Genetically designed biosensing systems for high-throughput screening of pharmaceuticals, clinical diagnostics, and environmental monitoring

The genetically-modified binding proteins calmodulin (CaM), the phosphate binding protein (PBP), the sulfate binding protein (SBP), and the galactose/glucose binding protein (GBP) have been successfully employed as biosensing elements for the detection of phenothiazines, phosphate, sulfate, and glucose, respectively. Mutant proteins containing unique cysteine residues were utilized in the site-specific labeling of environment-sensitive fluorescent probes. Changes in the environment of the probes upon ligand-induced conformational changes of the proteins result in changes in fluorescence intensity. Calibration plots for the respective analytes were generated that relate the concentration of analyte with a change in fluorescence intensity of the biosensing element. The assays were also characterized in terms of their selectivity and the stability of the binding protein. To illustrate the usefulness of the reagents in high-throughput analyses for application in drug discovery, point-of-care diagnostics, and environmental monitoring, the assays were evaluated on a novel system-the CD platform. This microfluidic compact disc-based platform utilizes centrifugal force to control the release, flow, and mixing of buffers, reagents, and analytes in channels and reservoirs contained on the microfabricated CD. Coupling of fluorescence detection on this system with the genetically designed reagents provides highly sensitive assays for microscale applications. Specifically, the labeled proteins were shown to be sensitive to increasing concentrations of analyte in mL, muL, nL, and pL volume samples, with limits of detection in the sub-micromolar range. in addition, soi-gel composites have been investigated as a means of entrapping the aforementioned biorecognition elements for the purpose of immobilizing the proteins on a sensing platform such as the CD. The advantages of the CD platform and its application in drug discovery and diagnostics will be discussed, along with preliminary experiments showing the response of PBP in sol-gel composites.