title: Directed self-assembly of gold binding polypeptide-protein A fusion proteins for development of gold nanoparticle-based SPR immunosensors.
abstract: 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.
authors.affiliation: Korea Food Research Institute, Seongnam, Gyeonggi-Do 463-746, Republic of Korea. shko7@kfri.re.kr
authors.forename: Sungho
authors.initials: S
authors.lastname: Ko
authors.forename: Tae Jung
authors.initials: TJ
authors.lastname: Park
authors.forename: Hyo-Sop
authors.initials: HS
authors.lastname: Kim
authors.forename: Jae-Ho
authors.initials: JH
authors.lastname: Kim
authors.forename: Yong-Jin
authors.initials: YJ
authors.lastname: Cho
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D015169:Colony Count, Microbial / Q000295:instrumentation; D003460:Crystallization / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry; D007118:Immunoassay / Q000295:instrumentation*; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D006046:Gold