RESUMO
Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.
Assuntos
DNA , Simulação de Dinâmica Molecular , Nanoporos , Transistores Eletrônicos , DNA/químicaRESUMO
We present a synthetic nanoscale piston that uses chemical energy to perform molecular transport against an applied bias. Such a device comprises a 13 by 5 nm protein cylinder, embedded in a biological membrane enclosing a single-stranded DNA (ssDNA) rod. Hybridization with DNA cargo rigidifies the rod, allowing for transport of a selected DNA molecule across the nanopore. A strand displacement reaction from ssDNA fuel on the other side of the membrane then liberates the DNA cargo back into solution and regenerates the initial configuration. The entropic penalty of ssDNA confinement inside the nanopore drives DNA transport regardless of the applied bias. Multiple automated and reciprocating cycles are observed, in which the DNA piston moves through the 10 nm length of the nanopore. In every cycle, a single DNA molecule is transported across the nanopore against an external bias force, which is the hallmark of biological transporters.
Assuntos
Nanoporos , Transporte Biológico Ativo , DNA/genética , DNA de Cadeia Simples , NanotecnologiaRESUMO
The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible.
Assuntos
Nanoporos , Aminoácidos/química , Hemoglobinas , Transporte de Íons , Simulação de Dinâmica MolecularRESUMO
Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76-81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.
Assuntos
Nanoporos , Engenharia de Proteínas , Proteínas/isolamento & purificação , Eletricidade , Eletroforese , Proteínas Fúngicas , Proteínas Hemolisinas , Humanos , Bicamadas Lipídicas , Dobramento de ProteínaRESUMO
Applications such as nucleic acid synthesis or next-generation sequencing involve repeated fluidic cycles with the same set of reagents. The large dead volumes present in external valves and pumps with relatively long supply lines mandate the inclusion of extensive rinsing steps in current protocols, resulting in the consumption of significant quantities of reagents. To allow for fast rinsing, to reduce reagent consumption, and to ensure high reagent purity, we propose a fluidic concept based on a hierarchical branching structure. The working principle comprises a 3D fluidic network of supply lines - one line per reagent - that ensures reagents to be provided up to the entrance of every single reaction cavity, called supercells. Because all reagents are always present inside or at the inlet of a supercell, the principle allows for very rapid reagent switching, while a continuous flow avoids cross contamination. Selection of a specific reagent to enter the supercells is controlled by adjusting the pressure over different supply lines. As the pressure is regulated by a single, external controller per reagent, no integrated valves are needed. The very small distances to the reaction cavities also results in the use of minimal reagent volumes and, hence, largely reduces operational costs. We demonstrated the working principle of this concept and show an average switching time of 0.23 ± 0.09 s for the current design at a flow rate of 10 nL s-1. We used a 10 × 10 matrix of supercells to validate the fluidic concept to be scalable towards a large number of reaction sites. In summary, we believe the presented fluidic 3D hierarchical concept allows designing flow cells that enable highly parallel, more cost-efficient, and faster work flows for applications requiring many reagent cycles.
RESUMO
Single-molecule nanopore electrophysiology is an emerging technique for the detection of analytes in aqueous solutions with high sensitivity. These detectors have proven applicable for the enzyme-assisted sequencing of oligonucleotides. There has recently been an increased interest in the use of nanopores for the fingerprinting of peptides and proteins, referred to as single-molecule nanopore spectrometry. However, the analysis of the resulting electrophysiology traces remains complicated due to the fast unassisted translocation of such analytes, usually in the order of micro- to milliseconds, and the small ion current signal produced (in the picoampere range). Here, we present the application of a generalized normal distribution function (gNDF) for the characterization of short-lived ion current signals (blockades). We show that the gNDF can be used to determine if the observed blockades have adequate time to reach their maximum current plateau while also providing a description of each blockade based on the open pore current (I O), the difference caused by the pore blockade (ΔI B), the position in time (µ), the standard deviation (σ), and a shape parameter (ß), leaving only the noise component. In addition, this method allows the estimation of an ideal range of low-pass filter frequencies that contains maximum information with minimal noise. In summary, we show a parameter-free and generalized method for the analysis of short-lived ion current blockades, which facilitates single-molecule nanopore spectrometry with minimal user bias.
RESUMO
Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin.
Assuntos
Actinas , Proteínas dos Microfilamentos , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , PolimerizaçãoRESUMO
Despite the broad success of biological nanopores as powerful instruments for the analysis of proteins and nucleic acids at the single-molecule level, a fast simulation methodology to accurately model their nanofluidic properties is currently unavailable. This limits the rational engineering of nanopore traits and makes the unambiguous interpretation of experimental results challenging. Here, we present a continuum approach that can faithfully reproduce the experimentally measured ionic conductance of the biological nanopore Cytolysin A (ClyA) over a wide range of ionic strengths and bias potentials. Our model consists of the extended Poisson-Nernst-Planck and Navier-Stokes (ePNP-NS) equations and a computationally efficient 2D-axisymmetric representation for the geometry and charge distribution of the nanopore. Importantly, the ePNP-NS equations achieve this accuracy by self-consistently considering the finite size of the ions and the influence of both the ionic strength and the nanoscopic scale of the pore on the local properties of the electrolyte. These comprise the mobility and diffusivity of the ions, and the density, viscosity and relative permittivity of the solvent. Crucially, by applying our methodology to ClyA, a biological nanopore used for single-molecule enzymology studies, we could directly quantify several nanofluidic characteristics difficult to determine experimentally. These include the ion selectivity, the ion concentration distributions, the electrostatic potential landscape, the magnitude of the electro-osmotic flow field, and the internal pressure distribution. Hence, this work provides a means to obtain fundamental new insights into the nanofluidic properties of biological nanopores and paves the way towards their rational engineering.
Assuntos
Nanoporos , Simulação por Computador , Íons , Nanotecnologia , Eletricidade EstáticaRESUMO
The ability to confine and to study single molecules has enabled important advances in natural and applied sciences. Recently, we have shown that unlabeled proteins can be confined inside the biological nanopore Cytolysin A (ClyA) and conformational changes monitored by ionic current recordings. However, trapping small proteins remains a challenge. Here, we describe a system where steric, electrostatic, electrophoretic, and electro-osmotic forces are exploited to immobilize a small protein, dihydrofolate reductase (DHFR), inside ClyA. Assisted by electrostatic simulations, we show that the dwell time of DHFR inside ClyA can be increased by orders of magnitude (from milliseconds to seconds) by manipulation of the DHFR charge distribution. Further, we describe a physical model that includes a double energy barrier and the main electrophoretic components for trapping DHFR inside the nanopore. Simultaneous fits to the voltage dependence of the dwell times allowed direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electro-osmotic flow on the protein (â 9 pN at -50 mV) to be retrieved. The observed binding of NADPH to the trapped DHFR molecules suggested that the engineered proteins remained folded and functional inside ClyA. Contact-free confinement of single proteins inside nanopores can be employed for the manipulation and localized delivery of individual proteins and will have further applications in single-molecule analyte sensing and enzymology studies.
Assuntos
Eletroforese , Nanoporos , Perforina/química , Engenharia de Proteínas , Tetra-Hidrofolato Desidrogenase/química , Eletricidade , Mutação/genética , NADP/química , Dobramento de Proteína , Tetra-Hidrofolato Desidrogenase/genética , Termodinâmica , Fatores de TempoRESUMO
Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.
Assuntos
DNA/análise , Nanotecnologia/métodos , Análise Espectral Raman/métodos , Adsorção , Nucleotídeos de Desoxiadenina/análise , Desoxicitidina Monofosfato/análise , Nucleotídeos de Desoxiguanina/análise , Nanoporos/ultraestrutura , Nanotecnologia/instrumentação , Análise Espectral Raman/instrumentaçãoRESUMO
Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.
Assuntos
Enzimas/química , Nanoporos , Nanotecnologia , Proteínas/química , Técnicas Biossensoriais , Membranas Artificiais , Nanotecnologia/instrumentação , Análise de Sequência de DNA , Imagem Individual de MoléculaRESUMO
Biological nanopores are nanoscale sensors employed for high-throughput, low-cost, and long read-length DNA sequencing applications. The analysis and sequencing of proteins, however, is complicated by their folded structure and non-uniform charge. Here we show that an electro-osmotic flow through Fragaceatoxin C (FraC) nanopores can be engineered to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. We further use the nanopore currents to discriminate peptide and protein biomarkers from 25 kDa down to 1.3 kDa including polypeptides differing by one amino acid. On the road to nanopore proteomics, our findings represent a rationale for amino-acid analysis of folded and unfolded polypeptides with nanopores.Biological nanopore-based protein sequencing and recognition is challenging due to the folded structure or non-uniform charge of peptides. Here the authors show that engineered FraC nanopores can overcome these problems and recognize biomarkers in the form of oligopeptides, polypeptides and folded proteins.
Assuntos
Biomarcadores/química , Venenos de Cnidários/química , Nanoporos , Peptídeos/química , Proteínas/química , DNA/química , OsmoseRESUMO
Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.
RESUMO
We present distinct asymmetric plasmon-induced noise properties of ionic transport observed through gold coated nanopores. We thoroughly investigated the effects of bias voltage and laser illumination. We show that the potential drop across top-coated silicon nanocavity pores can give rise to a large noise asymmetry (â¼2-3 orders of magnitude). Varying the bias voltage has an appreciable effect on the noise density spectra, typically in the Lorentzian components. The laser power is found to strongly affect the ionic noise level as well as the voltage threshold for light-induced noise generation. The asymmetric noise phenomenon is attributed to plasmon-induced interfacial reactions which promote light-induced charge fluctuation in the ion flow and allow voltage modulation of photo-induced carriers surmounting over such Schottky junctions. We further compare the ionic noise performances of gold nanocavities containing different material stacks, among which thermal oxide passivation of the silicon successfully mitigates the light-induced noise and is also fully CMOS-compatible. The understanding of the described noise characteristics will help to foster multiple applications using related structures including plasmonic-based sensing or plasmon-induced catalysis such as water splitting or solar energy conversion devices.