Measurement of the activity of HIV-1 PR in a nano-cavity. (a) 0 ng/mL HIV-1 PR; (b) 60 ng/mL HIV-1 PR; and (c) 300 ng/mL HIV-1 PR. (Left) Typical single-channel current recording trace segments after 55 min proteolytic reactions. Dashed lines represent the levels of zero current; (Right) the corresponding 3D plots of event count vs. residence time vs. blockage amplitude. Labels 1, 2, and 3 in Figs. 2a–c represent events attributed to the substrate, and the two degradation products, respectively. d) Plot of event frequency versus the concentration of HIV-1 PR. Labels 1 and 2 represent the substrate and the cleavage product, respectively. The experiments were performed by real-time monitoring of the substrate-protease interaction continuously for 1 h at −40 mV in 1 M NaCl solution buffered with 1 mM EDTA and 1 mM NaH2PO4 (pH 4.7). The concentration of the substrate peptide was 5 µM. Event counts and event frequency in Figs. 2a–d were calculated based on the last 5 min trace segment of a 60 min single channel recording.