our experimental conditions (633 nm

excitation, vibrational band at 220 cm�1 and an estimated

temperature of 298 K), the expected thermal value for IAS/IS is

approximately 0.28.

Fig. 1 shows the distribution of IAS/IS obtained using a

roughened silver electrode at �0.1 V immersed in 0.1 M KBr

and 5 mM of BG. The concentration of BG in Fig. 1 was high

enough to avoid the strong fluctuations in intensities observed

under the ‘‘single-molecule’’ regime. We will call this situation

of high concentration ‘‘average SERRS’’ conditions throughout

this work.

aDepartment of Chemistry, University of Victoria, P.O. Box 3065,Victoria, Canada V8W 3V6. E-mail: agbrolo@uvic.ca

b Laboratorio de Espectroscopia Molecular, Instituto de Quımica,Universidade de Sao Paulo, CEP 05513-970, Sao Paulo, Brazil.E-mail: mlatempe@iq.usp.br, fq.diego@gmail.com

cDepartamento de Quımica, Universidade Federal de Juiz de Fora,Campus Universitario s/n, CEP 36036-900, Juiz de Fora, Brazilw This article is part of a ChemComm web-based themed issue onSurface Enhanced Raman Spectroscopy.z Electronic supplementary information (ESI) available. See DOI:10.1039/c0cc05360k

ChemComm Dynamic Article Links

www.rsc.org/chemcomm COMMUNICATION

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 7158–7160 7159

In Fig. 1 the IAS/IS distribution presents a nearly Gaussian

behaviour under the average SERRS conditions, with an

average value centered at 0.15. The experimentally determined

average IAS/IS value (hIAS/ISi) from Fig. 1 is about half of that

predicted by eqn (1). In order to confirm that the hIAS/ISi fromthe SERRS experiments were not biased by our instrument,

the normal Raman spectra from CCl4 were obtained. The

IAS/IS for the B220 cm�1 band of CCl4 was determined to be

0.29, which is in reasonable agreement with eqn (1).

One of the features of eqn (1) is an equivalence between the

Stokes and the anti-Stokes cross sections (sAS = sS) under

non-resonance conditions. However, this equivalence is lost

under resonance conditions (sAS a sS).6,9 It is important to

notice in Fig. 1 that the Stokes intensities are actually

enhanced relative to the anti-Stokes when compared to the

non-resonant thermal equilibrium. This suggests that the

deviation of the hIAS/ISi must be attributed to differences in

the cross sections and not to an optical pumping mechanism.

A similar distribution to Fig. 1 was obtained for different

applied potentials within the range between �0.1 V to �0.4 V.

The dependence of the average SERRS of BG on the applied

potential is available in ESI.z The hIAS/ISi showed some

potential dependence, as presented in Fig. 2. These results

can be assigned to the effect of the applied potential on the

resonance conditions. The tuning of the SERS/SERRS inten-

sities by the applied potential is common in electrochemical

measurements.10 Here we show that the IAS/IS ratio can also

be controlled by varying the resonance conditions with the

potential. The variation in hIAS/ISi with the applied potential

is in good agreement with the resonance model previously

suggested by our group (see Fig. S2 in the ESIz).6

The deviation of the IAS/IS from the expected thermal

conditions (given by eqn (1)) has been previously quantified

using a K factor, defined as the ratio between the IAS/IS for the

SERS signal of interest and the IAS/IS for a reference

(unenhanced) system.6 Here we propose to define a new

quantity k by using as a reference the experimental hIAS/ISiof the average SERRS measurements instead (from Fig. 1).

The advantages of this new definition are: (1) the exact same

vibrational frequency is compared, minimizing even further

differences in instrument response; (2) as discussed above, the

hIAS/ISi values do not match the expected thermal behaviour

due to resonances. This intrinsic deviation is taken into

account with the new definition.

The fluctuations in SERRS intensities were recorded against

time for a roughened Ag electrode immersed in BG solutions

with concentrations lower than 20 nM. Strong fluctuations in

SERRS intensities were observed in these cases. The rough-

ened Ag electrode is not a very efficient SERS substrate, as

compared to aggregated silver colloids. Therefore, the surface

density of highly enhancing hot-spots on the surface is low,

allowing the observation of the SERRS intensity fluctuations

at higher concentrations.2a,7b

The single-molecule data (presented in ESIz) showed a few

spectra with the anti-Stokes side particularly enhanced with

respect to the Stokes side, with IAS/IS reaching values near 1,

i.e., k-values of about 7. However, the opposite behavior

(preferential enhancement on the Stokes side) was also

observed for some of the events. The wide variations in the

IAS/IS observed under the conditions of strong SERRS

intensity fluctuations relative to the average behaviour are

better captured on the histograms presented in Fig. 3.

In Fig. 3, the shape of the k-value distributions varies

significantly with the applied voltage. The Gaussian distri-

bution of the average SERRS (same conditions as Fig. 1) is

also plotted for a direct comparison.

The variation in k-values under single-molecule conditions

can be simply understood by considering the distribution of

hot-spots on a typical substrate that supports SERS. A roughened

electrode contains a variety of nanostructured environments

that support enhanced local fields for particular excitation

conditions. Each of these environments has its own resonance

Fig. 1 Distribution of IAS/IS for the 220 cm�1 vibrational band of

BG. 5 mM solution of BG in 0.1 M KBr.

Fig. 2 Dependence of the hIAS/ISi on the applied potential. The error

bars represent the standard deviation obtained from a Gaussian fit of

the distribution. The conditions were the same as in Fig. 1.

Fig. 3 Histograms of k-values for BG adsorbed on a roughened silver

electrode (grey bars). [BG] = 20 nM, 0.1 M KBr. (A) �0.1 V and (B)

�0.3 V. The shaded Gaussian (in red) in each graph is a distribution

under the average SERRS conditions (as in Fig. 1).

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7160 Chem. Commun., 2011, 47, 7158–7160 This journal is c The Royal Society of Chemistry 2011

energy (and linewidth). Under the average SERS conditions,

most of these hot-spots are populated by the molecular probe;

therefore, the average hIAS/ISi reflects the sum of the contri-

butions from all hot-spots. In the single-molecule case;

however, every time that a molecule visits a hot-spot, the local

resonance is revealed through how much the local IAS/ISdeviates from the average behaviour (this deviation is quantified

by the k-values). In a given moment, an individual molecule

might probe a hot-spot with resonance centered in the Stokes

side, leading to a low k-value, but later in time moves to

another hot-spot with resonance in the anti-Stokes side,

yielding a high k-value. The sum of all k-values in the single

molecule regime approaches unity, provided enough events are

monitored, as expected. Note that the resonance at the hot-spot

is probably a combination of electronic (charge transfer) and

plasmonic effects. The charge transfer contributions might

play an important role in the potential dependence of the

hIAS/ISi (Fig. 2), and the plasmonic resonances are well

accepted as the main contribution to the overall enhancement.

The relative contribution from each of these effects is not

relevant to the main point of the model, which is the link of the

variations in k to local resonance conditions that are probed

by a small number of molecules.

The k-value distributions show a log-normal shape, similar

to the distribution of ‘‘single-molecule’’ SERRS intensities.2,7b

This distribution reflects the various resonances of the

hot-spots probed in the regime of strong SERRS intensity

fluctuations. The log-normal shape is expected because when

by chance a molecule adsorbs on a hot-spot with a sharp

anti-Stokes resonance, the k-value will be much larger than

one, adding a tail to the distribution. At �0.1 V (Fig. 3A), a

significant fraction of the log-normal distribution overlaps

with the Gaussian distribution (from the average SERRS

measurements), with only 10% of the events having higher

IAS/IS than the average. As the potential swept towards

negative values, the ‘‘single-molecule’’ distributions change

drastically with respect to the average SERRS distribution,

extending towards higher k-values, indicating a preferential

enhancement on the anti-Stokes side for a few events.

However, it is also possible to observe components on the

distribution that show an enhancement of the Stokes scattering.

This can be qualitatively inferred by the distribution of events

from the log-normal plots that occur on each side of the

Gaussian distribution. A larger deviation between the distri-

butions from both situations (average SERRS and single-

molecule) is observed when the potential reaches �0.3 V

(Fig. 3B). In that case, the population of events of the log-

normal distribution on the region of enhanced anti-Stokes is

almost the same as the Stokes. As the potential is made more

negative (ESIz), the population of anti-Stokes enhanced events

decreases, but a few events with very high IAS/IS can still be

observed.

The shape of the distributions and their potential depen-

dence reflect the effect of the applied potential on both the

resonance conditions and the surface concentration. Although

the applied voltage might also affect the plasmonic properties,

this effect is expected to be small.11 The tuning of the surface

concentration with the applied potential has been investigated

under the conditions of strong SERRS intensity fluctuations.7b

The shape of the distribution was readily modified by the

potentials, following a trend that depended on the charge of

the adsorbed dye. For a given potential, however, the varia-

tion in k should be correlated to the local resonances of the

hot-spot. A comprehensive investigation of these resonances is

currently being carried out by our group.

In summary, the distribution of the anti-Stokes to the

Stokes ratio was investigated for BG adsorbed on a silver

electrode. A Gaussian distribution of the ratio was observed

for high concentrations of BG in solution, a condition that

allows the observation of the average SERRS behaviour of the

system. Under those conditions, the experimental average

ratios were lower than those expected by simple thermal

equilibration, indicating a resonance with the Stokes region.

The shape of the distribution was independent of the applied

potential. The hIAS/ISi values showed some potential depen-

dence, which supports a resonance model.

Strong fluctuations in the SERRS intensities at both the

Stokes and the anti-Stokes branches were observed when the

concentration of BG was lower than 20 nM. These fluctua-

tions have been linked to adsorption–desorption events

involving single-molecules. The distribution of the k-valuesin this case showed a log-normal shape, with a tail towards

values that indicate a preferential increase of the anti-Stokes

intensities. The shape of the distributions was dependent on

the applied voltage due to potential induced changes in both

the surface concentration and resonance conditions.

Notes and references

1 (a) S. M. Nie and S. R. Emery, Science, 1997, 275, 5303;(b) K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan,R. R. Dasari and M. S. Feld, Phys. Rev. Lett., 1997, 78, 1668;(c) A. Weiss and G. Haran, J. Phys. Chem. B, 2001, 105, 12348.

2 (a) E. C. Le Ru, P. G. Etchegoin and M. Meyer, J. Chem. Phys.,2006, 125, 204701; (b) J. P. Camden, J. A. Dieringer, Y. M. Wang,D. J. Masiello, L. D. Marks, G. C. Schatz and R. P. Van Duyne,J. Am. Chem. Soc., 2008, 130, 12616.

3 (a) P. G. Etchegoing, M. Meyer, E. Blackie and E. C. Le Ru, Anal.Chem., 2007, 79, 8411; (b) J. A. Dieringer, R. B. Lettan II,K. A. Scheidt and R. P. Van Duyne, J. Am. Chem. Soc., 2007,129, 16249.

4 K. Kneipp, Y. Wang, H. Kneipp, I. Itzkan, R. R. Dasari andM. S. Feld, Phys. Rev. Lett., 1996, 76, 2444.

5 C. M. Galloway, E. C. Le Ru and P. G. Etchegoin, Phys. Chem.Chem. Phys., 2009, 11, 7372.

6 (a) T. L. Haslett, L. Tay and M. Moskovits, J. Chem. Phys., 2000,113, 1641; (b) A. G. Brolo, A. C. Sanderson and A. P. Smith, Phys.Rev. B, 2004, 69, 045424.

7 (a) G. F. S. Andrade, G. A. Micke, M. F. M. Tavares andM. L. A. Temperini, J. Raman Spectrosc., 2004, 35, 1034;(b) D. P. Santos, G. F. S. Andrade, M. L. A. Temperini andA. G. Brolo, J. Phys. Chem. C, 2009, 113, 17737.

8 N.-H. Seong, Y. Fang and D. D. Dlott, J. Phys. Chem. C, 2009,113, 1445.

9 (a) E. C. Le Ru and P. G. Etchegoin, Faraday Discuss., 2006, 132,63; (b) R. C. Maher, C. M. Galloway, E. C. Le Ru, L. F. Cohenand P. G. Etchegoin, Chem. Soc. Rev., 2008, 37, 965.

10 (a) J. R. Lombardi, R. L. Birke, T. Lu and J. Xu, J. Chem. Phys.,1986, 84, 4174; (b) D. Y. Wu, J. F. Li, B. Ren and Z. Q. Tian,Chem. Soc. Rev., 2008, 37, 1025.

11 A. H. Ali and C. A. Foss Jr., J. Electrochem. Soc., 1999, 146, 628.

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