Enzymes immobilized in Langmuir-Blodgett films: Why determining the surface properties in Langmuir monolayer is important?

ABSTRACT In this review we discuss about the immobilization of enzymes in Langmuir-Blodgett films in order to determine the catalytic properties of these biomacromolecules when adsorbed on solid supports. Usually, the conformation of enzymes depends on the environmental conditions imposed to them, including the chemical composition of the matrix, and the morphology and thickness of the film. In this review, we show an outline of manuscripts that report the immobilization of enzymes as LB films since the 1980’s, and also some examples of how the surface properties of the floating monolayer prepared previously to the transfer to the solid support are important to determine the efficiency of the resulting device

N-Terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions

The N-terminus of the human dihydroorotate dehydrogenase (HsDHODH) has been described as important for the enzyme attachment in the inner mitochondrial membrane and possibly to regulate enzymatic activity. In this study, we synthesized the peptide acetyl-GDERFYAEHLMPTLQGLLDPESAHRL AVRFTSLGamide, comprising the residues 33-66 of HsDHODH N-terminal conserved microdomain. Langmuir monolayers and circular dichroism (CD) were employed to investigate the interactions between the peptide and membrane model, as micelles and monolayers of the lipids phosphatidylcholine (PC), 3-phosphatidylethanolamine (PE) and cardiolipin (CL). These lipids represent the major constituents of inner mitochondrial membranes. According to CD data, the peptide adopted a random structure in water, whereas it acquired α-helical structures in the presence of micelles. The π–A isotherms and polarization- modulated infrared reflection-absorption spectroscopy on monolayers showed that the peptide interacted with all lipids, but in different ways. In DPPC monolayers, the peptide penetrated into the hydrophobic region. The strongest initial interaction occurred with DPPE, but the peptide was expelled from this monolayer at high surface pressures. In CL, the peptide could induce a partial dissolution of the monolayer, leading to shorter areas at the monolayer collapse. These results corroborate the literature, where the HsDHODH microdomain is anchored into the inner mitochondrial membrane. Moreover, the existence of distinct conformations and interactions with the different membrane lipids indicates that the access to the enzyme active site may be controlled not only by conformational changes occurring at the microdomain of the protein, but also by some lipid-protein synergetic mechanism, where the HsDHODH peptide would be able to recognize lipid domains in the membrane. - See more at: http://www.eurekaselect.com/122062/article#sthash.1ZZbc7E0.dpu

Properties of lipophilic nucleoside monolayers at the air-water interface

The capability of self-assembly and molecular recognition of biomolecules is essential for many nanotechnological applications, as in the use of alkyl-modified nucleosides and oligonucleotides to increase the cellular uptake of DNA and RNA. in this study, we show that a lipophilic nucleoside, which is an isomer mixture of 2'-palmitoyluridin und 3'-palmitoyluridin, forms Langmuir monolayers and Langmuir-Blodgett films as a typical amphiphile, though with a smaller elasticity. the nucleoside may be incorporated into dipalmitoyl phosphatidyl choline (DPPC) monolayers that serve as a simplified cell membrane model. the molecular-level interactions between the nucleoside and DPPC led to a remarkable condensation of the mixed monolayer, which affected both surface pressure and surface potential isotherms. the morphology of the mixed monolayers was dominated by the small domains of the nucleoside. the mixed monolayers could be deposited onto solid substrates as a one-layer Langmuir Blodgett film that displayed UV-vis absorption spectra typical of aggregated nucleosides owing to the interaction between the nucleoside and DPPC. the formation of solid films with DNA building blocks in the polar heads may open the way for devices and sensors be produced to exploit their molecular recognition properties. (C) 2010 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Federal Ministry of Education and Research (BMBF)Universidade Federal de São Paulo, Dept Ciencias Exatas & Terra, Diadema, SP, BrazilUniv São Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilHumboldt Univ, Inst Chem, D-12489 Berlin, GermanyUniv Leipzig, Inst Med Phys & Biophys, D-01407 Leipzig, GermanyUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, Diadema, SP, BrazilFederal Ministry of Education and Research (BMBF): 0312027 CFederal Ministry of Education and Research (BMBF): D 0312018Web of Scienc

Information Visualization to Enhance Sensitivity and Selectivity in Biosensing

An overview is provided of the various methods for analyzing biosensing data, with emphasis on information visualization approaches such as multidimensional projection techniques. Emphasis is placed on the importance of data analysis methods, with a description of traditional techniques, including the advantages and limitations of linear and non-linear methods to generate layouts that emphasize similarity/dissimilarity relationships among data instances. Particularly important are recent methods that allow processing high-dimensional data, thus taking full advantage of the capabilities of modern equipment. In this area, now referred to as e-science, the choice of appropriate data analysis methods is crucial to enhance the sensitivity and selectivity of sensors and biosensors. Two types of systems deserving attention in this context are electronic noses and electronic tongues, which are made of sensor arrays whose electrical or electrochemical responses are combined to provide "finger print" information for aromas and tastes. Examples will also be given of unprecedented detection of tropical diseases, made possible with the use of multidimensional projection techniques. Furthermore, ways of using these techniques along with other information visualization methods to optimize biosensors will be discussed.FAPESPCNPqCAPESnBIONE

Interaction of polysaccharide-protein complex from Agaricus blazei with Langmuir and Langmuir-Blodgett films of phospholipids

The use of natural substances in health applications may be hampered by the difficulties in establishing the mechanisms of action, especially at molecular-level. the protein-polysaccharide complex extracted from the mushroom Agaricus blazei Murill, referred to as CAb, has been considered for treating various diseases with probable interaction with cell membranes. in this study, we investigate the interaction between CAb and a cell membrane model represented by a Langmuir monolayer of dimyristoyl phosphatidic acid (DMPA). CAb affects the structural properties of DMPA monolayers causing expansion and increasing compressibility. in addition, interaction with DMPA polar heads led to neutralization of the electrical double layer, yielding a zero surface potential at large areas per molecule. CAb remained at the interface even at high surface pressures, which allowed transfer of Langmuir-Blodgett (LB) films onto solid supports with the CAb-DMPA mixture. the mass transferred, according to quartz crystal microbalance (QCM) measurements, increased linearly with the number of deposited layers. With UV-vis absorption, fluorescence and FTIR spectroscopies, we confirmed that the LB films contain polysaccharides, proteins and DMPA. Therefore, the CAb biological action must be attributed not only to polysaccharides but also to proteins in the complex. (C) 2008 Elsevier Inc. All rights reserved.FAPESR IMMPFINEDConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Rede Biomat (Brazil)Universidade Federal de São Paulo, Diadema, SP, BrazilUniv São Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilUniv Fed Ceara, Dept Quim, Fortaleza, Ceara, BrazilUniversidade Federal de São Paulo, Diadema, SP, BrazilWeb of Scienc

Optimized architecture for Tyrosinase-containing Langmuir-Blodgett films to detect pyrogallol

The control of molecular architectures has been a key factor for the use of Langmuir-Blodgett (LB) films in biosensors, especially because biomolecules can be immobilized with preserved activity. In this paper we investigated the incorporation of tyrosinase (Tyr) in mixed Langmuir films of arachidic acid (AA) and a lutetium bisphthalocyanine (LuPc(2)), which is confirmed by a large expansion in the surface pressure isotherm. These mixed films of AA-LuPc(2) + Tyr could be transferred onto ITO and Pt electrodes as indicated by FTIR and electrochemical measurements, and there was no need for crosslinking of the enzyme molecules to preserve their activity. Significantly, the activity of the immobilised Tyr was considerably higher than in previous work in the literature, which allowed Tyr-containing LB films to be used as highly sensitive voltammetric sensors to detect pyrogallol. Linear responses have been found up to 400 mu M, with a detection limit of 4.87 x 10(-2) mu M (n = 4) and a sensitivity of 1.54 mu A mu M(-1) cm(-2). In addition, the Hill coefficient (h = 1.27) indicates cooperation with LuPc(2) that also acts as a catalyst. The enhanced performance of the LB-based biosensor resulted therefore from a preserved activity of Tyr combined with the catalytic activity of LuPc(2), in a strategy that can be extended to other enzymes and analytes upon varying the LB film architecture

Molecular-Level Modifications Induced by Photo-Oxidation of Lipid Monolayers Interacting with Erythrosin

Incorporation into cell membranes is key for the action of photosensitizers in photomedicine treatments, with hydroperoxidation as the prominent pathway of lipid oxidation. In this paper, we use Langmuir monolayers of 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC) as cell membrane models to investigate adsorption of the photosensitizer erythrosin and its effect on photoinduced lipid oxidation. From surface pressure isotherms and polarization-modulated infrared reflection–absorption spectroscopy (PM-IRRAS) data, erythrosin was found to adsorb mainly via electrostatic interaction with the choline in the head groups of both DOPC and DPPC. It caused larger monolayer expansion in DOPC, with possible penetration into the hydrophobic unsaturated chains, while penetration into the DPPC saturated chains was insignificant. Easier penetration is due to the less packed DOPC monolayer, in comparison to the more compact DPPC according to the monolayer compressibility data. Most importantly, light irradiation at 530 nm made the erythrosin-containing DOPC monolayer become less unstable, with a relative surface area increase of ca. 19%, in agreement with previous findings for bioadhesive giant vesicles. The relative area increase is consistent with hydroperoxidation, supporting the erythrosin penetration into the lipid chains, which favors singlet oxygen generation close to double bonds, an important requirement for photodynamic efficiency