Mann, Victor R.Manea, FrancescaBorys, Nicholas J.Ajo-Franklin, Caroline M.Cohen, Bruce E.2021-05-122021-05-122021Mann, Victor R., Manea, Francesca, Borys, Nicholas J., et al.. "Controlled and Stable Patterning of Diverse Inorganic Nanocrystals on Crystalline Two-Dimensional Protein Arrays." <i>Biochemistry,</i> 60, no. 13 (2021) American Chemical Society: 1063-1074. https://doi.org/10.1021/acs.biochem.1c00032.https://hdl.handle.net/1911/110508Controlled patterning of nanoparticles on bioassemblies enables synthesis of complex materials for applications in optics, nanoelectronics, and sensing. Biomolecular self-assembly offers molecular control for engineering patterned nanomaterials, but current approaches have been limited in their ability to combine high nanoparticle coverage with generality that enables incorporation of multiple nanoparticle types. Here, we synthesize photonic materials on crystalline two-dimensional (2D) protein sheets using orthogonal bioconjugation reactions, organizing quantum dots (QDs), gold nanoparticles (AuNPs), and upconverting nanoparticles along the surface-layer (S-layer) protein SbsB from the extremophile Geobacillus stearothermophilus. We use electron and optical microscopy to show that isopeptide bond-forming SpyCatcher and SnoopCatcher systems enable the simultaneous and controlled conjugation of multiple types of nanoparticles (NPs) at high densities along the SbsB sheets. These NP conjugation reactions are orthogonal to each other and to Au–thiol bond formation, allowing tailorable nanoparticle combinations at sufficient labeling efficiencies to permit optical interactions between nanoparticles. Fluorescence lifetime imaging of SbsB sheets conjugated to QDs and AuNPs at distinct attachment sites shows spatially heterogeneous QD emission, with shorter radiative decays and brighter fluorescence arising from plasmonic enhancement at short interparticle distances. This specific, stable, and efficient conjugation of NPs to 2D protein sheets enables the exploration of interactions between pairs of nanoparticles at defined distances for the engineering of protein-based photonic nanomaterials.engThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Chemical Society.Journal articlehttps://doi.org/10.1021/acs.biochem.1c00032