Split-FP Conjugated Metal Nanoparticle Raman Nanoprobes for Ultra-Sensitive Molecular Detection

Abstract

The recent convergence of biotechnology and nanotechnology now provides unique means to develop hybrid nanomaterials for advanced biosensing applications. In particular, the controlled assembly of metal gold (Au) and silver (Ag) nanoparticles using bio-inspired scaffolds is a promising approach for the design of novel Surface Enhanced Raman Spectroscopy (SERS) nanoprobes capable of single molecule detection sensitivity. Here, we propose a new design for colloidal Raman nanoprobes based on split-FP/metal nanoparticle hybrid composites. We use split fluorescence proteins (split-FPs) as both activatable Raman reporters and molecular glue to assemble Au or Ag nanoparticles into photonically-active SERS nanoclusters. We expressed recombinant split-FPs (sGFP, sYFP, sCFP) with metal binding domains and engineered their complementary peptide fragments for oriented and controlled grafting at the surface of Au and Ag nanoparticles. These split-FPs fragments were respectively coated on various size of metal nanoparticles (5-50nm diameter), and were characterized by electrophoresis, HPLC, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), and immunoblotting. In vitro, complementary metal nanoparticles functionalized with split-FP fragments can self-assemble and form nanoclusters as confirmed by electrophoresis and TEM measurements. To improve the complementation kinetics of split-FP/peptide pairs on nanoparticles, various point mutations were introduced in split-FPs. We further show that SERS signals from split-FPs are specifically detected upon biomolecular interactions of split-FP fragments and that the unique Raman signature of the FP chromophores is easily distinguished over other protein modes when bound to plasmonic nanomaterials. These results demonstrate the potential for highly selective and sensitive SERS detection based on split-FP/metal nanoparticle hybrid probes.