Department of Chemistry and Chemical Biology, ‡Chemical and Biological Engineering, §Biology, and ∥Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
Anal Chem. 2013 Oct 1;85(19):9356-62. doi: 10.1021/ac402242v. Epub 2013 Sep 11.
Rapid and highly sensitive detection of the carbohydrate components of glycoconjugates is critical for advancing glycobiology. Fluorescence (or Förster) resonance energy transfer (FRET) is commonly used in detection of DNA, in protein structural biology, and in protease assays but is less frequently applied to glycan analysis due to difficulties in inserting two fluorescent tags into small glycan structures. We report an ultrasensitive method for the detection and quantification of a chondroitin sulfate disaccharide based on FRET, involving a CdSe-ZnS core-shell nanocrystal quantum dot (QD) streptavidin conjugate donor and a Cy5 acceptor. The disaccharide was doubly labeled with biotin and Cy5. QDs then served to concentrate the target disaccharide, enhancing the overall energy transfer efficiency, with unlinked QDs and Cy5 hydrazide producing nearly zero background signal in capillary electrophoresis using laser-induced fluorescence detection with two different band-pass filters. This method is generally applicable to the ultrasensitive analysis of acidic glycans and offers promise for the high-throughput disaccharide analysis of glycosaminoglycans.
快速且高灵敏度地检测糖缀合物的碳水化合物组分对于推进糖生物学研究至关重要。荧光（或Förster）共振能量转移（FRET）广泛应用于 DNA 检测、蛋白质结构生物学和蛋白酶分析中，但由于难以将两个荧光标记物插入到小聚糖结构中，因此在糖分析中应用较少。我们报道了一种基于 FRET 的用于检测和定量软骨素硫酸盐二糖的超灵敏方法，涉及 CdSe-ZnS 核壳纳米晶体量子点（QD）链霉亲和素缀合物供体和 Cy5 受体。二糖通过生物素和 Cy5 进行双重标记。QD 然后用于浓缩目标二糖，提高整体能量转移效率，未连接的 QD 和 Cy5 酰肼在使用激光诱导荧光检测的毛细管电泳中产生几乎为零的背景信号，使用两种不同的带通滤波器。该方法通常适用于酸性聚糖的超灵敏分析，并为糖胺聚糖的高通量二糖分析提供了前景。
