Appendix 7—figure 1. Physical characteristics of timetraces.

(A, B) Scatter plots of the events detected for Kap95 (A) and BSA (B) indicating the distributions of event duration and the amount of photons within an event for the data shown in Figure 3C and D. Both distributions overlap, showing that the spike detection works equally for coated and open pores. (C, D) Plots of the diffusion coefficient vs. pore diameter for Kap95 and BSA, respectively. The diffusion coefficient is estimated by fluorescence correlation spectroscopy (FCS) analysis of the time traces obtained in the nanopore experiments at the highest protein concentration. The horizontal lines indicates the average diffusion coefficient and the width corresponds to twice the standard error of the mean. The average diffusion coefficient of both Kap95 and BSA shows no significant difference between open pores and Nsp1-coated pores. This indicates that interactions of the proteins with the Nsp1 mesh do not obstruct the diffusion. Alternatively, it is possible that the bound fraction is not detected in our experiments if it is close to the metal surface due to metal-induced quenching of the fluorescence signal. (E, F) Plots of the fluorescence lifetime vs. pore diameter for Kap95 and BSA, respectively. The fluorescence lifetime is calculated based on the individual time traces of the highest protein concentration. The mean (horizontal lines) with twice the standard error of the mean (width of the lines) gives an estimate of the spread. For both Kap95 and BSA, the average fluorescence lifetime is significantly lower than what is measured in open solution (black lines). This can be attributed to the influence of the nearby metal nanostructure on the radiative and non-radiative rates. The predicted lifetimes based on finite-difference time-domain (FDTD) simulations are shown as red crosses (compare Appendix 2—figure 4). Whereas for BSA the lifetime in Nsp1-coated pores and open pores does not differ significantly, there is a significant decrease of the fluorescence lifetime of Kap95 in Nsp1-coated pores compared to open pores. This suggests that Kap95 remains within the proximity of the pore for a longer time when Nsp1 is present.