The use of bio-inspired scaffolds to assemble nanomaterials into photonic nanostructures is a highly promising approach to design new nanoscale biosensors for biomedical applications. Here, we describe novel hybrid colloidal Raman nanoprobes based on the self-assembly of metal nanoparticles functionalized with split-fluorescent protein fragments (split-FPs). In our system, split-FPs serve as both unique Raman fingerprint reporters and molecular glue to assemble metal (Au, Ag) nanoparticles into photonically-active surface enhanced Raman scattering (SERS) nanoclusters. For oriented grafting at the surface of metal nanoparticles, we engineered recombinant split-FPs (sGFP, sYFPs, sCFPs) and their complementary peptide fragments with metal binding domains. Various sizes of metal nanoparticles (5-50 nm in diameter) were coated with these split-FPs fragments and characterized by electrophoresis, HPLC, DLS, TEM, and immunoblotting. <i>In vitro</i>, the self-assembly of complementary metal nanoparticles and the formation of nanoclusters were confirmed by electrophoresis and TEM measurements. Furthermore, collected SERS signals from these assembled nanoclusters display the unique Raman signature of FP chromophores confirming that the assembly process is driven by biomolecular interactions of the split-FP fragments and result in large electromagnetic field enhancements at stable plasmonic hotspots. We additionally performed 3D-FDTD simulations of these FP/metal nanoparticle hybrid nanoclusters to confirm the plasmonic coupling effects, to characterize the enhanced electromagnetic field and to optimize the spatial organization of nanoparticles within nanoclusters for maximal SERS responses. Finally, we show that these novel SERS nanoprobes can self-assemble at the plasma membrane of live cells as confirmed by scanning electron microscopy and by <i>in vivo</i> Raman microscopy imaging. These results denote that split-FP/metal nanoparticle hybrid nanoprobes hold great potential for highly selective and sensitive SERS detection of targeted cells.

Self-assembled split-FP/metal nanoclusters as Raman enhancers for molecular and cellular detection

251st National Meeting (2016)

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