Fluorescent protein-based biosensors

Kostrzynska, M., Leung, K.T., Lee, H. and Trevors, J.T. (2002) Green fluorescent protein-based biosensor for detecting SOS-inducing activity of genotoxic compounds. Journal of Microbiological Methods, 48, 43-51. 

Scognamiglio V, Staiano M, Rossi M, D Auria S. Protein-based biosensors for diabetic patients. Journal of Fluorescence 2004, 14, 491-498. 

A green fluorescent protein-based Pseudomonas fluorescens strain biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene transcriptional activator (Stiner and Halverson, 2002). Another microbial whole-cell biosensor, using Escherichia coli with the promoter luciferase luxAB gene, was developed for the determination of water-dissolved linear alkanes by luminescence (Sticher et al., 1997). The biosensor has been used to detect the bioavailable concentration of alkanes in heating oil-contaminated ground-water samples. 

The first FRET-based biosensors employing fluorescent proteins were developed over 10 years ago. These protease sensors consisted of a BFP donor fused to a GFP acceptor by a protease-sensitive linker [44, 119]. BFP and GFP have well separated emission spectra, resulting in little fluorescence bleed-through (Figs. 5.5A and 5.6A). This facilitates data analysis for FRET ratio imaging... 

Doi, N., and Yanagawa, H. (1999). Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution. FEBS Lett., 453, 305-307. 

Non-excitable cells with no obvious electrical activity play the same important role as excitable cells in cell-based biosensors. The changes in electrical signals, such as cell impedance, and nonelectrical parameters such as cell morphology, proliferation, metabolism, cell viability, pH, and extracellular analyte concentrations, can be measured upon chemical exposure and physical stimuli. Cells can also be genetically engineered to express reporters or biomarkers, such as the green fluorescence protein (GFP), upon specific stimulation. 

Aptamers have a strong potential for multiplex sensing of proteins. Thus, a chip-based biosensor was developed for specific detection and quantification of cancer-associated proteins in complex biological mixtures using immobilized fluorescently labeled DNA and RNA aptamers. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding [51]. 

Patra, D. (2010). Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly Biosensors and Bioelectronics 25, 1149-1154. 

In this FRET-based biosensor (a) one of the FPs is linked to the MRE and the other is linked to the analyte protein. When the sensory protein domain binds with the substrate, the donor and acceptor FPs are brought together, thus increasing the acceptor fluorescence intensity while reducing the donor fluorescence intensity. This strategy is commonly used to tag protein—protein interactions in live cells. 

Bimolecular fluorescence complementation (BiFC)-based biosensors have been used to visualize a variety of protein—protein interactions in live cells. In this type of biosensor, the FP that is split up and MRE are linked to one portion and the analyte protein is linked to the other portion. When the two proteins interact, the two fragments fuse together, refolding properly into its three-dimensional structure and producing a fluorescence signal. 

A single fluorescent protein coupled with an MRE makes up single FP-based biosensors. The MRE can be either exogenous or endogenous. Analyte binding to the MRE causes conformational changes of the fluorescent protein consequently altering its fluorescent properties. 

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