A reactive poly(ethylene glycol) layer to achieve specific surface plasmon resonance sensing with a high S/N ratio: The substantial role of a short underbrushed PEG layer in minimizing nonspecific adsorption

A reactive poly(ethylene glycol) (PEG)-brushed layer was constructed on a surface plasmon resonance (SPR) sensor chip using a heterobifunctional PEG possessing an acetal group at one end and a mercapto group at the other end (alpha-acetal-omega-mercapto-PEG). The density of the PEG brushed layer substantially increased with repetitive adsorption/rinse cycles of the PEG on the sensor chip, allowing dramatic reduction of nonspecific protein adsorption. Notably, formation of a short, filler layer of PEG (2 kDa) in the preconstructed longer PEG brushed layer (5 kDa) achieved almost complete prevention of nonspecific protein adsorption. The acetal group located at the distal end of the tethered PEG was converted to an aldehyde group by the acid treatment, followed by the installation of biocytin hydrazide through Schiff base formation. SPR sensing of streptavidin was done with a very high S/N ratio even in a proteinous medium using the biotinylated PEG (5 kDa) tethered chip with an inert filler layer of short PEG (2 kDa). Furthermore, the specific affinity of streptavidin for the biotinylated PEG was highly influenced by the length of the filler PEG and was significantly reduced when the length of the filler PEG was longer than that of the biotinylated PEG. This result clearly revealed the substantial importance of the steric factor on biospecific interaction at the distal end of tethered PEG on the sensor surface.