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Proceedings Paper

Development of a glucose binding protein biosensor
Author(s): M. Dweik; M. Milanick; S. Grant
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Paper Abstract

Glucose binding protein (GBP) is a monomeric periplasmic protein. It is synthesized in the cytoplasm of Escherichia coli which functions as a receptor for transport D-glucose. GBP binds glucose with high affinity. The binding mechanism is based on a hinge motion due to the protein conformational change. This change was utilized as an optical sensing mechanism by applying Fluorescence Resonance Energy Transfer (FRET). The wild-type GBP lacks cysteine in its structure, but by introducing a single cysteine at a specific site by site-directed mutagenesis, this ensured single-label attachment at specific sites with a fluorescent probe. The other sites were amino sites, which were labeled with second fluorophore. The near IR FRET pair, Alexa Fluor 680 (AF680) and Alexa Fluor 750(AF750), was utilized. The AF680 targeted the amine sites, which was the donor fluorophore, while the AF750 labeled the single cysteine site, which was the acceptor fluorophore. The sensing system strategy was based on the fluorescence changes of the probe as the protein undergoes a structural change upon binding. This biosensor had the ability to detect down to 10 uM concentrations of glucose. Next the probes were uploaded into red blood cells via hypo osmotic dialysis. The sensor responded to glucose while encapsulated with the red cells. These results showed the feasibility of an intracellular glucose biosensor.

Paper Details

Date Published: 4 October 2007
PDF: 8 pages
Proc. SPIE 6759, Smart Biomedical and Physiological Sensor Technology V, 67590I (4 October 2007); doi: 10.1117/12.732399
Show Author Affiliations
M. Dweik, Univ. of Missouri/Columbia (United States)
M. Milanick, Univ. of Missouri/Columbia (United States)
S. Grant, Univ. of Missouri/Columbia (United States)


Published in SPIE Proceedings Vol. 6759:
Smart Biomedical and Physiological Sensor Technology V
Brian M. Cullum; D. Marshall Porterfield, Editor(s)

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