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.     

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.     

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.     

A method for reproducibly preparing synthetic nanopores for resistive-pulse biosensors

There is increasing interest in using nanopores in synthetic membranes as resistive‐pulse sensors for biomedical analytes. Analytes detected with prototype artificial‐nanopore biosensors include drugs, DNA, proteins, and viruses. This field is, however, currently in its infancy. A key question that must be addressed in order for such sensors to progress from an interesting laboratory experiment to practical devices is: Can the artificial‐nanopore sensing element be reproducibly prepared? We have been evaluating sensors that employ a conically shaped nanopore prepared by the track‐etch method as the sensor element. We describe here a new two‐step pore‐etching procedure that allows for good reproducibility in nanopore fabrication. In addition, we describe a simple mathematical model that allows us to predict the characteristics of the pore produced given the experimental parameters of the two‐step etch. This method and model constitute important steps toward developing practical, real‐world, artificial‐nanopore biosensors.     

Biomolecule‐Functionalized Solid‐State Ion Nanochannels/Nanopores: Features and Techniques

Solid‐state ion nanochannels/nanopores, the biomimetic products of biological ion channels, are promising materials in real‐world applications due to their robust mechanical and controllable chemical properties. Functionalizations of solid‐state ion nanochannels/nanopores by biomolecules pave a wide way for the introduction of varied properties from biomolecules to solid‐state ion nanochannels/nanopores, making them smart in response to analytes or external stimuli and regulating the transport of ions/molecules. In this review, two features for nanochannels/nanopores functionalized by biomolecules are abstracted, i.e., specificity and signal amplification. Both of the two features are demonstrated from three kinds of nanochannels/nanopores: nucleic acid–functionalized nanochannels/nanopores, protein‐functionalized nanochannels/nanopores, and small biomolecule‐functionalized nanochannels/nanopores, respectively. Meanwhile, the fundamental mechanisms of these combinations between biomolecules and nanochannels/nanopores are explored, providing reasonable constructs for applications in sensing, transport, and energy conversion. And then, the techniques of functionalizations and the basic principle about biomolecules onto the solid‐state ion nanochannels/nanopores are summarized. Finally, some views about the future developments of the biomolecule‐functionalized nanochannels/nanopores are proposed.     

Solid‐State Nanopore Detection of Protein Complexes: Applications in Healthcare and Protein Kinetics

Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid‐state nanopores for the detection of gp120‐associated complexes are investigated. They exhibit monovalent and multivalent binding to anti‐gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein‐specific detection schemes, which are shown to be feasible in the realm of a ‘real‐world’ sample and an automated multidimensional method of detecting events.     

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.     

An Engineered ClyA Nanopore Detects Folded Target Proteins by Selective External Association and Pore Entry

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.     

Designing Nanofluidic Diode from a Hybrid-Bilayer Covalent Organic Framework: Molecular Simulation Investigation

Nanofluidic diodes are potentially useful in many important applications such as sensing, electronics, and energy conversion. However, the manufacturing of controllable nanopores for nanofluidic diodes is technically challenging. Herein, a nanofluidic diode is designed from a highly programmatic covalent organic framework (COF). Through molecular simulation, remarkable diode behavior is observed in a hybrid-bilayer COF but not in its constituent single-layer COFs. The rectification effect of ion current in the hybrid-bilayer COF is attributed to an asymmetric electrostatic potential across the COF nanopore. Furthermore, a synergistic effect of counterion is unraveled in the hybrid-bilayer COF, and the presence of counterion is found to reduce the entry barrier and facilitate ion transport. The performance of the hybrid-bilayer COF as a nanofluidic diode is comprehensively investigated by varying salt concentration, layer number, interlayer spacing, and slipping. This proof-of-concept simulation study demonstrates the feasibility of the hybrid-bilayer COF as a nanofluidic diode and the finding may stimulate the development of new nanofluidic platforms.     

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.     

DNA origami nanopores

We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.     

Surface engineering of synthetic nanopores by atomic layer deposition and their applications

In the past decade, nanopores have been developed extensively for various potential applications, and their performance greatly depends on the surface properties of the nanopores. Atomic layer deposition （ALD） is a new technology for depositing thin films, which has been rapidly developed from a niche technology to an established method. ALD films can cover the surface in confined regions even in nanoscale conformally, thus it is proved to be a powerful tool to modify the surface of the synthetic nanopores and also to fabricate complex nanopores. This review gives a brief introduction on nanopore synthesis and ALD fundamental knowledge, and then focuses on the various aspects of synthetic nanopores processing by ALD and their applications, including single-molecule sensing, nanofiuidic devices, nanostructure fabrication and other applications.     

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.     

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.     

Engineering Single Nanopores on Gold Nanoplates by Tuning Crystal Screw Dislocation

Compared with the large variety of solid gold nanostructures, synthetic approaches for their hollow counterparts are limited, largely confined to chemical and irradiation‐based etching of preformed nanostructures. In particular, the preparation of through nanopore structures is extremely challenging. Here, a unique strategy for direct synthesis of gold nanopores in solution without the need for sacrificial templates or postsynthesis processing is reported. By controlling the degree of crystal screw dislocation, a single through pore with diameter ranging from sub‐nanometer to tens of nanometers, in the center of large gold nanoplates, can be engineered with precision. Ionic current rectification behaviors are observed using the gold nanopore, potentially enabling new capabilities in biosensing, sequencing, and imaging.     

Layer-by-layer removal of insulating few-layer mica flakes for asymmetric ultra-thin nanopore fabrication

We demonstrate an elaborate method to controllably fabricate ultra-thin nanopores by layer-by-layer removal of insulating few-layer mica flakes with atomic force microscopy (AFM). The fabricated nanopores are geometrically asymmetric, like an inverted quadrangular frustum pyramid. The nanopore geometry can be engineered by finely tuning the mechanical load on the AFM tip and the scanning area. Particularly noteworthy is that the nanopores can also be fabricated in suspended few-layer mica membranes on a silicon window, and may find potential use as functional components in nanofluidic devices.      

Regulating Current Rectification and Nanoparticle Transport Through a Salt Gradient in Bipolar Nanopores

Tuning of ion and nanoparticle transport is validated through applying a salt gradient in two types of nanopores: the inner wall of a nanopore has bipolar charges and its outer wall neutral (type I), and both the inner and outer walls of a nanopore have bipolar charges (type II). The ion current rectification (ICR) behavior of these nanopores can be regulated by an applied salt gradient: if it is small, the degree of ICR in type II nanopore is more significant than that in type I nanopore; a reversed trend is observed at a sufficiently large salt gradient. If the applied salt gradient and electric field have the same direction, type I nanopore exhibits two significant features that are not observed in type II nanopore: (i) a cation‐rich concentration polarization field and an enhanced funneling electric field are present near the cathode side of the nanopore, and (ii) the magnitude of the axial electric field inside the nanopore is reduced. These features imply that applying a salt gradient to type I nanopore is capable of simultaneously enhancing the nanoparticle capture into the nanopore and reducing its translocation velocity inside, so that high sensing performance and resolution can be achieved.     

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.     

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.