Self-Assembly of a Monolayer Graphene Oxide Film Based on Surface Modification of Substrates and its Vapor-Phase Reduction

We have developed a novel method for fabricating a monolayer graphene oxide (GO) film that covers the whole substrate surface or patterned areas by chemically modifying the substrate surface. We discovered that partially surface-bound GO flakes are decomposed into surface-bound flakes and free flakes by sonication, the latter of which can be easily removed. This mechanism enables us to fabricate regularly patterned GO films from irregularly shaped GO flakes. We also found that the morphology and conduction properties of the monolayer GO films are improved by annealing in CH₄ atmosphere

Nanopatterning of Suspended Graphene Films by Local Catalytic Etching Using Atomic Force Microscopy Equipped with an Ag-Coated Probe

We have developed a novel technique for fabrication of nanopores in suspended graphene (SG) films using atomic force microscopy (AFM) equipped with a catalytic Ag-coated probe. By applying voltages between an Ag-coated tip and SG films in tapping mode AFM under ambient conditions at room temperature, Ag nanoparticles on the tip contact to the graphene surface, and simultaneously local Joule heating is generated at the tip–Ag nanoparticle–graphene contact. As a result, the SG films can be etched via oxidation assisted by the heated Ag catalytic nanoparticles, forming a nanopore. Because the hole shape and size on the SG films depend on the shape of the AFM tip, we can regulate the patterns on the graphene films by optional AFM tips

Substrate-Induced Structure and Molecular Dynamics in a Lipid Bilayer Membrane

The solid-substrate-dependent structure and dynamics of molecules in a supported lipid bilayer (SLB) were directly investigated via atomic force microscopy (AFM) and single particle tracking (SPT) measurements. The appearance of either vertical or horizontal heterogeneities in the SLB was found to be strongly dependent on the underlying substrates. SLB has been widely used as a biointerface with incorporated proteins and other biological materials. Both silica and mica are popular substrates for SLB. Using single-molecule dynamics, the fluidity of the upper and lower membrane leaflets was found to depend on the substrate, undergoing coupling and decoupling on the SiO₂/Si and mica substrates, respectively. The anisotropic diffusion caused by the locally destabilized structure of the SLB at atomic steps appeared on the Al₂O₃(0001) substrate because of the strong van der Waals interaction between the SLB and the substrate. Our finding that the well-defined surfaces of mica and sapphire result in asymmetry and anisotropy in the plasma membrane is useful for the design of new plasma-membrane-mimetic systems. The application of well-defined supporting substrates for SLBs should have similar effects as cell membrane scaffolds, which regulate the dynamic structure of the membrane

Fabrication of Au-Nanoparticle-Embedded Lipid Bilayer Membranes Supported on Solid Substrates

We fabricated gold nanoparticle (Au-NP)-embedded supported lipid bilayers (SLBs) by two methods. In the vesicle–vesicle fusion method, vesicles with hydrophobized Au-NPs are ruptured and fused on SiO₂/Si substrates. In the vesicle-membrane fusion method, SLBs without Au-NPs were preformed on the substrate and then vesicles with Au-NPs were fused into the preformed membranes. In the former method, Au-NP incorporation into the SLBs was observed as an increase in the membrane thickness in atomic force microscopy (AFM) images and directly observed by transmission electron microscopy. In the latter method, fusion of vesicles into the preformed membranes was confirmed by the fluorescent color change in the preformed membranes, and Au-NP incorporation was also confirmed by an increase in the membrane thickness in the AFM images. Key techniques for the successful vesicle-membrane fusion are hydrophobization of Au-NPs, approach control of vesicles by mixing the charged lipids, and destabilization of the lipid bilayers by adding lipids with a small polar headgroup

Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning

Exosomes are extracellular nanovesicles released from any cells and found in any body fluid. Because exosomes exhibit information of their host cells (secreting cells), their analysis is expected to be a powerful tool for early diagnosis of cancers. To predict the host cells, we extracted multidimensional feature data about size, shape, and deformation of exosomes immobilized on solid surfaces by atomic force microscopy (AFM). The key idea is combination of support vector machine (SVM) learning for individual exosome particles and their interpretation by principal component analysis (PCA). We observed exosomes derived from three different cancer cells on SiO₂/Si, 3-aminopropyltriethoxysilane-modified-SiO₂/Si, and TiO₂ substrates by AFM. Then, 14-dimensional feature vectors were extracted from AFM particle data, and classifiers were trained in 14-dimensional space. The prediction accuracy for host cells of test AFM particles was examined by the cross-validation test. As a result, we obtained prediction of exosome host cells with the best accuracy of 85.2% for two-class SVM learning and 82.6% for three-class one. By PCA of the particle classifiers, we concluded that the main factors for prediction accuracy and its strong dependence on substrates are incremental decrease in the PCA-defined aspect ratio of the particles with their volume

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO₂/Si₃N₄ septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO₂/Si₃N₄ septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins