Figure 1. Experimental principle and nanofabrication.

(A) Sketch of the experimental principle. Nanopores in a metal membrane block light from traversing if the pore diameter is small compared to the wavelength of light. The selectivity of Nsp1-coated metal nanopores is probed by measuring the translocation rate of fluorescently labeled proteins from the top reservoir (cis) to the detection (trans) side, where they rapidly diffuse out of the laser focus. Measurements on open pores (top) serve as a control where both the nuclear transport receptor (NTR) Kap95 and the inert protein probe BSA pass unhindered. Nsp1-coated pores are expected to block the translocation of BSA while still allowing Kap95 to translocate. Zooms at bottom right illustrate the passivation of open pores with (1-mercaptoundec-11-yl)hexa(ethylene glycol) (MUHEG) (top) and functionalization of the palladium surface with the FG-nucleoporin Nsp1 and 350 Da SH-PEG (bottom), achieved via thiol-palladium chemistry. (B) Fabrication of nanopores in a freestanding palladium membrane was performed by physical vapor deposition of palladium onto silicon nitride (SiN_x), reactive ion etching (RIE), and focused ion beam (FIB) milling of the nanopores. The palladium surface was then cleaned either with H₂O₂ or ethanol to remove contaminants before the functionalization step.