title: Reagentless optical sensing of glutamine using a dual-emitting glutamine-binding protein.
abstract: 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.
authors.affiliation: Department of Chemical and Biochemical Engineering, University of Maryland, 1000 Hilltop Circle, Baltimore County, Baltimore, MD 21250, USA.
authors.forename: Leah
authors.initials: L
authors.lastname: Tolosa
authors.forename: Xudong
authors.initials: X
authors.lastname: Ge
authors.forename: Govind
authors.initials: G
authors.lastname: Rao
mesh_terms: 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
chemical_list: D002352:Carrier Proteins; D002614:Chelating Agents; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005973:Glutamine; D012428:Ruthenium; C038518:acrylodan; D015081:2-Naphthylamine