Simple Fabrication of Solid-State Nanopores on a Carbon Film.

Solid-state nanopores are widely used as a platform for stochastic nanopore sensing because they can provide better robustness, controllable pore size, and higher integrability than biological nanopores. However, the fabrication procedures, including thin film preparation and nanopore formation, require advanced micro-and nano-fabrication techniques. Here, we describe the simple fabrication of solid-state nanopores in a commercially available material: a flat thin carbon film-coated micro-grid for a transmission electron microscope (TEM). We attempted two general methods for nanopore fabrication in the carbon film. The first method was a scanning TEM (STEM) electron beam method. Nanopores were fabricated by irradiating a focused electron beam on the carbon membrane on micro-grids, resulting in the production of nanopores with pore diameters ranging from 2 to 135 nm. The second attempt was a dielectric breakdown method. In this method, nanopores were fabricated by applying a transmembrane voltage of 10 or 30 V through the carbon film on micro-grids. As a result, nanopores with pore diameters ranging from 3.7 to 1345 nm were obtained. Since these nanopores were successfully fabricated in the commercially available carbon thin film using readily available devices, we believe that these solid-state nanopores offer great utility in the field of nanopore research.

Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy.

Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.

Biological Nanopore Probe: Probing of Viscous Solutions in a Confined Nanospace.

This paper describes a nanospace probing system constructed with a pore-forming toxin and a hairpin DNA (hpDNA) molecule. The single hpDNA molecule can be inserted and can move in the confined nanospace of the alpha-hemolysin (αHL) pore. The molecular motion of the hpDNA can be determined based on the fluctuation of the blocking current via channel current measurements. Using this system, we investigated the effect of viscosity of the aqueous solution in the macrospace (bulk) and in the confined nanospace with a small molecule (glycerol) and a polymer (PEG600). The molecular motion of the hpDNA in the nanospace differed in glycerol and PEG600 solutions, while the viscosity remained the same in the bulk solution. The fundamental factors for the viscosity in glycerol and PEG600 solutions are hydrogen bonding and the entanglement of polymer chains, respectively. This difference in factors becomes significant in confined nanospaces, and our system allows us to observe its effect. Additionally, we constructed a spatially resolved nanopore probe integrated into a gold nanoneedle. The αHL-hpDNA nanoprobe system was constructed with the nanoneedle and can be used to monitor the nanospace with nanometer spatial resolution.

Analysis of Pore Formation and Protein Translocation Using Large Biological Nanopores.

This paper describes the analysis of pore formation and detection of a single protein molecule using a large nanopore among five different pore-forming proteins. We demonstrate that the identification of appropriate pores for nanopore sensing can be achieved by classifying the channel current signals and performing noise analysis. Through these analyses, we selected a perforin nanopore from the membrane attack complex/perforin superfamily and attempted to use it to detect the granzyme B protein, a serine protease. As a result, we found that granzyme B might pass through the perforin nanopore if it adopts an unfolded structure. Our proposed analytical approach should be useful for exploring several types of nanopore as large biological nanopores other than α-hemolysin.