Monitoring Charge Flux to Quantify Unusual Ligand-Induced Ion Channel Activity for Use in Biological Nanopore-Based Sensors

The utility of biological nanopores for the development of sensors has become a growing area of interest in analytical chemistry. Their emerging use in chemical analysis is a result of several ideal characteristics. First, they provide reproducible control over nanoscale pore sizes with an atomic level of precision. Second, they are amenable to resistive-pulse type measurement systems when embedded into an artificial lipid bilayer. A single binding event causes a change in the flow of millions of ions across the membrane per second that is readily measured as a change in current with excellent signal-to-noise ratio. To date, ion channel-based biosensors have been limited to well-behaved proteins. Most demonstrations of using ion channels as sensors have been limited to proteins that remain in the open, conducting state, unless occupied by an analyte of interest. Furthermore, these proteins are nonspecific, requiring chemical, biochemical, or genetic manipulations to impart chemical specificity. Here, we report on the use of the pore-forming abilities of heat shock cognate 70 (Hsc70) to quantify a specific analyte. Hsc70 reconstitutes into phospholipid membranes and opens to form multiple conductance states specifically in the presence of ATP. We introduce the measurement of "charge flux" to characterize the ATP-regulated multiconductance nature of Hsc70, which enables sensitive quantification of ATP (100 μM-4 mM). We believe that monitoring protein-induced charge flux across a bilayer membrane represents a universal method for quantitatively monitoring ion-channel activity. This measurement has the potential to broaden the library of usable proteins in the development of nanopore-based biosensors.