Ultrasensitive Detection of Aß42 Seeds in Cerebrospinal Fluid with a Nanopipette-Based Real-Time Fast Amyloid Seeding and Translocation Assay.

In this work, early-stage Aß42 aggregates were detected using a real-time fast amyloid seeding and translocation (RT-FAST) assay. Specifically, Aß42 monomers were incubated in buffer solution with and without preformed Aß42 seeds in a quartz nanopipette coated with L-DOPA. Then, formed Aß42 aggregates were analyzed on flyby resistive pulse sensing at various incubation time points. Aß42 aggregates were detected only in the sample with Aß42 seeds after 180 min of incubation, giving an on/off readout of the presence of preformed seeds. Moreover, this RT-FAST assay could detect preformed seeds spiked in 4% cerebrospinal fluid/buffer solution. However, in this condition, the time to detect the first aggregates was increased. Analysis of Cy3-labeled Aß42 monomer adsorption on a quartz substrate after L-DOPA coating by confocal fluorescence spectroscopy and molecular dynamics simulation showed the huge influence of Aß42 adsorption on the aggregation process.

Investigation of α-Synuclein and Amyloid-ß(42)-E22Δ Oligomers Using SiN Nanopore Functionalized with L-Dopa.

Solid-state nanopores are an emerging technology used as a high-throughput, label-free analytical method for the characterization of protein aggregation in an aqueous solution. In this work, we used Levodopamine to coat a silicon nitride nanopore surface that was fabricated through a dielectric breakdown in order to reduce the unspecific adsorption. The coating of inner nanopore wall by investigation of the translocation of heparin. The functionalized nanopore was used to investigate the aggregation of amyloid-ß and α-synuclein, two biomarkers of degenerative diseases. In the first application, we demonstrate that the α-synuclein WT is more prone to form dimers than the variant A53T. In the second one, we show for the Aß(42)-E22Δ (Osaka mutant) that the addition of Aß(42)-WT monomers increases the polymorphism of oligomers, while the incubation with Aß(42)-WT fibrils generates larger aggregates.

Detection of Amyloid-ß Fibrils Using Track-Etched Nanopores: Effect of Geometry and Crowding.

Several neurodegenerative diseases have been linked to proteins or peptides that are prone to aggregate in different brain regions. Aggregation of amyloid-ß (Aß) peptides is recognized as the main cause of Alzheimer's disease (AD) progression, leading to the formation of toxic Aß oligomers and amyloid fibrils. The molecular mechanism of Aß aggregation is complex and still not fully understood. Nanopore technology provides a new way to obtain kinetic and morphological aspects of Aß aggregation at a single-molecule scale without labeling by detecting the electrochemical signal of the peptides when they pass through the hole. Here, we investigate the influence of nanoscale geometry (conical and bullet-like shape) of a track-etched nanopore pore and the effect of molecular crowding (polyethylene glycol-functionalized pores) on Aß fibril sensing and analysis. Various Aß fibril samples that differed by their length were produced by sonication of fibrils obtained in the presence of epigallocatechin gallate. The conical nanopore functionalized with polyethylene glycol (PEG) 5 kDa is suitable for discrimination of the fibril size from relative current blockade. The bullet-like-shaped nanopore enhances the amplitude of the current and increases the dwell time, allowing us to well discern the fibrils. Finally, the nanopore crowded with PEG 20 kDa enhances the relative current blockade and increases the dwell time; however, the discrimination is not improved compared to the "bullet-shaped" nanopore.

Polynucleotide differentiation using hybrid solid-state nanopore functionalizing with α-hemolysin.

We report results from full atomistic molecular dynamics simulations on the properties of biomimetic nanopores. This latter result was obtained through the direct insertion of an α-hemolysin protein inside a hydrophobic solid-state nanopore. Upon translocation of different DNA strands, we demonstrate here that the theoretical system presents the same discrimination properties as the experimental one obtained previously. This opens an interesting way to promote the stability of a specific protein inside a solid nanopore to develop further biomimetic applications for DNA or protein sequencing.

Influence of nanotube section on carboplatin confinement.

The confinement of anticancer carboplatin molecules (CBPT) in boron nitride nanotubes (BNNTs) with various sections was studied by means of density functional theory and molecular dynamic simulations. We show that the molecular insertion in BNNT is favored depending on the tube radius. The range of the energy adsorption varied from -1 eV to -2 eV depending on BNNT dimension. We also determined the critical diameter for the possible vectorization of the anticancer molecule. Indeed, the hydrophobicity of small BNNT radius R < 5.5 Å) is so large that CBPT encapsulation is impossible to achieve. On the contrary, a larger radius could offer an ideal situation to enhance drug delivery and allow a progressive release of the therapeutic near its target. Comparison with carbon nanotubes allowed us to draw conclusions on the best adapted nanovector for CBPT.

Voltage-activated transport of ions through single-walled carbon nanotubes.

Ionic transport through single-walled carbon nanotubes (SWCNTs) is promising for many applications but remains both experimentally challenging and highly debated. Here we report ionic current measurements through microfluidic devices containing one or several SWCNTs of diameter of 1.2 to 2 nm unexpectedly showing a linear or a voltage-activated I-V dependence. Transition from an activated to a linear behavior, and stochastic fluctuations between different current levels were notably observed. For linear devices, the high conductance confirmed with different chloride salts indicates that the nanotube/water interface exhibits both a high surface charge density and flow slippage, in agreement with previous reports. In addition, the sublinear dependence of the conductance on the salt concentration points toward a charge-regulation mechanism. Theoretical modelling and computer simulations show that the voltage-activated behavior can be accounted for by the presence of local energy barriers along or at the ends of the nanotube. Raman spectroscopy reveals strain fluctuations along the tubes induced by the polymer matrix but displays insufficient doping or variations of doping to account for the apparent surface charge density and energy barriers revealed by ion transport measurements. Finally, experimental evidence points toward environment-sensitive chemical moieties at the nanotube mouths as being responsible for the energy barriers causing the activated transport of ions through SWCNTs within this diameter range.