Urinary extracellular vesicles signature for diagnosis of kidney disease.

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.

Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes.

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.

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules.

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).

Suppression of thermally activated carrier transport in atomically thin MoS2 on crystalline hexagonal boron nitride substrates.

We present the temperature-dependent carrier mobility of atomically thin MoS2 field-effect transistors on crystalline hexagonal boron nitride (h-BN) and SiO2 substrates. Our results reveal distinct weak temperature dependence of the MoS2 devices on h-BN substrates. The room temperature mobility enhancement and reduced interface trap density of the single and bilayer MoS2 devices on h-BN substrates further indicate that reducing substrate traps is crucial for enhancing the mobility in atomically thin MoS2 devices.

Quantitative Raman spectrum and reliable thickness identification for atomic layers on insulating substrates.

We demonstrate the possibility in quantifying the Raman intensities for both specimen and substrate layers in a common stacked experimental configuration and, consequently, propose a general and rapid thickness identification technique for atomic-scale layers on dielectric substrates. Unprecedentedly wide-range Raman data for atomically flat MoS(2) flakes are collected to compare with theoretical models. We reveal that all intensity features can be accurately captured when including optical interference effect. Surprisingly, we find that even freely suspended chalcogenide few-layer flakes have a stronger Raman response than that from the bulk phase. Importantly, despite the oscillating intensity of specimen spectrum versus thickness, the substrate weighted spectral intensity becomes monotonic. Combined with its sensitivity to specimen thickness, we suggest this quantity can be used to rapidly determine the accurate thickness for atomic layers.

Higher cell stiffness indicating lower metastatic potential in B16 melanoma cell variants and in (-)-epigallocatechin gallate-treated cells.

PURPOSE: To understand how nanomechanical stiffness affects metastatic potential, we studied the relationship between cell migration, a characteristic of metastasis, and cell stiffness using atomic force microscopy (AFM), which can measure stiffness (elasticity) of individual living cells. METHODS: Migration and cell stiffness of three metastatic B16 melanoma variants (B16-F10, B16-BL6, and B16-F1 cells), and also effects of (-)-epigallocatechin gallate (EGCG), were studied using Transwell assay and AFM. RESULTS: Migration of B16-F10 and B16-BL6 cells was 3 and 2 times higher than that of B16-F1 cells in Transwell assay, and cell stiffness determined by AFM was also different among the three variants, although they have similar morphologies and the same growth rates: Means of Young's modulus were 350.8 ± 4.8 Pa for B16-F10 cells, 661.9 ± 16.5 Pa for B16-BL6 cells, and 727.2 ± 13.0 Pa for B16-F1 cells. AFM measurements revealed that highly motile B16-F10 cells have low cell stiffness, and low motile and metastatic B16-F1 cells have high cell stiffness: Nanomechanical stiffness is inversely correlated with migration potential. Treatment of highly motile B16-F10 cells with EGCG increased cell stiffness 2-fold and inhibited migration of the cells. CONCLUSIONS: Our study with AFM clearly demonstrates that cell stiffness is a reliable quantitative indicator of migration potential, and very likely metastatic potential, even in morphologically similar cells. And increased cell stiffness may be a key nanomechanical feature in inhibition of metastasis.

A quadruple-scanning-probe force microscope for electrical property measurements of microscopic materials.

Four-terminal electrical measurement is realized on a microscopic structure in air, without a lithographic process, using a home-built quadruple-scanning-probe force microscope (QSPFM). The QSPFM has four probes whose positions are individually controlled by obtaining images of a sample in the manner of atomic force microscopy (AFM), and uses the probes as contacting electrodes for electrical measurements. A specially arranged tuning fork probe (TFP) is used as a self-detection force sensor to operate each probe in a frequency modulation AFM mode, resulting in simultaneous imaging of the same microscopic feature on an insulator using the four TFPs. Four-terminal electrical measurement is then demonstrated in air by placing each probe electrode in contact with a graphene flake exfoliated on a silicon dioxide film, and the sheet resistance of the flake is measured by the van der Pauw method. The present work shows that the QSPFM has the potential to measure the intrinsic electrical properties of a wide range of microscopic materials in situ without electrode fabrication.

Angled long tip to tuning fork probes for atomic force microscopy in various environments.

We expand the range of applications of a tuning fork probe (TFP) in frequency-modulation atomic force microscopy (FM-AFM) by attaching a long metal tip at a certain angle. By the combined flexure of the metal tip and the tuning fork prong, this TFP can change the direction of the detectable force by switching the resonance frequency, which has not been realized with conventional TFPs with short tips. The oscillatory behavior of the tip apex of the TFP is predicted by computer simulations and is experimentally confirmed with scanning electron microscope. FM-AFM operations using this TFP are performed in various environments, i.e., in ultrahigh vacuum, air, and water. FM-AFM images obtained at an atomic step of highly oriented pyrolytic graphite in air show a clear difference depending on the excitation frequency. It is also revealed that the higher order flexural modes of this TFP are advantageous for FM-AFM in water due to the reduction in the degree of hydrodynamic damping.

Multiple-scanning-probe tunneling microscope with nanoscale positional recognition function.

Over the past decade, multiple-scanning-probe microscope systems with independently controlled probes have been developed for nanoscale electrical measurements. We developed a quadruple-scanning-probe tunneling microscope (QSPTM) that can determine and control the probe position through scanning-probe imaging. The difficulty of operating multiple probes with submicrometer precision drastically increases with the number of probes. To solve problems such as determining the relative positions of the probes and avoiding of contact between the probes, we adopted sample-scanning methods to obtain four images simultaneously and developed an original control system for QSPTM operation with a function of automatic positional recognition. These improvements make the QSPTM a more practical and useful instrument since four images can now be reliably produced, and consequently the positioning of the four probes becomes easier owing to the reduced chance of accidental contact between the probes.