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 CaCO(3) 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 approximately 2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (approximately 0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (approximately 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.
Original language | English |
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Pages (from-to) | 3229 - 3234 |
Number of pages | 6 |
Journal | Biosensors and Bioelectronics |
Volume | 24 |
Issue number | 11 |
DOIs | |
Publication status | Published - 15 Jul 2009 |
Keywords
- Galactose
- Capsules
- Nanomedicine
- Enzyme Activation
- Particle Size
- Glucose
- Protein Binding
- Biosensing Techniques
- Microscopy, Fluorescence, Multiphoton
- Microscopy, Fluorescence
- Enzyme Stability
- Kinetics
- Materials Testing
- Biomedical Engineering
- Nanostructures