Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein

Saxl, T. and Khan, F. and Matthews, D.R. and Zhi, Z.L. and Rolinski, O.J. and Ameer-Beg, S. and Pickup, J.C. (2009) Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein. Biosensors and Bioelectronics, 24 (11). pp. 3229-3234. ISSN 0956-5663

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Abstract

We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO3 as templates and encapsulated in alternating nano-layers of poly-l-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules. Addition of glucose increased the mean lifetime of GBP-badan by a maximum of 2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (3.1 ns) representing the closed form with bound glucose and where the lobes of GBP have closed round the dye creating a more hydrophobic environment. FLIM demonstrated that increasing glucose increased the fractional proportion of the long-lifetime component. We conclude that fluorescence lifetime-based glucose sensing using GBP encapsulated with nano-engineered layer-by-layer films is a glucose monitoring technology suitable for development in diabetes management.