Local nano-electrode fabrication utilizing nanofluidic and nano-electrochemical control.

Miniaturized systems have attracted much attention with the recent advances in microfluidics and nanofluidics. From the capillary electrophoresis, the development of glass-based microfluidic and nanofluidic technologies has supported advances in microfluidics and nanofluidics. Most microfluidic systems, especially nanofluidic systems, are still simple, such as systems constructed with simple straight nanochannels and bulk-scale electrodes. One of the bottlenecks to the development of more complicated and sophisticated systems is to develop the locally integrated nano-electrodes. However, there are still issues with integrating nano-electrodes into nanofluidic devices because it is difficult to fit the nano-electrode size into a nanofluidic channel at the nanometer level. In this study, we propose a new method for the fabrication of local nano-electrodes in nanofluidic devices with nanofluidic and nano-electrochemistry-based experiments. An electroplating solution was introduced to a nanochannel with control of the flow and the electroplating reaction, by which nano-electrodes were successfully fabricated. In addition, a nanofluidic device was available for nanofluidic experiments with the application of 200 kPa. This method can be applied to any electroplating material such as gold and copper. The local nano-electrode will make a significant contribution to the development of more complicated and sophisticated nanofluidic electrophoresis systems and to local electric detection methods for various nanofluidic devices.

Femtoliter-Droplet Mass Spectrometry Interface Utilizing Nanofluidics for Ultrasmall and High-Sensitivity Analysis.

In the fields of biology and medicine, comprehensive protein analysis at the single-cell level utilizing mass spectrometry (MS) with pL sample volumes and zmol to amol sensitivity is required. Our group has developed nanofluidic analytical pretreatment methods that exploit nanochannels for downsizing chemical unit operations to fL-pL volumes. In the field of analytical instruments, mass spectrometers have advanced to achieve ultrahigh sensitivity. However, a method to interface between fL-pL pretreatments and mass spectrometers without sample loss and dispersion is still challenging. In this study, we developed an MS interface utilizing nanofluidics to achieve high-sensitivity detection. After charging analyte molecules by an applied voltage through an electrode, the liquid sample was converted to fL droplets by a nanofluidic device. Considering the inertial force that acts on the droplets, the droplets were carried with a controlled trajectory, even in turbulent air flow, and injected into a mass spectrometer with 100% efficiency. A module for heat transfer was designed and constructed, by which all of the injected droplets were vaporized to produce gas-phase ions. The detection of caffeine ions was achieved at a limit of detection of 1.52 amol, which was 290 times higher than a conventional MS interface by electrospray ionization with sample dispersion combined with a similar mass spectrometer. Therefore, sensitivity that was 2 orders of magnitude higher could be realized due to the 100% sample injection rate. The present study provides a new methodology for the analysis of ultrasmall samples with high-sensitivity, such as protein molecules produced from a single cell.

Kinetics of Enzymatic Reactions at the Solid/Liquid Interface in Nanofluidic Channels.

Nanostructures can realize highly efficient reactions due to their structural advantages. However, the mechanism of accelerating enzyme reactions in a nanospace is still unknown from a kinetic perspective because it is difficult to control a well-defined nanospace, enzyme density, and reaction time. Here, we investigated kinetic parameters of an immobilized enzyme in micro- and nanochannels using nanofabrication, partial enzyme patterning, fluidic control, and a high sensitivity detection system. Devices with channel depths of 300 nm, 4.4 µm, and 13.6 µm were fabricated. Kinetic parameters were determined by the Michaelis-Menten model. Compared to the bulk reaction, all kcats for immobilized enzyme reactors were decreased, although the kcats were approximately the same for the immobilized enzyme reactors of different depths. An ultrafast enzyme reaction could overcome the drawback due to immobilization by an increase of the apparent [E]0 due to the decreased channel depth.

Super-Resolution Defocusing Nanoparticle Image Velocimetry Utilizing Spherical Aberration for Nanochannel Flows.

Understanding fluid flows and mass transport in nanospaces is becoming important with recent advances in nanofluidic analytical devices utilizing nanopores and nanochannels. In the present study, we developed a super-resolution and fast particle tracking method utilizing defocusing images with spherical aberration and demonstrated the measurement of nanochannel flow. Since the spherical aberration generates the defocusing nanoparticle image with diffraction rings, the position of fluorescent nanoparticles was determined from the radius of the diffraction ring. Effects of components of an optical system on the diffraction ring of the defocusing image were investigated and optimized to achieve the spatial resolution exceeding the optical diffraction limit. We found that there is an optimal magnitude of spherical aberration to enhance the spatial resolution. Furthermore, we confirmed that nanoparticles with diameters in the order of 101 nm, which is much smaller than the light wavelength, do not affect the defocusing images and the spatial resolution because such nanoparticles can be regarded as point light sources. At optimized conditions, we achieved a spatial resolution of 19 nm and a temporal resolution of 160 µs, which are sufficient for the nanochannel flow measurements. We succeeded in the measurement of pressure-driven flow in a nanochannel with a depth of 370 nm using 67 nm fluorescent nanoparticles. The measured nanoparticle velocities exhibited a parabolic flow profile with a slip velocity even at the hydrophilic glass surface but with an average velocity similar to the Hagen-Poiseuille law. The method will accelerate researches in the nanofluidics and other related fields.

Picoliter enzyme reactor on a nanofluidic device exceeding the bulk reaction rate.

Single-cell analyses have recently become important to understand cell heterogeneity, the mechanism of cell function, and diseases. In contrast to single-cell analyses that target nucleic acids, single-cell protein analyses still pose challenges. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale with femtoliter-picoliter volumes which are smaller than the volume of a single cell exploring ultimate analytical performances (e.g. single-cell target proteomics). However, single-cell shotgun proteomics, which is used to analyze even unknown proteins, is still challenging because there is no digestion column with picoliter volume. The issues were long reaction time (overnight) and much larger reaction volume (microliter) in the conventional bulk method. In this study, an ultra-fast picoliter enzyme reactor using a nanochannel was developed. A device with a channel depth of 300 nm and a volume of 32.4 pL was fabricated. To prevent the self-digestion of trypsin (enzyme), the picoliter enzyme reactor was prepared by immobilizing trypsinogen which was activated to trypsin by enterokinase. The enzyme density obtained by the trypsinogen immobilization process was 2.5 times higher than that obtained by the conventional trypsin immobilization process. Furthermore, the apparent enzyme concentration was 36 times higher due to an extremely high surface-to-volume ratio of the nanochannel, compared to the limit concentration in the bulk. Finally, the enzyme reaction in the picoliter enzyme reactor was accelerated 25 times compared to that in the bulk. Using the picoliter enzyme reactor, protein solution with picoliter volume will be digested without self-digestion and artificial modification, which will greatly contribute to single-cell shotgun proteomics.

Ultrasensitive detection of nonlabelled bovine serum albumin using photothermal optical phase shift detection with UV excitation.

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.

Femtoliter Volumetric Pipette and Flask Utilizing Nanofluidics.

Microfluidics has achieved integration of analytical processes in microspaces and realized miniaturized analyses in fields such as chemistry and biology. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale exploring ultimate analytical performances (e.g. immunoassay of a single-protein molecule). However, a sampling method is still challenging for nanofluidics despite its importance in analytical chemistry. In this study, we developed a femtoliter (fL) sampling method for volume measurement and sample transport. Traditionally, sampling has been performed using a volumetric pipette and flask. In this research, a nanofluidic device consisting of a femtoliter volumetric pipette and flask was fabricated on glass substrates. Since gravity, which is exploited in bulk fluidic operations, becomes less dominant than surface effects on the nanometer scale, fluidic operation of the femtoliter sampling was designed utilizing surface tension and air pressure control. The working principle of an 11 fL volumetric pipette and a 50 fL flask, which were connected by a nanochannel, was verified. It was found that evaporation of the sample solution by air flow was a significant source of error because of the ultra-small volumes being processed. Thus, the evaporation issue was solved by suppressing the air flow. As a result, the volumetric measurement error was decreased to ±0.06 fL (CV 0.6%), which is sufficiently low for use in nanofluidic analytical applications. This study will present a fundamental technology for the development of novel analytical methods for femtoliter volume samples such as single molecule analyses.

Femtoliter Gradient Elution System for Liquid Chromatography Utilizing Extended Nanofluidics.

A gradient system was developed for the separation of proteins on a femtoliter scale utilizing nanofluidic channels. In the history of chromatography, miniaturization of the separation column has been important for efficient separation and downsizing of instruments. Previously, our group developed a small and highly efficient chromatography system utilizing nanofluidic channels, although a flexible design of the gradient was difficult and separation of proteins was not achieved. Here, we propose a flexible gradient system using standard HPLC pumps and an auxiliary mixer with a simple sample injection system. In contrast to our previous sample injection system using pressure balance, the system enables a femtoliter-scale sample injection which is compatible with gradient elution using HPLC pumps. The system was carefully designed, verified for sample injection and gradient elution, and finally applied to the separation of proteins from model and real samples. This femtoliter-scale, efficient separation system will contribute to omics studies at the single-cell level.

Enzyme-linked immunosorbent assay utilizing thin-layered microfluidics.

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) is required for on-site clinical diagnosis. Previously, a microfluidic ELISA in which antibody-immobilized beads are packed in a microchannel for a high surface-to-volume (S/V) ratio was developed, but utilizing beads led to complicated fluidic operation. Recently, we have reported nanofluidic ELISA that utilizes antibody-immobilized glass nanochannels (102-103 nm) to achieve a high S/V ratio without beads, enabling even single-molecule detection, but it is not applicable to clinical diagnosis owing to its fL sample volume, much smaller than the nL-µL sample volume in clinical diagnosis. Here, we propose an antibody-immobilized, thin-layered microfluidic channel as a novel platform. Based on the method of nanofluidic ELISA, the channel width was expanded from 103 nm to 100 mm to expand the volume of the reaction field to 102 nL, while the channel depth (103 nm) was maintained to retain the high S/V ratio. A device design which incorporates a taper-shaped interface between the thin-layered channel and the microchannel for sample injection was proposed, and the uniform introduction of the sample into the high-aspect-ratio (width/depth ∼ 200) channel was experimentally confirmed. For the proof of concept, a thin-layered ELISA device with the same S/V ratio as the bead-based ELISA format was designed and fabricated. By measuring a standard C-reactive protein solution, the working principle was verified. The limit of detection was 34 ng mL-1, which was comparable to that of bead-based ELISA. We believe that the thin-layered ELISA can contribute to medicine and biology as a novel platform for sensitive and rapid ELISA.

Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices.

Analysis of proteins released from living single cells is strongly required in the fields of biology and medicine to elucidate the mechanism of gene expression, cell-cell communication and cytopathology. However, as living single-cell analysis involves fL sample volumes with ultra-small amounts of analyte, comprehensive integration of entire chemical processing for single cells and proteins into spaces smaller than single cells (pL) would be indispensable to prevent dispersion-associated analyte loss. In this study, we proposed and developed a living single-cell protein analysis device based on micro/nanofluidics and demonstrated analysis of cytokines released from living single B cells by enzyme-linked immunosorbent assay. Based on our integration method and technologies including top-down nanofabrication, surface modifications and pressure-driven flow control, we designed and prepared the device where pL-microfluidic- and fL-nanofluidic channels are hierarchically allocated for cellular and molecular processing, respectively, and succeeded in micro/nanofluidic control for manipulating single cells and molecules. 13-unit operations for pL-cellular processing including single-cell trapping and stimulation and fL-molecular processing including fL-volumetry, antigen-antibody reactions and detection were entirely integrated into a microchip. The results suggest analytical performances for countable interleukin (IL)-6 molecules at the limit of detection of 5.27 molecules and that stimulated single B cells secrete 3.41 IL-6 molecules per min. The device is a novel tool for single-cell targeted proteomics, and the methodology of device integration is applicable to other single-cell analyses such as single-cell shotgun proteomics. This study thus provides a general approach and technical breakthroughs that will facilitate further advances in micro/nanofluidics, single-cell life science research, and other fields.

A single-molecule ELISA device 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.

A Photothermal Spectrometer for Fast and Background-Free Detection of Individual Nanoparticles in Flow.

Sensitive detection and quantification of individual plasmonic nanoparticles is critical in a range of applications in the biological, nanomaterials, and analytical sciences. Although a wide range of techniques can be applied to the analysis of immobilized particles, high-throughput analysis of nanoscale species in flow is surprisingly underdeveloped. To address this shortcoming, we present an ultrasensitive, background-free technique based on the photothermal effect and termed differential detection photothermal interferometry (DDPI). We show, both theoretically and experimentally, that DDPI can specifically extract either the phase or amplitude of a photothermal signal. We then quantitatively detect 10 and 20 nm diameter gold nanoparticles at femtomolar concentrations and at linear flow speeds of 10 mm/s. In the case of 50 nm gold particles, we operate at an even higher linear flow speed of 100 mm/s, corresponding to an analyzed volume of more than 1 nL/s. This allows quantification of particle content at attomolar to femtomolar concentrations and counting rates between 0.1 and 400 particles per second. Finally, we confirm that the signal follows the size-dependent variations predicted by Mie theory.

Thermo-optical Characterization of Photothermal Optical Phase Shift Detection in Extended-Nano Channels and UV Detection of Biomolecules.

The expansion of microfluidics research to nanofluidics requires 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 (UV) 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 the 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 µM (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.

Integrated Microfluidic Platform with Multiple Functions To Probe Tumor-Endothelial Cell Interaction.

Interaction between tumor and endothelial cells could affect tumor growth and progression and induce drug resistance during cancer therapy. Investigation of tumor-endothelial cell interaction involves cell coculture, protein detection, and analysis of drug metabolites, which are complicated and time-consuming. In this work, we present an integrated microfluidic device with three individual components (cell coculture component, protein detection component, and pretreatment component for drug metabolites) to probe the interaction between tumor and endothelial cells. Cocultured cervical carcinoma cells (CaSki cells) and human umbilical vein endothelial cells (HUVECs) show higher resistance to chemotherapeutic agents than single-cultured cells, indicated by higher cell viability, increased expression of angiogenic proteins, and elevated level of paclitaxel metabolites under coculture conditions. This integrated microfluidic platform with multiple functions facilitates understanding of the interaction between tumor and endothelial cells, and it may become a promising tool for drug screening within an engineered tumor microenvironment.

From Extended Nanofluidics to an Autonomous Solar-Light-Driven Micro Fuel-Cell Device.

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 (µFC) and a photocatalytic micro fuel generator (µFG) integrated on a single microfluidic chip. The µFG produces hydrogen by photocatalytic water splitting under solar light. The hydrogen fuel is then consumed by the µFC to generate electricity. Importantly, the by-product water returns back to the photocatalytic µFG 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 µFG/µFC source achieves remarkable energy density of ca. 17.2 mWh cm-2 at room temperature.

On-Chip Step-Mixing in a T-Nanomixer for Liquid Chromatography in Extended-Nanochannels.

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 101-103 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 fluorescently labeled amino acids were separated in 16 s, followed by separation of 17 labeled amino acids in only 50 s with a plate height for most of the peaks of less than 1 µm.

Femtoliter high-performance liquid chromatography using extended-nano channels.

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 × 106 plates per m. However, the real theoretical plate number was limited to 103 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 × 104, 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.

Behavior of nanoparticles in extended nanospace measured by evanescent wave-based particle velocimetry.

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.

Dielectric constant of liquids confined in the extended nanospace measured by a streaming potential method.

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.

Extended-nanofluidics: fundamental technologies, unique liquid properties, and application in chemical and bio analysis methods and devices.

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.