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.
Keywords: Glutathione; Nanopore; Per-6-quaternary ammonium-β-cyclodextrin; Single-molecule chemistry; Single-molecule technique.
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