| Summary: | Neutral Red (NR), a eurhodin dye, is often used for staining living cells, but we demonstrated for the first time that NR can also serve as a CO<sub>2</sub> sensor, because of NR’s unique optical properties, which change with dissolved carbon dioxide (dCO<sub>2</sub>) concentrations. In the present study, optical sensitivity of NR was quantified as a function of changes in absorption and emission spectra to dCO<sub>2</sub> in a pH 7.3 buffer medium at eight dCO<sub>2</sub> concentrations. NR exhibited a response time of two minutes for equilibration in pure N<sub>2</sub> to 100% CO<sub>2</sub> with an ~200% percent change (%∆) in emission intensity and >400%∆ in absorbance, with full reversibility. Important to its application to biological systems, NR exhibited zero sensitivity to dissolved oxygen, which has routinely caused interference for CO<sub>2</sub> measurements. NR exhibited pH sensitive emission and excitation energies with dual excitation wavelengths at 455 nm and 540 nm, and a single emission at 640 nm. The CO<sub>2</sub> sensing properties of NR were benchmarked by a comparison to pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid trisodium salt) (HPTS). Future studies will evaluate the feasibility of NR as an intracellular in vivo <i>p</i>CO<sub>2</sub> sensor in aquatic organisms critically impacted by increasing global CO<sub>2</sub> levels.
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