Nanopore based detection of Bacillus thuringiensis HD-73 spores using aptamers and versatile DNA hairpins.

A versatile nanopore sensing platform to detect any aptamer using nanopores by designing DNA hairpins that are complementary to the aptamer is described. This platform can detect the presence of aptamer binding targets regardless of their size, which has been a major hurdle for nanopore detection systems. Moreover, the signal-to-noise ratio is increased by eliminating most of the unwanted substances from the sample via simple sample preparation steps. To detect Bacillus thuringiensis HD-73 spores using this sensing platform, DNA hairpins complementary to the target-specific aptamers were designed, and the hairpins were characterized using alpha-hemolysin nanopores after the reaction of spores and aptamers and subsequent reaction with the complementary DNA hairpins. The platform exhibited a detection limit as low as 1.2 × 101 CFU mL-1 and was compatible with a wide range of spore concentrations from 1.2 × 101 CFU mL-1 to 1.2 × 106 CFU mL-1 while it is still expandable to higher spore concentrations.

Automated lipid membrane formation using a polydimethylsiloxane film for ion channel measurements.

A black lipid membrane (BLM) is a powerful platform for studying the electrophysiology of cell membranes as well as transmembrane proteins. However, BLMs have disadvantages in terms of stability, accessibility, and transportability, which preclude their industrial applications. To resolve these issues, frozen membrane precursor (MP) was devised to improve the transportability and storability of BLMs. As described previously, MP is a storable and transportable platform that can be delivered to the point-of-use, where BLMs are automatically formed upon thawing at room temperature. However, MP has an inconsistent thinning-out time, ranging from 30 min to 24 h, as well as a low success rate of BLM formation (~27%), which make it undesirable for practical use. In our study, polydimethylsiloxane (PDMS) was introduced as a replacement for conventionally used Teflon film to control thinning-out time. As such, we used a PDMS thin-film, a porous-structured hydrophobic polymer, and squalene, a high viscosity solvent, to facilitate membrane formation, whereas the absorption rates of solvents were controlled to achieve consistent BLM formation time. We successfully reduced thinning-out time down to <1 h as well as enhanced the success rate of BLM formation to greater than 80%. Moreover, we demonstrated the feasibility of our platform for use in drug screening using gramicidin A and guanidine.