Maja Balarin

FT-IR spectroscopy of lipoproteins—A comparative study

FT-IR spectra, in the frequency region 4000-600 cm(-1), of four major lipoprotein classes: very low density lipoprotein (VLDL), low density lipoprotein (LDL) and two subclasses of high density lipoproteins (HDL(2) and HDL(3)) were analyzed to obtain their detailed spectral characterization. Information about the protein domain of particle was obtained from the analysis of amide I band. The procedure of decomposition and curve fitting of this band confirms the data already known about the secondary structure of two different apolipoproteins: apo A-I in HDL(2) and HDL(3) and apo B-100 in LDL and VLDL. For information about the lipid composition and packing of the particular lipoprotein the well expressed lipid bands in the spectra were analyzed. Characterization of spectral details in the FT-IR spectrum of natural lipoprotein is necessary to study the influence of external compounds on its structure.

Investigation of Spermatozoa and Seminal Plasma by Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectra of human spermatozoa and seminal plasma were recorded and analyzed. The procedure that was established for sample preparation enabled acquisition of reproducible spectra. The parameter I1087/I966 for controlling spectra reproducibility was defined. The assignment of bands was carried out using an empirical approach and the origin of the “sperm specific doublet”, the bands at 968 cm−1 and 981 cm−1, was determined. The principal component regression (PCR) algorithm was used to define the specific spectral regions correlating to characteristics of spermatozoa, such as concentration, straight-line velocity (VSL), and beat cross frequency (BCF). Then, simple spectral parameters, such as band intensities and band ratios, were tested to determine which one best correlates to characteristics of spermatozoa. The region of the amide I band, between 1700 cm−1 and 1590 cm−1, was defined as a specific spectral region that correlates to the concentration of spermatozoa. The parameter that gave the linear dependence to the concentration of spermatozoa was the intensity of the amide I band. For VSL, the bands between 1119 cm−1 and 943 cm−1 were defined as the specific spectral region. The relative amount of nucleic acids with respect to proteins showed linear dependence on the straight-line velocity of spermatozoa. BCF showed the best correlation to the bands between 3678 cm−1 and 2749 cm−1, which largely represent lipids and proteins. These results suggest that FT-IR spectroscopy can serve as an adjunct to conventional histopathology studies.

Porous Silicon Covered with Silver Nanoparticles as Surface-Enhanced Raman Scattering (SERS) Substrate for Ultra-Low Concentration Detection

Microporous and macro-mesoporous silicon templates for surface-enhanced Raman scattering (SERS) substrates were produced by anodization of low doped p-type silicon wafers. By immersion plating in AgNO3, the templates were covered with silver metallic film consisting of different silver nanostructures. Scanning electron microscopy (SEM) micrographs of these SERS substrates showed diverse morphology with significant difference in an average size and size distribution of silver nanoparticles. Ultraviolet–visible–near-infrared (UV-Vis-NIR) reflection spectroscopy showed plasmonic absorption at 398 and 469 nm, which is in accordance with the SEM findings. The activity of the SERS substrates was tested using rhodamine 6G (R6G) dye molecules and 514.5 nm laser excitation. Contrary to the microporous silicon template, the SERS substrate prepared from macro-mesoporous silicon template showed significantly broader size distribution of irregular silver nanoparticles as well as localized surface plasmon resonance closer to excitation laser wavelength. Such silver morphology has high SERS sensitivity that enables ultralow concentration detection of R6G dye molecules up to 10−15 M. To our knowledge, this is the lowest concentration detected of R6G dye molecules on porous silicon-based SERS substrates, which might even indicate possible single molecule detection.

Location of PRODAN in lipid layer of HDL particle: a Raman study

FT Raman spectroscopy has been applied to determine the location of PRODAN within HDL and to investigate its influence on the structure of the particle. The complex spectra of HDL and HDL labeled with PRODAN were divided into three regions according to the wave numbers, and adherent spectra were compared separately. Additionally, recorded spectra of protein and lipid fractions of HDL were used as a support for the assignment of particular vibrations in intact particles. In high frequency region, the shift in vibrational frequencies of CH3 groups but almost negligible shift of CH2 groups suggests that PRODAN is situated at the water/lipid interface in the vicinity of the protein. The statement is supported by the observed influence of PRODAN on particular lipid vibrations of phospholipids head-groups. In the fingerprint region, the influence of PRODAN is observed as the slight change in β-strand secondary structure of apolipoprotein and strongly reduced vibrations of the acyl chain in lipids. That additionally confirms that PRODAN mainly interacts with the lipid domain of the particle. In the low frequency region, the lack of change in Tyr Fermi resonance doublet and only slight differences in the pattern of CS and SS stretching vibrations in labeled HDL confirms that PRODAN has no influence on structure of apolipoprotein embedded in lipid domain. The main conclusions drawn from the vibrational spectra of HDL with and without PRODAN clearly confirm that PRODAN induces negligible changes in HDL structure and hence is reliable fluorescent label for the structural analysis.

Porous silicon by galvanostatic electrochemical anodisation of epitaxial silicon, polycrystalline silicon and silicon on insulator layers

Porous silicon (PSi) samples were prepared by galvanostatic electrochemical anodization of epitaxial silicon, polycrystalline silicon and silicon on insulator layers. Structural and optical properties of prepared samples were investigated by Raman and photoluminescence (PL) spectroscopy, field emission scanning electron microscopy (FE-SEM) and energy dispersive x-ray spectroscopy (EDS). Epitaxial silicon layers of n-type and {111} orientation grown on n-type {111} oriented silicon substrates were anodized as a function of concentration of 48 % HF in ethanol solution and anodization time. Electrical resistivities of the epitaxial layers and of the silicon substrate were ∼2 and ∼0.015 Ω cm, successively. Within the epitaxial layer and on the substrate surface, micro- and nano-pores of different sizes in dependence on HF concentration and anodization time were obtained. For anodization times longer than 30 min epitaxially grown layer detached from the substrate. A high density of micrometer sized pores with regions of three-dimensional photonic crystal expressed in an intersecting oriented macropore lattice on {111} oriented crystal was observed. After detaching the epitaxial layer, the black colored substrate consisted of fine nanometer sized cobweb-like silicon structures whose morphology and density depended on HF concentration and anodization time was found. The Raman spectra of such structures show broadened and red-shifted optical phonon band O(Γ), depending on the size of silicon nanostructures. The intensity of PL of such fine porous substrate shows the sensitivity on the level of the optical phonon confinement. Polycrystalline p-type silicon film were prepared by low pressure chemical vapor deposition (LPCVD) and anodized in aqueous hydrofluoric acid (HF)/ethanol electrolyte. The FE-SEM images showed preferential anodization and macro-PSi formation along grain boundaries. Weak PL signal was detected in all samples, while Raman measurements indicated minimal or no confinement effects. The anodization of silicon on insulator layers was performed by direct and alternating currents at 50 Hz. Raman spectra of DC samples showed no significant shift of c-Si peak while PL spectra showed intensive luminescence over the entire surface. When etched with AC, a very intensive PL was observed at the circular edge of the sample that exhibited micrometer sized island-like porous structures, while the center of the sample showed moderate PL signal similar to DC samples. The formation of such island-like structures was interpreted as stress due to difference of the piezoelectric effect of silicon and quartz layers (buried SiO2). Micro-Raman spectra of islands show strong phonon confinement in the range 1.4–3.5 nm.

W1244 The Role of Adenine and Guanine in Beneficial Effects of the Gastric Pentadecapeptide BPC 157 (PL 14736) On Ileoileal Anastomosis Healing in Rats Impaired with Systemic Corticosteroid Application. Raman Spectroscopy Study

Safe in inflammatory bowel disease (PL14736 Pliva, Croatia), no toxicity reported (Gastroenterology, 2005), gastric pentadecapeptide BPC 157 (BPC 157) heals the intestinal anastomosis (Surg Today, 2007). Raman spectroscopy was used to show the beneficial effect of BPC 157 on anastomosis healing in rats impaired with systemic corticosteroid application. Assessed were Raman spectroscopy, ileoileal anastomosis dehiscence, histology, biomechanical presentation (the volume (ml) (infunded through syringe-perfusion pump system (1ml /10 sec)) and the leakage pressure (mmHg) (catheter connected with chamber and monitor, at 10 cm proximal to anastomosis), on days 1, 2, 3, 4, 5 and 6. BPC 157 10µg, 6-alpha-methylprednisolone (MP) 1 mg, given alone or together /kg i.p. (or an equivolume of saline 5ml/kg). First application was after surgery and last was 24 h before sacrifice. Raman spectroscopy. Measurements used Perkin Elmer Spectrum GX FT Raman spectrometer with 4 cm-1 resolution. Excitation was NdYaG laser at 1064 nm wavelength and used laser power was 500 mW. Spectra were recorded in the area between 300 and 2300 cm-1, with 50 scans for better S/N ratio. Prior to mounting on a universal holder, the specimen was cut to a 0.3 x 0.3 cm piece. Presenting almost constant intensity for all investigated samples, all spectra were baseline corrected and normalized to band at 1450 cm-1. Raman spectroscopy. To point out an advanced healing rate the spectral areas from 510 to 560 cm-1, 800 to 1150 cm-1 and band at 1490 cm-1 were individually observed. We focused the band at 1490 cm-1, assigned to vibrations of adenine and guanine, to be indicative for increased tissue regeneration, since it fully correlates with the evidenced healing process. BPC 157 rats showed an increase throughout the 1-6 days following surgery ; MP-rats did not show before day 6. This healing delay is fully counteracted when rats received BPC 157 simultaneously with MP. Macroscopic and histology data support these findings: adhesion formation attenuated, blood vessels filled, mild passage obstruction only temporary ; anastomosis without leakage sustains markedly higher both volume and pressure, with consistent increase to healthy values ; decreased edema, granulocyte number and necrosis, along with granulation tissue, reticulin and collagen formation substantially increased, and finally epithelization increased (BPC 157) ; (MP)causing aggravation. These data signify the role of adenine and guanine in the anastomosis healing process, BPC 157 has a beneficial effect, MP negative effect, and counteracted corticosteroid impairment by BPC 157 application.