Stochastic Sensing of TNT with a Genetically Engineered Pore

Abstract

Engineered versions of the transmembrane protein pore alpha-hemolysin (alphaHL) can be used as stochastic sensing elements for the identification and quantification of a wide variety of analytes at the single-molecule level. Until now, nitroaromatic analytes have eluded detection by this approach. We now report that binding sites for nitroaromatics can be built within the lumen of the alphaHL pore from simple rings of seven aromatic amino acid side chains (Phe, Tyr or Trp). By monitoring the ionic current that passes through a single pore at a fixed applied potential, various nitroaromatics can be distinguished from TNT on the basis of the amplitude and duration of individual current-blocking events. Rings of less than seven aromatics bind the analytes more weakly; this suggests that direct aromatic-aromatic interactions are involved. The engineered pores should be useful for the detection of explosives and, in combination with computational approaches and structural analysis, they could further our understanding of noncovalent interactions between aromatic molecules.

Air-stable droplet interface bilayers on oil-infused surfaces

“…We show that air-stable DIBs still allow for the robust electrical characterization of ion channels inserted in the lipid bilayer. Previously, it has been demonstrated that black lipid membranes or DIBs can be used for biosensing (46)(47)(48)(49)(50), light sensing (26), microscale biobatteries (26), electrical circuits (51,52), and engineering tissue-like material (53). However, these suspended lipid bilayers have always been confined to fluid reservoirs (25,45).…”

Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

“…The versatility and specificity of stochastic sensing have been enhanced by introducing sensing elements (analyte binding sites) into the αHL pore by protein engineering, chemical modification, or the use of adapter molecules (1,4,5). Through such approaches, the stochastic detection of ions (1,6), small molecules (1,7,8), reactive molecules (9,10), and proteinligand interactions has been achieved (1,(11)(12)(13)(14). The stochastic detection of proteins has also been achieved with functionalized solid-state nanopores (15).…”

Engineering of bacterial toxins for research and medicine

“…Such engineered pores can be useful components to build sensors of cell-signaling molecules. It was also reported that binding sites for nitro-aromatics could be built within the lumen of the αHL pore, enabling the sensing of explosives such as TNT [160].…”