Directed self-assembly of gold binding polypeptide-protein A fusion proteins for development of gold nanoparticle-based SPR immunosensors

Effective immobilization of antibodies on a sensing platform and sensitivity enhancement are crucial in designing surface plasmon resonance (SPR) immunosensors. Colloidal gold nanoparticles (AuNPs) were directly assembled onto a surface of SPR Au chip via 2-aminoethanethiol for the enhancement of sensitivity as a label-free detection system. SEM image showed most AuNPs were uniformly distributed over the surface. A novel fusion protein was constructed by genetically fusing gold binding polypeptides (GBP) to protein A (ProA) as a crosslinker for effective immobilization of antibodies. The resulting GBP-ProA protein was directly self-immobilized onto both bare and AuNPs-assembled SPR chip surfaces via the GBP portion, followed by the oriented binding of human immunoglobulin G (hIgG) onto the ProA domain targeting the Fc region of antibodies and anti-hIgG in series. Furthermore, anti-Salmonella antibodies were immobilized onto both GBP-ProA layered chips for detection of Salmonella typhimurium. SPR analyses indicated the signal increases for successive binding of hIgG and anti-hIgG onto the GBP-ProA layered AuNPs-assembled chip were higher (about 92 and 30%, respectively) than that onto the identically treated bare chip. This signal enhancement in the AuNPs-assembled chip also caused a 10-fold increased sensitivity in detection of S. typhimurium compared to the bare one. These results demonstrate the direct assembly of AuNPs onto a SPR chip could enhance the signal in biomolecular interaction events, and the GBP-ProA protein could be a valuable crosslinker for simple and oriented immobilization of antibodies onto Au chip surfaces without any surface chemical modification.