Quantitative Real-Time Imaging of Protein-Protein Interactions by LSPR Detection with Micropatterned Gold Nanoparticles
Autor(en): | Bhagawati, Maniraj You, Changjiang Piehler, Jacob |
Stichwörter: | AFFINITY; BINDING; Chemistry; Chemistry, Analytical; FUNCTIONAL IMMOBILIZATION; HISTIDINE-TAGGED PROTEINS; LABEL-FREE DETECTION; LAYERS; MASS-TRANSPORT; POLY(ETHYLENE GLYCOL); RECEPTOR INTERACTIONS; SURFACE-PLASMON RESONANCE | Erscheinungsdatum: | 2013 | Herausgeber: | AMER CHEMICAL SOC | Journal: | ANALYTICAL CHEMISTRY | Volumen: | 85 | Ausgabe: | 20 | Startseite: | 9564 | Seitenende: | 9571 | Zusammenfassung: | 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 similar to 30 protein molecules within a diffraction-limited surface area. |
ISSN: | 00032700 | DOI: | 10.1021/ac401673e |
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geprüft am 02.05.2024