Ratiometric Oxygen Sensing with H-NOX Protein Conjugates.
Christopher M LemonDeirdre HanleyAllison E BatkaMichael A MarlettaPublished in: Inorganic chemistry (2022)
Ratiometric sensors are self-referencing constructs that are functional in cells and tissues, and the read-out is independent of sensor concentration. One strategy for ratiometric sensing is to utilize two-color emission, where one component possesses analyte-dependent emission and the other is independent of analyte concentration, serving as an internal standard. In this way, the intensity ratio of the two components is a quantitative measure of the analyte. In this study, protein-based ratiometric oxygen sensors are prepared using the heme nitric oxide/oxygen-binding protein (H-NOX) from the thermophilic bacterium Caldanaerobacter subterraneus . The native heme cofactor is replaced with a Pd(II) or Pt(II) porphyrin as the oxygen-responsive phosphor. Mutagenesis is performed to incorporate a cysteine residue on the protein surface for thiol/maleimide coupling of the oxygen-insensitive dye, which serves as a Förster resonance energy transfer (FRET) donor for the porphyrin. While both Pd(II)- and Pt(II)-based sensors are responsive over biologically relevant ranges, the Pd sensor exhibits greater sensitivity at lower oxygen concentrations. Together, these sensors represent a new class of protein-based ratiometric oxygen sensors, and the modular platform allows the oxygen sensitivity to be tailored for a specific application. This proof-of-principle study has identified the key considerations and optimal methodologies to develop and subsequently refine protein-based ratiometric oxygen sensors.
Keyphrases
- energy transfer
- quantum dots
- fluorescent probe
- living cells
- binding protein
- nitric oxide
- low cost
- sensitive detection
- hydrogen peroxide
- protein protein
- amino acid
- cancer therapy
- cell death
- photodynamic therapy
- induced apoptosis
- gene expression
- small molecule
- oxidative stress
- high intensity
- metal organic framework
- mass spectrometry
- signaling pathway