Protein Design Provides Lead(II) Ion Biosensors for Imaging Molecular Fluxes around Red Blood Cells

Abstract

Metalloprotein design and semiconductor nanoparticles have been combined to generate a reagent for selective fluorescence imaging of Pb(2+) ions in the presence red blood cells. A biosensor system based on semiconductor nanoparticles provides the photonic properties for small molecule measurement in and around red blood cells. Metalloprotein design was used to generate a Pb(2+) ion selective receptor from a protein that is structurally homologous to a protein used previously in this biosensing system. Parameters for the Pb(2+) ion binding site were derived from crystallographic structures of low molecular weight Pb(2+) ion complexes that contain a stereoactive lone pair. When the designed protein was produced and attached to ZnS-coated CdSe nanoparticles, two Pb(NO(3))(2)-associated binding events were observed (2-fold emission decrease; K(A1) = 1 x 10(9) M(-1); K(A2) = 3.5 x 10(6) M(-1)). The fluorescence response had a 100 pM Pb(NO(3))(2) detection limit, while no response was observed with Ca(2+) ions (10 mM), Zn(2+) ions (100 muM), or Cd(2+) ions (100 muM). Metal ion selectivity presumably comes from the coordination geometry selected to favor lone pair formation on Pb(2+) ions and electrostatically disfavor tetrahedral coordination. Replacement of ZnS-coated CdSe with ZnS-coated InGaP nanoparticles provided similar biosensors (100 pM limit of detection; K(A1) = 1 x 10(9) M(-1); K(A2) = 1 x 10(7) M(-1)) but with excitation/emission wavelengths longer than the major absorbance of red blood cell hemoglobin (>620 nm). The InGaP nanoparticle-based biosensors provided a 5 nM Pb(NO(3))(2) detection limit in the presence of red blood cells. The modularity of the biosensor system provides exchangeable Pb(2+) ion detection around red blood cells.