Asahi Nakahara

Real-time single-cell imaging of protein secretion

Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1β secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1β secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.

Hydrodynamic on-rail droplet pass filter for fully passive sorting of droplet-phase samples

A hydrodynamic droplet pass filter for droplet-phase sample sorting was developed in this study. Using only groove rails, without additional components or external controls, droplets were sorted based on their physical properties. This is the first report of a droplet pass filter used for effective sorting, and the sorting structure provides a novel fluidic component for fluidic circuits for many applications. Depending on the number of rails, we obtained high-pass, low-pass, band-pass, and multi band-pass filters for sorting droplet samples, and their filtration performance was controlled by varying the dimensions of the rail structures. We evaluated in detail the effect of the rail width on sorting, threshold size of droplets sorted into each rail, and capillary number-dependent sample sorting. Furthermore, band-pass droplet sorting, useful for sample quantification, was provided, and multi-step rail ways allowed multi band-pass droplet sorting that was independent of flow conditions. The proposed sorting method does not require any external systems or skilled operation, and thus, it is expected that the device can contribute to on-site sample treatment and analysis in various fields such as medical care or the military.

Selective droplet sampling using a minimum number of horizontal pneumatic actuators in a high aspect ratio and highly flexible PDMS device

This paper presents a droplet sampling device driven by horizontal pneumatic actuators. A high aspect ratio and highly flexible PDMS (polydimethylsiloxane) structure was proposed for carrying out larger number of sampling than the number of actuators. Large deformation of the actuators caused domino-deformation of parallel walls, and the deformations allowed for selective collection of target droplets. The dimensions of the PDMS structure and the ratio of resin to curing agent were optimized for efficient sampling under the low pressure applied to the actuators. Five sampling modes were achieved in the simple one-layer structure consisting of one inlet, four walls, one drain channel, and two pneumatic actuators, formed by single-step soft lithography process.

Single cell real time secretion assay using amorphous fluoropolymer microwell array

Real time assay of protein secretion from living single cells was realized by coupling total internal reflection fluorescence (TIRF) microscopy observation and an optically optimized microwell array device. This system was based on a sandwich immunosorbent assay. Individual cells were cultured on an inverted microscope in microwell arrays fabricated of an amorphous fluoropolymer, CYTOP, to be monitored by TIRF imaging. CYTOP have no interference for the TIRF imaging because the refractive index of CYTOP is almost same as that of water. Using this system, we could succeed in real time secretion assay of lipopolysaccharide stimulated mast cells and the secretion time course of IL-1β from a single MC/9 cell was obtained successfully.

XPS and NEXAFS studies of VUV/O3-treated aromatic polyurea and its application to microchip electrophoresis

In this study, the authors performed X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) studies of vacuum ultraviolet (VUV)/O₃-treated aromatic polyurea films to investigate their treatment effects. XPS and NEXAFS spectra indicate that the benzene ring was cleaved after treatment and that carboxyl, hydroxyl, ketone and aldehyde groups were formed at the cleaved sites. The VUV/O₃-treated polyurea film was applied to a polymethylmethacrylate (PMMA) microchip for microchip electrophoresis (MCE) of bovine serum albumin (BSA). Fast electro-osmotic mobility of 4.6×10(-4) cm²/V/s as well as reduction of the BSA adhesion was achieved. This functional surface is useful for high-speed MCE analysis.

Development of an electrohydrodynamic ion-drag micropump using three-dimensional carbon micromesh electrodes

An electrohydrodynamic (EHD) ion-drag micropump using three-dimensional carbon micromesh electrodes was developed. The carbon micromesh electrodes were created by the pyrolysis of SU-8 structures. The carbon electrodes and microchannel were formed on a quartz substrate, and the microchannel was sealed by an SU-8 slab structure. The pumping behaviors were evaluated using Fluorinert as a non-conductive sample solution. The maximum pressure and volume flow rate were approximately 23 Pa and 400 nL/min, respectively, under an applied voltage of 500 V.

MEMS LC microchip with low dispersion and low pressure drop turn structure using distribution controlled micro pillar array

MEMS liquid chromatography (LC) microchip with low dispersion and a low pressure drop turn structure was fabricated. In the turn structure, we used higher density pillar array at the inner side while lower density pillar array was used at the outer side to control inner and outer speed of a sample flow. An LC chip with an improved turn structure can obtain similar theoretical plate number (1233) compared to the previously proposed tapered turn (1135). The necessary inlet pressure is also reduced from 2.7 to 0.4 MPa under a flow rate of 10 μL/min. These results show the improved turn structure is applicable for fast analysis time under high flow rate conditions.

Matrix Arrangement of Three-Dimensional Sheath Flow for Multiple Component Nanofibers

Multiple core-sheath flow was realized using matrix arrangement of 3D sheath flows. The sheath flow was hydrodynamically formed in a flow shift area which has symmetrical microgrooves on channel walls. Vertical and horizontal alignments of the sample streams are a key element of matrix configuration. The flow shift areas were connected in parallel to achieve horizontal alignment of the sheath flow. The cascade connection of the flow shift areas is used for vertical alignment of the sheath flow. In order to achieve matrix arrangement of core-sheath flow, combination of the parallel and cascade connections was utilized. In this work, the horizontal and vertical configurations of the 2-sample sheath flow were demonstrated. Two streams of the vertically aligned 2-sample sheath flow were joined horizontally, and, as a result, 4-sample core-sheath flow of matrix configuration was obtained successfully.

PDMS-CYTOP hybrid structure microwell array chip for total internal reflection fluorescence microscopy

A microwell arrry device with a hybrid structure of amorphous perfluoroplymer, CYTOPTM, and polydimethylsiloxane (PDMS) which is specialized in total internal reflection fluorescence (TIRE) microscopy was developed. Since the CYTOP layer, whose refractive index is as same as that of water, is sandwiched between PDMS and glass substrate, leakage of incident light is avoided. PDMS layer upon the CYTOP enables high aspect ratio structures and easy integration of microfluidic components. Since direct bonding of CYTOP and PDMS is difficult to achieve, a chemical bonding method using aminosilane coupling reagent and 3-Glycoidoxypropyltriethoxysilane was applied. The bond strength was about 0.74 MPa. 24 × 50 microwell array was fabricated and their optical properties was evaluated. Measured TIRE microscopy results shows no interfere with incident light under the conventionally used conditions.

NEXAFS and XPS studies of VUV/O 3 treated aromatic polyurea for high-speed electrophoresis microchips

This research studies on VUV/O<inf>3</inf>-treated aromatic polyurea film from near-edge X-ray absorption fine structure (NEXAFS) and X-ray photoelectron spectroscopy (XPS) spectra. These spectra indicate that the benzene ring was cleaved after the treatment, and carboxyl and hydroxyl groups were formed at the cleaved sites. The VUV/O<inf>3</inf>-treated polyurea was applied to polymethylmethacrylate (PMMA) microchip for microchip electrophoresis (MCE) of bovine serum albumin (BSA). Fast electroosmotic mobility of 4.6×10⁻⁴ cm²V⁻¹s⁻¹ was realized as well as reduction of the BSA adhesion. This functional surface is useful for high speed MCE analysis.