Correction for ‘Integration of sequential analytical processes into sub-100 nm channels: volumetric sampling, chromatographic separation, and label-free molecule detection’ by Yoshiyuki Tsuyama et al., Nanoscale, 2021, 13, 8855–8863, https://doi.org/10.1039/D0NR08385B.

Despite antigen affinity of B cells varying from cell to cell, functional analyses of antigen-reactive B cells on individual B cells are missing due to technical difficulties. Especially in the field of autoimmune diseases, promising pathogenic B cells have not been adequately studied to date because of its rarity. In this study, functions of autoantigen-reactive B cells in autoimmune disease were analyzed at the single-cell level. Since topoisomerase I is a distinct autoantigen, we targeted systemic sclerosis as autoimmune disease. Decreased and increased affinities for topoisomerase I of topoisomerase I-reactive B cells led to anti-inflammatory and pro-inflammatory cytokine production associated with the inhibition and development of fibrosis, which is the major symptom of systemic sclerosis. Furthermore, inhibition of pro-inflammatory cytokine production and increased affinity of topoisomerase I-reactive B cells suppressed fibrosis. These results indicate that autoantigen-reactive B cells contribute to the disease manifestations in autoimmune disease through their antigen affinity.

<title>Abstract</title>Systemic sclerosis (SSc) is a chronic multisystem disorder characterized by fibrosis and autoimmunity. Interleukin (IL)-31 has been implicated in fibrosis and T helper (Th) 2 immune responses, both of which are characteristics of SSc. The exact role of IL-31 in SSc pathogenesis is unclear. Here we show the overexpression of IL-31 and IL-31 receptor A (IL-31RA) in dermal fibroblasts (DFs) from SSc patients. We elucidate the dual role of IL-31 in SSc, where IL-31 directly promotes collagen production in DFs and indirectly enhances Th2 immune responses by increasing pro-Th2 cytokine expression in DFs. Furthermore, blockade of IL-31 with anti-IL-31RA antibody significantly ameliorates fibrosis and Th2 polarization in a mouse model of SSc. Therefore, in addition to defining IL-31 as a mediator of fibrosis and Th2 immune responses in SSc, our study provides a rationale for targeting the IL-31/IL-31RA axis in the treatment of SSc.

In microfluidics, especially in nanofluidics, nanochannels with functionalized surfaces have recently attracted attention for use as a new tool for the investigation of chemical reaction fields. Molecules handled in the reaction field can reach the single–molecule level due to the small size of the nanochannel. In such surroundings, contamination of the channel surface should be removed at the single–molecule level. In this study, it was assumed that metal materials could contaminate the nanochannels during the fabrication processes; therefore, we aimed to develop metal-free fabrication processes. Fused silica channels 1000 nm-deep were conventionally fabricated using a chromium mask. Instead of chromium, electron beam resists more than 1000 nm thick were used and the lithography conditions were optimized. From the results of optimization, channels with 1000 nm scale width and depth were fabricated on fused silica substrates without the use of a chromium mask. In nanofluidic experiments, an oxidation reaction was observed in a device fabricated by conventional fabrication processes using a chromium mask. It was found that Cr6+ remained on the channel surfaces and reacted with chemicals in the liquid phase in the extended nanochannels; this effect occurred at least to the micromolar level. In contrast, the device fabricated with metal-free processes was free of artifacts induced by the presence of chromium. The developed fabrication processes and results of this study will be a significant contribution to the fundamental technologies employed in the fields of microfluidics and nanofluidics.

Miniaturization of column diameter in liquid chromatography is one of the major trends in separation sciences toward single-cell proteomics and metabolomics. Micro/nanoscale open tubular (OT) capillaries are promising tools for efficient separation analyses of the ultra-small volume of samples. However, highly sensitive and label-free on-column detection is still challenging for such ultra-small capillaries. In this study, we developed a photothermal detector using optical diffraction phenomena by a single nanocapillary. Our detection method realized concentration determination of unlabeled sample solutions in a nanocapillary with 460 nm inner diameter. The calculated limit of detection was 0.12 µM, which corresponds to 16 molecules in a detection volume of 0.23 fL. Furthermore, normal-phase chromatography was performed on a 12 cm long nanocapillary, and femtoliter sample injection, efficient separation, and label-free detection of dye molecules were demonstrated. Our photothermal detector will be widely used as a universal tool for chemical/biological analyses using capillaries with micro/nanoscale diameters.

The progress of nanotechnology has developed nanofluidic devices utilizing nanochannels with a width and/or depth of sub-100 nm (101 nm channels), and several experiments have been implemented in ultra-small spaces comparable to DNAs and proteins. However, current experiments utilizing 101 nm channels focus on a single function or operation; integration of multiple analytical operations into 101 nm channels using nanofluidic circuits and fluidic control has yet to be realized despite the advantage of nanochannels. Herein, we report the establishment of a label-free molecule detection method for 101 nm channels and demonstration of sequential analytical processes using integrated nanofluidic devices. Our absorption-based detection method called photothermal optical diffraction (POD) enables non-invasive label-free molecule detection in 101 nm channels for the first time, and the limit of detection (LOD) of 1.8 µM is achieved in 70 nm wide and deep nanochannels, which corresponds to 7.5 molecules in the detection volume of 7 aL. As a demonstration of sampling in 101 nm channels, aL-fL volumetric sampling is performed using 90 nm deep cross-shaped nanochannels and pressure-driven fluidic control from three directions. Finally, the POD and volumetric sampling are combined with nanochannel chromatography, and separation analysis in 101 nm channels is demonstrated. The experimental results reported in this paper will contribute to the advances in 101 nm fluidic devices which have the potential to provide a novel platform for chemical/biological analyses.

Liquids confined in 10-100 nm spaces show different liquid properties from those in the bulk. Proton transfer plays an essential role in liquid properties. The Grotthuss mechanism, in which charge transfer occurs among neighboring water molecules, is considered to be dominant in bulk water. However, the rotational motion and proton transfer kinetics have not been studied well, which makes further analysis difficult. In this study, an isotope effect was used to study the kinetic effect of rotational motion and proton hopping processes by measurement of the viscosity, proton diffusion coefficient, and the proton hopping activation energy. As a result, a significant isotope effect was observed. These results indicate that the rotational motion is not significant, and the decrease of the proton hopping activation energy enhances the apparent proton diffusion coefficient.

Valve technology is one of the important elements in micro or nanofluidic control. Many valve technologies have been realized for microfluidic control, by which the applications of microfluidic devices have expanded. However, valve technology for nanofluidic control (nanovalves) is still challenging due to the ultrasmall size (100 nm scale) of the nanochannels. We propose a novel nanovalve that utilizes nanobubbles. The entire nanochannel surface is made hydrophobic by surface modification. Nanobubbles are generated by light and removed in the nanochannels. The basic principle of nanobubble generation and removal was confirmed, and the valve function was confirmed by the introduction/cessation of pure water, which led to a change in the concentration of a fluorescent solution after mixing with the pure water. As a result, the high performance (70 ms response time and 400 kPa pressure capacity) of this simple nanovalve was demonstrated.

Nanofluidic devices have become a powerful tool for extremely precise analyses at a single-molecule/nanoparticle level. However, a simple and sensitive molecular detection method is essential for nanofluidic devices because of ultrasmall volume (fL-aL). One such technology is photothermal spectroscopy (PTS), which utilizes light absorption and thermal relaxation by target molecules. Recently, we developed a photothermal optical diffraction (POD) detection method as PTS for nanofluidic devices. However, the detectable concentration range was in the order of μM (102 to 104 molecules), and further improvement in detection performance is strongly required. Here, we demonstrate solvent-enhanced POD with optimized experimental conditions and show its capability of concentration determination of nonfluorescent molecules in nanochannels at a countable molecular level. A relationship between the POD signal and thermal/optical properties of solvents is elucidated. We estimate the diffraction factor and photothermal factor of the solvent enhancement effect by thermal simulations and theoretical calculations. Experimental results show good agreement with the prediction, and the detection performance of the POD is successfully improved. At the optimized condition, we demonstrate the concentration determination with the limit of detection of 75 nM, which corresponds to an average of 10 molecules in a detection volume of 0.23 fL. Our sensitive nonfluorescent molecule detection method will be applied to a wide range of chemical/biological analyses utilizing nanofluidics.

Column miniaturization of liquid chromatography is a major trend in separation sciences with the advent of single-cell proteomics and metabolomics. Nanochannel chromatography is one of the promising tools for single-cell analyses because it provides ultra-small sample volume and high separation efficiency. However, non-fluorescent molecular detection in such small channels is still quite difficult due to fL–aL sample volume, which hinders further miniaturization of nanochannels. In this study, we overcame the size limitation of nanochannel chromatography by our label-free molecular detection method: photothermal optical diffraction (POD), which utilizes the photothermal effect of analytes and optical diffraction by nanochannel. The combination of the nanochannel chromatography and the POD enables 1.8 fL sample separation and label-free molecule detection in nanochannels with 800 nm width and 300 nm depth at the optimized experimental conditions. The limit of detection is 5.4 zmol (3300 molecules), approximately 50 times lower than the conventional label-free detection method. Furthermore, the theoretical plate number is calculated to be 105 plates/m, and the separation performance is discussed. Our label-free detection method will be widely used as a universal detector for nanochannel chromatography.

Water inside and between cells with dimensions on the order of 101-103 nm such as synaptic clefts and mitochondria is thought to be important to biological functions, such as signal transmissions and energy production. However, the characterization of water in such spaces has been difficult owing to the small size and complexity of cellular environments. To this end, we proposed and fabricated a biomimetic nanospace exploiting nanofluidic channels with defined dimensions of hundreds of nanometers and controlled environments. A method of modifying a glass nanochannel with a unilamellar lipid bilayer was developed. We revealed that 2.1-5.6 times higher viscosity of water arises in a 200 nm sized biomimetic nanospace by interactions between water molecules and the lipid bilayer surface and significantly affects the molecular/ion transport that is required for the biological functions. The proposed method provides both a technical breakthrough and new findings to the fields of physical chemistry and biology.

Nanofluidics which integrates analytical systems in 101–103 nm space provides ultra-sensitive analyses at a single-cell and single-molecule level. One of the key technologies for nanofluidics is the ultra-sensitive detection method; however, the ultra-small volume at aL–fL scale makes it challenging. Recently, we have developed a non-fluorescent molecule detection method for nanofluidics called photo-thermal optical diffraction (POD) which utilizes the photo-thermal effect of target molecules and optical diffraction by a single nanochannel. To improve the performance of such diffraction-based detection methods, the design and optimization of optical diffraction are essential. However, it is unknown whether the optical diffraction by a single nanochannel follows general diffraction theory because liquid properties change in the ultra-small space. In this study, we elucidated optical diffraction by a single nanochannel from theoretical calculations and experiments. Our experiments revealed the effect of channel size, channel position, and solvents in the nanochannel, which showed good agreement with proposed theoretical calculations. We also revealed no or little change of refractive index of water in the nanochannel compared with that in the bulk. Finally, we confirmed that the POD signal was proportional to the diffracted light intensity, and the calculated limit of detection of POD was 7.0 × 10–7 RIU in a detection volume of 0.23 fL. Our theoretical calculations and experimental results can be widely applied to the design and optimization of detection methods using optical diffraction by nanochannels and nanostructures.

Ultrasensitive detection of nonlabelled bovine serum albumin is performed in micro/nanofluidic chips using a photothermal optical phase shift (POPS) detection system. Currently, micro- and nanofluidics allow the analysis of various single cells, and their targets of interest are shifting from nucleic acids to proteins. Previously, our group developed photothermal detection techniques for the sensitive detection of nonfluorescent molecules. For example, we developed a thermal lens microscope (TLM) with ultrahigh sensitivity at the single-molecule level and a POPS detector that is applicable to nanochannels smaller than the wavelength of light. The POPS detector also realized the detection of nonlabelled proteins in nanochannels, although its detection sensitivity is less than that of the TLM in microchannels due to insufficient background light reduction. To overcome this problem, we developed a new POPS detector using relay optics for further reduction of the background light. In addition, heat transfer from the sample solution to the nanochannel wall was thoroughly investigated to achieve ultrahigh sensitivity. The limit of detection (LOD) obtained with the new POPS detector is 30 molecules in 1.0 fL. Considering this LOD, the performance of the new POPS detector is comparable with that of the TLM. Owing to the applicability of the POPS detector for sensitive detection even in nanochannels or single-μm channels, which cannot be realized with the TLM, combinations of the POPS detector and separation techniques employing unique nanochannel properties will contribute to advances in single-cell proteomics in the future.

Detection and characterization of individual nanoparticles less than 100 nm are important for semiconductor manufacturing, environmental monitoring, biomedical diagnostics, and drug delivery. Photothermal spectroscopy is a light absorptiometry and promising method for detection and characterization because of its high sensitivity and selectivity compared with light scattering or electrical detection methods. However, the characterization of individual nanoparticles in liquids is still challenging for conventional photothermal detection methods. Here, we report a method for the ultrasensitive detection and accurate characterization of individual nanoparticles in liquids by photothermal optical diffraction, which utilizes enhancement of optical diffraction by a nanochannel after light absorption and heat generation of individual nanoparticles in the channel. Our method realized individual 20 nm Au nanoparticle detection with almost 100% detection efficiency by utilizing nanochannels, leading to concentration determination without a calibration curve. Furthermore, we measured individual nanoparticle size and discriminated 20 and 40 nm Au nanoparticles from their photothermal signals. Our photothermal-based nanoparticle detection method in nanochannels has a potential for a wide range of applications such as on-site evaluation of synthesized plasmonic nanoparticles and drug delivery particles.

Thermophysical properties are basis of physical chemistry and essential for both science and engineering. In this paper, we report a technique for precise measurement of thermal diffusion constant in micro/nanochannel using photothermal spectroscopy. As a result, accuracy of the measurement was dramatically improved to ±2% from our previous report (±17%).

This paper reports a novel label-free concentration determination method for nanofluidics: solvent-enhanced photothermal optical diffraction. Our method realized concentration determination with a detection limit of fewer than 10 molecules. Furthermore, we applied our method to normal-phase chromatography and demonstrated femtoliter sample separation and label-free detection in 102 nm channel. Our method will be widely used for various chemical/biological analyses utilizing nanofluidics.

Single molecule analysis is desired in many areas that require the analysis of ultra-small volume and/or extremely low concentration samples (e.g., single-cell biology, medicine diagnosis, virus detection, etc.). Due to the ultra-small volume or concentration, the sample contains only single or countable analyte molecules. Thus, specific single molecules should be precisely processed and detected for analysis. However, except nucleic acids, most molecules are difficult to amplify, and a new analytical methodology for specific single molecules is thus essential. For this, efficient chemical processing and detection, which are important analytical elements, should be developed. Here, we report a single-molecule ELISA (enzyme-linked immunosorbent assay) device utilizing micro/nanofluidic technology. Both chemical processing and detection were integrated into an ultra-small space (102 nm in size), and the integration allowed precise processing (∼100% capture) and detection of a specific single molecule (protein) for the first time. This new concept and enabling technology represent a significant innovation in analytical chemistry and will have a large impact on general biology and medicine.

Nanofluidics is gaining attention because it has unique liquid and fluidic properties that are not observed in microfluidics. It has been reported that many liquid properties change when the size of a fluidic channel is reduced below 500-800 nm. To discuss the underlying mechanism, information on the microscopic liquid structure must be obtained (e.g., by X-ray diffractometry). However, the very small volume (attoliters to femtoliters) of a nanochannel and the large volume of its glass substrate prevent measurement of signals from the nanochannel liquid. In this study, we report a novel nanofluidic device that can be used in conjunction with X-ray diffractometry to analyze the structure of water confined in nanochannels. Top-down and bottom-up micro- and nano-fabrication processes were established, and the substrate thickness of the measurement area was reduced to only 2.7 μm, which was almost 1000 times smaller than that of conventional substrates (millimeter scale). With this new device, X-ray diffraction signals were clearly observed in nanochannels 500 nm wide and deep. Based on the X-ray diffraction pattern, the radial distribution function was calculated, which showed a structure nearly similar to that of a bulk sample. Therefore, X-ray diffractometry in nanochannels was realized. This method will provide important information on how a liquid behaves when confined in a nanospace and contribute to chemistry and biology on scales of 10-100 nm (e.g., inter- and intra-cellular spaces). It is also important for designing chemical reactions and fluidic circuits in nanochannels for realizing highly functional devices.

A compact portable nanofluidic pump that enables precise manipulation of ultra-small amount of liquid is highly desirable for the trend in miniaturization. Here we develop an integrated micro/nanofluidic pumping device utilizing nanopillar structures with diminishing intervals and locally controlled evaporation. A continuous and steady flow rate of 2.7 pL/s was achieved for more than 12 h without external mechanical power source. The liquid transports in the nanofluidic channels were controllable by adjusting the temperature of evaporation surface. The picoliter-scale flow rates meet the demands for performing nanofluidic immunoassays and other interdisciplinary applications.

Objective. To determine the function and serum levels of soluble forms of programmed death 1 (sPD-1) and one of its ligands, soluble PD ligand 2 (sPD-L2), in patients with systemic sclerosis (SSc) and in a mouse model of topoisomerase I (topo I)-induced SSc.
Methods. Serum levels of sPD-1 and sPD-L2 in 91 patients with SSc were examined by enzyme-linked immunosorbent assay (ELISA). Expression of PD-1 and PD-L2 on T cells, B cells, and macrophages was quantified by flow cytometry. The effects of blockade of PD-1 and PD-L2 were analyzed by microfluidic ELISA (micro-ELISA), a technique that can measure very low amounts of cytokines. In addition, the effects of sPD-1 and sPD-L2 on disease progression were assessed in mice with topo I-induced SSc.
Results. Serum levels of sPD-1 and sPD-L2 were elevated in patients with SSc and correlated with the extent of fibrosis and immunologic abnormalities. Expression levels of PD-1 and PD-L2 were significantly elevated on SSc T cells, B cells, and macrophages. Micro-ELISA analysis of serum samples from patients with SSc showed that PD-L2 high B cells had higher levels of interleukin-10 (IL-10) production compared with PD-L2 low B cells, indicating that PD-L2 acts as a regulator of T cell cytokine production via cognate interactions with T cells and B cells. In mice with topo I-induced SSc, production of IL-10 by topo I-specific B cells in cultures with T cells and topo I protein was significantly higher than that by conventional B cells, and intraperitoneal injection of recombinant chimeric PD-1-Fc and PD-L2-Fc canceled these enhanced effects.
Conclusion. These results suggest that sPD-1 and sPD-L2 contribute to disease development in SSc via the regulation of cognate interactions with T cells and B cells.

We have developed dry etching process of lithium niobate (LN) wafer using neutral loop discharge reactive ion etching (NLD-RIE) to fabricate both micro- and nano-channels for investigating proton diffusion enhancement in ferroelectric nanochannels. We have also developed low-temperature direct bonding process between LN wafers. Two-hundred parallel nanochannel array of 200-nm deep and wide and 400-μm long connected to two microchannels (width: 500 μm, depth: 5.9 μm) at the both ends were fabricated. We have succeeded in measuring the proton diffusion coefficient as high as 1.2×10-8 m2/s.

Autonomous micro/nano mechanical, chemical, and biomedical sensors require persistent power sources scaled to their size. Realization of autonomous micro-power sources is a challenging task, as it requires combination of wireless energy supply, conversion, storage, and delivery to the sensor. Herein, we realized a solar-light-driven power source that consists of a micro fuel cell (mFC) and a photocatalytic micro fuel generator (mu FG) integrated on a single microfluidic chip. The mu FG produces hydrogen by photocatalytic water splitting under solar light. The hydrogen fuel is then consumed by the mu FC to generate electricity. Importantly, the by-product water returns back to the photocatalytic mu FG via recirculation loop without losses. Both devices rely on novel phenomena in extended-nano-fluidic channels that ensure ultra-fast proton transport. As a proof of concept, we demonstrate that mu FG/mu FC source achieves remarkable energy density of ca. 17.2 m Wh cm(-2) at room temperature.

The expansion of microfluidics research to nanofluidics requites absolutely sensitive and universal detection methods. Photothermal detection, which utilizes optical absorption and nonradiative relaxation, is promising for the sensitive detection of nonlabeled biomolecules in nanofluidic channels. We have previously developed a photothermal optical phase Shift,(POPS) detection method to detect nonfluorescent molecules sensitively, while a rapid decrease of the sensitivity in nanochannels and the introduction of an ultraviolet (DV) excitation system-were issues to be addressed. In the present study, our primary aim is to characterize the POPS signal in terms of the thermo-optical properties and quantitatively evaluate the causes-for the decrease in sensitivity. The UV excitation system is then introduced into the POPS detector to realize the sensitive detection Of nonlabeled biomolecules. The UV-POPS detection system is designed and constructed from scratch based on a symmetric microscope. The results of simulations and experiments reveal that the sensitivity decreases due to a reduction of the detection volume, dissipation of the heat, and cancellation of the changes in refractive indices. Finally, determination of the concentration of a nonlabeled protein (bovine serum albumin) is performed in a very thin 900 nm deep nanochannel. As a result, the limit of detection (LOD) is 2.3 mu m (600 molecules in the 440 attoliter detection volume), which is as low as that previously obtained for our visible POPS detector. UV-POPS detection is thus expected be a powerful technique for the study of biomolecules, including DNAs and proteins confined: in nanofluidic channels.

All of the process from sampling to detection could be performed in microliter order by using a new device, mu ELISA, which is equipped with microfluidics and a thermal lens microscope. However, as for handling of a small amount of patient sample in clinical sites, when the sample volume is much smaller, a preservation method should be investigated while considering the influences of the S/V ratio in small area. In this research, preservation stability for a small amount of patient sample was investigated, by analyzing CRP. As a result, the inner wall of the container, liquid volume, and preservation time are should be taken into account and evaluated in advance.

Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells. Indeed, analysis of a specific analyte might require the analysis of a single molecule or several molecules. Analytical processes usually include sampling, chemical processing, and detection. Although several papers have reported chemical processing and detection methods for single cells, a sampling method compatible with maintaining the viability of a single cell during sampling has yet to be developed. Here, we propose a femtoliter sampling method from a living single cell using micro/nanofluidic device technology. The sampling of 39 fL of cytoplasm from a single human aortic endothelial cell was demonstrated and its viability after sampling was confirmed.

Microfluidic immunoassays are expected to be the next-generation diagnosticmethods due to the small size of the reaction space and their high performance. Various tests are performed in clinics or for homemedical care. However, the analyticalmarkers are restricted in comparison to the clinical tests performed inmedical facilities. We developed a microfluidic ELISA (micro-ELISA) system, which utilized a microfluidic device for reactions and a thermal lens microscope (TLM) for detection. This system allowed a very sensitive and rapid assay due to the small size of the reaction space. In this study, to apply this systemfor clinic use, we investigated the detection of an unstable and low concentration (pg mL(-1)) marker in patient's plasma. Among the various markers, brain natriuretic peptide (BNP), a heart failure marker, was chosen because BNP is one of the difficult markers to handle in clinics and at home due to its instability (biological half-life: similar to 20 minutes); moreover, its activity in the patient's sample declines within several hours. To overcome these problems, we investigated the chemical procedure. A streptavidin-beads/biotin-antibody reaction was applied because of its high association constant, and a one-step pre-mixed method was developed for a facile process and higher sensitivity. However, commercial standard BNP with sodium azide cannot be used in this pre-mixed method because sodium azide will inhibit the activity of HRP. Therefore, the preparation and preservation stability of the standard sample was evaluated. To maintain the stability of BNP, a buffer solution containing aprotinin was used for dilution in our investigations. In addition, to reduce the influence of the viscosity changes in patient's blood samples, a dilution procedure was also considered. Based on these investigations, the quantitative analysis of BNP in only 10 mL of patient's plasma was successfully realized, whereas the conventional 96-well microtiter plate method required about 500 mL of plasma sample. Moreover, a superior performance was demonstrated in the limit of quantitation (3.61 pg mL(-1)) and short assay time (20 min for 1 sample and 70 min for 5 samples). An excellent correlation was shown between the conventional method used in the University of Tokyo Hospital and our micro-ELISA method.

A novel liquid chromatographic method utilizing the extended-nano space called extended-nano chromatography, encompassing the 10-1000nm region, has emerged recently. It utilizes an extended-nano fluidic channel, which is fabricated on a glass chip, as a separation column. The advantages of extended nano chromatography are that it uses extremely small sample volumes (attoliter to femtoliter) and demonstrate high separation efficiency. In this review, the fundamentals of extended-nano chromatography are summarized. Instrumentations to realize attoliter sample injections and sensitive detection methods are described. A fabrication method for nanochannel separation columns, including substrate bonding and surface modification, is also introduced. A highly efficient separation was performed within several seconds, as predicted by the theory. Future perspectives, including living single cell analysis and ultrahigh performance separation, are also discussed. (C) 2016 Elsevier B.V. All rights reserved.

An irreversible bonding method for bonding porous polycarbonate membranes to glass microfluidic devices is demonstrated. The membrane surfaces were modified with an ammonia solution that contained amino hydrophilic groups. Additionally, the glass substrates were terminated with hydroxyl groups after exposure to an oxygen plasma. Based on the dehydration reaction, reliable bonding between the glass and the porous membrane was achieved at 110 degrees C and was verified by the fluidic leakage tests for burst pressure (&gt; 500 kPa) and long-term durability (similar to 5 days). In particular, chemical modification by small ammonia molecules allowed bonding of the porous membranes without clogging to the pores. Therefore, this method has great potential for use in nanofluidic channels integrated with nanoporous membranes. Moreover, a simple disassembly strategy for the sandwich-structured microfluidic devices was proposed and realized for the reuse and recycling of glass substrates with microchannels in the event that the membrane morphology changes after long-term use. (C) The Author(s) 2017. Published by ECS.

The next-generation cooling devices are gradually being scaled to smaller than the size of high-performance microchips to enable local heat removal from small hot spots. Realization of a micro/nanofluidic heat pipe device is a challenging task, as it requires high condensation efficiency in an ultra-small space and sufficient liquid transport without employing any wick. Herein, we demonstrate a two-phase loop micro heat pipe device based on unique liquid properties in extended nanospace (10-1000 nm) to meet the growing demands of the miniaturization of electronics and optoelectronics. The device, which contains a small volume of liquid (tens of nanoliter) and does not require a wick, can be conveniently embedded in the microchip. The capillary condensation of water on nanopillars was investigated. The experimental results showed a significant enhancement of the condensation rate on nanopillars for a faster vapor-liquid phase transition. In addition, a streaming potential measurement was performed to evaluate the liquid transport during operation of the micro heat pipe device. This method enables the measurement of water flow rates through extended nanochannels without requiring probe molecules. The micro heat pipe device was verified to work properly. Finally, the cooling performance of the micro heat pipe device was quantitatively estimated, and improvements were proposed to achieve highly efficient cooling.

Despite of the progress made to engineer structured microtissues such as BioMEMS and 3D bioprinting, little control exists how microtissues transform as they mature, as the misbalance between cell generated forces and the strength of cell-cell and cell-substrate contacts can result in unintended tissue deformations and ruptures. To develop a quantitative perspective on how cellular contractility, scaffold curvature and cell-substrate adhesion control such rupture processes, human aortic smooth muscle cells were grown on glass substrates with submillimeter semichannels. We quantified cell sheet detachment from 3D confocal image stacks as a function of channel curvature and cell sheet tension by adding different amounts of Blebbistatin and TGF-beta to inhibit or enhance cell contractility, respectively. We found that both higher curvature and higher contractility increased the detachment probability. Variations of the adhesive strength of the protein coating on the substrate revealed that the rupture plane was localized along the substrate-extracellular matrix interface for non-covalently adsorbed adhesion proteins, while the collagen-integrin interface ruptured when collagen I was covalently crosslinked to the substrate. Finally, a simple mechanical model is introduced that quantitatively explains how the tuning of substrate curvature, cell sheet contractility and adhesive strength can be used as tunable parameters as summarized in a first semi-quantitative phase diagram. These parameters can thus be exploited to either inhibit or purposefully induce a collective detachment of sheet-like microtissues for the use in tissue engineering and regenerative therapies.
Statement of Significance
Despite of the significant progress in 3D tissue fabrication technologies at the microscale, there is still no quantitative model that can predict if cells seeded on a 3D structure maintain the imposed geometry while they form a continuous microtissue. Especially, detachment or loss of shape control of growing tissue is a major concern when designing 3D-structured scaffolds. Utilizing semi-cylindrical channels and vascular smooth muscle cells, we characterized how geometrical and mechanical parameters such as curvature of the substrate, cellular contractility, or protein-substrate adhesion strength tune the catastrophic detachment of microtissue. Observed results were rationalized by a theoretical model. The phase diagram showing how unintended tissue detachment progresses would help in designing of mechanically-balanced 3D scaffolds in future tissue engineering applications. (C) 2016 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Miniaturization of liquid chromatography separation columns is a key trend in chemical and biochemical areas, particularly in genomics; proteomics,"=and single-cell analysis. The work at this level relies upon a novel analytical platform that can deal with sample volumes that are much smaller than a cell. An extended-nanospace is within a scale of 10(1)-10(3) nm and defines the space between a single molecule and normal liquid. Our group has realized high-performance liquid chromatography (HPLC) separation in extended-nanospace with sample injections of hundreds of attoliters :and a separation efficiency of hundreds of thousands of plates/m that can overcome the limitations of a conventional packed column by a magnitude of several orders. However, gradient flow is needed to improve the separation performance, and in this work we present reversed-phase chromatography with step-mixing in extended-nanospace and describe its application. Six fluoresceritly labeled amino acids were separated in 16 s, separation of 17 labeled amino acids in only 50 s with a plate height for most of the peaks of less than 1 mu m.

The integration of microfluidics with living biological systems has paved the way to the exciting concept of "organs-on-a-chip," which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass-based devices have long been utilized in the field of microfluidics but the integration of alternative functional elements within multilayered glass microdevices, such as polymeric membranes, remains a challenge. To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimized on specialty low-temperature bonding equipment (mu TAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units ( EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650 degrees C) and quartz/fused silica bonding (1050 degrees C) processes, this method maintains the integrity and functionality of the membrane (T-g 150 degrees C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 h, indicating sufficient bond strength for long-term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)

Microfluidics has been used to perform various chemical operations for pL-nL volumes of samples, such as mixing, reaction and separation, by exploiting diffusion, viscous forces, and surface tension, which are dominant in spaces with dimensions on the micrometer scale. To further develop this field, we previously developed a novel microfluidic device, termed a microdroplet collider, which exploits spatially and temporally localized kinetic energy. This device accelerates a microdroplet in the gas phase along a microchannel until it collides with a target. We demonstrated 6000-fold faster mixing compared to mixing by diffusion; however, the droplet acceleration was not optimized, because the experiments were conducted for only one droplet size and at pressures in the 10-100 kPa range. In this study, we investigated the acceleration of a microdroplet using a high-pressure (MPa) control system, in order to achieve higher acceleration and kinetic energy. The motion of the nL droplet was observed using a high-speed complementary metal oxide semiconductor (CMOS) camera. A maximum droplet velocity of similar to 5 m/s was achieved at a pressure of 1-2 MPa. Despite the higher fluid resistance, longer droplets yielded higher acceleration and kinetic energy, because droplet splitting was a determining factor in the acceleration and using a longer droplet helped prevent it. The results provide design guidelines for achieving higher kinetic energies in the microdroplet collider for various microfluidic applications.

We demonstrated highly efficient solar hydrogen generation via water splitting by photovoltaic-photoelectrochemical (PV-PEC) tandem device based on GaAs/InGaAsP (PV cell) and WO3/BiVO4 core/shell nanorods (PEC cell). We utilized extremely thin absorber (ETA) concept to design the WO3/BiVO4 core/shell heterojunction nanorods and obtained the highest efficiencies of generation, separation and transfer of the photoinduced charge carriers that are possible for the WO3/BiVO4 material combination. The PV-PEC tandem shows stable water splitting photocurrent of 6.56 mA.cm(-2) under standard AM1.5G solar light that corresponds to the record solar-to-hydrogen (STH) conversion efficiency of 8.1%. (C) 2016 The Japan Society of Applied Physics

Extended-nano space, which is in 10-1000 nm scale, is a transitional region from single molecules to continuous fluid. Even though many specific effects are expected, device engineering of extended-nano space has not been developed so far due to the lack of basic technologies for fluidic engineering. Previously, our group established a strategy of device integration for microchemical systems called continuous flow chemical processing and applied the strategy to various analytical systems. In addition, we have succeeded in developments of basic technologies including fabrication, fluidic control and detection for extended-nano space to find very unique effects such as higher viscosity, lower dielectric constant and higher proton mobility. In this chapter, the uniqueness, device engineering of extended-nano space and its application to bioanalytical devices are introduced. Especially, we focus on an ultimate chromatography using extended-nano space and its innovative performances to break the limits of conventional technologies.

Microfluidic devices are downscaling to 10-100 nm space, which we call extended-nano space. Because the extended-nano space is a space to bridge isolated molecules and normal bulk fluid, new solution chemistry can be expected. However, it was difficult to investigate due to the ultra-small space. Our group developed fundamental technologies for the extended-nano fluidics such as nanofabrication and bonding for glass substrates, aL-fL pressure driven fluidic control, partial surface modification, and single molecule detection. Based on these technologies, many unique liquid properties were found such as viscosity increase, enhanced proton mobility, lower dielectric constant. In addition, the liquid property changes depended on channel size, channel shape, and kinds of liquid. New analytical and energy devices are created utilizing the unique properties in the extended-nano space. In this talk, fundamental technologies and unique liquid properties found in this space are mainly presented, which would have impact not only on chemistry and biology but also on semiconductor industry.

Single cell analysis has been of great interest in recent years. In particular, to achieve living single cell analysis is the ultimate goal to study the dynamic process of the single cell. However, single cell volume is pL in scale, and it is difficult to realize living single cell analysis, even by microfluidic technology (nL-sub nL). Herein, a novel microfluidic platform was developed by integrating a single cell chamber and an extended-nano channel (aL-fL volume). A single cell was isolated and cultured for more than 12 h by pressure-driven flow control. In addition, an electric resistance measurement method was developed to monitor the cell viability without fluorescence labeling. This platform will provide a new method for living single cell analysis by utilizing the novel analytical functions of the extended-nano space.

A high-performance liquid chromatography system with 35 fL sample volume was developed using extended-nano (10-1000 nm) fluidic channels. For many years, miniaturization and enhancement of separation performance have been important issues in separation science. Recently, we have reported an ultimate miniaturization of chromatography using extended-nano channels with extremely high separation efficiency of 7 x 10(6) plates per m. However, the real theoretical plate number was limited to 10(3) due to the short nanochannel length. In this paper, the theoretical plate number was dramatically increased by developing a new high-pressure system with a very long nanochannel. A separation experiment of two fluorescent dyes demonstrated that the theoretical plate number could be improved to 1.4 x 10(4), which is much higher than that with conventional HPLC. The theoretical plate number is also comparable to those of capillary monolithic columns. The extremely small sample volume of extended-nano chromatography could support innovative analytical techniques capable of analyzing a single living cell in the near future.

In this study, a microfluidic plasma-separation device that realizes the whole blood analysis of C-reactive protein (CRP) using one drop of blood is developed. A small, fast, and easy-to-operate blood-testing device is desirable for point-of-care testing, home medical care, and medical cost reduction. Recently, significant advances have been made in analytical instrumentation, and an enzyme-linked immunosorbent assay (ELISA) of a 1 mu L plasma sample has previously been realized. Moreover, recent developments in microfluidics have led to the miniaturization of analytical instruments. However, analytical centrifuges that are conventionally used for the separation of plasma and blood cells are very large and cumbersome to combine with microfluidic devices. Therefore, we propose a plasma-separation device combining microfluidics and membrane filtering. Herein, the proposed plasmaseparation device is applied to whole blood analysis using one drop of blood. Specifically, the device is designed and fabricated to realize high plasma-separation efficiency via the evaluation of fluidic resistance of a porous membrane. Then, the quality of the plasma separated using the device is evaluated and compared with that of the plasma separated using a conventional centrifuge. Our results reveal that the microfluidic plasma-separation device can efficiently provide a sample for the ELISA of CRP using one drop (50 mu L) of whole blood. The total analysis time including plasma separation and ELISA is approximately 25 min.

Microfluidic devices are downscaling to 10-100 nm space, which we call extended-nano space. Because the extended-nano space is a space to bridge isolated molecules and normal bulk fluid, new solution chemistry can be expected. However, it was difficult to investigate due to the ultra-small space. Our group developed fundamental technologies for the extended-nano fluidics such as nanofabrication and bonding for glass substrates, aL-fL pressure driven fluidic control, partial surface modification, and single molecule detection. Based on these technologies, many unique liquid properties were found such as viscosity increase, enhanced proton mobility, lower dielectric constant. In addition, the liquid property changes depended on channel size, channel shape, and kinds of liquid. New analytical and energy devices are created utilizing the unique properties in the extended-nano space. In this talk, fundamental technologies and unique liquid properties found in this space are mainly presented, which would have impact not only on chemistry and biology but also on semiconductor industry.

Micro- and nanofluidics has attracted much attention, particularly concerning single-cell analysis when small amounts of liquids are examined. In present work we successfully fabricated extended-nano channels that were more narrow and shorter (2 mm) as well as wider and longer (10 mm), and accomplished a reversed-phase HPLC separation of labeled amino acids on these channels after octadecylsilylation (ODS). The separation performance characteristics were compared for both types of nano spaces. At an equal amount of pressure, the longer extended-nano channels showed permeability that was one-order higher (K = 47 x 10(-14) m(2)) and separation impedance (E = 13) that was one-order lower than that of the shorter version. Also, the separation plate number for the longer channel was 4000 with a plate height of 2.5 mu m. Both channels have advantages for use in single-cell analysis. The longer channel can be applied for the separation of macromolecules (proteomics), while the short version is more applicable to small molecules (amino acids).

We aim to clarify the effects of size confinement, solvent, and deuterium substitution on keto-enol tautomerization of acetylacetone (AcAc) in solutions confined in 10-100 inn spaces (i.e., = extended; nanospaces) using H-1 NMR spectroscopy. The keto-enol equilibrium constants of AcAc [k(EQ) = [keto]/enol]) In various solvents confined in extended nanospaces of 200-3000 nm were examined using the area ratios of -CH3 peaks in keto, to enol forms. The results showed that the keto form of AcAc in hydrogen-bonded solvents such as water and ethanol increased, drastically with decreasing space sizes, below about 500 run, but the size confinement did not induce equilibrium shifts in aprotic solvents such as DMSO. The magnitudes of K-EQ enhancement were well correlated with solvent proton donicity. It followed from the determination of thermodynamic parameters that the stabilization of intermolecular interactions;between protons in water and carbonyl oxygen (C=O) in the keto form of AcAc were promoted by size-confinement, and that the keto form could be energetically and structurally favored in extended nanospaces vis-a-vis the bulk space. Furthermore) the measurements of deuterium dependence of the K-EQ. values verified that the nanoconfinement-induced shifts of keto-enol tautomerization of AcAc are attributable to high proton mobility via a proton hopping mechanism of the confined water.

In this work we used reversed-phase chromatography in extended-nano channels to separate amino acids. A hydrophobic surface modification of extended-nano channels was established. A sample mixture of fluorescein and sulforhodamine B (0.5 and 0.05 mM respectively) was used for the demonstration of a reversed-phase separation mode. A small amount of sample band (30 fL) was injected into the separation channel, and two compounds were successfully separated. The maximum theoretical plate number of sulforhodamine B was 300,000 plates/m. Two sets of 3 amino acids (3.75 mM each) were separated using 0.01 M citrate buffer (pH 5.5) with 0.01 M sodium perchlorate and 12 and 25% of acetonitrile as a mobile phase. A successful separation (320,000 plates/m with plate height of 3.2 mu m for serine) was accomplished. (C) 2015 Elsevier B.V. All rights reserved.

Currently, continuous culture/passage and cryopreservation are two major, well-established methods to provide cultivated mammalian cells for experiments in laboratories. Due to the lack of flexibility, however, both laboratory-oriented methods are unable to meet the need for rapidly growing cell-based applications, which require cell supply in a variety of occasions outside of laboratories. Herein, we report spontaneous packaging and hypothermic storage of mammalian cells under refrigerated (4 degrees C) and ambient conditions (25 degrees C) using a cell-membrane-mimetic methacryloyloxyethyl phosphorylcholine (MPC) polymer hydrogel incorporated within a glass microchip. Its capability for hypothermic storage of cells was comparatively evaluated over 16 days. The results reveal that the cytocompatible MPC polymer hydrogel, in combination with the microchip structure, enabled hypothermic storage of cells with quite high viability, high intracellular esterase activity, maintained cell membrane integrity, and small morphological change for more than 1 week at 4 degrees C and at least 4 days at 25 degrees C. Furthermore, the stored cells from the hydrogel and exhibited the ability to adhere to a surface and achieve confluence under standard cell culture conditions. Both hypothermic storage conditions are ordinary flexible conditions which can be easily established in places outside of laboratories. Therefore, cell packaging and storage using the hydrogel incorporated within the microchip would be a promising miniature and portable solution for flexible supply and delivery of small amounts of cells from bench to bedside.

Efficient photocatalytic water splitting requires effective generation, separation and transfer of photo-induced charge carriers that can hardly be achieved simultaneously in a single material. Here we show that the effectiveness of each process can be separately maximized in a nanostructured heterojunction with extremely thin absorber layer. We demonstrate this concept on WO3/BiVO4+CoPi core-shell nanostructured photoanode that achieves near theoretical water splitting efficiency. BiVO4 is characterized by a high recombination rate of photogenerated carriers that have much shorter diffusion length than the thickness required for sufficient light absorption. This issue can be resolved by the combination of BiVO4 with more conductive WO3 nanorods in a form of coreshell heterojunction, where the BiVO4 absorber layer is thinner than the carrier diffusion length while it's optical thickness is reestablished by light trapping in high aspect ratio nanostructures. Our photoanode demonstrates ultimate water splitting photocurrent of 6.72 mA cm(-2) under 1 sun illumination at 1.23 VRHE that corresponds to -90% of the theoretically possible value for BiVO4. We also demonstrate a self-biased operation of the photoanode in tandem with a double-junction GaAs/InGaAsP photovoltaic cell with stable water splitting photocurrent of 6.56 mA cm(-2) that corresponds to the solar to hydrogen generation efficiency of 8.1%.

Previously, we developed a new functional device (mu ELISA) by applying microfluidics and thermal-lens microscope. It has been clarified from our research that mu ELISA exhibits excellent performance for measuring human serum. However, when the analysis is performed for a very small amount of various patient samples in microliter order, differences in the composition or viscosity of each sample may effect the measurement values. In this research, the measurement conditions were determined for real patient serum by utilizing CRP. As a result, it has been confirmed that there is some effect that originates from some difference of each patient's serum. To obtain reliable measured values, it is necessary to dilute each sample by a buffer.

The transport and behavior of nanoparticles, viruses, and biomacromolecules in 10-1000 nm confined spaces (hereafter extended nanospaces) are important for novel analytical devices based on nanofluidics. This study investigated the concentration and diffusion of 64 nm nanoparticles in a fused-silica nanochannel of 410 nm depth, using evanescent wave-based particle velocimetry. We found that the injection of nanoparticles into the nanochannel by pressure-driven flow was significantly inhibited and that the nanoparticle diffusion was hindered anisotropically. A 0.2-pN repulsive force induced by the interaction between the nanoparticles and the channel wall is proposed as the dominant factor governing the behavior of nanoparticles in the nanochannel, on the basis of both experimental measurements and theoretical estimations. The results of this study will greatly further our understanding of mass transfer in extended nanospaces.

An extended nanospace (10-100 nm scale) makes it possible to induce unique physicochemical properties, because scientific and technological concepts in this region are shifted from the bulk condensed phase to single molecule, and from microfluidic technology to conventional nanotechnology, respectively. In this study, the molecular structure and dynamics of water and nonaqueous solvents confined in extended nanospaces on a fused-silica substrate were examined by using NMR chemical spectra, relaxation times, and so on. The results showed that the collective properties of molecular clusters with a size range from 10 to 100 nm in a liquid phase were characterized due to the effects of charged surface SiOH groups, and that unique properties differing from bulk water and surface-adsorbing water could appear in extended nanospaces. In particular, we found that (1) inhibition of molecular translational motions, (2) localization of proton charge distribution along a linear O center dot center dot center dot H-O hydrogen bonding chain, and (3) an enhancement of proton transfer of water due to the Grotthuss mechanism; ( SiO- center dot center dot center dot H+ center dot center dot center dot H2O) + H2O -&gt; SiO- +(H3O+ +H2O) -&gt; SiO- + (H2O + H3O+), were induced in extended nanospaces. Such changes appeared for sizes smaller than 800 nm. These results suggested that a proton transfer phase, in which water molecules are loosely coupled within about 50 nm from the surface, exists in extended nanospaces. This model could be qualitatively supported by a three-phase theory invoking the bulk, proton transfer, and surface-adsorbing phases.

Understanding liquid structure and the electrical properties of liquids confined in extended nanospaces (10-1000 nm) is important for nanofluidics and nanochemistry. To understand these liquid properties requires determination of the dielectric constant of liquids confined in extended nanospaces. A novel dielectric constant measurement method has thus been developed for extended nanospaces using a streaming potential method. We focused on the nonsteady-state streaming potential in extended nanospaces and successfully measured the dielectric constant of liquids within them without the use of probe molecules. The dielectric constant of water was determined to be significantly reduced by about 3 times compared to that of the bulk. This result contributes key information toward further understanding of the chemistry and fluidics in extended nanospaces.

The integration of microfluidics with living biological systems has paved the way to the exciting concept of "organs-on-a-chip", which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass based devices have long been utilised in the field of microfluidics but the integration of alternative functional elements within multi-layered glass microdevices, such as polymeric membranes, remains a challenge.
To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimised on specialty low-temperature bonding equipment (mu TAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units (EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650 degrees C) and quartz/fused silica bonding (1050 degrees C) processes, this method maintains the integrity and functionality of the membrane (T-g 150 degrees C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 hours, indicating sufficient bond strength for long term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days.

The surface modification is indispensable to facilitate new functional applications of micro/nanofluidics devices. Among many modification techniques developed so far, the photo-induced chemical modification is the most versatile method in terms of robustness, process simplicity, and feasibility of chemical functionality. In particular, the method is useful for closed spaces, such as post-bonded devices. However, the limitation by optical diffraction limit is still a challenging issue in scaling down the pattern sizes to nanoscale. Here, we demonstrated a novel surface modification on sub-100 nm scale utilizing the novel optical near-field (ONF) generated on nanostructures of photocatalyst (TiO2). The minimum pattern size of 40 nm, which was much smaller than diffraction limit, was achieved using a visible light source (488 nm) and a conventional irradiation setup. The controllability of pattern size by light intensity, the feasibility of functionality, and the non-contact working mode have impacts on surface patterning of post-bonded micro/nanofluidics devices. It is also worthy to note that our results verified for the first time the ONF on nanostructures of non-metal materials and its ability to manipulate the chemical reaction on nanoscale.

Nanostructured photoanodes based on well-separated and vertically oriented WO3 nanorods capped with extremely thin BiVO4 absorber layers are fabricated by the combination of Glancing Angle Deposition and normal physical sputtering techniques. The optimized WO3-NRs/BiVO4 photoanode modified with Co-Pi oxygen evolution co-catalyst shows remarkably stable photocurrents of 3.2 and 5.1 mA/cm(2) at 1.23 V versus a reversible hydrogen electrode in a stable Na2SO4 electrolyte under simulated solar light at the standard 1 Sun and concentrated 2 Suns illumination, respectively. The photocurrent enhancement is attributed to the faster charge separation in the electronically thin BiVO4 layer and significantly reduced charge recombination. The enhanced light trapping in the nanostructured WO3-NRs/BiVO4 photoanode effectively increases the optical thickness of the BiVO4 layer and results in efficient absorption of the incident light.

We demonstrate a new approach to plasmonic enhanced photocatalytic water splitting by developing a novel core-shell Ti@TiO2 brush nanostructure where an elongated Ti nanorod forms a plasmonic core that concentrates light inside of a nanotubular anodic TiO2 shell. Following the ubiquitous element approach aimed at providing an enhanced device functionality without the usage of noble or rare earth elements, we utilized only inexpensive Ti to create a complex Ti@TiO2 nanostructure with an enhanced UV and Vis photocatalytic activity that emerges from the interplay between the surface plasmon resonance in the Ti core, Vis light absorption in the Tirich oxide layer at the Ti/TiO2 interface and UV light absorption in the nanotubular TiO2 shell.

The small length scales that make microfluidics attractive are also the source of some very stringent constraints, especially with respect to the detection approach used. The low concentrations often analyzed in microfluidic devices require highly sensitive detection methods that are effective even in vanishingly small sample volumes. Over the years, many detection approaches have been developed for microfluidics. The majority of these methods rely upon optical phenomena, with the most common being fluorescence detection. Fluorescence detection is well suited to microfluidics because it is both flexible and sensitive; however, it does have shortcomings. Weak fluorescence of targets, autofluorescence of materials, and photobleaching are a few of the issues that have to be dealt with when working with fluorescence detection. Another option that eliminates all of these problems is thermal lens microscopy (TLM), a photothermal spectroscopy technique. TLM is a flexible, sensitive detection approach for nonfluorescent molecules that is capable of carrying out single-molecule detection to label-free in vivo quantification. Despite the potential benefits of TLM, it is still an underutilized detection approach. We hope this review will help broaden the use of TLM for microchip-based CE, as well as a host of other microfluidic applications.

We studied photocatalytic activity of highly porous nanotubular TiO2 films modified with nanoclusters of ubiquitous metal (Ti, Al, Zn, Sn, Cu, W) oxides prepared by chemical bath deposition and atomic layer deposition as well as nanoclusters of metal rich suboxides and mixed titania suboxides prepared by atomic layer deposition by following decomposition of methylene blue under simulated solar light. The mixed titania suboxide clusters constructed on the surface of TiO2 nanotubes by atomic layer deposition demonstrated significantly enhanced photocatalytic activity in comparison to the naked TiO2 nanotubes attributed to the better absorption of visible light due to the upward shift of the valence band near the TiO2 surface induced by the suboxide clusters that feature low valence states and metal-metal bonds. (C) 2014 The Ceramic Society of Japan. All rights reserved.

Engineering using liquids confined in channels 10-1000 nm in dimension, or "extended-nanofluidics," is the next target of microfluidic science. Liquid properties at this scale were unrevealed until recently because of the lack of fundamental technologies for investigating these ultrasmall spaces. In this article, the fundamental technologies are reviewed, and the emerging science and technology in the extended-nanospace are discussed.

The growing need to optimize immunoassay performance driven by interest in analyzing individual cells has resulted in a decrease in the amount of sample required. Miniaturized immunoassays that use ultra-small femtoliter to attoliter sample volumes, a range known as the extended nanospace, can satisfy this analytical need; however, capturing every targeted molecule without loss in extended nanochannels for subsequent detection remains challenging. This is the first report of a successful extended nanofluidics-based quantitative immunochemical reaction capable of high capture efficiency using a femtoliter-scale sample volume. A novel patterning method using a photolithographic technique with vacuum ultraviolet light and low-temperature (100 degrees C) bonding enables patterning of functional groups for antibody immobilization before bonding, resulting in an immunochemical reaction space of only 86 fL. Reaction rate analyses indicate a decrease in the required sample volume to 810 fL and improvement in the limit of detection to 3 zmol, 5-6 orders of magnitude better than possible with the microfluidic immunoassay format. Highly efficient (near 100%) immunochemical reactions on a seconds time scale are possible due to the nm-scale diffusion length, which should be advantageous for the analysis of ultra-low-volume samples.

The development of foot-and-mouth disease virus (FMDV) detection methods is crucial for animal food security, tackling regional FMDV epidemic, and global FMDV prognostic control. For these purposes, a fast and sensitive analysis method is required. In this study, we developed a microchip-based ELISA (enzyme-linked immunosorbent assay), micro-ELISA, to realize FMDV detection. Nickel(11) chelating chemistry was utilized to immobilize recombinant protein (antigen) on polystyrene micro-beads in order to determine FMDV antibodies in cattle serum samples. In addition, reaction protocol and conditions were investigated. As a result, the FMDV detection was successfully demonstrated with only a 10-mu L, sample volume in 25-minute assay time. Analytical sensitivity was evaluated by a maximum nominal positiveness percentage value (NPPV) of 303 and a dilution factor of 32x. The method's inter-run and intra-run CV (coefficients of variance) values were 15.5 and 17.1%, respectively, which were fully compatible with the OIE (World Organization for Animal Health) principle of validation of diagnosis assays for infectious diseases. The developed method should become a powerful tool for determining other animal contagious diseases and/or zoonosis.

Nanofluidics in 10(1) nm space, whose scale is comparable to the electric double layer (EDL) and the size of biomolecules, promises novel functional analytical devices. However, the detection, which is indispensable to the integrated chemical system, is still challenging in such an ultra-small space. Previously, we reported a differential interference contrast thermal lens microscope (DIC-TLM) based on the photothermal interferometry principle and succeeded in detection of nonfluorescent molecules in 10(2) nm spaces. However, the thermal diffusion into substrates becomes a problem for detection in 10(1) nm spaces. The DIC-TLM signals are significantly cancelled out in spaces much smaller than the confocal length (similar to 10(2) nm), which makes DIC-TLM detection in 10(1) nm space quite difficult. To overcome this problem, we propose a new channel structure that benefits the thermal diffusion and sensitivity enhancement in DIC-TLM by employing TiO2 as a substrate material for compensating the signal cancellation effect. As a result, DIC-TLM detection of nonfluorescent molecules (800 molecules) was successfully demonstrated in a nanochannel with a depth of 50 nm. The developed detection method will contribute to the functional nanofluidic devices utilizing 10(1) nm spaces.

The separation and sensitive detection of nonfluorescent molecules at the femtoliter (fL) scale has been achieved for the first time in a nanofluidic channel. Smaller sample volumes and higher separation efficiencies have been significant targets for liquid chromatography for many years. However, the use of packed columns hindered further miniaturization and improvement of separation efficiency. Our group recently developed a novel chromatographic method using an open nanofluidic channel to realize attoliter sample injection and a separation efficiency of several million plates per m. However, because of the extremely small optical path length, this detection method was limited to fluorescent molecules. Herein, we describe the combination of nanofluidic chromatography with differential interference contrast thermal lens microscopy (DIC-TLM), a sensitive detection method for nonfluorescent molecules developed by our group that has the ability to detect 0.61 zmol (370 molecules) with an optical path length of 350 nm. As a result, separation of a 21 fL sample containing 250 zmol was possible at the limit of detection (LOD).

We present a novel method to analyze clenbuterol based on a competitive microfluidic immunoassay scheme with a micro-ELISA system, and obtain a limit of detection that is less than 0.1 ng ml(-1) with a quantitative working range of 0.1 ng ml(-1) to 27.0 ng ml(-1). The approach was envisaged to be a promising method for efficient onsite clenbuterol control with good sensitivity and portability.

Understanding fluid flows in 10-1000 nm space, which we call extended nanospace, is important for novel nanofluidic devices in analytical chemistry. This study therefore developed a particle tracking velocimetry for measuring velocity distribution in nanochannel flows, by using the evanescent wave illumination. 64 nm fluorescent nanoparticles were used as flow tracer. The particle position was determined from fluorescent intensity by the evanescent wave field, with a spatial resolution smaller than light wavelengths. The time resolution of 260 mu s was achieved to make error by the Brownian diffusion of the tracer small to be neglected. An image processing by multitime particle tracking was established to detect the tracer nanoparticles of weak fluorescent intensity. Though the measurement region was affected by nonuniform particle distribution with the electrostatic interactions, pressure-driven flows of water in a nanochannel of 50 mu m width and 410 nm depth were successfully measured. The results of the velocity distribution in the depth-wise direction approximately showed agreement with the fluid dynamics with the bulk liquid properties from the macroscopic view, however, suggested slip velocities even in the hydrophilic channel. We suggest a possibility of appearance of molecular behavior in the fluid near the wall within 10 nm-order scale.

Understanding the properties of liquid confined in extended nanospaces (10-1000 nm) is crucial for nanofluidics. Because of the confinement and surface effects, water may have specific structures and reveals unique physicochemical properties. Recently, our group has developed a super resolution laser-induced fluorescence (LIF) technique to visualize proton distribution with the electrical double layer (EDL) in a fused-silica extended nanochannel (Kazoe, Y.; Mawatari, K.; Sugii, Y.; Kitamori, T. Anal. Chem. 2011, 83, 8152). In this study, based on the coupling of the Poisson-Boltzmann theory and site-dissociation model, the effect of specific water properties in an extended nanochannel on formation of EDL was investigated by comparison of numerical results with our previous experimental results. The numerical results of the proton distribution with a lower dielectric constant of approximately 17 were shown to be in good agreement with our experimental results, which confirms our previous observation showing a lower water permittivity in an extended nanochannel. In addition, the higher silanol deprotonation rate in extended nanochannels was also demonstrated, which is supported by our previous results of NMR and streaming current measurements. The present results will be beneficial for a further understanding of interfacial chemistry, fluid physics, and electrokinetics in extended nanochannels.

A technical bottleneck to the broadening of applications of glass nanofluidic chips is bonding, due to the strict conditions, especially the extremely high temperatures (similar to 1000 degrees C) and the high vacuum required in the current glass-to-glass fusion bonding method. Herein, we report a strong, nanostructure-friendly, and high pressure-resistant bonding method, performed at room temperature (RT, similar to 25 degrees C) for glass nanofluidic chips, using a one-step surface activation process with an O-2/CF4 gas mixture plasma treatment. The developed RT bonding method is believed to be able to conquer the technical bottleneck in bonding in nanofluidic fields.

Analytical technologies of ultrasmall volume liquid, in particular femtoliter to attoliter liquid, is essential for single-cell and single-molecule analysis, which is becoming highly important in biology and medical diagnosis. Nanofluidic chips will be a powerful tool to realize chemical processes for such a small volume sample. However, a technical challenge exists in fluidic control, which is femtoliter to attoliter liquid generation in air and handling for further chemical analysis. Integrating mechanical valves fabricated by MEMS (micro-electric mechanical systems) technology into nanofluidic channels is difficult. Here, we propose a nonmechanical valve, which is a Laplace nanovalve. For this purpose, a nanopillar array was embedded in a nanochannel using a two-step electron beam lithography and dry-etching process. The nanostructure allowed precise wettability patterning with a resolution below 100 nm, which was difficult by photochemical wettability patterning due to the optical diffraction. The basic principle of the Laplace nanovalve was verified, and a 1.7 fL droplet (water in air) was successfully generated and handled for the first time.

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath , Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4771968]

We report on a straightforward method for creating micropatterns of multiple biomolecules. The anti-fouling agent 2-methacryloyloxyethylphosphorylcholine (MPC) polymer and a photolabile linker (PL) were covalently linked to an amino-terminated silane surface. Patterns were generated by selective removal of the MPC polymer via UV irradiation. Multiple micropatterns of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) and rhodamine-labeled goat fragment antigen-binding fragments (FAB) were deposited on a same glass substrate. We also employed micropatterning of multiple biomolecules in that Texas red-labeled BSA and FITC-labeled rabbit anti-mouse IgG were placed inside a microchannel.

A new compact near-field desktop-sized diode laser thermal-lens microscope for analysis in microfluidics was proposed. A novel beam-alignment and detection systems provided high signal stability and, along with reduced number of optical elements rendered the instrument portable. The detection of nonfluorescent model species (Fe(II)-bathophenanthroline chelate) in water showed good linearity in the range of 5 x 10-9 to 1 x 10-4 M, and the limit of detection was 3.5 x 10-9 M, which corresponded to 3.5 x 10-7 absorbance units and provided a 20-fold enhancement in sensitivity compared with existing schematic.

Understanding fluid and interfacial properties in extended nanospace (10-1000 nm) is important for recent advances of nanofluidics. We studied properties of water confined in fused-silica nanochannels of 50-1500 nm sizes with two types of cross-section: (1) square channel of nanoscale width and depth, and (2) plate channel of microscale width and nanoscale depth. Viscosity and wetting property were simultaneously measured from capillary filling controlled by megapascal external pressure. The viscosity increased in extended nanospace, while the wetting property was almost constant. Especially, water in the square nanochannels had much higher viscosity than the plate channel, which can be explained considering loosely coupled water molecules by hydrogen bond on the surface within 24 nm. This study suggests specificity of fluids two-dimensionally confined in extended nanoscale, in which the liquid is highly viscous by the specific water phase, while the wetting dynamics is governed by the well-known adsorbed water layer of several-molecules thickness.

A palmtop-sized microfluidic cell culture system is presented. The system consists of a microfluidic device and a miniaturized infusion pump that possesses a reservoir of culture medium, an electrical control circuit, and an internal battery. The footprint of the system was downsized to 87 x 57 mm, which is, to the best of our knowledge, the smallest integrated cell culture system. Immortalized human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were cultured in the system. HMEC-1 in the system proliferated at the same speed as cells in a microchannel perfused by a syringe pump and cells in a culture flask. HUVEC in the system oriented along the direction of the fluid flow. Claudin-5, a tight junction protein, was localized along the peripheries of the HUVEC. We expect that the present system is applicable to various cell types as a stand-alone and easy-to-use system for microfluidic bioanalysis.

The rapidly developing interest in nanofluidics, which is used to examine liquids on an order that ranges from an attoliter to a femtoliter, correlates with the recent interest in decreased sample amounts, such as in the field of single-cell analysis. In this paper, we have succeeded in the normal phase separation of a 10(1) fL sample with a high number of theoretical plates (10(3) plates), and a fast separation (4 s), using a pressure-driven flow in extended nanochannels. A meander separation channel 1 mu m wide was introduced and showed no band broadening at the turns, unlike microchannels (10(1)-10(2) mu m wide), which is an important aspect in order to lengthen the channel in a space-limited micro/nanofluidic chip. Our device enables the fast separation of an ultra-small volume of sample with a high number of theoretical plates, and will serve as a general platform for the separation of aL to fL volumes of samples. (C) 2012 Elsevier B.V. All rights reserved.

The rapidly developing interest in nanofluidic analysis, which is used to examine liquids ranging in amounts from the attoliter to the femtoliter scale, correlates with the recent interest in decreased sample amounts, such as in the field of single-cell analysis. For general nanofluidic analysis, the fact that a pressure-driven flow does not limit the choice of solvents (aqueous or organic) is important. This study shows the first pressure-driven liquid chromatography technique that enables separation of atto- to femtoliter sample volumes, with a high separation efficiency within a few seconds. The apparent diffusion coefficient measurement of the unretentive sample suggests that there is no increase in the viscosity of toluene in the extended nanospace, unlike in aqueous solvents. Evaluation of the normal phase separation, therefore, should involve only the examination of the effect of the small size of the extended nanospace. Compared to a conventionally packed high-performance liquid chromatography column, the separation here results in a faster separation (4 s) by 2 orders of magnitude, a smaller injection volume (100 fL) by 9 orders, and a higher separation efficiency (440 000 plates/m) by 1 order. Moreover, the separation behavior agrees with the theory showing that this high efficiency was due to the small and controlled size of the separation channel, where the diffusion through the channel depth direction is fast enough to be neglected. Our chip-based platform should allow direct and real-time analysis or screening of ultralow volume of sample.

The rapidly developing interest in nanofluidics, which is used to examine liquids on an order that ranges from an attoliter to a femtoliter, correlates with the recent interest in decreased sample amounts, such as in the field of single-cell analysis. For general nanofluidic analysis, the fact that a pressure-driven flow does not limit the choice of solvents (aqueous or organic) is an important aspect. In this paper, an automated injection system using a pressure-driven flow for several hundred nanometer-sized channels (extended nanochannels) is described. By automatically, and independently, switching four pressure lines using solenoid valves controlled by a sequencer with a time resolution of 10 ms, 550aL sample band in minimum was reproducibly injected under normal phase conditions. The reproducibility of the band injection was improved by one order when compared with the previous injection method, which enables determination of time zero for injection. These facts are essential for the further band analysis in nanochannels, where diffusion is dominant. This injection system using pressure-driven flow can be used with any kind of solvent, which should make it a significant tool for nanofluidic applications, such as immunoassay. DNA analysis, and chromatography. (C) 2011 Elsevier B.V. All rights reserved.

This study reports a novel partial hydrophobic/hydrophilic modification using the optical near-field (ONF) induced photocatalytic reaction. Herein the achievement of sub-diffraction limit resolution by photo-induced method opens a new platform towards extended-nano (lOOmn- lOOOmn) fluidic devices, in which the partial surface modification is indispensible to exploit functional applications, yet difficult to realize due to the diffraction limit nature of light. It is also worthy to note that the simple experimental setup and feasible protocol in our method promise critical impacts 011 surface modification and the development of extended-nano devices.

Understanding fluid and interfacial properties in inter/intracellular space is crucial for biological system. Here, an in vitro method to simultaneously measure the viscosity and wetting property was developed using capillary filling controlled by MPa external pressure in bio-mimetic extended-nano space (10 1-103 nm) which mimicked inter/intracellular space (101-103 nm). Bio-mimetic extended-nanoconfinement effect on water properties was evaluated. It suggested that specificity of two-dimensionally nanoconfined channel, in which the viscosity showing much higher values than bulk, while wetting property is independent on bio-mimetic extended-nanoconfinement. This study will offer a deeper understanding of biological fluid and also contribute to biological system design.

We developed a strong bonding of glass nanofluidic chips at room temperature (RT) in ambient air, overcoming the strict requirements for glass fusion bonding conventionally performed at extremely high temperature (∼1,000 °C) in vacuum.

A water vapor containing plasma activated bonding process is developed to optimize the bonding quality of Si/Si and glass/glass bondings in air ambient. Sufficient bonding strength was achieved at room temperature with no heating process. Meanwhile, good bonding efficiency was realized owing to the appropriate water molecules adsorbed on the surfaces. The whole processes are performed in low-vacuum and ambient air environment. The water vapor containing plasma activated bonding is thus low-cost and environmentally friendly process. © 2012 IEEE.

Owing to the well-established nanochannel fabrication technology in 2D nanoscales with high resolution, reproducibility, and flexibility, glass is the leading, ideal, and unsubstitutable material for the fabrication of nanofluidic chips. However, high temperature (similar to 1,000 A degrees C) and a vacuum condition are usually required in the conventional fusion bonding process, unfortunately impeding the nanofluidic applications and even the development of the whole field of nanofluidics. We present a direct bonding of fused silica glass nanofluidic chips at low temperature, around 200 A degrees C in ambient air, through a two-step plasma surface activation process which consists of an O(2) reactive ion etching plasma treatment followed by a nitrogen microwave radical activation. The low-temperature bonded glass nanofluidic chips not only had high bonding strength but also could work continuously without leakage during liquid introduction driven by air pressure even at 450 kPa, a very high pressure which can meet the requirements of most nanofluidic operations. Owing to the mild conditions required in the bonding process, the method has the potential to allow the integration of a range of functional elements into nanofluidic chips during manufacture, which is nearly impossible in the conventional high-temperature fusion bonding process. Therefore, we believe that the developed low-temperature bonding would be very useful and contribute to the field of nanofluidics.

By combining cell technology and microchip technology, innovative cellular biochemical tools can be created from the microscale to the nanoscale for both practical applications and fundamental research. On the microscale level, novel practical applications taking advantage of the unique capabilities of microfluidics have been accelerated in clinical diagnosis, food safety, environmental monitoring, and drug discovery. On the other hand, one important trend of this field is further downscaling of feature size to the 10(1)-10(3) nm scale, which we call extended-nano space. Extended-nano space technology is leading to the creation of innovative nanofluidic cellular and biochemical tools for analysis of single cells at the single-molecule level. As a pioneering group in this field, we focus not only on the development of practical applications of cellular microchip devices but also on fundamental research to initiate new possibilities in the field. In this paper, we review our recent progress on tissue reconstruction, routine cell-based assays on microchip systems, and preliminary fundamental method for single-cell analysis at the single-molecule level with integration of the burgeoning technologies of extended-nano space.

Direct measurements of the saturated vapor pressures of water confined in extended nanospaces (10-100 nm scale) were realized using capillary evaporation phenomena. These results verified that the saturated vapor pressure was reduced with decreasing space sizes and that Kelvin's equation was applicable even in extended nanospaces.

We developed a method to measure μg/s order flow rate driven by MPa order pressure in extended nanospace (101-103 m). Highly pressurized mass flow rate was measured by an electric balance of 1 μg resolution. The obtained flow rate in 500 nm extended nanochannel showed lower value than predicted, which suggests specific fluid dynamics affected by structured water near channel surface and electric double layer in extended nanospace. This study is important to reveal basic science in the space and establish fundamental technology of extended nanofluidic systems for chemical analysis. Copyright © (2011) by the Chemical and Biological Microsystems Society.

We integrated the pre-preparation processes of single cell at picoliter scale on a quartz silica microchip. The processes contain single cell trap and separation into picoliter volume liquid, injection of cell lysis buffer of sub-picoliter volume, and injection of single cell lysate into the femtoliter volume space. Single cell was separated into 7 picoliter liquid by the use of chamber structure and chemically lysed with minimum dilution. Lysed sample was injected into the extended-nano channel by pressure. The developed method enabled manipulating samples extracted from single cell from picoliter to femtoliter scale and is useful for single-cell protein analysis. Copyright © (2011) by the Chemical and Biological Microsystems Society.

Ion behavior confined in extended nanospace (101-103 nm) is important to develop novel miniaturized systems for biochemical analysis and further applications. The present study developed a measurement technique of ion distribution in nanochannel using stimulated emission depletion microscopy to achieve a spatial resolution of 87 nm. Fluorescein was used for ratiometric measurement of pH by two-excitation wavelengths. The proton distribution in a 2D nanochannel of 410 nm width and 405 nm depth was firstly measured at an uncertainty of pH 0.05, which was strongly related to the electric double layer. This technique will greatly contribute to establish nanofluidics and nanochemistry. Copyright © (2011) by the Chemical and Biological Microsystems Society.

Highly efficient cell-free plasma separation from 200 mu L of human whole blood was realized via axial migration of blood cells and cross-flow filtration in a microchip. Although various analyses of small volumes of blood have been reported, a large volume of blood is necessary for obtaining blood cells and plasma for the conventional plasma separation technique of centrifugation. A highly efficient plasma separation method using small volumes of blood without hemolysis is an important issue. We developed a plasma separation method based on a microchip with a filter, which utilizes the axial migration of blood cells observed in blood vessels. Clogging and hemolysis on the filter can be prevented by the axial migration of the blood cells. Using this method, 65% of the plasma from 200 mu L of whole blood was successfully separated without hemolysis. When the plasma separation microchip interfaced with a micro-ELISA system was applied to C-reactive protein (CRP) analysis, the CRP concentration obtained by the microchip showed good correlation with that obtained by conventional centrifugation. Total analysis time, including plasma separation, was achieved in only 25 min.

Ion behavior confined in extended nanospace (10(1)-10(3) nm) is important for nanofluidics and nanochemistry with dominant surface effects. In this paper, we developed a new measurement technique of ion distribution in the nanochannel by super-resolution-laser-induced fluorescence. Stimulated emission depletion microscopy was used to achieve a spatial resolution of 87 nm higher than the diffraction limit. Fluorescein was used for ratiometric measurement of pH with two excitation wavelengths. The pH profile in a 2D nanochannel of 410 nm width and 405 nm depth was successfully measured at an uncertainty of 0.05. The excess protons, showing lower pH than the bulk, nonuniformly distributed in the nanochannel to cancel the negative charge of glass wall, especially when the electric double layer is thick compared to the channel size. The present study first revealed the ion distribution near the surface or in the nanochannel, which is directly related to the electric double layer. In addition, the obtained proton distribution is important to understand the nanoscale water structure between single molecules and continuum phase. This technique will greatly contribute to understanding the basic science in nanoscale and interfacial dynamics, which are strongly required to develop novel miniaturized systems for biochemical analysis and further applications.

Here we report a way to induce the visible response of non-doped TiO2 in the photocatalytic electrochemical water splitting, which is achieved by utilizing the optical near-field (ONF) generated on nanorod TiO2. The visible response is attributed to the ONF-induced phonon-assisted excitation process, in which TiO2 is excited by sub-bandgap photons via phonon energy. Our approach directly gets involved in the excitation process without chemical modification of materials; accordingly it is expected to have few drawbacks on the photocatalytic performance. This study may offer another perspective on the development of solar harvesting materials. (C) 2011 American Institute of Physics. [doi:10.1063/1.3663632]

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 x 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 mu l/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.

An ultrasensitive absorbance detector, the differential interference contrast thermal lens microscope (DIC-TLM), was employed for a chromatography system using silica nanochannel. Recently, separation of ultrasmall volume sample has been strongly required for single-cell biological and chemical analysis. Previously, we have developed a chromatography system using nanochannels of similar to 100 nm scale (extended nanochannels) fabricated on a silica substrate. The extended nanochromatography realized highly efficient separation of samples &lt;1 fL without packing materials. However, its detection method was limited to fluorescence method due to the small volume, and a new detector based on absorbance has been required. On the contrary, we have also developed DIC-TLM, a photothermal spectrometer based on absorption and thermal relaxation of sample for determination of concentration of nonfluorescent molecules in extended nanochannel. In this paper, we combined the extended nanochromatography and the DIC-TLM for separation and detection of nonfluorescent dyes. Particularly, basic performances of the DIC-TLM including quantitative performance and sensitivity were deliberated for injected samples of similar to fL volume.

Various separation processes have been integrated in microfluidics, such as capillary electrophoresis and chromatography, on a microchip. However, it is extremely difficult to separate a complicated biological system by conventional methods. Here, we report on a feasible structure and the culture condition of human renal proximal tubule epithelial cells (RPTECs), with the aim to construct a bioartificial renal tubule on a chip. Glass microchips and a polycarbonate membrane were sealed with no leakage after a surface modification. Furthermore, matrigel was selected as an optimized extracellular matrix (ECM) for cell-proliferation on the membrane. After culturing for 5 days, RPTECs reached confluent in the chip-membrane structure, which was confirmed by nuclei staining. So far, we have constructed the basic structure and cell proliferation circumstance for the future demonstration of the RPTECs separating function. This separation microdevice has promising potential to be applied as both a unit of a circulation cell culture system and a research platform of cell biology.

Isoelectric points in extended nanochannels (580-2720 nm) fabricated on fused-silica substrates were measured using the streaming current method. The isoelectric point obtained in a 2720 nm channel was almost the same as the isoelectric point reported for the bulk (2.6-3.2). However, the isoelectric point in the extended nanochannel (580 nm) was decreased to less than 2.0. This result provides important information for the modeling of ion transport in extended nanospace. (C) 2011 American Institute of Physics. [doi:10.1063/1.3644481]

Integrated microchemical systems on microchips, which are based on continuous microflows, are expected to become important tools for analysis and chemical synthesis applications for biological sciences and technologies. For these purposes, general integration concepts have been developed, including microunit operations (MUOs) and continuous-flow chemical processing (CFCP) to create fully functional systems for various chemical processing applications. The general methodology has enabled analysis, synthesis, and fabrication of chemical systems on microchips, and these microsystems have demonstrated superior performance (e. g., rapid, simple, easy operation, and highly efficient processing) compared to conventional methodologies. Microchemical technology has now entered the phase of practical application. In this review, we discuss the methods for integration of continuous flow-based chemical process on microchips, relevant technologies, and applications.

A portable, highly sensitive, and continuous ammonia gas monitoring system was developed with a microfluidic chip. The system consists of a main unit, a gas pumping unit, and a computer which serves as an operation console. The size of the system is 45 cm width x 30 cm depth x 30 cm height, and the portable system was realized. A highly efficient and stable extraction method was developed by utilizing an annular gas/liquid laminar flow. In addition, a stable gas/liquid separation method with a PTFE membrane was developed by arranging a fluidic network in three dimensions to achieve almost zero dead volume at the gas/liquid extraction part. The extraction rate was almost 100% with a liquid flow rate of 3.5 mu L/min and a gas flow rate of 100 mL/min (contact time of similar to 15 ms), and the concentration factor was 200 times by calculating the NH3 concentration (w/w unit) in the gas and liquid phases. Stable phase separation and detection was sustained for more than 3 weeks in an automated operation, which was sufficient for the monitoring application. The lower limit of detection calculated based on a signal-to-noise ratio of 3 was 84 ppt, which showed good detectability for NH3 analysis. We believe that our system is a very powerful tool for gas analysis due to the advantages of portable size, high sensitivity, and continuous monitoring, and it is particularly useful in the semiconductor field.

The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.

Integrated micro chemical systems on a chip have been attractive as evolutional tools for high speed, functional and compact instrumentation for chemical analysis, organic synthesis and many others. We have established micro unit operations (MUOs) and applied them to continuous flow chemical processing (CFCP). Recently, the space size of the system is further downscaled to 10(1)-10(2) nm, which we call extended nano space. We have developed fundamental research tools for the extended-nano space, located between conventional nanotechnology (10(0)-10(1) nm) and microtechnology (&gt;1 mu m) and have been studying this space as new research field. In this review, we focus on the methodology and application of integrated micro- and nano-chemical systems for chemical processing, organic synthesis and other applications.

Various micro cell culture systems have recently been developed. However, it is extremely difficult to recover cultured cells from a microchannel because the upper and lower substrates of a microchip are permanently combined. Therefore, we developed a cell culture and recovery system that uses a separable microchip with reversible combining that allows separation between closed and open channels. To realize this system, two problems related to microfluidic control-prevention of leakage and non-invasive recovery of cultured cells from the substrate-must be overcome. In the present study, we used surface chemistry modification to solve both problems. First, octadecyltrimethoxysilane (ODTMS) was utilized to control the Laplace pressure at the liquid/vapor phase interface, such that it was directed toward the microchannels, which suppressed leakage from the slight gap between two substrates. Second, a thermoresponsive polymer poly(N-isopropyl acrylamide) (PNIPAAm) was used to coat the surface of the ODTMS-modified microchannel by UV-mediated photopolymerization. PNIPAAm substrates are well known for controlled cell adhesion/detachment by alteration of temperature. Finally, the ODTMS- and PNIPAAm-modified separable microchips were subjected to patterning, and human arterial endothelial cells (HAECs) were cultured in the resulting microchannels with no leakage. After 96 h of the culture, the HAECs were detached from the microchips by decreasing the temperature and were then recovered from the microchannels. This study is the first to demonstrate the recovery of living cells cultured in a microchannel, and may be useful as a fundamental technique for vascular tissue engineering. (C) 2010 Elsevier Ltd. All rights reserved.

Microfluidic solvent extraction (SX) of metal ions from particle-laden aqueous solutions is demonstrated as an alternative to conventional solvent extraction for a system of industrial interest: extraction of Cu(2+) using 2-hydroxy-5-nonylacetophenone. In the presence of silica nanoparticles, bulk SX suffers from prolonged phase separation and, for hydrophobic particles, the formation of particle-stabilised emulsions, which can be indefinitely stable, leading to significant losses of valuable materials to the emulsion phase. In contrast, non-dispersive microfluidic SX can process fluids containing high particle concentrations (e.g. 61 g/L, 80 nm hydrophilic silica and 5 g/L, and 13 nm moderately hydrophobic silica). The SX was operated continuously for more than 7 h without blockage or failure of the microfluidic chip, in part due to the very short residence time of the silica nanoparticles in the aqueous phase. The microfluidic method is also able to access extraction kinetics for particle-laden systems, which cannot be obtained otherwise due to delayed phase separation. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.

Here we report the fabrication of TiO2 nanorod by using glancing angle deposition and confirm for the first time the novel way to induce the visible response of TiO2 in photoelectrochemical water splitting, that is achieved by the utilizing of optical near-field (ONF) generated on nanorod structures. The ONF allows the so-called phonon-assisted excitation process, which excites TiO2 with sub-bandgap photons via phonon energy, and hence induce the visible response. This study suggest that this kind of nanostructured photocatalyst is promising material for hydrogen production in fuel cell application. Moreover, we believe that the present approach can be applied not only to photocatalyst but also other solar-harvesting materials. Copyright © (2011) by the Chemical and Biological Microsystems Society.

Integration of chemical processes on a microchemical chip has gained much attention in the past decade, and the basic concepts of micro-integration and the supporting technologies have been intensively developed. As a result, many analytical and chemical synthesis applications were demonstrated. The superior performances were verified including shortening analysis time, decrease of sample and reagent volume, and easy chemical operations. Now, the micro-technologies are moving toward practical applications by establishing the systems in which the microchemical chip works as chemical central processing unit. Recently, as a new research field, integration is further proceeding to the 10(1)-10(3) nm scale, which we call extended nanospace. The extended nanospace locates the gap between the targets of conventional nanotechnology (10(0)-10(1) nm) and micro-technology (&gt;1 mu m), and the fluidics and chemistry have not been explored well due to a lack of fundamental technologies. For these purposes, many methodologies were established in recent years. Unique liquid properties were reported, which were quite different from those in microspace. Some properties can be expected by considering the characteristics of microspace and the downscaling, and the others are unexpected or are difficult to predict. These properties enabled new chemical operations which will be quite important as the next analytical technologies. Now, chemistry and fluidics in the extended nanospace are forming a new research field. In this review, we survey the fundamental technologies for extended nanospace researches and introduce several unique liquid properties. Finally, unique chemical operations are also illustrated leading to new analytical operations.

In order to tackle both regional and global foot-and-mouth disease virus (FMDV) epdimics, we hereby develop a rapid microfluidic thermal lens microscopic method to screen swine type O FMDV with good efficiency. The scheme has great merits in terms of field portability, sample volume, assay time, analytical sensitivity, and test reproducibility.

A thermal lens detection device was developed to realize an easy-to-use, portable and sensitive detector for nonfluorescent molecules. Two laser diodes (658 nm for excitation and 785 nm for probe) were made coaxial in an optical unit and were coupled to a single-mode optical fiber. On a microfluidic chip, a small holder for the optical fiber was fixed, and micro-lenses (numerical aperture of 0.2) were also integrated inside the holder. The micro-lenses were designed to realize an adequate chromatic aberration (50 mm), which was essential for sensitive thermal lens detection. Compared with conventional thermal lens detection systems which required very laborious and accurate optical alignment with the microchannel, the new device needed just attachment-detachment of the optical fiber, which was important for practical application. The lower limit of detection was 10 nM for nickel (II) phthalocyaninetetrasulfonic acid tetrasodium salt solutions (model sample), and the absorbance was 9 x 10(-6) AU. The absolute number of molecules detected was less than 200 zmol. The coefficient of variance for 5-time attachment-detachment of the optical probe was as small as 3.6%. The technical development allowed integration of the thermal lens detection devices inside a microsystem (e. g. enzyme-linked immuno-sorbent assay system), and practical microsystems were realized with sensitivities several-orders higher than absorptiometry.

We developed a novel microfluidic system, termed a micro-droplet collider, by utilizing the spatial-temporal localized liquid energy to realize chemical processes, which achieved rapid mixing between droplets having a large volume ratio by collision. In this paper, in order to clarify the characteristics of the micro-droplet collider, dynamics of droplet acceleration, stationary motion and collision in the gas phase in a microchannel were experimentally investigated with visualized images using a microscope equipped with a high-speed camera. The maximum velocity of 450 mm s(-1) and acceleration of 1500 m s(-2) of a 1.6 nL water droplet were achieved at an air pressure of 100 kPa. Measurement results of dynamic contact angles of droplets indicated that wettability of the surface played an important role in the stability of droplet acceleration and collision. We found that the bullet droplet penetrated into the target droplet at collision, which differed from bulk scale. The deformation of the droplet was strongly suppressed by the channel structure, thus stable collision and efficient utilization of the droplet energy were possible. These results are useful for estimating the localized energy, for improving the system in order to realize extreme performance, and for extending the applications of microfluidic devices.

Dynamics of droplet, which utilizes the spatial-temporal localized liquid energy to realize chemical processes in the gas phase in a microchannel, was experimentally investigated using a high-speed camera under the microscope observation. The velocity of 1.6 nL droplet became constant (220 mm s -1) due to the damping force after short from launcher by applying 20 kPa of air pressure. The droplet terminal velocities increased in proportion to the magnitude of the applied air pressure (50 to 200 kPa). The behaviors of droplet collision in a chamber seeded with fluorescent particles were investigated. The bullet droplet was penetrated into the target droplet although both droplets had the same cross section. The deformation is strongly suppressed by the channel structure, thus stable collision and efficient utilization of the droplet energy are possible. These results show usefulness to estimate the localized energy, to improve the system for realizing the extreme performance, and to extend the applications of the microfluidic devices.

We here report the new culturing method of vascular cells using a separable microchip whose substrates can be combined and separated reversibly. This separable microchip allowed to culture smooth muscle cells (SMCs) under the medium perfusion, and to subsequently recover the cultured cells from microchannel only by reducing the temperature. This system has a potential to produce micro scale vascular-mimetic tissue consisting of endothelial cells (ECs) and smooth muscle cells (SMCs), and will be useful for the regeneration of various tissues with multilayered complex structures.

The biological performances of a cell-containing phospholipid polymer hydrogel in bulk and miniaturized formats without an additional culture medium support were investigated and compared. The cell-containing hydrogel was formed spontaneously when solutions of commercial polyvinyl alcohol (PVA) and the phospholipid polymer poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)] (PMBV) suspended with cells in a cell culture medium are mixed together. Bulk and miniaturized hydrogels, with approximate thicknesses of 3.1 mm and 400 mu m, respectively, were prepared in a 96-well microplate and a glass microchip, respectively. In both cases, the hydrogels were homogeneous, and cells were spatially encapsulated. The long-term observation (4 and 8 days) of cell morphology suggested that cells were passively attached to the interface of the hydrogel but were unable to spread and flatten, which inhibited cell growth in both hydrogels. Viability evaluations revealed that cells in both hydrogel formats maintained the same high viability levels after long-term encapsulation. Cytotoxicity assays indicated that the cells in the miniaturized hydrogel maintained a high degree of correlation in cytotoxic sensitivity with the cells in the bulk hydrogel and a routine medium culture. The PMBV/PVA hydrogel not only provides a beneficial cyto-compatible microenvironment for long-term cell survival without an additional culture medium support but also creates a static condition for cell sustainment in a microchip similar to that in bulk. The uniform long-term performances of PMBV/PVA hydrogels in bulk and miniaturized formats make them ideal for the development of long-term, flexible, three-dimensional, living cell-based tools for routine cell-based assays and applications on bulk to microscale levels. (C) 2010 Elsevier Ltd. All rights reserved.

We introduce the liquid-liquid system to utilize both of the spatial and the temporal localized energy simultaneously by adding temporal localization to micro-fluidics. Liquid droplets in the gas phase confined in microchannels are used for spatial-temporal localization to realize various chemical processes. A highly accelerated droplet generates the high kinetic energy and momentum in the spatial localization. "Rapid" collision caused by the highly accelerated droplet enables the spatial and temporal localization of the high-energy, which induces the conversion and transfer of the high-energy. We refer to this new microfluidics system as a "micro droplet collider." We fabricated launchers for the droplet shot, tracks for the droplet run, and a chamber for the collision between two droplets on a microchip based on gas-liquid Laplace pressure. The acceleration of the droplet to the high velocity (10(6) mu m s(-1); 10(2) times faster velocity and 10(4) times higher energy than that of typical water-in-oil droplet format), rapid collision (&lt;100 mu s) at a small interface between droplets were achieved. Inelastic and minimally deformable collision was conducted, which enabled the utilization of the localized high-energy efficiently. As one application to chemical processes, we demonstrated rapid mixing (0.5 s) between two droplets (volume ratio; 1 (0.25 nl):10), a process that has not been achieved yet. Mixing time was 6,000 times faster than that of molecular diffusive mixing by utilizing the localized high-energy. The developed micro droplet collider is expected to contribute greatly to microfluidics and chemical processes on a microchip.

A photothermal detector, named differential interference contrast thermal lens microscope (DIC-TLM), was used to determine the concentration of a nonfluorescent solution in a nanochannel. This method exploits a local change in the refractive index of a solution caused by light absorption. A solution was introduced into a 100-nm scale channel by a pressure-driven nanofluidic control system and its concentration was determined by DIC-TLM. The limit of detection (LOD) was 2.4 mu M in a nanochannel that was 21 mu m wide and 500 nm deep. The LOD was 3 orders of magnitude smaller than that of conventional method. Moreover, the detection volume was accurately determined to be merely 0.25 fL by using a nanochannel with an optical path length of 500 nm. Based on these results, the number of detected molecules was calculated to be 390. In addition, the concentration of a solution in a nanochannel that was 790 nm wide and 500 nm deep could be determined. Finally, the relationship between sensitivity and channel size was investigated and the sensitivity was found to decrease with decreasing nanochannel size, which indicates that the changes in the refractive indices of water and silica cancel each other out. The DIC-TLM realizes sensitive detection of nonfluorescent species in nanochannels without requiring any special fabrication techniques. Therefore, DIC-TLM is expected to be a highly useful analytical technique in nanofluidics.

Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample/reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample/reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection &lt; 20 ng/ml) was verified. (C) 2010 American Institute of Physics. [doi:10.1063/1.3437592]

Recently, interest in single cell analysis has increased because of its potential for improving our understanding of cellular processes. Single cell operation and attachment is indispensable to realize this task. In this paper, we employed a simple and direct method for single-cell attachment and culture in a closed microchannel. The microchannel surface was modified by applying a nonbiofouling polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, and a nitrobenzyl photocleavable linker. Using ultraviolet (UV) light irradiation, the MPC polymer was selectively removed by a photochemical reaction that adjusted the cell adherence inside the microchannel. To obtain the desired single endothelial cell patterning in the microchannel, cell-adhesive regions were controlled by use of round photomasks with diameters of 10, 20, 30, or 50 mu m. Single-cell adherence patterns were formed after 12 h of incubation, only when 20 and 30 mu m photomasks were used, and the proportions of adherent and nonadherent cells among the entire UV-illuminated areas were 21.3%+/- 0.3% and 7.9%+/- 0.3%, respectively. The frequency of single-cell adherence in the case of the 20 mu m photomask was 2.7 times greater than that in the case of the 30 mu m photomask. We found that the 20 mu m photomask was optimal for the formation of single-cell adherence patterns in the microchannel. This technique can be a powerful tool for analyzing environmental factors like cell-surface and cell-extracellular matrix contact. (C) 2010 American Institute of Physics. [doi:10.1063/1.3494287]

A 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer hydrogel exhibits a remarkable ability to preserve cells on microfluidic chips without perfusion culture under cell-based assay conditions, with characteristics of maintenance of high cell viability, restraint of cell proliferation, and minimization of cellular function loss over a period of days and weeks. This would establish a revolutionary flexibility for cell-based applications.

A glass-fabricated microreactor was applied to the direct synthesis of hydrogen peroxide from hydrogen and oxygen. The direct synthesis reaction is a three-phase reaction, with gas (hydrogen and oxygen), liquid (reaction solution) and solid (catalyst) being involved. We designed an advanced microreactor in which an ideal gas-liquid distribution could be accomplished throughout the catalyst packed bed. We also set up a reaction system that enables the reaction operation at more than 2 MPa over 1 week. With these efforts, more than 3 wt% of hydrogen peroxide was successfully produced using the microreactor technology. (C) 2010 Elsevier B.V. All rights reserved.

We report herein the investigation of a novel non-adiabatic optical near-field (ONF) excitation on nanostructured TiO2 photo-anode. The usage of ONF allows the transition to the dipole forbidden phonon states, which is called the phonon-assisted ONF transition, and hence allows us to excite TiO2 by sub-bandgap photon. Here, we investigated the usage of ONF to induce the photocatalytic activity of TiO2 with visible light instead of conventional UV light. By introducing nanostructure into TiO 2 photo-anode to generate optical near-field at the surface of electrode, we confirmed the enhancement of photo current in the visible range, and this current is attributed to the phonon-assisted ONF excitation. This study should lead to a novel approach to excite TiO2 by sub-bandgap photon, consequently improve its visible-light response photocatalytic performance. It also suggest that this kind of nanostructured semiconductor photocatalyst have promising properties for hydrogen production from water splitting.

A novel detection system that combines the merits of open-sandwich (OS) enzyme-linked immunoadsorbent assay (ELISA) and a microfluidic sensor chip system, and which enables rapid and noncompetitive immunodetection of small antigens of less than 1000 in molecular weight, has been proposed. Equipped with a sensitive thermal lens microscope, a minute amount of the carboxyl-terminal peptide of human osteocalcin (BGP), a biomarker for bone metabolism, was quantified utilizing antigen-dependent stabilization of an antibody variable region (OS principle). In a short analysis time (similar to 12 min), we could attain a detection limit comparable to that of the microplate-based OS ELISA (1 mu g L(-1)). In addition, the effects of several pretreatments for serum-derived samples were investigated: an albumin absorption resin, addition of a protease inhibitor cocktail and heat treatment. Each pretreatment was found to be effective. Consequently, intrinsic BGP and its fragments could be detected in healthy human serum with a superior detection limit and working range compared to those of the conventional competitive ELISA method.

A liquid chromatography system, comprising a separation column with a width and depth of a few hundred nanometers, wits fabricated on a glass microchip (femto liquid chromatography, fLC). The size of this system was approximately 10(11) times smaller than that of a conventional LC system, the flow rate was subpicoliter/minute, and the injection volume was a few hundred attoliters. The fLC system did not require packing stationary phase and was capable of separating solutes with different molecular charges (fluorescein and sulforhodamine B) that could not be separated on a conventional LC column whose surface was covered with the same functional group as that of the column of the fLC system. ne fLC system represented herein overcomes limitations of conventional chromatography separation, namely, heterogeneity of the stationary phases and eddy diffusion. Scale-down of the chromatography system brought advantages not only in reduction of sample volume but also in separation efficiency. The fLC system can analyze a very small amount of sample with high efficiency and will be useful in analyzing small samples, such as single cells and synaptic clefts. fLC greatly influences and benefits various fields such as life sciences, medicine, environmental science, and manufacturing by the improvement of separation technology.

In a past decade, new research fields utilizing microfluidics have been formed. General microintegration methods were proposed, and the supporting fundamental technologies were widely developed. These methodologies made various applications in analytical and chemical synthesis fields, and their superior performances such as rapid, simple, and high efficient processing have been proved. Recently, the space is further downscaling to the 10(1)-10(2) nm scale (extended-nano space). The extended-nano space is located between conventional nanotechnology (10(0)-10(1) nm) and microtechnology (&gt;1 mu m), and the research tools are not well established. In addition, the extended-nano space is a transient space from single molecules to bulk condensed phase, and fluidics and chemistry are unknown. For these purposes, basic methodologies were developed, and new specific phenomena in fluidics and chemistry were found. These new phenomena were applied to unique chemical operations such as concentration and ion selection. The new research fields are now being created which are quite different with those in microspace. In this tutorial review, we focus on the basic researches in extended- nano space and survey the fundamental technologies for extended- nano space and reported specific liquid properties. Then, several unique chemical operations utilizing the properties are introduced. Finally, we show the future perspectives by showing the problems to be solved and illustrating the applications in development and in near future.

The extended-nanospace, a space on the scale of 10(1)-10(3) nm, is mostly unexplored due to the lack of sufficient experimental technology. Recently, the research of liquid properties in the extended-nanospace has gathered much interest, because the behavior of water molecules in this space is between that of liquid-like bulk phase water molecules and single molecules. Due to the large surface-to-volume ratio in the channel, the surface charge of the wall directly affects the water structure and ion distribution. The streaming potential/current measurement method, which is used to evaluate surface states directly, is an important and useful method to investigate the liquid properties. In this paper, we report a new method for measuring the streaming potential/current in size-controlled 2D extended-nanospace on glass substrate. Nano-in-microfluidic systems were fabricated on fused-silica glass substrates, and the liquid was air-driven using a pressure controller. An equivalent circuit of the detection system was designed to selectively detect the potential and current in the extended-nanospace. The basic measurement principle was verified using several different experiments. The absolute values obtained for the potential and current were also compared with the theoretical values for various channel sizes (360-1650 nm), and good agreement was observed for micrometre-scale channels. This technique will be valuable for the investigation of chemistry and fluidics in the extended-nanospace.

The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.

This report describes a direct approach for cell micropatterning in a closed glass microchannel. To control the cell adhesiveness inside the microchannel, the application of an external stimulus such as ultraviolet (UV) was indispensible. This technique focused on the use of a modified 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to be a non-biofouling compound that is a photocleavable linker (PL), to localize cells via connection to an amino-terminated silanized surface. Using UV light illumination, the MPC polymer was selectively eliminated by photochemical reaction that controlled the cell attachment inside the microchannel. For suitable cell micropatterning in a microchannel, the optimal UV illumination time and concentration for cell suspension were investigated. After selective removal of the MPC polymer through the photomask, MC-3T3 E1 cells and vascular endothelial cells (ECs) were localized only to the UV-exposed area. In addition, the stability of patterned ECs was also confirmed by culturing for 2 weeks in a microchannel under flow conditions. Furthermore, we employed two different types of cells inside the same microchannel through multiple removal of the MPC polymer. ECs and Piccells were localized in both the upper and down streams of the microchannel, respectively. When the ECs were stimulated by adenosine triphosphate (ATP), NO was secreted from the ECs and could be detected by fluorescence resonance energy transfer (FRET) in Piccells, which is a cell-based NO indicator. This technique can be a powerful tool for analyzing cell interaction research.

Recently, integrated chemical systems have been further downscaled to the 10(1)-10(3) nm scale, which we call extended-nano space. The extended-nano space is a transient space from single molecules to bulk condensed phase, and fluidics and chemistry have not been explored. One of the reasons is the lack of research tools for the extended-nano space, because the space locates the gap between the conventional nanotechnology (10(0)-10(1) nm) and microtechnology (&gt;1 mu m). For these purposes, basic methodologies were developed such as nanofabrication, fluidic control, detection methods, and surface modification methods. Especially, fluidic control is one of the important methods. By utilizing the methodologies, new specific phenomena in fluidics and chemistry were reported, and the new phenomena are increasingly applied to unique applications. Microfluidic technologies are now entering new research phase combined with the nanofluidic technologies. In this review, we mainly focus on pressure-driven or shear-driven extended-nano fluidic systems and illustrate the basic nanofluidics and the representative applications.

Solvent extraction of copper has been explored in a microfluidic chip (mu SX). The transfer efficiency and rate of phase separation in mu SX were compared to that achieved using conventional methods (bulk dispersion) both with and without fine silica particles present. Using the microfluidic approach, transfer efficiency was comparable to that achieved in conventional extraction. Phase separation is slow or totally arrested in bulk extraction, while instantaneous phase separation was achieved in mu SX, even at high particle concentrations.

A thermal lens microscope (TLM) with a new principle was developed to improve the detection limit of conventional TLM. The detection limit was decreased by introducing a differential interference contrast (DIC) method which realizes background-free photodetection. The new differential interference contrast thermal lens microscope (DIC-TLM) exploits phase contrast resulting from a photothermal effect instead of refraction used in conventional TLM. In order to produce high phase contrast, we fabricated a pair of DIC prisms with a large shear value of 5 mu m which is in accordance with the thermal diffusion length. First, we verified the principle of DIC-TLM. The background of TLM measurement was reduced to 1/100 by differential interference, and the signal-to-background (S/B) ratio was improved by I order of magnitude. The signal was confirmed to originate from phase contrast, and the expansion of the shear value was effective. Furthermore, we demonstrated counting of individual gold nanoparticles (5 nm) using DIC-TLM. The particles were counted with high signal-to-noise (S/N) ratio, and the S/N ratio was improved by 1 order of magnitude. Finally, we discuss the possibility of single molecule counting in a liquid.

Fused silica glass microchips have several attractive features for lab-on-a-chip applications; they can be machined with excellent precision down to nanospace; are stable; transparent and can be modified with a range of silanization agents to change channel surface properties. For immobilization, however, ligands must be added after bonding, since the harsh bonding conditions using heat or hydrofluoric acid would remove all prior immobilized ligands. For spatial control over immobilization, UV-mediated immobilization offers several advantages; spots can be created in parallel, the feature size can be made small, and spatial control over patterns and positions is excellent. However, UV sensitive groups are often based on hydrophobic chemical moieties, which unfortunately result in greater non-specific binding of biomolecules, especially proteins. Here, we present techniques in which any -CH(x) (x = 1,2,3) containing surface coating can be used as foundation for grafting a hydrophilic linker with a chemical anchor, a carboxyl group, to which proteins and amine containing molecules can be covalently coupled. Hence, the attractive features of many well-known protein and biomolecule repelling polymer coatings can be utilized while achieving site-specific immobilization only to pre-determined areas within the bonded microchips.

This paper reports on the development of a micro-potentiometric sensor based on external microelectrodes introduced into a microchip. We miniaturized reference and ion-selective electrodes (ISEs) and embedded them into a plastic (PDMS) microchip; the miniaturization of ISE was attained by using a monolithic capillary-based membrane. This sensor was applied to the detection of alkali ions (Na(+), K(+) and NH(4+)) in a microflow on the mu g/L level.

We developed a novel nanofluidic chip equipped with mercury microelectrodes, which enables electrochemical measurements to be made in 10-100 nm scale spaces (called extended nanospaces), and evaluated the performances. The effects of both space sizes and concentrations on the conductance (G) values of KCl solutions in extended nanospaces (216-5000 nm) were examined using impedance spectrometry. We found that the experimental G values in the extended nanospaces decreased non-linearly with decreasing KCl concentrations in the range of 10(-2) to 10(-7) M and could be explained by theoretical model taking account of surface charge density of on a glass surface. This was found to result from enhancement of proton concentrations of the confined solution owing to fast proton exchange between SiOH groups on surfaces and water. Moreover, the G values provided the specific resistance and capacitance of KCl solutions in the extended nanospaces. These results showed that the viscosity of KCl solutions increased by size-confinement and that the viscosity of solution in 216 rim-sized extended nanospaces became about 2.8 times as large as that of bulk solution. We concluded that the developed nanofluidic chip becomes a new experimental tool for demonstrating confinement-induced nanospatial electrochemical properties of liquids.

We fabricated an NMR cell equipped with 10-100 nm scale spaces oil a glass substrate (called extended nanospaces), and investigated molecular structure and dynamics of water confined in the extended nanospaces by (1)H NMR chemical shift (delta(H)) and (1)H and (2)H NMR spin-lattice relaxation rate ((1)H- and (2)H-1/T(1)), (1)H NMR spin-spin relaxation rate ((1)H-1/T(2)), and (1)H NMR rotating-frame spin-lattice relaxation rate ((1)H-1/T(1 rho)) measurements of H(2)O and (2)H(2)O. The delta(H) and (1)H- and (2)H-1/T(1) results showed that size-confinement produces slower translational motions and higher proton mobility of water, but does not affect the hydrogen-bonding structure and rotational motions. Such unique phenomena appeared in the space size of 40 to 800 nm. However, the (1)H-1/T(1) value at 40 nm was still different from that in 4 nm porous nanomaterial, because translational and rotational motions were inhibited for H(2)O molecules in the nanomaterial. By examining temperature- and deuterium-dependence of the (1)H-1/T(1) values, the molecular translational motions of the confined water were found to be controlled by protonic diffusion invoking a proton hopping pathway between adjacent water rather than hydrodynamic translational diffusion. Furthermore, we clarified that proton exchange between adjacent water molecules in extended nanospaces could be enhanced by the chemical exchange of protons between water and SiOH groups on glass surfaces, ( SiO(-)center dot center dot center dot H(+)center dot center dot center dot H(2)O) + H(2)O -&gt; SiO(-) + (H(3)O(+) + H(2)O) -&gt; SiO(-) + (H(2)O + H(3)O(+)), based on (1)H-1/T(2) measurements. An enhancement of proton exchange rate of water due to the reduction of space sizes was verified from the results of (1)H-1/T(1 rho) values, and the rate of water in the 100 nm sized spaces is larger by a factor of more than ten from that of bulk water. Such size-confinement effects were distinctly observed for hydrogen-bond solvents with strong proton-donating ability, while they did not appear for aprotic and nonpolar solvent cases. Based oil these NMR results, we suggested that ail intermediate phase, in which protons migrate through a hydrogen-bonding network and the water molecules are loosely coupled within 50 nm from the Surface, exists mainly in extended nanospaces. This model could be supported by a three-phase theory based oil the weight average of three phases invoking the bulk, adsorbed, and intermediate phases.

5 wt % hydrogen peroxide was achieved by direct synthesis from hydrogen and oxygen at room temperature and 1.0 MPa, in a glass-fabricated microreactor designated for gas-liquid-solid reaction with ideal gas-liquid distribution to the catalyst packed bed.

Fused silica microchips have several attractive features; they are stable, transparent and allow fabrication with immense precision, also of nanochannels, which make them good candidates for both micro- and nanofluidics. Although fused silica microchips have been around for years, the single most difficult fabrication step, thermal bonding, remains a major hurdle. However, thermal bonding can be mastered by observing some simple rules. We provide an illustrated, step-by-step guide on how to prepare glass and fused silica microchips, pointing out pitfalls and advising correct manners for successful thermal bonding.

The possible application of continuous scanning thermal lens microscopy (TLM) as alternative online biofilm observation method is studied. As biofilm is a heterogeneous sample, the influence of spatially limited thermal flow at the sample heterogeneities and the biofilm-environment border has to be considered. The influence of the edges on the lateral resolution with respect to scanning velocity during continuous scanning TLM was therefore evaluated. Lateral scanning experiments on 100 nm thin gold stripes showed that the maximum scan speed can be predicted from a time constant of a lock-in amplifier and the beamwidth. Since three-dimensional mapping is needed to fully characterize the biofilm structure, depth scanning experiments with stained 4 mu m thick polystyrene samples with the coaxial TLM setup were evaluated for signal width at full width at half maximum. Thus, a minimum step width for depth scanning of 10 mu m for observation has been acquired. A three-dimensional image of unstained biofilm grown in a flow chamber was acquired using continuous scanning TLM.

This report describes a new surface-treatment technique for cell micropatterning. Cell attachment was selectively controlled on the glass surface using a photochemical reaction. This strategy is based on combining 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to reduce nonspecific adsorption, and a photolabile linker (PI.) for selective cell patterning. The MPC polymer was coated directly on the glass surface using a straightforward surface modification method, and was removed by ultraviolet (UV) light illumination. All the surface mollification steps were evaluated using static water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), measurements of non-specific protein adsorption, and the cell attachment test. After selective cleavage of the MPC polymer through the photomask, cells attached only to the UV-illuminated region where the MPC polymer was removed, which made the hydrophilic surface relatively hydrophobic. Furthermore, the size of the MC-3T3 E1 cell patterns could be controlled by single cell level. Stability of the cell micropatterns was demonstrated by culturing MC-3T3 E1 cell patterns for 5 weeks on glass slide. The micropatterns were stable during culturing; cell viability also was verified. This method can be a powerful tool for cell patterning research. (c) 2008 Elsevier Ltd. All rights reserved.

We report on the first successful miniaturization of sandwich immunoassay into an extended nanospace channel for the detection of alpha-fetoprotein. A fused silica microchip with a nanochannel flanked by two microchannels, to facilitate liquid handling, was constructed using photo- and electron beam lithography, ICP and thermal bonding. Reagents could be selectively introduced into the nanochannel and the flow be stopped by using a pressure controller that alternatively opened and closed inlet and outlet ports. The construction and handling of the nano-in-microchip device for immunoassay is described.

A new method has been developed for liquid-liquid microextraction utilizing a circulation microchannel. A glass microchemical chip having a circular shallow microchannel in contact with a surrounding deeper microchannel was fabricated by a two-step photolithographic wet-etching technique. Surface modification reagent was selectively introduced to the shallow channel by utilizing capillary force, and the surface of the shallow channel was selectively made hydrophobic. With the aid of the hydrophobic/hydrophilic surface patterning, it was possible to keep organic solvent in the circular channel while the aqueous sample solution was continuously flowing in the deep channel. As a result, concentration extraction from sample solution to stationary extractant with a nanoliter scale volume became possible. Concentration extraction has been difficult in a multiphase continuous flow. Function of the newly developed microextraction system was verified with methyl red as a test sample, and concentration extraction to reach equilibrium was successfully carried out. A novel surface modification method utilizing frozen liquid as a masking material was also developed as a reverse process to make the shallow channel hydrophilic and the deep channel hydrophobic. Visualization of circulation motion inside the circular shallow channel induced by flow in the deep channel was observed with a particle tracing method.

Phase separation of gas-liquid and liquid-liquid microflows in microchannels were examined and characterized by interfacial pressure balance. We considered the conditions of the phase separation, where the phase separation requires a single phase flow in each output of the microchannel. As the interfacial pressure, we considered the pressure difference between the two phases due to pressure loss in each phase and the Laplace pressure generated by the interfacial tension at the interface between the separated phases. When the pressure difference between the two phases is balanced by the Laplace pressure, the contact line between the two phases is static. Since the contact angle characterizing the Laplace pressure is restricted to values between the advancing and receding contact angles, the Laplace pressure has a limit. When the pressure difference between the two phases exceeds the limiting Laplace pressure, one of the phases leaks into the output channel of the other phase, and the phase separation fails. In order to experimentally verify this physical picture, microchips were used having a width of 215 mu m and a depth of 34 mu m for the liquid-liquid microflows, a width of 100 mu m and a depth of 45 mu m for the gas-liquid microflows. The experimental results of the liquid-liquid microflows agreed well with the model whilst that of the gas-liquid microflows did not agree with the model because of the compressive properties of the gas phase and evaporation of the liquid phase. The model is useful for general liquid-liquid microflows in continuous flow chemical processing.

Nanoparticles are a key material in nanoscience and nanotechnology due to their unique physicochemical properties, so an analytical method is increasingly required. In the present research, we developed a method for individual nanoparticle detection by thermal lens microscopy and microfluidic chips. Pulsed signals were clearly observed, as nanoparticles were passing through the detection volume. The scale of the microfluidic channel was reduced from 100 to 1 mu m to improve the detection efficiency. As a result, a detection efficiency of 100% was demonstrated.

An automated full-range quantitation method for identifying d-methamphetamine in human hair using a microchip-based ELISA system (microELISA) in combination with a micropulverized extraction method for sample preparation has been developed. An antibody and a peroxidase-linked methamphetamine, both are commercially available, were used for the competitive ELISA assay. Method validation was carried Out using doped hair samples, and segmental analyses of real-case specimens were carried out by both microELISA and LC/MS/MS to verify the reliability and applicability of this new method. Due to the small size of the system and the lack of an evaporation process, sample preparation and quantitation can be accomplished easily and quickly (less than 30 min) in small-scale contamination-free environments. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

We have developed a novel, practical micro-ELISA system for sensitive and rapid allergy diagnosis. The enzymatic reactions occurred under stopped-flow conditions, resulting in both high precision and high sensitivity. A BSA-biotin-avidin linker was introduced for the immobilization of water-soluble allergens on polystyrene microbeads, enabling immobilization of allergens in sufficient density to provide high sensitivity. Evaluation of the system&apos;s performance showed a good detection limit (2 ng/mL) for total IgE measurement. In addition, a good correlation with a conventional method (CAP method) was demonstrated using human serum samples from 85 allergy patients. Importantly, sample volumes (5 mu L) were 10 times smaller and analysis time (12 min) was &gt; 20 times faster than the conventional method. All procedures were automatically regulated with our simple microfluidic system, and all the fluidic, optic and electronic components were integrated for portability. We believe that our system has the potential to become a very powerful tool, particularly for point-of-care diagnosis.

That focused arrays, even with a small set of ligands, provide more data than single point experiments is well established in the DNA microarray research field, but microarray technology has yet to be transferred to fused silica microchips. Fused silica microchips have several attractive features such as stability to pressure, solvents, acids and bases, and can be fabricated with minute dimensions, making them good candidates for nanofluidic research. However, due to harsh bonding conditions, DNA ligands must be immobilized after fabrication, thus preventing standard microarray spotting techniques from being used. In this paper, we provide tools for serial DNA immobilization in fused silica microchips using UV. We report the synthesis of a new UV-linker which was used to covalently couple functional DNA oligos to the inside of channels in fused silica microchips. With some simple modifications to our mask aligner, we were able to transfer OHP mask patterns, which allows the creation of basically any pattern in the channels. The functionality of the oligos was measured through the binding of fluorophore-labeled complementary target oligos. We examined parameters influencing DNA immobilization, and carry-over between spots after consecutive immobilizations inside the same channel. We also report the first successful multiple immobilizations of functional DNA oligos inside single channels of extended nanospace depth (460 nm).

Parallel multiphase microflows, which can integrate unit operations in a microchip under continuous flow conditions, are discussed. Fundamental physics, stabilization methods and some applications are shown.

This paper reports a novel distillation method in microchannels. For the gas-liquid separator in the evaporator, hydrophilic-hydrophobic patterned microchannel structure was utilized. In order to control condensation of the vapor, nanopillar structures having a capillary radius of 270 nm were fabricated after the separator. In the nanopillars, vapor pressure is lower than that at a flat liquid surface. An aqueous Solution of 9.0 wt % ethanol was used as a model sample, and concentration of the condensed liquid in the nanopillar was estimated as 19 wt %.

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 x 10(-4) mol L(-1) (Delta A5.2 x 10(-6) Abs.) for (+)-CSA and 8.4 x 10(-4) mol L(-1) (Delta A=5.0 x 10(-6) Abs.) for (-)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified.

A novel air-pressure-based nanofluidic control system was developed and its performance was examined. We found that the flow in a 100 nm scale nanochannel on a chip (called an extended nanospace channel) could be controlled within the pressure range of 0.003-0.4 MPa, flow rate range of 0.16-21.2 pL/min, and residence time range of 24 ms-32.4 s by using the developed nanofluidic control system. Furthermore, we successfully demonstrated an enzyme reaction in which the fluorogenic substrate TokyoGreen-beta-galactoside (TG-beta-gal) was hydrolyzed to the fluorescein derivative TokyoGreen (TG) and beta-galactose by the action of beta-galactosidase enzyme as a calalyst in a Y-shaped extended nanospace channel. The parameters for the reaction kinetics, such as K (m), V (max) and k (cat), were estimated for the nanofluidic reaction, and these values were compared with the results of bulk and microfluidic reactions. A comparison showed that the enzyme reaction rate in the Y-shaped extended nanospace channel increased by a factor of about two compared with the rates in the bulk and micro spaces. We thought that this nanospatial property resulted from the activated protons of water molecules in the extended nanospace. This assumption was supported by the result that the pH dependence of the maximum enzyme activity in the Y-shaped extended nanospace channel was slightly different from that in the bulk and micro spaces.

A novel micro-flow velocimeter, the flowing thermal lens micro-flow velocimeter (FTL-MFV) is developed in which a photothermally generated local refractive index change (a thermal lens) is utilized as a micro-tracer of flow. Flow velocity is measured from the time required for the thermal lens to travel between two points. Generation and detection of thermal lenses are carried out optically without contacting the flow. By choosing the wavelength of the excitation beam pulse so that it coincides with the absorption band of the solvent used, thermal lenses can be generated without adding anything to the liquid. Synchronous detection using a lock-in amplifier makes detection of thermal lens with a very small temperature rise possible. Thus, with the FTL-MFV, non-contact in situ measurement of flow can be carried out with only slight disturbance to the microfluid. In order to make the sensor small, optical fibers and SELFOC(TM) microlenses are used in focusing the excitation and probe beams. A dynamic range of 25-300 mu L/min is realized in measurement of flow rates in a microchannel. (C) 2008 Elsevier B.V. All rights reserved.

Thermal lens microscope (TLM) is a sensitive detection method for nonfluorescent molecules and widely applied to detection in a capillary or on a microchip. In this paper, we developed a flexible design tool for TLM systems to meet various applications utilizing a microspace. The TL effect was modeled, including signal processing, and calculated by combining fluidic dynamics and wave optics software. The coincidence of the calculations and experiments was investigated by measuring the effects of optical path length or focus positions of the excitation beams on TL signals which are quite difficult to calculate by a conventional method. Good agreement was shown and the applicability of the TLM design tool was verified.

Cell analysis and clinical diagnosis systems are now becoming the largest field of application for microchip-based analytical systems. Technological advantages include: small volume, fast analysis time, highly integrated analytical functions, easy operation and small size. For these purposes, basic methodologies for general micro-integration and basic technologies, including fluidic control and ultrasensitive detection, are required. In this review, we introduce our approach to the general integration of various analytical functions and the application of cell analysis systems with cultured cells in microchannels, as well as practical analytical systems for clinical diagnosis utilizing human serum samples.

A UV excitation thermal-lens microscope (UV-TLM) with an excitation beam wavelength of 266 nm and liquid chromatograph (LC) were connected to build a new system (LC/UV-TLM) for the separation and highly sensitive label-free determination of biomolecules, such as peptides. A variation in the composition of the mobile phase with time, as in gradient elution, is very commonly utilized in LC for improved separations. Thermal-lens signals are generally highly dependent on the properties of the media surrounding the photo-absorber. This effect must be carefully considered in such situations where the composition of a solution drastically changes in time. Therefore, we tested the developed LC/UV-TLM system operated in a gradient elution mode, by observing the effects of the variation in the solution composition upon TLM signal intensities. In water/acetonitrile gradient elution, no serious effects were observed as long as the acetonitrile content was less than 40% v/v. We then tested the developed system in the separation of a standard sample solution of mixed synthetic peptides; we found that the developed system was 10-times more sensitive than a conventional system with the spectrophotometer used as a detector.

A detection and fixation method of single and multiple gold nanoparticles on the wall of a microfluidic channel is demonstrated. A thermal lens microscope (TLM) with continuous-wave excitation (wavelength, 532 nm) and probe (wavelength, 670 nm) laser beams was used to realize the sensitive detection of heat generated by light absorption of individual gold natioparticles (50 nm in diameter); fixation of the individual nanoparticles was realized simultaneously. The fixation mechanism was investigated and attributed to an absorption-based optical force. In addition to single nanoparticle detection, multiple-nanoparticle detection and fixation was demonstrated. An acceleration of fixation was observed when the number of fixed particles was increased. TLM is expected to be a powerful tool for both the quantitative detection and precise fixation of individual nanoparticles.

A novel chiral detector, a circular-dichroism thermal lens microscope (CD-TLM), was developed to realize sensitive and selective detection of small volume chiral samples on a microchip. To realize chiral recognition on TLM, an excitation beam was phase-modulated at a frequency of 1.2 kHz, and left-circularly polarized light (LCPL) and right-circularly polarized light (RCPL) were generated. Then, the differential light absorption between LCPL and RCPL, which is the CD effect, was detected as thermal lens signal intensity and phase. As a standard sample, optically active tris(ethylenediamine)cobalt(III) [Co(en)(3)]I-3+(3)- aqueous solutions were used for performance evaluations. First, we verified the basic principle for selective chiral analysis by comparing the signals in intensity-modulation and phase-modulation modes of the excitation beam. Also, we found that the g-factor, which is significant for determining enantiomeric excess, agreed well with the value obtained by the CD spectrometer. The limit of detection (LOD) for enantiopure [Co-(en)(3)]I-3+(3)- was 6.3 x 10(-5) M (1.9 X 10(-7) abs) for (-)-Co(en)(3)(3+), and the sensitivity in absorbance units was more than 250 times higher than that in a CD spectrophotometer. Finally, we demonstrated enantiomeric excess determination on a microchip. The LOD was 1.7% (8.5 x 10(-7) abs) for (-)Co(en)(3)(3+) and at least one order superior to the LOD of a CD spectrometer. The applicability of CD-TLM for sensitive chiral analysis on a microchip was verified, and CD-TLM is expected to be promising for microchip-based chiral synthesis and analysis systems.

A new solvent extraction concentration method utilizing microchip technology has been developed. As an important application of this system, carbaryl determination with thermal lens microscope detection was demonstrated. Carbaryl pesticide was hydrolyzed in an alkaline medium to 1-naphthol, was coupled with diazotized trimethylaniline, and, then, was extracted to toluene as a colored azo dye. Two microchips with modified complex-shape microchannels were used for mixing, reaction, extraction, and detection. A thermal lens microscope was used for the detection of the colored product. Optimum conditions for aqueous phase and organic phase flow rates inside the microchannels were identified. The calibration line indicated good linearity for concentrations of 3.4 x 10(-7) to 3.5 x 10(-6) M and a detection limit of 7 x 10(-8) M was obtained. This limit of detection is at least two orders less than LODs for conventional spectrophotometric methods. The results with the present integrated system suggested there was a good potential for implementing an on-site carbaryl analysis system. (c) 2005 Elsevier B.V. All rights reserved.

A reflective thermal lens detection device was developed for realizing a portable and sensitive detector for a microsystem. An aluminum mirror was formed on the main plate of a microchip, and a reflected probe beam was detected with a single pick-up unit. The background signal due to light absorption of the aluminum mirror was 60 times reduced when the microchannel and the mirror were separated with an interval of 600 mm. The tilt angle of the microchip significantly affected the precision of the measurement. Then a quadrant photodiode was used to detect the center of gravity of the reflected probe beam to regulate the tilt angle within +/- 0.05 degrees, and this value was enough to achieve 1% CV (coefficient of variance) precision in the measurements. The limit of detection (LOD) was 60 nM for xylene cyanol solution, and the absorbance was 9.4 x 10(-6) AU. About 40 times higher sensitivity was obtained in comparison with a spectrophotometer.

Thermal lens microscope (TLM) is a kind of absorption spectrophotometry based on photothermal phenomena of non-fluorescent molecules. TLM has high sensitivity (single molecule concentration in fL detection volume) and wide applicability (non-fluorescent molecules). TLM was successfully applied to detection on microchip in clinical diagnosis, environmental analysis, single cell analysis and so on. The basic function of TLM is concentration determination in microspace. In addition, we have realized various functions on TLM for sensitive chiral analysis, individual nanoparticle counting and in situ flow sensing. In this presentation, we explain these functional TLMs for microchip chemistry.

Thermal lens microscope (TLM) is our original sensitive detector for non-fluorescent molecules in microspace. The principle is based on absorption of light followed by photothermal process. TLM has been successfully applied to sensitive detection on microchip, and TLM enabled various applications combined with microchip technologies. We are now developing HPLC microchips as one of the important separation techniques for analysis and synthesis. For HPLC microchip systems, direct and sensitive UV detection on microchip becomes key technology. Therefore, we extended applicability of TLM from visible to UV light absorbing samples by pulse UV laser excitation (UV-TLM). Quasi-continuous wave (QCW) method was applied for lock-in amplifier detection. By applying UV-TLM for biomolecules separation and detection, about two orders of higher sensitivity was achieved compared with UV spectrophotometer. For synthesis on microchip, recognition and detection of chiral samples become important in pharmaceutical field. Therefore, function of TLM was extended for selective detection of chiral samples by utilizing polarization modulation of excitation beam and resultant circular dichroism of sample (CD-TLM). The chirality of samples was detected selectively on microchip with two orders higher sensitivity than CD spectrophotometer. Finally, we explained the instrumentation using fiber optics and micro lens technology for achieving a miniaturized practical device.

Thermal lens microscope (TLM) is an ultra-sensitive method for detecting non-fluorescent samples in microspace. TLM is a kind of optical absorption spectrophotometry with comparable sensitivity to the laser induced fluorescence method. In this presentation, the applicability of TLM was extended from visible to ultraviolet (UV) light absorbing samples by UV laser excitation. The flow velocity was investigated for optimizing the sensitivity. The detection limit of 9.2 x 10-7 in absorbance unit was obtained and about 2 orders of magnitude superior to spectrophotometry: In addition, TLM was further developed for individual nanoparticles counting with visible laser excitation. Individual gold nanoparticles of 15 nm in diameter could be detected, and the detection limit of 5 nm, that was about 1 order lower in diameter than laser scattering method, was obtained.

A portable thermal lens spectrometer with a precise focusing system was developed. Astigmatism of the reflected excitation beam from the microchip was used for depth direction focusing. For width direction focusing, the scattering effect of the transmitted probe beam by a microchannel edge was used. The focusing system was evaluated with a 250 mum wide x 50 mum deep microchannel. Focusing resolutions for depth and width directions were 1 and 10 mum, respectively. The repeatability of the thermal lens signal (40 muM xylenecyanol solution) was proved to be similar to1% coefficient of variance when using these focusing methods. The limit of detection for a xylenecyanol solution was 30 nM, and the absorbance was 4.7 x 10(-6) AU. The sensitivity was 20-100 times higher than that obtained by spectrophotometry. In consequence, a practical thermal lens spectrometer was realized.