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Understanding α-synuclein aggregation is crucial in the context of Parkinson's disease. The objective of this study was to investigate the influence of aggregation induced by preformed seeding on the volume of oligomers during the early stages, using a label-free, single-molecule characterization approach. By utilizing nanopipettes of varying sizes, the volume of the oligomers can be calculated from the amplitude of the current blockade and pipette geometry. Further investigation of the aggregates formed over time in the presence of added seeds revealed an acceleration in the formation of large aggregates and the existence of multiple distinct populations of oligomers. Additionally, we observed that spontaneously formed seeds inhibited the formation of smaller oligomers, in contrast to the effect of HNE seeds. These results suggest that the seeds play a crucial role in the formation of oligomers and their sizes during the early stages of aggregation, whereas the classical thioflavin T assay remains negative.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Bioensaio , SementesRESUMO
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.
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Levodopa , Quartzo , Proteínas Amiloidogênicas , SementesRESUMO
Protein detection and identification at the single-molecule level are major challenges in many biotechnological fields. Solid-state nanopores have raised attention as label-free biosensors with high sensitivity. Here, we use solid-state nanopore sensing to discriminate two closely related proteins, α-thrombin and γ-thrombin. We show that aptamer functionalization improves protein discrimination thanks to a significant difference in the relative current blockade amplitude. To enhance discrimination, we postprocessed the signals using machine learning and training algorithms and we were able to reach an accuracy of 98.8% using seven features and ensemble methods.
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Técnicas Biossensoriais , Nanoporos , Nanotecnologia , Proteínas , TrombinaRESUMO
Amyloid fibrils are involved in several neurodegenerative diseases. However, because of their polymorphism and low concentration, they are challenging to assess in real-time with conventional techniques. Here, we present a new approach for the characterization of the intermediates: protofibrils and "end-off" aggregates which are produced during the amyloid formation. To do so, we have fashioned conical track-etched nanopores that are functionalized to prevent the fouling. Using these nanopores, we have followed the kinetic of amyloid growth to discriminate the different intermediates protofibrils and "end-off. Then, the nanopore was used to characterize the effect of promoter and inhibitor of the fibrillation process. Finally, we have followed in real-time the degradation of amyloid with peptase. Compare with the SiN nanopore, the track-etched one features exceptionally high success rate via functionalization and detection in "one-pot". Our results demonstrate the potential for a conical nanopore to be used as a routine technique for the characterization of the amyloid growth and/or degradation.
Assuntos
Amiloide/química , Lactoglobulinas/química , Nanoporos , Multimerização Proteica , Proteólise , Amiloide/antagonistas & inibidores , Curcumina/química , Sulfato de Dextrana/química , Etanol/química , Concentração de Íons de Hidrogênio , Cinética , Nanoporos/ultraestrutura , Pepsina A/química , Polietilenoglicóis/química , Multimerização Proteica/efeitos dos fármacos , Quercetina/químicaRESUMO
The understanding of the interactions between nanomaterials, biomolecules, and polyphenols is fundamental in food chemistry, toxicology, and new emerging fields, such as nanomedicine. Here, we investigated the effect of the resveratrol, a principal actor in drug-delivery application on the interaction between bovine serum albumin (BSA), employed as a vector for the delivery of polyphenol drugs, and gold nanoparticle (gNP), the most promising tool in theranostic applications. Through a combination of experimental techniques, which includes an initial evaluation by dynamic light scattering and surface plasmon resonance spectroscopy, we were able to evaluate the evolution of the gold nanoparticle aggregation with increasing ionic strength and the consequences of the BSA and resveratrol addition. To investigate the mechanisms of the interactions, we pursued at the single-molecule level using solid-state nanopore and fluorescence correlation spectroscopy. Our results show that without resveratrol, the BSA is adsorbed on the gNP in water or saline solution. In the presence of resveratrol, the BSA is normally absorbed on gNP in water, but the salt addition leads to its desorption. The resveratrol clearly plays a fundamental role, changing the protein behavior and making the BSA adsorption a reversible process in the presence of salt.
Assuntos
Adsorção/efeitos dos fármacos , Ouro , Nanopartículas Metálicas/química , Resveratrol/farmacologia , Soroalbumina Bovina , Difusão Dinâmica da Luz , Ouro/química , Ressonância de Plasmônio de SuperfícieRESUMO
Single conical nanopores were functionalised layer by layer with weak polyelectrolytes. We studied their influence on the ionic diode properties We have considered different couples of polyelectrolytes: poly-l-lysine/poly(acrylic acid) and poly(ethyleneimine)/poly(acrylic acid) as well as the influence of cross-linking. The results show that the nanopores decorated with poly(ethyleneimine)/poly(acrylic acid) exhibit an interesting behavior. Indeed, at pH 3, the nanopore is open only at the low salt concentration, while at pH 7, it is already open. The nanopores functionalized with poly-l-lysine/poly(acrylic acid) do not show an inversion of ionic transport properties with the pH as expected. After cross-linked to prevent large conformational changes, the ionic diode properties are dependent on the pH.
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We investigated ionic transport behavior in the case of uncharged conical nanopores. To do so, we designed conical nanopores using atomic layer deposition of Al2O3/ZnO nanolaminates and then coated these with trimethylsilane. The experimental results are supported by molecular dynamics simulations. The ionic transport reveals an unexpected behavior: (i) a current rectification and (ii) a constant conductance at low salt concentration which are usually reported for charged conical nanopore. To explain these results, we have considered different assumptions: (i) a default of functionalization, (ii) the adsorption anion and (iii) the slippage. The first one was refuted by the study of the poly-l-lysine transport through the nanopore. To verify the second assumption, we investigate the effect of pH on the current rectification and the molecular dynamics simulations. Finally our study demonstrates that the unexpected ionic transport is provided to a predominant effect of slippage due to the water organization at the solid/liquid interface.
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Solid-state nanopore technology for nanoparticle sensing is considered for the development of analytical tools to characterise their size, shape or zeta potential. In this field, it is crucial to understand how the nanopore inner surface influences the dynamic of nanoparticle translocation. Here, three single nanopores directly drilled in metal alloys (titanium nitride, titanium-tantalum and tantalum) are considered. The translocation of polystyrene nanoparticles coated with ssDNA is investigated by the resistive pulse method at different concentrations and voltages. The results show that the nanoparticle energy barrier for entrance into the pore decreases for nanopores that exhibits a higher surface energy and hydrophilicity, while the dwell time is found to depend on the nanopore surface state. Overall, this study demonstrates that the control of nanopore surface state must be taken into account for the resistive pulse experiments for nanoparticle detection.
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Biological channels have served as inspiration to design stimuli-response artificial nanopores. Here we propose an original approach to design a pH-gate nanopore based on polyethylenimine and chondroitin-4-sulfate (ChS) layer-by-layer self-assembly. This approach is interesting because it is rapid and permits monitoring in real time of functionalization. The study of ionic transport through these single nanopores reveals a selectivity on anions and pH-gate properties at low salt concentration. It is open at pH below 4 or 5 depending on salt concentration. These properties are explained by the modification of both charge and conformation of ChS as well as swelling of the polyelectrolyte complex.
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The fundamental understanding of the transport mechanisms of objects across a single nanopore is one key point to develop Coulter counters at the nanoscale for macromolecule or nanoparticle detection. In this area, nanoparticles have been less investigated than biomacromolecules such as DNA or proteins due to their self-aggregation in the presence of salts. In this work, the transport of modified latex nanoparticles across solid-state nanopores was investigated. To prevent their aggregation, their surface was modified with a low molecular weight single strand DNA coating. Then the coated nanoparticles were successfully detected across a single pore material in 200 mM NaCl buffer. The experimental capture rate was compared to that of the predictive model. It reveals that the nanoparticle entrance inside the nanopore is mainly governed by diffusion and required a weak energy. For relative current blockades, the predictive model should take into account both the nanopore shape and the additional charge due to ssDNA coating.
Assuntos
DNA de Cadeia Simples/química , Látex/química , Nanopartículas/química , Nanoporos , Adsorção , DifusãoRESUMO
Amphotericin B (AmB) is a well-known polyene which self-organizes into membrane cell in order to cause the cell death. Its specific action towards fungal cell is not fully understood but was proved to become from sterol composition. The mechanism was shown experimentally to require the formation of stable sterol/polyene couples which could then organize in a nanochannel. This would allow the leakage of ions responsible for the death of fungal cells, only. In this present study, we investigate the arrangement of AmB/sterols in biological membrane using molecular dynamic simulations in order to understand the role of the sterol structure on the antifungal action of the polyene. We show in particular that the nanochannels tend to close up when cell was composed with cholesterol (animal cell) due to strong interaction between amphotericin and sterol. On the other side, with ergosterol (fungal cell) the largest interactions between amphotericin and lipid membrane lead to the appearance of large hole that could favor the important leakage of ions and thus, the fungal cell death. This work appears as a good complement in the extensive studies linked to the understanding of the antifungal molecules in membrane cells.
Assuntos
Anfotericina B/química , Conformação Molecular , Simulação de Dinâmica Molecular , Esteróis/química , Membrana Celular/química , Ligação de HidrogênioRESUMO
For the past 2 decades, emerging single-nanopore technologies have opened the route to multiple sensing applications. Besides DNA sensing, the identification of proteins and amyloids is a promising field for early diagnosis. However, the influence of the interactions between the nanopore surface and proteins should be taken into account. In this work, we have selected three proteins (avidin, lysozyme, and IgG) that exhibit different affinities with the SiNx surface, and we have also examined lysozyme amyloid. Our results show that the piranha treatment of SiNx significantly decreases protein adsorption. Moreover, we have successfully detected all proteins (pore diameter 17 nm) and shown the possibility of discriminating between denatured lysozyme and its amyloid. For all proteins, the capture rates are lower than expected, and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that proteins interacting only with the nanopore surface wall stay long enough to be detected. For lysozyme amyloid, we show that the use of the nanopore is suitable for determining the number of monomer units even if only the proteins interacting with the nanopore are detected.
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Amiloide/análise , Avidina/análise , Imunoglobulina G/análise , Muramidase/análise , Compostos de Silício/química , Adsorção , Amiloide/química , Avidina/química , Técnicas Eletroquímicas , Imunoglobulina G/química , Cinética , Muramidase/química , Nanoporos/ultraestrutura , SoluçõesRESUMO
A water molecule is the foundation of life and is the primary compound in every living system. While many of its properties are understood in a bulk solvent, its behavior in a small hydrophobic nanopore still raises fundamental questions. For instance, a wetting/dewetting transition in a hydrophobic solid-state or a polymer nanopore occurs stochastically and can only be prevented by external physical stimuli. Controlling these transitions would be a primary requirement to improve many applications. Some biological channels, such as gramicidin A (gA) proteins, show a high rate of water and ion diffusion in their central subnanochannel while their external surface is highly hydrophobic. The diameter of this channel is significantly smaller than the inner size of the lowest artificial nanopore in which water drying occurs (i.e. 1.4 nm). In this paper, we propose an innovative idea to generate nanopore wetting as a result of which the application of an external field is no longer required. In a nanopore, the drying or wetting of the inner walls occurs randomly (in experiments and in simulations). However, we have shown how the confinement of gA, in a dried hydrophobic nanopore, rapidly generates a stable wetting of the latter. We believe that this simple idea, based on biomimetism, could represent a real breakthrough that could help to improve and develop new nanoscale applications.
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Biomimética , Nanoporos , Molhabilidade , Difusão , Interações Hidrofóbicas e HidrofílicasRESUMO
We investigate the influence of a nanopore surface state and the addition of Mg(2+) on poly-adenosine translocation. To do so, two kinds of nanopores with a low aspect ratio (diameter â¼3-5 nm, length 30 nm) were tailored: the first one with a negative charge surface and the second one uncharged. It was shown that the velocity and the energy barrier strongly depend on the nanopore surface. Typically if the nanopore and polyA exhibit a similar charge, the macromolecule velocity increases and its global energy barrier of entrance in the nanopore decreases, as opposed to the non-charged nanopore. Moreover, the addition of a divalent chelating cation induces an increase of energy barrier of entrance, as expected. However, for a negative nanopore, this effect is counterbalanced by the inversion of the surface charge induced by the adsorption of divalent cations.
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Nanoporos , Adenosina/química , Eletricidade , Magnésio/química , Nanoporos/ultraestrutura , Polímeros/química , Propriedades de SuperfícieRESUMO
The development of nanometric Coulter counters for nanoparticle detection is an attractive and promising field of research. In this work, we have studied the influence of the nanopore surface state on charged polymer nanoparticle translocations. To make this, the translocation of carboxylate modified polystyrene microspheres (diameter 40, 70 and 100 nm) has been investigated through two kinds of high aspect ratio nanopores (negative and uncharged). The latter were tailored by a single track-etched and atomic layer deposition technique. It was shown that the mobility and the energy barrier are strongly dependent on nanopore surface charge. Typically if the latter exhibits negative surface charge, the microsphere mobility increases and the global energy barrier of entrance inside the nanopore decreases with its diameter, converse to the uncharged nanopore.
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Biological ion channels present unique ionic properties. They can be highly permeable to ions, while selecting only one type of ions, without external energy supply. An important research field has been developed to transfer these properties to solid state nanoporous membranes in order to develop artificial biomimetic nanofilters. One of the promising ways to develop biomimetic structures is based on the direct insertion of the gramicidin A, i.e. an ionic channel, inside a nanopore. Experiments have recently proved the feasibility of such a hybrid membrane with very interesting results regarding the ionic selectivity. Here, we propose to interpret these experiments using theoretical molecular dynamic simulations which allow us to analyze more profoundly the structures of the proteins confined inside the nanopore and the relation between their conformation and the observed ionic properties.
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Biomimética , Nanoporos , Potássio/metabolismo , Difusão , Gramicidina/química , Gramicidina/metabolismo , Membranas Artificiais , Simulação de Dinâmica Molecular , Conformação Proteica , Eletricidade Estática , Especificidade por SubstratoRESUMO
The Aß42 aggregates with different structures and morphology was investigated through a single molecule label-free technique. To this end, the quartz nanopipettes were functionalized with polyethylene glycol. The set of Aß42- epigallocatechin-3-gallate fibrils with length (from 85 nm to 250 nm) obtained by sonication was detected. The comparison of experimental and computed value of the amplitude of relative current blockade using a geometrical model show that for fibrils longer than 80 nm, the discriminating parameter is their diameter. Then, non-fibril oligomers obtain from Aß42(Osaka) aggregation at different time seed was investigated. The analysis of the amplitude of relative current blockade shows that detected oligomers are smaller than 30 nm regardless the aggregation time. In addition, the wide distributions of the dwell time suggests the polymorph character of the sample.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Amiloide/químicaRESUMO
In this work, we aim to capture, detect and analysis at single molecule level Aß42 aggregates. To this end, two strategies of track-etched nanopore membranes functionalization were investigated. The first one uses an aptamer and requires only three steps, whereas the second strategy uses Lecanemab antibodies and requires six steps. Out of the two presented strategies, the second one was found to be the most suitable to detect Aß42 aggregates using a quick current-voltage readout. The resulting single nanopore was then upscale to multipore membranes to capture the Aß42 aggregates before analysis through them through a single-molecule approach. By comparing the species present in the retentate and filtrate, we confirmed the membrane's affinity for the larger Aß42 aggregates present in the sample. We found that chromatographic membranes combined with an ionic diode for binary on/off readout are powerful tools for detecting rare biomarkers before single molecule analysis.
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Doença de Alzheimer , Nanoporos , Humanos , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , OligonucleotídeosRESUMO
A hybrid nanoporous membrane made of a solid-state polymeric thin film in which an ion channel is confined is realized. The primary and extremely encouraging results obtained by confocal fluorescence spectroscopy and ion diffusion measurement demonstrate respectively that (i) the considered ion channel, that is, Gramicidin-A, can be confined selectively inside the nanopores and (ii) the ionic permeability of the membrane is enhanced. Atomistic molecular simulations are also reported and fruitfully compared to the experimental findings.
Assuntos
Materiais Biomiméticos/química , Gramicidina/química , Canais Iônicos/química , Membranas Artificiais , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Cristalização/métodos , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
The detection to α-synuclein (αS) assemblies as a biomarker of synucleinopathies is an important challenge for further development of an early diagnosis tool. Here, we present proof of concept real-time fast amyloid seeding and translocation (RT-FAST) based on a nanopipette that combines in one unique system a reaction vessel to accelerate the seed amplification and nanopore sensor for single-molecule αS assembly detection. RT-FAST allows the detection of the presence αS seeds WT and A53T variant in a given sample in only 90 min by adding a low quantity (35 µL at 100 nM) of recombinant αS for amplification. It also shows cross-seeding aggregation by adding mixing seeds A53T with WT monomers. Finally, we establish the dependence between the capture rate of aggregates by the nanopore sensor and the initial seed concentration from 200 pM to 2 pM, which promises further development toward a quantitative analysis of the initial seed concentration.