Newswise — Researchers in an inter - departmental collaboration at Oxford University have been working in the area of existing fluorescent probe technology to make sensors capable of detecting a wide range of compounds including contaminants and environmental pollutants.
Fluorescence Resonance Energy Transfer (FRET) is a process by which energy that would normally be emitted as a photon from an excited fluorophore can be directly transferred to a second fluorophore to excite one of its electrons. This, on decay, then generates an even longer wavelength photon. The extent of FRET is critically dependent on the distance between the two fluorophores as well as their spectral overlap. Thus FRET is a powerful reporter of the separation of the two fluorophores.
There is an increasing requirement to detect a wide range of compounds with high sensitivity and specificity. Molecular reporters using FRET had previously been demonstrated for calcium using calmodulin and a calmodulin-binding peptide as linkers between two fluorescent proteins. Calmodulin undergoes a conformational change in the presence of calcium and also binds to the adjacent peptide sequence to bring two fluorophores closer together, thus increasing the amount of FRET and shifting the emission towards a longer wavelength.
The new probes consist of two peptides, that normally bind together, attached at opposite ends of two fluorophors separated by a flexible peptide linker. In this configuration, the fluorophores are held in close proximity, allowing FRET to take place. In the presence of a target molecule which preferentially binds to one of the peptide sites, the link is broken allowing the fluorophores to spring apart with a concomitant reduction in FRET.
The range of probes that can be generated is therefore only limited by the range of interacting binding sites that can be characterised. A large number of antibody combinations or substrate binding sites are already known and further sites are being discovered daily.
These probes could be used in the detection of metabolites, drugs, contaminants, environmental pollutants and many others where high specificity and low detection limits are necessary. Usage in industry and in research is envisaged.
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