RESUMO
Ultrathin nanopore sensors allow single-molecule and polymer measurements at sub-microsecond time resolution enabled by high current signals (â¼10-30 nA). We demonstrate for the first time the experimental probing of the ultrafast translocation and folded dynamics of double-stranded DNA (dsDNA) through a nanopore at 10 MHz bandwidth with acquisition of data points per 25 ns (150 MB/s). By introducing a rigorous algorithm, we are able to accurately identify each current level present within translocation events and elucidate the dynamic folded and unfolded behaviors. The remarkable sensitivity of this system reveals distortions of short-lived folded states at a lower bandwidth. This work revisits probing of dsDNA as a model polymer and develops broadly applicable methods. The combined improvements in sensor signals, instrumentation, and large data analysis methods uncover biomolecular dynamics at unprecedentedly small time scales.
Assuntos
Nanoporos , Polímeros , Nanotecnologia/métodos , DNA/análiseRESUMO
We demonstrate DNA translocations through silicon nitride pores formed by simple chemical etching on glass substrates using microscopic amounts of hydrofluoric acid. DNA translocations and transmission electron microscopy (TEM) prove the fabrication of nanopores and allow their characterization. From ionic measurements on 318 chips, we report the effective pore diameters ranging from zero (pristine membranes) and sub-nm to over 100 nm, within 50 µm diameter membranes. The combination of ionic conductance, DNA current blockades, TEM imaging, and electron energy loss spectroscopy (EELS) provides comprehensive information about the pore area and number, from single to few pores, and pore structure. We also show the formation of thinned membrane regions as precursors of pores. The average pore density, about 5 × 10-4 pores/µm2, allows pore number adjustment statistically (0, 1, or more). This simple and affordable chemical method for making solid-state nanopores accelerates their adoption for DNA sensing and characterization applications.
Assuntos
Nanoporos , Compostos de Silício/química , DNA/química , Microscopia Eletrônica de Transmissão , ÍonsRESUMO
We fabricate on-chip solid-state nanofluidic-2D nanopore systems that can limit the range of motion for DNA in the sensing region of a nanopore. We do so by creating devices containing one or more silicon nitride pores and silicon nitride pillars supporting a 2D pore that orient DNA within a nanopore device to a restricted geometry, yet allow the free motion of ions to maintain a high signal-to-noise ratio. We discuss two concepts with two and three independent electrical connections and corresponding nanopore chip device architectures to achieve this goal in practice. Here, we describe device fabrication and transmission electron microscope (TEM) images, and provide simulated translocations based on the finite element analysis in 3D to demonstrate its merit. In both methods, there is a main 2D nanopore which we refer to as a "sensing" nanopore (monolayer MoS2 in this paper). A secondary layer is either an array of guiding pores sharing the same electrode pair as the sensing pore (Method 1) or a single, independently contacted, guiding pore (Method 2). These pores are constructed parallel to the "sensing" pore and serve as "guiding" elements to stretch and feed DNA into the atomically thin sensing pore. We discuss the practical implementation of these concepts with nanofluidic and Si-based technology, including detailed fabrication steps and challenges involved for DNA applications in solution.
Assuntos
Nanoporos , DNA , ÍonsRESUMO
Nanopore sensing is a powerful tool for the detection of biomolecules. Solid-state nanopores act as single-molecule sensors that can function in harsh conditions. Their resilient nature makes them attractive candidates for taking this technology into the field to measure environmental samples for life detection in space and water quality monitoring. Here, we discuss the fabrication of silicon nitride pores from â¼1.6 to 20 nm in diameter in 20-nm-thick silicon nitride membranes suspended on glass chips and their performance. We detect pure laboratory samples containing a single analyte including DNA, BSA, microRNA, TAT, and poly-D-lys-hydrobromide. We also measured an environmental (mixed-analyte) sample, containing Antarctic dirt provided by NASA Ames. For DNA measurements, in addition to using KCl and NaCl solutions, we used the artificial (synthetic) seawater, which is a mixture of different salts mimicking the composition of natural seawater. These samples were spiked with double-stranded DNA (dsDNA) fragments at different concentrations to establish the limits of nanopore sensitivity in candidate environment conditions. Nanopore chips were cleaned and reused for successive measurements. A stand-alone, 1-MHz-bandwidth Chimera amplifier was used to determine the DNA concentration in artificial seawater that we can detect in a practical time scale of a few minutes. We also designed and developed a new compact nanopore reader, a portable read-out device with miniaturized fluidic cells, which can obtain translocation data at bandwidths up to 100 kHz. Using this new instrument, we record translocations of 400 bp, 1000 bp, and 15000 bp dsDNA fragments and show discrimination by analysis of current amplitude and event duration histograms.
Assuntos
DNA/análise , Membranas Artificiais , Nanoporos , Água do Mar/análise , Compostos de Silício/química , Regiões Antárticas , DNA/química , Água do Mar/químicaRESUMO
Accurate and low-cost analysis of biomolecules is important for many applications. This work seeks to further improve the measurement bandwidths achievable with solid-state nanopores, which have emerged as an important platform for this analysis. We report single-stranded DNA translocation recordings at a bandwidth of 10 MHz copolymers of 80 (C20A20C20A20), 90 (C30A30C30), and 200 (C50A50C50A50) nucleotides through Si nanopores with effective diameters of 1.4-2.1 nm and effective membrane thicknesses 0.5-8.9 nm. By optimizing glass chips with thin nanopores and by integrating them with custom-designed amplifiers based on complementary metal-oxide-semiconductor technology, this work demonstrates detection of translocation events as brief as 100 ns with a signal-to-noise ratio exceeding seven at a measurement bandwidth of 10 MHz. We also report data robustness and variability across 13 pores of similar size and thickness, yielding a current blockade between 30 and 60% with a mean ionic current blockade (ΔI) of â¼3-9 nA and a characteristic dwell time of â¼2-21 ns per nucleotide. These measurements show that characteristic translocation rates are at least 10 times faster than previously recorded. We detect transient intraevent fluctuations, multiple current levels within translocation events, and variability of DNA translocation event signatures and durations.
Assuntos
DNA de Cadeia Simples/química , Vidro/química , Nanoporos , Metais/química , Óxidos/química , Semicondutores , Processamento de Sinais Assistido por ComputadorRESUMO
Despite the potential for nanopores to be a platform for high-bandwidth study of single-molecule systems, ionic current measurements through nanopores have been limited in their temporal resolution by noise arising from poorly optimized measurement electronics and large parasitic capacitances in the nanopore membranes. Here, we present a complementary metal-oxide-semiconductor (CMOS) nanopore (CNP) amplifier capable of low noise recordings at an unprecedented 10 MHz bandwidth. When integrated with state-of-the-art solid-state nanopores in silicon nitride membranes, we achieve an SNR of greater than 10 for ssDNA translocations at a measurement bandwidth of 5 MHz, which represents the fastest ion current recordings through nanopores reported to date. We observe transient features in ssDNA translocation events that are as short as 200 ns, which are hidden even at bandwidths as high as 1 MHz. These features offer further insights into the translocation kinetics of molecules entering and exiting the pore. This platform highlights the advantages of high-bandwidth translocation measurements made possible by integrating nanopores and custom-designed electronics.
Assuntos
DNA de Cadeia Simples/análise , Nanoporos , Semicondutores , NanotecnologiaRESUMO
Protein analysis using solid-state nanopores is challenging due to limitations in bandwidth and signal-to-noise ratio. Recent improvements of those two aspects have made feasible the study of small peptides using solid-state nanopores, which have an advantage over biological counterparts in tunability of the pore diameter. Here, we report on the detection and characterization of peptides as small as 33 amino acids. Silicon nitride nanopores with thicknesses less than 10 nm are used to provide signal-to-noise (S/N) levels up to S/N â¼ 10 at 100 kHz. We demonstrate differentiation of monomer and dimer forms of the GCN4-p1 leucine zipper, a coiled-coil structure well studied in molecular biology, and compare with the unstructured 33-residue monomer. GCN4-p1 is sequence segment associated with homodimerization of the transcription factor General Control Nonderepressible 4 (GCN4), which is involved in the control of amino acid synthesis in yeast. The differentiation between two oligomeric forms demonstrates the capabilities of improved solid-state nanopore platforms to extract structural information involving short peptide structures.
Assuntos
Nanoporos/ultraestrutura , Dobramento de Proteína , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Dimerização , Saccharomyces cerevisiae/químicaRESUMO
One primary goal in nanobiotechnology is designing new methodologies for molecular biomedical diagnosis at stages much earlier than currently possible and without use of expensive reagents and sophisticated equipment. In this work, we show the proof of principle for single-molecule detection of the nucleocapsid protein 7 (NCp7), a protein biomarker of the HIV-1 virus, using synthetic nanopores and the resistive-pulse technique. The biosensing mechanism relied upon specific interactions between NCp7 and aptamers of stem-loop 3 (SL3) in the packaging domain of the retroviral RNA genome. One critical step of this study was the choice of the optimal size of the nanopores for accurate, label-free determinations of the dissociation constant of the NCp7 protein-SL3 RNA aptamer complex. Therefore, we systematically investigated the NCp7 protein-SL3 RNA aptamer complex employing two categories of nanopores in a silicon nitride membrane: (i) small, whose internal diameter was smaller than 6 nm, and (ii) large, whose internal diameter was in the range of 7 to 15 nm. Here, we demonstrate that only the use of nanopores with an internal diameter that is smaller than or comparable with the largest cross-sectional size of the NCp7-SL3 aptamer complex enables accurate measurement of the dissociation constant between the two interacting partners. Notably, this determination can be accomplished without the need for prior nanopore functionalization. Moreover, using small solid-state nanopores, we demonstrate the ability to detect drug candidates that inhibit the binding interactions between NCp7 and SL3 RNA by using a test case of N-ethylmaleimide.
Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , HIV-1/isolamento & purificação , Nanoporos , Nanotecnologia/instrumentação , Produtos do Gene gag do Vírus da Imunodeficiência Humana/análise , Biomarcadores/análise , Desenho de Equipamento , Análise de Falha de Equipamento , HIV-1/metabolismoRESUMO
Using rational membrane protein design, we were recently able to obtain a ß-barrel protein nanopore that was robust under an unusually broad range of experimental circumstances. This protein nanopore was based upon the native scaffold of the bacterial ferric hydroxamate uptake component A (FhuA) of Escherichia coli. In this work, we expanded the examinations of the open-state current of this engineered protein nanopore, also called FhuA ΔC/Δ4L, employing an array of lipid bilayer systems that contained charged and uncharged as well as conical and cylindrical lipids. Remarkably, systematical single-channel analysis of FhuA ΔC/Δ4L indicated that most of its biophysical features, such as the unitary conductance and the stability of the open-state current, were not altered under the conditions tested in this work. However, electrical recordings at high transmembrane potentials revealed that the presence of conical phospholipids within the bilayer catalyzes the first, stepwise current transition of the FhuA ΔC/Δ4L protein nanopore to a lower-conductance open state. This study reinforces the stability of the open-state current of the engineered FhuA ΔC/Δ4L protein nanopore under various experimental conditions, paving the way for further critical developments in biosensing and molecular biomedical diagnosis.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Lipídeos/química , Nanoporos , Engenharia de Proteínas/métodos , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofísica/métodos , Técnicas Biossensoriais , Condutividade Elétrica , Eletrofisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Potenciais da Membrana , Conformação Molecular , Nanopartículas/química , Nanotecnologia/métodos , Estrutura Secundária de ProteínaRESUMO
Extensive engineering of protein nanopores for biotechnological applications using native scaffolds requires further inspection of their internal geometry and size. Recently, we redesigned ferric hydroxamate uptake component A (FhuA), a 22-ß-stranded protein containing an N-terminal 160-residue cork domain (C). The cork domain and four large extracellular loops (4L) were deleted to obtain an unusually stiff engineered FhuA ΔC/Δ4L nanopore. We employed water-soluble poly(ethylene glycols) and dextran polymers to examine the interior of FhuA ΔC/Δ4L. When this nanopore was reconstituted into a synthetic planar lipid bilayer, addition of poly(ethylene glycols) produced modifications in the single-channel conductance, allowing for the evaluation of the nanopore diameter. Here, we report that FhuA ΔC/Δ4L features an approximate conical internal geometry with the cis entrance smaller than the trans entrance, in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Further experiments with impermeable dextran polymers indicated an average internal diameter of ~2.4 nm, a conclusion we arrived at based upon the polymer-induced alteration of the access resistance contribution to the nanopore's total resistance. Molecular insights inferred from this work represent a platform for future protein engineering of FhuA that will be employed for specific tasks in biotechnological applications.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Nanoporos , Polímeros/metabolismo , Engenharia de Proteínas , Fenômenos Biofísicos , Dextranos/metabolismo , Escherichia coli , Ativação do Canal Iônico , Modelos Moleculares , Peso Molecular , Polietilenoglicóis/metabolismo , Deleção de Sequência , Solubilidade , Água/químicaRESUMO
Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids, the unfolding of proteins, and binding affinity of different ligands. By coupling these nanopores to the resistive-pulse technique, such measurements can be done under a wide variety of conditions and without the need for labeling. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.
Assuntos
Nanoporos , Proteínas/química , Adsorção , Compostos de Nitrogênio/química , Ligação Proteica , Proteínas/metabolismoRESUMO
Understanding the interactions between silicon-based materials and proteins from the bloodstream is of key importance in a myriad of realms, such as the design of nanofluidic devices and functional biomaterials, biosensors, and biomedical molecular diagnosis. By using nanopores fabricated in 20 nm-thin silicon nitride membranes and highly sensitive electrical recordings, we show single-molecule observation of nonspecific protein adsorption onto an inorganic surface. A transmembrane potential was applied across a single nanopore-containing membrane immersed into an electrolyte-filled chamber. Through the current fluctuations measured across the nanopore, we detected long-lived captures of bovine serum albumin (BSA), a major multifunctional protein present in the circulatory system. Based upon single-molecule electrical signatures observed in this work, we judge that the bindings of BSA to the nitride surface occurred in two distinct orientations. With some adaptation and further experimentation, this approach, applied on a parallel array of synthetic nanopores, holds potential for use in methodical quantitative studies of protein adsorption onto inorganic surfaces.