Surface-enhanced resonance Raman scattering (SERRS) as a tool for the studies of electron transfer proteins attached to biomimetic surfaces: Case of cytochrome c

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Electrochimica Acta xxx (2013) xxx– xxx

Contents lists available at ScienceDirect

Electrochimica Acta

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urface-enhanced resonance Raman scattering (SERRS) as a tool forhe studies of electron transfer proteins attached to biomimeticurfaces: Case of cytochrome c

gata Królikowska ∗

arsaw University, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland

r t i c l e i n f o

rticle history:eceived 23 May 2013eceived in revised form 20 August 2013ccepted 22 August 2013vailable online xxx

eywords:lectrochemical SERS

a b s t r a c t

This review presents the up-to-date knowledge collected on the redox and electron transfer (ET) prop-erties of cytochrome c (cyt c) attached to biomimetic organic films on electrodes with the aid, ofsurface-enhanced resonance Raman scattering (SERRS). Strategies to attach the cyt c to alkanethiol, self-assembled monolayers (SAMs) coated metals (Ag and Ag/Au hybrids) are described. General, featuresof SER(R)S spectroscopy and conditions for conducting a meaningful electrochemical SERRS (EC-SERRS)experiment are highlighted. Structural information on SAM and tethered cyt c envisaged with SER(R)Sis discussed. A great effort is made to demonstrate a key role of substrate selection, when collecting and

ERRSlkanethiol SAMs linkeriomimetic interphaseytochrome clectron transfer

comparing SERRS and electrochemical data. Influence of biomimetic linker on structure, orientation andelectronic properties of immobilized cyt c is discussed. A principle of probing heterogeneous ET withtime-resolved (TR) potential-jump SERRS is explained. Conclusions concerning the mechanism of theET for cyt c attached to alkanethiol SAM coated gold provided by the insight with SERRS spectroscopyare presented. A special attention is paid to the discussion of the effect of the electric field provided byalkanethiol SAM on protein ET properties and biological function.

© 2013 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Principles and methodology of SERS, RR and EC-SERRS techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Surface-enhanced Raman scattering (SERS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.1. Historical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.2. Features of SERS spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.3. Mechanism of surface enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.4. SERS active substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.2. Resonance Raman scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2.1. Brief theory of resonance Raman (RR) scattering and features of RR spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.3. Electrochemical SERS (EC-SERS) technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3.1. Electrical double layer (EDL) of EC-SERS system and effect of the polarity of the surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3.2. Effect of the electrode potential on surface enhancement in EC-SERS system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3.3. Solvent and electrolyte related effects in EC-SERS experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3.4. Design of the EC-SERS cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3.5. Substrate selection for EC-SERS system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Attachment of cytochrome c to alkanethiol coated electrode prior ET studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: A. Królikowska, Surface-enhanced resonanfer proteins attached to biomimetic surfaces: Case of cytochrome c, Electro

3.1. Strategies for an establishment of a communication between elec3.2. Cytochrome c – structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2.1. Structure of cyt c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2.2. Cytochrome c biological function . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Tel.: +48 22 822 02 11x520.E-mail address: [email protected]

013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2013.08.140

ce Raman scattering (SERRS) as a tool for the studies of electron trans-chim. Acta (2013), http://dx.doi.org/10.1016/j.electacta.2013.08.140

trode and redox biomolecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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3.3. Alkanethiol SAMs as linkage monolayers for biomolecules immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3.1. Concept of self-assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3.2. Strategies to immobilize cytochrome c on alkanethiol SAM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. EC-SERRS studies of cyt c attached to alkenethiol coated electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1. Principles of surface-enhanced resonance scattering (SERRS) experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2. Resonance Raman spectrum of cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3. Choice of a substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.3.1. Gold vs. silver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3.2. Flat vs. roughened . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3.3. Performance of SERRS and electrochemistry experiment on the same silver substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3.4. Hybrid Ag/Au systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.4. Information derived from (EC)-SERRS spectra of cytochrome c attached to thiol modified metal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.4.1. Heme structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.4.2. Redox group orientation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.4.3. SER(R)S studies of alkanethiol SAM induced effects on linked cyt c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.4.4. Effect of excessive amount of a bulk protein on heme electronic structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.5. Results of SERRS studies on redox and ET properties of cyt c tethered to alkanethiol coated metal substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.5.1. Stationary potential SERRS measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.5.2. Time-resolved potential-jump SERRS spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.5.3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Studies of redox proteins adsorbed on electrodes have gainedncreasing attention in recent years. In this area detailed knowledgef the mechanism and dynamics of the interfacial processes, par-icularly heterogeneous electron transfer (ET) reaction is essential.oating metals with a thin layer of organic film allows preventingenaturation of biomolecules and retaining their biological activity.elf-assembled monolayers (SAMs) of �- substituted alkanethiolsre ideal linkage monolayers for protein/enzyme binding, sincehey are firmly bound to the surface via chemisorption, interchainnteractions provide high degree of order and terminal group allowsontrolling the surface properties. Cytochrome c (cyt c) has beenemonstrated to be redox active in many systems involving goldnd silver coated with alkanethiol SAMs. Identification of the mech-nism of alkanethiol monolayer promoted ET for a simple redoxrotein, like cytochrome c, can allow recognition of the parame-ers governing interfacial charge transfer processes in general androvide the basis for understanding more complex systems. SERRSechnique, combining surface-enhanced Raman scattering (SERS)ith the resonance Raman (RR) spectroscopy, gives the opportu-ity to probe both structure of linkage alkanethiol monolayer andttached cyt c, as well as to recognize protein redox state, spinonfiguration and iron coordination pattern. Time-resolved mea-urements provide an insight into the dynamics of the interfaciallectron transfer.

In this review paper the potential of electrochemical EC-SERRSpectroscopy technique for probing ET properties of the surfaceonfined redox biomolecules in the systems mimicking biologicalnterfaces is presented. The first part is thus dedicated to the princi-les of SERS (surface-enhanced Raman scattering) and RR (resonanceaman) techniques and methodology of electrochemical EC-SERRS.he second part is introducing the cyt c and the concept of tether-ng the biomolecules to the �-substituted alkanethiols SAMs onilver/gold.

The third part describes the methodology of stationary potentialnd time-resolved EC-SERRS studies of cyt c attached to alka-ethiol coated metal electrodes and the information provided


y spectro-electrochemical experiment. It also summarizes thebtained results dealing with the redox behavior and ET kinetics forhe mammalian (horse heart) heme protein, cytochrome c, attachedo thiol linkage SAMs on metal substrates. A special attention is paid

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

to the electric field effects at the metal/SAM/cyt c interface on theimmobilized protein.

2. Principles and methodology of SERS, RR and EC-SERRStechniques

2.1. Surface-enhanced Raman scattering (SERS)

2.1.1. Historical backgroundIn 1974 a first report of the observation of unexpectedly

intense Raman scattering in the vicinity of a nanostructuredmetal surface was made by Fleischmann et al. [1], who studiedadsorption of pyridine on roughened Ag substrate. The nature ofthis phenomenon was initially not fully understood and ascribedto the increased metal surface area (due to roughening procedure),resulting in a higher pyridine surface coverage in comparison tothe smooth electrode. The enhancement factors were determinedindependently by groups of Van Duyne [2] and Creighton [3] toreach up to 105 for pyridine molecule and were related to surfaceproperties of roughened electrode. Mechanism responsible for thisgiant enhancement of Raman cross-sections is known generallyas surface-enhanced Raman scattering (SERS). During the nextmore than 30 years, the number of systems studied with SERShas grown drastically and this technique proved to be a powerfulanalytical tool for sensitive and selective studies of adsorbates onnoble metal nanostructures [4–7].

2.1.2. Features of SERS spectrumSERS spectrum can differ significantly from the normal Raman

(NR) scattering spectrum of the same molecule. The following spec-tral features allow us believe that we observe the spectrum of thesurface bound, not solution species [5,7]:

• frequency shift of the bands• changed relative intensities compared to the NR spectrum• appearance of additional modes: forbidden in normal Raman

spectrum and metal–molecule vibrations at low frequencies• increased depolarization ratios• broadening of the bandwidths


• dependence of the band intensity on excitation beam energy(similar to excitation profiles characteristic for resonance Raman(RR) effect) and applied potential

• strong distance dependence of the signal intensity


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Fig. 1. Theoretical simulations of the electromagnetic field enhancement around Agnanoparticles: of a triangular shape (SP resonance at 700 nm, top left), a dimer ofspherical nanoparticles (430 nm; bottom left), an ellipsoidal nanoparticle (695 nm;right). The intensity scale for top left applies also to the bottom left. (For interpreta-tion of the references to color in this figure legend, the reader is referred to the webversion of the article.)

ARTICLEA-21165; No. of Pages 44

A. Królikowska / Electroch

Most of these changes arises from metal–molecule interactionsesulting in breaking molecular symmetry, changed geometry andandomization of molecular orientation with respect to the metalurface, while the rest is related to the physical nature of the SERShenomenon.

.1.3. Mechanism of surface enhancementIntensity of the Raman scattering is proportional to the square

f the induced dipole moment (�), which in turn is dependentn the molecular polarizability (˛) and the amplitude of the inci-ent electric field (Ei). Hence, an increase in the magnitude of

and/or Ei will result in enhancement of the Raman scatter-ng. Thus, the enhancement factor of the SERS spectrum can benderstood as a product of two contributions: chemical and elec-romagnetic enhancement. This approach provides two classes of

odels explaining the origin of surface enhancement: electromag-etic (EM) model and chemical (charge transfer; CT) model, whichill be now briefly discussed.

.1.3.1. Electromagnetic enhancement. When an electromagneticave illuminates a metal surface, the magnitude of the field at the

urface is changed with respect to this observed in the far field. If theurface is rough, the wave may excite localized surface plasmonsLSPs) on the surface. Surface plasmons (SPs) are collective oscilla-ions of free electrons that propagate along the surface of a metalhen it is in contact with a dielectric interface. Localized charac-

er means their confinement to metallic nanostructure resulting innhancement of the electromagnetic field in vicinity of the surface8]. To predict the magnitude of the electromagnetic enhance-

ent: the size, shape and material of the nanoscale roughnesseatures must be taken into account [9]. These factors determine theesonant frequency of the conduction electrons in a metallic nano-tructure. When the frequency of the radiation incident on metallicanostructure matches this characteristic frequency, the electriceld drives the conduction electrons into collective oscillation.xcitation of LSPs outcomes at first in a selective absorption andcattering of the electromagnetic resonant radiation, next in theeneration of a large electric field at the surface roughness (see theheoretical simulations of electric field distribution shown in Fig. 1).lectromagnetic enhancement is applicable for the molecules ofaman scatterers confined within this electromagnetic field [10].

It provides an enhancement of the Raman signal of factor up to04 (with respect to NR intensity) [12].

As already mentioned the energy corresponding to resonancef LSPs will be substantially influenced by type of metal, size,ize distribution and shape of the nanostructures and the localnvironment. For noble metal substrates, when approximating theoughness feature with an isolated sphere model, an expressionor the electric field magnitude (E) at the surface of a sphere can beiven by [10]:

∝ E2i

∣∣∣ εm − ε0

εm − 2ε0

∣∣∣2(1)

here Ei is the incident field magnitude, εm is the wavelengthependent dielectric constant of the metal and ε0 is the dielectriconstant of the sphere surroundings. When εm = −2ε0, the magni-ude of E becomes very large. This means that the dielectric functionf the metal and the adjacent dielectric medium must be of oppositeign, with an absolute value of the former exceeding the latter forhe efficient field enhancement. The dielectric function of a metalan be further expressed as a complex one. The real part deter-ines the resonance condition for a plasmon, while the imaginary


art of metal dielectric constant accounts for the damping of thelasmon waves passing through a metal experienced due to Ohmic

osses and electron–core interactions. The imaginary part of theetal dielectric constant must be therefore close to zero, for the

Adapted from reference [11].

effective surface plasmons propagation. Metals, especially noblemetals such as gold and silver, exhibit a large negative real part ofthe dielectric constant along with a small imaginary part. There-fore at the interface between a noble metal and a dielectric, suchas glass or air, localized surface plasmon modes can exist in a visi-ble range. The two discussed conditions can be met simultaneouslypractically at any wavelength in the visible and near-IR region forthe silver. While for Au and Cu, the localized surface plasmon reso-nance (LSPR) condition related to negligible imaginary part of metaldielectric constant is fulfilled at wavelengths longer than 600 nm,which shifts away the purely real part resonant condition, fulfilledat 490 and 366 nm respectively for gold and copper. This explainswhy SERS effect for Au and Cu is observed only under red and near-IR lines excitation. The imaginary part of εm reflects the presence ofintra- and interband transitions. A strong increase of the measuredimaginary part of the dielectric function of gold, at the wavelengthsshorter than 550 nm is explained in terms of higher energy photonsthat can promote electrons of lower-lying bands into the conduc-tion band, which in a classical approach will correspond to excitingthe oscillations of bound electrons [13].

SERS investigations of colloidal systems revealed, for exam-ple, that only 0.01% of the adsorbed molecules contribute to theobserved SERS signal [14]. There are reports that dye moleculesmay preferentially adsorb at highly SERS active sites [15]. Amongthese, adsorption sites attributed to be responsible for electromag-netic enhancement are often called ‘hot spots’, as exhibiting local,exceptionally high enhancement of electromagnetic field in com-parison to the ‘cold zones’ [16]. Especially the molecules trapped atthe junction of the metal particles experience the extremely largefield and thus exhibit strong SERS signal [17,18] (see Fig. 1).

Thus EM model of SERS effect revealed the resonance nature ofthe phenomenon, explained enhancement of signal by 104 factorand long-range character of the effect (separation of the adsorbate


with the thin polymer layer from the metal surface conserves theenhancement). On the other hand, the issues of the potential depen-dence of SERS signal and significantly stronger enhancement for thefirst layer of the adsorbate remained unclear.





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ig. 2. Schematic representation of charge transfer (CT) model involving electron totential applied to the metal on Fermi level energy is also illustrated.

.1.3.2. Chemical enhancement. The additional effect contributingo SERS is chemical enhancement, also called the first layer effect.ts origin can be ascribed to specific interactions, i.e. electronicoupling between molecule and metal, leading to formation of aolecule–metal complex, followed by an increase of the Raman

ross-section of the adsorbed molecule within this complex. Onepproach states it relies on resonance Raman (RR) effect, basing onhe coincidence of incident photon energy and electronic transi-ion in the molecule, which results in significant increase of signalntensity. RR effect becomes operative due to shifted and broadenedlectronic levels in the adsorbed molecule (compared to the freene) or due to a new electronic transition in the metal–moleculeystem.

There are two possibilities of (charge transfer; CT) mechanism19]:

electron transfer from Fermi level (EF) of metal to nearly locatedcharge transfer level (ECT), composed of excited electronic stateof adsorbed molecule and LUMO (lowest unoccupied molecularorbital) level of metal (see the scheme in Fig. 2)electron transfer from adsorbed molecule energetic level (EM),which involves the transfer from HOMO (highest occupied molec-ular orbital) level of metal–molecule complex to metal Fermilevel (EF)

CT model explains the potential dependence of SERS intensity,haracteristic for a particular vibrational mode. Polarization of thelectrode shifts the Fermi level of the metal (see the Fig. 2) and thushe energy of the laser must be tuned to re-establish the resonanceondition.

In photon-driven charge transfer theory (PDCT) proposed bytto [20] the four steps can be distinguished (for the moleculepproximately neutral in a ground state):

1) photon annihilation, excitation of the electron (surface plasmoncreation)

2) tunneling of the electron into the electron affinity level of adsor-bate molecule

3) tunneling of the electron back from electron affinity level (with


changed normal coordinates of some internal molecular vibra-tions) to the metal

4) recombination of ‘electron–hole’ pair followed by Raman pho-ton emission

r from Fermi level of the metal (EF) to the CT level of the molecule. Influence of the

Alternatively an analogous roundtrip of a hole involving the holeaffinity level for anionic species (charged with approximately oneunit in a ground state) can be imagined.

This dynamical charge transfer is useful in order to explainextremely large surface enhancement factors in the case of singlemolecule SERS [21]. In reference [20] atomic scale surface rough-ness is discussed as a requirement necessary for the chemicalsurface enhancement in terms of PDCT theory.

Chemical effect is also often related to the existence on the sur-face of SERS ‘active sites’, which are the areas characterized by apresence of complexes of the adsorbate with metal adatoms ora few atoms metal clusters (often charged), which are respon-sible for the enhancement of SERS signal. These ‘active sites’can be introduced by displaced surface atoms, translocation ofcrystallographic plane, step edges, voids and slits [22,23]. Modelassuming metal-adsorbate interactions within such complexesexplains nicely the short range mechanism contribution to theoverall surface enhancement.

In general, the chemical enhancement is considered to pro-vide enhancement factors on the order of 10–102 [24]. Chargetransfer mechanism explains the potential dependence of the SERSspectrum (shift of the maximum band intensity with the variedpotential for different excitation lines) [25].

2.1.3.3. Total surface enhancement and enhancement factor. Thecontribution of both electromagnetic and chemical enhancementto the overall SERS intensity can be expressed approximately as:

ISERS ∝ GEM∑�,�

∣∣(˛��)nm∣∣2

(2)

where GEM is the electromagnetic enhancement factor for the elec-tromagnetic fields of both incident and scattered light, while thesum term (˛��)nm is the molecular specific, frequency dependentpolarizability. These two terms include all discussed here mech-anisms of the electromagnetic and chemical contributions to theenhancement.

The magnitude of the overall enhancement factor (EF) can bedetermined experimentally using the following expression [26]:[

ISERS/Nsurf]


EF = [INRS/Nvol

] , (3)

where ISERS and INRS are respectively intensities of the surface-enhanced Raman and normal Raman Stokes bands measured at a


ING Model

E

imica

sna

2actoin

2

cels[p

•

••••

2

2f

nftvwdib

(

watsqBf⟨wowtfe

M



ingle excitation wavelength and Nsurf and Nvol are respectively theumber of molecules bound to the enhancing metallic substratend in the excitation volume.

.1.3.4. The electromagnetic surface selection rule. The electric fieldt the metal surface is strongly anisotropic, which means that theomponent normal to the surface is significantly larger than theangential one, and therefore estimation of the average orientationf adsorbed molecules versus the metal surface from SERS spectrums possible [27]. Raman modes with the polarizability componentormal to the surface will exhibit the strongest enhancement.

.1.4. SERS active substratesThe most common metals used as substrates for SERS are

oinage metals, i.e. Ag, Au and Cu as they provide large surfacenhancement [28]. Even for Ag, Au and Cu supports there is anotherimitation: they must exhibit surface morphology with roughnesscale of 50–200 nm, prerequisite for high surface enhancement28]. These submicroscopic dimensions can be fabricated by variousreparation procedures:

electrochemical roughening with use of oxidation–reductioncycles [29]chemical etching [30]metal film coatings [31]chemical reduction of metal salts (colloids preparation) [32]formation of ordered nanostructured surfaces by means of elec-tron, ion, or light beam lithography and SPM (Scanning ProbeMicroscopy) lithography [28].

.2. Resonance Raman scattering

.2.1. Brief theory of resonance Raman (RR) scattering andeatures of RR spectrum

The selective enhancement of Raman vibrations can be achievedot only by SERS technique, but also by tuning the excitation laser

requency to match with the energy of an electronic transition inhe molecule, in so-called resonance Raman (RR) scattering. It pro-ides the enhancement often by more than six orders of magnitudeith respect to Raman scattering and allows the characterization ofilute solutions of chromophoric species. The change in the polar-

zability between the ground (g) and final (f) electronic states cane expressed as [33]:

˛��)gf

= 1�

∑e

⟨f∣∣��∣∣ e⟩ ⟨

e∣∣��∣∣ g⟩

�eg − �0 + i�e+

⟨f∣∣��∣∣ e⟩ ⟨

e∣∣��∣∣ g⟩

�ef + �0 + i�e,

(4)

here � and � denote Cartesian coordinates of the tensor, ��nd �� are the dipole moment operators, g> and f> are the ini-ial and final wave function and e> is a wave function of an excitedtate of half bandwidth � e and �eg and �ef are the transition fre-uencies. Separation of electrons and nuclei movement applyingorn–Oppenheimer approximation allows for separation of wave

unctions into vibrational and electronic parts:

f∣∣�∣∣ e⟩ =

⟨j∣∣Me∣∣ v

⟩and

⟨e∣∣�∣∣ g⟩ =

⟨v∣∣Me∣∣ i⟩ (5)

here i> and j> are initial and final vibrational wave functionsf the ground electronic state (g) and v> is a vibrational functionave function of the excited electronic state e. Me is the pure elec-

ronic transition moment between (g) and (e). As a weakly varyingunction of nuclear coordinates (Q), it may be expressed as Taylor


xpansion around the equilibrium position (0):

e = M0e +

(∂Me∂Q

)o

Q + ... (6)

PRESS Acta xxx (2013) xxx– xxx 5

where Q is a given normal mode of the molecule. Neglecting thenon-resonant terms the first two terms of the expansion will pro-vide the following expression for the polarizability:

= A + B (6a)

A = (M0e )

2 1�

∑v

⟨j |v〉

⟨v∣∣i⟩

�vi − �0 + i�v(6b)

B = M0e

(∂Me∂Q

)o

1�

∑v

⟨j∣∣Q ∣∣ v〉

⟨v∣∣i⟩ +

⟨j |v〉〈v

∣∣Q ∣∣ i⟩�vi − �0 + i�v

(6c)

The frequencies �vi refer to the transitions from the groundvibrational level v to the vibrational levels of resonant excited state.In order to obtain RR enhancement A- and/or B-term must be non-zero. For the A-term two conditions must be met:

• the electronic transition must be dipole electric allowed• the product of the vibrational overlap integrals must be non-zero

(so-called Frank–Condon factors, i.e.⟨j |v〉

⟨v∣∣i⟩ )

This second condition is met for the potential energy wells of dif-ferent shape for the ground and excited electronic state or when theequilibrium bond length for the excited state is shifted with respectto the ground state. Assuming lack of molecular symmetry changesthe displacement of equilibrium nuclear positions between theground and excited state occurs only for totally symmetric modes.Thus, the totally symmetric mode will be selectively enhanced in RRspectrum by A-term, dependent on the Franck–Condon overlaps.On the other hand, non-totally symmetric modes can gain intensityvia B-term enhancement. It is explained in terms of vibronic cou-pling, which is the coupling between the excitation of electronicstate with vibrations. Its magnitude is determined by the magni-tude of electronic transition moment (Me) and its modulation byoscillation

(∂Me/∂Q

). Paradoxically, this interrelation and influ-

ence of vibrational and electronic states was neglected within theBorn–Oppenheimer approximation.

Selective enhancement of the particular modes is typical for RRspectrum. Moreover, the maximum intensity of the given RR bandis dependent on the excitation line energy. Therefore so-called exci-tation profiles can be determined (plots of the intensity of the RRsignal as a function of energy of incident light), which shape shouldimitate the contour of electronic absorption band. Strong and longprogression of overtones are often observed in RR spectra and theratios of depolarization exhibit often anomalous or inversed polar-ization.

The disadvantage of RR spectroscopy is that it is actually lim-ited to chromophoric molecules and probes only this part of themolecule, especially the fragments exhibiting a large change ingeometry between the two electronic states. Indigenous fluores-cence or traces of fluorescent impurities can prevent the acquisitionof RR spectrum. Meaningful vibrational analysis requires extensiveisotope substitution data.

2.3. Electrochemical SERS (EC-SERS) technique

To have an insight into the redox and ET properties of the redoxactive proteins with SERS spectroscopy, a spectro-electrochemicalexperiment must be carried out. Performing electrochemical SERS(abbreviated further as EC-SERS) measurements and hence study-ing an effect of varying potential effect at molecular level requires


in situ SERS measurements under potential control. Varying apotential applied to the electrode (SERS active substrate) can affectboth the chemical and physical enhancement of the SERS signal,as it influences the position of the Fermi level of metal and the





F g the

(

dmSacecseeteo

2o

iwctfipciea[maftctatavgcwc

•

•

tF

ig. 3. Schematic diagrams of the electrochemical interfaces exhibiting SERS, showina) positive or (b) negative to the pzc (potential of zero charge) [34].

ielectric constant of the electrolyte. Moreover, all these featuresake EC-SERS greatly more complicated system than conventional

ERS, i.e. taken ex situ and/or at open circuit potential. Therefore few aspects of the EC-SERS, i.e.: properties of the electrochemi-al double layer, polarity of the surface, effect of potential on SERSnhancement, effect of electrolyte and solvent, design of EC-SERSell and choice of a substrate will be briefly discussed within thisection. The structure and content of this section is strongly influ-nced by an excellent tutorial review by Wu et al., devoted tolectrochemical surface-enhanced Raman spectroscopy of nanos-ructures [34]. However in this review some aspects have beenxpanded and presented from a more here discussed applicationriented perspective.

.3.1. Electrical double layer (EDL) of EC-SERS system and effectf the polarity of the surface

The key region when interfacial electrochemistry is discusseds the electrode/electrolyte interface [35]. This important region,

here charge transfer occurs and gradients in chemical and electri-al potentials arise, driving the electrochemical reactions is calledhe electric double layer (EDL). von Helmholtz in 1879 was therst who developed a model of EDL, wherein he proposed a sim-le charge separation at the interface [36]. In other words surfaceharge is balanced by a layer of oppositely charged ions arrangedn a rigid fashion and thus these two charged layers (one at thelectrode surface and second in solution) can be treated as two par-llel plates of the capacitor. At the beginning of 20th century Gouy37] and Chapman [38] independently proposed alternative EDL

odel, where the layer is no longer rigid, but the ions considereds point charges are free to move. So-called diffuse double layer isormed and the distribution of charges with distance from the elec-rode is predicted with the aid of Boltzmann’s law. Eventually Sternombined these two models proposing that to the double layer con-ribute a compact layer of ions next to the electrode, followed by

diffuse layer extending into bulk solution [39]. Across the EDLhe potential drop over the compact and diffuse layer results in therise of a huge, highly localized electric field, which can reach even aalue of 109 V m−1 [40]. As already discussed (see Section 2.1.3) it isenerally accepted that mainly electromagnetic field enhancementontributes to the amplification of Raman signal in SERS. Thereforehile EC-SERS experiment one has to keep in mind that for this

ase two types of electric field are present in the system:

alternating EM-field – used for the sample illumination andexcited at nanostructured substratestatic electrochemical field (EC-field) – co-existing in the electro-chemical system, mostly across the EDL


A schematic diagram showing the overall distribution of thesewo electromagnetic electric field contributions is presented inig. 3, as proposed by Wu et al. [34]. In the same figure, the effect of

coexistence of electromagnetic field and the electric field at the electrode potentials:

the surface polarity is also illustrated. In part a of Fig. 3, when thepotential of the electrode is positive with respect to the potential ofzero charge (pzc), the surface attracts the negatively charged oxy-gen of water molecules (assuming aqueous electrolyte solution),however if some other anionic species are present in the elec-trolyte they may move towards the surface and repel the surfacewater. In part a of Fig. 3, pyridine is shown as an example, whichtends to interact with the positively charged electrode via both �orbital and lone pair orbital on nitrogen atom, which results in atilted configuration of the molecule. When the potential is variedto the more negative value than pzc, binding of water via hydrogenatoms will be favored and other cations will approach the surface,as shown in Fig. 3b. For the pyridine this will result in preferen-tial adsorption exclusively via lone pair orbtital of nitrogen atom.This shows that for pyridine the change of the potential from pos-itive to negative, the orientation of the molecules changes fromtilted to vertical. Application of the strongly negative potential tothe electrode may result in gradual weakening of its interactionwith the surface, proceeding from chemisorption to physisorptionand eventual desorption of the molecules.

This latter example shows nicely an effect of the potentialinduced changes within the adsorbate, which will be reflectedin the SERS spectrum. The changes in molecular orientation canbe followed with monitoring the variation of SERS bands relativeintensities employing surface selection rules, while the strengthof the chemical interactions will be reflected in the shifts of thepositions of the SERS bands. This shows that for the EC-SERSthe chemical enhancement contribution becomes more significantthan for the regular SERS experiment when the potential is con-stant.

2.3.2. Effect of the electrode potential on surface enhancement inEC-SERS system

Moreover, the intensity of the SERS bands, meaning the magni-tude of the surface enhancement is also dependent on the appliedpotential. This may indisputably relate to the potential provokedchanges of the electrode surface coverage. A linear dependence ofthe SERS intensity on the surface coverage was obtained for the2-amino 5-nitro-pyridine (ANP) adsorbed on the roughened silver[41]. To minimize the effect of the irreproducibility of roughnessfabrication the surface charge was determined instantaneouslyafter taking SERS spectrum at a given potential by stepping toelectro-reduction potential of ANP. Nevertheless, silver is veryoften contaminated with oxide/hydroxide species, which questionsthe accuracy of determination of surface coverage of an adsorbate ofa choice. Studies by Stolberg et al. [42] on the relationship betweenSERS intensities and surface concentration for pyridine adsorbed on


roughened gold demonstrated there is no simple correlation. After-wards they proposed that the lack of relationship occurs since thedata obtained from a rough surface (SERS intensities) is comparedwith data obtained from a smooth surface (surface coverage).




A. Królikowska / Electrochimica Acta xxx (2013) xxx– xxx 7

Fig. 4. Dependence of the extrapolated (to smooth electrode surface) SERS intensityof the ring-breathing mode of pyrazine on the adsorbate surface coverage [43]. Thea

toSrmaoms

sibieicadi

Fpef

Fig. 6. Schematic diagrams of the photon-driven charge transfer (PDCT) from ametal electrode to adsorbed molecule in the EC-SERS system.Left: the conceptual model of the energy levels changed with the electrode potentialin the CT process.Right: relevant energy states involved in the electronic levels and the vibrationallevels in the CT process. Right bottom: the corresponding SERS intensity–potentialprofile.

pplied potentials are indicated on the graph.

In order to challenge this problem they developed a procedureo calculate the SERS intensity of a smooth surface from the databtained for a rough surface [43]. Studies of potential profiles ofERS spectra of pyrazine on smooth gold employing this procedureevealed that the changes of the SERS intensity of the ring-breathingode simply track the coverage variations up to ca. two-thirds of

monolayer. A nice linear relationship obtained between extrap-lated SERS intensity (for zero roughness) of the ring-breathingode of pyrazine and the surface coverage on unroughened gold is

hown in Fig. 4.However, even when working in a potential window corre-

ponding to undisturbed coverage of the adsorbate, still the SERSntensity is dependent on the applied potential. Moreover, theehavior of different modes is not the same, i.e. their maximum

ntensity is reached for different potential values. This can beasily seen in Fig. 5, which presents the variation of the SERSntensity for different modes of pyridine adsorbed on silver, uponhange of potential applied to the electrode. Bands marked with


, b and c (located respectively at 1006, 1035 and 3056 cm−1) areetectable at whole studied potential range and exhibit maximum

ntensity at less negative potential values than bands d, e and f

ig. 5. Potential dependence of the SERS intensity (exc = 514.5 nm) for modes ofyridine adsorbed on silver: a) 1006 cm−1, b) 1035 cm−1 c) 3056 cm−1, d) 1215 cm−1,) 1594 cm−1 and f) 623 cm−1. Potential was varied (at a rate of ∼1 V s−1) startingrom 0 V (vs. SCE) and each curve is an averaged signal of 10 potential scans [2].

Vi denotes the applied potential [34].

(at 1215, 1594 and 623 cm−1). The later become active at stronglynegative potential and reach maximum intensity at around −0.8 V(vs. saturated calomel electrode; SCE) [2].

The assignment of the five most prominent pyridine bands(below 1600 cm−1), under a C2v point group, is as follows:624 cm−1 – asymmetric ring deformation (�6a, the Wilson notation)1010 cm−1 – ring-breathing mode (�1), 1035 cm−1 – symmetric ringdeformation mode (�12), 1218 cm−1 – C–H in-plane bending mode(�9a), and 1598 cm−1 ring C–C stretching mode (�9a). For the neatliquid pyridine only the �1 and �12 modes are Raman active [44].According to the assignment of the pyridine SERS active modes, thisperformance suggests a contribution of chemical enhancement tothe change in the relative Raman intensities as a function of theelectrode potential [45].

The reason that the magnitude of the chemical enhancementchanges with the potential for a given system (metal/adsorbateand at fixed excitation wavelength) is that energy of Fermi levelis affected by the applied potential. In Fig. 6 the scheme illus-trating the response of the photon-driven charge transfer to thevarying potential at the metal/solution interphase of the EC-SERSsystem, introduced by Wu et al. [34] is given. On the left the up-shift or the down-shift of the Fermi level in the metal electrodewith the varying potential is shown. The HOMO and LUMO orbitalsof the adsorbed molecule are shown in the center. The g(Vi) onthe right denotes the molecular adsorption ground state, which


energy is potential dependent, while e(CT) stands for a photon-driven CT excited state formed from a filled level of the metal tothe LUMO of the adsorbate. Assuming that the energy position of


IN PRESSG Model

E

8 imica Acta xxx (2013) xxx– xxx

tSFtttiottcutivptcspo

(g

E

(

d

itDSoa

tac[wt

ω

wvTfpibntisire



he CT state is independent on the potential, the appearance ofERS intensity–potential profile seen in the right bottom of theig. 6 can be interpreted in terms of changed resonance condi-ion. Let one compare the difference between energy levels withinhe metal (on the left) and electronic states of the adsorbate (cen-er) and relevant vibrational levels of combined system (right side)nvolved in CT energy states at a given potential with the energyf the exciting photon. At the potential V1 the quantum of excita-ion energy (h�) is to small to create the CT states on the interface,herefore the changes of the SERS intensity are mainly due to thehemical bonding effect. When the potential is changed to V2, thep-shift of the Fermi level energy leads to the matching of the exci-ation energy of h� and the charge transfer energy, which resultsn a significant enhancement of the SERS signal of the relevantibrational modes due to resonance-like scattering. Change of theotential to more negative value V3 results in the further up-shift ofhe Fermi lever, which position no longer matches the resonanceondition. Consequently, the decrease of the enhancement by CTtates to the SERS intensity for this potential is observed (see theotential-dependent SERS intensity profile shown at right bottomf the Fig. 6).

Depending whether the CT takes place from metal to moleculeas in Fig. 6) or opposite direction, the resonance condition caniven by following formulas:

( e(CT)) − (EF(PZC) + eVi) = hv for the metal → molecule CT

(7)

EF(PZC) + eVi) − E( g(Vi)) = hv for the molecule → metal CT;

(8)

which clearly show the resonance nature and potential depen-ence of the PDCT enhancement.

Detailed potential and laser wavelength dependent SERS stud-es of spectral intensities behavior for pyridine on silver [25], alsoaking into account voltage-dependent coverage [46] and furtherFT calculations [45] demonstrated that for this system an EC-ERS potential dependence is quite complex. It is a combinationf chemical bonding, charge transfer and active sites effect, whichll contribute to the total chemical effect.

At the early stage of SERS measurements it was believed, thathe potential does not effect the EM mechanism of enhancementnd that the changes in the intensity profile not exhibiting stronghemical enhancement, reflect only the surface coverage changes47]. What can be undoubtedly stated, is that the applied potentialill influence the SPR frequency (ωp), which can be predicted with

he use of following expression:

p =√

4�ne2

mε0(9)

here e and m are the charge and mass of the electron, ε0 is theacuum permittivity and n is the number density of free electrons.he latter quantity is the one responsible for the change of the SPRrequency with potential, as nicely shown by Wu et al. [34]. Whenotential becomes negative (with respect to the pzc), it leads to an

ncreased polarizability due to extra electrons in the conductanceand, resulting in larger enhancement. Hence, an application of aegative potential to the electrode will increase and up-shift (inerms of frequency) the plasmon resonance band, whereas a pos-tive potential will damp and downshift it. Still, according to the


urface selection rules [27], only the chemical effect can change thentensity of the vibrational modes belonging to the same irreducibleepresentation, while it is not possible for purely electromagneticffect.

Fig. 7. a) Trans (T) and two gauche conformers ((G1) and (G2)) of CYS molecule onAg surface; SERS spectra of CYS on Ag soaked in: b) 1 M LiClO4 and c) water [49].

2.3.3. Solvent and electrolyte related effects in EC-SERSexperiment

To make the solvent being in contact with the adsorbate/metalinterface electrically conductive, a presence of electrolyte is nec-essary. Its type and concentration may also have a strong effecton EC-SERS conditions. First of all the dimensions of the EDL areconcentration dependent. High ionic strength may also screen theCoulombic attraction of the proteins exhibiting asymmetric chargedistribution, which may be the key factor for the attachment of theredox active molecules that cannot interact direct with the metalsubstrate. Some ions composing the electrolyte may also bind tothe metal surface and in a case of specific adsorption, it will resultin a shift of the SPR band [48]. Co-adsorption or competitive adsorp-tion of electrolyte ions in addition to the adsorbate is also possible.SERS studies of cysteamine (CYS) on Ag showed that at OCP (opencircuit potential) the type of electrolyte influences considerably theconformation of adsorbed CYS molecules (see Fig. 7a for schematicrepresentations of the conformers of CYS molecule on silver) [49].Hence, halides additionally stabilized the gauche (G) conformer bythe formation of hydrogen bonds with NH2 groups in addition tothe direct N· · ·Ag bond, while perchlorate stabilized trans (T) con-


former and there was a spectral evidence of its co-adsorption onthe Ag surface. SERS spectra of CYS soaked in 1 M LiClO4 and waterare compared respectively in Fig. 7b and c. An increased intensityratio of the C–S stretching vibrational modes of the T/G conformers


ING Model

E

imica

(tCi

ftVcdft

tw[saaoTab

amtpvraissofeo

2

pEusb

ioabs

omactcopt(d(m



marked in the spectra with the respective symbols) correspondingo their relative surface concentration and additional band due tolO4

− stretching mode (around 930 cm−1) can be seen after soakingn perchlorate solution.

This shows, that ClO4− anion competes with the amino groups

or adsorption sites, not occupied by sulfur and imposes conforma-ional change of the CYS molecules, already under OCP potentials.arying charging state of the surface will additionally compli-ate the conditions and a special emphasis should be thus put toistinguish between the intensity changes corresponding to con-ormational changes exclusively due to applied potential and dueo ion co-adsorption.

Type of the solvent may considerably change the properties ofhe EC-SERS system. The most common approach is an exchange ofater with some non-aqueous solvent: organic or ionic liquid one

50–52]. The advantages are the expansion of potential window anduppression of hydrogen evolution at negative potentials. Howevergain, the surface physico-chemical surface properties are changednd as refractive index increases (comparing to water), the redshiftf the surface plasmon resonance frequency should be expected.he severe limitation of the method is that spectral features of thedsorbate cannot be easily studied as they may be overwhelmedy the intense scattering signal from the solvent molecules [53].

The potential dependence of the EC-SERS intensities of Pyrdsorbed on Ag was demonstrated to be different for non-aqueousedium comparing to water, which evidences the changed poten-

ial induced orientation changes of the molecule [51]. Presence ofyridyne affected also the electrochemistry of the ionic liquid sol-ent (1-n-butyl-3-methylimidazolium hexafluorophosfate). Theseesults show that although for a non-aqueous solvent, the SERSctivity is maintained and oxidation–reduction process are inhib-ted under a wide range of potentials, the consciousness of carefulpectral data interpretation and influence of the solvent on thepectro-electrochemical behavior of the adsorbate is crucial. More-ver, the obstacle to perform EC-SERS in the non-aqueous mediaor some transition metals like Pt, Fe and Ni are their weak surfacenhancement factors, which may be an obstacle for the detectionf decent adsorbate SERS signal [53].

.3.4. Design of the EC-SERS cellFor the obtaining of both a reliable, strong SERS signal and

recise control of the electrochemical experiment a design of theC-SERS should be thoughtful. As mostly employed electrodessed for EC-SERS are opaque, a set-up of the cell suited for back-cattering Raman system will be here presented, described earliery Wu et al. [34].

For the aqueous electrolyte, the set-up is quite simple andnvolves a judicious placement of the three-electrode system inrder to allow run the electrochemical reaction and simultaneouslyssure the minimal loss of the SERS signal. The cell body should beuilt of a chemically inert material, as it is exposed to electrolyteolution. Various types of Teflon are strongly recommended.

Usually the working electrode (WE) is mounted at the bottomf the cell. This can be either a SERS active disc metal electrode oretal nanoparticles attached to a flat, conductive metallic (prefer-

bly noble metal) substrate (e.g. gold, platinum etc). Next an inertounter electrode (CE), which is typically a Pt wire ring, is placedo form a closed circuit and a reference electrode (RE) is used toontrol the potential of the working electrode. Usually an Ag/AgClr saturated calomel electrode (SCE) is used as RE. To control thelacement of the reference electrode relative to the working elec-rode a Luggin capillary can be used, which is a glass tube, with a fine


capillary) end. It allows probing precisely the applied potential, asue to small dimensions of the capillary tip and its close distancetwo times the capillary diameter) to the WE it allows to mini-

ize the ohmic drop across the electrolyte. An optically transparent


quartz or glass window should be used as a cover of the EC-SERS cell,the former especially when the UV laser is needed for the excitation.Use of a material, which is a good Raman scatterer (like some poly-carbonate or other polymers) for the cover is obviously pointless.

The glass/quartz coating has a multifunctional role:

• prevents the solution from evaporation under heating upon illu-mination with laser beam

• eliminates contamination of the solution and interference of theelectrochemical system with the ambient atmosphere (oxygenintroduction etc.)

• isolates solution in the cell from the Raman microscope objectiveto prevent etching of the objective lens by the chemically aggres-sive electrolyte (possible both by immersion and evaporation)

The overall scheme of the cell used by a group of Tian and Rencan be seen in Fig. 8a [54]. Described design of the cell allowsalso purging with gas and/or serving as a flow cell (gas/liquid inand out holes). This is especially important when performing theexperiment in non-aqueous system, for which the O-ring designshould be employed to provide water- and gas-tightness. In thiscase a water-free reference electrode is recommended to be used[55,56]. An exemplary sketch of the flow cell, originally developedby Chen et al. [57] for EC-IRRAS (electrochemical infrared reflec-tion absorption spectroscopy) in ATR (attenuated total reflection)configuration and further adapted by Zhang et al. [58] for EC-SERSis given in Fig. 8b. It consists of poly(chlorotrifluoroethylene) body(1), with a hole in the central part for mounting the cylindrical WE(2). The openings for inlet and outlet capillaries are located on twosides. The left-hand capillary goes to a four-way connector (3) withtwo ports for inlet capillaries and one for the RE, while the right-hand capillary is joint to the three-way connector (4) leading toboth the outlet capillary and the CE. A thin Au foil (thickness of50 �m) was used as a CE in this case instead of a metal wire. A flatquartz window (6) was mounted, separated by a thin Teflon filmspacer (5), and pressed against the cell body. The variable thicknessfrom 0.1 to 1 mm was used to adjust the thickness of the electrolytelayer above the WE. In a final step, a silicone rubber O-ring (7) waspressed against the quartz window by a stainless steel cover (8) tojoin the whole cell. Such design allows performance of the in situ EC-SERS studies under continuous flow conditions, with well-definedmass transport to/from the electrode. This in turn allows:

• time-resolved spectro-electrochemical measurements upon sud-den exchange of the electrolyte under potential control;

• spectro-electrochemical adsorption/stripping measurements ofnonvolatile species;

• modification of the electrode by chemical/electrochemicaladsorption/deposition in the flow cell prior to the actual measure-ments, enabling a direct evaluation of different electrocatalysts.

The latter cell design provides additional advantage of the effi-cient coupling of the EC-SERS cell and Raman instrument, due toworking with only a thin layer of the electrolyte, adjustable by theemployed spacer thickness. Presence of a quartz window and layerof the electrolyte within an optical pathway reduces the solid angleof the objective and hence the efficiency of the light collection isdeteriorated. The effect of the electrolyte layer thickness on theintensity of the collected Raman signal is shown in Fig. 9a. It can beseen that the change of the solution layer size from 0.2 to 2 mm out-comes in a decrease of the scattered light from 89% of the maximum


intensity to only 35% [59]. Introduction of a corrosion-protectiveglass cover results in a 50% loss of the signal (see the Fig. 9b). There-fore alternatively to insulating with a glass, Tian and Ren proposedwrapping the objective with a very thin and highly transparent





Fb tor forT stain

pt

22cS

1

2

3

4

finstoaae

ig. 8. a) Sketch of the typical EC-SERS [54];) Design of the flow EC-SERS cell [58]: (1) cell body; (2) WE; (3) four-way conneceflon membrane spacer; (6) quartz optical window; (7) silicone rubber O-ring; (8)

olyvinyl chloride (PVC) film (see Fig. 9c); which improves a lothe Raman signal loss that is now only 10% [59].

.3.5. Substrate selection for EC-SERS system

.3.5.1. General requirements for the SERS electrodes. There are a fewriteria which must be met for the substrate to name it ideal forERS spectroscopy [60,61]:

It should exhibit high SERS activity and therefore provide highsensitivity. By tailoring the plasmonic properties of the substrate,guided by the size of the nanostructures (more than 50 nm) andinterparticle spacing (less than 10 nm) one can tune the LSPRfrequency of the substrate to match the incident laser frequencyin order to maximize the enhancement.

It should provide a laterally uniform surface enhancement,so that the deviation in enhancement factor across the wholesurface is less than 20.

It should be characterized by a good stability and repro-ducibility. Long storage should remain the enhancement, whichdeviation should not be higher than 20% when comparing thesubstrates from different batches, prepared by the same protocol.

The cleanness of the substrate should be high enough to allowalso the studies of the weak adsorbates or even unknown sam-ples.

Regrettably, it is still very hard to meet all of the listed aboveeatures at once, therefore usually a choice of the SERS substrates a compromise, oriented toward a specific application. For theeeds of a quantitative SERS analysis, a uniform and reproducibleurface enhancement is the priority; while in a trace analysishe maximum enhancement is the main concern. For the studies


f the biomolecules, which is the case described here, a puritynd high enhancement of the substrate are the key factors for

decent interpretation, keeping in mind a complexity of thexamined biosystems. For the EC-SERS system, regardless on the

inlet capillaries and RE; (4) three-way connector for outlet capillaries and CE; (5)less steel cover; (9) incident laser beam; (10) microscope objective.

nature of the studies, elimination any surface impurities, whichmay interfere the detection of potential-dependent redox speciesis crucial. This can be achieved in different ways, i.e.: poten-tial cycling, hydrogen evolution, use of a strong adsorbate andits subsequent desorption or electrochemical oxidation/reductivedesorption.

2.3.5.2. EC-SERS substrates. For the purpose of the EC-SERS mea-surements, different strategies can be employed to fabricateworking electrodes, serving at the same time as a SERS active sub-strate. Here is a classification proposed by Wu et al. [34]:

(a) Electrochemical oxidation and reduction cycle(s) (ORC)(b) Assembly of metallic nanoparticles (NPs) on the electrically

conductive substrate, i.e. GC (glassy carbon), ITO (tin-dopedindium oxide), Au, Pt, Pd

(c) Deposition of the core–shell NPs – on an electric conductivesubstrate (i.e. GC, ITO, Au, Pt, Pd)

(d) Templated SERS substrates, with ordered arrays of metallicnanoparticles

The most common metals used as substrates for SERS arecoinage metals, i.e. Ag, Au and Cu as they provide large surfaceenhancement. Between the latter three metals, a superior oxidationresistance, high chemical resistance and large potential windowmake gold preferable working electrode material for electrochem-ical techniques. However, silver still remains the most frequent(EC)-SERS substrate, as it provides the strongest enhancement andallows to work using a numerous excitation wavelengths in thevisible range. Even for Ag and Au supports, a surface morphol-


ogy with roughness scale of 50–200 nm is the prerequisite forhigh surface enhancement, therefore, special procedures to pro-duce nanostructured electrodes for the purpose of EC-SERS activeelectrode preparations are required.


ARTICLE ING Model


A. Królikowska / Electrochimica

Fig. 9. a) An effect of the thickness of the solution layer between the optical windowand the working electrode on the Raman intensity [59].Sbw

asBaardoattmtpdr

2moTAte−

chematic diagrams of the optical configuration of a confocal Raman microscopeetween the electrode and the objective: (b) protected by a cell covering quartzindow; (c) with the objective wrapped with PVC film.

Among them, electrochemical roughening is the easiest onend can be applied to the bulk electrode. Typically, a metal rod isealed into an inert, tubular Teflon sheath forming a disk electrode.efore each use, its surface should be mechanically polished withlumina powders down to 0.3 �m, rinsed with ultra pure water,nd sonicated to remove alumina residuals. Several ORC cyclesesult in anodic dissolution of the metal, which atoms are nexteposited during the cathodic cycle, forming the rough featuresn the electrode surface. The most frequently employed oxidationnd reduction methods are the step and linear (in time) change ofhe potential (from the potential corresponding to metal oxidationo the one characteristic to ion reduction and reverse). This latter

ethod employs the cyclic voltammetry technique (CV); with whichhe roughness of the substrate can be controlled by the appliedotential range, number of the cycles and sweep rate. The proce-ures employed successfully in our lab for the silver and gold ORCoughening are given below [62,63].

.3.5.3. ORC roughening of silver and gold. Prior to SERS measure-ents polycrystalline silver and gold electrodes are roughened by

xidation–reduction cycling in 0.1 M KCl solution in a separate cell.he process is performed in a three-electrode system, with the


g/Au as WE and Pt as CE. All potentials are reported with respecto Ag/AgCl/1 M KClaq electrode, which is employed as a referencelectrode. Five cycles in which the potential is changed from +0.3 to0.3 V, at a sweep rate of 5 mV s−1 are applied in case of silver. The


color of the electrode after the whole procedure should be milky-brown for the best SERS activity. Polycrystalline gold electrodes arepolarized in a potential range from −0.5 to 1.2 V, at a scan rate of50 mV s−1. The number of the cycles is around 20–30-cycles for eachelectrode and a good judgment for the termination of the roughen-ing procedure is the brown (but not black) appearance of the gold.Each procedure is always terminated in the negative vortex poten-tial (a potential corresponding to reduction peak maximum shouldbe held for 30 s) to provide the fullest-possible reduction of theoxidized surface. ORC roughened substrates should be rinsed thor-oughly with the ultra pure water just after roughening procedureand stored in it prior to further use, but an immediate adsorptionof the analyte is strongly recommended.

Typical SEM (scanning electron microscopy) images of the mor-phology of Ag and Au obtained via described above ORC rougheningprocedures are presented in Fig. 10a. They show there is somesize dispersion across the electrode and it is expected to changefrom one electrode to another. This will result respectively innon-uniform distribution of surface enhancement factor and wors-ened reproducibility. Elemental analysis deduced from EDS (energydispersive spectroscopy) spectra taken for such ORC roughened elec-trodes (Fig. 10b) shows that although the electrodes are rinsed withwater, there is still some chlorine present at the surface. Chlorineis believed to exist in a form of chloride ions, which stabilize posi-tively charged metal clusters, during roughening procedure. It wasproposed that SERS active sites at rough silver surfaces are intro-duced by formation of silver Ag4

+ clusters [65]. The first steps of thereduction of Ag+ ion by hydrated electrons are well known [66]:

Ag+ + e−aq → Ag0 (10)

Ag0 + Ag+ → Ag2+ (11)

One can imagine that during the oxidation–reduction cycles(ORCs) applied in silver electrode roughening procedure furtheraggregation of Ag2

+ with silver atoms takes place, leading to pro-duction of Ag4

+ clusters. From two known stable forms of Ag4+

clusters: linear and pyramidal, this second was proposed as morerelevant to observe SERS modes [65] Moreover, it was evidenced forsilver colloids that Cl− ions stabilize Agn

+ complexes on the silversurface [67]. In here described roughening procedure, 0.1 M KCl isemployed as a supporting electrolyte. In spite of extensive rinsingof roughened substrate with water, preadsorbed during ORC pre-treatment Cl− ions remain at the surface even after chemisorptionof a strong adsorbate, as evidenced by SERS spectra of CYS on Ag[68].

2.3.5.4. Chemical synthesis of Ag/Au NPs. ORC roughened elec-trodes may suffer from irreproducibility and non-uniform surfaceenhancement spatial distribution, but they still exhibit high SERSactivity. Chemical reduction of metal salts is an alternative methodfor preparation of SERS active substrate. Citrate reduction of AgNO3[69] and HAuCl4 [70] and AgNO3 reduced with hydroxylamine [71]or sodium borohydride [32] can serve as the SERS active colloidexamples. The disadvantage of the metal colloids is their instabil-ity, resulting in aggregation of the nanoparticles upon time, leadingto loss of their SERS activity.

If one needs to tune the plasmon properties of the Ag/Aunanoparticles, numerous synthesis methods exist. They relymostly on the size- and shape-controlled synthesis of the metalparticles, which geometry deviates from the spherical, formedunder a thermodynamically controlled regime, which favors


isotropic growth. Among them, methods to produce anisotropicnanoparticles, i.e. nanoscale rods, disks, triangular prisms, mul-tipods, cubes and hollow metal spheres can be found [72–75].The size and growth of the anisotropic nanoparticles can be





ver; (b

rgsbtpt[tllTHhs

tIliabcdtSusp

2aeosA

Fig. 10. a) SEM images of ORC roughened gold and sil

egulated by different routes: capping agent guiding, seed mediatedrowth, light irradiated and microwave- and ultrasound-assistedynthesis. In the case of hollow metal spheres mostly the templateased methods are employed, e.g. galvanic replacement of cobaltemplate spheres with gold [76]. Playing with the geometricalroperties of such fabricated hollow gold nanospheres (HGNs),heir surface plasmon band can be tuned between 550 and 820 nm77]. UV–vis spectra of suspensions of three different batches (withhe observed colors of the solutions containing NPs given in theegend) of Au HGNs prepared according to this procedure in ouraboratory are shown in Fig. 11a. In part b of the same Figure theEM (transmission electron microscopy) image of the ‘dark blue’ AuGNs NPs is shown, which proves that large FWHM (full width atalf maximum) visible in the Vis spectrum corresponding to thisynthesis reflects somewhat broad distribution of the NPs size.

Next step prior the EC-SERS experiment is the deposition ofhe NPs from suspension onto the conductive support, such asTO, GC or non-roughened metal substrate (hence, SERS inactive),ike gold. A solid support requires thorough cleaning or polish-ng before NPs assembly. The deposition is spontaneous: placing

droplet of NPs suspensions, and leaving to dry in air or assistedy a vacuum pump [34]. In order to improve the deposition effi-iency the suspension containing NPs can be centrifuged beforeropping. Different approach is coating the substrate with the func-ional groups capable of bonding the colloidal metal. In Fig. 12 theEM image of the fabricated in our lab citrate capped gold HGNs,tilizing their electrostatic attraction by NH3

+ groups of the sub-trate, provided by 3-aminopropyl-trimethoxysilane (APTMS) isresented.

.3.5.5. Catalytic metals as SERS active substrates. The other met-ls, which attract the interest for EC-SERS studies, are these which


xhibit catalytical activity, like Pt or Pd. Unfortunately, typically NPsf Pt group or Fe group transition metals exhibit relatively weakurface enhancement, especially when compared to their Ag andu counterparts. Their electrochemical roughening is not trivial, as

) EDS spectra of ORC roughened gold and silver [64].

these metals tend to form a compact oxide layer, which preventsfurther surface oxidation. Thus, formation of the particles of thesize capable of SERS enhancement is nearly impossible.

Arvia and co-workers developed a unique roughening pro-cedure for Pt electrodes, involving periodic potential electro-reduction of a previously formed thick hydrous Pt oxide layer [79].This procedure was modified by Tian and co-workers in order tomaximize the SERS activity of roughened Pt electrodes [80]. Thedetails of ORC surface roughening procedure for some other popu-lar transition metals, leading to obtaining electrodes of high surfacearea and at least moderate SERS activity can be found in a reviewpaper by Tian et al. [28]. Fabrication of monometallic transitionmetals NPs as SERS substrates also does not provide satisfyingresults, since only the small size particles can be produced, e.g. forthe Pt and Pd NPs typically with a diameter below 20 nm [81], whichleads to only weak SERS enhancement. Some improvement in termsof SERS activity was achieved using special capping agent, allowingPt nanocubes [82] and Pd nanotriangles synthesis [83]. Better SERSperformance was possibly owing to the special effect of the sharpedges of the NPs. Further improvement of the surface enhancementfor the transition metal NPs is due to electrodeposition of a verythin layer (several atomic layers to several tens of atomic layers) oftransition metal (usually Pt group metal) on highly SERS active Agor Au to exploit the long-range effect of SERS [84,85]. Use for EC-SERS studies ultrathin platinum-group films on SERS active gold orsilver was demonstrated to be invaluable tool for exploring tech-nologically important heterogeneous catalytic processes, even atelevated temperatures [84]. It provides real-time vibrational spec-tral sequences on subsecond timescales, even for ambient-pressuregaseous (and liquid phase), which facilitates the understanding ofthe electrochemical interfaces and the mechanism of electrocat-alytic processes. Regrettably, the effect of the substrate metal (here:


gold or silver) on the physico-chemical properties of the overlayermetal (here Pt NPs) and the problem of stability and reversibilityof the thin metal layer is still a limitation which prevents a widerapplication of this method.



ARTICLE ING Model


A. Królikowska / Electrochimica

Fig. 11. a) Vis spectra of gold HGNs obtained in our lab by cobalt galvanic replace-ment (according to protocol given in [77]);b) Representative TEM image of “dark blue” NPs. (For interpretation of the refer-ences to color in this figure legend, the reader is referred to the web version of thearticle.)

Fig. 12. SEM image of the “pink” (see the legend in Fig. 11a) Au HGNs after theirimmobilization on solid substrate, with the use of APTMs (according to the protocolby Hajdukova et al. [78]), 24 h of dipping in NPs.(For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of the article.)


An excellent solution to overcome this problem was proposedby Tian and co-workers, who introduced the concept of borrow-ing SERS intensity by coating Au nanoparticles with a thin layerof transition metal [58]. For such composed core–shell NPs, theSERS activity arises from the gold core, while the chemical prop-erties are determined by the transition metal shell. The advantageof this strategy, comparing to metal NPs thin film electrodeposi-tion, is reduced risk of the pinholes presence in the transition metalshell. The enhancement factors are up to 104–105 [86,87], whichmeans that for example for [email protected] nm Pd the intensity of Ramansignal is 100 times higher than for an electrochemically roughenedPt electrode [86]. A scheme presenting a protocol of core–shell NPspreparation is shown in Fig. 13a. SEM images of [email protected] nm Pd NPsassembled at glassy carbon (GC) electrode are presented in Fig. 13b.The NPs deposition is proceeded by a simple drop coating of thecolloidal suspension, followed by drying. The results of the simula-tions of the local EM field distribution around the core–shell Au@Ptnanoparticle surface are reproduced in Fig. 13c [86]. NPs preparedaccording to this protocol were successfully employed for time-resolved, potential-controlled EC-SERS studies of CO chemisorptionat a Au Core–Pt shell [58].

2.3.5.6. Template-assisted fabrication of arrays of SERS active NPs.When not only high uniformity of the size and shape of the nanos-tructures, but also the controlled NPs interspacing is the priority,the template-assisted methods are required to supply the orderedarrays of NPs. Among the numerous template methods of SERSactive substrates fabrication two the most frequently employed areanodic aluminum oxide films (AAO) and nanosphere lithography(NSL).

2.3.5.6.1. Aluminum oxide films (AAO) method. AAO template isformed by anodic oxidation of previously electrochemically pol-ished aluminum foil. In a next step, the alternating current (AC) isapplied to deposit the SERS active metal into the pores of AAO mem-brane. In a final stage, the AAO matrix is etched in dilute phosphoricacid to ensure well-ordered metal nanorods protruding and enabletheir exposition. The consecutive steps of AAO template method areillustrated schematically in Fig. 14, together with the AFM imagesfollowing the partial removal of aluminum oxide, [28]. Resultingnanorods exhibit high SERS activity and high ordering, but fromthe point of view of EC-SERS experiment their severe limitation isthe difficulty of establishment the electric contact to control thepotential of the nanorods.

2.3.5.6.2. Nanosphere lithography (NSL). NSL method involvesdeposition onto the conductive substrate of the monodispersedsuspension of the polystyrene or SiO2 nanospheres, which self-assembly, producing a highly ordered pattern on the surface [88].In a next step, a thin layer of the SERS active metal is depositedon the polymer mask. In the case of the physical vapor deposition(PVD) of the metal, already this metal film over nanosphere (MFON)can serve as a SERS substrate [89]. However very often the spheresare removed by sonication in an appropriate solvent, to form theperiodic particle array, which in case of PVD results in triangularfootprint, as initially spheres exhibit hexagonal packing. This pro-cedure of NSL method was originally introduced by the group ofVan Duyne [90] and SEM images of its successful employment inour lab to produce AgFON and Ag triangular footprint can be seenin Fig. 15.

This method allows fabrication of nanostructured SERS sub-strates with a precise control over the shape, size and interparticlespacing. These goals can be achieved varying the angle betweenthe normal to the substrate and the deposited sputtered metal


beam [91], metal type, thickness of the deposited metal film andsize of the spheres [92], which in turn allows tuning the plas-monic properties of the SERS. The alternative approach to PVD iselectrodeposition of the metal film on nanosphere mask, launched


Please cite this article in press as: A. Królikowska, Surface-enhanced resonance Raman scattering (SERRS) as a tool for the studies of electron trans-fer proteins attached to biomimetic surfaces: Case of cytochrome c, Electrochim. Acta (2013), http://dx.doi.org/10.1016/j.electacta.2013.08.140




Fig. 13. a. Scheme of Au@Pd/Pt NPs fabrication and its assembly on solid electrode; b. SEM/TEM images of the [email protected] nm Pd at different magnification: (a), (b) and (c); c.Theoretical simulations of the electromagnetic field enhancement around Au@Pt core–shell and electric field plotted as a function of a distance from this spherical NP.

All adapted from [86].





Fig. 14. A) Schematic diagram of the fabrication process of metal nanorod arrays:(a) Anodic oxidation, (b) alternative current deposition of metal, (c) dissolution of the AAO layer to the emergence of the metal nanorods, (d) partial dissolution of the AAOlayer to the appearance of sufficient length of nanorods while they are standing on the surface, and (e) collapse of the metal nanorods on the AAO surface.T er:( m proo

A

ssodw

3e

3e

eEtege(

•

he corresponding AFM images at different stages of the dissolution of the AAO layB) at the initial stage, (C) at the intermediate stage with the nanorods having 100 nnto the surface.

dapted from [28].

uccessfully by Bartlett’s group [93]. In this case lifting off thephere mask leaves a thin metallic film, containing a regular hexag-nal array of uniform segment sphere voids. The comparison of theetails of these two NSL procedures and morphologies obtainedith each approach is shown in Fig. 16.

. Attachment of cytochrome c to alkanethiol coatedlectrode prior ET studies

.1. Strategies for an establishment of a communication betweenlectrode and redox biomolecule

Electrode surface can be considered as a source or sink oflectrons (charge) employed to drive electrochemical reactions.lectron transfer (ET) is a key step of almost every type of elec-rochemical reactions. Redox enzymes and proteins are capable oflectron exchange with suitably prepared electrode. A few strate-ies can be employed for coupling the redox center with thelectrode, which can be categorized according to the ET mechanism


see Fig. 17 to view a variety of ET mechanisms) [94]:

shuttle mechanism – based on a free-diffusing redox medi-ator: natural enzyme co-substrates were used in so-called

trusion over the surface, and (D) at the final stage after the collapse of the nanorods

first generation of amperometric biosensors; whereas in thesecond generation artificial redox mediators, mainly soluble low-molecular-weight metal complexes with reversible ET propertiesreplaced them

• via a sequence of electron-hopping reactions in a redox polymer– electrical wiring of redox-relay modified polymeric hydrogelsemployed; mediator is tightly retained at the surface and thisimproved design also belongs to the second generation of amper-ometric biosensors

• conducting polymer as molecular cable – electron is transferred viaconducting polymer chain; in this case again mediator is fixed atthe surface and this approach is qualified again as a subtype ofsecond generation amperometric biosensor

• direct electron transfer – occurs via a tunneling process betweenthe redox center of the biomolecule and a bare or monolayer-modified electrode; mechanism typical for third generation ofamperometric biosensors

• direct ET – electron tunneling between enzyme/protein anduncoated (bottom) or SAM-covered electrode (top)


The most attractive feature of direct ET (DET) is avoidingintermediate ET steps via self-exchange reactions. The limitationis that DET is only possible with these enzyme/molecules that areimmobilized in the first monolayer [95], which is a disadvantage


ARTICLE ING Model


16 A. Królikowska / Electrochimica

Fig. 15. SEM images of hexagonally packed polystyrene nanospheres (diameter4rs

wtfghcntcalDdp

3

cfchaw

3

g

76 nm) with 300 nm thick sputtered Ag film (top) and the same substrate afteremoval of the polystyrene mask by sonication in absolute ethanol (bottom). AgFONubstrates were prepared in our lab according to NSL protocol proposed in [90].

hen application as a sensor is considered, since it lowers sensi-ivity and stability of the sensor. In practice DET is only possibleor relatively small enzymes with an easily accessible prostheticroup like group of peroxidases, such as cytochrome c peroxidase,orseradish peroxidase or fungal peroxidase [95,96]. Cytochrome

is also an excellent object of ET studies, since its heme group isot buried deeply in the amino acid matrix. Moreover, cyt c struc-ure and redox properties were intensively studied and are wellharacterized. Biomolecules directly adsorbed on substrate suchs carbon or noble metals undergo at least partial denaturation,eading to electrode fouling and to deterioration of conditions forET. Chemical modification of electrode surface allows preventenaturation, limiting significantly direct interactions of therotein with substrate.

.2. Cytochrome c – structure and function

Cytochrome c (cyt c) belongs to the class of proteins known asytochromes, which can be defined as electron or proton trans-er proteins, possessing one or several heme groups. Cytochromesan be subdivided according to their heme iron coordination andeme type. Structure of cyt c, its biological function and strategies ofttachment to metal surface coated with – substituted akanethiolsill be characterized below.


.2.1. Structure of cyt cCytochromes c are electron transfer proteins, which heme

roup or groups are covalently bound to the protein by one or

PRESS Acta xxx (2013) xxx– xxx

more commonly two thioether bonds, involving thiol groups ofcysteine (Cys) residues. Heme c is a complex of iron-protoporhyrinIX (structure presented in Fig. 18a), where the suffix proto pointsat the presence of two vinyl substituents on the porphyrin ring(see Fig. 18b for a complete structure of heme c).

These vinyl side chains are the second constituent of thethioether link, which is formed in reaction with the thiol groupsof two cysteine residues. The single heme iron atom is chelated byfour nitrogen atoms of the four pyrrole rings, provided by porhyringroup. Two additional ligands can bind along the z-axis. The fifthligand for c type of cytochromes is always a nitrogen provided byhistidine (His) residue. Cyt c belongs to the class I hemes, accord-ing to heme coordination classes of cytochrome c proposed byAmbler [97]. Class I includes the low spin, soluble cytochrome c ofmitochondria and bacteria, with the heme-attachment site towardsthe N terminus and the sixth ligand provided by methionine [98]residue towards the C terminus.

The iron atom is in the ferrous (Fe2+) state in the reduced formand in the ferric (Fe3+) state in the oxidized form of the cytochrome,which are known as ferro- and ferricytochrome c, respectively. Theterm low spin and high spin refer to the spin state of the heme iron.Electronic configuration of iron atom (atomic number 26) is givenby: [Ar] 4s2 3d6. Fe2+ and Fe3+ ions correspond to [Ar] 3d6 and to [Ar]3d5 configurations, respectively. Iron is a transition metal capableof forming octahedral complexes with suitable ligands.

To describe the iron complex, the simplification resulting fromcrystal field theory is valid, which provides two main assumptions:

• all charges particles are considered as simple point charges• electron delocalization is not included

In octahedral symmetry d orbitals split in two sets with theenergy difference referred as 0. Energy is differentiated, becausetwo filled orbitals: dx2-y2 and d2

z undergo stronger repulsion withnegatively charged ligands than dxy, dxz and dyz orbitals. The rea-son is better coincidence of the lobes of dx2-y2 and d2

z orbitalswith the axes along which ligands are located, while dxy, dxz

and dyz orbital lobes point between these axes. Hence, the threeweaker repulsed orbitals posses lower energy than two others,which causes a split of initially energetically equivalent orbitals(see Fig. 19).

Occupation of the orbitals is determined by the comparison ofthe repulsion energy between electrons (denoted as P) and split-ting energy ( 0). When P is greater than 0 (weak ligand field) theelectrons will follow the Hund’s rule, while for P smaller than 0(strong ligand field) it is favored to first pair up the electrons in thelower energy orbitals, against the Hund’s rule. Complexes formedaccording to the Hund’s rule are known as high spin (HS), whereasthese with lower energy orbitals completely filled before popula-tion of the upper sets starts, are called the low spin (LS). Hence, itis the ligand field strength, which determines the electronic state.For cyt c the strength of the ligands is close to the crossover point,thus an equilibrium between low and high spin is attainable. Thedistribution of the electrons over the d orbitals for the LS and HSconfigurations of ferrous and ferric heme ion is presented in Fig. 20.

Jahn–Teller distortion along the z-axis of octahedral complex isalso possible. It results in a tetragonal complex, which d orbitalsare split in a more complex way. The energy of the dxy, dxz anddyz becomes diversified (increased energy of dxy) and d2

z orbitalhas decreased energy relative to dx2-y2 (see Fig. 21) and as a resultdifferent intermediate spin states are achievable.


Subject of this review is a horse heart ferricytochrome c, whichis a mitochondrial cytochrome c. The structure of this protein wasvery extensively studied and it has been thoroughly characterized.It is composed of 104 amino acids and covalently attached heme,






Fig. 16. Schematic diagram of the template-assisted methods NSL methods for highly ordered nanostructured SERS active substrates fabrication and the resulting morpholo-gies redrawn according to the published works of Van Duyne’s group and Bartlett’s group.

Adapted from [34].

Fig. 17. Schematic representation of possible ET mechanism between biomolecule and electrode [94]:a. Shuttle mechanism based on free-diffusing redox mediator.b. Electron hopping within redox polymer matrix (wired biomolecule).c. Conducting polymer chain.d. Direct ET – electron tunneling between enzyme/protein and uncoated (bottom) or SAM covered electrode (top).





orhyr

wsSNeaHotTimbaTsdd�tgt

Fae

Fig. 18. a. Structure of protop

ater-soluble and its molecular weight is around 12.5 kDa. Itshape is nearly spherical, with the dimensions of 30Å × 34Å × 30Å.olution structure of horse heart ferrocytochrome c, deduced fromMR (nuclear magnetic resonance) is presented in Fig. 22a [99]. Gen-ral structure of this biomolecule can be described as a one aminocid thick polypeptide layer surrounding heme prosthetic group.ydrophobic side chains of these amino acids create the interiorf this layer. Next layer consist of main polypeptide chain, withhe polar groups of amino acid residues exposed to the exterior.he secondary structure of reduced horse heart cyt c in solutions presented in Fig. 22b. The protein is mostly � – helical (four

ajor helices can be seen), however according to IR spectrum theands characteristic for extended � strand and � turns were foundnd 13% content of the structure was assigned as unordered [101].he amino acid sequence of the ferrocytochrome c with depictedecondary structure is presented in Fig. 22c [100]. IR spectroscopyemonstrated also, that horse heart cyt c undergoes redox depen-ent changes. Upon reduction, the band indicating presence of-helix experiences a shift to a higher wavenumber. This according


o 1H 2D-NMR spectroscopy, combined with the hybrid distanceeometry-simulation annealing calculations, was interpreted inerms of transformation of �-helix into 310-helix (right-handed

ig. 19. Splitting of the d orbitals in an octahedral crystal field. In the case of ironnd iron-protoporhyrin complexes, the d orbitals are filled with 5 (Fe3+) or 6 (Fe2+)lectrons.

in IX; b. Structure of heme c.

helical structure, the N–H group of an amino acid forms a hydrogenbond with the C O group of the amino acid three residues earlier),involving residues 60–63. NMR showed also, that residues 49–54 ofdissolved ferrocytochrome c display a helical structure, identical tothat of both redox forms of crystal structure of the protein, whereasdissolved ferricytochrome c posses in this segment two non-helicalturns, which are classified as turn and 310-helix. In general thestructure of the protein matrix of both redox states of cyt c in solu-tion and crystalline state is similar to high extent, however consid-erable differences in the details of the hydrogen-bonding networkare present [101–103]. Heme group of cyt c is located slightly off-center of the biomolecule. It is anchored to the protein via twothiother bonds to cysteine side chains (Cys-14 and Cys-17). This isquite unconventional way of the attachment of the heme prostheticgroup, which is typically bound simply by iron ligation, supportedby tertiary interactions. The role of covalent attachment is notcompletely understood, however a few possible explanations werepresented. Covalent linking could be responsible for restriction ofthe heme group orientation, which in the case of exclusive ligationfound in other heme proteins may exhibit orientational disorder[104,105]. XRD (X-ray diffraction) studies showed that two-armsof covalent connection can induce non-planar distortion of heme,maintained by the protein tertiary structure [106]. It was pro-posed that heme distortion modulates electron transfer functionthrough modification of redox potentials of the porphyrin ring andthe protein-binding properties with redox partner, which latter isalso controlled by thioether linkages, thus the information aboutheme oxidation is transferred via these two bonds. The role of hemecovalent attachment in structural organization of horse heart cyt cwas also examined, inspired by increased disorder of the structureobserved for apocytochrome c (lacking heme group). Studies werefocused on the function of planar rigidity in protein ordering, whichwas accomplished by replacement of the porphyrin group withtwo heme surrogates differing in planarity and flexibility [107].Surprisingly, the similarity of the two derivatives to porphyrincytochrome c in helical content and structural stability was estab-lished. These findings suggest that the common feature among thethree covalently attached groups, was their hydrophobicity, and itis the dominant factor responsible for stability of protein structuralorganization. CD (circular dichroism) spectra revealed that mixing of


two fragments of horse heart cyt c with heme increases the amountof the helical structure (from 8% to 22%), however this trend israther associated with an effect of the iron ligation than thioetherlinkage [108].





l spin

ar8ohtbfpte[

F((

Fig. 20. Occupation of the d orbitals and tota

Heme is not only covalently attached, but its iron is also lig-ted. Heme iron is coordinated by four nitrogen atoms of porhyrining and the two axial ligands are sulfur from methionine (Met-0) and nitrogen from histidine (His-18). The native configurationf both reduced and oxidized cyt c in aqueous, neutral solutioneme iron is the six-coordinated low spin (referred further in theext as 6cLS) [109]. The conformation of cyt c may change uponinding to chemically modified metal electrode, which will beurther described. Heme group is primarily buried in a proteinocket. However, about 4% of the heme atoms are in contact with


he solvent [110]. This is interpreted in terms of exposure of onedge of the heme, making it accessible to the solvent molecules111]. However, as already mentioned the interior of cyt c exhibits

ig. 21. Schematic representation of Jahn–Teller distortion of octahedral complextop right) into tetragonal one (top left) and concomitant splitting of 3d orbitalsbottom).

in Fe2+ and Fe3+ HS and LS heme complexes.

hydrophobic character. According to NMR experiments solving thestructure of cyt in aqueous solution, a few amino acid residues (con-stituting to hydrophobic heme cavity): tyrosine (Tyr), tryptophan(Trp) and phenylalanine (Phe) effectively separate heme iron fromwater molecules [102]. Presence of six water molecules in the hemecavity of both redox forms of cyt c was established with NMR spec-troscopy and five of them were found to preserve their positionupon redox process [112].

Amino acids location with respect to the heme redox center isas follows: residues 1–47 are positioned on the histidine (His-18)side of heme, called the right side; while 48–91 residues are situ-ated at the methionine (Met-80) side, called the left side and finally92–104 segment goes back to the right side [113]. Asymmetriccharge distribution across the cyt c surface is an intriguing fea-ture (see Fig. 23). Cyt c possesses 21 positively charged residues(19 lysines (Lys) and 2 arginines [111], shown in red and orange inFig. 23, respectively), which cover most of the area of the proteinsurface, including the proximity of the heme edge. Negative chargeis introduced by 12 residues (10 glutamate (Glu) and 2 aspartate(Asp), shown in blue and green in Fig. 23, respectively), situatedopposite the heme edge, thus neutralizing the cationic residuesplaced in the backside of cyt c. Uncompensated charge of the sixLys groups, near the exposed heme edge, plays important role in cytc binding to biological partners, introduced in the section dealingwith the biological function of the protein.

3.2.2. Cytochrome c biological functionCytochrome c can be found loosely associated with the inner

membrane of the mitochondrion. Its role is the transport of elec-trons in the mitochondrial respiratory chain. The respiratory chainemploys the oxidative phosphorylation process, which in generalcan be described as a metabolic pathway that uses energy releasedby the oxidation of nutrients to produce adenosine triphosphate(ATP). It includes multistep thermodynamically driven electrontransport chains, which employ sets of linked multi-subunit pro-tein complexes within mitochondria (in eukaryotes), containinga variety of metal redox cofactors and mobile electron carriers,which function is shuttling of electrons among former species


[114,115]. The role of electron carriers is performed by smallorganic molecules (e.g. ubiquionone/ubihydroquinone) or smallproteins (molecular weight of 10–15 kDa) like cyt c or iron–sulfurproteins [114,115]. Electrons are transferred from electron donors





Fig. 22. Solution structure of horse heart ferrocytochrome obtained from NMR (1GIW pdb file) [99]:a. Amino acid backbone and heme prosthetic group shown in CPK scheme colors;bc ]. (Fort

troae

Fti((r

. Secondary structure of the protein;. Amino acid sequence of cyt c acquired from reversed phase chromatography [100o the web version of the article.)

o electron acceptors such as oxygen, in redox reactions and


eleased energy is consumed to form ATP. Electron transfers duringxidative phosphorylation are coupled with proton translocationcross the inner mitochondrial membrane. Proton movement gen-rates energy in the form of a proton gradient and an electrical

ig. 23. Charge distribution across ferricytochrome c at neutral pH (solution struc-ure obtained from 1GIW pdb file with RasMol software). Heme group is notedn magenta. Positively charged residues are depicted as red (lysines) and orangearginines). Negatively charged residues are shown in blue (glutamates) and greenaspartates).(For interpretation of the references to color in this figure legend, theeader is referred to the web version of the article.)

interpretation of the references to color in this figure legend, the reader is referred

potential across the membrane. The flow back of the protonsacross the membrane lowers this gradient and thus supplies theenergy driving the synthesis of ATP by a large enzyme called ATPsynthase. This energy is used to convert adenosine diphosphate(ADP) into ATP through a phosphorylation reaction (addition ofa phosphate group PO4

3− to ADP molecule). The general schemeof eukariotic electron transport chain during oxidative phospho-rylation is presented in Fig. 24. The role of cyt c is a transfer ofthe electrons between the so-called complex III and IV. Complex IIIconsists of cytochrome c reductase, known also as Q-cytochromec or cytochrome bc1 complex. Cytochrome c oxidase (complex IV)accepts the four electrons transferred by heme group of cytochromec from complex III.

3.3. Alkanethiol SAMs as linkage monolayers for biomoleculesimmobilization

The subject of this review is characterization of ET propertiesof cytochrome c, immobilized on gold and silver substrates. Themost straightforward procedure for binding a biological moleculeis physical adsorption at the metal surface, which involves immobi-lization directly on unmodified metal substrate via hydrophobic orelectrostatic interactions. The simplicity of this strategy offers lackof manipulation with the protein sample, but on the other handthe mechanism of the adsorption is complex. Even if we considera single, well-defined protein adsorbing at a uniform, well-definedsurface a substantial number of processes is associated with theinitial adsorption [116]:

• lateral motion of the protein molecules


• induced dissociation of the neighboring native or altered proteinmolecule from the surface to the solution

• reorientation and conformation change but with preservation ofbiological activity





hosphS

••

•

•

•

sitfstaa

3

nbcdmml

Fig. 24. Scheme of eukaryotic oxidative pource: Wikipedia.

denaturation and activity lossexchange with a protein molecule in solution

The complexity of the processes coupled with the physicaladsorption of the proteins at the metal surface introduces severaldrawbacks:instability of the protein-metal binding which results in exchangeevents or even reversibility of the adsorption process [117]non-specific adsorption which introduces large distribution ofavailable orientations or totally random orientation of immobi-lized protein molecules, which usually blocks their active site orligand-binding site [88]denaturation and loss of biological activity due to high surfaceenergy of a solid/gas interphase [118]

Therefore a common strategy to attach biomolecules to metalurface is substrate modification with molecules capable of bind-ng via interactions or reactions with specific moieties present onhe biomolecule surface. Selected molecular coating must meetollowing criteria to be suitable as linkage monolayer: stability,urface structure uniformity and relative ease of varying the func-ionalities. Self-assembled monolayers (SAMs) of �-terminatedlkanethiolates are probably one of the best surface modificationsccomplishing these requirements.

.3.1. Concept of self-assemblySelf-assembly can be defined as a process of spontaneous orga-

ization of complex, more ordered structures from the primaryuilding units. As a result the atoms, molecules, biomolecules etc.ombine together, forming structures with fewer degrees of free-


om. The simplest case of self-assembly is so-called self-assembledonolayers (SAMs). Being more specific, for a layer of adsorbedaterial to be classified as a self-assembled monolayer, the fol-

owing conditions must be met:

orylation – overview of the ET pathway.

• the layer of material must be only of a single molecule thickness• the layer of material must be continuous• the material must be bound to the surface by chemical bonds• the formation of the layer must be self-limiting in the presence

of excess reagent• the layer must be well-ordered

Typical SAM elements are head-group, chain/backbone andend-group (see Fig. 25 for details). Every constituent of the mono-layer has its own function. SAMs are formed spontaneously by theadsorption of a surfactant molecules on the substrate due to highand specific affinity of the head-group to this substrate. The back-bones are responsible for the intermolecular interactions, crucialfor the ordering of the monolayer, leading to formation of closelypacked and highly ordered structure. Finally the end-group pro-vides the capability to control surface properties, mainly due togroup specific interactions.

Alkanethiols are widely known to form SAMs on silver, gold andcopper substrates [119]. The thiol head-group anchors the moleculeto the metal surface via metal–sulfur bond, created in a processof chemisorption. The SAM structure is further stabilized by thevan der Waals forces occurring between hydrocarbon backbones ofneighboring molecules. The terminal group determines the func-tional properties of the monolayer. It was demonstrated that theend-group is responsible for the physico-chemical properties ofthe monolayer [120,121]. Hence, the CH3 or CF3 end-group makesthe SAM hydro- and metalophobic and highly anti-adherent, whileintroduction of –OH, –COOH or NH2 groups result in an increasedhydrophilicity and strong binding properties toward metal ions


and biomolecules. All the building units of the alkanethiol SAMstogether with their functions are presented in the Fig. 26.

The advantages of alkanethiol SAMs as a platform for linkingbiomolecules are listed below [122]:





F ion of

a

••

•

•

•

•

i

•

•

•

Fr

ig. 25. The scheme illustrating the process of self-assembly performed by immersnd their functions are indicated.

simplicity in forming ordered and stable monolayersintroduction of micro-environment mimicking biological mem-branes (the alkyl chains imitate to some extent the ion barrierproperties of the hydrocarbon chains in lipid bilayers) suitablefor biomolecules immobilizationcontrolled surface properties (hydrophilicity, hydrophobicity,charge) by choosing SAM functional groupminimal amount of the biomolecules (monolayer) is necessaryfor attachment on SAMtime stability, enabling performing a series of reliable experi-mentssimplicity in providing data about immobilized biomolecules onmolecular level

The limitations of the alkanethiol SAMs based biomoleculesmmobilization are as follows [122]:

high sensitivity of immobilized proteins/enzymes to pH, ionicstrength and temperature changes, which slight variation maylead to a loss of biological activitysome SAMs provide insufficient chemical stability, since mono-


layer may be e.g. chemically oxidized during the experimentinduced electric field and monolayer desorption may complicatepotential application as a biosensor

ig. 26. The basic units of the alkanethiol SAM on gold and their function; X and Yepresent different end-groups.

the substrate in a surface-active material solution. The fundamental SAM elements

• highly surface energetic hydrophobic SAMs surface may accumu-late contaminations, blocking the active sites

The strategies have been developed, which offer improvementsovercoming some of the limitations listed above. The problem ofthe changes in pH, ionic strength or particular additives that maycause biomolecule desorption can be solved using covalent cou-pling procedures, which details can be found in a review paper byJonkheijm et al. [123]. The most common method to covalentlyattach proteins to surfaces employs coupling between the aminegroup of the lysine side chain and surface-bound N-Ethyl-N-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide(NHS) activated carboxylic groups, forming stable peptide bonds. Itwas demonstrated that incorporation of osmolytes (organic com-pounds affecting osmosis) or kosmotropes (organic compoundscontributing to the stability and structure of water–water inter-actions) into alkanethiolates makes the surface partially proteinresistant [124]. SAMs presenting oligo(ethylene glycol) (OEG)groups were demonstrated to prevent the non-specific adsorptionof proteins [125]. Phosphate terminated SAMs were on the otherhand shown to provide a biocompatible micro-environment forthe protein adsorption, being an alternative to negatively chargedcarboxyl groups tailored alkanethiolates [126,127]. Susceptibilityof the thiolate SAMs to photooxidation can be also taken as anadvantage and utilized to selectively pattern SAMs by lithographictechniques [128,129].

Although alkanethiol SAMs provide some drawbacks, the gainedbenefit still places them as first of choice for linking biomoleculesto the metal substrate. Next sections of this review paper will bededicated to the (EC)-SERRS studies of the cytochrome c attachedto alkanethiol SAMs on metal supports.

3.3.2. Strategies to immobilize cytochrome c on alkanethiol SAMMain interest of this review paper is ET between cytochrome

c and alkanethiol modified metal surface, therefore strategies fortailoring alkanethiol monolayer on Ag/Au with cyt c will be herediscussed. As already mentioned, cyt c exhibits asymmetric distri-


bution of charge and posses a patch of positively charged lysineresidues. This area of the protein is an excellent binding domainbased on electrostatic attraction between protein and anionicdocking sites of natural cyt c redox partners or, in general, with





Fig. 27. Different modes of cyt c immobilization with aid of �-terminated alkanethiol SAMs [131]:a. Anionic termination (e.g. –COO− , –SO3

− , –PO3−) for electrostatic binding with lysines (blue).

b. Methyl end-groups for hydrophobic binding via the peptide segment 81–85 (yellow).c ovalend on of to

nic[meSl[[tialprwmi(

gbaardeb

4e

4e

tnc

. Carboxylate end-groups cross-linked to amino groups of the lysine residues for c. Pyridinyl end-groups for coordinative binding to the heme [110].(For interpretatif the article.)

egatively charged surfaces [130]. Although Ag electrode surfaces positively charged above the pzc (−0.9 V vs. Ag/AgCl [131]) thisharge is overcompensated by either specific adsorption of anions132,133] or formation of negatively charged layer provided by

onolayer [134,135], thus enabling attachment of cyt c due tolectrostatic forces (see Fig. 27a to view electrostatic binding toAM/metal interface). The most common cyt c linkage mono-ayers are SAMs carrying carboxylate (–COO−) functional groups134–136], but sulfonate (–SO3

−) [137,138] and phosphate (–PO3−)

139] were also successfully used. Alternative approach for cyt cethering to SAM coated electrode is a hydrophobic patch involv-ng amino acid residues 81–85, neighboring with lysines sectionnd placed above the heme plane, i.e. on the side of axial Met-80igand. A possible role of this fragment is stabilization of the com-lex formed due to driving electrostatic forces between cyt c and itsedox partners (bc1 complex and COx). Evidence for binding of cyt cith n-alkanethiols monolayers on Ag by hydrophobic interactions,ost likely via partial penetration of the peptide segment 81–85

nto the monolayers was shown with aid of Raman spectroscopy[140]; Fig. 27b).

Cross-linking of the amino groups of the protein and carboxylroups of the SAM, resulting in the formation of covalent amideond is also easily attainable [141] (see Fig. 27c). Finally, cyt cttachment can be achieved by SAM providing a competitive lig-nd for native heme iron ligands, cabable of coordination to theedox center. Monolayers carrying N-ligand end-groups, like piry-ynyl functionalized alkanethiols were demonstrated to exchangeffectively Met-80 ligand, thus providing direct molecular wiringetween redox center and metal electrode [142,143] (see Fig. 27d).

. EC-SERRS studies of cyt c attached to alkenethiol coatedlectrodes

.1. Principles of surface-enhanced resonance scattering (SERRS)xperiment


Surface enhancement decays strongly with the distance fromhe surface to the analyzed adsorbed molecules. Therefore, one isot able to observe the bands of the biomolecule attached to thiol-oated surface unless we use the one containing chromophore like

t binding.he references to color in this figure legend, the reader is referred to the web version

cyt c. This approach overcomes the obstacle of the decaying surfaceenhancement at a distance provided by a molecular spacer, whenusing the excitation wavelength near the electronic transition in thesurface attached molecule and thus combining SERS and resonanceRaman (RR) effects into so-called SERRS (surface-enhanced reso-nance Raman scattering). SERRS is actually limited to the moleculespossessing chromophore, but it is very useful in studies of the chro-mophoric biomolecules attached to metal via biocompatible spacermolecules [144,145]. SERRS provides additional 103–104 enhance-ment in comparison to standard SERS experiment. The additionaladvantages of SERRS are its high sensitivity (down to 10−8 M) andselectivity (probing only chromophore group).

4.2. Resonance Raman spectrum of cytochrome c

In Fig. 28 the near UV and visible absorption spectrum of fer-rocytochrome c is presented. It exhibits Soret band in the near UVand Q-bands in the visible region. Assuming idealized D4h symme-try of iron-protoporphyrin IX constituent of heme c and ignoringthe peripheral constituents, 81 chromophore normal modes can bedistinguished [146]:

�in−plane = 7A1g + 6A2g + 7B1g + 7B2g + 14 Eu

�out−of −plane = 2A1u + 5A2u + 4B1u + 3B2u + 6Eg

Since the transition dipole moments of hemes lie in the por-phyrin plane, resonance enhancement is only expected for thegerade in-plane vibrational modes [147]. Upon Soret band excita-tion (ca. 413 nm), mainly the totally symmetric A1g modes will gainintensity, due to A-term enhancement. When Q-band excitationline (ca. 514.5 nm) is used, the intensity of A1g modes decreasesand alternatively the non-totally symmetric modes B1g, A2g andB2g gain intensity through the B-term enhancement mechanism[148,149]. Frequencies of resonance Raman (RR) marker bands ofcyt c allow to probe the spin configuration, redox state and ligation


pattern of heme iron [149,150], as they are influenced by theinteratomic distances within the heme group, which are differentfor various electronic structures of the redox center. Calculationof the distance between the atoms is based on applying empirical





A

rv(

�

wv

5Si

assscfrlefiai

4

4

eki

Fig. 29. Scheme of contributions to in situ Raman signal of cyt c immobilized on

Fig. 28. Absorption spectrum of cyt c Fe2+.dapted from [152].

elationship between observed experimental frequency of theibration in RR spectrum (�RR) and the distance between the atomsr) determined by different technique:

RR = K(A − r) (12)

here K and A are factors found experimentally for a givenibration [151].

Measurements performed with the excitation wavelength14.5 nm, pre-resonant with Q-band, increase the contribution ofERS in SERRS effect (and applicability of surface selection rules)n comparison to 413 nm, where RR effect is dominant.

Performing in situ SERRS measurements of cyt c anchored tolkanethiol SAM one has to deal with the possibility of differentcattering contributions by various components of the examinedystem in the overall Raman signal (see Fig. 29 for schematic repre-entation). First of all certainly the SERRS signal of the SAM attachedyt c will be recorded, however the risk of the RR contributionrom the solvated solution protein molecules should be minimizededucing the protein solution used for immobilization to micromo-ar range (10−6 M). The other solution is transfer of the SAM coatedlectrode with the attached cyt c to a protein free electrolyte andurther performing of the EC-SERRS under these conditions. Typ-cally in the SERRS spectrum also the SERS signal of the linkagelkanethiol monolayer is also observed, from which the protein-nduced structural changes within SAM can be deduced.

.3. Choice of a substrate

.3.1. Gold vs. silver


As already mentioned, selection of a proper substrate for SERSxperiment, regarding the aim of the measurements is one of theey factors. In the case of SERRS spectroscopy another restrictions that only metal supports that exhibit SPR in the visible region

alkanethiol SAM coated Ag. Representation of different scattering mechanisms fromdifferent elements of the studied system [64].

can be considered. In practice this limits the choice to use of Agand Au substrates. Au supports provide high biocompatibility andeasiness of electrochemical studies of redox active biomolecules inlarge potential window [153]. On the other hand, silver is consid-ered as toxic and instable, but it exhibits superior optical propertiescomparing to gold, naming higher surface enhancement and SPRtunable from near UV to infrared [154]. In contrast, wavelengthdependence of the SPR for gold imposes use of wavelengths inthe red or even infrared region in order to obtain SERS activity,which makes this metal not suitable for combining SERS and RRfor chromophores absorbing out of this region. Heme of cyt c isunfortunately absorbing mostly in the blue and violet region (seethe UV–vis spectrum in Fig. 28) and exhibits only some weakerRR effect at green wavelengths. Therefore, initial SERRS and EC-SERRS spectroscopy investigations of cyt c attached to alkanethiolcoated metal were limited to the silver substrates, mostly colloidalor ORC roughened and its further correlation with the ET proper-ties of deduced from electrochemical experiments for the proteinimmobilized in similar systems but on the flat gold supports. Nextsections of this review will be dedicated to the description of possi-ble risks resulting from such approach and presentation of differentsupport architectures allowing better selective enhancement ofcyt c SERRS modes and more direct analysis of interfacial electrontransfer processes analogous to these on flat gold substrates.

4.3.2. Flat vs. roughenedA common approach in the initial studies on ET properties of

cyt c attached to thiol SAM-modified gold was cyclic voltammetry(CV) experiment in order to obtain kinetics data for cyt immo-bilized on SAM-covered Au, whereas SERRS, revealing structuralinformation, deal with the protein anchored to thiol SAMs onAg. Therefore, one on the topics studied in our laboratory wascomparison of the electrochemically roughened silver and goldplates with atomically flat Au(1 1 1), as substrates for the thiol self-assembly [155]. We co-adsorbed two �-functionalized aliphatic


thiols: 2-mercaptoetanosulfonate (MES) and mercaptoundecanol(MUL) on electrochemically roughened gold and silver, expect-ing to form SAMs capable of selective binding cyt c only to MEScomponent. Next we compared the composition and structure of





Fig. 30. (a) Composition of the MES–MUL SAMs calculated from the electrochemical desorption data as a function of molar fraction of MES in solutions used for monolayerdeposition.T( tions ua positi

tftrtS

tamatrs�dpcrmaft(r

rend line: 4th order polynomial fit;b) Surface coverage for MES and MUL as a function of molar fraction of MES in soludapted from [155], c) STM images of Au(1 1 1) of different magnification before de

hese two-component SAMs on these two substrates with thoseormed by analogous protocol but on Au(1 1 1) substrates. Quantita-ive and qualitative examination of the mixed SAMs was performedespectively with reductive desorption technique and STM (scanningunneling microscopy) imaging on flat Au(1 1 1) substrates and withERS spectroscopy on ORC roughened Ag and Au.

From the charge values obtained during thiol reductive desorp-ion on Au(1 1 1), the surface molar fraction of MES was calculated,pplying the Faraday law, and plotted as a function of the MESolar fraction in the solution used for SAM formation. These results

re displayed in Fig. 30a. Error bars represent the standard devia-ion of the values of molar fractions determined from the CV curvesecorded for the six SAMs deposited from the solutions for eachtudied thiol concentration. In Fig. 30b the total surface coverage

and the respective values for both components of binary SAMs,etermined from the area under the reductive desorption peak, areresented. As seen in Fig. 30b the two plots representing the singleomponent surface concentration crossover when the MES molaratio in the solution used for SAM preparation was about 0.7. Thiseans that at this molar ratio the surface is covered with equal

mounts of both components. The same conclusion may be reached


rom the plot in Fig. 30a. The obtained results pointed at MUL ashe preferably adsorbed component in the whole studied bulksolution) concentration range. According to the electrochemicalesults for MUL dominant solutions MES was adsorbed in fractions

sed for monolayer deposition; total surface coverage plotted for comparison; bothon of a thiol, acquired with 1.5 V bias and current set point of 50 nA [64].

significantly lower than its contributions in the bulk or there wereno peaks in the CV curves indicating its presence on the surface(for �MES < 0.4). The morphology of the studied for this set of datagold substrate is shown in two STM images in Fig. 30c. It exhibitsterrace-step features characteristic of atomically flat Au(1 1 1).

The SERS spectra of SAMs grown on ORC roughened Ag and Ausupports from single or two-component thiol solutions of varyingmolar ratios of MES and MUL were also recorded. Collected spectrawere expected to bring information about the SAM composition asa function of the molar ratios of MES and MUL in solutions, used forthe SAMs preparation. For more quantitative analysis of the SAMscomposition, the relative intensity ratios of the bands chosen asMES and MUL exclusive markers were plotted as a function of theMES molar fraction in the solution, used for the SAM formation.Results obtained for Au- and Ag-roughened substrates are pre-sented in Fig. 31a and b, respectively. The plots show the relativeintensities of the MES and MUL marker bands obtained in threeseparate experiment runs. In each run, a fresh SAM sample wasmeasured at five different points. Colors indicate separate sampleseries while error bars show the standard deviations (STD) foundwithin one sample. All the three series represented by points of dif-


ferent color align, suggesting similar change trends and the pointscattering (sample to sample reproducibility) for most samplesdoes not exceed the STD (error bars) obtained for a single sample.The polynomial trend line is to guide the eye and was fitted to the





Fig. 31. Relative intensities of the SERS MUL marker band, normalized to the sum of the MES and MUL marker plotted as a function of the molar fraction of MES in thes s. Trec om lef

wpobptcfqastt

olution used for the SAM preparation on ORC roughened Au (a) and Ag (b) support) SEM images showing differences in the morphology for ORC roughened Au (bott

eight-average of all the experimental series. The results indicatereferential MES adsorption on a rough Ag support. The plateauccurring for MES dominant solutions (�MES > 0.5) reaches theand intensity of the one-component MES monolayer, being aerfect illustration of the strong preference of MES adsorption inhis case (see Fig. 31b). This conclusion may be questionable if oneonsiders the differences in the Raman scattering cross-sectionsor various vibrational transitions, bringing difficulty to the reliableuantitative comparison of different Raman bands. Nevertheless,


s here analyzed bands were assigned to the same vibration (C–Stretching) for the MES and MUL molecules, we may safely assumehe similarity of the Raman scattering cross-sections. Hence, therend deduced from the analysis of the SERS intensities of these

nd lines: 4th order polynomial fits. Both adapted from [155].t) and Ag (bottom right) substrates [64].

bands corresponds to changes of the relative surface compositionof the SAM with varying contribution of the components in theself-assembly solution. Gold is known to provide weaker surfaceenhancement of the Raman scattering than silver. Therefore,greater errors and lower reproducibility of the spectra are foundin the results shown in Fig. 31a, in comparison with the similarplots for the Ag-supported SAMs (Fig. 31b). As in the case of theAg substrates a correlation between the MES concentration in thesolution used for the SAMs deposition and the intensity ratio of


the MES–MUL marker bands can be seen, although in this case theMES preferential adsorption cannot be so clearly found, and a morelinear trend line fits the experimental data. Differences in mixedthiol composition observed for roughened Ag and Au substrates


IN PRESSG Model

E

imica Acta xxx (2013) xxx– xxx 27

mt

ot[ocsnlsiAtt[ttptCttappas

temtcitciS

4t

acwuSibrwoaiUadccaete[

Fig. 32. Comparison of CV curves (scan rate of 100 mV s−1) for 0.5 mM bovine heartcyt c in 10 mM phosphate buffer solution (pH 7.00) with 0.05 M NaClO4 obtained



ay arise from slightly different substrate morphology for thesewo metals, shown in Fig. 31c.

Substrate dependent structural and conformation propertiesf the monolayer were also previously found by our group forhe single component thioglycolic acid (TGA) SAMs on Ag and Au63]. More interestingly, the reversed preference of adsorption ofne thiol from the MES–MUL mixture on the rough support asompared to the flat one (Au(1 1 1)) needs some comment. SERSpectra are recorded at the nanostructured surfaces that containanoclusters of various sizes. It was reported that SAMs on col-

oidal Au nanoparticles differ strongly from SAMs on planar Auupport and these differences were interpreted in terms of changesn intermolecular interactions between adsorbed molecules [156].s proved by IR measurements the aliphatic chains of long length

hiols adsorbed on nanometer sized gold clusters are ordered closeo the surface, but become more disordered at the opposite end157]. In the case of MUL molecules, gauche defects near the OHerminal groups may cause lateral H-bonding among these groupso be less extensive in comparison to the SAMs on the ‘flat’ sup-orts. It is known that the contribution of the hydrogen bonds tohe enthalpy of adsorption of �-terminated thiols is high [129,158].onsidering that preferential adsorption and phase segregation inhiol SAMs are likely to be the results of thermodynamically con-rolled process (at room temperature) [159], one may expect thatdsorption of MUL is no longer preferred at the SERS active sup-orts. Giving this literature background, we can suggest that thishenomenon is related to MES–MES and MUL–MUL interactionsnd the impact of structure imposed on the SAM by a structuredubstrate.

This somewhat unexpected finding is extremely important forhose combining Raman spectroscopy on rough supports with thelectrochemical experiments performed on macroscopically flatetals, aimed at determination of electron transfer rates for pro-

eins adsorbed on mixed SAMs, among them here discussed cyt. Therefore experimentalists should be aware of possible strongnfluence of the used substrate (Ag vs. Au, smooth vs. rough) onhe structure and composition of the formed linkage SAM, espe-ially when comparing the results obtained for different substrates,mposed by specific analytical methods (e.g. electrochemistry andERRS).

.3.3. Performance of SERRS and electrochemistry experiment onhe same silver substrate

The results discussed in the previous section prove that reli-ble investigation of electronic properties of cyt c adsorbed athemically modified metal support, aiming at their correlationith the structural information provided by SERRS studies, requiresing of electrodes prepared in a manner enabling performance ofERRS and electrochemistry for the same substrate. Electrochem-cal measurements for roughened, polycrystalline support woulde difficult and ambiguous for several reasons, listed below. First,oughness of the electrode is problematic for cyclic voltammetry,hich like all electrochemical techniques is employed preferably

n smooth surfaces. Next, electrochemical roughening procedurend previous polishing of the electrode is expected to introducenhomogeneity, especially in the case of silver surface [160,161].se of non-smooth substrate will very likely provide a variety of thevailable adsorption sites for linkage monolayers, which may hin-er the interpretation of the electrochemical data for the attachedyt c. Use of silver introduces additional obstacles to the electro-hemical experiment, mentioned previously. However, a few yearsgo a group of van der Zwan introduced a new method of Ag


lectrodes preparation, which allows their simultaneous applica-ion as SER(R)S active platforms and substrate for performance oflectrochemistry of cyt c immobilized on alkanethiolate coatings162]. A first step was an introduction of novel polishing procedure

at Ag (solid line) and Au (dashed line) disk electrodes chemically modified with4-mercaptopyridine [162].

involving the aluminum oxide lapping film and a shortimmersion time of the electrode in the promoter solution (4-mercaptopyridine). Such prepared Ag electrodes were foundappropriate to perform direct electrochemistry of solution cyt c,providing voltammetric responses essentially identical to thoseobtained on gold electrodes under diffusion conditions (comparethe CV curves reproduced in Fig. 32).

The electrochemical pretreatment of the polished silver wasclaimed to worsen the voltammetric response of cyt c at Ag/PySHelectrode considerably, in terms of smaller current peaks and lowsignal-to-background level compared with that obtained in theabsence of electrochemical pretreatment. Next step was ex situroughening of the polished to a mirror like appearance silver elec-trodes, employing a modified procedure of this proposed by Rothet al. [163]. The reports on successfully combined voltammetry andSERRS studies of cyt c immobilized on 4-mercaptopyridine [164]and 11-mercapto-1-undecanoic acid (MUA) [165] coated silverelectrodes prepared according to the procedure described abovehave appeared. Furthermore, the formal reduction potential valuederived by both techniques for mercaptopyridine was identical[164].

For the cyt c on polished prior roughening Ag electrodes coatedwith MUA, the nearly ideal Nernstian behavior of the adsorbed pro-tein at pH 7 was derived from the treatment of EC-SERRS data (seeFig. 33) [165]. In fact, the formal potential value obtained from thepotential-controlled EC-SERRS experiment for this substrate is infine agreement with the values reported for native cyt c adsorbedon gold electrodes chemically modified with the same carboxylicterminated alkanethiol (MUA) [166,167] and altered by only 10 mVin comparison for that determined from CV curve recorded forsuch treated silver electrodes. Such insignificant potential shiftcan be attributed to the local heating of the system upon irradi-ation with laser beam during SERRS experiment. The absence ofa strong voltammetric response characteristic of the native redoxcouple cyt c3+/cyt c2+ upon increase of the pH of the workingsolution from 7.0 to 9.0 is in good correlation with the devia-tion from the ideal Nernstian behavior expected for a one-electroncouple deduced from EC-SERRS spectra (see again the Fig. 33 forcomparison). The complementary of the results provided by Zwan


and co-workers by cyclic voltammetry and Raman spectroscopyshould be emphasized. This is a remarkable outcome, which high-lights the advantages of operating with these two techniquessimultaneously.


ARTICLE ING Model



Fig. 33. Nernstian plots of Ag/MUA/cyt c at pH 7.0 (�) and pH 9.0 (�), resultingfa

4

tdSaaittAotS2

FA

rom EC-SERRS data (exc = 413.1 nm). The linear fit of the data at pH 7.0 is given as dotted line (the intercept at −56 mV) is shown [165].

.3.4. Hybrid Ag/Au systemsThe plasmonic properties of the metallic substrate can be fur-

hermore tuned by changing the surface morphology and theielectric constant of the surrounding medium. The red-shift ofPR peak for metal NPs is expected for increasing diameter andspect ratio, defined as the ratio between the longer and the shorterxis of the particle. Further red-shift is predicted for the increas-ng dielectric constant of the surrounding medium [168]. Due tohe interparticle plasmonic coupling, aggregated metal NPs exhibitypically higher field enhancement than isolated nanostructures.ccording to the mentioned above rule of a thumb, SPR positionf the NPs aggregates is also considerably red-shifted, comparing


o isolated ones [169,170]. Keeping in mind these features of theERS active nanostructures the group of Hilderbrandt developed in009 novel hybrid Au–Ag supports, employing transfer of plasmon

Fig. 34. Scheme of the set-up of the hybrid Ag–Au substrate d

ig. 35. SEM top views of different regions of SERS active hybrid Ag–Au supports:) electrochemically roughened Ag and B) Au overlayer on Ag C) SEM side view of the Ag


resonance excitation from the Ag to the Au coating, possible dueto the presence of the thin dielectric layer between the two met-als [171]. General design of this device is given in the scheme inFig. 34. The more detailed structure of the hybrid Ag–Au systememployed for cyt c studies is as follows. The bottom layer consistsof a mechanically polished and electrochemically roughened Agelectrode, on which subsequently an amino-1-undecanethiol (AUT)SAM was deposited. Next, the adsorbed AuCl4− ions were adsorbedat SAM-modified Ag electrode and electrochemically reduced toform a gold coating. In a final step to guarantee the biocompatibilityof the substrate, the mixed SAM of 11-mercapto-undecanoic acid(MUA) and 11-mercapto-1-unodecanol (MUL) was formed on thetop gold layer. SEM images showing the morphology of the roughsilver bottom layer, gold overlayer and a section through hybrid lay-ered Ag–Au electrode are shown respectively in Fig. 35A–C [171].

Yeast cyt c was chosen as a test molecule to study the EC-SERRSand electrochemical performance of this novel device [171]. It wasdemonstrated that this hybrid Ag–Au support is suitable for SE(R)RSspectroscopy in the entire visible spectral region and the surfaceenhancement is similar to this for pure Ag substrates. Furthermore,its electrochemical properties are basically the same as those of apure Au electrode. These results show that the device combinesthe optical properties of Ag with the chemical advantages of Au.Successful transfer of plasmonic properties of the Ag system toupper gold layer secures the high SER(R)S sensitivity, while dis-playing the electrochemical stability and higher biocompatibility ofgold. Stationary and time-resolved SERRS studies of interfacial ETtransfer processes were successfully performed for cyt c on hybridAg–SAM1–Au–SAM2 (SAM1 and SAM2 were respectively –NH2 and–COOH terminated) by Sezer et al. [173].

4.4. Information derived from (EC)-SERRS spectra of cytochromec attached to thiol modified metal


4.4.1. Heme structureSimilar like for the RR spectroscopy, the SERRS spectra of

cyt c attached to Ag (or Ag–Au hybrid structures) provide the

esigned for SER(R)S studies of different analytes [172].

–Au junction [171].





Fc

idctSp

toletrFsoArp

ap5wttott

tslfifhbHp

Fig. 37. EC-SERRS of cyt c electrostatically adsorbed on MES SAM on roughened Agat pH 7.0 (exc = 514.5 nm).

in addition to B1 6cLS ferric ion. Therefore, not only the info onoxidation state of heme iron, but also on coordination state, spinconfiguration and type of heme-pocket ligation are provided bySERRS spectra. An exemplary set of SERRS spectra corresponding

ig. 36. SERRS (excited with 413 nm) spectra of the reduced and oxidized form ofyt c attached to alkanethiol coated Ag [172].

nformation on the electronic state of the heme. Hence, the oxi-ation state, spin configuration and ligation pattern on the redoxenter iron atom can be deduced from the SERRS data. In Fig. 36he SERRS spectra excited with the 413 nm line, resonant withoret band, showing the marker bands of iron oxidation state areresented.

The bands labeling given here in the legend and for fur-her discussed spectra is according to the complete assignmentf the RR spectra via enzymatic reconstitution with isotopicallyabeled hemes [149]. EC-SERRS spectra collected for cyt c adsorbedlectrostatically pH 7.0 at mercaptoethanosulfonate SAM on elec-rochemically roughened Ag at a gradually varying potential, in aange corresponding to protein reduction/oxidation are shown inig. 37 [138]. Preservation of protein electroactivity upon electro-tatic immobilization on MES SAM on Ag is evidenced by the setf the presented spectra. Application of potential of −500 mV (vs.g/AgCl) allows safely assume that adsorbed cyt c is completelyeduced [141,167]. Markers in the SERRS spectrum taken at thisotential confirm total reduction of cyt c.

At potential of 0 mV and higher striking changes become notice-ble in cyt c SERRS spectrum, confirming the oxidation of therotein. SERRS spectra shown in Fig. 37 were excited with the14.5 nm line and it can be seen that this line being pre-resonantith the Q-bands results in quite different intensity pattern, than

his observed for Soret band excitation (413 nm), however the posi-ion of the bands is conserved. This feature of the signal dependencen energy of the incident light is typical for the spectra with con-ribution of RR enhancement. More straightforward comparison ofhe RR spectra of cyt c excited at 413 and 514.5 nm is given in Fig. 38.

In Fig. 37, also the bands sensitive to the heme ligation pat-ern are marked. The native conformational state of cyt c isix-coordinated low spin (6cLS) with His-18 and Met-80 as axialigands, denoted as B1. Exposure of cyt c to too strong electrostaticeld or some other factors may lead to conformational conversion

rom B1 to non-native B2 spin states of heme iron: five-coordinatedigh spin (5cHS) due to loss of Met-80 native axial ligand or 6cLS/HS


is-His due to occupancy of the vacant position left by Met-80 byis-26 or His-33 residue [167]. It can be seen in the Fig. 37 thatotential induced oxidation of cyt c attached to MES coated Ag is

Bottom: SERS spectrum of MES SAM grown for 2 h from 10 mM MES aqueous solu-tion. Above: EC-SERRS spectra taken at OCP and at applied potential of MES SAMtransferred to 10−6 M cyt c solution of pH 7.0 [138].

concomitant with appearance of portion of Fe3+ B2 5cHS species,


Fig. 38. RR spectra of ferrocytochrome c taken with Soret resonant (top) and Qpre-resonant (bottom) excitation lines [147].


ARTICLE ING Model



Fb(

ttcimt

4

tetSsvmawpspooitatdtoolB

ig. 39. SERRS spectra of different redox, spin, and coordination states of cyt c immo-ilized on COO− terminated SAM measured with 413 nm excitation: (a) B1[6cLS]Red,b) B1[6cLS]Ox, (c) B2[5cHS]Ox, and (d) B2[6cLS]Ox [131].

o altered heme ligation pattern is shown in Fig. 39. The oxida-ion state (Red and Ox denote respectively reduced and oxidize),oordination type and spin configuration are given schematicallyn the legend of the figure. For a more detailed of RR and SERRS cyt c

arker bands, especially of non-native states, the reader is referredo the papers by Hildebrandt’s group [174,175].

.4.2. Redox group orientationStrong contribution of electromagnetic enhancement through

he surface plasmon resonance effect for 514.5 nm excitationxtends applicability of surface selection rules in the interpreta-ion of SERRS spectrum of cyt c. Thus, intensification of particularERRS bands is accomplished via the individual components of thecattering tensor depending on the direction of the electric fieldector and the orientation of the heme plane with respect to theetal surface. When idealized D4h symmetry of porhyrin group is

ssumed all Raman active in-plane A1g, B1g, A2g and B2g modes wille enhanced when each mode involves a polarization componenterpendicular to the surface [27,176]. This condition is fulfilled irre-pective of the symmetry labels when the heme plane is orientederpendicular to the metal surface, while for parallel alignmentf the heme plane with respect to the surface only the intensityf the A1g modes will be selectively enhanced [177]. The reasons that both totally symmetric modes (e.g. A1g mode �4) and non-otally symmetric modes (e.g. B1g mode �10) involve movementlong the x- and y-axes (in the plane of heme group), while theotally symmetric (A1g) modes involve also motion along the zirection [152,178]. Hence, the variations of the relative intensity ofhe A1g/B1g modes provide information about dynamics of protein


rientation [179]. The supplied information shows only the trendf the changes of the heme plane orientation, however the abso-ute orientation cannot be accurately determined. Increase of the1g/A1g ratio, interpreted in terms of the surface selections rules,


implies reorientation of the plane of the heme ring to an increasedangular position relative to the metal surface plane. Selecting themodes for the relative intensity analysis, one has to keep in mindthat this comparison makes sense only when comparing the specieswith the same coordination and spin configuration of the hemeiron. In Fig. 40, the changes in B1g/A1g relative intensity ratio for�10 and �4 bands corresponding to “straightening” of the hemeplane upon reduction of redox center are shown. These orientationchanges, concomitant to redox-linked changes can be explainedin terms of adapting the more perpendicular orientation, which isconsidered as the one facilitating ET between the cyt c and metalelectrode.

Strongly resonant Soret band excitated SERRS of cyt c, taken at413 nm provide the highest sensitivity for monitoring the redox-linked structural changes, but are insensitive to the here discussedprotein reorientation. In contrast, SERRS spectra excited with sub-stantially less resonant Q-band at 514.5 nm provide much weakersurface enhancement, but are sensitive both to the relative orienta-tion of the heme plane and to the redox state and the heme-pocketstructure.

4.4.3. SER(R)S studies of alkanethiol SAM induced effects onlinked cyt c

This review is devoted to the ET properties of cyt c attached to�-terminated alkanethiols SAMs on Ag and Au supports, studiedwith SERRS and EC-SERRS spectroscopy. Therefore, in this section acloser look will be taken at the issue of alkanethiol �-functionalizedcoatings on metal acting as linkage monolayers to bind cyt c and therisks carried by direct interaction of the biomolecule with the metalsubstrate or unwilling effects on native cyt c structure, induced bypresence of the alkanethiol SAM. SAMs structural properties antic-ipated to influence the ET properties of the anchored cyt c will bealso shortly discussed.

4.4.3.1. SAM defects of various origin. Although alkanethiol SAMs onAu/Ag are known to form monolayers of high order and quality, theyare not free of defects. A scheme presenting an overview of the rea-sons of possible SAM defects is shown in Fig. 41. The most commonexternal factors responsible for presence of defects are cleanness ofthe substrate, method of preparation of the substrate and purity ofsolvents and reagents. All of them can disturb the kinetics of self-assembly and affect a final structure. They are easy to minimize oreliminate by careful control of experimental procedure.

As can be seen in Fig. 41, already a polycrystalline metal supportmay lead to many substrate related defects, hence the nanos-trustured one may be also expected to bring some disorder intomonolayer structure. Once again, these negative effects can be min-imized by meaningful substrate preparation protocol. Moleculardefects can also appear in the SAM when the degree of molecu-lar organization is poor: aliphatic chains are not fully extended orthe tilt angle is not uniform across the monolayer. Even the mono-layers of n-alkanethiols are disordered at the room temperature,since the methyl terminal groups introduce a number of gauchedefects [180]. Another important class of the defects are coveragedefects [181], which arise from the lack of the monolayer. They canbe divided in two main classes:

• single site defect/pinhole – a void site where the substrate isexposed to solution, which size can vary from one atom to >103

gold atoms• collapsed site – takes place when substrate–solution spacing lies

below thickness of the full monolayer, thus allowing molecules


to physisorb

Elimination of these defects would be crucial to study the intrin-sic mechanism of the ET of cyt c across the metal/alkanethiol






Fig. 40. SERRS spectra collected with 514.5-nm excitation (Q-band) for reduced and oxidized form of cyt c, exhibiting changed B1g/A1g intensity ratio, corresponding to moreperpendicular orientation of heme with respect to the metal surface for ferricytochrome c [172].

Fig. 41. Schematic illustration of the potential factors leading to defects of the SAM formed on polycrystalline substrate. A dark line at the metal/SAM interface is to guidethe eye to follow the changing substrate topography [119].


ARTICLE ING Model



Fig. 42. Ex situ SERS spectra (exc = 647.1 nm) of MSA SAM on roughened Ag formedfae

iw

4cvouispvhvplbmcoesitf

or:. 48 h in 10 mM aqueous solution, b. 3 h in 1 mM ethanolic solution, c. 48 h in 10 mMthanolic solution [183].

nterphase, as for the defects rich SAMs the electron transfers occursas found to occur primarily through the defects [182].

.4.3.2. Influence of the SAM low surface coverage on attached cyt properties. The coverage effect is very often related to the sol-ent used for the self-assembly. However, the influence of a solventn the self-assembly process seems to be complex and poorlynderstood. A common assumption is that the solvent–substrate

nteractions can hinder the adsorption rate of alkanethiols, as theolvent molecules have to be place-exchanged from the surfacerior to thiol adsorption. It is evident that the choice of the sol-ent is of great importance with respect to the formed SAM quality,owever full description of the complex interactions between sol-ent, surface and adsorbate during SAM formation has not beenrovided yet [119]. Our group studied this aspect for cyt c immobi-

ized at dicarboxyl tailored SAMs on ORC roughened Ag, introducedy mercaptosuccinic acid (MSA, called also thiomalic acid), whicholecular formula is shown in inset in Fig. 42. SERRS experiments

onfirmed successful electrostatic attachment of cyt c on MSA SAMsn silver formed from aqueous solution at neutral pH [183]. How-ver, a negative potential has to be applied to obtain native protein


tructure. In the case of covalent bonding, native 6cLS configurations preserved. Although MSA is water-soluble as other short �-erminated thiols, it is a common strategy to dissolve it in ethanol,or preparation of linkage monolayer [98], which encouraged us


to investigate the influence of the solvent on the monolayer struc-ture.

Spectrum a in Fig. 42 shows the typical SERS pattern for MSASAM formed from aqeuous solution (48 h from 10 mM MSA solu-tion) [184], indicating that MSA molecules chemisorb through thiolmoiety, with carboxylic group in proximity to the sulfur dissociatedand the second one protonated. SERS spectra b and c in Fig. 42 com-pare the monolayers incubated for 3 h from 1 mM and for 48 h from10 mM ethanolic solutions. The most striking difference betweenthe spectra b and c in Fig. 42 is weak enhancement of the �(C-COOH) band (around 910 cm−1) and high intensity of the bandsabout 1150, 1390, and 1537 cm−1 in the spectrum of the monolayergrown for short time and from more dilute MSA solution (spec-trum b in Fig. 42). These two experimental factors may result inlower surface coverage of MSA on silver, which leaves more spatialfreedom to MSA molecules for monolayer exhibiting spectrum inFig. 42b. Consequently, selective surface enhancement or lack ofenhancement of particular vibrations may occur. Moreover, in thecase of low surface coverage the probability of interactions with sil-ver of both carboxylic groups in deprotonated forms increases. Thismay be further confirmed by high intensity of the 1390 cm−1 band,assigned to �s(COO−) and high-frequency shift of the band ascribedto COO deformation, from 821 cm−1 (see Fig. 42c) for higher cover-age, to 834 cm−1 (see Fig. 42b) for the lower coverage.

More interestingly, for MSA monolayers on Ag formed for shorttime from diluted ethanolic solution undesirable oxidation of theiron occurs in the case of electrostatic immobilization, as evidencedby cyt c spectrum attached under these conditions (data here notshown) [184], very likely due to incomplete blocking of the metalsurface by MSA. This effect is rather related to the low surface cov-erage due to short incubation time than solvent itself, howeverfor MSA SAMs on Ag grown from aqueous solution, only less pro-nounced restructuring of monolayer was observed within first 3 hof self-assembly [184]. Structure of the protein layer is improved inthe case of covalent attachment of the cyt c and application of extranegative charge to the electrode. More compact SAM structure,achieved by application of longer time and higher concentrationof thiol self-assembly solution also results in binding of cyt c in itsnative form of heme pocket (reduced 6cLS).

4.4.3.3. Alkanethiol SAMs as insulating barriers for electron transfer– chain length dependence. A densely packed alkanethiol SAM mayact as a barrier between the electrode surface and the electroactivespecies in solution. Distinctive feature of the EDL structure rep-resentative for such insulating barrier is shift of both IHP (innerHelmholtz plane) and OHP (outer Helmholtz plane) away from themetal–solution interface (see Fig. 43a to view EDL model proposedfor Au-S(CH2)nCOOH – electrolyte interface). The increased dis-tance of the planes from the electrode surface results in a linearpotential drop within the aliphatic chain layer [185] (see Fig. 43b).The ET rate is strongly dependent on the thickness of the insulat-ing layer (exponential variation with alkyl chain length). Electrontransfer in this case is expected to proceed via electron tunnelingthrough the SAM insulator, which reduces the absolute ET rate toa level at which diffusion limitations are either greatly diminishedor totally eliminated [186].

Additional advantages is that occurrence of the ET takes placewith the redox couple from a few to tens of Angstroms fromthe metal surface, which decreases the participation of specificadsorption, double-layer corrections and image charge effects incomparison to measurements for bare electrodes. Thus, SAMs


of derivatized alkanethiols on gold electrodes can be considereduseful tunneling barriers, which allow ultrafast heterogeneouselectron tunneling measurements. A more detailed description ofthe number of methylene groups effect on the ET mechanism for





Fig. 43. a. Schematic representation of EDL at the interface between Au-S(CH2)nCOOH and aqueous electrolyte. Ionizable carboxylic groups are representedas HA (protonated) and A− (dissociated) were assumed to be in direct contact withthe water molecules (open circles), while the electrolyte ions are located at the OHPand further away in the diffuse layer [187]. b. The potential profile across the inter-face: the solid line shows a linear potential drop within the alkyl chain of the SAM,wbp

tr

4pccwptHmtw(

�

tlrtei

hile the dotted line represents possible dipoles associated with the Au–S and S–Conds. Em is potential drop in the monolayer part, Es is potential drop in a solutionart [185].

he case of attached cyt c will be given in further sections of thiseview.

.4.3.4. Effect of electric field provided by the alkanethiol SAM. Aotential drop across the electrode/SAM/cyt c/solution interfacean be estimated using a simple electrostatic model. The modelonsidering the interfacial potential changes for electrodes coatedith monolayers of electroactive molecules, was originally pro-osed by Smith and White [188] and adapted for the system withhe protein adsorbed on SAM coated electrodes by Murgida andildebrandt [131,167] (see Fig. 44). The main assumption of thisodel is presence of the net charge densities on the metal (�M), at

he carboxylate/protein interface (�C), and in the redox center (�RC)hich are compensated by the charge density in the bulk solution�S), to fulfill electroneutrality condition, i.e.:

M + �C + �RC + �S = 0 (13)

Positive and negative charges of amino acid side chains insidehe cyt c are assumed to cancel, so the protein matrix and the mono-ayer are treated as dielectric continua. Consequently, in these two


egions the potential profile exhibits linear variation with poten-ial. Beyond the plane of the redox center, the potential decaysxponentially, consistent with the Gouy–Chapman theory. Suchnhomogeneous charge distribution is also observed within the

Fig. 44. Schematic representation of the potential distribution at the Ag/SAM/cytc/solution interface (see the text for symbols explanation) [131].

lipid bilayers. The potential drop at the redox site ERC decreaseswith SAM thickness dC [131]. The electric field strength EF at theprotein-binding site is determined by following parameters:

EF (dC ) = ε0εS�ERC − �C − �RCε0εC

, (14)

where εS and εC denote respectively the dielectric constants of thesolution and the SAM, and � stands for the inverse Debye length. Forcarboxylate terminated SAMs the electric field strength at the cyt cbinding site was estimated to be in the order of 109 [V m−1] [131],which value is similar to that predicted for biological membranesin the proximity of charged lipid headgroups [189]. Mimickingimportant features of the lipid bilayers (potential distribution, elec-tric field strength) and close structural resemblance (hydrophobiccore and the charged terminal groups) imply that alkanethiolateSAM coated electrodes can also be considered as simple models forbiomembranes. Higher field strengths are predicted for phosphateand sulfate SAMs, since their �C is noticeably larger, whereas thefield strength is distinctly reduced in the case of hydrophobic head-groups, which provide �C = 0 C m−2. Thus, it can be expected thatthe magnitude of the electric field dependent on the terminal groupof the linkage SAM and its distance from the metal surface will havean effect on the coordination pattern of the heme pocket. Indeed,a cleavage of Fe–(Met-80) bond deduced from the SERRS spectra(5cHS markers) was reported for cyt c anchored to carboxylate ter-minated alkanethiolate (HS-(CH2)n-COOH)SAMs on Ag [167]. It waspartially assisted with the occupation of the free sixth coordinationsite by water molecule or by a new ligand, most likely His-33, togive a B2 six-coordinated high spin (6cHS) or a six-coordinated lowspin (6cLS) configuration. For 11 and more ethylene groups in thealkyl chain there was no contribution from 5cHS and other substateforms (only native protein denoted as B1 in 6cLS His/Met state wasdetected), while for n < 11 the content of new conformational states(represented as B2) increased with the alkyl chain length decrease.Relative contribution of 5cHS in the sum of B1 and B2 states satis-fied the same relationship, however for n = 6 the 5cHS conformationwas the only non-native substate, while for n = 3 the 6cLS appeared


and for n = 2 in addition 5cLS was present. These structural changeswere claimed to be induced by the electric field at the anionic bind-ing site of the monolayer. Obviously the strength of the electric fieldwill be larger for a shorter distance between the electrode and the


IN PRESSG Model

E

3 imica Acta xxx (2013) xxx– xxx

Scbbphb

noS

4ae(aoeaaiaigueacacwirmtS

tMtfoohBTtItF

ctct

4e

isAifia

Fig. 45. Ex situ SERRS spectra (exc = 514.5 nm) of 10−4 M cyt c of pH 7 anchored elec-trostatically to mixed MES/MEL SAMs formed from ethanolic solutions (ctotal = 1 mM)with varying MES mole fraction in solution (�sol

MES) on roughened Ag at pH 7.0. MES,

MEL and cyt c bands are marked with red, green and blue respectively. OverlappingMES and MEL bands are indicated with orange. �sol

MESis given in magenta. Light blue

and light green bars mark the B and A modes [190].(For interpretation of the


4 A. Królikowska / Electroch

AM binding site, which is responsible for the observed transitionalhanges. The postulated mechanism of these is that electrostaticinding salt bridges formed between lysine residues of the cyt cinding site and the anionic groups of the linkage monolayer sup-ort the interfacial electric field and perturb the structure of theeme pocket, which eventually results in dissociation of the Met-80ond from the heme iron [131].

Certainly, the strength of the electric field provided by the alka-ethiol linker and its end-group will also affect the ET propertiesf the attached cyt c, but a closer look at these aspect made withERRS spectroscopy will be given in following sections.

.4.3.5. Influence of monolayer composition on protein orientationnd its effect on ET properties. SERRS measurement for cyt c attachedlectrostatically to mixed SAMs of 2-mercaptoethanosulfonateMES) and 2-mercaptoethanol (MEL), formed through co-dsorption on electrochemically roughened Ag were conducted byur group [190]. Spectra were taken both in situ (in presence ofxcessive protein) and ex-situ (after removal from cyt c solutionnd rinsing with buffer) and for two cyt c concentration of 10−4

nd 10−6 M at pH 7.0. The concentration in a range of micromoless satisfactory to get a complete monolayer and sufficient to obtain

SERRS signal exclusively for adsorbed cyt c, but a part of our stud-es was related to the reported in the literature similar system onold substrate, where cyt c concentration in a range of 10−4 M wassed [191]. Moreover, this range of concentration is often used forlectrochemical studies of cyt c, which brought our interest to maken effort to examine the properties of protein adsorbed under theseonditions. We were aware that especially for in situ experiment;

contribution of RR signal of solution protein to SERRS spectrumannot be totally neglected. On the other hand, the intensity patterne observed was changed for SERRS experiments with 10−4 M cyt c

n comparison to volumetric sample (data here not shown) and theelative intensities in the SERRS spectra of cyt c depended on theonolayer composition. For these two reasons we are convinced

hat spectra taken in situ for 10−4 M were dominated by signal ofAM bound protein.

The SERRS spectra collected ex situ for cyt immobilized elec-rostatically using 1 × 10−4 M aqueous solution of pH 7 on binary

ES/MEL SAMs are shown in Fig. 45. Alteration of heme orienta-ion, following changes of the monolayer composition was deducedrom the dependence of intensity ratio of B1g to A1g bands of cyt cn SAM composition, reproduced in Fig. 46a. Lower molar fractionf MES in solution resulted in more perpendicular orientation ofeme with respect to the metal surface (indicated by increasing1g to A1g ratio), which should facilitate the electron transfer (ET).he conclusions made for silver surface correlate very well withhe results obtained for gold coated with mixed MES/MEL SAM bymabayashi et al. [191], who observed increasing kET (ET rate) onhe SAMs, with the increasing contribution of MEL component (seeig. 46 to compare with the trend obtained by them).

Hence, too high negative charge provided by linkage monolayeran deteriorate ET kinetics of tethered cyt c. Similar findings forhe in situ experiment with use of 10−6 M cyt c solution prove thatontrol of the surface composition of the linkage monolayer allowso govern the redox center orientation and hence the ET process.

.4.4. Effect of excessive amount of a bulk protein on hemelectronic structure

Moreover, on the course of the SERRS experiments describedn the previous section a comparison of ex situ and in situ SERRSpectra of cyt c (10−4 M solution) attached to MES/MEL coated


g showed differences of the heme state [190]. Oxidation ofron and conversion of native six-coordinated low spin (6cLS) tove-coordinated high spin (5cHS) under in situ conditions wasscribed respectively to presence of surface Ag4

+ species and/or

1g 1g

references to color in this figure legend, the reader is referred to the web version ofthe article.)

sandwiching protein multilayer by silver clusters. Changes withinheme electronic structure were eliminated during in situ experi-ment with 10−6 M cyt c solution. These results show importanceof protein multilayer formation for properties of electroactivefirst layer. Demonstration of serious differences between theheme properties (redox and configuration state, ligation pat-tern of iron) of adsorbed cyt c in presence and absence of bulkprotein is an important result for the researchers working withthe cyt c immobilized on alkanethiolate SAMs, as many elec-trochemical experiments are conducted under one of these tworegimes.

4.5. Results of SERRS studies on redox and ET properties of cyt ctethered to alkanethiol coated metal substrates

Discussing the EC-SERRS studies on cyt c attached to bio-mimetic interphase, here metal: (Ag or Ag–Au hybrids) coated withfunctionalized alkanethiolate SAMs one must distinguish two typesof the EC-SERRS experiments which can be performed and providedifferent information on a studied system. These are:


• stationary potential-dependent SERRS spectroscopy• time-resolved potential-jump SERRS spectroscopy





F action sol

(b of mi

pa

4

scsTa

•••

pgaiitlriassmrpsdsp

NpcpF

lngm

ig. 46. a. Intensity ratio of �13(B1g) to �4(A1g) SERRS modes of cyt c vs. MES mole frex situ experiment with 10−4 cyt c solution) [190].. ET rate (kET) of cyt c vs. MES mole fraction in solution (�sol

MES) used for preparation

Within this section their principles, supplied information andrimarily insight provided by their means on ET properties of cyt cttached to coated electrodes will be presented.

.5.1. Stationary potential SERRS measurementsThis type of SERRS measurements involves collection of SERRS

pectra, measured at different stationary potentials. As already dis-ussed in Sections 4.4.1 and 4.4.2, SERRS spectrum is sensitive totructural and/or electronic properties of the immobilized protein.herefore the following information can be deduced from station-ry potentials SERRS

heme structureconformational and redox equilibriaorientational distribution

An example of potential induced, corresponding to change ofrotein redox state alteration of orientational distribution of hemeroup, derived from B1g to A1g SERRS modes intensity ratio waslready given in Section 4.4.2 (see Fig. 40), but probably the mostnteresting topic to study by stationary potential EC-SERRS is prob-ng the conformational and redox equilibria of cyt c attachedo coated metal electrodes and studies of effect of alkanethio-ate SAM structural properties on them. Such SERRS examinationelies usually on spectro-electrochemical titrations, which resultsn quantification of unknown relative contribution of a given redoxnd/or conformational form of cyt c heme, presumed from SERRSpectra, as a function of the potential. Aiming at this, carefulelection of the SERRS marker bands and cautious fitting the experi-ental data to extract the spectral components indicative of a given

edox and/or conformational state are the key steps. And exam-le of the titration curves obtained by Hildebrant’s group [172]howing the potential-dependent relative concentration of oxi-ized (blue curve) and reduced cyt c (red curve) and correspondingtationary EC-SERRS spectra, displaying the details of the fittingrocedure are presented in Fig. 47.

When plotting potential vs. log [Red]/[Ox], one can validate theernstian behavior of the studied system and the apparent redoxotential of the bound cyt c (Eapp) can be determined from the inter-eption. An exemplary set of data showing both analyzed stationaryotential EC-SERRS spectra and obtained Nernst plot is shown inig. 48.

More systematic SERRS studies how does the thickness of the


inkage SAM, achieved by use of the �-carboxyl terminated alka-ethiols Cn-SAMs with varying n, being the number of methyleneroups (general formula: SH-(CH2)COOH), on Ag affects the deter-ined apparent redox potentials (Eapp) of bound electrostatically

in solution (�MES

) used for preparation of mixed MES/MEL SAMs on roughened Ag

xed MES/MEL SAMs on Au(1 1 1) [191].

cyt c was performed by Murgida and Hildebrandt [167]. The valueof Eapp for B1 species displayed a steady downshift from 244 mV(vs. NHE; normal hydrogen electrode) for n = 1 to 202 mV for n = 15.When comparing a potential drop across monolayer, correspond-ing to potential difference given by Eapp − E0 and further denotedas ERC to potential value for cyt c in solution (E0

S = 257 mV vs. NHE[109]), one obtains:

ERC = E − E0S + RT

nFln

(CB1(red)

CB1(ox)

)(15)

where R, T, n and F denote gas constant, temperature, number ofelectrons involved in ET and Faraday constant, respectively. It wasshown that both ERC and Eapp decrease nonlinearly with the lengthof the SAM [167]. It was also demonstrated that these shifts reflectonly the distance-dependent interfacial potential drop which canbe quantitatively described by a simple electrostatic model pre-sented already in the Section 4.4.3 dealing with the effect of electricfield provided by the alkanethiol SAM. In contrast to electrostaticadsorption, covalently tethered cyt c (B1) exhibits a non-idealelectrochemical behavior [193]. Most possible, the cross-linkingreactions engage various lysine residues leading to a distributionof orientations that may result in electrochemically less active orinactive species.

EC-SERRS gives also insight into conformational equilibrium ofcyt c attached to different �-substituted alkanethiol SAMs. Theconformational changes involve mostly appearance of non-nativeB2 states, which involves the loss of the heme axial Met-80 lig-and is removed, leading to a coordination equilibrium between afive-coordinated and a six-coordinated species, in which this axialcoordination site remains vacant or is occupied by a histidine (His-33 or His-26). It was evidenced that both type of terminal group andlength of aliphatic strongly influence the content of the B2-species.

On carboxyl terminated Cn-SAMs, the total amount of B2 speciesmeasured at Eapp increases from 0% for n = 15 and 10 up to 75% forn = 1 (see Fig. 49). This finding can be understood on the basis ofan electrostatic model that assumes a linear variation of the poten-tial across the SAM and up to the redox site and a Gouy–Chapmandistribution for the region beyond the redox site [167].

Assuming a constant ionic strength, the magnitude of theelectric field sensed by the adsorbed cyt c increases with theshift of the electrode potential relative to the potential of zerocharge, increased charge density on the SAM surface (dependenton SAM terminal group), and shortening of the thiol aliphatic chain.


Depending on these parameters, the magnitude of the field mayvary between 108 and 3 × 109 V m−1. The plot in Fig. 49 shows thatconformational B2/B1 equilibrium sensitively depends on the elec-tric field experienced by the adsorbed cyt c. Even larger portions





Fig. 47. Left: Redox titration curves obtained basing on SERRS spectra of cyt c shownin the right panel.Right: Experimental SERRS spectra (excited with 413 nm) of cyt c adsorbed on coatedelectrodes at different potentials. The titration curves and component spectra of thevarious redox state species are given in different colors: blue – cyt ; red – cyt[i

oS[s

ssipBde

Fig. 48. Nernst plot obtained from the quantitative treatment of the SERRS spectra

(compare Fig. 51 for cyt c bound to negatively charged SAMs on Agand lipid) [195]. Accordingly, it is likely that B2 state may also beformed when cyt c binds to the inner mitochondrial membrane,including its main lipid component, the anionic cardiolipin. Under

Ox Red

172].(For interpretation of the references to color in this figure legend, the readers referred to the web version of the article.)

f oxidized B2 species than these for short carboxylate terminatedAMs are found on phosphate and sulfate terminated monolayers194], which is in good agreement with a predicted electric fieldtrength influence.

Moreover, redox and structural equilibria of cyt c in electro-tatic complexes were determined by Hilderbrandt’s group fromtationary potential EC-SERRS spectra [150,167]. Certainly, againt employed the component analysis, to determine quantitatively


otential-dependent equilibrium of the reduced and oxidized1 and B2 forms. The results of this analysis shown in Fig. 50emonstrate these conformational changes are reversible, whichvidences they do not involve a protein denaturation. Due to

(excited with 413 nm) of cyt c on alkanethiol SAM coated Ag, as a function of theelectrode potential. The component spectra of the various redox state species aregiven with solid (cytRed) and dotted (cytOx) lines [192].

considerable structural changes within the heme pocket andthe ligand exchange, the redox potentials of the B2 species aredownshifted up to ca. 400 mV with respect to the native 6cLSB1 conformation. Interestingly, reduced form of the immobilizedprotein shifts the equilibrium nearly quantitatively towards the B1form, independent of the chain length of the alkanethiol SAM linker.

The transition to B2 species, accompanied by the strongdownshift of the redox potential occurs also when cyt c bindsto liposomes of negatively charged phospholipids, in particularat low protein/lipid ratios equivalent to high local electric fields


Fig. 49. Relative concentration of the B2 state of ferric cyt c as a function of theSH-Cn-COOH chain length and the electric field strength, calculated assuming elec-trostatic model [167].





Fc

R

prpeoppi[cipvbf

4

ctonwadsab�tte

Fig. 51. Relative contributions of the B2 states for ferric cyt c bound to dioleoyl-phosphatidylglycerol (DOPG) vesicles as a function of the protein/lipid ratio,determined by RR spectroscopy (top) [195] and for cyt c attached to coated Ag elec-trodes as a function of the electric field strength, determined by SERRS spectroscopy

ig. 50. Schematic representation of the redox and conformational equilibria of cyt in electrostatic complexes [150,167].

eprinted from [192].

hysiological conditions, this conformational transition would beelated to a changed protein function, since due to the low redoxotential, state B2 cannot be any more reduced by complex III,liminating the cyt c ability to serve as an electron carrier. On thether hand, loss of the Met-80 ligand results in strongly increasederoxidase activity [196], which may account for the cyt c catalyticroperties toward peroxidation of cardiolipinand and the resultant

ncreased permeability of the inner mitochondrial membrane197]. This process is believed to be functional for the release ofytochrome c to the cytosol, where the protein may be involvedn caspase-dependent apoptosis [198]. As the local electric fieldromotes the conformational conversion to B2 state, exhibitingery low redox potential and increased peroxidase activity, it maye the factor triggering the switch of cyt c from redox to peroxidaseunction under physiological conditions.

.5.2. Time-resolved potential-jump SERRS spectroscopyTo investigate in fact the dynamics of the heme group of the cyt

during interfacial ET process and provide kinetic data, it is essen-ial to probe within a time scale of this process. Therefore a needf use a time-resolved EC-SERRS spectroscopy (TR EC-SERRS) isecessary to examine the electron transfer dynamics. A pioneeringork in this field for the TR EC-SERRS studies of ET reaction of cyt c

ttached to silver electrodes was done by Hildebrandt, who intro-uced the concept of the time-resolved potential-jump (EC) SERRSpectroscopy [132]. In this approach, the redox-linked processesre provoked by a rapid potential jump and further monitoredy SERRS spectroscopy after a delay time ı for the probe interval


t. The synchronization is possible due to a mechanical chopper,riggering the potential jump via a photodiode and hence gatinghe exciting continuous-wave (CW) laser beam. After the probevent, the potential is reset to its initial value (Ei). The following

(bottom) [167]. The solid lines are included to guide the eyes.

Reprinted from [199].

relaxation processes that restore thermodynamic equilibrium atthe final potential (Ef) are eventually probed by SERRS. Restorationof disturbed initial equilibrium allows a continuous repetitionof the sequence of potential jumps and probe events. Use of CWlaser, together with a rotating electrode as SERRS substrate enablesavoidance of unwanted photo-induced degradation and desorptionprocesses of sensitive biological material (cyt c), quite likely whenpulsed laser excitation and stationary electrodes are used. Thescheme showing the principles of TR SERRS experiment, SERRSspectra corresponding to the potential jump and the analyzed dataafter varying delay times are shown in Fig. 52. Original set-up wasfurther modified by a group of Hildebrandt and Murgida in twoaspects [200]. First, mechanical chopper was replaced with an elec-tronically controlled intensity modulator equipped with a pulseamplifier, which allowed fine-tuning of �t down to the nanosec-ond time scale. Second, synchronization and timing of the potentialjumps, delay times ı, and measuring intervals was achieved withuse of a multichannel pulse delay generator. Further improvementby Kranich et al. [201] involving use of two consecutive laser inten-sity modulators resulted in obtaining a time of response of 20 ns.


Summarizing, in TR EC-SERRS experiment, potential jumps ofvariable height and duration are applied to trigger the electrontransfer (ET) reaction. Next, the SERRS spectra are taken after vari-able delay times following each jump. This provides an insight into:





Fig. 52. Scheme of the time-resolved SERRS experiment for a potential jump from reductive to oxidizing potential [199]. Left: temporal relationship between potential jump,concentration profile, and data accumulation.Middle: SERRS spectra taken at the initial potential Ei (top), the final potential Ef (bottom), and after a delay time ı following the potential jump from Ei to Ef .T cyt c, rR e potl

•••

tsetigt((pEcp

i

F2w

he red and blue lines refer to the component spectra of the reduced and oxidized

ight: results of the analysis showing the relaxation of the reduced cyt c following thegend, the reader is referred to the web version of the article.)

ET process kineticsprotein dynamicsredox center reorientation

Depending whether one is interested in redox and conforma-ional equilibria or heme group reorientation disentangling, onehould use respectively Soret band (413 nm) or Q-band (514.5 nm)xcited TR SERRS in order to get the required information. The fit-ing procedure of the individual species to the measured spectranvolves obligatory background subtraction and treatment by sin-le band (514.5 nm) or component analysis (413 nm) [202]. Theime evolutions of SERRS marker band of oxidized state of cyt cmeasured with the 413 nm) and B1g to A1g modes of ferric cyt cmeasured with 514.5 nm), monitoring respectively the kinetics ofrotein reduction and reorientation (as potentials jump was fromi = 250 mV to Ef = 150 mV vs. NHE) for cyt c adsorbed electrostati-


ally at carboxyl terminated C15-SAM and C5-SAM coated silver areresented in Fig. 53a and b.

At SAMs of mercaptocarboxylic containing 15 methylene groupsn the alkyl chain (C15-SAM), potential jump-induced protein

ig. 53. Time evolution of the SERRS �10(B1g)/�4(A1g) intensity ratio for ferric cyt adsorbed50 to 150 mV (vs. NHE). SERRS spectra were excited with 541.5 nm (green) [201]. The insith 413 nm excitation (violet).(For interpretation of the references to color in this figure

espectively.ential jump accumulation.(For interpretation of the references to color in this figure

reorientation, as probed by TR SERRS experiments with Q-bandexcitation, is much faster than electron tunneling (compare greenand violet curves in Fig. 53a). While when decreasing number ofmethylene groups, the rate of protein reorientation decreases untilit approaches the rate of electron tunneling, which takes place formercaptohexanoic acid (C5-SAM), as can be seen in Fig. 53b.

Therefore, two different regimes for the interfacial electrontransfer of the attached cyt c should be distinguished:

• at long distances, for carboxyl terminated alkanethiol SAMs with10 or more CH2 groups, ET is controlled exclusively by electrontunneling,

• at distances shorter than for n = 10 orientational changes of thetethtered protein appear to be rate-limiting.

The more precise analysis of the derived TR SERRS spectra allows


extracting the kinetic data, when one assumes a correct one-steprelaxation process, according to the experimental conditions andresults. Usually the initial and final potential values correspond tothe heme redox process and next the spectral signatures indicative

at pH 7.0 on (a) C15 and (b) C5 COOH terminated SAMs after a potential jump fromets show the kinetics of cyt reduction on the same SAMs as monitored by TR SERRS

legend, the reader is referred to the web version of the article.)





Fig. 54. Rate constants for reorientation [110] and reduction (blue) of cyt c immobi-lized on Ag electrodes coated with COOH terminated SAMs of different chain lengths,determined by TR SERRS spectroscopy. The upper and bottom axis indicate respec-tively the distance from the electrode surface and the electric field strength at theSAM–cyt c interface, as estimated on the basis of an electrostatic model. The straightline represents the exponential distance dependence of electron tunneling, extrapo-lated from rate constants determined for C15-SAM and C10-SAM. The dotted lines arett

R

oftt

evTct0i

�

or

wbFdbwbpecpor

db

Fig. 55. Apparent rate constant of ET at zero driving force as a function of the elec-tric field strength for cyt c adsorbed on Ag electrodes with different coatings. SAMdenotes the alkanethiol linker with a different functionalization, while Cn refers tothe carboxyl terminated SAMs [192].

o guide the eyes.(For interpretation of the references to color in this figure legend,he reader is referred to the web version of the article.)

eprinted from [199].

f individual species should be distinguished and analyzed. Hence,or example when only B1 species were detected in the SERRS spec-ra, the one-step relaxation being the ET of the native state B1 ofhe adsorbed cyt c can be examined. Then one obtains [200]:

[B1red]ı′ [B1red]0

= exp(−t�

), (16)

with �[B1red]ı′ and �[B1red]0 denoting concentration differ-nces of the reduced form of B1 with respect to the equilibriumalues at the final potential Ef for ı′ = ı + �t/2 and t = 0, respectively.he � (sometimes referred also as krelax) stands for the relaxationonstant. If the final potential was set equal to the redox poten-ial so that the relative equilibrium concentration at Ef of B1red is.5 and the ET rate constants (kET) for oxidation and reduction are

dentical then one acquires:

= 12kET

(17)

Thus when plotting ln(�[B1red]t = ı/�[B1red]t = 0) as a functionf time: the relaxation constant, and hence the related kET can beeadily determined.

When plotting rates of reorientation and reduction (determinedith TR SERRS) for cyt c immobilized at Ag modified with car-

oxyl terminated alkanethiol SAMS of varying chain length (seeig. 54, upper axis), one can see again more clearly that at a shortistance from the electrode; the orientation changes of the immo-ilized protein slow down dramatically and become rate-limiting,hile for the thick SAMs the measured ET rates are determined

y electron tunneling probabilities [199]. Again applying the sim-le electrostatic model the distance dependence of the interfaciallectric field can be predicted and as can be seen from relaxationonstants dependence (see Fig. 54, bottom axis) the interfacial ETrocess of immobilized cyt c may be alternatively classified in termsf a low-field (long distances) and a high-field (short distances)


egime.Expanded view of the apparent ET rates for B1 state of cyt c

etermined with aid of TR SERRS for coatings different than car-oxyl terminated SAMs, shown in Fig. 55, yields a uniform picture

Fig. 56. Overpotential dependencies of electron tunneling rates for cyt c attachedto COOH terminated C15-SAM on Ag (top left) and hybrid Au–Ag (bottom left).Bottom panel: modified Marcus equation including metal specific electric fielddependent retardation of electron tunneling [205].





tron t

onrSBt

tmtgft[AtlcanpaiiapSti

4

acteoFvrTep

biomolecules one has to carefully choose a SER(R)S active substrate,

Fig. 57. Schematic representation of the proposed mechanism of elec

f the heterogeneous redox reaction controlled by electron tun-eling at long distances (low field) and by electric field dependenteorientation at short distances (high field). For SO4-Ag and PO3-AMs coatings, which provide considerably increased amount of2 species, the apparent ET rates are much lower than for COOHerminated coatings at comparable distances.

What is worth emphasis, is that the overpotential- andemperature-dependence TR SERRS measurements for COOH ter-

inated C15-SAM on silver are in fine agreement with the Marcusheory for long-range electron tunneling [203]. The derived reor-anization energy from these studies is distinctly lower than thator cyt c in solution [204], demonstrating a strongly reduced con-ribution of the solvent reorganization for the immobilized protein203]. However, when the substrate was changed from Ag to hybridu–Ag (see the Section 4.3.4 for details) much weaker overpoten-

ial dependence was found [205], providing physically meaninglessow reorganization energy values. In attempt to resolve this dis-repancy, it was proposed that also under low-field regime there isn electric field effect on the free-energy term controlling the tun-eling rate. Under assumption of a local electric field strength beingroportional to the difference between actual electrode potentialnd metallic system specific pzc, an empirical linear correction wasncluded in a free-energy term of the Marcus equation, resultingn electric field dependent retardation of electron tunneling. Thisllowed a reasonable explanation of incontinency of ET rates over-otential dependencies on Ag and Au–Ag hybrid observed with TRERRS [205]. The metal specific overpotential dependencies andhe modified Marcus equation describing their behavior are givenn Fig. 56.

.5.3. ConclusionsGathering all the information provided by stationary potential

nd time-resolved SERRS measurements of cyt c for Ag electrodesoated with COOH terminated alkanethiol SAMs summarized inhe preceding sections, one can derive the simplified scheme of thelectron transfer (ET) reaction between cyt- and the correspondingxidase embedded into the mitochondrial membrane given inig. 57. The first step is the formation of an electrostatic complexia the positively charged area of cyt c around the partially exposed


edox center and the negatively charged domain of the oxidase.his electrostatic complex is not necessarily optimized for thelectron transfer from cyt c to the CuA (binuclear copper center),rimary electron acceptor of the enzyme. Hence, the second step of

ransfer reaction between cyt c and cytochrome c oxidase (CcO) [147].

the mechanism may involve the reorganization of the electrostaticcomplex (reorientation of the heme redox center). At sufficientlylow local electric field, this reorganization will proceed rapidly,resulting in efficient electron tunneling. The charge transfer reac-tion is fast despite the long distance and extremely low drivingforce (close to 0 eV) due to the considerably decreased reorga-nization energy in the electrostatic complex, as suggested by TRSERRS results. The close to zero driving force for the forward redoxreaction implies the high probability of efficiently proceeding,competing back electron transfer. On the other hand, oxidationof cyt c at low electric fields may trigger a fast conformationaltransition to the B2 form, which redox potential is lowered enoughto prevent re-reduction of the carrier. The terminal step wouldbe the dissociation of cyt c from the binding site, which returnsrapidly to the B1 state in a released form in solution. This mech-anism would secure an efficient and unidirectional inter-proteinreaction.

5. Summary

In this review (EC)-SERRS spectroscopy was presented as apowerful tool for the studies of redox active proteins immobi-lized on metallic substrates. The provided information is limitednot only to Faradaic processes, as for traditional electrochemi-cal methods, but it additionally offers a simultaneous insight intostructural and orientational parameters, also under a dynamicrange (time-resolved SERRS technique). Presented here results forthe cytochrome c (cyt c), being a model redox protein, demon-strated a potential of EC-SERRS technique to monitor protein redoxactivity, solve the heterogeneous interfacial electron transfer (ET)and indicate the parameters governing the ET process. For cyt cattached to chemically modified metal supports, the strength ofthe electric field at the interface is the key parameter controllingthe ET properties. Its second function is switching the protein func-tion from the redox to the peroxidase one, which latter is one ofthe key events preceding apoptosis. Aiming to study electrical per-formance of the metal supports with different immobilized redox


metal type, bio-mimicking coating of the metal support and adjustthe spectro-electrochemical experiment conditions to the partic-ular needs, utilizing the flexibility of the EC-SERRS spectroscopytechnique.


ING Model

E

imica

R



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ING Model

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Documents

Surface-enhanced resonance Raman scattering (SERRS) as a tool for the studies of electron transfer proteins attached to biomimetic surfaces: Case of cytochrome c