期刊
INORGANIC CHEMISTRY
卷 61, 期 27, 页码 10521-10532出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.inorgchem.2c01430
关键词
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资金
- Miller Institute at UC Berkeley
- National Institutes of Health (NIH) [R01GM127854]
Ratiometric sensors are a type of self-referencing constructs that can function in cells and tissues, and their read-out is not affected by sensor concentration. This study developed protein-based ratiometric oxygen sensors using heme nitric oxide/oxygen-binding protein (H-NOX) and oxygen-responsive fluorescent probes. By modifying the amino acid residues on the protein surface, the oxygen sensitivity of the sensor can be adjusted.
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 Fo''rster 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.
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