The design and characterization of multifunctional aptamer nanopore sensors

Aptamer-modified nanomaterials provide a simple, yet powerful sensing platform when combined with resistive pulse sensing technologies. Aptamers adopt a more stable tertiary structure in the presence of a target analyte, which results in a change in charge density and velocity of the carrier particle. In practice the tertiary structure is specific for each aptamer and target, and the strength of the signal varies with different applications and experimental conditions. Resistive pulse sensors (RPS) have single particle resolution, allowing for the detailed characterization of the sample. Measuring the velocity of aptamer-modified nanomaterials as they traverse the RPS provides information on their charge state and densities. To help understand how the aptamer structure and charge density effects the sensitivity of aptamer-RPS assays, here we study two metal binding aptamers. This creates a sensor for mercury and lead ions that is capable of being run in a range of electrolyte concentrations, equivalent to river to seawater conditions. The observed results are in excellent agreement with our proposed model. Building on this we combine two aptamers together in an attempt to form a dual sensing strand of DNA for the simultaneous detection of two metal ions. We show experimental and theoretical responses for the aptamer which creates layers of differing charge densities around the nanomaterial. The density and diameter of these zones effects both the viability and sensitivity of the assay. While this approach allows the interrogation of the DNA structure, the data also highlight the limitations and considerations for future assays

Diffusion and Trapping of Single Particles in Pores with Combined Pressure and Dynamic Voltage

In this article we report resistive-pulse experiments with polystyrene particles whose transport through pores is controlled by modulating the driving voltage during the process of translocation. Balancing electric and hydrostatic forces acting on the particles allowed us to observe a random walk of single particles in a pore for tens of seconds and to quantify their diffusion coefficient using two methods. The first approach is based on the mean square displacement and requires passage of multiple particles for a range of diffusion times. The diffusion coefficient of individual particles was determined based on the variance of their local diffusion velocities. The developed methods for measuring the diffusion coefficient in pores are applicable to particles of different sizes, do not require fluorescence labeling, and are entirely based on ion current recordings. In addition, application of a modulating voltage signal together with rising edge triggers enabled transporting the same particle back and forth in the pore without letting the particle leave the pore

Concentration-Polarization-Induced Precipitation and Ionic Current Oscillations with Tunable Frequency

At the nanoscale, charges present at the surfaces of liquid–solid interfaces greatly influence the properties of ions and molecules present in the solution, and can lead to nanoscale effects such as ion selectivity, ion current rectification, and modulation of local ionic concentrations. Concentration polarization is another nanoscale phenomenon whereby ion concentrations are enriched at one opening of an ion-selective nanopore and depleted at the other. We show that when a nanopore is in contact with a weakly soluble salt present at a concentration below its solubility product, concentration polarization can lead to locally enhanced ionic concentrations and precipitation of the salt. Formed precipitates partially or fully occlude the nanopore’s opening as indicated by a measured transient decrease of the nanopore’s conductance. We have identified experimental conditions at which the locally created precipitate is either pushed through or dissolved, clearing the pore entrance and allowing the precipitation reaction to occur again. The dynamic process of precipitate formation and dissolution is observed as ion current fluctuations and oscillations with frequencies reaching 200 Hz. The frequency of the system operation exceeds other nanopore-based oscillators by 2 orders of magnitude, which we believe stems from the 30 nm length of the pores examined here, versus ∼10 μm long pores reported before

Viscosity and Conductivity Tunable Diode-like Behavior for Meso- and Micropores

Rectifying pores, which transport ions mainly in one direction blocking the ionic flow in the other, were shown to be important in the preparation of chemical sensors, components of ionic circuits, and mimics of biological channels. Ionic rectification has been shown with various engineered systems, but pores with similar opening diameters often rectify to a various uncontrolled extent. In this Letter we present a system of single meso-pores, whose current–voltage curves and rectification can be tuned with great precision via viscosity and conductivity gradients of solutions placed on both sides of the membrane. The mechanism of rectification is based on electroosmotically induced flow, which fills the entire volume of the pore with a single solution from either side of the membrane. The highly predictable rectifying system can find various applications, including measuring viscosity of unknown media and tuning electrokinetic passage of particles

Direction Dependence of Resistive-Pulse Amplitude in Conically Shaped Mesopores

Conically shaped pores such as glass pipets as well as asymmetric pores in polymers became an important analytics tool used for the detection of molecules, viruses, and particles. Electrokinetic or pressure driven passage of single particles through a single pore causes a transient change of the transmembrane current, called a resistive-pulse, whose amplitude is the measure of the particle volume. The shape of the pulse reflects the pore topography, and in a conical pore, resistive pulses have a shape of a tick point. Passage of particles in both directions was reported to produce pulses of the same amplitude and shapes that are mirror images of each other. In this manuscript we identify conditions at which the amplitude of resistive-pulses in a conical mesopore is direction dependent. Neutral particles entering the pore from the larger entrance of a conical pore, called the base, block the current to a larger extent than the particles traveling in the opposite direction. Negatively charged particles on the other hand size larger when being transported in the direction from tip to base. The findings are explained via voltage-regulated ionic concentrations in the pore such that for one voltage polarity a weak depletion zone is formed, which increases the current blockage caused by a particle. For the opposite polarity, an enhancement of ionic concentrations was predicted. The findings reported here are of crucial importance for the resistive-pulse technique, which relates the current blockage with the size of the passing object

Voltage-Induced Modulation of Ionic Concentrations and Ion Current Rectification in Mesopores with Highly Charged Pore Walls

It is believed that ion current rectification (ICR), a property that assures preferential ionic transport in one direction, can only be observed in nanopores when the pore size is comparable to the thickness of the electric double layer (EDL). Rectifying nanopores became the basis of biological sensors and components of ionic circuits. Here we report that appreciable ICR can also occur in highly charged conical, polymer mesopores whose tip diameters are as large as 400 nm, thus over 100-fold larger than the EDL thickness. A rigorous model taking into account the surface equilibrium reaction of functional carboxyl groups on the pore wall and electroosmotic flow is employed to explain that unexpected phenomenon. Results show that the pore rectification results from the high density of surface charges as well as the presence of highly mobile hydroxide ions, whose concentration is enhanced for one voltage polarity. This work provides evidence that highly charged surfaces can extend the ICR of pores to the submicron scale, suggesting the potential use of highly charged large pores for energy and sensing applications. Our results also provide insight into how a mixture of ions with different mobilities can influence current–voltage curves and rectification

DNA-Modified Polymer Pores Allow pH- and Voltage-Gated Control of Channel Flux

Biological channels embedded in cell membranes regulate ionic transport by responding to external stimuli such as pH, voltage, and molecular binding. Mimicking the gating properties of these biological structures would be instrumental in the preparation of smart membranes used in biosensing, drug delivery, and ionic circuit construction. Here we present a new concept for building synthetic nanopores that can simultaneously respond to pH and transmembrane potential changes. DNA oligomers containing protonatable A and C bases are attached at the narrow opening of an asymmetric nanopore. Lowering the pH to 5.5 causes the positively charged DNA molecules to bind to other strands with negative backbones, thereby creating an electrostatic mesh that closes the pore to unprecedentedly high resistances of several tens of gigaohms. At neutral pH values, voltage switching causes the isolated DNA strands to undergo nanomechanical movement, as seen by a reversible current modulation. We provide evidence that the pH-dependent reversible closing mechanism is robust and applicable for nanopores with opening diameters of up to 14 nm. The concept of creating an electrostatic mesh may also be applied to different organic polymers

Probing Porous Structure of Single Manganese Oxide Mesorods with Ionic Current

Characterization of materials in confined spaces, rather than attempting to extrapolate from bulk material behavior, requires the development of new measurement techniques. In particular, measurements of individual meso- or nanoscale objects can provide information about their structure which is unavailable by other means. In this report, we perform measurements of ion currents through a few hundred nanometer long MnO₂ rods deposited in single polymer pores. The recorded current confirms an existence of a meshlike character of the MnO₂ structure and probes the effective size of the mesh voids and the polarity of surface charges. The recorded ion current through deposited MnO₂ structure also suggests that the signal is mostly due to metal cations and not to protons. This is the first time that ionic current measurements have been used to characterize mesoporous structure of this important electrode material

Polarization of Gold in Nanopores Leads to Ion Current Rectification

Biomimetic nanopores with rectifying properties are relevant components of ionic switches, ionic circuits, and biological sensors. Rectification indicates that currents for voltages of one polarity are higher than currents for voltages of the opposite polarity. Ion current rectification requires the presence of surface charges on the pore walls, achieved either by the attachment of charged groups or in multielectrode systems by applying voltage to integrated gate electrodes. Here we present a simpler concept for introducing surface charges via polarization of a thin layer of Au present at one entrance of a silicon nitride nanopore. In an electric field applied by two electrodes placed in bulk solution on both sides of the membrane, the Au layer polarizes such that excess positive charge locally concentrates at one end and negative charge concentrates at the other end. Consequently, a junction is formed between zones with enhanced anion and cation concentrations in the solution adjacent to the Au layer. This bipolar double layer together with enhanced cation concentration in a negatively charged silicon nitride nanopore leads to voltage-controlled surface-charge patterns and ion current rectification. The experimental findings are supported by numerical modeling that confirm modulation of ionic concentrations by the Au layer and ion current rectification even in low-aspect ratio nanopores. Our findings enable a new strategy for creating ionic circuits with diodes and transistors

Anomalous Mobility of Highly Charged Particles in Pores

Single micropores in resistive-pulse technique were used to understand a complex dependence of particle mobility on its surface charge density. We show that the mobility of highly charged carboxylated particles decreases with the increase of the solution pH due to an interplay of three effects: (i) ion condensation, (ii) formation of an asymmetric electrical double layer around the particle, and (iii) electroosmotic flow induced by the charges on the pore walls and the particle surfaces. The results are important for applying resistive-pulse technique to determine surface charge density and zeta potential of the particles. The experiments also indicate the presence of condensed ions, which contribute to the measured current if a sufficiently high electric field is applied across the pore