Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein.
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.
Metabolic Unit, King's College London School of Medicine, Guy's Hospital, London, UK. tania.saxl@kcl.ac.uk
Tania
T
Saxl
Faaizah
F
Khan
Daniel R
DR
Matthews
Zheng-Liang
ZL
Zhi
Olaf
O
Rolinski
Simon
S
Ameer-Beg
John
J
Pickup
D001698:Biomedical Engineering / Q000379:methods; D015374:Biosensing Techniques / Q000379:methods*; D002214:Capsules; D004789:Enzyme Activation; D004795:Enzyme Stability; D005690:Galactose / Q000032:analysis* / Q000737:chemistry*; D005947:Glucose / Q000032:analysis* / Q000737:chemistry*; D007700:Kinetics; D008422:Materials Testing; D008856:Microscopy, Fluorescence; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D050997:Nanomedicine / Q000379:methods; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D010316:Particle Size; D011485:Protein Binding
D002214:Capsules; D005947:Glucose; D005690:Galactose