Chemically modified solid-state nanopores

Nanopores are extremely sensitive single-molecule sensors. Recently, electron beams have been used to fabricate synthetic nanopores in thin solid-state membranes with subnanometer resolution. Here we report a new class of chemically modified nanopore sensors. We describe two approaches for monolayer coating of nanopores: (1) self-assembly from solution, in which nanopores approximately 10 nm diameter can be reproducibly coated, and (2) self-assembly under voltage-driven electrolyte flow, in which we are able to coat 5 nm nanopores. We present an extensive characterization of coated nanopores, their stability, reactivity, and pH response.     

Stochastic sensing of proteins with receptor-modified solid-state nanopores

Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.     

Detection of nucleosomal substructures using solid-state nanopores

Histone proteins assemble onto DNA into nucleosomes that control the structure and function of eukaryotic chromatin. More specifically, the structural integrity of nucleosomes regulates gene expression rates and serves as an important early marker for cell apoptosis. Nucleosomal (sub)structures are however hard to detect and characterize. Here, we show that solid-state nanopores are well suited for fast and label-free detection of nucleosomes and its histone subcomplexes. (Nucleo-)protein complexes are individually driven through the nanopore by an applied electric field, which results in characteristic conductance blockades that provide quantitative information on the molecular size of the translocating complex. We observe a systematic dependence of the conductance blockade and translocation time on the molecular weight of the nucleosomal substructures. This allows discriminating and characterizing these protein and DNA-protein complexes at the single-complex level. Finally, we demonstrate the ability to distinguish nucleosomes and dinucleosomes as a first step toward using the nanopore platform to study chromatin arrays.     

Fabrication of nanopores for biomacromolecule detection

Nanopores embedded in a thin membrane with diameter below 10 nm are suitable for the biomacromolecule detection. For such purpose, in this study, we developed a technique of how to obtain small nanopores in silicon nitride films using a focused-ion-beam (FIB) system. By changing the process parameters, such as the beam current, the film thickness of the membrane and the ion beam exposure time, the diameter of the nanopore can be tuned. Under an optimized condition, high quality nanopores with diameter as low as 6 nm was fabricated on a 7 nm thick membrane. Our result suggests that FIB direct writing technique might be a suitable approach for biomacromolecule detector fabrication.     

Translocation of single-wall carbon nanotubes through solid-state nanopores

We report the translocation of individual single-wall carbon nanotubes (SWNTs) through solid-state nanopores. Single-strand DNA oligomers are used to both disperse the SWNTs in aqueous solution and to provide them with a net charge, allowing them to be driven through the nanopores by an applied electric field. The resulting temporary interruptions in the measured nanopore conductance provide quantitative information on the diameter and length of the translocated nanotubes at a single-molecule level. Furthermore, we demonstrate that the technique can be utilized to monitor bundling of SWNT in solution by using complementary nucleotides to induce tube-tube agglomeration.     

Nanometer-thin solid-state nanopores by cold ion beam sculpting

Recent work on protein nanopores indicates that single molecule characterization (including DNA sequencing) is possible when the length of the nanopore constriction is about a nanometer. Solid-state nanopores offer advantages in stability and tunability, but a scalable method for creating nanometer-thin solid-state pores has yet to be demonstrated. Here we demonstrate that solid-state nanopores with nanometer-thin constrictions can be produced by "cold ion beam sculpting," an original method that is broadly applicable to many materials, is easily scalable, and requires only modest instrumentation.     

SEM-induced shrinking of solid-state nanopores for single molecule detection

We have investigated the mechanism by which the diameter of solid-state nanopores is reduced by a scanning electron microscope. The process depends on beam parameters such as the accelerating voltage and electron flux and does not involve simple electron-beam-induced deposition of hydrocarbon contaminants. Instead, it is an energy-dependent process that involves material flow along the surface of the nanopore membrane. We also show that pores fabricated in this manner can detect double stranded DNA.     

The passage of homopolymeric RNA through small solid-state nanopores

Solid-state nanopores are widely acknowledged as tools with which to study local structure in biological molecules. Individual molecules are forced through a nanopore, causing a characteristic change in an ionic current that depends on the molecules' local diameter and charge distribution. Here, the translocation measurements of long (~5-30 kilobases) single-stranded poly(U) and poly(A) molecules through nanopores ranging from 1.5 to 8 nm in diameter are presented. Individual molecules are found to be able to cause multiple levels of conductance blockade upon traversing the pore. By analyzing these conductance blockades and their relative incidence as a function of nanopore diameter, it is concluded that the smallest conductance blockades likely correspond to molecules that translocate through the pore in predominantly head-to-tail fashion. The larger conductance blockades are likely caused by molecules that arrive at the nanopore entrance with many strands simultaneously. These measurements constitute the first demonstration that single-stranded RNA can be captured in solid-state nanopores that are smaller than the diameter of double-stranded RNA. These results further the understanding of the conductance blockades caused by nucleic acids in solid-state nanopores, relevant for future applications, such as the direct determination of RNA secondary structure.     

Modeling the conductance and DNA blockade of solid-state nanopores

We present measurements and theoretical modeling of the ionic conductance G of solid-state nanopores with 5-100 nm diameters, with and without DNA inserted into the pore. First, we show that it is essential to include access resistance to describe the conductance, in particular for larger pore diameters. We then present an exact solution for G of an hourglass-shaped pore, which agrees very well with our measurements without any adjustable parameters, and which is an improvement over the cylindrical approximation. Subsequently we discuss the conductance blockade ΔG due to the insertion of a DNA molecule into the pore, which we study experimentally as a function of pore diameter. We find that ΔG decreases with pore diameter, contrary to the predictions of earlier models that forecasted a constant ΔG. We compare three models for ΔG, all of which provide good agreement with our experimental data.     

Pressure-induced solid-state lattice mending of nanopores by pulse laser annealing

This paper presents the pressure-induced solid-state lattice mending of nanopores in single-crystal copper by femtosecond laser annealing processes. The microscopic mechanism of lattice mending is investigated by a modified continuum-atomistic modeling approach. Three typical lattice mending phases, including (i) the incubation of dislocation nucleation, (ii) plastic deformation under the combined effect of pressure and atomic thermal diffusion, and (iii) lattice recovery and reconstruction, are characterized via the microscopic structure changes and transient thermodynamic trajectories. The simulation results reveal that the structural mending of a pore is originated in heterogeneous nucleation of dislocations from the pore surface. The shear-induced multiple lattice glides are found to significantly contribute to the mending of a nanopore in the process of solid-state structural mending. The mending rates of two different modes, the pressure-induced and the classical unsteady-state atomic diffusion, are estimated and found to be very different from each other, by an order of 10(4). In addition, the location of the pore is also found to significantly influence the annealing threshold. Since the largest amplitude of the pressure wave is built up at a characteristic depth of approximately 45?nm below the irradiated surface, the shock wave will directly impinge on the pore and induce a fast solid-state lattice mending if the pore is located within the lower limit of the range of the characteristic depth. Furthermore, it is also interesting to note that the mending of a nanopore close to the characteristic depth by annealing fluence is generally lower than that of a pore near the surface. These results provide vital insights of photomechanical interactions with the microstructure of metallic solid, and the proposed approach can be further considered and enhanced to predict the mending depth for various defects in the future.     

Salt dependence of ion transport and DNA translocation through solid-state nanopores

We report experimental measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concentrations from 1 M to 1 muM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-mum-long dsDNA for 50 mM to 1 M salt concentrations. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concentration of 370 +/- 40 mM.     

Large apparent electric size of solid-state nanopores due to spatially extended surface conduction

Ion transport through nanopores drilled in thin membranes is central to numerous applications, including biosensing and ion selective membranes. This paper reports experiments, numerical calculations, and theoretical predictions demonstrating an unexpectedly large ionic conduction in solid-state nanopores, taking its origin in anomalous entrance effects. In contrast to naive expectations based on analogies with electric circuits, the surface conductance inside the nanopore is shown to perturb the three-dimensional electric current streamlines far outside the nanopore in order to meet charge conservation at the pore entrance. This unexpected contribution to the ionic conductance can be interpreted in terms of an apparent electric size of the solid-state nanopore, which is much larger than its geometric counterpart whenever the number of charges carried by the nanopore surface exceeds its bulk counterpart. This apparent electric size, which can reach hundreds of nanometers, can have a major impact on the electrical detection of translocation events through nanopores, as well as for ionic transport in biological nanopores.     

Rapid and precise scanning helium ion microscope milling of solid-state nanopores for biomolecule detection

We report the formation of solid-state nanopores using a scanning helium ion microscope. The fabrication process offers the advantage of high sample throughput along with fine control over nanopore dimensions, producing single pores with diameters below 4 nm. Electronic noise associated with ion transport through the resultant pores is found to be comparable with levels measured on devices made with the established technique of transmission electron microscope milling. We demonstrate the utility of our nanopores for biomolecular analysis by measuring the passage of double-strand DNA.     

Fabrication of a one-dimensional array of nanopores horizontally aligned on a Si substrate

A one-dimensional array of nanopores horizontally aligned on a silicon substrate was successfully fabricated by anodic aluminum oxidation (AAO) using a modified two-step procedure. SEM pictures show clear nanostructures of well-aligned one-dimensional nanopore arrays without cracks at the interfaces of the sandwiched structures. The processes are compatible with the planar silicon integrated circuit processing technology, promising for applications in nanoelectronics. The formation mechanism of a single nanopore array on Si substrates was also discussed.     

Fabrication of nanopores in a 100-nm thick Si3N4 membrane

Textured alumina films have been used to fabricate nanoscale pores in Si3N4 membranes. A few nanometer-thick alumina layer was used as a masking material for nanopore fabrication, and the pattern was transferred into a 100-nm thick, 200 microm x 200 microm Si3N4 membrane by reactive ion etching (RIE). The nanopores were found to be concentrated in a approximately 150-microm diameter region at the center of the membrane.     

Fabrication and characterization of a solid-state nanopore with self-aligned carbon nanoelectrodes for molecular detection

Stochastic molecular sensors based on resistive pulse nanopore modalities are envisioned as facile DNA sequencers. However, recent advances in nanotechnology fabrication have highlighted promising alternative detection mechanisms with higher sensitivity and potential single-base resolution. In this paper we present the novel self-aligned fabrication of a solid-state nanopore device with integrated transverse graphene-like carbon nanoelectrodes for polyelectrolyte molecular detection. The electrochemical transduction mechanism is characterized and found to result primarily from thermionic emission between the two transverse electrodes. Response of the nanopore to Lambda dsDNA and short (16-mer) ssDNA is demonstrated and distinguished.     

Fabrication of precision fiber-optic time delays with in situ monitoring for subpicosecond accuracy

We have developed a technique to produce precise fiber-optic time delays with subpicosecond accuracy and <0.1-dB loss by heating and stretching optical fiber in a fusion splicer. A fiber Mach-Zehnder interferometer allows in situ measurement of these precise delays using a simple alignment process and requiring only a weak optical signal. To demonstrate this capability, we assembled a six-stage feed-forward delay line that can be used to generate 64 optical pulses with 9.5 +/- 0.8-ps pulse spacings and 4.8-dB total insertion loss.     

Fabrication and characterization of solid-state nanopore arrays for high-throughput DNA sequencing

We report the fabrication and characterization of uniformly sized nanopore arrays, integrated into an optical detection system for high-throughput DNA sequencing applications. Nanopore arrays were fabricated using focused ion beam milling, followed by?TiO(2) coating using atomic layer deposition. The?TiO(2) layer decreases the initial pore diameter down to the sub-10?nm range, compatible with the requirements for nanopore-based sequencing using optical readout. We find that the?TiO(2) layers produce a lower photoluminescence background as compared with the more widely used Al(2)O(3) coatings. The functionality of the nanopore array was demonstrated by the simultaneous optical detection of DNA-quantum dot conjugates, which were electro-kinetically driven through the nanopores. Our optical scheme employs total internal reflection fluorescence microscopy to illuminate a wide area of the?TiO(2)-coated membrane. A highly parallel system for observing DNA capture events in a uniformly sized 6?×?6 nanopore array was experimentally realized.     

Solid-state nanopores

The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring nanopores, but advances in technology now allow artificial solid-state nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.