Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes.

Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD (7-nitrobenz-2-oxa-1,3-diazole)-labeled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original fluorescence loss in electrobleaching (FLIE) assay. As expected, the reorganization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with fluorescence loss in photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar to that obtained by other methods reported in the literature. This versatile and selective bleaching procedure appears reliable to explore important biological and pharmacological issues.

Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.

Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.

Electrocatalytic (Bio)Nanostructures Based on Polymer-Grafted Platinum Nanoparticles for Analytical Purpose.

Functionalized platinum nanoparticles (PtNPs) possess electrocatalytic properties toward H2O2 oxidation, which are of great interest for the construction of electrochemical oxidoreductase-based sensors. In this context, we have shown that polymer-grafted PtNPs could efficiently be used as building bricks for electroactive structures. In the present work, we prepared different 2D-nanostructures based on these elementary bricks, followed by the subsequent grafting of enzymes. The aim was to provide well-defined architectures to establish a correlation between their electrocatalytic properties and the arrangement of building bricks. Two different nanostructures have been elaborated via the smart combination of surface initiated-atom transfer radical polymerization (SI-ATRP), functionalized PtNPs (Br-PtNPs) and Langmuir-Blodgett (LB) technique. The first nanostructure (A) has been elaborated from LB films of poly(methacrylic acid)-grafted PtNPs (PMAA-PtNPs). The second nanostructure (B) consisted in the elaboration of polymer brushes (PMAA brushes) from Br-PtNPs LB films. In both systems, grafting of the glucose oxidase (GOx) has been performed directly to nanostructures, via peptide bonding. Structural features of nanostructures have been carefully characterized (compression isotherms, neutron reflectivity, and profilometry) and correlated to their electrocatalytic properties toward H2O2 oxidation or glucose sensing.

Rational design of a redox-labeled chiral target for an enantioselective aptamer-based electrochemical binding assay.

A series of redox-labeled L-tyrosinamide (L-Tym) derivatives was prepared and the nature of the functional group and the chain length of the spacer were systematically varied in a step-by-step affinity optimization process of the tracer for the L-Tym aptamer. The choice of the labeling position on L-Tym proved to be crucial for the molecular recognition event, which could be monitored by cyclic voltammetry and is based on the different diffusion rates of free and bound targets in solution. From this screening approach an efficient electroactive tracer emerged. Comparable dissociation constants Kd were obtained for the unlabeled and labeled targets in direct or competitive binding assays. The enantiomeric tracer was prepared and its enantioselective recognition by the corresponding anti-D-Tym aptamer was demonstrated. The access to both enantiomeric tracer molecules opens the door for the development of one-pot determination of the enantiomeric excess when using different labels with well-separated redox potentials for each enantiomer.

Kinetic rotating droplet electrochemistry: a simple and versatile method for reaction progress kinetic analysis in microliter volumes.

Here, we demonstrate a new generic, affordable, simple, versatile, sensitive, and easy-to-implement electrochemical kinetic method for monitoring, in real time, the progress of a chemical or biological reaction in a microdrop of a few tens of microliters, with a kinetic time resolution of ca. 1 s. The methodology is based on a fast injection and mixing of a reactant solution (1-10 µL) in a reaction droplet (15-50 µL) rapidly rotated over the surface of a nonmoving working electrode and on the recording of the ensuing transient faradaic current associated with the transformation of one of the components. Rapid rotation of the droplet was ensured mechanically by a rotating rod brought in contact atop the droplet. This simple setup makes it possible to mix reactants efficiently and rotate the droplet at a high spin rate, hence generating a well-defined hydrodynamic steady-state convection layer at the underlying stationary electrode. The features afforded by this new kinetic method were investigated for three different reaction schemes: (i) the chemical oxidative deprotection of a boronic ester by H2O2, (ii) a biomolecular binding recognition between a small target and an aptamer, and (iii) the inhibition of the redox-mediated catalytic cycle of horseradish peroxidase (HRP) by its substrate H2O2. For the small target/aptamer binding reaction, the kinetic and thermodynamic parameters were recovered from rational analysis of the kinetic plots, whereas for the HRP catalytic/inhibition reaction, the experimental amperometric kinetic plots were reproduced from numerical simulations. From the best fits of simulations to the experimental data, the kinetics rate constants primarily associated with the inactivation/reactivation pathways of the enzyme were retrieved. The ability to perform kinetics in microliter-size samples makes this methodology particularly attractive for reactions involving low-abundance or expensive reagents.

Simple and highly enantioselective electrochemical aptamer-based binding assay for trace detection of chiral compounds.

A new electrochemical methodology is reported for monitoring in homogeneous solution the enantiospecific binding of a small chiral analyte to an aptamer. The principle relies on the difference of diffusion rates between the targeted molecule and the aptamer/target complex, and thus on the ability to more easily electrochemically detect the former over the latter in a homogeneous solution. This electrochemical detection strategy is significant because, in contrast to the common laborious and time-consuming heterogeneous binding approaches, it is based on a simple and fast homogeneous binding assay which does not call for an aptamer conformational change upon ligand binding. The methodology is here exemplified with the specific chiral recognition of trace amounts of l- or d-tyrosinamide by a 49-mer d- or l-deoxyribooligonucleotide receptor. Detection as low as 0.1% of the minor enantiomer in a nonracemic mixture can be achieved in a very short analysis time (<1 min). The assay finally combines numerous attractive features including simplicity, rapidity, low cost, flexibility, low volume samples (few microliters), and homogeneous format.

Investigations of a prion infectivity assay to evaluate methods of decontamination.

Prions are unique infectious agents which have been shown to be transmitted iatrogenically through contaminated surfaces. Surface contamination is a concern on reusable medical devices and various industrial surfaces, but there is currently no standard, accepted model to evaluate surface prion decontamination. In this report, a set of both in vitro and in vivo methods were investigated based on the contamination of surface through artificial exposure to infected brain. An in vitro surface contamination protocol was developed with subsequent biochemical detection of the prion protein (PrPres). In parallel, the in vivo investigations included the contamination of different types of surface materials (stainless steel or plastic wires) with different prion strains (scrapie strain adapted to hamsters 263K or bovine spongiform encephalopathy strain adapted to mouse 6PB1). The in vivo models with various prion strains and brain homogenate dilutions reproducibly transmitted the disease and a relationship was established between the infectivity titre, the transmission rate and the incubation period. Moreover, the in vivo models were studied for their ability to demonstrate the efficacy of heat and chemical-based decontamination methods, with similar results. The in vivo scrapie method described is proposed as a standard to evaluate existing and developing prion decontamination technologies.