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.

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.

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.

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 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.

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.

Pneumatic handling of droplets on-demand on a microfluidic device for seamless processing of reaction and electrophoretic separation.

Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.

Nanofluidic single-molecule sorting of DNA: a new concept in separation and analysis of biomolecules towards ultimate level performance.

Separation and separation-based analysis of biomolecules are fundamentally important techniques in the field of biotechnology. These techniques, however, depend on stochastic processes that intrinsically involve uncertainty, and thus it is not possible to achieve 100% separation accuracy. Theoretically, the ultimate resolution and sensitivity should be realized in a single-molecule system because of the deterministic nature of single-molecule manipulation. Here, we have proposed and experimentally demonstrated the concept of a 'single-molecule sorter' that detects and correctly identifies individual single molecules, realizing the ultimate level of resolution and sensitivity for any separation-based technology. The single-molecule sorter was created using a nanofluidic network consisting of a single inlet channel that branches off into multiple outlet channels. It includes two major functional elements, namely a single-molecule detection and identification element and a flow path switching element to accurately separate single molecules. With this system we have successfully demonstrated the world's first single-molecule sorting using DNA as a sample molecule. In the future, we hope to expand the application of such devices to comprehensive sorting of single-proteins from a single cell. We also believe that in addition to the single-molecule sorting method reported here, other types of single-molecule based processes will emerge and find use in a wide variety of applications.

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.

An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models.

Conventional cell-based assays in life science and medical applications can be difficult to maintain functionally over long periods. Microfluidics is an emerging technology with potential to provide integrated environments for cell maintenance, continuous perfusion, and monitoring. In this study, we developed an integrated microfluidic device with on-chip pumping and detection functionalities. The microfluidic structure in the device is divided into two independent channels separated by a semipermeable membrane on which cells are inoculated and cultured. Perfusion and fluorescence measurements of culture media for each channel can be conducted by the on-chip pumping system and optical fiber detection system. Performance of the device was examined through long-term culture and monitoring of polarized transport activity of intestinal tissue models (Caco-2 cells). The cells could be cultured for more than two weeks, and monolayer transport of rhodamine 123 was successfully monitored by on-line fluorescent measurement. This device may have applications in toxicity testing and drug screening.

Stable immobilization of rat hepatocytes as hemispheroids onto collagen-conjugated poly-dimethylsiloxane (PDMS) surfaces: importance of direct oxygenation through PDMS for both formation and function.

The highly oxygen-permeable material, poly-dimethylsiloxane (PDMS), has the potential to be applied to cell culture microdevices, but cell detachment from PDMS has been a major problem. In this study, we demonstrate that a combination of collagen covalently immobilized PDMS and an adequate oxygen supply enables the establishment of a stable, attached spheroid (hemispheroid) culture of rat hepatocytes. The bottom PDMS surfaces were first treated with oxygen plasma, then coupled with aminosilane followed by a photoreactive crosslinker, and they were finally reacted with a collagen solution. X-ray photoelectron spectroscopy (XPS) and contact angle measurements showed that the covalent immobilization of collagen on the surface occurred only where the crosslinker had been introduced. On the collagen-conjugated PDMS surface, rat hepatocytes organized themselves into hemispheroids and maintained the viability and a remarkably high albumin production at least for 2 weeks of culture. In contrast, hepatocytes on the other types of PDMS surfaces formed suspended spheroids that had low albumin production. In addition, we showed that blocking the oxygen supply through the bottom PDMS surface inhibited the formation of hemispheroids and the augmentation of hepatocellular function. These results show that appropriate surface modification of PDMS is a promising approach towards the development of liver tissue microdevices.

Polymerase chain reaction-based biochemical logic gate coupled with cell-free transcription-translation of green fluorescent protein as a report gate.

Polymerase chain reaction-based biochemical logic gates were designed for AND, OR, NOT, and AND-NOT operations, whose output signal is reported by coupled cell-free transcription-translation of green fluorescent protein.

Enhanced maintenance and functions of rat hepatocytes induced by combination of on-site oxygenation and coculture with fibroblasts.

In in vivo liver tissue, each hepatocyte has intimate interactions not only with adjacent hepatocytes but also with nonparenchymal cells in a three-dimensional (3D) manner. We recently reported that hepatic function is highly maintained on collagen covalently immobilized poly-dimethylsiloxane (PDMS) membranes through which oxygen is supplied directly to the cells. In this study, to further enhance performances of hepatocytes culture, we investigated cocultivation of rat hepatocytes with a mouse fibroblast, NIH/3T3 (3T3) in the same PDMS membranes. Various functions of hepatocytes were better maintained on the membrane at remarkably higher levels, particularly albumin secretion on such coculture was about 20 times higher than that in conventional coculture on tissue-culture-treated polystyrene (TCPS) surfaces. The remarkable functional enhancements are likely to be explained by the net growth of hepatocytes (from 1.2- to 1.4-fold inoculated number) and very intimate contact between hepatocytes and 3T3 cells in almost continuous double-layered structures under the adequate oxygen supply. The results demonstrate that simultaneous realization of different requirements toward mimicking in vivo liver tissue microstructure is effective in improving performance of hepatocytes culture system.

Evaluation of cell-free protein synthesis using PDMS-based microreactor arrays.

A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.

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.

Active immobilization of biomolecules on a hybrid three-dimensional nanoelectrode by dielectrophoresis for single-biomolecule study.

We propose and experimentally demonstrate a method of active immobilization for biomolecules on a three-dimensional nanometre-scale electrode (3D nanoelectrode) using dielectrophoresis to immobilize the biomolecules at predetermined locations for single-biomolecule study. We have developed a novel two-step fabrication process for obtaining a 3D nanoelectrode having a sharp top, which is necessary for immobilizing a single biomolecule at a single point. The first step is to fabricate the backbone structure, which is rigid and defines the shape of the 3D nanoelectrode. It was fabricated with diamond-like carbon (DLC) obtained using focused ion beam assisted chemical vapour deposition followed by post-plasma etching, which reshapes the DLC structure. The second step coats the DLC structure with a thin layer of aluminium, which supplies electrical conductivity to the DLC structure. By applying a high frequency (of the order of megahertz) and high intensity (greater than or equal to a few megavolts per metre) electric field using the 3D nanoelectrodes, the generated dielectrophoresis attracted and then immobilized target biomolecules onto the tops of 3D nanoelectrodes, as a demonstration of active immobilization of biomolecules.

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.