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

Abstract

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.

Ligand-Binding Proteins: Structure, Stability and Practical Application

“…Though the most preferable approaches for analyte registration are fluorescent methods, intrinsic protein fluorescence is not suitable as cells and living tissues are completely impervious to UV-light. Rational introduction of FRET-compatible protein pairs and environmentally sensitive dyes into different sites on the ligand-binding protein, as well as use of surface plasmon resonance methods, have been successful approaches to the design of biosensor systems (Ge et al, 2004;Khan et al, 2008Khan et al, , 2010de Lorimier et al, 2002;Okada et al, 2009;Thomas et al, 2006). Changing the affinity and specificity of ligand-binding proteins has been achieved by the re-engineering of the protein's ligand-binding site.…”

The construction of a glucose-sensing luciferase

“…As such, it has been expected to be a suitable sensing element for blood glucose monitoring. Fluorescent-based glucose-sensing systems have been developed using GGBP conjugated with an environmentally sensitive fluo- rophore or fluorophores, which change their fluorescent intensity with a GGBP conformational change (Tolosa et al, 1999;Salins et al, 2001;de Lorimier et al, 2002;Thomas et al, 2006;Der and Dattelbaum, 2008), as well as other PBP-based fluorescence sensors (de Lorimier et al, 2002;Chino et al, 2007;Sakaguchi et al, 2007). There have also been several reports about the design of FRET-based methods using a GGBP modified with two fluorophores (Ye and Schultz, 2003;Ge et al, 2004) or fused with two different fluorescent proteins, CFP and YFP (Fehr et al, 2003;Deuschle et al, 2005;de Lorimier et al, 2006;Khan et al, 2008), which allow the glucose sensing not only in vitro but also in mammalian cells, as well as the real-time monitoring.…”

Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

“…Environmentally sensitive or solvatochromic fluorophores have a fluorescence that is dependent on the solvent polarity-low in a polar/hydrophilic solvent or environment but high in a hydrophobic/water-excluded environment. 15,21,22,[26][27][28][29][30] In our research, we have used the solvatochromic fluorophore, badan (6-bromo-acetyl-2-dimethylaminonaphthalene; Figure 2), which we covalently attached to cysteine mutations near the GBP binding site (Figure 1). Native GBP does not contain any thiol groups, so that a site-directed mutagenesis can be used to substitute the thiol-containing amino acid cysteine at a chosen site, allowing covalent linkage of a thiolreactive fluorophore at only this position.…”