Monitoring disulfide bonds making and breaking in biological nanopore at single molecule level

Monitoring subtle changes in ionic current flow through a nanopore could be applied to observe single molecule reaction. Here, we introduced cysteine to substitute for lysine at position 238 constructing a mutant aerolysin K238C. It could be regarded as a nanoreactor to efficiently visualize chemical bonds making and breaking. The compound 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was selected as a reactant coming into collisions on the interface of the pore to occur a reversible reaction. Our results showed that the mutant aerolysin could respond to three molecules of DTNB simultaneously and reflect corresponding levels with distinguishable current signals. Therefore, this method constitutes a simple, generic tool for monitoring single molecule reaction, which evokes a guidance for the mutant aerolysin towards the application of tracking other more reactions at single molecule level.