Aggregation and Oligomerization Characterization of ß-Lactoglobulin Protein Using a Solid-State Nanopore Sensor.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (ßLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing ßLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the ßLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, ßLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

Chemical Imaging of RNA-Tau Amyloid Fibrils at the Nanoscale Using Tip-Enhanced Raman Spectroscopy.

In the presence of cofactors, tau protein can form amyloid deposits in the brain which are implicated in many neurodegenerative disorders. Heparin, lipids, and RNA are used to recreate tau aggregates in vitro from recombinant protein. However, the mechanism of interaction of these cofactors and the interactions between cofactors and tau are poorly understood. Herein, we use tip-enhanced Raman spectroscopy (TERS) to visualize the spatial distribution of adenine, protein secondary structure, and amino acids (arginine, lysine and histidine) in single polyadenosine (polyA)-induced tau fibrils with nanoscale spatial resolution (<10-20 nm). Based on reference unenhanced and surface-enhanced Raman spectra, we show that the polyA anionic cofactor is incorporated in the fibril structure and seems to be superficial to the ß-sheet core, but nonetheless enveloped within the random-coiled fuzzy coat. TERS images also prove the colocalization of positively charged arginine, lysine, and histidine amino acids and negatively charged polyA, which constitutes an important step forward to better comprehend the action of RNA cofactors in the mechanism of formation of toxic tau fibrils. TERS appears as a powerful technique for the identification of cofactors in individual tau fibrils and their mode of interaction.

Optically Active Polyoxometalate-Based Silica Nanohelices: Induced Chirality from Inorganic Nanohelices to Achiral POM Clusters.

In order to investigate the principle of chiral induction from nanometric silica helices to polyoxometalate (POM) clusters, a series of optically active silica POM-based nanohelices (NANOPOMs) have been prepared by electrostatic grafting and direct adsorption of α-Keggin polyoxometalate [α-PW12 O40 ]3- to well-defined left- and right-handed silica nanohelices. UV/Vis, Raman, DRIFT, TEM, HR-TEM, EDS and circular dichroism (CD) spectroscopy were used to characterize these NANOPOMs, and confirm the presence of POM clusters as well as their interactions with the helical support. The optical activity of the left-handed and right-handed NANOPOMs has been proven by CD spectroscopy. Their CD spectra are mirror images of one another, showing cotton effects at around 214 and 276 nm, this last contribution corresponding to the oxygen-to-tungsten charge-transfer bands of Keggin polyoxoanions. The CD signal of POM clusters is strongly enhanced for NANOPOMs built by adsorption of POM onto silica nanohelices, indicating a better induced optical activity to POM clusters. These nanohelices are stable, recoverable and active catalysts in the oxidation of sulfides. To the best of our knowledge, the present research represents the first examples of optically active POM-containing silica nanohelices in which achiral POM clusters have been grafted onto silica nanohelices, and display chiroptical effects.

PIP2 Phospholipid-Induced Aggregation of Tau Filaments Probed by Tip-Enhanced Raman Spectroscopy.

The morphology and secondary structure of peptide fibers formed by aggregation of tubulin-associated unit (Tau) fragments (K18), in the presence of the inner cytoplasmic membrane phosphatidylinositol component (PIP2 ) or heparin sodium (HS) as cofactors, are determined with nanoscale (<10 nm) spatial resolution. By means of tip-enhanced Raman spectroscopy (TERS), the inclusion of PIP2 lipids in fibers is determined based on the observation of specific C=O ester vibration modes. Moreover, analysis of amide I and amide III bands suggests that the parallel ß-sheet secondary structure content is lower and the random coil content is higher for fibers grown from the PIP2 cofactor instead of HS. These observations highlight the occurrence of some local structural differences between these fibers. This study constitutes the first nanoscale structural characterization of Tau/phospholipid aggregates, which are implicated in deleterious mechanisms on neural membranes in Alzheimer's disease.

Direct Observation of Siloxane Chirality on Twisted and Helical Nanometric Amorphous Silica.

Synthesis of chiral inorganic or hybrid nanomaterials through sol-gel transcription of chiral organic templates has attracted a great deal of interest for more than a decade. However, the chiral nature of these inorganic matrices has never been directly observed. For the first time, we report a direct evaluation of chirality on noncrystalline silica chiral nanoribbons by vibrational circular dichroism (VCD) measurements. Strong Cotton effect around 1150-1000 cm-1 from Si-O-Si asymmetric stretching vibration was observed. Surprisingly, calcination of these hybrid nanoribbons doubled the intensity of Cotton effects. On the basis of transmission electron microscopy observations, IR, VCD, NMR, and Raman spectroscopies, we demonstrate that the silica chirality originates from twisted siloxane network composed of chiral arrangement of the Si-O-Si bonds. Our findings clearly prove the presence of chiral organization of amorphous silica network, making them very promising chiral platforms for chiral recognition, optical applications, or asymmetric catalysis.

Tip-Enhanced Raman Spectroscopy to Distinguish Toxic Oligomers from Aß1-42 Fibrils at the Nanometer Scale.

For the first time, natural Aß1-42 fibrils (WT) implicated in Alzheimer's disease, as well as two synthetic mutants forming less toxic amyloid fibrils (L34T) and highly toxic oligomers (oG37C), are chemically characterized at the scale of a single structure using tip-enhanced Raman spectroscopy (TERS). While the proportion of TERS features associated with amino acid residues is similar for the three peptides, a careful examination of amide I and amide III bands allows us to clearly distinguish WT and L34T fibers organized in parallel ß-sheets from the small and more toxic oG37C oligomers organized in anti-parallel ß-sheets.

Nanoscale Chemical Imaging of Amyloid Fibrils in Water Using Total-Internal-Reflection Tip-Enhanced Raman Spectroscopy.

Total-internal-reflection tip-enhanced Raman spectroscopy (TIR-TERS) imaging of amyloid-ß (Aß1-42-L34T) fibrils is performed with nanoscale spatial resolution in water, using TERS tips fabricated by bipolar electrodeposition. Ideal experimental parameters are corroborated by both theoretical simulations and TIR-TERS measurements. TIR-TERS imaging reveals the predominant parallel ß-sheet secondary structure of Aß1-42-L34T fibrils as well as the nanoscale spatial distribution of tyrosine, histidine, and phenylalanine aromatic amino acids. Their proportion in TERS spectra can be qualitatively explained by the combined effect of their localization in the Aß1-42-L34T fibril structure and their molecular orientation with respect to the excitation laser light polarization. Conclusions drawn from the TERS experiments in water corroborate and significantly enrich our previous study in ambient air, thus confirming that hydration has only a marginal impact on the structure of such amyloid fibrils. This first TIR-TERS study in liquid opens fascinating perspectives for future applications in biology.

Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores.

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Sequential chiral induction between organic and inorganic supramolecular helical assemblies for the in situ formation of chiral carbon dots.

Self-organised helical bilayers of dicationic gemini surfactants confined in helical silica nanospace were transformed in situ to carbon dots (CDots) via pyrolysis. These water-dispersible CDots exhibit electronic absorption spanning the UV and visible range and possess symmetrical circular dichroism (CD) signals, the sign of which depends on the handedness of the helices.

Toward a better understanding of ferric-oxalate complex photolysis: The role of the aqueous/air interface of droplet.

In this work, the photo reactivity of ferric oxalate (Fe(III)-Ox) complex in atmospheric particles was investigated. Raman spectroscopy was used to explore the mechanism and kinetics of Fe(III)-Ox photolysis occurring at the aqueous/gas interface, inside the droplet and in bulk solution. Ferrous carbonate (FeCO3) was detected indicating that carbonate ion (CO32-) formed inside the droplets would compete with oxalate ligands for iron complexation. A higher concentration of photoproduct Fe(II)-Ox was observed at the surface and inside of the droplets than in bulk solution. In particular, Fe(III)-Ox on the droplet surface was quickly reduced with light and Fe(II)-Ox concentration gradually decreased with irradiation time. The evolution of Fe(II)-Ox concentration was similar inside the droplet and in bulk solution with a trend of first increasing and then gradually decreasing during irradiation time. Although FeCO3 would hinder Fenton intermediate reaction, the photolysis rate of Fe(III)-Ox in droplets was almost two orders of magnitude times faster than that observed during bulk experiment. In general, the photolysis mechanism and kinetics of Fe(III)-Ox in aqueous/air interface, inside of droplet and bulk solution were distinct, and the production of oxide species from the atmospheric Fe(III)-Ox droplets was underestimated.

Total Internal Reflection Tip-Enhanced Raman Spectroscopy of Tau Fibrils.

Total internal reflection tip-enhanced Raman spectroscopy (TIR-TERS) has recently emerged as a promising technique for noninvasive nanoscale chemical characterization of biomolecules. We demonstrate that the TERS enhancement achieved in this experimental configuration is nearly 30 times higher than that in linear polarization and 8 times higher than that in radial polarization using traditional bottom-illumination geometry. TIR-TERS is applied to the study of Tau amyloid fibrils formed with the human full-length Tau protein mixed with heparin. This technique reveals the possibility to perform TERS imaging with 1-4 nm axial and 5-10 nm lateral spatial optical resolution. In these Tau/heparin fibrils, spectral signatures assigned to aromatic amino acid residues (phenylalanine, histidine, and tyrosine) and nonaromatic ones (e.g., cysteine, lysine, arginine, asparagine, and glutamine) are distinctly observed. Amide I and amide III bands can also be detected. In a fibril portion, it is shown that antiparallel ß-sheets and fibril core ß-sheets are abundant and are often localized in amino acid-rich regions where parallel ß-sheets and random coils are present in lower proportions. This first TIR-TERS study on a nonresonant biological sample paves the way for future nanoscale chemical and structural characterization of biomolecules using this performant and original technique.

Determining nanocapillary geometry from electrochemical impedance spectroscopy using a variable topology network circuit model.

Solid-state nanopores and nanocapillaries find increasing use in a variety of applications including DNA sequencing, synthetic nanopores, next-generation membranes for water purification, and other nanofluidic structures. This paper develops the use of electrochemical impedance spectroscopy to determine the geometry of nanocapillaries. A network equivalent circuit element is derived to include the effects of the capacitive double layer inside the nanocapillaries as well as the influence of varying nanocapillary radius. This variable topology function is similar to the finite Warburg impedance in certain limits. Analytical expressions for several different nanocapillary shapes are derived. The functions are evaluated to determine how the impedance signals will change with different nanocapillary aspect ratios and different degrees of constriction or inflation at the capillary center. Next, the complex impedance spectrum of a nanocapillary array membrane is measured at varying concentrations of electrolyte to separate the effects of nanocapillary double layer capacitance from those of nanocapillary geometry. The variable topology equivalent circuit element model of the nanocapillary is used in an equivalent circuit model that included contributions from the membrane and the measurement apparatus. The resulting values are consistent with the manufacturer's specified tolerances of the nanocapillary geometry. It is demonstrated that electrochemical impedance spectroscopy can be used as a tool for in situ determination of the geometry of nanocapillaries.

Graphite-type activated carbon from coconut shell: a natural source for eco-friendly non-volatile storage devices.

Carbon from biomass as an active material for supercapacitor electrodes has attracted much interest due to its environmental soundness, abundance, and porous nature. In this context, activated carbon prepared from coconut shells via a simple activation process (water or steam as activation agents) was used as an active material in electrodes for eco-friendly supercapacitors. X-ray diffraction (XRD), Raman spectroscopy, conductivity, scanning electron microscopy (SEM), N2 sorption and thermogravimetry coupled to mass spectrometry (TGA-MS) studies revealed that activated carbon produced by this approach exhibit a graphitic phase, a high surface area, and large pore volume. The energy storage properties of activated carbon electrodes correlate with the morphological and structural properties of the precursor material. In particular, electrodes made of activated carbon exhibiting the largest Brunauer-Emmett-Teller (BET) surface area, i.e. 1998 m2 g-1, showed specific capacitance of 132.3 F g-1 in aqueous electrolyte (1.5 M H2SO4), using expanded graphite sheets as current collector substrates. Remarkably, this sample in a configuration with ionic liquid (1-methyl-1-propy-pyrrolizinium bis(fluorosulfonyl)mide) (MPPyFSI) as electrolyte and a polyethylene separator displayed an outstanding storage capability and energy-power handling capability of 219.4 F g-1 with a specific energy of 92.1 W h kg-1 and power density of 2046.9 W kg-1 at 1 A g-1 and maintains ultra-high values at 30 A g-1 indicating the ability for a broad potential of energy and power related applications. To the best of our knowledge, these values are the highest ever reported for ionic liquid-based supercapacitors with activated carbon obtained from the biomass of coconut shells.

In vitro formation of amyloid from alpha-synuclein is dominated by reactions at hydrophobic interfaces.

Most in vitro investigations of alpha-Synuclein (alphaSyn) aggregation and amyloidogenesis use agitation in the presence of air and/or Teflon to accelerate kinetics. The effect of the agitation is implicitly or explicitly attributed to mass transfer or fibril fragmentation. This paper evaluates these hypotheses by agitating alphaSyn under typical amyloidogenic conditions with controlled numbers of balls made of polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), and borosilicate glass with no headspace. Amyloid was assayed using thioflavin T fluorescence and atomic force microscopy. The observed kinetics were proportional to the PTFE surface area; the effects of PMMA and glass balls were negligible by comparison. No amyloid was observed to form in the absence of mixing balls. Agitation with only air also showed accelerated kinetics but different aggregate morphology. The results indicate that the mechanism active in agitation experiments is dominated by reactions at the hydrophobic-water interface. Of the mass transfer, fragmentation, and hydrophobic interface hypotheses, only the last is capable of explaining the data. Condition and sequence determinants of amyloidogenic propensity that have thus far been reported must be reinterpreted as being reflective of partitioning to hydrophobic-water interfaces. Comparable hydrophobic interfaces are not found in vivo.

Promoting the Insertion of Molecular Hydrogen in Tetrahydrofuran Hydrate With the Help of Acidic Additives.

Among hydrogen storage materials, hydrogen hydrates have received a particular attention over the last decades. The pure hydrogen hydrate is generated only at extremely high-pressure (few thousands of bars) and the formation conditions are known to be softened by co-including guest molecules such as tetrahydrofuran (THF). Since this discovery, there have been considerable efforts to optimize the storage capacities in hydrates through the variability of the formation condition, of the cage occupancy, of the chemical composition or of the hydrate structure (ranging from clathrate to semi-clathrate). In addition to this issue, the hydrogen insertion mechanism plays also a crucial role not only at a fundamental level, but also in view of potential applications. This paper aims at studying the molecular hydrogen diffusion in the THF hydrate by in-situ confocal Raman microspectroscopy and imaging, and at investigating the impact of strong acid onto this diffusive process. This study represents the first report to shed light on hydrogen diffusion in acidic THF-H2 hydrate. Integrating the present result with those from previous experimental investigations, it is shown that the hydrogen insertion in the THF hydrate is optimum for a pressure of ca. 55 bar at 270 K. Moreover, the co-inclusion of perchloric acid (with concentration as low as 1 acidic molecules per 136 water molecules) lead to promote the molecular hydrogen insertion within the hydrate structure. The hydrogen diffusion coefficient-measured at 270 K and 200 bar-is improved by a factor of 2 thanks to the acidic additive.

Total Internal Reflection Tip-Enhanced Raman Spectroscopy of Cytochrome c.

Surface and tip-enhanced Raman spectroscopies in total internal reflection (TIR-SERS and TIR-TERS) are used to characterize the oxidation, spin, and ligation state of cytochrome c (Cc) molecules electrostatically bound on a hydrophilic thiol self-assembled monolayer. TIR-SERS spectra of this model hemoprotein show marker bands typical of the 6cLS ferric state of Cc. The performances of the TIR-TERS technique as a function of the incidence angle are described, showing in particular a significant electromagnetic enhancement of the Raman signal under p-polarized light excitation. TIR-TERS spectra of Cc confirm the 6cLS ferric state assignment deduced from TIR-SERS spectra, thus demonstrating the possibility of probing with nanoscale spatial resolution the 6cLS oxidized form of Cc that is potentially implicated in cell apoptotic processes. The minimal far-field contribution of the sample in TIR-TERS also offers promising perspectives for future nanoscale chemical characterizations of photosensitive biomolecules in complex biological media.

Single-molecule protein unfolding in solid state nanopores.

We use single silicon nitride nanopores to study folded, partially folded, and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of beta-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins. Our results show that the nanopore translocation signals are sensitive enough to distinguish the folding state of a protein and distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain that transiently stalls in the nanopore due to the primary sequence of charges.

Remote surface enhanced Raman spectroscopy imaging via a nanostructured optical fiber bundle.

Remote surface enhanced Raman spectroscopy (SERS) imaging of an adsorbed monolayer was demonstrated through a nanostructured array of conical tips inscribed onto the distal face of a 30 cm optical fiber bundle. Despite intense Raman signal from the germanium oxide doped fibers, the Raman signal of an adsorbed monolayer of a reference compound (benzene thiol) was detected in the fingerprint region. This opens up the possibility of local remote imaging through an optical fiber that embeds a SERS active platform.

Information-theoretical analysis of time-correlated single-photon counting measurements of single molecules.

Time-correlated single photon counting allows luminescence lifetime information to be determined on a single molecule level. This paper develops a formalism to allow information theory analysis of the ability of luminescence lifetime measurements to resolve states in a single molecule. It analyzes the information content of the photon stream and the fraction of that information that is relevant to the state determination problem. Experimental losses of information due to instrument response, digitization, and different types of background are calculated and a procedure to determine the optimal value of experimental parameters is demonstrated. This paper shows how to use the information theoretical formalism to evaluate the number of photons required to distinguish dyes that differ only by lifetime. It extends this idea to include distinguishing molecular states that differ in the electron transfer quenching or resonant energy transfer and shows how the differences between the lifetime of signal and background can help distinguish the dye position in an excitation beam.

Separation and analysis of dynamic Stokes shift with multiple fluorescence environments: coumarin 153 in bovine beta-lactoglobulin A.

We use time-dependent fluorescence Stokes shift (TDFSS) information to study the fluctuation rates of the lipocalin, beta-lactoglobulin A in the vicinity of an encapsulated coumarin 153 molecule. The system has three unique dielectric environments in which the fluorophore binds. We develop a method to decompose the static and dynamic contributions to the spectral heterogeneity. This method is applied to temperature-dependent steady-state fluorescence spectra providing us with site-specific information about thermodynamic transitions in beta-lactoglobulin. We confirm previously reported transitions and discuss the presence of an unreported transition of the central calyx at 18 degrees C. Our method also resolves the contributions to the TDFSS from the coumarin 153 centrally located in the calyx of beta-lactoglobulin despite overlapping signals from solvent exposed dyes. Our experiments show dynamics ranging from 3-12,00 ps. The analysis shows a decrease in the encapsulated dye's heterogeneity during the relaxation, which is taken as evidence of the breakdown of linear response.