Quantifying Short-Lived Events in Multi-State Ionic Channel Measurements

Abstract

We present a new technique that considerably improves the resolution and accuracy of single molecule measurements with nanopores. Molecular interactions with nanopores are characterized by electrical measurements of discrete changes in the channel conductance. By representing physical components of the system with electrical equivalents, we used circuit theory to model response of the system to a stimulus (e.g., a molecule entering the channel). This allowed us to characterize short-lived events where the ionic current does not reach a steady state value, and were previously not analyzed. Applying this technique to measurements of poly(ethylene glycol) (PEG) molecules with the a-hemolysin (aHL) nanopore resulted in remarkable improvements in accuracy and the number of detected events. When measuring polydisperse PEG (mean molecular weights of 400 g/mol and 600 g/mol), the new method recovered z 18-fold more events per unit time, compared with existing techniques, and discriminated molecules with as few as 8 monomers (PEG8). We validated the measurement of PEG with an aHL nanopore using results from a recently published study (Balijepalli et. al, J Am Chem Soc, 135: 7064, 2013) that refined a previous analytical theory with molecular dynamics simulations. Fitting this model to the newly obtained experimental data resulted in excellent agreement of both the blockade depth (the ratio of the ionic current when a molecule occupies the pore to the open channel current) and the residence times of the molecule in the channel, over the entire measurement range (PEG8 to PEG19). Finally, we applied the new analysis technique to recover the sequence of a known DNA strand with 26 bases, from a published ionic current trace (Manrao et. al, Nat. Biotechnol. 30: 349, 2012). The technique detected systematic fluctuations in the ionic current that were as small as 0.9 5 0.04 pA.