Physical Chemistry Chemical Physics

This paper is published as part of a PCCP Themed Issue on:

New Frontiers in Surface-Enhanced Raman Scattering

Guest Editor: Pablo Etchegoin

Editorial

Quo vadis surface-enhanced Raman scattering? Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b913171j

Perspective

Surface-enhanced Raman spectroscopy of dyes: from single molecules to the artists canvas Kristin L. Wustholz, Christa L. Brosseau, Francesca Casadio and Richard P. Van Duyne, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904733f

Communication

Tip-enhanced Raman scattering (TERS) of oxidised glutathione on an ultraflat gold nanoplate Tanja Deckert-Gaudig, Elena Bailo and Volker Deckert, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904735b

Papers

Self-assembly of , -aliphatic diamines on Ag nanoparticles as an effective localized surface plasmon nanosensor based in interparticle hot spots Luca Guerrini, Irene Izquierdo-Lorenzo, José V. Garcia-Ramos, Concepción Domingo and Santiago Sanchez-Cortes, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904631c

Single-molecule vibrational pumping in SERS C. M. Galloway, E. C. Le Ru and P. G. Etchegoin, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904638k

Silver nanoparticles self assembly as SERS substrates with near single molecule detection limit Meikun Fan and Alexandre G. Brolo, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904744a

Gated electron transfer of cytochrome c6 at biomimetic interfaces: a time-resolved SERR study Anja Kranich, Hendrik Naumann, Fernando P. Molina-Heredia, H. Justin Moore, T. Randall Lee, Sophie Lecomte, Miguel A. de la Rosa, Peter Hildebrandt and Daniel H. Murgida, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904434e

Investigation of particle shape and size effects in SERS using T-matrix calculations Rufus Boyack and Eric C. Le Ru, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b905645a

Plasmon-dispersion corrections and constraints for surface selection rules of single molecule SERS spectra S. Buchanan, E. C. Le Ru and P. G. Etchegoin, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b905846j

Redox molecule based SERS sensors Nicolás G. Tognalli, Pablo Scodeller, Victoria Flexer, Rafael Szamocki, Alejandra Ricci, Mario Tagliazucchi, Ernesto J. Calvo and Alejandro Fainstein, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b905600a

Controlling the non-resonant chemical mechanism of SERS using a molecular photoswitch Seth Michael Morton, Ebo Ewusi-Annan and Lasse Jensen, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904745j

Interfacial redox processes of cytochrome b562 P. Zuo, T. Albrecht, P. D. Barker, D. H. Murgida and P. Hildebrandt, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904926f

Surface-enhanced Raman scattering of 5-fluorouracil adsorbed on silver nanostructures Mariana Sardo, Cristina Ruano, José Luis Castro, Isabel López-Tocón, Juan Soto, Paulo Ribeiro-Claro and Juan Carlos Otero, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b903823j

SERS imaging of HER2-overexpressed MCF7 cells using antibody-conjugated gold nanorods Hyejin Park, Sangyeop Lee, Lingxin Chen, Eun Kyu Lee, Soon Young Shin, Young Han Lee, Sang Wook Son, Chil Hwan Oh, Joon Myong Song, Seong Ho Kang and Jaebum Choo, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904592a

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Nanospheres of silver nanoparticles: agglomeration, surface morphology control and application as SERS substrates Xiao Shuang Shen, Guan Zhong Wang, Xun Hong and Wei Zhu, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904712c

SERS enhancement by aggregated Au colloids: effect of particle size Steven E. J. Bell and Maighread R. McCourt, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b906049a

Towards a metrological determination of the performance of SERS media R. C. Maher, T. Zhang, L. F. Cohen, J. C. Gallop, F. M. Liu and M. Green, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904621f

Ag-modified Au nanocavity SERS substrates Emiliano Cortés, Nicolás G. Tognalli, Alejandro Fainstein, María E. Vela and Roberto C. Salvarezza, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904685m

Surface-enhanced Raman scattering studies of rhodanines: evidence for substrate surface-induced dimerization Saima Jabeen, Trevor J. Dines, Robert Withnall, Stephen A. Leharne and Babur Z. Chowdhry, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b905008f

Characteristics of surface-enhanced Raman scattering and surface-enhanced fluorescence using a single and a double layer gold nanostructure Mohammad Kamal Hossain, Genin Gary Huang, Tadaaki Kaneko and Yukihiro Ozaki, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b903819c

High performance gold nanorods and silver nanocubes in surface-enhanced Raman spectroscopy of pesticides Jean Claudio Santos Costa, Rômulo Augusto Ando, Antonio Carlos Sant Ana, Liane Marcia Rossi, Paulo Sérgio Santos, Márcia Laudelina Arruda Temperini and Paola Corio, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904734d

Water soluble SERS labels comprising a SAM with dual spacers for controlled bioconjugation C. Jehn, B. Küstner, P. Adam, A. Marx, P. Ströbel, C. Schmuck and S. Schlücker, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b905092b

Chromic materials for responsive surface-enhanced resonance Raman scattering systems: a nanometric pH sensor Rômulo A. Ando, Nicholas P. W. Pieczonka, Paulo S. Santos and Ricardo F. Aroca, Phys. Chem. Chem. Phys., 2009 DOI: 10.1039/b904747f

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Chromic materials for responsive surface-enhanced resonance Raman

scattering systems: a nanometric pH sensorw

Romulo A. Ando,a Nicholas P. W. Pieczonka,b Paulo S. Santosa

and Ricardo F. Aroca*b

Received 9th March 2009, Accepted 22nd June 2009

First published as an Advance Article on the web 29th July 2009

DOI: 10.1039/b904747f

The use of chromic materials for responsive surface-enhanced resonance Raman scattering

(SERRS) based nanosensors is reported. The potential of nano-chromic SERRS is demonstrated

with the use of the halochrome methyl yellow to fabricate an ultrasensitive pH optical sensor.

Some of the challenges of the incorporation of chromic materials with metal nanostructures are

addressed through the use of computational calculations and a comparison to measured SERRS

and surface-enhanced Raman scattering (SERS) spectra is presented. A strong correlation

between the measured SERRS and the medium’s proton concentration is demonstrated for the

pH range 2–6. The high sensitivity achieved by the use of resonance Raman conditions is shown

through responsive SERRS measurements from only femtolitres of volume and with the

concentration of the reporting molecules approaching the single molecule regime.

Introduction

Surface-enhanced Raman scattering (SERS) has been an

active field of research for over 30 years.1 It is now an

established plasmon driven phenomenon that has demonstrated

single molecule sensitivity.2,3 The focus has now turned towards

realizing this potential in a host of possible applications.4,5 The

near-field nature of SERS makes the process an ideal basis for

extremely sensitive optical sensors, capable of responding to

changes to nanometric environments. For instance, there is a

strong desire for sensors capable of probing internal environments

non-intrusively.6–9 An optical probe that could report pH, for

example, could be used to monitor the pH inside living cells

and help differentiate cancerous cells from healthy ones.10

We report here that through the incorporation of chromic

materials it is possible to create functional SERS sensors that

are capable of responding to changes to the conditions of a

local environment. Chromic materials can undergo a reversible

change to their electronic configuration upon exposure to an

external stimulus such as heat, radiation, or pressure.11 In this

work we exploit the fact that the perturbed and unperturbed

species have distinct electronic absorptions that can be

successfully probed with resonance Raman scattering (RRS)

and, more importantly here, surfaced-enhanced RRS (SERRS).

RRS is a process whereby the Raman scattering efficiency of a

molecular system can be increased by orders of magnitude

when a wavelength of excitation resonant with an absorption

band of the target is used.12 This large difference in cross

section allows a minority resonant species to be easily

distinguished from that of a majority non-resonant species.13

Under the conditions of SERRS, this effect is magnified so

that, even at trace levels, the signal of the resonant species can

be recorded. Hence, the combination of chromic materials

with SERRS allows for probes which are extremely sensitive to

changes in the local environment. This approach is a template

that can be used in conjunction with a wide range of suitable

chromic materials, with the reporting system chosen to give

response to a desired stimulus (Fig. 1).

Fig. 1 A schematic for the incorporation of chromic materials into

SERRS sensors, shown here for a halochromic based system.

a Instituto de Quımica USP, Sao Paulo, Av. Prof. Lineu Prestes,Sao Paulo, SP 05508-000, Brazil

b Chemistry & Biochemistry, University of Windsor, Windsor,Ontario, Canada N9B 3P4. E-mail: raroca1@cogeco.ca

w Electronic supplementary information (ESI) available: TheoreticalRaman calculations. See DOI: 10.1039/b904747f

This journal is �c the Owner Societies 2009 Phys. Chem. Chem. Phys., 2009, 11, 7505–7508 | 7505

PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

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In this study, we show the effectiveness of the incorporation

of chromic materials into SERRS systems by demonstrating

the ultrasensitive detection of protonation using silver

nanostructures and 4-(dimethylamino)azobenzene (methyl

yellow or MY), a molecule with well known halochromic

properties. The distinct SERRS profiles of the neutral and

protonated species allow for the observation of spectral

changes directly correlated to the proton concentration of

the nanometric environment for pH values between the limits

of 2 and 6. The enhanced Raman cross section achieved

with SERRS provides a successful optical pH probe down

to femtolitre volumes and with very low concentrations

of the reporting molecule (approaching the single molecule

regime).

Results and discussion

MY is a substituted azobenzene derivative with two available

protonation sites, namely the dimethylamino group and the

azo bond. The dispersion of MY’s Raman intensity near

electronic resonances has been previously reported, and the

RRS spectra were shown to be very sensitive to protonation

and deprotonation, with each species having characteristic

vibrational profiles.14 The neutral species is characterized

by a broad electronic absorption band at ca. 435 nm, while

the corresponding protonated species (P-MY) shows an

absorption band with a maximum at ca. 515 nm.14

Each species has a very distinct dispersion profile of the

Raman intensity near resonance, which in turn produces

characteristic vibrational Raman spectra. These distinct

spectral differences in the near-resonance and resonance

regions can be exploited as reporting markers with chromic

materials.

The unique properties of chromic materials are a function of

their electronic states. One obstacle to a more effective usage is

the need to understand what possible impact the interaction

with the underlying nanostructure will have on the electronic

properties and the characteristic Raman signature of the

reporting system. To address these questions, high level

quantum chemical calculations have proven in the past to be

extremely helpful.15 Additionally, it has been shown that

incorporating Ag+ in the calculated structure can be a good

approximation to the influence of the silver surface on the

properties of an analyte.16

The theoretical MY–Ag Raman spectrum was calculated by

DFT methods with the MY interacting with a Ag+ through

the azo bond. The computed spectrum shows a great agreement

with the experimental SERRS spectrum (Fig. 2). When one

compares the SERRS spectrum of the neutral species to that

of the previously reported resonance Raman,14 there are no

significant changes in the frequencies of the bands, suggesting

a weak interaction between the molecule and the Ag surface.

Contrarily, the SERRS spectrum of the protonated species

shows noticeable differences from the previously reported

Raman signature of P-MY. While the bands of the protonated

species are present and dominate the SERRS spectrum, the

relative intensities are different. Again QM calculations assist

in providing a rationale for these observed perturbations. The

calculated spectrum of the P-MY–Ag structure shows an

intensity profile that is in good agreement with the recorded

SERRS (Fig. 2) and indicates that the interaction with the

silver surface is most likely occurring at the azo moiety.

In addition, the electronic absorption of each species

appears to be redshifted as a result of interaction with the

silver surface, as evidenced by signature resonance Raman

bands and the appearance of overtones in the SERRS

spectrum (Fig. 3) for both the neutral (measured with a

514.5 nm excitation line) and the protonated species (measured

with a 633 nm excitation line). The calculated HOMO–LUMO

gap for both MY–Ag and P-MY–Ag were found to be

redshifted relative to that of the same structure without silver,

suggesting that it is the interaction with the surface that is the

source of these redshifts in these absorption bands. It is

important for the sensor’s effectiveness to verify that the

probing excitation is in resonance with an absorption

band for this system. Here, the presence of overtones and

combination bands for both species (a feature generally only

seen under resonance conditions) supports this redshift of the

electronic absorption, and provides indirect evidence that

the resonance between the 633 nm excitation line and the

electronic transition needed for RRS of the protonated species

is present.

Fig. 2 SERRS of MY and protonated-MY (P-MY) (10�5 M with Ag

colloids); compared to the theoretical Raman spectra of species

complexed with a Ag+ through the azo moiety.

Fig. 3 SERRS spectra of P-MY–Ag and MY–Ag. The expanded

region highlights the overtone and combination bands for each

species.

7506 | Phys. Chem. Chem. Phys., 2009, 11, 7505–7508 This journal is �c the Owner Societies 2009

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For the pH range investigated here, it is the SERRS signal

of the minority protonated species, which is the most sensitive

to changes due to pH, that is targeted.

This is accomplished by acquiring spectra with the

633 nm excitation line, which is resonant with P-MY–Ag.

For calibration of the pH response of this system, ensemble

measurements were taken from a 3 mL, 10�5 M MY sample

using a f/15 lens.

From this system, several bands characteristic of the neutral

and protonated species could be followed (particularly,

1140 cm�1 for MY and 1280 cm�1 for P-MY) to monitor

the variation in pH. In addition, we can use the band at

ca. 1000 cm�1, assigned to the breathing mode of the benzene

ring, as an internal standard, since this mode has been shown

to be insensitive to pH changes and is present in the spectrum

of both species. The relative intensity variations of these bands

clearly follows the pH changes without any modification to the

Raman frequencies over a range of pH values [Fig. 4(a)]. Also,

when the intensity of the 1170 cm�1 band was referenced to the

internal standard band of 1000 cm�1, an easily fitted titration

plot was found [Fig. 4(b)].

The pKa of the protonated MY in aqueous solution is ca. 3.

Therefore, at a pH value of 6, the majority of all MY in the

sample is of the neutral species and this is reflected in

the SERS spectrum recorded with the 633 nm laser line. As

the proton concentration is increased, the relative concentra-

tion of P-MY is incrementally increased and though its relative

concentration could be minute, the cross sectional difference

of RRS to RS (and accordingly SERRS to SERS) allows the

detection of small changes in the pH value. For example, again

using the pKa of 3 for P-MY, at pH 6 there are ca. 1 � 103 MY

for each P-MY, while at pH 4 the number is ca. 10 MY for one

P-MY. The signature spectrum of the P-MY SERRS begins to

appear even at pH = 6 when only 1 in 103 molecules are

protonated (Fig. 4), and the sensitivity to pH change can be

incrementally monitored up to the point where the spectrum

is completely dominated by the P-MY species (pH = 2).

Additionally, the process is reversible. The signature spectra

and the relative intensities of the representative species vary

accordingly with changes in pH through successive additions

of NaOH or HCl.

A lower concentration and much smaller volume study was

undertaken to determine the sensitivity and the limit of detec-

tion which could be achieved for this system. Measurements

were recorded from 200 mL of 1 mM solutions of MY using

a 65� immersion objective, giving a collection volume of

B10 fL. Responsive, pH distinct SERRS measurements could

be easily recorded from these extremely small volumes (Fig. 5),

demonstrating the extreme sensitivity of this system to changes

in the local environment. An interesting aspect of these

measurements is that the SERRS spectrum acquired from a

sample measuring a pH of 2 would have no more than a few

hundred P-MY molecules contributing. While not in the scope

of this work, these results suggest a possible way to achieve

appropriate analyte concentrations for SM-SERRS (single

molecules-SERS) studies through the careful adjustment of

the pH.

Experimental

Materials

The plasmon enhancing substrates used were silver nano-

particles of 30–80 nm diameter prepared through citrate

reduction in an aqueous solution.17

Preparation

Samples for the reference ensemble SER(R)S measurements

were prepared by the addition of 20 mL of the 10�3 M solution

of MY to a solution of Ag colloids, making up a final volume

Fig. 4 (a) SERRS/SERS spectra of MY in solutions of varying pH

values. All spectra were recorded with a 633 nm excitation laser line.

(b) Titration plot: the relative intensity ratio is between the P-MY

band at 1170 cm�1 and the ring mode at 1000 cm�1.

Fig. 5 pH dependent SERRS spectra taken from B10 fL of the

200 mL solution of 1 mM MY. (Inset picture shows the sample

arrangement used to collect fL SERRS/SERS).

This journal is �c the Owner Societies 2009 Phys. Chem. Chem. Phys., 2009, 11, 7505–7508 | 7507

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of 2 mL and consequently a final analyte concentration

of 10�5 M. Lower concentration samples of 10�6 M MY were

prepared similarly using a 10�4 M MY solution. The pH

values of the samples were adjusted by the addition of HCl

and NaOH to the desired level.

SER(R)S measurements

All Raman spectra were acquired with a Renishaw inVia with

either a 633 nm HeNe laser or a 514.5 nm Ar+ ion laser.

Ensemble SERRS and SERS measurements were collected

from a 3 mL quartz cuvette with a f/15 lens. Low volume,

low concentration SERRS signals were collected with a

63� immersion objective (0.9 numerical aperture, N. A.)

immersed directly in 200 mL of solution. The volume probed

by the objective was less than 10 fL.

pH measurements

All pH measurements were recorded with a micro pH

electrode (Lazar PHR-146).

Conclusions

The success of MY as a chromic SERRS material opens up

several areas for future work. MY has shown to be not only a

sensitive probe of the local nanometric environment but also a

very stable reporter molecule for protonation, one of the most

fundamental and basic kinds of chemical reactions. These

results are a prelude towards the possible use of SERRS for

the monitoring of a variety of individual reactions (protonation,

complexation, adduct formation, etc.), and extending this tool

to observe single molecular chemistry.

This study demonstrates that the use of chromic materials

for SERRS nanometric sensors has great potential. The

same concepts explored here could be readily applied to any

number of chromic systems. With the increased sophistication

of Raman instrumentation, it is easy to envision how

this can be extended to multiplex systems,18 where multiple

excitation lines could probe many resonant chromic species

and thereby monitor multiple environmental variables

simultaneously.19

Acknowledgements

Financial assistance from NSERC of Canada is gratefully

acknowledged. RA Ando acknowledges CNPq (201506/

2007-6). We would also like to thank Daniel J. Ross for his

assistance with the theoretical computations.

Notes and references

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12 N. P. W. Pieczonka, P. G. Goulet and R. F. Aroca, in SurfaceEnhanced Raman Scattering Physics and Applications,ed. K. Kneipp, M. Moskovits and H. Kneipp, Springer-Verlag,Berlin, 2006, vol. 103, pp. 197–216.

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