|Type of Publication:||Article|
|Journal:||Journal of the American Chemical Society||Volume:||133|
|Month:||NOV 9 2011|
PT: J; TC: 8; UT: WOS:000296312200063
There is significant interest in developing new detection platforms for characterizing glycosylated proteins, despite the lack of easily synthesized model glycans or high affinity receptors for this analytical problem. In this work, we demonstrate a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni(2+) complexes that act as fluorescent quenchers for semiconducting carbon nonotubes (SWNTs) embedded,in a chitosan hydrogel spot to measure binding kinetics of model glycans. We examine, as model glycans, both free and streptavidin-tethered biotinylated monosaccharides. Two higher-affined glycan lectin pairs are explored : fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (K(D)) for these pairs, as free glycans (106 and 19 mu M, respectively) and streptavidin-tethered (142 and 50 mu M respectively) were found. The absolute detection limit for the current platform was found to be 2 fig of glycosylated protein or 100 ng of free glycan to 20 mu g of lectin. Glycan; detection (GlcNAc-streptavidin at 10 mu M) is demonstrated at single nanotube level as well by monitoring the fluorescence from individual SWNT sensors tethered to GafD lectin. Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (K(D) of 250 +/- 10 mu M). We are also able to identify the locations of "strong transducers" on the basis of dissociation constant (four sensors with K(D) 5% quench response). We report the key finding that the brightest SWNTs are not the best transducers of glycan binding. SWNTs ranging in intensity between 50 and 75% of the maximum show the greatest response. The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a high throughput Method to profile glycans without protein labeling or glycan liberation pretreatment steps.
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