Publications by authors named "Noritada Kaji"

128 Publications

Rapid Discrimination of Extracellular Vesicles by Shape Distribution Analysis.

Anal Chem 2021 05 28;93(18):7037-7044. Epub 2021 Apr 28.

The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan.

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.
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http://dx.doi.org/10.1021/acs.analchem.1c00258DOI Listing
May 2021

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

Nanotechnology 2021 Apr 1;32(25). Epub 2021 Apr 1.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan.

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 realizedannealed 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.
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http://dx.doi.org/10.1088/1361-6528/abef2cDOI Listing
April 2021

Development of an immuno-wall device for the rapid and sensitive detection of EGFR mutations in tumor tissues resected from lung cancer patients.

PLoS One 2020 16;15(11):e0241422. Epub 2020 Nov 16.

Department of Respiratory Medicine, Graduate School of Medicine, Nagoya University, Nagoya, Japan.

Detecting molecular targets in specimens from patients with lung cancer is essential for targeted therapy. Recently, we developed a highly sensitive, rapid-detection device (an immuno-wall device) that utilizes photoreactive polyvinyl alcohol immobilized with antibodies against a target protein via a streptavidin-biotin interaction. To evaluate its performance, we assayed epidermal growth factor receptor (EGFR) mutations, such as E746_A750 deletion in exon 19 or L858R substitution in exon 21, both of which are common in non-small cell lung cancer and important predictors of the treatment efficacy of EGFR tyrosine kinase inhibitors. The results showed that in 20-min assays, the devices detected as few as 1% (E746_A750 deletion) and 0.1% (L858R substitution) of mutant cells. Subsequent evaluation of detection of the mutations in surgically resected lung cancer specimens from patients with or without EGFR mutations and previously diagnosed using commercially available, clinically approved genotyping assays revealed diagnostic sensitivities of the immuno-wall device for E746_A750 deletion and L858R substitution of 85.7% and 87.5%, respectively, with specificities of 100% for both mutations. These results suggest that the immuno-wall device represents a good candidate next-generation diagnostic tool, especially for screening of EGFR mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241422PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668601PMC
December 2020

Determination of three antiepileptic drugs in pharmaceutical formulations using microfluidic chips coupled with light-emitting diode induced fluorescence detection.

Spectrochim Acta A Mol Biomol Spectrosc 2021 Feb 3;246:119021. Epub 2020 Oct 3.

Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Moto-oka 744, Nishi-ku, Fukuoka-shi, Fukuoka 819-0395, Japan.

In this study, a facile, sensitive, and precise lab-on-a-chip electrophoretic method coupled with light-emitting diode induced fluorescence (LED-IF) detection was developed to assay three antiepileptic drugs, namely, vigabatrin, pregabalin, and gabapentin, in pharmaceutical formulations. The analytes were derivatised offline for the first time with fluorescine-5-isothiocyanate (FITC) to yield highly fluorescent derivatives with λ of 490/520nm. The FITC-labelled analytes were injected, separated, and quantitated by a microfluidic electrophoresis device using fluorescence detection. The labelled analytes were monitored using a blue LED-IF system. The separation conditions were significantly optimised adding specific concentrations of heptakis-(2,6-di-O-methyl)-β-cyclodextrin (HDM-β-CD) and methylcellulose to the buffer solution (40mM borate buffer). HDM-β-CD acted as a selective host for the studied antiepileptic drugs, rendering a high separation efficiency. Methylcellulose was used as an efficient dynamic coating polymer to prevent the labelled drugs from being adsorbed on the inner surfaces of the poly (methylmethacrylate) microchannels. A laboratory-prepared ternary mixture of the three antiepileptic drugs was separated within 100s with acceptable resolution between all the three analytes (R>3) and a high number of theoretical plates (N) for each analyte (N≈10 plates/m). The sensitivity of the method was enhanced approximately 80-fold by stacking to yield a detection limit below 0.6ngmL in the concentration range of 2.0-200.0ngmL. The method was successfully validated for analysing the studied drugs in their pharmaceutical formulations.
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http://dx.doi.org/10.1016/j.saa.2020.119021DOI Listing
February 2021

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

Micromachines (Basel) 2020 Jun 24;11(6). Epub 2020 Jun 24.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

There are growing interests in mechanical rupture-based antibacterial surfaces with nanostructures that have little toxicity to cells around the surfaces; however, current surfaces are fabricated via top-down nanotechnologies, which presents difficulties to apply for bio-surfaces with hierarchal three-dimensional structures. Herein, we developed ZnO/SiO nanowire structures by using bottom-up approaches and demonstrated to show mechanical rupture-based antibacterial activity and compatibility with human cells. When were cultured on the surface for 24 h, over 99% of the bacteria were inactivated, while more than 80% of HeLa cells that were cultured on the surface for 24 h were still alive. This is the first demonstration of mechanical rupture-based bacterial rupture via the hydrothermally synthesized nanowire structures with antibacterial activity and cell compatibility.
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http://dx.doi.org/10.3390/mi11060610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345559PMC
June 2020

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

Anal Chem 2020 07 9;92(13):9132-9137. Epub 2020 Jun 9.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

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.
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http://dx.doi.org/10.1021/acs.analchem.0c01417DOI Listing
July 2020

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

Anal Chem 2020 02 22;92(3):2483-2491. Epub 2020 Jan 22.

Health Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Hayashi-cho 2217-14 , Takamatsu 761-0395 , Japan.

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).
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http://dx.doi.org/10.1021/acs.analchem.9b03939DOI Listing
February 2020

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

Anal Chem 2019 10 11;91(20):12890-12899. Epub 2019 Sep 11.

Department of Biomolecular Engineering, Graduate School of Engineering , Nagoya University , Furo-cho , Chikusa-ku, Nagoya 464-8603 , Japan.

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.
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http://dx.doi.org/10.1021/acs.analchem.9b02818DOI Listing
October 2019

Geometry-Based Self-Assembly of Histone-DNA Nanostructures at Single-Nucleotide Resolution.

ACS Nano 2019 07 25;13(7):8155-8168. Epub 2019 Jun 25.

Biological and Environmental Science and Engineering Division , King Abdullah University of Science and Technology (KAUST) , Thuwal 23955-6900 , Saudi Arabia.

Histones are basic protein monomers capable of interacting with DNA, providing the mechanism of DNA compaction inside the cell nucleus. The well-ordered assembly process of histone and DNA is a potential candidate as the approach for building DNA-protein nanostructures. Here, utilizing the sequence-independent histone-DNA interaction, we present an approach to self-assemble histones and single-stranded DNA (ssDNA) to form well-defined histone-DNA (sHD) nanoparticles and their multidimensional cross-linked complexes (cHD). By using various molecular biology and microscopy techniques, we elucidate the structure of these complexes, and we show that they are formed at carefully controlled conditions of temperature, ionic strength, concentration, and incubation time. We also demonstrate using a set of ssDNA molecular rulers and a geometric accommodation model that the assembly of sHD and cHD particles proceeds with precise geometry so that the number of ssDNA in these particles can be programmed by the length of ssDNA. We further show that the formation of cHD amplifies the effect of the length of ssDNA on the self-assembly, allowing for distinguishing ssDNA of different lengths at single nucleotide resolution. We envision that our geometry-directed approach of self-assembling histone-DNA nanostructures and the fundamental insights can serve as a structural platform to advance building precisely ordered DNA-protein nanostructures.
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http://dx.doi.org/10.1021/acsnano.9b03259DOI Listing
July 2019

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

Anal Chem 2019 05 3;91(10):6514-6521. Epub 2019 May 3.

Department of Biomolecular Engineering, Graduate School of Engineering , Nagoya University , Furo-cho, Chikusa-ku , Nagoya , 464-8603 , Japan.

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.
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http://dx.doi.org/10.1021/acs.analchem.8b05538DOI Listing
May 2019

Engineering Nanowire-Mediated Cell Lysis for Microbial Cell Identification.

ACS Nano 2019 02 13;13(2):2262-2273. Epub 2019 Feb 13.

Health Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Takamatsu 761-0395 , Japan.

Researchers have demonstrated great promise for inorganic nanowire use in analyzing cells or intracellular components. Although a stealth effect of nanowires toward cell surfaces allows preservation of the living intact cells when analyzing cells, as a completely opposite approach, the applicability to analyze intracellular components through disrupting cells is also central to understanding cellular information. However, the reported lysis strategy is insufficient for microbial cell lysis due to the cell robustness and wrong approach taken so far ( i. e., nanowire penetration into a cell membrane). Here we propose a nanowire-mediated lysis method for microbial cells by introducing the rupture approach initiated by cell membrane stretching; in other words, the nanowires do not penetrate the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and flexible nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediated lysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible with the loop-mediated isothermal amplification (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells. This approach allows researchers to develop a microfluidic analytical platform not only for microbial cell identification including drug- and heat-resistance cells but also for on-site detection without any contamination.
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http://dx.doi.org/10.1021/acsnano.8b08959DOI Listing
February 2019

"New Insights and Concepts of Biological Sciences Based on Cell and Biomolecule Analysis".

Anal Sci 2019 ;35(1)

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University.

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http://dx.doi.org/10.2116/analsci.GE1901DOI Listing
February 2019

Development of a microdevice for facile analysis of theophylline in whole blood by a cloned enzyme donor immunoassay.

Lab Chip 2019 01;19(2):233-240

Graduate School of Chemical Sciences and Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan.

We have developed a microdevice for therapeutic drug monitoring. In this device, dispensing of sample and reagent was accomplished by simple manual operation of a syringe. Moreover, for a simple and rapid measurement, we used cloned enzyme donor immunoassay as a detection principle. These features and the reagent that is enclosed in microdevice beforehand make it possible to complete the facile analysis. In this paper, our model analyte was 1,3-dimethylxanthine (theophylline), a kind of bronchodilator. The fluorescence measurement of theophylline in whole blood was achieved with the limit of detection of 0.73 μg mL-1. This microdevice provides rapid analysis (4 min), requires only a small volume of sample (2 μL) and features simple operation; hence, it is readily applicable to point of care testing.
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http://dx.doi.org/10.1039/c8lc01105bDOI Listing
January 2019

PM Particle Detection in a Microfluidic Device by Using Ionic Current Sensing.

Anal Sci 2018 Dec 16;34(12):1347-1349. Epub 2018 Nov 16.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University.

We have demonstrated a PM analysis method that adds information on the number concentration and size by using microfluidic-based ionic current sensing with a bridge circuit. The bridge circuit allows for suppression of the background current and the detection of small PM particles, even if a relatively large micropore is used. This is the first demonstration of the detection of PM particles via ionic current sensing; our method enables analyses of both the number concentration and size.
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http://dx.doi.org/10.2116/analsci.18C018DOI Listing
December 2018

Quantitative Evaluation of Dielectric Breakdown of Silicon Micro- and Nanofluidic Devices for Electrophoretic Transport of a Single DNA Molecule.

Micromachines (Basel) 2018 Apr 13;9(4). Epub 2018 Apr 13.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

In the present study, we quantitatively evaluated dielectric breakdown in silicon-based micro- and nanofluidic devices under practical electrophoretic conditions by changing the thickness of the insulating layer. At higher buffer concentration, a silicon nanofluidic device with a 100 nm or 250 nm silicon dioxide layer tolerated dielectric breakdown up to ca. 10 V/cm, thereby allowing successful electrophoretic migration of a single DNA molecule through a nanochannel. The observed DNA migration behavior suggested that parameters, such as thickness of the insulating layer, tolerance of dielectric breakdown, and bonding status of silicon and glass substrate, should be optimized to achieve successful electrophoretic transport of a DNA molecule through a nanopore for nanopore-based DNA sequencing applications.
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http://dx.doi.org/10.3390/mi9040180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187859PMC
April 2018

Biomolecular recognition on nanowire surfaces modified by the self-assembled monolayer.

Lab Chip 2018 10;18(21):3225-3229

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Molecular recognition is one of the key factors in designing biosensors due to which nanowires functionalized with molecular recognition have attracted a lot of attention as promising candidates for nanostructures embedded in biosensors. However, the difficulty in real-world applications with analytical specificity is that molecular recognition on nanowires mainly depends on antibody modification with multistep modification procedures. Furthermore, the antibody modification suffers from nonspecific adsorption of undesired proteins in body fluid on the nanowires, which causes false responses and lowers sensitivity. Herein, we propose biomolecular recognition using surface-modified nanowires via thiolated 2-methacryloxyethyl phosphorylcholine (MPC-SH). MPC-SH enables self-assembled monolayer (SAM) modification, which contributes to the reduction of nonspecific adsorption of biomolecules onto the nanowires, and the specific capture of a target protein is attained in the presence of calcium ions. Our concept demonstrates the recognition of the biomarker protein on nanowire surfaces modified by MPC-SH SAM with a single step modification procedure.
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http://dx.doi.org/10.1039/c8lc00438bDOI Listing
October 2018

Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells.

Sci Rep 2018 04 30;8(1):6765. Epub 2018 Apr 30.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.

Hepatocellular carcinoma (HCC) is a typical hyper-vascular tumor, so the understanding the mechanisms of angiogenesis in HCC is very important for its treatment. However, the influence of the exosomes secreted from HCC cells (HCC-exosomes) on angiogenesis remains poorly understood. We herein examined the effects of the exosomes secreted from HepG2 cells (HepG2-exosomes) on the lumen formation of human umbilical vein endothelial cells (HUVECs) by the imaging of angiogenesis. The degree of lumen formation of HUVECs was dependent on the number of HepG2-exosomes. The HepG2-exosomes expressed NKG2D, an activating receptor for immune cells, and HSP70, a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor (VEGF) receptor. In addition, the HepG2-exosomes contained several microRNAs (miRNAs) reported to exist in the serum of HCC patients. These results suggest that the HCC-exosomes play an important role in angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment.
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http://dx.doi.org/10.1038/s41598-018-24563-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928189PMC
April 2018

Robust Ionic Current Sensor for Bacterial Cell Size Detection.

ACS Sens 2018 03 5;3(3):574-579. Epub 2018 Mar 5.

Health Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , Takamatsu 761-0395 , Japan.

Ionic current sensing methods are useful tools for detecting sub- to several-micron scale particles such as bacteria. However, conventional commercially available ionic current sensing devices are not suitable for on-site measurement use because of inherent limitations on their robustness. Here, we proposed a portable robust ionic current sensor (Robust-ICS) using a bridge circuit that offers a high signal-to-noise (S/N) ratio by suppressing background current. Because the Robust-ICS can tolerate increased noise in current sensing, a simple, lightweight electromagnetic shield can be used and measurements under large electromagnetic noise conditions can be made. The weight of the device was lowered below 4 kg and outdoor particle detection measurements were completed successfully. Accuracy of size detection of Staphylococcus aureus ( S. aureus) was equivalent to that obtained by SEM imaging.
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http://dx.doi.org/10.1021/acssensors.8b00045DOI Listing
March 2018

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires.

Sci Adv 2017 12 15;3(12):e1701133. Epub 2017 Dec 15.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer.
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http://dx.doi.org/10.1126/sciadv.1701133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744465PMC
December 2017

Discriminating single-bacterial shape using low-aspect-ratio pores.

Sci Rep 2017 12 12;7(1):17371. Epub 2017 Dec 12.

The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan.

Conventional concepts of resistive pulse analysis is to discriminate particles in liquid by the difference in their size through comparing the amount of ionic current blockage. In sharp contrast, we herein report a proof-of-concept demonstration of the shape sensing capability of solid-state pore sensors by leveraging the synergy between nanopore technology and machine learning. We found ionic current spikes of similar patterns for two bacteria reflecting the closely resembled morphology and size in an ultra-low thickness-to-diameter aspect-ratio pore. We examined the feasibility of a machine learning strategy to pattern-analyse the sub-nanoampere corrugations in each ionic current waveform and identify characteristic electrical signatures signifying nanoscopic differences in the microbial shape, thereby demonstrating discrimination of single-bacterial cells with accuracy up to 90%. This data-analytics-driven microporescopy capability opens new applications of resistive pulse analyses for screening viruses and bacteria by their unique morphologies at a single-particle level.
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http://dx.doi.org/10.1038/s41598-017-17443-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727063PMC
December 2017

Understanding the formation mechanism of lipid nanoparticles in microfluidic devices with chaotic micromixers.

PLoS One 2017 28;12(11):e0187962. Epub 2017 Nov 28.

Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Kita-ku, Sapporo, Japan.

Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 μm and 31 μm, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 μm chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP size. The critical ethanol concentration range was estimated to be 60-80% ethanol. Ten nanometer-sized tuning of LNPs was achieved for the optimum residence time at the critical concentration using the microfluidic devices with chaotic mixer structures. The residence times at the critical concentration necessary to control the LNP size were 10, 15-25, and 50 ms time-scales for 30, 40, and 50 nm-sized LNPs, respectively. Finally, we proposed the LNP formation mechanism based on the determined LNP formation behavior and the critical ethanol concentration. The precise size-controlled LNPs produced by the microfluidic devices are expected to become carriers for next generation nanomedicines and they will lead to new and effective approaches for cancer treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187962PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705116PMC
December 2017

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

J Am Chem Soc 2017 10 29;139(40):14137-14142. Epub 2017 Sep 29.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University , Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

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 × 10 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.
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http://dx.doi.org/10.1021/jacs.7b06440DOI Listing
October 2017

Nanostructures Integrated with a Nanochannel for Slowing Down DNA Translocation Velocity for Nanopore Sequencing.

Anal Sci 2017 ;33(6):735-738

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University.

Here, we developed a device integrated with a nanochannel and nanostructures to slow DNA translocation velocity. We found that translocation velocity of a single DNA molecule inside a nanochannel was decreased by pre-elongating it using some nanostructures, such as a shallow channel or nanopillars. This decrease of the translocation velocity was associated with the DNA mobility change, which is an intrinsic parameter of DNA molecules and unaffected by an electric field.
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http://dx.doi.org/10.2116/analsci.33.735DOI Listing
August 2018

Effect of DNA Methylation on the Velocity of DNA Translocation through a Nanochannel.

Anal Sci 2017 ;33(6):727-730

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University.

Here, we report the effect of DNA methylation on the velocity of DNA translocation through a nanochannel, as determined by measuring differences in translocation velocities between methylated and non-methylated DNA molecules. We found that the velocity of translocation of methylated DNA was faster than that of non-methylated DNA, which we attributed to variation in the coefficients of diffusion and friction with the nanochannel wall, due to the increased molecular weight and stiffness, respectively, of methylated DNA.
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http://dx.doi.org/10.2116/analsci.33.727DOI Listing
August 2018

Stacking-cyclodextrin-microchip electrokinetic chromatographic determination of gabapentinoid drugs in pharmaceutical and biological matrices.

J Chromatogr A 2017 Jun 26;1503:65-75. Epub 2017 Apr 26.

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt.

A facile, rapid, and highly sensitive microchip-based electrokinetic chromatographic method was developed for the simultaneous analysis of two gabapentinoid drugs, gabapentin (GPN) and pregabalin (PGN). Both drugs were first reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) via nucleophilic substitution reactions to yield highly fluorescent products with λ 470/540nm. Analyses of both fluorescently labeled compounds were achieved within 200s in a poly(methyl methacrylate) (PMMA) microchip with a 30mm separation channel. Optimum separation was achieved using a borate buffer (pH 9.0) solution containing methylcellulose and β-cyclodextrin (β-CD) as buffer additives. Methylcellulose acted as a dynamic coating to prevent adsorption of the studied compounds on the inner surfaces of the microchannels, while β-CD acted as a pseudo-stationary phase to improve the separation efficiency between the labeled drugs with high resolution (Rs>7). The fluorescence intensities of the labeled drugs were measured using a light emitting diode-induced fluorescence detector at 540nm after excitation at 470nm. The sensitivity of the method was enhanced 14- and 17-fold for PGN and GPN, respectively by field-amplified stacking relative to traditional pinched injection so that it could quantify 10ngmL for both analytes, with a detection limit lower than 3ngmL. The developed method was efficiently applied to analyze PGN and GPN in their pharmaceutical dosage forms and in biological fluids. The extraction recoveries of the studied drugs from plasma and urine samples were more than 89% with%RSD values lower than 6.2.
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http://dx.doi.org/10.1016/j.chroma.2017.04.049DOI Listing
June 2017

A millisecond micro-RNA separation technique by a hybrid structure of nanopillars and nanoslits.

Sci Rep 2017 03 8;7:43877. Epub 2017 Mar 8.

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan.

A millisecond micro-RNA separation of a mixture of total RNA and genomic DNA, extracted from cultured HeLa cells, was successfully achieved using a hybrid structure of nanopillars and nanoslits contained inside a microchannel. The nanopillars, 250-nm in diameter and 100-nm in height, were fabricated with a 750-nm space inside the nanoslits, which were 100-nm in height and 25-μm in width; the nanopillars were then applied as a new sieve matrix. This ultra-fast technique for the separation of miRNA can be an effective pretreatment for semiconductor nanopore DNA sequencing, which has an optimum reading speed of 1 base/ms to obtain effective signal-to-noise ratio and discriminate each base by ion or tunneling current during the passage of nucleic acids.
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http://dx.doi.org/10.1038/srep43877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341051PMC
March 2017

Fabrication and Evaluation of Microfluidic Immunoassay Devices with Antibody-Immobilized Microbeads Retained in Porous Hydrogel Micropillars.

Methods Mol Biol 2017 ;1547:49-56

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.

Due to the inherent characteristics including confinement of molecular diffusion and high surface-to-volume ratio, microfluidic device-based immunoassay has great advantages in cost, speed, sensitivity, and so on, compared with conventional techniques such as microtiter plate-based ELISA, latex agglutination method, and lateral flow immunochromatography. In this paper, we explain the detection of C-reactive protein as a model antigen by using our microfluidic immunoassay device, so-called immuno-pillar device. We describe in detail how we fabricated and used the immuno-pillar devices.
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http://dx.doi.org/10.1007/978-1-4939-6734-6_4DOI Listing
February 2018

Identifying DNA methylation in a nanochannel.

Sci Technol Adv Mater 2016 11;17(1):644-649. Epub 2016 Oct 11.

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan; ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Nagoya, Japan; Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan.

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.
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http://dx.doi.org/10.1080/14686996.2016.1223516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102024PMC
October 2016

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

Sci Technol Adv Mater 2016 4;17(1):618-625. Epub 2016 Oct 4.

Department of Neurosurgery, Nagoya University Graduate School of Medicine , Nagoya , Japan.

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 (), of which 83-90% are found to be the mutation. Detection of the 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 mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the 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 mutation in routine clinical practice.
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http://dx.doi.org/10.1080/14686996.2016.1227222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101859PMC
October 2016

Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating.

Sci Rep 2016 08 17;6:31642. Epub 2016 Aug 17.

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.
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http://dx.doi.org/10.1038/srep31642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987677PMC
August 2016
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