Journal: Analytical Chemistry

Article Title: Continuous, Low Latency Estimation of the Size and Shape of Single Proteins from Real-Time Nanopore Data

doi: 10.1021/acs.analchem.5c04044

Figure Lengend Snippet: Estimating the volume and shape of proteins during nanopore data acquisition. (A) Schematic illustration of the nanopore recording setup and of the real-time data analysis approach for protein characterization. The electrolyte contains 2 M KCl with 10 mM HEPES buffered at pH 7.4. Two Ag/AgCl electrodes apply a potential difference of −100 mV across a nanopore with a diameter of 20 nm and a length of 30 nm (with negative polarity applied to the top). (B) Representative current recording of protein translocations through a nanopore (top), with resistive pulses detected instantaneously by the TSW algorithm as indicated by green pulses (bottom). (C) Principle of determining shape and volume of proteins from I min and I max . Top: translocation of different proteins with their shapes represented as a sphere (streptavidin), oblate (IgG), and prolate (Tg). Bottom: Representative current pulses generated from protein translocations and their corresponding histogram, where I min and I max are the minimum and maximum current blockades of the single resistive pulse. The ratio between the magnitude of I min and I max determines the shape m of proteins. Here, m is defined as the axis ratio b / a of an ellipsoid of revolution with semiaxes a , a , b ; m < 1 corresponds to an oblate shape, while m > 1 indicates a prolate shape. (D) Estimation of shape ( m ) and volume ( V ) fro m the cumulative residence time of detected resistive pulses during recording for a protein modeled as an oblate shape IgG. The arrows represent that the estimated volume and shape stabilize at V ref = 332 nm 3 and m ref = 0.46 after a cumulative residence time of 18 ms. Here, only the resistive pulses with dwell times greater than 150 μs were analyzed. Data were acquired at a sampling rate of 500 kHz and a bandwidth of 50 kHz. This measurements were performed independently of those in C.

Article Snippet: We used Ag/AgCl pellet electrodes (Warner Instruments) to monitor the ionic currents during nanopore experiments using a patch-clamp amplifier (AxonPatch 200B, Molecular Devices) in voltage clamp mode with a 100 kHz lowpass Bessel filter.

Techniques: Translocation Assay, Generated, Sampling