RESUMO
Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of â¼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (â¼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.
Assuntos
Citocromos c/fisiologia , Transporte Proteico/fisiologia , Constrição , Citocromos c/química , Eletricidade , Modelos Moleculares , Nanoporos , Dobramento de Proteína , Desdobramento de Proteína , Compostos de Silício/química , TermodinâmicaRESUMO
The use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter, we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers, which exhibit deleterious leakage under similar conditions. Furthermore, insertion and functional analysis of channels such as α-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that α-hemolysin channels maintain their structural integrity at 2 M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.
Assuntos
Bicamadas Lipídicas , Polímeros , Guanidina , Proteínas HemolisinasRESUMO
We have recently shown that nanopore zero-mode waveguides are effective tools for capturing picogram levels of long DNA fragments for single-molecule DNA sequencing. Despite these key advantages, the manufacturing of large arrays is not practical due to the need for serial nanopore fabrication. To overcome this challenge, we have developed an approach for the wafer-scale fabrication of waveguide arrays on low-cost porous membranes, which are deposited using molecular-layer deposition. The membrane at each waveguide base contains a network of serpentine pores that allows for efficient electrophoretic DNA capture at picogram levels while eliminating the need for prohibitive serial pore milling. Here, we show that the loading efficiency of these porous waveguides is up to 2 orders of magnitude greater than their nanopore predecessors. This new device facilitates the scaling-up of the process, greatly reducing the cost and effort of manufacturing. Furthermore, the porous zero-mode waveguides can be used for applications that benefit from low-input single-molecule real-time sequencing.
Assuntos
DNA/química , Nanoporos/ultraestrutura , Nanotecnologia/instrumentação , Análise de Sequência de DNA/instrumentação , Asbestos Serpentinas/química , DNA/isolamento & purificação , Desenho de Equipamento , Membranas Artificiais , PorosidadeRESUMO
We present the first detailed experimental observation and analysis of nanoparticle electrophoresis through a nanochannel obtained with synchronous high-bandwidth electrical and camera recordings. Optically determined particle diffusion coefficients agree with values extracted from fitting electrical transport measurements to distributions from 1D Fokker-Planck diffusion-drift theory. This combined tracking strategy enables optical recognition and electrical characterization of nanoparticles in solution, which can have a broad range of applications in biology and materials science.
Assuntos
Eletroforese/instrumentação , Dispositivos Lab-On-A-Chip , Nanopartículas/análise , Difusão , Dimetilpolisiloxanos/química , Desenho de Equipamento , Corantes Fluorescentes/análise , Nanotecnologia/instrumentação , Óptica e Fotônica/instrumentação , Tamanho da Partícula , Gravação em VídeoRESUMO
Freestanding graphene membranes are unique materials. The combination of atomically thin dimensions, remarkable mechanical robustness, and chemical stability make porous and non-porous graphene membranes attractive for water purification and various sensing applications. Nanopores in graphene and other 2D materials have been identified as promising devices for next-generation DNA sequencing based on readout of either transverse DNA base-gated current or through-pore ion current. While several ground breaking studies of graphene-based nanopores for DNA analysis have been reported, all methods to date require a physical transfer of the graphene from its source of production onto an aperture support. The transfer process is slow and often leads to tears in the graphene that render many devices useless for nanopore measurements. In this work, we report a novel scalable approach for site-directed fabrication of pinhole-free graphene nanomembranes. Our approach yields high quality few-layer graphene nanomembranes produced in less than a day using a few steps that do not involve transfer. We highlight the functionality of these graphene devices by measuring DNA translocation through electron-beam fabricated nanopores in such membranes.
Assuntos
Grafite/química , Grafite/síntese química , Membranas Artificiais , Nanopartículas/química , Nanotecnologia/métodos , DNA/química , Condutividade Elétrica , Íons , NanoporosRESUMO
High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 µs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ~50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of µ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.
Assuntos
Técnicas Biossensoriais/métodos , Potenciais da Membrana , Nanoporos , Proteínas/química , Sequência de Aminoácidos , Háfnio/química , Membranas Artificiais , Dados de Sequência Molecular , Óxidos/química , Permeabilidade , Proteínas/análise , Compostos de Silício/químicaRESUMO
We report on DNA translocations through nanopores created in graphene membranes. Devices consist of 1-5 nm thick graphene membranes with electron-beam sculpted nanopores from 5 to 10 nm in diameter. Due to the thin nature of the graphene membranes, we observe larger blocked currents than for traditional solid-state nanopores. However, ionic current noise levels are several orders of magnitude larger than those for silicon nitride nanopores. These fluctuations are reduced with the atomic-layer deposition of 5 nm of titanium dioxide over the device. Unlike traditional solid-state nanopore materials that are insulating, graphene is an excellent electrical conductor. Use of graphene as a membrane material opens the door to a new class of nanopore devices in which electronic sensing and control are performed directly at the pore.
Assuntos
DNA/química , Membranas Artificiais , Nanoestruturas , Transporte BiológicoRESUMO
We present experimental and theoretical results on the voltage-dependent escape dynamics of DNA hairpins of different orientations threaded into an alpha -hemolysin channel. Using a coarse-grained formulation, we map the motion of the polymer in the pore to that of a biased single-particle random walk along the translocation coordinate. By fitting the escape probability distributions obtained from theory to experimental data, we extract the voltage-dependent diffusion constants and bias-induced velocities. Using our two-parameter theory, we obtain excellent agreement with experimentally measured escape time distributions. Further, we find that the ratio of mean escape times for hairpins of different orientations is strongly voltage dependent, with the ratio of 3' - to 5' -threaded DNA decreasing from approximately 1.7 to approximately 1 with increasing assisting voltages V(a) . We also find that our model describes 5' -threaded DNA escape extremely well, while providing inadequate fits for 3' escape. Finally, we find that the escape times for both orientations are equal for high assisting voltages, suggesting that the interactions of DNA with the alpha -hemolysin channel are both orientation and voltage dependent.
Assuntos
Biofísica/métodos , DNA/química , Proteínas Hemolisinas/química , Polímeros/química , Difusão , Modelos Químicos , Modelos Moleculares , Modelos Estatísticos , Conformação Molecular , Conformação de Ácido Nucleico , Probabilidade , Conformação Proteica , Eletricidade Estática , Fatores de TempoRESUMO
Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.
Assuntos
Técnicas Biossensoriais/métodos , Nanoporos , Temperatura , Proteínas Virais/metabolismo , Biopolímeros/análise , Lipídeos/química , Peptídeos/metabolismo , Estabilidade ProteicaRESUMO
Molybdenum disulfide (MoS2) flakes can grow beyond the edge of an underlying substrate into a planar freestanding crystal. When the substrate edge is in the form of an aperture, reagent-limited nucleation followed by edge growth facilitate direct and selective growth of freestanding MoS2 membranes. We have found conditions under which MoS2 grows preferentially across micrometer-scale prefabricated solid-state apertures in silicon nitride membranes, resulting in sealed membranes that are one to a few atomic layers thick. We have investigated the structure and purity of our membranes by a combination of atomic-resolution transmission electron microscopy, elemental analysis, Raman spectroscopy, photoluminescence spectroscopy, and low-noise ion-current recordings through nanopores fabricated in such membranes. Finally, we demonstrate the utility of fabricated ultrathin nanopores in such membranes for single-stranded DNA translocation detection.
Assuntos
Membranas Artificiais , Molibdênio/química , Nanoporos , DNA de Cadeia Simples/química , Silício/química , Sulfatos/químicaRESUMO
We present a study of double- and single-stranded DNA transport through nanopores fabricated in ultrathin (2-7 nm thick) freestanding hafnium oxide (HfO2) membranes. The high chemical stability of ultrathin HfO2 enables long-lived experiments with <2 nm diameter pores that last several hours, in which we observe >50 000 DNA translocations with no detectable pore expansion. Mean DNA velocities are slower than velocities through comparable silicon nitride pores, providing evidence that HfO2 nanopores have favorable physicochemical interactions with nucleic acids that can be leveraged to slow down DNA in a nanopore.
Assuntos
DNA/química , Háfnio/química , Nanoporos , Óxidos/química , Transporte Biológico , Biopolímeros/química , Técnicas Biossensoriais , DNA de Cadeia Simples , Teste de Materiais , Membranas Artificiais , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanotecnologia/métodos , Politetrafluoretileno/química , Compostos de Silício/química , Propriedades de SuperfícieRESUMO
Nanopores in thin solid-state membranes are used to rapidly analyze individual RNA/drug complexes. The interactions of a truncated A-site RNA model of the prokaryotic ribosome with aminoglycoside antibiotics are characterized by passing individual molecules through a 3-3.5 nm diameter pore fabricated in a 8-10 nm thick silicon nitride membrane. Complexes of the A-site RNA with aminoglycosides can be distinguished from unbound A-site based on the ion current signatures produced as they pass through the nanopores. Counting the fraction of free and drug-bound molecules affords label-free drug-RNA binding isotherms consistent with literature reports and with data generated using independent fluorescence-based assays. Our measurements are supported by molecular dynamics simulations, which illustrate the relationship between the ionic current and complexation of the A-site RNA with paramomycin, a prototypical aminoglycoside antibiotic.
Assuntos
Eletroporação/métodos , Membranas Artificiais , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Paromomicina/química , RNA/química , Antibacterianos/química , Sítios de Ligação , Difusão , Teste de Materiais , Porosidade , SemicondutoresRESUMO
Gold nanoparticle (NP) mono- and multilayers were constructed on gold surfaces using coordination chemistry. Hydrophilic Au NPs (6.4 nm average core diameter), capped with a monolayer of 6-mercaptohexanol, were modified by partial substitution of bishydroxamic acid disulfide ligand molecules into their capping layer. A monolayer of the ligand-modified Au NPs was assembled via coordination with Zr4+ ions onto a semitransparent Au substrate (15 nm Au, evaporated on silanized glass and annealed) precoated with a self-assembled monolayer of the bishydroxamate disulfide ligand. Layer-by-layer construction of NP multilayers was achieved by alternate binding of Zr4+ ions and ligand-modified NPs onto the first NP layer. Characterization by atomic force microscopy (AFM), ellipsometry, wettability, transmission UV-vis spectroscopy, and cross-sectional transmission electron microscopy showed regular growth of NP layers, with a similar NP density in successive layers and gradually increased roughness. The use of coordination chemistry enables convenient step-by-step assembly of different ligand-possessing components to obtain elaborate structures. This is demonstrated by introducing nanometer-scale vertical spacing between a NP layer and the gold surface, using a coordination-based organic multilayer. Electrical characterization of the NP films was carried out using conductive AFM, emphasizing the barrier properties of the organic spacer multilayer. The results exhibit the potential of coordination self-assembly in achieving highly controlled composite nanostructures comprising molecules, NPs, and other ligand-derivatized components.