. Effect of pH and SAM composition on the apparent redox rate of YCyt
Biomimetic systems employed for biotechnological applications i.e. as biosensors or bio fuel cells, require initial formation of conducting support/protein complexes with controlled properties. The specific interaction of the protein with the support determines important qualities of the device such as electrical communication, long-term stability and catalytic efficiency. In this respect the system parameters have to be chosen in a way that high protein loading on the support is achieved while protein denaturation upon adsorption is prevented. The conditions on the surface have to be adjusted in such a way that the desired surface reaction of the protein i.e. electron transfer to either the electrode or a second redox partner, is still guaranteed. Hence the choice of support, its functionlisation as well as the right adjustment of solution parameters play a crucial role in the rational design of these support/protein constructs.
Optical spectroscopy on surface bound proteins can give insight into the molecular processes that occur at the protein/support interface and thus represent a powerful addition to electrochemical methods that monitor the integrated response of surface active species only. However, optical spectroscopy in general lacks surface sensitivity which is necessary to detect the small amounts of proteins that are attached to the surface. Surface sensitivity can be obtained if the conducting support is able to create surface plasmon resonances (SPRs) generated by the resonant interaction of light with the free electron gas of a metal support. The SPR enhances the electromagnetic field in close vicinity of the metal surface which in turn amplifies the optical signal of surface bound molecules.
The most widely used spectroscopic technique that makes use of plasmonically enhanced electric fields is
The choice of support is a clear limitation in SER(R) spectroscopy as only metal supports that exhibit SPRs in the visible region can be considered. This narrows possible choices down to silver (Ag) and gold (Au) supports. While Au is a relative biocompatible metal, which is commonly used in electrochemical investigations of redox enzymes (Leger & Bertrand, 2008), Ag is generally considered as toxic and chemically instable. However, regarding its optical properties Ag is clearly superior to Au since it exhibits higher surface enhancement and its SPR can be tuned from near UV to infrared (Le Ru & Etchegoin, 2009). The SER activity of Au on the other hand is restricted to wavelengths larger than 520 nm which makes this metal not suitable for SERR spectroscopic investigations of chromophores that absorb in the blue and violet region.
Within the limits given by the metal support the position and magnitude of an SPR can be furthermore tuned by changing the surface morphology and the dielectric constant of the surrounding medium. Monodisperse nanoparticles show defined SPRs normally in the size range between 10 and 100 nm. As a rule of thumb the SPR position for nanoparticles is red-shifted with increasing diameter and aspect ratio, which is defined as the ratio between the longer and the shorter axis of the particle. It is further red-shifted with increasing dielectric constant of the surrounding medium (Kelly et al. 2003; Link & El-Sayed, 1999). Metal aggregates generally exhibit higher field enhancement than isolated nanostructures due to plasmonic interaction between the nanoscopic subunits. Moreover their SPR position is significantly red-shifted (Lal et al., 2008; Prodan et al., 2003).
For biological applications the influence of the metal on the biomolecule has to be considered as the interaction with bare metal surfaces usually denaturates proteins. In order to retain the native structure of the protein upon adsorption the metal has to be coated with a biocompatible material. Such coatings can be made of
In SERR spectroscopy of protein cofactors the following steps have to be considered: First the support material for optical amplification has to be chosen and its SPR has to be tuned in respect to the protein cofactor under investigation. Second, a surface functionalisation has to be chosen that guarantees protein adsorption in a defined orientation and preservation of the protein´s native state. Third, solution parameters like pH and ionic strength have to be adjusted for optimum functionality of the protein in respect to i.e. electrical communication and/or catalytic activity.
Heme cofactors are found in a variety of proteins with a broad range of functions. One of its main functions is found in electron transport reactions as the central Fe-ion of the heme can easily be reduced or oxidised. Hemes show strong absorption in the Soret region around 410 nm which makes them highly visible in RR spectroscopy (Siebert & Hildebrandt, 2008) Excitation with a 413 nm line of a Krypton ion laser is in this case the method of choice to selectively monitor the structural state of the heme. The vibrational pattern obtained in this way is characteristic for the redox and conformational state of the heme. Thus redox reactions and/or changes in axial ligation of the heme iron can be precisely monitored. The latter allows distinguishing heme cofactors of different protein complexes and, even more important, following denaturation processes that go along with changes of the axial ligation pattern. As a drawback SERR analysis of heme cofactors requires surface enhancement upon violet light excitation which can only be achieved using Ag as amplifying support. Several aspects have to be taken into account for designing the appropriate SER active support:
The enhancement of the Raman scattered light
Hence maximum SERR intensity is expected when the SPR maximum lies somewhere in between λexc and λRa.
Additionally, the Raman enhancement
Monolayers of organic molecules represent the most versatile biocompatible coating material. Attachment of these layers to nanostructured surfaces changes the particle size and the dielectric constant of the surrounding medium and thus results in a shift in SPR that has to be taken into account in the design of smart SERS active supports. Moreover, the organic coating separates the protein from the metal surface which, according to equation 2, can decrease the Raman signal enhancement drastically.
If the SER active support can be utilized as a working electrode in an electrochemical cell, redox titrations of surface bound proteins can be monitored by potential dependent SER(R) spectroscopy. The redox state of the protein cofactor is in this case followed as a function of applied potential. Moreover electrochemical methods can be employed on the same system that give additional and often complementary information.
Time resolved SER(R) spectroscopy (Tr-SER(R)S) can be used to study the electron transfer dynamics of redox proteins on SER active electrodes. Here the redox equilibrium is disturbed by a potential jump of the working electrode. The relative contribution of the reduced and oxidised state of the protein, as it reaches its new equilibrium, can be followed with SERS as a function of time subsequent to the jump. This allows determining the apparent redox rate of the metal/protein complex also as a function of the reaction´s driving force.
3. SERR active biomimetic systems
As pointed out in section 2, Ag is the only support that can be used as optical amplifier for SERR spectroscopic investigations of heme protein cofactors. However the quantitative yield of the amplification at violet light excitation can change drastically as a function of Ag morphology. In this section several Ag architectures are presented, that can be chosen in respect to the intended application (Figure 1). We start with size-tuned nanoparticles followed by rough electrodes for spectro-electrochemical applications. Last but not least we will introduce multilayered hybrid Ag-Au systems that aim to combine the optical properties of Ag with the chemical advantages of Au.
3.1. Tunable and biocompatible Ag nanoparticles
Although the SERS effect was discovered on rough electrodes (Jeanmaire & van Duyne 1977), nanoparticles (NPs) were the first systems extensively investigated on their electric field enhancement properties. Isolated monodisperse NP ensembles exhibit very sharp SPRs which can be seen as a resonance absorption in the UV-vis spectrum. The frequency of the SPR maximum strongly depends on the particle’s size, its shape and the dielectric properties of the environment. The frequency dependent enhancement factors of spherical and ellipsoidal particles can be determined analytically using Mie scattering theory (Zeman & Schatz, 1987). In general, experimentally determined SPR positions are in very good agreement with the theoretical predictions (Link & El-Sayed, 1999).
AuNPs are widely used for SER investigations since they can be synthesized very homogeneous in size, exhibit a high stability and are commercially available. The use of Ag NPs is less common since they have to be prepared individually and it is much more difficult to obtain a narrow size distribution (Le Ru & Etchegoin, 2009). For selective enhancement of heme cofactors AgNPs are designed with SPRs that match exactly the Soret band transition of the heme (Sivanesan et al, 2011).
3.1.1. Size adjustement
Small NPs can be prepared by reducing Ag+ ions with borohydride in the presence of citrate. The resulting NPs are quite spherical with an average size of 12 nm and an aspect ratio, defined as the longer
3.1.2. Surface functionalisation
The surface of the NPs is initially covered by citrate ions which are known to cause severe protein denaturation (Hildebrandt & Stockburger, 1986). The citrate ions can be replaced by SAMs of ω-functionalised mercaptoalkanes via ligand exchange (Bonifacio et al., 2004). These SAMs have shown to significantly improve the structural integrity of redox proteins on metal surfaces (Tarlov & Bowden, 1991). SAMs are commercially available with a variety of alkane chain length and functionlisation groups. The latter is chosen individually in respect to the target protein.
Successful replacement of citrate by MUA is accompanied by a red shift in the plasmon resonance of ca. 14 nm. Subsequent binding of Cyt
If the monolayer coverage is complete and homogeneous, the vibrational pattern of Cyt c in the SERR spectrum is similar to its RR spectrum indicating that the protein retains in its native structural conformation upon immobilisation. Damaged SAMs or incomplete SAM coverage, as obtained i.e. if too low SAM concentrations are used during NP preparation, alter the SERR spectrum as can be seen i.e. by the formation of an additional band at 1490 cm-1 (Figure 3) which is attributed to a non native 5 coordinated high spin state where the 6th axial ligand is missing (Oellerich et al. 2002). At least an excess ratio of 6:1 MUA:NP concentration is needed to obtain exclusively native protein on the surface. However the formation of a biocompatible layer is done on the expense of SERR intensity which drops by roughly one order of magnitude due to the increasing distance of the heme cofactor from the Ag surface by ca. 2 nm.
The SERR intensity of the protein shows a strong dependence on the SPR position as can be seen in figure 2 C. A clear intensity maximum is seen for the NP ensemble with a maximum SPR at 413 nm which matches both, the laser line (413 nm) and the molecular absorption maximum of oxidised Cyt c (410 nm), best. NPs with higher or lower SPR maximum show much lower SERR intensities. This clearly demonstrates that efficient enhancement is only achieved in the very narrow frequency range where laser excitation, NP plasmon resonance and molecular absorption of the chromophore overlap.
If the particle size and the Cyt
3.2. Spectro-electrochemistry of heme proteins on rough Ag electrodes
Enhanced electromagnetic fields via surface plasmon resonances can also be generated by nanostructured electrodes. These electrodes can additionally function as working electrodes in an electrochemical cell. Thus by applying an external potential the metal support may serve as an electron supply or sink for electron transfer reactions of immobilised redox proteins.
A nanostructured electrode can be seen as an ensemble of connected nanoparticles. Due to the plasmonic coupling of the individual nano-units the intensity and position of the electrode’s SPR is significantly altered as a function of surface morphology. Lithographic methods can be used to create highly periodic nanostructures with distinct plasmonic properties (Haynes & Van Duyne, 2001); the setup of such devices is, however, very expensive. Electrochemical methods on the other hand can be performed at much lower costs but this goes along with a less defined nanostructured surface and, as a consequence, much broader plasmonic resonance. In Figure 1 B the coral like structure of an electrochemically roughened Ag electrode can be seen. The surface roughness can be approximated by a random nanostructure. For such systems an almost continuous surface enhancement is predicted over the entire optical range (Sanchez-Gil & Garcia-Ramos, 1998). Surprisingly the SERR intensity of heme proteins adsorbed on these surfaces is quite reproducible. Nevertheless it is not possible to determine which part of the surface is responsible for the obtained SERR intensity. It might very well be that the measured signal is achieved from a small fraction of proteins localised so called
Biocompatible surface functionalisation of roughened Ag electrodes can be achieved by exposing the electrode to a solution containing ω-functionalised mercaptoalkanes for several hours. The choice of the SAM´s solvent exposed headgroup as well as proper adjustment of buffer solution parameters have a strong influence on the protein’s adsorption efficiency but also on its electrochemical and catalytic performance on the surface. We will demonstrate this on two different heme proteins;
HSO on the other hand is an enzyme that catalyses the oxidation of sulphite to sulphate, which is the terminal reaction in the oxidative degradation of the sulphur-containing amino acids cysteine and methionine (Garrett et al., 1998). HSO contains three sub-domains. The large central domain harbours the catalytic center, made of a
At ambient pH YCyt
3.2.1. Selective protein binding
High quality SERR spectra of YCyt
An inverse behaviour is observed for HSO, where only on amino terminated SAMs a SERR spectrum can be recorded (Figure 4 C,D). The heme is in both proteins differently coordinated as the 6th ligand is a methionine for YCyt
3.2.2. Redox properties and electron transfer dynamics
The electron transfer properties of redox proteins on surfaces are strongly influenced by the binding strength of the protein to the SAM and hence are a function of the local charge density at the SAM/protein interface (Ly et al., 2011). If the charge density of the SAM headgroup is too high the flexibility of the protein is largely restrained which can lead to a significant decrease of the redox rate
|SAM||kredox / s-1|
|pH 7||pH 6|
The local charge density is expected to have an even stronger impact if the direction of the protein´s dipole moment is significantly different from the direction of the most efficient electron transfer pathway. This is the case for HSO where the Cyt
|I / mM||E0 / V||kredox / s-1||kcat /s-1|
Increasing the ionic strength allows rotational diffusion of the protein on the surface which leads to a significant increase in redox rate (Table 2). The highest change in redox rate though is observed between 5 mM and 150 mM buffer concentration which is accompanied by a change in the Cyt
Flexibility of the Cyt
In order to get efficient catalytic turnover rates, experimental conditions have to be optimised in respect to both the intramolecular- and the heterogeneous electron transfer rates. This requires that the Cyt
3.3. Hybrid Ag-Au systems
In the system described in section 3.2 Ag electrodes function as both, optical amplifier and redox partner. While optical amplification is unambiguously preferred for Ag its chemical properties are rather poor as Ag is generally assumed to be bio-incompatible and easy to oxidise. In this respect Au is preferable since it is inert and its higher thiol affinity works in favour of well defined organic layer formation by mercaptoalkane derivatives. However, Au is not capable to provide surface enhancement at wavelengths lower than 520 nm. Ag-Au hybrid electrodes where optical amplification is provided by Ag but surface reactions take place at an Au surface could be able to combine the advantages of both metals. Hence we describe a procedure to create such multilayered hybrid supports and discuss the parameters that are responsible for the optical and chemical performance of the device in respect to bioelectronic applications:
The first layer consists of a rough Ag electrode as described in section 3.2. In order to minimise the contact between the biomolecule and the Ag surface a dielectric spacer (
Finally an Au island film is formed on top of the
3.3.1. Protein binding strategies
The electrical communication between Cyt c bound to the Au islands and the working electrode remains generally intact which makes it possible to perform redox titrations by potential controlled SERR spectroscopy. In Figure 6 C the molar fractions of the reduced and oxidised Cyt c species as a function of applied potential are shown. A sharp redox transition can be seen at the characteristic potential for the native MUA bound Cyt
In contrast drastic differences for Ag and Au supports can be observed for immobilisation strategies that include direct thiol binding of proteins via cysteine residues. YCyt
3.3.2. Parameters for optical amplification in multilayerd systems
In this section the parameters that are responsible for surface enhancement at the Au surface in Ag-S-Au electrodes are discussed in more detail.
If one takes a hybrid electrode with a dielectric spacer of
A crucial role for the magnitude of the induced enhancement in such multilayered systems is played by the dielectric spacers that are placed in between and on top of the metal films. These two dielectric layers will be referred as the
For the enhancement factor directly at the Ag surface we adopt the value of EF(0)= 8∙104 at 413 nm excitation from AgNP aggregates (Hildebrandt & Stockburger 1986). Correlation of this enhancement factor to measured Cyt
Variation of the thickness of the inner dielectric layer in Ag-S-Au systems reveals a different behaviour: Here a very weak distance dependence of Δlog(EF) = 0.02 nm-1 is determined from measurements with SiO2 coatings of different thickness (Figure 6 B). Most likely this discrepancy from the theory occurs due to the fact that the inner spacer layer is situated between two metals (Ag and Au). Such an effect was observed in calculations for spherical Ag-SAM-Au core shell particles (David et al., 2010) and most likely can be transferred to nanostructured layered devices. For practical applications in SERR spectroscopy of biomimetic systems this result can have an enormous impact, since it allows separating sensitive biological probes from harmful optical amplifiers much more than so far expected from theory.
3.3.3. Electron transfer properties
In section 3.3.1 it was shown that Cyt c bound to Au surfaces in Ag-S-Au devices still has an intact electrical communication. We will further analyse this aspect by comparing the redox rates of Cyt
First, the tilt angle with respect to the surface normal is higher on gold (~ 30° (Porter et al., 1987)) as compared to silver (~15°, Laibinis et al., 1991), which results in a slightly shorter tunnelling distance for electrons through SAMs on Au surfaces.
Second the potential of zero charge EPZC is different for both metals. The difference between the applied potential and the potential of zero charge ΔE = E-EPZC influences the charge density of the metal and hence also the deprotonation grade of the COOH/COO- SAM. It is thus, directly and indirectly, responsible for the electric field strength at the SAM/protein interface. On Ag-MUA we measured a value of EPZC=-0.45 V (Feng et al., 2008) whereas on Au-MUA EPZC = 0 V was determined (Ramirez et al., 2007). The latter value is close to the Cyt
If we assume that the use of MHDA instead of MUA does not change the overall trend in EPZC of SAM coated Ag and Au surfaces, both effects are most likely responsible for the faster rates observed on Ag-AUT-Au-MHDA as compared to Ag-MHDA (table 3). However, in this case a modification of EPZC in respect to pure Au surfaces has to be considered since Ag indirectly influences the charge density on the gold island film. We have indeed measured a value of ca. EPZC=-0.2 V for Ag-AUT-Au-MUA systems (Sezer et al., 2010) which lies between the two values of the respective pure metals.
The driving force for the electron transfer process can be increased by applying a higher overpotential η, which is defined as the difference between the applied potential
For MUA used as outer SAM the redox rates of Cyt
|kredox (Ag-AUT-Au) / s-1||kredox (Ag)|
|kredox (Ag-AUT-Au) / s-1|
Nevertheless for MHDA as outer layer the redox dynamics is clearly limited by the electron tunnelling rate through the outer layer and hence can be analysed in respect to heterogeneous electron transfer theory. In order to determine the electron transfer rate from measured redox rates, the contribution of the back reaction has to be taken into account. At zero driving force (η = 0 V) we have
In order to elucidate the role of the metal support in electron transfer reactions the measured values of
where kB and T stand for the Boltzmann constant and temperature respectively. λ denotes the reorganisation energy of the redox process. On the basis of equation 4 the data for Ag-MHDA was previously fitted with a reorganisation energy of λ= 0.22 eV (Murgida & Hildebrandt, 2002). The same approach for multilayered Ag-Au systems yields a lower value of λ = 0.15 eV. For Au λ would approach a value close to zero. It is highly unlikely that the reorganisation energy is so drastically altered by the support material. Hence these findings can only be rationalised by considering a dependence of the electron transfer on the magnitude and sign of the metal charge, which, given their different potential of zero charge, will be different for Ag, Ag-AUT-Au and Au in the investigated potential range.
The data in figure 8 suggests that a process other than electron tunnelling becomes rate limiting if the applied potential is more negative than
The functional dependence of
a good fit to the measured
In this book chapter different plasmonic support materials were described in respect to their applicability for biomimetic systems. Fine tuning of the optical amplification parameters allows employing
The highest control over frequency dependent optical amplification is achieved with nanoparticles, which are designed for selective enhancement of protein heme cofactors in solution. Biocompatible coatings can be applied to the particles on the expense of SERR intensity but with a significant gain in protein structure preservation.
While those nanoparticles provide a very flexible tool to enhance specific cofactors in solution or on biological interfaces, the study of redox processes additionally requires simultaneous control of the supports electrical potential. This is achieved using nanostructured Ag electrodes or newly developed Ag-S-Au supports. The latter have Ag as a bulk support and Au as solution facing reaction surface and thus combine the broad optical amplification properties of Ag with the superior chemical characteristics of Au. The multilayer structure of the Ag-S-Au hybrid supports allows furthermore increasing the distance of the protein from the harmful Ag surface without loosing significant Raman signal intensity.
It is further demonstrated how the change of the interfacial conditions can increase electrical communication and catalytic efficiency of immobilized proteins while at the same time sufficient signal enhancement is provided for spectroscopic analysis. It turns out that, rather than the amount of immobilised protein, its flexibility on the surface is crucial for the electron transfer and catalytic performance of the device. Protein flexibility on SAM coated metal surfaces can be influenced by adjusting the local charge distribution at the Protein/SAM interface as demonstrated i.e. by the increase in electron transfer rate of HSO as a function of buffer ionic strength. Also the choice of metal support can be crucial for the electrical performance of biomimetic devices as different metals i.e., Ag, Au and Ag-S-Au, show a different electron transfer rate dependence on the applied overpotential. This different behaviour is attributed to a yet not fully understood electric field dependence arising from the metal specific potential of zero charge.
We would like to thank Peter Hildebrandt for his great support and Alois Weidinger for last minute corrections. Financial support by the Fonds der Chemie, the DFG (Unicat) and the National Science Foundation of China (J.J.F.; NSFC No. 20905021) is gratefully acknowledged. Big thanks also to our colleagues Khoa Ly, Jacek Kozuch, Diego Millo and Anna Fischer who contributed to this work.