Measuring and modeling the fraction of tagged events as a function of TNFα-to-scaffold/fusion ratio, X. (a) EMSA assay shows increasing fraction of target-bound scaffold/fusion molecules as TNFα protein concentration increases, using a 309 bp/PNA-affibody detection reagent (Methods, Supplementary Fig. S27). (b) All-event scatter plot of max δG versus duration for reagents with varying X. Each reagent was measured sequentially on the same 25–30 nm diameter pore (P29, Supplementary Table S52), with buffer only flushing and recording between reagent pairs. The number of events and recording periods for each reagent are: (X = 0) N = 518 in 10 min; (X = 12.5) N = 1222 in 10 min; (X = 6) N = 1336 in 10 min; (X = 3) N = 1565 in 10 min; (X = 1) N = 819 in 10 min; (X = 0.5) N = 350 in 4 min; and (X = 0.25) N = 690 in 10 min. Using the X = 0 reagent to establish the tagging criteria max δG > 3.56 nS (dashed line), all X > 0 values were positive with 99% confidence above a false positive of 2%, with the exception of X = 0.25 which was not positive. The time to positive (TTP) values: X = 12.5 in 6 sec; X = 6 in 5 sec; X = 3 in 10 sec; X = 1 in 41 sec; and X = 0.5 in 2.3 min. (c) The positively detected data in (b) were subsequently modeled using the three-parameter model Ftag(X), resulting in parameter values (α, q1, q2) = (0.35, 0.17, 0.11) and NRMS fitting error of 3.8% (Supplementary Table S53).