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.

Ubiquitous element approach to plasmonic enhanced photocatalytic water splitting: the case of Ti@TiO2 core-shell nanostructure.

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 Ti-rich oxide layer at the Ti/TiO2 interface and UV light absorption in the nanotubular TiO2 shell.

Enzyme-linked immunosorbent assay using thin-layered microfluidics with perfect capture of the target protein.

We developed a process for enzyme-linked immunosorbent assay on a glass microchip via the use of a thin-layered microfluidic channel. This channel possesses a high aspect ratio (width/depth ∼200) and has an antibody layer immobilized directly on the channel surface. A depth of several microns and an excessive width and length (mm scale) of the channel provide a large-volume capacity (102 nL) and maximum capture efficiency of the analyte for a high level of detection sensitivity (102 pg mL-1). The developed reusable immunosensor has demonstrated high-performance characteristics by requiring less than 50 µL of sample and providing analysis in less than 25 min. This new method could impact the development of point-of-care devices for biomedical applications.

Nanofluidic analytical system integrated with nanochannel open/close valves for enzyme-linked immunosorbent assay.

There have been significant advances in the field of nanofluidics, and novel technologies such as single-cell analysis have been demonstrated. Despite the evident advantages of nanofluidics, fluid control in nanochannels for complicated analyses is extremely difficult because the fluids are currently manipulated by maintaining the balance of driving pressure. To address this issue, the use of valves will be essential. Our group previously developed a nanochannel open/close valve utilizing glass deformation, but this has not yet been integrated into nanofluidic devices for analytical applications. In the present study, a nanofluidic analytical system integrated with multiple nanochannel open/close valves was developed. This system consists of eight pneumatic pumps, seven nanochannel open/close valves combined with piezoelectric actuators, and an ultra-high sensitivity detector for non-fluorescent molecules. For simultaneous actuation of multiple valves, a device holder was designed that prevented deformation of the entire device caused by operating the valves. A system was subsequently devised to align each valve and actuator with a precision of better than 20 µm to permit the operation of valves. The developed analytical system was verified by analyzing IL-6 molecules using an enzyme-linked immunosorbent assay. Fluid operations such as sample injection, pL-level aliquot sampling and flow switching were accomplished in this device simply by opening/closing specific valves, and a sample consisting of approximately 1500 IL-6 molecules was successfully detected. This study is expected to significantly improve the usability of nanofluidic analytical devices and lead to the realization of sophisticated analytical techniques such as single-cell proteomics.

Metal-Free Fabrication of Fused Silica Extended Nanofluidic Channel to Remove Artifacts in Chemical Analysis.

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.

Surface Patterning of Closed Nanochannel Using VUV Light and Surface Evaluation by Streaming Current.

In nanofluidics, surface control is a critical technology because nanospaces are surface-governed spaces as a consequence of their extremely high surface-to-volume ratio. Various surface patterning methods have been developed, including patterning on an open substrate and patterning using a liquid modifier in microchannels. However, the surface patterning of a closed nanochannel is difficult. In addition, the surface evaluation of closed nanochannels is difficult because of a lack of appropriate experimental tools. In this study, we verified the surface patterning of a closed nanochannel by vacuum ultraviolet (VUV) light and evaluated the surface using streaming-current measurements. First, the C18 modification of closed nanochannels was confirmed by Laplace pressure measurements. In addition, no streaming-current signal was detected for the C18-modified surface, confirming the successful modification of the nanochannel surface with C18 groups. The C18 groups were subsequently decomposed by VUV light, and the nanochannel surface became hydrophilic because of the presence of silanol groups. In streaming-current measurements, the current signals increased in amplitude with increasing VUV light irradiation time, indicating the decomposition of the C18 groups on the closed nanochannel surfaces. Finally, hydrophilic/hydrophobic patterning by VUV light was performed in a nanochannel. Capillary filling experiments confirmed the presence of a hydrophilic/hydrophobic interface. Therefore, VUV patterning in a closed nanochannel was demonstrated, and the surface of a closed nanochannel was successfully evaluated using streaming-current measurements.

Fabrication of Infrared-Compatible Nanofluidic Devices for Plasmon-Enhanced Infrared Absorption Spectroscopy.

Nanofluidic devices have offered us fascinating analytical platforms for chemical and bioanalysis by exploiting unique properties of liquids and molecules confined in nanospaces. The increasing interests in nanofluidic analytical devices have triggered the development of new robust and sensitive detection techniques, especially label-free ones. IR absorption spectroscopy is one of the most powerful biochemical analysis methods for identification and quantitative measurement of chemical species in the label-free and non-invasive fashion. However, the low sensitivity and the difficulties in fabrication of IR-compatible nanofluidic devices are major obstacles that restrict the applications of IR spectroscopy in nanofluidics. Here, we realized the bonding of CaF2 and SiO2 at room temperature and demonstrated an IR-compatible nanofluidic device that allowed the IR spectroscopy in a wide range of mid-IR regime. We also performed the integration of metal-insulator-metal perfect absorber metamaterials into nanofluidic devices for plasmon-enhanced infrared absorption spectroscopy with ultrahigh sensitivity. This study also shows a proof-of-concept of the multi-band absorber by combining different types of nanostructures. The results indicate the potential of implementing metamaterials in tracking several characteristic molecular vibrational modes simultaneously, making it possible to identify molecular species in mixture or complex biological entities.

Detachable glass micro/nanofluidic device.

Glass is one of the most ideal materials for micro/nanofluidic devices due to its excellent optical transparency, resistance to a wide range of solvents and reagents, and easy to modify surfaces by silane-coupling reagents. From a practical point of view, glass is a hard material and is suitable for real applications. One of the advantages of glass is its reusability; however, this reusability is difficult to realize in certain conditions. Washing or re-modification of micro/nanofluidic channels is sometimes difficult due to the ultrasmall space in these channels. If the glass devices are detachable, it is easy to access the channel surface, and the channels can be cleaned and re-modified. When the substrates are bonded again, the devices are fabricated easily without repeating laborious and expensive micro/nano-fabrication processes. This technology gives researchers and users a choice of glass substrates in fundamental research studies and real-time applications. In this study, we propose a detachable glass micro/nanofluidic device by our low temperature bonding method. The surface bonding energy is controlled to realize both high pressure capacity for micro/nanofluidics and easy separation of glass substrates without fracturing. As a result, at least four times detaching and bonding is confirmed.

Parallel multiphase nanofluidics utilizing nanochannels with partial hydrophobic surface modification and application to femtoliter solvent extraction.

In the field of microfluidics, utilizing parallel multiphase flows with immiscible liquid/liquid or gas/liquid interfaces along a microchannel has achieved the integration of various chemical processes for analyses and syntheses. Recently, our group has developed nanofluidics that exploits 100 nm nanochannels to realize ultra-small (aL to fL scale) and highly efficient chemical operations. Novel applications such as single-molecule analyses and single-cell omics are anticipated. However, the formation of parallel multiphase flows in a nanochannel remains challenging. To this end, here we developed a novel method for nanoscale partial hydrophobic surface modification of a nanochannel utilizing a focused ion beam. Hydrophobic and hydrophilic areas could be patterned beside one another even in a 60 nm glass nanochannel. Because this patterning maintained the liquid/liquid interface in the nanochannel based on the difference in wettability, stable aqueous/organic parallel two-phase flow in a 40 fL nanochannel was realized for the first time. Utilizing this flow, nanoscale unit operations involving phase confluence, extraction and phase separation were integrated to demonstrate solvent extraction of a lipid according to the Bligh-Dyer method, which is a broadly used pretreatment process in lipidomics. We accomplished the separation of a lipid and an amino acid in a sample volume of 4 fL (250 times smaller than the pL volume of a single cell) with a processing time of 1 ms (10 000 times faster than that in a microchannel). This study therefore provides a technological breakthrough that advances the field of nanofluidics to allow multiphase chemical processing at fL volumes.