PEGylation of concanavalin A to improve its stability for an in vivo glucose sensing assay

Abstract

Competitive binding assays utilizing concanavalin A (ConA) have the potential to be the basis of improved continuous glucose monitoring devices. However, the efficacy and lifetime of these assays have been limited, in part, by ConA's instability due to its thermal denaturation in the physiological environment (37 °C, pH 7.4, 0.15 M NaCl) and its electrostatic interaction with charged molecules or surfaces. These undesirable interactions change the constitution of the assay and the kinetics of its behavior over time, resulting in an unstable glucose response. In this work, poly(ethylene glycol) (PEG) chains are covalently attached to lysine groups on the surface of ConA (i.e., PEGylation) in an attempt to improve its stability in these environments. Dynamic light scattering measurements indicate that PEGylation significantly improved ConA's thermal stability at 37 °C, remaining stable for at least 30 days. Furthermore, after PEGylation, ConA's binding affinity to the fluorescent competing ligand previously designed for the assay was not significantly affected and remained at ~5.4 × 10(6) M(-1) even after incubation at 37 °C for 30 days. Moreover, PEGylated ConA maintained the ability to track glucose concentrations when implemented within a competitive binding assay system. Finally, PEGylation showed a reduction in electrostatic-induced aggregation of ConA with poly(allylamine), a positively charged polymer, by shielding ConA's charges. These results indicate that PEGylated ConA can overcome the instability issues from thermal denaturation and nonspecific electrostatic binding while maintaining the required sugar-binding characteristics. Therefore, the PEGylation of ConA can overcome major hurdles for ConA-based glucose sensing assays to be used for long-term continuous monitoring applications in vivo.

Figure 1

Decrease in the fluorescence intensity of fluorescamine in the presence of PEGylated ConA compared to unmodified ConA, indicating the degree of PEGylation.

Figure 2

Z-average particle size of unmodified ConA compared to PEGylated ConA (top) in response to incubation at 37 °C, over the course of 30 days. In the bottom panel, PEGylated ConA is rescaled to show its average particle size. The error bars show the standard deviation for three different runs.

Figure 3

Average PDI of unmodified and PEGylated ConA at 37 °C for 30 days. The error bars show the standard deviation for three different runs.

Figure 4

Stability of the binding affinity of PEGylated ConA to the competing ligand. The error bars show the standard deviation for three different runs.

Figure 5

Glucose response of fluorescence anisotropy assay using PEGylated ConA (1 μM PEGylated ConA and 200 nM APTS-MT). Solid line: best fit to experimental data using the competitive binding equation. The error bars show the standard deviation for three different runs.

Figure 6

Scattering effect due electrostatic interaction between unmodified ConA (top) and PEGylated ConA (bottom) with PAH⁺. The error bars show the standard deviation for three different runs.

Figure 7

Aggregate formation (“white particulate”) during unmodified ConA/PAH⁺ electrostatic interaction (left) versus PEGylated ConA/PAH⁺ (right).