Fig. 3: Measurement of •OH by 1-NP in vitro.

a Normalized absorption and b nomalied fluorescence (FL) spectra of 1-NP upon incubation with different concentration •OH (0, 1, 2, 5, 10, 20, 50, 100, 150, 200, 250 μM Fe²⁺ + 1 mM H₂O₂) at r.t. for 3 min. c Plot of the normalized FL₇₈₀/FL₁₁₁₃ values versus the concentration of •OH show a linear relationship ranging from 0.05-20 μM of •OH. The limit of detection (LOD) was determined to be ~3.69 nM by using the 3δ/k method. d Photoacoustic (PA) images at 755 and 905 nm, and ratiometric PA₇₅₅/PA₉₀₅ images of 1-NP (56/1.65/20 μΜ 1-Br-Et/NIR775/IR1048) upon incubation with the indicated concentration of •OH at r.t. for 3 min. e Plot of the normalized PA₇₅₅/PA₉₀₅ values versus the concentration of •OH show a linear relationship between 1 and 20 μM of •OH. The LOD was determined to be ~0.24 μM by using the 3δ/k method. f Plot of the normalized PA₇₅₅/PA₉₀₅ ratios versus the normalized FL₇₈₀/FL₁₁₁₃ ratios shows a strong correlation (Pearson’s r = −0.994) between them upon incubation of 1-NP with the indicated concentration of •OH. g Normalized UV-Vis-NIR absorption spectra, h normalized FL₇₈₀/FL₁₁₁₃ values and i normalized PA₇₅₅/PA₉₀₅ values of 1-NP after incubation with •OH and other representative reactive oxygen species (ROS) at r.t. for 24 h. Inset: PA images at 755 nm and 905 nm, and ratiometric PA₇₅₅/PA₉₀₅ images of 1-NP following indicated treatment. 1: •OH (200 µM Fe²⁺ + 1 mM H₂O₂); 2: ¹O₂ (1 mM H₂O₂ + 1 mM ClO⁻); 3: 300 µM t-BuOOH; 4: 1 mM ClO⁻; 5: 300 µM CuOOH; 6: 1 mM H₂O₂; 7: O₂^·- (100 µM xanthine + 22 mU xanthine oxidase (XO)); 8: ONOO⁻ (1 mM NaNO₂ + 1 mM H₂O₂); 9: Control. Data are presented as mean ± s.d. (n = 3 independent samples). Source data are provided as a Source Data file.