The multiplexed biosensing of target molecules with high specificity and accuracy is of fundamental importance in both biological research and medical diagnostics. In this paper, the working range of the recent nanopore-DNA carrier based method is extended by introducing a two-step assay using specific DNA aptamers. A signal translation step allows for binding of the target in physiological conditions before the nanopore measurements. Using protein encoded DNA carriers, the simultaneous detection of three targets spanning several orders of magnitude in molecular weight is demonstrated. The single-molecule method may be integrated into nanopore sensing devices for future applied research and point-of-care applications.