Microfluidic vascular formation model for assessing angiogenic capacities of single islets.

Pancreatic islet transplantation presents a promising therapy for individuals suffering from type 1 diabetes. To maintain the function of transplanted islets in vivo, it is imperative to induce angiogenesis. However, the mechanisms underlying angiogenesis triggered by islets remain unclear. In this study, we introduced a microphysiological system to study the angiogenic capacity and dynamics of individual islets. The system, which features an open-top structure, uniquely facilitates the inoculation of islets and the longitudinal observation of vascular formation in in vivo like microenvironment with islet-endothelial cell communication. By leveraging our system, we discovered notable islet-islet heterogeneity in the angiogenic capacity. Transcriptomic analysis of the vascularized islets revealed that islets with high angiogenic capacity exhibited upregulation of genes related to insulin secretion and downregulation of genes related to angiogenesis and fibroblasts. In conclusion, our microfluidic approach is effective in characterizing the vascular formation of individual islets and holds great promise for elucidating the angiogenic mechanisms that enhance islet transplantation therapy.

Vasculature-on-a-Chip with a Porous Membrane Electrode for In Situ Electrochemical Detection of Nitric Oxide Released from Endothelial Cells.

Vasculature-on-a-chip is a microfluidic cell culture device used for modeling vascular functions by culturing endothelial cells. Porous membranes are widely used to create cell culture environments. However, in situ real-time measurements of cellular metabolites in microchannels are challenging. In this study, a novel microfluidic device with a porous membrane electrode was developed for the in situ monitoring of nitric oxide (NO) released by endothelial cells in real time. In this system, a porous Au membrane electrode was placed directly beneath the cells for in situ and real-time measurements of NO, a biomarker of endothelial cells. First, the device was electrochemically characterized to construct a calibration plot for NO. Next, NO released by human umbilical vein endothelial cells under l-arginine stimulation was successfully quantified. Furthermore, the changes in NO release with culture time (in days) using the same sample were successfully recorded by exploiting minimally invasive measurements. This is the first report on the combination of a microfluidic device and porous membrane electrode for the electrochemical analysis of endothelial cells. This device will contribute to the development of organ-on-a-chip technology for real-time in situ cell analyses.

Batteryless wireless magnetostrictive Fe30Co70/Ni clad plate for human coronavirus 229E detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been garnered increasing for its rapid worldwide spread. Each country had implemented city-wide lockdowns and immigration regulations to prevent the spread of the infection, resulting in severe economic consequences. Materials and technologies that monitor environmental conditions and wirelessly communicate such information to people are thus gaining considerable attention as a countermeasure. This study investigated the dynamic characteristics of batteryless magnetostrictive alloys for energy harvesting to detect human coronavirus 229E (HCoV-229E). Light and thin magnetostrictive Fe-Co/Ni clad plate with rectification, direct current (DC) voltage storage capacitor, and wireless information transmission circuits were developed for this purpose. The power consumption was reduced by improving the energy storage circuit, and the magnetostrictive clad plate under bending vibration stored a DC voltage of 1.9 V and wirelessly transmitted a signal to a personal computer once every 5 min and 10 s under bias magnetic fields of 0 and 10 mT, respectively. Then, on the clad plate surface, a novel CD13 biorecognition layer was immobilized using a self-assembled monolayer of -COOH groups, thus forming an amide bond with -NH2 groups for the detection of HCoV-229E. A bending vibration test demonstrated the resonance frequency changes because of HCoV-229E binding. The fluorescence signal demonstrated that HCoV-229E could be successfully detected. Thus, because HCoV-229E changed the dynamic characteristics of this plate, the CD13-modified magnetostrictive clad plate could detect HCoV-229E from the interval of wireless communication time. Therefore, a monitoring system that transmits/detects the presence of human coronavirus without batteries will be realized soon.

Fabrication of High-Density Vertical Closed Bipolar Electrode Arrays by Carbon Paste Filling Method for Two-Dimensional Chemical Imaging.

In this study, a carbon paste filling method was proposed as a simple strategy for fabricating high-density bipolar electrode (BPE) arrays for bipolar electrochemical microscopy (BEM). High spatiotemporal resolution imaging was achieved using the fabricated BPE array. BEM, which is an emerging microscopic system in recent years, achieves label-free and high spatiotemporal resolution imaging of molecular distributions using high-density BPE arrays and electrochemiluminescence (ECL) signals. We devised a simple method to fabricate a BPE array by filling a porous plate with carbon paste and succeeded in fabricating a high-density BPE array (15 µm pitch). After a detailed observation of the surface of the BPE array using a scanning electron microscope, the basic electrochemical and ECL emission characteristics were evaluated using potassium ferricyanide solution as a sample solution. Moreover, inflow imaging of the sample molecules was conducted to evaluate the imaging ability of the prepared BPE array. In addition, Prussian Blue containing carbon ink was applied to the sample solution side of the BPE array to provide catalytic activity to hydrogen peroxide, and the quantification and inflow imaging of hydrogen peroxide by ECL signals was achieved. This simple fabrication method of the BPE array can accelerate the research and development of BEM. Furthermore, hydrogen peroxide imaging by BEM is an important milestone for achieving bioimaging with high spatiotemporal resolution such as biomolecule imaging using enzymes.

High-Sensitivity Amperometric Dual Immunoassay Using Two Cascade Reactions with Signal Amplification of Redox Cycling in Nanoscale Gap.

Here, we report a high-sensitivity dual immunoassay using Lumulus amebocyte lysate (LAL) and blood coagulation cascade reactions with redox cycling in a nanoscale-gap electrode. Endotoxin and factor XIa were used as the label molecules for the immunoassay of two types of analytes to induce the LAL and coagulation cascade reactions, respectively, when each corresponding analyte existed in the sample solution. In addition to the signal amplification by the cascade reactions, we employed redox cycling in a nanoscale gap to achieve a highly sensitive assay. The nanoscale-gap electrode amplifies the amperometric signals from p-aminophenol liberated from artificial substrates in the final steps of the cascade reactions. First, the cross reaction between the LAL and coagulation cascade reactions was investigated. The results indicated that these cascade reactions did not efficiently proceed in a single solution owing to the cross reaction. Therefore, we selected to induce two cascade reactions in different solutions by bisecting the beads after the immunocomplex formation on the beads. The cross reactions of factor XIa with the LAL cascade reaction and of endotoxin with the coagulation cascade reaction were investigated. The effects of these cross reactions were revealed to be negligible by bisecting the beads before inducing the cascade reactions. Finally, a dual immunoassay for goat and human immunoglobulin G was performed, for which the limits of detection were 70 pg/mL (470 fmol/L) and 1.0 ng/mL (6.6 pmol/L), respectively. Thus, our dual immunoassay provides a sensitive platform for clinical diagnosis requiring detection of multiple analytes.

Highly Sensitive Electrochemical Immunoassay Using Signal Amplification of the Coagulation Cascade.

Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys-p-nitroaniline (FPK-pNA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.

Nanoscale Visualization of Morphological Alteration of Live-Cell Membranes by the Interaction with Oligoarginine Cell-Penetrating Peptides.

The interactions between the cell membrane and biomolecules remain poorly understood. For example, arginine-rich cell-penetrating peptides (CPPs), including octaarginines (R8), are internalized by interactions with cell membranes. However, during the internalization process, the exact membrane dynamics introduced by these CPPs are still unknown. Here, we visualize arginine-rich CPPs and cell-membrane interaction-induced morphological changes using a system that combines scanning ion-conductance microscopy and spinning-disk confocal microscopy, using fluorescently labeled R8. This system allows time-dependent, nanoscale visualization of structural dynamics in live-cell membranes. Various types of membrane remodeling caused by arginine-rich CPPs are thus observed. The induction of membrane ruffling and the cup closure are observed as a process of endocytic uptake of the peptide. Alternatively suggested is the concave structural formation accompanied by direct peptide translocation through cell membranes. Studies using R8 without fluorescent labeling also demonstrate a non-negligible effect of the fluorescent moiety on membrane structural alteration.

Micropipet-Based Navigation in a Microvascular Model for Imaging Endothelial Cell Topography Using Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) has enabled cell surface topography at a high resolution with low invasiveness. However, SICM has not been applied to the observation of cell surfaces in hydrogels, which can serve as scaffolds for three-dimensional cell culture. In this study, we applied SICM for imaging a cell surface in a microvascular lumen reconstructed in a hydrogel. To achieve this goal, we developed a micropipet navigation technique using ionic current to detect the position of a microvascular lumen. Combining this navigation technique with SICM, endothelial cells in a microvascular model and blebs were visualized successfully at the single-cell level. To the best of our knowledge, this is the first report on visualizing cell surfaces in hydrogels using a SICM. This technique will be useful for furthering our understanding of the mechanism of intravascular diseases.

Bioimaging using bipolar electrochemical microscopy with improved spatial resolution.

In this study, we developed bipolar electrochemical microscopy (BEM) using a closed bipolar electrode (cBPE) array with an electrochemiluminescence (ECL) detecting system. Because cBPEs are not directly connected to a detector, high spatio-temporal resolution imaging can be achieved by fabricating a microelectrode array in which each electrode point is arranged in a short interval. A cBPE array with individual cBPEs arranged in 41 µm intervals was successfully fabricated by depositing gold in the pores of a track-etched membrane using electroless plating. Using BEM with the cBPE array, which has a higher density of electrode points than the conventional multi-electrode array, we effectively demonstrated the imaging of [Fe(CN)6]3- diffusion and the respiratory activity of MCF-7 spheroids with high spatio-temporal resolution.

Oxygen consumption rate of tumour spheroids during necrotic-like core formation.

Hypoxia is one of the major hallmarks of solid tumours and is associated with the poor prognosis of various cancers. A multicellular aggregate, termed a spheroid, has been used as a tumour model with a necrotic-like core for more than 45 years. Oxygen metabolism in spheroids has been studied using phosphorescence quenching and oxygen-sensitive electrodes. However, these conventional methods require chemical labelling and physical insertion of the electrode into each spheroid, which may be functionally and structurally disruptive. Scanning electrochemical microscopy (SECM) can non-invasively analyse oxygen metabolism. Here, we used SECM to investigate whether the changes of the internal structure of spheroids affect the oxygen metabolism. We investigated the oxygen consumption rate (OCR) of MCF-7 breast tumour spheroids with and without a necrotic-like core. A numerical simulation was used to describe a method for estimating the OCR of spheroids that settled at the bottom of the conventional culture plates. The OCR per spheroid volume decreased with increasing spheroid radius, indicating the limitation of the oxygen supply to the core of the MCF-7 spheroid. Formation of the necrotic-like core did not affect the oxygen metabolism significantly, implying that the core had minimal contribution to the OCR even before necrosis occurred. OCR analysis using SECM non-invasively monitors the change of oxygen metabolism in tumour spheroids. The approach is promising to evaluate various three-dimensional culture models.

High-Resolution Electrochemical Mapping of the Hydrogen Evolution Reaction on Transition-Metal Dichalcogenide Nanosheets.

High-resolution scanning electrochemical cell microscopy (SECCM) is used to image and quantitatively analyze the hydrogen evolution reaction (HER) catalytically active sites of 1H-MoS2 nanosheets, MoS2 , and WS2 heteronanosheets. Using a 20 nm radius nanopipette and hopping mode scanning, the resolution of SECCM was beyond the optical microscopy limit and visualized a small triangular MoS2 nanosheet with a side length of ca. 130 nm. The electrochemical cell provides local cyclic voltammograms with a nanoscale spatial resolution for visualizing HER active sites as electrochemical images. The HER activity difference of edge, terrace, and heterojunction of MoS2 and WS2 were revealed. The SECCM imaging directly visualized the relationship of HER activity and number of MoS2 nanosheet layers and unveiled the heterogeneous aging state of MoS2 nanosheets. SECCM can be used for improving local HER activities by producing sulfur vacancies using electrochemical reaction at the selected region.

Site-Specific Cytosol Sampling from a Single Cell in an Intact Tumor Spheroid Using an Electrochemical Syringe.

A multicellular tumor aggregate, known as a spheroid, is an indispensable tool to study cancer biology. Owing to its three-dimensional organization, a spheroid exhibits an inherent gradient of nutrients, oxygen, and metabolites within itself. The spheroid provides culture conditions that resemble the microenvironment of certain cancer cells and causes these cells to acquire characteristics relevant to tumors in our body. However, site-specific gene expression analysis in an intact spheroid with single-cell resolution has not been explored. Recently, some types of electrochemical syringes were developed to extract cellular materials from living single cells for transcriptomic analysis. Here, we investigated whether an electrochemical syringe could be used to evaluate site-specific gene expression in a spheroid. A small amount of cytosol (roughly 540-1480 fL, less than the volume of a single cell) was successfully collected from the first, second, and third layers of the spheroid using an electrochemical syringe without causing damage to the spheroid architecture. We found that the CCNB1 and CCNA2 expression levels were different between the surface and the average of the entire spheroid, indicating that there are heterogeneous cellular functions across different regions of the spheroid. This method provides opportunities to improve our understanding of spatial gene expression of single cells in a three-dimensional environment.

A highly sensitive endotoxin sensor based on redox cycling in a nanocavity.

We report a highly sensitive and rapid electrochemical method for the detection of endotoxin, based on a Limulus amebocyte lysate (LAL) assay using redox cycling at a pair of electrodes in a nanocavity for electrochemical signal amplification. We have previously developed Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as a substrate for the amperometric LAL assay, and in this work, Z-Leu-Gly-Arg-aminomethylferrocene (LGR-AMF) was newly prepared. They were examined as substrates for a LAL-based endotoxin assay using a nanocavity device. During the last step of the endotoxin-induced LAL cascade reaction, pAP or AMF is generated from the substrate, which can be detected electrochemically with efficient signal amplification by redox cycling between the two electrodes in the nanocavity. A device with a 190 nm-high nanocavity was fabricated by photolithography. With the fabricated device in model assay solutions prepared by mixing LGR-pAP and pAP, we demonstrated that pAP could be quantitatively detected from the difference in oxidation potentials between LGR-pAP and pAP. For LGR-AMF and AMF, a difference in the formal potential of 0.1 V was obtained which was considered to be insufficient to distinguish AMF from LGR-AMF. However, we showed for the first time that analytes such as AMF can be detected by differences in diffusion coefficients between the analyte and coexisting molecules (such as LGR-AMF) using a device with high redox-cycling efficiency. Next, the endotoxin assay was performed using the fabricated nanocavity device. Using this method, endotoxin was detected at concentrations as low as 0.2 and 0.5 EU L-1 after LAL reaction times of 1 h and 30 min, respectively, using the LGR-pAP substrate. However, the endotoxin assay using LGR-AMF was not successful because the clotting enzyme did not react with LGR-AMF. This problem might be solved by further design of the substrate. Our nanocavity device represents an effective platform for the simple and rapid detection of endotoxin with high sensitivity.

High Speed Scanning Ion Conductance Microscopy for Quantitative Analysis of Nanoscale Dynamics of Microvilli.

Observation of nanoscale structure dynamics on cell surfaces is essential to understanding cell functions. Hopping-mode scanning ion conductance microscopy (SICM) was used to visualize the topography of fragile convoluted nanoscale structures on cell surfaces under noninvasive conditions. However, conventional hopping mode SICM does not have sufficient temporal resolution to observe cell-surface dynamics in situ because of the additional time required for performing vertical probe movements of the nanopipette. Here, we introduce a new scanning algorithm for high speed SICM measurements using low capacitance and high-resonance-frequency piezo stages. As a result, a topographic image is taken within 18 s with a 64 × 64 pixel resolution at 10 × 10 µm. The high speed SICM is applied to the characterization of microvilli dynamics on surfaces, which shows clear structural changes after the epidermal growth factor stimulation.

Development of Oxygen Consumption Analysis with an on-Chip Electrochemical Device and Simulation.

The O2 consumption rate of embryos has been attracting much attention as a key indicator of cell metabolisms and development. In this study, we propose an on-chip device that enables the accurate, easy, and noninvasive measurement of O2 consumption rates of single embryos. Pt electrodes and micropits for embryo settlement were fabricated on Si chips via microfabrication techniques. The configuration of the device enables the detection of O2 concentration profiles surrounding the embryos by settling embryos into the pits with a mouth pipet. Moreover, as the detection is based on an electrochemical method, the influence of O2 consumption on the electrodes was also considered. By using a simulator (COMSOL Multiphysics), we estimated the O2 concentration profiles in the device with and without the effects of the electrodes. Based on the simulation results, we developed a normalization process to calculate the precise O2 consumption rate of the sample. Finally, using both the measurement system and the algorithm for the analysis, the respiratory activities of mouse embryos were successfully measured.

Electrochemicolor Imaging Using an LSI-Based Device for Multiplexed Cell Assays.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)-based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.

Carbon nanotubes embedded in embryoid bodies direct cardiac differentiation.

We embedded carbon nanotubes (CNTs) in mouse embryoid bodies (EBs) for modulating mechanical and electrical cues of the stem cell niche. The CNTs increased the mechanical integrity and electrical conductivity of the EBs. Measured currents for the unmodified EBs (hereafter, EBs) and the EBs-0.25 mg/mL CNTs were 0.79 and 26.3 mA, respectively, at voltage of 5 V. The EBs had a Young's modulus of 20.9 ± 6.5 kPa, whereas the Young's modulus of the EB-0.1 mg/mL CNTs was 35.2 ± 5.6 kPa. The EB-CNTs also showed lower proliferation and greater differentiation rates compared with the EBs as determined by the expression of pluripotency genes and the analysis of EB sizes. Interestingly, the cardiac differentiation of the EB-CNTs was significantly greater than that of the EBs, as confirmed by high-throughput gene analysis at day 5 of culture. Applying electrical stimulation to the EB-CNTs specifically enhanced the cardiac differentiation and beating activity of the EBs.

Micro/nanoelectrochemical probe and chip devices for evaluation of three-dimensional cultured cells.

Herein, we present an overview of recent research progress in the development of micro/nanoelectrochemical probe and chip devices for the evaluation of three-dimensional (3D) cultured cells. First, we discuss probe devices: a general outline, evaluation of O2 consumption, enzyme-modified electrodes, evaluation of endogenous enzyme activity, and the collection of cell components from cell aggregates are discussed. The next section is focused on integrated chip devices: a general outline, electrode array devices, smart electrode array devices, droplet detection of 3D cultured cells, cell manipulation using dielectrophoresis (DEP), and electrodeposited hydrogels used for fabrication of 3D cultured cells on chip devices are discussed. Finally, we provide a summary and discussion of future directions of research in this field.

Continuous collection and simultaneous detection of picoliter volume of nucleic acid samples using a mille-feuille probe.

Investigation of the positional heterogeneity of messenger RNA (mRNA) expression in tissues requires a technology that facilitates analysis of mRNA expression in the selected single cells. We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control. The MP was used for continuous collection of different nucleic acid samples and sequential evaluation of gene expression with mRNA barcoding tags. First, we found that the aqueous phases could be laminated into five individual layers and separated by the plugs of the organic phases in a nanopipette when the salt (THATPBCl) concentration in the organic phase was 100 mM. Second, the aspiration rate of the MP was stabilized and the velocity of the aqueous phase in the MP was lowered at higher THATPBCl concentrations in the organic phase. This was because the force during ingression of the aqueous phase into the organic - phase-filled nanopipette induced an electro-osmotic flow between the inside wall of the nanopipette and THATPBCl in the organic phase. Third, inclusion of mRNA barcoding tags in the MP facilitated complementary DNA construction and sequential analysis of gene expression. This technique has potential to be applicable to RNA sequencing from different cell samples across the life sciences. Graphical abstract We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control.

3D electrochemical and ion current imaging using scanning electrochemical-scanning ion conductance microscopy.

Local cell-membrane permeability and ionic strength are important factors for maintaining the functions of cells. Here, we measured the spatial electrochemical and ion concentration profile near the sample surface with nanoscale resolution using scanning electrochemical microscopy (SECM) combined with scanning ion-conductance microscopy (SICM). The ion current feedback system is an effective way to control probe-sample distance without contact and monitor the kinetic effect of mediator regeneration and the chemical concentration profile. For demonstrating 3D electrochemical and ion concentration mapping, we evaluated the reaction rate of electrochemical mediator regeneration on an unbiased conductor and visualized inhomogeneous permeability and the ion concentration 3D profile on a single fixed adipocyte cell surface.