Figure 3. In vitro Characterization of pnGFP-Ultra.

(A) Dynamic range (expressed as ΔF/F₀) of pnGFP-Ultra and relevant mutants towards ONOO^–. F₀ is the initial fluorescence intensity. ΔF is the final fluorescent intensity (after treatment) minus F₀. Samples were treated with 100 μM ONOO⁻ for 1 hour.

(B) Dynamic range (ΔF/F₀) of pnGFP-Ultra and relevant mutants towards H₂O₂. Samples were treated with 100 μM H₂O₂ for 1 hour.

(C) Selectivity of pnGFP-Ultra and its mutants. Arbitrarily defined selectivity is calculated as the fold of fluorescence enhancement by ONOO⁻ / fold of fluorescence enhancement by H₂O₂.

(D) Response of pnGFP-Ultra post 1-h incubation with a panel of redox-active chemicals: (1) ^•OtBu (1 mM Fe²⁺ and 100 μM HOOtBu), (2) 100 μM HOOtBu, (3) ^•OH (1 mM Fe²⁺ and 100 μM H₂O₂), (4) 5 mM oxidized glutathione, (5) 100 μM O₂^•−, (6) 100 μM NOC-7 (^•NO donor), (7) 100 μM HOCl, (8) 100 μM NaHS (H₂S donor), (9) 5 mM L-cysteine, (10) 1 mM DL-homocysteine, (11) 1 mM vitamin C, (12) 100 μM H₂O₂, (13) 1 mM H₂O₂, (14) 100 μM ONOO⁻, (15) Tris buffer.

(E) Fluorescence responses of pnGFP-Ultra (0.2 μM) to nanomolar to high micromolar concentrations of ONOO⁻. The half maximal response concentration (EC₅₀) was determined to be 19.5 μM.

(F) Linear fluorescence responses of pnGFP-Ultra (0.2 μM) to nanomolar to low micromolar concentrations of ONOO⁻. The limit of detection was determined to be 122 nM with a signal-to-noise ratio (S/N) of 3.

Data in all panels represent the mean ± SD of triplicates.