High-bandwidth protein analysis using solid-state nanopores

Abstract

High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ~50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.

Figure 1

Protein detection using small solid-state nanopores. (a) Space-filling models of ProtK molecules electrophoretically driven through a 5-nm-diameter pore (electrodes not drawn to scale). (Inset) TEM image of a HfO₂ pore used in these experiments. (b) Continuous 0.5 s current versus time trace of a 69 nM ProtK solution at V = −125 mV (data sampled at 4.19 MHz and digitally low-pass-filtered at 250 kHz). Concatenated set of analyzed events is shown on right (black), along with OpenNanopore rectangular fits (red). Dwell times for a selection of magnified events are indicated. To see this figure in color, go online.

Figure 2

(a and b) Snapshot current traces for RNase (a) and ProtK (b) at various voltages in the range +200 mV to −200 mV ([KCl] = 1 M, pH 8.1, T = 25°C, low-pass filtered at 125 kHz for presentation only). (Insets) Current spikes for V < 0, indicating positive charges for both proteins. (c and d) Dwell time versus fractional current scatterplots for ProtK and RNase at the voltages indicated. Detected events become faster with voltage, and bandwidth limitations are clearly seen by arrows I (red) and II (green), where the ProtK population is more cut off by the 2.5 μs time resolution at −150 mV than at −125 mV, respectively. To see this figure in color, go online.

Figure 3

(a) Normalized mean capture rates in a 5.2-nm-diameter HfO₂ pore as a function of V. (b) Corresponding Smoluchowski-based capture radii for both proteins (see text). To see this figure in color, go online.

Figure 4

(a and b) Dwell-time distributions for ProtK (a) and RNase (b) at selected voltages, along with fits to Eq. 1 with D and v as free parameters (black line) and constrained fits for bulk D₀ values (dashed red line). Missed regimes shaded red in distributions. (c) Diffusion coefficients (D) obtained for the proteins from the fits to Eq. 1. (d) Drift velocity (v) versus voltage (V) for ProtK, RNase, and 100 bp dsDNA. Linear fits are used to extract electrophoretic mobility (μ) values, as indicated in legend. DNA data are scaled by 0.5×, and dashed line represents resolution limits of our system (see text). To see this figure in color, go online.

Figure 5

Scatterplots of in-pore electrophoretic mobilities, calculated for each experiment as μ^∗ = vh_eff/ΔV, versus in-pore diffusion coefficients, D (where D and v, are extracted from two-parameter fits to Eq. 1; see text). Compiled results for experiments in different HfO₂ and SiN pores and different voltages are shown. Apart from an outlier for RNase at −75 mV (dashed circle), the data suggest voltage-independent values of μ and D in the range tested. To see this figure in color, go online.

Figure 6

Volumetric measurement of proteins using nanopores. (a) Fractional current amplitude distributions for RNase and ProtK in an HfO₂ pore at −125 mV. (b) Fractional current blockage for the same proteins in an SiN pore (upper), and PDB-based cartoons of the proteins and their corresponding Vorlume-based solvent-accessible volumes. When the proteins are mixed at equal stoichiometry, a third peak with a deeper fractional blockage emerges, corresponding to an RNase/ProtK complex (red, lower). To see this figure in color, go online.