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Nanopores are powerful tools for single-molecule sensing of biomolecules and nanoparticles. The signal coming from the molecule to be analyzed strongly depends on its interaction with the narrower section of the nanopore (constriction) that may be tailored to increase sensing accuracy. Modifications of nanopore constriction have also been commonly used to induce electroosmosis, that favors the capture of molecules in the nanopore under a voltage bias and independently of their charge. However, engineering nanopores for increasing both electroosmosis and sensing accuracy is challenging. Here it is shown that large electroosmotic flows can be achieved without altering the nanopore constriction. Using continuum electrohydrodynamic simulations, it is found that an external charged ring generates strong electroosmosis in cylindrical nanopores. Similarly, for conical nanopores it is shown that moving charges away from the cone tip still results in an electroosmotic flow (EOF), whose intensity reduces increasing the diameter of the nanopore section where charges are placed. This paradigm is applied to engineered biological nanopores showing, via atomistic simulations and experiments, that mutations outside the constriction induce a relatively intense electroosmosis. This strategy provides much more flexibility in nanopore design since electroosmosis can be controlled independently from the constriction, which can be optimized to improve sensing accuracy.
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Prion-like protein aggregation is characteristic of numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. This process involves the formation of aggregates ranging from small and potentially neurotoxic oligomers to highly structured self-propagating amyloid fibrils. Various approaches are used to study protein aggregation, but they do not always provide continuous information on the polymorphic, transient, and heterogeneous species formed. This review provides an updated state-of-the-art approach to the detection and characterization of a wide range of protein aggregates using nanopore technology. For each type of nanopore, biological, solid-state polymer, and nanopipette, discuss the main achievements for the detection of protein aggregates as well as the significant contributions to the understanding of protein aggregation and diagnostics.
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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.
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Enfermedad de Parkinson , alfa-Sinucleína , Bioensayo , SemillasRESUMEN
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 , Cuarzo , Proteínas Amiloidogénicas , SemillasRESUMEN
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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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Amiloide/químicaRESUMEN
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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Enfermedad de Alzheimer , Nanoporos , Humanos , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , OligonucleótidosRESUMEN
Bio-based ceramic membranes were elaborated from kaolinite clays, coconut husks and eggshells to retain E. coli bacteria present in water intended for human consumption. Their characterization and removal performances are investigated in this work. These bio-ceramic membranes were obtained by heating the formulation containing 75% clay, 15% coconut husk and 10% eggshell at 900 °C or 1000 °C, at different temperature rates, to give S1, S2 and S3 materials. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), mercury porosimetry and scanning electron microscopy (SEM) were used to characterize these membranes. Water flux density, bacterial removal and biofouling were also assessed. Water flux density was shown to depend on material porosity. Bacteria retention was 90% (with 1 log-removal) for S1, 80% (with 0.7 log-removal) for S2 and 100% (with 3.3 log-removal) for S3. Membranes S1 and S2 presented reversible biofouling, while no fouling was evidenced for S3 in the tested conditions. This work shows that the best bio-ceramic membrane in terms of bacterial removal and flux density was S3. Its water flux density was 2123 ± 72 L/h/m2 at an initial pressure of 0.2 bar. This material is particularly interesting because its production protocol is quite simple, fast and without the addition of chemical additives. Moreover, it can be used to efficiently remove bacteria from drinking water.
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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.
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Nanoporos , alfa-Sinucleína , Agregado de Proteínas , Levodopa , Péptidos beta-Amiloides/metabolismo , Heparina , Amiloide/metabolismoRESUMEN
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.
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The Aß(1-42) aggregation is a key event in the physiopathology of Alzheimer's disease (AD). Exogenous factors such as environmental pollutants, and more particularly pesticides, can corrupt Aß(1-42) assembly and could influence the occurrence and pathophysiology of AD. However, pesticide involvement in the early stages of Aß(1-42) aggregation is still unknown. Here, we employed conical track-etched nanopore in order to analyse the Aß(1-42) fibril formation in the presence of pyrimethanil, a widely used fungicide belonging to the anilinopyrimidine class. Our results evidenced a pro-aggregating effect of pyrimethanil on Aß(1-42). Aß(1-42) assemblies were successfully detected using conical nanopore coated with PEG. Using an analytical model, the large current blockades observed (>0.7) were assigned to species with size close to the sensing pore. The long dwell times (hundreds ms scale) were interpreted by the possible interactions amyloid/PEG using molecular dynamic simulation. Such interaction could leave until splitting phenomena of the dimer structure. Our work also evidences that the pyrimethanil induce an aggregation of Aß(1-42) mechanism in two steps including the reorganization prior the elongation phase.
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Fungicidas Industriales , Nanoporos , Péptidos beta-Amiloides , Fungicidas Industriales/toxicidad , Fragmentos de Péptidos , PirimidinasRESUMEN
The switching of "ON" and "OFF" states of an ionic diode is investigated by considering a conical nanopore partially functionalized two polyelectrolyte (PE) layers via layer-by-layer deposition. Through observing the inversion of its rectification behavior, we demonstrate the function of the PE bilayers in ionic transport regulation. The ionic diode exhibits an ultrahigh ion rectification at a low level of pH. In an aqueous NaCl solution at pH 2, for example, the ratio of the current at "ON" state and that at "OFF" state can be about 800 and 200 for 1 and 100 mM, respectively. This remarkable gating behavior can be explained by the anion-pump-induced ion accumulation in the neutral region as well as the depletion zone at the interface. Our results further demonstrate the possibility of achieving an ultrahigh rectification in an ionic diode having a unipolar-like configuration.
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In two decades, the solid state and polymer nanopores became attractive method for the protein sensing with high specificity and sensitivity. They also allow the characterization of conformational changes, unfolding, assembly and aggregation as well the following of enzymatic reaction. This review aims to provide an overview of the protein sensing regarding the technique of detection: the resistive pulse and ionic diodes. For each strategy, we report the most significant achievement regarding the detection of peptides and protein as well as the conformational change, protein-protein assembly and aggregation process. We discuss the limitations and the recent strategies to improve the nanopore resolution and accuracy. A focus is done about concomitant problematic such as protein adsorption and nanopore lifetime.
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Nanoporos , Adsorción , Péptidos , Polímeros , ProteínasRESUMEN
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.
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Enfermedad de Alzheimer , Nanoporos , Amiloide , Péptidos beta-Amiloides , Humanos , CinéticaRESUMEN
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 Biosensibles , Nanoporos , Nanotecnología , Proteínas , TrombinaRESUMEN
TiO2 nanoparticles known as E171 are one controversial food additive due to its potential toxicity. In this work, the main hypothesis is that the proteins adsorbed on the TiO2 nanoparticles prevent their aggregation and favor the cell penetration. To do so, the TiO2 nanoparticles were coated with gelatin and ß-lactoglobulin to reach interfacial concentrations about 0.25 mg/mg and 0.32 mg/mg, respectively. The measurement of NP size showed that the protein coating improve the colloidal stability of TiO2 nanoparticles. The FTIR analysis suggests that the ß-lactoglobulin structure is modified after adsorption. The penetration of TiO2 penetration inside human intestinal epithelial cells was shown and quantify by using confocal Raman microscopy. The promoting role of the protein coating on the cell penetration was demonstrated for both the gelatin and ß-lactoglobulin. Finally, the results allow establishing a correlation between the ability of proteins to prevent NP aggregation and the cell penetration.
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Titanio/química , Adsorción , Coloides/química , Aditivos Alimentarios , Gelatina/química , Humanos , Lactoglobulinas/química , Nanopartículas/química , Tamaño de la PartículaRESUMEN
Solid-state nanopores are a promising platform for characterizing proteins. In order to improve their lifetime and prevent fouling, Polyethylene Glycol (PEG) grafting is one of the most efficient and low-cost solutions. Different models to calculate the PEG thickness do not consider their interaction with the nanopore inner surface nor the effect of confinement. Here, we investigate by molecular dynamic simulation the PEG conformation inside a nanopore in the case of hydrophobic and hydrophilic nanopores. Our results reveal that the nanopore inner surface plays a role in the PEG organization and, thus, in the speed of the salt constituent. The resulting pair interaction between PEG and its environment clearly shows a more important affinity for K+ compared to Li+ cations.
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The modification of the inner nanopore wall by polymers is currently used to change the specific properties of the nanosystem. Among them, the polyethylene glycol (PEG) is the most used to prevent the fouling and ensure the wettability. However, its properties depend mainly on the chain structure that is very difficult to estimate inside this confined space. Combining experimental and simulation approaches, we provide an insight to the consequence of the PEG presence inside the nanopore on the nanopore properties. We show, in particular, that the cation type in the electrolyte, together with the type of electrolyte (water or urea), is at the origin of the ion transport modification in the nanopore.
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Synthetic polymers are widely employed for bone tissue engineering due to their tunable physical properties and biocompatibility. Inherently, most of these polymers display poor antimicrobial properties. Infection at the site of implantation is a major cause for failure or delay in bone healing process and the development of antimicrobial polymers is highly desired. In this study, silver nanoparticles (AgNps) were synthesized in polycaprolactone (PCL) solution by in-situ reduction and further extruded into PCL/AgNps filaments. Customized 3D structures were fabricated using the PCL/AgNps filaments through 3D printing technique. As demonstrated by scanning electron microscopy, the 3D printed scaffolds exhibited interconnected porous structures. Furthermore, X-ray photoelectron spectroscopy analysis revealed the reduction of silver ions. Transmission electron microscopy along with energy-dispersive X-ray spectroscopy analysis confirmed the formation of silver nanoparticles throughout the PCL matrix. In vitro enzymatic degradation studies showed that the PCL/AgNps scaffolds displayed 80% degradation in 20â¯days. The scaffolds were cytocompatible, as assessed using hFOB cells and their antibacterial activity was demonstrated on Escherichia coli. Due to their interconnected porous structure, mechanical and antibacterial properties, these cytocompatible multifunctional 3D printed PCL/AgNps scaffolds appear highly suitable for bone tissue engineering.
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Nanopartículas del Metal , Ingeniería de Tejidos , Antibacterianos/farmacología , Poliésteres , Impresión Tridimensional , Plata/farmacología , Andamios del TejidoRESUMEN
Single nanopore is a powerful platform to detect, discriminate and identify biomacromolecules. Among the different devices, the conical nanopores obtained by the track-etched technique on a polymer film are stable and easy to functionalize. However, these advantages are hampered by their high aspect ratio that avoids the discrimination of similar samples. Using machine learning, we demonstrate an improved resolution so that it can identify short single- and double-stranded DNA (10- and 40-mers). We have characterized each current blockade event by the relative intensity, dwell time, surface area and both the right and left slope. We show an overlap of the relative current blockade amplitudes and dwell time distributions that prevents their identification. We define the different parameters that characterize the events as features and the type of DNA sample as the target. By applying support-vector machines to discriminate each sample, we show accuracy between 50% and 72% by using two features that distinctly classify the data points. Finally, we achieved an increased accuracy (up to 82%) when five features were implemented.