期刊
BIOSENSORS & BIOELECTRONICS
卷 63, 期 -, 页码 53-60出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2014.07.015
关键词
Glucose sensing; Competitive binding; Fluorescence anisotropy; Concanavalin A; Affinity; Biosensor
类别
资金
- National Institute of Diabetes, Digestive and Kidney Disease (NIDDK) of the National Institutes of Health [R01DK09510]
- Whitaker International Program in the form of a summer grant
Competitive binding assays based on the lectin Concanavalin A (ConA) have displayed significant potential to serve in continuous glucose monitoring applications. However, to date, this type of fluorescent, affinity-based assay has yet to show the stable, glucose predictive capabilities that are required for such an application. This instability has been associated with the extensive crosslinking between traditionally-used fluorescent ligands (presenting multiple low-affinity moieties) and ConA (presenting multiple binding sites) in free solution. The work herein introduces the design and synthesis of a new type of fluorescent ligand that can avoid this aggregation and allow the assay to be sensitive across the physiologically relevant glucose concentration range. This fluorescent ligand (APTS-MT) presents a single high-affinity trimannose moiety that is recognized by ConA's full binding site and a fluorophore that can effectively track the ligand's equilibrium binding via fluorescent anisotropy. This is confirmed by comparing its measured fluorescent lifetime to experimentally-determined rotational correlation lifetimes of the free and bound populations. Using an assay comprised of 200 nM APTS-MT and 1 mu M ConA, the fluorescence anisotropy capably tracks the concentration of monosaccharides that are known to bind to ConA's primary binding site, and the assay displays a MARD of 6.5% across physiologically relevant glucose concentrations. Ultimately, this rationally-designed fluorescent ligand can facilitate the realization of the full potential of ConA-based glucose sensing assays and provide the basis for a new set of competing ligands to be paired with ConA. (C) 2014 Published by Elsevier B.V.
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