Applications of Infrared and Raman Microspectroscopy of Cells and Tissue in Medical Diagnostics: Present Status and Future Promises

This paper summarizes the progress achieved over the past fifteen years in applying vibrational (Raman and IR) spectroscopy to problems of medical diagnostics and cellular biology. During this time, a number of research groups have verified the enormous information content of vibrational spectra; in fact, genomic, proteomic, and metabolomic information can be deduced by decoding the observed vibrational spectra. This decoding process is aided enormously by the availability of high-power computer workstations and advanced algorithms for data analysis. Furthermore, commercial instrumentation for the fast collection of both Raman and infrared microspectral data has rendered practical the collection of images based solely on spectral data. The progress in the field has been manifested by a steady increase in the number and quality of publications submitted by established and new research groups in vibrational biological and biomedical arenas.

Thymoquinoneβ-Cyclodextrin Nanoparticles System: A Preliminary Study

Thymoquinone is a natural product, the main constituent ofNigella sativaseeds, which exhibits anti-inflammatory and anticancer activities. Among several existing molecules capable of forming an inclusion compound structure, cyclodextrins are applied in the pharmaceutical industry either to increase solubility of hydrophobic molecules or to protect molecules from inactivation or degradation.β-Cyclodextrin is currently the most common cyclodextrin in pharmaceutical formulations and probably the best studied in humans. In order to study the properties of inclusion compounds based on cyclodextrins and thymoquinone Fourier Transform Infrared (FTIR), Ultraviolet-Visible, Positron Annihilation Lifetime (PAL) Spectroscopies and calorimetric studies by Differential Scanning Calorimetry (DSC) were used. The obtained results indicate the formation of a 1 : 1 inclusion compound between cyclodextrin and thymoquinone. PALS and DSC measurements also provided evidence of the inclusion compound's activity.

Structural Perturbation of Superfolder GFP in the Presence of Guanidine Thiocyanate

The guanidine-thiocyanate-induced denaturation-renaturation of sfGFP was studied. It was shown that the disruption of sfGFP native structure occurs in the range of guanidine thiocyanate concentrations from 0.5 to 2.5 M. This process was accompanied by simultaneous changes of all recorded parameters. It was found that the small guanidine thiocyanate concentrations (less then 0.1-0.2 M) triggered local structural disturbances in protein which result in significant decrease of chromophore and tryptophan fluorescence intensity and change of protein visible absorption spectrum.

A novel opto-mechanical system constituted by LEDs to employment in photobiostimulation: Clinical application in optical therapy

Optical techniques of photobiostimulation, which use transducer as lasers and LEDs, have been employed in the treatment of several diseases. The laser systems usually irradiate in a reduced area of the target biological tissue, presenting high cost of acquisition. Devices with a LED of “Surface Mount Device” (SMD) type have high angle of light emission, implying in intense decrease of the density of optical potency. Furthermore, the use of this device requires the utilization of a cooling system, which provokes an increase in the cost of this product. In the present work, it is suggested the application of a novel opto-mechanical device constituted by usual LEDs with package dimension of 5 mm of diameter, which presents significant efficient and very low cost. The arrangement of the geometric configuration of these LEDs is elaborated in such way that all units emit photons in a unique section of an specific area. An opto-mechanical system was developed, which includes seven LEDs, being that six of these LEDs are disposed in a specific angle around a unique central LED. It is important to notice that all the opto-mechanical system with seven LEDs emit light in a unique area. Besides, a methodology was employed to allow the verification of the distribution of light intensity upon this respective area. This arrangement allows that this novel device propitiate measurements of the degree of homogeneity of the light intensity in specific areas, which are accessed by the seven light emitting units. The potency employing LEDs in the red (visible) and near infrared regions were also measured, being that the result demonstrated the capability of clinical application in optical therapy of photobiostimulation, which was also used in a pilot test of clinical application.

Spectroscopic (FT-IR, Raman, NMR) and DFT Quantum Chemical Studies on Phenoxyacetic Acid and Its Sodium Salt

FT-IR, Raman, and NMR spectra of phenoxyacetic acid and its sodium salt were recorded and analyzed. Optimized geometrical structures of studied compounds were calculated by B3LYP/6-311++ method. The atomic charges were calculated by Mulliken, NPA (natural population analysis), APT (atomic polar tensor), MK (Merz-Singh-Kollman method), and ChelpG (charges from electrostatic potentials using grid-based method) methods. Geometric as well as magnetic aromaticity indices, dipole moments, and energies were also calculated. The theoretical wavenumbers and intensities of IR spectra as well as chemical shifts in and NMR spectra were obtained. The calculated parameters were compared with experimental characteristics of these molecules.

Analysis of protein from nasopharyngeal carcinoma cell line CNE2 irradiated by X-ray using Raman spectroscopy

Our work aims to explore Raman spectroscopy (RS) to study the effects of total protein of nasopharyngeal carcinoma (NPC) cell line CNE2 treated by different doses of X-ray irradiation. The total protein is extracted from the cell line after irradiation with no incubation (0 h) and with incubation for 72 h, respectively. Both paired-samplesttest and support vector machine (SVM) are employed for statistical analysis of Raman spectrum. The results show that several Raman bands assigned to total protein can be detected; and the X-ray irradiation have various effects on different components in total protein, especially for the dose higher than 6 Gy, and also associated with the incubation time after irradiation. The differences of content between 0 h groups and 72 h groups appear at bands of C–S stretching, C–H stretching, O=C–O−stretching and L-phenylalanine. It indicated no significant diversification on structures of protein, while the content may be varied during irradiation. Our spectroscopic results reveal that RS analysis of total protein of cell line irradiated by X-ray, in conjunction with mathematical statistical model, can be a potential method to explore the Raman characterization target of cell radiosensitivity, and then for making known the mechanism of radiosensitivity of tumor further.

Impact ofβ-Turn Sequence onβ-Hairpin Dynamics Studied with Infrared-Detected Temperature Jump

Folding dynamics forβ-structure loss and disordered structure gain were studied in a modelβ-hairpin peptide based on Cochran’s tryptophan zipper peptide Trpzip2, but with an altered Thr-Gly (TG) turn sequence, that is, SWTWETGKWTWK, using laser-induced temperature-jump (T-jump) kinetics with IR detection. As has been shown previously, the TG turn sequence reduces the thermodynamicβ-hairpin stability as compared to the Asn-Gly sequence used in Trpzip2 (TZ2-NG). In this study, we found that the TG-turn slows down the overall relaxation dynamics as compared to TZ2-NG, which were studied at higher temperatures where the time constants show little difference between relaxation of theβ-strand and the disordered conformation. These time constants become equivalent at lower temperatures for TZ2-TG than was seen for TZ2-NG. The correlation of thermodynamic stability and rates of relaxation suggests that the change from NG to TG turn results in a slowing of folding, lowerkf, with less change of the unfolding rate,ku, assuming two state behavior at higher temperatures.

A Comparison between Vanadyl, Vanadate, and Decavanadate Effects in Actin Structure and Function: Combination of Several Spectroscopic Studies

The studies about the interaction of actin with vanadium are seldom. In the present paper the effects of vanadyl, vanadate, and decavanadate in the actin structure and function were compared. Decavanadate clearly interacts with actin, as shown byV51-NMR spectroscopy. Decavanadate interaction with actin induces protein cysteine oxidation and vanadyl formation, being both prevented by the natural ligand of the protein, ATP. Monomeric actin (G-actin) titration with vanadyl, as analysed by EPR spectroscopy, indicates a 1 : 1 binding stoichiometry and akdof 7.5 μM. Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with aIC50of 68 and 300 μM, respectively, as analysed by light-scattering assays. However, only vanadyl induces G-actin intrinsic fluorescence quenching, which suggests the presence of vanadyl high-affinity actin-binding sites. Decavanadate increases (2.6-fold) actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Finally, both vanadium species increasedε-ATP exchange rate (k=6.5×10−3and4.47×10−3 s−1for decavanadate and vanadyl, resp.). Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl.

Pt(II) Complexes with Linear Diamines—Part I: Vibrational Study of Pt-Diaminopropane

A conformational analysis of the Pt(dap)Cl2complex (-diaminopropane) was performed by vibrational spectroscopy (FTIR, Raman, and INS), coupled to quantum mechanical methods within the density functional theory (DFT) and effective core potential (ECP) approaches. A complete spectral assignment of the system was achieved, due to the combined use of all available vibrational spectroscopic techniques. A good agreement was found between experimental and theoretical results, as well as with reported data for analogous complexes (e.g., cisplatin).

SERS Microspectroscopy of Biomolecules on Dried Ag Colloidal Drops

We report the application of dried Ag hydroxylamine-reduced colloidal drops to surface-enhanced (resonance) Raman scattering (SE(R)RS) study of biomolecules using Raman microspectroscopy. 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP), amino acid tryptophan, and phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) served as testing biomolecules. Ag colloid/biomolecule drop dried on glass support forms a ring in the edge part of the drop in which almost all nanoparticles are clustered. This specific drying process promotes adsorption of the studied biomolecules in highly enhancing sites (“hot spots”) as well as concentrates them in the ring. We were able to obtain SE(R)RS spectra from the ring that cannot be acquired directly from Ag colloidal solutions (SERRS spectrum of1×10−10 M TMPyP by 1 s accumulation time, SERS spectrum of2×10−7 M DSPC). Despite the spectral irreproducibility and problems with spurious bands in some cases, SERS microspectroscopy of studied biomolecules using dried Ag colloid/adsorbate systems improves SERS applicability and sensitivity in comparison to measurements directly from Ag colloidal solution.