Fabrication of Metallized Nanopores in Silicon Nitride Membranes for Single‐Molecule Sensing

The fabrication and characterization of a metallized nanopore structure for the sensing of single molecules is described. Pores of varying diameters (>10 nm) are patterned into free‐standing silicon nitride membranes by electron‐beam lithography and reactive ion etching. Structural characterization by transmission electron microscopy (TEM) and tomography reveals a conical pore shape with a 40° aperture. Metal films of Ti/Au are vapor deposited and the pore shape and shrinking are studied as a function of evaporated film thickness. TEM tomography analysis confirms metalization of the inner pore walls as well as conservation of the conical pore shape. In electrical measurements of the transpore current in aqueous electrolyte solution, the pores feature very low noise. The applicability of the metallized pores for stochastic sensing is demonstrated in real‐time translocation experiments of single λ‐DNA molecules. We observe exceptionally long‐lasting current blockades with a fine structure of distinct current levels, suggesting an attractive interaction between the DNA and the PEGylated metallic pore walls.     

Surface Modification of Solid‐State Nanopores for Sticky‐Free Translocation of Single‐Stranded DNA

Nanopore technology is one of the most promising approaches for fast and low‐cost DNA sequencing application. Single‐stranded DNA detection is primary objective in such device, while solid‐state nanopores remain less explored than their biological counterparts due to bio‐molecule clogging issue caused by surface interaction between DNA and nanopore wall. By surface coating a layer of polyethylene glycol (PEG), solid‐state nanopore can achieve long lifetime for single‐stranded DNA sticky‐free translocation at pH 11.5. Associated with elimination of non‐specific binding of molecule, PEG coated nanopore presents new surface characteristic as less hydrophility, lower 1/f noise, and passivated surface charge responsiveness on pH. Meanwhile, conductance blockage of single‐stranded DNA is found to be deeper than double‐stranded DNA, which can be well described by a string of blobs model for a quasi‐equilibrium state polymer in constraint space.     

Hybrid pore formation by directed insertion of α-haemolysin into solid-state nanopores

Most experiments on nanopores have concentrated on the pore-forming protein α-haemolysin (αHL) and on artificial pores in solid-state membranes. While biological pores offer an atomically precise structure and the potential for genetic engineering, solid-state nanopores offer durability, size and shape control, and are also better suited for integration into wafer-scale devices. However, each system has significant limitations: αHL is difficult to integrate because it relies on delicate lipid bilayers for mechanical support, and the fabrication of solid-state nanopores with precise dimensions remains challenging. Here we show that these limitations may be overcome by inserting a single αHL pore into a solid-state nanopore. A double-stranded DNA attached to the protein pore is threaded into a solid-state nanopore by electrophoretic translocation. Protein insertion is observed in 30-40% of our attempts, and translocation of single-stranded DNA demonstrates that the hybrid nanopore remains functional. The hybrid structure offers a platform to create wafer-scale device arrays for genomic analysis, including sequencing.     

Flossing DNA in a Dual Nanopore Device

Solid‐state nanopores are a single‐molecule technique that can provide access to biomolecular information that is otherwise masked by ensemble averaging. A promising application uses pores and barcoding chemistries to map molecular motifs along single DNA molecules. Despite recent research breakthroughs, however, it remains challenging to overcome molecular noise to fully exploit single‐molecule data. Here, an active control technique termed “flossing” that uses a dual nanopore device is presented to trap a proteintagged DNA molecule and up to 100's of back‐and‐forth electrical scans of the molecule are performed in a few seconds. The protein motifs bound to 48.5 kb λ‐DNA are used as detectable features for active triggering of the bidirectional control. Molecular noise is suppressed by averaging the multiscan data to produce averaged intertag distance estimates that are comparable to their known values. Since nanopore feature‐mapping applications require DNA linearization when passing through the pore, a key advantage of flossing is that trans‐pore linearization is increased to >98% by the second scan, compared to 35% for single nanopore passage of the same set of molecules. In concert with barcoding methods, the dual‐pore flossing technique could enable genome mapping and structural variation applications, or mapping loci of epigenetic relevance.     

Recapturing and trapping single molecules with a solid-state nanopore

The development of solid-state nanopores, inspired by their biological counterparts, shows great potential for the study of single macromolecules. Applications such as DNA sequencing and the exploration of protein folding require control of the dynamics of the molecule's interaction with the pore, but DNA capture by a solid-state nanopore is not well understood. By recapturing individual molecules soon after they pass through a nanopore, we reveal the mechanism by which double-stranded DNA enters the pore. The observed recapture rates and times agree with solutions of a drift-diffusion model. Electric forces draw DNA to the pore over micrometer-scale distances, and upon arrival at the pore, molecules begin translocation almost immediately. Repeated translocation of the same molecule improves measurement accuracy, offers a way to probe the chemical transformations and internal dynamics of macromolecules on sub-millisecond time and sub-micrometre length scales, and demonstrates the ability to trap, study and manipulate individual macromolecules in solution.     

Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes

Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state nanopores.     

Slowing DNA translocation in a solid-state nanopore

Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/micros translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.     

Nanopore Biosensors: Monitoring of an ATP‐Binding Aptamer and its Conformational Changes Using an α‐Hemolysin Nanopore (Small 1/2011)

An aptamer is a specific oligonucleotide sequence that spontaneously forms a secondary structure capable of selectively binding an analyte. An aptamer’s conformation is the key to specific binding of a target molecule, even in the case of very closely related targets. Nanopores are a sensitive tool for the single‐molecule analysis of DNA, peptides, and proteins transporting through the pore. Herein, a single α‐hemolysin natural nanopore is utilized to sense the conformational changes of an adenosine 5’‐triphosphate (ATP)‐binding aptamer (ABA). The known DNA sequence of the ABA is used as a model to develop real‐time monitoring of molecular conformational changes that occur by binding targets. The native, folded ABA structure has a nanopore unfolding time of 4.17 ms, compared with 0.29 ms for the ABA:ATP complex. A complementary 14‐mer strand, which binds the ABA sequence in the key nucleic acids responsible for folding, forms linear duplex DNA, resulting in a nanopore transit time of 0.50 ms and a higher capture probability than that of the folded ABA oligomer. Competition assays between the ABA:ATP and ABA:reporter complexes are carried out, and the results suggest that the ABA:ATP complex is formed preferentially. The nanopore allows for the detection of an ABA in its folded, ATP‐bound, and linear conformations.     

Programmed Synthesis of Freestanding Graphene Nanomembrane Arrays

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.     

An All‐Plastic Field‐Effect Nanofluidic Diode Gated by a Conducting Polymer Layer

The design of an all‐plastic field‐effect nanofluidic diode is proposed, which allows precise nanofluidic operations to be performed. The fabrication process involves the chemical synthesis of a conductive poly(3,4‐ethylenedioxythiophene) (PEDOT) layer over a previously fabricated solid‐state nanopore. The conducting layer acts as gate electrode by changing its electrochemical state upon the application of different voltages, ultimately changing the surface charge of the nanopore. A PEDOT‐based nanopore is able to discriminate the ionic species passing through it in a quantitative and qualitative manner, as PEDOT nanopores display three well‐defined voltage‐controlled transport regimes: cation‐rectifying, non‐rectifying, and anion rectifying regimes. This work illustrates the potential and versatility of PEDOT as a key enabler to achieve electrochemically addressable solid‐state nanopores. The synergism arising from the combination of highly functional conducting polymers and the remarkable physical characteristics of asymmetric nanopores is believed to offer a promising framework to explore new design concepts in nanofluidic devices.     

Reduction of DNA Folding by Ionic Liquids and Its Effects on the Analysis of DNA–Protein Interaction Using Solid‐State Nanopore

DNA folding is not desirable for solid‐state nanopore techniques when analyzing the interaction of a biomolecule with its specific binding sites on DNA since the signal derived from the binding site could be buried by a large signal from the folding of DNA nearby. To resolve the problems associated with DNA folding, ionic liquids (ILs), which are known to interact with DNA through charge–charge and hydrophobic interactions are employed. 1‐n‐butyl‐3‐methylimidazolium chloride (C₄mim) is found to be the most effective in lowering the incident of DNA folding during its translocation through solid‐state nanopores (4–5 nm diameter). The rate of folding signals from the translocation of DNA–C₄mim is decreased by half in comparison to that from the control bare DNA. The conformational changes of DNA upon complexation with C₄mim are further examined using atomic force microscopy, showing that the entanglement of DNA which is common in bare DNA is not observed when treated with C₄mim. The stretching effect of C₄mim on DNA strands improves the detection accuracy of nanopore for identifying the location of zinc finger protein bound to its specific binding site in DNA by lowering the incident of DNA folding.     

Automated Fabrication of 2‐nm Solid‐State Nanopores for Nucleic Acid Analysis

We demonstrate the automated and reproducible fabrication of sub‐2‐nm nanopores in 10‐nm thick silicon nitride membranes, through controlled dielectric breakdown in solution. Our results reveal that under the appropriate conditions, nanopores can be fabricated with a size no larger than 2.0 ± 0.5‐nm in diameter for a sample of N = 23 nanopores, with an average and standard deviation of 1.3 ± 0.6‐nm. The dimensions of these nanopores are confirmed by using individual translocating DNA molecules as molecular rulers. We show that a 2.0‐nm and a 2.1‐nm diameter nanopore are capable of distinguishing single‐stranded DNA versus double‐stranded DNA, and that a 2.4‐nm diameter nanopore can be used to investigate the overstretching transition in short dsDNA fragments. These results highlight the reliability and precision of the automated fabrication of nanopores via controlled dielectric breakdown, showing great promise for the manufacturing of future nanopore‐based technologies.     

Optically Controlled Pore Formation in Self‐Sealing Giant Porphyrin Vesicles

Efforts to develop self‐contained microreactors and artificial cells have been limited by difficulty in generating membranes that can be robustly and repeatedly manipulated to load and release cargo from phospholipid compartments. Here we describe a purely optical method to form pores in a membrane generated from porphyrin‐phospholipid conjugates electro‐assembled into microscale giant porphyrin vesicles and manipulated using confocal microscopy. The pores in the membrane resealed within a minute allowing for repeated pore formation with precise spatial and temporal control and optical gating to allow selective diffusion of biomolecules across the membrane. Temporal control of pore formation was illustrated by performing sequential DNA hybridization reactions. A biotin‐avidin based strategy was developed to selectively attach enzymes to the interior of the vesicle, demonstrating spatial control and the potential of giant porphyrin vesicles as versatile microreactors.     

Multi‐Responsive and Logic Controlled Release of DNA‐Gated Mesoporous Silica Vehicles Functionalized with Intercalators for Multiple Delivery

Novel DNA‐gated mesoporous silica nanoparticle (MSN) vehicles functionalized with disulfide‐linked acridinamine intercalators are constructed for multi‐responsive controlled release. The DNA‐gated MSN vehicles release cargo encapsulated in the MSN pores under different stimuli, including disulfide reducing agents, elevated temperature, and deoxyribonuclease I (DNase I), for codelivery of drugs and DNA/genes in different forms. Furthermore, the cascade release of encapsulated and intercalative drugs is controlled by AND logic gates in combination of dual stimuli. The ingeniously designed DNA‐gated MSN vehicles integrates multiple responses and AND logic gate operations into a single smart nanodevice not only for codelivery of drugs and DNA/genes but also for cascade release of two drugs and has promising biological applications to meet diverse requirements of controlled release.     

Electrophoretic time-of-flight measurements of single DNA molecules with two stacked nanopores

Electrophoretic transport through a solid-state nanodevice comprised of two stacked nanopore sensors is used to determine the free-solution mobility of DNA molecules based on their "time-of-flight" between the two pores. Mobility measurements are possible at very low (100 pM) DNA concentration and for low as well as high salt concentrations (here 30 mM and 1 M KCl). The mechanism of DNA transport through the device is elucidated by statistical analysis, showing the free-draining nature of the translocating DNA polymers and a barrier-dominated escape through the second pore. Furthermore, consecutive threading of single molecules through the two pores can be used to gain more detailed information on the dynamics of the molecules by correlation analysis, which also provides a direct electrical proof for translocation.     

Tunable Confinement for Bridging Single‐Cell Manipulation and Single‐Molecule DNA Linearization

DNA linearization by nanoconfinement has offered a new avenue toward large‐scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single‐cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic‐length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub‐millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single‐cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp‐long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single‐cell, single‐genome analysis.     

Fast DNA translocation through a solid-state nanopore

We report experiments and modeling of translocation of double-strand DNA through a siliconoxide nanopore. Long DNA molecules with different lengths ranging from 6500 to 97000 base pairs have been electrophoretically driven through a 10 nm pore. We observe a power-law caling of the translocation time with the length, with an exponent of 1.27. This nonlinear scaling is strikingly different from the well-studied linear behavior observed in similar experiments performed on protein pores. We present a theoretical model where hydrodynamic drag on the ection of the polymer outside the pore is the dominant force counteracting the electrical driving force. We show that this applies to our experiments, and we derive a power-law scaling with an exponent of 1.22, in good agreement with the data.     

Assessing graphene nanopores for sequencing DNA

Using all-atom molecular dynamics and atomic-resolution Brownian dynamics, we simulate the translocation of single-stranded DNA through graphene nanopores and characterize the ionic current blockades produced by DNA nucleotides. We find that transport of single DNA strands through graphene nanopores may occur in single nucleotide steps. For certain pore geometries, hydrophobic interactions with the graphene membrane lead to a dramatic reduction in the conformational fluctuations of the nucleotides in the nanopores. Furthermore, we show that ionic current blockades produced by different DNA nucleotides are, in general, indicative of the nucleotide type, but very sensitive to the orientation of the nucleotides in the nanopore. Taken together, our simulations suggest that strand sequencing of DNA by measuring the ionic current blockades in graphene nanopores may be possible, given that the conformation of DNA nucleotides in the nanopore can be controlled through precise engineering of the nanopore surface.     

DNA molecules and configurations in a solid-state nanopore microscope

A nanometre-scale pore in a solid-state membrane provides a new way of electronically probing the structure of single linear polymers, including those of biological interest in their native environments. Previous work with biological protein pores wide enough to let through and sense single-stranded DNA molecules demonstrates the power of using nanopores, but many future tasks and applications call for a robust solid-state pore whose nanometre-scale dimensions and properties may be selected, as one selects the lenses of a microscope. Here we demonstrate a solid-state nanopore microscope capable of observing individual molecules of double-stranded DNA and their folding behaviour. We discuss extensions of the nanopore microscope concept to alternative probing mechanisms and applications, including the study of molecular structure and sequencing.