Resistive Pulse Sensing on a Capillary-Assisted Microfluidic Platform for On-Site Single-Particle Analyses.

Capillary-assisted flow is valuable for utilizing microfluidics-based electrical sensing platforms at on-site locations by simplifying microfluidic operations and system construction; however, incorporating capillary-assisted flow in platforms requires easy microfluidic modification and stability over time for capillary-assisted flow generation and sensing performance. Herein, we report a capillary-assisted microfluidics-based electrical sensing platform using a one-step modification of polydimethylsiloxane (PDMS) with polyethylene glycol (PEG). As a model of electrical sensing platforms, this work focused on resistive pulse sensing (RPS) using a micropore in a microfluidic chip for label-free electrical detection of single analytes, and filling the micropore with an electrolyte is the first step to perform this RPS. The PEG-PDMS surfaces remained hydrophilic after ambient storage for 30 d and assisted in generating an electrolyte flow for filling the micropore with the electrolyte. We demonstrated the successful detection and size analysis of micrometer particles and bacterial cells based on RPS using the microfluidic chip stored in a dry state for 30 d. Combining this capillary-assisted microfluidic platform with a portable RPS system makes on-site detection and analysis of single pathogens possible.

Quantitative Evaluation of Interleukin-4 by Immunowall Devices Made of Gelatin Methacryloyl Hydrogel.

Immunoassays, which use antigen-antibody reactions, are the primary techniques used to selectively quantify specific disease markers in blood. Conventional immunoassays, such as the microplate-based enzyme-linked immunosorbent assay (ELISA) and paper-based immunochromatography, are widely used, but they have advantages and disadvantages in terms of sensitivity and operating time. Therefore, in recent years, microfluidic-chip-based immunoassay devices with high sensitivity, rapidity and simplicity, which are compatible with whole blood assays and multiplex assays, have been actively investigated. In this study, we developed a microfluidic device using gelatin methacryloyl (GelMA) hydrogel to form a wall-like structure in a microfluidic channel and perform immunoassays inside the wall-like structure, which can be used for rapid and highly sensitive multiplex assays with extremely small sample amounts of ~1 µL. The characteristics of GelMA hydrogel, such as swelling rate, optical absorption and fluorescence spectra, and morphology, were carefully studied to adapt the iImmunowall device and immunoassay. Using this device, a quantitative analysis of interleukin-4 (IL-4), a biomarker of chronic inflammatory diseases, was performed and a limit of detection (LOD) of 0.98 ng/mL was achieved with only 1 µL sample and 25 min incubation time. The superior optical transparency over a wide range of wavelengths and lack of autofluorescence will help to expand the application field of the iImmunowall device, such as to a simultaneous multiple assay in a single microfluidic channel, and provide a fast and cost-effective immunoassay method.

Enzyme kinetics in confined geometries at the single enzyme level.

The effects of increased confinement on the catalytic rates of individual enzyme molecules were studied at the single molecule level using femtolitre chambers and molecular crowders. According to the increase of confinement, from micro to nanometer cubic space in the chambers, the hydrolysis rate of ß-galactosidase (ß-gal) decreased to one-tenth of the rate in bulk. When molecular crowders suppressed the diffusion rates that reduced the collision chance of an enzyme and a substrate, the hydrolysis rate also decreased, which happened also in the case of femtolitre chambers. However, their kinetic trend was different especially from the viewpoint of the diffusion rates in diffusion-limited space. These data suggested that cell or organelle-scale confined environments might affect the kinetics of biochemical reactions and emphasized the importance of understanding enzyme kinetics in the in vivo environment.

Label-Free Cancer Stem-like Cell Assay Conducted at a Single Cell Level Using Microfluidic Mechanotyping Devices.

The mechanical phenotype of cells is an intrinsic property of individual cells. In fact, this property could serve as a label-free, non-destructive, diagnostic marker of the state of cells owing to its remarkable translational potential. A microfluidic device is a strong candidate for meeting the demand of this translational research as it can be used to diagnose a large population of cells at a single cell level in a high-throughput manner, without the need for off-line pretreatment operations. In this study, we investigated the mechanical phenotype of the human colon adenocarcinoma cell, HT29, which is known to be a heterogeneous cell line with both multipotency and self-renewal abilities. This type of cancer stem-like cell (CSC) is believed to be the unique originators of all tumor cells and may serve as the leading cause of cancer metastasis and drug resistance. By combining consecutive constrictions and microchannels with an ionic current sensing system, we found a high heterogeneity of cell deformability in the population of HT29 cells. Moreover, based on the level of aldehyde dehydrogenase (ALDH) activity and the expression level of CD44s, which are biochemical markers that suggest the multipotency of cells, the high heterogeneity of cell deformability was concluded to be a potential mechanical marker of CSCs. The development of label-free and non-destructive identification and collection techniques for CSCs has remarkable potential not only for cancer diagnosis and prognosis but also for the discovery of a new treatment for cancer.

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.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA.

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realizedin-situannealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System.

Conformational transitions from secondary (e.g., B- to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several single-molecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil-globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression.

Mechanical Low-Pass Filtering of Cells for Detection of Circulating Tumor Cells in Whole Blood.

The detection of circulating tumor cells (CTCs) from liquid biopsies using microfluidic devices is attracting a considerable amount of attention as a new, less-invasive cancer diagnostic and prognostic method. One of the drawbacks of the existing antibody-based detection systems is the false negatives for epithelial cell adhesion molecule detection of CTCs. Here we report a mechanical low-pass filtering technique based on a microfluidic constriction and electrical current sensing system for the novel CTC detection in whole blood without any specific antigen-antibody interaction or biochemical modification of the cell surface. The mechanical response of model cells of CTCs, such as HeLa, A549, and MDA-MB-231 cells, clearly demonstrated different behaviors from that of Jurkat cells, a human T-lymphocyte cell line, when they passed through the 6-µm wide constriction channel. A 6-µm wide constriction channel was determined as the optimum size to identify CTCs in whole blood with an accuracy greater than 95% in tens of milliseconds. The mechanical filtering of cells at a single cell level was achieved from whole blood without any pretreatment (e.g., dilution of lysing) and prelabeling (e.g., fluorophores or antibodies).

Micro- and Nanopillar Chips for Continuous Separation of Extracellular Vesicles.

Micro- and nanopillar chips are widely used to separate and enrich biomolecules, such as DNA, RNA, protein, and cells, as an analytical technique and to provide a confined nanospace for polymer science analyses. Herein, we demonstrated a continuous accurate and precise separation technique for extracellular vesicles (EVs), nanometer-sized vesicles (typically 50-200 nm) currently recognized as novel biomarkers present in biofluids, based on the principle of electroosmotic flow-driven deterministic lateral displacement in micro- and nanopillar array chips. Notably, the easy-to-operate flow control afforded by electroosmotic flow allowed nanoparticles 50-500 nm in size, including EVs, to be precisely separated and enriched in a continuous manner. By observation of the flow behavior of nanoparticles, we found that electroosmotic flow velocity in the nanopillar arrays did not solely depend on counterion mobility on the surface of nanopillar chips, but rather showed a parabolic flow profile. This hydrodynamic pressure-free and easy-to-use separation and enrichment technique, which requires only electrode insertion into the reservoirs and electric field application, may thus serve as a promising technique for future precise and accurate EV analysis, reflecting both size and composition for research and potential clinical diagnostic applications.

Microfluidic Mechanotyping of a Single Cell with Two Consecutive Constrictions of Different Sizes and an Electrical Detection System.

The mechanical properties of a cell, which include parameters such as elasticity, inner pressure, and tensile strength, are extremely important because changes in these properties are indicative of diseases ranging from diabetes to malignant transformation. Considering the heterogeneity within a population of cancer cells, a robust measurement system at the single cell level is required for research and in clinical purposes. In this study, a potential microfluidic device for high-throughput and practical mechanotyping were developed to investigate the deformability and sizes of cells through a single run. This mechanotyping device consisted of two different sizes of consecutive constrictions in a microchannel and measured the size of cells and related deformability during transit. Cell deformability was evaluated based on the transit and on the effects of cytoskeleton-affecting drugs, which were detected within 50 ms. The mechanotyping device was able to also measure a cell cycle without the use of fluorescent or protein tags.

Substantial Expansion of Detectable Size Range in Ionic Current Sensing through Pores by Using a Microfluidic Bridge Circuit.

Measuring ionic currents passing through nano- or micropores has shown great promise for the electrical discrimination of various biomolecules, cells, bacteria, and viruses. However, conventional measurements have shown there is an inherent limitation to the detectable particle volume (1% of the pore volume), which critically hinders applications to real mixtures of biomolecule samples with a wide size range of suspended particles. Here we propose a rational methodology that can detect samples with the detectable particle volume of 0.01% of the pore volume by measuring a transient current generated from the potential differences in a microfluidic bridge circuit. Our method substantially suppresses the background ionic current from the µA level to the pA level, which essentially lowers the detectable particle volume limit even for relatively large pore structures. Indeed, utilizing a microscale long pore structure (volume of 5.6 × 104 aL; height and width of 2.0 × 2.0 µm; length of 14 µm), we successfully detected various samples including polystyrene nanoparticles (volume: 4 aL), bacteria, cancer cells, and DNA molecules. Our method will expand the applicability of ionic current sensing systems for various mixed biomolecule samples with a wide size range, which have been difficult to measure by previously existing pore technologies.

Identifying DNA methylation in a nanochannel.

DNA methylation is a stable epigenetic modification, which is well known to be involved in gene expression regulation. In general, however, analyzing DNA methylation requires rather time consuming processes (24-96 h) via DNA replication and protein modification. Here we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level without any protein modifications by measuring the contracted length and relaxation time of DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower contraction rate). The present methodology offers a promising way to identify DNA methylation without any protein modification at a single DNA molecule level within 2 h.

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas.

World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69-80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83-90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 µl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.

Arrangement of a nanostructure array to control equilibrium and nonequilibrium transports of macromolecules.

Exploiting the nonequilibrium transport of macromolecules makes it possible to increase the separation speed without any loss of separation resolution. Here we report the arrangement of a nanostructure array in microchannels to control equilibrium and nonequilibrium transports of macromolecules. The direct observation and separation of macromolecules in the nanopillar array reported here are the first to reveal the nonequilibrium transport, which has a potential to overcome the intrinsic trade-off between the separation speed and resolution.

Reduced plasma glucose by asparagine synthetase knockdown in the mouse liver.

Expression of the asparagine synthetase gene is dependent on extracellular glucose concentration. This gene was knocked-down in livers of male Balb/c mice by a hydrodynamic tail vein injection of small interfering RNA (siRNA) against the gene. This knockdown resulted in a significant decrease in plasma glucose concentration. These results suggested that asparagine synthetase is a novel protein that regulates plasma glucose levels.

Spatiotemporal visualization of subcellular dynamics of carbon nanotubes.

To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards.

Micro- and nanochamber array system for single enzyme assays.

Arrays of small reaction containers, ranging from 624 femtoliters (10-15 L) to 270 attoliters (10-18 L), for capturing a single enzyme molecule and measuring the activity were developed along with a new reversible sealing system based on a pneumatic valve actuator made of polydimethylsiloxane (PDMS). The valve was actuated by PBS solution, effectively preventing evaporation of the solution from the micro- and nanochambers and allowing the assay to be performed over a long period of time. The hydrolysis rates of ß-D-galactosidase (ß-gal), kcat, were decreased according to the decrease of the chamber size, and the overall tendency seems to be symmetrically related to the specific surface area of the chambers even under the prevented condition of non-specific adsorption. The spatial localization of the protons in the chambers, which might could affect the dissociation state of the proteins, was also investigated to explain the decrease in the hydrolysis rate. The developed chamber system developed here may be useful for artificially reproducing the confined intracellular environment and molecular crowding conditions.

Antioxidants encapsulated milk-derived exosomes for functional food development.

Reactive oxygen species are known to be involved in various diseases, and antioxidant ingredients are expected to essentially prevent diseases and contribute to improving health. However, antioxidants are easily degraded by enzymes before being absorbed in the intestine, so a means of transport that prevents their degradation in the body is necessary. Exosomes, which play an important role in communication between individual cells, have attracted attention as a new transport carrier of miRNA and DNA, but not yet fully exploited in food research. More recently, exosomes extracted from bovine milk began to be widely used as a cost-effective transport carrier not in clinical medicine but also in functional food materials. To develop practical applications as carriers for functional foods, systematic studies are necessary to clarify the introduction efficiency and the properties of encapsulated substances. In this study, we applied electroporation and incubation to encapsulate antioxidants into the exosomes and studied the encapsulation efficiency into the exosomes and the anticancer activity.

Inkjet injection of DNA droplets for microchannel array electrophoresis.

We demonstrated DNA droplets could be injected with an inkjet injector for microchannel array electrophoresis and attained high throughput analysis of biomolecules. This injection method greatly reduced both analysis time and sample amount, compared with a conventional microchip electrophoresis method, and allowed high parallelization of a microchannel array on a small substrate. Since we do not need to use complicated electric programs or microchannel design, our injection method should facilitate omics analyses and contribute to high performance clinical assays.

A DNA microarray-based analysis of the host response to a nonviral gene carrier: a strategy for improving the immune response.

The purpose of this study was to investigate the host response to systemically administered lipid nanoparticles (NPs) encapsulating plasmid DNA (pDNA) in the spleen using a DNA microarray. As a model for NPs, we used a multifunctional envelope-type nano device (MEND). Microarray analysis revealed that 1,581 of the differentially expressed genes could be identified by polyethylene glycol (PEG)-unmodified NP using a threefold change relative to the control. As the result of PEGylation, the NP treatment resulted in the reduction in the expression of most of the genes. However, the expression of type I interferon (IFN) was specifically increased by PEGylation. Based on the microarray and a pathway analysis, we hypothesize that PEGylation inhibited the endosomal escape of NP, and extended the interaction of toll-like receptor-9 (TLR9) with CpG-DNA accompanied by the production of type I IFN. This hypothesis was tested by introducing a pH-sensitive fusogenic peptide, GALA, which enhances the endosomal escape of PEGylated NP. As expected, type I IFN was reduced and interleukin-6 (IL-6) remained at the baseline. These findings indicate that a carrier design based on microarray analysis and the manipulation of intracellular trafficking constitutes a rational strategy for reducing the host immune response to NPs.