Figure 3. Experimental workflow for measuring the selectivity of Nsp1-coated nanopores.

(A, B) Two pores of approximately 48 nm diameter were coated with (1-mercaptoundec-11-yl)hexa(ethylene glycol) (MUHEG) (A) or functionalized with Nsp1 (B). Pore dimensions were measured from transmission electron microscopy (TEM) micrographs. (C, D) Fluorescence time traces recorded for the open and Nsp1-coated pores that are shown in (A) and (B) for Kap95–Alexa647 at 100 nM (red) and BSA–Alexa488 at 250 nM (blue). Both proteins were present at the same time. Whereas the Kap95 signal is comparable between the open and Nsp1-coated pores, a clear decrease of the BSA event rate is evident for the Nsp1-coated pore. (E, F) Measured event rates (top panel) resulting from the analyte concentrations for the different conditions probed sequentially during the experiment (bottom panel; see Appendix 4 for details). The concentrations used in the time traces shown in (C) and (D) are indicated with an asterisk. (G) In order to compare the different conditions, the obtained event rates are normalized to the respective protein concentration and corrected for the labeling degree (white dots in G). Bars indicate the average normalized event rates ${\displaystyle k_{\mathrm{K}\mathrm{a}\mathrm{p}95}}$ or ${\displaystyle k_{\mathrm{B}\mathrm{S}\mathrm{A}}}$ of the pore. Error bars represent the standard error of the mean. (H) The selectivity was calculated as the ratio of the average normalized event rates ${\displaystyle \frac{k_{\mathrm{K}\mathrm{a}\mathrm{p}95}}{k_{\mathrm{B}\mathrm{S}\mathrm{A}}}}$. Errors are propagated from the data shown in (G). The data show a clear selectivity of the Nsp1-coated pore compared to the open pore.