Fig. 5: Design and characterization of dual-locked fluorogenic probes.

a The synthesis route of CyNA_P-SS-FK. b Activation mechanism of the dual-locked molecular probe CyNA_P-SS-FK for LM3 cell detection. c Fluorescence spectra of CyNA_P-SS-FK (20 µM) after incubation with/without GSH, Cat B, or GSH and Cat B at 37 °C in PBS buffer (pH 7.4). Fluorescence excitation at 580 nm. d Fluorescence intensity of CyNA_P-SS-FK (40 µM) after incubation with the indicated enzymes, metal ions (60 µM), ROS (60 µM) in PBS (10 mM, pH 7.4) at 37 °C. Mg²⁺, magnesium chloride; Ca²⁺, calcium chloride; ClO^-, sodium hypochlorite; H₂O₂, hydrogen peroxide, SO₄^2-, sodium sulfate; GSH, glutathione; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride; Cas-3, caspase 3; Galactosidase; Cat B, Cathepsin B. (n =  3, mean ± s.d.). Two-tailed Student’s t test; GSH and Cat B treated group versus other groups. NS, no statistically significant differences. e Mean NIRF intensities of HCC-LM3 after incubation with CyNA_P-SS-FK (with or without BSO) in the panel h (n =  4, mean ± s.d.). n refers to cell imaging experiment repeat times. (Scale bar, 5 μm). Two-tailed Student’s t test; Control versus different incubation time. NS, no statistically significant differences. f Representative TEM image and DLS result of CyNA_P-SS-FK before and after incubation with GSH and Cat B in buffer solution (scale bar, 150 nm). g Schematic illustration of the sensing mechanism of CyNA_P-SS-FK in living cells. h Representative confocal microscopy images of LM3 cells incubated with CyNA_P-SS-FK (25 μM) for 0, 4, 8 and 12 h (with or without BSO), respectively. Blue and red fluorescence indicate a cell nucleus stained with DAPI and the signals from CyNA_P-SS-FK, respectively. NIRF images acquired at excitation at 594 nm. The experiments in c, d, f and h were repeated independently three times with similar results. Source data are provided as a Source Data file.