Translocation Speed Research Articles

Detection of Single Proteins with a General Nanopore Sensor.

Single protein sensing based on solid-state nanopores is promising but challenging, because the fast translocation velocity of a protein is beyond the bandwidth of nanopore instruments. To decelerate the translocation speed, here, we employed a common protein cross-link interaction to achieve a general and robust nanopore sensing platform for single-molecule detection of protein. Benefiting from the EDC/NHS coupling interaction between nanopore and proteins, a 10-fold decrease in speed has been achieved. The clearly distinguishable current signatures further reveal that the anisotropictranslocationof a protein, which are horizontal, vertical, and flipping transit inside nanopore confinement. This strategy provides a general platform for rapid detection of proteins as well as exploring fundamental protein dynamics at the single-molecule level.

Short-chain oligonucleotide detection by glass nanopore using targeting-induced DNA tetrahedron deformation as signal amplifier

Glass capillary nanopore has been developed as a promising sensing platform for bioassay with single-molecule resolution. Although the diameter of glass capillary nanopore can be easily tuned, direct event-readouts of small biomacromolecules, like short-chain oligonucleotide fragments (within ∼20 nucleotides) remain great challenge, which limited by the configuration of the conical-shaped nanopore and the instrumental temporal resolution. Here, we exploit a smart strategy for glass nanopore detection of short-chain oligonucleotides by using relatively big-sized tetrahedral DNA nanostructures as a signal amplifier, which can amplify the signals and retard the translocation speed meanwhile. The tetrahedral DNA nanostructure with a hairpin loop sequence in one edge, undergoes a shape transformation upon the complementary combination of the target oligonucleotides, in which the presence of short-chain target oligonucleotide can be readout due to obvious variation in amplitude of ion current pulse that caused by volume change of the DNA tetrahedral. Therefore, this strategy is promising for extending glass nanopore sensing platform for sensitive detection of short-chain oligonucleotides.

Ultralow-aspect-ratio micropore sensor for a particle translocation: Critical role of the access region

In this paper, we analyze particle translocation through micropores with low aspect ratios. Micropores with diameters of 11.6 μm and various lengths (1000 nm, 500 nm, 100 nm, and 50 nm) are fabricated on a silicon nitride membrane via laser machining at a low aspect ratio. After measuring the translocation signals of a 5.4- μm particle, we find that the effects of the access region become more significant than those of the pore region for detecting signals at low aspect ratios. To clarify this mechanism, finite element method simulation and mathematical analysis are performed and we describe the relationship between an inhomogeneous electric field and the critical role of the access region. Additionally, we detect the translocation signal of a double particle. As a result, we achieve a decrease in translocation speed and improve signal detection at an ultralow aspect ratio, demonstrating the potential to detect the shapes of small particles directly.

DNA translocation through a nanopore in an ultrathin self-assembled peptide membrane

Here, we explore the possibility of using peptide-based materials as a membrane in solid-state nanopore devices in an effort to develop a sequence-specific, programmable biological membrane platform. We use a recently developed tyrosine-mediated self-assembly peptide sheet. At the air/water interface, the 5mer peptide YFCFY self-assembles into a uniform and robust two-dimensional (2D) structure, and the peptide sheet is easily transferred to a low-noise glass substrate. The thickness of the peptide membrane can be adjusted to approximately 5 nm (or even to 2 nm) by an etching process, and the diameters of the peptide nanopores can be precisely controlled using a focused electron beam with an attuned spot size. The ionic current noise of the peptide nanopore is comparable to those of typical silicon nitride nanopores or multilayer 2D materials. Using this membrane, we successfully observe translocation of 1000 bp double-stranded DNA with a sufficient signal-to-noise ratio of ∼30 and an elongated translocation speed of ∼1 bp μs−1. Our results suggest that the self-assembled peptide film can be used as a sensitive nanopore membrane and employed as a platform for applying biological functionalities to solid-state substrates.

Discrimination of single‐stranded DNA homopolymers by sieving out G‐quadruplex using tiny solid‐state nanopores

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single-base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G-quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20-nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105-nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore-based DNA sequencing at single-base level.

Mucus penetrating properties of soft, distensible lipid nanocapsules

Designing nanomaterials to release their drug pay-load upon exposure to an exogenous trigger can help to direct drug delivery, but how the triggered release, which often modifies the nanomaterial properties, influences the biological fate of these systems is currently unknown. The aim of this study was to investigate how the triggered drug release from PEG coated, soft, 50 nm distensible lipid nanocapsules (LNC) influenced their diffusion across a mucus barrier. The translocation speed of the non-triggered LNC across a 35 µm thick purified gastric mucin (PGM) barrier was 3 times faster (30.08 ± 2.49 × 10−10 cm2 s−1) compared to equivalent-sized negatively charged polystyrene particles (9.87 ± 0.61 × 10−10 cm2 s−1, p < 0.05). In cystic fibrosis mucus (CFM), harvested from patient primary cells, the non-triggered LNC translocation speed was similar to the PGM, but the polystyrene particle diffusion was so slow it could not be measured. The trigger induced LNC distension process had no effect on the particle diffusion rate in both PGM and CFM (p > 0.05) in a static mucus barrier, but when shear was applied to the barrier the distended LNCs diffused more slowly (3.97 ± 1.38 × 10−8 cm2 s−1, p < 0.05) compared to the non-distended materials (4.94 ± 0.04 × 10−8 cm2 s−1). This data suggested the rapid mucus penetration of the distended LNCs, despite their increased size, was a consequence of their capacity to take a less tortuous path through the barrier, i.e., they experienced less steric hinderance, compared to the non-distended LNC.

Molecular Dynamics Investigation of Polylysine Peptide Translocation through MoS2 Nanopores.

Solid-state nanopores (SSN) made of two-dimensional materials such as molybdenum disulfide (MoS2) have emerged as candidate devices for biomolecules sequencing. SSN sequencing is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels, in response to an external voltage applied across the membrane. Although several experiments on DNA translocation through SSNs have been performed in the past decade, translocation of proteins has been less studied, partly due to small protein size and detection limits. Moreover, the threading of proteins through nanopore channels is challenging, because proteins can exhibit neutral global charge and not be sensitive to the electric field. In this paper, we investigate the translocation of lysine residues and a model protein with polylysine tags through MoS2 nanoporous membranes using molecular dynamics simulations. Adding lysine tags to biological peptides is the method proposed here to promote the entrance of proteins through SSN. Specifically, we study the relationship existing between the translocation events and the ionic conductance signal drops. We show that individual lysine residues translocate easily through MoS2 nanopores, but the translocation speed is extremely fast, which leads to indiscernible ionic conductance drops. To reduce the translocation speed, we demonstrate that increasing the thickness of the membrane from single-layer to bilayer MoS2 reveals a stepwise process of translocation with discernible conductance drops that could be measured experimentally. Finally, a study of the threading of proteins with polylysine tags through MoS2 nanopores is presented. The addition of the positively charged tag to the neutral protein allows the threading and full translocation of the protein through the pore (at least two lysine residues are necessary in this case to observe translocation) and a similar sequence of translocation events is detected, independently of the tag length.

Revealing the Dynamics of Single-Molecule Reactions in a Single-Molecule Nanoreactor

The dynamics of bond breaking and formation at the single-molecule level are of both fundamental importance and practical significance. Tremendous efforts have been made over the last decade toward the visualization of such transient phenomenon, with quite a few amazing single-molecule techniques being designed and developed. However, it remains a major challenge to realize the truly single-molecule (cascade) reactions largely due to the stochastic nature of, e.g. molecular motions. Inspired by the enzymatic biosynthesis, the authors consider that a nanoscale confined space, or nanoreactor, must be essential to the highly ordered single-molecule reactions. Moreover, an ideal nanoreactor also needs to possess a well defined structure that is of comparable size to the individual reactant molecules. Hence, the rationally-mutated biological nanopores may be applied as a new brand of single-molecule nanoreactors, where the entry of reactants and exit of products are voltage-driven. Herein, a mutant areolysin was adopted to study the unique confinement induced selectivity of nanopore reactor. In addition to regio- or stereo-selectivity, it is hypothesized that the mismatch between the bond formation kinetics of reactant molecules and their translocation speeds can offer an extra level of control on reaction selectivity. To prove this concept, the reaction dynamics of three constitutional isomers, 2-, 3-, and 4-mercaptobenzoic acid, at seven identical reaction sites (i.e. at the same position on each monomer) were examined in this work. Surprisingly, those tiny structural variations of three isomers could be easily distinguished by not only the different current blockades caused by each tethered molecule, but also the most frequent and prolonged current levels as well. This observation shed light on the transient dynamics of single-molecule reactions within a single-molecule nanoreactor, providing a crucial first step to the ultimate objective of 100% yield of targeted product.

Electric-Field-Driven Translocation of ssDNA through Hydrophobic Nanopores

The accurate sequencing of DNA using nanopores requires control over the speed of DNA translocation through the pores and also of the DNA conformation. Our studies show that ssDNA translocates through hourglass-shaped pores with hydrophobic constriction regions when an electric field is applied. The constriction provides a barrier to translocation and thereby slows down DNA movement through the pore compared with pores without the constriction. We show that ssDNA moves through these hydrophobic pores in an extended conformation and therefore does not form undesirable secondary structures that may affect the accuracy of partial current blockages for DNA sequencing.

Determining the effects of DNA sequence on Hel308 helicase translocation along single-stranded DNA using nanopore tweezers.

Motor enzymes that process nucleic-acid substrates play vital roles in all aspects of genome replication, expression, and repair. The DNA and RNA nucleobases are known to affect the kinetics of these systems in biologically meaningful ways. Recently, it was shown that DNA bases control the translocation speed of helicases on single-stranded DNA, however the cause of these effects remains unclear. We use single-molecule picometer-resolution nanopore tweezers (SPRNT) to measure the kinetics of translocation along single-stranded DNA by the helicase Hel308 from Thermococcus gammatolerans. SPRNT can measure enzyme steps with subangstrom resolution on millisecond timescales while simultaneously measuring the absolute position of the enzyme along the DNA substrate. Previous experiments with SPRNT revealed the presence of two distinct substates within the Hel308 ATP hydrolysis cycle, one [ATP]-dependent and the other [ATP]-independent. Here, we analyze in-depth the apparent sequence dependent behavior of the [ATP]-independent step. We find that DNA bases at two sites within Hel308 control sequence-specific kinetics of the [ATP]-independent step. We suggest mechanisms for the observed sequence-specific translocation kinetics. Similar SPRNT measurements and methods can be applied to other nucleic-acid-processing motor enzymes.

Silicon nitride nanopore created by dielectric breakdown with a divalent cation: deceleration of translocation speed and identification of single nucleotides.

Nanopore DNA sequencing with a solid-state nanopore requires deceleration of the ultrafast translocation speed of single-stranded DNA (ssDNA). We report an unexpected phenomenon: controlled dielectric breakdown (CBD) with a divalent metal cation, especially Ca2+, provides a silicon nitride nanopore with the ability to decelerate ssDNA speed to 100 μs per base even after solution replacement. This speed is two orders of magnitude slower than that for CBD with a conventional monovalent metal cation. Temperature dependence experiments revealed that the enthalpic barrier for a nanopore created via CBD with Ca2+ is 25-30kBT, comparable to that of a biological nanopore. The slowing effect originates from the strong interaction between ssDNA and divalent cations, which were coated on the sidewall of the nanopore during the CBD process. In addition, we found that the nanopore created via CBD with Ca2+ can decelerate the speed of even single-nucleotide monomers, dNMPs, to 0.1-10 ms per base. The four single nucleotides could be statistically identified according to their blockade currents. Our approach is simple and practical because it simultaneously allows nanopore fabrication, ssDNA deceleration and the identification of nucleotide monomers.

Sculpturing wafer-scale nanofluidic devices for DNA single molecule analysis.

We present micro- and nanofluidic devices with 3D structures and nanochannels with multiple depths for the analysis of single molecules of DNA. Interfacing the nanochannels with graded and 3D inlets allows the improvement of the flow and controls not only the translocation speed of the DNA but also its conformation inside the nanochannels. The complex, multilevel, multiscale fluidic circuits are patterned in a simple, two-minute imprinting step. The stamp, the key of the technology, is directly milled by focused ion beam, which allows patterning nanochannels with different cross sections and depths, together with 3D transient inlets, all at once. Having such a variety of structures integrated in the same sample allows studying, optimizing and directly comparing their effect on the DNA flow. Here, DNA translocation is studied in long (160 µm) and short (5-40 µm) nanochannels. We study the homogeneity of the stretched molecules in long, meander nanochannels made with this technology. In addition, we analyze the effect of the different types of inlet structures interfacing short nanochannels. We observe pre-stretching and an optimal flow, and no hairpin formation, when the inlets have gradually decreasing widths and depths. In contrast, when the nanochannels are faced with an abrupt transition, we observe clogging and hairpin formation. In addition, 3D inlets strongly decrease the DNA molecules' speed before they enter the nanochannels, and help capturing more DNA molecules. The robustness and versatility of this technology and DNA testing results evidence the potential of imprinted devices in biomedical applications as low cost, disposable lab-on-a-chip devices.

Challenges of Single-Molecule DNA Sequencing with Solid-State Nanopores.

A powerful DNA sequencing tool with high accuracy, long read length and high-throughput would be required more and more for decoding the complicated genetic code. Solid-state nanopore has attracted many researchers for its promising future as a next-generation DNA sequencing platform due to the processability, the robustness and the large-scale integratability. While the diverse materials have been widely explored for a solid-state nanopore, silicon nitride (Si3N4) is especially preferable from the viewpoint of mass production based on semiconductor fabrication process. Here, as a nanopore sensing mechanism, we focused on the ionic blockade current method which is the most developed technique. We also highlight the main challenges of Si3N4 nanopore-based DNA sequencer that should be addressed: the fabrication of ultra-small nanopore and ultra-thin membrane, the modulation of DNA translocation speed and the detection of base-specific signals. In this chapter, we discuss the recent progress relating to solid-state nanopore DNA sequencing, which helps to provide a comprehensive information about the current technical situation.

Electric-Field-Driven Translocation of ssDNA through Hydrophobic Nanopores.

The accurate sequencing of DNA using nanopores requires control over the speed of DNA translocation through the pores and also of the DNA conformation. Our studies show that ssDNA translocates through hourglass-shaped pores with hydrophobic constriction regions when an electric field is applied. The constriction provides a barrier to translocation and thereby slows down DNA movement through the pore compared with pores without the constriction. We show that ssDNA moves through these hydrophobic pores in an extended conformation and therefore does not form undesirable secondary structures that may affect the accuracy of partial current blockages for DNA sequencing.

Controlling DNA translocation speed through graphene nanopore via plasmonic fields

We propose a novel plasmonic-based method for controlling translocation speed of DNA molecule through graphene nanopore. Dynamic properties of a double-stranded DNA molecule passage through a graphene nanopore are investigated by employing molecular dynamics simulation. Also, the effect of plasmonic fields parallel to the graphene plane on the translocation speed of the DNA molecule is studied. The DNA translocation speed is calculated for different values of confinement, spectral width, and power of the plasmonic field. Results show the potential of the method for controlling translocation speed of DNA via surface plasmons in graphene nanopore. The plasmon field power, confinement depth, and spectral width can increase translocation time of DNA up to 107, 62 and 15 %. Also, strong plasmon field can trap the DNA molecule in the nanopore. The suggested method can be utilized to solve the fast-translocation challenge of the nanopore DNA sequencers.

Translocation of polyampholytes and intrinsically disordered proteins⋆.

Polyampholytes are polymers carrying electrical charges of both signs along their backbone. We consider synthetic polyampholytes with a quenched random charge sequence and intrinsically disordered proteins, which have a well-defined charge sequence and behave like polyampholytes in the denaturated state. We study their translocation driven by an electric field through a pore. The role of disorder along the charge sequence of synthetic polyampholytes is analyzed. We show how disorder slows down the translocation dynamics. For intrinsically disordered proteins, the translocation vs. rejection rates by the pore depends on which end is engaged in the translocation channel. We discuss the rejection time, the blockade time distributions and the translocation speed for the charge sequence of two specific intrinsically disordered proteins differing in length and structure.

Nitrogen Doping of Carbon Nanoelectrodes for Enhanced Control of DNA Translocation Dynamics.

Controlling the dynamics of DNA translocation is a central issue in the emerging nanopore-based DNA sequencing. To address the potential of heteroatom doping of carbon nanostructures and for achieving this goal, herein, we carry out atomistic molecular dynamics simulations for single-stranded DNAs translocating between two pristine or doped carbon nanotube (CNT) electrodes. Specifically, we consider the substitutional nitrogen doping of capped CNT (capCNT) electrodes and perform two types of molecular dynamics simulations for the entrapped and translocating single-stranded DNAs. We find that the substitutional nitrogen doping of capCNTs facilitates and stabilizes the edge-on nucleobase configurations rather than the original face-on ones and slows down the DNA translocation speed by establishing hydrogen bonds between the N dopant atoms and nucleobases. Due to the enhanced interactions between DNAs and N-doped capCNTs, the duration time of nucleobases within the nanogap was extended by up to ∼300%. Given the possibility to be combined with the extrinsic light or gate voltage modulation methods, the current work demonstrates that the substitutional nitrogen doping is a promising direction for the control of DNA translocation dynamics through a nanopore or nanogap, based of carbon nanomaterials.

Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis.

Selectivity and sensitivity are two key parameters utilized to describe the performance of a sensor. In order to investigate selectivity and sensitivity of the aerolysin nanosensor, we manipulated its surface charge at different locations via single site-directed mutagenesis. To study the selectivity, we replaced the positively charged R220 at the entrance of the pore with negatively charged glutamic acid, resulting in barely no current blockages for sensing negatively charged oligonucleotides. For the sensitivity, we substituted the positively charged lumen-exposed amino acid K238 located at trans-ward third of the β-barrel stem with glutamic acid. This leads to a surprisingly longer duration time at +140 mV, which is about 20 times slower in translocation speed for Poly(dA)4 compared to that of wild-type aerolysin, indicating the stronger pore-analyte interactions and enhanced sensitivity. Therefore, it is both feasible and understandable to rationally design confined biological nanosensors for single molecule detection with high selectivity and sensitivity.

Actuating Single Nano‐Oscillators with Light

AbstractThermoresponsive polymers can be used to rapidly actuate individual nanoparticles forming nanomechanical oscillators. Such systems are realized here with poly(N‐isopropylacrylamide) (PNIPAM)‐coated gold nanoparticles deposited on gold films, which have a strong optical response that can be dynamically tracked. Changes in temperature surrounding the nanoparticles result in the contraction or expansion of the PNIPAM coating, thereby displacing the gold nanoparticles up and down on the gold mirror. Particle oscillation is optically monitored via the spectral position of the coupled plasmon modes, achieving sub‐millisecond translocation speeds when actuated by light via plasmonic heating. These fast nano‐oscillators can also generate forces exceeding 1 nN with efficiencies already exceeding 1%, which makes them ideal prototypes for nanomachines.

Single-Molecule Nanomechanics of Kinesin and Kinesin-Family Proteins

There are roughly 45 kinesin superfamily members coded by the human genome, and 25 by the fruit fly genome: these motor proteins can be classified into at least 14 different subtypes. An individual cell typically expresses up to a dozen-or-more different versions at any given time. Although the nanomechanical properties of the founding member of the superfamily, kinesin-1, are well established, it's been an ongoing challenge to understand the physiology of the other kinesin members, and in particular, how that physiology ties into their varied cellular functions. It's become clear that different kinesin motors exhibit radically different nanomechanical properties, including wide variations in translocation speed, processivity, randomness, and response to load. These properties dictate not only the diverse range of behaviors exhibited by individual motors, but also the behavior of motors working in ensembles, for example, when multiple motors are attached to a common cargo. For a number of years, my lab has been using single-molecule optical trapping to characterize the nanomechanical properties of kinesin family motors in detail, including Eg-5 (kinesin-5), KIF17 and KIF3AB (kinesin-2), and KIF15 (kinesin-12), as well as variants of kinesin-1. A common feature that has emerged is that different kinesin motors can all be modeled by a similar four-state, biochemical cycle involving a partial docking of the neck linker. This minimal model accounts quantitatively for forward stepping, backward stepping, and eventual microtubule release, adjusting only the transition rates among states for different motors. In particular, the substantial nanomechanical differences exhibited by two kinesin-2 motors, KIF17 and KIF3AB, which act jointly to drive intraflagellar transport, can explain in vivo data showing that these motors “trade off” while transporting vesicular cargo along the length of cilium (Prevo at el., 2015, Nat. Cell. Biol. 17:1546-45; Milic et al., 2017 Proc. Nat. Acad. Sci. USA 114:E6830-E6838).