A long-wavelength fluorescent glucose biosensor based on bioconjugates of galactose/glucose binding protein and Nile Red derivatives.
BACKGROUND
Fluorescent biosensors based on galactose/glucose binding protein (GGBP) and environmentally sensitive derivatives of the phenoxazine dye Nile Red are described. These biosensors are proposed as the sensing platform for a minimally invasive, continuous glucose monitoring system that can be implanted under the skin and read transdermally using an external fluorometer.


METHODS
To construct the biosensors, the thiol-reactive Nile Red derivatives INR and IANR were prepared and conjugated to GGBP proteins possessing cysteine mutations that were designed for optimal site-specific fluorophore attachment. The attachment sites were selected to maximize the local environment change for attached dyes between the bound and unbound conformations of GGBP.


RESULTS
Fluorescence responses at the selected cysteine sites of GGBP upon binding to glucose showed that the conjugates typically yielded fluorescence emission around 640-650 nm with up to 50% changes in fluorescence intensity. Conjugate E149C/A213C/L238S INR GGBP also displayed glucose binding in the human physiological range (K (D) = 7.4 mM).


CONCLUSIONS
The phenoxazine derivatives fluoresced at longer wavelengths (>600 nm) approaching the near-infrared spectral window, where interference from scattering and tissue absorbance are minimal. Ultimately, we expect that monitoring systems based on GGBP and longwavelength dyes will be implanted for up to 6 months and can be used to transmit information through the skin to an external monitor.
Thomas
K Joseph
KJ
BD Technologies, Research Triangle Park, North Carolina 27709, USA.
Sherman
Douglas B
DB
Amiss
Terry J
TJ
Andaluz
Sandra A
SA
Pitner
J Bruce
JB
D015374:Biosensing Techniques; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005947:Glucose / Q000032:analysis* / Q000737:chemistry*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010078:Oxazines; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D010078:Oxazines; C509064:mglB protein, E coli; D005947:Glucose; C044808:nile red