In vitro GST detection with NI9 and the detection mechanism for NI-series probes. (a) Time-dependent fluorescence spectra of NI9 (20 μM) in HEPES buffer (20 mM, 0.5% DMSO, pH 7.4) upon addition of GSTs (12.5 μg/mL) over the course of 25 min at 37°C in the presence of GSH (2 mM). λex = 445 nm. (b) A full set of control experiments on examining the cause of fluorescence enhancement. EA = ethacrynic acid (GSTs were preincubated with 200 μM EA for 30 min before addition of GSH and NI9 sequentially); deac-GST = deactivated GSTs (12.5 μg/mL) by pretreatment at 100°C for 10 min; GSSG = oxidized glutathione (2 mM); NAC = N-acetylcysteine (2 mM); Cys = L-cysteine (2 mM); Hcy = L-homocysteine (2 mM). λex/em = 445/560 nm. (c) Schematic for the proposed GST activity detection mechanism of NI-series probes.