Hainer Wackerbarth

Novel Time-Resolved Surface-Enhanced (Resonance) Raman Spectroscopic Technique for Studying the Dynamics of Interfacial Processes: Application to the Electron Transfer Reaction of Cytochrome c at a Silver Electrode

A novel approach for time-resolved (TR) surface-enhanced (resonance) Raman (SE(R)R) spectroscopy is presented for probing potential-dependent processes of molecules adsorbed on a silver electrode. TR SE(R)R spectroscopy offers the unique advantage of providing structural and kinetic data exclusively of the adsorbed species and their reactions. These processes are initiated by a rapid potential jump and monitored by SE(R)R spectroscopy after a delay time δ for the probe interval Δt. The synchronization is achieved by a mechanical chopper that triggers the potential jump via a photodiode and gates the exciting continuous-wave laser beam. After the probe event, the potential is reset to its initial value. Thus, the original equilibrium is restored to allow a continuous repetition of the sequence of potential jumps and probe events. During the entire experiment, the detection system, a liquid nitrogen-cooled charge-coupled device (CCD) detector, is active so that the signal-to-noise ratio (SNR) can be iteratively improved. This mode of detection does not limit the time resolution, so that the present approach allows TR SE(R)R experiments down to the microsecond time scale without lowering the SNR. The possibilities and limitations of this method are discussed. As an example we present preliminary results of a TR SERR study on yeast iso-1 cytochrome c (Cyt-c) adsorbed on a Ag electrode by applying a potential jump from −0.4 V to +0.05 V (vs. saturated calomel electrode). The experiments are carried out with a rotating electrode to avoid photoinduced degradation and desorption processes. The SERR spectra, which were measured with delay times between 45 to 175 ms, were analyzed quantitively in terms of the various states of the adsorbed Cyt-c that are formed in this potential range. The results show that under these conditions the relaxation processes include the electron transfer of the adsorbed Cyt-c to the electrode and a subsequent conformational transition. The analysis of the data reveals a heterogenous oxidation rate constant of 10.3 s−1 and rate constant for the conformational transition of 4.3 s−1, supporting the view that the biological electron transfer of Cyt-c is coupled with conformational transitions.

Detection of explosives based on surface-enhanced Raman spectroscopy

In this study we present a device based on surface-enhanced Raman scattering (SERS) for the detection of airborne explosives. The explosives are resublimated on a cooled nanostructured gold substrate. The explosives trinitrotoluene (TNT) and triacetone triperoxide (TATP) are used. The SERS spectrum of the explosives is analyzed. Thus, TNT is deposited from an acetonitrile solution on the gold substrate. In the case of TATP, first the bulk TATP Raman spectrum was recorded and compared with the SERS spectrum, generated by deposition out of the gas phase. The frequencies of the SERS spectrum are hardly shifted compared to the spectrum of bulk TATP. The influence of the nanostructured gold substrate temperature on the signals of TATP was studied. A decrease in temperature up to 200 K increased the intensities of the TATP bands in the SERS spectrum; below 200 K, the TATP fingerprint disappeared.

Conformational equilibria and dynamics of cytochrome c induced by binding of sodium dodecyl sulfate monomers and micelles

Circular dichroism, nuclear magnetic resonance, electron paramagnetic resonance, UV-vis absorption, and resonance Raman (RR) spectroscopic techniques were employed to study protein and heme structural changes of cytochrome c (Cyt-c) induced by sodium dodecyl sulfate (SDS) monomers and micelles via hydrophobic and electrostatic interactions, respectively. Both modes of interactions cause the transition to the conformational state B2, which is implicated to be involved in the physiological processes of Cyt-c. At sub-micellar concentrations of SDS, specific binding of only ca. three SDS monomers, which is likely to occur at the hydrophobic peptide segment 81–85, is sufficient for a complete conversion to a B2 state in which Met80 is replaced by His33 (His26). These heme pocket structural changes are not linked to secondary structure changes of the protein brought about by nonspecific binding of SDS monomers in different regions of the protein. Upon binding of micelles, B2 high-spin species can also be stabilized by electrostatic interactions. In addition, the micelle interaction domain is located on the front surface of Cyt-c, which includes a ring-like arrangement of lysine residues appropriate for binding one micelle. According to freeze-quench RR and stopped-flow experiments, state B2 is formed on the long millisecond timescale and reveals a complex dependence on the SDS concentration that can be interpreted in terms of competitive binding of monomers and micelles.

Molecular effects of high‐pressure processing on food studied by resonance Raman

Pressurization may cause unwanted side effects including color or texture changes of fish and meat. The color changes of poultry, pork, and smoked salmon were studied by CIE L*, a*, b* system, and resonance Raman (RR). High‐pressure processing (HPP) of pork and chicken meat resulted in significant color modification at pressures higher than 270 and 280 MPa, respectively. RR spectra were taken after a high‐pressure treatment of pork meat. According to the RR‐data, deoxymyoglobin is the dominating myoglobin species in pork meat. High‐pressure treatment causes conformational changes resulting in a stabile nonnative ferrous myoglobin species while the ferrous myoglobin state is maintained. High‐pressure treatment causes a decrease of the relative RR intensities of astaxanthin by salmon as probed with 514 nm. RR spectra excited at 413 nm revealed a heterogeneous broadening of astaxanthin bands accompanied by the formation of deoxymyoglobin or deoxyhemoglobin. The broadening is interpreted as the degradation products of astaxanthin. Obviously, the high‐pressure treatment of smoked salmon triggers redox processes of astaxanthin and the heme protein.

Preparation and Characterization of Multilayer Coated Microdroplets : Droplet Deformation Simultaneously Probed by Atomic Force Spectroscopy and Optical Detection

We report the preparation and characterization of multilayer coated droplets in an emulsion. Stability and control of mass transport across the interface are the key issues for such coated microdroplets. Shelf life of cosmetic, pharmaceutical, and food formulations can be improved by increasing stability. Moreover, such emulsions have potential applications in drug delivery and storage. A primary oil-in-water emulsion with caseinate as an emulsifier was prepared. On the basis of attractive electrostatic interactions, polyelectrolytes with opposite charges were added layer by layer. The oil droplets (particle size around 10 microm) were successively coated with casein, pectin, whey proteins, pectin, whey proteins, and pectin. Laser diffraction spectroscopy, particle charge measurements, and confocal laser scanning microscopy were applied to characterize the multilayer droplets. The complementary results indicate that the inner layers merge and the packing density of the interface increases. AFM-induced mechanical compression of single oil droplets coated with casein and pectin is monitored by an inverted optical microscope, and simultaneously AFM force curves are recorded. Thus, the deformation of the droplet is reflected by its lateral expansion and the force curve. Force volume imaging is applied to probe the lateral distribution of mechanical properties of the droplet.

Fabrication and characterization of homogeneous surface-enhanced Raman scattering substrates by single pulse UV-laser treatment of gold and silver films.

The fabrication of SERS-active substrates, which offer high enhancement factors as well as spatially homogeneous distribution of the enhancement, plays an important role in the expansion of surface-enhanced Raman scattering (SERS) spectroscopy to a powerful, quantitative, and noninvasive measurement technique for analytical applications. In this paper, a novel method for the fabrication of SERS-active substrates by laser treatment of 20, 40, and 60 nm thick gold and of 40 nm thick silver films supported on quartz glass is presented. Single 308 nm UV-laser pulses were applied to melt the thin gold and silver films. During the cooling process of the noble metal, particles were formed. The particle size and density were imaged by atomic force microscopy. By varying the fluence, the size of the particles can be controlled. The enhancement factors of the nanostructures were determined by recording self-assembled monolayers of benzenethiol. The intensity of the SERS signal from benzenethiol is correlated to the mean particle size and thus to the fluence. Enhancement factors up to 10(6) with a high reproducibility were reached. Finally we have analyzed the temperature dependence of the SERS effect by recording the intensity of benzenethiol vibrations from 300 to 120 K. The temperature dependence of the SERS effect is discussed with regard to the metal properties.

Electron transfer behaviour of biological macromolecules towards the single-molecule level

Redox metalloproteins immobilized on metallic surfaces in contact with aqueous biological media are important in many areas of pure and applied sciences. Redox metalloprotein films are currently being addressed by new approaches where biotechnology including modified and synthetic proteins is combined with state-of-the-art physical electrochemistry with emphasis on single-crystal, atomically planar electrode surfaces, in situ scanning tunnelling microscopy (STM) and other surface techniques. These approaches have brought bioelectrochemistry important steps forward towards the nanoscale and single-molecule levels. We discuss here these advances with reference to two specific redox metalloproteins, the blue single-copper protein Pseudomonas aeruginosa azurin and the single-haem protein Saccharomyces cerevisiae yeast cytochrome c, and a short oligonucleotide. Both proteins can be immobilized on Au(111) by chemisorption via exposed sulfur-containing residues. Voltammetric, interfacial capacitance, x-ray photoelectron spectroscopy and microcantilever sensor data, together with in situ STM with single-molecule resolution, all point to a coherent view of monolayer organization with protein electron transfer (ET) function retained. In situ STM can also address the microscopic mechanisms for electron tunnelling through the biomolecules and offers novel notions such as coherent multi-ET between the substrate and tip via the molecular redox levels. This differs in important respects from electrochemical ET at a single metal/electrolyte interface. Similar data for a short oligonucleotide immobilized on Au(111) show that oligonucleotides can be characterized with comparable detail, with novel perspectives for addressing DNA electronic conduction mechanisms and for biological screening towards the single-molecule level.

Challenge of false alarms in nitroaromatic explosive detection--a detection device based on surface-enhanced Raman spectroscopy.

A challenge in the detection of explosives is the differentiation between explosives and contaminants. Synthetic musk-containing perfumes can cause false alarms, as these perfumes are nitroaromatic compounds, which can be mistaken for trinitro toluene (TNT) by some detectors. We present a detection principle based on surface-enhanced Raman scattering (SERS). A stream of the airborne compounds is focused and resublimated on a cooled nanostructured gold surface. We recorded high-resolution SERS spectra of TNT, musk xylene, and musk ketone. The nitroaromatic compounds can be identified unambiguously by their SERS spectra. Even the dominant bands containing nitro-group scissoring and symmetric stretching modes are significantly shifted by the difference in molecular structure.

Temperature- and pressure-dependent midinfrared absorption cross sections of gaseous hydrocarbons

Transmittance spectra of four gaseous hydrocarbons were measured by using a Fourier transform infrared spectrometer. The analyzed substances are propane, n-butane, ethanol, and iso-octane (2,2,4-trimethyl-pentane). Mixtures of hydrocarbons and nitrogen were prepared and analyzed in an optical cell between 298 and 473 K at pressures up to 1800 kPa. Molecule specific absorption cross sections were calculated for different temperatures and pressures that are relevant for technical absorption measurements. Dependences of the spectral absorption cross sections, as well as the integrated absorption cross sections on temperature and pressure, were investigated.

Electron transfer and redox metalloenzyme catalysis at the single-molecule level

Voltammetry based on single-crystal, atomically-planar metal electrodes is novel in bioelectrochemistry. Together with in situ scanning tunneling microscopy (STM) directly in aqueous buffer, single-crystal voltammetry has disclosed new detail in molecular adsorption and interfacial electron transfer (ET). Image interpretation requires, however, theoretical support, as STM represents both electronic and topographic features. Molecules with accessible redox levels offer other insight into electron tunneling mechanisms, addressed in detail for ET metalloproteins. We present here in situ STM of the blue redox metalloenzyme copper nitrite reductase (Achromobacter xylosoxidans, Ax CuNiR) on Au(111) electrode surfaces modified by a self-assembled cysteamine monolayer. Ax CuNiR displays strong nitrite reduction waves in this environment. Ax CuNiR/cysteamine/Au(111) surfaces were imaged at KNO2 concentrations where most of the enzyme is in the enzyme-substrate bound state. Molecular resolution for both cysteamine/Au(111) and Ax CuNiR/cysteamine/Au(111) electrode surfaces was achieved. The enzyme coverage is about 1.5 × 10−13 mol cm−2, which is low compared with an ideal close-packed monolayer. The adlayer behaves as an assembly of individual molecules, reflected in distributions of molecular appearance, although a number of molecules do show the triangular shape of the trimeric Ax CuNiR structure. The apparent average molecular height is about 11 A. This suggests that details of electronic structures and larger assemblies are needed to disentangle enzyme mechanisms at the single-molecule level.