We developed a generalized technique to characterize polymer-nanopore interactions via single channel ionic current measurements. Physical interactions between analytes, such as DNA, proteins or synthetic polymers, and a nanopore cause multiple discrete states in the ionic current. We modeled the transitions of the ionic current to individual states with an equivalent electrical circuit of the nanopore system, which allowed us to describe the system response. This enables the estimation of short-lived states in single-molecule nanopore data that are presently not characterized by existing analysis techniques. Our approach considerably improves the range and resolution of single-molecule characterization with nanopores. For example, we characterized the residence times of molecules in the nanopore that are three times shorter than those estimated with existing algorithms. Because the molecule’s residence time follows an exponential distribution, we recover nearly 20-fold more events per unit time that can be used for analysis. Furthermore, the measurement range was extended from 11 monomers to as few as 8. Finally, we apply this technique to recover a known sequence of single stranded DNA from previously published ion channel recordings, identifying discrete current states with sub-picoampere resolution.

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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.