The heme protein cytochrome c adsorbed to a two-layer gold surface modified with a self-assembled monolayer of 2-mercaptoethanol was analyzed using a two-dimensional (2D) heterospectral correlation analysis that combined surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced Raman spectroscopy (SERS). Stepwise increasing electric potentials were applied to alter the redox state of the protein and to induce conformational changes within the protein backbone. We demonstrate herein that 2D heterospectral correlation analysis is a particularly suitable and useful technique for the study of heme-containing proteins as the two spectroscopies address different portions of the protein. Thus, by correlating SERS and SEIRAS data in a 2D plot, we can obtain a deeper understanding of the conformational changes occurring at the redox center and in the supporting protein backbone during the electron transfer process. The correlation analyses are complemented by molecular dynamics calculations to explore the intramolecular interactions.

Time-resolved surface-enhanced IR-absorption spectroscopy triggered by electrochemical modulation has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. Single bands isolated from a broad band in the amide I region using phase-sensitive detection were attributed to different redox centers. Their absorbances changing on the millisecond timescale could be fitted to a model based on protonation-dependent chemical reaction kinetics established previously. Substantial conformational changes of secondary structures coupled to redox transitions were revealed.

We demonstrate that double-layered stacks of gold and insulator nanoparticles arranged on a flat gold surface dramatically enhance the sensitivity in absorption infrared microscopy. Through morphological variations of the nanoparticles, the frequency of the plasmon resonances can be tuned to match the frequency of the molecular vibration in the mid-infrared region. The results show that the nanostructures enhance the absorption signal of the molecules by a factor of up to ~2.2 × 10(6), while preserving their characteristic line-shape remarkably well.

An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.

In this short summary we summarize some of the latest developments in vibrational spectroscopic tools applied for the sensing of (small) molecules and biomolecules in a label-free mode of operation. We first introduce various concepts for the enhancement of InfraRed spectroscopic techniques, including the principles of Attenuated Total Reflection InfraRed (ATR-IR), (phase-modulated) InfraRed Reflection Absorption Spectroscopy (IRRAS/PM-IRRAS), and Surface Enhanced Infrared Reflection Absorption Spectroscopy (SEIRAS). Particular attention is put on the use of novel nanostructured substrates that allow for the excitation of propagating and localized surface plasmon modes aimed at operating additional enhancement mechanisms. This is then be complemented by the description of the latest development in Surface- and Tip-Enhanced Raman Spectroscopies, again with an emphasis on the detection of small molecules or bioanalytes.

We use chemical reaction kinetics to explore the stepwise electron and proton transfer reactions of cytochrome c oxidase (CcO) from R. sphaeroides. Proton transport coupled to electron transport (ET) is investigated in terms of a sequence of protonation-dependent second-order redox reactions. Thereby, we assume fixed rather than shifting dissociation constants of the redox sites. Proton transport can thus be simulated particularly when separate proton uptake and release sites are assumed rather than the same proton pump site for every ET step. In order to test these assumptions, we make use of a model system introduced earlier, which allows us to study direct ET of redox enzymes by electrochemistry. A four-electron transfer model of CcO had been developed before, according to which electrons are transferred from the electrode to Cu A . Thereafter, electrons are transferred along the sequence heme a, heme a 3 and Cu B . In the present investigation, we consider protonation equilibria of the oxidised and reduced species for each of the four centres. Moreover, we add oxygen/H 2 O as the terminal (fifth) redox couple including protonation of reduced oxygen to water. Finally we arrive at a kinetic model comprising five protonation-dependent redox couples. The results from the simulations are compared with experimental data obtained in the absence and presence of oxygen. As a result, we can show that proton transport can be modelled in terms of protonation-dependent redox kinetics.

Surface-enhanced Raman spectroscopy (SERS) is an analytical technique exploiting plasmonic effects that enhance sensitivity significantly, compared to conventional Raman spectroscopy. Progress in nanotechnology led to new fabrication methods for nanostructures and nanoparticles over the last decade. Besides increased comprehension of mechanisms that cause the signal enhancement, computational methods have been developed to tailor analytespecific nanostructures efficiently. The ability to control the size, shape, and material of surfaces has facilitated the widespread application of SERS in biomedical analytics and clinical diagnostics. In this review, a brief excerpt of such SERS applications is shown, with special focus on cancer diagnostics, glucose detection and in vivo imaging applications. Simulation techniques are discussed to show that electro-dynamic theory can be used to predict the characteristics of nanostructure arrangements. Different fabrication methods, such as nanoparticle synthesis, their immobilization and lithographic methods are reviewed in brief.

Here we present a surface based on double-layered nanoparticle stacks suitable for spectro-electrochemical applications. The structure is formed on a continuous gold layer by a two-dimensional periodic array of stacks of gold and tantalum pentoxide nanodisks. Reflection spectra in the visible wavelength region showed the multiple-resonant nature of surface plasmon (SP) excitations in the nanostructure, which is in good agreement with simulations based on a finite-difference-time-domain method. The multiple SP resonances can be tuned to various wavelength regions, which are required for simultaneous enhancement at excitation and emission wavelengths. Cyclic voltammetry measurements on the nanostructure proved the applicability of electrochemical methods involving interfacial redox processes.