A Long-Wavelength Fluorescent Glucose Biosensor Based on Bioconjugates of Galactose/Glucose Binding Protein and Nile Red Derivatives

Abstract

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.