Polarization Modulation Infrared Reflection Absorption Spectroscopy Spectra Research Articles

Incorporation of acridine orange and methylene blue in Langmuir monolayers mimicking releasing nanostructures

The efficiency of methylene blue (MB) and acridine orange (AO) for photodynamic therapy (PDT) is increased if encapsulated in liposomes. In this paper we determine the molecular-level interactions between MB or AO and mixed monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) and cholesterol (CHOL) using surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). To increase liposome stability, the effects from adding the surfactants Span® 80 and sodium cholate were also studied. Both MB and AO induce an expansion in the mixed monolayer, but this expansion is less significant in the presence of either Span® 80 or sodium cholate. The action of AO and MB occurred via coupling with phosphate groups of DPPC or DPPG. However, the levels of chain ordering and hydration of carbonyl and phosphate in headgroups depended on the photosensitizer and on the presence of Span® 80 or sodium cholate. From the PM-IRRAS spectra, we inferred that incorporation of MB and AO increased hydration of the monolayer headgroup, except for the case of the monolayer containing sodium cholate. This variability in behaviour offers an opportunity to tune the incorporation of AO and MB into liposomes which could be exploited in the release necessary for PDT.

Mechanisms of alamethicin ion channel inhibition by amiloride in zwitterionic tethered lipid bilayers

The effect of amiloride, an acid-sensing ion channel (ASIC) blocker, on the formation of alamethicin ion channels in tethered zwitterionic phospholipid bilayers was investigated using electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). EIS measurements show a decrease in conductivity indicating that ion transported across the alamethicin-rich phospholipid membrane is inhibited by the presence of amiloride. The PM-IRRAS spectra indicate that amiloride has no effect on the secondary structure of the alamethicin peptide. The IR data suggests that amiloride does not block ion translocation through the alamethicin ion pore but prevents the insertion of alamethicin peptides into the biomembrane suppressing the formation of ion channels. The mechanism responsible for amiloride inhibition is explained in terms of the electrostatic interactions between the molecule and membrane surface. Amiloride is positively charged at physiological pH levels invoking a repulsive force on the positive pole of the alamethicin molecular dipole. These repulsive interactions prevent the insertion of the alamethicin peptides into the hydrophobic core of the lipid bilayer. The present paper offers a molecular view of the interactions that occur between amiloride, an ion channel inhibitor, with the lipids and ion channel forming peptides in model tBLMs.

Sulphate sensing in self-assembled monolayers by surface infrared and Raman spectroscopy techniques

In this paper we demonstrate the applicability of surface vibrational spectroscopy techniques in sensing sulphate anions, using self-assembled monolayers of hydrogen bonding receptors. Three uncharged, diamidocarbazole receptors were synthesised and attached to gold and silver surfaces via one or two disulphide arms. Their ability to bind anions on the metal surfaces was investigated with polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and surface enhanced Raman scattering (SERS) spectroscopy. Contrary to other techniques applied in optical sensors, such as UV–vis absorption, fluorescence or surface plasmon resonance, surface vibrational spectroscopy techniques used in this work not only display spectral changes proportional to the concentration of sulphate, but also provide an insight into the state of the receptor monolayer and its interactions with the analyte. While SERS measurements confirm the formation of anion-receptor complexes on metal surfaces, PM-IRRAS spectra were further employed to quantify the optical response of the sensor. The system can detect SO42− anions within a concentration range of 10-6 – 10-1 M (in acetonitrile). An effect of the hydrogen bonds formed at the binding site of the diamidocarbazole molecules on the symmetry of sulphate anions is observed, which ultimately proves the anion-receptor binding.

Adsorption and enzyme activity of asparaginase at lipid Langmuir and Langmuir-Blodgett films

In this present work, the surface activity of the enzyme asparaginase was investigated at the air-water interface, presenting surface activity in high ionic strengths. Asparaginase was incorporated in Langmuir monolayers of the phospholipid dipalmitoylphosphatidylcholine (DPPC), forming a mixed film, which was characterized with surface pressure-area isotherms, surface potential-area isotherms, polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The adsorption of the enzyme at the air-water interface condensed the lipid monolayer and increased the film compressibility at high surface pressures. Amide bands in the PM-IRRAS spectra were identified, with the CN and CO dipole moments lying parallel to monolayer plane, revealing the structuring of the enzyme into α-helices and β-sheets. The floating monolayers were transferred to solid supports as Langmuir-Blodgett (LB) films and characterized with fluorescence spectroscopy and atomic force microscopy. Catalytic activities of the films were measured and compared to the homogenous medium. The enzyme accommodated in the LB films preserved more than 78% of the enzyme activity after 30days, in contrast for the homogeneous medium, which preserved less than 13%. The method presented in this work not only allows for an enhanced catalytic activity, but also can help explain why certain film architectures exhibit better performance.

Controlling the molecular architecture of lactase immobilized in Langmuir-Blodgett films of phospholipids to modulate the enzyme activity

In this present work, the adsorption of the enzyme lactase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid (DMPA) was investigated and characterized with surface pressure-area isotherms, surface potential-area isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). The adsorption of the enzyme at the air-water interface expanded the lipid monolayer and increased the film compressibility at high surface pressures. Amide bands in the PM-IRRAS spectra were identified, with the CN and CO dipole moments lying parallel to the monolayer plane, revealing that the structuring of the enzyme into β-sheets was kept in the mixed monolayer. The enzyme-lipid films were transferred from the floating monolayer to solid supports as Langmuir-Blodgett (LB) films and characterized with fluorescence spectroscopy and atomic force microscopy. The catalytic activity of the films was measured and compared to the homogenous medium. The enzyme accommodated in the LB films preserved more than 80% of the enzyme activity after 20days, in contrast for the homogeneous medium, which preserved less than 60% of the enzyme activity. The method presented in this present work not only allows for an enhanced catalytic activity toward lactose, but also can help explain why certain film architectures exhibit better performance.

Rhodanese incorporated in Langmuir and Langmuir–Blodgett films of dimyristoylphosphatidic acid: Physical chemical properties and improvement of the enzyme activity

Preserving the catalytic activity of enzymes immobilized in bioelectronics devices is essential for optimal performance in biosensors. Therefore, ultrathin films in which the architecture can be controlled at the molecular level are of interest. In this work, the enzyme rhodanese was adsorbed onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid and characterized by surface pressure-area isotherms, polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The incorporation of the enzyme (5% in mol) in the lipid monolayer expanded the film, providing small surface domains, as visualized by BAM. Also, amide bands could be identified in the PM-IRRAS spectra, confirming the presence of the enzyme at the air–water interface. Structuring of the enzyme into α-helices was identified in the mixed monolayer and was preserved when the film was transferred from the liquid interface to solids supports as Langmuir–Blodgett (LB) films. The enzyme-lipid LB films were then characterized by fluorescence spectroscopy, PM-IRRAS, and atomic force microscopy. Measurements of the catalytic activity towards cyanide showed that the enzyme accommodated in the LB films preserved more than 87% of the enzyme activity in relation to the homogeneous medium. After 1 month, the enzyme in the LB film maintained 85% of the activity in contrast to the homogeneous medium, which 24% of the enzyme activity was kept. The method presented in this work not only points to an enhanced catalytic activity toward cyanide, but also may explain why certain film architectures exhibit an improved performance.

Langmuir and Langmuir–Blodgett films of lipids and penicillinase: Studies on adsorption and enzymatic activity

Bioelectronic devices, such as biosensors, can be constructed with enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is fundamental for optimal performance. In this sense, nanostructured films in which molecular architectures can be controlled are of interest. In this present work, the adsorption of the enzyme penicillinase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid was investigated and characterized with surface pressure–area isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). The incorporation of the enzyme in the lipid monolayer not only caused the film to expand, but also could be identified through amide bands in the PM-IRRAS spectra, with the CN and CO dipole moments being identified, lying parallel to monolayer plane. Structuring of the enzyme into α-helices was identified in the mixed enzyme–phospholipid monolayer and preserved when transferred to solid as a Langmuir–Blodgett (LB) film. The enzyme–lipid LB films were then characterized with PM-IRRAS, atomic force microscopy and fluorescence spectroscopy. Measurements of the catalytic activity showed that the enzyme accommodated in the LB films preserved 76% of the enzyme activity in relation to the homogeneous medium. The method presented here not only allows for enhanced catalytic activity toward penicillin, but also can be useful to explain why certain film architectures exhibit better enzyme activity.

Functionalized Hydrogen-Bonding Self-Assembled Monolayers Grafted onto SiO2 Substrates

A novel urea coupling agent possessing a vinyl-terminal group and trimethoxysilyl anchoring group was synthesized and grafted onto SiO(2)/Au substrates. This ureido coupling agent exhibits a good capacity to directly yield homogeneous SAMs with a surface smoothing. Polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) was used to monitor these SAMs. Indeed, the different functional groups (alkyl chain, urea, and vinyl) of this coupling agent were clearly observed in the PM-IRRAS spectra. Chemical modifications of the terminal function for the covalent immobilization of biomolecules were monitored by PM-IRRAS for the first time. We have demonstrated the successful reactions of the conversion of the vinyl-terminated SAMs successively into SAM-COOH and SAM-NHS without any degradation of the monolayer. The reactivity of activated esters was successfully investigated in order to immobilize the protein A.

Application of PM-IRRAS to study thin films on industrial and environmental samples

Polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS) was employed to analyze two unique samples: (1) an industrially prepared alkoxysilane-pretreated aluminum alloy (AA6111) in the absence and presence of a ~600-nm-thick lubricant coating and (2) a chemical warfare agent simulant, triethyl phosphate (TEP), on glass. For the pretreated aluminum samples, PM-IRRAS spectra were analyzed for three distinct regions; the SiO stretching vibration around 1120 cm(-1), the NH(2) bending mode at ~1600 cm(-1) and the CH stretching region around 2900 cm(-1). Our results showed that increasing the curing temperature (from 55 to 100 °C) improved the overall extent of cross-linking within the siloxane network. In addition, the spectra of lubricant (top coating) and the underlying siloxane layer for the aluminum samples with lubricant were collected for the same sample. Our results show that the nature of the siloxane film remains intact and unaltered after deposition of the lubricant top-coat. For detection of TEP on glass, the band at 1268 cm(-1), corresponding to the P═O vibration, was monitored. A droplet of TEP solution in dichloromethane was deposited on glass. After solvent evaporation had occurred, the intensity of the P═O vibration band was used to construct calibration curves to determine the experimental limit of detection, which was found to be ~200 μg for TEP on glass.

Determination of molecular groups involved in the interaction of annexin A5 with lipid membrane models at the air–water interface

Annexin A5 (AnxA5) is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. In this paper, we used polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS), Brewster angle microscopy (BAM), and ellipsometry to investigate changes both in the structure of AnxA5 and phospholipid head groups associated with membrane binding. We found that the secondary structure of AnxA5 in the AnxA5/Ca(2+)/lipid ternary complex is conserved, mainly in alpha-helices and the average orientation of the alpha-helices of the protein is slightly tilted with respect to the normal to the phospholipid monolayer. Upon interaction between AnxA5 and phospholipids, a shift of the nu(as) PO(2)(-) band is observed by PMIRRAS. This reveals that the phosphate group is the main group involved in the binding of AnxA5 to phospholipids via Ca(2+) ions, even when some carboxylate groups are accessible (PS). PMIRRAS spectra also indicate a change of carboxylate orientation in the aspartate and glutamate residues implicated in the association of the AnxA5, which could be linked to the 2D crystallization of protein under the phospholipid monolayer. Finally, we demonstrated that the interaction of AnxA5 with pure carboxylate groups of an oleic acid monolayer is possible, but the orientation of the protein under the lipid is completely different.

The influence of surface defects on methanol decomposition on Pd(111) studied by XPS and PM-IRAS

Methanol adsorption/desorption and its time- and temperature-dependent decomposition on well-annealed and defect-rich (ion-bombarded) Pd(1 1 1) were examined by X-ray photoelectron spectroscopy (XPS) and polarization-modulation infrared reflection absorption spectroscopy (PM-IRAS). Annealing CH3OH multilayers from 100 to 700 K mainly resulted in CH3OH desorption. Dehydrogenation to CO was a minor path and only trace amounts of carbon or carbonaceous species (CHx; x=0–3) were produced, i.e. C–O bond scission was very limited. By contrast, an exposure of 5 × 10−7 mbar CH3OH at 300 K produced CHx (∼0.3 ML) on both surfaces but the rate of formation was not considerably enhanced by surface defects. On well-annealed Pd(1 1 1) isolated carbon atoms were identified by XPS in the early stages of carbon deposition, with carbon diffusion leading to the growth of carbon clusters in the later stages. Since carbon(aceous) species may either originate from C–O bond scission within methanol (or CHxO) or from a consecutive dissociation of the dehydrogenation product CO, analogous experiments were also carried out with CO. PM-IRAS spectra up to 170 mbar CO, acquired using a UHV-high-pressure cell, did not show any indications of CO dissociation, excluding CO as source of carbonaceous deposits.

Orthorhombic structure of cadmium behenate monolayers on the water surface of a Langmuir trough detected by polarization modulation infrared spectroscopy

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was applied to investigate the structure of behenic acid monolayers on the surface of aqueous Cd 2+ sub-phase in a Langmuir trough at the room temperature of 293 K. The PM-IRRAS spectra were recorded at representative points throughout 0.401∼0.100 nm 2 and the surface-pressure area isotherms were recorded during compression to these points. It is found that compressed cadmium behenate (CdB) monolayers possess an orthorhombic unit cell. On the other hand, single monolayers of CdB deposited on CaF 2 substrates at 20 mN m −1 possessed a triclinic parallel packing.