Pim Kinase Inhibitors Evaluated with a Single‐Molecule Engineered Nanopore Sensor

Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label‐free determination of inhibition constants. The procedure circumvents many sources of bias or false‐positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.     

Nanosensors lost in space. A random walk study of single molecule detection with single-nanopore sensors

Nanopores by providing single molecule detection and manipulation are lately in the forefront of life science and nanotechnology research. While single nanopore sensors can detect the residence of even one molecule or nanoparticle within the nanopore, the analytical significance of this process is often misunderstood. A fundamental problem of nanosensors is that their sensing zone is generally infinitesimal with respect of the probed sample volume. Consequently, the probability to have in extremely diluted solutions target molecules or nanoparticles encountering the nanosensor is low. Thus, eventhough the sensor by itself has single molecule detection capability the average time frame in which this occurs is by far not irrelevant for the analysis. In this paper we report on random walk simulations to determine the average time (encounter time) needed by a single molecule to encounter a single nanopore sensor. By assigning the simulation environment with real space and time values a semi-empirical equation for expressing the average encounter time in purely diffusive systems is provided. We also show that random walk simulations can be adapted to evaluate the encounter time in the presence of an external force field acting on the target molecule. As practically relevant application the case of electrophoretically driving DNA strands towards the nanopore sensor is presented and a semi-empirical equation for the encounter time is provided.     

Analyte‐Triggered DNA‐Probe Release from a Triplex Molecular Beacon for Nanopore Sensing

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex‐forming molecular beacon and a stem‐forming DNA component that is modified with a host–guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α‐hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point‐of‐care diagnostics with a portable nanopore kit in the future.     

Protein Detection Through Single Molecule Nanopore

A single nanopore represents a versatile single-molecule probe that can be employed to reveal several important features of proteins, such as physical structure, backbone flexibility, mechanical stability, their folding state, binding affinity to other interacting ligands and enzymatic activity. In this review, we summarize the development and current research related to the field of protein detection by nanopore, as well as a few examples of the pioneer work on protein detection. We first discuss the principle of electrical detection with nanopores and how this technique provides information from current traces. Then the development from peptide detection with biological nanopore to protein detection through solid-state nanopore is described. Finally, we prospect the measurement of protein shape and construction using nanopore technology for the applications in life research area.     

Selectively detecting attomolar concentrations of proteins using gold lined nanopores in a nanopore blockade sensor

Disease diagnosis at earlier stages requires the development of ultrasensitive biosensors for detecting low-abundance biomarkers in complex biological fluids within a reasonable time frame. Here, we demonstrate the development of an ultrasensitive nanopore blockade biosensor that can rapidly diagnose a model protein biomarker, prostate-specific antigen (PSA) with high selectivity. The solid-state nanopores have gold located only along the length of the nanopore whilst the rest of the membrane is silicon nitride. The orthogonal use of materials allows nanopore arrays with a different surface chemistry inside the nanopore relative to the rest of the membrane to be fabricated. The importance of this differential surface chemistry is it can improve the detection limit of nanopore blockade sensors in quantitative analysis. Based on such functionalized nanopore devices, nanopore blockade sensors lower the limit of detection by an order of magnitude and enable ultrasensitive detection of PSA as low as 80 aM. The findings from this study open new opportunities for nanopore sensors in further developments including optical detection and ultralow detection limit biosensing at complex biological fluids.

Selective detection of attomolar proteins was achieved using gold lined nanopores in a nanopore blockade sensor.     

Biosensing with conically shaped nanopores and nanotubes

In this review we consider recent results from our group that are directed towards developing "smart" synthetic nanopores that can mimic the functions of biological nanopores (transmembrane proteins). We first discuss the preparation and characterization of conical nanopores synthesized using the track-etch process. We then consider the design and function of conical nanopores that can rectify the ionic current that flows through these pores under an applied transmembrane potential. Finally, two types of sensors that we have developed with these conical nanopores are described. The first sensor makes use of molecular recognition elements that are bound to the nanopore mouth to selectively block the nanopore tip, thus detecting the presence of the analyte. The second sensor makes use of conical nanopores in a resistive-pulse type experiment, detecting the analyte via transient blockages in ionic current.     

Discrimination of single‐stranded DNA homopolymers by sieving out G‐quadruplex using tiny solid‐state nanopores

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single‐base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G‐quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20‐nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105‐nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore‐based DNA sequencing at single‐base level.     

Electrochemical Quantitative Evaluation of the Surface Charge of a Poly(1-Vinylimidazole) Multilayer Film and Application to Nanopore pH Sensor

Nanopore pH sensing is based on the interaction between the surface charge of the nanopore and ions passing through the nanopore. The nanopore surface charge can be derived from the acid-base dissociation equilibrium of the modified polyelectrolyte. Various polyelectrolytes have been selected based on the acid dissociation constant of the monomer units, and various techniques have been applied to modify nanopores. However, they have been developed without clear guidelines for characterizing the surface modification status or surface charge. One reason has been the difficulty in accurately estimating the surface charge of nanopores in solution. Thus, in this study, the dissociation constant (pKa_app) of the surface charge of a modified polyelectrolyte nanopore was quantitatively estimated via electrochemical measurements. Previously, the modification status of nanopores has been evaluated using the ion current response. In addition, we monitored in real-time the polyelectrolyte modification status using a quartz crystal microbalance (QCM). Some polyelectrolytes were difficult to immobilize directly on the nanopore surface, and those polymers could be effectively modified by the layer-by-layer (LbL) technique. Therefore, we produced a guideline for the fabrication of a nanopore sensor for pH measurements under physiological conditions by quantitative evaluation of the pKa_app via electrochemical methods, the monitoring of the modification status by QCM, and the development of an effective modification method via the LbL technique.     

A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin

Nanopore emerged as a powerful single‐molecule technique over the past two decades, and has shown applications in the stochastic sensing and biophysical studies of individual molecules. Here, we report a versatile strategy for nanopore sensing by employing the combination of aptamers and host–guest interactions. An aptamer is first hybridized with a DNA probe which is modified with a ferrocene?cucurbit[7]uril complex. The presence of analytes causes the aptamer–probe duplex to unwind and release the DNA probe which can quantitatively produce signature current events when translocated through an α‐hemolysin nanopore. The integrated use of magnetic beads can further lower the detection limit by approximately two to three orders of magnitude. Because aptamers have shown robust binding affinities with a wide variety of target molecules, our proposed strategy should be universally applicable for sensing different types of analytes with nanopore sensors.     

A storable encapsulated bilayer chip containing a single protein nanopore

A robust, portable chip containing a single protein nanopore would be a significant development in the practical application of stochastic sensing technology. Here, we describe a chip in which a single alpha-hemolysin (alphaHL) pore in a planar phospholipid bilayer is sandwiched between two layers of agarose gel. These encapsulated nanopore chips remain functional after storage for weeks. The detection of the second messenger inositol 1,4,5-trisphosphate (IP3) was demonstrated with a chip containing a genetically engineered alphaHL pore as the sensor element.     

Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.     

The effect of secondary structures on the generation of characteristic events during the translocation of DNA hybrid through α-hemolysin

Nanopore has been developed to be a powerful,single-molecule analytical tool for sensing ions,small organic molecules and biomacromolecules such as proteins and DNAs.Generally,the identity of the analyte can be revealed by current amplitude changes and mean dwell time of the analyte binding events.In some cases,generation of highly characteristic current events affords an alternative way of analyte determination with high confidence level.However,we found that secondary structures in DNA/RNA hybrids might severely hinder the generation of signature events during their translocation through?-hemolysin nanopore.In this report,we propose a strategy to add a certain concentration of urea in the buffer solution for single channel recordings and validate that low concentration of urea can effectively denature the secondary structures in DNA hybrids and recover the generation of signature events.This finding might be useful in other secondary structure-related nanopore sensing activities.     

Modeling electrochemical deposition inside nanotubes to obtain metal-semiconductor multiscale nanocables or conical nanopores

Nanocables with a radial metal-semiconductor heterostructure have recently been prepared by electrochemical deposition inside metal nanotubes. First, a bare nanoporous polycarbonate track-etched membrane is coated uniformly with a metal film by electroless deposition. The film forms a working electrode for further deposition of a semiconductor layer that grows radially inside the nanopore when the deposition rate is slow. We propose a new physical model for the nanocable synthesis and study the effects of the deposited species concentration, potential-dependent reaction rate, and nanopore dimensions on the electrochemical deposition. The problem involves both axial diffusion through the nanopore and radial transport to the nanopore surface, with a surface reaction rate that depends on the axial position and the time. This is so because the radial potential drop across the deposited semiconductor layer changes with the layer thickness through the nanopore. Since axially uniform nanocables are needed for most applications, we consider the relative role of reaction and axial diffusion rates on the deposition process. However, in those cases where partial, empty-core deposition should be desirable (e.g., for producing conical nanopores to be used in single nanoparticle detection), we give conditions where asymmetric geometries can be experimentally realized.     

Protein detection by nanopores equipped with aptamers

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.     

Boronic Acid-containing Stimuli-responsive Polymers Modified Nanopores for Label-free Dual-signal-output Detection of Glucose

Herein, a dual-signal-output glass nanopore system was developed for sensing glucose. Upon binding glucose, the boronic acid-containing stimuli-responsive polymer underwent a wettability switch and pKa shift. When the polymer was immobilized on the inner wall of nanopore, the nanopore offered a sensitive method for evaluating glucose by ion rectification. Besides, due to the electrostatic assembly of cationic pyrene onto the glucose-bound anionic polymer, the pyrene excimer emission was observed. By separating the substrate binding and fluorescence reporting process, this work avoided the fluorescence labeling of the target and nanopore, thus simplifying the design of dual-signal-output nanopore for potential point-of-care test.     

Chaperone-assisted translocation of a polymer through a nanopore

Using Langevin dynamics simulations, we investigate the dynamics of chaperone-assisted translocation of a flexible polymer through a nanopore. We find that increasing the binding energy ε between the chaperone and the chain and the chaperone concentration N(c) can greatly improve the translocation probability. Particularly, with increasing the chaperone concentration a maximum translocation probability is observed for weak binding. For a fixed chaperone concentration, the histogram of translocation time τ has a transition from a long-tailed distribution to a gaussian distribution with increasing ε. τ rapidly decreases and then almost saturates with increasing binding energy for a short chain; however, it has a minimum for longer chains at a lower chaperone concentration. We also show that τ has a minimum as a function of the chaperone concentration. For different ε, a nonuniversal dependence of τ on the chain length N is also observed. These results can be interpreted by characteristic entropic effects for flexible polymers induced by either the crowding effect from a high chaperone concentration or the intersegmental binding for the high binding energy.     

Pressure-dependent ion current rectification in conical-shaped glass nanopores

Ion current rectification that occurs in conical-shaped glass nanopores in low ionic strength solutions is shown to be dependent on the rate of pressure-driven electrolyte flow through the nanopore, decreasing with increasing flow rate. The dependence of the i-V response on pressure is due to the disruption of cation and anion distributions at equilibrium within the nanopore. Because the flow rate is proportional to the third power of the nanopore orifice radius, the pressure-driven flow can eliminate rectification in nanopores with radii of ～200 nm but has a negligible influence on rectification in a smaller nanopore with a radius of ～30 nm. The experimental results are in qualitative agreement with predictions based on finite-element simulations used to solve simultaneously the Nernst-Planck, Poisson, and Navier-Stokes equations for ion fluxes in a moving electrolyte within a conical nanopore.     

Investigation of entrance effects on particle electrophoretic behavior near a nanopore for resistive pulse sensing

Resistive pulse sensing using solid-state nanopores provides a unique platform for detecting the structure and concentration of molecules of different types of analytes in an electrolyte solution. The capture of an entity into a nanopore is subject not only to the electrostatic force but also the effect of electroosmotic flow originating from the charged nanopore surface. In this study, we theoretically analyze spherical particle electrophoretic behavior near the entrance of a charged nanopore. By investigating the effects of pore size, particle–pore distance, and salt concentration on particle velocity, we summarize dominant mechanisms governing particle behavior for a range of conditions. In the literature, the Helmholtz–Smoluchowski equation is often adopted to evaluate particle translocation by considering the zeta potential difference between the particle and nanopore surfaces. We point out that, due to the difference of the electric field inside and outside the nanopore and the influence from the existence of the particle itself, the zeta potential of the particle, however, needs to be at least 30% higher than that of the nanopore to allow the particle to enter into the nanopore when its velocity is close to zero. Accordingly, we summarize the effective salt concentrations that enable successful particle capture and detection for different pore sizes, offering direct guidance for nanopore applications.     

Single‐Molecule Determination of the Isomers of d‐Glucose and d‐Fructose that Bind to Boronic Acids

Monosaccharides, such as d ‐glucose and d ‐fructose, exist in aqueous solution as an equilibrium mixture of cyclic isomers and can be detected with boronic acids by the reversible formation of boronate esters. The engineering of accurate, discriminating and continuous monitoring devices relies on knowledge of which cyclic isomer of a sugar binds to a boronic acid receptor. Herein, by monitoring fluctuations in ionic current, we show that an engineered α‐hemolysin (αHL) nanopore modified with a boronic acid reacts reversibly with d ‐glucose as the pyranose isomer (α‐d ‐glucopyranose) and d ‐fructose as either the furanose (β‐d ‐fructofuranose) or the pyranose (β‐d ‐fructopyranose). Both of these binding modes contradict current binding models. With this knowledge, we distinguished the individual sugars in a mixture of d ‐maltose, d ‐glucose, and d ‐fructose.     

Evaluation of nanopores as candidates for electronic analyte detection

In an effort to increase throughput and decrease the cost of electrophoretic separation of DNA and proteins, various groups are developing highly parallel, miniaturized separation devices based on capillaries etched into silicon, glass or plastic substrates. To date, these miniaturized devices have relied on optical detectors, thus placing a lower limit on instrument size, and complicating the incorporation of an entire DNA analyzer instrument on a chip. To address this limitation, we are evaluating nanopores as candidate Coulter counters for purely electronic detection of analytes in miniaturized electrophoresis and similar separation devices. To establish feasibility of this detection scheme, we have investigated the detection sensitivity of a nanopore sensor through experiments with the alpha-hemolysin (alpha-HL) ion channel, and through a Monte Carlo (MC) model of polymer capture rate with a cylindrical nanopore under an applied voltage. Experimental and model results are extrapolated to predict the capture rate of synthetic pores operating at higher voltages than presently achievable with protein pores.