Figure 2. Characterization of pH/H₂O₂-activatable NIR fluorescent nanoprobes.

(A) High dye loading in nanoprobes results in quenching of their fluorescence properties, creating an ‘OFF’ state. (B) H₂O₂-triggered fluorescence activation of dual-responsive nanoprobes is enhanced at low pH. (C) Transmission electron micrographs of the nanoprobes in different environments (pH 7.4, pH 7.4 + H₂O₂, pH 6.0, pH 6.0 + H₂O₂). [H₂O₂] = 0.25 mM, T = 37°C, incubation time: 2 h. (D) Number of nanoprobes over time in different environments (pH 7.4, pH 7.4 + H₂O₂, pH 6.0, pH 6.0 + H₂O₂) represented as percent of initial DLS count rate. [H₂O₂] = 0.25 mM, T = 37°C. (E) Fluorescence microscopic images of the nanoprobes before (left) and after (right) activation. [H₂O₂] = 0.25 mM, pH = 6, incubation time: 1 h. (F) Silenced (‘OFF’ state, green trace) and activated (‘ON’ state, red trace) nanoprobes possess different emission maxima. (G) H₂O₂-triggered (0 - 1 mM) fluorescence signal acquired using a single filter set (λ_ex = 745 ± 15 nm, λ_em = 800 ± 10 nm) consists of a mixture of both ‘OFF’ and ‘ON’ state emission (raw signal, yellow). The NPs' distinct spectral fingerprints allow quantitative decomposition of the mixed raw signal into its individually resolved fluorescence constituents, i.e., ‘OFF’ state (green) and ‘ON’ state signal (red), using multispectral acquisition (λ_ex = 745 ± 15 nm, emission scanned from 790 to 850 nm) and software-based spectral unmixing algorithms. Higher concentrations of H₂O₂ yield faster and greater fluorescence activation.