Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein.
The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.
Hsieh
Helen V
HV
Biosense, BD Technologies, 21 Davis Drive, PO Box 12016, Research Triangle Park, NC 27709, USA. helen_v_hsieh@bd.com
Pfeiffer
Zachary A
ZA
Amiss
Terry J
TJ
Sherman
Douglas B
DB
Pitner
J Bruce
JB
D019943:Amino Acid Substitution; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005947:Glucose / Q000032:analysis* / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods
D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D005947:Glucose