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.

A Layer-by-Layer Approach To Retain a Fluorescent Glucose Sensing Assay within the Cavity of a Hydrogel Membrane

“…41,42 In this way, PEGylated-TRITC-ConA was formed with an average of ∼5 mPEG chains grafted per ConA monomer. 42 …”

“…31,39,40 Thus, we recently reported the use of a new type of competing ligand, aminopyrene trisulfonate mannotetraose (APTS-MT), which presents a single high-affinity trimannose moiety that utilizes the full binding site of ConA and thus avoids aggregation. 41 Additionally, we demonstrated that tethering of poly(ethylene glycol) (PEG) to ConA (i.e., PEGylation) improved ConA’s thermal stability and resistance to electrostatic binding. 42 Finally, we demonstrated improved FRET efficiency leading to enhanced sensitivity to changes in glucose levels.…”

Long term response of a Concanavalin-A based fluorescence glucose sensing assay

“…Calcium chloride dihydrates (CaCl 2 ) was purchased from Mallinckrodt Chemical Inc (St. Louis, MO) and sodium chloride (NaCl) from J.T.Baker (Center Valley, PA). The competing ligand, amino-pyrene trisulfonate mannotetraose (APTS-MT) was synthesized with Dr. Vigh at Texas A&M University as previously described [9]. A 0.2 μm syringe filter from VWR (Radnor, PA) was used to filter all solutions before each experiment.…”

“…The FA assay was expected to maintain its response to glucose under thermal conditions as the addition of PEG chains should provide thermal stability of ConA. Also, the use of mannotetraos se should furth her prolong the assay's sensing capabilitie es as its been s shown to resul lt in a non-agg gregative assay [9]. …”

“…Mannotetraose, which has previously been shown to achieve high affinity with ConA by binding to its full binding site, was chosen as the competing ligand for this study [9]. Furthermore, by fluorescently tagging mannotetraose, a transduction mechanism can be generated via fluorescence anisotropy.…”