Overcoming the aggregation problem : a new type of fluorescent ligand for ConA-based glucose sensing

Cummins, Brian M. and Li, Mingchien and Locke, Andrea K. and Birch, David J.S. and Vigh, Gyula and Cote, Gerard L. (2015) Overcoming the aggregation problem : a new type of fluorescent ligand for ConA-based glucose sensing. Biosensors and Bioelectronics, 63. pp. 53-60. ISSN 0956-5663

[img] PDF (Cummins-et-al-BAB-2015-overcoming-the-aggregation-problem)
Cummins_et_al_BAB_2015_overcoming_the_aggregation_problem.pdf
Accepted Author Manuscript
License: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 logo

Download (1MB)

    Abstract

    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 μM 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.