Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome <i>c</i> Oxidase from <i>Paracoccus denitrificans</i>

A novel oxo state of cytochrome <i>c</i> oxidase from <i>Paracoccus denitrificans</i> generated by successive addition of excess H₂O₂ and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H₂O₂-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm^–1, indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm^–1 showed a high-spin to low-spin transition of heme <i>a</i>₃ upon generation of the F state that was not altered by addition of ammonia. Removal of H₂O₂ by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme <i>a</i>₃ into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme <i>a</i>₃. Only for the ammonia-created state was a band at 612 nm observed in the UV–visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) <i>Proc. Natl. Acad. Sci. U.S.A. 108</i>, 3964–3969] that addition of ammonia creates a novel oxo intermediate state called P_N where ammonia binds to Cu_B once the oxo intermediate F state has been formed

Magnetic Silver Hybrid Nanoparticles for Surface-Enhanced Resonance Raman Spectroscopic Detection and Decontamination of Small Toxic Molecules

Magnetic hybrid assemblies of Ag and Fe₃O₄ nanoparticles with biocompatibly immobilized myoglobin (Mb) were designed to detect and capture toxic targets (NO₂^–, CN^–, and H₂O₂). Mb was covalently attached to chitosan-coated magnetic silver hybrid nanoparticles (M-Ag-C) <i>via</i> glutaraldehyde that serves as a linker for the amine groups of Mb and chitosan. As verified by surface-enhanced resonance Raman (SERR) spectroscopy, this immobilization strategy preserves the native structure of the bound Mb as well as the binding affinity for small molecules. On the basis of characteristic spectral markers, binding of NO₂^–, CN^–, and H₂O₂ could be monitored and quantified, demonstrating the high sensitivity of this approach with detection limits of 1 nM for nitrite, 0.2 μM for cyanide, and 10 nM for H₂O₂. Owing to the magnetic properties, these particles were collected by an external magnet to achieve an efficient decontamination of the solutions as demonstrated by SERR spectroscopy. Thus, the present approach combines the highly sensitive analytical potential of SERR spectroscopy with an easy approach for decontamination of aqueous solutions with potential applications in food and in environmental and medical safety control

Complementary Surface-Enhanced Resonance Raman Spectroscopic Biodetection of Mixed Protein Solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome <i>b</i>₅, whereas functionalization with SiO₂ amplifies only the spectra of the cationic protein cytochrome <i>c</i>. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy

Induced Surface Enhancement in Coral Pt Island Films Attached to Nanostructured Ag Electrodes

Coral Pt islands films are deposited via electrochemical reduction on silica-coated nanostructured Ag electrodes. From these devices surface-enhanced (resonance) Raman [SE­(R)­R] signals of molecules exclusively attached to Pt are obtained with intensity up to 50% of the value determined for Ag. SE­(R)­R spectroscopic investigations are carried out with different probe molecules, silica-coating thicknesses, and excitation lines. Additionally, field enhancement calculations on Ag–SiO₂–Pt support geometries are performed to elucidate the influence of the Pt island film nanostructure on the observed Raman intensities. It is concluded that the nonperfect coating of the Pt island film promotes the efficiency of the induced Pt SER activity. Comparison with similar measurements on Ag–SiO₂–Au electrodes further suggests that the chemical nature of the deposited metal island film plays a minor role for the SE­(R)­R intensity

Polarization- and Wavelength-Dependent Surface-Enhanced Raman Spectroscopy Using Optically Anisotropic Rippled Substrates for Sensing

Anisotropic Ag nanoparticle arrays were created by metal evaporation on rippled silicon templates for sensing of molecules with surface-enhanced Raman spectroscopy. Our results show that these substrates can be used for analysis of complex molecular mixtures and discrimination of solvent molecules. These properties are due to their polarization and wavelength dependency that provide enhancement in a wide spectral range. The dielectric function parallel and perpendicular to the long axis of the nanostructures was determined via ellipsometry yielding two different plasmonic resonances. Polarized surface-enhanced raman scattering (SERS) was subsequently measured as a function of the polarization angle θ for a 4-mercaptobenzonitrile self-assembled monolayer covalently attached to the Ag surface. For 514 nm excitation a cos² θ-dependence and for 647 nm excitation a sin² θ-dependency were found, with the maxima expressing the resonances perpendicular and parallel to the ripples, respectively. Those results open the path for using such a substrate as a chemical sensor providing strong enhancement in a broad range of laser wavelengths on only one sensing surface and increasing the specificity by matching resonant Raman conditions