The energetics of silent translocation through a biological nanopore.

Abstract

Electrophoretically driving molecules through the lumen of a nanopore can produce transient channel conductance fluctuations by occlusion of ionic flow. However, many molecules do not possess the necessary size, shape, charge, or binding affinity to produce a detectable electrical signature. For cases where transport is quick (e.g., <20 μs), or transport is exceedingly rare, or critical interaction with the pore is lacking, the resulting current recordings appear silent. Within the performance range of common electrophysiological amplifiers, these recordings show no excess noise and no momentary electrical blockage. In general, there is no electrical way to discern if translocation occurs. Here, we report on the electrically silent passage of fluorescent dye molecules through the alpha hemolysin (αHL) nanopore. Simultaneous single-molecule fluorescence and electrical measurements in bilayers formed on microcavity electrode arrays (MECA-opto chips) demonstrate that translocation does, indeed, take place, albeit infrequently. We describe the methodology for detecting fluorophore transport, report observations of silent translocations as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurements of the translocation rate as a function of applied potential permit estimation of the energy barrier for transport through the pore, as well as the effective charge on the dye. The energetics of silent translocation can only be revealed using measurement techniques that complement traditional electrical recording.