Correction to Nanopore Impedance Spectroscopy Reveals Electrical Properties of Single Nanoparticles for Detecting and Identifying Pathogenic Viruses.

[This corrects the article DOI: 10.1021/acsomega.3c00628.].

Nanopore Impedance Spectroscopy Reveals Electrical Properties of Single Nanoparticles for Detecting and Identifying Pathogenic Viruses.

In the conventional nanopore method, direct current (DC) is used to study molecules and nanoparticles; however, it cannot easily discriminate between materials with similarly sized particles. Herein, we developed an alternating current (AC)-based nanopore method to measure the impedance of a single nanoparticle and distinguish between particles of the same size based on their material characteristics. We demonstrated the performance of this method using impedance measurements to determine the size and frequency characteristics of various particles, ranging in diameter from 200 nm to 1 µm. Furthermore, the alternating current method exhibited high accuracy for biosensing applications, identifying viruses with over 85% accuracy using single-particle measurement and machine learning. Therefore, this novel nanopore method is useful for applications in materials science, biology, and medicine.

Measurement of low-grade inflammation of the esophageal mucosa with electrical conductivity shows promise in assessing PPI responsiveness in patients with GERD.

A device that can easily measure electrical impedance might be a helpful tool for investigating the pathophysiology of gastroesophageal reflux disease. The first aim of this study was to validate our newly developed bioelectrical admittance measurement (BAM) through in vitro experimentation. The second aim was to investigate whether evaluation of BAM by this measurement differed between patients with heartburn according to their response to proton pump inhibitor (PPI) therapy. Caco-2 cell monolayers and three-dimensional tissues were examined by BAM using a frequency response analyzer. BAM was also used to measure the impedance through cell layers. Subsequently, BAM was performed during endoscopy in 41 patients experiencing heartburn without esophageal mucosal breaks. After 2-wk administration of 20-mg rabeprazole twice daily, patient responses to PPI were classified as "good" or "poor" according to their clinical course. In each patient, histological alterations and gene expression levels of inflammation mediators and tight junction proteins were evaluated. Impedance profiles indicated that monolayer Caco-2 cells on top of eight-layered normal human dermal fibroblasts had the highest magnitude of impedance over the range of frequencies. In vivo results revealed that patients with good responses to PPI displayed significantly higher admittance. Severity of low-grade inflammation was significantly associated with esophageal wall admittance. Moreover, esophageal wall admittance may be more closely related to basal zone hyperplasia than dilatation of intercellular spaces. Thus, BAM may be able to detect abnormalities in the subepithelial layer of the esophagus.NEW & NOTEWORTHY Bioelectrical admittance measurement is a new method to evaluate esophageal mucosal permeability vertically during upper gastrointestinal endoscopy. Measurement of low-grade inflammation of the esophageal mucosa with electrical conductivity shows promise in assessing proton pump inhibitor responsiveness in patients with gastroesophageal reflux disease. As various gastrointestinal diseases are associated with changes in mucosal permeability, bioelectrical admittance measurement is expected to be clinically applied to therapeutic decision-making for these diseases in the future.

One-Dimensional Flow of Bacteria on an Electrode Rail by Dielectrophoresis: Toward Single-Cell-Based Analysis.

Many applications in biotechnology and medicine, among other disciplines, require the rapid enumeration of bacteria, preferably using miniaturized portable devices. Microfluidic technology is expected to solve this miniaturization issue. In the enumeration of bacteria in microfluidic devices, the technique of aligning bacteria in a single line prior to counting is the key to an accurate count at single-bacterium resolution. Here, we describe the numerical and experimental evaluation of a device utilizing a dielectrophoretic force to array bacteria in a single line, allowing their facile numeration. The device comprises a channel to flow bacteria, two counter electrodes, and a capture electrode several microns or less in width for arranging bacteria in a single line. When the capture electrode is narrower than the diameter of a bacterium, the entrapment efficiency of the one-dimensional array is 80% or more within 2 s. Furthermore, since some cell-sorting applications require bacteria to move against the liquid flow, we demonstrated that bacteria can move in a single line in the off-axial direction tilted 30° from the flow direction. Our findings provide the basis for designing miniature, portable devices for evaluating bacteria with single-cell accuracy.

Optical Etching to Pattern Microstructures on Plastics by Vacuum Ultraviolet Light.

We proposed and demonstrated an optical dry etching method for transferring a pattern on a photomask to a surface of plastics by decomposing the irradiated area using the high energy of vacuum ultraviolet light (VUV) at room temperature and pressure. Two kinds of wavelengths of 160 nm and 172 nm were used as the vacuum ultraviolet light, and the patterning performances for polymethyl methacrylate (PMMA) and polycarbonate (PC) were compared. As a result, it was revealed that proportional relationships were obtained between the etching rate and the irradiation dose for both wavelengths, and the cross-sectional profiles were anisotropic. In addition, both PMMA and PC were etched at a wavelength of 160 nm, whereas PC could not be etched at a wavelength of 172 nm, suggesting that it correlates with the bond dissociation energies of the molecular bonds of the materials and the energies of the photons. Furthermore, by combining this method with the optical bonding method that we had previously developed to bond surfaces irradiated with VUV, we have demonstrated a method for fabricating microfluidic devices by irradiating only with VUV. This paper shows that this technique is a new microfabrication method suitable for simple and mass production of plastic materials.

Single-Molecule Detection of DNA in a Nanochannel by High-Field Strength-Assisted Electrical Impedance Spectroscopy.

Many researchers have fabricated micro and nanofluidic devices incorporating optical, chemical, and electrical detection systems with the aim of achieving on-chip analysis of macromolecules. The present study demonstrates a label-free detection of DNA using a nanofluidic device based on impedance measurements that is both sensitive and simple to operate. Using this device, the electrophoresis and dielectrophoresis effect on DNA conformation and the length dependence were examined. A low alternating voltage was applied to the nanogap electrodes to generate a high intensity field (>0.5 MV/m) under non-faradaic conditions. In addition, a 100 nm thick gold electrode was completely embedded in the substrate to allow direct measurements of a solution containing the sample passing through the gap, without any surface modification required. The high intensity field in this device produced a dielectrophoretic force that stretched the DNA molecule across the electrode gap at a specific frequency, based on back and forth movements between the electrodes with the DNA in a random coil conformation. The characteristics of 100 bp, 500 bp, 1 kbp, 5 kbp, 10 kbp, and 48 kbp λ DNA associated with various conformations were quantitatively analyzed with high resolution (on the femtomolar level). The sensitivity of this system was found to be more than about 10 orders of magnitude higher than that obtained from conventional linear alternating current (AC) impedance for the analysis of bio-polymers. This new high-sensitivity process is expected to be advantageous with regard to the study of complex macromolecules and nanoparticles.

Fabrication of an Anti-Reflective and Super-Hydrophobic Structure by Vacuum Ultraviolet Light-Assisted Bonding and Nanoscale Pattern Transfer.

The application of subwavelength, textured structures to glass surfaces has been shown to reduce reflectivity and also results in self-cleaning due to super-hydrophobicity. However, current methods of producing such textures are typically either expensive or difficult to scale up. Based on prior work by the authors, the present study employed a combination of vacuum ultraviolet (VUV) light-assisted bonding and release agent-free pattern transfer to fabricate a moth-eye texture on a glass substrate. This was accomplished by forming a cyclic olefin polymer mold master with a moth-eye pattern, transferring this pattern to a polydimethylsiloxane (PDMS) spin coating, activating both the PDMS and a glass substrate with VUV light, and then bonding the PDMS to the glass before releasing the mold. Atomic force microscopy demonstrated that the desired pattern was successfully replicated on the PDMS surface with a high degree of accuracy, and the textured glass specimen exhibited approximately 3% higher transmittance than untreated glass. Contact angle measurements also showed that the hydrophobicity of the textured surface was significantly increased. These results confirm that this new technique is a viable means of fabricating optical nanostructures via a simple, inexpensive process.

Damage-less Handling of Exosomes Using an Ion-depletion Zone in a Microchannel.

Exosomes are of increasing research interest because they are integral to cell-cell communication and are implicated in various disease states. Here, we investigated the utility of using an ion-depletion zone in a microfluidic device to concentrate exosomes from the culture media of four types of cell lines. Furthermore, we evaluated the extent of damage to the exosomes following concentration by an ion-depletion zone microchannel device compared with exosomes concentrated by a conventional ultra-centrifugation technique. Our results conclusively demonstrate that significantly less damage is incurred by exosomes following passage through and concentration by the ion-depleted zone microchannel device compared to concentration by ultra-centrifugation. Our findings will help extend the utility of exosomes to various applications.

Quantification of Virus Particles Using Nanopore-Based Resistive-Pulse Sensing Techniques.

Viruses have drawn much attention in recent years due to increased recognition of their important roles in virology, immunology, clinical diagnosis, and therapy. Because the biological and physical properties of viruses significantly impact their applications, quantitative detection of individual virus particles has become a critical issue. However, due to various inherent limitations of conventional enumeration techniques such as infectious titer assays, immunological assays, and electron microscopic observation, this issue remains challenging. Thanks to significant advances in nanotechnology, nanostructure-based electrical sensors have emerged as promising platforms for real-time, sensitive detection of numerous bioanalytes. In this paper, we review recent progress in nanopore-based electrical sensing, with particular emphasis on the application of this technique to the quantification of virus particles. Our aim is to provide insights into this novel nanosensor technology, and highlight its ability to enhance current understanding of a variety of viruses.

A Novel Fabrication Technique for Liquid-Tight Microchannels by Combination of a Paraffin Polymer and a Photo-Curable Silicone Elastomer.

The development and growth of microfluidics has been mainly based on various novel fabrication techniques for downsizing and integration of the micro/nano components. Especially, an effective fabrication technique of three-dimensional structures still continues to be strongly required in order to improve device performance, functionality, and device packing density because the conventional lamination-based technique for integrating several two-dimensional components is not enough to satisfy the requirement. Although three-dimensional printers have a high potential for becoming an effective tool to fabricate a three-dimensional microstructure, a leak caused by the roughness of a low-precision structure made by a 3D printer is a critical problem when the microfluidic device is composed of several parts. To build a liquid-tight microchannel on such a low-precision structure, we developed a novel assembly technique in which a paraffin polymer was used as a mold for a microchannel of photo-curable silicone elastomer on a rough surface. The shape and roughness of the molded microchannel was in good agreement with the master pattern. Additionally, the seal performance of the microchannel was demonstrated by an experiment of electrophoresis in the microchannel built on a substrate which has a huge roughness and a joint.

Nonlinear electrical impedance spectroscopy of viruses using very high electric fields created by nanogap electrodes.

Our living sphere is constantly exposed to a wide range of pathogenic viruses, which can be either known, or of novel origin. Currently, there is no methodology for continuously monitoring the environment for viruses in general, much less a methodology that allows the rapid and sensitive identification of a wide variety of viruses responsible for communicable diseases. Traditional approaches, based on PCR and immunodetection systems, only detect known or specifically targeted viruses. We here describe a simple device that can potentially detect any virus between nanogap electrodes using nonlinear impedance spectroscopy. Three test viruses, differing in shape and size, were used to demonstrate the general applicability of this approach: baculovirus, tobacco mosaic virus (TMV), and influenza virus. We show that each of the virus types responded differently in the nanogap to changes in the electric field strength, and the impedance of the virus solutions differed depending both on virus type and virus concentration. These preliminary results show that the three virus types can be distinguished and their approximate concentrations determined. Although further studies are required, the proposed nonlinear impedance spectroscopy method may achieve a sensitivity comparable to that of more traditional, but less versatile, virus detection systems.

Single molecular level analysis and processing in nanochannels.

Recent advances in nanofluidic technologies have enabled devices to be fabricated that integrate nanochannels with at least one dimension smaller than several hundred nanometers. Since this dimension is close to the sizes of biomolecules such as DNA, proteins, and other biomolecules, it opens up new scientific and technological fields based on analysis and manipulation of single molecules. This paper reviews the current state of knowledge regarding single molecular level analysis and processing in nanochannels, particularly experimental findings in this area. While there have been many theoretical and molecular simulation studies, this paper surveys experimental studies. Following a brief survey of techniques for fabricating nanochannels, we review fundamental studies of single molecule behavior and manipulation in nanochannels. We then discuss important transport phenomena of single molecules in nanochannels. Finally, the emerging challenge of electrical single-molecule detection and its possible applications are highlighted.

Nanoscale etching and flattening of metals with ozone water.

Etchants used for metal etching are generally harmful to the environment. We propose an environmentally friendly method that uses ozone water to etch metals. We measured the dependencies of ozone water etching on the temperature and ozone concentration for several metals and evaluated the surface roughness of the etched surfaces. The etching rate was proportional to the dissolved ozone concentration, and the temperature and the surfaces were smoothed by etching.

Modification of the glass surface property in PDMS-glass hybrid microfluidic devices.

This paper presents a simple method to change the hydrophilic nature of the glass surface in a poly(dimethylsiloxane) (PDMS)-glass hybrid microfluidic device to hydrophobic by an extra-heating step during the fabrication process. Glass substrates bonded to a native or oxygen plasma-treated PDMS chip having microchambers (12.5 mm diameter, 110 µm height) were heated at 200°C for 3 h, and then the hydrophobicity of the glass surfaces on the substrate was evaluated by measuring the contact angle of water. By the extra-heating process, the glass surfaces became hydrophobic, and its contact angle was around 109°, which is nearly the same as native PDMS surfaces. To demonstrate the usefulness of this surface modification method, a PDMS-glass hybrid microfluidic device equipped with microcapillary vent structures for pneumatic manipulation of droplets was fabricated. The feasibility of the microcapillary vent structures on the device with the hydrophobic glass surfaces are confirmed in practical use through leakage tests of the vent structures and liquid handling for the electrophoretic separation of DNA molecules.

Application of cell-free expression of GFP for evaluation of microsystems.

Coupled cell-free transcription-translation (CFTT) of green fluorescent protein (GFP) has been applied as a reporter system to microfluidic chip-related technologies. In polymerase chain reaction (PCR)-based biomolecular logic gate system, in which addition of primer set and amplification of PCR product represent input and output signal respectively, GFP gene was inserted in the template DNA, which was then amplified, transcribed and translated to GFP. The green fluorescence reported as if the amplification has occurred or not, that is, the fluorescence reports positive output signal. CFTT of GFP was also adopted to evaluate on-chip capillary electrophoresis (CE)-based DNA fractionation, which was developed to isolate single DNA species from reaction mixture of DNA ligase-catalyzed DNA-assembly. As a model system, GFP gene was inserted in the target DNA fragment. The collected fraction was amplified with PCR and subjected to a CFTT system, and green fluorescence was observed showing that the fractionation was successful. These results showed that CFTT of GFP is a useful tool to verify, estimate, and monitor microfluidic chip-related technologies in which cell-free protein synthesis is involved.

Electroactive microwell arrays for highly efficient single-cell trapping and analysis.

We present a novel method, implemented in the form of a microfluidic device, for arraying and analyzing large populations of single cells. The device contains a large array of electroactive microwells where manipulation and analysis of large population of cells are carried out. On the device, single cells can be actively trapped in the microwells by dielectrophoresis (DEP) and then lysed by electroporation (EP) for subsequent analysis of the confined cell lysates. The DEP force in the selected dimensions of the microwells could achieve efficient trapping in nearly all the microwells (95%) in less than three minutes. Moreover, the positions of the cells in the microwells are maintained even when unstable flow of liquid is applied. This makes it possible to exchange the DEP buffer to a solution that will be subsequently used for stimulating or analyzing the trapped cells. After closing the microwells, EP is conducted to lyse the trapped cells by applying short electric pulses. Tight enclosure is critical to prevent dilution, diffusion and cross contamination of the cell lysates. We demonstrated the feasibility of our approach with an enzymatic assay measuring the intracellular-galactosidase activity. The use of this method should greatly help analysis of large populations of cells at the single-cell level. Furthermore, the method offers rapidity in the trapping and analysis of multiple cell types in physiological conditions that will be important to ensure the relevance of single cell analyses.

A microfluidic in situ analyzer for ATP quantification in ocean environments.

We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M.

A rapid method for optimizing running temperature of electrophoresis through repetitive on-chip CE operations.

In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method.

An electroactive microwell array for trapping and lysing single-bacterial cells.

Interest in single-cell analysis has increased because it allows to understand cell metabolism and characterize disease states, cellular adaptation to environmental changes, cell cycles, etc. Here, the authors propose a device to electrically trap and lyse single-bacterial cells in an array format for high-throughput single-cell analysis. The applied electric field is highly deformed and concentrated toward the inside of the microwell structures patterned on the planar electrode. This configuration effectively generates dielectrophoretic force to attract a single cell per well. The microwell has a comparable size to the target bacterial cell making it possible to trap single cells by physically excluding additional cells. Inducing highly concentrated electric potential on the cell membrane can also effectively lyse the trapped single-bacterial cells. The feasibility of the authors' approach was demonstrated by trapping and lysing Escherichia coli cells at the single-cell level. The present microwell array can be used as a basic tool for individual bacterial cell analysis.