Discrimination between Alpha-Synuclein Protein Variants with a Single Nanometer-Scale Pore.

Alpha-synuclein is one of several key factors in the regulation of nerve activity. It is striking that single- or multiple-point mutations in the 140-amino-acid-long protein can change its structure, which leads to the protein's aggregation and fibril formation (which is associated with several neurodegenerative diseases, , Parkinson's disease). We recently demonstrated that a single nanometer-scale pore can identify proteins based on its ability to discriminate between protease-generated polypeptide fragments.

Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore.

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues.

On possible trypsin-induced biases in peptides analysis with aerolysin nanopore.

Nanopore-based single-molecule analysis technique is a promising approach in the field of proteomics. In this Technical Brief, the interaction between the biological nanopore of Aerolysin (AeL) and peptides is investigated, focusing on potential biases depending on the AeL activation protocol. Our results reveal that residual trypsin, which may be unintentionally introduced in analyte solution when using a classical AeL activation protocol, can induce a significant formation of shorter peptides by enzymatic degradation of longer ones, which may lead to unwanted effects and/or misinterpretations.

Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin.

Pore-forming toxins as tools for polymer analytics: From sizing to sequencing.

We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade.

Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications.

The nanopore electrical approach is a breakthrough in single molecular level detection of particles as small as ions, and complex as biomolecules. This technique can be used for molecule analysis and characterization as well as for the understanding of confined medium dynamics in chemical or biological reactions. Altogether, the information obtained from these kinds of experiments will allow us to address challenges in a variety of biological fields.

Dynamics of a polyelectrolyte through aerolysin channel as a function of applied voltage and concentration.

We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged.

Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore.

There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues.

Tailoring Nanofluid Thermophysical Profile through Graphene Nanoplatelets Surface Functionalization.

In this study, the effect of chemical surface functionalization through oxidation of exfoliated graphite nanoplatelets in the transport properties of their aqueous nanofluids has been analyzed. With this objective, thermal conductivity and viscoelastic properties have been determined for original and oxidized nanoplatelets. The results show that the functionalization completely changes the internal structure of the suspension, which is reflected in shifts of even orders of magnitude on viscosity, yield stress, or storage or loss moduli.

Tuning the electrical conductivity of exfoliated graphite nanosheets nanofluids by surface functionalization.

The electrical conductivity of exfoliated graphite in water nanofluids has been experimentally determined, and compared with the same property when the dispersed nanosheets have been oxidized. The effect of oxidation on this property is different if compared with the case of sintered dry nanosheets. In any case, for the sintered raw material the conduction behaves as expected in a metal, while for the nanofluid it shows values and trends typical of a weak electrolyte solution.

Probing driving forces in aerolysin and α-hemolysin biological nanopores: electrophoresis versus electroosmosis.

The transport of macromolecules through nanopores is involved in many biological functions and is today at the basis of promising technological applications. Nevertheless the interpretation of the dynamics of the macromolecule/nanopore interaction is still misunderstood and under debate. At the nanoscale, inside biomimetic channels under an external applied voltage, electrophoresis, which is the electric force acting on electrically charged molecules, and electroosmotic flow (EOF), which is the fluid transport associated with ions, contribute to the direction and magnitude of the molecular transport.

Evidence of viscoplastic behavior of exfoliated graphite nanofluids.

The rheological behavior of ethylene glycol-based nanofluids containing exfoliated graphite nanoplatelets has been carried out using a cone-plate Physica MCR rheometer. Initial experiments based on flow curves were carried out, the flow curves were based on the controlled shear stress model, these tests show that the studied nanofluids present non-Newtonian shear thinning behavior with yield stress. Furthermore, linear viscoelastic experiments were conducted in order to determine the viscoelastic behavior: using strain sweep and frequency sweep tests the storage and loss modulus were determined.

Temperature Effect on Ionic Current and ssDNA Transport through Nanopores.

We have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge.

To Model Chemical Reactivity in Heterogeneous Emulsions, Think Homogeneous Microemulsions.

Two important and unsolved problems in the food industry and also fundamental questions in colloid chemistry are how to measure molecular distributions, especially antioxidants (AOs), and how to model chemical reactivity, including AO efficiency in opaque emulsions. The key to understanding reactivity in organized surfactant media is that reaction mechanisms are consistent with a discrete structures-separate continuous regions duality. Aggregate structures in emulsions are determined by highly cooperative but weak organizing forces that allow reactants to diffuse at rates approaching their diffusion-controlled limit.

Evidence of unfolded protein translocation through a protein nanopore.

Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein-ligand complexes. This single-molecule sensor for biomedical and biotechnological applications is promising but until now direct proof of protein translocation through a narrow channel is lacking. Here, we report the translocation of a chimera molecule through the aerolysin nanopore in the presence of a denaturing agent, guanidium chloride (1.

Protein unfolding through nanopores.

In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation.

Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling.

The enzymatic degradation of long polysaccharide chains is monitored by nanopore detection. It follows a Michaelis-Menten mechanism. We measure the corresponding kinetic constants at the single molecule level.

On the formation of a third, nanostructured domain in ionic liquids.

The study of solid-fluid transitions in fluorinated ionic liquids using differential scanning calorimetry, rheology, and molecular modeling techniques is an essential step toward the understanding of their dynamics and the thermodynamics and the development of potential applications. Two fluorinated ionic liquids were studied: 1-hexyl-3-methylimidazolium perfluorobutanesulfonate, HMIm(PFBu)SO3, and tetrabutylammonium perfluorobutanesulfonate, NB4(PFBu)SO3. The experimental calorimetric and rheological data were analyzed taking into account the possible mesoscale structure of the two fluorinated ionic liquids.

Rheological and volumetric properties of TiO2-ethylene glycol nanofluids.

Homogeneous stable suspensions obtained by dispersing dry TiO2 nanoparticles in pure ethylene glycol were prepared and studied. Two types of nanocrystalline structure were analyzed, namely anatase and rutile phases, which have been characterized by scanning electron microscopy. The rheological behavior was determined for both nanofluids at nanoparticle mass concentrations up to 25%, including flow curves and frequency-dependent storage and loss moduli, using a cone-plate rotational rheometer.

Sensing proteins through nanopores: fundamental to applications.

Proteins subjected to an electric field and forced to pass through a nanopore induce blockades of ionic current that depend on the protein and nanopore characteristics and interactions between them. Recent advances in the analysis of these blockades have highlighted a variety of phenomena that can be used to study protein translocation and protein folding, to probe single-molecule catalytic reactions in order to obtain kinetic and thermodynamic information, and to detect protein-antibody complexes, proteins with DNA and RNA aptamers, and protein-pore interactions. Nanopore design is now well controlled, allowing the development of future biotechnologies and medicine applications.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to β-cyclodextrin (MBP-βCD) under native conditions.

Single molecule detection of glycosaminoglycan hyaluronic acid oligosaccharides and depolymerization enzyme activity using a protein nanopore.

Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides.

DNA unzipping and protein unfolding using nanopores.

We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system.