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.

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.

How environmental solution conditions determine the compaction velocity of single DNA molecules.

Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg(2+) for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA-protein interactions in vivo.

Direct conversion of silver complexes to nanoscale hexagonal columns on a copper alloy for plasmonic applications.

We introduced a novel method for the rapid synthesis of silver nanohexagonal thin columns from an aqueous mixture of sodium thiosulfate (Na2S2O3) and silver chloride (AgCl) simply added to a phosphor bronze substrate. The reaction is based on galvanic displacement and the products are potentially useful for plasmonic applications.

Spatial exosome analysis using cellulose nanofiber sheets reveals the location heterogeneity of extracellular vesicles.

Extracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 µL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

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.

Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip.

Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method.

Immuno-pillar chip: a new platform for rapid and easy-to-use immunoassay.

We present a new rapid and easy-to-use immunoassay chip which we have named the immuno-pillar chip. It has hydrogel pillars, fabricated inside a microchannel, with many antibody molecules immobilized onto 1 µm diameter polystyrene beads. To evaluate the chip performance, we applied it to the sandwich assay of C-reactive protein (CRP), α-fetoprotein (AFP) and prostate-specific antigen (PSA), a cardiac and inflammation marker, tumors and prostate cancer markers, respectively. For detection of disease markers, we confirmed the chip provides rapid analysis (total assay time of about 4 min) with high sensitivity, it is easy-to-use (no special skills are needed), and it uses small volumes of the sample and reagent (0.25 µL each). Moreover, multiplex assay for three biomarkers was also possible. Additionally, the immuno-pillar chip has a big advantage of having hardly any influence on the assay results even if the introduction quantities of the sample or reagents are different.

Developing spray-freeze-dried particles containing a hyaluronic acid-coated liposome-protamine-DNA complex for pulmonary inhalation.

The liposome-protamine-DNA complex (LPD) is an effective cationic carrier of various nucleic acid constructs such as plasmid DNA and small interfering RNA (siRNA). Hyaluronic acid coated on LPD (LPDH) reduces cytotoxicity and maintains the silencing effect of LPD-encapsulated siRNA. Herein, we aim to develop LPD- or LPDH-containing spray-freeze-dried particles (SFDPs) for therapeutic delivery of siRNA to the lungs. LPD- or LPDH-containing SFDPs (LPD- or LPDH-SFDPs) were synthesized and their structure and function as gene carriers were evaluated using physical and biological methods. The particle size of LPDH, but not of LPD, was constant after re-dispersal from the SFDPs and the amount of siRNA encapsulated in LPDH was larger than that in LPD after re-dispersal from the SFDPs. The in vitro pulmonary inhalation properties of LPDH-SFDPs and LPD-SFDPs were almost the same. The cytotoxicity of LPDH-SFDPs in human umbilical vein endothelial cells (HUVEC) was greatly decreased compared with that of LPD-SFDPs. In addition, Bcl-2 siRNA in LPDH-SFDPs had a significant gene silencing effect in human lung cancer cells (A549), whereas Bcl-2 siRNA in LPD-SFDPs had little effect. These results indicate that compared with LPD, LPDH is more useful for developing SFDPs for siRNA pulmonary inhalation.

A Novel Treatment with Stem Cells from Human Exfoliated Deciduous Teeth for Hypoxic-Ischemic Encephalopathy in Neonatal Rats.

Recently, cell therapy has been developed as a novel treatment for perinatal hypoxic-ischemic encephalopathy (HIE), which is an important cause of neurological disorder and death, and stem cells from human exfoliated deciduous teeth (SHED) express early markers for mesenchymal and neuroectodermal stem cells. We investigated the treatment effect of SHED for HIE in neonatal rats. Seven-day-old rats underwent ligation of the left carotid artery and were exposed to 8% hypoxic treatment. SHED (1 × 105 cells) were injected via the right external jugular vein 24 h after the insult. The effect of intravenous administration of SHED cells was evaluated neurologically and pathophysiologically. In the evaluation of engraftment using quantum dots 655, only a few SHED were detected in the injured cortex. In the immunohistological evaluation 24 h after injection, the numbers of positive cells of active caspase-3 and anti-4 hydroxynonenal antiserum were lower in the SHED group than in the vehicle group. The number of Iba-1+ cells in the cortex was higher in the SHED group. However, the proportion of M1 microglia (Iba-1+/ED-1+) was significantly decreased, whereas M2 microglia (Iba-1+/CD206+) tended to increase in the SHED group. In the behavioral tests performed 5 months after hypoxic treatment, compared to the vehicle group, the SHED group showed significant elongation of the endurance time in the rotarod treadmill test, significantly ameliorated proportion of using the impaired hand in the cylinder test, significantly lower ratio of right/left front paw area in gait analysis, and significantly higher avoidance rate in the active avoidance test. In the in vitro experiment with cultured neurons exposed to oxygen-glucose deprivation, we confirmed the neuroprotective effect of the condition medium of SHED. These results suggested that intravenous administration of SHED exerted a treatment effect both histologically and functionally, possibly via a paracrine effect.

Carbohydrate-protein interactions investigated on plastic chips statically coated with hydrophobically modified hydroxyethylcellulose.

We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from alpha1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC.

Exceeding 20,000-fold concentration of protein by the on-line isotachophoresis concentration in poly(methyl methacrylate) microchip.

In this research, a simple on-line microchip gel electrophoresis with ITP was applied for the concentration and separation of BSA and its immunoassay complex with mAb in a single cross form PMMA microchip. We investigated the ITP concentration effect in PMMA MCE using combination of leading electrolytes, terminating electrolytes and other factors. We realized an ITP-based concentration and separation of BSA and its immunoassay complexes in standard cross-channel microchip gel electrophoresis, which exceeded 2000-fold concentration of BSA immunocomplex using Tris-H3PO4 as a leading electrolyte and Tris-gamma-amino butyric acid as a terminating electrolyte. In addition, we also realized concentration of BSA sample in water, which was more than 20 000-fold and was the result of the concentration effect from combining ITP and the sample stacking techniques.

Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping.

We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.

Rinse and evaporation coating of poly(methyl methacrylate) microchip for separation of sodium dodecyl sulfate-protein complex.

We developed a novel channel wall coating on a poly(methyl methacrylate) (PMMA) microchip using methylcellulose (MC) as a coating reagent to suppress electroosmotic flow (EOF) following the strong analytes adsorption via hydrophobic interaction with channel walls of PMMA. Our coating was obtained by first rinsing channel walls with MC-containing aqueous solution followed by evaporation. The coating made the hydrophilic channel wall lowering EOF by two orders of magnitude (1.2 x 10(-5)cm(2)V(-1)s(-1)) as well as reducing the hydrophobic adsorption. On the coated channel walls, we successfully separated sodium dodecyl sulfate-protein complexes with high reproducibility and efficiency using dextran as a lower viscosity protein separation medium.

A viscosity-tunable polymer for DNA separation by microchip electrophoresis.

A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25 degrees C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17-19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis.

Quantitative determination of amino acids in functional foods by microchip electrophoresis.

Microchip electrophoresis (MCE), a first-generation micrototal analysis system, has emerged during the miniaturization phase of food analysis. Based on the micellar electrokinetic chromatography mode, a simple and fast MCE method with light emitting diode-induced fluorescence detection was developed for quantitative analysis of amino acids in three different kinds of functional foods, viz. sports beverages, jelly-form beverages, and tablet-form functional foods. In contrast to the glass microchip, we improved the separation of amino acids on a poly(methyl methacrylate) (PMMA) chip by addition of cationic starch derivatives. 4-fluoro-7-nitro-2,1,3-benzoxadiazole, which has a short labeling time for amino acids, was used as the fluorescently labeled dye. This MCE method takes less than 10 min of total analysis time including sample preparation and analysis of amino acids in functional foods on a PMMA chip. The results show that this approach has the potential to be a fast and simple method for amino acid analysis in functional foods.

Poly(methylmethacrylate) microchip electrophoresis of proteins using linear-poly(acrylamide) solutions as separation matrix.

Poly(methylmethacrylate) (PMMA) microchip electrophoresis of sodium dodecyl sulfate-protein complexes (SDS-PC) using linear-poly(acrylamide) (L-PA) as a separation matrix was investigated. Prior to electrophoresis, channel walls of PMMA were modified with methylcellulose (MC) to prevent adsorption between channel walls and SDS-PC. Size-based protein separation (SBPS) was successfully performed using the MC-coated microchips with Ferguson plot-fittings. The entangled L-PA solution provided high resolution of peaks of SDS-PC when the concentration of L-PA was increased. Some investigations into the separation mechanism, such as the plot of the logarithm of mobility of each SDS-PC versus the logarithm of the molecular weight of the complex exhibiting linear behavior, indicated that the separation mechanism was dependent on mass discrimination, in accordance with Ogston model.

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

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

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

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 λex/em 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 ß-cyclodextrin (ß-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 ß-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-1 for both analytes, with a detection limit lower than 3ngmL-1. 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.