Immobilization of lutetium bisphthalocyanine in nanostructured biomimetic sensors using the LbL technique for phenol detection

This study describes the development of amperometric sensors based on poly(allylamine hydrochloride) (PAH) and lutetium bisphthalocyanine (LuPc(2)) films assembled using the Layer-by-Layer (LbL) technique. The films have been used as modified electrodes for catechol quantification. Electrochemical measurements have been employed to investigate the catalytic properties of the LuPc(2) immobilized in the LbL films. By chronoamperometry, the sensors present excellent sensitivity (20 nA mu M(-1)) in a wide linear range (R(2) = 0.994) up to 900 mu M and limit of detection (s/n = 3) of 37.5 x 10(-8) M for catechol. The sensors have good reproducibility and can be used at least for ten times. The work potential is +0.3 V vs. saturated calomel electrode (SCE). In voltammetry measurements, the calibration curve shows a good linearity (R(2) = 0.992) in the range of catechol up to 500 mu M with a sensitivity of 90 nA mu M(-1) and LD of 8 mu M. (C) 2011 Elsevier B.V. All rights reserved.

Calcium Carbonate Particle Growth Depending on Coupling among Adjacent Layers in Hybrid LB/LbL Films

There are practical and academic situations that justify the study of calcium carbonate crystallization and especially of systems that are associated with organic matrices and a confined medium. Despite the fact that many different matrices have been studied, the use of well-behaved, thin organic films may provide new knowledge about this system. In this work, we have studied the growth of calcium carbonate particles on well-defined organic matrices that were formed by layer-by-layer (LbL) polyelectrolyte films deposited on phospholipid Langmuir-Blodgett films (LB). We were able to change the surface electrical charge density of the LB films by changing the proportions of a negatively charged lipid, the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid (DMPA), and a zwitterionic lipid. dimyristoyl-sn-glycero-phosphatidylethanolamine (DMPE). This affects the subsequent polyelectrolyte LbL film deposition, which also changes the the nature of the bonding (electrostatic interaction or hydrogen bonding). This approach allowed for the formation of calcium carbonate particles of different final shapes, roughnesses, and sizes. The masses of deposited lipids, polyelectrolytes, and calcium cabonate were quantified by the quartz crystal microbalance technique. The structures of obtained particles were analyzed by scanning electron microscopy.

Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers. whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms. Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC). the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property. (C) 2009 Elsevier B.V. All rights reserved.

Physical Vapor Deposited Thin Films of Lignins Extracted from Sugar Cane Bagasse: Morphology, Electrical Properties, and Sensing Applications

The concern related to the environmental degradation and to the exhaustion of natural resources has induced the research on biodegradable materials obtained from renewable sources, which involves fundamental properties and general application. In this context, we have fabricated thin films of lignins, which were extracted from sugar cane bagasse via modified organosolv process using ethanol as organic solvent. The films were made using the vacuum thermal evaporation technique (PVD, physical vapor deposition) grown up to 120 nm. The main objective was to explore basic properties such as electrical and surface morphology and the sensing performance of these lignins as transducers. The PVD film growth was monitored via ultraviolet-visible (UV-vis) absorption spectroscopy and quartz crystal microbalance, revealing a linear relationship between absorbance and film thickness. The 120 nm lignin PVD film morphology presented small aggregates spread all over the film surface on the nanometer scale (atomic force microscopy, AFM) and homogeneous on the micrometer scale (optical microscopy). The PVD films were deposited onto Au interdigitated electrode (IDE) for both electrical characterization and sensing experiments. In the case of electrical characterization, current versus voltage (I vs V) dc measurements were carried out for the Au IDE coated with 120 nm lignin PVD film, leading to a conductivity of 3.6 x 10(-10) S/m. Using impedance spectroscopy, also for the Au IDE coated with the 120 nm lignin PVD film, dielectric constant of 8.0, tan delta of 3.9 x 10(-3)) and conductivity of 1.75 x 10(-9) S/m were calculated at 1 kHz. As a proof-of-principle, the application of these lignins as transducers in sensing devices was monitored by both impedance spectroscopy (capacitance vs frequency) and I versus time dc measurements toward aniline vapor (saturated atmosphere). The electrical responses showed that the sensing units are sensible to aniline vapor with the process being reversible. AFM images conducted directly onto the sensing units (Au IDE coated with 120 nm lignin PVD film) before and after the sensing experiments showed a decrease in the PVD film roughness from 5.8 to 3.2 nm after exposing to aniline.

Hyaluronidase Behavior at the Air/Liquid and Air/Lipid Interfaces and Improved Enzymatic Activity by Its Immobilization in a Biomembrane Model

Bovine testicular hyalurphidase (BT-HAase), a tetrameric enzyme responsible for randomly hyaluronic acid, catalytic hydrolysis, was successfully immobilized on Langmuir- Blodgett films prepared with the sodium salt of dihexadacylphosphoric acid, (DHP-Zn(II)) ending with dipalmitoylphosphatidylcholine, DPPC. Data of protein, adsorption at the air-liquid interface by means of pendant drop shipe analysis and interaction of the protein with Langmuir monolayers of DPPC, using a Langmuir trough, have provided information. about the conditions to be used in the protein immobilization. The dynamic surface pressure curves obtained from pendant drop experiments for the enzyme in buffer solutions indicate that, within the range of concentration investigated in this study, the enzyme exhibits the largest induction time at 5 mu g L(-1) attributed to diffusion processes. Nevertheless, it seems that, at this concentration, the most probable conformation should be the one which occupies the smallest area at pi -> 0. The surface pressure (pi) area curves obtained for BT-HAase and mixed DPPC- BT-HAase monolayers reveal the presence of the enzyme at the air-lipid interface up to 45 mN m(-1). Tests of enzymatic activity, using hyaluronic acid, HA, as the substrate, showed an increase of activity compared to the homogeneous medium. A simplified model of protein insertion into the lipid matrix is used to explain the obtained results.

Chitosan as a Bioadhesive Agent Between Porphyrins and Phospholipids in a Biomembrane Model

Porphyrins are currently used in photodynamic therapy as photosensitizers. In this paper we studied the interaction of two charged porphyrins, 5, 10, 15, 20-mesotetrakis(N-metyl-4-pyridyl) porphyrin, (TMPyP/chloride salt) cationic, and 5, 10, 15, 20-meso-tetrakis(sulfonatophenyl) porphyrin, (TPPS(4)/sodium salt) anionic, nanoassembled in phospholipid Langmuir monolayers and Langmuir-Blodgett films. Furthermore, we used chitosan to mediate the interaction between the porphyrins and the model membrane, aiming to understand the role of the polysaccharide in a molecular level. The effect of the interaction of the photosensitizers on the fluidity of the lipid monolayer was investigated by using dilatational surface elasticity. We also used photoluminescence (PL) spectroscopy to identify the porphyrins adsorbed in the phospholipid films. We observed an expansion of the monolayer promoted by the adsorption of the porphyrins into the lipid-air interface which was more pronounced in the case of TMPyP, as a consequence of a strong electrostatic interaction with the anionic monolayer. The chitosan promoted a higher adsorption of the porphyrins on the phospholipid monolayers and enabled the porphyrin to stay in its monomeric form (as confirmed by PL spectroscopy), thus demonstrating that chitosan can be pointed out as a potential photosensitizer delivery system in photodynamic therapy.

Strategies to Optimize Biosensors Based on Impedance Spectroscopy to Detect Phytic Acid Using Layer-by-Layer Films

Impedance spectroscopy has been proven a powerful tool for reaching high sensitivity in sensor arrays made with nanostructured films in the so-called electronic tongue systems, whose distinguishing ability may be enhanced with sensing units capable of molecular recognition. In this study we show that for optimized sensors and bio-sensors the dielectric relaxation processes involved in impedance measurements should also be considered, in addition to an adequate choice of sensing materials. We used sensing units made from layer-by-layer (LbL) films with alternating layers of the polyeletrolytes, poly(allylamine) hydrochloride (PAH) and poly(vinyl sulfonate) (PVS), or LbL films of PAH alternated with layers of the enzyme phytase, all adsorbed on gold interdigitate electrodes. Surprisingly, the detection of phytic acid was as effective in the PVS/PAH sensing system as with the PAH/phytase system, in spite of the specific interactions of the latter. This was attributed to the dependence of the relaxation processes on nonspecific interactions such as electrostatic cross-linking and possibly on the distinct film architecture as the phytase layers were found to grow as columns on the LbL film, in contrast to the molecularly thin PAH/PVS films. Using projection techniques, we were able to detect phytic acid at the micromolar level with either of the sensing units in a data analysis procedure that allows for further optimization.

Electrostatic Interactions Are Not Sufficient to Account for Chitosan Bioactivity

Recent studies involving chitosan interacting with phospholipid monolayers that mimic cell membranes have brought molecular-level evidence for some of the physiological actions of chitosan, as in removing a protein from the membrane. This interaction has been proven to be primarily of electrostatic origin because of the positive charge OF chitosan in low pH solutions, but indirect evidence has also appeared of the presence of hydrophobic interactions. In this study, we provide definitive proof that model membranes are not affected merely by the charges in the amine groups of chitosan. Such a proof was obtained by comparing surface pressure and surface potential isotherms of dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) monolayers incorporating either chitosan or poly(allylamine hydrochloride) (PAH). As the latter is also positively charged and With the same charged Functional group as chitosan, similar effects should be observed in case the electrical charge was the only relevant parameter. Instead, we observed a large expansion in the surface pressure isotherms upon interaction with chitosan, whereas PAH had much smaller effects. Of particular relevance for biological implications, chitosan considerably reduced the monolayer elasticity, whereas PAH had almost no effect. it is clear therefore that chitosan action depends strongly either on its functional uncharged groups and/or on its specific conformation in solution.

Ultrafast dynamics of bis (n-butylimido) perylene thin films excited by two-photon absorption

We report a pump-probe study of the two-photon induced reflectivity changes in bis (n-butylimido) perylene thin films. To enhance the two-photon excitation we deposited bis (n-butylimido) perylene films on top of gold nanoislands. The observed transient response in the reflectivity spectrum of bis (n-butylimido) perylene is due to a depletion of the molecule`s ground state and excited state absorption.

Enzyme activity of horseradish peroxidase immobilized in chitosan matrices in alternated layers

The successful immobilization of enzymes such as horseradish peroxidase (HRP) in solid films is essential for applications in sensors and for fundamental studies aimed at identifying possible biotechnological devices. In this study we show that HRP can be immobilized in alternated layers with chitosan as the template material. The activity of HRP in HRP/chitosan films was preserved for several weeks, and could be detected optically upon monitoring the reaction with pyrogallol. The morphology of the film displayed stripes that disappeared after reaction with pyrogallol. Though the activity in the HRP/chitosan film was lower than in a homogeneous solution or in an LB film investigated earlier, the response was linear for a considerable period of time, which may be advantageous for sensing hydrogen peroxide. (C) 2009 Elsevier B.V. All rights reserved.

Synthesis of azopolymers with controlled structure and photoinduced birefringence in their LB films

The molecular architecture of azopolymers may be controlled via chemical synthesis and with selection of a suitable film-forming method, which is important for improving their properties for practical uses. Here we address the main challenge of combining the photoinduced birefringence features of azopolymers with the higher thermal and mechanical stabilities of poly(methyl methacrylate) (PMMA) using Atom Transfer Radical Polymerization (ATRP) to synthesize diblock- and triblock-copolymers of an azomonomer and the monomer methyl methacrylate. Langmuir-Blodgett (LB) films made with the copolymers mixed with cadmium stearate displayed essentially the same optically induced birefringence characteristics, in terms of maximum and residual birefringence and time for writing, as the mixed LB films with the homopolymer poly[4-(N-ethyl-N-(2-methacryloxyethyl))amino-2`-chloro-4`-nitroazobenzene] (HPDR13), also synthesized via ATRP. In fact, the controlled architecture of HPDR13 chains led to Langmuir films that could be more closely packed and reach higher collapse pressures than the corresponding films obtained with HPDR13-conv synthesized via conventional radicalar polymerization. This allowed LB films to be fabricated from neat HPDR13, which was not possible with HPDR13-conv. The enhanced organization in the LB films produced with controlled azopolymer chains, however, led to a smaller free volume available for isomerization of the azochromophores, thus yielding a lower photoinduced birefringence than in the HPDR13-conv films. The combination of ATRP synthesis and LB technology is then promising to obtain optical storage in films with improved thermal and mechanical processabilities, though a further degree of control must be sought to exploit film organization while maintaining the necessary free volume in the films. (C) 2008 Elsevier Ltd. All rights reserved.

Detection of phenolic compounds using impedance spectroscopy measurements

Langmuir-Blodgett (LB) and layer-by-layer films (LbL) of a PPV (p-phenylenevinylene) derivative, an azo compound and tetrasulfonated phthalocyanines were successfully employed as transducers in an ""electronic tongue"" system for detecting trace levels of phenolic compounds in water. The choice of the materials was based on their distinct electrical natures, which enabled the array to establish a fingerprint of very similar liquids. Impedance spectroscopy measurements were taken in the frequency range from 10 Hz to 1 MHz, with the data analysed with principal component analysis (PCA). The sensing units were obtained from five-layer LB films of (poly[(2-methoxy-5-n-hexyloxy)-p-phenylenevinylene]), OC(1)OC(18)-PPV (poly(2-methoxy,5-(n-octadecyl)-p-phenylenevinylene)), DR (HEMA-co-DR13MA (poly-(hydroxyethylmethacrylate-co-[4`-[[2-(methacryloyloxy)-ethyl]ethylamino]-2-chloro-4-nitroazobenzene]))) and five-bilayer LbL films of tetrasulfonated metallic phthalocyanines deposited onto gold interdigitated electrodes. The sensors were immersed into phenol, 2-chloro-4-methoxyphenol, 2-chlorophenol and 3-chlorophenol (isomers) solutions at 1 x 10(-9) mol L(-1), with control experiments carried out in ultra pure water. Samples could be distinguished if the principal component analysis (PCA) plots were made with capacitance values taken at 10(3) Hz, which is promising for detection of trace amounts of phenolic pollutants in natural water.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

This paper describes the preparation of a biomimetic Langmuir-Blodgett film of tyrosinase incorporated in a lipidic layer and the use of lutetium bisphthalocyanine as an electron mediator for the voltammetric detection of phenol derivatives, which include one monophenol (vanillic acid), two diphenols (catechol and caffeic acid) and two triphenols (gallic acid and pyrogallol). The first redox process of the voltammetric responses is associated with the reduction of the enzymatically formed o-quinone and is favoured by the lutetium bisphthalocyanine because significant signal amplification is observed, while the second is associated with the electrochemical oxidation of the antioxidant and occurs at lower potentials in the presence of an electron mediator. The biosensor shows low detection limit (1.98 x 10(-6)-27.49 x 10(-6) M), good reproducibility, and high affinity to antioxidants (Km in the range of 62.31-144.87 mu M). The excellent functionality of the enzyme obtained using a biomimetic immobilisation method, the selectivity afforded by enzyme catalysis, the signal enhancement caused by the lutetium bisphthalocyanine mediator and the increased selectivity of the curves due to the occurrence of two redox processes make these sensors exceptionally suitable for the detection of phenolic compounds. (C) 2010 Elsevier B.V. All rights reserved.