Pulse-driven magnetoimpedance sensor detection of biomagnetic fields in musculatures with spontaneous electric activity

We describe analysis and control of 50S ribosomal subunits by a solid-state 45nm diameter nanopore incorporated in a microfluidic chip. When used as a resistive pulse sensor, translocation of single 50S subunits through the nanopore produces current blockades that have a linear dependence on applied voltage. Introduction of individual subunits into the fluidic channel shows a threshold behavior that allows controlled entry of individual 50S ribosomal subunits. The incorporation of nanopores into a larger optofluidic chip system opens possibilities for electrical and optical studies of single ribosomes in well-defined and rapidly variable chemical environments.     

An ON/OFF biosensor based on blockade of ionic current passing through a solid-state nanopore

Single nanopores have attracted interest for their use as biosensing devices. In general, methods involve measuring ionic current blockades associated with translocation of analytes through the nanopore, but the detection of such short time lasting events requires complex equipment and setup that are critical for convenient routine biosensing. Here we present a novel biosensing concept based on a single nanopore in a silicon nitride membrane and two anchor-linked DNA species that forms trans-pore hybrids, realizing a stable blockade of ionic current through the pore. Molecular recognition events affecting the DNA hybrids cause a pore opening and the consequent establishment of an ionic current. In the present implementation of the device, we constructed a magnetic bead/streptavidin/biotin-DNA1/DNA2-biotin/streptavidin/Quantumdot-cluster complex (where DNA1 is a mismatched reverse complement of DNA2) through a sub-micrometric pore and monitored DNA strand displacement events occurring after addition of an oligonucleotide complementary to DNA2. The electric and mechanical aspects of the novel device, as well as its potential in biosensing are discussed.     

Mechanisms of pressure-induced water infiltration process through graphene nanopores

Understanding the mechanism of water infiltration through nanopores is essential for wide applications ranging from membrane separation to gene therapy. In this paper, the molecular dynamics simulation method is used to investigate the pressure-assisted water transport process through graphene nanopores. Various factors including the hydrophobicity of nanopore surface, nanopore dimension, temperature as well as external electric field that affect water in permeation into graphene nanopores are discussed. It is found that classic Laplace-Young equation fails and the relationship between pressure and diameter (D) does not follow the 1/D dependence as the characteristic dimension of a nanopore is sufficiently small (smaller than 1?nm). The critical pressure significantly depends on both the pore length and electric field as D is smaller than 5?nm. Besides, enhancing temperature and electric field intensity are obviously beneficial for water infiltration through those nanopores with a diameter smaller than 5?nm.     

The electromechanics of DNA in a synthetic nanopore

We have explored the electromechanical properties of DNA on a nanometer-length scale using an electric field to force single molecules through synthetic nanopores in ultrathin silicon nitride membranes. At low electric fields, E < 200 mV/10 nm, we observed that single-stranded DNA can permeate pores with a diameter >/=1.0 nm, whereas double-stranded DNA only permeates pores with a diameter >/=3 nm. For pores <3.0 nm diameter, we find a threshold for permeation of double-stranded DNA that depends on the electric field and pH. For a 2 nm diameter pore, the electric field threshold is approximately 3.1 V/10 nm at pH = 8.5; the threshold decreases as pH becomes more acidic or the diameter increases. Molecular dynamics indicates that the field threshold originates from a stretching transition in DNA that occurs under the force gradient in a nanopore. Lowering pH destabilizes the double helix, facilitating DNA translocation at lower fields.     

Cheminformatics methods for novel nanopore analysis of HIV DNA termini

BACKGROUND: Channel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis. RESULTS: HMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus.     

Single polypeptide detection using a translocon EXP2 nanopore

DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore-forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL) at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000–70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.     

Nanopore cheminformatics

A cheminformatics method is described for classification, and biophysical examination, of individual molecules. A novel molecular detector is used--one based on current blockade measurements through a nanometer-scale ion channel (alpha-hemolysin). Classification results are described for blockades caused by DNA molecules in the alpha-hemolysin nanopore detector, with signal analysis and pattern recognition performed using a combination of methods from bioinformatics and machine learning. Due to the size of the alpha-hemolysin protein channel, the blockade events report on one DNA molecule at a time, which enables a variety of reproducible, single-molecule biophysical experiments. To capture the full sensitivity of the nanopore detector's blockade signal, Hidden Markov Models (HMMs) were used with Expectation/Maximization for denoising and for associating a feature vector with the ionic current blockade of each captured DNA molecule. Support Vector Machines (SVMs) that employ novel kernel designs were then used as discriminators. With SVM training performed off-line, and economical HMM processing on-line, blockade classification was possible during capture. HMMs were also used in conjunction with a time-domain finite state automaton (off-line) for feature discovery and kinetics analysis. Analysis of the DNA data indicates a variety of binding (DNA-protein), fraying, and conformational shifts that are consistent with data obtained from thermodynamic analyses (melting curves), X-ray crystallography, and NMR studies. The software tools are designed for analysis of generic blockades in ionic channels, including those in other biological pore-forming toxins, other biological channels in general, and semiconductor-based channels.     

DNA translocation governed by interactions with solid-state nanopores

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.     

Voltage-Driven Translocation of DNA through a High Throughput Conical Solid-State Nanopore

Nanopores have become an important tool for molecule detection at single molecular level. With the development of fabrication technology, synthesized solid-state membranes are promising candidate substrates in respect of their exceptional robustness and controllable size and shape. Here, a 30–60 (tip-base) nm conical nanopore fabricated in 100 nm thick silicon nitride (Si₃N₄) membrane by focused ion beam (FIB) has been employed for the analysis of λ-DNA translocations at different voltage biases from 200 to 450 mV. The distributions of translocation time and current blockage, as well as the events frequencies as a function of voltage are investigated. Similar to previously published work, the presence and configurations of λ-DNA molecules are characterized, also, we find that greater applied voltages markedly increase the events rate, and stretch the coiled λ-DNA molecules into linear form. However, compared to 6–30 nm ultrathin solid-state nanopores, a threshold voltage of 181 mV is found to be necessary to drive DNA molecules through the nanopore due to conical shape and length of the pore. The speed is slowed down ∼5 times, while the capture radius is ∼2 fold larger. The results show that the large nanopore in thick membrane with an improved stability and throughput also has the ability to detect the molecules at a single molecular level, as well as slows down the velocity of molecules passing through the pore. This work will provide more motivations for the development of nanopores as a Multi-functional sensor for a wide range of biopolymers and nano materials.     

Microscopic Kinetics of DNA Translocation through synthetic nanopores

We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 x 2.6 nm(2) cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.     

"DNA-Dressed NAnopore" for complementary sequence detection

Single molecule electrical sensing with nanopores is a rapidly developing field with potential revolutionary effects on bioanalytics and diagnostics. The recent success of this technology is in the simplicity of its working principle, which exploits the conductance modulations induced by the electrophoretic translocation of molecules through a nanometric channel. Initially proposed as fast and powerful tools for molecular stochastic sensing, nanopores find now application in a range of different domains, thanks to the possibility of finely tuning their surface properties, thus introducing artificial binding and recognition sites. Here we show the results of DNA translocation and hybridization experiments at the single molecule level by a novel class of selective biosensor devices that we call "DNA-Dressed NAnopore" (DNA(2)), based on solid state nanopore with large initial dimensions, resized and activated by functionalization with DNA molecules. The presented data demonstrate the ability of the DNA(2) to selectively detect complementary target sequences, that is to distinguish between molecules depending on their affinity to the functionalization. The DNA(2) can thus constitute the basis for the design of integrable parallel devices for mutation DNA analysis, diagnostics and bioanalytic investigations.     

Inspection of the Engineered FhuA ΔC/Δ4L Protein Nanopore by Polymer Exclusion

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-β-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.     

Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.     

Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.     

DNA Strands Attached Inside Single Conical Nanopores: Ionic Pore Characteristics and Insight into DNA Biophysics

Single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume. Tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution. Here, we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single-stranded DNA molecules. We find that the DNA occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte. The results are explained by the ionic strength dependence of the persistence length of DNA. At low KCl concentrations (10 mM) the molecules assume an extended and rigid conformation, thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted DNA at high salt concentrations. Attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species.     

Logic Gate Operation by DNA Translocation through Biological Nanopores

Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs “1” and “0” as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND) operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.     

Probing distance and electrical potential within a protein pore with tethered DNA

DNA molecules tethered inside a protein pore can be used as a tool to probe distance and electrical potential. The approach and its limitations were tested with alpha-hemolysin, a pore of known structure. A single oligonucleotide was attached to an engineered cysteine to allow the binding of complementary DNA strands inside the wide internal cavity of the extramembranous domain of the pore. The reversible binding of individual oligonucleotides produced transient current blockades in single channel current recordings. To probe the internal structure of the pore, oligonucleotides with 5' overhangs of deoxyadenosines and deoxythymidines up to nine bases in length were used. The characteristics of the blockades produced by the oligonucleotides indicated that single-stranded overhangs of increasing length first approach and then thread into the transmembrane beta-barrel. The distance from the point at which the DNA was attached and the internal entrance to the barrel is 43 A, consistent with the lengths of the DNA probes and the signals produced by them. In addition, the tethered DNAs were used to probe the electrical potential within the protein pore. Binding events of oligonucleotides with an overhang of five bases or more, which threaded into the beta-barrel, exhibited shorter residence times at higher applied potentials. This finding is consistent with the idea that the main potential drop is across the alpha-hemolysin transmembrane beta-barrel, rather than the entire length of the lumen of the pore. It therefore explains why the kinetics and thermodynamics of formation of short duplexes within the extramembranous cavity of the pore are similar to those measured in solution, and bolsters the idea that a "DNA nanopore" provides a useful means for examining duplex formation at the single molecule level.     

Molecular dynamics simulations of DNA within a nanopore: arginine-phosphate tethering and a binding/sliding mechanism for translocation

Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore α-hemolysin (αHL). In the present study, we have developed simplified models of the wild-type αHL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for αHL and its mutants.     

Hydroxymethyluracil modifications enhance the flexibility and hydrophilicity of double-stranded DNA

Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here, we report the results of nanopore translocation experiments and molecular dynamics simulations that provide insight into the impact of this modification on the structure and dynamics of DNA. When transported through ultrathin solid-state nanopores, short DNA fragments containing thymine modifications were found to exhibit distinct, reproducible features in their transport characteristics that differentiate them from unmodified molecules. Molecular dynamics simulations suggest that 5-hydroxymethyluracil alters the flexibility and hydrophilicity of the DNA molecules, which may account for the differences observed in our nanopore translocation experiments. The altered physico-chemical properties of DNA produced by the thymine modifications may have implications for recognition and processing of such modifications by regulatory DNA-binding proteins.     

Integrated nanopore sensing platform with sub-microsecond temporal resolution

Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1 MHz, which to our knowledge is the highest bandwidth nanopore recording to date. We demonstrate transient signals as brief as 1 μs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.