FRET probes consisting

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

The selection of extrinsic fluorescent probe is driven by the consideration of which biological macromolecule or lipid is to be labelled, the requirement for compatibility between the intended fluorescent probe (in terms of solubility in water, pH sensitivity and so on) and the properties of the molecule to be labelled. Also, choice of the fluorescent probe should be consistent with experimental objectives. For instance, FRET experiments require that extrinsic donor and acceptor fluorophores should be properly matched for their capacity to participate in the FRET effect (see Section 4.5.4). 

Figure 7.. Schematic diagram of our FRET nano probes. It consists of several components, a) Melles-Griot green laser pointer (18 Lab 181), b) neutral density filter, c) plano-convex lens, d) optical fiber, e) sample holder, fi 532 nm cut-off filter, g) optical fiber, h) ocean optics QE65000 spectrometer. (Reprinted from Nanotechnology, Paresh Chandra Ray, Gopala K Darbha, Anandhi Ray, William Hardy and Joshua Walker, Nanotechnology, 2007, 18, 375504).

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