The influence of fluoroquinolone drugs on the bacterial growth of S. epidermidis utilizing the unique potential of vibrational spectroscopy

Increasing resistance of many antibiotics has made the design of new drugs necessary. To assist a target-oriented search for new structures and for the elucidation of the mode of action of existing drugs, powerful analytical techniques are required. In this work, vibrational spectroscopy is used to shed more light on the as-yet elusive interaction of gyrase inhibitors of the fluoroquinolone type with their biological target inside the Gram-positive bacterium Staphylococcus epidermidis by investigating whole-cell changes that occur as a result of the presence of the drug moxifloxacin. IR absorption and Raman spectra with excitation off resonance (lambda exc = 532 nm) and in resonance with the biological targets DNA and the aromatic amino acids of gyrase (lambda exc = 244 nm) were recorded for unperturbed bacteria and bacteria in varying drug concentrations (0.08, 0.16, 0.27, and 0.62 microg moxifloxacin/mL bacterial culture). The spectral changes caused by the action of the drug were analyzed with the help of statistical methods, such as hierarchical cluster analysis (HCA), principal component analysis (PCA), and Fisher's linear discriminant analysis (LDA) combined with variable selection. The wavenumbers mostly affected by the action of the drug could be assigned to protein and DNA moieties, supporting the proposed mechanisms of a tertiary complex of the fluoroquinolone, the enzyme gyrase, and DNA.     

SERS: a versatile tool in chemical and biochemical diagnostics

Raman spectroscopy is a valuable tool in various research fields. The technique yields structural information from all kind of samples often without the need for extensive sample preparation. Since the Raman signals are inherently weak and therefore do not allow one to investigate substances in low concentrations, one possible approach is surface-enhanced (resonance) Raman spectroscopy. Here, rough coin metal surfaces enhance the Raman signal by a factor of 10⁴–10¹⁵, depending on the applied method. In this review we discuss recent developments in SERS spectroscopy and their impact on different research fields.     

Raman Spectrum of Er-Y-codoped ZrO2 and Fluorescence Properties of Er3+

Er-Y-codoped ZrO2 mixed oxides with monoclinic, tetragonal and cubic structures were prepared by a sol-gel method. The crystal structure of ZrO2 matrix and the effect of the ZrO2 phases on the fluorescence properties of Er3+ were studied using Raman spectroscopy. The results indicated that the fluorescence properties of Er3+ depend on its local ZrO2 crystal structures. As ZrO2 matrix transferred from monoclinic to tetragonal and cubic phase, the Raman and fluorescence bands of Er3+ decreased in intensities and tended to form a single peak. With 632.8 nm excitation, the bands between 640 and 680 nm were attributed to the fluorescence of Er3+ in the ZrO2 environment. However, only the fluorescence was observed and no Raman spectra were seen under 514.5 nm excitation, while only Raman spectra were observed under 325 nm excitation. UV Raman spectroscopy was found to be more sensitive in the surface region while the information provided by XRD mainly came from the bulk. The phase with lower symmetry forms more easily on the surface than in the bulk.     

Excitation wavelength dependent surface-enhanced Raman spectra of a dipping film of azobenzene-containing long-chain fatty acid on a silver mirror

Surface-enhanced Raman scattering (SERS) of dipping films of azobenzene-containing long-chain fatty acids, nAmH (n=8, 12, m=3, 5), on silver mirrors measured with a wide range of excitation wavelengths in the 457.9-1064 nm region is reported. The obtained Raman spectra show great SERS effect even with the 1064 nm excitation, and the excitation with 457.9, 476.5, and 488.0 nm gives surface-enhanced resonance Raman scattering (SERRS) due to the resonance effect of the symmetry-forbidden n-pi* transition of the azo group. Of particular note in the present study is that the SERS spectra with the excitation in the 532-1064 nm region yield Raman bands whose frequencies are almost identical to those bands in Raman spectra of nAmH in solid state while the SERRS spectra with the excitation in the 457.9-514.5 nm region show not only a set of bands which correspond to those of nAmH in the solid state but also a set of bands whose frequencies show a significant shift from those of the bands of nAmH in the solid state. These observations lead us to conclude that there are two kinds of molecular aggregates in the dipping films of azobenzene-containing long-chain fatty acid in which azobenzene moieties are condensed to form small bundles.     

In situ UV resonance Raman micro-spectroscopic localization of the antimalarial quinine in cinchona bark

Deep UV resonance Raman micro-spectroscopy (lambda(exc) = 244 nm) was applied for a highly sensitive, selective, and gentle localization of the antimalarial quinine in situ in cinchona bark. The high potential of the method was demonstrated by the detection of small amounts of the alkaloid in the plant material without any further sample preparation, where conventional (non-resonant) Raman microscopy was unsuccessful due to a strong fluorescence background. The resonance Raman spectrum of cinchona bark corresponds well with that of quinine; it can be distinguished from its diastereomer quinidine via the mode at 831 cm(-1), which is shifted to 843 cm(-1) in the case of quinidine. This vibration involves a bending motion within the side chain around the chiral center of quinine. Vibrations belonging to the quinoline ring (important for its antimalarial activity in forming pi-pi-interactions to hemozoin) and the vinyl group are resonantly enhanced in the UV Raman spectra. A convincing mode assignment is derived by means of a combination of NIR Raman spectroscopy and DFT calculations. The Raman spectra of quinine in cinchona bark are modeled by considering a hydrous environment that causes a shift of the band at 1362 compared with 1371 cm(-1) in anhydrous quinine. This intense vibration is therefore sensitive to the presence of an aqueous environment and is assigned mostly to a stretching motion within the quinoline ring. The presented results nicely show the sensitivity of Raman spectroscopy to monitor subtle differences within the molecular structure and the influence of a biological relevant hydrous environment and trace low concentrated pharmaceutical relevant active agents in plant material.     

Methods for extracting biochemical information from bacterial Raman spectra: focus on a group of structurally similar biomolecules--fatty acids

In this paper we explore the possibilities of Raman spectroscopy in order to deduce information on the fatty acid composition of bacterial cells. Therefore, representative strains of two bacterial taxa were each cultured in different conditions and in parallel analyzed by Raman spectroscopy and gaschromatographic FAME analysis. Raman spectra of pure fatty acids were recorded and used as reference spectra. The culturing conditions for each strain could be easily distinguished by the fatty acid information retrieved from bacterial Raman spectra. Chemometric techniques such as EMSC and PCA allowed to extract information about groups of fatty acids, that was consistent with the results from FAME analysis. Although the information retrieved from Raman spectroscopy is not as refined as that from FAME analysis, the presented methods could be useful to obtain basic information on the fatty acid present in bacteria when performing Raman spectroscopic analysis for fast whole cell profiling, which provides information for different types of cell components (fatty acids, amino acids, primary metabolites, etc.).     

Raman spectroscopic identification of usnic acid in hydrothermal minerals as a potential Martian analogue

Raman spectroscopy using 785 nm excitation was tested as a nondestructive method for determining the presence of the potential biomarker, usnic acid, in experimentally prepared mineral matrices. Investigated samples consisting of usnic acid mixed with powdered hydrothermal minerals, gypsum and calcite were studied. Various concentrations of usnic acid in the mineral matrix were studied to determine the detection limits of this biomarker. Usnic acid was mixed with gypsum (respectively, calcite) and covered by a UV-transparent crystal of gypsum (CaSO(4) x 2 H(2)O), thereby creating artificial inclusions similar to those which could be present in Martian minerals. A Raman usnic acid signal at the concentration level as low as 1 g kg(-1) was obtained in the powdered mineral matrix and 5 g kg(-1) when analyzed through the monocrystal. The number of registered usnic acid key Raman bands was dependent on the particular mineral matrix. If a similar concentration of usnic acid could persist in Martian samples, then Raman spectroscopy will be able to identify it. Obtained results will aid both in situ Raman analyses on Mars and on Earth.     

The electrooxidation of small organic molecules on platinum nanoparticles supported on gold: influence of platinum deposition procedure

The electrocatalytic properties of small platinum nanoparticles were investigated for the oxidation of CO, methanol, and formic acid using voltammetry, chronoamperometry, and surface-enhanced Raman spectroscopy. The particles were generated by galvanostatic deposition of platinum on a polished gold surface from an H₂PtCl₆ containing electrolyte and ranged between 10 and 20 nm in diameter for low platinum surface concentrations, 10 and 120 nm for medium concentrations, and full Pt monolayers for high concentrations. CO stripping and bulk CO oxidation experiments on the particles up to 120 nm in diameter displayed pronounced structural effects. The CO oxidation current-time transients show a current decay for low platinum coverages and a current maximum for medium and high coverages. These results were also observed in the literature for particles of 2- to 5-nm size and agglomerates of these particles. The similarities between the literature and our results, despite large differences in particle size and morphology, suggest that particle structure and morphology are also very important catalytic parameters. Surface-enhanced Raman spectroscopy data obtained for the oxidation of CO on the Pt-modified Au electrodes corroborate this conclusion. A difference in the ratio between CO adsorbed in linear- and bridge-bonded positions on the Pt nanoparticles of different sizes demonstrates the influence of the surface morphology. The oxidation activity of methanol was found to decrease with the particle size, while the formic acid oxidation rate increases. Again, a structural effect is observed for particles of up to ca. 120 nm in diameter, which is much larger than the particles for which a particle size effect was reported in the literature. The particle shape effect for the methanol oxidation reaction can be explained by a reduction in available “ensemble sites” and a reduction in the mobility of CO formed by decomposition of methanol. As formic acid does not require Pt ensemble sites, decreasing the particle size, and thus, the relative number of defects, increases the reaction rate. Dedicated to Prof. Dr. Teresa Iwasita on the occasion of her 65th birthday in recognition of her numerous contributions to interfacial electrochemistry.     

Spatial distribution of heme species in erythrocytes infected with Plasmodium falciparum by use of resonance Raman imaging and multivariate analysis

The multivariate algorithm hierarchical cluster analysis is applied to sets of resonance Raman spectra collected from human erythrocytes infected with the malaria parasite Plasmodium falciparum. The images obtained yield information about the distribution of hemoglobin and hemozoin (or malaria pigment) within the parasitized cells and about their molecular structure. This method has the advantage of conveying more information than other imaging approaches based on resonance Raman spectroscopy, and it is a promising tool to study the hemozoin formation process and its interaction with antimalarial drugs within unstained, well-preserved parasites.     

The study of resonance Raman scattering spectrum on the surface of Cu nanoparticles with ultraviolet excitation and density functional theory

Cu colloid was prepared by oxidation-reduction; it was relatively steady in fixed conditions, with size about 10-30 nm. The Raman spectrum of p-hydroxybenzoic acid (PHBA) in Cu colloid solution with the ultraviolet (UV) excitation at 325 nm, was obtained, even it is usually difficult to obtain Raman signals in Ag or Au in the UV region. It was found that the Raman signal intensities result from the resonance enhanced of surface plasmon resonance of Cu nanoparticles excited at 325 nm. The adsorption behavior of PHBA on the Cu nanoparticles was studied by combining with density functional theory (DFT); it was found that the calculated Raman frequencies were in good agreement with experimental value. So one can conclude that the simplified model is probably reasonable to describe some resonance Raman experiments.     

A new approach for the detection of a nonfluorescent compound by CE-resonance Raman spectroscopy based on the sweeping-MEKC mode

A CE-resonance Raman spectroscopy (CE-RRS) method based on MEKC and sweeping-MEKC modes is described. A nonfluorescent compound, malachite green (MG), and a doubled Nd:YAG laser (532 nm, 300 mW) were selected as model compound and light source, respectively. In order to carry out a quantitative analysis of MG, a monochromator (effective bandwidth, 0.4 nm) was used to collect the specific Raman line at 1616 cm(-1) (N-phi and C-C stretch, corresponding to 582 nm when the wavelength of the exciting source was 532 nm). As a result, the LOD for MG was 10 ppm, based on the MEKC/RRS mode. This could be improved to 5 ppb when the sweeping-MEKC/RRS mode was applied. Furthermore, with the addition of nano-size silver colloids to the CE buffer the detection limits can be further improved, but the data obtained with surface-enhanced resonance Raman spectroscopy (SERRS) are less useful for quantitative purposes.     

UV-resonance raman thermal unfolding study of Trp-cage shows that it is not a simple two-state miniprotein

Trp-cage, a synthetic 20 residue polypeptide, is proposed to be an ultrafast folding synthetic miniprotein which utilizes tertiary contacts to define its native conformation. We utilized UV resonance Raman spectroscopy (UVRS) with 204 and 229 nm excitation to follow its thermal melting. Our results indicate that Trp-cage melting is complex, and it is not a simple two-state process. Using 204 nm excitation we probe the peptide secondary structure and find the Trp-cage's alpha-helix shows a broad melting curve where on average four alpha-helical amide bonds melt upon a temperature increase from 4 to 70 degrees C. Using 229 nm excitation we probe the environment of the Trp side chain and find that its immediate environment becomes more compact as the temperature is increased from 4 to 20 degrees C; however, further temperature increases lead to exposure of the Trp to water. The chi(2) angle of the Trp side chain remains invariant throughout the entire temperature range. Previous kinetic results indicated a single-exponential decay in the 4-70 degrees C temperature range, suggesting that Trp-cage behaves as a two-state folder. However, this miniprotein does not show clear two-state behavior in our steady-state studies. Rather it shows a continuous distribution of steady-state spectral parameters. Only the alpha-helix melting curve even hints of a cooperative transition. Possibly, the previous kinetic results monitor only a small region of the Trp-cage which locally appears two-state. This would then argue for spatially decoupled folding even for this small peptide.     

Au nanorod arrays tailored for surface-enhanced Raman spectroscopy.

We have demonstrated surface-enhanced Raman spectroscopy (SERS) on arrays of Au nanorods aligned in line by a dynamic oblique deposition technique. For light polarized along the major axes of the nanorods, the plasma resonance of the Au nanorods has been tuned to a wavelength suitable for Raman spectroscopy. Raman scattering on the discrete nanorods is significantly enhanced compared with that on semicontinuous Au films. Since the preparation process is physically bottom-up, it is robust in its selection of the materials and is useful for providing SERS sensors at low cost.     

Total internal reflection Raman spectroscopy

Total internal reflection (TIR) Raman spectroscopy is an experimentally straightforward, surface-sensitive technique for obtaining chemically specific spectroscopic information from a region within approximately 100-200 nm of a surface. While TIR Raman spectroscopy has long been overshadowed by surface-enhanced Raman scattering, with modern instrumentation TIR Raman spectra can be acquired from sub-nm thick films in only a few seconds. In this review, we describe the physical basis of TIR Raman spectroscopy and illustrate the performance of the technique in the diverse fields of surfactant adsorption, liquid crystals, lubrication, polymer films and biological interfaces, including both macroscopic structures such as the surfaces of leaves, and microscopic structures such as lipid bilayers. Progress, and challenges, in using TIR Raman to obtain depth profiles with sub-diffraction resolution are described.     

ULTRAVIOLET RESONANCE RAMAN SPECTROSCOPY OF BACTERIORHODOPSIN

Ultraviolet resonance Raman spectra of bacteriorhodopsin have been obtained using 229 nm excitation from a hydrogen-shifted neodymium yttrium aluminum garnet (Nd: YAG) laser. High signal-to-noise spectra are observed exhibiting vibrational bands at 762, 877, 1011, 1175, 1356, 1552 and 1617 cm-1 which are assigned to scattering from tryptophan and tyrosine side chains. This demonstrates the feasibility of using UV resonance Raman spectroscopy to monitor aromatic amino acid structural changes during the bacteriorhodopsin photocycle.     

Red-excitation resonance Raman analysis of the nu(Fe=O) mode of ferryl-oxo hemoproteins

The Raman excitation profile of the nuFe O mode of horseradish peroxidase compound II exhibits a maximum at 580 nm. This maximum is located within an absorption band with a shoulder assignable to an oxygen-to-iron charge transfer band on the longer wavelength side of the alpha-band. Resonance Raman bands of the nuFe O mode of various ferryl-oxo type hemoproteins measured at 590 nm excitation indicate that many hemoproteins in the ferryl-oxo state have an oxygen-to-iron charge transfer band in the visible region. Since this red-excited resonance Raman technique causes much less photochemical damage in the proteins relative to blue-excited resonance Raman spectroscopy, it produces a higher signal-to-noise ratio and thus represents a powerful tool for investigations of ferryl-oxo intermediates of hemoproteins.     

UV resonance Raman spectroscopic monitoring of supramolecular complex formation: peptide recognition in aqueous solution

The formation of a supramolecular complex between a tetrapeptide and an artificial receptor , is monitored at submillimolar concentrations in water by UV resonance Raman spectroscopy. Using 275 nm excitation, we selectively probe the carboxylate binding site (CBS) within the receptor, a moiety which is very efficient in binding the carboxy terminus of peptides in aqueous media. Complexation of the receptor with the tetrapeptide involves the formation of a H-bond enforced ion pair, resulting in significant changes in the corresponding UV resonance Raman spectra. Our qualitative interpretation is based on experimental reference and calculated Raman spectra on model systems. First preliminary calculations show that for a quantitative analysis, also the distinct contributions of multiple CBS conformers must be considered in addition to the H-bond induced changes upon complexation.     

A UV resonance Raman (UVRR) spectroscopic study on the extractable compounds of Scots pine (Pinus sylvestris) wood. Part I: lipophilic compounds

The wood resin in Scots pine (Pinus sylvestris) stemwood and branch wood were studied using UV resonance Raman (UVRR) spectroscopy. UVRR spectra of the sapwood and heartwood hexane extracts, solid wood samples and model compounds (six resin acids, three fatty acids, a fatty acid ester, sitosterol and sitosterol acetate) were collected using excitation wavelengths of 229, 244 and 257 nm. In addition, visible Raman spectra of the fatty and resin acids were recorded. Resin compositions of heartwood and sapwood hexane extracts were determined using gas chromatography. Raman signals of both conjugated and isolated double bonds of all the model compounds were resonance enhanced by UV excitation. The oleophilic structures showed strong bands in the region of 1660-1630 cm(-1). Distinct structures were enhanced depending on the excitation wavelength. The UVRR spectra of the hexane extracts showed characteristic bands for resin and fatty acids. It was possible to identify certain resin acids from the spectra. UV Raman spectra collected from the solid wood samples containing wood resin showed a band at approximately 1650 cm(-1) due to unsaturated resin components. The Raman signals from extractives in the resin rich branch wood sample gave even more strongly enhanced signals than the aromatic lignin.     

Applicability of surface-enhanced resonance Raman scattering for the direct discrimination of ballpoint pen inks

In situ surface-enhanced resonance Raman spectroscopy (SERRS) with excitation at 685 nm is suitable for the direct discrimination of blue and black ballpoint pen inks on paper. For black inks, shorter excitation wavelengths can also be used. For blue inks, SERRS at 514.5 and 457.9 nm does not provide adequate discriminative power. At these excitation wavelengths, the SERRS signals of the Methyl Violet derivatives present in inks easily dominate the overall spectrum because of resonance enhancement and preferential interaction with silver sol particles. At 685 nm, this problem is not encountered as the Methyl Violet derivatives do not show resonance enhancement, while other components may still exhibit resonance. Thirteen blue and thirteen black ink lines were examined. For the blue and black inks, on the basis of the 685 nm SERR spectra, eight and six groups of spectra, respectively, could be distinguished. This discrimination largely agrees with information from thin layer chromatography (TLC) experiments, although some differences in group compositions are found. The in situ SERR spectra show good repeatability with regard to the Raman frequencies, band shapes and relative intensities of the spectral bands. However, absolute intensities cannot be used for discrimination purposes.     

UV Raman spectroscopic study on TiO2. I. Phase transformation at the surface and in the bulk

Phase transformation of TiO2 from anatase to rutile is studied by UV Raman spectroscopy excited by 325 and 244 nm lasers, visible Raman spectroscopy excited by 532 nm laser, X-ray diffraction (XRD), and transmission electron microscopy (TEM). UV Raman spectroscopy is found to be more sensitive to the surface region of TiO2 than visible Raman spectroscopy and XRD because TiO2 strongly absorbs UV light. The anatase phase is detected by UV Raman spectroscopy for the sample calcined at higher temperatures than when it is detected by visible Raman spectroscopy and XRD. The inconsistency in the results from the above three techniques suggests that the anatase phase of TiO2 at the surface region can remain at relatively higher calcination temperatures than that in the bulk during the phase transformation. The TEM results show that small particles agglomerate into big particles when the TiO2 sample is calcined at elevated temperatures and the agglomeration of the TiO2 particles is along with the phase transformation from anatase to rutile. It is suggested that the rutile phase starts to form at the interfaces between the anatase particles in the agglomerated TiO2 particles; namely, the anatase phase in the inner region of the agglomerated TiO2 particles turns out to change into the rutile phase more easily than that in the outer surface region of the agglomerated TiO2 particles. When the anatase particles of TiO2 are covered with highly dispersed La2O3, the phase transformation in both the bulk and surface regions is significantly retarded, owing to avoiding direct contact of the anatase particles and occupying the surface defect sites of the anatase particles by La2O3.