The optical response of isolated holes in 20 nm thin gold is probed as a function of alkanethiol CH(3)(CH2)x SH (x epsilon in 1-15) and protein adsorption using dark-field spectroscopy. We establish that the plasmon excitations of single and short-range ordered 60 nm holes exhibit similar E-field decay lengths delta approximately 10-20 nm and that a single hole can be used to resolve the successive adsorption of a protein (biotin-BSA) and its interaction partner (neutravidin). The data confirm the localized character of the hole plasmon and demonstrate that its applicability for bio/chemosensing is similar to that of particle plasmons.