Reagentless optical sensing of glutamine using a dual-emitting glutamine-binding protein.
Glutamine is a major source of nitrogen and carbon in cell culture media. Thus, glutamine monitoring is important in bioprocess control. Here we report a reagentless fluorescence sensing for glutamine based on the Escherichia coli glutamine-binding protein (GlnBP) that is sensitive in the submicromolar ranges. The S179C variant of GlnBP was labeled at the -SH and N-terminal positions with acrylodan and ruthenium bis-(2,2'-bipyridyl)-1,10-phenanthroline-9-isothiocyanate, respectively. The acrylodan emission is quenched in the presence of glutamine while the ruthenium acts as a nonresponsive long-lived reference. The apparent binding constant, K'(d), of 0.72 microM was calculated from the ratio of emission intensities of acrylodan and ruthenium (I(515)/I(610)). The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of +/-0.02 microM glutamine. Dual-frequency ratiometric sensing was also demonstrated. Finally, the extraordinary sensitivity of GlnBP allows for dilution of the sample, thereby eliminating the effects of background fluorescence from the culture media.
Tolosa
Leah
L
Department of Chemical and Biochemical Engineering, University of Maryland, 1000 Hilltop Circle, Baltimore County, Baltimore, MD 21250, USA.
Ge
Xudong
X
Rao
Govind
G
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005973:Glutamine / Q000032:analysis* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D012428:Ruthenium / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D002614:Chelating Agents; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005973:Glutamine; D012428:Ruthenium; C038518:acrylodan; D015081:2-Naphthylamine