Nanopore Applications Research Articles

Gold nanorod translocation through a solid-state nanopore

Recently, nanopores have been used in an essential technique for detecting single molecule with high sensitivity. The initial application of nanopores to DNA and RNA sequencing has been expanded to sensing proteins and nanoparticles, including Bovine serum albumin, silica nanoparticles, polystyrene beads, and others. In our study, for the first time, a positively charged gold nanorod was investigated using a solid-state nanopore device. Various gold nanorods passed through the nanopore with different current blockages and duration times, providing a measurement of the nanorod diameter, length, and charge. Our findings indicate that nanopore sensing might be a new method for characterizing the size, shape, and charge of nanoparticles.

A Miniaturized Single Channel Amplifier for Various Different Electrophysiology Setup

Different electrophysiology setups allow single ion channel recordings using Lipid Bilayer Membrane (BLM) for protein insertion. Ion channels are transmembrane proteins involved in many biological process of cell life. They are used as target for drug discovery and single-molecule sensors. Synthetic nanopores can also be used as nano-coulter counters and very promising transducers for DNA sequencing. Complex protocols are under investigation to assembly ion channels on reconstituted lipid membranes. They use different setups, ranging from suspended BLM, microfluidic and lab-on-a-chip devices, planar patch clamp, tip-dip method. These studies, combined to new emerging nanopore applications, require high sensitive and high resolution instrumentation, able to detect small currents (in the pA range) over large bandwith (up to 100kHz), providing flexible voltage stimuli generation. We present a compact and complete system for single ion channels and synthetic nanopores experiment, useful for scientists and students for academic purposes, composed by:- a miniaturized low noise single channel voltage-clamp amplifier and digitizer integrated into a single ASIC, with direct connection to a computer USB port;- a software interface for real time data display and analysis featuring flexible voltage stimuli control;- customised electrode interfaces for various different electrophysiology platforms and microfluidic devices.As a proof of concept, we demonstrate the system performances using different test proteins (KcsA, Gramicidin, Alpha-hemolysin) embedded into reconstituted lipid membranes. The acquired data are presented.

Dynamic Monte Carlo Simulation of Coupled Transport through a Narrow Multiply-Occupied Pore

Dynamic Monte Carlo simulations are used to study coupled transport (cotransport) through subnanometer-diameter pores. In this classic Hodgkin–Keynes mechanism, an ion species uses the large flux of an abundant ion species to move against its concentration gradient. The efficiency of cotransport is examined for various pore parameters so that synthetic nanopores can be engineered to maximize this effect. In general, the pore must be narrow enough that ions cannot pass each other and the charge of the pore large enough to attract many ions so that they exchange momentum. Cotransport efficiency increases as pore length increases, but even very short pores exhibit cotransport, in contradiction to the usual perception that long pores are necessary. The parameter ranges where cotransport occurs is consistent with current and near-future synthetic nanopore geometry parameters, suggesting that cotransport of ions may be a new application of nanopores.

A Spike-Like Ionic Current Behavior via Graphene Nanopore

Ionic current characterization is critical for the application of nanopores with sub 5 nm as bio medical sensors and devices. Here, we demonstrate an eccentric ionic current behavior in graphene nanopore fabricated by high resolution transmission electron microscopy (HR-TEM). A spike-like current enhancement is shown in the absence of any bio molecule or nanoparticle in the LaCl3and KCl solution. By tuning the hydrophobicity of the graphene surface, the spikes diminish in the current recordings acquired in graphene nanopore after 20 seconds plasma etching. We consider that the hydrophocity-induced nanobubble is present in the nanopore area, leading to the currents change as the bubbles deformation due to the voltage driven electrostatic forces on the transported ions surrounding the bubble surface.

Stretching and Controlled Motion of Single-Stranded DNA in Locally Heated Solid-State Nanopores

Practical applications of solid-state nanopores for DNA detection and sequencing require the electrophoretic motion of DNA through the nanopores to be precisely controlled. Controlling the motion of single-stranded DNA presents a particular challenge, in part because of the multitude of conformations that a DNA strand can adopt in a nanopore. Through continuum, coarse-grained and atomistic modeling, we demonstrate that local heating of the nanopore volume can be used to alter the electrophoretic mobility and conformation of single-stranded DNA. In the nanopore systems considered, the temperature near the nanopore is modulated via a nanometer-size heater element that can be radiatively switched on and off. The local enhancement of temperature produces considerable stretching of the DNA fragment confined within the nanopore. Such stretching is reversible, so that the conformation of DNA can be toggled between compact (local heating is off) and extended (local heating is on) states. The effective thermophoretic force acting on single-stranded DNA in the vicinity of the nanopore is found to be sufficiently large (4-8 pN) to affect such changes in the DNA conformation. The local heating of the nanopore volume is observed to promote single-file translocation of DNA strands at transmembrane biases as low as 10 mV, which opens new avenues for using solid-state nanopores for detection and sequencing of DNA.

Nanopore Analytical Chemistry

There is a plenty of biological nano-scale devices in nature,such as ion channels and protein pores,which inspired people to design and fabricate biomimetic systems(solid-state,hybrid nanopores and nano-channels) for analytical and sensing applications.Nanopore Analytical Chemistry designed based on these nano-devices are important for biosensors,nanofluidic devices,molecular filtration,synthetic biology,detection of single molecules and DNA sequence.In this paper,the history,classification,fundamental,applications and respects of nanopore analytical chemistry,especially recent advances of nanopore applications in DNA sequence and detection of proteins have been reviewed.

Detecting single-abasic residues within a DNA strand immobilized in a biological nanopore using an integrated CMOS sensor

In this paper, we demonstrate the application of a novel current-measuring sensor (CMS) customized for nanopore applications. The low-noise CMS is fabricated in a 0.35μm CMOS process and is implemented in experiments involving DNA captured in an α-hemolysin (α-HL) nanopore. Specifically, the CMS is used to build a current amplitude map as a function of varying positions of a single-abasic residue within a homopolymer cytosine single-stranded DNA (ssDNA) that is captured and held in the pore. Each ssDNA is immobilized using a biotin-streptavidin linkage. Five different DNA templates are measured and compared: one all-cytosine ssDNA, and four with a single-abasic residue substitution that resides in or near the ~1.5nm aperture of the α-HL channel when the strand is immobilized. The CMOS CMS is shown to resolves the ~5Å displacements of the abasic residue within the varying templates. The demonstration represents an advance in application-specific circuitry that is optimized for small-footprint nanopore applications, including genomic sequencing.

An area-efficient low-noise CMOS DNA detection sensor for multichannel nanopore applications

Abstract In this paper, an area-efficient low-noise DNA detection sensor is presented for multichannel nanopore applications. A resistive-feedback transimpedance amplifier (rf-TIA) is typically employed as a DNA detection sensor to amplify ionic current variations through a nanopore channel and convert it to a useful voltage range. However, big feedback resistors occupy large areas and limit the number of the DNA detection sensors that can be integrated on a given chip area for multichannel nanopore sensing. In this work, we propose a novel pseudo-resistor technique to drastically reduce the feedback resistor size. We will first review conventional pseudo resistors and then describe the new technique using a deep N-well NMOS transistor that is free from body effect. A DNA detection sensor, adopting the novel pseudo resistor, is fabricated in a 0.35 μm CMOS process and is tested using an α-hemolysin (α-HL) protein nanopore for detection individual molecules of single-stranded DNA (ssDNA) that pass through the 1.5 nm-diameter aperture. The active die area is reduced by almost 90% allowing a near 10-fold increase in the number of channels on a silicon chip. In future work, the refined CMOS DNA detection sensor arrays will functionalize multichannel nanopores formed in integrated microfluidic devices for high-throughput DNA analysis.

Integrated nanopore sensing platform with sub-microsecond temporal resolution

Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1 MHz, which to our knowledge is the highest bandwidth nanopore recording to date. We demonstrate transient signals as brief as 1 μs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.

Slowing down DNA Translocation through a Nanopore in Lithium Chloride

The charge of a DNA molecule is a crucial parameter in many DNA detection and manipulation schemes such as gel electrophoresis and lab-on-a-chip applications. Here, we study the partial reduction of the DNA charge due to counterion binding by means of nanopore translocation experiments and all-atom molecular dynamics (MD) simulations. Surprisingly, we find that the translocation time of a DNA molecule through a solid-state nanopore strongly increases as the counterions decrease in size from K(+) to Na(+) to Li(+), both for double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). MD simulations elucidate the microscopic origin of this effect: Li(+) and Na(+) bind DNA stronger than K(+). These fundamental insights into the counterion binding to DNA also provide a practical method for achieving at least 10-fold enhanced resolution in nanopore applications.

Nanopores: The Passage of Homopolymeric RNA through Small Solid‐State Nanopores (Small 15/2011)

The cover picture, prepared by Gary M. Skinner, depicts a single molecule of the RNA homopolymer poly(U) (green), as it is electrically driven through a molecular-sized (diameter ≈ 2 nm), solid-state nanopore etched into a 20-nm-thick silicon nitride membrane. The highly flexible molecule ‘crowds’ the pore entrance, in some cases preventing translocation and instead forming a coiled structure that leads to significant blockage of the ionic current passing through the nanopore. Many biotechnological applications of nanopores require them to operate on this size-scale, and therefore our understanding of how biopolymers behave under such conditions is critical. For more information, please read the Full Paper “The Passage of Homopolymeric RNA through Small Solid-State Nanopores” by N. H. Dekker and co-workers, beginning on page 2217.

Functionalizing a Nanopore with Nano-Electrodes Towards the Control of the Translocation of DNA with Single Base Resolution

Recently, application of nanopores to low-cost DNA sequencing has attracted great interest as there is great need to reduce the cost of sequencing a whole human genome to $1000. A key issue in the field of nanopore DNA sequencing is to control the DNA translocation. Here we will report the development of what we call a “DNA transistor”: a nanopore-based electrical device for controlling the translocation of DNA with single base resolution. The key part of this device is a free standing membrane, within which multiple layers of electrically addressable metal electrodes separated by dielectric layers are embedded. A 1-5 nanometer size pore is made through the membrane. We demonstrated that such a device is electrically viable for the electrode layer or the spacing dielectric layer as thin as 3 nm. Confirming the basic function of the device, induced electrical signals on the nano-electrodes by the translocating DNA, as well as the modulation of DNA translocation speed by the voltage bias applied on the nano-electrodes are also observed. Our ongoing experiments test if the modulated electrical field can trap or translocate DNA at a single base resolution.

Forces affecting double-stranded DNA translocation through synthetic nanopores

One of the recent applications of nanopores is to use them as detectors/analyzers for bio-molecules and nanopore based sequencing has been studied to quickly sequence DNA. In this paper, three categories of forces proposed in the literature to oppose the electrical driving forces in the DNA translocation process are analyzed, (1) the entropic forces of DNA uncoiling/recoiling at the pore entrance/exits, (2) the viscous drag acting on the blob like DNA outside the nanopore, and (3) the viscous drag acting on the linear DNA inside the nanopore. The magnitudes of these forces are calculated based on the parameters used in experiments and it is shown that the first two of the aforementioned categories of forces are usually small compared to the electrical driving force, while the last one is of the same order as the electrical driving force. To evaluate the viscous drag force acting on the linear DNA inside the nanopore, a hydrodynamic model based on the lubrication approximation is used to calculate the flow field and the viscous drag force acting on a DNA immobilized in a nanopore. This model is validated by good agreement with the experimental data for the tethering force used to immobilize a DNA inside the nanopore.

On-chip patch-clamp sensor for solid-state nanopore applications

An integrated low-noise patch-clamp sensor is implemented for single-molecule DNA analysis using a solid-state nanopore. By adopting an instrumentation amplifier and a compensation switch for the headstage, the input-offset voltage and the deleterious effects of input parasitic capacitances are minimised. This high-sensitivity sensor amplifier was fabricated in a 0.35 µm CMOS process and tested with the solid-state nanopore also developed.

DNA nanowire translocation phenomena in nanopores

One recent application of nanopores is to use them as detectors and analyzers for fast DNA sequencing. To better understand the DNA electrokinetic transport through a nanopore, a hydrodynamic model is developed to investigate the flow field, the resistive forces acting on the DNA, the DNA velocity and the ionic current through the nanopore. The numerical results reveal the relation between the DNA velocity and various parameters such as nanopore surface charge and solution concentration. The model is validated by comparing the numerical results with the experimental data for both DNA velocity and ionic current through the nanopore.

Fabrication and investigation of single track-etched nanopore and its applications

Solid-state nanopores have been widely used as building blocks for nanofluidic devices and circuits from both fundamental and application aspects. In this review, we sketch the recent research progress in our group on the fabrication and application of single nanopores in PET membrane by track-etching method. The asymmetric ion transport properties in the conical nanopore are intensively studied both experimentally and theoretically. Based on the understanding of the particular ion transport behavior on nano-scale, some applications are present, including the pressure-driven streaming current and synthetic nanopore–DNA system.

Direct Probing of DNA/Nanopore Interactions Using Optical Tweezers

Solid-state nanopores can be used to analyze the structure of long double-stranded DNA molecules and to probe their interactions with proteins. This method utilizes the native biopolymers' charge to electrically draw the molecules from the cis to the trans side of the pore. In small pores (<5 nm) the transport time of the biopolymer is determined by a balance of the electrical field and the frictional force resulting from interactions with the pore walls and hydrodynamic drag. Despite the central importance of biopolymer dynamics in virtually all nanopore applications, to date there have been no direct measurements of force in small pores during biopolymer transport. Here, we use optical tweezers to dynamically manipulate a λ-DNA molecule threaded through a <5nm pore while simultaneously recording force and ionic current. To characterize the interaction strength in the pore, we measure force/velocity profiles as a function of the applied voltage and ionic strength. By comparing experiments using different-sized pores, we quantify the relative contribution of interactions to the overall translocation dynamics. These measurements provide basic insight into the principles governing translocation in the interaction-dominated regime.

Aptamer-Encoded Nanopore For Single-Molecule Detection In Sensing Of Biomedical And Bioterrorist Agents

Various solid nanopores have been constructed with the advantage of changeable pore size over the protein nanopore. In principle, these solid nanopores, once functionalized in the lumen, should be able to capture individual target molecules. One should be able to observe this process from the binding-produced discrete current blocks. However, direct observation of single molecule binding to solid nanopores has been rarely reported. Here we for the first time report the aptamer encoded nanopore for single-molecule biosensing. Aptamers are short DNA or RNA sequences that can fold into specific conformations to bind their target proteins with high affinity and high selectivity. Comparing with antibodies, aptamers are durable in severe environment such as high temperature and extreme pH values. In particular, aptamers are advantageous in nanopore applications due to their smaller volume than their target proteins, allowing target-generated current block to be visualized. In this report, we demonstrate an aptamer-encoded nanopore for single molecule detection of IgE and bioterrorist agent ricin.

DNA Translocation Governed by Interactions with Solid-State Nanopores

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7–5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150–3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

Fabrication and Application of Nanopores using TEM, STEM and Ion Beams

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008