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.