Unbiased Data Analysis for the Parameterization of Fast Translocation Events through Nanopores.

Single-molecule nanopore electrophysiology is an emerging technique for the detection of analytes in aqueous solutions with high sensitivity. These detectors have proven applicable for the enzyme-assisted sequencing of oligonucleotides. There has recently been an increased interest in the use of nanopores for the fingerprinting of peptides and proteins, referred to as single-molecule nanopore spectrometry. However, the analysis of the resulting electrophysiology traces remains complicated due to the fast unassisted translocation of such analytes, usually in the order of micro- to milliseconds, and the small ion current signal produced (in the picoampere range). Here, we present the application of a generalized normal distribution function (gNDF) for the characterization of short-lived ion current signals (blockades). We show that the gNDF can be used to determine if the observed blockades have adequate time to reach their maximum current plateau while also providing a description of each blockade based on the open pore current (I O), the difference caused by the pore blockade (ΔI B), the position in time (µ), the standard deviation (σ), and a shape parameter (ß), leaving only the noise component. In addition, this method allows the estimation of an ideal range of low-pass filter frequencies that contains maximum information with minimal noise. In summary, we show a parameter-free and generalized method for the analysis of short-lived ion current blockades, which facilitates single-molecule nanopore spectrometry with minimal user bias.

Strategies for enzymological studies and measurements of biological molecules with the cytolysin A nanopore.

Pore-forming toxins are used in a variety of biotechnological applications. Typically, individual membrane proteins are reconstituted in artificial lipid bilayers where they form water-filled nanoscale apertures (nanopores). When a voltage is applied, the ionic current passing through a nanopore can be used for example to sequence biopolymers, identify molecules, or to study chemical or enzymatic reactions at the single-molecule level. Here we present strategies for studying individual enzymes and measuring molecules, also in highly complex biological samples such as blood.