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.     

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.     

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.     

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.     

Controlling DNA Tug‐of‐War in a Dual Nanopore Device

Methods for reducing and directly controlling the speed of DNA through a nanopore are needed to enhance sensing performance for direct strand sequencing and detection/mapping of sequence‐specific features. A method is created for reducing and controlling the speed of DNA that uses two independently controllable nanopores operated with an active control logic. The pores are positioned sufficiently close to permit cocapture of a single DNA by both pores. Once cocapture occurs, control logic turns on constant competing voltages at the pores leading to a “tug‐of‐war” whereby opposing forces are applied to regions of the molecules threading through the pores. These forces exert both conformational and speed control over the cocaptured molecule, removing folds and reducing the translocation rate. When the voltages are tuned so that the electrophoretic force applied to both pores comes into balance, the life time of the tug‐of‐war state is limited purely by diffusive sliding of the DNA between the pores. A tug‐of‐war state is produced on 76.8% of molecules that are captured with a maximum two‐order of magnitude increase in average pore translocation time relative to the average time for single‐pore translocation. Moreover, the translocation slow‐down is quantified as a function of voltage tuning and it is shown that the slow‐down is well described by a first passage analysis for a 1D subdiffusive process. The ionic current of each nanopore provides an independent sensor that synchronously measures a different region of the same molecule, enabling sequential detection of physical labels, such as monostreptavidin tags. With advances in devices and control logic, future dual‐pore applications include genome mapping and enzyme‐free sequencing.     

Thermal unfolding of proteins probed at the single molecule level using nanopores

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.     

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

The function of a protein is determined by the composition of amino acids and is essential to proteomics. However, protein sequencing remains challenging due to the protein's irregular charge state and its high‐order structure. Here, a proof of principle study on the capability of protein sequencing by graphene nanopores integrated with atomic force microscopy is performed using molecular dynamics simulations. It is found that nanopores can discriminate a protein sequence and even its protonation state at single‐residue resolution. Both the pulling forces and current blockades induced by the permeation of protein residues are found to be highly correlated with the type of amino acids, which makes the residues identifiable. It is also found that aside from the dimension, both the conformation and charge state of the residue can significantly influence the force and current signal during its permeation through the nanopore. In particular, due to the electro‐osmotic flow effect, the blockade current for the double‐protonated histidine is slightly smaller than that for single‐protonated histidine, which makes it possible for discrimination of different protonation states of amino acids. The results reported here present a novel protein sequencing scheme using graphene nanopores combined with nanomanipulation technology.     

Thermally controlled permeation of ionic molecules through synthetic nanopores functionalized with amine-terminated polymer brushes

We present temperature-dependent ionic transport through an array of nanopores (cylindrical and conical) and a single conical nanopore functionalized with amine-terminated poly(N-isopropylacrylamide) [PNIPAAM-NH(2)] brushes. For this purpose, nanopores are fabricated in heavy ion irradiated polyethylene terephthlate (PET) membranes by a controlled chemical track-etching technique, which leads to the generation of carboxyl (COOH) groups on the pore surface. End-functionalized polymer chains are immobilized onto the inner pore walls via a 'grafting-to' approach through the covalent linkage of surface COOH moieties with the terminal amine groups of the PNIPAAM molecules by using carbodiimide coupling chemistry. The success of the chemical modification reaction is corroborated by measuring the permeation flux of charged analytes across the multipore membranes in an aqueous solution, and for the case of single conical pore by measuring the current-voltage (I-V) characteristics, which are dictated by the electrostatic interaction of the charged pore surface with the mobile ions in an electrolyte solution. The effective nanopore diameter is tuned by manipulating the environmental temperature due to the swelling/shrinking behaviour of polymer brushes attached to the inner nanopore walls, leading to a decrease/increase in the ionic transport across the membrane. This process should permit the thermal gating and controlled release of ionic drug molecules through the nanopores modified with thermoresponsive polymer chains across the membrane.     

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.     

Plasmonic‐Nanopore Biosensors for Superior Single‐Molecule Detection

Plasmonic and nanopore sensors have separately received much attention for achieving single‐molecule precision. A plasmonic “hotspot” confines and enhances optical excitation at the nanometer length scale sufficient to optically detect surface–analyte interactions. A nanopore biosensor actively funnels and threads analytes through a molecular‐scale aperture, wherein they are interrogated by electrical or optical means. Recently, solid‐state plasmonic and nanopore structures have been integrated within monolithic devices that address fundamental challenges in each of the individual sensing methods and offer complimentary improvements in overall single‐molecule sensitivity, detection rates, dwell time and scalability. Here, the physical phenomena and sensing principles of plasmonic and nanopore sensing are summarized to highlight the novel complementarity in dovetailing these techniques for vastly improved single‐molecule sensing. A literature review of recent plasmonic nanopore devices is then presented to delineate methods for solid‐state fabrication of a range of hybrid device formats, evaluate the progress and challenges in the detection of unlabeled and labeled analyte, and assess the impact and utility of localized plasmonic heating. Finally, future directions and applications inspired by the present state of the art are discussed.     

Zero‐Depth Interfacial Nanopore Capillaries

High‐fidelity analysis of translocating biomolecules through nanopores demands shortening the nanocapillary length to a minimal value. Existing nanopores and capillaries, however, inherit a finite length from the parent membranes. Here, nanocapillaries of zero depth are formed by dissolving two superimposed and crossing metallic nanorods, molded in polymeric slabs. In an electrolyte, the interface shared by the crossing fluidic channels is mathematically of zero thickness and defines the narrowest constriction in the stream of ions through the nanopore device. This novel architecture provides the possibility to design nanopore fluidic channels, particularly with a robust 3D architecture maintaining the ultimate zero thickness geometry independently of the thickness of the fluidic channels. With orders of magnitude reduced biomolecule translocation speed, and lowered electronic and ionic noise compared to nanopores in 2D materials, the findings establish interfacial nanopores as a scalable platform for realizing nanofluidic systems, capable of single‐molecule detection.     

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.     

Modifying the surface charge of single track-etched conical nanopores in polyimide

Chemical modification of nanopore surfaces is of great interest as it means that the surface composition is no longer fixed by the choice of substrate material, even to the point where large biomolecules can be attached to the pore walls. Controlling nanopore transport characteristics is one important application of surface modification which is very relevant given the significant interest in sensors based on the transport of ions and molecules through nanopores. Reported here is a method to change the surface charge polarity of single track-etched conical nanopores in polyimide, which also has the potential to attach more complex molecules to the carboxyl groups on the nanopore walls. These carboxyl groups were converted into terminal amino groups, first by activation with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) followed by the covalent coupling of ethylenediamine. This results in a changed surface charge polarity. Regeneration of a carboxyl-terminated surface was also possible, by reaction of the amino groups with succinic anhydride. The success of these reactions was confirmed by measurements of the pore's pH sensitive current-voltage (I-V) characteristics before and after the chemical modification, which depend on surface charge. The?permselectivity of the pores also changed accordingly with the modification.     

Solid-state nanopore channels with DNA selectivity

Solid-state nanopores have emerged as possible candidates for next-generation DNA sequencing devices. In such a device, the DNA sequence would be determined by measuring how the forces on the DNA molecules, and also the ion currents through the nanopore, change as the molecules pass through the nanopore. Unlike their biological counterparts, solid-state nanopores have the advantage that they can withstand a wide range of analyte solutions and environments. Here we report solid-state nanopore channels that are selective towards single-stranded DNA (ssDNA). Nanopores functionalized with a 'probe' of hair-pin loop DNA can, under an applied electrical field, selectively transport short lengths of 'target' ssDNA that are complementary to the probe. Even a single base mismatch between the probe and the target results in longer translocation pulses and a significantly reduced number of translocation events. Our single-molecule measurements allow us to measure separately the molecular flux and the pulse duration, providing a tool to gain fundamental insight into the channel-molecule interactions. The results can be explained in the conceptual framework of diffusive molecular transport with particle-channel interactions.     

Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores

Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of beta-human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.     

Detecting single stranded DNA with a solid state nanopore

Voltage biased solid-state nanopores are used to detect and characterize individual single stranded DNA molecules of fixed micrometer length by operating a nanopore detector at pH values greater than approximately 11.6. The distribution of observed molecular event durations and blockade currents shows that a significant fraction of the events obey a rule of constant event charge deficit (ecd) indicating that they correspond to molecules translocating through the nanopore in a distribution of folded and unfolded configurations. A surprisingly large component is unfolded. The result is an important milestone in developing solid-state nanopores for single molecule sequencing applications.     

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.     

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.     

Electrical characterization of protein molecules by a solid-state nanopore

The authors measured ionic current blockages caused by protein translocation through voltage-biased silicon nitride nanopores in ionic solution. By calculating the mean amplitude, time duration, and the integral of current blockages, they estimated the relative charge and size of protein molecules at a single molecule level. The authors measured the change in protein charge of bovine serum albumin (BSA) protein induced by pH variation. They also confirmed that BSA molecules indeed traverse nanopores using an improved chemiluminescent analysis. They demonstrated that a larger protein fibrinogen could be distinguished from BSA by a solid-state nanopore measurement.