Internal response correction for fluorescent whole-cell biosensors.
Whole-cell biosensors based on reporter genes are finding a variety of applications in analytical chemistry. Despite their ability to selectively recognize the analyte in a complex mixture, few applications of such sensing devices to real sample analysis are reported. This is mainly due to nonspecific effects on the biosensor response caused by components of the sample matrix and by environmental changes. To overcome this problem, a bacterial biosensor with an internal correction mechanism of the analytical response was developed by introducing an additional reporter gene that provides a reference signal of the analytical performance of the biosensor. The first reporter (GFPuv), expressed in response to the concentration of L-arabinose, provides the analytical signal; the second reporter (EYFP), constitutively expressed if a constant amount of IPTG is added to each sample, was used as an internal reference. By inducing the biosensor with varying amounts of L-arabinose and a constant amount of IPTG, it was possible to obtain a dose-response curve for L-arabinose, together with a constant production of EYFP, which allowed for a dynamic evaluation of the metabolic activity of the cell. When tested in nonoptimal conditions (e.g., in the presence of either ethanol or deoxycholic acid at toxic concentrations), the presence of the internal reference system corrected the analytical response due to nonspecific interferences.
Mirasoli
Mara
M
Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy.
Feliciano
Jessika
J
Michelini
Elisa
E
Daunert
Sylvia
S
Roda
Aldo
A
D001089:Arabinose / Q000032:analysis; D001419:Bacteria / Q000166:cytology / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D007544:Isopropyl Thiogalactoside; D008660:Metabolism; D012015:Reference Standards; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D007544:Isopropyl Thiogalactoside; D001089:Arabinose