Raman Spectra and Ancient Life: Vibrational ID Profiles of Fossilized (Bone) Tissues.

Raman micro-spectroscopy is a non-destructive and non-contact analytical technique that combines microscopy and spectroscopy, thus providing a potential for non-invasive and in situ molecular identification, even over heterogeneous and rare samples such as fossilized tissues. Recently, chemical imaging techniques have become an increasingly popular tool for characterizing trace elements, isotopic information, and organic markers in fossils. Raman spectroscopy also shows a growing potential in understanding bone microstructure, chemical composition, and mineral assemblance affected by diagenetic processes. In our lab, we have investigated a wide range of different fossil tissues, mainly of Mesozoic vertebrates (from Jurassic through Cretaceous). Besides standard spectra of sedimentary rocks, including pigment contamination, our Raman spectra also exhibit interesting spectral features in the 1200-1800 cm-1 spectral range, where Raman bands of proteins, nucleic acids, and other organic molecules can be identified. In the present study, we discuss both a possible origin of the observed bands of ancient organic residues and difficulties with definition of the specific spectral markers in fossilized soft and hard tissues.

Analysis of Iron Complexes of Tannic Acid and Other Related Polyphenols as Revealed by Spectroscopic Techniques: Implications in the Identification and Characterization of Iron Gall Inks in Historical Manuscripts.

In this work, a structural analysis of the polyphenol complexes with iron and copper at several conditions is reported. The investigated polyphenols were tannic acid, gallic acid, pyrogallol, and syringic acid, being components and molecular models of the gallnuts usually employed in the past in fabrication of iron gall inks (IGIs). Commercial tannic acid extracted from gallnuts, which is a complex mixture of different gallotannins and simpler galloylglucoses, was also employed in this analysis. This analysis comprised the use of Raman, Fourier-transform infrared (FTIR), UV-vis absorption, and fluorescence spectroscopy. The complexation of iron with these molecules leads to a strong change in color due to the deep restructuring of the polyphenol that can be clearly seen by Raman and FTIR spectra. Three main Raman bands appeared at 1450-1490 cm-1 (ν1), 1320-1345 cm-1 (ν2), and 400-650 cm-1 (ν3), which are characteristic of the metal complexes. The structural changes of the polyphenol complexes with iron were also investigated at different pHs and different polyphenol/iron stoichiometries. Other effects of the interaction of polyphenols with iron are the pH decrease of the mixture upon metal complexation and fluorescence quenching induced by the interaction of iron. This quenching is important since it facilitates the Raman inspection of manuscripts since polyphenols show a strong fluorescence emission that overlaps the Raman spectrum. Furthermore, DFT calculations were performed for the first time on the gallic acid complex with iron in order to elaborate a detailed assignment of the vibrational modes of polyphenols and their metal complexes, something that was missed in previous applications of Raman to IGIs.

Vibrational Study (Raman, SERS, and IR) of Plant Gallnut Polyphenols Related to the Fabrication of Iron Gall Inks.

FT-Raman, FTIR, and SERS spectra of the structurally related gallnut polyphenols tannic acid, gallic acid, pyrogallol, and syringic acid are reported in this work aiming at performing a comparative assignation of the bands and finding specific marker features that can identify these compounds in complex polyphenol mixtures. Tannic and gallic acids are the principal components in oak gallnuts, and they can be found in iron gall inks. The different functional groups existing in these molecules and their spatial distribution lead to slight changes of the vibrations. The Raman spectra are dominated by bands corresponding to the ring vibrations, but the substituents in the ring strongly affect these vibrations. In contrast, the FTIR spectra of these molecules are dominated by the peripheral oxygen-containing substituents of the aromatic ring and afford complementary information. SERS spectroscopy can be used to analyze trace amounts of these compounds, but the spectra of these polyphenols show strong changes in comparison with the Raman spectra, indicating a strong interaction with the metal. The most significant modification observed in the SERS spectra of these compounds is the weakening of the benzene 8a ring vibration and the subsequent intensification of the 19a mode of the benzene ring. This mode is also more intense in the FTIR spectra, and its intensification in the SERS spectra could be related to a drastic change in the molecular polarizability associated with the interaction of the polyphenol with the metal in Ag NPs.

Vibrational characterization of the pesticide molecule Tebuconazole.

Pesticide use worldwide exhibits a positive effect on agricultural production while it may negatively affect organisms living in soil, water or the air. Importantly, numerous negative health effects also occur in humans exposed to (accumulated) pesticides or their metabolites over a long period of time. To prevent both environmental catastrophes and adverse human health impacts, initial studies of the selected pesticides need to be performed together with the constant post-approval control; risk assessment analysis and on site monitoring have to be continuously carried out. Given this, Raman spectroscopy, especially surface-enhanced Raman spectroscopy (SERS), during the last decade has become a powerful analytical technique since it can offer quick, selective, and in situ detection of selected pollutants found in analyzed samples at very low concentrations. Moreover, the structural changes caused by the pollutant-biomacromolecule interaction can also be recognized in the molecule-specific Raman spectral signatures of biomolecules. In this study, we report a vibrational characterization of the fungicide molecule Tebuconazole (TB) which is listed to be a possible carcinogen. Even though its international and common use there is no evidence about the use of Raman/SERS spectroscopy to detect it sensitively and selectively as well as to analyse its impacts on biological systems. Therefore, we have recorded and calculated Raman and infrared spectra of TB. Furthermore, SERS spectra of TB were also registered and comprehensively analysed in view of the employed SERS substrates, dependence on the excitation wavelengths and pH of the analysed molecular systems. The molecule of TB interacts preferentially through the triazole moiety with the colloidal metal nanoparticles (NPs) whereas the silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride resulted to be the most effective ones. Consequently, the limit of detection was determined to be 1.4 µM≈430 ppb. The present paper thus could serve significantly for further investigations focused on both conducting vibrational analyses of structurally related molecules as well as providing a more precise explanation of the mechanism of action of TB and its influence on biological macromolecules.

Structural Lesions of Proteins Connected to Lipid Membrane Damages Caused by Radical Stress: Assessment by Biomimetic Systems and Raman Spectroscopy.

Model systems constituted by proteins and unsaturated lipid vesicles were used to gain more insight into the effects of the propagation of an initial radical damage on protein to the lipid compartment. The latter is based on liposome technology and allows measuring the trans unsaturated fatty acid content as a result of free radical stress on proteins. Two kinds of sulfur-containing proteins were chosen to connect their chemical reactivity with membrane lipid transformation, serum albumins and metallothioneins. Biomimetic systems based on radiation chemistry were used to mimic the protein exposure to different kinds of free radical stress and Raman spectroscopy to shed light on protein structural changes caused by the free radical attack. Among the amino acid residues, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting that metal-Cys clusters are good interceptors of reactive species in metallothioneins, together with disulfides moieties in serum albumins. Met is another important site of the attack, in particular under reductive conditions. Tyr and Phe are sensitive to radical stress too, leading to electron transfer reactions or radical-induced modifications of their structures. Finally, modifications in protein folding take place depending on reactive species attacking the protein.

Structural analysis of the neuropeptide substance P by using vibrational spectroscopy.

Substance P (SP) is one of the most studied peptide hormones and knowing the relationship between its structure and function may have important therapeutic applications in the treatment of a variety of stress-related illnesses. In order to obtain a deeper insight into its folding, the effects of different factors, such as pH changes, the presence of Ca2+ ions, and the substitution of the Met-NH2 moiety in the SP structure, was studied by Raman and infrared spectroscopies. SP has a pH-dependent structure. Under acidic-neutral conditions, SP possesses a prevalent ß-sheet structure although also other secondary structure elements are present. By increasing pH, a higher orderliness in the SP secondary structure is induced, as well as the formation of strongly bound intermolecular ß-strands with a parallel alignment, which favour the self-assembly of SP in ß-aggregates. The substitution of the Met-NH2 moiety with the acidic functional group in the SP sequence, giving rise to a not biologically active SP analogue, results in a more disordered folding, where the predominant contribution comes from a random coil. Conversely, the presence of Ca2+ ions affects slightly but sensitively the folding of the polypeptide chain, by favouring the α-helical content and a different alignment of ß-strands; these are structural elements, which may favour the SP biological activity. In addition, the capability of SERS spectroscopy to detect SP in its biologically active form was also tested by using different metal nanoparticles. Thanks to the use of silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride, SP can be detected at very low peptide concentration (~ 90 nM). However, the SERS spectra cannot be obtained under alkaline conditions since both the formation of SP aggregates and the lack of ion pairs do not allow a strong enough interaction of SP with silver NPs. Graphical abstract.

Benefits of hypericin transport and delivery by low- and high-density lipoproteins to cancer cells: From in vitro to ex ovo.

Lipoproteins are very attractive natural-based transport systems suitable for applications in diagnostics and cancer therapy. Low- and high-density lipoproteins (LDL, HDL) were selected for hypericin (hyp) delivery in cancer cells. Hyp was used, as it is a well-known model for hydrophobic molecules, in order to estimate the LDL and HDL transport efficacy. We applied fluorescence techniques, absorption and Raman spectroscopy to characterize the state and alteration of LDL and HDL in the absence and presence of hyp. The fluorescence intensity of hyp loaded in lipoproteins was two times weaker in HDL than LDL. We demonstrated that there are faster redistribution kinetics of hyp from HDL than from LDL. As a consequence, hyp uptake by glioma and breast cancer cells was driven more via endocytosis when hyp was delivered by LDL than by HDL. Hyp induced photodynamic action was stronger when hyp was delivered by HDL than LDL. Ex ovo hyp fluorescence pharmacokinetics demonstrated differences in biodistributions of hyp in lipoproteins topical applications. However, hyp was successfully delivered to cancer cells grafted on quail's chorioallantoic membrane. The results presented in this paper could provide strategies to develop adequate and targeted anticancer therapy.

Electrochemically deposited silver detection substrate for surface-enhanced Raman spectroscopy cancer diagnostics.

Surface-enhanced Raman spectroscopy (SERS) of blood plasma on an electrochemically prepared silver surface has been studied as a label-free, noninvasive diagnostic test for colorectal cancer. Indium tin oxide glass substrates were modified with 0.01 mol dm - 3 silver nitrate using the pulsed double-potentiostatic method. The prepared silver substrates were tested with Rhodamine 6G as a model analyte and the surface with the highest signal enhancement was selected. This silver dendritic surface was used as a diagnostic substrate for SERS measurements of human blood plasma. A group of oncological patients with declared colorectal carcinoma (n = 15) and the control group of healthy volunteers (n = 15) were compared. The biomolecular changes in chemical composition in the cancer samples were detected by statistical processing of the resulting SERS spectra. About 94% specificity and 100% sensitivity were achieved for the analysis by the ratio of the SERS peak intensity at 725 cm - 1 for adenine to the peak intensity at 638 cm - 1 for tyrosine and 100% specificity and sensitivity by using principal component analysis. This method of SERS diagnostics of colorectal cancer, which does not require the nanoparticle preparation, mixing, and incubation of plasma with a colloidal solution as in conventional tests, is a rapid, inexpensive method, which could be introduced as a primary diagnostic test.

Negligible interaction of [Ru(Phen)3]2+ with human serum albumin makes it promising for a reliable invivo assessment of the tissue oxygenation.

The interaction between a ruthenium - based water soluble oxygen probe ([Ru(Phen)3]2+, phen - phenanthroline) and human serum albumin (HSA) was investigated with the aim of describing the influence of HSA on the [Ru(Phen)3]2+ luminescence properties. Nowadays, several oxygen sensitive luminescent probes are used to determine the oxygen level in different compartments of living organisms. However, they can interact, depending on their hydrophilic/hydrophobic characters, with various serum proteins, and/or lipids, during their utilization for invivo oxygen measurement. Since HSA is the most abundant serum protein in most biological organisms, its presence may affect the spectral properties of the employed probes and, consequently, the determination of the oxygen concentration. Having this in mind, we have applied several spectroscopic and calorimetric techniques to study [Ru(Phen)3]2+ - HSA mixtures. Only a negligible effect of HSA on the absorption and luminescence spectra of [Ru(Phen)3]2+ was observed. In addition, differential scanning calorimetric studies showed that [Ru(Phen)3]2+ does not significantly influence HSA thermal stability. Importantly, [Ru(Phen)3]2+ retained a reliable luminescence lifetime sensitivity to the oxygen concentration in solutions supplemented with HSA and in U87 MG cancer cells. Finally, the biodistribution of [Ru(Phen)3]2+ in the presence of serum proteins in the blood stream of chick embryo's chorioallantoic membrane (CAM) was investigated. Fast [Ru(Phen)3]2+ and similar extravasations were observed in the presence or absence of CAM-serum. We can conclude that HSA-[Ru(Phen)3]2+ complex interaction does not significantly influence the potential of [Ru(Phen)3]2+ to be a suitable candidate for a reliable oxygen probe in living organisms.

Deeper insights into the drug defense of glioma cells against hydrophobic molecules.

By means of fluorescence microscopy the intracellular distribution of fluorescent drugs with different hydrophobicity (quinizarin, emodin and hypericin) was studied. Selective photoactivation of these drugs in precisely defined position (nuclear envelope) allowed moderately hydrophobic emodin enter the nucleus. Highly hydrophobic hypericin was predominantly kept in the membranes with no fluorescence observed in the nucleus. The redistribution of quinizarin, emodin and hypericin between lipids, proteins and DNA was studied in solutions and cells. Based on these results was proposed theoretical model of hydrophobic drugs' nuclear internalization after photo-activation. Molecular docking models showed that hypericin has the strongest affinity to P-glycoprotein involved in the cell detoxification. Presence of 10 µM quinizarin, emodin or hypericin increased P-glycoprotein function in U87 MG cells. Moreover, emodin pretreatment allowed quinizarin nuclear internalization without photo-activation, which was not the case for hypericin. The synergy of such pretreatment and photo-activation should lessen the drug doses with simultaneous increase of drug efficacy triggering cell apoptosis/necrosis.

Effect of metal-liquid interface composition on the adsorption of a cyanine dye onto gold nanoparticles.

Synthesis of asymmetric nanoparticles, such as gold nanorods, with tunable optical properties providing metal structures with improved SERS performance is playing a critical role in expanding the use of SERS to imaging and sensing applications. However, the synthetic methods usually require surfactants or polymers as shape-directing agents. These chemicals normally remain firmly bound to the metal after the synthesis, preventing the direct adsorption of a large number of potential analytes and often hampering the chemical functionalization of the surface unless extended, and critical for the nanoparticle stability, postremoval steps were performed. For this reason, it is of great importance for the full exploitation of these nanostructures to gain a deeper insight into the dependence of the analyte-metal interaction to the metal-liquid interface composition. In this article, we investigated in detail the role played by each component of the gold nanorod (GNR) interface in the adsorption of indocyanine green (ICG) as a probe molecule. Citrate-reduced gold nanospheres were used as a model substrate since the negative citrate anions adsorbed onto the metal surface can be easily displaced by those chemicals usually involved in the GNR synthesis, allowing the GNR-like interface composition to be progressively rebuilt and modified at will on the citrate-capped nanoparticles. The obtained results provide a meticulous description of the role played by each individual component of the metal-liquid interface on the ICG interaction with the metal, illustrating how apparently minor experimental changes can dramatically modify the affinity and optical properties of the ICG probe adsorbed onto the nanoparticle.

Spectroscopic and molecular modeling studies on the binding of the flavonoid luteolin and human serum albumin.

Luteolin (LUT) is a polyphenolic compound, found in a variety of fruits, vegetables, and seeds, which has a variety of pharmacological properties. In the present contribution, binding of LUT to human serum albumin (HSA), the most abundant carrier protein in the blood, was investigated with the aim of describing the binding mode and parameters of the interaction. The application of circular dichroism, UV-Vis absorption, fluorescence, Raman and surface-enhanced Raman scattering spectroscopy combined with molecular modeling afforded a clear picture of the association mode of LUT to HSA. Specific interactions with protein amino acids were evidenced. LUT was found to be associated in subdomain IIA where an interaction with Trp-214 is established. Hydrophobic and electrostatic interactions are the major acting forces in the binding of LUT to HSA. The HSA conformations were slightly altered by the drug complexation with reduction of alpha-helix and increase of beta-turns structures, suggesting a partial protein unfolding. Also the configuration of at least two disulfide bridges were altered. Furthermore, the study of molecular modeling afforded the binding geometry.