Fabien Morote

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

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.

Oxidation of Langmuir–Blodgett films of monounsaturated lipids studied by atomic force microscopy

In this work, we studied the stability in time of Langmuir-Blodgett films of POPC and OPPC, two unsaturated phospholipids with similar chains, differing by the relative position of these chains on the glycerol backbone. These films, transferred from the air-water interface onto freshly cleaved mica, were characterised by Atomic Force Microscopy (AFM) giving information on their topography at a lateral and perpendicular resolution in the nm range. AFM images (obtained in tapping mode) of freshly transferred films are homogenous, in agreement with the fact that these two lipids are in a liquid-expanded phase under our experimental conditions. After two days, small domains are observed, higher than the surrounding phase of about 0.8 nm in both types of samples. These domains are not observed if the samples are kept under vacuum, or if LB films are made of saturated phospholipids, suggesting that they are due to the local oxidation of POPC or OPPC, the oxidation being slightly more pronounced in the last case. Their dispersion in LB films suggests that oxidation occurs at different points at the same time, likely in areas presenting a loose packing or a defect. The local increase of thickness could be due to the reversal of the oxidised chain, raising the oxidised lipid above the surrounding phase.

Optimization of Spirulina biofilm for in-situ heavy metals detection with microfluidic-acoustic sensor and AFM

Arthrospira platensis, called spirulina (Sp), is known to bind a wide range of heavy metals [1]. We propose an innovative approach, based on Spirulina as bioreceptor combined with highly sensitive Love wave platform for fast detection of heavy metals in solution. Our goal is to optimize the biofunctionalization of the sensor surface with microalgae, based on realtime responses of the acoustic sensor during Spirulina immobilization then heavy metals detection, combined with atomic force microscopy characterization to improve understanding of interaction phenomena. Both methods have proved the efficiency of a microfluidic chip to control the hydrodynamical flow, resulting in a biofunctional layer of microalgae. This work is an application of three generations of PDMS chips already manufactured in IMS of Bordeaux1. In particular, the protocol is optimized from that previously proposed for E. coli bacteria [2], by using the real-time oscillating frequency due to mass loading during layers deposition : the microalgae is fixed onto the sensor surface coated with a polyelectrolyte multilayer (PEM), and the bioreceptor immobilization response time is greatly reduced using the microfluidic set up.

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.