title: Quantum dot-fluorescent protein FRET probes for sensing intracellular pH.
abstract: Intracellular pH (pH(i)) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pH(i) of intact cells and subcellular compartments. Here we describe a nanoparticle-based ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging. We found that Förster resonance energy transfer between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Atlanta, Georgia 30332, United States.
authors.forename: Allison M
authors.initials: AM
authors.lastname: Dennis
authors.forename: Won Jong
authors.initials: WJ
authors.lastname: Rhee
authors.forename: David
authors.initials: D
authors.lastname: Sotto
authors.forename: Steven N
authors.initials: SN
authors.lastname: Dublin
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry*; D057054:Molecular Imaging; D038761:Photobleaching; D045663:Quantum Dots
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins