Development of Neutral Red as a pH/pCO2 Luminescent Sensor for Biological Systems

Abstract

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 CO2 sensor, because of NR’s unique optical properties, which change with dissolved carbon dioxide (dCO2) concentrations. In the present study, optical sensitivity of NR was quantified as a function of changes in absorption and emission spectra to dCO2 in a pH 7.3 buffer medium at eight dCO2 concentrations. NR exhibited a response time of two minutes for equilibration in pure N2 to 100% CO2 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 CO2 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 CO2 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 pCO2 sensor in aquatic organisms critically impacted by increasing global CO2 levels.