Fig. 6: Pro-catalyst as a signal module for transmembrane fluorescence catalysis.

a Schematic diagram of the pro-catalyst dipyridylamine used for transmembrane fluorescence catalysis. H⁺-mediated triplex formation translocates the pro-catalyst dipyridylamine, tagged on the 5'-terminus of the TD, into the membrane interior in the presence of Zn²⁺, where it catalyzes the conversion of APTS into HPTS. b Normalized fluorescence spectra of APTS (Em 390 nm) and HPTS (Em 520 nm). c Fluorescence kinetic profiles showing HPTS generation inside LUVs (Ex: 415 nm, Em: 510 nm) at an external pH of 7.5 (red line) and pH 5.5 after adding dilute HCl (aq) (green line), and control vesicles without TD incubated at an external pH of 5.5 (black line). APTS, 250 μM; Zn²⁺, 250 μM; TD, 10 μM; Aux₁₁, 50 μM; 1 mM DOPC lipid. Fluorescence kinetic curves showing dependency on TD concentration (d) and APTS substrate concentration (e). TD: 0-10 μM; APTS: 0.1–1 mM; TD = 1:5 molar ratio. Data in (c–e) are presented as mean ± s.d. (n = 3 independent samples). f–i Time-dependent confocal fluorescence imaging illustrating HPTS fluorescence enhancement over time (f) with concurrent attenuation of APTS fluorescence (g). Quantitative intensity analysis (h) and fluorescence intensity histogram comparison at 0 and 60 min (i). Data in (h, i) are presented as mean ± s.d., (n = 3 independent GUVs in h, and n = 6 independent GUVs in i, two-sided Student’s t-test); Scale bar (f, g), 5.0 μm. Two-dimensional flow cytometry scatter plots of FSC-A versus HPTS-A, showing signal transduction at t = 0 (j) and t = 150 min (k), with a corresponding histogram of HPTS fluorescence (l). m Time-dependent changes in mean HPTS fluorescence (green), mean FSC intensity (blue), and mean SSC intensity (yellow) within vesicles. a.u. arbitrary units.