Enhanced Nanoparticle Sensing in a Highly Viscous Nanopore.

Slowing down translocation dynamics is a crucial challenge in nanopore sensing of small molecules and particles. Here, it is reported on nanoparticle motion-mediated local viscosity enhancement of water-organic mixtures in a nanofluidic channel that enables slow translocation speed, enhanced capture efficiency, and improved signal-to-noise ratio by transmembrane voltage control. It is found that higher detection rates of nanoparticles under larger electrophoretic voltage in the highly viscous solvents. Meanwhile, the strongly pulled particles distort the liquid in the pore at high shear rates over 103 s-1 which leads to a counterintuitive phenomenon of slower translocation speed under higher voltage via the induced dilatant viscosity behavior. This mechanism is demonstrated as feasible with a variety of organic molecules, including glycerol, xanthan gum, and polyethylene glycol. The present findings can be useful in resistive pulse analyses of nanoscale objects such as viruses and proteins by allowing a simple and effective way for translocation slowdown, improved detection throughput, and enhanced signal-to-noise ratio.

Protocol for preparation of solid-state multipore osmotic power generators.

Nanopore is an emerging energy-harvesting device that can create electricity directly from salt solutions. Here, we present a protocol for the preparation and structure optimization of solid-state multipore osmotic power generators. We describe steps for sculpting multiple pores at well-defined positions in a thin SiNx membrane using electron-beam lithography. We also detail an imprinting technique to form polydimethylsiloxane blocks with fluidic channels bonded to the multipore membrane. This approach facilitates repeated liquid-exchange processes involved in ionic current measurements. For complete details on the use and execution of this protocol, please refer to Tsutsui et al.1.

Regulating Nonlinear Ion Transport through a Solid-State Pore by Partial Surface Coatings.

Using functional nanofluidic devices to manipulate ion transport allows us to explore the nanoscale development of blue energy harvesters and iontronic building blocks. Herein, we report on a method to alter the nonlinear ionic current through a pore by partial dielectric coatings. A variety of dielectric materials are examined on both the inner and outer surfaces of the channel with four different patterns of coated or uncoated surfaces. Through controlling the specific part of the surface charge, the pore can behave like a resistor, diode, and bipolar junction transistor. We use numerical simulations to find out the reason for the asymmetric ion transport in the pore and illustrate the relationship between specifically charged surfaces and electroosmotic flow. These findings help understand the role of the corresponding surface composition in ion transport, which provides a direct approach to modify the electroosmotic-flow-driven ionic current rectification in the channel-based device via dielectric coatings.

Ionic Signal Amplification of DNA in a Nanopore.

Ionic signal amplification is a key challenge for single-molecule analyses by solid-state nanopore sensing. Here, a permittivity gradient approach for amplifying ionic blockade characteristics of DNA in a nanofluidic channel is reported. The transmembrane ionic current response is found to change substantially through modifying the liquid permittivity at one side of a pore with an organic solvent. Imposing positive liquid permittivity gradients with respect to the direction of DNA electrophoresis, this study observes the resistive ionic signals to become larger due to the varying contributions of molecular counterions. On the contrary, negative gradients render adverse effects causing conductive ionic current pulses upon polynucleotide translocations. Most importantly, both the positive and negative gradients are demonstrated to be capable of amplifying the ionic signals by an order of magnitude with a 1.3-fold difference in the transmembrane liquid dielectric constants. This phenomenon allows a novel way to enhance the single-molecule sensitivity of nanopore sensing that may be useful in analyzing secondary structures and genome sequence of DNA by ionic current measurements.

Interference of electrochemical ion diffusion in nanopore sensing.

Stable and fast-responding ionic current is a prerequisite for reliable measurements of small objects with a nanopore. Here, we report on the interference of ion diffusion kinetics at liquid-electrode interfaces in nanopore sensing. Using platinum as electrodes, we observed a slow and large decrease in the ionic current through a nanopore in a salt solution suggestive of the considerable influence of the growing impedance at the liquid-metal interfaces via Cottrell diffusion. When detecting nanoparticles, the resistive pulses became weaker following the steady increase in the resistance at the partially polarizable electrodes. The interfacial impedance was also demonstrated to couple with the nanopore chip capacitance thereby degraded the temporal resolution of the ionic current measurements in a time-varying manner. These findings can be useful for choosing the suitable size and material of electrodes for the single-particle and -molecule analyses by ionic current.

Dependence of Molecular Diode Behaviors on Aromaticity.

A molecule-scale diode is an essential component for the concept of molecular electronics. Here we report on heterogeneous contact-mediated rectifying behavior in single-molecule junctions. We performed massive current versus voltage characteristics measurements of metal-molecule-metal structures under stretching by a mechanical break junction method. In-situ deformations of the molecular bridges were revealed to induce stochastic switching of the rectifying direction to varying rectification ratio derived from the induced asymmetry in the contact motifs at the molecule termini. Aromatic molecules were found to enable stronger rectifications via the more pronounced Fermi pinning effect to shift the molecular orbital levels by the applied voltage. Dissimilar anchoring groups also served to stabilize the single-molecule diode properties by bestowing a chemically defined difference in the electronic coupling strengths at the electrode-molecule links. The present findings provide a guide to design diodes with the smallest and simplest structures.

Ionic heat dissipation in solid-state pores.

Energy dissipation in solid-state nanopores is an important issue for their use as a sensor for detecting and analyzing individual objects in electrolyte solution by ionic current measurements. Here, we report on evaluations of heating via diffusive ion transport in the nanoscale conduits using thermocouple-embedded SiNx pores. We found a linear rise in the nanopore temperature with the input electrical power suggestive of steady-state ionic heat dissipation in the confined nanospace. Meanwhile, the heating efficiency was elucidated to become higher in a smaller pore due to a rapid decrease in the through-water thermal conduction for cooling the fluidic channel. The scaling law suggested nonnegligible influence of the heating to raise the temperature of single-nanometer two-dimensional nanopores by a few kelvins under the standard cross-membrane voltage and ionic strength conditions. The present findings may be useful in advancing our understanding of ion and mass transport phenomena in nanopores.

Deep Learning-Enhanced Nanopore Sensing of Single-Nanoparticle Translocation Dynamics.

Noise is ubiquitous in real space that hinders detection of minute yet important signals in electrical sensors. Here, the authors report on a deep learning approach for denoising ionic current in resistive pulse sensing. Electrophoretically-driven translocation motions of single-nanoparticles in a nano-corrugated nanopore are detected. The noise is reduced by a convolutional auto-encoding neural network, designed to iteratively compare and minimize differences between a pair of waveforms via a gradient descent optimization. This denoising in a high-dimensional feature space is demonstrated to allow detection of the corrugation-derived wavy signals that cannot be identified in the raw curves nor after digital processing in frequency domains under the given noise floor, thereby enabled in-situ tracking to electrokinetic analysis of fast-moving single- and double-nanoparticles. The ability of the unlabeled learning to remove noise without compromising temporal resolution may be useful in solid-state nanopore sensing of protein structure and polynucleotide sequence.

Detecting Single Molecule Deoxyribonucleic Acid in a Cell Using a Three-Dimensionally Integrated Nanopore.

Amplification-free genome analysis can revolutionize biology and medicine by uncovering genetic variations among individuals. Here, the authors report on a 3D-integrated nanopore for electrolysis to in situ detection of single-molecule DNA in a cell by ionic current measurements. It consists of a SiO2 multipore sheet and a SiNx nanopore membrane stacked vertically on a Si wafer. Single cell lysis is demonstrated by 106  V m-1 -level electrostatic field focused at the multinanopore. The intracellular molecules are then directly detected as they move through a sensing zone, wherein the authors find telegraphic current signatures reflecting folding degrees of freedom of the millimeter-long polynucleotides threaded through the SiNx nanopore. The present device concept may enable on-chip single-molecule sequencing to multi-omics analyses at a single-cell level.

Salt Gradient Control of Translocation Dynamics in a Solid-State Nanopore.

Tuning capture rates and translocation time of analytes in solid-state nanopores are one of the major challenges for their use in detecting and analyzing individual nanoscale objects via ionic current measurements. Here, we report on the use of salt gradient for the fine control of capture-to-translocation dynamics in 300 nm sized SiNx nanopores. We demonstrated a decrease up to a factor of 3 in the electrophoretic speed of nanoparticles at the pore exit along with an over 3-fold increase in particle detection efficiency by subjecting a 5-fold ion concentration difference across the dielectric membrane. The improvement in the sensor performance was elucidated to be a result of the salt-gradient-mediated electric field and electroosmotic flow asymmetry at nanochannel orifices. The present findings can be used to enhance nanopore sensing capability for detecting biomolecules such as amyloids and proteins.

Inertial focusing and zeta potential measurements of single-nanoparticles using octet-nanochannels.

Capture-to-translocation dynamics control is an important issue for single-particle and -molecule analyses by resistive pulse waveforms. Here, we report on regulated motions for accurate zeta-potential assessments of single nanoscale objects passing through an octet-nanochannel. We observed ionic spike signals consisting of eight consecutive sub-pulses signifying the ion blockage at the eight sensing zones in series upon electrophoretic translocation of individual nanoparticles. We find an exponential decrease to saturation of the channel-to-channel translocation duration as a nanobead moves forward, reflecting the more restricted radial motion degrees of freedom via inertial effects at the downstream side of the octet channel. This finding enabled a protocol for single-nanoparticle zeta potential estimation impervious to the uncertainty stemming from the stochastic nature of the translocation dynamics. The multi-channel approach presented in this study may be used as a useful tool for analyzing particles and molecules of variable sizes.

Rapid Discrimination of Extracellular Vesicles by Shape Distribution Analysis.

A rapid and simple cancer detection method independent of cancer type is an important technology for cancer diagnosis. Although the expression profiles of biological molecules contained in cancer cell-derived extracellular vesicles (EVs) are considered candidates for discrimination indexes to identify any cancerous cells in the body, it takes a certain amount of time to examine these expression profiles. Here, we report the shape distributions of EVs suspended in a solution and the potential of these distributions as a discrimination index to discriminate cancer cells. Distribution analysis is achieved by low-aspect-ratio nanopore devices that enable us to rapidly analyze EV shapes individually in solution, and the present results reveal a dependence of EV shape distribution on the type of cells (cultured liver, breast, and colorectal cancer cells and cultured normal breast cells) secreting EVs. The findings in this study provide realizability and experimental basis for a simple method to discriminate several types of cancerous cells based on rapid analyses of EV shape distributions.

Effect of Electrolyte Concentration on Cell Sensing by Measuring Ionic Current Waveform through Micropores.

Immunostaining has been widely used in cancer prognosis for the quantitative detection of cancer cells present in the bloodstream. However, conventional detection methods based on the target membrane protein expression exhibit the risk of missing cancer cells owing to variable protein expressions. In this study, the resistive pulse method (RPM) was employed to discriminate between cultured cancer cells (NCI-H1650) and T lymphoblastoid leukemia cells (CCRF-CEM) by measuring the ionic current response of cells flowing through a micro-space. The height and shape of a pulse signal were used for the simultaneous measurement of size, deformability, and surface charge of individual cells. An accurate discrimination of cancer cells could not be obtained using 1.0 × phosphate-buffered saline (PBS) as an electrolyte solution to compare the size measurements by a microscopic observation. However, an accurate discrimination of cancer cells with a discrimination error rate of 4.5 ± 0.5% was achieved using 0.5 × PBS containing 2.77% glucose as the electrolyte solution. The potential application of RPM for the accurate discrimination of cancer cells from leukocytes was demonstrated through the measurement of the individual cell size, deformability, and surface charge in a solution with a low electrolyte concentration.

Dielectric Coatings for Resistive Pulse Sensing Using Solid-State Pores.

The present study reports on the systematic characterization of the effectiveness of dielectric coating to tailor capture-to-translocation dynamics of single particles in solid-state pores. We covered the surface of SiNx membranes with SiO2, HfO2, Al2O3, TiO2, or ZnO, which allowed us to change the ζ-potential at the pore wall, reflecting the isoelectric points of these coating materials. Resistive pulse measurements of negatively charged polystyrene beads elucidated more facile electrophoretic capture of the particles and slower translocation motions in the channel under more negative electric potential at the oxide surface. These findings provide a guide to engineer pore wall surface for optimizing the translocation dynamics for efficient sensing of particles and molecules.

Quasi-Stable Salt Gradient and Resistive Switching in Solid-State Nanopores.

Understanding and control of ion transport in a fluidic channel is of crucial importance for iontronics. The present study reports on quasi-stable ionic current characteristics in a SiNx nanopore under a salinity gradient. An intriguing interplay between electro-osmotic flow and local ion density distributions in a solid-state pore is found to induce highly asymmetric ion transport to negative differential resistance behavior under a 100-fold difference in the cross-membrane salt concentrations. Meanwhile, a subtle change in the salinity gradient profile led to observations of resistive switching. This peculiar characteristic was suggested to stem from quasi-stable local ion density around the channel that can be switched between two distinct states via the electro-osmotic flow under voltage control. The present findings may be useful for neuromorphic devices based on micro- and nanofluidic channels.

Digital Pathology Platform for Respiratory Tract Infection Diagnosis via Multiplex Single-Particle Detections.

The variability of bioparticles remains a key barrier to realizing the competent potential of nanoscale detection into a digital diagnosis of an extraneous object that causes an infectious disease. Here, we report label-free virus identification based on machine-learning classification. Single virus particles were detected using nanopores, and resistive-pulse waveforms were analyzed multilaterally using artificial intelligence. In the discrimination, over 99% accuracy for five different virus species was demonstrated. This advance is accessed through the classification of virus-derived ionic current signal patterns reflecting their intrinsic physical properties in a high-dimensional feature space. Moreover, consideration of viral similarity based on the accuracies indicates the contributing factors in the recognitions. The present findings offer the prospect of a novel surveillance system applicable to detection of multiple viruses including new strains.

Machine learning-driven electronic identifications of single pathogenic bacteria.

A rapid method for screening pathogens can revolutionize health care by enabling infection control through medication before symptom. Here we report on label-free single-cell identifications of clinically-important pathogenic bacteria by using a polymer-integrated low thickness-to-diameter aspect ratio pore and machine learning-driven resistive pulse analyses. A high-spatiotemporal resolution of this electrical sensor enabled to observe galvanotactic response intrinsic to the microbes during their translocation. We demonstrated discrimination of the cellular motility via signal pattern classifications in a high-dimensional feature space. As the detection-to-decision can be completed within milliseconds, the present technique may be used for real-time screening of pathogenic bacteria for environmental and medical applications.

Nano-corrugated Nanochannels for In Situ Tracking of Single-Nanoparticle Translocation Dynamics.

Dynamic motions of materials in liquid present a wealth of information concerning their physical properties. While fluorescence microscopy has been widely utilized for single-particle observations, the method cannot be used for characterizing fast motions of nanoscale objects due to the limited spatiotemporal resolution. Here, we report on a nanostructure strategy for nanoscale tracking of single nanoparticles. We fabricated a straight conduit in a SiO2 layer on a Si wafer with lithographically defined 30 nm-sized protrusions formed on the side walls. We performed resistive pulse measurements at a 1 MHz sampling rate wherein we found n-stepped current traces signifying n number of nanoparticles moving concurrently inside the nanochannel. Ensemble average of the ionic current signals revealed a peculiar feature reflecting the slightly stronger ion blockage at the nanoconstrictions between the protrusions, thereby proving the ability of nano-corrugation as physical gates to signify the precise positions of objects inside the nanofluidic channel. This in situ tracking approach elucidated steady-state motions of the nanoparticles moving at a constant speed under the counter-balanced electrophoretic and viscous drag forces, which also allowed estimations of their surface charge densities. The present method can be utilized as a speedometer for nanoscale objects of virtually any size as long as they are able to be put through the sensing zones with potential applications for single-molecule time-of-flight mass spectrometry.

Electroosmosis-Driven Nanofluidic Diodes.

Fundamental understanding of ion transport in a fluidic channel is of critical importance for realizing iontronics. Here we report on asymmetric ion transport in a low thickness-to-diameter aspect ratio nanopore. Under uniform salt concentration conditions, the cross-pore ionic current showed ohmic characteristics with no bias polarity dependence. In stark contrast, despite the weak ion selectivity expected for the relatively large nanopores employed, we observed diode-like behavior when a salt gradient was imposed across the thin membrane. This unexpected result was attributed to the electroosmotic flow that served to modulate the access resistance through dragging the condensed ions into or out of the nanopore orifices. The simple mechanism was also revealed to be effective in fluidic channels of various size from micro- to nanoscale enabling rectification of the property engineering by the pore geometries. The present findings allow for novel designs of artificial ion channel building blocks.