Christine Grauby-Heywang

Characterization of hemicyanine Langmuir–Blodgett films by picosecond time-resolved fluorescence

Hemicyanine Langmuir-Blodgett films have been elaborated and characterized using stationary and time-resolved spectroscopic techniques. Depending on the experimental conditions, especially the pH of the water subphase, the absorption spectra of the films indicate the presence of non-fluorescent H-aggregates in the monolayer. Time-resolved fluorescence measurements revealed three mono-exponential decay times: a very short one (20-23 ps) attributed to an excited intramolecular charge transfer state and two longer ones (100-120 ps and 400-590 ps) attributed to the photoisomerization of the chromophores.

High sensitive mesoporous TiO2-coated Love wave device for heavy metal detection

This work deals with the design of a highly sensitive whole cell-based biosensor for heavy metal detection in liquid medium. The biosensor is constituted of a Love wave sensor coated with a polyelectrolyte multilayer (PEM). Escherichia coli bacteria are used as bioreceptors as their viscoelastic properties are influenced by toxic heavy metals. The acoustic sensor is constituted of a quartz substrate with interdigitated transducers and a SiO2 guiding layer. However, SiO2 shows some degradation when used in a saline medium. Mesoporous TiO2 presents good mechanical and chemical stability and offers a high active surface area. Then, the addition of a thin titania layer dip-coated onto the acoustic path of the sensor is proposed to overcome the silica degradation and to improve the mass effect sensitivity of the acoustic device. PEM and bacteria deposition, and heavy metal influence, are real time monitored through the resonance frequency variations of the acoustic device. The first polyelectrolyte layer is inserted through the titania mesoporosity, favouring rigid link of the PEM on the sensor and improving the device sensitivity. Also, the mesoporosity of surface increases the specific surface area which can be occupied and favors the formation of homogeneous PEM. It was found a frequency shift near -20±1 kHz for bacteria immobilization with titania film instead of -7±3 kHz with bare silica surface. The sensitivity is highlighted towards cadmium detection. Moreover, in this paper, particular attention is given to the immobilization of bacteria and to biosensor lifetime. Atomic Force Microscopy characterizations of the biosurface have been done for several weeks. They showed significant morphological differences depending on the bacterial life time. We noticed that the lifetime of the biosensor is longer in the case of using a mesoporous TiO2 layer.

Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria

The engineering of nanomaterials, because of their specific properties, is increasingly being developed for commercial purposes over the past decades, to enhance diagnosis, cosmetics properties as well as sensing efficiency. However, the understanding of their fate and thus their interactions at the cellular level with bio-organisms remains elusive. Here, we investigate the size- and charge-dependence of the damages induced by silica nanoparticles (SiO2-NPs) on Gram-negative Escherichia coli bacteria. We show and quantify the existence of a NPs size threshold discriminating toxic and inert SiO2-NPs with a critical particle diameter (Φc) in the range 50nm-80nm. This particular threshold is identified at both the micrometer scale via viability tests through Colony Forming Units (CFU) counting, and the nanometer scale via atomic force microscopy (AFM). At this nanometer scale, AFM emphasizes the interaction between the cell membrane and SiO2-NPs from both topographic and mechanical points of view. For SiO2-NPs with Φ>Φc no change in E. coli morphology nor its outer membrane (OM) organization is observed unless the NPs are positively charged in which case reorganization and disruption of the OM are detected. Conversely, when Φ

Study of the interaction of β-cyclodextrin with phospholipid monolayers by surface pressure measurements and fluorescence microscopy

The interaction of beta-cyclodextrin (beta-CD) with different lipids has been studied, using Langmuir monolayers kept at constant surface pressure or constant spreading surface. Results show that beta-CD, injected beneath the monolayer, is able to desorb unsaturated palmitoyloleoylphosphatidylcholine (POPC) and sphingomyelin (SM) under specific experimental conditions. In this last case, SM monolayers, labeled with the fluorescent NBD-PC probe, were also observed by fluorescence microscopy, before and after beta-CD injection. Images show that SM monolayers are more homogeneous after beta-CD injection, because of the lipid desorption. At last, it seems that lipid desorption occurs only in a restricted surface pressure range, depending on the lipid.

Influence of oxidized lipids on palmitoyl-oleoyl-phosphatidylcholine organization, contribution of Langmuir monolayers and Langmuir–Blodgett films

In this work, we studied the interaction of two oxidized lipids, PoxnoPC and PazePC, with POPC phospholipid. Mean molecular areas obtained from (π-A) isotherms of mixed PoxnoPC-POPC and PazePC-POPC monolayers revealed different behaviors of these two oxidized lipids: the presence of PoxnoPC in the monolayers induces their expansion, mean molecular areas being higher than those expected in the case of ideal mixtures. PazePC-POPC behave on the whole ideally. This difference can be explained by a different conformation of oxidized lipids. Moreover the carboxylic function of PazePC is protonated under our experimental conditions, as shown by (π-A) isotherms of PazePC at different pH values. Both oxidized lipids induce also an increase of the monolayer elasticity, PoxnoPC being slightly more efficient than PazePC. These monolayers were transferred from the air-water interface onto mica supports for a study by AFM. AFM images are on the whole homogenous, suggesting the presence of only one lipid phase in both cases. However, in the case of PazePC-POPC monolayers, AFM images show also the presence of areas thicker of 7nm to 10nm than the surrounding lipid phase, probably due to the local formation of multilayer systems induced by compression.

Complex behavior of hemicyanine in Langmuir-Blodgett films revealed by surface pressure measurements and fluorescence microscopy

We studied in this work the organization of a hemicyanine dye in Langmuir monolayers and Langmuir-Blodgett (LB) films. Surface pressure measurements revealed the complex behaviour of this molecule at the air-water interface, since three different π-A compression isotherms were obtained under similar experimental conditions. The probability to get one type of isotherm as compared to the others depends on the amount of salts added to adjust the subphase pH, these salts acting as “structure breakers”. Absorption spectra of hemicyanine LB films reveals under specific experimental conditions the presence of H-aggregates into the films, molecules in aggregates being characterized by a particular orientation as compared to surrounding monomers.Their fluorescence images are also different according to corresponding π-A isotherms and pH subphase, showing domains with specific patterns and organization.

Raman study of the puroindoline-a/lysopamitoylphosphatidylcholine interaction in free standing black films.

The conformation of puroindoline-a (PIN-a), a protein extracted from wheat endosperm, in free-standing black films has been studied using confocal Raman spectroscopy. This protein is characterized by the presence in its sequence of a unique tryptophan (Trp)-rich domain and of five disulfide bridges stabilizing its three-dimensional structure. PIN-a is able to form free-standing films, which are very stable in time, because of its remarkable surface-active properties. These films become black in a few hours and are characterized by the presence of numerous aggregates. Their Raman spectra show major modifications of the PIN-a structure as compared to the solid form, such as the formation of beta-sheets or unordered structures, the modification of the environment of the Trp domain and, the conformation of disulfide bridges. These modifications are in agreement with an unfolding of the protein at the interfaces of the film and suggest that the Trp domain is involved in the aggregation. We have also studied the influence of increasing amounts of lysopalmitoylphosphatidylcholine (LPC) into the films. The direct observation of these mixed films shows that LPC inhibits the formation of PIN-a aggregates and that the conformation of PIN-a is strongly correlated to the LPC/PIN-a molar ratio. Raman spectroscopy also shows that PIN-a disturbs the highly organized arrangement of LPC molecules in the film.

Artificial Iono‐ and Photosensitive Membranes Based on an Amphiphilic Aza‐Crown‐Substituted Hemicyanine

Artificial iono- and photosensitive membranes based on an amphiphilic aza-crown-substituted hemicyanine are assembled on liquid and solid supports and their aggregation behaviour, which is influenced by the binding of metal cations and surface density, is studied. The photoinduced charge-transfer properties of an analogous non-amphiphilic hemicyanine in solution are also demonstrated. An asymmetric sandwich dimer model is proposed and existence of such dimers in solution is evidenced by transient absorption and fluorescence anisotropy experiments. Changes in absorption and emission spectra, as well as compression isotherms of the amphiphile observed in the presence of cations, are discussed in terms of 2D molecular reorganisation. Surface-pressure-controlled reversible excimer formation at the air-water interphase and excimer-type emission of Langmuir-Blodgett films in the presence of cations are demonstrated and are discussed on the basis of fibre-optic fluorimetry and fluorescence microscopy results.

Photosensitized Degradation of Model Lipid Membranes based on 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC)

In this work, we study the interaction of a well-known photosensitizer, MePha, with models of biological membrane (Langmuir monolayers and Langmuir–Schaeffer planar bilayers) based on one of the most important natural lipid, POPC, for the subsequent investigation of photodestruction processes in a context of photodynamic therapy treatment. Changes of macroscopic properties and morphology of POPC/MePha model membranes upon irradiation by visible light are recorded by means of contact angle measurements and atomic force microscopy, demonstrating clearly the possibility to use these methods for the study of photodestruction of artificial lipid membranes on solid substrates, but also for a comparative study of the efficiency of novel photosensitizers.

Detrimental impact of silica nanoparticles on the nanomechanical properties of Escherichia coli , studied by AFM

Despite great innovative and technological promises, nanoparticles (NPs) can ultimately exert an antibacterial activity by affecting the cell envelope integrity. This envelope, by conferring the cell its rigidity and protection, is intimately related to the mechanical behavior of the bacterial surface. Depending on their size, surface chemistry, shape, NPs can induce damages to the cell morphology and structure among others, and are therefore expected to alter the overall mechanical properties of bacteria. Although Atomic Force Microscopy (AFM) stands as a powerful tool to study biological systems, with high resolution and in near physiological environment, it has rarely been applied to investigate at the same time both morphological and mechanical degradations of bacteria upon NPs treatment. Consequently, this study aims at quantifying the impact of the silica NPs (SiO2-NPs) on the mechanical properties of E. coli cells after their exposure, and relating it to their toxic activity under a critical diameter. Cell elasticity was calculated by fitting the force curves with the Hertz model, and was correlated with the morphological study. SiO2-NPs of 100 nm diameter did not trigger any significant change in the Young modulus of E. coli, in agreement with the bacterial intact morphology and membrane structure. On the opposite, the 4 nm diameter SiO2-NPs did induce a significant decrease in E. coli Young modulus, mainly associated with the disorganization of lipopolysaccharides in the outer membrane and the permeation of the underlying peptidoglycan layer. The subsequent toxic behavior of these NPs is finally confirmed by the presence of membrane residues, due to cell lysis, exhibiting typical adhesion features.