Transduction of Glycan鈥揕ectin Binding Using Near-Infrared Fluorescent Single-Walled Carbon Nanotubes for Glycan Profiling

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• 作者：Nigel F. Reuel ; Jin-Ho Ahn ; Jong-Ho Kim ; Jingqing Zhang ; Ardemis A. Boghossian ; Lara K. Mahal ; Michael S. Strano
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：November 9, 2011
• 年：2011
• 卷：133
• 期：44
• 页码：17923-17933
• 全文大小：1235K
• 年卷期：v.133,no.44(November 9, 2011)
• ISSN：1520-5126

文摘

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 Ni²⁺ complexes that act as fluorescent quenchers for semiconducting single-walled carbon nanotubes (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鈥搇ectin 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 渭M, respectively) and streptavidin-tethered (142 and 50 渭M respectively) were found. The absolute detection limit for the current platform was found to be 2 渭g of glycosylated protein or 100 ng of free glycan to 20 渭g of lectin. Glycan detection (GlcNAc-streptavidin at 10 渭M) is demonstrated at the 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 卤 10 渭M). We are also able to identify the locations of 鈥渟trong transducers鈥?on the basis of dissociation constant (four sensors with K_D < 10 渭M) or overall signal modulation (eight sensors with >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鈥搇ectin transducers as a high throughput method to profile glycans without protein labeling or glycan liberation pretreatment steps.