A fluorescence lifetime-based fibre-optic glucose sensor using glucose/galactose-binding protein

Alternative, non-electrochemistry-based technologies for continuous glucose monitoring are needed for eventual use in diabetes mellitus. As part of a programme investigating fluorescent glucose sensors, we have developed fibre-optic biosensors using glucose/galactose binding protein (GBP) labelled with the environmentally sensitive fluorophore, Badan. GBP-Badan was attached via an oligohistidine-tag to the surface of Ni-nitrilotriacetic acid (NTA)-functionalized agarose or polystyrene beads. Fluorescence lifetime increased in response to glucose, observed by fluorescence lifetime imaging microscopy of the GBP-Badan-beads. Either GBP-Badan agarose or polystyrene beads were loaded into a porous chamber at the end of a multimode optical fibre. Fluorescence lifetime responses were recorded using pulsed laser excitation, high speed photodiode detection and time-correlated single-photon counting. The maximal response was at 100 mM glucose with an apparent K-d of 13 mM (agarose) and 20 mM (polystyrene), and good working-day stability was demonstrated. We conclude that fluorescence lifetime fibre-optic glucose sensors based on GBP-Badan are suitable for development as clinical glucose monitors

A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration.

Both lung disease and elevation of blood glucose are associated with increased glucose concentration (from 0.4 to ~4.0 mM) in the airway surface liquid (ASL). This perturbation of ASL glucose makes the airway more susceptible to infection by respiratory pathogens. ASL is minute (~1 μl/cm(2)) and the measurement of glucose concentration in the small volume ASL is extremely difficult. Therefore, we sought to develop a fluorescent biosensor with sufficient sensitivity to determine glucose concentrations in ASL in situ. We coupled a range of environmentally sensitive fluorophores to mutated forms of a glucose/galactose-binding protein (GBP) including H152C and H152C/A213R and determined their equilibrium binding properties. Of these, GBP H152C/A213R-BADAN (Kd 0.86 ± 0.01 mM, Fmax/F0 3.6) was optimal for glucose sensing and in ASL increased fluorescence when basolateral glucose concentration was raised from 1 to 20 mM. Moreover, interpolation of the data showed that the glucose concentration in ASL was increased, with results similar to that using glucose oxidase analysis. The fluorescence of GBP H152C/A213R-BADAN in native ASL from human airway epithelial cultures in situ was significantly increased over time when basolateral glucose was increased from 5 to 20 mM. Overall our data indicate that this GBP is a useful tool to monitor glucose homoeostasis in the lung

Fluorescence intensity- and lifetime-based glucose sensing using glucose/galactose-binding protein

We review recent progress in our laboratories towards developing in vivo glucose sensors for diabetes that are based on fluorescence labelling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labelling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber optic-based glucose sensor using this technology is being tested