Controlling Electroosmosis in Nanopores Without Altering the Nanopore Sensing Region.

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 favours 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 we show that large electroosmotic flows can be achieved without altering the nanopore constriction. Using continuum electrohydrodynamic simulations, we found that an external charged ring generates strong electroosmosis in cylindrical nanopores. Similarly, for conical nanopores we show that moving charges away from the cone tip still results in an electroosmotic flow, whose intensity reduces increasing the diameter of the nanopore section where charges are placed. We applied this paradigm 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. This article is protected by copyright. All rights reserved.

Hydrophobically gated memristive nanopores for neuromorphic applications.

Signal transmission in the brain relies on voltage-gated ion channels, which exhibit the electrical behaviour of memristors, resistors with memory. State-of-the-art technologies currently employ semiconductor-based neuromorphic approaches, which have already demonstrated their efficacy in machine learning systems. However, these approaches still cannot match performance achieved by biological neurons in terms of energy efficiency and size. In this study, we utilise molecular dynamics simulations, continuum models, and electrophysiological experiments to propose and realise a bioinspired hydrophobically gated memristive nanopore. Our findings indicate that hydrophobic gating enables memory through an electrowetting mechanism, and we establish simple design rules accordingly. Through the engineering of a biological nanopore, we successfully replicate the characteristic hysteresis cycles of a memristor and construct a synaptic device capable of learning and forgetting. This advancement offers a promising pathway for the realization of nanoscale, cost- and energy-effective, and adaptable bioinspired memristors.

Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force.

Nanopores have recently been used to identify and fingerprint proteins. However, because proteins, unlike DNA, do not have a uniform charge, the electrophoretic force cannot in general be used to translocate or linearize them. Here we show that the introduction of sets of charges in the lumen of the CytK nanopore spaced by ~1 nm creates an electroosmotic flow that induces the unidirectional transport of unstructured natural polypeptides against a strong electrophoretic force. Molecular dynamics simulations indicate that this electroosmotic-dominated force has a strength of ~20 pN at -100 mV, which is similar to the electric force on single-stranded DNA. Unfolded polypeptides produce current signatures as they traverse the nanopore, which may be used to identify proteins. This approach can be used to translocate and stretch proteins for enzymatic and non-enzymatic protein identification and sequencing.

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB.

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins.

Surface-particle interactions control the escape time of a particle from a nanopore-gated nanocavity system: a coarse grained simulation.

Nanopores and nanocavities are promising single molecule tools for investigating the behavior of individual molecules within confined spaces. For single molecule analysis, the total duration of time the analyte remains within the pore/cavity is highly important. However, this dwell time is ruled by a complex interplay among particle-surface interactions, external forces on the particle and Brownian diffusion, making the prediction of the dwell time challenging. Here, we show how the dwell time of an analyte in a nanocavity that is connected to the external environment by two nanopore gates depends on the sizes of the nanocavity/nanopore, as well as particle-wall interactions. For this purpose, we used a coarse-grained model that allowed us to simulate hundreds of individual analyte trajectories within a nanocavity volume. We found that by increasing the attraction between the particle and the wall, the diffusion process transforms from a usual 3D scenario (repulsive wall) to a 2D motion along the cavity surface (highly attractive wall). This results in a significant reduction of the average dwell time. Additionally, the comparison of our results with existing theories on narrow escape problem allowed us to quantify the reliability of theory derived for ideal conditions to geometries more similar to actual devices.

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

DNA compaction and dynamic observation in a nanopore gated sub-attoliter silicon nanocavity.

Confinement of biopolymers inside volumes with micro- or nanoscale lateral dimensions is ubiquitous in nature. Investigating the behavior of biopolymers in a confined environment is essential to improve our basic understanding in life sciences. In this work, we present a nanopore gated sub-attoliter silicon nanocavity device, which allows DNA compaction similar to that in virus capsids. Single DNA molecules can be electrically driven into the nanocavity, and then get compacted inside the nanocavity under certain conditions. The dynamic fluctuations of the compacted DNA can be monitored via ionic current measurements. The mechanism for the DNA compaction is elucidated by varying the DNA length or concentration, voltage polarity, nanocavity dimensions and ionic strength. Furthermore, Brownian dynamics simulations reveal the dynamic fluctuations of the compacted DNA, which are reflected in the measured ionic current. Our nanocavity device is anticipated to provide a controlled environment in extremely small volumes for investigating the physics of confined biopolymers.

Geometrically Induced Selectivity and Unidirectional Electroosmosis in Uncharged Nanopores.

Selectivity toward positive and negative ions in nanopores is often associated with electroosmotic flow, the control of which is pivotal in several micro-nanofluidic technologies. Selectivity is traditionally understood to be a consequence of surface charges that alter the ion distribution in the pore lumen. Here we present a purely geometrical mechanism to induce ionic selectivity and electroosmotic flow in uncharged nanopores, and we tested it via molecular dynamics simulations. Our approach exploits the accumulation of charges, driven by an external electric field, in a coaxial cavity that decorates the membrane close to the pore entrance. The selectivity was shown to depend on the applied voltage and becomes completely inverted when reversing the voltage. The simultaneous inversion of ionic selectivity and electric field direction causes a unidirectional electroosmotic flow. We developed a quantitatively accurate theoretical model for designing pore geometry to achieve the desired electroosmotic velocity. Finally, we show that unidirectional electroosmosis also occurs in much more complex scenarios, such as a biological pore whose structure presents a coaxial cavity surrounding the pore constriction as well as a complex surface charge pattern. The capability to induce ion selectivity without altering the pore lumen shape or the surface charge may be useful for a more flexible design of selective membranes.

Conical nanopores highlight the pro-aggregating effects of pyrimethanil fungicide on Aß(1-42) peptides and dimeric splitting phenomena.

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.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Base flow decomposition for complex moving objects in linear hydrodynamics: Application to helix-shaped flagellated microswimmers.

The motion of microswimmers in complex flows is ruled by the interplay between swimmer propulsion and the dynamics induced by the fluid velocity field. Here we study the motion of a chiral microswimmer whose propulsion is provided by the spinning of a helical tail with respect to its body in a simple shear flow. Thanks to an efficient computational strategy that allowed us to simulate thousands of different trajectories, we show that the tail shape dramatically affects the swimmer's motion. In the shear dominated regime, the swimmers carrying an elliptical helical tail show several different Jeffery-like (tumbling) trajectories depending on their initial configuration. As the propulsion torque increases, a progressive regularization of the motion is observed until, in the propulsion dominated regime, the swimmers converge to the same final trajectory independently on the initial configuration. Overall, our results show that elliptical helix swimmer presents a much richer variety of trajectories with respect to the usually studied circular helix tails.

Analytical Model for Particle Capture in Nanopores Elucidates Competition among Electrophoresis, Electroosmosis, and Dielectrophoresis.

The interaction between nanoparticles dispersed in a fluid and nanopores is governed by the interplay of hydrodynamical, electrical, and chemical effects. We developed a theory for particle capture in nanopores and derived analytical expressions for the capture rate under the concurrent action of electrical forces, fluid advection, and Brownian motion. Our approach naturally splits the average capture time in two terms, an approaching time due to the migration of particles from the bulk to the pore mouth and an entrance time associated with a free-energy barrier at the pore entrance. Within this theoretical framework, we described the standard experimental condition where a particle concentration is driven into the pore by an applied voltage, with specific focus on different capture mechanisms: under pure electrophoretic force, in the presence of a competition between electrophoresis and electroosmosis, and finally under dielectrophoretic reorientation of dipolar particles. Our theory predicts that dielectrophoresis is able to induce capture for both positive and negative voltages. We performed a dedicated experiment involving a biological nanopore (α-hemolysin) and a rigid dipolar dumbbell (realized with a ß-hairpin peptide) that confirms the theoretically proposed capture mechanism.

Native-state fingerprint on the ubiquitin translocation across a nanopore.

We study the translocation of the ubiquitin molecule (Ubq) across a channel with a double section which constitutes a general feature of several transmembrane nanopores such as the α-hemolysin (αHL). Our purpose is to establish the structure-dependent character of the Ubq translocation pathway. This implies to find the correspondence, if any, between the translocational unfolding steps and the Ubq native state. For this reason, it is convenient to apply a coarse-grained computational approach, where the protein is described only by the backbone and the force field only exploits the information contained in the native state (in the spirit of Go-like models, or native-centric models). The αHL-like pore is portrayed as two coaxial confining cylinders: a larger one for the vestibule and a narrower one for the barrel (or stem). Such simplified approach allows a large number of translocation events to be collected by limited computational resources. The co-translocational unfolding of Ubq is described via a few collective variables that characterize the translocation progress. We find two translocation intermediates (stalled conformations) that can be associated with specific unfolding stages. In particular, in the earliest step, the strand S5 unfolds and enters the pore. This step splits the native conformation into two structural clusters packing against each other in the Ubq fold. A second stall occurs when the hairpin of the N terminal engages the stem region.

Confinement effects on the dynamics of a rigid particle in a nanochannel.

The transport of nanoparticles in confined geometries plays a crucial role in several technological applications ranging from nanopore sensors to filtration membranes. Here we describe a Brownian approach to simulate the motion of a rigid-body nanoparticle of an arbitrary shape under confinement. A quaternion formulation is used for the nanoparticle orientation, and the corresponding overdamped Langevin equation, completed by the proper fluctuation-dissipation relation, is derived. The hydrodynamic mobility matrix is obtained via dissipative particle dynamics simulation equipped with a new method for enforcing the no-slip boundary condition for curved moving solid-liquid interfaces. As an application, we analyzed the motion of a nanoparticle in a cylindrical channel under the action of external fields. We show that both axial effective diffusion and rotational diffusion decrease with confinement.

Reversible Cavitation-Induced Junctional Opening in an Artificial Endothelial Layer.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.

Translocation intermediates of ubiquitin through an α-hemolysin nanopore: implications for detection of post-translational modifications.

Nanopore based sensors constitute a promising approach to single molecule protein characterization being able, in principle, to detect sequences, structural elements and folding states of proteins and polypeptide chains. In narrow nanopores, one of the open issues concerns the coupling between unfolding and translocation. Here, we studied the ubiquitin translocation in an α-hemolysin nanopore, the most widely used pore for nanopore sensing, via all-atom molecular dynamics simulations. We completely characterize the co-translocational unfolding pathway finding that robust translocation intermediates are associated with the rearrangement of secondary structural elements, as also confirmed by coarse grained simulations. An interesting recurrent pattern is the clogging of the α-hemolysin constriction by an N-terminal ß-hairpin. This region of ubiquitin is the target of several post-translational modifications. We propose a strategy to detect post-translational modifications at the N-terminal using the α-hemolysin nanopore based on the comparison of the co-translocational unfolding signals associated with modified and unmodified proteins.

Insights into protein sequencing with an α-Hemolysin nanopore by atomistic simulations.

Single molecule protein sequencing would represent a disruptive burst in proteomic research with important biomedical impacts. Due to their success in DNA sequencing, nanopore based devices have been recently proposed as possible tools for the sequencing of peptide chains. One of the open questions in nanopore protein sequencing concerns the ability of such devices to provide different signals for all the 20 standard amino acids. Here, using equilibrium all-atom molecular dynamics simulations, we estimated the pore clogging in α-Hemolysin nanopore associated to 20 different homopeptides, one for each standard amino acid. Our results show that pore clogging is affected by amino acid volume, hydrophobicity and net charge. The equilibrium estimations are also supported by non-equilibrium runs for calculating the current blockades for selected homopeptides. Finally, we discuss the possibility to modify the α-Hemolysin nanopore, cutting a portion of the barrel region close to the trans side, to reduce spurious signals and, hence, to enhance the sensitivity of the nanopore.

Charge polarization, local electroneutrality breakdown and eddy formation due to electroosmosis in varying-section channels.

We characterize the dynamics of an electrolyte embedded in a varying-section channel under the action of a constant external electrostatic field. By means of molecular dynamics simulations we determine the stationary density, charge and velocity profiles of the electrolyte. Our results show that when the Debye length is comparable to the width of the channel bottlenecks a concentration polarization along with two eddies sets inside the channel. Interestingly, upon increasing the external field, local electroneutrality breaks down and charge polarization sets leading to the onset of net dipolar field. This novel scenario, that cannot be captured by the standard approaches based on local electroneutrality, opens the route for the realization of novel micro and nano-fluidic devices.

Amyloid Growth, Inhibition, and Real-Time Enzymatic Degradation Revealed with Single Conical Nanopore.

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.