Surface Plasmon Resonance Monitoring of Mono-Rhamnolipid Interaction with Phospholipid-Based Liposomes.

The interactions of mono-rhamnolipids (mono-RLs) with model membranes were investigated through a biomimetic approach using phospholipid-based liposomes immobilized on a gold substrate and also by the multiparametric surface plasmon resonance (MP-SPR) technique. Biotinylated liposomes were bound onto an SPR gold chip surface coated with a streptavidin layer. The resulting MP-SPR signal proved the efficient binding of the liposomes. The thickness of the liposome layer calculated by modeling the MP-SPR signal was about 80 nm, which matched the average diameter of the liposomes. The mono-RL binding to the film of the phospholipid liposomes was monitored by SPR and the morphological changes of the liposome layer were assessed by modeling the SPR signal. We demonstrated the capacity of the MP-SPR technique to characterize the different steps of the liposome architecture evolution, i.e., from a monolayer of phospholipid liposomes to a single phospholipid bilayer induced by the interaction with mono-RLs. Further washing treatment with Triton X-100 detergent left a monolayer of phospholipid on the surface. As a possible practical application, our method based on a biomimetic membrane coupled to an SPR measurement proved to be a robust and sensitive analytical tool for the detection of mono-RLs with a limit of detection of 2 µg mL-1.

Development of enteric polymer-based microspheres by spray-drying for colonic delivery of Lactobacillus rhamnosus GG.

Antibiotics are well-known disruptive elements of the intestinal microbiota and antibiotic-associated diarrhea appeared as the most common complication related with post-antibiotic dysbiosis. Lactobacillus rhamnosus GG (LGG) strain is very effective in preventing antibiotic-associated diarrhea in children and adults. However, as any probiotics, it is concerned by the loss of viability during storage and gastrointestinal transit. The aim of this study was to develop an encapsulation system suitable for the specific colonic delivery of LGG strain after oral administration. For this purpose, spray-dried Eudragit® S100 microparticles encapsulating LGG bacteria were developed by using an aqueous based spray-drying approach, avoiding the use of organic solvents. Carbohydrates were added to the formulation since they are widely used as protective agents of bacteria against the harmful effect of dehydration stress. Here, both Surface Enhanced Raman Scattering (SERS) and conventional plate count methods showed that carbohydrates increased the survival ratio of bacteria after spray-drying from 3 to more than 50%. Moreover, these protective agents ensured low residual moisture content thus providing great stability of the cells in the spray-dried powder during storage. Significant improvement of the cell viability in simulated gastro intestinal fluid (SGIF) was observed for encapsulated cells as compared with free LGG bacteria for which no viable cell was detectable after 1 h incubation in gastric fluid only. As a consequence, 4.5 × 107 CFU/g of encapsulated LGG were found viable after incubation of microparticles 1 h in Simulated Gastric Fluid followed by 6 h in Simulated Intestinal Fluid, corresponding to less than 3 log reduction of viable cells during the 7 h incubation in Simulated Gastro Intestinal Fluid. These results attested that the developed encapsulation system is suitable for its use as a bacteria carrier for specific colonic delivery.

Voltammetric glucose biosensor based on glucose oxidase encapsulation in a chitosan-kappa-carrageenan polyelectrolyte complex.

In this work, a new design of voltammetric glucose biosensor, based on the encapsulation of glucose oxidase (GOx) in a chitosan/κ­carrageenan (CHIT/CAR) polyelectrolyte complex (PEC) using a simple coacervation process is presented. A conductometric monitoring of this is performed. Spectroscopic and morphological characterization of the PEC film encapsulating GOx is carried out. Compared to biosensors based on a chitosan film, a more sensitive voltammetric detection of glucose is obtained. Using square wave voltammetry (SWV), the CHIT/CAR PEC based biosensor exhibits a wide linearity range from 5 µM to 7 mM glucose with a detection limit of 5 µM. Excellent selectivity against ascorbic acid, uric acid and urea is observed and the applicability of the biosensor for glucose detection in spiked saliva samples was demonstrated.

Elaboration of PLLA-based superparamagnetic nanoparticles: characterization, magnetic behaviour study and in vitro relaxivity evaluation.

Oleic acid-coated magnetite has been encapsulated in biocompatible magnetic nanoparticles (MNP) by a simple emulsion evaporation method. The different parameters influencing the particles size were studied. Between these parameters, the stirring speed and the polymer concentration were found to influence positively or negatively, respectively, the MNP size which varied between 320 and 1500nm. The magnetite encapsulation efficacy was about than 90% yielding a high magnetite loading of up to 30% (w/w). X-ray diffraction showed that magnetite crystalline pattern was not modified after emulsification and solvent evaporation. The X-ray photoelectron spectroscopy (XPS) results indicated the presence of less than 0.1% of iron atoms at the nanoparticles surface. Vibration simple magnetometer (VSM) showed a superparamagnetic behaviour of the MNP and a saturation magnetization increasing with the increased magnetite amount used in formulation. Moreover, T(1) and T(2) relaxivities of MNP (4.7T, 20 degrees C) were 1.7+/-0.1 and 228.3+/-13.1s(-1)mM(-1), respectively, rendering them in the same category of known negative contrast agents which shorten the T(2) relaxation time. Therefore, by using an appropriate anticancer drug in their formulation, these magnetic nanoparticles can present a promising mean for simultaneous tumor imaging, drug delivery and real time monitoring of therapeutic effect.

Evaluation of endothelial cell adhesion onto different protein/gold electrodes by EIS.

To study cell attachment to biomaterials, several proteins such as fibronectin, collagen IV, heparin, immunoglobulin G, and albumin have been deposited onto polystyrene adsorbed on a self-assembled monolayer (silane or thiol) on glass or gold, respectively. The different steps of this multilayer assembly have been characterized by electrochemical impedance spectroscopy (EIS). These data are compared to those of adhesion rate, viability percentage, and cytoskeleton labeling for a better understanding of the cell adhesion process to each protein. All the proteins are endothelial cell adhering biomolecules but not with the same features. A linear relationship has been established between adhesion rate and resistance of the endothelial cell/protein interface for all negatively charged proteins.

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy.

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.

Anticancer drug detection using a highly sensitive molecularly imprinted electrochemical sensor based on an electropolymerized microporous metal organic framework.

A simple and highly sensitive approach for the detection of the anti-neoplastic drug gemcitabine is presented, based on a one-step electropolymerized molecularly imprinted microporous-metal-organic-framework. The sensitive layer was prepared by electropolymerization of the aniline moieties of p-aminothiophenol- gold nanoparticles on the surface of gold electrodes tethered with p-aminothiophenol, in the presence of gemcitabine as a template molecule. Experimental parameters that control the performance of the sensor were investigated and optimized. Under optimal conditions a calibration curve was obtained in the linear range from 3.8 fM to 38 nM with a limit of detection of 3 fM. The obtained imprinted sensor has the advantages of easy manufacture, high sensitivity and selectivity and good reproducibility. Furthermore the feasibility of the proposed technique has been investigated on spiked serum samples and infusion solution containing gemcitabine.

Development of a conductometric phosphate biosensor based on tri-layer maltose phosphorylase composite films.

A conductometric biosensor for phosphate detection was developed using maltose phosphorylase (MP) from recombinant Escherichia coli immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin (BSA). The process parameters for the fabrication of the mono-enzymatic sensor and various experimental variables such as the enzyme loading, time of immobilization in saturated GA vapour, working buffer solution and temperature were investigated with regard to their influence on sensitivity, detection limit, dynamic range, operational and storage stability. The biosensor can work well at the temperature between 20 degrees C and 50 degrees C, and reach 90% of steady-state conductance in about 10s. The sensor has two linear ranges, one is from 1.0 microM to 20 microM phosphate with a detection limit of 1.0 microM, and the other is between 20 microM and 400 microM phosphate. When stored in citrate buffer (0.1M, pH 6.0) at 4 degrees C, the biosensor showed good stability over two months. No obvious interference from other anionic species like SO(4)(2-), Cl(-), NO(3)(-), NO(2)(-) and HCO(3)(-) was detected. The biosensor is suitable for use in real water samples.