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A photo-responsive nanovesicle is fabricated by polyion complex (PIC) formation between poly(ethylene glycol) (PEG)-block-polypeptides and photo-reactive oligodeoxynucleotides (PROs)/anti-sense oligonucleotides (ASOs). The ultraviolet (UV) light triggers reversible crosslinking between PROs and ASOs in the vesicular membrane, providing the nanovesicle with switchable stability under physiological conditions. The resulting nanovesicle allows efficient cellular internalization, leading to significant UV-triggered gene knockdown in cultured cells.

Congenital disorders characterized by the quantitative and qualitative reduction in the number of functional nephrons are the primary cause of chronic kidney disease (CKD) in children. We aimed to describe the alteration of urinary extracellular vesicles (uEVs) associated with decreased renal function during childhood. By nanoparticle tracking analysis and quantitative proteomics, we identified differentially expressed proteins in uEVs in bilateral renal hypoplasia, which is characterized by a congenitally reduced number of nephrons. This expression signature of uEVs reflected decreased renal function in CKD patients by congenital anomalies of the kidney and urinary tract or ciliopathy. As a proof-of-concept, we constructed a prototype ELISA system that enabled the isolation of uEVs and quantitation of expression of molecules representing the signature. The system identified decreased renal function even in its early stage. The uEVs signature could pave the way for non-invasive methods that can complement existing testing methods for diagnosing kidney diseases.

To explore minimally invasive and hypodermic access for in-body sensing, polymer microneedles of 2 mm in length have been studied. Sharper tips are desirable to reduce pain, however, the mechanical strength of such tips is low. To design polymer microneedles that do not plastically deform, the deformation behavior of poly(L-lactic acid) microneedles with different tip diameters was investigated by simulation and experiment. It was found that the plastic deformation of the tip may increase the insertion force. The minimum insertion force was approximately 200 mN for microneedles with a tip diameter of 80 μm, which were inserted without plastic deformation.

In this study, we report an opto-microfluidic platform integrated with dark-field light scattering imaging and fluorescence microspectroscopy to investigate the temperature-dependent behavior of nanoliposomes. This newly developed experimental platform enabled both in situ measurements of the temperature of the sample solution and observation of individual nanoliposomes. As a proof-of-concept, the temperature-dependent interaction between 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) nanoliposomes and a poly(ethylene glycol) (PEG) grafted surface was investigated using this opto-microfluidic platform. Temperature-dependent detachment behavior of DLPC nanoliposomes, which was because of the phase transition of DLPC, was observed. The opto-microfluidic platform for individual nanoparticle measurements may become a powerful tool for investigating temperature-dependent physicochemical properties of nanoparticles in aqueous solution.

The micromolding process using biocompatible thermoplastic polymers is highly attractive as a fabrication process of microdevices in biomedical applications. In this study, we investigated the effect of the thermal history in the micromolding process on the crystallinity of semi-crystalline polymers, such as poly (L-lactic acid) (PLLA), during their crystallization from the amorphous and molten states. In particular, the thermal history in the micromolding process using poly(dimethylsiloxane) replica mold embedded with a thermocouple was recorded. The crystallinity of PLLA constructs fabricated using the micromolding process was measured via wide-angle X-ray scattering, and crystallization kinetics was analyzed based on the Kolmogorov&ndash, Johnson&ndash, Mehl&ndash, Avrami equation. A crystallization rate of k = 0.061 min&minus, n was obtained in the micromolding process of PLLA crystallization from the amorphous state, accompanied by the quenching operation, forming a large number of crystal nuclei. Finally, the fabrication of PLLA microneedles was performed using micromolding processes with different thermal histories. The information about the thermal history during the micromolding process is significant in the development of polymer microdevices to achieve better material properties.

Attoliter-volume and air-sealed reactor array for fast and robust single-molecule enzyme assay., Single-molecule digital enzyme assay using micron-sized droplet array is a promising analysis method to quantify biomolecules at extremely low concentrations. However, multiplex digital enzyme assays are still difficult to access because the best buffer conditions can vary largely among enzymes. In addition, the best conditions for flurogenic compounds to retain high quantum efficiency and to avoid leakage into the oil phase can be also very different. In this study, digital enzyme assay was performed using an array of nanometer-sized droplets of 200 aL volume, termed ‘nanocell’. Due to the small reaction volume, nanocell enhanced the accumulation rate of fluorescent products by a factor of 100 when compared with micron-sized reactors. Nanocell also enabled oil-free sealing of reactors: when flushed with an air flow, nanocell displayed water droplets under air, allowing enzymes to catalyze the reaction at the same rate as in oil-sealed reactors. Dual digital enzyme assay was also demonstrated using β-galactosidase and alkaline phosphatase (ALP) at pH 7.4, which is far from the optimum condition for ALP. Even under such a non-optimum condition, ALP molecules were successfully detected. Nanocell could largely expand the applicability of digital bioassay for enzymes under non-optimum conditions or enzymes of low turnover rate. The sealing of the reactor with air would also expand the applicability, allowing the use of fluorescent dyes that leak into oil.

A novel on-chip integrated high-voltage (HV) generator is realized by our unique MEMS post-processing method with the standard CMOS process on a silicon-on-insulator (SOI). Foundry-made charge-pump elements with standard 5-V transistors on an SOI are isolated with deep reactive ion etching and isotropic etching. This method facilitated the successful operation of a HV-requiring MEMS device, the nominal working voltage of which is greater than 50 V, and by using the silicon device, the driving of an electroosmotic flow micropump was demonstrated. The measured output was proportional to the number of pump stages, and it was observed as 95.2 V with 20 stages. The post-processed CMOS-on-SOI can sustain voltages up to 800 V, and various MEMS with very high voltages can be driven using this technology.

Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy.

The application of chip-based microcapillary electrophoresis (µCE) to determine the electrophoretic mobility of molecules and particles has been intensively studied in the last two decades. Balancing the hydrostatic pressure between both ends of the microchannel is essential for free-zone electrophoresis and highly accurate measurement. This balancing operation appears simple on a macroscale (e.g., > 10−3 m); however, on a microscale (e.g., 10−6–10−3 m), it is not straightforward because of the complexity of the interface dynamics at the meniscus. The hydrostatic pressure flow is unstable because of the small size of the microchannel, which is smaller than a single droplet of water. In this study, a µCE chip design was proposed by adding an extra bypass channel to balance the fluid level of the two open reservoirs and inhibit the generation of hydrostatic pressure flow within the microchannel. The fluid behaviors in the microchannel and current and voltage (I–V) characterization were experimentally studied. In addition, a numerical simulation of the electroosmotic flow and hydrostatic flow in the µCE chip was performed. The comparison between the µCE chip with and without the bypass channel showed that the bypass channel did not produce a disturbance in the microchannel for the electrophoretic measurement. The simple microchannel design enabled autonomous compensation of the hydrostatic pressure from the instability of the meniscus, and thus improved the usability of the chip-based µCE chip and the accuracy in the electrophoretic measurement.

Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA)-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing) a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density), ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era.

We have developed an advanced microintaglio printing method for fabricating fine and high-density micropatterns and applied it to the microarraying of RNA molecules. The microintaglio printing of RNA reported here is based on the hybridization of RNA with immobilized complementary DNA probes. The hybridization was controlled by switching the RNA conformation via the temperature, and an RNA microarray with a diameter of 1.5 µm and a density of 40,000 spots/mm(2) with high contrast was successfully fabricated. Specifically, no size effects were observed in the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude. Additionally, we have developed a microintaglio printing method for transcribed RNA microarrays on demand using DNA-immobilized magnetic beads. The beads were arrayed on wells fabricated on a printing mold and the wells were filled with in vitro transcription reagent and sealed with a DNA-immobilized glass substrate. Subsequently, RNA was in situ synthesized using the bead-immobilized DNA as a template and printed onto the substrate via hybridization. Since the microintaglio printing of RNA using DNA-immobilized beads enables the fabrication of a microarray of spots composed of multiple RNA sequences, it will be possible to screen or analyze RNA functions using an RNA microarray fabricated by temperature-controlled microintaglio printing (TC-µIP).

A microcapillary chip-based particle electrophoresis system developed for characterizing extracellular vesicles (EVs) is described. So far, it is technologically difficult to analyze or identify a heterogeneous population of particles ranging from several tens to one hundred nanometers, and hence, there is a growing demand for a new analytical method of nanoparticles among researchers working on extracellular vesicles. The analytical platform presented in this chapter allows detection of individual nanoparticles or nanovesicles of less than 50 nm in diameter and enables the characterization of nanoparticles based on multiple indexes such as concentration, diameter, zeta potential, and surface antigenicity. This platform will provide a useful and easy-to-use solution for obtaining both quantitative and qualitative information on EV samples used in research and development of exosome biology and medicine.

In this review, we discuss the progress of emerging dry processes for nanoscale fabrication. Experts in the fields of plasma processing have contributed to addressing the increasingly challenging demands in achieving atomic-level control of material selectivity and physicochemical reactions involving ion bombardment. The discussion encompasses major challenges shared across the plasma science and technology community. Focus is placed on advances in the development of fabrication technologies for emerging materials, especially metallic and intermetallic compounds and multiferroic, and two-dimensional (2D) materials, as well as state-of-the-art techniques used in nanoscale semiconductor manufacturing with a brief summary of future challenges.

A microcapillary chip-based particle electrophoresis system developed for characterizing extracellular vesicles (EVs) is described. So far, it is technologically difficult to analyze or identify a heterogeneous population of particles ranging from several tens to one hundred nanometers, and hence, there is a growing demand for a new analytical method of nanoparticles among researchers working on extracellular vesicles. The analytical platform presented in this chapter allows detection of individual nanoparticles or nanovesicles of less than 50 nm in diameter and enables the characterization of nanoparticles based on multiple indexes such as concentration, diameter, zeta potential, and surface antigenicity. This platform will provide a useful and easy-to-use solution for obtaining both quantitative and qualitative information on EV samples used in research and development of exosome biology and medicine.

Arraying individual extracellular vesicles (EVs) on a chip is expected one of the promising approaches for investigating their inherent properties. In this study, we immobilized individual EVs on a surface using a nanopatterned tethering chip-based versatile platform. A microfluidic device was used to ensure soft, reproducible exposure of the EVs over the whole chip surface. The device is incorporated with a high-density nanoarray chip patterned with 200-nm diameter nanospots composed of polyethylene glycol (PEG)-lipid conjugate brushes. We present a procedure adopted for fabricating high-density PEG-lipid modified nanospots (200 nmϕ, 5.0 × 105 spots/mm2 in 2 × 2 mm2 area). This procedure involves nanopatterning using electron beam lithography, followed by multistep selective chemical modification. Aqueous treatment of a silane coupling agent, used as a linker between PEG-lipid molecules and the silicon surface, was the key step that enabled surface modification using a nanopatterned resist film as a mask. The nanoarray chip was removed from the device for subsequent measurements such as atomic force microscopy (AFM). We developed a prototype device and individually immobilized EVs derived from different cell lines (Sk-Br-3 and HEK293) on tethering nanospots. We characterized EV's morphology using AFM and showed the possibility of evaluating the deformability of EVs using the aspect ratio as an indicator.