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

Brian M. Cummins, Mingchien Li, Andrea K. Locke, David J.S. Birch, Gyula Vigh, Gerard L. Cote

Research output: Contribution to journalArticle

10 Citations (Scopus)
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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.
Original languageEnglish
Pages (from-to)53-60
Number of pages8
JournalBiosensors and Bioelectronics
Volume63
Early online date11 Jul 2014
DOIs
Publication statusPublished - 15 Jan 2015

Keywords

  • affinity
  • glucose sensing
  • competitive binding
  • fluorescence anisotropy
  • concanavalin A

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