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.

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.

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.

Molecular profiling of extracellular vesicles via charge-based capture using oxide nanowire microfluidics.

Extracellular vesicles (EVs) have shown promising features as biomarkers for early cancer diagnoses. The outer layer of cancer cell-derived EVs consists of organotropic metastasis-induced membrane proteins and specifically enriched proteoglycans, and these molecular compositions determine EV surface charge. Although many efforts have been devoted to investigating the correlation between EV subsets obtained through density-, size-, and immunoaffinity-based captures and expressed membrane proteins, understanding the correlation between EV subsets obtained through surface charge-based capture and expressed membrane proteins is lacking. Here, we propose a methodology to profile membrane proteins of EV subsets obtained through surface charge-based capture. Nanowire-induced charge-based capture of EVs and in-situ profiling of EV membrane proteins are the two key methodology points. The oxide nanowires allowed EVs to be obtained through surface charge-based capture due to the diverse isoelectric points of the oxides and the large surface-to-volume ratios of the nanowire structures. And, with the ZnO nanowire device, whose use does not require any purification and concentration processes, we demonstrated the correlation between negatively-charged EV subsets and expressed membrane proteins derived from each cell. Furthermore, we determined that a colon cancer related membrane protein was overexpressed on negatively charged surface EVs derived from colon cancer cells.

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.

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.

Mechanical Rupture-Based Antibacterial and Cell-Compatible ZnO/SiO2 Nanowire Structures Formed by Bottom-Up Approaches.

There are growing interests in mechanical rupture-based antibacterial surfaces with nanostructures that have little toxicity to cells around the surfaces; however, current surfaces are fabricated via top-down nanotechnologies, which presents difficulties to apply for bio-surfaces with hierarchal three-dimensional structures. Herein, we developed ZnO/SiO2 nanowire structures by using bottom-up approaches and demonstrated to show mechanical rupture-based antibacterial activity and compatibility with human cells. When Escherichia coli were cultured on the surface for 24 h, over 99% of the bacteria were inactivated, while more than 80% of HeLa cells that were cultured on the surface for 24 h were still alive. This is the first demonstration of mechanical rupture-based bacterial rupture via the hydrothermally synthesized nanowire structures with antibacterial activity and cell compatibility.

Solid-State Nanopore Time-of-Flight Mass Spectrometer.

Analysis of field-controlled dynamics of ionized substances in a vacuum enables mass spectroscopy of particles and molecules. Analogously, here we report that nanoscale tracking of electrophoretically driven fast motions of single nanoparticles allows label-free and nondestructive detection of their mass in liquid. We fine-traced the time-dependent positions of space-filtered regular motions of particles passed through a thin solid-state nanopore by dissecting the associated ionic blockade phenomena under a scope of multiphysics simulations. Characterizing the viscous-drag-mediated exponential decay in the electrophoretic speed of particles ejected into an electrolyte solution from the nanochannel, we demonstrated the discrimination of nanoparticles by the femtogram mass difference. The present method is viable for mass measurement of virtually any object that can be put through the sensing zone, the sensor capability of which may find many applications such as pathogen screening and proteomics.

High-throughput single nanoparticle detection using a feed-through channel-integrated nanopore.

The outstanding sensitivity of solid-state nanopore sensors comes at a price of low detection efficiency due to the lack of active means to transfer objects into the nanoscale sensing zone. Here we report on a key technology for high-throughput single-nanoparticle detection which exploits mutual effects of microfluidics control and multipore electrophoresis in nanopore-in-channel units integrated on a thin Si3N4 membrane. Using this novel nanostructure, we demonstrated a proof-of-concept for influenza viruses via hydropressure regulation of mass transport in the fluidic channel for continuous feeding of biosamples into the effective electric field extending out from the nanopores, wherein the feed-through mechanism allowed us to selectively detect charged objects in physiological media such as human saliva. With the versatility of nanopore sensing technologies applicable to analytes of virtually any size from cells to polynucleotides, the present integration strategy may open new avenues for practical ultrasensitive bioanalytical tools.

Back-Side Polymer-Coated Solid-State Nanopore Sensors.

We systematically investigated the influence of polymer coating on temporal resolution of solid-state nanopores. We fabricated a Si3N4 nanopore integrated with a polyimide sheet partially covering the substrate surface. Upon detecting the nanoparticles dispersed in an electrolyte buffer by ionic current measurements, we observed a larger resistive pulse height along with a faster current decay at the tails under larger coverage of the polymeric layer, thereby suggesting a prominent role of the water-touching Si3N4 thin film as a significant capacitor serving to retard the ionic current response to the ion blockade by fast translocation of particles through the nanopores. From this, we came up with back-side polymer-coated chip designs and demonstrated improved pore sensor temporal resolution by developing a nanopore with a thick polymethyl-methacrylate layer laminated on the bottom surface. The present findings may be useful in developing integrated solid-state nanopore sensors with embedded nanochannels and nanoelectrodes.

High-throughput single-particle detections using a dual-height-channel-integrated pore.

We report a proof-of-principle demonstration of particle concentration to achieve high-throughput resistive pulse detections of bacteria using a microfluidic-channel-integrated micropore. We fabricated polymeric nanochannels to trap micrometer-sized bioparticles via a simple water pumping mechanism that allowed aggregation-free size-selective particle concentration with negligible loss. Single-bioparticle detections by ionic current measurements were then implemented through releasing and transporting the thus-collected analytes to the micropore. As a result, we attained two orders of magnitude enhancement in the detection throughput by virtue of an accumulation effect via hydrodynamic control. The device concept presented may be useful in developing nanopores and nanochannels for high-throughput single-particle and -molecule analyses.

Electric field interference and bimodal particle translocation in nano-integrated multipores.

Parallel integration of multiple channels is a fundamental strategy for high-throughput particle detection in solid-state nanopores wherein understanding and control of crosstalk is an important issue for the post resistive pulse analysis. Here we report on a prominent effect of cross-channel electric field interference on the ionic current blockade by nanoparticles in nano-spaced pore arrays in a thin Si3N4 membrane. We systematically investigated the variations in resistive pulse profiles in multipore systems of various inter-channel distances. Although each pore acted independently when they were formed at excessively far distances, we observed significant cross-pore electrostatic interactions under close-integration that led the multipores to virtually act as a single-pore of equivalent area. As a result of the interference, the resistive pulse height demonstrated bimodal distributions due to the pronounced particle trajectory-dependence of the ionic blockade effects. Most importantly, the overcrowded multi-channel structure was found to deliver significant crosstalk with serious degradation of the sensor sensitivity to particle sizes. The present results provide a guide to design multipore structures regarding the trade-off between the detection throughput and sensor sensitivity.

Silicon substrate effects on ionic current blockade in solid-state nanopores.

We investigated the roles of silicon substrate material compositions in ionic current blockade in solid-state nanopores. When detecting single nanoparticles using an ionic current in a Si3N4 nanopore supported on a doped silicon wafer, resistive pulses were found to be blunted significantly via signal retardation due to predominant contributions of large capacitance at the ultrathin membrane. Unexpectedly, in contrast, changing the substrate material to non-doped silicon led to the sharpening of the spike-like signal feature, suggesting a better temporal resolution of the cross-channel ionic current measurements by virtue of the thick intrinsic semiconductor layer that served to diminish the net chip capacitance. The present results suggest the importance of the choice of Si compositions regarding the capacitance effects to attain better spatiotemporal resolution in solid-state nanopore sensors.

Identifying Single Particles in Air Using a 3D-Integrated Solid-State Pore.

Solid-state micro- and nanopores are a versatile sensor platform capable of detecting single particles in electrolyte solution by cross-pore ionic current. Here we report on a use of this technology to identify airborne particulate matter. The detection concept lies in an electrophoretic control of air-floating particles captured in liquid to deliver them into a pore detector via microfluidic channels. We demonstrate resistive pulse measurements to machine-learning-based discriminations of intragranular contents of cypress and cedar pollens at a single-particle level. This all-electrical-sensor technique would pave a new venue toward real-time monitoring of single particles and molecules in air.

Engineering Nanowire-Mediated Cell Lysis for Microbial Cell Identification.

Researchers have demonstrated great promise for inorganic nanowire use in analyzing cells or intracellular components. Although a stealth effect of nanowires toward cell surfaces allows preservation of the living intact cells when analyzing cells, as a completely opposite approach, the applicability to analyze intracellular components through disrupting cells is also central to understanding cellular information. However, the reported lysis strategy is insufficient for microbial cell lysis due to the cell robustness and wrong approach taken so far ( i. e., nanowire penetration into a cell membrane). Here we propose a nanowire-mediated lysis method for microbial cells by introducing the rupture approach initiated by cell membrane stretching; in other words, the nanowires do not penetrate the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and flexible nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediated lysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible with the loop-mediated isothermal amplification (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells. This approach allows researchers to develop a microfluidic analytical platform not only for microbial cell identification including drug- and heat-resistance cells but also for on-site detection without any contamination.