Competitive binding assays comprised of the protein Concanavalin A (ConA) have shown potential for use in continuous glucose monitoring devices. However, its time-dependent, thermal instability can impact the lifetime of these ConA based assays. In an attempt to design sensors with longer in vivo lifetimes, different groups have immobilized the protein to various surfaces. For example, Ballerstadt et al. have shown that immobilizing ConA onto the interior of a micro-dialysis membrane and allowing dextran to be freely suspended within solution allowed for successful in vivo glucose sensing up to 16 days. This work explores the glucose response of an assay comprised of modified ConA and a single fluorescently labeled competing ligand in free solution to increase the in vivo sensing lifetime without immobilization,. The behavior of this assay in the presence of varying glucose concentrations is monitored via fluorescence anisotropy over a 30 day period.
Fluorescent glucose sensing technologies have been identified as possible alternatives to current continuous glucose monitoring approaches. We have recently introduced a new, smart fluorescent ligand to overcome the traditional problems of ConA-based glucose sensors. For this assay to be translated into a continuous glucose monitoring device where both components are free in solution, the molecular weight of the smart fluorescent ligand must be increased. We have identified ovalbumin as a naturally-occurring glycoprotein that could serve as the core-component of a 2nd generation smart fluorescent ligand. It has a single asparagine residue that is capable of displaying an N-linked glycan and a similar isoelectric point to ConA. Thus, binding between ConA and ovalbumin can potentially be monovalent and sugar specific. This work is the preliminary implementation of fluorescently-labeled ovalbumin in the ConA-based assay. We conjugate the red-emitting, long-lifetime azadioxatriangulenium (ADOTA+) dye to ovalbumin, as ADOTA have many advantageous properties to track the equilibrium binding of the assay. The ADOTA-labeled ovalbumin is paired with Alexa Fluor 647-labeled ConA to create a Förster Resonance Energy Transfer (FRET) assay that is glucose dependent. The assay responds across the physiologically relevant glucose range (0-500 mg/dL) with increasing intensity from the ADOTA-ovalbumin, showing that the strategy may allow for the translation of the smart fluorescent ligand concept into a continuous glucose monitoring device.
The ability of people with diabetes to both monitor and regulate blood sugar levels is limited by the conventional “finger-prick” test that provides intermittent, single point measurements. Toward the development of a continuous glucose monitoring (CGM) system, the lectin, Concanavalin A (ConA), has been utilized as a component in a Förster resonance energy transfer (FRET), competitive glucose binding assay. Recently, to avoid reversibility problems associated with ConA aggregation, a suitable competing ligand labeled with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) has been engineered. However, its ability to function as part of a glucose sensing assay is compromised due to the negative charge (at physiological pH) of native ConA that gives rise to non-specific binding with other ConA groups as well as with electrostatically charged assay-delivery carriers. To minimize these undesirable interactions, we have conjugated ConA with monomethoxy-poly(ethylene glycol) (mPEG) (i.e. “PEGylation”). In this preliminary research, fluorescently-labeled ConA was successfully PEGylated with mPEG-Nhydroxylsuccinimide( succinimidyl carbonate) (mPEG-NHS(SC)). The FRET response of APTS-labeled competing ligand (donor) conveyed an increase in the fluorescence intensity with increasing glucose concentrations.
Competitive binding assays based on the protein Concanavalin A (ConA) have been proposed as potential sensors for
continuous glucose monitoring applications. However, ConA-based assays in the literature have primarily displayed a
lack of sensitivity or a lack of repeatability in their glucose response. This work explores this apparent trade-off by
separating the measured glucose response into the recognition and fluorescence transduction mechanisms. The
recognition responses are modeled for typical competing ligands/assays used in the literature, and they are combined
with an optimized fluorescence approach to yield expected fluorescent glucose responses. Because aggregation is
known to increase the apparent affinity between multivalent ligands and multivalent receptors, preliminary models are
generated for assays that were initially optimized with multivalent ligands but increase in affinity over time. These
models accurately predict the low sensitivity for monovalent ligands and the lack of repeatability in the responses with
multivalent ligands as seen in the literature. This subsequently explains the aforementioned trade-off no matter the
A fluorescence polarization (FP) assay was developed to determine concentrations of glucose using concanavalin A
(ConA) and fluorescently-labeled dextran. Predictive FP responses to glucose were elicited for different assay
configurations using mathematical modeling and displayed herein. Using 4 kDa FITC-dextran, we predicted a change of
0.120 P units from 0 mg/dL glucose to 500 mg/dL. This shows the potential that a homogenous, reproducible FP assay
can be engineered to measure glucose concentrations using tetrameric ConA and 4k kDa FITC-dextran.
Our lab group is currently developing a fluorescent competitive binding assay between the Alexa fluor 647 labeled
lectin, Concanavalin A, and highly structured glycosylated dendrimers to be sensitive to varying levels of glucose.
Previously, this chemistry has elicited a high sensitivity to additions of physiological concentrations of glucose.
However, the exact mechanism behind the sensing has not yet been well understood. This work presents a conceptual
model of the response in which competitive binding results in different distributions of aggregates size to varying
amounts of glucose. Preliminary experiments were performed by using Numerical Tracking Analysis (NTA) which
correlates the movement of particles, positioned by light scattering, to the equivalent Brownian motion associated with
particles of a certain spherical diameter. Using this method, the sensing chemistry was exposed to two different glucose
concentrations and histograms of the size distribution for glucose concentrations were obtained. Herein the aggregation
profile, mean aggregate size, and the number of aggregates (aggregates per mL) for two glucose concentrations are
displayed, showing a correlation between the aggregation and glucose concentration.