A monochromophoric approach to succinct ratiometric fluorescent probes without probe-product crosstalk

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational development of ratiometric probes with zero probe-product crosstalk remains challenging. Harnessing the recent progress on photophysical modulation of xanthenoid fluorochromes, we propose a powerful and versatile probe design principle, that is, “bridging-group modification,” and developed totalROX, a robust probe for monitoring the total cellular oxidative capacity. First, totalRox affirmatively detected the complete set of biorelevant highly oxidative species: per-acids (2 e⁻), radicals (1 e⁻), nitrosative (NO⁺) species, and singlet oxygen (¹O₂). Nonoxidative or mildly oxidative pro-oxidants, for example, O₂^•–, H₂O₂, NO, ONOO^–, and ClO^– were not detected. Second, the absorption/fluorescence maxima of the probe (totalROX, λ_abs/λ_em = 425/525 nm) and the detection product (Ox670, λ_abs/λ_em = 650/675 nm) were separated by ca. 225/150 nm, respectively, which eliminated probe-product crosstalk. Third, it renders the ratiometric signal with a single chromophore and is structurally succinct. TotalROX allowed quantitative analysis and was more sensitive than Amplex Red and CellROX Deep Red, two commercial probes for cell oxidative species. Bioimaging potentials of totalROX for monitoring cell redox status were exemplified in three different cell lines.