Single-molecule transformation and analysis of glutathione oxidized and reduced in nanopore

The determination of glutathione reduced (GSH) or oxidized (GSSG) in bulk solution has been reported previously. However, it is critically important to set up a simple and label-free method to recognize GSSG and GSH selectively and dynamically, especially at a single-molecule level. Here we report a novel nanopore method to recognize GSSG based on a newly synthesized per-6-quaternary ammonium-β-cyclodextrin (p-QABCD), which is used as both the molecular adaptor of protein nanopore and the recognizing element of GSSG. Distinct current signature is observed upon GSSG binding in a mutant protein nanopore (M113R RL2)₇ equipped with p-QABCD, while there is no signal for GSH. Thus GSSG in the mixture can be selectively detected in the concentration range of 6.00-90.0μM. Furthermore, the conversion between GSH and GSSG both in bulk solution and in nanochannel can be continuously monitored in real time and in situ. The label-free method provides a possibility to investigate enzymatic activity as well as its activators or inhibitors related to the transformation between GSH and GSSG.