title: Quantitative real-time imaging of protein-protein interactions by LSPR detection with micropatterned gold nanoparticles.
abstract: Localized surface plasmon resonance (LSPR) offers powerful means for sensitive label-free detection of protein-protein interactions in a highly multiplexed format. We have here established self-assembly and surface modification of plasmonic nanostructures on solid support suitable for quantitative protein-protein interaction analysis by spectroscopic and microscopic LSPR detection. These architectures were obtained by layer-by-layer assembly via electrostatic attraction. Gold nanoparticles (AuNP) were adsorbed on a biocompatible amine-terminated poly(ethylene glycol) (PEG) polymer brush and further functionalized by poly-l-lysine graft PEG (PLL-PEG) copolymers. Stable yet reversible protein immobilization was achieved via tris(nitrilotriacetic acid) groups incorporated into the PLL-PEG coating. Thus, site-specific immobilization of His-tagged proteins via complexed Ni(II) ions was achieved. Functional protein immobilization on the surface was confirmed by real-time detection of LSPR scattering by reflectance spectroscopy. Association and dissociation rate constants obtained for a reversible protein-protein interaction were in good agreement with the data obtained by other surface-sensitive detection techniques. For spatially resolved detection, AuNP were assembled into micropatterns by means of photolithographic uncaging of surface amines. LSPR imaging of reversible protein-protein interactions was possible in a conventional wide field microscope, yielding detection limits of ∼30 protein molecules within a diffraction-limited surface area.
authors.affiliation: Department of Biology, University of Osnabrück , Barbarastrasse 11, 49076 Osnabrück, Germany.
authors.forename: Maniraj
authors.initials: M
authors.lastname: Bhagawati
authors.forename: Changjiang
authors.initials: C
authors.lastname: You
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
mesh_terms: D000588:Amines / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry*; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D055672:Static Electricity; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D013499:Surface Properties; D013997:Time Factors
chemical_list: D000588:Amines; D055767:Immobilized Proteins; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D006639:Histidine; D006046:Gold