title: Overcoming the aggregation problem: a new type of fluorescent ligand for ConA-based glucose sensing.
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.
authors: Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA; Department of Physics, University of Strathclyde, Glasgow G1 1XQ, UK. Electronic address: bmcummins@ncsu.edu.
authors: Brian M
authors: BM
authors: Cummins
authors: Department of Chemistry, Texas A&M University, College Station, TX 77843, USA.
authors: Mingchien
authors: M
authors: Li
authors: Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA.
authors: Andrea K
authors: AK
authors: Locke
authors: Department of Physics, University of Strathclyde, Glasgow G1 1XQ, UK.
authors: David J S
authors: DJS
authors: Birch
authors: Department of Chemistry, Texas A&M University, College Station, TX 77843, USA.
authors: Gyula
authors: G
authors: Vigh
authors: Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA.
authors: Gerard L
authors: GL
authors: Coté
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry; D005454:Fluorescence Polarization; D005947:Glucose / Q000737:chemistry / Q000302:isolation & purification*; D008024:Ligands
chemical_list: D008024:Ligands; D003208:Concanavalin A; D005947:Glucose
keywords: Affinity; Biosensor; Competitive binding; Concanavalin A; Fluorescence anisotropy; Glucose sensing